Write Answers for the questions from the assigned Chapters. NOT from outside sources and NO plagiarism!!!!! The answers should be in detail!!!

Answer ONLY from the assigned chapters and NO outside sources!!!!!!!!!!

Chapter 10

1. What physiological/morphological changes occur to the spermatid during each phase of differentiation?

2. In detail, describe the cycle of seminiferous epithelium.

3. How is spermatozoal viability judged?

Chapter 11

1. What is the flehmen response? Why is it important for reproduction?

2. Understand the influence steroid exposure to a growing fetus in utero would have on postpubescent reproductive behavior.

3. Biochemically, how is an erection achieved?

 

Chapter 12

1. How does semen deposition within the female reproductive tract vary among species?

2. How is semen lost once inside the female tract?

3. What is the difference between the rapid and sustained transport phases?

4. What are the two types of cervical mucus? Which types aids in semen transport to the site of fertilization?

5. What is capacitation? Why is it important for fertility?

6. What is the cortical reaction?

7. Why is decondensation important for pronuclei formation?

8. What is superfecundation?

Chapter 13

1. What are the four steps that must be achieved before an embryo can successfully attach to the uterus?

2. Define the following terms: conceptus, fetus, fetus, syngamy, zygote, blastomere, blastocyst, and morula.

3. What are the four extraembryonic membranes and how are each formed?

4. What is Maternal Recognition of Pregnancy? Specifically, what does it do? How does this differ amongst species?

5. What is superovulation?

6. What is hCG? What is its mechanism of action?

Answer ONLY from the assigned chapters and NO outside sources!!!!!!!!!!

Chapter 14

1. Define chorionic villus

2. What are the four types of placenta classified according to chorionic villi distribution? Give examples of species where each would be found. Please make sure to review figures 14-1 to 14.3.

3. What are the three types of placenta classified according to number of placental layers that separate the fetal blood from the maternal blood? Give examples of species where each would be found.

4. What is the proposed purpose of the binucleate giant cells?

5. What is eCG? What does it do in the pregnant mare?

6. What is hCG? What does it do in the pregnant woman?

7. What hormone initiates the cascade of events resulting in parturition? (Hint: Figure 14-14)

Chapter 15

1. What are the four major puerperium events?

2. What kind of mammary growth occurs during birth and puberty?

3. What kind of mammary growth occurs during puberty and pregnancy?

4. What kind of mammary growth occurs during pregnancy?

5. How does an increase in suckling affect lactation? How does a decrease in suckling affect lactation?

Chapter 16

1. How is the first day of the menstrual cycle defined?

2. What major events occur during the phases of the ovarian cycle? Uterine cycle?

3. What are the endometrial changes that occur during the menstrual cycle?

4. How does estradiol and progesterone affect mood/emotional status throughout the menstrual cycle?

5. What are the three fundamental forms of birth control?

6. How does steroidal contraception work? What are the delivery methods?

7. What is IVF? What are the two methods? How do these methods differ? Who would be good candidates for each?

8. How is semen evaluated for fertility?

9. How is hyperstimulation of the ovary achieved for follicle collection?

10. How does reproductive aging in men and women compare and contrast?

The Puerperium & Lactation
Parturition
Fetal Attachment & Gestation
Early Embryogenesis &
Maternal Recognition of Pregnancy
Ovulation & Fertilization
Cyclicity
Regulation of
Reproduction
Tract Function
Puberty
Prenatal
Development
Spermatogenesis
Regulation of
Reproduction
Tract Function
Puberty
Prenatal
Development
Take Home Message
In the adult male, GnRH, LH and testosterone are secreted in pulses that occur eve1y
several hours. Follicle stimulating hormone is released in smaller pulses of longer duration.
Spermatozoa are produced by the testes by a process called spermatogenesis that requires 5
to 9 weeks, depending on the species. The number of sperm produced each day is indepen-
dent of the number ejaculated. Spermatogenesis is a process involving sequential mitotic
and meiotic divisions and concludes after differentiation of spherical spermatids into highly
specialized spermatozoa. Spermatozoa are released continually from the seminiferous epi-
thelium in post-pubertal males.
Endocrine Control/Regulation
is Different than in the Female
Before spemmtozoa can be produced, certain
endocrine requirements must be met. They are: I)
adequate secretion of GnRH from the hypothalamus;
2) FSH and LH secretion from the anterior lobe of the
pituitary and 3) secretion of gonadal steroids (testos-
terone and estradiol). Recall from Chapter 6 that the
hypothalamus in the male does not develop a surge
center. The discharge ofGnRH from the hypothalamus
in the male occurs in frequent, intermittent episodes that
occur throughout the day and night. These short-lived
bursts of GnRH last for only a few minutes and cause
discharges of LH that fo llow almost immediately after
the GnRH episode. The episodes of LH last from l 0
to 20 minutes and occur between 4 to 8 times every 24
hours. Concentrations ofFSH are lower, but the pulses
are oflonger duration than LH because of the relatively
constant secretion of inhibin by the adult testis and the
longer half-life of FSH (See Figure 10-1 ).
Luteinizing hormone acts on the Leydig cells
within the testes. These cells, named after the Gem1an
anatomist Franz von Leydig, are analogous to the cells
of the theca intema of antral follicles in the ovmy . They
contain membrane-bound receptors for LH. When LH
binds to their receptors, Leydig cells synthesize pro-
gesterone, most of which is converted to testosterone.
Blood LH is elevated for about 30 to 75 minutes. The
Leydig cells synthesize and secrete testosterone less
than 30 minutes after the onset of an LH episode. The
response (testosterone secretion) by Leydig cells is
short and secretion is pulsatile, lasting for a period of
20 to about 60 minutes (See Figure I 0-2).
Successful testis function requires:
• pulsatile GnRH secretion (every 3-6/trs)
• high concentrations of testosterone in
the seminiferous tubule
•low concentrations oftestosterone in a
systemic blood
• adequate LH receptors in Leydig cells
Pulsatile discharge of LH is important for
nomml testicular function. The pulsati le nature ofLH
secretion prevents sustained concentrations of LH to
which the Leydig cells become refractory (unrespon-
sive or not yielding to treatment). A refractory condi-
tion is thought to be caused by reduction in the number
of LH receptors in the Leydig cells. As a result oflow
receptor numbers, reduced secretion of testosterone by
Leydig cells follows. Pulsatile LH secretions optimize
LH receptor numbers and testosterone secetion by
Leydig cells.
Normally, intratesticular concentrations of
testosterone are 100-500 times higher than that of
systemic blood. High concentrations of testosterone
are required for normal spermatogenesis. When tes-
tosterone from the testis mixes with peripheral blood,
Production of normal numbers of fertile spermatozoa requires:
• endocrine regulation of the testis
• mitotic divisions of spermatogonia
• meiotic divisions resulting in haploid spermatids
• morphologic transformation of spermatids into spermatozoa
Ve
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.ir

The Puerperium & Lactation
Parturition
Fetal Attachment & Gestation
Early Embryogenesis &
Maternal Recognition of Pregnancy
Ovulation & Fertilization
Cyclicity
Regulation of
Reproduction
Tract Function
Puberty
Prenatal
Development
Spermatogenesis
Regulation of
Reproduction
Tract Function
Puberty
Prenatal
Development
Take Home Message
In the adult male, GnRH, LH and testosterone are secreted in pulses that occur eve1y
several hours. Follicle stimulating hormone is released in smaller pulses of longer duration.
Spermatozoa are produced by the testes by a process called spermatogenesis that requires 5
to 9 weeks, depending on the species. The number of sperm produced each day is indepen-
dent of the number ejaculated. Spermatogenesis is a process involving sequential mitotic
and meiotic divisions and concludes after differentiation of spherical spermatids into highly
specialized spermatozoa. Spermatozoa are released continually from the seminiferous epi-
thelium in post-pubertal males.
Endocrine Control/Regulation
is Different than in the Female
Before spemmtozoa can be produced, certain
endocrine requirements must be met. They are: I)
adequate secretion of GnRH from the hypothalamus;
2) FSH and LH secretion from the anterior lobe of the
pituitary and 3) secretion of gonadal steroids (testos-
terone and estradiol). Recall from Chapter 6 that the
hypothalamus in the male does not develop a surge
center. The discharge ofGnRH from the hypothalamus
in the male occurs in frequent, intermittent episodes that
occur throughout the day and night. These short-lived
bursts of GnRH last for only a few minutes and cause
discharges of LH that fo llow almost immediately after
the GnRH episode. The episodes of LH last from l 0
to 20 minutes and occur between 4 to 8 times every 24
hours. Concentrations ofFSH are lower, but the pulses
are oflonger duration than LH because of the relatively
constant secretion of inhibin by the adult testis and the
longer half-life of FSH (See Figure 10-1 ).
Luteinizing hormone acts on the Leydig cells
within the testes. These cells, named after the Gem1an
anatomist Franz von Leydig, are analogous to the cells
of the theca intema of antral follicles in the ovmy . They
contain membrane-bound receptors for LH. When LH
binds to their receptors, Leydig cells synthesize pro-
gesterone, most of which is converted to testosterone.
Blood LH is elevated for about 30 to 75 minutes. The
Leydig cells synthesize and secrete testosterone less
than 30 minutes after the onset of an LH episode. The
response (testosterone secretion) by Leydig cells is
short and secretion is pulsatile, lasting for a period of
20 to about 60 minutes (See Figure I 0-2).
Successful testis function requires:
• pulsatile GnRH secretion (every 3-6/trs)
• high concentrations of testosterone in
the seminiferous tubule
•low concentrations oftestosterone in a
systemic blood
• adequate LH receptors in Leydig cells
Pulsatile discharge of LH is important for
nomml testicular function. The pulsati le nature ofLH
secretion prevents sustained concentrations of LH to
which the Leydig cells become refractory (unrespon-
sive or not yielding to treatment). A refractory condi-
tion is thought to be caused by reduction in the number
of LH receptors in the Leydig cells. As a result oflow
receptor numbers, reduced secretion of testosterone by
Leydig cells follows. Pulsatile LH secretions optimize
LH receptor numbers and testosterone secetion by
Leydig cells.
Normally, intratesticular concentrations of
testosterone are 100-500 times higher than that of
systemic blood. High concentrations of testosterone
are required for normal spermatogenesis. When tes-
tosterone from the testis mixes with peripheral blood,
Production of normal numbers of fertile spermatozoa requires:
• endocrine regulation of the testis
• mitotic divisions of spermatogonia
• meiotic divisions resulting in haploid spermatids
• morphologic transformation of spermatids into spermatozoa
Ve
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10
204 Spermatogenesis
Figure 10-1. Relationship Between GnRH, LH and FSH in the Male
Cl)
1:
0
E 111 LH :1.. 1: oo .s:::·-…
-ot..s
0 :1..
ot:
-CI) .Ou
Cl) 1:
> 0 ·.p u
t..s
Cl)
LH LH
/\ G n RH
LH
GnRH causes the release of LH and
FSH. Episodes of all three hormones
occur between 4 and 8 times in 24
hours. The lower FSH profile, when
compared to LH , is due to inhibin
secretion by Sertoli cells. Also, the
greater duration of the FSH episode
is probably due to its longer half-life
(1 00 min) when compared to LH
0 3 6 9 12 IS (30 min).
Time (hours)
it is diluted over 500 times. This is important because
it keeps systemic concentrations well below that which
would cause down-regulation of the GnRH/LH feed-
back system. For example, if LH pulses were long
(hours), Leydig cells would secrete testosterone for
hours rather than minutes. This would likely result in
a metabolic overload for testosterone clearance and tes-
tosterone would exert a sustained negative feedback on
the GnRH neurons in the hypothalamus. The net effect
would be significantly reduced LH secretion, followed
by severely reduced testosterone secretion.
The role of the pulsatile nature of testosterone
is not f·ully understood. It is thought that a chronically
high systemic concentration of testosterone removes
the negative feedback on FSH. Sertoli cell function is
FSH dependent. Thus, their function is compromised
when FSH is reduced. The periodic reduction in tes-
tosterone removes the negative feedback on FSI-1 (See
Figure 10-3 ).
In addition to secretion of testosterone by the
Leydig cells, the testes also secrete estradiol and other
estrogens. The stallion and the boar secrete large
amounts of estrogens (both free and in conjugated
form). In fact, urinary estrogens in the male are signifi-
cantly higher than urinary estrogens in pregnant mares
and sows. These high concentrations of estradiol seem
to be of little consequence, s ince they are secreted as
molecules with low physiologic activity.
Ley dig cells are the male equivalent
ofthefol/icular theca interna cells.
Sertoli cells are the male equivalent
of the follicular granulosa! cells.
Figure 10-2. Typical Peripheral Concentrations of Blood LH
and Testosterone (T) in the Male
6
-E 4 -DO r:::
J: 2 …..
0
T
T
3 6 9
Time (hours)
T
12
IS
Q) LH is elevated for a period r::: of 0.5 to 1.25 hours, while
10 o-s..- the subsequent testoster-Q) E ._._ one (T) episode lasts for 1111:).0
0 c: 0.5 to 1.5 hours . … _
5 Ill Q)
1-
Spermatogenesis 205
Figure 10-3. Interrelationships Among Hormones Produced by Sertoli Cells,
Leydig Cells, the Hypothalamus and the Anterior Lobe of Pituitary
soox
dilution
by systemic
circulat ion
\
LH
The Sertoli cells secrete
inh ib in that exerts a
negative feedback on
the anterior lobe of the
pituitary to directly sup-
press FSH secretion.
( Blue spheres = spe r matogonia ; Re d sphe res = primary spermatocytes;
Brown spheres = secondary spermatocytes; Black spheres = spermatids
Testosterone (T) secreted by the Leydig cells is trans- LH binds to receptors in the interstitial
ported into the Sertoli cells where it is converted to cells of Leydig and FSH binds to Sertoli
dihydrotestosterone (DHT) and also estradiol (E2 ). cells. Leydig cells secrete testosterone
Testosterone and E2 are transported by the blood to that is transported to the adjacent vas-
the hypothalamus where they exert a negative feed- culature and the Sertoli cells where Tis
back on the GnRH neurons. converted to DHT.
Ve
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10
204 Spermatogenesis
Figure 10-1. Relationship Between GnRH, LH and FSH in the Male
Cl)
1:
0
E 111 LH :1.. 1: oo .s:::·-…
-ot..s
0 :1..
ot:
-CI) .Ou
Cl) 1:
> 0 ·.p u
t..s
Cl)
LH LH
/\ G n RH
LH
GnRH causes the release of LH and
FSH. Episodes of all three hormones
occur between 4 and 8 times in 24
hours. The lower FSH profile, when
compared to LH , is due to inhibin
secretion by Sertoli cells. Also, the
greater duration of the FSH episode
is probably due to its longer half-life
(1 00 min) when compared to LH
0 3 6 9 12 IS (30 min).
Time (hours)
it is diluted over 500 times. This is important because
it keeps systemic concentrations well below that which
would cause down-regulation of the GnRH/LH feed-
back system. For example, if LH pulses were long
(hours), Leydig cells would secrete testosterone for
hours rather than minutes. This would likely result in
a metabolic overload for testosterone clearance and tes-
tosterone would exert a sustained negative feedback on
the GnRH neurons in the hypothalamus. The net effect
would be significantly reduced LH secretion, followed
by severely reduced testosterone secretion.
The role of the pulsatile nature of testosterone
is not f·ully understood. It is thought that a chronically
high systemic concentration of testosterone removes
the negative feedback on FSH. Sertoli cell function is
FSH dependent. Thus, their function is compromised
when FSH is reduced. The periodic reduction in tes-
tosterone removes the negative feedback on FSI-1 (See
Figure 10-3 ).
In addition to secretion of testosterone by the
Leydig cells, the testes also secrete estradiol and other
estrogens. The stallion and the boar secrete large
amounts of estrogens (both free and in conjugated
form). In fact, urinary estrogens in the male are signifi-
cantly higher than urinary estrogens in pregnant mares
and sows. These high concentrations of estradiol seem
to be of little consequence, s ince they are secreted as
molecules with low physiologic activity.
Ley dig cells are the male equivalent
ofthefol/icular theca interna cells.
Sertoli cells are the male equivalent
of the follicular granulosa! cells.
Figure 10-2. Typical Peripheral Concentrations of Blood LH
and Testosterone (T) in the Male
6
-E 4 -DO r:::
J: 2 …..
0
T
T
3 6 9
Time (hours)
T
12
IS
Q) LH is elevated for a period r::: of 0.5 to 1.25 hours, while
10 o-s..- the subsequent testoster-Q) E ._._ one (T) episode lasts for 1111:).0
0 c: 0.5 to 1.5 hours . … _
5 Ill Q)
1-
Spermatogenesis 205
Figure 10-3. Interrelationships Among Hormones Produced by Sertoli Cells,
Leydig Cells, the Hypothalamus and the Anterior Lobe of Pituitary
soox
dilution
by systemic
circulat ion
\
LH
The Sertoli cells secrete
inh ib in that exerts a
negative feedback on
the anterior lobe of the
pituitary to directly sup-
press FSH secretion.
( Blue spheres = spe r matogonia ; Re d sphe res = primary spermatocytes;
Brown spheres = secondary spermatocytes; Black spheres = spermatids
Testosterone (T) secreted by the Leydig cells is trans- LH binds to receptors in the interstitial
ported into the Sertoli cells where it is converted to cells of Leydig and FSH binds to Sertoli
dihydrotestosterone (DHT) and also estradiol (E2 ). cells. Leydig cells secrete testosterone
Testosterone and E2 are transported by the blood to that is transported to the adjacent vas-
the hypothalamus where they exert a negative feed- culature and the Sertoli cells where Tis
back on the GnRH neurons. converted to DHT.
Ve
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206 Spermatogenesis
Sertoli cells convert testosterone to estradiol
utilizing a mechanism identical to the granulosa) cells
ofthe antral follicle in the female. The exact role of
estradiol in male reproduction is poorly understood,
but there is little doubt that this hormone has a
negative feedback role on the hypothalamus.
Testosterone and estradiol in the blood act on the
hypothalamus and exert a negative feedback on the
secretion of GnRH and, in tum, LH and FSH are
reduced. Therefore, high concentrations of estradiol
result in suppression of GnRH and LH discharges
(See Figure 1 0-3). In addition to
terone to estradiol, Sertoli cells also secrete mh1bm
that, as in the female, suppresses FSH secretion from
the anterior lobe of the pituitary. The importance
of inhibin and suppressed FSH release is not clear
in the male.
The goals of spermatogenesis are to:
• provide a continual supply of
male gametes (up to decades)
through stem cell renewal
• provide genetic diversity
• provide billions of sperm each
day (domestic animals) to maxi-
mize reproduction by both
natural service and artificial
insemination
• provide an immunologically
privileged site where developing
germ cells are not destroyed by
the immune system
Figure 10-4. Scanning Electron Micrograph of Testicular Parenchyma
in the Stallion
(Courtesy of Dr. Larry Texas The A:nerican Society
for Reproductive Med1cme. Fertil. and Stenl., 1978. 29.208-215)
Seminiferous tubules (ST) containing developing germ cells (GC) are surrounded by a basement
Flagella (F) from developing spermatids can be observed protruding into of some tubules. The rntersttt1al
compartment contains Leydig cells (LC), blood vessels (BV) and connective t1ssue (CT).
Spermatogenesis 207
Spermatogenesis =proliferation + meiosis + differentiation
Spermatogenesis is the Process of
Producing Spermatozoa
Spermatogenesis takes place entirely within
the seminiferous tubules (See Figure 1 0-4) and consists
of all cell divisions and morphologic changes that oc-
cur to developing gem1 cells. (See Figures I 0-5 and
3-16).
The process of spermatogenesis can be subdi-
vided into three phases. The first phase, designated the
proliferation phase, consists of all mitotic divisions of
spermatogonia. Several generations of A-spermatogo-
nia undergo mitotic divisions, generating a large number
of B-spermatogonia (See Figure I 0-5). An important
part of the proliferation phase is stem cell renewal.
Loss of intercellular bridges allows some spennatogo-
nia to revert to stem cells (spetmatogonial stem cells)
providing continual renewal of these stem cells from
which new spermatogonia can develop.
The meiotic phase begins with primary
spetmatocytes. During meiosis I, genetic diversity is
guaranteed by DNA replication and crossing over dur-
ing the production of secondary spermatocytes. From a
genetic perspective no two sperm are identical. Conclu-
sion of the meiotic phase (the second meiotic division)
produces haploid (lN) spermatids.
The third or final phase of spermatogenesis is
the differentiation phase. No further cell divisions
take place during this phase. The differentiation phase
has commonly been referred to as “spermiogenesis” in
reproductive physiology li terature. During the differ-
entiation phase, a spherical undifferentiated spermatid
Figure 10-5. Typical Sequence of Spermatogenesis in Mammals
Spermatogonia (A1-At, I and B) undergo a series of mitotic divisions (Mit) and the last mitotic division gives
rise to primary spermatocytes that enter meiosis. This series of mitotic divisions allows for continual prolif-
eration of spermatogonia and replacement of A1 spermatogonia.
I Proliferation I
Basement membrane
Spermatogonia (A2) D
Spermatogonia (A3) IJ Number of divisions depends on
species
Spermatogonia ( I ) IJI
<( Spermatogonia (B) m I Differentiation I Lumen After meiosis, haploid spherical spermatids differentiate into spermatozoa. Meiosis and differentiation take place in the adluminal compartment. Notice that each generation of cells is attached by intercellular cyto- plasmic bridges. Thus, each generation divides synchronously in cohorts. Some cells (black) degenerate during the process. Numbers indicate the theoretical number of cells generated by each division. Ve tB oo ks .ir 206 Spermatogenesis Sertoli cells convert testosterone to estradiol utilizing a mechanism identical to the granulosa) cells ofthe antral follicle in the female. The exact role of estradiol in male reproduction is poorly understood, but there is little doubt that this hormone has a negative feedback role on the hypothalamus. Testosterone and estradiol in the blood act on the hypothalamus and exert a negative feedback on the secretion of GnRH and, in tum, LH and FSH are reduced. Therefore, high concentrations of estradiol result in suppression of GnRH and LH discharges (See Figure 1 0-3). In addition to terone to estradiol, Sertoli cells also secrete mh1bm that, as in the female, suppresses FSH secretion from the anterior lobe of the pituitary. The importance of inhibin and suppressed FSH release is not clear in the male. The goals of spermatogenesis are to: • provide a continual supply of male gametes (up to decades) through stem cell renewal • provide genetic diversity • provide billions of sperm each day (domestic animals) to maxi- mize reproduction by both natural service and artificial insemination • provide an immunologically privileged site where developing germ cells are not destroyed by the immune system Figure 10-4. Scanning Electron Micrograph of Testicular Parenchyma in the Stallion (Courtesy of Dr. Larry Texas The A:nerican Society for Reproductive Med1cme. Fertil. and Stenl., 1978. 29.208-215) Seminiferous tubules (ST) containing developing germ cells (GC) are surrounded by a basement Flagella (F) from developing spermatids can be observed protruding into of some tubules. The rntersttt1al compartment contains Leydig cells (LC), blood vessels (BV) and connective t1ssue (CT). Spermatogenesis 207 Spermatogenesis =proliferation + meiosis + differentiation Spermatogenesis is the Process of Producing Spermatozoa Spermatogenesis takes place entirely within the seminiferous tubules (See Figure 1 0-4) and consists of all cell divisions and morphologic changes that oc- cur to developing gem1 cells. (See Figures I 0-5 and 3-16). The process of spermatogenesis can be subdi- vided into three phases. The first phase, designated the proliferation phase, consists of all mitotic divisions of spermatogonia. Several generations of A-spermatogo- nia undergo mitotic divisions, generating a large number of B-spermatogonia (See Figure I 0-5). An important part of the proliferation phase is stem cell renewal. Loss of intercellular bridges allows some spennatogo- nia to revert to stem cells (spetmatogonial stem cells) providing continual renewal of these stem cells from which new spermatogonia can develop. The meiotic phase begins with primary spetmatocytes. During meiosis I, genetic diversity is guaranteed by DNA replication and crossing over dur- ing the production of secondary spermatocytes. From a genetic perspective no two sperm are identical. Conclu- sion of the meiotic phase (the second meiotic division) produces haploid (lN) spermatids. The third or final phase of spermatogenesis is the differentiation phase. No further cell divisions take place during this phase. The differentiation phase has commonly been referred to as "spermiogenesis" in reproductive physiology li terature. During the differ- entiation phase, a spherical undifferentiated spermatid Figure 10-5. Typical Sequence of Spermatogenesis in Mammals Spermatogonia (A1-At, I and B) undergo a series of mitotic divisions (Mit) and the last mitotic division gives rise to primary spermatocytes that enter meiosis. This series of mitotic divisions allows for continual prolif- eration of spermatogonia and replacement of A1 spermatogonia. I Proliferation I Basement membrane Spermatogonia (A2) D Spermatogonia (A3) IJ Number of divisions depends on species Spermatogonia ( I ) IJI <( Spermatogonia (B) m I Differentiation I Lumen After meiosis, haploid spherical spermatids differentiate into spermatozoa. Meiosis and differentiation take place in the adluminal compartment. Notice that each generation of cells is attached by intercellular cyto- plasmic bridges. Thus, each generation divides synchronously in cohorts. Some cells (black) degenerate during the process. Numbers indicate the theoretical number of cells generated by each division. Ve tB oo ks .ir 10 208 Spermatogenesis undergoes a remarkable transformation that results in the production of a fully differentiated, highly special- ized spermatozoon containing a head (nuclear material), a flagellum including a midpiece (with a mitochondrial helix) and a principal piece. The most immature genn cells (spem1atogonia) are located at the periphe1y of a seminiferous tubule near the basement membrane. As these germ cells prolifer- ate, they move toward the lumen. The cell types in the seminiferous epithelium are illustrated in Figure 10-5. Developing germ cells are connected by intercellular bridges. Groups of spem1atogonia, spermatocytes or spenn atids are connected by intercellular bridges, so that the cytoplasm of an entire cohort (groups of cells of the same type) is interconnected. The exact number of genn cells that are interconnected is not !mown, but might approach 50. The significance of these intercel- lular bridges is not fully understood. However, they undoubtedly provide communication between cells that contributes to synchronized development of a cohort. Proliferation Generates Spermatogonia That are Committed to Become More Advanced Cell Types The most primitive cells encountered in the seminiferous epithelium are the spemmtogonia. These specialized diploid (2N chromosomal content) cells are located in the basal compartment of the semi- niferous epithelium. Spermatogonia undergo several mitotic divisions with the last division resulting in primary spermatocytes (See Figure I 0-5). There are three types of spermatogonia: A-spermatogonia, !- spermatogonia (intem1ediate) and B-spermatogonia. A-spemmtogonia undergo several mitotic divisions in which they progress mitotically from A, through A4• A pool of stem cells is also maintained so that the process can continue indefinitely. Stem cells divide mitotically to provide a continual source of A-spermatogonia al- lowing spermatogenesis to continue without interrup- tion for years. The mechanism for the renewal of stem cells is not understood. Meiotic Divisions Produce Haploid Spermatids During spermatogenesis the number of chro- mosomes in the gamete is reduced to the haploid state. This is accomplished by meiosis. The mitotic divisions of 8-spennatogonia result in the fonnation of primmy spermatocytes . These primary spermatocytes imme- diately enter the first meiotic prophase. As you will recall fi·om your previous courses, the meiotic prophase consists of five stages: preleptotene, leptotene, zygo- tene, pachytene and diplotene. Each of these stages represents a different step in the progression of DNA synthesis and replication. Primary spermatocytes must progress through these five steps before the first meiotic division can occur. The important event of the prelepto- tene phase is complete DNA replication forming tetrads without separation. These tetrads then fuse at random points known as chiasmata and crossing-over ofDNA material later takes place. The term "crossing-over" refers to segments of one chromosome crossing-over to a homologous chromosome when the chromatids separate. Crossing-over results in a random assort- ment of different segments of each clu·omosome. Thus, prophase of the first meiotic division insures genetic heterogeneity and that each secondary spem1atocyte and subsequently each spermatid will be genetically unique. Prophase of the first meiotic division is a relatively long process. In fact, the lifespan of the pri- maiy spermatocyte is the longest of all germ cell types found in the seminiferous epithelium. For example, in the bull the lifespan of the primmy spermatocyte is 18 to 19 days. The total duration of spennatogenesis in bulls is 61 days. Thus, prophase of the first meiotic division (primary spem1atocyte) is about 30% of the time required for the entire spermatogenic process. The secondary spermatocyte resulting from the first meiotic division of a primary spermatocyte is short-lived. It exists for only l.l to 1.7 days depending on the species. The secondary spennatocyte rapidly undergoes the second meiotic division, resulting in haploid spherical spennatids. Differentiation Produces a Highly Sophisticated, Self-Propelled Package of Enzymes and DNA The role of a spennatozoon is to deliver the male 's genetic material to an oocyte during fertilization. To form cells that are capable of fertilization, spherical spermatids undergo a series of changes in which the nucleus becomes highly condensed, the acrosome is formed and the cell becomes potentially motile. The abi lity to swim (motility) requires the development of a flagellum and a metabolic " powerplant" known as the mitochondrial helix. Differentiation consists ofthe: • Golgi phase • cap phase • acrosomal phase • maturation phase Spermatogenesis 209 Figure 10-6. The Golgi Phase of Spermatid Differentiation 0 0 e \ The newly formed sper- Small vesicles of the Golgi Vesicle fusion continues until matid is almost perfectly fuse, giving rise to larger a large acrosomic vesicle is spherical and has a well secretory granules called formed containing a dense developed Golgi appa- pro-acrosomic granules. acrosomic granule. The proxi- ratus. The centrioles start to mi- mal centriole (PC) gives rise grate to a position beneath to the attachment point of the the nucleus that is opposite tail . The distal centriole (DC) the acrosomic vesicle. gives rise to the developing The Golgi phase = acrosomic vesicle formation The Golgi phase is characterized by the first steps in the development of the acrosome. The newly fom1ed spermatid contains a large, highly-developed Golgi apparatus located near the nucleus that consists of many small vesicles (See Figure 1 0-6). The Golgi apparatus is not unique to the spermatid, but is the intracellular "packaging" system in a ll secretory cells. In a spermatid, the Golgi will give rise to an important subcellular organelle known as the acrosome. First, proacrosomic vesicles are formed and these fuse, gen- erating a larger vesicle that resides on one side of the nucleus. This vesicle is called the acrosomic vesicle and contains a dense acrosomic granule (See Figure I 0-6). Smaller Golgi vesicles are continually added to the larger vesicle increasing its size. While the acrosomic vesicle is being fom1ed, the centrioles migrate from the cytoplasm to the base of the nucleus (See Figure 1 0-6). The proximal centriole will give ri se to an implantation apparatus that allows the flagellum to be anchored to the nucleus (See Figure I 0-9). The distal centriole gives rise to the develop- ing axoneme. The axoneme is the central portion of a flagellum, in this case the sperm tail. axoneme (central portion of the tail) inside the cytoplasm of the spermatid. The cap phase = acrosomic vesicle spreading over the nucleus During the cap phase the acrosome fonns a distinct, easily recognized cap over the anterior portion of the nucleus (See Figure I 0-7). The Golgi now has performed its function by packaging the acrosomal con- tents and membranes and moves away from the nucleus toward the caudal end of the spem1atid and eventually disappears. The primitive flagellum (tail), fom1ed fi·om the distal centriole, begins to project from the spem1atid toward the lumen ofthe seminiferous tubule. The acrosomal phase =nuclear and cytoplasmic elongation During the acrosomal phase the acrosome continues to spread until it covers about two-thirds of the anterior nucleus (See Figures 10-7 and I 0-8). The nucleus begins to elongate. A unique system of mi- crotubules known as the manchette develops near the area of the posterior nucleus. Portions of the manchette attach to the region of the nucleus just posterior to the 101 Ve tB oo ks .ir 10 208 Spermatogenesis undergoes a remarkable transformation that results in the production of a fully differentiated, highly special- ized spermatozoon containing a head (nuclear material), a flagellum including a midpiece (with a mitochondrial helix) and a principal piece. The most immature genn cells (spem1atogonia) are located at the periphe1y of a seminiferous tubule near the basement membrane. As these germ cells prolifer- ate, they move toward the lumen. The cell types in the seminiferous epithelium are illustrated in Figure 10-5. Developing germ cells are connected by intercellular bridges. Groups of spem1atogonia, spermatocytes or spenn atids are connected by intercellular bridges, so that the cytoplasm of an entire cohort (groups of cells of the same type) is interconnected. The exact number of genn cells that are interconnected is not !mown, but might approach 50. The significance of these intercel- lular bridges is not fully understood. However, they undoubtedly provide communication between cells that contributes to synchronized development of a cohort. Proliferation Generates Spermatogonia That are Committed to Become More Advanced Cell Types The most primitive cells encountered in the seminiferous epithelium are the spemmtogonia. These specialized diploid (2N chromosomal content) cells are located in the basal compartment of the semi- niferous epithelium. Spermatogonia undergo several mitotic divisions with the last division resulting in primary spermatocytes (See Figure I 0-5). There are three types of spermatogonia: A-spermatogonia, !- spermatogonia (intem1ediate) and B-spermatogonia. A-spemmtogonia undergo several mitotic divisions in which they progress mitotically from A, through A4• A pool of stem cells is also maintained so that the process can continue indefinitely. Stem cells divide mitotically to provide a continual source of A-spermatogonia al- lowing spermatogenesis to continue without interrup- tion for years. The mechanism for the renewal of stem cells is not understood. Meiotic Divisions Produce Haploid Spermatids During spermatogenesis the number of chro- mosomes in the gamete is reduced to the haploid state. This is accomplished by meiosis. The mitotic divisions of 8-spennatogonia result in the fonnation of primmy spermatocytes . These primary spermatocytes imme- diately enter the first meiotic prophase. As you will recall fi·om your previous courses, the meiotic prophase consists of five stages: preleptotene, leptotene, zygo- tene, pachytene and diplotene. Each of these stages represents a different step in the progression of DNA synthesis and replication. Primary spermatocytes must progress through these five steps before the first meiotic division can occur. The important event of the prelepto- tene phase is complete DNA replication forming tetrads without separation. These tetrads then fuse at random points known as chiasmata and crossing-over ofDNA material later takes place. The term "crossing-over" refers to segments of one chromosome crossing-over to a homologous chromosome when the chromatids separate. Crossing-over results in a random assort- ment of different segments of each clu·omosome. Thus, prophase of the first meiotic division insures genetic heterogeneity and that each secondary spem1atocyte and subsequently each spermatid will be genetically unique. Prophase of the first meiotic division is a relatively long process. In fact, the lifespan of the pri- maiy spermatocyte is the longest of all germ cell types found in the seminiferous epithelium. For example, in the bull the lifespan of the primmy spermatocyte is 18 to 19 days. The total duration of spennatogenesis in bulls is 61 days. Thus, prophase of the first meiotic division (primary spem1atocyte) is about 30% of the time required for the entire spermatogenic process. The secondary spermatocyte resulting from the first meiotic division of a primary spermatocyte is short-lived. It exists for only l.l to 1.7 days depending on the species. The secondary spennatocyte rapidly undergoes the second meiotic division, resulting in haploid spherical spennatids. Differentiation Produces a Highly Sophisticated, Self-Propelled Package of Enzymes and DNA The role of a spennatozoon is to deliver the male 's genetic material to an oocyte during fertilization. To form cells that are capable of fertilization, spherical spermatids undergo a series of changes in which the nucleus becomes highly condensed, the acrosome is formed and the cell becomes potentially motile. The abi lity to swim (motility) requires the development of a flagellum and a metabolic " powerplant" known as the mitochondrial helix. Differentiation consists ofthe: • Golgi phase • cap phase • acrosomal phase • maturation phase Spermatogenesis 209 Figure 10-6. The Golgi Phase of Spermatid Differentiation 0 0 e \ The newly formed sper- Small vesicles of the Golgi Vesicle fusion continues until matid is almost perfectly fuse, giving rise to larger a large acrosomic vesicle is spherical and has a well secretory granules called formed containing a dense developed Golgi appa- pro-acrosomic granules. acrosomic granule. The proxi- ratus. The centrioles start to mi- mal centriole (PC) gives rise grate to a position beneath to the attachment point of the the nucleus that is opposite tail . The distal centriole (DC) the acrosomic vesicle. gives rise to the developing The Golgi phase = acrosomic vesicle formation The Golgi phase is characterized by the first steps in the development of the acrosome. The newly fom1ed spermatid contains a large, highly-developed Golgi apparatus located near the nucleus that consists of many small vesicles (See Figure 1 0-6). The Golgi apparatus is not unique to the spermatid, but is the intracellular "packaging" system in a ll secretory cells. In a spermatid, the Golgi will give rise to an important subcellular organelle known as the acrosome. First, proacrosomic vesicles are formed and these fuse, gen- erating a larger vesicle that resides on one side of the nucleus. This vesicle is called the acrosomic vesicle and contains a dense acrosomic granule (See Figure I 0-6). Smaller Golgi vesicles are continually added to the larger vesicle increasing its size. While the acrosomic vesicle is being fom1ed, the centrioles migrate from the cytoplasm to the base of the nucleus (See Figure 1 0-6). The proximal centriole will give ri se to an implantation apparatus that allows the flagellum to be anchored to the nucleus (See Figure I 0-9). The distal centriole gives rise to the develop- ing axoneme. The axoneme is the central portion of a flagellum, in this case the sperm tail. axoneme (central portion of the tail) inside the cytoplasm of the spermatid. The cap phase = acrosomic vesicle spreading over the nucleus During the cap phase the acrosome fonns a distinct, easily recognized cap over the anterior portion of the nucleus (See Figure I 0-7). The Golgi now has performed its function by packaging the acrosomal con- tents and membranes and moves away from the nucleus toward the caudal end of the spem1atid and eventually disappears. The primitive flagellum (tail), fom1ed fi·om the distal centriole, begins to project from the spem1atid toward the lumen ofthe seminiferous tubule. The acrosomal phase =nuclear and cytoplasmic elongation During the acrosomal phase the acrosome continues to spread until it covers about two-thirds of the anterior nucleus (See Figures 10-7 and I 0-8). The nucleus begins to elongate. A unique system of mi- crotubules known as the manchette develops near the area of the posterior nucleus. Portions of the manchette attach to the region of the nucleus just posterior to the 101 Ve tB oo ks .ir 21 0 Spermatogenesis Figure 10-7. The Cap, Acrosomal and Maturation Phases of Spermatid Differentiation The Cap Phase The Acrosomal Phase The Maturation Phase II<=.,;,-- Posrnucleor ---MI C:!p Acrosome Head Principle piece A The Golgi migrates toward the caudal part of the cell. The distal centriole (DC) forms the axoneme (AX) or flagellum that projects away from the nucleus toward the lumen of the seminiferous tubule. B The acrosomic vesicle flattens and begins to form a distinct cap consisting of an outer acrosomal membrane (OAM), an inner acrosomal membrane (lAM) and the acrosomal contents (enzymes). A The spermatid nucleus begins to elongate and the acrosome eventually covers the majority of the anterior nucleus. The man- chette forms in the region of the caudal half of the nucleus and extends down toward the developing flagellum. B The neck and the annulus are formed and the later will become the juncture between the middle piece and the princi- pal piece. Notice that all components of the developing spermatid are completely surrounded by a plasma membrane. M = mitochondria. AandB Mitochondria form a spiral assembly around the flagellum that defines the middle piece. The postnuclear cap is formed from the manchette microtubules. The annulus forms the juncture between the middle piece and the principal piece. Figure 10-8. The Head of the Bovine Spermatozoon (Courtesy of Dr. R.G. Saacke, Virginia Polytechnic Institute and State University wi th permission from John R. Wiley and Sons, Inc. Am. J. Anat. 115:143) Plasma me mbrane Postnuclear cap acrosome (See Figure I 0-7). Some of the microtu- bules of the manchette w ill become the postnuclear cap. During the acrosomal phase, spem1atids become deeply embedded in Sertoli cells with their tails pro- truding toward the lumen of the seminiferous tubule (See Figure 10-4). The maturation phase =final assembly that forms a spermatozoon During the maturation phase microtubules of the manchette direct the fonnation of the postnuclear cap. Mitochondria migrate toward and cluster around the flagellum in the region posterior to the nucleus. Mitochondria are quickly assembled around the flagel- lum from the base of the nucleus to the anterior one- third of the tail. They are assembled in a spiral fashion (See Figure 10-9) and fom1 the middle piece in fiilly differentiated spermatozoa. Dense outer fibers of the flagellum and the fibrous sheath are produced and final assembly is complete. It should be emphasized that, as in any cell, the entire spermatozoon is covered with a plasma membrane. Integrity of the plasma membrane is required for the survival and function of spem1atozoa as you w ill see later in the chapter. Spermatogenesis 211 Spermatozoa = head + tail Head= nucleus +acrosome +post- nuclear cap Tail = middle piefe +principal piece + tenmnal p1ece Finally, release of spermatozoa from the Sertoli cells into the lumen of the seminiferous tubule occurs. This release is referred to as spermiation and is analo- gous to ovulation in the female, except that spermiation occurs continuously throughout the testis. The head of a mammalian spermatozoon has a shape characteristic for each species. In domestic mam- mals the nucleus is oval and fl attened and is surrounded by a nuclear membrane. The chromatin is compacted and is almost inert because it is highly keratinized. Ke- ratinoid proteins (hair, claws, hoofs and feathers) have a high degree of disulfide cross- linking and are quite insoluble. During spenniogenesis, nuclear histones of the haploid spem1 nucleus are replaced by protamines. Protamines are small, arginine-rich nuclear proteins thought to be essential for DNA condensation. The sufihydryl groups of protamines fonn d isulfide bonds. These bonds are the basis for nuclear condensation that results in a highly compact, stable nucleus that forms the spenn head. At this point in spenniogenesis, transcrip- tion and translation stops because the "transcriptional machinery" can no longer access nuclear DNA. Most of the "translational machinery" has been partitioned and lost within the residual cytoplasm ofthe spennatid. The DNA within the spenn head remains fundamentally inert unti l the time of fertilization. The inert nature of the DNA is thought to be a mechanism to prevent damage to the DNA between spem1iation and ferti lization. At fertilization, the process is reversed because the disulfide cross-links within the sperm nucleus are reduced by glu- tathione in the cytoplasm of the oocyte. The protamines are replaced with histones from the oocyte cytoplasm resulting in nuclear decondensation and fonnation of the male pronucleus (See Figure 12-8). Thus, the process of nuclear condensation, characterized by a high degree of keratinization and DN A stability is reversed only after the spem1 enters the oocyte cytoplasm. The anterior two-thirds of the nucleus is cov- ered by the acrosome. The acrosome is a membrane- bound lysosome that contains hydrolytic enzymes. These enzymes, acrosin, hyaluronidase, zona lysin, esterases and acid hydro lases, are required for penetra- tion ofthe cellular investments and the zona pellucida of the ovulated oocyte. During ferti lization the acrosome undergoes an ordered, highly specialized exocytosis, known as the acrosome reaction, that allows release of the enzymes that are packaged in it to digest or penetrate Ve tB oo ks .ir 21 0 Spermatogenesis Figure 10-7. The Cap, Acrosomal and Maturation Phases of Spermatid Differentiation The Cap Phase The Acrosomal Phase The Maturation Phase II<=.,;,-- Posrnucleor ---MI C:!p Acrosome Head Principle piece A The Golgi migrates toward the caudal part of the cell. The distal centriole (DC) forms the axoneme (AX) or flagellum that projects away from the nucleus toward the lumen of the seminiferous tubule. B The acrosomic vesicle flattens and begins to form a distinct cap consisting of an outer acrosomal membrane (OAM), an inner acrosomal membrane (lAM) and the acrosomal contents (enzymes). A The spermatid nucleus begins to elongate and the acrosome eventually covers the majority of the anterior nucleus. The man- chette forms in the region of the caudal half of the nucleus and extends down toward the developing flagellum. B The neck and the annulus are formed and the later will become the juncture between the middle piece and the princi- pal piece. Notice that all components of the developing spermatid are completely surrounded by a plasma membrane. M = mitochondria. AandB Mitochondria form a spiral assembly around the flagellum that defines the middle piece. The postnuclear cap is formed from the manchette microtubules. The annulus forms the juncture between the middle piece and the principal piece. Figure 10-8. The Head of the Bovine Spermatozoon (Courtesy of Dr. R.G. Saacke, Virginia Polytechnic Institute and State University wi th permission from John R. Wiley and Sons, Inc. Am. J. Anat. 115:143) Plasma me mbrane Postnuclear cap acrosome (See Figure I 0-7). Some of the microtu- bules of the manchette w ill become the postnuclear cap. During the acrosomal phase, spem1atids become deeply embedded in Sertoli cells with their tails pro- truding toward the lumen of the seminiferous tubule (See Figure 10-4). The maturation phase =final assembly that forms a spermatozoon During the maturation phase microtubules of the manchette direct the fonnation of the postnuclear cap. Mitochondria migrate toward and cluster around the flagellum in the region posterior to the nucleus. Mitochondria are quickly assembled around the flagel- lum from the base of the nucleus to the anterior one- third of the tail. They are assembled in a spiral fashion (See Figure 10-9) and fom1 the middle piece in fiilly differentiated spermatozoa. Dense outer fibers of the flagellum and the fibrous sheath are produced and final assembly is complete. It should be emphasized that, as in any cell, the entire spermatozoon is covered with a plasma membrane. Integrity of the plasma membrane is required for the survival and function of spem1atozoa as you w ill see later in the chapter. Spermatogenesis 211 Spermatozoa = head + tail Head= nucleus +acrosome +post- nuclear cap Tail = middle piefe +principal piece + tenmnal p1ece Finally, release of spermatozoa from the Sertoli cells into the lumen of the seminiferous tubule occurs. This release is referred to as spermiation and is analo- gous to ovulation in the female, except that spermiation occurs continuously throughout the testis. The head of a mammalian spermatozoon has a shape characteristic for each species. In domestic mam- mals the nucleus is oval and fl attened and is surrounded by a nuclear membrane. The chromatin is compacted and is almost inert because it is highly keratinized. Ke- ratinoid proteins (hair, claws, hoofs and feathers) have a high degree of disulfide cross- linking and are quite insoluble. During spenniogenesis, nuclear histones of the haploid spem1 nucleus are replaced by protamines. Protamines are small, arginine-rich nuclear proteins thought to be essential for DNA condensation. The sufihydryl groups of protamines fonn d isulfide bonds. These bonds are the basis for nuclear condensation that results in a highly compact, stable nucleus that forms the spenn head. At this point in spenniogenesis, transcrip- tion and translation stops because the "transcriptional machinery" can no longer access nuclear DNA. Most of the "translational machinery" has been partitioned and lost within the residual cytoplasm ofthe spennatid. The DNA within the spenn head remains fundamentally inert unti l the time of fertilization. The inert nature of the DNA is thought to be a mechanism to prevent damage to the DNA between spem1iation and ferti lization. At fertilization, the process is reversed because the disulfide cross-links within the sperm nucleus are reduced by glu- tathione in the cytoplasm of the oocyte. The protamines are replaced with histones from the oocyte cytoplasm resulting in nuclear decondensation and fonnation of the male pronucleus (See Figure 12-8). Thus, the process of nuclear condensation, characterized by a high degree of keratinization and DN A stability is reversed only after the spem1 enters the oocyte cytoplasm. The anterior two-thirds of the nucleus is cov- ered by the acrosome. The acrosome is a membrane- bound lysosome that contains hydrolytic enzymes. These enzymes, acrosin, hyaluronidase, zona lysin, esterases and acid hydro lases, are required for penetra- tion ofthe cellular investments and the zona pellucida of the ovulated oocyte. During ferti lization the acrosome undergoes an ordered, highly specialized exocytosis, known as the acrosome reaction, that allows release of the enzymes that are packaged in it to digest or penetrate Ve tB oo ks .ir [10 212 Spermatogenesis Figure 10-9. The Tail of the Bovine . . (Courtesy of Dr. R.G. Saacke, Virginia Polytechnic Institute and State Umvers1ty w1th perm1ss1on from John R. Wiley and Sons, Inc. Am. J. Anat. 115:163) I \ Capitulum Coarse outer fibers Mitochondrial helix Longitudinal elements Axoneme (9 dublets + 2 central fibers) Coarse outer fibers Fibrous helix piece Principal piece • • .J the zona pellucida. These reactions will be described in more detail in Chapter 12. Acrosomal morphology var- ies among species, but in the boar, ram, bull and stallion the acrosome is similar to that shown in Figure 10-8 . The membrane component posterior to the acrosome is the postnuclear cap. The sperm tail is a self-powered flagellum. The tai l is composed of the capitulum, the middle piece, the principal piece and the terminal piece. The capitulum fits into the implantation socket, a depression in the posterior nucleus. The anterior por- tion of the tail consists of laminated columns that give the neck region flexibility when it becomes motile, so the tail can move laterally from side-to-side during the flagellar beat. The axonemal component of the tail originates fi·om the distal centriole and is composed of 9 pairs of microhibules that are ananged radially around two central filaments. Surrounding this 9+9+2 arrangement ofmicrotubules are 9 coarse fibers that are unique to the flagellum of spennatozoa. This arrange- ment ofhibules in the tail ofspern1atozoa is illustrated in Figure 10-9. The mitochondrial sheath is arranged in a heli- cal pattem (See Figure 1 0-9) around the outer coarse fibers of the tail and contributes to the middle piece. The annulus demarcates the j uncture between the middle piece and the principal piece. The principal piece makes up the majority of the tail and continues almost to the end of the flagellum, where only the microh1bules end in the terminal piece. Spermatozoa are Released Continually into the Lumen of the Seminiferous Tubules One of the major differences between gamete production in the female and the male is that the fe- male's gamete supply is produced entirely before birth. After puberty, she begins to produce oocytes that will undergo meiosis and ovulate every 3-4 weeks. Thus, maturation, meiosis and release of female gametes is pulsatile. In contrast, the male produces gametes continually and unifom1ly throughout his reproductive lifespan. An exception to this is the seasonal breeder that produces spermatozoa during the breeding season only. Understanding the mechanisms responsible for the continual production of spermatozoa by the semi- niferous epithelium represents a major challenge for sh1dents of reproductive physiology. Appreciating the spermatogenic process is necessary for a complete understanding of reproductive physiology. But the importance of this understanding Spermatogenesis 213 goes beyond the academic. From a clinical perspective, evaluation of sperm numbers in the ejaculate does not always accurately reflect nonnal or abnonnal spermato- genesis. Therefore, the fate of males being evaluated is often fraught with error and thus bad decisions are made. One needs to understand that there is a 2 to 4 week delay before the effects of deleterious events (heat stress, shipping, fever, exposure to certain toxins) can be observed by monitoring changes in ejaculated spenn. Furthermore, 6 to 12 weeks are required before restora- tion of normal spermatogenesis can be accomplished after these events. Therefore, clinical interpretations of ejaculate characteristics requires specific knowledge of the timing of spermatogenesis in the species being evaluated. Seasonal spermatogenesis requires that the genninal epithelium "him-on" and "tum-off" as a function of environmental influences. More and more emphasis is being placed on "saving and managing" endangered species. For these efforts to be successful, the timing of spermatogenesis and sperm producing potential must be understood so that sufficient male gametes are avai lable for reproductive manipulation (artificial insemination, in vitro fertil ization, etc.). As of yet, a practical, cost-effective contraceptive is not available for men. We need to leam how to temporarily "tum-off" and later "turn-on" spennatogenesis without altering the behavior of the male. Our ability to manipu- late male gamete production will play a major part in the abi lity to manipulate reproduction in the future. In order to comprehend the cycle of the seminiferous epithelium you must first understand: • cellular generations • stages of the cycle • duration of one cycle • how the cycle is repeated The cycle of the seminiferous epithelium is the progression through a complete series of cellular associations (stages) at one location along a seminifer- ous tubule. The time required for this progression is the duration of the cycle of the seminiferous epithelium and is unique for each species. Germ cell generations are cells of the same type located at one site within the seminiferous epithelium. 10 Ve tB oo ks .ir [10 212 Spermatogenesis Figure 10-9. The Tail of the Bovine . . (Courtesy of Dr. R.G. Saacke, Virginia Polytechnic Institute and State Umvers1ty w1th perm1ss1on from John R. Wiley and Sons, Inc. Am. J. Anat. 115:163) I \ Capitulum Coarse outer fibers Mitochondrial helix Longitudinal elements Axoneme (9 dublets + 2 central fibers) Coarse outer fibers Fibrous helix piece Principal piece • • .J the zona pellucida. These reactions will be described in more detail in Chapter 12. Acrosomal morphology var- ies among species, but in the boar, ram, bull and stallion the acrosome is similar to that shown in Figure 10-8 . The membrane component posterior to the acrosome is the postnuclear cap. The sperm tail is a self-powered flagellum. The tai l is composed of the capitulum, the middle piece, the principal piece and the terminal piece. The capitulum fits into the implantation socket, a depression in the posterior nucleus. The anterior por- tion of the tail consists of laminated columns that give the neck region flexibility when it becomes motile, so the tail can move laterally from side-to-side during the flagellar beat. The axonemal component of the tail originates fi·om the distal centriole and is composed of 9 pairs of microhibules that are ananged radially around two central filaments. Surrounding this 9+9+2 arrangement ofmicrotubules are 9 coarse fibers that are unique to the flagellum of spennatozoa. This arrange- ment ofhibules in the tail ofspern1atozoa is illustrated in Figure 10-9. The mitochondrial sheath is arranged in a heli- cal pattem (See Figure 1 0-9) around the outer coarse fibers of the tail and contributes to the middle piece. The annulus demarcates the j uncture between the middle piece and the principal piece. The principal piece makes up the majority of the tail and continues almost to the end of the flagellum, where only the microh1bules end in the terminal piece. Spermatozoa are Released Continually into the Lumen of the Seminiferous Tubules One of the major differences between gamete production in the female and the male is that the fe- male's gamete supply is produced entirely before birth. After puberty, she begins to produce oocytes that will undergo meiosis and ovulate every 3-4 weeks. Thus, maturation, meiosis and release of female gametes is pulsatile. In contrast, the male produces gametes continually and unifom1ly throughout his reproductive lifespan. An exception to this is the seasonal breeder that produces spermatozoa during the breeding season only. Understanding the mechanisms responsible for the continual production of spermatozoa by the semi- niferous epithelium represents a major challenge for sh1dents of reproductive physiology. Appreciating the spermatogenic process is necessary for a complete understanding of reproductive physiology. But the importance of this understanding Spermatogenesis 213 goes beyond the academic. From a clinical perspective, evaluation of sperm numbers in the ejaculate does not always accurately reflect nonnal or abnonnal spermato- genesis. Therefore, the fate of males being evaluated is often fraught with error and thus bad decisions are made. One needs to understand that there is a 2 to 4 week delay before the effects of deleterious events (heat stress, shipping, fever, exposure to certain toxins) can be observed by monitoring changes in ejaculated spenn. Furthermore, 6 to 12 weeks are required before restora- tion of normal spermatogenesis can be accomplished after these events. Therefore, clinical interpretations of ejaculate characteristics requires specific knowledge of the timing of spermatogenesis in the species being evaluated. Seasonal spermatogenesis requires that the genninal epithelium "him-on" and "tum-off" as a function of environmental influences. More and more emphasis is being placed on "saving and managing" endangered species. For these efforts to be successful, the timing of spermatogenesis and sperm producing potential must be understood so that sufficient male gametes are avai lable for reproductive manipulation (artificial insemination, in vitro fertil ization, etc.). As of yet, a practical, cost-effective contraceptive is not available for men. We need to leam how to temporarily "tum-off" and later "turn-on" spennatogenesis without altering the behavior of the male. Our ability to manipu- late male gamete production will play a major part in the abi lity to manipulate reproduction in the future. In order to comprehend the cycle of the seminiferous epithelium you must first understand: • cellular generations • stages of the cycle • duration of one cycle • how the cycle is repeated The cycle of the seminiferous epithelium is the progression through a complete series of cellular associations (stages) at one location along a seminifer- ous tubule. The time required for this progression is the duration of the cycle of the seminiferous epithelium and is unique for each species. Germ cell generations are cells of the same type located at one site within the seminiferous epithelium. 10 Ve tB oo ks .ir 214 Spermatogenesis Within any given microscopic cross-section of a seminiferous tubule, one can observe four or five concentric "layers" of germ cells. Cells in each layer comprise a generation. A generation is a cohort of cells that develops as a synchronous group. Each generation of cells (each concentric layer) has a similar appear- ance and function. Cross-sections along the length of a seminiferous tubule will have a different appear- ance but the entire cross-section at a given location will usually appear similar. For example, while view- ing cross-section I (stage I) in Figure l 0-10, you will observe four generations of genu cells. Each generation will give rise to a succeeding, more advanced genera- tion. Observe in Figure 10-10 that there is a generation of A -spermatogonia near the basement membrane in the section of the h1bule labeled Stage I. Just above the A -spermatogonia is a young generation of primary sper- matocytes. Above it lies a third generation consisting of more mahlre prima1y spermatocytes. Finally, near the lumen, is a fomth generation of cells. This generation consists of spherical immarure spennatids. Remember that the more immah1re cell types are generally located near the basement membrane (basal compartment) and the more advanced cell types reside in the adluminal compartment. In cross-section IV (stage IV) of Figure 10- 10, there are five generations of genn cells. You will observe a generation of A-spermatogonia, one genera- tion of intermediate spermatogonia, one generation of primary spermatocytes, one generation of secondary Figure 10-10. Associations of Developing Germ Cells That Represent Various Stages of the Cycle of the Seminiferous Epithelium VIII ll ill IV V VI VII VITI Stage I A stage I tubule consists of 1 generation of A- spermatogonia, 2 generations of primary sperma- tocytes ( 1 o cyte) and 1 generation of immature sper- matids ('Tid). \.. At any given cross-sectioned loca- tion along a seminiferous tubule, one can observe different stages of the cycle of the seminiferous epithelium. In this example, we see three stages (I, IV, and VIII). 'gonia = spermatogonium 1 o cyte = primary spermatocyte 2° cyte = secondary spermatocyte 'Tid = immature spermatid 'Tid-m =mature spermatid Stage IV Stage VIII A stage IV tubule con- sists of 2 generations of spermatogonia (A+I), 1 generation of primary spermatocytes (1 o cyte ), 1 generation of second- ary spermatocytes (2° cyte) and 1 generation of immature spermatids ('Tid). A stage VIII tubule consists of 2 genera- tions of spermatogonia (A+B), 1 genera- tion of primary spermatocytes (1° cyte) and 2 generations of spermatids ('Tid). The young generation of spermatids ('Tid) have formed only a few days earlier and are quite immature. The second generation of spermatids are mature ('Tid-m) and are about to be released into the lumen. Spermatogenesis 215 Figure 10-11. Cycle of the Seminiferous Epithelium in the Bull (Modified from Amann, R.P. Am. J. Anat. 110:69) Spermiation Lumenl 61.days 5 ---- ---- c 0 •.t; f C1) c C1) C) --C1) u 4 3 2 ...... __ _ --- -- I ---- ---- "(- -- -- VIII 13.5 • Horizontal axis - Stage of cycle and days spent in each stage. • Vertical axis = Cell generations in each stage i.e. type of cell seen from the basal level to the luminal level within a cross section of a seminiferous tubule. • Horizontal line= Developmental pathway from spermatogonia to spermatozoa (61 days). • The release of spermatozoa from the Sertoli cells occurs in stage VIII and is called spermiation. It occurs 61 days after A-spermatogonia are formed at the beginning of Stage Ill. Ocell division (mitotic for 'gonia, meiotic for primary and secondary 'cytes). • In the bull, it takes about 4.5 cycles of the seminiferous epithelium to complete spermatogenesis (4.5 cycles x 13.5 days/cycle = 61 days). spennatocytes and one generation of spermatids. The spermatids in stage IV are elongated and, thus are more advanced than the spemmtids in stage I. In cross-section VIII (stage VIII), there are also five generations of gem1 cells. Observe two generations of spermatogonia (one generation of A and one genera- tion of B-spermatogonia), one generation of primary spem1atocytes and two generations of spermatids. One generation of spermatids is rather immahlre and spheii- cal, while the more advanced generation consists of mature spemmtids ready for release from Sertoli cells into the lumen of the seminiferous tubule. At one instance in time, three cross-sections at d ifferent locations along the seminiferous tubule show different generations of cells . Cells in each section are actively engaged in spennatogenesis, but only one cross- section (VIII) is ready to release spermatozoa into the lumen. Thus, along the length of any seminiferous tu- bule there are only certain zones (cross-sections) where spermatozoa are released at any given point in time. All other zones or stages are preparing to release spenna- tozoa, but the cells in those zones have not reached the appropriate stage of maturity for spermiation to occur. 10 Ve tB oo ks .ir 214 Spermatogenesis Within any given microscopic cross-section of a seminiferous tubule, one can observe four or five concentric "layers" of germ cells. Cells in each layer comprise a generation. A generation is a cohort of cells that develops as a synchronous group. Each generation of cells (each concentric layer) has a similar appear- ance and function. Cross-sections along the length of a seminiferous tubule will have a different appear- ance but the entire cross-section at a given location will usually appear similar. For example, while view- ing cross-section I (stage I) in Figure l 0-10, you will observe four generations of genu cells. Each generation will give rise to a succeeding, more advanced genera- tion. Observe in Figure 10-10 that there is a generation of A -spermatogonia near the basement membrane in the section of the h1bule labeled Stage I. Just above the A -spermatogonia is a young generation of primary sper- matocytes. Above it lies a third generation consisting of more mahlre prima1y spermatocytes. Finally, near the lumen, is a fomth generation of cells. This generation consists of spherical immarure spennatids. Remember that the more immah1re cell types are generally located near the basement membrane (basal compartment) and the more advanced cell types reside in the adluminal compartment. In cross-section IV (stage IV) of Figure 10- 10, there are five generations of genn cells. You will observe a generation of A-spermatogonia, one genera- tion of intermediate spermatogonia, one generation of primary spermatocytes, one generation of secondary Figure 10-10. Associations of Developing Germ Cells That Represent Various Stages of the Cycle of the Seminiferous Epithelium VIII ll ill IV V VI VII VITI Stage I A stage I tubule consists of 1 generation of A- spermatogonia, 2 generations of primary sperma- tocytes ( 1 o cyte) and 1 generation of immature sper- matids ('Tid). \.. At any given cross-sectioned loca- tion along a seminiferous tubule, one can observe different stages of the cycle of the seminiferous epithelium. In this example, we see three stages (I, IV, and VIII). 'gonia = spermatogonium 1 o cyte = primary spermatocyte 2° cyte = secondary spermatocyte 'Tid = immature spermatid 'Tid-m =mature spermatid Stage IV Stage VIII A stage IV tubule con- sists of 2 generations of spermatogonia (A+I), 1 generation of primary spermatocytes (1 o cyte ), 1 generation of second- ary spermatocytes (2° cyte) and 1 generation of immature spermatids ('Tid). A stage VIII tubule consists of 2 genera- tions of spermatogonia (A+B), 1 genera- tion of primary spermatocytes (1° cyte) and 2 generations of spermatids ('Tid). The young generation of spermatids ('Tid) have formed only a few days earlier and are quite immature. The second generation of spermatids are mature ('Tid-m) and are about to be released into the lumen. Spermatogenesis 215 Figure 10-11. Cycle of the Seminiferous Epithelium in the Bull (Modified from Amann, R.P. Am. J. Anat. 110:69) Spermiation Lumenl 61.days 5 ---- ---- c 0 •.t; f C1) c C1) C) --C1) u 4 3 2 ...... __ _ --- -- I ---- ---- "(- -- -- VIII 13.5 • Horizontal axis - Stage of cycle and days spent in each stage. • Vertical axis = Cell generations in each stage i.e. type of cell seen from the basal level to the luminal level within a cross section of a seminiferous tubule. • Horizontal line= Developmental pathway from spermatogonia to spermatozoa (61 days). • The release of spermatozoa from the Sertoli cells occurs in stage VIII and is called spermiation. It occurs 61 days after A-spermatogonia are formed at the beginning of Stage Ill. Ocell division (mitotic for 'gonia, meiotic for primary and secondary 'cytes). • In the bull, it takes about 4.5 cycles of the seminiferous epithelium to complete spermatogenesis (4.5 cycles x 13.5 days/cycle = 61 days). spennatocytes and one generation of spermatids. The spermatids in stage IV are elongated and, thus are more advanced than the spemmtids in stage I. In cross-section VIII (stage VIII), there are also five generations of gem1 cells. Observe two generations of spermatogonia (one generation of A and one genera- tion of B-spermatogonia), one generation of primary spem1atocytes and two generations of spermatids. One generation of spermatids is rather immahlre and spheii- cal, while the more advanced generation consists of mature spemmtids ready for release from Sertoli cells into the lumen of the seminiferous tubule. At one instance in time, three cross-sections at d ifferent locations along the seminiferous tubule show different generations of cells . Cells in each section are actively engaged in spennatogenesis, but only one cross- section (VIII) is ready to release spermatozoa into the lumen. Thus, along the length of any seminiferous tu- bule there are only certain zones (cross-sections) where spermatozoa are released at any given point in time. All other zones or stages are preparing to release spenna- tozoa, but the cells in those zones have not reached the appropriate stage of maturity for spermiation to occur. 10 Ve tB oo ks .ir [ 10 216 Spermatogenesis = specific cellular associations duration= time required for completion of one stage (cell association) Cvcle =progression through sequence of all stages Cvcle dumtion = time required to complete one cycle Figure 10-12. The Cycle of Seminiferous Epithelium is Analogous to a University (Modified from Johnson, 1991) Every year, freshmen (spermatogonia) enter and seniors (spermatozoa) graduate. How- ever four years are required for a freshmen to through the various classes become a graduating senior. Each class 1s analogous to a generation of germ cells found in the seminiferous epithelium. Senior I ('tid) I ... ______________ Junior (Secondary 'cyte) I I .............................. Graduation (Spermiation) """4 years \ , \ Sophomore (Primary 'cyte) ® ..... 0 Freshman ('gonia) , ______________ _ Fall semester ------------- .... Spring semester I year \ I , "Flunk-out" Stages of the cycle are arbitrarily defined cellular associations that transition one to the next at predictable intervals. As previously explained, sections or zones along a seminiferous tubule contain different cellular associations. These cellular associations, or stages of the cycle of the seminifet·ous epithelium, have been defined arbitrarily by researchers who have made thou- sands and thousands of observations of the seminiferous epithelium using light microscopy. If you were to microscopically scan a number of tubules in the testicular parenchyma, you would see tubule cross-sections that contain exactly the same cell types and relationships as other tubules. In fact, with enough observation you would begin to encounter dif- ferent cross-sections with definable cellular composi- tions at predictable frequencies. For the purposes of this text, we will describe eight stages in the cycle of the seminiferous epithelium, even though other schemes are available with as many as 14 stages. Figure 10-11 illustrates the cellular composi- tion of each stage of the seminiferous epithelium. For example, stage I contains one generation of A-sper- matogonia, two generations of primary spermatocytes and one generation of spennatids. By scanning from the basement membrane (bottom of diagram) toward the lumen, you can quickly detennine which cell types are present at each of the eight stages. Lifespan of cells and duration oftlte cycle vary among species. The entire progression of one cycle of the seminiferous epithelium from stage I through stage VIII requires 13.5 days in the bull (for other species see Table 1 0-1 ). That is, if you could observe one cross-section of a seminiferous tubule continually, starting at the beginning of stage I, it would require 13.5 days before you would observe spem1iation (the end of stage VIII). After spem1iation (end of stage VIII), the cross section you were observing would again have the same cellular association as it did on the day you s tarted watching (stage 1). Thus, one cycle of the seminiferous epithe- lium would have been completed. The complete process ofspennatogenesis from A-spennatogonia to the fonnation of fully differentiated spermatozoa takes 61 days in the bull. During the 61 days, cells at a given area ofthe seminiferous epithelium proceed through 4.5 cycles of the seminiferous epithe- lium (13 .5 days/cycle X 4.5 cycles = 61 days). Spermatogenesis 217 This process is analogous to a traditional uni- versity. Every year a new class of freshmen enters the university in the fall. These freshmen are analogous to committed A-spemmtogonia entering the spemlato- genic pathway. The freshmen (A-spermatogonia) un- dergo noticeable changes during the first year, and after one year they become sophomores. Sophomores are analogous to primaty spem1atocytes. The sophomores (primary spermatocytes) also undergo maturational changes and become juniors (secondary spermatocytes; although they actually are short-lived). Finally, they become seniors (spennatids) and graduate after four years (See Figure 10-12). The cycle of the seminiferous epithelium is almost identical in concept to the university situation, except the school year is only 13.5 days (1 cycle of the seminiferous epithelium in the bull). Every 13.5 days a new generation of freshmen (A-spermatogonia) enter and a generation of seniors (spermatids) gradu- ate. Graduation by the seniors is analogous to sperm- iation. Remember, it takes four years to graduate from the university. Similarly, it takes 4.5 cycles for an A-spermatogonium (freshman) to become a fully dif- ferentiated spermatozoon (senior). A major difference between the university example and the actual cycle of the seminiferous epithelium is that the germinal elements have different lifespans. For example, a primary spem1atocyte exists for about 21 days while a secondary spermatocyte exists for only 1. 7 days in the bull. In the university, freshmen, sophomores, juniors and seniors have similar lifespans (assuming a basal academic perfonnance ). There is another major difference between the university analogy and what actually takes place in the genninal epithelium. Spennatogonia (freshmen), primary (sophomores) and secondary (juniors) spenna- tocytes all divide and generate many spennatids. For example each incoming freshmen (A-gonia) could theo- retically produce 256 seniors (spennatids). Obviously, such multiplication does not take place with university students. In the university, a significant proportion of entering freshmen "flunk-out" and never graduate, so there are always more freshmen than graduating seniors. Similarly, during spennatogenesis many proliferating spennatogonia die and never become primary spennato- cytes. Therefore, the numberofprimruy spermatocytes generated per conm1itted A-spem1atogonium is closer to 20-30 than the theoretical 64 as depicted in Figure I 0-5. There also is death of primary spem1atocytes, although most spherical spennatids do form a sper- matozoon. In contrast from a university where each student can choose their pace throughout the years, to amass 120 credits, in the testis of a given species, the pace through spemmtogenesis is essentially identical and is not affected by environment. 10 Ve tB oo ks .ir [ 10 216 Spermatogenesis = specific cellular associations duration= time required for completion of one stage (cell association) Cvcle =progression through sequence of all stages Cvcle dumtion = time required to complete one cycle Figure 10-12. The Cycle of Seminiferous Epithelium is Analogous to a University (Modified from Johnson, 1991) Every year, freshmen (spermatogonia) enter and seniors (spermatozoa) graduate. How- ever four years are required for a freshmen to through the various classes become a graduating senior. Each class 1s analogous to a generation of germ cells found in the seminiferous epithelium. Senior I ('tid) I ... ______________ Junior (Secondary 'cyte) I I .............................. Graduation (Spermiation) """4 years \ , \ Sophomore (Primary 'cyte) ® ..... 0 Freshman ('gonia) , ______________ _ Fall semester ------------- .... Spring semester I year \ I , "Flunk-out" Stages of the cycle are arbitrarily defined cellular associations that transition one to the next at predictable intervals. As previously explained, sections or zones along a seminiferous tubule contain different cellular associations. These cellular associations, or stages of the cycle of the seminifet·ous epithelium, have been defined arbitrarily by researchers who have made thou- sands and thousands of observations of the seminiferous epithelium using light microscopy. If you were to microscopically scan a number of tubules in the testicular parenchyma, you would see tubule cross-sections that contain exactly the same cell types and relationships as other tubules. In fact, with enough observation you would begin to encounter dif- ferent cross-sections with definable cellular composi- tions at predictable frequencies. For the purposes of this text, we will describe eight stages in the cycle of the seminiferous epithelium, even though other schemes are available with as many as 14 stages. Figure 10-11 illustrates the cellular composi- tion of each stage of the seminiferous epithelium. For example, stage I contains one generation of A-sper- matogonia, two generations of primary spermatocytes and one generation of spennatids. By scanning from the basement membrane (bottom of diagram) toward the lumen, you can quickly detennine which cell types are present at each of the eight stages. Lifespan of cells and duration oftlte cycle vary among species. The entire progression of one cycle of the seminiferous epithelium from stage I through stage VIII requires 13.5 days in the bull (for other species see Table 1 0-1 ). That is, if you could observe one cross-section of a seminiferous tubule continually, starting at the beginning of stage I, it would require 13.5 days before you would observe spem1iation (the end of stage VIII). After spem1iation (end of stage VIII), the cross section you were observing would again have the same cellular association as it did on the day you s tarted watching (stage 1). Thus, one cycle of the seminiferous epithe- lium would have been completed. The complete process ofspennatogenesis from A-spennatogonia to the fonnation of fully differentiated spermatozoa takes 61 days in the bull. During the 61 days, cells at a given area ofthe seminiferous epithelium proceed through 4.5 cycles of the seminiferous epithe- lium (13 .5 days/cycle X 4.5 cycles = 61 days). Spermatogenesis 217 This process is analogous to a traditional uni- versity. Every year a new class of freshmen enters the university in the fall. These freshmen are analogous to committed A-spemmtogonia entering the spemlato- genic pathway. The freshmen (A-spermatogonia) un- dergo noticeable changes during the first year, and after one year they become sophomores. Sophomores are analogous to primaty spem1atocytes. The sophomores (primary spermatocytes) also undergo maturational changes and become juniors (secondary spermatocytes; although they actually are short-lived). Finally, they become seniors (spennatids) and graduate after four years (See Figure 10-12). The cycle of the seminiferous epithelium is almost identical in concept to the university situation, except the school year is only 13.5 days (1 cycle of the seminiferous epithelium in the bull). Every 13.5 days a new generation of freshmen (A-spermatogonia) enter and a generation of seniors (spermatids) gradu- ate. Graduation by the seniors is analogous to sperm- iation. Remember, it takes four years to graduate from the university. Similarly, it takes 4.5 cycles for an A-spermatogonium (freshman) to become a fully dif- ferentiated spermatozoon (senior). A major difference between the university example and the actual cycle of the seminiferous epithelium is that the germinal elements have different lifespans. For example, a primary spem1atocyte exists for about 21 days while a secondary spermatocyte exists for only 1. 7 days in the bull. In the university, freshmen, sophomores, juniors and seniors have similar lifespans (assuming a basal academic perfonnance ). There is another major difference between the university analogy and what actually takes place in the genninal epithelium. Spennatogonia (freshmen), primary (sophomores) and secondary (juniors) spenna- tocytes all divide and generate many spennatids. For example each incoming freshmen (A-gonia) could theo- retically produce 256 seniors (spennatids). Obviously, such multiplication does not take place with university students. In the university, a significant proportion of entering freshmen "flunk-out" and never graduate, so there are always more freshmen than graduating seniors. Similarly, during spennatogenesis many proliferating spennatogonia die and never become primary spennato- cytes. Therefore, the numberofprimruy spermatocytes generated per conm1itted A-spem1atogonium is closer to 20-30 than the theoretical 64 as depicted in Figure I 0-5. There also is death of primary spem1atocytes, although most spherical spennatids do form a sper- matozoon. In contrast from a university where each student can choose their pace throughout the years, to amass 120 credits, in the testis of a given species, the pace through spemmtogenesis is essentially identical and is not affected by environment. 10 Ve tB oo ks .ir 218 Spermatogenesis Table 10-1. Duration of the Stages of the Cycle of the Seminiferous Epithelium in Various Species Stage Bull Ram Boar Stallion Rabbit I 4.2 2.2 1.1 2.0 3.1 II 1.2 1.1 1.4 1.8 1.5 Ill 2.7 1.9 0.4 0.4 0.8 IV 1.7 1.1 1.2 1.9 1.2 v 0.2 0.4 0.8 0.9 0.5 VI 0.8 1.3 1.6 1.7 1.7 VII 1.1 1.1 1.0 1.6 1.3 VIII 1.6 1.0 0.8 1.9 0.9 TOTAL A 13.5 10.1 8.3 12.2 11 .0 SPERMATOGENESIS8 61 47 39 55 48 ATotal days required for 1 cycle of the seminiferous epithelium BApproximate days to complete spermatogenesis (sperm.atogonia to spermatozoa) The spermatogenic wave is the sequential ordering of stages along the length of the seminiferous tubule. The duration of each stage of the cycle of the seminiferous epithelium varies with species, as does the length of the cycle of the seminiferous epithelium. Variations in stage, cycle length and total time required for spemmtogenesis are presented in Table 1 0-l. The spermatogenic wave refers to the differ- ences at any given instant in time along the length of the seminiferous tubule. Imagine that you could run down the lumen of the seminiferous tubule. As you run down the tubule, you will encounter zones that are near spem1iation (stage VIII). The distance between these spermiation sites is relatively constant. During the wave, each stage of the seminiferous epithelium transitions to a successively more advanced stage. For example, a stage I tubule will later become a stage II and stage II will later become a III and so on. Thus, the site of spermiation along the tubule is constantly changing, creating a "wave" ofspenn release down the length of the tubule. This "wave" is like the wave con- ducted by football fans in a stadium. When the fans stand up, they mimic spenniation. They sit back down and don't stand up again until it's their tum again. The time spent sitting (stages I-VII) is much longer than the time spent standing. As the wave in the stadium continues, repeated standing and sitting takes place at a relatively constant rate. So does spenniation. The physiologic importance of the spermatogenic wave is to provide a relatively constant supply of spem1atozoa to the epididymis, creating a pool for ejaculation. Figure 10-13. Scrotal Circumference Measurements are Good Indicators of Sperm Producing Ability (Photograph courtesy of Select Sires, Inc. Plain City, Ohio, www.selectsires.com) Accurate scrotal circumference measurements require that both testicles be pushed ventrally by applying pressure to the spermatic cord. A specially designed tape is then placed around the scrotum at its widest point and a measure- ment is taken (in this case, 40cm). Spermatogenesis 219 Table 10-2. Testicular Characteristics and Sperm Production Estimates of Sexually Mature Mammals SBecies Gross weight of SBerm Broduced Daily Baired testes Ber gram of SBermatozoal (grams) testicular Barenchyma Broduction Beef Bull 650 11x1 06 6x109 Boar 750 23x106 16x109 Cat 21 16x106 32x1 06 Dairy Bull 725 12x106 7.5x109 Dog (16 kg body weight) 31 17x106 0.50x109 Man 35 4x106 0.13x109 Rabbit 6 25x106 0.20x109 Ram* 550 21x106 10x109 Rooster*** 25 100x106 2.5x109 Stallion** 340 16x1 06 5x1 09 *in breeding season (shortening-day length), **in breeding season (increasing day l!:mgth), · ***varies greatly with management and strain Daily sperm production (DSP) is defined as the total number of spennatozoa produced per day by both testicles of the male. Accurate measurement of DSP requires removal of all or a portion of the testicle and thus, DSP cannot be measured using non- invasive techniques . However, noninvasive measures such as total number of spermatozoa ejaculated into an artificial vagina with daily ejaculations for 2-3 weeks gives a good estimate of DSP. Interspecies variation in testicular weights, sperm produced per gram of testicular parenchyma ·and daily sperm production is presented in Table 10-2. The number of spermatozoa produced per day per gram of testicular parenchyma is referred to as efficiency of sperm production. Daily sperm production is dependent, at least in part, on the number of Sertoli cells populating the testes. For example, the higher the number of Sertoli cells, the higher the spem1atozoal production rates. Numbers of Sertoli cells also have been positively correlated with spermatogonial and spermatid numbers. Testicular Size is a Good Estimator of Sperm Producing Ability To detennine a given male 's sperm producing capability, it is necessary to collect ejaculates from the animal for a period of time. This enables one to ac- curately estimate how many spem1atozoa the animal can produce per unit time. If collection of semen is not possible, a good estimate ofspenn producing capability can be made by measuring the circumference of both testicles (See Figure 1 0- I 3 ). The greater the testicular circumference, the greater the sperm producing capabil- ity, in other words, "the bigger the factory, the greater the output." Because of the non-pendular scrotum in the boar and stallion, scrotal width or length is used as the measurement. Assuming that a male can develop an erect penis, mount and ejaculate in the female, his potential fertility is determined by: • his sperm producing ability • the viability of his spermatozoa • the number of morphologically abnor- mal spermatozoa that he ejaculates • the number of functionally normal spermatozoa that he ejaculates Spermatozoal Viability is Judged by Evaluating Motility Even though a male can produce large quanti- ties of spem1atozoa, it is important that these spem1 are alive and highly motile. M oti lity is generally de- scribed as the ability of spe1m to swim progressively forward. Moti lity is the most commonly used assess- ment of viability. It is expressed as an estimate of the percentage of sperm that are swimming in a linear fashion with in a given environment as determined mi- croscopically. Unfortunately, the relationship between percentage of motile sperm and fertility is not a good one. However, if few spennatozoa within a series of Ve tB oo ks .ir 218 Spermatogenesis Table 10-1. Duration of the Stages of the Cycle of the Seminiferous Epithelium in Various Species Stage Bull Ram Boar Stallion Rabbit I 4.2 2.2 1.1 2.0 3.1 II 1.2 1.1 1.4 1.8 1.5 Ill 2.7 1.9 0.4 0.4 0.8 IV 1.7 1.1 1.2 1.9 1.2 v 0.2 0.4 0.8 0.9 0.5 VI 0.8 1.3 1.6 1.7 1.7 VII 1.1 1.1 1.0 1.6 1.3 VIII 1.6 1.0 0.8 1.9 0.9 TOTAL A 13.5 10.1 8.3 12.2 11 .0 SPERMATOGENESIS8 61 47 39 55 48 ATotal days required for 1 cycle of the seminiferous epithelium BApproximate days to complete spermatogenesis (sperm.atogonia to spermatozoa) The spermatogenic wave is the sequential ordering of stages along the length of the seminiferous tubule. The duration of each stage of the cycle of the seminiferous epithelium varies with species, as does the length of the cycle of the seminiferous epithelium. Variations in stage, cycle length and total time required for spemmtogenesis are presented in Table 1 0-l. The spermatogenic wave refers to the differ- ences at any given instant in time along the length of the seminiferous tubule. Imagine that you could run down the lumen of the seminiferous tubule. As you run down the tubule, you will encounter zones that are near spem1iation (stage VIII). The distance between these spermiation sites is relatively constant. During the wave, each stage of the seminiferous epithelium transitions to a successively more advanced stage. For example, a stage I tubule will later become a stage II and stage II will later become a III and so on. Thus, the site of spermiation along the tubule is constantly changing, creating a "wave" ofspenn release down the length of the tubule. This "wave" is like the wave con- ducted by football fans in a stadium. When the fans stand up, they mimic spenniation. They sit back down and don't stand up again until it's their tum again. The time spent sitting (stages I-VII) is much longer than the time spent standing. As the wave in the stadium continues, repeated standing and sitting takes place at a relatively constant rate. So does spenniation. The physiologic importance of the spermatogenic wave is to provide a relatively constant supply of spem1atozoa to the epididymis, creating a pool for ejaculation. Figure 10-13. Scrotal Circumference Measurements are Good Indicators of Sperm Producing Ability (Photograph courtesy of Select Sires, Inc. Plain City, Ohio, www.selectsires.com) Accurate scrotal circumference measurements require that both testicles be pushed ventrally by applying pressure to the spermatic cord. A specially designed tape is then placed around the scrotum at its widest point and a measure- ment is taken (in this case, 40cm). Spermatogenesis 219 Table 10-2. Testicular Characteristics and Sperm Production Estimates of Sexually Mature Mammals SBecies Gross weight of SBerm Broduced Daily Baired testes Ber gram of SBermatozoal (grams) testicular Barenchyma Broduction Beef Bull 650 11x1 06 6x109 Boar 750 23x106 16x109 Cat 21 16x106 32x1 06 Dairy Bull 725 12x106 7.5x109 Dog (16 kg body weight) 31 17x106 0.50x109 Man 35 4x106 0.13x109 Rabbit 6 25x106 0.20x109 Ram* 550 21x106 10x109 Rooster*** 25 100x106 2.5x109 Stallion** 340 16x1 06 5x1 09 *in breeding season (shortening-day length), **in breeding season (increasing day l!:mgth), · ***varies greatly with management and strain Daily sperm production (DSP) is defined as the total number of spennatozoa produced per day by both testicles of the male. Accurate measurement of DSP requires removal of all or a portion of the testicle and thus, DSP cannot be measured using non- invasive techniques . However, noninvasive measures such as total number of spermatozoa ejaculated into an artificial vagina with daily ejaculations for 2-3 weeks gives a good estimate of DSP. Interspecies variation in testicular weights, sperm produced per gram of testicular parenchyma ·and daily sperm production is presented in Table 10-2. The number of spermatozoa produced per day per gram of testicular parenchyma is referred to as efficiency of sperm production. Daily sperm production is dependent, at least in part, on the number of Sertoli cells populating the testes. For example, the higher the number of Sertoli cells, the higher the spem1atozoal production rates. Numbers of Sertoli cells also have been positively correlated with spermatogonial and spermatid numbers. Testicular Size is a Good Estimator of Sperm Producing Ability To detennine a given male 's sperm producing capability, it is necessary to collect ejaculates from the animal for a period of time. This enables one to ac- curately estimate how many spem1atozoa the animal can produce per unit time. If collection of semen is not possible, a good estimate ofspenn producing capability can be made by measuring the circumference of both testicles (See Figure 1 0- I 3 ). The greater the testicular circumference, the greater the sperm producing capabil- ity, in other words, "the bigger the factory, the greater the output." Because of the non-pendular scrotum in the boar and stallion, scrotal width or length is used as the measurement. Assuming that a male can develop an erect penis, mount and ejaculate in the female, his potential fertility is determined by: • his sperm producing ability • the viability of his spermatozoa • the number of morphologically abnor- mal spermatozoa that he ejaculates • the number of functionally normal spermatozoa that he ejaculates Spermatozoal Viability is Judged by Evaluating Motility Even though a male can produce large quanti- ties of spem1atozoa, it is important that these spem1 are alive and highly motile. M oti lity is generally de- scribed as the ability of spe1m to swim progressively forward. Moti lity is the most commonly used assess- ment of viability. It is expressed as an estimate of the percentage of sperm that are swimming in a linear fashion with in a given environment as determined mi- croscopically. Unfortunately, the relationship between percentage of motile sperm and fertility is not a good one. However, if few spennatozoa within a series of Ve tB oo ks .ir 220 Spermatogenesis Figure 10-14. Some Common Abnormalities in Bovine Sperm as Observed With Differential-Interference Contrast Microscopy (Courtesy of R.G. Saacke, Virginia Polytechnic Institute and State University) Head Abnormalities Crater Defect (Nuclear Vacuoles) Tapered Heads Ruffled Acrosome Knobbed Acrosome Tail Abnormalities Coiled Tail Double Midpiece Folded Tail Detached Head Note: A vast amount of information is available for bulls because of intense scrutiny given to abnormal sperm by commercial AI organizations. For details on the incidence, causes and their effects on fertility of abnormal sperm shown here (and other types as well) see Barth and Oko, 1998 in the Key References section at the end of the chapter. Most descriptions in the bull apply to other mammals as well. ejaculates are motile, the assumption can be made correctly that spenn in the ejaculate are not alive and therefore cannot fertilize the egg. There are many ways to tell if a spennatozoon is alive. These include oxygen consumption, exclusion of certain dyes by the plasma membrane (live-dead stains) and examination by flow cytometry. However, the simplest and most common is to determine if a cell moves forward in a progressive manner (motile) when examined at 37°C. Evaluating motility at temperatures below 3 7oc is not a good practice because motility stops at about l8°C. The use of a phase-contrast microscope (essential to clearly visualize spenn) and a heated stage (to allow sperm to display their potential to swim) is the most practical way to evaluate motility of sperm. Decisions about motility should never be based solely on one ejaculate. There are Many Types of Abnormal Spermatozoa As you might imagine, a process that poten- tially produces up to 20 billion spenn per day (over 200,000 per second) will have enors. These errors are expressed as abnormal spermatozoa some of which can be detected on the basis of abnomml shape. Mor- phologically abnom1al spem1 can be defined as any shape characteristic deviating from normal. Every ejaculate will contain between 5 and 15% abnormal sperm and these levels are generally considered ac- ceptable. Reduced fertility may result when morpho- logically abnormal sperm exceed 20% of sperm in the ejaculate. Some morphologic abnonnalities have a severe effect on fertility while others have little or no effect. In general, morphologic abnormalities either originate in the testes because of faulty differentiation or in the epididymis because of faulty epididymal transit and/or maturation. The latter results in the presence of cytoplasmic droplets (See Chapter 3 ). Morphologically abnormal sperm of testicular origin are generally classi- fied as either head abnormalities or tail abnonnalities. Potential fertil ity of the male can be related to the percentage of morphologically abnormal spenn within an ejaculate. Some common abnonnalities in bull spem1 are shown in Figure 10-14. Some abnor- malities are heritable and result in sterility. Males possessing these abnormalities should be eliminated from the gene pool. Evaluation of the proportion of abnormal sperm in an ejaculate requires a microscope. For most laboratories, a phase-contrast microscope and a skilled observer will yield satisfactory diagnoses. For laboratories examining large numbers of ejaculates, a differential-interference contrast microscope is preferred because of the high resolution and the cellular detail generated with this optical system. A differential-interference contrast microscope transfonns gradients in intracellular density into an optical image that appears as a relief or an indentation in the cell. Thus, abnormalities of both the head and tai l can be observed and quantitated with a high degree of preci- sion. All ofthe micrographs presented in Figure 10-14 were generated with a differential-interference contrast microscope. A description of each type of abnormality that one can encounter within a series of ejaculates is beyond the scope of this text. It must be recognized that morphologic ab- normalities represent only one characteristic among a myriad of possibilities for abnormal function. For example, abnormal nuclear composition (faulty DNA), abnormal biochemical composition, surface protein deficiency and faulty response to stimuli within the female tract represent only a few possibilities that may limit the function of spermatozoa. Artificial Insemination is the Single Most Important Physiologic Technology Ever Devised for Accelerating Genetic Improvement The components of artificial insemination (AI) in the fact box, will be presented in the chapters that discuss the physiology of each process. For example, collection of semen involves behavioral issues requir- ing specific stimuli for mounting and ejaculation (See Chapter II). Preservation and extension of semen is an issue associated with providing an optimum in-vitro environment to preserve sperm viability (Current Chap- ter). Finally, insemination of the female delivers sperm Spermatogenesis 221 to the female reproductive tract so that adequate num- bers are present and fertilization can be accomplished (See Chapter 12). Successful AI can be accomplished in any species provided the criteria below are met. The majm· steps of artificial insemination are: • collection of semen from the male (See Chapter 11) • preservation and extension of sperm (See Below) • insemination of the female (See Chapter 12) Artificial insemination is a common practice in some species. For example, over 7 million dairy cows and about 2 million beef cows are artificially inseminated annually in the United States. All turkey hens in commercial flocks (over 300 million) are artifi- cially inseminated because the toms have such a broad breast that they cannot mount and copulate. The use of AI in swine has exploded during the past 10 years. Approximately 85% of all female swine are artificially inseminated in the United States. This means that about 5 million females are artificially inseminated each year. Typically, each female receives about 2.3 inseminations per year in order to deliver two litters per year. This means that in the swine industry there are about 11.3 million artificial inseminations per year. This means that nearly 120 million pigs are s ired by artificial in- semination in the United States each year. It should be emphasized that widespread ap- plication of artificial insemination allows for intense and relatively rapid genetic selection that significantly improves production efficiency in dairy, beef, poultry and swine. Improvements in animal efficiency result in a wide variety ofhighly affordable animal products to the consumer. Artificial insemination is also common for horses. In addition, many species in zoos have been artificially inseminated to avoid inbreeding and facili- tate reproduction in exotic and endangered species. In 2002 the first baby elephant was produced by artificial insemination at the National Zoo in Washington D.C. Artificial insemination is used routinely in assisted reproductive techniques in humans, allowing pregnan- cies to occur that otherwise would not be possible (See Chapter 16). 10] Ve tB oo ks .ir 220 Spermatogenesis Figure 10-14. Some Common Abnormalities in Bovine Sperm as Observed With Differential-Interference Contrast Microscopy (Courtesy of R.G. Saacke, Virginia Polytechnic Institute and State University) Head Abnormalities Crater Defect (Nuclear Vacuoles) Tapered Heads Ruffled Acrosome Knobbed Acrosome Tail Abnormalities Coiled Tail Double Midpiece Folded Tail Detached Head Note: A vast amount of information is available for bulls because of intense scrutiny given to abnormal sperm by commercial AI organizations. For details on the incidence, causes and their effects on fertility of abnormal sperm shown here (and other types as well) see Barth and Oko, 1998 in the Key References section at the end of the chapter. Most descriptions in the bull apply to other mammals as well. ejaculates are motile, the assumption can be made correctly that spenn in the ejaculate are not alive and therefore cannot fertilize the egg. There are many ways to tell if a spennatozoon is alive. These include oxygen consumption, exclusion of certain dyes by the plasma membrane (live-dead stains) and examination by flow cytometry. However, the simplest and most common is to determine if a cell moves forward in a progressive manner (motile) when examined at 37°C. Evaluating motility at temperatures below 3 7oc is not a good practice because motility stops at about l8°C. The use of a phase-contrast microscope (essential to clearly visualize spenn) and a heated stage (to allow sperm to display their potential to swim) is the most practical way to evaluate motility of sperm. Decisions about motility should never be based solely on one ejaculate. There are Many Types of Abnormal Spermatozoa As you might imagine, a process that poten- tially produces up to 20 billion spenn per day (over 200,000 per second) will have enors. These errors are expressed as abnormal spermatozoa some of which can be detected on the basis of abnomml shape. Mor- phologically abnom1al spem1 can be defined as any shape characteristic deviating from normal. Every ejaculate will contain between 5 and 15% abnormal sperm and these levels are generally considered ac- ceptable. Reduced fertility may result when morpho- logically abnormal sperm exceed 20% of sperm in the ejaculate. Some morphologic abnonnalities have a severe effect on fertility while others have little or no effect. In general, morphologic abnormalities either originate in the testes because of faulty differentiation or in the epididymis because of faulty epididymal transit and/or maturation. The latter results in the presence of cytoplasmic droplets (See Chapter 3 ). Morphologically abnormal sperm of testicular origin are generally classi- fied as either head abnormalities or tail abnonnalities. Potential fertil ity of the male can be related to the percentage of morphologically abnormal spenn within an ejaculate. Some common abnonnalities in bull spem1 are shown in Figure 10-14. Some abnor- malities are heritable and result in sterility. Males possessing these abnormalities should be eliminated from the gene pool. Evaluation of the proportion of abnormal sperm in an ejaculate requires a microscope. For most laboratories, a phase-contrast microscope and a skilled observer will yield satisfactory diagnoses. For laboratories examining large numbers of ejaculates, a differential-interference contrast microscope is preferred because of the high resolution and the cellular detail generated with this optical system. A differential-interference contrast microscope transfonns gradients in intracellular density into an optical image that appears as a relief or an indentation in the cell. Thus, abnormalities of both the head and tai l can be observed and quantitated with a high degree of preci- sion. All ofthe micrographs presented in Figure 10-14 were generated with a differential-interference contrast microscope. A description of each type of abnormality that one can encounter within a series of ejaculates is beyond the scope of this text. It must be recognized that morphologic ab- normalities represent only one characteristic among a myriad of possibilities for abnormal function. For example, abnormal nuclear composition (faulty DNA), abnormal biochemical composition, surface protein deficiency and faulty response to stimuli within the female tract represent only a few possibilities that may limit the function of spermatozoa. Artificial Insemination is the Single Most Important Physiologic Technology Ever Devised for Accelerating Genetic Improvement The components of artificial insemination (AI) in the fact box, will be presented in the chapters that discuss the physiology of each process. For example, collection of semen involves behavioral issues requir- ing specific stimuli for mounting and ejaculation (See Chapter II). Preservation and extension of semen is an issue associated with providing an optimum in-vitro environment to preserve sperm viability (Current Chap- ter). Finally, insemination of the female delivers sperm Spermatogenesis 221 to the female reproductive tract so that adequate num- bers are present and fertilization can be accomplished (See Chapter 12). Successful AI can be accomplished in any species provided the criteria below are met. The majm· steps of artificial insemination are: • collection of semen from the male (See Chapter 11) • preservation and extension of sperm (See Below) • insemination of the female (See Chapter 12) Artificial insemination is a common practice in some species. For example, over 7 million dairy cows and about 2 million beef cows are artificially inseminated annually in the United States. All turkey hens in commercial flocks (over 300 million) are artifi- cially inseminated because the toms have such a broad breast that they cannot mount and copulate. The use of AI in swine has exploded during the past 10 years. Approximately 85% of all female swine are artificially inseminated in the United States. This means that about 5 million females are artificially inseminated each year. Typically, each female receives about 2.3 inseminations per year in order to deliver two litters per year. This means that in the swine industry there are about 11.3 million artificial inseminations per year. This means that nearly 120 million pigs are s ired by artificial in- semination in the United States each year. It should be emphasized that widespread ap- plication of artificial insemination allows for intense and relatively rapid genetic selection that significantly improves production efficiency in dairy, beef, poultry and swine. Improvements in animal efficiency result in a wide variety ofhighly affordable animal products to the consumer. Artificial insemination is also common for horses. In addition, many species in zoos have been artificially inseminated to avoid inbreeding and facili- tate reproduction in exotic and endangered species. In 2002 the first baby elephant was produced by artificial insemination at the National Zoo in Washington D.C. Artificial insemination is used routinely in assisted reproductive techniques in humans, allowing pregnan- cies to occur that otherwise would not be possible (See Chapter 16). 10] Ve tB oo ks .ir 10 222 Spermatogenesis Immediately after collection, the following information is needed: • ejaculate volume • concentration of spermatozoa in the ejaculate (sperm/mL ejaculate) • percentage of motile sperm In Vitro Preservation is Obligatory for Successful AI After semen has been collected from the male, in vitro preservation of sperm for a period of time must be accomplished before successful delivery ofspenn to the female can take place. Preservation and dilution of sperm requires an environment that minimizes death of spem1. It also requires )mow ledge about the volume of the ejaculate, the concentration of sperm in the ejaculate and their motility. Having the above information is necessary to determine the appropriate dilution rate of the spenn so multiple females can be inseminated with sperm from the same ejaculate. Where multiple females are to be inseminated, one must know the concentration ofspenn in the ejaculate so that each female can be inseminated with a threshold number (minimum number) of spenna- tozoa to maximize the probability of a pregnancy. Evaluation of Semen is Needed Before Dilution Immediately after collection of the ejaculate seminal evaluation is conducted. First, ejaculate vol- ume must be determined. Second, the percentage of sperm displaying progressive motility (swimming in a linear fashion) is estimated by viewing live smears at 37°C with a phase-contrast microscope. Third, the concentration of spermatozoa in the ejaculate is de- termined by comparing optical density of a standard dilution of neat semen with a reference standard. The greater the spenn concentration, the greater the optical density. The sperm concentration is determined from a standard curve where optical density is plotted against concentration. The ejaculate volume and concentration of spennatozoa are important elements of seminal evalu- ation because the volume multiplied by the concentra- tion equals the total number of sperm in the ejaculate as shown in the equation below. Total Sperm in Ejac. = Ejac. Vol. x Sperm/ml Knowing the total number of sperm in the ejaculate enables the laboratory technician to determine how many insemination doses are potentially avai lable within each ejaculate. A high percentage of motile sperm (60% or more) indicates good quality. An ejaculate containing few motile sperm (less than 50%) is a candidate for discard especially if sperm are to be frozen and later thawed to inseminate females. Information for determining the number of insemination doses contained in a typical ejaculate for the bull is presented below. These calculations apply in principle to other species except that the values (vol- ume, concentration and motility) may vary significantly from spec ies-to-species and from male-to-male. Ejaculate volume = 6 ml Sperm concentration = 1.0 X I 09 sperm/ml (I billion) Total sperm in ejaculate = 6 ml x I .Ox I 09sperm/m i = 6x I 09 (6 billion) Progressive motility = 70% Total motile sperm= 6.0xl09 x.7 = 4.2x I09 motile sperm/ejaculate Desired concentration = 15x I 06/dose ( I insemination) Number of doses= 4.2x I 09/15x I 06 = 280 doses To determine the number of doses a single ejaculate will generate, one must divide the total number of sperm by the desired number of spenn in each dose. For example, the ejaculate illustrated above contains 4.2 billion motile sperm. If a dose of semen is intended to contain 15 million motile spenn ( 15x I 06) then we divide 4.2x I 09 sperm by 15x 1 06 sperm. By the computation in the box above, this ejaculate will produce 280 doses (units) of semen after di lution. Good seminal extenders must: • be isotonic • be good buffers • minimize cold damage ("cold shock'') • provide appropriate nutrients • prevent microbial growth • maintain viability • be relatively low in cost Seminal Extenders Extend Both Sperm Viability and Numbers After it has been determined that the ejaculate is of sufficient quali ty (volume,% motile spermatozoa and concentration of spermatozoa) the sperm must be preserved so that they can be used to inseminate females over an extended period of time (e.g. several days to one week). To inseminate many females with a single ejaculate the neat semen must be extended so that each inseminate dose contains less sperm than the entire ejaculate. Typically, the solution into which spermatozoa are diluted is referred to as an extender because it not only "extends" the number of sperm in the original ejaculate, but it "extends" their functional life. Extenders may be purchased from commercial sources or they can be prepared in the laboratory. The Extender Must be Isotonic You will recall from your basic biology class that when a cell is in an isotonic solution there is no net movement of water into or out of the cell. A hy- potonic solution is a solution in which the medium contains fewer osmotically active particles than the cell and water rushes into the cell and the cell membrane ruptures (cell lysis). In contrast, a hyper·tonic solution contains more osmotically active particles than does the inside of the cell and water moves out of the cell and it dehydrates. Providing the proper osmotic balance of the seminal extender is obligatmy for survival of spennatozoa. An Extender Must Buffer and Protect A buffer is a material that prevents marked changes in pH (hydrogen ion concentration). Extremes in pH, both acidic and alkaline, are damaging to all cells including spemmtozoa. The cell membrane of a spem1atozoon is quite sensitive to sudden drops in temperature ("cold shock"). Care must be taken to prevent sudden declines in tem- perature so that the cell membrane and motile apparatus of the sperm do not become damaged. In neat semen, particular care must be taken to prevent damage to the spermatozoa. The design of the artificial vagina is important so that "cold shock" can be prevented (See Chapter I I). Slow, controlled cooling of sperm is important because it lowers the temperahtre gradually and minimizes stresses on the cell membrane. A low storage temperature reduces metabolism by about 50% for each 1 O"C decline. Spem1 are analogous to a battery. They have no option but to "run down." Unfortu- nately, recharging sperm cells after ejaculation is not possible. Spermatogenesis 223 Where the goal is to extend the semen for a sustained period oftime (I week to years), a cryopro- tectant in the extender is required. Cryoprotectants are materials that protect the cells against cold damage that would occur between 0 and -50"C. These compounds protect spem1 membranes by minimizing ice crystal for- mation within the cell. In general, cyroprotectants can be classified as cell-permeating (glycerol, DMSO) and non-permeating (milk protein and egg yolk lipoprotein). Depending on species, one or a combination of types of cryoprotectants may be optimum. Common cryopro- tectants are glycerol and dimethyl sulfoxide (DMSO) with glycerol being the dominant ctyoprotectant for frozen sperm. Physiologic fluids are used frequently as extender ingredients. These include hen's egg yolk and cow's milk. These provide macromolecules that minimize cold damage and provide nutrients. The rate of temperature decline and ultimate storage temperature are important depending on spe- cies. For example, a slow decline in temperature is important in the bull and the stall ion but is of much less importance to the dog and human. The influence of holding temperatures for unfrozen spetm also vaty among species. For example, bull and stallion semen can be stored effectively at 5"C while boar semen re- quires 18"C for best preservation. These differences are due, at least in part, to differences in lipid composition of the spenn membranes. Spermatozoa have no anabolic capabi lity. In other words, spennatozoa are incapable of synthesizing materials for energy and repair. Therefore, the viability of sperm is totally dependent on the environment in which they are suspended. Nutrients need to be sup- plied in adequate quantities so that metabolism can be maintained for the appropriate duration of time. The major nutrients for spenn metabolism are fructose and glucose. Sperm are capable of converting glucose to fructose and metabolizing it to fuel their motility. Ejaculated Semen is Not Sterile Bacteria are present in the sheath and on the penis of the male and occasionally in the urethra and vesicular glands and therefore semen contains a variety of microorganisms. Seminal plasma and extender are ideal mediums for microbial growth and steps must be taken to mi nimize this growth. Antibiotics typically are added to the neat semen and extender to prevent micro- bial growth. Antibiotics such as penicillin, liquamycin, linco-spectin and streptomycin may be added in some combination to neat semen and to extenders. Preservation of spermatozoa can be accom- plished using two methods. For relatively short term use, fresh liquid semen is used after the semen has been extended. In most species, liquid semen can be cooled Ve tB oo ks .ir 10 222 Spermatogenesis Immediately after collection, the following information is needed: • ejaculate volume • concentration of spermatozoa in the ejaculate (sperm/mL ejaculate) • percentage of motile sperm In Vitro Preservation is Obligatory for Successful AI After semen has been collected from the male, in vitro preservation of sperm for a period of time must be accomplished before successful delivery ofspenn to the female can take place. Preservation and dilution of sperm requires an environment that minimizes death of spem1. It also requires )mow ledge about the volume of the ejaculate, the concentration of sperm in the ejaculate and their motility. Having the above information is necessary to determine the appropriate dilution rate of the spenn so multiple females can be inseminated with sperm from the same ejaculate. Where multiple females are to be inseminated, one must know the concentration ofspenn in the ejaculate so that each female can be inseminated with a threshold number (minimum number) of spenna- tozoa to maximize the probability of a pregnancy. Evaluation of Semen is Needed Before Dilution Immediately after collection of the ejaculate seminal evaluation is conducted. First, ejaculate vol- ume must be determined. Second, the percentage of sperm displaying progressive motility (swimming in a linear fashion) is estimated by viewing live smears at 37°C with a phase-contrast microscope. Third, the concentration of spermatozoa in the ejaculate is de- termined by comparing optical density of a standard dilution of neat semen with a reference standard. The greater the spenn concentration, the greater the optical density. The sperm concentration is determined from a standard curve where optical density is plotted against concentration. The ejaculate volume and concentration of spennatozoa are important elements of seminal evalu- ation because the volume multiplied by the concentra- tion equals the total number of sperm in the ejaculate as shown in the equation below. Total Sperm in Ejac. = Ejac. Vol. x Sperm/ml Knowing the total number of sperm in the ejaculate enables the laboratory technician to determine how many insemination doses are potentially avai lable within each ejaculate. A high percentage of motile sperm (60% or more) indicates good quality. An ejaculate containing few motile sperm (less than 50%) is a candidate for discard especially if sperm are to be frozen and later thawed to inseminate females. Information for determining the number of insemination doses contained in a typical ejaculate for the bull is presented below. These calculations apply in principle to other species except that the values (vol- ume, concentration and motility) may vary significantly from spec ies-to-species and from male-to-male. Ejaculate volume = 6 ml Sperm concentration = 1.0 X I 09 sperm/ml (I billion) Total sperm in ejaculate = 6 ml x I .Ox I 09sperm/m i = 6x I 09 (6 billion) Progressive motility = 70% Total motile sperm= 6.0xl09 x.7 = 4.2x I09 motile sperm/ejaculate Desired concentration = 15x I 06/dose ( I insemination) Number of doses= 4.2x I 09/15x I 06 = 280 doses To determine the number of doses a single ejaculate will generate, one must divide the total number of sperm by the desired number of spenn in each dose. For example, the ejaculate illustrated above contains 4.2 billion motile sperm. If a dose of semen is intended to contain 15 million motile spenn ( 15x I 06) then we divide 4.2x I 09 sperm by 15x 1 06 sperm. By the computation in the box above, this ejaculate will produce 280 doses (units) of semen after di lution. Good seminal extenders must: • be isotonic • be good buffers • minimize cold damage ("cold shock'') • provide appropriate nutrients • prevent microbial growth • maintain viability • be relatively low in cost Seminal Extenders Extend Both Sperm Viability and Numbers After it has been determined that the ejaculate is of sufficient quali ty (volume,% motile spermatozoa and concentration of spermatozoa) the sperm must be preserved so that they can be used to inseminate females over an extended period of time (e.g. several days to one week). To inseminate many females with a single ejaculate the neat semen must be extended so that each inseminate dose contains less sperm than the entire ejaculate. Typically, the solution into which spermatozoa are diluted is referred to as an extender because it not only "extends" the number of sperm in the original ejaculate, but it "extends" their functional life. Extenders may be purchased from commercial sources or they can be prepared in the laboratory. The Extender Must be Isotonic You will recall from your basic biology class that when a cell is in an isotonic solution there is no net movement of water into or out of the cell. A hy- potonic solution is a solution in which the medium contains fewer osmotically active particles than the cell and water rushes into the cell and the cell membrane ruptures (cell lysis). In contrast, a hyper·tonic solution contains more osmotically active particles than does the inside of the cell and water moves out of the cell and it dehydrates. Providing the proper osmotic balance of the seminal extender is obligatmy for survival of spennatozoa. An Extender Must Buffer and Protect A buffer is a material that prevents marked changes in pH (hydrogen ion concentration). Extremes in pH, both acidic and alkaline, are damaging to all cells including spemmtozoa. The cell membrane of a spem1atozoon is quite sensitive to sudden drops in temperature ("cold shock"). Care must be taken to prevent sudden declines in tem- perature so that the cell membrane and motile apparatus of the sperm do not become damaged. In neat semen, particular care must be taken to prevent damage to the spermatozoa. The design of the artificial vagina is important so that "cold shock" can be prevented (See Chapter I I). Slow, controlled cooling of sperm is important because it lowers the temperahtre gradually and minimizes stresses on the cell membrane. A low storage temperature reduces metabolism by about 50% for each 1 O"C decline. Spem1 are analogous to a battery. They have no option but to "run down." Unfortu- nately, recharging sperm cells after ejaculation is not possible. Spermatogenesis 223 Where the goal is to extend the semen for a sustained period oftime (I week to years), a cryopro- tectant in the extender is required. Cryoprotectants are materials that protect the cells against cold damage that would occur between 0 and -50"C. These compounds protect spem1 membranes by minimizing ice crystal for- mation within the cell. In general, cyroprotectants can be classified as cell-permeating (glycerol, DMSO) and non-permeating (milk protein and egg yolk lipoprotein). Depending on species, one or a combination of types of cryoprotectants may be optimum. Common cryopro- tectants are glycerol and dimethyl sulfoxide (DMSO) with glycerol being the dominant ctyoprotectant for frozen sperm. Physiologic fluids are used frequently as extender ingredients. These include hen's egg yolk and cow's milk. These provide macromolecules that minimize cold damage and provide nutrients. The rate of temperature decline and ultimate storage temperature are important depending on spe- cies. For example, a slow decline in temperature is important in the bull and the stall ion but is of much less importance to the dog and human. The influence of holding temperatures for unfrozen spetm also vaty among species. For example, bull and stallion semen can be stored effectively at 5"C while boar semen re- quires 18"C for best preservation. These differences are due, at least in part, to differences in lipid composition of the spenn membranes. Spermatozoa have no anabolic capabi lity. In other words, spennatozoa are incapable of synthesizing materials for energy and repair. Therefore, the viability of sperm is totally dependent on the environment in which they are suspended. Nutrients need to be sup- plied in adequate quantities so that metabolism can be maintained for the appropriate duration of time. The major nutrients for spenn metabolism are fructose and glucose. Sperm are capable of converting glucose to fructose and metabolizing it to fuel their motility. Ejaculated Semen is Not Sterile Bacteria are present in the sheath and on the penis of the male and occasionally in the urethra and vesicular glands and therefore semen contains a variety of microorganisms. Seminal plasma and extender are ideal mediums for microbial growth and steps must be taken to mi nimize this growth. Antibiotics typically are added to the neat semen and extender to prevent micro- bial growth. Antibiotics such as penicillin, liquamycin, linco-spectin and streptomycin may be added in some combination to neat semen and to extenders. Preservation of spermatozoa can be accom- plished using two methods. For relatively short term use, fresh liquid semen is used after the semen has been extended. In most species, liquid semen can be cooled Ve tB oo ks .ir 224 Spermatogenesis Table 10-3. Offspring Ratios of Spermatozoa Sorted for the X and Y Chromosome Species Sorted for Y Chromosome Sorted for X Chromosome %Male %Female %Male %Female Cattle 81 Rabbit 81 Swine 75 and stored at near freezing temperature ( 5°C) for several days to about one week. In swine, 17 -18"C is optimum. When widespread distribution and long-term usage is a requirement, frozen semen is the prefened method of preservation. When frozen semen is used, careful attention to freezing and thawing techniques must be practiced. Freezing and thawing compromises sper- matozoal viability in all species. However, the degree to which viability and fertility are affected depends on the individual male and species. Sex of the Conceptus is Determined by the Sperm Because Each Spermatozoon Contains Either an X or a Y Chromosome As you already know, each secondary sperma- tocyte produces two haploid daughter spennatids. Each spermatid contains either an X or a Y chromosome. Sperm containing the X chromosome that fertilize an oocyte will generate a female. Spenn containing the Y chromosome will generate a male (See Figure 10-15). The desire to separate the X and Y bearing spenn is driven by the fact that one sex has signifi- cantly more economic value than the other in certain species. For example, in the dairy industry, bull calves are of little value since about 80% of all cows in the U.S. (and higher on a worldwide basis) are artificially inseminated. Thus, relatively few bulls are required to inseminate the cows in the national dairy herd. The cow is the primary income generator for a dairy business. It would be advantageous to have a high percentage of female offspring since lactation is limited to the female. In other food producing animals, it might be more desirable to produce higher percentage males since these animals grow faster and have more desired meat characteristics. The X and Y chromosome contain differ- ent quantities of DNA. For example, an X bearing spenn contains 2.8-4.2% more DNA (depending on the species) than does a Y bearing sperm. Based on this difference, it is possible to separate the X and Y bearing spenn into two subpopulations. The separation 19 11 89 19 6 94 25 10 90 procedure requires the uptake of a DNA stain or dye (called a flourochrome) into both living and dead sper- matozoa. Those sperm that contain the X chromosome "take-up" more DNA dye than do sperm containing the Y chromosome. Vital dyes used to stain sperm produce emissions oflight at a specific wavelength when excited or activated by light at a specific wavelength. The technology utilized for separation of X and Y bearing spennatozoa is referred to as flow cytometry (sometimes called "cell sorting"). Figure I 0-15 high- lights the major steps for separation of the X and Y bearing spem1atozoa using flow cytometry. Experimental evidence clearly shows the success of this technology for separating the X and Y bearing spermatozoa from common mammals and most experiments have yielded 80-90% successful separa- tion for either males or females in cattle, swine and rabbits (See Table 1 0-3). Several factors have limited the efficiency of this technology, but the application is now widespread in dairy cattle and is feasible in many other species. Regardless of the problems associated with separating the X andY bearing spermatozoa, the tech- nology is now available through artificial insemination organizations. Thus, it is reasonable to expect that separation of X andY bearing spermatozoa eventually will be commonplace. Manipulation of the sex ratio under controlled conditions could greatly impact the efficiency offood animal production. Spermatogenesis 225 Figure 10-15. Major Steps for Separation of X andY Bearing Spermatozoa by Flow Cytometry I' Step 1 X and Y bearing spermatozoa are produced by the testis and ejaculated by the male. Step 2 Ejaculated spermatozoa are treated with a fl uorescent DNA dye. X bearing sperm absorb more dye than Y beanng. sperm. They therefore emit more intense light when exc1ted by a laser. Sperm also are treated with a dye that greatly suppresses the signal from dead sperm. Dead sperm are therefore identified and rejected. Step 3 Once enter the flow cytometer chamber, they smgle-file through a small nozzle. At a region just out- Side the nozzle, a laser beam excites the fluorescent dye in sper£!1. Eac.h sperm emits light at a wavelength and that IS directly related to quantity of DNA. X- live sperm produce more intensity. A light sensing dev1ce 1s coupled to a computer that determines the intensity of light emission by each sperm and the order of passage of each sperm through a column below the nozzle. When the sperm pass by charged plates, they are assigned either a positive or negative charge depending on their DNA content or Y chromosome). When the microdroplet containing a s1ngle sperm passes through an electromagnetic field the computer applies an appropriate charge and directs the (and to one side or the other. Dead sperm are discarded 1nto the center tube. Thus, at the conclusion of the separation process there are three vessels that contain sperm. One contains a high proportion of X one contains a high proportion of Y chromosome bearing and one contains dead sperm. Ve tB oo ks .ir 224 Spermatogenesis Table 10-3. Offspring Ratios of Spermatozoa Sorted for the X and Y Chromosome Species Sorted for Y Chromosome Sorted for X Chromosome %Male %Female %Male %Female Cattle 81 Rabbit 81 Swine 75 and stored at near freezing temperature ( 5°C) for several days to about one week. In swine, 17 -18"C is optimum. When widespread distribution and long-term usage is a requirement, frozen semen is the prefened method of preservation. When frozen semen is used, careful attention to freezing and thawing techniques must be practiced. Freezing and thawing compromises sper- matozoal viability in all species. However, the degree to which viability and fertility are affected depends on the individual male and species. Sex of the Conceptus is Determined by the Sperm Because Each Spermatozoon Contains Either an X or a Y Chromosome As you already know, each secondary sperma- tocyte produces two haploid daughter spennatids. Each spermatid contains either an X or a Y chromosome. Sperm containing the X chromosome that fertilize an oocyte will generate a female. Spenn containing the Y chromosome will generate a male (See Figure 10-15). The desire to separate the X and Y bearing spenn is driven by the fact that one sex has signifi- cantly more economic value than the other in certain species. For example, in the dairy industry, bull calves are of little value since about 80% of all cows in the U.S. (and higher on a worldwide basis) are artificially inseminated. Thus, relatively few bulls are required to inseminate the cows in the national dairy herd. The cow is the primary income generator for a dairy business. It would be advantageous to have a high percentage of female offspring since lactation is limited to the female. In other food producing animals, it might be more desirable to produce higher percentage males since these animals grow faster and have more desired meat characteristics. The X and Y chromosome contain differ- ent quantities of DNA. For example, an X bearing spenn contains 2.8-4.2% more DNA (depending on the species) than does a Y bearing sperm. Based on this difference, it is possible to separate the X and Y bearing spenn into two subpopulations. The separation 19 11 89 19 6 94 25 10 90 procedure requires the uptake of a DNA stain or dye (called a flourochrome) into both living and dead sper- matozoa. Those sperm that contain the X chromosome "take-up" more DNA dye than do sperm containing the Y chromosome. Vital dyes used to stain sperm produce emissions oflight at a specific wavelength when excited or activated by light at a specific wavelength. The technology utilized for separation of X and Y bearing spennatozoa is referred to as flow cytometry (sometimes called "cell sorting"). Figure I 0-15 high- lights the major steps for separation of the X and Y bearing spem1atozoa using flow cytometry. Experimental evidence clearly shows the success of this technology for separating the X and Y bearing spermatozoa from common mammals and most experiments have yielded 80-90% successful separa- tion for either males or females in cattle, swine and rabbits (See Table 1 0-3). Several factors have limited the efficiency of this technology, but the application is now widespread in dairy cattle and is feasible in many other species. Regardless of the problems associated with separating the X andY bearing spermatozoa, the tech- nology is now available through artificial insemination organizations. Thus, it is reasonable to expect that separation of X andY bearing spermatozoa eventually will be commonplace. Manipulation of the sex ratio under controlled conditions could greatly impact the efficiency offood animal production. Spermatogenesis 225 Figure 10-15. Major Steps for Separation of X andY Bearing Spermatozoa by Flow Cytometry I' Step 1 X and Y bearing spermatozoa are produced by the testis and ejaculated by the male. Step 2 Ejaculated spermatozoa are treated with a fl uorescent DNA dye. X bearing sperm absorb more dye than Y beanng. sperm. They therefore emit more intense light when exc1ted by a laser. Sperm also are treated with a dye that greatly suppresses the signal from dead sperm. Dead sperm are therefore identified and rejected. Step 3 Once enter the flow cytometer chamber, they smgle-file through a small nozzle. At a region just out- Side the nozzle, a laser beam excites the fluorescent dye in sper£!1. Eac.h sperm emits light at a wavelength and that IS directly related to quantity of DNA. X- live sperm produce more intensity. A light sensing dev1ce 1s coupled to a computer that determines the intensity of light emission by each sperm and the order of passage of each sperm through a column below the nozzle. When the sperm pass by charged plates, they are assigned either a positive or negative charge depending on their DNA content or Y chromosome). When the microdroplet containing a s1ngle sperm passes through an electromagnetic field the computer applies an appropriate charge and directs the (and to one side or the other. Dead sperm are discarded 1nto the center tube. Thus, at the conclusion of the separation process there are three vessels that contain sperm. One contains a high proportion of X one contains a high proportion of Y chromosome bearing and one contains dead sperm. Ve tB oo ks .ir 10 226 Spermatogenesis Further PHENOMENA for Fertility Spermatozoa of the American opossum are ejaculated in doublets. They are formed in the seminiferous epithelium as single cells with an acrosome. During epitlidymal tran- sit the acrosome of two spermatozoa attach to each other, so that a pair of spermatozoa exists. These doublets apparently have more progressive motility than do single cells. When motility ceases they apparently separate. In Greek Mythology, when Priapus was in the womb of his mother Aphrodite, Hem put a spell on him to make him ugly. When he was bom, he was of small stature am/ very ugly but possessed an extremely large penis that was always erect. The name Priapus gave rise to the medical term "priapism", which is defined as a persistent (sometimes painful) erection of the penis, associated with some form of pathology (blood clot in cavernous tissue) and not sexual e.'Ccita- tion. Priapus became known as the Greek God of Fertility in most species, including plants, animals and humans. As the source of fertility, statues of Priapus were kept itt gardens to ensure fertile crops am/ to scare away thieves. He has also been thought to be a cm·e for impotence. In some regions of the world, testes are prized as gourmet treats. In Japan, tes- ticles of dolphins m·e highly valued hors d'oeuvres. In Spain, bull testicles are served at social eJ,ents surrounding the occasion of a bullfight. Bull testicles are also consumed by hungry American cowboys at castration time. In all cases, they are cooked. The bulls at a leading AI organization produce a lot of sperm. The annual semen production from the bulls collected is as follows: • 42-43,000 ejaculations per year • 205 trillion spermatozoa per year • 454 lbs. (206kg) neat semen per year • 10,282,759 0.5-ml straws per year • 12,196lbs. (6.1 tons) of extended semen pe1· year It is rumored that during the early stages of Christianity, the church had succeeded in getting the pagans to give up worship of all the old gods e.'Ccept P1·iapus (the Greek God of Fertility with a huge penis). No matter what the threats or enticements were, the loyal worshipers of Priapus would not give up reverence for their favorite god. The expression of this unwavering reverence included the baking of bread in the shape of a penis 011 eveiJ' available celebrat01y occasion, including church holidays. Un- able to dissuade the people from this rather un-Christian practice, the wise church fa- thers sanctified the loaves, providing each had three crosses carved into its top. This was the reported beginning of hot crossed buns. The ancient Greeks thought that sperma- tozoa from the left testicle produced girls and spermatozoa from the right testicle pro- duced boys. This myth apparently stood the "test of time" because as late as the 1700s, French noblemen would have their left testicle removed in an attempt to sire boys only. The author proposes that the modem day declaration by males, "I would give my left testicle for a ---", is a sexist comment that devalues the left testicle because it was once thought to produce females only. Have you ever heard a male say he was willing to give-up his right testicle for something? Lazzaro Spal/anzani was a mathematician and He was also a priest who conducted experiments with sperm and eggs. His religious beliefs prevented him from collecting and working with human sperm. He wondered, tltouglt, if eve1y 1m- man sperm in an ejaculate hat! a soul and, if so, what happened to the millions of souls in wasted semen. If eve1y sperm had its own soul, then masturbation and contraception were serious sins . . In Spallanzani's era (1700s) many biologists believed in "re- mote fertilization," in which the egg could be stimulated to develop without contact with semen. They thought if an egg were exposed to invisible "spermatic vapor" it would develop into an emb1yo. Since "spermatic vapor," like a ghost, could not be seen there was some wony that this ghostly vapm; once released from an ejaculate, might waft-up the legs of some unsuspecting female, causing an unwanted pregnancy. No one knows how many 1mman·ied women of Spallanzani's era may have credited their pregnancies to "spermatic vapor. " Spa/lanzmzi beliel'ed in these sperm ghosts . but wanted to test his belief. He attached fi'eshly laid toad eggs to a watch glass and inverted it over another watch glass con- taining toad seminal fluid. He thus had an enclosed system in which the eggs and semina/fluid were separated, and where the invisible "spermatic vapor" could migrate to stimulate the eggs. Not/zing happened. But when the eggs were mixed directly into semina/fluid, the physical contact produced tadpoles. How ditl Spallanzani obtain frog semen? He dressed male frogs in tiny taf- feta trousers and placed them with a female frog (without clothes). He watelproofed the pants with a light coating of candle wax. Aroused, the males mounted the females and ejaculated in their pants. Spallanzani then collected the semen. These experi- ments might have been the forerunners to in vitro fertilization because tadpoles developed. Spallanzani was one oftlte first scientists to achieve artificial collection of semen in a laboratory under controlled conditions. Spermatogenesis 221 Kev References Amann, R.P. 1999. "Cryopreservation of sperm" in Encvc/opedia o(Reproduction Vol. 1 p 773-783. Knobil, E. and J.D. Neill (eds). Academic Press, San Diego. ISBN 0- 12-227021 -5. Barth, A.D. and R.J. Oko. 1989. Abnormal MomholofJY o{Bovine Spermatozoa. Iowa State University Press, Ames. ISBN 0-8138-01 12-5. Dadoune, J.P. and A. Demoulin. 1993. "Stmcture and fu nctions of the testis" in Reproduction in Mammals and Man . C. Thibault, M.C. Levasseur and R. H. F. Hunter, eds. Ellipses, Paris. ISBN 2-7298-9354-7. Ericsson, R.J. and S.A. E1i csson. 1999. "Sex ratios" in Encvc/opedia o(Reproduction. Vol. 4 p 431 -436. Knobil, E. and J.D. Nei ll (eds.) Academic Press, San Diego ISBN 0- 12-227024-X. Hess, R.A. 1999. "Spermatogenesis overview" in En- cvc/opedia o(Reproduction. Vol. 4 p 539-545. Knobil, E. and J.D. Nei ll (eds). Academic Press, San Diego. ISBN 0- 12-227024-X. Johnson, L. I 991. "Spermatogenes is" in Reproduction in Domestic Animals 4th Edition, P.T. Cupps, ed. Aca- demic Press, Inc. San Diego. ISBN 0-12-1 9657 5-9. Johnson, L. T.A. McGowen, G.E. Keillor. 1999. "The Testis, overview" in Encvc/opedia o{Reproduction, Vol. 4 p 769-783. Knobil and NeiiJ , eds. Academic Press, San Diego. ISBN 0-12-227024-X. Lamming, G.E. ed. 1990. Marshal/'s PhvsiolofJY of Reproduction. Fourth Edition Vol. 2: Reproduction in the Male. Churchill Livingstone, New York. ISBN 0-443-0 1968-1 . Russel, L. D. and M. D. Griswold, eds. 1993. The Serto/i Ce/1. Cache River Press, Clearwater. ISBN 0-9627422-0-1-X. 10 Ve tB oo ks .ir 10 226 Spermatogenesis Further PHENOMENA for Fertility Spermatozoa of the American opossum are ejaculated in doublets. They are formed in the seminiferous epithelium as single cells with an acrosome. During epitlidymal tran- sit the acrosome of two spermatozoa attach to each other, so that a pair of spermatozoa exists. These doublets apparently have more progressive motility than do single cells. When motility ceases they apparently separate. In Greek Mythology, when Priapus was in the womb of his mother Aphrodite, Hem put a spell on him to make him ugly. When he was bom, he was of small stature am/ very ugly but possessed an extremely large penis that was always erect. The name Priapus gave rise to the medical term "priapism", which is defined as a persistent (sometimes painful) erection of the penis, associated with some form of pathology (blood clot in cavernous tissue) and not sexual e.'Ccita- tion. Priapus became known as the Greek God of Fertility in most species, including plants, animals and humans. As the source of fertility, statues of Priapus were kept itt gardens to ensure fertile crops am/ to scare away thieves. He has also been thought to be a cm·e for impotence. In some regions of the world, testes are prized as gourmet treats. In Japan, tes- ticles of dolphins m·e highly valued hors d'oeuvres. In Spain, bull testicles are served at social eJ,ents surrounding the occasion of a bullfight. Bull testicles are also consumed by hungry American cowboys at castration time. In all cases, they are cooked. The bulls at a leading AI organization produce a lot of sperm. The annual semen production from the bulls collected is as follows: • 42-43,000 ejaculations per year • 205 trillion spermatozoa per year • 454 lbs. (206kg) neat semen per year • 10,282,759 0.5-ml straws per year • 12,196lbs. (6.1 tons) of extended semen pe1· year It is rumored that during the early stages of Christianity, the church had succeeded in getting the pagans to give up worship of all the old gods e.'Ccept P1·iapus (the Greek God of Fertility with a huge penis). No matter what the threats or enticements were, the loyal worshipers of Priapus would not give up reverence for their favorite god. The expression of this unwavering reverence included the baking of bread in the shape of a penis 011 eveiJ' available celebrat01y occasion, including church holidays. Un- able to dissuade the people from this rather un-Christian practice, the wise church fa- thers sanctified the loaves, providing each had three crosses carved into its top. This was the reported beginning of hot crossed buns. The ancient Greeks thought that sperma- tozoa from the left testicle produced girls and spermatozoa from the right testicle pro- duced boys. This myth apparently stood the "test of time" because as late as the 1700s, French noblemen would have their left testicle removed in an attempt to sire boys only. The author proposes that the modem day declaration by males, "I would give my left testicle for a ---", is a sexist comment that devalues the left testicle because it was once thought to produce females only. Have you ever heard a male say he was willing to give-up his right testicle for something? Lazzaro Spal/anzani was a mathematician and He was also a priest who conducted experiments with sperm and eggs. His religious beliefs prevented him from collecting and working with human sperm. He wondered, tltouglt, if eve1y 1m- man sperm in an ejaculate hat! a soul and, if so, what happened to the millions of souls in wasted semen. If eve1y sperm had its own soul, then masturbation and contraception were serious sins . . In Spallanzani's era (1700s) many biologists believed in "re- mote fertilization," in which the egg could be stimulated to develop without contact with semen. They thought if an egg were exposed to invisible "spermatic vapor" it would develop into an emb1yo. Since "spermatic vapor," like a ghost, could not be seen there was some wony that this ghostly vapm; once released from an ejaculate, might waft-up the legs of some unsuspecting female, causing an unwanted pregnancy. No one knows how many 1mman·ied women of Spallanzani's era may have credited their pregnancies to "spermatic vapor. " Spa/lanzmzi beliel'ed in these sperm ghosts . but wanted to test his belief. He attached fi'eshly laid toad eggs to a watch glass and inverted it over another watch glass con- taining toad seminal fluid. He thus had an enclosed system in which the eggs and semina/fluid were separated, and where the invisible "spermatic vapor" could migrate to stimulate the eggs. Not/zing happened. But when the eggs were mixed directly into semina/fluid, the physical contact produced tadpoles. How ditl Spallanzani obtain frog semen? He dressed male frogs in tiny taf- feta trousers and placed them with a female frog (without clothes). He watelproofed the pants with a light coating of candle wax. Aroused, the males mounted the females and ejaculated in their pants. Spallanzani then collected the semen. These experi- ments might have been the forerunners to in vitro fertilization because tadpoles developed. Spallanzani was one oftlte first scientists to achieve artificial collection of semen in a laboratory under controlled conditions. Spermatogenesis 221 Kev References Amann, R.P. 1999. "Cryopreservation of sperm" in Encvc/opedia o(Reproduction Vol. 1 p 773-783. Knobil, E. and J.D. Neill (eds). Academic Press, San Diego. ISBN 0- 12-227021 -5. Barth, A.D. and R.J. Oko. 1989. Abnormal MomholofJY o{Bovine Spermatozoa. Iowa State University Press, Ames. ISBN 0-8138-01 12-5. Dadoune, J.P. and A. Demoulin. 1993. "Stmcture and fu nctions of the testis" in Reproduction in Mammals and Man . C. Thibault, M.C. Levasseur and R. H. F. Hunter, eds. Ellipses, Paris. ISBN 2-7298-9354-7. Ericsson, R.J. and S.A. E1i csson. 1999. "Sex ratios" in Encvc/opedia o(Reproduction. Vol. 4 p 431 -436. Knobil, E. and J.D. Nei ll (eds.) Academic Press, San Diego ISBN 0- 12-227024-X. Hess, R.A. 1999. "Spermatogenesis overview" in En- cvc/opedia o(Reproduction. Vol. 4 p 539-545. Knobil, E. and J.D. Nei ll (eds). Academic Press, San Diego. ISBN 0- 12-227024-X. Johnson, L. I 991. "Spermatogenes is" in Reproduction in Domestic Animals 4th Edition, P.T. Cupps, ed. Aca- demic Press, Inc. San Diego. ISBN 0-12-1 9657 5-9. Johnson, L. T.A. McGowen, G.E. Keillor. 1999. "The Testis, overview" in Encvc/opedia o{Reproduction, Vol. 4 p 769-783. Knobil and NeiiJ , eds. Academic Press, San Diego. ISBN 0-12-227024-X. Lamming, G.E. ed. 1990. Marshal/'s PhvsiolofJY of Reproduction. Fourth Edition Vol. 2: Reproduction in the Male. Churchill Livingstone, New York. ISBN 0-443-0 1968-1 . Russel, L. D. and M. D. Griswold, eds. 1993. The Serto/i Ce/1. Cache River Press, Clearwater. ISBN 0-9627422-0-1-X. 10 Ve tB oo ks .ir The Puerperium & Lactation Parturition Fetal Attachment & Gestation Early Embryogenesis & Maternal Recognition of Pregnancy Ovulation & Fertilization Cyclicity Regulation of Reproduction Tract Function Puberty Prenatal Development ... , ... , \ I Spermatogenesis Regulation of Reproduction Tract Function Puberty Prenatal Development Take Home Message Reproductive behavior is an obligatmy component oftlze reproductive process. It consists ofprecopulatory, copulatory and postcopulatory stages. In the female, sexual receptivity occurs only during estrus and is characterized by distinct behavior and mating posture (lm·- dosis). In the male, reproductive behavior can occur potentially any time. Sexual arousal in the male involves a cascade of endocrine and neural events that result in erection oftlte penis, mounting oftlze sexually receptive female, intromission and ejaculation. Erection of the penis involves specific neural and biochemical events that culminate in penile vasodila- tion. Ejaculation is a reflex that is initiated by stimulation of the glans penis and concludes with expulsion ofsemen Reproductive behavior has evolved as one of the strongest drives in the animal kingdom and usually takes precedence over all other forms of activity such as eating, resting and s leeping. The purpose of reproduc- tive behavior is to promote the opporhmity for copula- tion and thus increase the probability that the spem1 and the egg will meet. The ultimate goals of copulation are pregnancy, successful embryogenesis and parturition. Reproductive behavior in the male consists of three distinct stages: • the precopulatory stage • the copulatory stage • the postcopulatory stage Reproductive behavior in the male can be di- vided into three distinct stages. These stages are : the precopulatory stage; the copulatory stage; and the postcopulatory stage. The specific events that occur during each of these stages are presented in Figure 11-1 . Reproductive behavior in the female can be considered to serve the following functions: • attractivity • proceptivity • receptivity Figure 11-1. Stages of Male Reproductive Behavior and Specific Events in Each Stage Precopulatory Behavior Search for sexual partner • Courtship - Sexual arousal - Erection - Penile protrusion • .. Copulatory Behavior Mounting Intromission Ejaculation Postcopulatory Behavior I Dismount I - I T I Refractory period - I Memory I Ve tB oo ks .ir The Puerperium & Lactation Parturition Fetal Attachment & Gestation Early Embryogenesis & Maternal Recognition of Pregnancy Ovulation & Fertilization Cyclicity Regulation of Reproduction Tract Function Puberty Prenatal Development ... , ... , \ I Spermatogenesis Regulation of Reproduction Tract Function Puberty Prenatal Development Take Home Message Reproductive behavior is an obligatmy component oftlze reproductive process. It consists ofprecopulatory, copulatory and postcopulatory stages. In the female, sexual receptivity occurs only during estrus and is characterized by distinct behavior and mating posture (lm·- dosis). In the male, reproductive behavior can occur potentially any time. Sexual arousal in the male involves a cascade of endocrine and neural events that result in erection oftlte penis, mounting oftlze sexually receptive female, intromission and ejaculation. Erection of the penis involves specific neural and biochemical events that culminate in penile vasodila- tion. Ejaculation is a reflex that is initiated by stimulation of the glans penis and concludes with expulsion ofsemen Reproductive behavior has evolved as one of the strongest drives in the animal kingdom and usually takes precedence over all other forms of activity such as eating, resting and s leeping. The purpose of reproduc- tive behavior is to promote the opporhmity for copula- tion and thus increase the probability that the spem1 and the egg will meet. The ultimate goals of copulation are pregnancy, successful embryogenesis and parturition. Reproductive behavior in the male consists of three distinct stages: • the precopulatory stage • the copulatory stage • the postcopulatory stage Reproductive behavior in the male can be di- vided into three distinct stages. These stages are : the precopulatory stage; the copulatory stage; and the postcopulatory stage. The specific events that occur during each of these stages are presented in Figure 11-1 . Reproductive behavior in the female can be considered to serve the following functions: • attractivity • proceptivity • receptivity Figure 11-1. Stages of Male Reproductive Behavior and Specific Events in Each Stage Precopulatory Behavior Search for sexual partner • Courtship - Sexual arousal - Erection - Penile protrusion • .. Copulatory Behavior Mounting Intromission Ejaculation Postcopulatory Behavior I Dismount I - I T I Refractory period - I Memory I Ve tB oo ks .ir I 230 Reproductive Behavior Precopulatory, copulatory and postcopulatory behaviors in the female can be considered as serving the functions of: attractivity, proceptivity and receptiv- ity. Attractivity refers to behaviors and other signals that serve to attract males. This can include postures, vocalizations, behaviors and chemical cues such as pheromones that attract the male to approach and en- gage in precopulatory behavior. Proceptivity refers to the behaviors exhibited by females toward males that stimulate the male to copulate or that reinitiate sexual behavior after copulation. For example, head butting of the male and mounting the male are two of the most common preceptive behaviors exhibited by females. Proceptivity may also include behaviors among fe- males, such as female-female mounting that sexually stimulate males. Finally, r·eceptivity is the copulatory behavior of females that ensures insemination. This may include the immobility or standing response (lor- dosis) as well as tail deviation or backing-up toward the male. As you have already learned, sexual activity of the postpubertal female is confined to estrus (heat). This short period of sexual receptivity limits the time during which precopulatory behavior occurs in most females. In contrast, the male is potentially capable of initiating reproductive behavior at any time after puberty. The initiation of courtship-specific behavior is generally under the influence of the female. She will send subtle, or sometimes overt signals to the male (attractivity) to initiate courtship behavior. Fac- tors such as sexual signaling pheromones, vocaliza- tion, increased physical activity and subtle postural changes are signals provided by the female that will initiate more intense courtship behavior on the part of the male. In addition, it has been hypothesized that female-female (proceptivity) interactions such as ho- mosexual mounting activity among cattle may serve as signals to initiate male-female courtship behavior. In general, the postpubertal male is almost constantly searching for signals sent by the female to indicate that she is sexually receptive. Identification of a sexual partner probably requires mostofthe senses (olfactory, visual, auditory and tactile). The relative importance of these sensory stimuli has not been described critically in most spe- CleS. Females of almost all species appear to show a marked increase in general physical activity as they come into estrus (See Figure 11-2). Elevated physical activity is generally manifested by increased locomotion. In addition, milling around, exploration, increased vocalization and agonistic behavior towards other females can be observed. In almost all species studied, including humans, there is a marked increase 1/) a.. w I- I/) 1/) a.. w I- I/) 1/) a.. w I- I/) 1/) a.. w I- I/) Figure 11-2. Relationship Between Physical Activity and Reproductive Cycles in Various Female Mammals !cows I Estrus Estrus sows Estrus Estrus RATS Estrus Estrus !woMEN! Menses Menses Physical activity increases significantly around the time of estrus and/or ovulation. in physical activity that accompanies the time of ovula- tion. Presumably, this physical activity is associated with searching for a mate. This increased physical activity can be measured by equipping females with pedometers. Pedometers are devices that monitor and quantitate steps taken by the animal and are currently used in commercial dairy enterprises for detection of estrus. Courtship-specific behavior is initiated after a sexual partner has been identified. Once a sexual partner has been identified, a series of highly specific courtship behaviors begin. Courtship-specific behaviors include sniffing of the vulva by the male, urination by the female in the pres- ence of the male, exhibiting flehmen behavior (See Figure 11 -5), chin resting, circling and increased pho- nation. In many species the sense of vision appears to be the most important with regard to sexual arousal in the male. This should not be interpreted to mean that other stimuli, such as auditory or olfactmy are not important. Copulatmy behavior varies significantly among species with regard to duration. Lordosis (mating posture) by the female (re- ceptivity) triggers significant sexual arousal behavior on the part of the male. Once the male discovers that the female will display lordosis, he becomes sexually stimulated. It should be emphasized that lordosis is a highly specific female motor response associated with the "willingness" to mate. Sexual arousal is followed by erection and penile protrusion. Following exposure to the appropriate stimuli, erection and protmsion of the penis occur. These highly specific motor events are controlled by the central nervous system. The mechanisms of penile protrusion and erection will be presented later. Typical behavior dming search, courtship and sexual arousal for domestic animals is presented in Table 11 -1 . Reproductive Behavior 231 After significant sexual stimulation, mount- ing, intromission and ejaculation follow. In general, mammals can be classified as sustained copulators or short copulators. The bull, ram, buck and tom are short copulators while the boar, dog and camelids are sustained copulators. The stallion is intem1ediate with regard to duration of copulation. Mounting behavior generally requires immobi- lization of the female and elevation ofthe front legs of the male to straddle the caudal region of the female (See Figure 11-1 0). Intromission is entrance of the penis into the vagina. Ejaculation is expulsion of semen from the penis into the female reproductive tract. Copulatory behavior on the part of the male is learned. Past sexual experiences are important in order for the male to develop appropriate reproductive behavior. For example, negative experiences during the precopulatory and copulatory stages will generally result in less enthusiasm on the part of the male. From a practical standpoint, management of the breeding male should always be directed towards providing the male with totally positive stimul i. U ti lizing non-estrus females to collect semen fi·om stallions, boars, rams and bulls should be avoided because these females do not willingly stand to be mounted. Injmy to both the female and the male can occur under these circumstances. Postcopulatmy behavior is a period of refractivity. Postcopulatory behavior involves dismounting and a period during which either the male, the female or both will not engage in copulatory behavior. This refractory period is a period of time during which a second copulation will not take place. Memory is important in both a positive and negative way. Positive mating experiences promote reproductive behavior and negative inhibit reproductive behavior. When semen is collected for artificial insemination, it is important to re- duce the duration of the refi·actmy period when multiple ejaculations need to be collected in the shortest possible time. Techniques to reduce the refractory period will be presented later in the chapter. Both males and females often display specific postcopulatory behavior such as vocal emissions, genital grooming, changing postural relationships and various tacti le behaviors, such as licking and nuzzling. Ve tB oo ks .ir I 230 Reproductive Behavior Precopulatory, copulatory and postcopulatory behaviors in the female can be considered as serving the functions of: attractivity, proceptivity and receptiv- ity. Attractivity refers to behaviors and other signals that serve to attract males. This can include postures, vocalizations, behaviors and chemical cues such as pheromones that attract the male to approach and en- gage in precopulatory behavior. Proceptivity refers to the behaviors exhibited by females toward males that stimulate the male to copulate or that reinitiate sexual behavior after copulation. For example, head butting of the male and mounting the male are two of the most common preceptive behaviors exhibited by females. Proceptivity may also include behaviors among fe- males, such as female-female mounting that sexually stimulate males. Finally, r·eceptivity is the copulatory behavior of females that ensures insemination. This may include the immobility or standing response (lor- dosis) as well as tail deviation or backing-up toward the male. As you have already learned, sexual activity of the postpubertal female is confined to estrus (heat). This short period of sexual receptivity limits the time during which precopulatory behavior occurs in most females. In contrast, the male is potentially capable of initiating reproductive behavior at any time after puberty. The initiation of courtship-specific behavior is generally under the influence of the female. She will send subtle, or sometimes overt signals to the male (attractivity) to initiate courtship behavior. Fac- tors such as sexual signaling pheromones, vocaliza- tion, increased physical activity and subtle postural changes are signals provided by the female that will initiate more intense courtship behavior on the part of the male. In addition, it has been hypothesized that female-female (proceptivity) interactions such as ho- mosexual mounting activity among cattle may serve as signals to initiate male-female courtship behavior. In general, the postpubertal male is almost constantly searching for signals sent by the female to indicate that she is sexually receptive. Identification of a sexual partner probably requires mostofthe senses (olfactory, visual, auditory and tactile). The relative importance of these sensory stimuli has not been described critically in most spe- CleS. Females of almost all species appear to show a marked increase in general physical activity as they come into estrus (See Figure 11-2). Elevated physical activity is generally manifested by increased locomotion. In addition, milling around, exploration, increased vocalization and agonistic behavior towards other females can be observed. In almost all species studied, including humans, there is a marked increase 1/) a.. w I- I/) 1/) a.. w I- I/) 1/) a.. w I- I/) 1/) a.. w I- I/) Figure 11-2. Relationship Between Physical Activity and Reproductive Cycles in Various Female Mammals !cows I Estrus Estrus sows Estrus Estrus RATS Estrus Estrus !woMEN! Menses Menses Physical activity increases significantly around the time of estrus and/or ovulation. in physical activity that accompanies the time of ovula- tion. Presumably, this physical activity is associated with searching for a mate. This increased physical activity can be measured by equipping females with pedometers. Pedometers are devices that monitor and quantitate steps taken by the animal and are currently used in commercial dairy enterprises for detection of estrus. Courtship-specific behavior is initiated after a sexual partner has been identified. Once a sexual partner has been identified, a series of highly specific courtship behaviors begin. Courtship-specific behaviors include sniffing of the vulva by the male, urination by the female in the pres- ence of the male, exhibiting flehmen behavior (See Figure 11 -5), chin resting, circling and increased pho- nation. In many species the sense of vision appears to be the most important with regard to sexual arousal in the male. This should not be interpreted to mean that other stimuli, such as auditory or olfactmy are not important. Copulatmy behavior varies significantly among species with regard to duration. Lordosis (mating posture) by the female (re- ceptivity) triggers significant sexual arousal behavior on the part of the male. Once the male discovers that the female will display lordosis, he becomes sexually stimulated. It should be emphasized that lordosis is a highly specific female motor response associated with the "willingness" to mate. Sexual arousal is followed by erection and penile protrusion. Following exposure to the appropriate stimuli, erection and protmsion of the penis occur. These highly specific motor events are controlled by the central nervous system. The mechanisms of penile protrusion and erection will be presented later. Typical behavior dming search, courtship and sexual arousal for domestic animals is presented in Table 11 -1 . Reproductive Behavior 231 After significant sexual stimulation, mount- ing, intromission and ejaculation follow. In general, mammals can be classified as sustained copulators or short copulators. The bull, ram, buck and tom are short copulators while the boar, dog and camelids are sustained copulators. The stallion is intem1ediate with regard to duration of copulation. Mounting behavior generally requires immobi- lization of the female and elevation ofthe front legs of the male to straddle the caudal region of the female (See Figure 11-1 0). Intromission is entrance of the penis into the vagina. Ejaculation is expulsion of semen from the penis into the female reproductive tract. Copulatory behavior on the part of the male is learned. Past sexual experiences are important in order for the male to develop appropriate reproductive behavior. For example, negative experiences during the precopulatory and copulatory stages will generally result in less enthusiasm on the part of the male. From a practical standpoint, management of the breeding male should always be directed towards providing the male with totally positive stimul i. U ti lizing non-estrus females to collect semen fi·om stallions, boars, rams and bulls should be avoided because these females do not willingly stand to be mounted. Injmy to both the female and the male can occur under these circumstances. Postcopulatmy behavior is a period of refractivity. Postcopulatory behavior involves dismounting and a period during which either the male, the female or both will not engage in copulatory behavior. This refractory period is a period of time during which a second copulation will not take place. Memory is important in both a positive and negative way. Positive mating experiences promote reproductive behavior and negative inhibit reproductive behavior. When semen is collected for artificial insemination, it is important to re- duce the duration of the refi·actmy period when multiple ejaculations need to be collected in the shortest possible time. Techniques to reduce the refractory period will be presented later in the chapter. Both males and females often display specific postcopulatory behavior such as vocal emissions, genital grooming, changing postural relationships and various tacti le behaviors, such as licking and nuzzling. Ve tB oo ks .ir I Ill 232 Reproductive Behavior Table 11-1. Typical Behavior During Search, Courtship and Consummation by Female and Male Domestic Animals Species Search Cow Increased locomotion, increased vocalization, twitching & elevation of the tail Mare Increased locomotion, tail erected ("flagging") Ewe Short period of restlessness ram "seeking" Sow Mild restlessness Bitch Roaming Queen Vocalization (calling) Species Search Bull Approach sexually active group of females testing for lordosis, flelm1en Stallion Visual search, flehmen Sniffing and licking of ana-genital region, nudging ewe, flehmen Moving among females Roaming around territory Prowling FEMALE Courtship Increased grooming, mounting attempts with other females Urination stance, urination in presence of stallion Urination in presence of ram Immobile stance limnobile stance Crouching, affectionate head rubbing, rolling MALE Courtship Nuzzling and licking of perineal region: chin resting, testing for lordosis High degree of excitement Neck outstretched and head held horizontally Nuzzling, grinding of teeth, foams at mouth Sniffing, licking of the vulva Biting queen on dorsal neck Consummation Homosexual mounting & immobile stance (standing to be mounted) Presents hindquarters to male, clitoral exposure by labial eversion, pulsatile contractions of labia Immobile stance Immobile stance Tail deflected to one side Urination in presence of male affectionate head rubbing Elevation of rear quarters and hyper- extension ofback (lordosis), presentation of vulva, tai l deviation Consummation Penile protrusion w ith dribbling of seminal fluid with few sperm- atozoa, erection and attempted mounts Penile protrusion with no preejaculatmy expulsion of seminal fluid Repeated dorsal retraction of scrotum, penile protrusion with no dribbling of seminal fluid Penile protrusion, shallow pelvic thrusts, attempted mounting Erection, protrusion of penis, mounting Mounting Reproductive Behavior is Programmed During Prenatal Development During embryogenesis, sexual differentiation occurs, during which the brain is programmed to be either male or female. Recent findings suggest that the very early embryo is neutral with regard to sex (gender). Under the influence of extremely small quantities of estradiol the brain becomes feminized. Feminiza- tion is the development of female-like behavior. As you learned in Chapter 6, during fetal development, a.-fetoprotein is produced that prevents most fetal and maternal estradiol from crossing the blood-brain barrier and entering the brain. When a.-fetoprotein prevents estradiol from entering the brain, the embryo becomes "fully feminized," because it has not been exposed to estrogen (See Chapter 6). Alpha-fetoprotein does not bind to testosterone, which can then enter the brain and be converted to estradiol. In developing males this high concentration of estradiol in the brain causes defemini- zation and masculinization of the brain. Defeminiza- tion reduces the likelihood that the animal will express female-like behavior postpubertally. Masculinization results in the potential of the animal to develop male- like behavior after puberty. Sex differences in specific brain structures for the control of reproductive behavior have been observed. For example, in the male, the preoptic area Reproductive Behavior 233 of the hypothalamus is larger than in females. In the male, the size of neurons, the neuron nuclei and the dendritic arborizations are greater. In the female, the ventromedial hypothalamus is more important with regard to reproductive behavior. In most mammals, reproductive behaviors are sexually differentiated. For example, mounting, erection and ejaculation are typically male behaviors, while standing to be mounted (lordosis), crouching and increased locomotion are typically female behaviors. These behaviors are endocrine controlled. For example, sequential treatment with progesterone and estradiol induces sexual receptivity in ovariectomized females and testosterone will restore reproductive behavior in castrated males. In some species, injections of testos- terone into castrated females will even induce male-like reproductive behavior. Female fetuses exposed to androgens prenatally will display significantly reduced female behavior (defeminized) and acquire male-like behavior postnatally (masculinized). In contrast, males exposed to estrogen or progesterone prena- tally are unaffected. A classic example illustrating the behavioral manifestations of prenatal exposure to andro- gens is the freemartin heifer. As previously discussed (See Chapter 4), this animal has abnormal development of the reproductive tract for two reasons. First, from a genetic perspective, freemartins are chimeras that are XX/XY and therefore they have an ovotestis. Second, Figure 11-3. Influence of Various Steroid Treatments Upon Reproductive Behavior PRENATAL Fetus + E2 ------• f Estrous behavior + male-like behavior Fetus + Testosterone f Estrous behavior + male-like behavior 'b Fetus + E2 or P4 No effect (normal 'b behavior) 'b Fetus+ Testosterone - No effect (normal 'b behavior) POSTNATAL No estrous behavior Estrous behavior ------+- Maximum estrous behavior + E2 -------+ P4 and E2 + Testosterone Male-like behavior ----------.- f Sexual behavior + Testosterone ----• Ovaries removed (ovariectomy) Sexual behavior restored Sexual behavior restored removed (orchidectomy) Ve tB oo ks .ir I Ill 232 Reproductive Behavior Table 11-1. Typical Behavior During Search, Courtship and Consummation by Female and Male Domestic Animals Species Search Cow Increased locomotion, increased vocalization, twitching & elevation of the tail Mare Increased locomotion, tail erected ("flagging") Ewe Short period of restlessness ram "seeking" Sow Mild restlessness Bitch Roaming Queen Vocalization (calling) Species Search Bull Approach sexually active group of females testing for lordosis, flelm1en Stallion Visual search, flehmen Sniffing and licking of ana-genital region, nudging ewe, flehmen Moving among females Roaming around territory Prowling FEMALE Courtship Increased grooming, mounting attempts with other females Urination stance, urination in presence of stallion Urination in presence of ram Immobile stance limnobile stance Crouching, affectionate head rubbing, rolling MALE Courtship Nuzzling and licking of perineal region: chin resting, testing for lordosis High degree of excitement Neck outstretched and head held horizontally Nuzzling, grinding of teeth, foams at mouth Sniffing, licking of the vulva Biting queen on dorsal neck Consummation Homosexual mounting & immobile stance (standing to be mounted) Presents hindquarters to male, clitoral exposure by labial eversion, pulsatile contractions of labia Immobile stance Immobile stance Tail deflected to one side Urination in presence of male affectionate head rubbing Elevation of rear quarters and hyper- extension ofback (lordosis), presentation of vulva, tai l deviation Consummation Penile protrusion w ith dribbling of seminal fluid with few sperm- atozoa, erection and attempted mounts Penile protrusion with no preejaculatmy expulsion of seminal fluid Repeated dorsal retraction of scrotum, penile protrusion with no dribbling of seminal fluid Penile protrusion, shallow pelvic thrusts, attempted mounting Erection, protrusion of penis, mounting Mounting Reproductive Behavior is Programmed During Prenatal Development During embryogenesis, sexual differentiation occurs, during which the brain is programmed to be either male or female. Recent findings suggest that the very early embryo is neutral with regard to sex (gender). Under the influence of extremely small quantities of estradiol the brain becomes feminized. Feminiza- tion is the development of female-like behavior. As you learned in Chapter 6, during fetal development, a.-fetoprotein is produced that prevents most fetal and maternal estradiol from crossing the blood-brain barrier and entering the brain. When a.-fetoprotein prevents estradiol from entering the brain, the embryo becomes "fully feminized," because it has not been exposed to estrogen (See Chapter 6). Alpha-fetoprotein does not bind to testosterone, which can then enter the brain and be converted to estradiol. In developing males this high concentration of estradiol in the brain causes defemini- zation and masculinization of the brain. Defeminiza- tion reduces the likelihood that the animal will express female-like behavior postpubertally. Masculinization results in the potential of the animal to develop male- like behavior after puberty. Sex differences in specific brain structures for the control of reproductive behavior have been observed. For example, in the male, the preoptic area Reproductive Behavior 233 of the hypothalamus is larger than in females. In the male, the size of neurons, the neuron nuclei and the dendritic arborizations are greater. In the female, the ventromedial hypothalamus is more important with regard to reproductive behavior. In most mammals, reproductive behaviors are sexually differentiated. For example, mounting, erection and ejaculation are typically male behaviors, while standing to be mounted (lordosis), crouching and increased locomotion are typically female behaviors. These behaviors are endocrine controlled. For example, sequential treatment with progesterone and estradiol induces sexual receptivity in ovariectomized females and testosterone will restore reproductive behavior in castrated males. In some species, injections of testos- terone into castrated females will even induce male-like reproductive behavior. Female fetuses exposed to androgens prenatally will display significantly reduced female behavior (defeminized) and acquire male-like behavior postnatally (masculinized). In contrast, males exposed to estrogen or progesterone prena- tally are unaffected. A classic example illustrating the behavioral manifestations of prenatal exposure to andro- gens is the freemartin heifer. As previously discussed (See Chapter 4), this animal has abnormal development of the reproductive tract for two reasons. First, from a genetic perspective, freemartins are chimeras that are XX/XY and therefore they have an ovotestis. Second, Figure 11-3. Influence of Various Steroid Treatments Upon Reproductive Behavior PRENATAL Fetus + E2 ------• f Estrous behavior + male-like behavior Fetus + Testosterone f Estrous behavior + male-like behavior 'b Fetus + E2 or P4 No effect (normal 'b behavior) 'b Fetus+ Testosterone - No effect (normal 'b behavior) POSTNATAL No estrous behavior Estrous behavior ------+- Maximum estrous behavior + E2 -------+ P4 and E2 + Testosterone Male-like behavior ----------.- f Sexual behavior + Testosterone ----• Ovaries removed (ovariectomy) Sexual behavior restored Sexual behavior restored removed (orchidectomy) Ve tB oo ks .ir 234 Reproductive Behavior androgen exposure per se causes abnonnal develop- ment of the female tract. In addition, the freemartin displays more male-like behavior than do her normal heifer counterparts. Figure 11-3 summarizes the influ- ence of reproductive steroids on behavior in the male and the female. The presence of gonadal steroids (estradiol and testosterone) is obligatory for normal reproduc- tive behavior in both the male and the female. For example, ovariectomized females display no estrous behavior (See Figure 11-3 ). Likewise, castrated males have significantly reduced reproductive behav- ior. However, the abolition of reproductive behavior depends on the duration of time between castration and the opportunity to copulate. For example, males that have reached puberty and established a sustained pattern of reproductive behavior require a longer period of time between abolition of sexual behavior after castration than do males that have not estab- lished a sustained pattern of reproductive behavior. Females will display male reproductive be- havior following injections of testosterone. When ovariectomized females receive injections of estradiol, estrous behavior is reestablished, but at a less than maximum level. Among farm animals, ovariectomized females that are tTeated fi rst with pro- gesterone (to mimic the luteal phase of the cycle) and then treated with estradiol display maximum estrous behavior. In other species, estradiol must precede progesterone to produce maximal behavior. It is not clear why progesterone "priming" of the central ner- vous system for maximal stimulation is necessary. It would be logical to propose that progesterone promotes upregulation of estradiol receptors in the brain. Ova- riectomized females that are treated with testosterone develop male-like behavior. They will even develop secondary sex characteristics (reduced pitch of voice, hump on the back of the neck and atrophy of the female reproductive tract). Figure 11-4. Hypothetical NeNous Pathway Eliciting Reproductive-Specific Motor Behavior • Visual • Olfactory • Auditory • Tactile • Estrogen receptors • t E2 -+ t increased nerve excitability • Neurons produce behavior specific peptides OC = Optic Chiasm AL = Anterior Lobe of Pituitary PL = Posterior Lobe of Pituitary • "Receiving zone" for hypothalamic peptides • Speeds up impulses and mounting Spinal cord • Generates signals to specific muscles fo r lordosis and mounting Specific muscles responsible for lordosis and Reproductive Behavior is Controlled by the Central Nervous System The neural pathways and key anatomical com- ponents for the control of reproductive behavior are pre- sented in Figure 11 -4. Reproductive behavior can take place only if the nemons in the hypothalamus have been sensitized to respond to sensory signals. Testosterone in the male is aromatized to esh·adiol in the brain and estradiol promotes reproductive behav ior. Recall that testosterone is produced in small episodes every 4 to 6 hours. Therefore, there is a relatively constant supply of testosterone and thus estradiol, to the hypothalamus in the male. This allows the male to initiate reproductive behavior at any time. In contrast, the female experi- ences high esh·adiol during the follicular phase only and will display sexual receptivity during estrus only. Figure 11-4 outlines a generic neural pathway for sexual behavior. Under the influence of estrogen, sensory inputs such as olfaction, audition, vision and tactility send neural messages to the hypothalamus. These neurons synapse directly on neurons in the ven- h·omedial hypothalamus as well as the preoptic and anterior hypothalamic regions. These sensory inputs cause neurons in the hypothalamus to release behavior specific peptides that serve as neurotransmitters. These neurotransmitters act on neurons in the midbrain. The neurons in the midbrain serve as receiving zones for the peptides produced by the hypothalamic neurons. The midbrain h·anslates neuropeptide signals released by hypothalamic neurons into a fast response. Neu- rons in the midbrain synapse with neurons in the brain stem (medulla). These nervous s ignals are integrated in the medulla. From the medulla, nerve tracts extend to the spinal cord where the nerves synapse with mo- tor neurons that innervate muscles that cause lordosis and mounting. It should be emphasized that the model presented in Figure 11-4 does not account for all of the nerve pathways involved in reproductive behavior. Reproductive behavior is initiated by: • olfaction • vision • audition • tactility The primary sensory inputs for reproductive behavior are olfaction, audition, vision and tactility. The degree to which these sensory inputs influence repro- ductive behavior, particularly precopulatmy behavior, varies significantly among species. Reproductive Behavior 235 Figure 11-5. Flehmen Response in the Stallion and Bull and the Vomeronasal Pathway 0;2/ Fluids Nasopalatine Fluids duct The flehmen response involves curling of the upper lip so that airflow through the nasal pas- sages is restricted. A subatmospheric pres- sure is thus created in the nasopalatine duct. Therefore, flu ids can be aspirated through the duct and into the sensory surfaces of the vomeronasal organ. Arrows in the bull indicate the approximate openings of the nasopalatine ducts. (Photo of stallion courtesy of Dr. A T1bary, Washington State University, College of Veterinary Medicine; Photo of bull courtesy of Select Sires, Inc. www.selectsires.com) Ve tB oo ks .ir 234 Reproductive Behavior androgen exposure per se causes abnonnal develop- ment of the female tract. In addition, the freemartin displays more male-like behavior than do her normal heifer counterparts. Figure 11-3 summarizes the influ- ence of reproductive steroids on behavior in the male and the female. The presence of gonadal steroids (estradiol and testosterone) is obligatory for normal reproduc- tive behavior in both the male and the female. For example, ovariectomized females display no estrous behavior (See Figure 11-3 ). Likewise, castrated males have significantly reduced reproductive behav- ior. However, the abolition of reproductive behavior depends on the duration of time between castration and the opportunity to copulate. For example, males that have reached puberty and established a sustained pattern of reproductive behavior require a longer period of time between abolition of sexual behavior after castration than do males that have not estab- lished a sustained pattern of reproductive behavior. Females will display male reproductive be- havior following injections of testosterone. When ovariectomized females receive injections of estradiol, estrous behavior is reestablished, but at a less than maximum level. Among farm animals, ovariectomized females that are tTeated fi rst with pro- gesterone (to mimic the luteal phase of the cycle) and then treated with estradiol display maximum estrous behavior. In other species, estradiol must precede progesterone to produce maximal behavior. It is not clear why progesterone "priming" of the central ner- vous system for maximal stimulation is necessary. It would be logical to propose that progesterone promotes upregulation of estradiol receptors in the brain. Ova- riectomized females that are treated with testosterone develop male-like behavior. They will even develop secondary sex characteristics (reduced pitch of voice, hump on the back of the neck and atrophy of the female reproductive tract). Figure 11-4. Hypothetical NeNous Pathway Eliciting Reproductive-Specific Motor Behavior • Visual • Olfactory • Auditory • Tactile • Estrogen receptors • t E2 -+ t increased nerve excitability • Neurons produce behavior specific peptides OC = Optic Chiasm AL = Anterior Lobe of Pituitary PL = Posterior Lobe of Pituitary • "Receiving zone" for hypothalamic peptides • Speeds up impulses and mounting Spinal cord • Generates signals to specific muscles fo r lordosis and mounting Specific muscles responsible for lordosis and Reproductive Behavior is Controlled by the Central Nervous System The neural pathways and key anatomical com- ponents for the control of reproductive behavior are pre- sented in Figure 11 -4. Reproductive behavior can take place only if the nemons in the hypothalamus have been sensitized to respond to sensory signals. Testosterone in the male is aromatized to esh·adiol in the brain and estradiol promotes reproductive behav ior. Recall that testosterone is produced in small episodes every 4 to 6 hours. Therefore, there is a relatively constant supply of testosterone and thus estradiol, to the hypothalamus in the male. This allows the male to initiate reproductive behavior at any time. In contrast, the female experi- ences high esh·adiol during the follicular phase only and will display sexual receptivity during estrus only. Figure 11-4 outlines a generic neural pathway for sexual behavior. Under the influence of estrogen, sensory inputs such as olfaction, audition, vision and tactility send neural messages to the hypothalamus. These neurons synapse directly on neurons in the ven- h·omedial hypothalamus as well as the preoptic and anterior hypothalamic regions. These sensory inputs cause neurons in the hypothalamus to release behavior specific peptides that serve as neurotransmitters. These neurotransmitters act on neurons in the midbrain. The neurons in the midbrain serve as receiving zones for the peptides produced by the hypothalamic neurons. The midbrain h·anslates neuropeptide signals released by hypothalamic neurons into a fast response. Neu- rons in the midbrain synapse with neurons in the brain stem (medulla). These nervous s ignals are integrated in the medulla. From the medulla, nerve tracts extend to the spinal cord where the nerves synapse with mo- tor neurons that innervate muscles that cause lordosis and mounting. It should be emphasized that the model presented in Figure 11-4 does not account for all of the nerve pathways involved in reproductive behavior. Reproductive behavior is initiated by: • olfaction • vision • audition • tactility The primary sensory inputs for reproductive behavior are olfaction, audition, vision and tactility. The degree to which these sensory inputs influence repro- ductive behavior, particularly precopulatmy behavior, varies significantly among species. Reproductive Behavior 235 Figure 11-5. Flehmen Response in the Stallion and Bull and the Vomeronasal Pathway 0;2/ Fluids Nasopalatine Fluids duct The flehmen response involves curling of the upper lip so that airflow through the nasal pas- sages is restricted. A subatmospheric pres- sure is thus created in the nasopalatine duct. Therefore, flu ids can be aspirated through the duct and into the sensory surfaces of the vomeronasal organ. Arrows in the bull indicate the approximate openings of the nasopalatine ducts. (Photo of stallion courtesy of Dr. A T1bary, Washington State University, College of Veterinary Medicine; Photo of bull courtesy of Select Sires, Inc. www.selectsires.com) Ve tB oo ks .ir 236 Reproductive Behavior The Olfactory and Vomeronasal Systems Respond to Pheromones that Trigger Reproductive Behavior Secretions from the female reproductive tract serve to sexually stimulate and attract the male to the female. Vaginal and urinary secretions from females in estrus smell different to the male than secretions from females not in estrus. There is good scientific evidence that females produce pheromonal substances that are identifiable both within species and among species. However, their action is species specific. Recall that a phe1·omone is a volatile substance secreted or released to the outside of the body and perceived by the olfac- tory system and/or activated by the vomeronasal organ. Releasing pheromones can cause specific behavior in the recipient. Pheromones can also be priming phero- mones that have physiologic rather than behavioral effects on the recipient. Males also produce sex pheromones that attract and stimulate females. Among food producing animals, the best documentation for a male sex pheromone is in swine. Boars produce specific substances that cause sows and gilts to become sexually aroused when they are in estms. Two sexual attractants are produced by boars. One of these attractants is a preputial pouch secretion. The second pheromonal-like substance is present in saliva secreted by the submaxillary salivary glands. During sexual excitement and precopulatory interac- tions, the boar produces copious quantities of foamy saliva. The active components in saliva are the androgen metabolites 3a.-androstenol and 5a.-androstenone. Both compounds have a musk-like odor. It has been demonstrated that dogs have the ability to identifY cows in estrus by olfactory discrimi- nation. In addition, rats can be trained to press a lever in response to air bubbled through urine from cows in estms. Rats did not press the lever when air was bubbled through urine fi·om nonestrous cows. Clearly, urine from cows in estrus contains a material that can be identified by olfaction by other species (dogs and rats). Figure 11-6. "Warm-Up" Stalls Used for Stimulating Sexual Behavior in Bulls Providing Semen for Artificial Insemination Bulls waiting to be ejaculated (arrows) watch mounting and ejaculatory behavior of another bull. Such a practice "prestimulates" bulls and reduces stimulation time when they enter the collection arena. It also in- creases sperm harvest. A false-mount is being performed by the bull mounting the stimulus animal (SA). (Photo courtesy of Select Sires, Inc., www.selectsires.com) Flehmen Behavior is a Close-Range Investigative Behavior Some pheromones appear to be less volati le and need to be detected by the vomeronasal organ in the bull, ram, stallion and to some extent, the boar. The male needs to closely approach the source of pheromones and he will nuzzle the genital region of the female. The vomeronasal organ (See Figure 11-5) is an accessory olfactory organ. It is connected to two small openings in the anterior roof of the mouth just behind the upper lip. Fluid-borne, less volatile chemicals can enter the vomeronasal organ through the oral cavity by means of the nasopalatine (incisive) ducts. Many species, such as bulls, rams and stallions, perfom1 a special investigative maneuver when in close proxim- ity to a female. Vaginal secretions and urine evoke an investigative behavior known as the flehmen response. Flehmen behavior allows less volatile materials to be "examined" by sensory neurons in the vomeronasal organ. Flehmen behavior is characterized by head el- evation and curling of the upper lip (See Figure 11-5). Curling of the upper lip closes the nostrils and allows a negative pressure to forn1 in the nasopalatine duct. Thus, less volatile materials (like mucous and urine) can be aspirated through the duct into the vomeronasal organ where they can be "evaluated" by sensory neurons in the organ. Olfactory bulbectomy in goats inhibits the flehmen response. Flehmen behavior in males is likely to be performed whether the material is from an estrus or nonestrus female. It is believed that the fleh- men behavior is used to help a male identifY mating opportunities. Flehmen is occasionally performed by females during sexual encounters with males. Cows will frequently perform the maneuver when sniffing other cows that are in estrus or proestrus. As in the male, females will display flelunen to novel compmmds, including fluids associated with the placenta, newborn animals and other volatile materials. Flehmen is fre- quently displayed by post-parturient females as they make identity discriminations between their own versus other's neonates. Auditory stimulation can serve as a long-range signal. In many species, sexual readiness is accompa- nied by some fom1 of unique vocalization or "mating calls". For example, cows are known to increase their bellowing during the time of estrus. Sows display a characteristic grunting sound associated with estrus. Queens often "yeow" repeatedly to call the tom. By Reproductive Behavior 237 comparison, mares and ewes are relatively silent. El- evated vocalization serves to alert or send a signal to males that sexual readiness is imminent. The auditory stimulus is more useful in long-range discrimination, rather than close discrimination. The classic example of reproductive driven vocalization is bugling of the bull elk during rut (the breeding season). Visual signals are valuable for close encounters. All females display a fonn of sexual postur- ing that can be perceived by males. While posturing can be quite subtle, especially to human observers, the identification of postures probably takes place easily among members of the same species. Tactile stimulation is generally the final stimulus before copulation. Almost all males experience a degree of sexual stimulation when they observe mating behavior among other individuals of the same species. It is well documented that in bulls, visual observation of mating behavior enhances sexual stimulation. This observa- tion has led to the common practice of placing bulls used for artificial insemination in "warm-up" stalls (See Figure 11-6). Bulls are brought to the "warm-up" stalls and are allowed to observe the mounting behav- ior and collection of semen from other bulls prior to entering the collection area themselves. This causes an elevated level of sexual excitement and reduces the time required for final sexual stimulation and collection of semen. This is important because labor requirements for semen collection are significant. This procedure is also important because it tends to increase spem1 concentration in the ejaculate. Tactile stimuli from males appears to be im- portant in evoking sexual postures or standing postures by females. For example, biting on the neck and the withers of mares by stallions appears to be important for sexual stimulation. Biting of the neck of the queen by the tom is also a characteristic reproductive behav- ior among cats. Rubbing of the flanks and genitalia of mares, whether done by the stallion or by a human handler, evokes behavior signals of estrus from the mare that othe1wise would not be displayed. Chin resting by a bull on the back of a cow just prior to mounting may have some stimulatory effect on the cow. Ve tB oo ks .ir 236 Reproductive Behavior The Olfactory and Vomeronasal Systems Respond to Pheromones that Trigger Reproductive Behavior Secretions from the female reproductive tract serve to sexually stimulate and attract the male to the female. Vaginal and urinary secretions from females in estrus smell different to the male than secretions from females not in estrus. There is good scientific evidence that females produce pheromonal substances that are identifiable both within species and among species. However, their action is species specific. Recall that a phe1·omone is a volatile substance secreted or released to the outside of the body and perceived by the olfac- tory system and/or activated by the vomeronasal organ. Releasing pheromones can cause specific behavior in the recipient. Pheromones can also be priming phero- mones that have physiologic rather than behavioral effects on the recipient. Males also produce sex pheromones that attract and stimulate females. Among food producing animals, the best documentation for a male sex pheromone is in swine. Boars produce specific substances that cause sows and gilts to become sexually aroused when they are in estms. Two sexual attractants are produced by boars. One of these attractants is a preputial pouch secretion. The second pheromonal-like substance is present in saliva secreted by the submaxillary salivary glands. During sexual excitement and precopulatory interac- tions, the boar produces copious quantities of foamy saliva. The active components in saliva are the androgen metabolites 3a.-androstenol and 5a.-androstenone. Both compounds have a musk-like odor. It has been demonstrated that dogs have the ability to identifY cows in estrus by olfactory discrimi- nation. In addition, rats can be trained to press a lever in response to air bubbled through urine from cows in estms. Rats did not press the lever when air was bubbled through urine fi·om nonestrous cows. Clearly, urine from cows in estrus contains a material that can be identified by olfaction by other species (dogs and rats). Figure 11-6. "Warm-Up" Stalls Used for Stimulating Sexual Behavior in Bulls Providing Semen for Artificial Insemination Bulls waiting to be ejaculated (arrows) watch mounting and ejaculatory behavior of another bull. Such a practice "prestimulates" bulls and reduces stimulation time when they enter the collection arena. It also in- creases sperm harvest. A false-mount is being performed by the bull mounting the stimulus animal (SA). (Photo courtesy of Select Sires, Inc., www.selectsires.com) Flehmen Behavior is a Close-Range Investigative Behavior Some pheromones appear to be less volati le and need to be detected by the vomeronasal organ in the bull, ram, stallion and to some extent, the boar. The male needs to closely approach the source of pheromones and he will nuzzle the genital region of the female. The vomeronasal organ (See Figure 11-5) is an accessory olfactory organ. It is connected to two small openings in the anterior roof of the mouth just behind the upper lip. Fluid-borne, less volatile chemicals can enter the vomeronasal organ through the oral cavity by means of the nasopalatine (incisive) ducts. Many species, such as bulls, rams and stallions, perfom1 a special investigative maneuver when in close proxim- ity to a female. Vaginal secretions and urine evoke an investigative behavior known as the flehmen response. Flehmen behavior allows less volatile materials to be "examined" by sensory neurons in the vomeronasal organ. Flehmen behavior is characterized by head el- evation and curling of the upper lip (See Figure 11-5). Curling of the upper lip closes the nostrils and allows a negative pressure to forn1 in the nasopalatine duct. Thus, less volatile materials (like mucous and urine) can be aspirated through the duct into the vomeronasal organ where they can be "evaluated" by sensory neurons in the organ. Olfactory bulbectomy in goats inhibits the flehmen response. Flehmen behavior in males is likely to be performed whether the material is from an estrus or nonestrus female. It is believed that the fleh- men behavior is used to help a male identifY mating opportunities. Flehmen is occasionally performed by females during sexual encounters with males. Cows will frequently perform the maneuver when sniffing other cows that are in estrus or proestrus. As in the male, females will display flelunen to novel compmmds, including fluids associated with the placenta, newborn animals and other volatile materials. Flehmen is fre- quently displayed by post-parturient females as they make identity discriminations between their own versus other's neonates. Auditory stimulation can serve as a long-range signal. In many species, sexual readiness is accompa- nied by some fom1 of unique vocalization or "mating calls". For example, cows are known to increase their bellowing during the time of estrus. Sows display a characteristic grunting sound associated with estrus. Queens often "yeow" repeatedly to call the tom. By Reproductive Behavior 237 comparison, mares and ewes are relatively silent. El- evated vocalization serves to alert or send a signal to males that sexual readiness is imminent. The auditory stimulus is more useful in long-range discrimination, rather than close discrimination. The classic example of reproductive driven vocalization is bugling of the bull elk during rut (the breeding season). Visual signals are valuable for close encounters. All females display a fonn of sexual postur- ing that can be perceived by males. While posturing can be quite subtle, especially to human observers, the identification of postures probably takes place easily among members of the same species. Tactile stimulation is generally the final stimulus before copulation. Almost all males experience a degree of sexual stimulation when they observe mating behavior among other individuals of the same species. It is well documented that in bulls, visual observation of mating behavior enhances sexual stimulation. This observa- tion has led to the common practice of placing bulls used for artificial insemination in "warm-up" stalls (See Figure 11-6). Bulls are brought to the "warm-up" stalls and are allowed to observe the mounting behav- ior and collection of semen from other bulls prior to entering the collection area themselves. This causes an elevated level of sexual excitement and reduces the time required for final sexual stimulation and collection of semen. This is important because labor requirements for semen collection are significant. This procedure is also important because it tends to increase spem1 concentration in the ejaculate. Tactile stimuli from males appears to be im- portant in evoking sexual postures or standing postures by females. For example, biting on the neck and the withers of mares by stallions appears to be important for sexual stimulation. Biting of the neck of the queen by the tom is also a characteristic reproductive behav- ior among cats. Rubbing of the flanks and genitalia of mares, whether done by the stallion or by a human handler, evokes behavior signals of estrus from the mare that othe1wise would not be displayed. Chin resting by a bull on the back of a cow just prior to mounting may have some stimulatory effect on the cow. Ve tB oo ks .ir 238 Reproductive Behavior Penile Erection and Protrusion Completes the Pr·ecopulatory Phase of Reproductive Behavior When sexual receptivity of a female is es- tablished and sufficient arousal is accomplished in the male, erection and protrusion of the penis ensue . Successful penile erection requires a complex series of neural and vasomotor (blood vessel) reactions. Erection of the penis is necessary for copulation and deposition of semen in the female reproductive h·act. Erection is characterized by a marked increase in the rigidity of the penis. The increased rigidity is the result of a marked increase in arterial inflow of blood when compared to the venous outflow of blood. Erection requires that blood be trapped within the cavernous sinuses of the penis. Increased blood flow to the pe- nis is brought about by vasodilation of the arterioles supplying it. In the bull, ram and boar erection not only involves increased blood flow and a subsequent Figure 11-7. Steps in Penile Erection as They Relate to Cavernous Blood Pressure and Contraction of the Bulbospongiosus and Ischiocavernosus Muscles ";) :I E E -f :s Ul Ul f D. "C 0 0 a5 Ill :s 0 c Q) u Sexual arousal (visual, tactile, olfactory) (Modified from Beckett, et al. 1972. Bioi. of Reprod. 7:359) . .•. ,ljJJ..:::: of bulbospongiosus --- - - ------ --41'1-___,- , . A.\1 Contractions of ischiocavernosus t Blood flow to cavernous tissue + + venous outflow Vasodilation of helicine arteries (tblood flow) 7 Time (seconds) Cavernous pressure increase in pressure, but a simultaneous relaxation ofthe reh·actor penis muscles. Thus, erection and protrusion also involve straightening of the penis to eliminate the sigmoid flexure. The penis of the bull, boar and ram is fibroelastic in nature and therefore does not increase significantly in diameter during erection and protrusion. In contrast, the penis of the stallion increases signifi- cantly in diameter during erection. The stallion has a retractor penis muscle that, as in other species, relaxes during erection. However, the stallion does not have a sigmoid flexure. Engorgement with blood plays a much more significant role in the highly vascular penis of the stallion, dog and man than in the bu11, ram, boar and camelids. Erection of the penis requires: • elevated arterial blood inflow • dilation of corporal sinusoids • restricted venous outflow • elevated intrapenile pressure • relaxation of the retractor . penis muscle Contractions of the ischiocavernosus muscles cause compression of the penile veins. This compres- sion causes blockage of venous retum thus enabling the cavem ous tissue to retain blood for maintenance of an erection. As you will recall, the ischiocavernosus muscles surround the two crura. Intem1ittent contrac- tions of the muscles creates a pump-like action at the base of the penis. These contractions result in a buildup of blood within the corpus cavemosum of the penis and exceptionally high pressures result. For example, during the final stages of erection, the pressures within the cavernous tissue of the goat penis can reach 7,000 nun Hg (See Figme 11-7). When the penis is flaccid, pressures within the corpus cavernosum are only 19 mm Hg. Pressures in the bull penis are around 1,700 mm Hg during peak erection and about 30 mm Hg when the cavernous spaces are collapsed. Figure 11-7 summarizes the steps of penile erection and intrapenile pressures as they relate to contraction of the ischiocav- emosus and bulbospongiosus muscles. One of the most publicized phannaceuticals ever introduced is a material called Sildenafil Citrate (Viagra®). This pharmaceutical provides a therapy for erectile dysfunction in men. Erectile dysfunction is defined as the inability to achieve and maintain a penile erection (tumescence). Reports indicate that 10% of men between the ages 40 and 70 years old are affl icted Reproductive Behavior 239 Figure 11-8. Basic Steps in the Erectile Process STEP I Erotogenic stimuli cause sensory nerves to fire r STEP 2 Sensory nerves activate "Reproductive Behavior Center" in hypothalamus- (See Figure 11-4) I ... STEP 3 Stimulation of parasympathetic nerves that innervate peni le arterioles STEP4 Parasympathetic ne rve te rminals release nitric oxide (NO) - (See Figure 11-9) STEP 5 Nitric oxide init iates biochemical cascade that causes erection - (See Figure 11 -9) by complete erectile failure. Other reports have esti- mated that up to 30 million men in the United States may have some fonn of erectile dysfunction. Erectile dysfunction is rare among domestic animals because such males are rapidly eliminated from the gene pool by artificial selection (culling) or by natural selection (no erection-no copulation-no offspring). Erection of the Penis Requires Sensory Input and a Local Vascular Response As mentioned earlier in the chapter, penile erec- tion is a complex series of neural and vasomotor events. These events can be broadly subdivided into a nervous component (cerebral and spinal) and a local vascular component within the penis. The nervous component is arousal-driven. For example, there must be ap- propriate sensory stimuli (tactile, visual, auditory and olfactory) in order for the central nervous system to be appropriately stimulated so that efferent neural events Ve tB oo ks .ir 238 Reproductive Behavior Penile Erection and Protrusion Completes the Pr·ecopulatory Phase of Reproductive Behavior When sexual receptivity of a female is es- tablished and sufficient arousal is accomplished in the male, erection and protrusion of the penis ensue . Successful penile erection requires a complex series of neural and vasomotor (blood vessel) reactions. Erection of the penis is necessary for copulation and deposition of semen in the female reproductive h·act. Erection is characterized by a marked increase in the rigidity of the penis. The increased rigidity is the result of a marked increase in arterial inflow of blood when compared to the venous outflow of blood. Erection requires that blood be trapped within the cavernous sinuses of the penis. Increased blood flow to the pe- nis is brought about by vasodilation of the arterioles supplying it. In the bull, ram and boar erection not only involves increased blood flow and a subsequent Figure 11-7. Steps in Penile Erection as They Relate to Cavernous Blood Pressure and Contraction of the Bulbospongiosus and Ischiocavernosus Muscles ";) :I E E -f :s Ul Ul f D. "C 0 0 a5 Ill :s 0 c Q) u Sexual arousal (visual, tactile, olfactory) (Modified from Beckett, et al. 1972. Bioi. of Reprod. 7:359) . .•. ,ljJJ..:::: of bulbospongiosus --- - - ------ --41'1-___,- , . A.\1 Contractions of ischiocavernosus t Blood flow to cavernous tissue + + venous outflow Vasodilation of helicine arteries (tblood flow) 7 Time (seconds) Cavernous pressure increase in pressure, but a simultaneous relaxation ofthe reh·actor penis muscles. Thus, erection and protrusion also involve straightening of the penis to eliminate the sigmoid flexure. The penis of the bull, boar and ram is fibroelastic in nature and therefore does not increase significantly in diameter during erection and protrusion. In contrast, the penis of the stallion increases signifi- cantly in diameter during erection. The stallion has a retractor penis muscle that, as in other species, relaxes during erection. However, the stallion does not have a sigmoid flexure. Engorgement with blood plays a much more significant role in the highly vascular penis of the stallion, dog and man than in the bu11, ram, boar and camelids. Erection of the penis requires: • elevated arterial blood inflow • dilation of corporal sinusoids • restricted venous outflow • elevated intrapenile pressure • relaxation of the retractor . penis muscle Contractions of the ischiocavernosus muscles cause compression of the penile veins. This compres- sion causes blockage of venous retum thus enabling the cavem ous tissue to retain blood for maintenance of an erection. As you will recall, the ischiocavernosus muscles surround the two crura. Intem1ittent contrac- tions of the muscles creates a pump-like action at the base of the penis. These contractions result in a buildup of blood within the corpus cavemosum of the penis and exceptionally high pressures result. For example, during the final stages of erection, the pressures within the cavernous tissue of the goat penis can reach 7,000 nun Hg (See Figme 11-7). When the penis is flaccid, pressures within the corpus cavernosum are only 19 mm Hg. Pressures in the bull penis are around 1,700 mm Hg during peak erection and about 30 mm Hg when the cavernous spaces are collapsed. Figure 11-7 summarizes the steps of penile erection and intrapenile pressures as they relate to contraction of the ischiocav- emosus and bulbospongiosus muscles. One of the most publicized phannaceuticals ever introduced is a material called Sildenafil Citrate (Viagra®). This pharmaceutical provides a therapy for erectile dysfunction in men. Erectile dysfunction is defined as the inability to achieve and maintain a penile erection (tumescence). Reports indicate that 10% of men between the ages 40 and 70 years old are affl icted Reproductive Behavior 239 Figure 11-8. Basic Steps in the Erectile Process STEP I Erotogenic stimuli cause sensory nerves to fire r STEP 2 Sensory nerves activate "Reproductive Behavior Center" in hypothalamus- (See Figure 11-4) I ... STEP 3 Stimulation of parasympathetic nerves that innervate peni le arterioles STEP4 Parasympathetic ne rve te rminals release nitric oxide (NO) - (See Figure 11-9) STEP 5 Nitric oxide init iates biochemical cascade that causes erection - (See Figure 11 -9) by complete erectile failure. Other reports have esti- mated that up to 30 million men in the United States may have some fonn of erectile dysfunction. Erectile dysfunction is rare among domestic animals because such males are rapidly eliminated from the gene pool by artificial selection (culling) or by natural selection (no erection-no copulation-no offspring). Erection of the Penis Requires Sensory Input and a Local Vascular Response As mentioned earlier in the chapter, penile erec- tion is a complex series of neural and vasomotor events. These events can be broadly subdivided into a nervous component (cerebral and spinal) and a local vascular component within the penis. The nervous component is arousal-driven. For example, there must be ap- propriate sensory stimuli (tactile, visual, auditory and olfactory) in order for the central nervous system to be appropriately stimulated so that efferent neural events Ve tB oo ks .ir I 240 Reproductive Behavior Figure 11-9. Vascular and Biochemical Contra! of an Erection (Modified from Korenman. 1998. Am. J. Med. 105.135.) Superficial dorsal vein vein · Erect Penis Arte rlol inflow Internal pudendal -- . Circ umflex vein Emissory --+ Cavernosal artery Flaccid Penis vein PDEs + Inhibi tion Erect Penis Sinusoid smooth muscle relaxes I ERElJoNI Anatomy The shaft of the penis consists of two dorso-lateral corpora caver- nosa and the corpus spongiosum. Arteria l blood is supplied by the in- ternal pudendal artery that supplies the dorsal and deep cavernosal ar- teries. Corporal sinusoids are sup- plied by helicine arteries. The deep dorsal vein and superficial dorsal vein drain the erectile tissues. Flaccid penis The sinusoids are flattened be- cause adrenergic nerves secrete norepinepherine that causes vaso- constriction. Blood flow to the cav- ernous tissue therefore is quite low for the majority of the time. Since no erotogenic stimuli are pres- ent, nonadrenergic noncholinergic (NANC) parasympathetic neurons do not fire and thus do not release nitric oxide (NO). Therefore, vaso- constriction takes precedence over vasodilation. Erect penis When erotogenic stimuli are pres- ent the NANC neurons fire and release nitric oxide (NO) from their terminals. When NO is released, it activ ates an enzyme called guanylate cyclase. This enzyme converts guanylate tri phosphate (GTP) to cyclic guanyosine mono- phosphate (cGMP) and causes the smooth muscle of the corporal sinusoids to relax (vasodilatation). The cavernous sinusoids engorge with blood and intracorporal pres- sure increases dramatically. This compresses the venules through which blood exits the penis. Blood is then trapped within the penis causing an erection. Reproductive Behavior 241 can cause an erection. These extrinsic stimuli are called erotogenic stimuli. As shown in Figure 11-4, these stimuli cause afferent sensory nerves to fire. Their tern1inals synapse with neurons in the so-called "behav- ior center" in the hypothalamus. These hypothalamic neurons synapse with parasympathetic and sympathetic efferent neurons that control penile vascular smooth muscle (vascular tone). The basic steps in the erectile process are outlined in Figure 11 -8. Mounting postures and characteri stics of copulatory behavior for various species are presented in Figures 11- I 0 and I I -l I. The purpose of mounting is for the male to position himself so that intromission can occur. Intromission is the successful entrance of the penis into the vagina. Following intromission, ejaculation takes place in response to sensory stimula- tion of the glans penis. The time of ejaculation relative to intromission varies significantly among species (See Figures 11- 10, I 1- 11 and 11-12). For example, in the bull and the ram ejaculation occurs within one or two seconds after intromission. In these species ejaculation is stimulated by the warm temperature of th e vagina. Vag inal pressure is relatively unimportant in inducing ejaculation in the ram and bull. In contrast, the boar may have a sustained ejaculation for periods of up to 30 minutes. The stallion has a mating duration of between 30 seconds and one minute. The llama and the dog are perhaps the most sustained copulators with reports of copulation occuring continually for up to 50 minutes. Erection is caused by the firing ofnonadrener- gic, noncholonergic (NAN C) parasympathetic neurons that release nitric oxide (NO), a gas, from their ter- minals. N itric oxide is the principal neurotransmitter that "dr ives" the erecti le process. Nitric oxide causes its effect by stimulating an enzyme, guanylate cyclase, to convert guany late triphosphate (GTP) to cyclic guanosine monophosphate ( cGMP). Cyclic guanosine monophosphate causes corporal smooth muscle relax- ation (vasodilation) and an erection results. Under nonerotogenic conditions, cGMP is acted upon by PDE5 (Phosphodiesterase 5) and this enzyme promotes the conversion of cGMP to GMP. This breakdown causes increased vascular tone result- ing in outflow of blood from the corpora cavernosa and loss of an erection. Sildenafil blocks the action of PDE5 thus prolonging the vasodilation effect of cGMP and an erection develops that can be maintained for a sustained period of time. It should be emphasized that without nitric oxide production by the parasympathetic nerve terminals Sildenafil can have no effect because nitric oxide must be present for cGMP to be produced. The usual flaccid state of the penis (contracted corporal arteries) results from a tonic contraction of the arterial and corporal smooth muscles mediated by sympathetic adrenergic neurons. Such vasoconstriction keeps pe- nile blood flow to a minimum under non-erotogenic conditions. When the corporal smooth muscles relax because of cGMP, the resistance to blood flow by the penile arterioles and corporal sinusoids decreases and blood flow to the penis triples or quadruples when the appropriate erotogenic stimuli are present. When an erection occurs, the sinusoid pressure is so great that the emissary veins are collapsed. Therefore, blood cannot return through them because venous outflow is blocked. Penile erection can be maintained for as long as vasodi- lation of the corporal smooth muscle takes place. TI1ese reactions are summarized in Figure l l-9. Ejaculation is a simple neural reflex caused by: • intromission • stimulation of the glans penis • forceful muscle contraction Ejaculation is defined as the reflex expulsion of spermatozoa and seminal plasma from the male repro- ductive tract. The basic mechanism for ejaculation of semen is quite s imilar among all mammals. Expulsion of semen is the result of sensory stimulation, primarily to the glans penis, that causes a series of coordinated muscular contractions. Once intromission has been achieved, reflex impulses are initiated. These neural impulses are derived mainly from sensory nerves in the glans penis. Upon threshold stimulation, impulses are transmitted from the glans penis by way of the internal pudendal nerve to the lumbosacral region of the spinal cord (See Figure 11 -13). The sensory impulses result in fi ring of nerves in the spinal cord and the forcing of semen into the urethra is accompl ished by nerves in the hypogastric plexus that innervate the target muscles. Of primary importance for ejaculation are the urethralis muscle (that sun ounds the pelvic uretlu-a), the ischiocavernosus and the bulbospongiosus muscles. Copulatory behavior includes: • mounting • intromission • ejaculation Figure l l -13 summarizes the nerve pathways resulting in emission and ejaculation. It should be emphasized that emission is defined as the movement of seminal fluids from the accessory sex glands into the pelvic uretlu-a so they can mix with spennatozoa. Emission occurs before and during ejaculation. In some Ve tB oo ks .ir I 240 Reproductive Behavior Figure 11-9. Vascular and Biochemical Contra! of an Erection (Modified from Korenman. 1998. Am. J. Med. 105.135.) Superficial dorsal vein vein · Erect Penis Arte rlol inflow Internal pudendal -- . Circ umflex vein Emissory --+ Cavernosal artery Flaccid Penis vein PDEs + Inhibi tion Erect Penis Sinusoid smooth muscle relaxes I ERElJoNI Anatomy The shaft of the penis consists of two dorso-lateral corpora caver- nosa and the corpus spongiosum. Arteria l blood is supplied by the in- ternal pudendal artery that supplies the dorsal and deep cavernosal ar- teries. Corporal sinusoids are sup- plied by helicine arteries. The deep dorsal vein and superficial dorsal vein drain the erectile tissues. Flaccid penis The sinusoids are flattened be- cause adrenergic nerves secrete norepinepherine that causes vaso- constriction. Blood flow to the cav- ernous tissue therefore is quite low for the majority of the time. Since no erotogenic stimuli are pres- ent, nonadrenergic noncholinergic (NANC) parasympathetic neurons do not fire and thus do not release nitric oxide (NO). Therefore, vaso- constriction takes precedence over vasodilation. Erect penis When erotogenic stimuli are pres- ent the NANC neurons fire and release nitric oxide (NO) from their terminals. When NO is released, it activ ates an enzyme called guanylate cyclase. This enzyme converts guanylate tri phosphate (GTP) to cyclic guanyosine mono- phosphate (cGMP) and causes the smooth muscle of the corporal sinusoids to relax (vasodilatation). The cavernous sinusoids engorge with blood and intracorporal pres- sure increases dramatically. This compresses the venules through which blood exits the penis. Blood is then trapped within the penis causing an erection. Reproductive Behavior 241 can cause an erection. These extrinsic stimuli are called erotogenic stimuli. As shown in Figure 11-4, these stimuli cause afferent sensory nerves to fire. Their tern1inals synapse with neurons in the so-called "behav- ior center" in the hypothalamus. These hypothalamic neurons synapse with parasympathetic and sympathetic efferent neurons that control penile vascular smooth muscle (vascular tone). The basic steps in the erectile process are outlined in Figure 11 -8. Mounting postures and characteri stics of copulatory behavior for various species are presented in Figures 11- I 0 and I I -l I. The purpose of mounting is for the male to position himself so that intromission can occur. Intromission is the successful entrance of the penis into the vagina. Following intromission, ejaculation takes place in response to sensory stimula- tion of the glans penis. The time of ejaculation relative to intromission varies significantly among species (See Figures 11- 10, I 1- 11 and 11-12). For example, in the bull and the ram ejaculation occurs within one or two seconds after intromission. In these species ejaculation is stimulated by the warm temperature of th e vagina. Vag inal pressure is relatively unimportant in inducing ejaculation in the ram and bull. In contrast, the boar may have a sustained ejaculation for periods of up to 30 minutes. The stallion has a mating duration of between 30 seconds and one minute. The llama and the dog are perhaps the most sustained copulators with reports of copulation occuring continually for up to 50 minutes. Erection is caused by the firing ofnonadrener- gic, noncholonergic (NAN C) parasympathetic neurons that release nitric oxide (NO), a gas, from their ter- minals. N itric oxide is the principal neurotransmitter that "dr ives" the erecti le process. Nitric oxide causes its effect by stimulating an enzyme, guanylate cyclase, to convert guany late triphosphate (GTP) to cyclic guanosine monophosphate ( cGMP). Cyclic guanosine monophosphate causes corporal smooth muscle relax- ation (vasodilation) and an erection results. Under nonerotogenic conditions, cGMP is acted upon by PDE5 (Phosphodiesterase 5) and this enzyme promotes the conversion of cGMP to GMP. This breakdown causes increased vascular tone result- ing in outflow of blood from the corpora cavernosa and loss of an erection. Sildenafil blocks the action of PDE5 thus prolonging the vasodilation effect of cGMP and an erection develops that can be maintained for a sustained period of time. It should be emphasized that without nitric oxide production by the parasympathetic nerve terminals Sildenafil can have no effect because nitric oxide must be present for cGMP to be produced. The usual flaccid state of the penis (contracted corporal arteries) results from a tonic contraction of the arterial and corporal smooth muscles mediated by sympathetic adrenergic neurons. Such vasoconstriction keeps pe- nile blood flow to a minimum under non-erotogenic conditions. When the corporal smooth muscles relax because of cGMP, the resistance to blood flow by the penile arterioles and corporal sinusoids decreases and blood flow to the penis triples or quadruples when the appropriate erotogenic stimuli are present. When an erection occurs, the sinusoid pressure is so great that the emissary veins are collapsed. Therefore, blood cannot return through them because venous outflow is blocked. Penile erection can be maintained for as long as vasodi- lation of the corporal smooth muscle takes place. TI1ese reactions are summarized in Figure l l-9. Ejaculation is a simple neural reflex caused by: • intromission • stimulation of the glans penis • forceful muscle contraction Ejaculation is defined as the reflex expulsion of spermatozoa and seminal plasma from the male repro- ductive tract. The basic mechanism for ejaculation of semen is quite s imilar among all mammals. Expulsion of semen is the result of sensory stimulation, primarily to the glans penis, that causes a series of coordinated muscular contractions. Once intromission has been achieved, reflex impulses are initiated. These neural impulses are derived mainly from sensory nerves in the glans penis. Upon threshold stimulation, impulses are transmitted from the glans penis by way of the internal pudendal nerve to the lumbosacral region of the spinal cord (See Figure 11 -13). The sensory impulses result in fi ring of nerves in the spinal cord and the forcing of semen into the urethra is accompl ished by nerves in the hypogastric plexus that innervate the target muscles. Of primary importance for ejaculation are the urethralis muscle (that sun ounds the pelvic uretlu-a), the ischiocavernosus and the bulbospongiosus muscles. Copulatory behavior includes: • mounting • intromission • ejaculation Figure l l -13 summarizes the nerve pathways resulting in emission and ejaculation. It should be emphasized that emission is defined as the movement of seminal fluids from the accessory sex glands into the pelvic uretlu-a so they can mix with spennatozoa. Emission occurs before and during ejaculation. In some Ve tB oo ks .ir ' [ill 242 Reproductive Behavior Figure 11-10. Characteristics of Copulation, Site of Seminal Deposition and Number of Ejaculations to Satiation and Exhaustion in the Ram, Bull, Stallion and Boar Mating pair Photos of: Deposition Ejaculations Ejaculations to Satiation to Exhaustion 1 to 2 sec- .8 to 1 ml e xt e r n a I onds (1 pel- (.1 to 2ml) cervical os 1 to 3 sec- 3-5ml fornix vagina onds (1 pel- (.5 to 12ml) commences that is ac- companied by somnolence) 75-120ml 200-250ml external cer- v ical as but semen enters uterus at high pressure cervix and uterus 10 30 to 40 20 60 to 80 3 20 3 8 Ram/Ewe-courtesy of Drs. G.S. Lewis and J.B. Taylor. U.S. Sheep Experimental Station http://pwa.ars.usda.gov/dubois!index Bull/Cow-courtesy of Dr. L.S. Katz, Rutgers University Stallion/Mare-courtesy of Dr. A. Tibary, Washington State University, College of Veterinary Medicine Reproductive Behavior 243 Figure 11-11. Characteristics of Copulation, Site of Seminal Deposition and Number of Ejaculations to Satiation and Exhaustion in the Camel and Llama Duration of Volume of Site of Average Maximum Mating pair Copulation Ejaculate Semen Number of Number of (Range) Deposition Ejaculations Ejaculations to Satiation to Exhaustion 6-20 minutes, 3-8m I Partly intrauter- 23 matings Data not extension of ine, partly intrac- in 24 hr available neck, straining erv ical , some of the body, intravaginal multiple ejacu- l a t ions per copulation 20 - 30 min - 1-5ml intrauterine Data not Data not u tes, bo d y available available tremors and pelvic thrusts (Photos courtesy of Dr. A. Tibary, Washington State University, College of Veterinary Medicine) species, such as the boar, stallion and dog, emission oc- curs in a sequence resulting in an ej aculate that consists of various fluid fractions (See Chapter I 2). Postcopulatory behavior involves refractivity and recovery. Following ejaculation, all males experience a refractory per iod before a second ejaculation can occur. The length oftime of this refractory period depends on several factors. These factors are; degree of sexual rest prior to copulation, age of the male, species of the male, degree of female novelty and number of previous ejaculations. The postcopulatory refractory period is sometimes erroneously refeiTed to as sexual exhaustion. The refractory period should be considered as part of satiation rather than exhaustion. With natural service, it is quite nonnal for a male to copulate repeatedly with the same female. For example, a stall ion will breed a mare in heat 5 to I 0 times during one estrus period. Rams are noted to remate with the same ewe 4 to 5 times. Bulls also remate with estrous cows repeatedly. In fact, it has been noted in most species that if more than one female is in heat at the same time, some males will generally copulate preferentially with one and sometimes will not copulate with a second female. Boars nonnally serve sows several times over a period of 1 to 2 days. Sexual satiation refers to a condition in which fi1rther stimul i will not cause immediate responsive- ness or motivation under a given set of stimulus con- ditions. Restimulation may occur after the refractory period. Figures I I - 1 0 and I 1- I I compare the normal number of ejaculations to satiety and the number of ejaculations to exhaustion among species. Exhaustion is the condition whereby no further sexual behavior can be induced even if sufficient stimuli are present. As you can see from Figures 11-1 0 and 11-11 , there is a large variation in the behavioral reserves (the behavioral capacity, or libido) among species. There is also a large variation in libido within species. For example, beef bulls have significantly lower behav- ioral reserves than dairy bulls. While the factors that control the degree of reproductive behavior among males are poorly understood, they are almost certainly governed by genetic factors as well as environmental factors. Reproductive behavior can be enhanced by: • introducing novel stimulus animals • changing stimulus settings Ve tB oo ks .ir ' [ill 242 Reproductive Behavior Figure 11-10. Characteristics of Copulation, Site of Seminal Deposition and Number of Ejaculations to Satiation and Exhaustion in the Ram, Bull, Stallion and Boar Mating pair Photos of: Deposition Ejaculations Ejaculations to Satiation to Exhaustion 1 to 2 sec- .8 to 1 ml e xt e r n a I onds (1 pel- (.1 to 2ml) cervical os 1 to 3 sec- 3-5ml fornix vagina onds (1 pel- (.5 to 12ml) commences that is ac- companied by somnolence) 75-120ml 200-250ml external cer- v ical as but semen enters uterus at high pressure cervix and uterus 10 30 to 40 20 60 to 80 3 20 3 8 Ram/Ewe-courtesy of Drs. G.S. Lewis and J.B. Taylor. U.S. Sheep Experimental Station http://pwa.ars.usda.gov/dubois!index Bull/Cow-courtesy of Dr. L.S. Katz, Rutgers University Stallion/Mare-courtesy of Dr. A. Tibary, Washington State University, College of Veterinary Medicine Reproductive Behavior 243 Figure 11-11. Characteristics of Copulation, Site of Seminal Deposition and Number of Ejaculations to Satiation and Exhaustion in the Camel and Llama Duration of Volume of Site of Average Maximum Mating pair Copulation Ejaculate Semen Number of Number of (Range) Deposition Ejaculations Ejaculations to Satiation to Exhaustion 6-20 minutes, 3-8m I Partly intrauter- 23 matings Data not extension of ine, partly intrac- in 24 hr available neck, straining erv ical , some of the body, intravaginal multiple ejacu- l a t ions per copulation 20 - 30 min - 1-5ml intrauterine Data not Data not u tes, bo d y available available tremors and pelvic thrusts (Photos courtesy of Dr. A. Tibary, Washington State University, College of Veterinary Medicine) species, such as the boar, stallion and dog, emission oc- curs in a sequence resulting in an ej aculate that consists of various fluid fractions (See Chapter I 2). Postcopulatory behavior involves refractivity and recovery. Following ejaculation, all males experience a refractory per iod before a second ejaculation can occur. The length oftime of this refractory period depends on several factors. These factors are; degree of sexual rest prior to copulation, age of the male, species of the male, degree of female novelty and number of previous ejaculations. The postcopulatory refractory period is sometimes erroneously refeiTed to as sexual exhaustion. The refractory period should be considered as part of satiation rather than exhaustion. With natural service, it is quite nonnal for a male to copulate repeatedly with the same female. For example, a stall ion will breed a mare in heat 5 to I 0 times during one estrus period. Rams are noted to remate with the same ewe 4 to 5 times. Bulls also remate with estrous cows repeatedly. In fact, it has been noted in most species that if more than one female is in heat at the same time, some males will generally copulate preferentially with one and sometimes will not copulate with a second female. Boars nonnally serve sows several times over a period of 1 to 2 days. Sexual satiation refers to a condition in which fi1rther stimul i will not cause immediate responsive- ness or motivation under a given set of stimulus con- ditions. Restimulation may occur after the refractory period. Figures I I - 1 0 and I 1- I I compare the normal number of ejaculations to satiety and the number of ejaculations to exhaustion among species. Exhaustion is the condition whereby no further sexual behavior can be induced even if sufficient stimuli are present. As you can see from Figures 11-1 0 and 11-11 , there is a large variation in the behavioral reserves (the behavioral capacity, or libido) among species. There is also a large variation in libido within species. For example, beef bulls have significantly lower behav- ioral reserves than dairy bulls. While the factors that control the degree of reproductive behavior among males are poorly understood, they are almost certainly governed by genetic factors as well as environmental factors. Reproductive behavior can be enhanced by: • introducing novel stimulus animals • changing stimulus settings Ve tB oo ks .ir 244 Reproductive Behavior Figure 11-12. Copulation in the Dog First Stage Coitus (1-2 min) The Turn (2-5 sec) The male and female remain "tied" together be- cause the bulbus glandis of the penis remains engorged with blood after the turn. Contractions of the muscles near the base of the penis prevent venous outflow of blood from the bulbus glandis. Also, the sphincter muscles of the vulva constrict thus compressing the dorsal veins of the penis preventing blood from leaving. (Figures modified from Grandage. 1972. Vet. Rec. 91:141) The vascu lature of the dog penis has been in- jected with latex and the tissue dissolved away leaving cast of the vascu lature. Red vessels are arteries and the blue vessels are veins. IL=IIeum, MCA=Medial Caudal Artery, LCA=Lateral Caudal Artery, IS=Ischium , A=Acetabulum , CS=Corpus Spong iousum, CC=Corpus Caver- nosum, DPA=Dorsal Penile Artery, DPV=Dorsal Penile Vein, OP=Os Penis, BG=Bulbus Glandis, PLG=Pars Longa Glandis, PA=Prostatic Artery, I P=lnternal Pudendal Artery, IIA=InternallliacArtery (Specimen courtesy of the Worthman Veterinary Anatomy Teach- ing Museum, College of Veterinary Medicine, Washington State University. Specimen prepared by Dr. R.P. Worthman) First Stage Coitus The male mounts the female in a manner typical of a quadraped. The female holds the tail to one side and the penis is introduced into the vagina by a few thrusting movements. This stage of copula- tion lasts for only 1-2 minutes. The first and second fractions of semen are ejaculated during the first stage coitus. The Turn This is the transition between first stage and sec- ond stage coitus. Shortly after ejaculation, the dog dismounts and turns around while lifting one hind leg over the bitch. Second Stage Coitus After the turn, the animals stand with their hind quarters in contact and their heads facing opposite directions. The third fraction of semen is ejaculated during this stage. Second stage coitus may last from 5-45 minutes. It is believed that the purpose of second stage coitus is to encourage uterine rather than vaginal insemination. Turning around discourages detumescence of the penis and there- fore maintains high intravaginal pressure. The dog steadily ejaculates up to 30-ml of seminal fluid that is delivered through the cervix into the uterus. This phenomenon tends to force the sperm-rich fraction into the uterus. The copulatory behavior described here is perfectly natural. Unfortunately this behavior is often interpreted as being unnatural and attempts to break the "tie" are often made by the uninformed. Such intervention compromises fertility because delivery of semen to the uterus over a sustained period of time is reduced. Reproductive Behavior 245 Figure 11-13. Major Steps in Ejaculation Afferent Sensory stimulat ion of glans penis (temperature and pressure) Int romission Reproductive Behavior and Spermatozoal Output can be Manipulated The degree of novelty of both the copulatmy partner and the copulatmy environment can be of great importance when managing reproductive behavior in breeding males. U nder condi tions of artificial insemina- tion, where repeated seminal collection is necessary to maximize the harvest of spermatozoa, understanding the influence of novelty and mating situations is important. The "Coolidge Effect" is defined as the restoration of mating behavior in males (that have reached sexual satiation) when the original female is replaced by a novel female . In other words, a sexually satiated male can be restimulated if exposed to a novel female. (For derivation of the term "Coolidge Effect" see Further Phenomena for Fertility) Semen collection in bull studs can occur as frequently as 4 to 6 ejaculations per week. In order for this collection frequency to be successful, the male 0 Sudden and pow erful contraction of urethralis, bulbospongiosus and ischiocavernosus m uscles 0 Expulsion of semen must first be sexually stimulated. Sexual stimulation is defined as the presentation of a stimulus situation that will achieve mounting and ejaculation. The purpose of sexual stimulation is to obtain ejaculation or mating in the shmtest time possible so that manpower involved in managing the mating of animals can be minimized. There are three approaches used to re-induce sexual stimulation in bulls used for artificial insemination. These approaches are: to introduce a novel stimulus animal; to change the stimulus setting; or both. Pre- sentation of novel stimulus animals reinitiates sexual behavior after sexual satiation in bulls (See Figure 11 - 14, "Novel Fema les"). A second approach to achieve sexual stimulation after satiation is to present familiar stimulus animals in new stimulus situations . In other words, changing the location or setting has a stimulatmy effect on the satiated male (See Figure 11-14 "New Lo- cation"). In cases where sexual stimulation is difficult to achieve, presenting a novel stimulus animal, coupled with changing locations, often has positive effects. Ve tB oo ks .ir 244 Reproductive Behavior Figure 11-12. Copulation in the Dog First Stage Coitus (1-2 min) The Turn (2-5 sec) The male and female remain "tied" together be- cause the bulbus glandis of the penis remains engorged with blood after the turn. Contractions of the muscles near the base of the penis prevent venous outflow of blood from the bulbus glandis. Also, the sphincter muscles of the vulva constrict thus compressing the dorsal veins of the penis preventing blood from leaving. (Figures modified from Grandage. 1972. Vet. Rec. 91:141) The vascu lature of the dog penis has been in- jected with latex and the tissue dissolved away leaving cast of the vascu lature. Red vessels are arteries and the blue vessels are veins. IL=IIeum, MCA=Medial Caudal Artery, LCA=Lateral Caudal Artery, IS=Ischium , A=Acetabulum , CS=Corpus Spong iousum, CC=Corpus Caver- nosum, DPA=Dorsal Penile Artery, DPV=Dorsal Penile Vein, OP=Os Penis, BG=Bulbus Glandis, PLG=Pars Longa Glandis, PA=Prostatic Artery, I P=lnternal Pudendal Artery, IIA=InternallliacArtery (Specimen courtesy of the Worthman Veterinary Anatomy Teach- ing Museum, College of Veterinary Medicine, Washington State University. Specimen prepared by Dr. R.P. Worthman) First Stage Coitus The male mounts the female in a manner typical of a quadraped. The female holds the tail to one side and the penis is introduced into the vagina by a few thrusting movements. This stage of copula- tion lasts for only 1-2 minutes. The first and second fractions of semen are ejaculated during the first stage coitus. The Turn This is the transition between first stage and sec- ond stage coitus. Shortly after ejaculation, the dog dismounts and turns around while lifting one hind leg over the bitch. Second Stage Coitus After the turn, the animals stand with their hind quarters in contact and their heads facing opposite directions. The third fraction of semen is ejaculated during this stage. Second stage coitus may last from 5-45 minutes. It is believed that the purpose of second stage coitus is to encourage uterine rather than vaginal insemination. Turning around discourages detumescence of the penis and there- fore maintains high intravaginal pressure. The dog steadily ejaculates up to 30-ml of seminal fluid that is delivered through the cervix into the uterus. This phenomenon tends to force the sperm-rich fraction into the uterus. The copulatory behavior described here is perfectly natural. Unfortunately this behavior is often interpreted as being unnatural and attempts to break the "tie" are often made by the uninformed. Such intervention compromises fertility because delivery of semen to the uterus over a sustained period of time is reduced. Reproductive Behavior 245 Figure 11-13. Major Steps in Ejaculation Afferent Sensory stimulat ion of glans penis (temperature and pressure) Int romission Reproductive Behavior and Spermatozoal Output can be Manipulated The degree of novelty of both the copulatmy partner and the copulatmy environment can be of great importance when managing reproductive behavior in breeding males. U nder condi tions of artificial insemina- tion, where repeated seminal collection is necessary to maximize the harvest of spermatozoa, understanding the influence of novelty and mating situations is important. The "Coolidge Effect" is defined as the restoration of mating behavior in males (that have reached sexual satiation) when the original female is replaced by a novel female . In other words, a sexually satiated male can be restimulated if exposed to a novel female. (For derivation of the term "Coolidge Effect" see Further Phenomena for Fertility) Semen collection in bull studs can occur as frequently as 4 to 6 ejaculations per week. In order for this collection frequency to be successful, the male 0 Sudden and pow erful contraction of urethralis, bulbospongiosus and ischiocavernosus m uscles 0 Expulsion of semen must first be sexually stimulated. Sexual stimulation is defined as the presentation of a stimulus situation that will achieve mounting and ejaculation. The purpose of sexual stimulation is to obtain ejaculation or mating in the shmtest time possible so that manpower involved in managing the mating of animals can be minimized. There are three approaches used to re-induce sexual stimulation in bulls used for artificial insemination. These approaches are: to introduce a novel stimulus animal; to change the stimulus setting; or both. Pre- sentation of novel stimulus animals reinitiates sexual behavior after sexual satiation in bulls (See Figure 11 - 14, "Novel Fema les"). A second approach to achieve sexual stimulation after satiation is to present familiar stimulus animals in new stimulus situations . In other words, changing the location or setting has a stimulatmy effect on the satiated male (See Figure 11-14 "New Lo- cation"). In cases where sexual stimulation is difficult to achieve, presenting a novel stimulus animal, coupled with changing locations, often has positive effects. Ve tB oo ks .ir II 246 Reproductive Behavior Figure 11-14. Introduction of Novel Females and a Change of Locations has a Positive Effect on Mounting Behavior (Hypothetical examples, not experimental data) Familiar Female A familiar female may stimulate a lllllllll ------ "------lll ----------" ----- ----1 bull to mount about 12 times in an 8 112 Mounts I hour period. SS= sexual satiation I I I I I I I I I 0 I 2 3 4 5 6 7 8 Time (h) Familiar Female and New Location Bulls can be restimulated to mount + New location + New location (after satiation) by changing the ! lllllllll ---·;.II IIlli --- Jiilll stimulus setting (new location). This induces more total mounts (18 liB Mounts I mounts) than the familiar female (12 I I mounts). 0 I 2 3 4 5 6 7 8 Time(h) Novel Females When the novel females (1-5) are introduced after a period of sexual llillllll Ji IIlli _,[ 1111 --- - I satiation, mounting behavior is stimulated beyond that realized with change of location and exposure to a single familiar female (24 mounts I I I I I I I I I 0 I 2 3 4 5 6 7 Time (h) There has been little research conducted on the effect of introducing novel animals upon stimulation of mounting behavior in the female. However, it has been shown that dairy cows will mount novel cows with a greater frequency than they do familiar cows. As you might expect, the effect of novelty is confounded with the stage of the cycle. Sexual preparation prolongs sexual stimulation and increases spermatozoa per ejaculation. In order to maximize the output of spermatozoa per ejaculate, sexual preparation is necessary. Sexual preparation is extending the period of sexual stimula- tion beyond that needed for mounting and ejaculation. 8 vs. 18 and 12 respectively). Sexual preparation prolongs the precopulatory stage of reproductive behavior. The purpose of sexual prepara- tion is to collect semen containing the greatest possible number of spemmtozoa per ejaculation. Figure ll-15 illustrates the physiologic mechanisms believed to be responsible for enhancing spermatozoal numbers in the ejaculate. Three approaches are used to sexually prepare a male. These are: false-mounting, restraint and false-mounting plus restraint. Sexual preparation may include: • false-mounting • restraint • false-mounting plus restraint Reproductive Behavior 24 7 Figure 11-15. Major Steps in Sexual Preparation Resulting in Transport of Spermoatozoa from the Tail of the Epididymis into the Pelvic Urethra Sensory stimulation (optic, olfactory, tactile and auditory) Afferent Transport of spermatozoa into an ejaculatory position • Stimulation of nerves in the supraoptic and paraventricular nuclei 0 Contractions of smooth muscle in distal tail of epididymis and ductus deferens [ill I Ve tB oo ks .ir II 246 Reproductive Behavior Figure 11-14. Introduction of Novel Females and a Change of Locations has a Positive Effect on Mounting Behavior (Hypothetical examples, not experimental data) Familiar Female A familiar female may stimulate a lllllllll ------ "------lll ----------" ----- ----1 bull to mount about 12 times in an 8 112 Mounts I hour period. SS= sexual satiation I I I I I I I I I 0 I 2 3 4 5 6 7 8 Time (h) Familiar Female and New Location Bulls can be restimulated to mount + New location + New location (after satiation) by changing the ! lllllllll ---·;.II IIlli --- Jiilll stimulus setting (new location). This induces more total mounts (18 liB Mounts I mounts) than the familiar female (12 I I mounts). 0 I 2 3 4 5 6 7 8 Time(h) Novel Females When the novel females (1-5) are introduced after a period of sexual llillllll Ji IIlli _,[ 1111 --- - I satiation, mounting behavior is stimulated beyond that realized with change of location and exposure to a single familiar female (24 mounts I I I I I I I I I 0 I 2 3 4 5 6 7 Time (h) There has been little research conducted on the effect of introducing novel animals upon stimulation of mounting behavior in the female. However, it has been shown that dairy cows will mount novel cows with a greater frequency than they do familiar cows. As you might expect, the effect of novelty is confounded with the stage of the cycle. Sexual preparation prolongs sexual stimulation and increases spermatozoa per ejaculation. In order to maximize the output of spermatozoa per ejaculate, sexual preparation is necessary. Sexual preparation is extending the period of sexual stimula- tion beyond that needed for mounting and ejaculation. 8 vs. 18 and 12 respectively). Sexual preparation prolongs the precopulatory stage of reproductive behavior. The purpose of sexual prepara- tion is to collect semen containing the greatest possible number of spemmtozoa per ejaculation. Figure ll-15 illustrates the physiologic mechanisms believed to be responsible for enhancing spermatozoal numbers in the ejaculate. Three approaches are used to sexually prepare a male. These are: false-mounting, restraint and false-mounting plus restraint. Sexual preparation may include: • false-mounting • restraint • false-mounting plus restraint Reproductive Behavior 24 7 Figure 11-15. Major Steps in Sexual Preparation Resulting in Transport of Spermoatozoa from the Tail of the Epididymis into the Pelvic Urethra Sensory stimulation (optic, olfactory, tactile and auditory) Afferent Transport of spermatozoa into an ejaculatory position • Stimulation of nerves in the supraoptic and paraventricular nuclei 0 Contractions of smooth muscle in distal tail of epididymis and ductus deferens [ill I Ve tB oo ks .ir 248 Reproductive Behavior False mounting consists of manually deviat- ing the penis during a mount so that intromission can- not occur. If intromission does not occur, ejaculation usually does not occur. Restraint prevents the male from mounting even though he wishes to do so. Gen- erally, restraint is for two to tlu·ee minutes within two or three feet of the stimulus animal. A combination of false mounting and restraint will result in the greatest improvement of spemmtozoal output. In dairy bulls, the recommended procedures for sexual preparation are: one false mount followed by two minutes of restraint, followed by two additional false mounts before each ejaculation. In beef bulls, sexual preparation involves three false mounts with no restraint. In general, beef bulls have lower behavioral reserves (libido) than dairy bulls and thus have a less rigorous sexual preparation regimen. While sexual preparation is taking place, re- lease of oxytocin from the posterior pituitary occurs. Oxytocin causes contraction of the smooth musculature surrounding the tail of the epididymis and the ductus deferens. These contractions transport spem1atozoa from the tail of the epididymis into the duchts deferens and eventually into the pelvic urethra. Once spem1 gain entrance into the pelvic urethra, they begin to mix with secretions from the accessory sex glands. Homosexual-like Behavior Homosexual-like behavior is common among domestic animals and is particularly common in cattle. The tenn homosexuality implies a sexual preference for same-sex partners. In animals, there is not a preference, but rather indiscriminate orientation or same-sex di- rected behavior. Thus, an alternative term that is appli- cable to sub-primate animals would be homosexual-like behavior. Cows and bulls exhibit strong homosexual- like behavior. Similar behavior is seen in sheep and dogs and to a Jesser extent in swine and horses. Such behavior has profound usefulness for detecting cattle in estrus. When a female stands to be mounted by another cow, this alerts the management team that the cow is in estrus and artificial insemination can be performed. A favorite question of managers and stu- dents of reproductive physiology alike is, "What is the evolutionary advantage of animals displaying this kind of behavior?" While a definitive answer is not known, two theories exist to explain female-female mounting behavior in cattle. The first explanation theorizes that cows mounting each other provide a visual signal that attracts a bull to the cow in estrus. In other words, when a bull sees cows mounting each other he will investigate and if the cow is in standing estrus, he will breed her. The second theory explaining the evolution of homosexual-like behavior among cows involves inad- vertent genetic selection by man for this behavior. It has been proposed that cattle of European descent were se- lected by humans for their estrous behavior. In Medieval Europe, cattle husbandry involved the use of a few cows by each peasant farmer for three purposes: draft, milk and meat. Peasant f.·mners could not afford to maintain a bull for breeding purposes since the bulls gave no milk, gave birth to no calves and had obnoxious behavior that made them unsuitable for everyday management. In addition, most bulls apparently were owned by wealthy land holders who probably controlled the breeding, as well as the financial aspects of cattle management. Since most cows were kept in groups without intact males, the herdsmen needed some sign to tell him when his cows should be bred. Obviously, the cow that showed the most intense mounting behavior was the one most likely to be observed by the peasant and most likely to be bred by the nobleman's bull. Those that showed little mount- ing behavior did not become pregnant in a reasonable amount of time. This theory suggests that cows with a high degree of mounting behavior were inadvertently selected because they were noticed by man and offered a greater opporhmity to become pregnant. Thus, this behavioral trait was transmitted to their offspring. Artificial Insemination Requires an Understanding of Reproductive Behavior and Physiology There are two fundamental ways to collect semen from the male. The preferred method utilizes an artificial vagina or a device that simulates vaginal conditions of a female in estrus. The second method relies on electrical stimulation of the accessory sex glands and the pelvic urethra and this method is called electroejaculation. Electroejaculation is generally used in males of high genetic value that cannot physically perform mounting and ejaculation. In the beef industry, electroejaculation is used in range bulls. Typical artificial vaginas for domestic animals are shown are Figure 11-I 6. In general, artificial vaginas consist of an outer casing fashioned of reinforced rubber and a liner that is generally made of rubber that can be lubricated. Tempera hire and pressure are controlled by the water that is p laced between the casing and the liner. One end of the artificial vagina is attached to a funnel- like cone that in tum is attached to a collection vessel, usually a nonbreakable graduated test tube. From a behavioral perspective, males that are to be collected with an artificial vagina need some form of training. Males with previous sexual experience will readily mount a surrogate animal (artificial animal or "dummy"). The degree to which animals will mount Reproductive Behavior 249 Figure 11-16. Artificial Vaginas for Various Animals Outer casing l -- Warm water Rubber liner - Warm water Rubber collection funnel I -· ; - .--......... The typical artificial vagina consists of a sturdy outer casing, a rubber liner, a chamber fi lled with warm water, a rubber collection funnel and a collection tube. tube The artificial vagina for the stallion consists of a leather outer casing (C) equiped with a port to drain water (arrow). The collection vessel (CV) and the protective covering (PC) are shown. Ide- ally, ejaculation takes place in the collection cone (CC) so that most of the semen will drain directly into the collection vessel. (Artificial vagina courtesy of Northwest Equine Reproduction Laboratory, University of Idaho, www.avs.uidaho.edu/nerl) The artificial vagina for the bull consists of a black casing (C), a rubber liner (RL) a collection cone (CC) and a collection vessel (CV). Water is placed between the casing and the liner. The proper tem- perature is critical for successful ejaculation in the bull . While not shown in the photograph a protec- tive covering is placed over the cone and collection vessel to prevent cold shock of the semen. The artificial vagina for the boar consists of a bulb that can apply pressure to the artificial vagina. High pressure is obligatory for stimulation of the glans pen is and ejaculation in the boar. The artificial vagina for the boar also consists of an outer casing (C), a liner (L) and a protective covering (PC) that houses the collection vessel. (Photograph courtesy of MinitOb Germany, www.minitilb.de) The artificial vagina for collection of semen from rams and bucks consists of a rubber casing (C) with a valve (arrow) through which water can be added or emptied, a rubber liner and a collection vessel (CV). The protective covering (PC) is shown above the artificial vagina. (Photograph courtesy of MinitOb Germany, www.minitilb.de) Ve tB oo ks .ir 248 Reproductive Behavior False mounting consists of manually deviat- ing the penis during a mount so that intromission can- not occur. If intromission does not occur, ejaculation usually does not occur. Restraint prevents the male from mounting even though he wishes to do so. Gen- erally, restraint is for two to tlu·ee minutes within two or three feet of the stimulus animal. A combination of false mounting and restraint will result in the greatest improvement of spemmtozoal output. In dairy bulls, the recommended procedures for sexual preparation are: one false mount followed by two minutes of restraint, followed by two additional false mounts before each ejaculation. In beef bulls, sexual preparation involves three false mounts with no restraint. In general, beef bulls have lower behavioral reserves (libido) than dairy bulls and thus have a less rigorous sexual preparation regimen. While sexual preparation is taking place, re- lease of oxytocin from the posterior pituitary occurs. Oxytocin causes contraction of the smooth musculature surrounding the tail of the epididymis and the ductus deferens. These contractions transport spem1atozoa from the tail of the epididymis into the duchts deferens and eventually into the pelvic urethra. Once spem1 gain entrance into the pelvic urethra, they begin to mix with secretions from the accessory sex glands. Homosexual-like Behavior Homosexual-like behavior is common among domestic animals and is particularly common in cattle. The tenn homosexuality implies a sexual preference for same-sex partners. In animals, there is not a preference, but rather indiscriminate orientation or same-sex di- rected behavior. Thus, an alternative term that is appli- cable to sub-primate animals would be homosexual-like behavior. Cows and bulls exhibit strong homosexual- like behavior. Similar behavior is seen in sheep and dogs and to a Jesser extent in swine and horses. Such behavior has profound usefulness for detecting cattle in estrus. When a female stands to be mounted by another cow, this alerts the management team that the cow is in estrus and artificial insemination can be performed. A favorite question of managers and stu- dents of reproductive physiology alike is, "What is the evolutionary advantage of animals displaying this kind of behavior?" While a definitive answer is not known, two theories exist to explain female-female mounting behavior in cattle. The first explanation theorizes that cows mounting each other provide a visual signal that attracts a bull to the cow in estrus. In other words, when a bull sees cows mounting each other he will investigate and if the cow is in standing estrus, he will breed her. The second theory explaining the evolution of homosexual-like behavior among cows involves inad- vertent genetic selection by man for this behavior. It has been proposed that cattle of European descent were se- lected by humans for their estrous behavior. In Medieval Europe, cattle husbandry involved the use of a few cows by each peasant farmer for three purposes: draft, milk and meat. Peasant f.·mners could not afford to maintain a bull for breeding purposes since the bulls gave no milk, gave birth to no calves and had obnoxious behavior that made them unsuitable for everyday management. In addition, most bulls apparently were owned by wealthy land holders who probably controlled the breeding, as well as the financial aspects of cattle management. Since most cows were kept in groups without intact males, the herdsmen needed some sign to tell him when his cows should be bred. Obviously, the cow that showed the most intense mounting behavior was the one most likely to be observed by the peasant and most likely to be bred by the nobleman's bull. Those that showed little mount- ing behavior did not become pregnant in a reasonable amount of time. This theory suggests that cows with a high degree of mounting behavior were inadvertently selected because they were noticed by man and offered a greater opporhmity to become pregnant. Thus, this behavioral trait was transmitted to their offspring. Artificial Insemination Requires an Understanding of Reproductive Behavior and Physiology There are two fundamental ways to collect semen from the male. The preferred method utilizes an artificial vagina or a device that simulates vaginal conditions of a female in estrus. The second method relies on electrical stimulation of the accessory sex glands and the pelvic urethra and this method is called electroejaculation. Electroejaculation is generally used in males of high genetic value that cannot physically perform mounting and ejaculation. In the beef industry, electroejaculation is used in range bulls. Typical artificial vaginas for domestic animals are shown are Figure 11-I 6. In general, artificial vaginas consist of an outer casing fashioned of reinforced rubber and a liner that is generally made of rubber that can be lubricated. Tempera hire and pressure are controlled by the water that is p laced between the casing and the liner. One end of the artificial vagina is attached to a funnel- like cone that in tum is attached to a collection vessel, usually a nonbreakable graduated test tube. From a behavioral perspective, males that are to be collected with an artificial vagina need some form of training. Males with previous sexual experience will readily mount a surrogate animal (artificial animal or "dummy"). The degree to which animals will mount Reproductive Behavior 249 Figure 11-16. Artificial Vaginas for Various Animals Outer casing l -- Warm water Rubber liner - Warm water Rubber collection funnel I -· ; - .--......... The typical artificial vagina consists of a sturdy outer casing, a rubber liner, a chamber fi lled with warm water, a rubber collection funnel and a collection tube. tube The artificial vagina for the stallion consists of a leather outer casing (C) equiped with a port to drain water (arrow). The collection vessel (CV) and the protective covering (PC) are shown. Ide- ally, ejaculation takes place in the collection cone (CC) so that most of the semen will drain directly into the collection vessel. (Artificial vagina courtesy of Northwest Equine Reproduction Laboratory, University of Idaho, www.avs.uidaho.edu/nerl) The artificial vagina for the bull consists of a black casing (C), a rubber liner (RL) a collection cone (CC) and a collection vessel (CV). Water is placed between the casing and the liner. The proper tem- perature is critical for successful ejaculation in the bull . While not shown in the photograph a protec- tive covering is placed over the cone and collection vessel to prevent cold shock of the semen. The artificial vagina for the boar consists of a bulb that can apply pressure to the artificial vagina. High pressure is obligatory for stimulation of the glans pen is and ejaculation in the boar. The artificial vagina for the boar also consists of an outer casing (C), a liner (L) and a protective covering (PC) that houses the collection vessel. (Photograph courtesy of MinitOb Germany, www.minitilb.de) The artificial vagina for collection of semen from rams and bucks consists of a rubber casing (C) with a valve (arrow) through which water can be added or emptied, a rubber liner and a collection vessel (CV). The protective covering (PC) is shown above the artificial vagina. (Photograph courtesy of MinitOb Germany, www.minitilb.de) Ve tB oo ks .ir 250 Reproductive Behavior Figure 11-17. Surrogate Stimulus Animals for Semen Collection "Phantom" for Stallion Semen Collection In general, males of most species can be trained to mount and ejaculate using surrogate stimulus animals. A surrogate stimulus animal provides ease of cleaning and minimizes the risk of injury and disease transmission. Further, surrogate stimulus animals do not require feed, hous- ing and labor for maintenance as does a live stimulus animal. The use of artificial stimulus animals requires previous training of the male. Once the male has been trained he will gener- ally mount the "dummy" readily. The size can be adjusted easily to accomodate various males. Mobile surrogate stimulus animals are used for collection of semen in bulls because the location can be changed with ease. The surrogate stimulus animal used to collect semen from the stallion is generally referred to as a "phantom". The "phan- tom" contains a biting belt (arrow) to provide the stallion with a surface to bite during mounting thus providing a means for natural behavior. All of the devices shown have a built-in artificial vagina in which the temperature and pressure can be controlled. (Photographs courtesy of MinitOb Germany, www.minitDb.de) dummies depends on the amount of training provided. A surrogate stimulus animal provides the advantage of safety, reduced expense and they can be designed to accomodate males of various stature. The disadvantage of using surrogate stimulus animal is that changing lo- cations and teasers is difficult. Figure 11-17 illustrates examples of surrogate animals for semen collection. Sometimes it is difficult to train animals to mount either a stimulus animal or a surrogate stimulus animal. In this event, semen can be collected by plac- ing a condom-like structure inside the vagina of the female in estrus. When the male mounts the female and ejaculates, the semen is deposited inside the vessel. Such techniques are valuable when animals have not been adequately trained. The design of an artificial vagina should accomplish the following: • provide a suitable environment for stimulation of the glans penis • provide an environment that prevents damage to the penis • provide an environment that maxi- mizes sperm recove1y and minimizes sperm insult Further PHENOMENA for Fertility One day President and Mrs. Coolidge were visiting a government farm. Soon after their arrival they were taken off on sepa- rate tours. Wizen M rs. Coolidge passed the chicken pens, she paused to ask the man in charge if the rooster copulated more than once each day. "Dozens of times," was the reply. "Please tell that to the President, " Mrs. Coolidge requested. Wizen the President passed the pens am/ was told about the rooste1; he asked, "Same hen eve1y day?" "Oft no, Mr. President, a different one each time. " The President nodded slowly and then said, "Please tell that to Mrs. Coolidge." The praying mantis has mwsual reproduc- tive As soon as the male mounts the female and accomplishes intromission, the f emale bites his head off. She imme- diately eats the top half of his body while intromission is still taking place. The rea- son for this behavior is because ejaculation is permanently inhibited in the male and can take place only after the head has been removed. It is not known whether the slang phrase "bite-your-head-off' was derived from this behavior. Roman snails shoot love darts at one an- other before copulation to determine if they are both members of the same species. Some male insects (certain flies and mos- quitoes) have evolved mmsual adaptations to ins me that their genetics will be passed on. Males have a sharp, specialized penis that can enter a pupa. The male insemi- nates the rmbom female. When a grey squirrel comes into estrus, up to a dozm males noisily chase her through the trees. This chase is necessary, because the female will not ovulate without it. Reproductive Behavior 251 To mate, the queen bee leaves the hive and pelforms a mating flight in an area where drones are congregated. The fastest drone is the first to copulate with the queen. Copu- lation is a11 hz-jfight event that lasts from 1 to 3 seconds. Wizen the copulating bees separate, the entire male genitalia is ripped from the male and stays with the queen. The male soon dies am/ another male will then mate with the queen. Up to 17 matings in one mati11g flight have been observed. Females of some species are quite choosy about who gets to fertilize their eggs. In these cases, mate choice is determined by nuptial gifts presented by the male. The female black-tipped hangffy accepts nuptial gifts in the form of food in exchange for copulation. Wizen edible food is presented by the male, the duration of copu/atio11 is depende11t on the size of the gift. If the gift is small and can be consumed in 5 minutes or less, the female will not allow mating. If the gift is large (cannot be consumed in 20 minutes), the female will allow mating to take place. If the gift provides a meal of only 12 minutes she will/eave the gift-giver prematurely and seek another gift-giver as a mate. Satin bowerbirds build their nests only with blue objects. Males gather blue flowers, pen caps, berries and ribbons and arrange them under bushes or in other cozy spots. If a female "likes" what she sees, she will choose the nest's decorator as Iter mate. A male newt begins his courtship by jump- ing on the back of the female and rubbing his jaw against her snout. This releases a scent that drives the female newt "crazy with desire. " When female rhinoceri are in heat they will run away from a male, then suddenly tum and fight him horn-to-horn, sometimes for longer than a day. Only if he is fit enough to pursue will she submit. There are no "wimp genes" in the rhinocerous gene pool. [li[] I Ve tB oo ks .ir 250 Reproductive Behavior Figure 11-17. Surrogate Stimulus Animals for Semen Collection "Phantom" for Stallion Semen Collection In general, males of most species can be trained to mount and ejaculate using surrogate stimulus animals. A surrogate stimulus animal provides ease of cleaning and minimizes the risk of injury and disease transmission. Further, surrogate stimulus animals do not require feed, hous- ing and labor for maintenance as does a live stimulus animal. The use of artificial stimulus animals requires previous training of the male. Once the male has been trained he will gener- ally mount the "dummy" readily. The size can be adjusted easily to accomodate various males. Mobile surrogate stimulus animals are used for collection of semen in bulls because the location can be changed with ease. The surrogate stimulus animal used to collect semen from the stallion is generally referred to as a "phantom". The "phan- tom" contains a biting belt (arrow) to provide the stallion with a surface to bite during mounting thus providing a means for natural behavior. All of the devices shown have a built-in artificial vagina in which the temperature and pressure can be controlled. (Photographs courtesy of MinitOb Germany, www.minitDb.de) dummies depends on the amount of training provided. A surrogate stimulus animal provides the advantage of safety, reduced expense and they can be designed to accomodate males of various stature. The disadvantage of using surrogate stimulus animal is that changing lo- cations and teasers is difficult. Figure 11-17 illustrates examples of surrogate animals for semen collection. Sometimes it is difficult to train animals to mount either a stimulus animal or a surrogate stimulus animal. In this event, semen can be collected by plac- ing a condom-like structure inside the vagina of the female in estrus. When the male mounts the female and ejaculates, the semen is deposited inside the vessel. Such techniques are valuable when animals have not been adequately trained. The design of an artificial vagina should accomplish the following: • provide a suitable environment for stimulation of the glans penis • provide an environment that prevents damage to the penis • provide an environment that maxi- mizes sperm recove1y and minimizes sperm insult Further PHENOMENA for Fertility One day President and Mrs. Coolidge were visiting a government farm. Soon after their arrival they were taken off on sepa- rate tours. Wizen M rs. Coolidge passed the chicken pens, she paused to ask the man in charge if the rooster copulated more than once each day. "Dozens of times," was the reply. "Please tell that to the President, " Mrs. Coolidge requested. Wizen the President passed the pens am/ was told about the rooste1; he asked, "Same hen eve1y day?" "Oft no, Mr. President, a different one each time. " The President nodded slowly and then said, "Please tell that to Mrs. Coolidge." The praying mantis has mwsual reproduc- tive As soon as the male mounts the female and accomplishes intromission, the f emale bites his head off. She imme- diately eats the top half of his body while intromission is still taking place. The rea- son for this behavior is because ejaculation is permanently inhibited in the male and can take place only after the head has been removed. It is not known whether the slang phrase "bite-your-head-off' was derived from this behavior. Roman snails shoot love darts at one an- other before copulation to determine if they are both members of the same species. Some male insects (certain flies and mos- quitoes) have evolved mmsual adaptations to ins me that their genetics will be passed on. Males have a sharp, specialized penis that can enter a pupa. The male insemi- nates the rmbom female. When a grey squirrel comes into estrus, up to a dozm males noisily chase her through the trees. This chase is necessary, because the female will not ovulate without it. Reproductive Behavior 251 To mate, the queen bee leaves the hive and pelforms a mating flight in an area where drones are congregated. The fastest drone is the first to copulate with the queen. Copu- lation is a11 hz-jfight event that lasts from 1 to 3 seconds. Wizen the copulating bees separate, the entire male genitalia is ripped from the male and stays with the queen. The male soon dies am/ another male will then mate with the queen. Up to 17 matings in one mati11g flight have been observed. Females of some species are quite choosy about who gets to fertilize their eggs. In these cases, mate choice is determined by nuptial gifts presented by the male. The female black-tipped hangffy accepts nuptial gifts in the form of food in exchange for copulation. Wizen edible food is presented by the male, the duration of copu/atio11 is depende11t on the size of the gift. If the gift is small and can be consumed in 5 minutes or less, the female will not allow mating. If the gift is large (cannot be consumed in 20 minutes), the female will allow mating to take place. If the gift provides a meal of only 12 minutes she will/eave the gift-giver prematurely and seek another gift-giver as a mate. Satin bowerbirds build their nests only with blue objects. Males gather blue flowers, pen caps, berries and ribbons and arrange them under bushes or in other cozy spots. If a female "likes" what she sees, she will choose the nest's decorator as Iter mate. A male newt begins his courtship by jump- ing on the back of the female and rubbing his jaw against her snout. This releases a scent that drives the female newt "crazy with desire. " When female rhinoceri are in heat they will run away from a male, then suddenly tum and fight him horn-to-horn, sometimes for longer than a day. Only if he is fit enough to pursue will she submit. There are no "wimp genes" in the rhinocerous gene pool. [li[] I Ve tB oo ks .ir 252 Reproductive Behavior During courtship the female balloon fly will eat the male if given the chance. To achieve copulation and keep from getting eaten, the male will present the female with a balloon-shaped cocoon as a "present". Unwrapping this "present" keeps the female occupied long enough for the male to mate her and fly off. When box turtles copulate, the male mounts the female and remains in an upright posi- tion in order to facilitate insemination. The pair may remain in this position for hours to ensure adequate insemination. At the conclusion of the event the female will sud- denly move away, sometimes causing the male to fall precariously on his hack where he may remain until his death if he can't right himself. Most frogs and toads copulate in the dark. They are often so eager to mate that the male will try to momzt anything that passes by. They have been observed keeping a .firm grip on strange objects and even other small animals in the hope that they might turn out to he females. The long neck of the giraffe plays an im- portant role in their reproductive First the male samples the urine to ascertain whether she is in estrus. If so, the two giraffes then indulge in a form of sexual preparation by entwining and rubbing their necks together. Physiologically, this behav- ior is like a false-mount and no doubt causes the release of oxytocin that moves sperm in the distal tail of the epididymis into an ejaculatory position. The pressure within the penis of the bull at the time of ejaculation is equivalent to 10 times the pressure within a normal vehicle tire. Key References Albright, J.L., and C.W. Arave. 1997. The Behaviour o(Cattle. CAB International, Wellingford, UK. ISBN 0-85199-1 96-3. Craig, J. V. 1981. Domestic Animal Behavior: causes and implications (or animal care and management. Prentice-Hall, Inc. New Jersey. ISBN 0-13-218339- 0. Evans, H.E. 1993. Anatomv o( the Do[, 3rd Edition. W.B.Saunders Co. Philadelphia. ISBN 0-721 6- 3200-9. Grandage, J. 1972. "The erect dog penis: a paradox of flexible rigidity." Vet Rec: 9 1:14 1- 147. Hart, Benjamin L. 1985. The Behavior o[Domestic Animals. W.H. Freeman and Co., New York. ISBN 0-7167-1595-3. Houpt, K.A . 1998. Domestic Animal Behavior for Veterinarians and Animal Scientists. 3rd Edition. Iowa State University Press, ISBN 0-8138-1061 -2. Katz, L.S. and T.J. McDonald. 1992. "Sexual Behavior of farm animals" in Repoduction in Farm Animals : Science, Application and Models. Theriogenology 38:240-254. Korenman, S.G. 1998. "New insights into erectile dys- function: a practical approach." Am. J. Med. 105:135- 144. Signoret, J.P. and J. Balthazart. 1993 "Sexual behavior" in Reproduction in Mammals and Man . C. Thibault, M.C. Levasseur and R.I-I.F. I-I under, eds. Ell ipses, Paris. ISBN 2-7298-9354-7. Tibary, A. and A. Anouassi. 1997. Theriogenolorsy in Camelidae. United Arab Emirates. Ministry of Cul- ture and Information. Publication authorization No. 3849/1116. ISBN 9981-801-32-1. Reproductive Behavior 253 ill] I Ve tB oo ks .ir 252 Reproductive Behavior During courtship the female balloon fly will eat the male if given the chance. To achieve copulation and keep from getting eaten, the male will present the female with a balloon-shaped cocoon as a "present". Unwrapping this "present" keeps the female occupied long enough for the male to mate her and fly off. When box turtles copulate, the male mounts the female and remains in an upright posi- tion in order to facilitate insemination. The pair may remain in this position for hours to ensure adequate insemination. At the conclusion of the event the female will sud- denly move away, sometimes causing the male to fall precariously on his hack where he may remain until his death if he can't right himself. Most frogs and toads copulate in the dark. They are often so eager to mate that the male will try to momzt anything that passes by. They have been observed keeping a .firm grip on strange objects and even other small animals in the hope that they might turn out to he females. The long neck of the giraffe plays an im- portant role in their reproductive First the male samples the urine to ascertain whether she is in estrus. If so, the two giraffes then indulge in a form of sexual preparation by entwining and rubbing their necks together. Physiologically, this behav- ior is like a false-mount and no doubt causes the release of oxytocin that moves sperm in the distal tail of the epididymis into an ejaculatory position. The pressure within the penis of the bull at the time of ejaculation is equivalent to 10 times the pressure within a normal vehicle tire. Key References Albright, J.L., and C.W. Arave. 1997. The Behaviour o(Cattle. CAB International, Wellingford, UK. ISBN 0-85199-1 96-3. Craig, J. V. 1981. Domestic Animal Behavior: causes and implications (or animal care and management. Prentice-Hall, Inc. New Jersey. ISBN 0-13-218339- 0. Evans, H.E. 1993. Anatomv o( the Do[, 3rd Edition. W.B.Saunders Co. Philadelphia. ISBN 0-721 6- 3200-9. Grandage, J. 1972. "The erect dog penis: a paradox of flexible rigidity." Vet Rec: 9 1:14 1- 147. Hart, Benjamin L. 1985. The Behavior o[Domestic Animals. W.H. Freeman and Co., New York. ISBN 0-7167-1595-3. Houpt, K.A . 1998. Domestic Animal Behavior for Veterinarians and Animal Scientists. 3rd Edition. Iowa State University Press, ISBN 0-8138-1061 -2. Katz, L.S. and T.J. McDonald. 1992. "Sexual Behavior of farm animals" in Repoduction in Farm Animals : Science, Application and Models. Theriogenology 38:240-254. Korenman, S.G. 1998. "New insights into erectile dys- function: a practical approach." Am. J. Med. 105:135- 144. Signoret, J.P. and J. Balthazart. 1993 "Sexual behavior" in Reproduction in Mammals and Man . C. Thibault, M.C. Levasseur and R.I-I.F. I-I under, eds. Ell ipses, Paris. ISBN 2-7298-9354-7. Tibary, A. and A. Anouassi. 1997. Theriogenolorsy in Camelidae. United Arab Emirates. Ministry of Cul- ture and Information. Publication authorization No. 3849/1116. ISBN 9981-801-32-1. Reproductive Behavior 253 ill] I Ve tB oo ks .ir The Puerperium & Lactation Parturition Fetal Attachment & Gestation Early Embryogenesis & Maternal Recognition of Pregnancy Cyclicity Regulation of Reproduction Tract Function Puberty Prenatal Development , " \ Spermatogenesis Regulation of Reproduction Tract Function Puberty Prenatal Development Take Home Message Following insemination, viable spermatozoa that are retained in the female repro- ductive tract must: 1) transverse the cervix, 2) he transported through the uterus to the oviduct, 3) rmdeJ'gO capacitation, 4) him/ to tlze oocyte, 5) rmdeJ'gO the acrosome reaction and 6) penetrate the zona p ellucid a and fuse with the oocyte plasma membrane. After fu- sion with the plasma membrane, tlze f ertilizing spermatozoon enters the oocyte cytoplasm and its nucleus decondenses. Tlze male pronucleus is formed. This signifies successful fertilization. Following deposition of semen during copulation, spermatozoa are exposed to a series of differen t environments that s ignificantly al- ter their numbers and their function. After their depos it ion , spermatozoa are lost from the fe- male reproductive tract by retrograde transport and many are phagocytized by leukocytes within the female tract. The remaining spermatozoa must traverse the cervix, enter and traverse the uterus and enter the oviduct. They must undergo ca- pacitation before they can fe rti li ze the oocyte. When sperm encounter the egg they undergo the acrosome reaction and fertilization takes place. This series of events is summarized in Figure 12- 1. Figure 12-1. Major Sequence of Events Following Deposition of Spermatozoa in Female Tract • Fertilization • acrosome reaction • spermatozoon penetrates oocyte • male and female pronuclei form 0 Oviduct • docking to oviductal cells • capacitation completed • hyperactive motility 0 Uterus • capacitation initiated • phagocytosis 0 Immediate Transport • retrograde loss • phagocytosis into cervix/uterus \ • Cervix • "privileged path- ways" • removal of non- motile sperm • removal of some abnormalities Ve tB oo ks .ir The Puerperium & Lactation Parturition Fetal Attachment & Gestation Early Embryogenesis & Maternal Recognition of Pregnancy Cyclicity Regulation of Reproduction Tract Function Puberty Prenatal Development , " \ Spermatogenesis Regulation of Reproduction Tract Function Puberty Prenatal Development Take Home Message Following insemination, viable spermatozoa that are retained in the female repro- ductive tract must: 1) transverse the cervix, 2) he transported through the uterus to the oviduct, 3) rmdeJ'gO capacitation, 4) him/ to tlze oocyte, 5) rmdeJ'gO the acrosome reaction and 6) penetrate the zona p ellucid a and fuse with the oocyte plasma membrane. After fu- sion with the plasma membrane, tlze f ertilizing spermatozoon enters the oocyte cytoplasm and its nucleus decondenses. Tlze male pronucleus is formed. This signifies successful fertilization. Following deposition of semen during copulation, spermatozoa are exposed to a series of differen t environments that s ignificantly al- ter their numbers and their function. After their depos it ion , spermatozoa are lost from the fe- male reproductive tract by retrograde transport and many are phagocytized by leukocytes within the female tract. The remaining spermatozoa must traverse the cervix, enter and traverse the uterus and enter the oviduct. They must undergo ca- pacitation before they can fe rti li ze the oocyte. When sperm encounter the egg they undergo the acrosome reaction and fertilization takes place. This series of events is summarized in Figure 12- 1. Figure 12-1. Major Sequence of Events Following Deposition of Spermatozoa in Female Tract • Fertilization • acrosome reaction • spermatozoon penetrates oocyte • male and female pronuclei form 0 Oviduct • docking to oviductal cells • capacitation completed • hyperactive motility 0 Uterus • capacitation initiated • phagocytosis 0 Immediate Transport • retrograde loss • phagocytosis into cervix/uterus \ • Cervix • "privileged path- ways" • removal of non- motile sperm • removal of some abnormalities Ve tB oo ks .ir 256 Sperm in the Female Tract In some animals (cow, sheep, rabbit, primates, dog and cat), the male ejaculates the semen into the cranial vagina. In others, (pigs, horses and camelids) semen is either deposited directly into the cervix (pig) or is squirted through the cervical lumen during copula- tion (horse). In the dog, pig and the horse most of the ejaculate gains entrance into the uterine lumen. The stallion ejaculates in a series of ' jets" in which a spenn-rich fraction is ejaculated first in 3-4 high pressure squirts. This fraction contains about 80% of the spermatozoa. The last 5 to 8 "jets" are of lower pressure and contain fewer spem1. The seminal plasma in the final "jets" is highly viscous and may serve to minimize retrograde sperm loss rrom the mare's tract. Because of the large volume (200 to 400 ml) of boar ejaculate, most of the semen flows from the cervix into the uterine lumen. As in the stallion, the boar ejaculates a series of seminal fractions with dif- ferent characteristics as ejaculation progresses. The first fraction consists of accessory fluids and gelatinous coagulum. This fraction contains few sperm. The second fraction is rich in spennatozoa and this sperm- rich fraction is followed by a final fraction that fonns a gelatinous coagulum that resembles rice pudding. This coagulum reduces retrograde spenn loss. Immediately after insemination, semen undergoes varying degrees of retrograde transport (from the cervix towards the vulva). In the dog semen is ejaculated in tlu·ee frac- tions. The first, is a pre-sperm fraction that is thought to originate from the prostate. The volume of the pre- spenn fraction is usually small but can range from 0.5 to 5ml. This pre-sperm fraction (clear and acellular) is ejaculated in conjunction with pelvic thrusting by the male during "first stage coitus." The second, a sperm rich traction, is between 1 and 4 ml and is opalescent in color and contains between 300 million and 2 billion sperm. The final fraction originating from the prostate ranges in volume from I to 80ml. The first two frac- tions are ejaculated without visible force. However, the third fraction is ejaculated in surges of prostatic fluid that squirt into the vagina of the bitch during "second stage coitus." Because of the " tie" (See Chapter 11) most of this fraction is forced cranially into the uterus and is believed to "push" the sperm-rich fraction ahead of it into the uterus. Ejaculate volumes in the tom turkey average only 0.2 to 0.3ml with a range of 0.1 to 0. 7ml and it is therefore difficult to evaluate whether the ejaculate consists of multiple fractions. The degree to which spemmtozoa are lost from the female tract depends upon the physical nature of the ejaculate and the site of seminal deposition. In some species, the seminal plasma contains coagulat- ing protein(s) that form a conspicuous vaginal plug to prevent spermatozoa from undergoing retrograde flow to the exterior. Female rodents (mice and rats) have a relatively solid vaginal plug that is externally visible following copulation. The presence of the vaginal plug can be used to detem1ine when mating occurred. Domestic animals do not have a conspicuous vaginal plug. Spel'lnatozoa are lost from the female tract by: • phagocytosis by neutrophils • retrograde transport When the female reproductive tract is under the influence of estradiol during estrus, neutrophi ls (powerful phagocytic white blood cells) sequester in the mucosa of the tract, especially in the vagina and utems. These neutrophils are poised to attack foreign materials that are introduced into the female reproductive tract at insemination. lt should be recognized that, in addition to spennatozoa, microorganisms are introduced into the tract duting copulation. Thus, the neutrophil population is important in preventing these microorganisms from colonizing the female tract. From an immunologic perspective, spermatozoa are foreign to the female. As a result, neutrophils actively phagocytize spem1atozoa. They do not discriminate between live and dead spem1. In fact, a single neutrophil is capable of engulfing sev- eral motile spermatozoa (See Figure 12-2). Studies have shown that within 6 to 12 hours after the introduction of spennatozoa into the uterus, there is a large migration of neutrophils from the uter- ine mucosa into the uterine lumen (See Figure 12-2). While leukocyte infiltration is an important contributor to post-insemination spem1atozoal losses, this infiltra- tion is important for the prevention of reproductive tract infection. Spermatozoal transport consists of a rapid phase and a sustained phase. Among the least understood phenomena in reproductive physiology are factors that regulate Joss of spermatozoa from the female tract. The ability of the female to retain viable spermatozoa may influence the fertility of a given mating. Transport of spem1atozoa following copulation can be divided into two phases. These are the rapid transport phase and ilie sustained transport phase. Within a few minutes after copula- tion, spem1atozoa can be found in the oviducts. The rapid phase of transport was once considered to be important because it delivered spem1atozoa to the site of the fertilization very shortly after copulation, where they "postured" themselves for the arrival of oocytes. However, further research has shown that spermatozoa arriving in the oviducts within minutes after copulation were not viable. The functional importance of the rapid phase of spe1m transport is not obvious. It may simply represent a burst of transport activity brought about by contraction of the muscularis of the female tract in conjunction with copulation. Cl) .Cc E Cl) E _ ..... :CCIJ C.,5 0 I.. I.. Cl) -4ol-4ol Z .5 CIJ.C ·.p 111 Qj a:: Q Figure 12-2. Leukocyte Infiltration Helps Prevent Reproductive Tract Infections Insemination 12 Within 6-12 hours af- ter the introduction of sperm into the uterus, there is a large infiltra- tion of neutrophils from the uterine mucosa into the uterine lumen. 24 36 48 Time- (Hrs) SH 0 ST ' l Three leukocytes (A,B and C) phagocytizing sperm. Sperm heads (SH) can be observed in the cytoplasm of the leukocytes. A sperm tail (ST) can also be seen protruding from the leukocyte (Micrograph courtesy of R.G. Saacke, Virginia Polytechnic Institute and State University, Blacksburg) Sperm in the Female Tract 257 The more important component of transport is the sustained phase in which spermatozoa are trans- ported to the oviducts in a " trickle-like" effect from so-called reservoirs in the cervix and the uterotubal junction. During the sustained transport phase, spem1 move into the isthmus and attach to the oviductal epi- thelium. Spem1 can attach to the epithelium along the entire oviduct. However, spenn temporatily "dock" to the epithelium of the lower isthmus near the uterotubal junction because this is the first oviductal region they encounter. Spem1 "docking" is crucial to spenn survival because it elicits a signal cascade in the sperm that pro- motes viability. Without "docking", spem1 die within 6-1 0 hours after insemination. Rapid transport of spermatozoa is primarily the result of elevated tone and motility of the muscularis ofthefemale tract As you already lrnow, estradiol is high during the follicular phase when insemination occms. Estra- diol stimulates contractions of the muscularis, particu- larly the myometrium. Also, prostaglandins in semen (PGF2a. and PGE1) cause increased tone and motility of the uterus and/or the oviduct. Intermittent contractions of the muscularis propel spennatozoa in both a cranial and a caudal direction. Fluids secreted into the lumen of the female tract also serve as a vehicle for transport. Control of directionality, while not understood, is prob- ably under the collective influence of muscular contrac- tions and fluid distribution and characteristics. In addition to alteration of tract motility, seminal plasma from boars has been shown by Ger- man researchers to advance the time of ovulation in gilts. For example, when seminal plasma was in.fi.1sed into the right uterine born, ovulation occurred about ll hours earlier in the right ovary than in the left ovary. The left uterine hom did not receive seminal plasma. The specific material in boar seminal plasma inducing early ovulation has not been identified, but it appears to be a protein. Identification of these factors could provide an avenue to control more precisely the time of ovulation in swine. A similar phenomenon occurs in camelids where seminal plasma components have been shown to cause ovulation. The cervix is a major barrier to sperma- tozoal transport and it can also serve as a reservoir for spermatozoa. Ve tB oo ks .ir 256 Sperm in the Female Tract In some animals (cow, sheep, rabbit, primates, dog and cat), the male ejaculates the semen into the cranial vagina. In others, (pigs, horses and camelids) semen is either deposited directly into the cervix (pig) or is squirted through the cervical lumen during copula- tion (horse). In the dog, pig and the horse most of the ejaculate gains entrance into the uterine lumen. The stallion ejaculates in a series of ' jets" in which a spenn-rich fraction is ejaculated first in 3-4 high pressure squirts. This fraction contains about 80% of the spermatozoa. The last 5 to 8 "jets" are of lower pressure and contain fewer spem1. The seminal plasma in the final "jets" is highly viscous and may serve to minimize retrograde sperm loss rrom the mare's tract. Because of the large volume (200 to 400 ml) of boar ejaculate, most of the semen flows from the cervix into the uterine lumen. As in the stallion, the boar ejaculates a series of seminal fractions with dif- ferent characteristics as ejaculation progresses. The first fraction consists of accessory fluids and gelatinous coagulum. This fraction contains few sperm. The second fraction is rich in spennatozoa and this sperm- rich fraction is followed by a final fraction that fonns a gelatinous coagulum that resembles rice pudding. This coagulum reduces retrograde spenn loss. Immediately after insemination, semen undergoes varying degrees of retrograde transport (from the cervix towards the vulva). In the dog semen is ejaculated in tlu·ee frac- tions. The first, is a pre-sperm fraction that is thought to originate from the prostate. The volume of the pre- spenn fraction is usually small but can range from 0.5 to 5ml. This pre-sperm fraction (clear and acellular) is ejaculated in conjunction with pelvic thrusting by the male during "first stage coitus." The second, a sperm rich traction, is between 1 and 4 ml and is opalescent in color and contains between 300 million and 2 billion sperm. The final fraction originating from the prostate ranges in volume from I to 80ml. The first two frac- tions are ejaculated without visible force. However, the third fraction is ejaculated in surges of prostatic fluid that squirt into the vagina of the bitch during "second stage coitus." Because of the " tie" (See Chapter 11) most of this fraction is forced cranially into the uterus and is believed to "push" the sperm-rich fraction ahead of it into the uterus. Ejaculate volumes in the tom turkey average only 0.2 to 0.3ml with a range of 0.1 to 0. 7ml and it is therefore difficult to evaluate whether the ejaculate consists of multiple fractions. The degree to which spemmtozoa are lost from the female tract depends upon the physical nature of the ejaculate and the site of seminal deposition. In some species, the seminal plasma contains coagulat- ing protein(s) that form a conspicuous vaginal plug to prevent spermatozoa from undergoing retrograde flow to the exterior. Female rodents (mice and rats) have a relatively solid vaginal plug that is externally visible following copulation. The presence of the vaginal plug can be used to detem1ine when mating occurred. Domestic animals do not have a conspicuous vaginal plug. Spel'lnatozoa are lost from the female tract by: • phagocytosis by neutrophils • retrograde transport When the female reproductive tract is under the influence of estradiol during estrus, neutrophi ls (powerful phagocytic white blood cells) sequester in the mucosa of the tract, especially in the vagina and utems. These neutrophils are poised to attack foreign materials that are introduced into the female reproductive tract at insemination. lt should be recognized that, in addition to spennatozoa, microorganisms are introduced into the tract duting copulation. Thus, the neutrophil population is important in preventing these microorganisms from colonizing the female tract. From an immunologic perspective, spermatozoa are foreign to the female. As a result, neutrophils actively phagocytize spem1atozoa. They do not discriminate between live and dead spem1. In fact, a single neutrophil is capable of engulfing sev- eral motile spermatozoa (See Figure 12-2). Studies have shown that within 6 to 12 hours after the introduction of spennatozoa into the uterus, there is a large migration of neutrophils from the uter- ine mucosa into the uterine lumen (See Figure 12-2). While leukocyte infiltration is an important contributor to post-insemination spem1atozoal losses, this infiltra- tion is important for the prevention of reproductive tract infection. Spermatozoal transport consists of a rapid phase and a sustained phase. Among the least understood phenomena in reproductive physiology are factors that regulate Joss of spermatozoa from the female tract. The ability of the female to retain viable spermatozoa may influence the fertility of a given mating. Transport of spem1atozoa following copulation can be divided into two phases. These are the rapid transport phase and ilie sustained transport phase. Within a few minutes after copula- tion, spem1atozoa can be found in the oviducts. The rapid phase of transport was once considered to be important because it delivered spem1atozoa to the site of the fertilization very shortly after copulation, where they "postured" themselves for the arrival of oocytes. However, further research has shown that spermatozoa arriving in the oviducts within minutes after copulation were not viable. The functional importance of the rapid phase of spe1m transport is not obvious. It may simply represent a burst of transport activity brought about by contraction of the muscularis of the female tract in conjunction with copulation. Cl) .Cc E Cl) E _ ..... :CCIJ C.,5 0 I.. I.. Cl) -4ol-4ol Z .5 CIJ.C ·.p 111 Qj a:: Q Figure 12-2. Leukocyte Infiltration Helps Prevent Reproductive Tract Infections Insemination 12 Within 6-12 hours af- ter the introduction of sperm into the uterus, there is a large infiltra- tion of neutrophils from the uterine mucosa into the uterine lumen. 24 36 48 Time- (Hrs) SH 0 ST ' l Three leukocytes (A,B and C) phagocytizing sperm. Sperm heads (SH) can be observed in the cytoplasm of the leukocytes. A sperm tail (ST) can also be seen protruding from the leukocyte (Micrograph courtesy of R.G. Saacke, Virginia Polytechnic Institute and State University, Blacksburg) Sperm in the Female Tract 257 The more important component of transport is the sustained phase in which spermatozoa are trans- ported to the oviducts in a " trickle-like" effect from so-called reservoirs in the cervix and the uterotubal junction. During the sustained transport phase, spem1 move into the isthmus and attach to the oviductal epi- thelium. Spem1 can attach to the epithelium along the entire oviduct. However, spenn temporatily "dock" to the epithelium of the lower isthmus near the uterotubal junction because this is the first oviductal region they encounter. Spem1 "docking" is crucial to spenn survival because it elicits a signal cascade in the sperm that pro- motes viability. Without "docking", spem1 die within 6-1 0 hours after insemination. Rapid transport of spermatozoa is primarily the result of elevated tone and motility of the muscularis ofthefemale tract As you already lrnow, estradiol is high during the follicular phase when insemination occms. Estra- diol stimulates contractions of the muscularis, particu- larly the myometrium. Also, prostaglandins in semen (PGF2a. and PGE1) cause increased tone and motility of the uterus and/or the oviduct. Intermittent contractions of the muscularis propel spennatozoa in both a cranial and a caudal direction. Fluids secreted into the lumen of the female tract also serve as a vehicle for transport. Control of directionality, while not understood, is prob- ably under the collective influence of muscular contrac- tions and fluid distribution and characteristics. In addition to alteration of tract motility, seminal plasma from boars has been shown by Ger- man researchers to advance the time of ovulation in gilts. For example, when seminal plasma was in.fi.1sed into the right uterine born, ovulation occurred about ll hours earlier in the right ovary than in the left ovary. The left uterine hom did not receive seminal plasma. The specific material in boar seminal plasma inducing early ovulation has not been identified, but it appears to be a protein. Identification of these factors could provide an avenue to control more precisely the time of ovulation in swine. A similar phenomenon occurs in camelids where seminal plasma components have been shown to cause ovulation. The cervix is a major barrier to sperma- tozoal transport and it can also serve as a reservoir for spermatozoa. Ve tB oo ks .ir I I 12 258 Sperm in the Female Tract Figure 12-3. Spermatozoa Travel Through "Privileged Pathways" in the Cow During estrus secretion of mucins from the apical port1on of the cervical mucosa produces sheets of viscous mucus. Se- cretion is toward the lumen and flows in a caudal direction. Less viscous sialomucins are produced in the basal crypts of the cervix. Spermatozoa found in the basal regions are orientated in the sam_e direction and traverse the cerv1x toward the uterus through these "privileged pathways" (PP) of low viscosity sialomucin. (Modified from Mullins and Sa a eke 1989, Anat. Rec. 225:1 06) To vagina Following copulation in the cow and ewe and, to some degree, the mare, spenn atozoa must negotiate the highly convoluted system of grooves within the cervix (See Figure 12-3). During estrus, the cervix produces mucus. In the cow cervical mucus consists of two types. One type is a sialomucin, a mucus of low viscosity. It is produced by cells in the basal areas of the cervical c1ypts (See Figure 12-3). A second type, sulfomucin is produced in the apical portions of the cervical epithelium covering the tips of the cervical folds. This type of mucus is quite viscous. The pro- duction of two types of mucus (one of low viscosity and one ofhigh viscosity) creates two distinct environ- ments within the cervix. Spermatozoa encountering the viscous sulfomucin are washed out of the tract. Those that encounter the low viscosity sialomucin in the environment of the crypts of the cervix swim into it. Thus, the low viscosity environment of the deeper cervical crypts creates "privileged pathways" through which spermatozoa can move. The ability of spennatozoa to traverse these "privileged pathways" is thought to depend on their ability to swim through the basal channels (crypts) of the cervix and the associated low viscosity mucus. ln this context, the cervix may be a filter that eliminates non-motile spermatozoa. The specific role of the cer- Sperm in the Female Tract 259 vix in spermatozoal transport and/or retention awaits further clarification in the sow and the mare, where a high proportion of spermatozoa are ejaculated into the uterus. Spermatozoa must reside in the female tract before they acquire maximum fertility. As you recall from Chapter 3, spem1atozoa acquire maturity during epididymal transit. However, the maturational changes that occur in the epididymis do not render spem1atozoa completely fertile. For maximum fertility to be achieved, spermatozoa must reside in the female reproductive tract for a minimum period of time. During the time in the female repro- ductive tract, some spem1atozoa will undergo changes that allow them to become fertile. These changes are referred to as spennatozoal capacitation (See Figure 12-4). The site for capacitation varies among species. In species where spem1atozoa are deposited in the cra- nial vagina, capacitation may begin as spenn ascend and pass through the cervix. In species where semen is Figure 12-4. Conceptual Version of Mammalian Capacitation Epididymal Ejaculated Capacitated ....., The plasma mem- brane of epididymal spermatozoa con- tains a complement of surface molecules (proteins and carbo- hydrates) illustrated here as yellow T's. plasma + tract The surface molecules in epididymal sperm become coated with seminal plasma proteins (orange halos) that mask portions of the membrane molecules. When sperm are ex- posed to the female tract environment, these seminal plasma coatings, along with some of the surface molecules , are re- moved, thus exposing portions of the mol- ecules that can bind to the zona pellucida of the oocyte. Ve tB oo ks .ir I I 12 258 Sperm in the Female Tract Figure 12-3. Spermatozoa Travel Through "Privileged Pathways" in the Cow During estrus secretion of mucins from the apical port1on of the cervical mucosa produces sheets of viscous mucus. Se- cretion is toward the lumen and flows in a caudal direction. Less viscous sialomucins are produced in the basal crypts of the cervix. Spermatozoa found in the basal regions are orientated in the sam_e direction and traverse the cerv1x toward the uterus through these "privileged pathways" (PP) of low viscosity sialomucin. (Modified from Mullins and Sa a eke 1989, Anat. Rec. 225:1 06) To vagina Following copulation in the cow and ewe and, to some degree, the mare, spenn atozoa must negotiate the highly convoluted system of grooves within the cervix (See Figure 12-3). During estrus, the cervix produces mucus. In the cow cervical mucus consists of two types. One type is a sialomucin, a mucus of low viscosity. It is produced by cells in the basal areas of the cervical c1ypts (See Figure 12-3). A second type, sulfomucin is produced in the apical portions of the cervical epithelium covering the tips of the cervical folds. This type of mucus is quite viscous. The pro- duction of two types of mucus (one of low viscosity and one ofhigh viscosity) creates two distinct environ- ments within the cervix. Spermatozoa encountering the viscous sulfomucin are washed out of the tract. Those that encounter the low viscosity sialomucin in the environment of the crypts of the cervix swim into it. Thus, the low viscosity environment of the deeper cervical crypts creates "privileged pathways" through which spermatozoa can move. The ability of spennatozoa to traverse these "privileged pathways" is thought to depend on their ability to swim through the basal channels (crypts) of the cervix and the associated low viscosity mucus. ln this context, the cervix may be a filter that eliminates non-motile spermatozoa. The specific role of the cer- Sperm in the Female Tract 259 vix in spermatozoal transport and/or retention awaits further clarification in the sow and the mare, where a high proportion of spermatozoa are ejaculated into the uterus. Spermatozoa must reside in the female tract before they acquire maximum fertility. As you recall from Chapter 3, spem1atozoa acquire maturity during epididymal transit. However, the maturational changes that occur in the epididymis do not render spem1atozoa completely fertile. For maximum fertility to be achieved, spermatozoa must reside in the female reproductive tract for a minimum period of time. During the time in the female repro- ductive tract, some spem1atozoa will undergo changes that allow them to become fertile. These changes are referred to as spennatozoal capacitation (See Figure 12-4). The site for capacitation varies among species. In species where spem1atozoa are deposited in the cra- nial vagina, capacitation may begin as spenn ascend and pass through the cervix. In species where semen is Figure 12-4. Conceptual Version of Mammalian Capacitation Epididymal Ejaculated Capacitated ....., The plasma mem- brane of epididymal spermatozoa con- tains a complement of surface molecules (proteins and carbo- hydrates) illustrated here as yellow T's. plasma + tract The surface molecules in epididymal sperm become coated with seminal plasma proteins (orange halos) that mask portions of the membrane molecules. When sperm are ex- posed to the female tract environment, these seminal plasma coatings, along with some of the surface molecules , are re- moved, thus exposing portions of the mol- ecules that can bind to the zona pellucida of the oocyte. Ve tB oo ks .ir 260 Sperm in the Female Tract deposited into the mid-cervix (sow) or caudal cervix (mare) and immediately enters the uterus, capacitation is probably initiated within the utems and completed in the isthmus of the oviduct as is the case with all spe- cies. All spermatozoa are not capacitated at the same rate. Instead, they are capacitated over a relatively long period of time (several hours). Capacitation can occur in fluids other than those found in the luminal compartment of the female reproductive tract. For example, in vitro capacitation has been accomplished in a wide variety of species using blood serum, a variety of commercial tissue culture media, Krebs Ringer solution and Tyrodes solution. No single in vitro environment will support capacitation for all species. There is little doubt that the plasma mem- brane of the sperm (particularly the head) un- dergoes marked biochemical changes during ca- pacitation. During mixing of sperm with seminal plasma the sperm become coated with various proteins. The coating of seminal plasma proteins is "stripped" away by the female tract environment. The exact nature of the "stripping process" of capacitation is not understood. An important concept with regard to capacita- tion is that the process can be reversed by rehtrning capacitated spern1atozoa to seminal plasma. For exam- ple, when capacitated spermatozoa are removed from the female reproductive tract and rehlrned to seminal plasma, they become decapacitated and require ad- ditional capacitation time in the female reproductive tract before they can regain their fertility. It appears that the seminal plasma components coat the plasma membrane with surface substances that prevent or inhibit interaction of spermatozoa with the egg. Fertilization is a Complex Process and Involves a Cascade of Events The process of fertilization involves a series of specific interactions between spennatozoa and the oocyte. These are outlined in Figure 12-5. Acquisition of hyperactive motility occurs in the oviduct. In the oviduct, as capacitation is completed, the motility patterns ofspennatozoa become hyperac- tive. The motility pattem changes from a progressive, linear motility in which they swim in a relatively straight line (like an Olympic swinuner), into a fren- zied, dancing motion that is not linear and is localized Figure 12-5. Postcapacitation Sequence of Events Leading to Fertilization Hyperactive motility .. Binding to zona pellucida .. Acrosomal reaction .. Penetration of zona pellucida .. Sperm-oocyte membrane fusion + Sperm engulfed .. Decondensation of sperm nucleus .. Formation of male pronucleus in a small area (like dancers in a disco). Hyperactive motility occurs throughout the oviduct and is thought to be brought about by specific molecules produced by the epithelium there. Hyperactive motility is thought to facilitate sperm-oocyte contact. Binding to the zona pellucida requires specific zona-binding proteins on the spermatozoal membrane. Spermatozoa are known to contain specific proteins on their plasma membrane surfaces overlying the acrosome that bind specifically to zona pellucida proteins. These zona binding proteins on the plasma membrane must be exposed during the capacitation process before binding to the zona pellucida can occur. Before zona binding can be understood fully, the mo- lecular makeup of the zona must be described. The zona pellucida of the oocyte consists of three glycoproteins. These glycoproteins have been named zona proteins 1, 2 and 3 (ZPl, ZP2 and ZP3). Zona proteins 1 and 2 are structural proteins providing the struchlral integrity of the zona. Zona protein 3 is much like a receptor for a honnone. It binds to proteins on the spern1atozoal membrane. Binding of spennato- zoa to the zona pellucida is believed to require between 10,000 and 50,000 ZP3 molecules. The cmTent under- standing is that the sperm plasma membrane contains two zona binding sites. The fi rst binding site, referred to as the primary zona binding region is responsible for adherence of spermatozoa to the zona pellucida. The second binding site on the spem1atozoal plasma membrane is believed to be acrosome reaction promot- ing ligand. When binding occurs between this region and the ZP3 molecule, a signal transduction occurs. This is much like a typical hormone-receptor binding complex. Binding initiates the acrosomal reaction. The relationship between ZP3 and the spennatozoal plasma membrane during binding is illustrated in Figure 12-6. Sperm in the Female Tract 261 The acrosomal reaction is an orderly fusion of the spermatozoal plasma membrane and the outer acrosomal membrane. The purpose of the acrosomal reaction is twofold. First, the reaction enables spennatozoa to penetrate the zona pellucida. Second, it modifies the equatorial segment so that it can later fuse with the plasma membrane of the oocyte . The acrosomal reaction begins when the plasma membrane of the spermatozoon forms multiple fusion sites with the outer acrosomal membrane. When the two membranes fi.1se, many small vesicles are formed (See F igure 12-7) and this process is called vesicula- tion. After vesiculation has occurred, the acrosomal contents are dispersed and the sperm nucleus is left Figure 12-6. Zona Binding by Sperm and Initiation of the Acrosomal Reaction Proposed model for zona binding and the initiation of the acrosom a I reac- tion in mammalian spermatozoa. The sperm plasma mem- brane overlying the acrosome contains two receptor-like re- gions. The first, called the zona binding re- gion (ZBR), reacts with ZP3 to cause physical attachment of the sperm to the zona pellucida. A second membrane region , the acrosome reac- tion promoting region (ARPR), also binds to ZP3 and initiates the acrosome reacti on by causing the sperm plasma membrane to fuse (arrows) to the outer acrosomal membrane. ARPR = Acrosome Reaction Promoting Region lAM = Inner Acrosomal Membrane OAM = Outer Acrosomal Membrane ZBR = Zona Binding Region Sperm plasma me mbrane Surface of --+-- zona pe llucida OAM lAM Ve tB oo ks .ir 260 Sperm in the Female Tract deposited into the mid-cervix (sow) or caudal cervix (mare) and immediately enters the uterus, capacitation is probably initiated within the utems and completed in the isthmus of the oviduct as is the case with all spe- cies. All spermatozoa are not capacitated at the same rate. Instead, they are capacitated over a relatively long period of time (several hours). Capacitation can occur in fluids other than those found in the luminal compartment of the female reproductive tract. For example, in vitro capacitation has been accomplished in a wide variety of species using blood serum, a variety of commercial tissue culture media, Krebs Ringer solution and Tyrodes solution. No single in vitro environment will support capacitation for all species. There is little doubt that the plasma mem- brane of the sperm (particularly the head) un- dergoes marked biochemical changes during ca- pacitation. During mixing of sperm with seminal plasma the sperm become coated with various proteins. The coating of seminal plasma proteins is "stripped" away by the female tract environment. The exact nature of the "stripping process" of capacitation is not understood. An important concept with regard to capacita- tion is that the process can be reversed by rehtrning capacitated spern1atozoa to seminal plasma. For exam- ple, when capacitated spermatozoa are removed from the female reproductive tract and rehlrned to seminal plasma, they become decapacitated and require ad- ditional capacitation time in the female reproductive tract before they can regain their fertility. It appears that the seminal plasma components coat the plasma membrane with surface substances that prevent or inhibit interaction of spermatozoa with the egg. Fertilization is a Complex Process and Involves a Cascade of Events The process of fertilization involves a series of specific interactions between spennatozoa and the oocyte. These are outlined in Figure 12-5. Acquisition of hyperactive motility occurs in the oviduct. In the oviduct, as capacitation is completed, the motility patterns ofspennatozoa become hyperac- tive. The motility pattem changes from a progressive, linear motility in which they swim in a relatively straight line (like an Olympic swinuner), into a fren- zied, dancing motion that is not linear and is localized Figure 12-5. Postcapacitation Sequence of Events Leading to Fertilization Hyperactive motility .. Binding to zona pellucida .. Acrosomal reaction .. Penetration of zona pellucida .. Sperm-oocyte membrane fusion + Sperm engulfed .. Decondensation of sperm nucleus .. Formation of male pronucleus in a small area (like dancers in a disco). Hyperactive motility occurs throughout the oviduct and is thought to be brought about by specific molecules produced by the epithelium there. Hyperactive motility is thought to facilitate sperm-oocyte contact. Binding to the zona pellucida requires specific zona-binding proteins on the spermatozoal membrane. Spermatozoa are known to contain specific proteins on their plasma membrane surfaces overlying the acrosome that bind specifically to zona pellucida proteins. These zona binding proteins on the plasma membrane must be exposed during the capacitation process before binding to the zona pellucida can occur. Before zona binding can be understood fully, the mo- lecular makeup of the zona must be described. The zona pellucida of the oocyte consists of three glycoproteins. These glycoproteins have been named zona proteins 1, 2 and 3 (ZPl, ZP2 and ZP3). Zona proteins 1 and 2 are structural proteins providing the struchlral integrity of the zona. Zona protein 3 is much like a receptor for a honnone. It binds to proteins on the spern1atozoal membrane. Binding of spennato- zoa to the zona pellucida is believed to require between 10,000 and 50,000 ZP3 molecules. The cmTent under- standing is that the sperm plasma membrane contains two zona binding sites. The fi rst binding site, referred to as the primary zona binding region is responsible for adherence of spermatozoa to the zona pellucida. The second binding site on the spem1atozoal plasma membrane is believed to be acrosome reaction promot- ing ligand. When binding occurs between this region and the ZP3 molecule, a signal transduction occurs. This is much like a typical hormone-receptor binding complex. Binding initiates the acrosomal reaction. The relationship between ZP3 and the spennatozoal plasma membrane during binding is illustrated in Figure 12-6. Sperm in the Female Tract 261 The acrosomal reaction is an orderly fusion of the spermatozoal plasma membrane and the outer acrosomal membrane. The purpose of the acrosomal reaction is twofold. First, the reaction enables spennatozoa to penetrate the zona pellucida. Second, it modifies the equatorial segment so that it can later fuse with the plasma membrane of the oocyte . The acrosomal reaction begins when the plasma membrane of the spermatozoon forms multiple fusion sites with the outer acrosomal membrane. When the two membranes fi.1se, many small vesicles are formed (See F igure 12-7) and this process is called vesicula- tion. After vesiculation has occurred, the acrosomal contents are dispersed and the sperm nucleus is left Figure 12-6. Zona Binding by Sperm and Initiation of the Acrosomal Reaction Proposed model for zona binding and the initiation of the acrosom a I reac- tion in mammalian spermatozoa. The sperm plasma mem- brane overlying the acrosome contains two receptor-like re- gions. The first, called the zona binding re- gion (ZBR), reacts with ZP3 to cause physical attachment of the sperm to the zona pellucida. A second membrane region , the acrosome reac- tion promoting region (ARPR), also binds to ZP3 and initiates the acrosome reacti on by causing the sperm plasma membrane to fuse (arrows) to the outer acrosomal membrane. ARPR = Acrosome Reaction Promoting Region lAM = Inner Acrosomal Membrane OAM = Outer Acrosomal Membrane ZBR = Zona Binding Region Sperm plasma me mbrane Surface of --+-- zona pe llucida OAM lAM Ve tB oo ks .ir 262 Sperm in the Female Tract Figure 12-7. Schematic Illustration of the Acrosomal Reaction Acrosomal ---+f- contents Outer acrosomal membrane Inner acrosomal membrane Post nuclear cap Plasma membrane Before acrosome reaction During acrosome reaction lt===tt-- Fusion After acrosome reaction protein Before Acrosomal Reaction Before the reaction begins, all mem- branes of the head are intact. During Acrosomal Reaction During the reaction, the plas- ma membrane overlying the acrosomal membrane begins to fuse with the outer acrosom- al membrane. The fusion of the two membranes leads to vesiculation that creates pores through which the acrosomal enzymes can pass. This al- lows the sperm to penetrate through the zona pellucida. After Acrosomal Reaction After the reaction, the vesicles are sloughed , leavi ng the inner acrosom- al membrane, the equatorial seg- ment and the post nuclear cap intact. with the inner acrosomal membrane surrounding it. Vesiculation characterizes the acrosomal reaction and morphologically distinguishes it from a damaged acrosome. Damage to the acrosome membrane and plasma membrane is in-eversible. Damage to these membranes is brought about by changes in osmotic pressure, sudden cooling, sudden heating or marked changes in pH. Damage to the membranes causes premature loss of acrosomal contents and such sperm cannot accomplish fertilization. 1, Release of acrosomal enzymes allows the spermatozoon to digest its way through the zona pellucida. The penetration of the zona pellucida by a spermatozoon is believed to be a rapid process and probably takes no more than a few minutes. Following attachment to the zona pellucida, the acrosome reaction allows the release of a variety of enzymes. Acrosin is one enzyme that is released from spem1atozoa during the acrosomal reaction. It hydrolyzes zona proteins as well as enhances the sperm's ability to bind to the zona. In the inactive form, acrosin is known as proacrosin which has a strong affinity for the zona. Thus, proacrosin aids in binding the spem1atozoon to the zona as the acrosomal reaction proceeds. As proacrosin is converted to acrosin, the sperm begins to penetrate and make its way through the zona pellucida. The mechanical force generated by the flagellar action of the tail may be sufficient to maintain spenn head contact with the zona pellucida. It is important to note that the acrosomal reaction allows the sperma- tozoon to digest a small hole through the zona through which it can pass. Placing a hot marble on the surface of a block of chilled butter would be an appropriate anal- ogy. The hot marble would move through the butter in a small regional hole, but the butter in most of the block would be unchanged. This small regional dissolution leaves the zona predominately intact. Maintenance of an intact zona pellucida is important because it prevents blastomeres in the early embryo from separating during embryogenesis. Fertilization requires fusion ofthe equatorial segment and the oocyte plasma membrane. Sperm in the Female Tract 263 When the spermatozoon completely penetrates the zona and reaches the perivitelline space (the space between the zona and the oocyte plasma membrane), it settles into a bed of microvilli formed from the oo- cyte plasma membrane. The plasma membrane of the oocyte fuses with the membrane of the equatorial segment and the fertilizing spennatozoon is engulfed. The actual fusion of the oocyte plasma membrane with the equatorial segment is believed to be brought about by a so-called fusion protein located on this portion of the membrane . Prior to the acrosome reaction, this fus ion protein is inactive. After vesiculation and release of the acrosomal contents, the fusion protein is Figure 12-8. Illustration of Sperm-Oocyte Fusion Before membrane fusion During membrane fusion After membrane fusion When the spermatozoon completely penetrates the zona and reaches the perivitelline space, it set- tles into a bed of microvilli fo rmed by the oocyte plasma membrane. The cortica l granules have migrated to the periphery of the oocyte . The plasma membrane of the oocyte fuses with the equatorial segment and the fertilizi ng sper- matozoon is engulfed. The cortica l granu le membrane fuses with the oocyte plasma mem- brane a nd the cortical contents a re released into to perivitell ine space by exocytosis. After the fusion between the membrane of th e equatoria l segment and the oocyte plasma mem- brane occurs, the nucle- us of the spermatozoon is within the cytoplasm. The sperm nuclear mem- bra ne disappears and the nucle us of the sperm decondenses. 12 Ve tB oo ks .ir 262 Sperm in the Female Tract Figure 12-7. Schematic Illustration of the Acrosomal Reaction Acrosomal ---+f- contents Outer acrosomal membrane Inner acrosomal membrane Post nuclear cap Plasma membrane Before acrosome reaction During acrosome reaction lt===tt-- Fusion After acrosome reaction protein Before Acrosomal Reaction Before the reaction begins, all mem- branes of the head are intact. During Acrosomal Reaction During the reaction, the plas- ma membrane overlying the acrosomal membrane begins to fuse with the outer acrosom- al membrane. The fusion of the two membranes leads to vesiculation that creates pores through which the acrosomal enzymes can pass. This al- lows the sperm to penetrate through the zona pellucida. After Acrosomal Reaction After the reaction, the vesicles are sloughed , leavi ng the inner acrosom- al membrane, the equatorial seg- ment and the post nuclear cap intact. with the inner acrosomal membrane surrounding it. Vesiculation characterizes the acrosomal reaction and morphologically distinguishes it from a damaged acrosome. Damage to the acrosome membrane and plasma membrane is in-eversible. Damage to these membranes is brought about by changes in osmotic pressure, sudden cooling, sudden heating or marked changes in pH. Damage to the membranes causes premature loss of acrosomal contents and such sperm cannot accomplish fertilization. 1, Release of acrosomal enzymes allows the spermatozoon to digest its way through the zona pellucida. The penetration of the zona pellucida by a spermatozoon is believed to be a rapid process and probably takes no more than a few minutes. Following attachment to the zona pellucida, the acrosome reaction allows the release of a variety of enzymes. Acrosin is one enzyme that is released from spem1atozoa during the acrosomal reaction. It hydrolyzes zona proteins as well as enhances the sperm's ability to bind to the zona. In the inactive form, acrosin is known as proacrosin which has a strong affinity for the zona. Thus, proacrosin aids in binding the spem1atozoon to the zona as the acrosomal reaction proceeds. As proacrosin is converted to acrosin, the sperm begins to penetrate and make its way through the zona pellucida. The mechanical force generated by the flagellar action of the tail may be sufficient to maintain spenn head contact with the zona pellucida. It is important to note that the acrosomal reaction allows the sperma- tozoon to digest a small hole through the zona through which it can pass. Placing a hot marble on the surface of a block of chilled butter would be an appropriate anal- ogy. The hot marble would move through the butter in a small regional hole, but the butter in most of the block would be unchanged. This small regional dissolution leaves the zona predominately intact. Maintenance of an intact zona pellucida is important because it prevents blastomeres in the early embryo from separating during embryogenesis. Fertilization requires fusion ofthe equatorial segment and the oocyte plasma membrane. Sperm in the Female Tract 263 When the spermatozoon completely penetrates the zona and reaches the perivitelline space (the space between the zona and the oocyte plasma membrane), it settles into a bed of microvilli formed from the oo- cyte plasma membrane. The plasma membrane of the oocyte fuses with the membrane of the equatorial segment and the fertilizing spennatozoon is engulfed. The actual fusion of the oocyte plasma membrane with the equatorial segment is believed to be brought about by a so-called fusion protein located on this portion of the membrane . Prior to the acrosome reaction, this fus ion protein is inactive. After vesiculation and release of the acrosomal contents, the fusion protein is Figure 12-8. Illustration of Sperm-Oocyte Fusion Before membrane fusion During membrane fusion After membrane fusion When the spermatozoon completely penetrates the zona and reaches the perivitelline space, it set- tles into a bed of microvilli fo rmed by the oocyte plasma membrane. The cortica l granules have migrated to the periphery of the oocyte . The plasma membrane of the oocyte fuses with the equatorial segment and the fertilizi ng sper- matozoon is engulfed. The cortica l granu le membrane fuses with the oocyte plasma mem- brane a nd the cortical contents a re released into to perivitell ine space by exocytosis. After the fusion between the membrane of th e equatoria l segment and the oocyte plasma mem- brane occurs, the nucle- us of the spermatozoon is within the cytoplasm. The sperm nuclear mem- bra ne disappears and the nucle us of the sperm decondenses. 12 Ve tB oo ks .ir 264 Sperm in the Female Tract activated, enabling the sperm membrane to fuse or bind with the oocyte membrane. This process is illustrated in Figure 12-8. The cortical reaction prevents penetration by additional spermatozoa. After membrane fusion, the oocyte undergoes a series of changes that prepare it for early embryogenesis. The most easily recognizable is the cortical reaction. During the first and second meiotic divisions of oogen- esis, small, dense granules called cortical granules move to the periphery of the oocyte cytoplasm. The contents of the cortical granules consist ofmucopolysaccharides, proteases, plasminogen activator, acid phosphatase and peroxidase. After membrane fusion between the oocyte and spem1atozoon, the cortical granules undergo exocy- tosis and their contents are released into the perivitelline space (See Figure 12-8). Exocytosis of the cortical granules results in the zona block, a process whereby the zona pellucida undergoes biochemical changes so that further spem1 cannot penetrate it. Polyspermy is prevented by the zona block. Polyspermy is the fertilization of an oocyte by more than one spermatozoon which results in embryo death. In addition to alteration of the zona pellucida, the cortical reaction is believed to reduce the ability of the oocyte plasma membrane to fuse with additional spermatozoa, thus causing the vitelline block, another mechanism that prevents polyspem1y. Some species have both a zona block as well as a vitelline block, while others have either a zona or a vitelline block. Pronuclei formation allows the male and female DNA to form a single nucleus. After the sperm nucleus has entered the cy- toplasm of the egg, it becomes the male pronucleus. Before the pronucleus can be formed, however, the nucleus of the sperm must undergo marked changes within the oocyte cytoplasm. As you will recall, one of the maturational changes that occurs in the epididymis is the acquisition of large numbers of disulfide cross- links in the sperm nucleus. Thus, the nucleus of the mammalian spetm is almost inert. The keratinoid-like quality of insolubility is considered to be important during exposure to the female tract environment, spem1 transport and penetration through the zona pellucida. After the fertilizing spermatozoon enters the oocyte cy- toplasm the nucleus must "decondense" so that the male chromosomes may pair up with the chromosomes of the female pronucleus. The decondensation of the sperm nucleus requires the reduction of the many disulfide cross-links. In the cytoplasm of the oocyte, disulfide cross-links in the spenn nucleus are reduced quickly. The primary reducing agent is glutathione. When disulfide bond reduction occurs, the sperm nucleus decondenses and the nuclear material is available for interaction with the female nuclear material. The final step of fertilization is the fusion of the male and female pronuclei. This fusion is refened to as syngamy. Fol- lowing syngamy, the zygote enters the first stages of embryogenesis that are described in Chapter 13. The Fertile Period Varies Significantly Among Mammalian Females The fertile life-span of spenn after deposition in the female reproductive tract varies immensely among species. For example, fertility of spem1atozoa is re- tained for four to five years in certain reptiles. Among mammals, batspem1atozoa remain viable after insemi- nation in the female tract for up to 4-5 months before the female ovulates. In general, retention of fertilizing capacity among domestic animals and humans lasts only a few days. Values in Table 12-1 document the variation in fertilizing ability in the fema le tract among various domestic species and women. In most domestic species the period of estrus is less than 24 hours. In other words, copulation must take place within a time-period that is close to ovulation. In contrast, spem1 can remain viable for as long as 5 to 6 days before ovulation in women. Another example of a sustained fertile period is the bitch. Ovulation takes place over about a three day period after the onset of sexual receptivity. Fertilization can be accomplished as long as six days after the onset of sexual receptivity. It should be pointed out that in a multiparous species like Table 12-1. Maximal Duration of Fertilizing Ability of Sperm Within the Female Reproductive Tract of Various Species Species Fertile Life (days) Bitch 9-11 Camelids (camel, llama, alpaca) 4-5 Cow 1.5-2 Mare Woman 5-6 the dog, several males can sire offspring because the bitch may be bred by several males during her relatively long estrus. Spennatozoa from all males are eligible to fertilize oocytes. This phenomenon is called superfe- cundation. Thus, it is not uncommon to observe litters that have different breeds of puppies. It should be emphasized that the long fertile period in women coupled with a high frequency of copulation predisposes humans to unwanted pregnan- cies and a high global birth rate. Since the woman does not have a definite period of sexual receptivity, copulation taking place within 5-6 days of ovulation can result in a pregnancy. Where a poor understanding of the cycle exists, the probability of pregnancy becomes quite high because almost 20% of the menstrual cycle has the potential to generate a pregnancy. The question is often asked as to whether the number of copulations can influence the chance of preg- nancy within a given mating period. In spontaneous ovulators the answer is "probably not". In induced ovu- Jators (especially in felids ), there appears to be a thresh- old number of copulations required to optimize GnRH and LH release. The chance of ovulation and therefore pregnancies are related to copulation frequency. In humans, the probability of conception (pregnancy) is about 0.33 per cycle. This means if mating takes place among fertile individuals there is a one-in-three chance that the woman will become pregnant every cycle (if sexual intercourse takes place within 2 days of ovula- tion as Figure 12-9 shows). It is like a batting average. Sperm in the Female Tract 265 If your favorite baseball player had a batting average of 0.333 for the season, he had a one in three chance to get a base-hit during each at-bat. Each at-bat is equivalent to the fertile period of an estrous or menstrual cycle. On average, your favorite hitter needs 3 at-bats to get a hit (a pregnancy). It makes no difference how many times the batter swings (number of copulations) during each "at-bat," his batting average will still be 0.333. Similarly, assuming a threshold number of sperm are deposited during the first copulation, the number of copulations during each fertile period (an "at bat") will not influence the probability of pregnancy because the fi rst copulation fills the oviductal reservoir and will not allow more sperm to populate the reservoir. Batting Averages and Pregnancies are Similar: • Each "at-bat"= 1 opportunity to achieve pregnancy • The batting average =probability of becoming pregnant • A swing = 1 mating • A good "at-bat"= litany swings (but depletes extragonadal reserves) Figure 12-9. Probability of Conception When Copulation Occurred on Specific Days Relative of Ovulation in Women Conception can occur wit hi n a 6-day window prior to ovulation . At 5 days prior to ovula- tion, the probability of conception was 0.11 and the prob- ability increases to about 0.33 two days before ovu- lation. (From Wilcox et a l. 1995. NEJM 333:1517) .4 ..0 >– .3 Rl u ..0 c 0 Rl lo. c Q. l).() .2 ., Cl)
Cl) lo.
._,Q.
Rl ….
E 0 .I ·.p
VI w
-6 -5 -4 -3 -2 -I
Day of Copulation
Relative to Ovulation
0
Ve
tB
oo
ks
.ir

264 Sperm in the Female Tract
activated, enabling the sperm membrane to fuse or bind
with the oocyte membrane. This process is illustrated
in Figure 12-8.
The cortical reaction prevents
penetration by additional spermatozoa.
After membrane fusion, the oocyte undergoes a
series of changes that prepare it for early embryogenesis.
The most easily recognizable is the cortical reaction.
During the first and second meiotic divisions of oogen-
esis, small, dense granules called cortical granules move
to the periphery of the oocyte cytoplasm. The contents
of the cortical granules consist ofmucopolysaccharides,
proteases, plasminogen activator, acid phosphatase and
peroxidase. After membrane fusion between the oocyte
and spem1atozoon, the cortical granules undergo exocy-
tosis and their contents are released into the perivitelline
space (See Figure 12-8). Exocytosis of the cortical
granules results in the zona block, a process whereby
the zona pellucida undergoes biochemical changes so
that further spem1 cannot penetrate it. Polyspermy is
prevented by the zona block.
Polyspermy is the fertilization of an oocyte by
more than one spermatozoon which results in embryo
death. In addition to alteration of the zona pellucida,
the cortical reaction is believed to reduce the ability of
the oocyte plasma membrane to fuse with additional
spermatozoa, thus causing the vitelline block, another
mechanism that prevents polyspem1y. Some species
have both a zona block as well as a vitelline block, while
others have either a zona or a vitelline block.
Pronuclei formation allows the male
and female DNA to form a single
nucleus.
After the sperm nucleus has entered the cy-
toplasm of the egg, it becomes the male pronucleus.
Before the pronucleus can be formed, however, the
nucleus of the sperm must undergo marked changes
within the oocyte cytoplasm. As you will recall, one of
the maturational changes that occurs in the epididymis
is the acquisition of large numbers of disulfide cross-
links in the sperm nucleus. Thus, the nucleus of the
mammalian spetm is almost inert. The keratinoid-like
quality of insolubility is considered to be important
during exposure to the female tract environment, spem1
transport and penetration through the zona pellucida.
After the fertilizing spermatozoon enters the oocyte cy-
toplasm the nucleus must “decondense” so that the male
chromosomes may pair up with the chromosomes of the
female pronucleus. The decondensation of the sperm
nucleus requires the reduction of the many disulfide
cross-links. In the cytoplasm of the oocyte, disulfide
cross-links in the spenn nucleus are reduced quickly.
The primary reducing agent is glutathione. When
disulfide bond reduction occurs, the sperm nucleus
decondenses and the nuclear material is available for
interaction with the female nuclear material. The final
step of fertilization is the fusion of the male and female
pronuclei. This fusion is refened to as syngamy. Fol-
lowing syngamy, the zygote enters the first stages of
embryogenesis that are described in Chapter 13.
The Fertile Period Varies Significantly Among
Mammalian Females
The fertile life-span of spenn after deposition in
the female reproductive tract varies immensely among
species. For example, fertility of spem1atozoa is re-
tained for four to five years in certain reptiles. Among
mammals, batspem1atozoa remain viable after insemi-
nation in the female tract for up to 4-5 months before
the female ovulates. In general, retention of fertilizing
capacity among domestic animals and humans lasts
only a few days. Values in Table 12-1 document the
variation in fertilizing ability in the fema le tract among
various domestic species and women.
In most domestic species the period of estrus is
less than 24 hours. In other words, copulation must take
place within a time-period that is close to ovulation. In
contrast, spem1 can remain viable for as long as 5 to 6
days before ovulation in women. Another example of
a sustained fertile period is the bitch. Ovulation takes
place over about a three day period after the onset of
sexual receptivity. Fertilization can be accomplished as
long as six days after the onset of sexual receptivity. It
should be pointed out that in a multiparous species like
Table 12-1. Maximal Duration of
Fertilizing Ability of Sperm Within the Female
Reproductive Tract of Various Species
Species Fertile Life (days)
Bitch 9-11
Camelids (camel, llama, alpaca) 4-5
Cow 1.5-2
Mare
Woman 5-6
the dog, several males can sire offspring because the
bitch may be bred by several males during her relatively
long estrus. Spennatozoa from all males are eligible to
fertilize oocytes. This phenomenon is called superfe-
cundation. Thus, it is not uncommon to observe litters
that have different breeds of puppies.
It should be emphasized that the long fertile
period in women coupled with a high frequency of
copulation predisposes humans to unwanted pregnan-
cies and a high global birth rate. Since the woman
does not have a definite period of sexual receptivity,
copulation taking place within 5-6 days of ovulation can
result in a pregnancy. Where a poor understanding of
the cycle exists, the probability of pregnancy becomes
quite high because almost 20% of the menstrual cycle
has the potential to generate a pregnancy.
The question is often asked as to whether the
number of copulations can influence the chance of preg-
nancy within a given mating period. In spontaneous
ovulators the answer is “probably not”. In induced ovu-
Jators (especially in felids ), there appears to be a thresh-
old number of copulations required to optimize GnRH
and LH release. The chance of ovulation and therefore
pregnancies are related to copulation frequency. In
humans, the probability of conception (pregnancy) is
about 0.33 per cycle. This means if mating takes place
among fertile individuals there is a one-in-three chance
that the woman will become pregnant every cycle (if
sexual intercourse takes place within 2 days of ovula-
tion as Figure 12-9 shows). It is like a batting average.
Sperm in the Female Tract 265
If your favorite baseball player had a batting average of
0.333 for the season, he had a one in three chance to get
a base-hit during each at-bat. Each at-bat is equivalent
to the fertile period of an estrous or menstrual cycle.
On average, your favorite hitter needs 3 at-bats to get
a hit (a pregnancy). It makes no difference how many
times the batter swings (number of copulations) during
each “at-bat,” his batting average will still be 0.333.
Similarly, assuming a threshold number of sperm are
deposited during the first copulation, the number of
copulations during each fertile period (an “at bat”) will
not influence the probability of pregnancy because the
fi rst copulation fills the oviductal reservoir and will not
allow more sperm to populate the reservoir.
Batting Averages and Pregnancies
are Similar:
• Each “at-bat”= 1 opportunity to achieve
pregnancy
• The batting average =probability of
becoming pregnant
• A swing = 1 mating
• A good “at-bat”= litany swings (but
depletes extragonadal reserves)
Figure 12-9. Probability of Conception When Copulation Occurred on
Specific Days Relative of Ovulation in Women
Conception can
occur wit hi n a
6-day window prior
to ovulation . At 5
days prior to ovula-
tion, the probability
of conception was
0.11 and the prob-
ability increases
to about 0.33 two
days before ovu-
lation.
(From Wilcox et a l. 1995. NEJM 333:1517)
.4
..0 >- .3 Rl u ..0 c 0 Rl lo. c Q. l).() .2 ., Cl)
Cl) lo.
._,Q.
Rl ….
E 0 .I ·.p
VI w
-6 -5 -4 -3 -2 -I
Day of Copulation
Relative to Ovulation
0
Ve
tB
oo
ks
.ir

12
266 Sperm in the Female Tract
Delivery of Semen to the Proper Anatomical
Region of the Female Tract is Required for
Successful Artificial Insemination
It had been erroneously assumed for years
that most spermatozoa ascend toward the oviduct soon
after they are deposited in the cow uterus by artificial
insemination. However, recent studies have shown that
a high proportion of spermatozoa deposited in the uterus
of the cow or ewe are lost from the tract by retrograde
transport. In most cows, over 60% of spennatozoa
artificially inseminated into the uterus are lost to the
exterior of the tract within 12 hours after deposition.
Given these findings, a logical interpretation would be
that artificial insemination of spermatozoa deep into
the uterus would result in reduced retrograde loss.
This assumption is not true because when spem1 are
deposited deep into both uterine horns (as opposed to
the uterine body) the degree of spenn recovered from
the vagina (an indication of retrograde loss) is quite
similar between the two sites of deposition (See Figure
12-l 0). However, when sperm are deposited in the mid-
cervix, a significantly higher degree of retrograde loss
of spennatozoa is encountered (See Figure 12-1 0).
Spermatozoa deposited into only one uterine
horn of the cow experience intercomual transport. That
is, when spem1atozoa are deposited into one uterine
horn (either right or left), they subsequently are redis-
tributed so that both uterine homs eventually contain
substantial numbers of spermatozoa. This phenomenon
also occurs in swine. In cows, fertility is not compro-
mised and in some studies is enhanced when spenn are
deposited within the uterine body or in the right and
left uterine homs.
The important message from the above discus-
sion is that when artificial insemination is perfonned in
the cow and semen is deposited into the cervix, a greater
proportion of spermatozoa are lost to the exterior than
when deposition is in the uterus. Thus, when the in-
semination procedure involves cervical deposition (a
serious technique eiTor), fertility may be compromised
because of greater spermatozoal loss.
Artificial Insemination Techniques in
Domestic Species
Artificial insemination technique requires
that spermatozoa be deposited in the reproductive
tract of the female by artificial means. In general,
semen is delivered using a pipette to penetrate and
bypass the cervix (See Figure I 2-1 I). This type
of insemination is referred to as transcervical in-
semination. In the sow, the insemination pipette is
positioned within the cervix and semen is delivered
into the cranial half of the cervix and flows directly
into the uterine horns. This type of insemination is
refeiTed to as intracervical insemination (See Figure
12-12). In dogs and cats semen is deposited in the cra-
nial vagina. This type of insemination is referred to as
intravaginal insemination (See Figure 12- I 2).
In cases where sperm are in very limited supply,
surgical insemination can be performed by exterioriz-
ing the reproductive tract and injecting sperm directly
into the uterus or uterotubal junction region. Also, use
of laparoscopy enables insemination to be performed
without laparotomy (an abdominal incision). In bulls,
X-Y sorted semen are in short-supply. Therefore, a
teclmique has been developed to “thread” the tip of an
insemination pipette through the cervix to the uterotubal
junction. Such a technique has been reported to gener-
ate excellent results.
Sperm in the Female Tract 267
Figure 12-10. Insemination into the Uterine Horns
Can Reduce Sperm Loss
“0
(!J
22-
ffi 18
“‘V 0 Body “V 0 Com”‘l
u
J!
E
1…
(!J
Cl..
–
14-
–
V) 10
–
(!J
> 6-
:l
E
:l u 2
70 –
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(!J > 0 u
(!J
0:::
so –
E
1…
40 –
(!J
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30 –
20 –
Ill
‘”3
E 10 -:l
m
0
If
.:..::
u n r 0
0
r
2 3 4 5 6
Hours After Insemination
1/
_., .:.:.:. ‘—
r
n
2 3 4 5 6
Hours After Insemination
7 8
7 8
Cumulative percentage of sperm recovered
from the vagina of heifers during an 8 hour
period after insemination. In one group of
heifers (green bar), sperm was deposited in
the uterine body. In the second group (bur-
gundy bar), sperm were deposited deep into
each uterine horn. The cumulative percent
of sperm recovered from the vagina did not
differ between the two treatment groups.
(Modified from Gallahger and Senger, 1989, J. Reprod. Fert.
86:19)
Cumulative percentage of sperm recovered
from the vagina of heifers during an 8 hour
period after insemination. In one group of
heifers (blue bar) sperm were deposited in
the cervix, wh ile in the second group (bur-
gundy bar) sperm were deposited in the
uterine horns. A significantly higher number
of sperm were found in the vagina of the
animals that were inseminated at midcervix
indicating retrograde sperm transport.
(Modified from Gallagher and Senger, 1989, J. Reprod. Fert.
86:19)
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12
266 Sperm in the Female Tract
Delivery of Semen to the Proper Anatomical
Region of the Female Tract is Required for
Successful Artificial Insemination
It had been erroneously assumed for years
that most spermatozoa ascend toward the oviduct soon
after they are deposited in the cow uterus by artificial
insemination. However, recent studies have shown that
a high proportion of spermatozoa deposited in the uterus
of the cow or ewe are lost from the tract by retrograde
transport. In most cows, over 60% of spennatozoa
artificially inseminated into the uterus are lost to the
exterior of the tract within 12 hours after deposition.
Given these findings, a logical interpretation would be
that artificial insemination of spermatozoa deep into
the uterus would result in reduced retrograde loss.
This assumption is not true because when spem1 are
deposited deep into both uterine horns (as opposed to
the uterine body) the degree of spenn recovered from
the vagina (an indication of retrograde loss) is quite
similar between the two sites of deposition (See Figure
12-l 0). However, when sperm are deposited in the mid-
cervix, a significantly higher degree of retrograde loss
of spennatozoa is encountered (See Figure 12-1 0).
Spermatozoa deposited into only one uterine
horn of the cow experience intercomual transport. That
is, when spem1atozoa are deposited into one uterine
horn (either right or left), they subsequently are redis-
tributed so that both uterine homs eventually contain
substantial numbers of spermatozoa. This phenomenon
also occurs in swine. In cows, fertility is not compro-
mised and in some studies is enhanced when spenn are
deposited within the uterine body or in the right and
left uterine homs.
The important message from the above discus-
sion is that when artificial insemination is perfonned in
the cow and semen is deposited into the cervix, a greater
proportion of spermatozoa are lost to the exterior than
when deposition is in the uterus. Thus, when the in-
semination procedure involves cervical deposition (a
serious technique eiTor), fertility may be compromised
because of greater spermatozoal loss.
Artificial Insemination Techniques in
Domestic Species
Artificial insemination technique requires
that spermatozoa be deposited in the reproductive
tract of the female by artificial means. In general,
semen is delivered using a pipette to penetrate and
bypass the cervix (See Figure I 2-1 I). This type
of insemination is referred to as transcervical in-
semination. In the sow, the insemination pipette is
positioned within the cervix and semen is delivered
into the cranial half of the cervix and flows directly
into the uterine horns. This type of insemination is
refeiTed to as intracervical insemination (See Figure
12-12). In dogs and cats semen is deposited in the cra-
nial vagina. This type of insemination is referred to as
intravaginal insemination (See Figure 12- I 2).
In cases where sperm are in very limited supply,
surgical insemination can be performed by exterioriz-
ing the reproductive tract and injecting sperm directly
into the uterus or uterotubal junction region. Also, use
of laparoscopy enables insemination to be performed
without laparotomy (an abdominal incision). In bulls,
X-Y sorted semen are in short-supply. Therefore, a
teclmique has been developed to “thread” the tip of an
insemination pipette through the cervix to the uterotubal
junction. Such a technique has been reported to gener-
ate excellent results.
Sperm in the Female Tract 267
Figure 12-10. Insemination into the Uterine Horns
Can Reduce Sperm Loss
“0
(!J
22-
ffi 18
“‘V 0 Body “V 0 Com”‘l
u
J!
E
1…
(!J
Cl..
–
14-
–
V) 10
–
(!J
> 6-
:l
E
:l u 2
70 –
“0 60 -e
(!J > 0 u
(!J
0:::
so –
E
1…
40 –
(!J
Cl..
V)
30 –
20 –
Ill
‘”3
E 10 -:l
m
0
If
.:..::
u n r 0
0
r
2 3 4 5 6
Hours After Insemination
1/
_., .:.:.:. ‘—
r
n
2 3 4 5 6
Hours After Insemination
7 8
7 8
Cumulative percentage of sperm recovered
from the vagina of heifers during an 8 hour
period after insemination. In one group of
heifers (green bar), sperm was deposited in
the uterine body. In the second group (bur-
gundy bar), sperm were deposited deep into
each uterine horn. The cumulative percent
of sperm recovered from the vagina did not
differ between the two treatment groups.
(Modified from Gallahger and Senger, 1989, J. Reprod. Fert.
86:19)
Cumulative percentage of sperm recovered
from the vagina of heifers during an 8 hour
period after insemination. In one group of
heifers (blue bar) sperm were deposited in
the cervix, wh ile in the second group (bur-
gundy bar) sperm were deposited in the
uterine horns. A significantly higher number
of sperm were found in the vagina of the
animals that were inseminated at midcervix
indicating retrograde sperm transport.
(Modified from Gallagher and Senger, 1989, J. Reprod. Fert.
86:19)
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268 Sperm in the Female Tract
Figure 12-11. Artificial Insemination Technique in the Cow and Mare
Semen
Inseminating pipette
Hand grasping cervix
Cow
The radiographs above are from extirpated cow reproductive tracts (dorsal view). In cornual
insemination, one-half of the semen is deposited in each uterine horn. In both examples, the in-
seminant volume is 0.5-ml. Cornual insemination minimizes the possibility of cervical deposition
that results in significant retrograde loss of spermatozoa (See Figure 12-3). RUL= Right Uterine
Lumen; LUL= Left Uterine Lumen; RO= right ovary; LO= left ovary; S= semen; AIS= artificial in-
semination syringe; CX= cervix
Mare l
Vagina
In the mare, the gloved lubricated hand is inserted directly into the vagina and the index finger is
used to guide the insemination pipette into the cervical lumen. A marker (arrow) is used to gauge
the depth of insemination.
I
Sperm in the Female Tract 269
Figure 12-12 Artificial Insemination Technique in the Sow and Bitch
Sow
of an sow repr?ducti.ve tracts (dorsal view). An artificial insemination pipette
(AIP).consrsts of a sprral trp that rs desrgned so that it can snugly penetrate the interdigitating
promrnences. (lOP) of. the ce’:’rx (CX). In the photograph to the right, about 80-ml of radiopaque
contrast medrum was rnfused rnto the reproductive tract to mimic the inseminant (I). Notice that the
semen. within both uterine horns. High volumes (about 80-ml) are necessary
to maxrmrze rn sows. The vagina (V) and the urinary bladder (UB) can be visualized.
LUL= Left Uterrne Lumen; RUL= Right Uterine Lumen.
Bitch
Cervix
Uterine
body
The vulva is elevated manually so that the ventral “tilt” of the vestibule is removed. This allows
the insemination pipette to be inserted with relative ease. The hindquarters of the bitch should be
elevated for about 5 minutes after deposition of the semen to allow pooling in the cranial vagina
and caudal cervix.
12
Ve
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268 Sperm in the Female Tract
Figure 12-11. Artificial Insemination Technique in the Cow and Mare
Semen
Inseminating pipette
Hand grasping cervix
Cow
The radiographs above are from extirpated cow reproductive tracts (dorsal view). In cornual
insemination, one-half of the semen is deposited in each uterine horn. In both examples, the in-
seminant volume is 0.5-ml. Cornual insemination minimizes the possibility of cervical deposition
that results in significant retrograde loss of spermatozoa (See Figure 12-3). RUL= Right Uterine
Lumen; LUL= Left Uterine Lumen; RO= right ovary; LO= left ovary; S= semen; AIS= artificial in-
semination syringe; CX= cervix
Mare l
Vagina
In the mare, the gloved lubricated hand is inserted directly into the vagina and the index finger is
used to guide the insemination pipette into the cervical lumen. A marker (arrow) is used to gauge
the depth of insemination.
I
Sperm in the Female Tract 269
Figure 12-12 Artificial Insemination Technique in the Sow and Bitch
Sow
of an sow repr?ducti.ve tracts (dorsal view). An artificial insemination pipette
(AIP).consrsts of a sprral trp that rs desrgned so that it can snugly penetrate the interdigitating
promrnences. (lOP) of. the ce’:’rx (CX). In the photograph to the right, about 80-ml of radiopaque
contrast medrum was rnfused rnto the reproductive tract to mimic the inseminant (I). Notice that the
semen. within both uterine horns. High volumes (about 80-ml) are necessary
to maxrmrze rn sows. The vagina (V) and the urinary bladder (UB) can be visualized.
LUL= Left Uterrne Lumen; RUL= Right Uterine Lumen.
Bitch
Cervix
Uterine
body
The vulva is elevated manually so that the ventral “tilt” of the vestibule is removed. This allows
the insemination pipette to be inserted with relative ease. The hindquarters of the bitch should be
elevated for about 5 minutes after deposition of the semen to allow pooling in the cranial vagina
and caudal cervix.
12
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270 Sperm in the Female Tract
Further
PHENOMENA
for Fertility
Some species have delayed fertilization. This
is a process whereby the male inseminates
the female and spermatozoa remain viable
in the female tract for a sustained period
of time. When a rooster inseminates a hen
she can lay fertile eggs for over 20 days.
Sperm are stored in special utero-vaginal
glands. Some bats mate in the autumn
before hibernation. The female does not
ovulate until spring. Sperm are stored in
her tract during the The fertilizing
life of bat sperm is reported to range from
68 to 198 days depending on the species of
bat. Snakes are reported to store sperm that
are fertile for up to 6 years.
The bifurcation of the glans penis of the
opossum led to the widespread Appalachian
folk belief that opossums mated through
the nose, with one fork of the glans penis
penetrating each nostril. Little scientific
consideration was given to the issues of
sperm transport.
Male mammals deliver sperm to the fe-
male in seminal plasma. However, many
lower forms of animals make use of special
packages for delivering spermatozoa to the
female reproductive tract. These packages
are called spermatoplwres. These sper-
matophores are produced within the male
reproductive tract and are stored there until
copulation. In some cephalopods (octopus
and squid) the male deposits the spermato-
plzore in the female tract or into the buccal
cavity (cheek pouch), from which it can
be conveniently transferred to the female
tract. In some annelids, spermatophores are
“injected” subcutaneously, after which the
spermatozoa spread throughout the
body before contacting eggs.
A Spermatozoon Race
by Cheryl A. Dudley
Half frenzied, thick and slick
and treacherous, through vast dark tunnels,
as motile and pmetratingly
zona-bmmd as any race ever,
none other is so victim-laden,
so masked by drunken seizures
or pleasures of full-bodied assaults,
the tadpoles’ mad dash
is like an escaped madman,
a drowner driven to o>..ygen,
thejoumey a seas-width heat
to life or death
When they jolted over the barrier
site didn ‘t realize a race was on,
yet in her own primordial way
site cheered for them, provided secret
privileged pathways through crypts
too difficult for most, whose dead,
flat-floating bodies cluttered the way.
The lone victor slithered through, sensed
the trophy ahead-the zona seducing him to dip
in her warm waters, melt into her soft globe.
(The courtship was only long enough for him
to work his way through her pellucida.)
A quivering union formed
primitive cords that proliferated
time and time and time again,
swelling to fill the primed pear-palmed
womb where the victor celebrated,
And a genesis began.
Cheryl Diu/ley typed the 1″ Edition of Path-
wavs to Pregnancv and Parturition from the
author’s dictation. Site has since graduated
Cum laude in Euglislt from the University of
ldalro and is now a graduate student in the
Department of English at that university.
Motility of trout spermatozoa is induced by
the fresh water into which it is ejaculated.
Motility lasts for only about 30 seconds.
During tltis time the sperm must locate a
single tiny hole in the egg (called a micro-
pyle) through which it enters before fertiliza-
tion can occur. All this happens while beillg
swept about by moving
Kev References
Anderson, G.B., 1991. “Fertil ization, early develop-
ment and embryo transfer” in Reproduction in Domes-
tic Animals. 4th Edition. P.T. Cupps, ed. Academic
Press. New York. ISBN 0- 12- 196575-9.
Crozet, N. 1993. “Fertilization in-vivo and in-vitro”
in Reproduction in Mammals and Man. C. Thibault,
M.C. Levasseur and R.H.F. Hunter, eds. Ellipses, Paris.
ISBN 2-7298-9354-7.
Flowers, W.L. 1999. “Artifi cial insemination in ani-
mals” in Encvclopedia o(Reproduction, Vol. I p291-
30 l. Knobil, E. and J.D. Neill, eds. Academic Press,
San Diego. ISBN 0-1 2-227021-5.
Mullins, K.J. and R.G. Saacke. 1989. “Study of the
functional anatomy of bovine cervical mucosa with
special reference to mucus secretion and sperm trans-
port.” Anal. Rec. 225: I 06-Il7.
Yanagimachi, R. 1996. “Mammalian fertilization” in
Phvsiolo’S)l o(Reproduction, 2nd Edition. Vol. I p 189-
3I 8. E. Knobil and J.D. Neill, eds. Raven Press, Ltd.,
New York. ISBN 0-7817-0086-8.
Sperm in the Female Tract 271
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270 Sperm in the Female Tract
Further
PHENOMENA
for Fertility
Some species have delayed fertilization. This
is a process whereby the male inseminates
the female and spermatozoa remain viable
in the female tract for a sustained period
of time. When a rooster inseminates a hen
she can lay fertile eggs for over 20 days.
Sperm are stored in special utero-vaginal
glands. Some bats mate in the autumn
before hibernation. The female does not
ovulate until spring. Sperm are stored in
her tract during the The fertilizing
life of bat sperm is reported to range from
68 to 198 days depending on the species of
bat. Snakes are reported to store sperm that
are fertile for up to 6 years.
The bifurcation of the glans penis of the
opossum led to the widespread Appalachian
folk belief that opossums mated through
the nose, with one fork of the glans penis
penetrating each nostril. Little scientific
consideration was given to the issues of
sperm transport.
Male mammals deliver sperm to the fe-
male in seminal plasma. However, many
lower forms of animals make use of special
packages for delivering spermatozoa to the
female reproductive tract. These packages
are called spermatoplwres. These sper-
matophores are produced within the male
reproductive tract and are stored there until
copulation. In some cephalopods (octopus
and squid) the male deposits the spermato-
plzore in the female tract or into the buccal
cavity (cheek pouch), from which it can
be conveniently transferred to the female
tract. In some annelids, spermatophores are
“injected” subcutaneously, after which the
spermatozoa spread throughout the
body before contacting eggs.
A Spermatozoon Race
by Cheryl A. Dudley
Half frenzied, thick and slick
and treacherous, through vast dark tunnels,
as motile and pmetratingly
zona-bmmd as any race ever,
none other is so victim-laden,
so masked by drunken seizures
or pleasures of full-bodied assaults,
the tadpoles’ mad dash
is like an escaped madman,
a drowner driven to o>..ygen,
thejoumey a seas-width heat
to life or death
When they jolted over the barrier
site didn ‘t realize a race was on,
yet in her own primordial way
site cheered for them, provided secret
privileged pathways through crypts
too difficult for most, whose dead,
flat-floating bodies cluttered the way.
The lone victor slithered through, sensed
the trophy ahead-the zona seducing him to dip
in her warm waters, melt into her soft globe.
(The courtship was only long enough for him
to work his way through her pellucida.)
A quivering union formed
primitive cords that proliferated
time and time and time again,
swelling to fill the primed pear-palmed
womb where the victor celebrated,
And a genesis began.
Cheryl Diu/ley typed the 1″ Edition of Path-
wavs to Pregnancv and Parturition from the
author’s dictation. Site has since graduated
Cum laude in Euglislt from the University of
ldalro and is now a graduate student in the
Department of English at that university.
Motility of trout spermatozoa is induced by
the fresh water into which it is ejaculated.
Motility lasts for only about 30 seconds.
During tltis time the sperm must locate a
single tiny hole in the egg (called a micro-
pyle) through which it enters before fertiliza-
tion can occur. All this happens while beillg
swept about by moving
Kev References
Anderson, G.B., 1991. “Fertil ization, early develop-
ment and embryo transfer” in Reproduction in Domes-
tic Animals. 4th Edition. P.T. Cupps, ed. Academic
Press. New York. ISBN 0- 12- 196575-9.
Crozet, N. 1993. “Fertilization in-vivo and in-vitro”
in Reproduction in Mammals and Man. C. Thibault,
M.C. Levasseur and R.H.F. Hunter, eds. Ellipses, Paris.
ISBN 2-7298-9354-7.
Flowers, W.L. 1999. “Artifi cial insemination in ani-
mals” in Encvclopedia o(Reproduction, Vol. I p291-
30 l. Knobil, E. and J.D. Neill, eds. Academic Press,
San Diego. ISBN 0-1 2-227021-5.
Mullins, K.J. and R.G. Saacke. 1989. “Study of the
functional anatomy of bovine cervical mucosa with
special reference to mucus secretion and sperm trans-
port.” Anal. Rec. 225: I 06-Il7.
Yanagimachi, R. 1996. “Mammalian fertilization” in
Phvsiolo’S)l o(Reproduction, 2nd Edition. Vol. I p 189-
3I 8. E. Knobil and J.D. Neill, eds. Raven Press, Ltd.,
New York. ISBN 0-7817-0086-8.
Sperm in the Female Tract 271
Ve
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oo
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.ir

The Puerperium & Lactation
Parturition
Ovulation & Fertilization
Cyclicity
Regulation of
Reproduction
Tract Function
Puberty
Prenatal
Development
, …
Spermatogenesis
Regulation of
Reproduction
Tract Function
Puberty
Prenatal
Development
– – ————. …
Take Home Message
A successful pregnancy requires that the preattachment embryo develop into a
blastocyst, hatch from tlze zona pellucida and develop a functional trophoblast. The
early embryo must secrete materials that prevent luteolysis or that enhance luteal
jimction to maintain pregnancy.
Before describing the important events of early
embryogenesis, several potentially confusing terms
with overlapping meanings need to be defined. These
terms have subtly different uses depending on the
species and the context in which they are used. After
syngamy (fusion of the male and female pronuclei),
the zygote becomes an embryo. An embryo is defined
as an organism in the early stages of development. In
general, an embryo has not acquired an anatomical form
that is readily recognizable in appearance as a member
of the specific species. For example, at early stages of
development, the pig embryo cannot be distinguished
from the cow embryo except by skilled embryolo-
gists. As a matter of fact, at certain stages, the human
embryo cannot be distinguished from the embryos of
lower species.
A fetus is defined as a potential offspring that is
still within the uterus, but is generally recognizable as
a member of a given species. Most physiologists think
of a fetus as the more advanced form of an embryo.
The tenm embryo, conceptus and fetus are often used
interchangeably to describe the developing organism.
But, it should be recognized that each term has a dis-
tinct meaning and students of reproductive physiology
are encouraged to use the term that most accurately
describes the developing organism.
A conceptus is defined as the product of con-
ception. It includes: 1) the embryo during the early
embryonic stage, 2) the embryo and extraembryonic
membranes during the preimplantation stage and 3) the
fetus and placenta during the post-attachment phase.
After fertilization, four important develop-
mental events must occur before the embryo attaches
to the uterus. Only after these milestones are achieved
will the embryo be eligible to develop a more intimate,
semipermanent relationship with the utems.
Four steps must be achieved before the
embryo can attach to the uterus. They are:
• development within the confines
of the zona pellucida
• hatching ofthe blastocyst from
the zona pellucida
• maternal recognition ofpregnancy
• formation of the extraembryonic
membranes
The presence of male and female pronuclei
within the cytoplasm of the oocyte characterizes a de-
velopmental stage of the newly fertilized oocyte. When
male and female pronuclei can be observed, the cell is
called an ootid (See Figure 13-1 ). The ootid is one of
the largest single cells in the body and is characterized
by having an enonnous cytoplasmic volume relative to
nuclear volume. This characteristic is important, since
subsequent cell divisions within the confines of the zona
pellucida will involve partitioning of the cytoplasm into
smaller and smaller cellular units (See Figure 13- 1 ).
Following fusion of the male and female pro-
nuclei, the single-celled embryo, now called a zygote,
undergoes a series of mitotic divisions called cleav-
age divisions. The first cleavage division generates a
two-celled embryo, the cells of which are called bias-
to meres. Each blastomere in the two-celled embryo
is about the same size and represents a lmost exactly
one-half of the single-celled zygote. Each blastomere
undergoes subsequent divisions, yielding 4, 8 and then
I 6 daughter cells.
In the early stages of embryogenesis, each blas-
tomere has the potential to develop into separate healthy
offspring. Identical twins are derived from blastomeres
of a two-celled embryo that divide independently to fonn
two separate embryos. Blastomeres fi·om the 2-, 4-,
Ve
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oo
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.ir

The Puerperium & Lactation
Parturition
Ovulation & Fertilization
Cyclicity
Regulation of
Reproduction
Tract Function
Puberty
Prenatal
Development
, …
Spermatogenesis
Regulation of
Reproduction
Tract Function
Puberty
Prenatal
Development
– – ————. …
Take Home Message
A successful pregnancy requires that the preattachment embryo develop into a
blastocyst, hatch from tlze zona pellucida and develop a functional trophoblast. The
early embryo must secrete materials that prevent luteolysis or that enhance luteal
jimction to maintain pregnancy.
Before describing the important events of early
embryogenesis, several potentially confusing terms
with overlapping meanings need to be defined. These
terms have subtly different uses depending on the
species and the context in which they are used. After
syngamy (fusion of the male and female pronuclei),
the zygote becomes an embryo. An embryo is defined
as an organism in the early stages of development. In
general, an embryo has not acquired an anatomical form
that is readily recognizable in appearance as a member
of the specific species. For example, at early stages of
development, the pig embryo cannot be distinguished
from the cow embryo except by skilled embryolo-
gists. As a matter of fact, at certain stages, the human
embryo cannot be distinguished from the embryos of
lower species.
A fetus is defined as a potential offspring that is
still within the uterus, but is generally recognizable as
a member of a given species. Most physiologists think
of a fetus as the more advanced form of an embryo.
The tenm embryo, conceptus and fetus are often used
interchangeably to describe the developing organism.
But, it should be recognized that each term has a dis-
tinct meaning and students of reproductive physiology
are encouraged to use the term that most accurately
describes the developing organism.
A conceptus is defined as the product of con-
ception. It includes: 1) the embryo during the early
embryonic stage, 2) the embryo and extraembryonic
membranes during the preimplantation stage and 3) the
fetus and placenta during the post-attachment phase.
After fertilization, four important develop-
mental events must occur before the embryo attaches
to the uterus. Only after these milestones are achieved
will the embryo be eligible to develop a more intimate,
semipermanent relationship with the utems.
Four steps must be achieved before the
embryo can attach to the uterus. They are:
• development within the confines
of the zona pellucida
• hatching ofthe blastocyst from
the zona pellucida
• maternal recognition ofpregnancy
• formation of the extraembryonic
membranes
The presence of male and female pronuclei
within the cytoplasm of the oocyte characterizes a de-
velopmental stage of the newly fertilized oocyte. When
male and female pronuclei can be observed, the cell is
called an ootid (See Figure 13-1 ). The ootid is one of
the largest single cells in the body and is characterized
by having an enonnous cytoplasmic volume relative to
nuclear volume. This characteristic is important, since
subsequent cell divisions within the confines of the zona
pellucida will involve partitioning of the cytoplasm into
smaller and smaller cellular units (See Figure 13- 1 ).
Following fusion of the male and female pro-
nuclei, the single-celled embryo, now called a zygote,
undergoes a series of mitotic divisions called cleav-
age divisions. The first cleavage division generates a
two-celled embryo, the cells of which are called bias-
to meres. Each blastomere in the two-celled embryo
is about the same size and represents a lmost exactly
one-half of the single-celled zygote. Each blastomere
undergoes subsequent divisions, yielding 4, 8 and then
I 6 daughter cells.
In the early stages of embryogenesis, each blas-
tomere has the potential to develop into separate healthy
offspring. Identical twins are derived from blastomeres
of a two-celled embryo that divide independently to fonn
two separate embryos. Blastomeres fi·om the 2-, 4-,
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27 4 Early Embryogenesis and Maternal Recognition of Pregnancy
Perivitelline
space
Pronudeii
Figure 13-1. Preattachment Development of the Embryo
J•t and 2nd
.;:…,;;,.._– Polar
bodies
Zona
pellucida
/
2-celled embryo
4-8 cell
Early blastocyst
Trophoblast
r
Hatched blastocyst
Blastocyst
Cells of the morula continue to
divide and a blastocyst devel-
ops. It consists of an inner cell
mass (ICM), a cavity called the
blastocoele and a single layer
of cells called the trophoblast.
Finally, the rapidly growing blas-
tocyst “hatches” from the zona
pellucida and forms a “hatched”
blastocyst that is free-floating
within the uterus.
Early Embryogenesis and Maternal Recognition of Pregnancy 275
Figure 13-2. Transition of a Morula into an Early Blastocyst
e;t.’i!’l
Tight junctions
Gap junctions
Tight junctions form between the outer
cells of the morula. Gap junctions form
between the inner cells thus creating two
groups of cells. Sodium is pumped into
the intercellular spaces by the outer cells
of the morula and water follows osmoti-
cally. Therefore, fluid begins to accumu-
late within the morula.
Early blastocyst
8- and 16- celled embryos are totipotent. Totipo-
tency is a tenn used to describe the ability of a single
cell (blastomere) to give rise to a complete, fully
fanned individual. Identical twins can be artificially
produced in the laboratory by separating individual
blastomeres, placing each blastomere inside a surrogate
zona pellucida and allowing it to develop within the
uterus of a host female. The individual blastomeres
isolated from 4- and 8- celled stages can develop into
normal embryos in the rabbit (doe), mare, cow and
ewe. Totipotency has not been demonstrated when
whole blastomeres beyond the 16-cell stage are used.
Recently, nuclei from somatic cells from adult cattle,
sheep, goats, horses, swine, cats and dogs have been
transplanted into enucleated oocytes. These oocytes
have developed into nomml offspring, although suc-
cess rates are low (< 5%). Therefore, it appears that all cells may have the potential for totipotency if exposed to the appropriate environmental conditions. As fluid accumulates, the outer cells be- come flattened and a cavity known as the blastocoele is formed. The gap junctions connecting the inner cells of the morula allow these cells to polarize as a group. As a result two separate cellular components emerge. These are, the inner cell mass (ICM) and the trophoblast. The mitotic divisions of each blastomere gener- ally occur simultaneously but are unique in that with each division, two cells are produced (from each blas- tomere) but there is no net change in cytoplasmic mass. The unique mitotic divisions are called cleavage divi- sions and occur between the 1-cell and the blastocyst stages. As a result of the cleavage divisions an embryo gains cell number but still contains the same total mass of cytoplasm it had when it was a 1-cell zygote. All of the cleavage divisions take place inside the zona pellucida that maintains a fixed volume throughout the process. When a sol id ball of cells is formed and individual blastomeres can no longer be counted ac- curately, the early embryo is called a morula (See Figure 13-1 ). When the morula is formed, the outer cells begin to be compacted more than the cells in the center. Thus, during the morula stage, cells begin to separate into two distinct populations, the inner and Ve tB oo ks .ir 27 4 Early Embryogenesis and Maternal Recognition of Pregnancy Perivitelline space Pronudeii Figure 13-1. Preattachment Development of the Embryo J•t and 2nd .;:...,;;,.._-- Polar bodies Zona pellucida / 2-celled embryo 4-8 cell Early blastocyst Trophoblast r Hatched blastocyst Blastocyst Cells of the morula continue to divide and a blastocyst devel- ops. It consists of an inner cell mass (ICM), a cavity called the blastocoele and a single layer of cells called the trophoblast. Finally, the rapidly growing blas- tocyst "hatches" from the zona pellucida and forms a "hatched" blastocyst that is free-floating within the uterus. Early Embryogenesis and Maternal Recognition of Pregnancy 275 Figure 13-2. Transition of a Morula into an Early Blastocyst e;t.'i!'l Tight junctions Gap junctions Tight junctions form between the outer cells of the morula. Gap junctions form between the inner cells thus creating two groups of cells. Sodium is pumped into the intercellular spaces by the outer cells of the morula and water follows osmoti- cally. Therefore, fluid begins to accumu- late within the morula. Early blastocyst 8- and 16- celled embryos are totipotent. Totipo- tency is a tenn used to describe the ability of a single cell (blastomere) to give rise to a complete, fully fanned individual. Identical twins can be artificially produced in the laboratory by separating individual blastomeres, placing each blastomere inside a surrogate zona pellucida and allowing it to develop within the uterus of a host female. The individual blastomeres isolated from 4- and 8- celled stages can develop into normal embryos in the rabbit (doe), mare, cow and ewe. Totipotency has not been demonstrated when whole blastomeres beyond the 16-cell stage are used. Recently, nuclei from somatic cells from adult cattle, sheep, goats, horses, swine, cats and dogs have been transplanted into enucleated oocytes. These oocytes have developed into nomml offspring, although suc- cess rates are low (< 5%). Therefore, it appears that all cells may have the potential for totipotency if exposed to the appropriate environmental conditions. As fluid accumulates, the outer cells be- come flattened and a cavity known as the blastocoele is formed. The gap junctions connecting the inner cells of the morula allow these cells to polarize as a group. As a result two separate cellular components emerge. These are, the inner cell mass (ICM) and the trophoblast. The mitotic divisions of each blastomere gener- ally occur simultaneously but are unique in that with each division, two cells are produced (from each blas- tomere) but there is no net change in cytoplasmic mass. The unique mitotic divisions are called cleavage divi- sions and occur between the 1-cell and the blastocyst stages. As a result of the cleavage divisions an embryo gains cell number but still contains the same total mass of cytoplasm it had when it was a 1-cell zygote. All of the cleavage divisions take place inside the zona pellucida that maintains a fixed volume throughout the process. When a sol id ball of cells is formed and individual blastomeres can no longer be counted ac- curately, the early embryo is called a morula (See Figure 13-1 ). When the morula is formed, the outer cells begin to be compacted more than the cells in the center. Thus, during the morula stage, cells begin to separate into two distinct populations, the inner and Ve tB oo ks .ir I ! 276 Early Embryogenesis and Maternal Recognition of Pregnancy Table 13-1 Timing of preattachment embryogenesis relative to ovulation within of various species. values are in the oviduct. Bold values in the shaded box are m the uterus; (-)=no data. 2-cell 4-cell 8-cell Morula Hatching Sgecies 13-15d bitch* 3-?d 7-12d 9-11d 24h 1.5d 3d 4-7d cow 4-10d 7-Sd 24h 1.3d 2.5d 3-4d ewe 4-5d 6-Sd 7-Sd 24h 1.5d 3d mare 5d 8d 10-12d queen 1.0d 2d 3.5d 4-5d 6d sow 14-16h 5-6d 24h 2d 3d 4d 5d woman *Recall from Figure 7-4 that ovulation and fertilization occur during a 6-7 day period during estrus. outer cells. During this transition, there is expression of genes involved in a.dheswn, molecule transport (including ions) and mtra/mter ce_ll communication. This is accompanied by asymmetric divisions of cells that are thought to sequester differ- entiation factors in the outer layer and stem cell factors in the inner cell mass. Cells in the inner portion of the morula develop gap junctions (See Figure 13-2) that allow for intercellular communication and may en- able the inner cells to remain in a defined cluster. The outer cells ofthe morula develop cell-to-cell adhesions known as tight junctions (See Figure 13-2). tight junctions are believed to alter the permeability of the outer cells. After the tight junctions are formed, fluid begins to accumulate inside the embryo. This fluid accumulation is believed to be brought about by an active sodium pump in the outer cells of the morula that pump sodium ions into the center portion of the Figure 13-3. Schematic Illustration of Preattachment Embryo Development Four-celled stage stage Ootid Fertilization Early Embryogenesis and Maternal Recognition of Pregnancy 277 morula. This buildup of ions causes the ionic concen- tration of the fluid sunounding the inner cells of the momla to increase. As the ionic strength inside the momla increases, water diffuses through the zona pel- Iucida into the embryo and begins to form a fluid filled cavity (See Figure 13-2) called a blastocoele. Hatching of the blastocyst is governed by three forces. They are: • growth and .fluid accumulation within the blastocyst • production of enzymes by the trophoblastic cells • contraction of the blastocyst When a distinct cavity is recognizable, the embryo is called a blastocyst. Because of the nature of the tight junctions (found in the outer cells) and the gap junctions (found among the inner cells), the embryo becomes partitioned into two distinct cellular populations. These are !mown as the inner ceiJ mass and the trophoblast. The inner cell mass will give rise to the body of the embtyo. The trophoblastic cells wi ll eventually give rise to the chorion. The chorion will become the fetal component of the placenta that will be described later. As the blastocyst continues to undergo mitosis, fluid continues to fill the blastocoele and the pressure within the embryo increases. Concurrent with growth and fluid accumulation is the production of proteolytic enzymes by the trophoblastic cells. These enzymes weaken the zona pellucida so that it ruptures easily as growth of the blastocyst continues. Finally, the blasto- cyst itself begins to contract and relax. Such behavior causes intermittent pressure pulses. These pressure pulses coupled with continued growth and enzymatic degradation cause the zona pellucida to ruphtre. When a small crack or fissure in the zona pel- lucida develops, the cells of the blastocyst squeeze out of the opening, escaping from their confines (See Figure I 3- I). The blastocyst now becomes a free-floating embtyo within the lumen of the uterus and is totally de- pendent on the uterine environment for survival. In this context, early embtyo survival is dependent on adequate luteal function, adequate progesterone synthesis and responsiveness of the utems to progesterone. Figure 13-3 illustrates the anatomical location of the various preattachment stages of the embtyo. The timing and species variation is presented in Table I 3-1 . Development of the Extraembryonic Membranes Represents an "Explosion" of Embryonic Tissue Growth Prior to Attachment After hatching, the conceptus undergoes mas- sive growth. For example, in the cow at day 13 the blastocyst is about 3 mm in diameter. During the next four days, the cow blastocyst will become 250 mm in length (about the vertical length of the printed portion of thi s page) and will appear as a filamentous thread. By day I 8 of gestation, the blastocyst occupies space in both uterine horns. While the blastocyst of the cow (and the ewe) grows quite rapidly during this early pre- attachment stage, the development of the pig blastocyst is even more dramatic. On day 10 of pregnancy, pig blastocysts are 2 mm spheres. During the next 24 to 48 hours, these 2 mm blastocysts will grow to about 200 mm in length (about the width of the printed portion of this page). This means that the blastocyst is growing at a rate of 4 to 8 mm per hour. By day 16, the pig blastocyst reaches lengths of 800 to I 000 mm. Mammalian embryos can be subdivided into two primary groups. In the first group (that includes most domestic animals), the preattachment period within the uterus is long (several weeks). During this time, extensive extraembtyonic membranes form by a folding process that generates the amnion, chorion and allantochorion. In the second group (primates) the blastocyst implants very soon after it enters the uterus. The extraembryonic membranes fonn after implanta- tion or attachment. In this text, we will deal exclusively with the first group. For details about implantation of the human blastocyst please consult the reference by Larsen in Key References. The extraembry onic membranes of the preattachment embryo consist of the: • yolk sac • chorion • amnion • allantois The dramatic growth of the conceptus is due largely to the development of a set of membranes called the extraembryonic membranes. The pig, sheep and cow are characterized as having filamentous or Ve tB oo ks .ir I ! 276 Early Embryogenesis and Maternal Recognition of Pregnancy Table 13-1 Timing of preattachment embryogenesis relative to ovulation within of various species. values are in the oviduct. Bold values in the shaded box are m the uterus; (-)=no data. 2-cell 4-cell 8-cell Morula Hatching Sgecies 13-15d bitch* 3-?d 7-12d 9-11d 24h 1.5d 3d 4-7d cow 4-10d 7-Sd 24h 1.3d 2.5d 3-4d ewe 4-5d 6-Sd 7-Sd 24h 1.5d 3d mare 5d 8d 10-12d queen 1.0d 2d 3.5d 4-5d 6d sow 14-16h 5-6d 24h 2d 3d 4d 5d woman *Recall from Figure 7-4 that ovulation and fertilization occur during a 6-7 day period during estrus. outer cells. During this transition, there is expression of genes involved in a.dheswn, molecule transport (including ions) and mtra/mter ce_ll communication. This is accompanied by asymmetric divisions of cells that are thought to sequester differ- entiation factors in the outer layer and stem cell factors in the inner cell mass. Cells in the inner portion of the morula develop gap junctions (See Figure 13-2) that allow for intercellular communication and may en- able the inner cells to remain in a defined cluster. The outer cells ofthe morula develop cell-to-cell adhesions known as tight junctions (See Figure 13-2). tight junctions are believed to alter the permeability of the outer cells. After the tight junctions are formed, fluid begins to accumulate inside the embryo. This fluid accumulation is believed to be brought about by an active sodium pump in the outer cells of the morula that pump sodium ions into the center portion of the Figure 13-3. Schematic Illustration of Preattachment Embryo Development Four-celled stage stage Ootid Fertilization Early Embryogenesis and Maternal Recognition of Pregnancy 277 morula. This buildup of ions causes the ionic concen- tration of the fluid sunounding the inner cells of the momla to increase. As the ionic strength inside the momla increases, water diffuses through the zona pel- Iucida into the embryo and begins to form a fluid filled cavity (See Figure 13-2) called a blastocoele. Hatching of the blastocyst is governed by three forces. They are: • growth and .fluid accumulation within the blastocyst • production of enzymes by the trophoblastic cells • contraction of the blastocyst When a distinct cavity is recognizable, the embryo is called a blastocyst. Because of the nature of the tight junctions (found in the outer cells) and the gap junctions (found among the inner cells), the embryo becomes partitioned into two distinct cellular populations. These are !mown as the inner ceiJ mass and the trophoblast. The inner cell mass will give rise to the body of the embtyo. The trophoblastic cells wi ll eventually give rise to the chorion. The chorion will become the fetal component of the placenta that will be described later. As the blastocyst continues to undergo mitosis, fluid continues to fill the blastocoele and the pressure within the embryo increases. Concurrent with growth and fluid accumulation is the production of proteolytic enzymes by the trophoblastic cells. These enzymes weaken the zona pellucida so that it ruptures easily as growth of the blastocyst continues. Finally, the blasto- cyst itself begins to contract and relax. Such behavior causes intermittent pressure pulses. These pressure pulses coupled with continued growth and enzymatic degradation cause the zona pellucida to ruphtre. When a small crack or fissure in the zona pel- lucida develops, the cells of the blastocyst squeeze out of the opening, escaping from their confines (See Figure I 3- I). The blastocyst now becomes a free-floating embtyo within the lumen of the uterus and is totally de- pendent on the uterine environment for survival. In this context, early embtyo survival is dependent on adequate luteal function, adequate progesterone synthesis and responsiveness of the utems to progesterone. Figure 13-3 illustrates the anatomical location of the various preattachment stages of the embtyo. The timing and species variation is presented in Table I 3-1 . Development of the Extraembryonic Membranes Represents an "Explosion" of Embryonic Tissue Growth Prior to Attachment After hatching, the conceptus undergoes mas- sive growth. For example, in the cow at day 13 the blastocyst is about 3 mm in diameter. During the next four days, the cow blastocyst will become 250 mm in length (about the vertical length of the printed portion of thi s page) and will appear as a filamentous thread. By day I 8 of gestation, the blastocyst occupies space in both uterine horns. While the blastocyst of the cow (and the ewe) grows quite rapidly during this early pre- attachment stage, the development of the pig blastocyst is even more dramatic. On day 10 of pregnancy, pig blastocysts are 2 mm spheres. During the next 24 to 48 hours, these 2 mm blastocysts will grow to about 200 mm in length (about the width of the printed portion of this page). This means that the blastocyst is growing at a rate of 4 to 8 mm per hour. By day 16, the pig blastocyst reaches lengths of 800 to I 000 mm. Mammalian embryos can be subdivided into two primary groups. In the first group (that includes most domestic animals), the preattachment period within the uterus is long (several weeks). During this time, extensive extraembtyonic membranes form by a folding process that generates the amnion, chorion and allantochorion. In the second group (primates) the blastocyst implants very soon after it enters the uterus. The extraembryonic membranes fonn after implanta- tion or attachment. In this text, we will deal exclusively with the first group. For details about implantation of the human blastocyst please consult the reference by Larsen in Key References. The extraembry onic membranes of the preattachment embryo consist of the: • yolk sac • chorion • amnion • allantois The dramatic growth of the conceptus is due largely to the development of a set of membranes called the extraembryonic membranes. The pig, sheep and cow are characterized as having filamentous or Ve tB oo ks .ir I ill] 278 Early Embryogenesis and Maternal Recognition of Pregnancy Figure 13-4. Schematic Diagram Illustrating the Typical Development of Extraembryonic Membranes in Mammals (This developmental sequence must occur before attachment to the endometrium can take place) The hatched blastocyst consists of the inner cell mass (ICM), the tro- phoblast and the blastocoele. Very early in embryonic development, the primitive endoderm (blue layer) begins to form beneath the inner cell mass and grows downward form- ing a lining on the inner surface of the trophoblast. At the same time, the mesoderm (red layer) begins to develop between the primitive endoderm and the embryo. Trophoblast Mesoderm Primitive Endoderm Trophoblast Chorion The mesoderm now completely surrounds the yolk sac and the developing allantois. The al- lantois is a diverticulum from the primitive gut that collects embryonic wastes. The meso- derm continues to fuse with the cells of the trophectoderm to form the chorion. The amnionic folds continue to grow upward around the embryo. When the primitive endo- derm completes its growth, it forms a cavity called a yolk sac. This cav ity does not contain yolk but is so named because it is analogous to the yolk sac in avian embryos. The mesoderm continues to grow, forming a sac that surrounds the yolk sac and pushes against the trophectoderm (previously the tro- phoblastic cells). The newly formed mesodermal sac pushes against the trophectoderm and begins to fold upward forming "wing-like" structures called amnionic folds. Primitive Endoderm Trophectoderm Mesoderm Primitive Gut The yolk sac begins tore- gress but the allantois con- tinues to grow and expand. The amnionic folds almost completely surround the embryo. The leading edg- es of the amnionic folds will eventually fuse. .. I The amnionic folds have completely fused resulting in the formation of a double sac around the embryo. The inner sac con- sists of troph ectoderm and mesoderm and is called the amnion. It creates the amnionic cavity. The chorion completely surrounds the entire conceptus. The al- lantois continues to expand and begins to fill-in the spaces of the cavity. Eventu- ally, the allantois and the chorion will fuse forming the allantochorion. The yolk sac continues to regress. Early Embryogenesis and Maternal Recognition of Pregnancy 279 threadlike blastocysts prior to attachment. In the mare, however, blastocysts do not change into a threadlike structm e but remain spherical. Formation of the extraembryonic membranes is an obligatory step in the acquisition ofthe embryo's ability to attach to the uterus of the dam. The extra- embryonic membranes are a set of four anatomically distinct membranes that originate from the trophoblast, endodenn, mesoderm and the embryo. The trophoblast, along with the primitive en- doderm and mesoderm, give rise to the chorion and the amnion (See Figure 13-4). The yolk sac develops from the primitive endodenn. The chorion will eventu- ally attach to the uterus, while the amnion will provide a fluid-filled protective sac for the developing fetus. As the hatched blastocyst begins to grow, it develops an additional layer just beneath, but in contact with the inner cell mass. This layer of cells is called the primitive endoderm (See Figure 13-4) and will continue to grow in a downward direction, eventually lining the trophoblast. At the same time the primitive endoderm is growing to become the inside lining of the trophoblast, it also fonns an evagination at the ventral portion of the inner cell mass. This evagination forms the yolk sac (See Figure 13-4). The yolk sac in domestic animal embryos is a transient extraembryonic membrane that regresses in size as the conceptus develops. In spite of its regression, you will recall (See Chapter 4) that the yolk sac conh·ibutes the primitive genn cells that migrate to the genital ridge. As the blastocyst continues to expand, the newly formed double membrane (the trophoblast and mesodenn ) becomes the chorion. As it develops, the chorion pushes upward in the dorsolateral region of the conceptus and begins to surround it. As the chorion be- gins to send "wing-like" projections above the embryo, the amnion begins to fmm (See Figure 13-4 ). When the chorion fuses over the dorsal portion ofthe embryo, it then forms a complete sac around the embryo. This sac is the amnion. The amnion is filled with fluid and serves to hydraulically protect the embryo from me- chanical perturbations. The amnionic fluid serves as an anti-adhesion material to prevent tissues in the rapidly developing embryo from adhering to each other. The amnionic vesicle can be palpated in the cow between days 30 and 45 and feels like a small, turgid balloon inside the uterus. The embryo, however, is quite fragile during this early period and amnionic vesicle palpation should be performed with caution. During the same time that the amnion is developing, a small evagination from the posterior region of the primitive gut begins to form (See Figure 13-4 ). This sac-like evagination is referred to as the aiJantois. The allantois is a fluid-filled sac that collects liquid waste from the embryo. As the embryo grows, the allantois continues to expand and eventually will make contact with the chorion. When the allantois reaches a certain volume, it presses against the chorion and eventually fuses with it. When fusion takes place the two membranes are called the aiJantochorion (See Figure 13-4 ). The allantochorionic membrane is the fetal conh·ibution to the placenta and will provide the surface for attachments to the endometrium. Details about the anatomy and function of the placenta will be presented in Chapter 14. In most species, the conceptus must p rovide a timely biochemical signal or the pregnancy will terminate. In order for the events of early embryogenesis to continue into an established pregnancy, luteolysis must be prevented. Progesterone must be maintained at sufficiently high levels so that embryogenesis and attachment of the developing conceptus to the endo- metrium can take place. The embryo enters the uterus between days 2 and 5 after ovulation (See Table 13-1 and F igure 13-3). The critical series of events by which the conceptus initially signals its presence to the dam and enables pregnancy to continue is referred to as maternal r·ecognition of pregnancy. If an adequate signal is not delivered in a timely manner, the dam will experience luteolysis, progesterone concenh·ations will decline and pregnancy will be tenninated. Recognition factors as they relate to the critical recognition period are presented in Table 13-2. Maternal recognition of pregnancy must occur prior to luteolysis. Recall from Chapter 9 that the corpus luteum of ruminants produces oxytocin that stimulates endo- metrial cells to synthesize PGF2a . The production of PGF2a is dependent upon a threshold number of oxytocin receptors that are synthesized by endometrial cells at a critical time during the estrous cycle. When these receptors are available in sufficient numbers, pulsatile secretion ofPGF2a occurs in response to lutea l oxytocin secretion and luteolysis follows (See Figure 13-5). Clearly, this mechanism must be prevented if a successful pregnancy is to proceed. Ve tB oo ks .ir I ill] 278 Early Embryogenesis and Maternal Recognition of Pregnancy Figure 13-4. Schematic Diagram Illustrating the Typical Development of Extraembryonic Membranes in Mammals (This developmental sequence must occur before attachment to the endometrium can take place) The hatched blastocyst consists of the inner cell mass (ICM), the tro- phoblast and the blastocoele. Very early in embryonic development, the primitive endoderm (blue layer) begins to form beneath the inner cell mass and grows downward form- ing a lining on the inner surface of the trophoblast. At the same time, the mesoderm (red layer) begins to develop between the primitive endoderm and the embryo. Trophoblast Mesoderm Primitive Endoderm Trophoblast Chorion The mesoderm now completely surrounds the yolk sac and the developing allantois. The al- lantois is a diverticulum from the primitive gut that collects embryonic wastes. The meso- derm continues to fuse with the cells of the trophectoderm to form the chorion. The amnionic folds continue to grow upward around the embryo. When the primitive endo- derm completes its growth, it forms a cavity called a yolk sac. This cav ity does not contain yolk but is so named because it is analogous to the yolk sac in avian embryos. The mesoderm continues to grow, forming a sac that surrounds the yolk sac and pushes against the trophectoderm (previously the tro- phoblastic cells). The newly formed mesodermal sac pushes against the trophectoderm and begins to fold upward forming "wing-like" structures called amnionic folds. Primitive Endoderm Trophectoderm Mesoderm Primitive Gut The yolk sac begins tore- gress but the allantois con- tinues to grow and expand. The amnionic folds almost completely surround the embryo. The leading edg- es of the amnionic folds will eventually fuse. .. I The amnionic folds have completely fused resulting in the formation of a double sac around the embryo. The inner sac con- sists of troph ectoderm and mesoderm and is called the amnion. It creates the amnionic cavity. The chorion completely surrounds the entire conceptus. The al- lantois continues to expand and begins to fill-in the spaces of the cavity. Eventu- ally, the allantois and the chorion will fuse forming the allantochorion. The yolk sac continues to regress. Early Embryogenesis and Maternal Recognition of Pregnancy 279 threadlike blastocysts prior to attachment. In the mare, however, blastocysts do not change into a threadlike structm e but remain spherical. Formation of the extraembryonic membranes is an obligatory step in the acquisition ofthe embryo's ability to attach to the uterus of the dam. The extra- embryonic membranes are a set of four anatomically distinct membranes that originate from the trophoblast, endodenn, mesoderm and the embryo. The trophoblast, along with the primitive en- doderm and mesoderm, give rise to the chorion and the amnion (See Figure 13-4). The yolk sac develops from the primitive endodenn. The chorion will eventu- ally attach to the uterus, while the amnion will provide a fluid-filled protective sac for the developing fetus. As the hatched blastocyst begins to grow, it develops an additional layer just beneath, but in contact with the inner cell mass. This layer of cells is called the primitive endoderm (See Figure 13-4) and will continue to grow in a downward direction, eventually lining the trophoblast. At the same time the primitive endoderm is growing to become the inside lining of the trophoblast, it also fonns an evagination at the ventral portion of the inner cell mass. This evagination forms the yolk sac (See Figure 13-4). The yolk sac in domestic animal embryos is a transient extraembryonic membrane that regresses in size as the conceptus develops. In spite of its regression, you will recall (See Chapter 4) that the yolk sac conh·ibutes the primitive genn cells that migrate to the genital ridge. As the blastocyst continues to expand, the newly formed double membrane (the trophoblast and mesodenn ) becomes the chorion. As it develops, the chorion pushes upward in the dorsolateral region of the conceptus and begins to surround it. As the chorion be- gins to send "wing-like" projections above the embryo, the amnion begins to fmm (See Figure 13-4 ). When the chorion fuses over the dorsal portion ofthe embryo, it then forms a complete sac around the embryo. This sac is the amnion. The amnion is filled with fluid and serves to hydraulically protect the embryo from me- chanical perturbations. The amnionic fluid serves as an anti-adhesion material to prevent tissues in the rapidly developing embryo from adhering to each other. The amnionic vesicle can be palpated in the cow between days 30 and 45 and feels like a small, turgid balloon inside the uterus. The embryo, however, is quite fragile during this early period and amnionic vesicle palpation should be performed with caution. During the same time that the amnion is developing, a small evagination from the posterior region of the primitive gut begins to form (See Figure 13-4 ). This sac-like evagination is referred to as the aiJantois. The allantois is a fluid-filled sac that collects liquid waste from the embryo. As the embryo grows, the allantois continues to expand and eventually will make contact with the chorion. When the allantois reaches a certain volume, it presses against the chorion and eventually fuses with it. When fusion takes place the two membranes are called the aiJantochorion (See Figure 13-4 ). The allantochorionic membrane is the fetal conh·ibution to the placenta and will provide the surface for attachments to the endometrium. Details about the anatomy and function of the placenta will be presented in Chapter 14. In most species, the conceptus must p rovide a timely biochemical signal or the pregnancy will terminate. In order for the events of early embryogenesis to continue into an established pregnancy, luteolysis must be prevented. Progesterone must be maintained at sufficiently high levels so that embryogenesis and attachment of the developing conceptus to the endo- metrium can take place. The embryo enters the uterus between days 2 and 5 after ovulation (See Table 13-1 and F igure 13-3). The critical series of events by which the conceptus initially signals its presence to the dam and enables pregnancy to continue is referred to as maternal r·ecognition of pregnancy. If an adequate signal is not delivered in a timely manner, the dam will experience luteolysis, progesterone concenh·ations will decline and pregnancy will be tenninated. Recognition factors as they relate to the critical recognition period are presented in Table 13-2. Maternal recognition of pregnancy must occur prior to luteolysis. Recall from Chapter 9 that the corpus luteum of ruminants produces oxytocin that stimulates endo- metrial cells to synthesize PGF2a . The production of PGF2a is dependent upon a threshold number of oxytocin receptors that are synthesized by endometrial cells at a critical time during the estrous cycle. When these receptors are available in sufficient numbers, pulsatile secretion ofPGF2a occurs in response to lutea l oxytocin secretion and luteolysis follows (See Figure 13-5). Clearly, this mechanism must be prevented if a successful pregnancy is to proceed. Ve tB oo ks .ir 280 Early Embryogenesis and Maternal Recognition of Pregnancy Figure 13-5. IFN-'t From the Conceptus Prevents Luteolysis in the Cow and Ewe Blastocyst Uterine tissue (uterus) to maternal blood Blastocyst Uterine gland J IFN-1: is secreted by the trophoblastic cells of the blastocyst (cow and ewe). IFN-1: acts on the endometrial cells of the uterus to inhibit the production of oxytocin receptors so that oxytocin cannot stimulate PGF2u syn- thesis. In addition, IFN-1: causes secretion of proteins from the uterine glands. The arrows from the uterine glands indicate the movement of products that are secreted into the uterine lumen to nourish the conceptus. Finally, IFN-1: can leave the uterus via the uterine vein to affect the ovary and circulating immune cells. In the ewe and cow, the blastocyst secretes materials that block the synthesis of uterine oxytocin receptors. ln the ewe and the cow the free-floating blasto- cyst produces specific proteins that provide the signal for prevention ofluteolysis. The specific proteins were once called ovine trophoblastic pt·otein 1 ( oTP-1) and bovine trophoblastic protein 1 (bTP- I). Both of these proteins belong to a class of materials known as interferons. Interferons are cytokines (immune cell hormones) secreted by many cell types, including leukocytes, fibroblasts, lymphocytes, and trophoblastic cells that are best known for their ability to inhibit virus replication. Because h·ophoblastic proteins ( oTP-1 and bTP-l) constitute a separate class of interferons, they are now referred to as ovine Interferon 1: (oiFN-1:) and bovine Inte.-feron 1: (biFN-1:). The use of the Greek letter r designates the trophoblastic origin of these proteins. A relatively small protein (18 ,000 to 20,000 daltons), olFN-1: is produced by the trophoblastic cells of the blastocyst and is present in the uterus from about day 13 to 21 after ovulation. Secretion of progesterone by the corpus luteum is not be enhanced by oiFN-1: and Early Embryogenesis and Maternal Recognition of Pregnancy 281 Figure 13-6. Estradiol Reroutes PGF2a to Prevent Luteolysis in the Sow Non-pregnant cycling sow {endocrine secretion of PGF2a) ' ' t Pregnant sow {exocrine secretion of PGF2cr) Blastocyst Oxytocin ... ' ' , \ , ' , ' , ' I ' , '. (CL CL CL CL I Luteolysis I In the non-pregnant sow, oxytocin from the endometrium, poste rior pituitary lobe and CL promotes PGF2" synthesis by the uterine e n- dometrium. PGF2a diffuses by conce ntration gradient towards the endometrial capillarie s where it drains into the uterine vein, is trans- ported to the ovary and caus es luteolysis . therefore it is not luteotrophic. Instead, oiFN-1: binds to the endometrium and inhibits oxytocin receptor synthe- sis by endometrial cells. Figure I 3-5 summarizes the proposed effect of oiFN-1: and blFN-1: on endometrial production of oxytocin receptors. In addition to block- ing oxytocin receptor synthesis, IFN-1: also binds to the apical portion (See Figure 13-5) of the uterine glands and promotes protein synthesis believed to be critical to preimplantation emb1y onic survival. Ongoing research suggests that IFN-T stimu- lates circulating inmmne cells of the dam to produce a family of proteins involved in immune response to invading viral pathogens. The presence of these blood proteins at days I 7-20 after insemination indicates that In the pregnant sow, the blastocyst produces estra- diol that causes the PGF2" to be rerouted into the ute rine lumen , where it is destroyed, thus prevent- ing luteolysis. Like the cycling cow, oxytocin is a lso produced by the CL and pos te rior pituitary lobe in the pregna nt sow. a concephls is present in the uterus. Females that do not show elevated levels of these blood proteins at days 17-20 would not be pregnant. Therefore, the absence of IFN-1: induced blood proteins has potential for identify- ing non-pregnant cows. Identification of non-pregnant cows at days 17-20 would allow earlier re-insemination of open cows to achieve a pregnancy sooner than pos- sible using other cuiTently available diagnostic tests. It should be emphasized that identification of JFN-1: induced blood proteins is not a pregnancy test. An early pregnancy test (day 17-20) would be oflittle value because a significant proportion (20-40%) of day I 7 emb1yos would fail to survive until term. Ve tB oo ks .ir 280 Early Embryogenesis and Maternal Recognition of Pregnancy Figure 13-5. IFN-'t From the Conceptus Prevents Luteolysis in the Cow and Ewe Blastocyst Uterine tissue (uterus) to maternal blood Blastocyst Uterine gland J IFN-1: is secreted by the trophoblastic cells of the blastocyst (cow and ewe). IFN-1: acts on the endometrial cells of the uterus to inhibit the production of oxytocin receptors so that oxytocin cannot stimulate PGF2u syn- thesis. In addition, IFN-1: causes secretion of proteins from the uterine glands. The arrows from the uterine glands indicate the movement of products that are secreted into the uterine lumen to nourish the conceptus. Finally, IFN-1: can leave the uterus via the uterine vein to affect the ovary and circulating immune cells. In the ewe and cow, the blastocyst secretes materials that block the synthesis of uterine oxytocin receptors. ln the ewe and the cow the free-floating blasto- cyst produces specific proteins that provide the signal for prevention ofluteolysis. The specific proteins were once called ovine trophoblastic pt·otein 1 ( oTP-1) and bovine trophoblastic protein 1 (bTP- I). Both of these proteins belong to a class of materials known as interferons. Interferons are cytokines (immune cell hormones) secreted by many cell types, including leukocytes, fibroblasts, lymphocytes, and trophoblastic cells that are best known for their ability to inhibit virus replication. Because h·ophoblastic proteins ( oTP-1 and bTP-l) constitute a separate class of interferons, they are now referred to as ovine Interferon 1: (oiFN-1:) and bovine Inte.-feron 1: (biFN-1:). The use of the Greek letter r designates the trophoblastic origin of these proteins. A relatively small protein (18 ,000 to 20,000 daltons), olFN-1: is produced by the trophoblastic cells of the blastocyst and is present in the uterus from about day 13 to 21 after ovulation. Secretion of progesterone by the corpus luteum is not be enhanced by oiFN-1: and Early Embryogenesis and Maternal Recognition of Pregnancy 281 Figure 13-6. Estradiol Reroutes PGF2a to Prevent Luteolysis in the Sow Non-pregnant cycling sow {endocrine secretion of PGF2a) ' ' t Pregnant sow {exocrine secretion of PGF2cr) Blastocyst Oxytocin ... ' ' , \ , ' , ' , ' I ' , '. (CL CL CL CL I Luteolysis I In the non-pregnant sow, oxytocin from the endometrium, poste rior pituitary lobe and CL promotes PGF2" synthesis by the uterine e n- dometrium. PGF2a diffuses by conce ntration gradient towards the endometrial capillarie s where it drains into the uterine vein, is trans- ported to the ovary and caus es luteolysis . therefore it is not luteotrophic. Instead, oiFN-1: binds to the endometrium and inhibits oxytocin receptor synthe- sis by endometrial cells. Figure I 3-5 summarizes the proposed effect of oiFN-1: and blFN-1: on endometrial production of oxytocin receptors. In addition to block- ing oxytocin receptor synthesis, IFN-1: also binds to the apical portion (See Figure 13-5) of the uterine glands and promotes protein synthesis believed to be critical to preimplantation emb1y onic survival. Ongoing research suggests that IFN-T stimu- lates circulating inmmne cells of the dam to produce a family of proteins involved in immune response to invading viral pathogens. The presence of these blood proteins at days I 7-20 after insemination indicates that In the pregnant sow, the blastocyst produces estra- diol that causes the PGF2" to be rerouted into the ute rine lumen , where it is destroyed, thus prevent- ing luteolysis. Like the cycling cow, oxytocin is a lso produced by the CL and pos te rior pituitary lobe in the pregna nt sow. a concephls is present in the uterus. Females that do not show elevated levels of these blood proteins at days 17-20 would not be pregnant. Therefore, the absence of IFN-1: induced blood proteins has potential for identify- ing non-pregnant cows. Identification of non-pregnant cows at days 17-20 would allow earlier re-insemination of open cows to achieve a pregnancy sooner than pos- sible using other cuiTently available diagnostic tests. It should be emphasized that identification of JFN-1: induced blood proteins is not a pregnancy test. An early pregnancy test (day 17-20) would be oflittle value because a significant proportion (20-40%) of day I 7 emb1yos would fail to survive until term. Ve tB oo ks .ir 282 Early Embryogenesis and Maternal Recognition of Pregnancy Figure 13-7. Transuterine Migration of the Equine Conceptus Oviduct Oviduct . . ; · Ovary Uterus • \ r Ovary Cervix Each black sphere represents a "stopping spot" in which the conceptus will spend between 5 and 20 minutes. The migration of the conceptus probably distributes pregnancy factors (white lines) over a wide surface of the endometrium. In the sow, estradiol reroutes PGF2a secreted by the endometrium. In the sow, two major differences exist in maternal recognition of pregnancy, compared to the ewe and cow. First, the concephts of the pig produces estradiol that serves as the signal for maternal recog- nition of pregnancy. Second, PGF2a is produced in significant quantities, but is rerouted into the uterine lumen. The conceptus begins to secrete estradiol This uterus is from a mare at day 14 of pregnancy. The uterus has been incised on the dorsal surface to expose the spherical conceptus (C). This speci- men shows the conceptus and uterus on the last day (day 14) of the uterine migration phenomenon. (Photograph courtesy of Dr. O.J. Ginther, Reproductive Biology of/he Mare) between days II and 12 after ovulation. The production of estrogen does not inhibit the production ofPGF2m but causes the PGF2a to be secreted in a different direction than in the cycling sow. The direction of secretion is away from the submucosal capillaries and toward the uterine lumen. Luminal PGF2a has little access to the circulation and thus cannot cause luteolysis. The pre- cise mechanism whereby the rerouting ofPGF2a occurs is not completely understood. However, it is believed that estrogen causes increased receptor production for prolactin in the endometrium. Prolactin changes the ionic flux for calcium. This is thought to promote the Table 13-2. Pregnancy recognition factors, critical days of pregnancy recogni tion and time of conceptus attachment in mammals SQecies Pregnancy Critical Period Time of Recognition Factors for Recognition Attachment (days after ovulation) (days after ovulation) Bitch none needed Cow biFN-1: (bTP-1) 15-16 18-22 Ewe oiFN-1: (oTP-1) 13-14 15-18 Mare 3 Proteins/Estrogens = ? 12-14 36-38 Queen none needed Sow Estradiol (E2) 11-12 14-18 Woman hCG 7-12 9-12 Early Embryogenesis and Maternal Recognition of Pregnancy 283 e .. .e: u. "' a. ., 0 0 iii Figure 13-8. Maternal Recognition Must Occur Prior to Luteolysis 350 300· 250 · 200 ISO 100 50 ' c ·3 £ c B c 8 u c 13 p. 14 Lutco lysis PGF2a 15 16 17 D-.y of estrous cycle Critical period Conceptus d.tm orlts presence 18 PGF2a 8 -7 - 6 5 - 4 - 2 19 f .. .:. u c e u .. e Q. ., 0 0 iii 20 13 16 17 19 20 O;ay of estrous cycle Comparison between the endocrine condition of the female (timing shown here is for the cow) with no conceptus present and with conceptus present. Notice that in the pregnant animal (conceptus pres- ent), episodes of PGF2u that cause luteolysis do not occur. These are blocked because endometrial oxytocin receptor synthesis is blocked. This is cal led maternal recognition. Maternal recognition must oc- cur prior to the onset of luteolysis if the pregnancy is to be maintained. exocrine secretion of PGF2a (into the uterine lumen) rather than an endocrine secretion (into the uterine vas- culature). Porcine conceptuses produce intetferons, but these materials do not affect corpora lutea longevity or function. Production ofE2 by the porcine conceptus not only serves as the matemal signal to prevent luteolysis, but also probably serves to stimulate contractions of the myometrium to distribute conceptuses with the proper spacing along the uterine horn. Another important feature of maternal recog- nition of pregnancy in the sow is that there must be at least two concephtses present in each uterine horn for pregnancy to be maintained. If conceptuses are not present in one uterine horn, PGF2a will be secreted in an endocrine fashion, luteolysis will occur and the pregnancy will be tenn inated. Figure 13-6 summarizes the proposed mechanism for matemal recognition of pregnancy in the sow. The equine conceptus must make extensive contact with the endometrial smface to initiate and complete maternal recognition of pregnancy . In the mare, the presence of the conceptus prevents luteolysis. Also, in the presence of the con- ceptus, endometrial production of PGF2a is significantly reduced. A unique feature of matemal recognition of pregnancy in the mare is that the conceptus is translo- cated over the endometrial surface by uterine contrac- tions. The conceph1s is moved from one uterine horn to the other. This movement must occur between 12 and 14 times per day during days 12, 13 and 14 of preg- nancy in order to inhibit PGF2a (See Figure 13-7). The intrauterine movement of the equine conceptus appears necessary because the conceptus does not elongate as in other species. Therefore, there is less contact between the conceptus and the endometrial surface . In other words, the movement of the conceptus is probably necessary to distribute pregnancy recognition factors to the endometrial cells. Like the other species, the conceph1s of the horse produces proteins that apparently have some ef- fect on the recognition of pregnancy (See Table 13-2). However, the specific roles are yet unknown. In the woman, maternal recognition of pregnancy is provided by a hormone called human chorionic gonadotropin (hCG). At about the time of implantation (day 7-9 after ovulation) the human concephts begins to secrete a hormone called human chorionic gonadotropin (hCG). This is an LH-like hormone that acts on the corpus luteum to inhibit intraovarian luteolysis (See Chapter 9). The precise mechanism whereby hCG blocks luteolysis is not known. Regardless, the luteotrophic effect ofhCG is sufficient to allow for implantation and maintenance of pregnancy. Maternal recognition of pregnancy in the dog and the cat probably does not require a signal from the conceptus. Ve tB oo ks .ir 282 Early Embryogenesis and Maternal Recognition of Pregnancy Figure 13-7. Transuterine Migration of the Equine Conceptus Oviduct Oviduct . . ; · Ovary Uterus • \ r Ovary Cervix Each black sphere represents a "stopping spot" in which the conceptus will spend between 5 and 20 minutes. The migration of the conceptus probably distributes pregnancy factors (white lines) over a wide surface of the endometrium. In the sow, estradiol reroutes PGF2a secreted by the endometrium. In the sow, two major differences exist in maternal recognition of pregnancy, compared to the ewe and cow. First, the concephts of the pig produces estradiol that serves as the signal for maternal recog- nition of pregnancy. Second, PGF2a is produced in significant quantities, but is rerouted into the uterine lumen. The conceptus begins to secrete estradiol This uterus is from a mare at day 14 of pregnancy. The uterus has been incised on the dorsal surface to expose the spherical conceptus (C). This speci- men shows the conceptus and uterus on the last day (day 14) of the uterine migration phenomenon. (Photograph courtesy of Dr. O.J. Ginther, Reproductive Biology of/he Mare) between days II and 12 after ovulation. The production of estrogen does not inhibit the production ofPGF2m but causes the PGF2a to be secreted in a different direction than in the cycling sow. The direction of secretion is away from the submucosal capillaries and toward the uterine lumen. Luminal PGF2a has little access to the circulation and thus cannot cause luteolysis. The pre- cise mechanism whereby the rerouting ofPGF2a occurs is not completely understood. However, it is believed that estrogen causes increased receptor production for prolactin in the endometrium. Prolactin changes the ionic flux for calcium. This is thought to promote the Table 13-2. Pregnancy recognition factors, critical days of pregnancy recogni tion and time of conceptus attachment in mammals SQecies Pregnancy Critical Period Time of Recognition Factors for Recognition Attachment (days after ovulation) (days after ovulation) Bitch none needed Cow biFN-1: (bTP-1) 15-16 18-22 Ewe oiFN-1: (oTP-1) 13-14 15-18 Mare 3 Proteins/Estrogens = ? 12-14 36-38 Queen none needed Sow Estradiol (E2) 11-12 14-18 Woman hCG 7-12 9-12 Early Embryogenesis and Maternal Recognition of Pregnancy 283 e .. .e: u. "' a. ., 0 0 iii Figure 13-8. Maternal Recognition Must Occur Prior to Luteolysis 350 300· 250 · 200 ISO 100 50 ' c ·3 £ c B c 8 u c 13 p. 14 Lutco lysis PGF2a 15 16 17 D-.y of estrous cycle Critical period Conceptus d.tm orlts presence 18 PGF2a 8 -7 - 6 5 - 4 - 2 19 f .. .:. u c e u .. e Q. ., 0 0 iii 20 13 16 17 19 20 O;ay of estrous cycle Comparison between the endocrine condition of the female (timing shown here is for the cow) with no conceptus present and with conceptus present. Notice that in the pregnant animal (conceptus pres- ent), episodes of PGF2u that cause luteolysis do not occur. These are blocked because endometrial oxytocin receptor synthesis is blocked. This is cal led maternal recognition. Maternal recognition must oc- cur prior to the onset of luteolysis if the pregnancy is to be maintained. exocrine secretion of PGF2a (into the uterine lumen) rather than an endocrine secretion (into the uterine vas- culature). Porcine conceptuses produce intetferons, but these materials do not affect corpora lutea longevity or function. Production ofE2 by the porcine conceptus not only serves as the matemal signal to prevent luteolysis, but also probably serves to stimulate contractions of the myometrium to distribute conceptuses with the proper spacing along the uterine horn. Another important feature of maternal recog- nition of pregnancy in the sow is that there must be at least two concephtses present in each uterine horn for pregnancy to be maintained. If conceptuses are not present in one uterine horn, PGF2a will be secreted in an endocrine fashion, luteolysis will occur and the pregnancy will be tenn inated. Figure 13-6 summarizes the proposed mechanism for matemal recognition of pregnancy in the sow. The equine conceptus must make extensive contact with the endometrial smface to initiate and complete maternal recognition of pregnancy . In the mare, the presence of the conceptus prevents luteolysis. Also, in the presence of the con- ceptus, endometrial production of PGF2a is significantly reduced. A unique feature of matemal recognition of pregnancy in the mare is that the conceptus is translo- cated over the endometrial surface by uterine contrac- tions. The conceph1s is moved from one uterine horn to the other. This movement must occur between 12 and 14 times per day during days 12, 13 and 14 of preg- nancy in order to inhibit PGF2a (See Figure 13-7). The intrauterine movement of the equine conceptus appears necessary because the conceptus does not elongate as in other species. Therefore, there is less contact between the conceptus and the endometrial surface . In other words, the movement of the conceptus is probably necessary to distribute pregnancy recognition factors to the endometrial cells. Like the other species, the conceph1s of the horse produces proteins that apparently have some ef- fect on the recognition of pregnancy (See Table 13-2). However, the specific roles are yet unknown. In the woman, maternal recognition of pregnancy is provided by a hormone called human chorionic gonadotropin (hCG). At about the time of implantation (day 7-9 after ovulation) the human concephts begins to secrete a hormone called human chorionic gonadotropin (hCG). This is an LH-like hormone that acts on the corpus luteum to inhibit intraovarian luteolysis (See Chapter 9). The precise mechanism whereby hCG blocks luteolysis is not known. Regardless, the luteotrophic effect ofhCG is sufficient to allow for implantation and maintenance of pregnancy. Maternal recognition of pregnancy in the dog and the cat probably does not require a signal from the conceptus. Ve tB oo ks .ir I '@] 284 Early Embryogenesis and Maternal Recognition of Pregnancy In the bitch, the CL of pregnancy and the CL of the cycle have similar lifespans. Therefore, under nom1al cyclic conditions, the CL is long-lived. When luteolysis does occur it is near the end of the nonnal gestation period. In other words, the period of diestrus is quite similar to the gestation period and thus, the corpus luteum is not lysed under normal conditions until the gestation period is complete. As you recall, the queen is an induced ovulator. If mating does not occur, corpora lutea are not formed and a "post estrous" period of several days (8-l 0) exists before another estrus. In the queen that has been bred, a CL forms and the duration is the same as gestation (about 60 days). Like the bitch, a signal from the con- ceptus is not needed because corpora lutea are not lysed before a pregnancy is established. Please see Chapter 7 for graphic illustrations of this concept. A successful pregnancy requires maintenance of high blood progesterone concentrations. Regardless of whether or not specific preg- nancy recognition signals are provided, progesterone concentrations in the blood of the dam must be main- tained at sufficiently high concentrations so that the conceptus will grow and develop. The extraembryonic membranes will form an attachment with the endome- trium to provide a semipermanent link between the dam and the fetus. This semipennanent linlc is known as the placenta and will be discussed in the next chapter. Embryo Transfer Technology Provides Avenues for Reproductive and Genetic Enhancement Embryo transfer requires a set of procedures that allows removal of pre-attachment embryos from the reproductive tract of a donor female and transfers them into the reproductive tract of a recipient female. Embryo transfer is a valuable production and research technique. It is commercially available in some species to increase the productivity of females with desired traits. The first successful embryo transfer procedure was performed in a rabbit in 1 890. Since that time embryo transfer techniques have been used in many species and countless offspring have been produced using this technique. In principle, emb1yo transfer can be perfonned in any mammalian species. How- ever, its widest application is in cattle and more em- bryos are transferred in this species per year than in all other species combined. The main advantage of emb1yo transfer in cattle is to amplify the number of offspring that donor females with desired genetic traits can pro- duce. With embryo transfer, a single donor cow is ca- pable of producing 10 to 20 offspring annually. Embryo transfer has been a contributor to assisted reproductive technology in humans. Human embryos derived from in vitro fertilization currently exceed 100,000 on a worldwide basis. Futhermore, .embryo transfer is an important technique used to enhance reproduction in endangered species. The advantages of embryo transfer are: • circumvention of seasonal reproduction • enhanced generation of offspl'ing in monotocous species • assisted reproduction for infertility in humans • enhanced reproductive potential of endangered species • enhanced genetic diversity across a wide geographical region (ship embryos rather than animals) A major advantage of embryo transfer is the ability to transport germ plasm from one geographical area to another. For example, embryos collected in North America can be shipped to any country in the world. This is particularly important in large animals (cows, horses, exotic species) because transportation of the animal over long distances is inefflcient, expensive and can transmit diseases. Embryo transfer offers sig- nificant biosecurity advantages over animal transport. In addition to the above contributions, emb1yo transfer is an essential step in many experimental techniques in the production of clones and transgenic animals. Successji1l embryo transfer involves: • synchronizing the cycles of donors and recipients • superovulation (hyperstimulation of the ovaries) of the donor • artificial insemination of the donor female • recovery of embryos from the donor • maintenance of viable embryos in vitro • transfer of embryos to recipient females ------------------------........... Early Embryogenesis and Maternal Recognition of Pregnancy 285 Synchronization of Donor and Recipient Cycles is Obligatory for Successful Embryo Transfer In order for emb1yos from the donor to develop within the recipient, the stage of the donor's cycle must be coincident with that of the recipient (See Figure I 3- 9). For example, if a 7-day embryo is to be transferred into a recipient, she must be in the seventh day of her estrous cycle. This allows for the appropriate uterine environment, maternal recognition of pregnancy and establishment of appropriate embryonic development and attachment to the uterus. Methods for synchroniza- tion of estrous are presented in Chapter 9. Superovulation Results from Hyperstimulation of the Ovaries with Gonadotropins Superovulation is the treatment of a female with gonadotropins (typically FSH) to increase the number of oocytes that are selected to domi- nant follicles and to ovulate (See Figure 13-9). Among monotocous animals, superovulation is used to increase the number of potential offspring from donor females possessing traits of high economic value. Superovu- lation is also used in humans (even though only one offspring is usually des ired) to compensate for low success rates with a single embryo transfer. In monotocous species, ovulation rates of 5- l 0 times normal occur. In polytocous species, ovulation rates of only 2-3 times normal are achieved. There is a wide variation in the individual's response to gonadotro- pin stimulation. Because a commercial embryo transfer indusny exists in cattle, there are significant data avail- able describing this variation. For example, a typical response in cattle would be 8 to I 0 ovulations, produc- ing 5 to 7 viable embtyos. But, about 30% of the cows respond by producing one or fewer viable embryos. About 2% of the cows may produce as many as 30 emb1yos or more. The physiologic reasons for this wide variation in ovarian response to hyperstimulation are not known. Recovery of oocy tes from ovaries can be accomplished by: • surgically exposing the ovary and aspirating follicles • non-surgically aspirating follicles utilizing ultrasonography • aspirating follicles postmortem in an abattoir Recovery of Embryos from the Donor Females may be Accomplished in Several Ways Most fi·equently, donor females are bred uti- lizing artificial insemination with semen fi·om a male possessing highly desired traits. After insemination emb1yos can be recovered by a variety of methods. ' Recovery of embryos from the oviduct requires surge1y in all species. Recovery of embryos from the uterus is accompl ished surgically in small species and non-surgically in large species. In cows and mares transrectal palpation and introduction of catheters for removal of embryos by flushing with various culture media is a routine procedure (See Figure I 3-9). Oocytes can be recovered directly from the ovary using aspiration with a hypodem1ic needle. In horses and cattle, a conm1on teclmique for recovery of oocytes by aspiration involves inserting a needle through the wall of the vagina and with the use of ul trasonography, identifying dominant foll icles and aspirating the oocytes into a special apparatus (See Figure I 3- I 0). The purpose of folli cular aspiration is to recover oocytes from dominant fo llicles and perform in vitro fertilization (See Figure 13- I 0). In the case of the postmortem recovery, large numbers of ovaries are available fi·om cattle immediately after exsanguination fi·om slaughter fac ilities. Oocytes remain viable for relatively long periods after exsanguination, typically 9-12 hours in most species. Therefore these serve as valuable sources of oocytes for experimental purposes. Even though cows have not received ovarian stimula- tion by gonadotropins numerous antral foll icles are normally present on ovaries and provide a ready source of viable oocytes for in vitro fertilization procedures. Embryo Viability Must be Maintained In Vitro In order for embryos to be transferred success- fully into recipient females they must be stored in an environment that maintains viability. The conditions for maintenance of viable embryos include: maintenance of appropriate temperature (near or at body temperature), exposure to the appropriate ahnospheric environment (5% C02 and 5-8% 0 2), pH slightly above neutral and the absence of microorganisms. A culh1re medium should also contain the appropriate ionic configuration and the appropriate energy sources for metabolism and growth by the young embryo. Embryos can be fi·ozen successfully for long term storage. Ve tB oo ks .ir I '@] 284 Early Embryogenesis and Maternal Recognition of Pregnancy In the bitch, the CL of pregnancy and the CL of the cycle have similar lifespans. Therefore, under nom1al cyclic conditions, the CL is long-lived. When luteolysis does occur it is near the end of the nonnal gestation period. In other words, the period of diestrus is quite similar to the gestation period and thus, the corpus luteum is not lysed under normal conditions until the gestation period is complete. As you recall, the queen is an induced ovulator. If mating does not occur, corpora lutea are not formed and a "post estrous" period of several days (8-l 0) exists before another estrus. In the queen that has been bred, a CL forms and the duration is the same as gestation (about 60 days). Like the bitch, a signal from the con- ceptus is not needed because corpora lutea are not lysed before a pregnancy is established. Please see Chapter 7 for graphic illustrations of this concept. A successful pregnancy requires maintenance of high blood progesterone concentrations. Regardless of whether or not specific preg- nancy recognition signals are provided, progesterone concentrations in the blood of the dam must be main- tained at sufficiently high concentrations so that the conceptus will grow and develop. The extraembryonic membranes will form an attachment with the endome- trium to provide a semipermanent link between the dam and the fetus. This semipennanent linlc is known as the placenta and will be discussed in the next chapter. Embryo Transfer Technology Provides Avenues for Reproductive and Genetic Enhancement Embryo transfer requires a set of procedures that allows removal of pre-attachment embryos from the reproductive tract of a donor female and transfers them into the reproductive tract of a recipient female. Embryo transfer is a valuable production and research technique. It is commercially available in some species to increase the productivity of females with desired traits. The first successful embryo transfer procedure was performed in a rabbit in 1 890. Since that time embryo transfer techniques have been used in many species and countless offspring have been produced using this technique. In principle, emb1yo transfer can be perfonned in any mammalian species. How- ever, its widest application is in cattle and more em- bryos are transferred in this species per year than in all other species combined. The main advantage of emb1yo transfer in cattle is to amplify the number of offspring that donor females with desired genetic traits can pro- duce. With embryo transfer, a single donor cow is ca- pable of producing 10 to 20 offspring annually. Embryo transfer has been a contributor to assisted reproductive technology in humans. Human embryos derived from in vitro fertilization currently exceed 100,000 on a worldwide basis. Futhermore, .embryo transfer is an important technique used to enhance reproduction in endangered species. The advantages of embryo transfer are: • circumvention of seasonal reproduction • enhanced generation of offspl'ing in monotocous species • assisted reproduction for infertility in humans • enhanced reproductive potential of endangered species • enhanced genetic diversity across a wide geographical region (ship embryos rather than animals) A major advantage of embryo transfer is the ability to transport germ plasm from one geographical area to another. For example, embryos collected in North America can be shipped to any country in the world. This is particularly important in large animals (cows, horses, exotic species) because transportation of the animal over long distances is inefflcient, expensive and can transmit diseases. Embryo transfer offers sig- nificant biosecurity advantages over animal transport. In addition to the above contributions, emb1yo transfer is an essential step in many experimental techniques in the production of clones and transgenic animals. Successji1l embryo transfer involves: • synchronizing the cycles of donors and recipients • superovulation (hyperstimulation of the ovaries) of the donor • artificial insemination of the donor female • recovery of embryos from the donor • maintenance of viable embryos in vitro • transfer of embryos to recipient females ------------------------........... Early Embryogenesis and Maternal Recognition of Pregnancy 285 Synchronization of Donor and Recipient Cycles is Obligatory for Successful Embryo Transfer In order for emb1yos from the donor to develop within the recipient, the stage of the donor's cycle must be coincident with that of the recipient (See Figure I 3- 9). For example, if a 7-day embryo is to be transferred into a recipient, she must be in the seventh day of her estrous cycle. This allows for the appropriate uterine environment, maternal recognition of pregnancy and establishment of appropriate embryonic development and attachment to the uterus. Methods for synchroniza- tion of estrous are presented in Chapter 9. Superovulation Results from Hyperstimulation of the Ovaries with Gonadotropins Superovulation is the treatment of a female with gonadotropins (typically FSH) to increase the number of oocytes that are selected to domi- nant follicles and to ovulate (See Figure 13-9). Among monotocous animals, superovulation is used to increase the number of potential offspring from donor females possessing traits of high economic value. Superovu- lation is also used in humans (even though only one offspring is usually des ired) to compensate for low success rates with a single embryo transfer. In monotocous species, ovulation rates of 5- l 0 times normal occur. In polytocous species, ovulation rates of only 2-3 times normal are achieved. There is a wide variation in the individual's response to gonadotro- pin stimulation. Because a commercial embryo transfer indusny exists in cattle, there are significant data avail- able describing this variation. For example, a typical response in cattle would be 8 to I 0 ovulations, produc- ing 5 to 7 viable embtyos. But, about 30% of the cows respond by producing one or fewer viable embryos. About 2% of the cows may produce as many as 30 emb1yos or more. The physiologic reasons for this wide variation in ovarian response to hyperstimulation are not known. Recovery of oocy tes from ovaries can be accomplished by: • surgically exposing the ovary and aspirating follicles • non-surgically aspirating follicles utilizing ultrasonography • aspirating follicles postmortem in an abattoir Recovery of Embryos from the Donor Females may be Accomplished in Several Ways Most fi·equently, donor females are bred uti- lizing artificial insemination with semen fi·om a male possessing highly desired traits. After insemination emb1yos can be recovered by a variety of methods. ' Recovery of embryos from the oviduct requires surge1y in all species. Recovery of embryos from the uterus is accompl ished surgically in small species and non-surgically in large species. In cows and mares transrectal palpation and introduction of catheters for removal of embryos by flushing with various culture media is a routine procedure (See Figure I 3-9). Oocytes can be recovered directly from the ovary using aspiration with a hypodem1ic needle. In horses and cattle, a conm1on teclmique for recovery of oocytes by aspiration involves inserting a needle through the wall of the vagina and with the use of ul trasonography, identifying dominant foll icles and aspirating the oocytes into a special apparatus (See Figure I 3- I 0). The purpose of folli cular aspiration is to recover oocytes from dominant fo llicles and perform in vitro fertilization (See Figure 13- I 0). In the case of the postmortem recovery, large numbers of ovaries are available fi·om cattle immediately after exsanguination fi·om slaughter fac ilities. Oocytes remain viable for relatively long periods after exsanguination, typically 9-12 hours in most species. Therefore these serve as valuable sources of oocytes for experimental purposes. Even though cows have not received ovarian stimula- tion by gonadotropins numerous antral foll icles are normally present on ovaries and provide a ready source of viable oocytes for in vitro fertilization procedures. Embryo Viability Must be Maintained In Vitro In order for embryos to be transferred success- fully into recipient females they must be stored in an environment that maintains viability. The conditions for maintenance of viable embryos include: maintenance of appropriate temperature (near or at body temperature), exposure to the appropriate ahnospheric environment (5% C02 and 5-8% 0 2), pH slightly above neutral and the absence of microorganisms. A culh1re medium should also contain the appropriate ionic configuration and the appropriate energy sources for metabolism and growth by the young embryo. Embryos can be fi·ozen successfully for long term storage. Ve tB oo ks .ir 13 286 Early Embryogenesis and Maternal Recognition of Pregnancy Figure 13-9. Major Steps of Embryo Transfer in Mammals-Cow Model Sychronization of recipients with donor Donor Donor Goal: To synchronize the donor and recipient to be in the same stage of the estrous cycle. Reason: To prepare the uterus of the recipient to support preattachment em- bryogenesis. How: Treat recipient with hormonal regime that induces estrus to occur at the same time as the donor. Goai:To hyperstimulate ovaries with gonadotropins. Reason: To provide higher than normal numbers of follicles that reach domi- nance and ovulate. How: Inject donor with gonadotropins to hyperstimulate foll icu lar development. Generally, FSH (or one of its analogs) is used. Ovary A- Hyperstimulated ovary. There are 9 follicles visible in this ovary. The donor is in estrus. Ovary B- 1 day after estrus. There are 9 corpora hemorrhag ica vi sible on this specimen. Goal: To generate the best fertilization rates and genetic combinations possible. Reason: Enhance rate of genetic progress. How: Utilize highly fertile semen and well- trained, experienced inseminators. AlP = AI Pipette, S = Semen, RO = Right Ovary, LO = Left Ovary, RUH = Right Uterine Horn, LUH = Left Uterine Horn (Ovarian specimens courtesy of Dr. B.R. Lindsey) Early Embryogenesis and Maternal Recognition of Pregnancy 287 Recovery and indentification of viable embryos Donor Goal: To nonsurgically collect (flush) embryos from the donor for transfer. Reason: To recover viable embryos. Retrieval of embryos Foley cathe ter in uterus How: Before the procedure is started a local anesthetic is injected to cause relaxation of the rectum. At day 6-8 a specialized catheter is inserted into the uterus. The catheter has a small balloon that can be inflated to prevent retrograde flow of the flush ing medium. A flush- ing medium is then introduced into the uterus, lavaged and then returned through the catheter to a col lec- tion vessel. The ovary in the photo has ten-7 day CL. (Ovarian specimens courtesy of Dr. B.R. Lindsey) Transfer of viable embryos into synchronized recipients Recipient Pipette containing e mbryo Hand grasping cervix -------' Goal: To deposit a potentially viable embryo into the uterine horn of each recipient. Reason: To achieve pregnancy in each recipient. How: A single embryo is placed into the uterine horn using a transfer pipette. Note that both the donor (step 4) and recipient here have CL at similar stages of leutinization. Thus, the uterine environment in the donor and recipient are quite similar. Ve tB oo ks .ir 13 286 Early Embryogenesis and Maternal Recognition of Pregnancy Figure 13-9. Major Steps of Embryo Transfer in Mammals-Cow Model Sychronization of recipients with donor Donor Donor Goal: To synchronize the donor and recipient to be in the same stage of the estrous cycle. Reason: To prepare the uterus of the recipient to support preattachment em- bryogenesis. How: Treat recipient with hormonal regime that induces estrus to occur at the same time as the donor. Goai:To hyperstimulate ovaries with gonadotropins. Reason: To provide higher than normal numbers of follicles that reach domi- nance and ovulate. How: Inject donor with gonadotropins to hyperstimulate foll icu lar development. Generally, FSH (or one of its analogs) is used. Ovary A- Hyperstimulated ovary. There are 9 follicles visible in this ovary. The donor is in estrus. Ovary B- 1 day after estrus. There are 9 corpora hemorrhag ica vi sible on this specimen. Goal: To generate the best fertilization rates and genetic combinations possible. Reason: Enhance rate of genetic progress. How: Utilize highly fertile semen and well- trained, experienced inseminators. AlP = AI Pipette, S = Semen, RO = Right Ovary, LO = Left Ovary, RUH = Right Uterine Horn, LUH = Left Uterine Horn (Ovarian specimens courtesy of Dr. B.R. Lindsey) Early Embryogenesis and Maternal Recognition of Pregnancy 287 Recovery and indentification of viable embryos Donor Goal: To nonsurgically collect (flush) embryos from the donor for transfer. Reason: To recover viable embryos. Retrieval of embryos Foley cathe ter in uterus How: Before the procedure is started a local anesthetic is injected to cause relaxation of the rectum. At day 6-8 a specialized catheter is inserted into the uterus. The catheter has a small balloon that can be inflated to prevent retrograde flow of the flush ing medium. A flush- ing medium is then introduced into the uterus, lavaged and then returned through the catheter to a col lec- tion vessel. The ovary in the photo has ten-7 day CL. (Ovarian specimens courtesy of Dr. B.R. Lindsey) Transfer of viable embryos into synchronized recipients Recipient Pipette containing e mbryo Hand grasping cervix -------' Goal: To deposit a potentially viable embryo into the uterine horn of each recipient. Reason: To achieve pregnancy in each recipient. How: A single embryo is placed into the uterine horn using a transfer pipette. Note that both the donor (step 4) and recipient here have CL at similar stages of leutinization. Thus, the uterine environment in the donor and recipient are quite similar. Ve tB oo ks .ir , I ' ' 288 Early Embryogenesis and Maternal Recognition of Pregnancy Figure 13-10. Oocyte Collection from Ovarian Follicles for In Vitro Fertilization A hypodermic needle is in- serted into the follicle and the follicular fluid is aspirated and then forcefully returned to the follicle. This is repeated 2-3 times to dislodge the oocytes. Direct Follicle Aspiration Prior to performing the procedure, mares are injected with propantheline bromide (a sedative) to relax the rectum. The lubricated ultrasound transducer is inserted into the vagina and held in the fornix vagina. The ovary is transrecta lly positioned against the dorsal vaginal wall directly over the transducer head so that the follicle can be visualized. The hypodermic needle is advanced through the vagina l wall into the antral follicle. Fol- licular fluid containing the oocyte is aspirated under constant vacuum (Graphic modified with permission from Ultrasonic Imaging and Animal Reproduction: Horses Book 2. 1995 by O.J. Ginther). Transvaginal Aspiration in the Mare Vaginal wall Power to source Follicular fluid and oocyte Aspirated oocytes Embryos transferred to recipient female (See Figure 13-9) Oocytes placed in cultur-e vessel with capacitated spermatozoa Embryos cultured to the appropriate stage for transfer 16-gauge hypodermic needle Early Embryogenesis and Maternal Recognition of Pregnancy 289 Transfer of Emb1·yos can be Accomplished Surgically or Non-Surgically In general, embryos can be transferred non- surgically into the recipients in almost any species. This is because the embryos can be recovered from the donor at a stage that allows them to be transferred directly into the uterus of the synchronized recipient. Transferring the embryos into the uterus involves passing a pipette through the vagina and cervix and depositing the embryos into the appropriate uterine hom (ipsilateral to the CL). The zona pellucida is an important component of the early embryo. First, it houses the blastomeres so that they do no separate and can develop together to fom1 an embryo. Equally important is the fact that the zona pellucida is impermeable to most viruses. This not only protects the embryo from viral infection under natural conditions but prevents disease transmission via the embryo after transfer. Embryo transfer procedures have become veiy successful. In commercial embryo transfer-programs with cattle, pregnancy rates of 70% with unfrozen embryos and 65% with frozen embryos have been accomplished routinely. In humans, 30% pregnancy rates are accomplished. It should be emphasized that in young human couples having regular copulatory pattems, the pregnancy rates per reproductive cycle are only about 35%. What this means is, it takes an aver- age of3.3 cycles for healthy, fertile couples to achieve a pregnancy. Further PHENOMENA for Fertility Some species have delayed implantation (attachment to the uterus) in which a viable embryo floats within the uterus for a sus- tained period of time. Martens (a mink-like animal) copulate in July or August and the embryo develops to the blastocyst stage, but attachment does not occur u11ti/ February. The young are bom about 26-30 days after attachment. The presence of the marsupial embryo with- in the uterus does not interrupt the estrous cycle. Therefore, pregnancy recognition in this species is apparently not caused by a substance(s) produced by the emb1yo. In- stead, the semipermanent attachment of the prematurely bom fetus to the teat provides a pregnancy recognition mechanism, because it arrests cyclicity. The female nine-banded armadillo has several unique features. First, the female has a simplex uterus (like primates), in spite of being a primitive life form. She has no vagina, but retains a urogenital sinus. She spontaneously ovulates a single oocyte and mates in the The emb1yo enters embryonic diapause (delayed attachment) for about 3 to 4 months. Soon after implan- tation, cells of the inner cell mass give rise to four separate identical embryos. Thus, the female armadillo gives birth to identi- cal quadruplets. The genetic implications of identical offspring in this species are not known. The human blastocyst (along with guinea pigs, hedgehogs and chimpamees) first at- taches to the endometrial epithelium, passes through and becomes completely imbedded. Thus, the embryo is isolated from the uterine lumen. Knowledge oft/tis phenomenon led to the term "implantation". True implanta- tion does not occur in domestic animals. 13 Ve tB oo ks .ir , I ' ' 288 Early Embryogenesis and Maternal Recognition of Pregnancy Figure 13-10. Oocyte Collection from Ovarian Follicles for In Vitro Fertilization A hypodermic needle is in- serted into the follicle and the follicular fluid is aspirated and then forcefully returned to the follicle. This is repeated 2-3 times to dislodge the oocytes. Direct Follicle Aspiration Prior to performing the procedure, mares are injected with propantheline bromide (a sedative) to relax the rectum. The lubricated ultrasound transducer is inserted into the vagina and held in the fornix vagina. The ovary is transrecta lly positioned against the dorsal vaginal wall directly over the transducer head so that the follicle can be visualized. The hypodermic needle is advanced through the vagina l wall into the antral follicle. Fol- licular fluid containing the oocyte is aspirated under constant vacuum (Graphic modified with permission from Ultrasonic Imaging and Animal Reproduction: Horses Book 2. 1995 by O.J. Ginther). Transvaginal Aspiration in the Mare Vaginal wall Power to source Follicular fluid and oocyte Aspirated oocytes Embryos transferred to recipient female (See Figure 13-9) Oocytes placed in cultur-e vessel with capacitated spermatozoa Embryos cultured to the appropriate stage for transfer 16-gauge hypodermic needle Early Embryogenesis and Maternal Recognition of Pregnancy 289 Transfer of Emb1·yos can be Accomplished Surgically or Non-Surgically In general, embryos can be transferred non- surgically into the recipients in almost any species. This is because the embryos can be recovered from the donor at a stage that allows them to be transferred directly into the uterus of the synchronized recipient. Transferring the embryos into the uterus involves passing a pipette through the vagina and cervix and depositing the embryos into the appropriate uterine hom (ipsilateral to the CL). The zona pellucida is an important component of the early embryo. First, it houses the blastomeres so that they do no separate and can develop together to fom1 an embryo. Equally important is the fact that the zona pellucida is impermeable to most viruses. This not only protects the embryo from viral infection under natural conditions but prevents disease transmission via the embryo after transfer. Embryo transfer procedures have become veiy successful. In commercial embryo transfer-programs with cattle, pregnancy rates of 70% with unfrozen embryos and 65% with frozen embryos have been accomplished routinely. In humans, 30% pregnancy rates are accomplished. It should be emphasized that in young human couples having regular copulatory pattems, the pregnancy rates per reproductive cycle are only about 35%. What this means is, it takes an aver- age of3.3 cycles for healthy, fertile couples to achieve a pregnancy. Further PHENOMENA for Fertility Some species have delayed implantation (attachment to the uterus) in which a viable embryo floats within the uterus for a sus- tained period of time. Martens (a mink-like animal) copulate in July or August and the embryo develops to the blastocyst stage, but attachment does not occur u11ti/ February. The young are bom about 26-30 days after attachment. The presence of the marsupial embryo with- in the uterus does not interrupt the estrous cycle. Therefore, pregnancy recognition in this species is apparently not caused by a substance(s) produced by the emb1yo. In- stead, the semipermanent attachment of the prematurely bom fetus to the teat provides a pregnancy recognition mechanism, because it arrests cyclicity. The female nine-banded armadillo has several unique features. First, the female has a simplex uterus (like primates), in spite of being a primitive life form. She has no vagina, but retains a urogenital sinus. She spontaneously ovulates a single oocyte and mates in the The emb1yo enters embryonic diapause (delayed attachment) for about 3 to 4 months. Soon after implan- tation, cells of the inner cell mass give rise to four separate identical embryos. Thus, the female armadillo gives birth to identi- cal quadruplets. The genetic implications of identical offspring in this species are not known. The human blastocyst (along with guinea pigs, hedgehogs and chimpamees) first at- taches to the endometrial epithelium, passes through and becomes completely imbedded. Thus, the embryo is isolated from the uterine lumen. Knowledge oft/tis phenomenon led to the term "implantation". True implanta- tion does not occur in domestic animals. 13 Ve tB oo ks .ir 13 290 Early Embryogenesis and Maternal Recognition of Pregnancy In rodents, a successful pregnancy can be terminated if an alien male (one that did not cause the pregnancy) shows up and hangs- out with thepregnantfemale. This is known as the uBruce Effect". The Apostlebird of Eastern Australia de- rived its name from the fact that it does everything in groups of twelve. During the mating season, nests are built on horizontal branches oftrees. The females lay eggs in each nests. All members share the task ofbzcubating the eggs am/rearing the young. A pair of Indian Pythons have been obsen,ed copulating for 180 days. After copulation, the male garter snake plugs the female's cloaca with a material made from renal secretions. This natural chastity belt prevents any further sexual activity, insuring that the offspring are sired by the first male to breed her. Cantharidin is derived from beetles known as "blister beetles". The material has been erroneously nicknamed "Spanish Fly". This material developed a reputation as being a "medical wonder" including being a powerful sexual stimulant. Canthal'idin irritates the urogenital tract, causing a tin- gling and burning sensation that is felt in both the male genitalia am/female genitalia because of vasodilation. This vasodilation of the labia made women more aware of their genitals and it was thought to build erotic passion and cause sexual excitement. Occasionally, cantharidin caused persistent erections (priapism) in males. Priapism was generally not associated with sexual pleasure and could cause vascular damage to the penis. Cantharidin has been illegal since the 1800 and is currelltly not for sale over-the-counter. In significant doses, cantharidin can cause health p1·oblems. It has been reported in the French literature that "Spanish Fly" had bem incorporated into a plate of pears that was consumed by the groom on his wedding night. uW/zen the night came, the husband embraced his wife so much that she began to suffer ex- haustion." These delights quickly changed to misfortune because "the man began to experience the effects of cantharidin in- flammation by midnight. He had difficulty urinating, saw a discharge from his penis, became frightened and fainted more than once. Considerable effort was made to re- store his health. " The Chinese apparently have been search- ing throughout the course of histmy for a Viagra-like compound. For example, ashes from homets or wasps' nests were mixed with water and wine and ingested. This mix- ture was also applied to the penis foJ' sexual stimulation to cure erectile dysfunction and to increase daily sperm output. Dragonflies and silkworms were believed to increase penile turgidity and prevent ejaculation. The latter effect was believed to lengthen the duration of copulation. Scale insects and stinkbugs were considered by the Chinese as aphrodisiacs. Consump- tion of scale insects was also believed to be a cure for amenorrhea. The Chinese believed egg cases from the praying mantis had several beneficial effects such as prevention of nocturnal emissions, premature ejaculation, male weakness and impotence. The word "aphrodisiac" is derived from the name of the Greek goddess of love, Aphrodite. In 1848, a physician named Frederick Hollick published a book entitled, The Male Generative Organs-Health and Disease (rom Infancv to Old Age that undoubtedly received more attention than the reproductive physiology books of the day. It was marketed uFor Every Man's Private Use". Not only did this book deal with the anatomy and physiology of the male genitalia, it dealt extensively with recipes and concoctions that would facilitate male genital function. Early Embryogenesis and Maternal Recognition of Pregnancy 291 Based 011 clay tablets dated 12th Century B. C., it was found that castration was the pwzislzmentfor several male sex offenders. Hence, they apparently knew that the testes were the source of mating behavior in hu- man males. Castration (peJfonned without anesthesia) was likely the first survivable surge1y in humans. Aristotle drew an analogy between the epididymis/ductus deferens, testis am/ a string being helcl tight by an at- taclzed rock. Aristotle thought that the function of the testis was only as a weight (like a rock attached to a string) to keep the "kinks" out of the ductus deferens. Peppermint shrimp begin their life as males, but most change into a female-with a slight twist. The 'remale" shrimp maintain theil"' male ducts, produce sperm ami fertilize other female-phase shrimp even when incubating their own emb1yos. They can do it all. On average, the bilaterally castrated man lives 12 years longer than intact men. The possible reason? There is no energy spent t1ying to copulate. The energy spent copu- lating is minuscule compared to the energy expended trying to convince the female part- ner to copulate. If no testes are available, there is no energy e.Y:penditure. In Cephalopods (squids, cuttlefishes and octopi) the male deposits a special spemz package called a spermatophore in the female body cavity by way of an artificial penis. This artificial penis is known as a hectocotylus and it is a specially modified tentacle. Some species have developed a detachable penis that they cattleave behind in the female's body. Spiders (arachnids) also have an artificial penis. In their case it is a leg that doubles as a penis and is known scientifically as a maxillmy palp. It is not known whether the detachable penis has the ability to grow back. Key References Bazer, F. W., T.L. Ott and T.E. Spencer. 1994. "Preg- nancy recognition in ruminants, pigs and horses: signals from the trophoblast." Theriogenology. 41 :79. Flint, A.P.F. 1995. "Interferon, the oxytocin receptor and the maternal recognition of pregnancy in nuninants and non-ruminants: A comparative approach." Reprod. Fertil. Dev. 7:313. Ginther, 0 .1. 1992. Reproductive Biolo'ty a [the Mare. 2nd Edition. Equiservices, Cross Plains, WI. Library of Congress Catalog No. 9 1-075595. Larsen, W.J. I 993. Human Embrvology. Churchill Livingstone, New York. ISBN 0-443-08724-5. Mirando, M.A. , M.U. Zumcu, K.G. Carnahan and T.E. Ludwig. 1996. "A role for oxytocin during luteolysis and early pregnancy in swine." Reprod. Dam. Anim. 31:455. Ott, T.L. and C.A. Gifford. 2010. "Effects of early con- ceptus signals on circulating immune cells: lessons from domestic ruminants." Am J. Reprod. lmmunol.l -9. Roberts, R.M., D.W. Leaman and J.C. Cross. 1992. " Role of interferons in maternal recognition of preg- nancy in ruminants" in P. S.E.B.M 200:7. Thatcher, W.W., C.R. Staples, G. Danet-Desnoyers, B. Oldick and E.P. Schmitt. 1994. "Embryo health and mortality in sheep and cattle." J. Anim. Sci. 72 (suppl. 3):16. Spencer, T.E. 1998. "Pregnancy, maternal recognition of' in Encvc/opedia o(Reproduction, Vol 3, pl006- 10 15. Knobil, E. and J.D. Neill, eds. Academic Press, San Diego. ISBN 0- 12-227023-1 . Seidel, G.E. 1998. "Embryo transfer" in Encvclopedia o(Reproduction, Vol 1, p1037- 1042. Knobil, E. and J.D. Neill, eds. Academic Press, San Diego. ISBN 0-12-227021-5. 13 Ve tB oo ks .ir 13 290 Early Embryogenesis and Maternal Recognition of Pregnancy In rodents, a successful pregnancy can be terminated if an alien male (one that did not cause the pregnancy) shows up and hangs- out with thepregnantfemale. This is known as the uBruce Effect". The Apostlebird of Eastern Australia de- rived its name from the fact that it does everything in groups of twelve. During the mating season, nests are built on horizontal branches oftrees. The females lay eggs in each nests. All members share the task ofbzcubating the eggs am/rearing the young. A pair of Indian Pythons have been obsen,ed copulating for 180 days. After copulation, the male garter snake plugs the female's cloaca with a material made from renal secretions. This natural chastity belt prevents any further sexual activity, insuring that the offspring are sired by the first male to breed her. Cantharidin is derived from beetles known as "blister beetles". The material has been erroneously nicknamed "Spanish Fly". This material developed a reputation as being a "medical wonder" including being a powerful sexual stimulant. Canthal'idin irritates the urogenital tract, causing a tin- gling and burning sensation that is felt in both the male genitalia am/female genitalia because of vasodilation. This vasodilation of the labia made women more aware of their genitals and it was thought to build erotic passion and cause sexual excitement. Occasionally, cantharidin caused persistent erections (priapism) in males. Priapism was generally not associated with sexual pleasure and could cause vascular damage to the penis. Cantharidin has been illegal since the 1800 and is currelltly not for sale over-the-counter. In significant doses, cantharidin can cause health p1·oblems. It has been reported in the French literature that "Spanish Fly" had bem incorporated into a plate of pears that was consumed by the groom on his wedding night. uW/zen the night came, the husband embraced his wife so much that she began to suffer ex- haustion." These delights quickly changed to misfortune because "the man began to experience the effects of cantharidin in- flammation by midnight. He had difficulty urinating, saw a discharge from his penis, became frightened and fainted more than once. Considerable effort was made to re- store his health. " The Chinese apparently have been search- ing throughout the course of histmy for a Viagra-like compound. For example, ashes from homets or wasps' nests were mixed with water and wine and ingested. This mix- ture was also applied to the penis foJ' sexual stimulation to cure erectile dysfunction and to increase daily sperm output. Dragonflies and silkworms were believed to increase penile turgidity and prevent ejaculation. The latter effect was believed to lengthen the duration of copulation. Scale insects and stinkbugs were considered by the Chinese as aphrodisiacs. Consump- tion of scale insects was also believed to be a cure for amenorrhea. The Chinese believed egg cases from the praying mantis had several beneficial effects such as prevention of nocturnal emissions, premature ejaculation, male weakness and impotence. The word "aphrodisiac" is derived from the name of the Greek goddess of love, Aphrodite. In 1848, a physician named Frederick Hollick published a book entitled, The Male Generative Organs-Health and Disease (rom Infancv to Old Age that undoubtedly received more attention than the reproductive physiology books of the day. It was marketed uFor Every Man's Private Use". Not only did this book deal with the anatomy and physiology of the male genitalia, it dealt extensively with recipes and concoctions that would facilitate male genital function. Early Embryogenesis and Maternal Recognition of Pregnancy 291 Based 011 clay tablets dated 12th Century B. C., it was found that castration was the pwzislzmentfor several male sex offenders. Hence, they apparently knew that the testes were the source of mating behavior in hu- man males. Castration (peJfonned without anesthesia) was likely the first survivable surge1y in humans. Aristotle drew an analogy between the epididymis/ductus deferens, testis am/ a string being helcl tight by an at- taclzed rock. Aristotle thought that the function of the testis was only as a weight (like a rock attached to a string) to keep the "kinks" out of the ductus deferens. Peppermint shrimp begin their life as males, but most change into a female-with a slight twist. The 'remale" shrimp maintain theil"' male ducts, produce sperm ami fertilize other female-phase shrimp even when incubating their own emb1yos. They can do it all. On average, the bilaterally castrated man lives 12 years longer than intact men. The possible reason? There is no energy spent t1ying to copulate. The energy spent copu- lating is minuscule compared to the energy expended trying to convince the female part- ner to copulate. If no testes are available, there is no energy e.Y:penditure. In Cephalopods (squids, cuttlefishes and octopi) the male deposits a special spemz package called a spermatophore in the female body cavity by way of an artificial penis. This artificial penis is known as a hectocotylus and it is a specially modified tentacle. Some species have developed a detachable penis that they cattleave behind in the female's body. Spiders (arachnids) also have an artificial penis. In their case it is a leg that doubles as a penis and is known scientifically as a maxillmy palp. It is not known whether the detachable penis has the ability to grow back. Key References Bazer, F. W., T.L. Ott and T.E. Spencer. 1994. "Preg- nancy recognition in ruminants, pigs and horses: signals from the trophoblast." Theriogenology. 41 :79. Flint, A.P.F. 1995. "Interferon, the oxytocin receptor and the maternal recognition of pregnancy in nuninants and non-ruminants: A comparative approach." Reprod. Fertil. Dev. 7:313. Ginther, 0 .1. 1992. Reproductive Biolo'ty a [the Mare. 2nd Edition. Equiservices, Cross Plains, WI. Library of Congress Catalog No. 9 1-075595. Larsen, W.J. I 993. Human Embrvology. Churchill Livingstone, New York. ISBN 0-443-08724-5. Mirando, M.A. , M.U. Zumcu, K.G. Carnahan and T.E. Ludwig. 1996. "A role for oxytocin during luteolysis and early pregnancy in swine." Reprod. Dam. Anim. 31:455. Ott, T.L. and C.A. Gifford. 2010. "Effects of early con- ceptus signals on circulating immune cells: lessons from domestic ruminants." Am J. Reprod. lmmunol.l -9. Roberts, R.M., D.W. Leaman and J.C. Cross. 1992. " Role of interferons in maternal recognition of preg- nancy in ruminants" in P. S.E.B.M 200:7. Thatcher, W.W., C.R. Staples, G. Danet-Desnoyers, B. Oldick and E.P. Schmitt. 1994. "Embryo health and mortality in sheep and cattle." J. Anim. Sci. 72 (suppl. 3):16. Spencer, T.E. 1998. "Pregnancy, maternal recognition of' in Encvc/opedia o(Reproduction, Vol 3, pl006- 10 15. Knobil, E. and J.D. Neill, eds. Academic Press, San Diego. ISBN 0- 12-227023-1 . Seidel, G.E. 1998. "Embryo transfer" in Encvclopedia o(Reproduction, Vol 1, p1037- 1042. Knobil, E. and J.D. Neill, eds. Academic Press, San Diego. ISBN 0-12-227021-5. 13 Ve tB oo ks .ir

The Puerperium &

Lactation

Early Embryogenesis &
Maternal Recognition of Pregnancy

Ovulation & Fertilization

Cyclicity

Regulation of
Reproduction

Tract Function

Puberty

Prenatal

Development

Spermatogenesis

Regulat ion of
Reproduction

Tract Function
Puberty
Prenatal
Development

Take Home Message
Gestation is the period of time that a female is pregnant. During gestation, the

placenta forms a major organ of pregnancy that provides an inteJface for metabolic
exchange between the dam and the f etus. Placentas are described mmplwlogically
according to the distribution of villi on the chorionic smface ami the degree of separa..:.
tion between matemal and fetal blood. The placenta is also an endocrine organ that
secretes hormones responsible for: 1) maintenance of pregnancy; 2) stimulation of the
matemal mammmy gland and 3) ensures fetal growth. Parturition is brought about
by secretion of fetal corticoitls and requires removal of the progesterone block. Par-
turition consists of three stages. They are: 1) initiation of myometrial contractions;
2) expulsion ofthe f etus and 3) e.:\:pulsion oftlzefetalmembranes.

The word gestation literally means “the act
of carry ing or being carried”. Thus, gestation means
the action or process of carrying or being carried’ in
the uterus between conception and birth . · Gest ation
and pregnancy are synonymous and thus, gestation
length means the length of pregnancy. Attachment
of the conceph1s to form an intimate, but temporary,
relationship with the uterus is an evolutionary step that
provides significant advantage to the conceph1s . The
phenomenon of intrauterine development ensures that
the developing conceptus will receive adequate nutri-
tion and protection during its development. In contrast,
lower fon11S of animals lay eggs (oviparous). The
survival of potential offspring of oviparous animals
is jeopardized because the female cannot completely
protect the eggs from environmental and predatmy dan-
ger. Thus, from an evolutionary perspective, eutherian
mammals (mammals with a placenta), are “equipped

”

with an in-utero protection mechanism that is highly
successful after the placenta is formed.

The final prepartum steps of
reproduction are:

• formation of a placenta

• acquisition of endocrine function of
the placenta

• initiation of parturition

The term implantation is often used to mean
attachment of the placental membranes to the endo-
metrium in most animals. Achmlly, true implantation
is a phenomenon in humans in which the conceptus
“buries” itself into the uterine endometrium. The con-
ceptus temporarily disappears beneath the surface. In

most other species, the conceph1s does not truly implant,
but rather attaches to the endometrial surface and neve

r

disappears from the luminal compartment.

The placenta is an organ of metabolic inter-
change between the conceph1s and the dam. It is also
an endocrine organ. The placenta is composed of a fetal
component derived fi·om the chorion and a maternal
component derived from modifications of the uterine
endometrium. The discrete regions of contact between
the chorion and the endometr ium form specific zones
of metabolic exchange. The placenta also produces
a variety of hormones . This endocrine function is
important for the maintenance of pregnancy and the
induction of parh1rition.

Parturition (giving birth to young) is the ste

p

in the reproductive process that immediately precedes
lactation, uterine repair and return to cyclicity. It is
ini tiated by the feh1s and involves a complex cascade
of endocrine events that promote myometrial contrac-
tions, dilation of the cervix, expulsion of the feh1s and
expulsion of the extraembryonic membranes.

Placentas Have Different Distributions
of Chorionic Villi

As you have learned in the previous chapter, the
conceptus consists of the embryo and the extraembry-
onic membranes (amnion, allantois and chorion). The
chorion is the fetal contribution to the placenta. The
functional uni t of the fetal placenta is the chorionic
villus. The chorionic villus is an “exchange apparatus”
and provides increased surface area so that exchange
is maximized. Chorionic villi are small, finger- like
projections that are on the surface of the chorion. These
tiny villi protrude away from the chorion toward the
uterine endometrium. Placentas are classified according
to the distribution of chorionic vill i on their surfaces,

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The Puerperium & Lactation
Early Embryogenesis &
Maternal Recognition of Pregnancy
Ovulation & Fertilization
Cyclicity
Regulation of
Reproduction
Tract Function
Puberty
Prenatal
Development
Spermatogenesis
Regulat ion of
Reproduction
Tract Function
Puberty
Prenatal
Development
Take Home Message
Gestation is the period of time that a female is pregnant. During gestation, the
placenta forms a major organ of pregnancy that provides an inteJface for metabolic
exchange between the dam and the f etus. Placentas are described mmplwlogically
according to the distribution of villi on the chorionic smface ami the degree of separa..:.
tion between matemal and fetal blood. The placenta is also an endocrine organ that
secretes hormones responsible for: 1) maintenance of pregnancy; 2) stimulation of the
matemal mammmy gland and 3) ensures fetal growth. Parturition is brought about
by secretion of fetal corticoitls and requires removal of the progesterone block. Par-
turition consists of three stages. They are: 1) initiation of myometrial contractions;
2) expulsion ofthe f etus and 3) e.:\:pulsion oftlzefetalmembranes.
The word gestation literally means “the act
of carry ing or being carried”. Thus, gestation means
the action or process of carrying or being carried’ in
the uterus between conception and birth . · Gest ation
and pregnancy are synonymous and thus, gestation
length means the length of pregnancy. Attachment
of the conceph1s to form an intimate, but temporary,
relationship with the uterus is an evolutionary step that
provides significant advantage to the conceph1s . The
phenomenon of intrauterine development ensures that
the developing conceptus will receive adequate nutri-
tion and protection during its development. In contrast,
lower fon11S of animals lay eggs (oviparous). The
survival of potential offspring of oviparous animals
is jeopardized because the female cannot completely
protect the eggs from environmental and predatmy dan-
ger. Thus, from an evolutionary perspective, eutherian
mammals (mammals with a placenta), are “equipped”
with an in-utero protection mechanism that is highly
successful after the placenta is formed.
The final prepartum steps of
reproduction are:
• formation of a placenta
• acquisition of endocrine function of
the placenta
• initiation of parturition
The term implantation is often used to mean
attachment of the placental membranes to the endo-
metrium in most animals. Achmlly, true implantation
is a phenomenon in humans in which the conceptus
“buries” itself into the uterine endometrium. The con-
ceptus temporarily disappears beneath the surface. In
most other species, the conceph1s does not truly implant,
but rather attaches to the endometrial surface and never
disappears from the luminal compartment.
The placenta is an organ of metabolic inter-
change between the conceph1s and the dam. It is also
an endocrine organ. The placenta is composed of a fetal
component derived fi·om the chorion and a maternal
component derived from modifications of the uterine
endometrium. The discrete regions of contact between
the chorion and the endometr ium form specific zones
of metabolic exchange. The placenta also produces
a variety of hormones . This endocrine function is
important for the maintenance of pregnancy and the
induction of parh1rition.
Parturition (giving birth to young) is the step
in the reproductive process that immediately precedes
lactation, uterine repair and return to cyclicity. It is
ini tiated by the feh1s and involves a complex cascade
of endocrine events that promote myometrial contrac-
tions, dilation of the cervix, expulsion of the feh1s and
expulsion of the extraembryonic membranes.
Placentas Have Different Distributions
of Chorionic Villi
As you have learned in the previous chapter, the
conceptus consists of the embryo and the extraembry-
onic membranes (amnion, allantois and chorion). The
chorion is the fetal contribution to the placenta. The
functional uni t of the fetal placenta is the chorionic
villus. The chorionic villus is an “exchange apparatus”
and provides increased surface area so that exchange
is maximized. Chorionic villi are small, finger- like
projections that are on the surface of the chorion. These
tiny villi protrude away from the chorion toward the
uterine endometrium. Placentas are classified according
to the distribution of chorionic vill i on their surfaces,
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294 Placentation, Gestation and

Parturition

giving each placental type a distinct anatomical appear-
ance. Placentas may also be classified by number of
tissue layers separating maternal and fetal blood.

Placentas are classified acc01·ding to the
distribution of chorionic viili. These
classifications are:

• diffuse
• zonary
• discoid
• cotyledonary

The diffi.tse placenta of the pig has a velvet-like
surface with many closely spaced chorionic villi that are
distributed over the entire surface of the chorion (See
Figure 14-1 ). Initial attachment occurs around day

12

and is well established by day 18 to 20 after ovulation
(See Chapter 13).

Diffuse placentas have uniform
distribution of chorionic villi that
cover the swface of the chorion.

Example= pig

The mare placenta is also classified as diffuse,
however it is characterized by having many specialized
“microzones” of chorionic villi known as microcoty-
ledons (See Figure 14-1 ). These microcotyledons are
microscopically discrete regions at the fetal-maternal
interface. As in the pig, they are also distributed over
the entire chorionic surface.

The mare placenta also contains unique tran-
sitory structures known as endometrial cups. These
are discrete areas that range from a few millimeters
to several centimeters in diameter. The endometrial
cups are of both trophoblastic and endometrial origin.
There are 5 to I 0 endometrial cups distributed over the
surface of the placenta (See Figure 14-6). Endometrial
cups produce equine chorionic gonadotropin (eCG

)

and develop between days 35 and 60 of pregnancy.
Following day 60, the endometrial cups are sloughed
into the uterine lumen and are no longer functional.
Attachment of the conceptus to the endometrium is
initiated at about day 24 and becomes well established
by 36 to 38 days (See Chapter 13).

Zonary placentas have a band-like
zone of chorionic villi.

Example = dogs and cats

The zonary placenta (found in dogs and cats)
includes a prominent region of exchange that fonns
a broad zone around the chorion near the middle of
the conceptus (See F igure 14-2). A second region
consists of a highly pigmented ring at either end of the
central zone. This pigmented zone consists of small
hematomas (blood clots). The pigmented zone is also
refetTed to as the paraplacenta and is thought to be
important in iron transport from the dam to the fehts.
The function of this zone is not well understood. A
third region is the transparent zone on the distal ends
of the chorion that has poor vascularity. This zone may
be involved in absorption of materials directly from the
uterine lumen.

Discoid placentas form a
regionalized disc.

Example = rodents and primates

The discoid placenta (See Figure 14-2) is found
in rodents and primates. It is characterized by having
one or two distinct adj acent discs. These discs contain
chorionic vi lli that interface with the endometrium
and provide the region for gas, nutrient and metabolic
waste exchange.

Cotyledonary placentas have nu-
merous, discrete button-like
structures called cotyledons.

Example = ruminants

Ruminants have a cotyledonary placenta (See
Figure 14-3). A cotyledon is defined as a placental
unit of trophoblastic origin cons isting of abundant
blood vessels and connective tissue. In sheep, there
are between 90 and 100 cotyledons distributed across
the surface of the chorion and, in cattle, 70 to 1

20

cotyledons have been observed. The placentome (point
of interface) in the cotyledonary placenta consists of
a fetal cotyledon contributed by the chorion and a
maternal cotyledon , originating from the caruncular
regions of the u terus. At about day 16 in sheep and
day 25 in cattle the chorion initiates attachment to the
cm·uncles of the uterus. Prior to this time the placenta
is essentially diffi.tse. During the formation of the
placentomes, chorionic v illi protrude into crypts in the
caruncular tissue. This relationship .lli not implantation
but an anatomically specialized forn1 of attachment.
Attachment is well established by day 30 in ewes and
day 40 in cows (See Chapter 13).

In the cow, the placentomes form a convex
structure, whi le in the ewe they are concave (See
Figure 14-3). During gestation, the cotyledons will

increase many-fold in diameter. In fact, cotyledons in
the cow near the end of gestation may measure 5 to
6 centimeters in diameter. Such growth provides
enormous surface area to support placental transfer
of nutrients from the dam and metabolic wastes from
the fetus.

Placental Classification by Microscopic
Appearance is Based on the Number of
Placental Layers that Separate the Fetal

Blood from the Maternal Blood

The nomenclature for describing placental in-
timacy is derived by first describing the tissues of the
maternal placenta in the prefix of the word. The tissues
of the fetal placenta constitute the suffix. Exchange can
occur through as many as six tissue layers and as few
as three. The name of the prefix and suffix of each type
of placenta changes depending on the number of tissue
layers that exist.

Prefix =maternal side Suffix =fetal side
“epithelia” “chorial”

epitheliochorial

Placentation, Gestation and Parturition 295

cells originate from trophoblast cells and are thotwht
to be fanned continuously throughout gestation. Bi-
nucleate giant cells constitute around 20% of the fetal
placenta. During development, the binucleate giant
cells migrate from the chorionic epithelium and invade
the endometrial epithelium (See Figure 14-4). The
binucleate giant cells are believed to transfer complex
molecules from the fetal to the maternal p lacenta.
There is evidence that they secrete placental lactogen.
Also, these cells secrete pregnancy specific protein
B (PSPB) that are also called pregnancy associated
glycoproteins (PAG). These proteins are unique to
pregnancy in ruminants. The binucleate giant cells
are also important sites of steroidogenesis, secreting
progesterone and estradiol. These cells will no doubt
emerge as increasingly important “players” in the func-
tion of the ruminant placenta with further research.

I Endotheliochorial = 5 layers

I

The endotheliochorial placenta is character-

ized as having complete erosion of the endometrial
epithelium and underlying interstitium. T hus, maternal
capillaries are directly exposed to epithelial cells of the
chorion (See Figure 14-5). The chorionic epithelium
packs around the vessels on the maternal side. Note in
Figure 14-5 that this type of placenta is more intimate

I Epitheliochorial 6 layers I than the epitheliochoriat placenta because the en dome– _ trial epithelium no longer exists. Dogs and cats possess ..__ _ _ ____ _ _ _ _____ ____ _. endotheliochorial placentation.
The epitheliochorial placenta (See Figure

14-5) is the least intimate among the placental types.
In the epitheliochorial placenta, both the endometrial
epithelium (maternal side) and epithelium of the chori-
onic villi are intact. In other words, there is a complete
intact layer of epithelium in both the maternal and fetal
components. The epitheliochorial placenta is found in
the sow and the mare. Recall that the placentas of the
sow and the mare are diffitse and villi occupy a large
proportion of the surface area of the chorion.

Ruminants also have an epitheliochorial pla-
centa. However, the endometrial epithelium transiently
erodes and then regrows, causing intennittent exposure
of the maternal capillaries to the chorionic epithelium.
This type of placenta has been tenned syndesmocho-
rial.

In addition to the feature of partial erosion
of the endometrial epithelium, a unique cell type is
found in the ruminant placenta. These cells are called
binucleate giant cells. As their name implies, they are
characterized as being quite large and have two nuclei.
Binucleate giant cells appear at about day 14 in the
sheep and between days 18 and 20 in the cow. These

I Hemochorial = 3 layers I
The hemochorial placenta (See Figure 14-5)

is characterized as having the chorionic epithelium in
direct apposition to maternal pools ofblood. Thus, nu-
trients and gases are exchanged directly from maternal
blood and must move tlu-ough only tlu-ee tissue layers.
This highly intimate relationship is found in primates
and rodents (See Figure 14-5).

The Placenta Regulates the Exchange
Between the Fetus and Dam

Placental exchange involves a number of
mechanisms found in other tissues. These are simple
diffusion, facilitated diffusion and active tr a nsport.
Gases and water pass from high to low concentrations
by simple diffusion. The p lacenta contains act ive
transport pumps for sodium and potassium, as well as
calcium. Glucose and other metabolically important
materials such as amino acids are transported by facili-
tated di ffusion uti lizing specific carrier molecules.

141

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294 Placentation, Gestation and Parturition
giving each placental type a distinct anatomical appear-
ance. Placentas may also be classified by number of
tissue layers separating maternal and fetal blood.
Placentas are classified acc01·ding to the
distribution of chorionic viili. These
classifications are:
• diffuse
• zonary
• discoid
• cotyledonary
The diffi.tse placenta of the pig has a velvet-like
surface with many closely spaced chorionic villi that are
distributed over the entire surface of the chorion (See
Figure 14-1 ). Initial attachment occurs around day 12
and is well established by day 18 to 20 after ovulation
(See Chapter 13).
Diffuse placentas have uniform
distribution of chorionic villi that
cover the swface of the chorion.
Example= pig
The mare placenta is also classified as diffuse,
however it is characterized by having many specialized
“microzones” of chorionic villi known as microcoty-
ledons (See Figure 14-1 ). These microcotyledons are
microscopically discrete regions at the fetal-maternal
interface. As in the pig, they are also distributed over
the entire chorionic surface.
The mare placenta also contains unique tran-
sitory structures known as endometrial cups. These
are discrete areas that range from a few millimeters
to several centimeters in diameter. The endometrial
cups are of both trophoblastic and endometrial origin.
There are 5 to I 0 endometrial cups distributed over the
surface of the placenta (See Figure 14-6). Endometrial
cups produce equine chorionic gonadotropin (eCG)
and develop between days 35 and 60 of pregnancy.
Following day 60, the endometrial cups are sloughed
into the uterine lumen and are no longer functional.
Attachment of the conceptus to the endometrium is
initiated at about day 24 and becomes well established
by 36 to 38 days (See Chapter 13).
Zonary placentas have a band-like
zone of chorionic villi.
Example = dogs and cats
The zonary placenta (found in dogs and cats)
includes a prominent region of exchange that fonns
a broad zone around the chorion near the middle of
the conceptus (See F igure 14-2). A second region
consists of a highly pigmented ring at either end of the
central zone. This pigmented zone consists of small
hematomas (blood clots). The pigmented zone is also
refetTed to as the paraplacenta and is thought to be
important in iron transport from the dam to the fehts.
The function of this zone is not well understood. A
third region is the transparent zone on the distal ends
of the chorion that has poor vascularity. This zone may
be involved in absorption of materials directly from the
uterine lumen.
Discoid placentas form a
regionalized disc.
Example = rodents and primates
The discoid placenta (See Figure 14-2) is found
in rodents and primates. It is characterized by having
one or two distinct adj acent discs. These discs contain
chorionic vi lli that interface with the endometrium
and provide the region for gas, nutrient and metabolic
waste exchange.
Cotyledonary placentas have nu-
merous, discrete button-like
structures called cotyledons.
Example = ruminants
Ruminants have a cotyledonary placenta (See
Figure 14-3). A cotyledon is defined as a placental
unit of trophoblastic origin cons isting of abundant
blood vessels and connective tissue. In sheep, there
are between 90 and 100 cotyledons distributed across
the surface of the chorion and, in cattle, 70 to 120
cotyledons have been observed. The placentome (point
of interface) in the cotyledonary placenta consists of
a fetal cotyledon contributed by the chorion and a
maternal cotyledon , originating from the caruncular
regions of the u terus. At about day 16 in sheep and
day 25 in cattle the chorion initiates attachment to the
cm·uncles of the uterus. Prior to this time the placenta
is essentially diffi.tse. During the formation of the
placentomes, chorionic v illi protrude into crypts in the
caruncular tissue. This relationship .lli not implantation
but an anatomically specialized forn1 of attachment.
Attachment is well established by day 30 in ewes and
day 40 in cows (See Chapter 13).
In the cow, the placentomes form a convex
structure, whi le in the ewe they are concave (See
Figure 14-3). During gestation, the cotyledons will
increase many-fold in diameter. In fact, cotyledons in
the cow near the end of gestation may measure 5 to
6 centimeters in diameter. Such growth provides
enormous surface area to support placental transfer
of nutrients from the dam and metabolic wastes from
the fetus.
Placental Classification by Microscopic
Appearance is Based on the Number of
Placental Layers that Separate the Fetal
Blood from the Maternal Blood
The nomenclature for describing placental in-
timacy is derived by first describing the tissues of the
maternal placenta in the prefix of the word. The tissues
of the fetal placenta constitute the suffix. Exchange can
occur through as many as six tissue layers and as few
as three. The name of the prefix and suffix of each type
of placenta changes depending on the number of tissue
layers that exist.
Prefix =maternal side Suffix =fetal side
“epithelia” “chorial”
epitheliochorial
Placentation, Gestation and Parturition 295
cells originate from trophoblast cells and are thotwht
to be fanned continuously throughout gestation. Bi-
nucleate giant cells constitute around 20% of the fetal
placenta. During development, the binucleate giant
cells migrate from the chorionic epithelium and invade
the endometrial epithelium (See Figure 14-4). The
binucleate giant cells are believed to transfer complex
molecules from the fetal to the maternal p lacenta.
There is evidence that they secrete placental lactogen.
Also, these cells secrete pregnancy specific protein
B (PSPB) that are also called pregnancy associated
glycoproteins (PAG). These proteins are unique to
pregnancy in ruminants. The binucleate giant cells
are also important sites of steroidogenesis, secreting
progesterone and estradiol. These cells will no doubt
emerge as increasingly important “players” in the func-
tion of the ruminant placenta with further research.
I Endotheliochorial = 5 layers I
The endotheliochorial placenta is character-
ized as having complete erosion of the endometrial
epithelium and underlying interstitium. T hus, maternal
capillaries are directly exposed to epithelial cells of the
chorion (See Figure 14-5). The chorionic epithelium
packs around the vessels on the maternal side. Note in
Figure 14-5 that this type of placenta is more intimate
I Epitheliochorial 6 layers I than the epitheliochoriat placenta because the en dome– _ trial epithelium no longer exists. Dogs and cats possess ..__ _ _ ____ _ _ _ _____ ____ _. endotheliochorial placentation.
The epitheliochorial placenta (See Figure
14-5) is the least intimate among the placental types.
In the epitheliochorial placenta, both the endometrial
epithelium (maternal side) and epithelium of the chori-
onic villi are intact. In other words, there is a complete
intact layer of epithelium in both the maternal and fetal
components. The epitheliochorial placenta is found in
the sow and the mare. Recall that the placentas of the
sow and the mare are diffitse and villi occupy a large
proportion of the surface area of the chorion.
Ruminants also have an epitheliochorial pla-
centa. However, the endometrial epithelium transiently
erodes and then regrows, causing intennittent exposure
of the maternal capillaries to the chorionic epithelium.
This type of placenta has been tenned syndesmocho-
rial.
In addition to the feature of partial erosion
of the endometrial epithelium, a unique cell type is
found in the ruminant placenta. These cells are called
binucleate giant cells. As their name implies, they are
characterized as being quite large and have two nuclei.
Binucleate giant cells appear at about day 14 in the
sheep and between days 18 and 20 in the cow. These
I Hemochorial = 3 layers I
The hemochorial placenta (See Figure 14-5)
is characterized as having the chorionic epithelium in
direct apposition to maternal pools ofblood. Thus, nu-
trients and gases are exchanged directly from maternal
blood and must move tlu-ough only tlu-ee tissue layers.
This highly intimate relationship is found in primates
and rodents (See Figure 14-5).
The Placenta Regulates the Exchange
Between the Fetus and Dam
Placental exchange involves a number of
mechanisms found in other tissues. These are simple
diffusion, facilitated diffusion and active tr a nsport.
Gases and water pass from high to low concentrations
by simple diffusion. The p lacenta contains act ive
transport pumps for sodium and potassium, as well as
calcium. Glucose and other metabolically important
materials such as amino acids are transported by facili-
tated di ffusion uti lizing specific carrier molecules.
141
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14

296 Placentation, Gestation and Parturition

Figure 14-1. The Diffuse Placenta

Sow

r

Endometrium

=—

The diffuse placenta of the sow consists of
many chorionic villi distributed over the entire
surface of the chorion. They penetrate into
the endometrium forming the fetal-maternal
interface. Vessels from each chorionic vil-
lus merge and eventually form large vessels
that enter the umbilical cord. A= Allantois,
AC= Allantochorion, AM= Amnionic Cavity,
E= Endometrium, M=

Myometrium

Mare

Endometrium
Myometrium

The diffuse placenta of the mare consists
of many microcotyledons distributed over
the entire surface of the chorion. These mi-
crocotyledons are the site of fetal-maternal
exchange. A= Allantois, AC= Allantochorion,
AM= Amnionic Cavity, E= Endometrium,
M= Myometrium, YS= Yolk Sac

Placentation, Gestation and Parturition 297

Figure 14-2. The Zonary and Discoid Placentas

AC

YS …..-“‘

PZ

Bitch

The zonary placenta consists of three distinct zones;
a transfer zone (TZ), a pigmented zone (PZ) and a
relatively nonvascular zone, the allantochorion (AC).
In the zonary placenta, a band of tissue forms around
the conceptus where nutrient transfer occurs. The
pigmented zone (PZ) or paraplacenta represents local
regions of maternal hemorrhage and necrosis.
A= Allantois, AC= Allantochorion, AM= Amnionic Cavity,
E= Endometrium, M= Myometrium, YS= Yolk Sac

Primates

The discoid placenta consists of a round patch of chori-
onic tissue that forms the fetal-maternal interface. Ves-
sels from the exchange zone merge to form the umbilical
vessels that supply the fetus with blood. The vasculature
of the chorion (within the disc) is immersed in pools of
blood where metabolic exchange takes place.

A= Allantois, AC = Allantochorion,
AM= Amnionic Cavity, E = Endometrium,
EZ = Exchange Zone, M = Myometrium

14
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14
296 Placentation, Gestation and Parturition
Figure 14-1. The Diffuse Placenta
Sow
r
Endometrium
=—

The diffuse placenta of the sow consists of
many chorionic villi distributed over the entire
surface of the chorion. They penetrate into
the endometrium forming the fetal-maternal
interface. Vessels from each chorionic vil-
lus merge and eventually form large vessels
that enter the umbilical cord. A= Allantois,
AC= Allantochorion, AM= Amnionic Cavity,
E= Endometrium, M= Myometrium

Mare
Endometrium
Myometrium
The diffuse placenta of the mare consists
of many microcotyledons distributed over
the entire surface of the chorion. These mi-
crocotyledons are the site of fetal-maternal
exchange. A= Allantois, AC= Allantochorion,
AM= Amnionic Cavity, E= Endometrium,
M= Myometrium, YS= Yolk Sac
Placentation, Gestation and Parturition 297
Figure 14-2. The Zonary and Discoid Placentas
AC
YS …..-“‘
PZ
Bitch
The zonary placenta consists of three distinct zones;
a transfer zone (TZ), a pigmented zone (PZ) and a
relatively nonvascular zone, the allantochorion (AC).
In the zonary placenta, a band of tissue forms around
the conceptus where nutrient transfer occurs. The
pigmented zone (PZ) or paraplacenta represents local
regions of maternal hemorrhage and necrosis.
A= Allantois, AC= Allantochorion, AM= Amnionic Cavity,
E= Endometrium, M= Myometrium, YS= Yolk Sac
Primates
The discoid placenta consists of a round patch of chori-
onic tissue that forms the fetal-maternal interface. Ves-
sels from the exchange zone merge to form the umbilical
vessels that supply the fetus with blood. The vasculature
of the chorion (within the disc) is immersed in pools of
blood where metabolic exchange takes place.
A= Allantois, AC = Allantochorion,
AM= Amnionic Cavity, E = Endometrium,
EZ = Exchange Zone, M = Myometrium
14
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II

298 Placentation, Gestation and Parturition

Figure 14-3. The Cotyledonary Placenta

Convex
(cow, giraffe)

In the photograph above, the fetal membranes and
the fetal cotyledons (FC) can be visualized. The
membrane labeled AC is the allantochorion. The
umbilical cord, (UC-arrow) of the fetus receives
blood vessels (BV) from the fetal cotyledons (FC).
Glycogen plaques (GP) can be visualized on the
surface of the chorion and the amnion. These
plaques are localized squamous proliferations
called verrucae.

Concave
(sheep, goat)

The cotyledonary placenta is characterized by numerous “button-like” structures distributed across the surface
of the chorion . These are called fetal cotyledons. When they jo in with the maternal caruncle they form a
placentome. Aconvex cotyledon becomes covered with the chorion. Many finger-like villi (red) originating
from the chorionic tissue protrude toward the lumen of the uterus. In the concave cotyledon, the chorionic
tissue pushes inward, forming a concave interface between the chorion and the maternal caruncle.

Placentation, Gestation and Parturition 299

Figure 14-3. The Cotyledonary Placenta

The diagram in the upper left illustrates the distribution
of the extraembryonic membranes prior to complete at-
tachment. The extraembryonic membranes consist of the
amnion (blue sac), yolk sac (YS) and the allantois (A).
Even though the fetus is located in one uterine horn, the
chorion invades the contralateral uterine horn and forms
placentomes.

Cow

Some fetal cotyledons (FC) have been partially separated
from maternal cotyledons (MC). The chorion (C) is the
outer fetal membrane. Arrows indicate the border of the
amnion (A). The myometrium (M) is indicated by the ar-
rows. Notice that the fetal cotyledon (FC) is attached to
the surface of the caruncle creating a convex cotyledon.
E= Endometrium

Ewe-A
The chorion can be seen entering the placentome (P).
The chorionic stalk (CS) contains the fetal vasculature.

Ewe-8

)

A portion of the chorion has been incised so that the fetal
vasculature can be visualized clearly. The fetal vessels
(arrow) and chorionic tissue “push” into the caruncular
tissue forming a concave cotyledon. A set of arteries (A)
and veins (V) emerge from each cotyledon and eventually
merge in the umbilical cord (UC). P= Placentoma

Ewe-C
A concave placentoma is clearly visible. The chorionic
stalk is draped over the needle holder. Notice the vessels
(arrows) within the chorionic tissue. The reddish-beige
tissue is the maternal cotyledon (MC) that is covered by
the allantochorion. The dark tissue in the center (arrows)
is the fetal component of the placentome.

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298 Placentation, Gestation and Parturition
Figure 14-3. The Cotyledonary Placenta
Convex
(cow, giraffe)
In the photograph above, the fetal membranes and
the fetal cotyledons (FC) can be visualized. The
membrane labeled AC is the allantochorion. The
umbilical cord, (UC-arrow) of the fetus receives
blood vessels (BV) from the fetal cotyledons (FC).
Glycogen plaques (GP) can be visualized on the
surface of the chorion and the amnion. These
plaques are localized squamous proliferations
called verrucae.
Concave
(sheep, goat)
The cotyledonary placenta is characterized by numerous “button-like” structures distributed across the surface
of the chorion . These are called fetal cotyledons. When they jo in with the maternal caruncle they form a
placentome. Aconvex cotyledon becomes covered with the chorion. Many finger-like villi (red) originating
from the chorionic tissue protrude toward the lumen of the uterus. In the concave cotyledon, the chorionic
tissue pushes inward, forming a concave interface between the chorion and the maternal caruncle.
Placentation, Gestation and Parturition 299
Figure 14-3. The Cotyledonary Placenta
The diagram in the upper left illustrates the distribution
of the extraembryonic membranes prior to complete at-
tachment. The extraembryonic membranes consist of the
amnion (blue sac), yolk sac (YS) and the allantois (A).
Even though the fetus is located in one uterine horn, the
chorion invades the contralateral uterine horn and forms
placentomes.
Cow
Some fetal cotyledons (FC) have been partially separated
from maternal cotyledons (MC). The chorion (C) is the
outer fetal membrane. Arrows indicate the border of the
amnion (A). The myometrium (M) is indicated by the ar-
rows. Notice that the fetal cotyledon (FC) is attached to
the surface of the caruncle creating a convex cotyledon.
E= Endometrium
Ewe-A
The chorion can be seen entering the placentome (P).
The chorionic stalk (CS) contains the fetal vasculature.
Ewe-8
)
A portion of the chorion has been incised so that the fetal
vasculature can be visualized clearly. The fetal vessels
(arrow) and chorionic tissue “push” into the caruncular
tissue forming a concave cotyledon. A set of arteries (A)
and veins (V) emerge from each cotyledon and eventually
merge in the umbilical cord (UC). P= Placentoma
Ewe-C
A concave placentoma is clearly visible. The chorionic
stalk is draped over the needle holder. Notice the vessels
(arrows) within the chorionic tissue. The reddish-beige
tissue is the maternal cotyledon (MC) that is covered by
the allantochorion. The dark tissue in the center (arrows)
is the fetal component of the placentome.
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300 Placentation, Gestation and Parturition

Glucose is the major source of energy for the
fetus. The majority of glucose is derived from the ma-
temal circulation. Near the end of gestation, glucose
consumption by the fetus is exceptionally high and
can lead to a metabolic drain of glucose away from the
dam. Such a glucose drain favors the development of
ketosis in the dam. Ketosis results from the metabo-
lism of body fat that generate ketones for energy when
glucose is limited. Periparturient ketosis is common
in dairy cows where postpartum metabolic demands
are exceptionally high because of high milk produc-
tion. Some materials cannot be transported across the
placenta. With the exception of some immunoglobu-
lins, matemal proteins do not cross the placental banier.
Immunoglobulins can be transported from the matemal
to the fetal side in a hemochorial or an endotheliochorial
placenta. However, the fetus synthesizes the majority
of its own proteins from amino acids contributed by
the dam. Nutritionally-based lipids do not cross the
placenta. Instead, the placenta hydrolyzes triglycer-
ides and matemal phospholipids and synthesizes new
lipid materials to be used by the fetus. Large peptide
honnones such as thyroid stimulating hom1one, adrenal
cortical stimulating hormone, growth honnone, insu-
lin and glucagon do not cross the placenta. Smaller
molecular weight hormones such as steroids, thyroid
hormone and the catecholamines (epinephrine and
norepinephrine) cross the placenta with relative ease.
Vitamins and minerals are transfened to the fetus at

variable rates. Fat soluble vitamins do not cross the
placenta with ease, while water soluble vitamins (Band
K) pass across the placenta with relative ease. Nutrients
are also transferred by pinocytosis and phagocytosis.
Areolae from the chorion form over the openings of
the uterine glands and are thought to absorb secretions
from these glands.

Of significant importance is the ability of the
placenta to transfer toxic and potentially pathogenic ma-
terials. Many toxic substances easily cross the placental
banier. These include ethyl alcohol, lead, phosphorus
and mercmy. Also, opiate drugs and numerous common
phmmaceuticals such as barbiturates and antibiotics can
cross the placental banier. Some substances may be
highly teratogenic. Teratogenic means inducing ab-
normal development (birth defects). These substances
include LSD, amphetamines, lithium, diethylstilbestrol
and thalidomide. It is well documented that these ma-
terials induce abnormal embtyonic development and
cause serious birth defects.

It is known that a wide range of microorgan-
isms can contaminate the fetus. Viruses can cross the
placental banier with ease and thus many viral diseases
can be transmitted from the dam to the fetus. Such
human diseases as German measles, Herpes virus and
HIV can be transmitted from the pregnant mother to the
fetus. Bacteria such as syphilis can also be transmitted
to the fetus.

Figure 14-4. The Migration of Binucleate Giant Cells
in the Ruminant Placenta

r::

Fetal
(chorion)

Maternal
(endometrial
epithelium)

Basement membrane

Maternal circulation

Binucleate giant cells
(BNGC) migrate from
the chorion to the en-
dometrial epithelium
in ruminants. These
cells are thought to se-
crete placental lactogen
and pregnancy specific
protein B.

(www. biotracking. com)

Placentation, Gestation and Parturition 301

Figure 14-5. Placental Classification Based on Separation
Between Fetal and Maternal Blood Supplies

Fetal
(chorion)
Maternal
(endometrial
epithelium)

Fetal
(chori on)

Fetal
(chorion)

Maternal
(endometrium)

Epitheliochorial

Endotheliochorial

Hemochorial

Epithel iochorial
(pigs, horses and ruminants)

6. Chorionic capillaries
5. Chorionic interstitium
4. Chorionic epithelium
3. Endometrial epithelium
2. Endometrial interstitium
1. Endometrial capillaries

Endotheliochorial
(dogs and cats)

5. Chorionic capillaries
4. Chorionic interstitium
3. Chorionic epithelium
2. Endometrial interstitium
1. Endometrial capillaries

Hemochorial
(primates and rodents)

3. Chorionic capillaries
2. Chorionic interstitium
1. Chorionic epithelium
RBC= Red blood cell

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300 Placentation, Gestation and Parturition
Glucose is the major source of energy for the
fetus. The majority of glucose is derived from the ma-
temal circulation. Near the end of gestation, glucose
consumption by the fetus is exceptionally high and
can lead to a metabolic drain of glucose away from the
dam. Such a glucose drain favors the development of
ketosis in the dam. Ketosis results from the metabo-
lism of body fat that generate ketones for energy when
glucose is limited. Periparturient ketosis is common
in dairy cows where postpartum metabolic demands
are exceptionally high because of high milk produc-
tion. Some materials cannot be transported across the
placenta. With the exception of some immunoglobu-
lins, matemal proteins do not cross the placental banier.
Immunoglobulins can be transported from the matemal
to the fetal side in a hemochorial or an endotheliochorial
placenta. However, the fetus synthesizes the majority
of its own proteins from amino acids contributed by
the dam. Nutritionally-based lipids do not cross the
placenta. Instead, the placenta hydrolyzes triglycer-
ides and matemal phospholipids and synthesizes new
lipid materials to be used by the fetus. Large peptide
honnones such as thyroid stimulating hom1one, adrenal
cortical stimulating hormone, growth honnone, insu-
lin and glucagon do not cross the placenta. Smaller
molecular weight hormones such as steroids, thyroid
hormone and the catecholamines (epinephrine and
norepinephrine) cross the placenta with relative ease.
Vitamins and minerals are transfened to the fetus at
variable rates. Fat soluble vitamins do not cross the
placenta with ease, while water soluble vitamins (Band
K) pass across the placenta with relative ease. Nutrients
are also transferred by pinocytosis and phagocytosis.
Areolae from the chorion form over the openings of
the uterine glands and are thought to absorb secretions
from these glands.
Of significant importance is the ability of the
placenta to transfer toxic and potentially pathogenic ma-
terials. Many toxic substances easily cross the placental
banier. These include ethyl alcohol, lead, phosphorus
and mercmy. Also, opiate drugs and numerous common
phmmaceuticals such as barbiturates and antibiotics can
cross the placental banier. Some substances may be
highly teratogenic. Teratogenic means inducing ab-
normal development (birth defects). These substances
include LSD, amphetamines, lithium, diethylstilbestrol
and thalidomide. It is well documented that these ma-
terials induce abnormal embtyonic development and
cause serious birth defects.
It is known that a wide range of microorgan-
isms can contaminate the fetus. Viruses can cross the
placental banier with ease and thus many viral diseases
can be transmitted from the dam to the fetus. Such
human diseases as German measles, Herpes virus and
HIV can be transmitted from the pregnant mother to the
fetus. Bacteria such as syphilis can also be transmitted
to the fetus.
Figure 14-4. The Migration of Binucleate Giant Cells
in the Ruminant Placenta
r::
Fetal
(chorion)
Maternal
(endometrial
epithelium)
Basement membrane
Maternal circulation
Binucleate giant cells
(BNGC) migrate from
the chorion to the en-
dometrial epithelium
in ruminants. These
cells are thought to se-
crete placental lactogen
and pregnancy specific
protein B.
(www. biotracking. com)
Placentation, Gestation and Parturition 301
Figure 14-5. Placental Classification Based on Separation
Between Fetal and Maternal Blood Supplies
Fetal
(chorion)
Maternal
(endometrial
epithelium)
Fetal
(chori on)
Fetal
(chorion)
Maternal
(endometrium)
Epitheliochorial
Endotheliochorial
Hemochorial
Epithel iochorial
(pigs, horses and ruminants)
6. Chorionic capillaries
5. Chorionic interstitium
4. Chorionic epithelium
3. Endometrial epithelium
2. Endometrial interstitium
1. Endometrial capillaries
Endotheliochorial
(dogs and cats)
5. Chorionic capillaries
4. Chorionic interstitium
3. Chorionic epithelium
2. Endometrial interstitium
1. Endometrial capillaries
Hemochorial
(primates and rodents)
3. Chorionic capillaries
2. Chorionic interstitium
1. Chorionic epithelium
RBC= Red blood cell
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302 Placentation, Gestation and Parturition

The Placenta is a Major Endocrine Organ
During Pregnancy

In addition to serving as a metabolic exchange
organ, the placenta serves as a transitory endocrine or-
gan. Hormones from the placenta gain access to both
the fetal and the matemal circulation.

The placenta secretes hormones that can:
• stimulate ovarian function
• maintain pregnancy
• influence fetal

growth

• stimulate mammary function
• assist in parturition

The placenta of the mare produces a gonado-
tropin called equine chorionic gonadotropin (eCG).
Equine chorionic gonadotropin is also called pregnant
mare’s serum gonadotropin (PMSG). Equine cho-
rionic gonadotropin is produced by the endometrial
cups of the placenta. Endomeh·ial cups are a transient
placental endocrine gland. They begin producing eCG
at the time of attachment of the conceptus to the endo-
metrium. The relationship between the fom1ation of
the endometrial cups in the mare and the synthesis of
eCG is presented in Figure 14-6. As you can see, the
production of eCG is closely related to the weight of
the endometrial cups.

Equine chorionic gonadotropin acts as a lu-
teotropin and provides a stimulus for maintenance of the
primary cm·pus luteum. The primary corpus luteum
in the mare is defined as the corpus luteum fom1ed
from the ovulated follicle. In addition, eCG is respon-
sible for controlling the formation and maintenance of
supplementary (accessory) corpora lutca. As eCG
increases, the pregnant mare will often ovulate, thus
generating accessory corpora lutea. The eCG-induced
ovulations occur between days 40 and 70 of preg-
nancy. Luteinization (promoted by eCG) also occurs
in antral follicles that do not ovulate. Thus, eCG has a
significant positive impact on the ability of the ovary
to produce progesterone. Indeed, if one examines the
progesterone profile, it can be seen that there is a close
relationship between the concentrations of proges-
terone and the production of accessory corpora lutea
(See Figure 14-7).

In addition to its luteotropic action, eCG has
powerful FSH-like actions when administered to fe-
males of other species. In fact, eCG will cause marked
follicular development in most species. It is used com-
monly to induce superovulation where embryo transfer
is performed (cow, sheep, rabbit). In mares, however,
eCG does not exert significant FSH-like action.

–‘E
Db c .._,
l!l u
Cll

Figure 14-6. Production of
Equine Chorionic Gonadotropin
(eGG) is Closely Related to the

Weight of the
Endometrial Cups
(Modified from Ginther,

Reproductive Biologv of the Mare)

175 10

ISO 9

125 I 0
I
I
I 100 I 7
I
I

75 I 6 I
I
I

50 I 5

4 25 I —-
40 60 80 100 120 140 160 18 0 200

Days of Gestation

Endometrial cups (EC) are seen here in
a U-shaped configuration. The fetus (F)
is surrounded by the amnion (not visible).
The membrane indicated by arrows is the
allantochorion . This specimen was re-
moved from a mare at 50 days of gestation.
(Photograph courtesy of Dr. O.J. Ginther from Reproductive
Biology of the Mare. 2nd Ed.)

,…,
Ill a.
:I u
iii
‘i: …
Ill
E
0
‘C c w ….
0
…..
J:

Placentation, Gestation and Parturition 303

Figure 14-7. Luteal Progesterone Output During the First Half
of Gestation in the Mare

(Modified from Ginther, Reproductive Biologv of the Mare)

Progesterone { P4) from the primary corpus
luteum increases rapidly after ovulation and
then decreases (hatched region) . Without
eCG, P4 would continue to decrease {dashed
line) and the pregnancy would terminate.

Ill c
0 -:p
ns
:1.. ns -4J c c

:1.. Cl)
Cl) u

-4J c ns 0 :ru
Cl) Cl)
> c ·.p 0 ns :1..

Q) Cl) -4J cc: Ill Cl)
b.O
0
:1..

Q.

‘ ,,

Upon stimulation by eCG, the primary CL is
stimulated and P4 in the maternal blood again
increases. If eCG were not produced, P4
would continue to decrease (dashed line).

As eCG continues to increase, accessory CL
develop and P4 increases until about day 100.
After day 100, the placenta assumes the
major P4 producing ro le.

0 30 60 90 120 ISO 180 2 10 240 270

Days of Gestation

Figure 14-8. The Production of hCG and Progesterone During
Gestation in the Pregnant Woman

Human chorionic gonadotropin peaks at about 2.5
months of gestation and then declines. This period of
time is critical for maintenance of pregnancy because
the corpus luteum assumes primary responsibility for
progesterone secretion.

Ovarian P4

hCG

2 3 4

At about 2 .5 to 3 months of the placenta
begins to assume the primary responsibility for proges-
terone secretion and continues this role until the time of
parturition . hCG increases slightly between months 6
and 9 because of the increased placental mass.

Parturition
Placental P4

5 6 7 8 9

Months of Gestation

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‘ I I
302 Placentation, Gestation and Parturition
The Placenta is a Major Endocrine Organ
During Pregnancy
In addition to serving as a metabolic exchange
organ, the placenta serves as a transitory endocrine or-
gan. Hormones from the placenta gain access to both
the fetal and the matemal circulation.
The placenta secretes hormones that can:
• stimulate ovarian function
• maintain pregnancy
• influence fetal growth
• stimulate mammary function
• assist in parturition
The placenta of the mare produces a gonado-
tropin called equine chorionic gonadotropin (eCG).
Equine chorionic gonadotropin is also called pregnant
mare’s serum gonadotropin (PMSG). Equine cho-
rionic gonadotropin is produced by the endometrial
cups of the placenta. Endomeh·ial cups are a transient
placental endocrine gland. They begin producing eCG
at the time of attachment of the conceptus to the endo-
metrium. The relationship between the fom1ation of
the endometrial cups in the mare and the synthesis of
eCG is presented in Figure 14-6. As you can see, the
production of eCG is closely related to the weight of
the endometrial cups.
Equine chorionic gonadotropin acts as a lu-
teotropin and provides a stimulus for maintenance of the
primary cm·pus luteum. The primary corpus luteum
in the mare is defined as the corpus luteum fom1ed
from the ovulated follicle. In addition, eCG is respon-
sible for controlling the formation and maintenance of
supplementary (accessory) corpora lutca. As eCG
increases, the pregnant mare will often ovulate, thus
generating accessory corpora lutea. The eCG-induced
ovulations occur between days 40 and 70 of preg-
nancy. Luteinization (promoted by eCG) also occurs
in antral follicles that do not ovulate. Thus, eCG has a
significant positive impact on the ability of the ovary
to produce progesterone. Indeed, if one examines the
progesterone profile, it can be seen that there is a close
relationship between the concentrations of proges-
terone and the production of accessory corpora lutea
(See Figure 14-7).
In addition to its luteotropic action, eCG has
powerful FSH-like actions when administered to fe-
males of other species. In fact, eCG will cause marked
follicular development in most species. It is used com-
monly to induce superovulation where embryo transfer
is performed (cow, sheep, rabbit). In mares, however,
eCG does not exert significant FSH-like action.
–‘E
Db c .._,
l!l u
Cll
Figure 14-6. Production of
Equine Chorionic Gonadotropin
(eGG) is Closely Related to the
Weight of the
Endometrial Cups
(Modified from Ginther,
Reproductive Biologv of the Mare)
175 10
ISO 9
125 I 0
I
I
I 100 I 7
I
I
75 I 6 I
I
I
50 I 5
4 25 I —-
40 60 80 100 120 140 160 18 0 200
Days of Gestation
Endometrial cups (EC) are seen here in
a U-shaped configuration. The fetus (F)
is surrounded by the amnion (not visible).
The membrane indicated by arrows is the
allantochorion . This specimen was re-
moved from a mare at 50 days of gestation.
(Photograph courtesy of Dr. O.J. Ginther from Reproductive
Biology of the Mare. 2nd Ed.)
,…,
Ill a.
:I u
iii
‘i: …
Ill
E
0
‘C c w ….
0
…..
J:
Placentation, Gestation and Parturition 303
Figure 14-7. Luteal Progesterone Output During the First Half
of Gestation in the Mare
(Modified from Ginther, Reproductive Biologv of the Mare)
Progesterone { P4) from the primary corpus
luteum increases rapidly after ovulation and
then decreases (hatched region) . Without
eCG, P4 would continue to decrease {dashed
line) and the pregnancy would terminate.
Ill c
0 -:p
ns
:1.. ns -4J c c
:1.. Cl)
Cl) u
-4J c ns 0 :ru
Cl) Cl)
> c ·.p 0 ns :1..
Q) Cl) -4J cc: Ill Cl)
b.O
0
:1..
Q.
‘ ,,
Upon stimulation by eCG, the primary CL is
stimulated and P4 in the maternal blood again
increases. If eCG were not produced, P4
would continue to decrease (dashed line).
As eCG continues to increase, accessory CL
develop and P4 increases until about day 100.
After day 100, the placenta assumes the
major P4 producing ro le.
0 30 60 90 120 ISO 180 2 10 240 270
Days of Gestation
Figure 14-8. The Production of hCG and Progesterone During
Gestation in the Pregnant Woman
Human chorionic gonadotropin peaks at about 2.5
months of gestation and then declines. This period of
time is critical for maintenance of pregnancy because
the corpus luteum assumes primary responsibility for
progesterone secretion.
Ovarian P4
hCG
2 3 4
At about 2 .5 to 3 months of the placenta
begins to assume the primary responsibility for proges-
terone secretion and continues this role until the time of
parturition . hCG increases slightly between months 6
and 9 because of the increased placental mass.
Parturition
Placental P4
5 6 7 8 9
Months of Gestation
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14

304 Placentation, Gestation and Parturition

The second major gonadotropin of placental
origin is human chorionic gonadotropin (hCG). This
hormone is not only found in the human but in many
other primates. Often hCG (and eCG) may simply be
referred to as “CG”. It originates from the trophoblas-
tic cells of the chorion and is secreted as soon as the
blastocyst hatches from the zona pellucida. Human
chorionic gonadotropin can be detected in the blood
and urine of the pregnant woman as early as days 8 to
1 0 of gestation. It increases rapidly in the urine of the
pregnant woman, reaching a maximum value at about
2.5 months (See Figure 14-8). Its presence in the urine
constitutes the basis for over-the-counter pregnancy
diagnosis kits.

The primary role of hCG during early preg-
nancy is to provide a luteotropic stimulus for the
ovulatory corpus luteum as it transitions into the CL of
pregnancy. Luteal LH receptors also bind hCG resulting
in sustained progesterone production. Administration
of hCG to non-primate females can cause ovulation.
In fact, hCG is used commonly to induce ovulation in
superovulation protocols.

The Placenta Secretes

Progesterone

and

Estrogens

Progesterone is obligatory for early embry-
onic development because it provides the stimulus for
elevated secretion by the endometrial glands. High
progesterone is also responsible for the so-called “pro-
gesterone block” that inhibits myometrial

contractions.

Progesterone increases in the blood of the pregnant
female and peaks at different stages of gestation for
different species. The absolute levels of progesterone
also vary significantly among species (See Figure 14-9).
While progesterone is always produced by the corpus
luteum in early pregnancy, the role of the corpus luteum
in maintenance of pregnancy varies among species. In
some species (ewe, mare and woman), the corpus luteum
is not needed for the entire gestational period because
the placenta takes over production of progesterone. For
example, in the ewe the corpus 1uteum is responsible
for initial production of progesterone, but the placenta
assumes responsibility for its production after only 50
days of gestation (See Table 14-1 ). In other species
(sow or rabbit), lutectomy (surgical removal of corpora
lutea) will terminate pregnancy regardless of when this
occurs during gestation. Lutectomy in the cow up to
8 months of gestation will result in abortion. It should
be pointed out that even though the placenta takes over
for the corpus luteum of pregnancy, the corpus luteum
secretes progesterone throughout gestation.

In addition to progesterone, estradiol also is an
important product of the placenta, particularly during
the last part of gestation. In fact, the peak of estradiol
in most species signals the early preparttu·ient period.
The profiles of estradiol during gestation are presented
in the subsequent section on parhrrition.

Cea·tain Placental Hormones
Stimulate Mammaa·y Function of the Dam

and Fetal Growth

The placenta is known to produce a polypep-
tide hom1one known as placental lactogen that is also
called somatomammotropin. Placental lactogens
have been found in rats, mice, sheep, cows and humans.
They are believed to be similar to growth hormone, thus
promoting the growth of the fehts. Placental lactogen
also stimulates the mammary gland (lactogenic) of the
dam. The degree to which fetal somatotropic (growth)
versus lactogenic effects occur depends on the species
(See Figure 14-10). For example, in the ewe ovine
placental lactogen (oPL) has a more potent lactogenic
activity than somatotropic activity. A similar condition
exists in humans, but not in the cow. Placentallactogens
have been shtdied most intensely in the ewe. They are
produced and secreted by the binucleate giant cells of
the placenta. The secretory products of the binucleate
cells are transferred into the maternal circulation.

It is hypothesized that the sire may have an
effect on the degree to which the fehts can produce
placental lactogen. Such an effect could cause elevated
concentrations of placental lactogen by the ferns. In-
creased placental lactogen secretion would cause
enhanced stimulation of the maternal manunary gland
and thus promote elevated milk production. This theory
suggests that it might be possible for the sire to influence
fetal placental lactogen and enhance milk production in
the dam. This sire-on-fetus-hypothesis has not been
tested critically, but could hold promise for the genetic
improvement in dairy, beef cattle and goats.

Placental relaxin is secreted in humans, mares,
cats, dogs, pigs, rabbits and monkeys. Its function is to
cause softening and “relaxation” ofthe pelvic ligaments
to facilitate expulsion of the ferns. The stimulus for
relaxin secretion is not known. Relaxin is not present
in the bovine placenta during any stage of gestation. It
is likely (with the exception of the rabbit) that relaxin,
during the time of parrnrition, originates from both the
ovary and the placenta. The role of relaxin is therefore
questionable in the cow. Maternal blood relaxin levels
are the basis for a commercial pregnancy diagnostic test
at about 30 days of gestation in the bitch.

Placentation, Gestation and Parturition 305

Figure 14-9. Progesterone Profiles in Various Pregnant Females
so (P = Parturition) -E 40 -..

00
1: 30 –

“C 20
0
0
iil 10

t e 2 3 4 Months of Gestation

“1 – 100 E -..
00
1: – 20

“C
0

..5!
al 10

®
t e 2 4 6 Months of Gestation 8 10 II

14-1. Length and Time of Placental Takeover for Progesterone Production in
Vanous Spec1es

SPECIES

Alpaca
Bitch
Camel
Cow
Ewe
Goat
Llama
Mare
Queen
Rabbit
Sow
Woman

GESTATION
LENGTH

11.4 mo
2 mo (65 days)

12.3 mo
9 mo
5 rna
5 mo

11.3 mo
11 mo
2 mo (65 days)
1 mo

3.8 mo
9mo

TIME OF PLACENTAL
TAKEOVER

11.4 mo (none)
2mo (none)

12.3 mo (none)
6-8 mo

50 days
5 mo (none)

11.3 mo (none)
70 days

2 mo (none)
1 mo (none)

3.8 mo (none)
60-70 days

14
Ve
tB
oo
ks
.ir

14
304 Placentation, Gestation and Parturition
The second major gonadotropin of placental
origin is human chorionic gonadotropin (hCG). This
hormone is not only found in the human but in many
other primates. Often hCG (and eCG) may simply be
referred to as “CG”. It originates from the trophoblas-
tic cells of the chorion and is secreted as soon as the
blastocyst hatches from the zona pellucida. Human
chorionic gonadotropin can be detected in the blood
and urine of the pregnant woman as early as days 8 to
1 0 of gestation. It increases rapidly in the urine of the
pregnant woman, reaching a maximum value at about
2.5 months (See Figure 14-8). Its presence in the urine
constitutes the basis for over-the-counter pregnancy
diagnosis kits.
The primary role of hCG during early preg-
nancy is to provide a luteotropic stimulus for the
ovulatory corpus luteum as it transitions into the CL of
pregnancy. Luteal LH receptors also bind hCG resulting
in sustained progesterone production. Administration
of hCG to non-primate females can cause ovulation.
In fact, hCG is used commonly to induce ovulation in
superovulation protocols.
The Placenta Secretes Progesterone
and Estrogens
Progesterone is obligatory for early embry-
onic development because it provides the stimulus for
elevated secretion by the endometrial glands. High
progesterone is also responsible for the so-called “pro-
gesterone block” that inhibits myometrial contractions.
Progesterone increases in the blood of the pregnant
female and peaks at different stages of gestation for
different species. The absolute levels of progesterone
also vary significantly among species (See Figure 14-9).
While progesterone is always produced by the corpus
luteum in early pregnancy, the role of the corpus luteum
in maintenance of pregnancy varies among species. In
some species (ewe, mare and woman), the corpus luteum
is not needed for the entire gestational period because
the placenta takes over production of progesterone. For
example, in the ewe the corpus 1uteum is responsible
for initial production of progesterone, but the placenta
assumes responsibility for its production after only 50
days of gestation (See Table 14-1 ). In other species
(sow or rabbit), lutectomy (surgical removal of corpora
lutea) will terminate pregnancy regardless of when this
occurs during gestation. Lutectomy in the cow up to
8 months of gestation will result in abortion. It should
be pointed out that even though the placenta takes over
for the corpus luteum of pregnancy, the corpus luteum
secretes progesterone throughout gestation.
In addition to progesterone, estradiol also is an
important product of the placenta, particularly during
the last part of gestation. In fact, the peak of estradiol
in most species signals the early preparttu·ient period.
The profiles of estradiol during gestation are presented
in the subsequent section on parhrrition.
Cea·tain Placental Hormones
Stimulate Mammaa·y Function of the Dam
and Fetal Growth
The placenta is known to produce a polypep-
tide hom1one known as placental lactogen that is also
called somatomammotropin. Placental lactogens
have been found in rats, mice, sheep, cows and humans.
They are believed to be similar to growth hormone, thus
promoting the growth of the fehts. Placental lactogen
also stimulates the mammary gland (lactogenic) of the
dam. The degree to which fetal somatotropic (growth)
versus lactogenic effects occur depends on the species
(See Figure 14-10). For example, in the ewe ovine
placental lactogen (oPL) has a more potent lactogenic
activity than somatotropic activity. A similar condition
exists in humans, but not in the cow. Placentallactogens
have been shtdied most intensely in the ewe. They are
produced and secreted by the binucleate giant cells of
the placenta. The secretory products of the binucleate
cells are transferred into the maternal circulation.
It is hypothesized that the sire may have an
effect on the degree to which the fehts can produce
placental lactogen. Such an effect could cause elevated
concentrations of placental lactogen by the ferns. In-
creased placental lactogen secretion would cause
enhanced stimulation of the maternal manunary gland
and thus promote elevated milk production. This theory
suggests that it might be possible for the sire to influence
fetal placental lactogen and enhance milk production in
the dam. This sire-on-fetus-hypothesis has not been
tested critically, but could hold promise for the genetic
improvement in dairy, beef cattle and goats.
Placental relaxin is secreted in humans, mares,
cats, dogs, pigs, rabbits and monkeys. Its function is to
cause softening and “relaxation” ofthe pelvic ligaments
to facilitate expulsion of the ferns. The stimulus for
relaxin secretion is not known. Relaxin is not present
in the bovine placenta during any stage of gestation. It
is likely (with the exception of the rabbit) that relaxin,
during the time of parrnrition, originates from both the
ovary and the placenta. The role of relaxin is therefore
questionable in the cow. Maternal blood relaxin levels
are the basis for a commercial pregnancy diagnostic test
at about 30 days of gestation in the bitch.
Placentation, Gestation and Parturition 305
Figure 14-9. Progesterone Profiles in Various Pregnant Females
so (P = Parturition) -E 40 -..
00
1: 30 –
“C 20
0
0
iil 10
t e 2 3 4 Months of Gestation
“1 – 100 E -..
00
1: – 20
“C
0
..5!
al 10
®
t e 2 4 6 Months of Gestation 8 10 II
14-1. Length and Time of Placental Takeover for Progesterone Production in
Vanous Spec1es
SPECIES
Alpaca
Bitch
Camel
Cow
Ewe
Goat
Llama
Mare
Queen
Rabbit
Sow
Woman
GESTATION
LENGTH
11.4 mo
2 mo (65 days)
12.3 mo
9 mo
5 rna
5 mo
11.3 mo
11 mo
2 mo (65 days)
1 mo
3.8 mo
9mo
TIME OF PLACENTAL
TAKEOVER
11.4 mo (none)
2mo (none)
12.3 mo (none)
6-8 mo
50 days
5 mo (none)
11.3 mo (none)
70 days
2 mo (none)
1 mo (none)
3.8 mo (none)
60-70 days
14
Ve
tB
oo
ks
.ir

306 Placentation, Gestation and Parturition

Figure 14-10. Placental Lactogen in Blood Near
Termination of Gestation

(From Martal in Reproduction in Man and Mammals)
Woman

4000_1
‘5:b c – 600 c

CIJ

Q
Ewe

0 Somatotropic activity
0 Lactogenic activity

!)0
0 …. u
Ill

…J
iii …. c

CIJ u
Ill
0::

500
400
300
200
100 Cow

0
270 120 ISO 270

Day of Gestation

Parturition is a Complex Cascade of
Physiologic Events

Rat

12

The fetus triggers the onset of parh1rition by
initiating a cascade of complex endocrine/biochemical
events. The fetal hypothalamo-pihlitary-adrenal axis is
obligatory for the initiation of parturition. During the
conclusion of gestation, fetal mass approaches the in-
herent space limitations of the uterus. This space limita-
tion has been considered by some to be the stimulus that
causes adrenal corticotropin (ACTH) to be secreted
by the fetal pih1itary. The fetal pituitary then stimulates
secretion of adrenal corticoids from the fetal adrenal
cortex. The elevation of fetal corticoids initiates a
cascade of events that cause dramatic changes in the
endocrine condition of the dam. These endocrine
changes cause two major events to occur: 1) removal of
the myometrial “progesterone block,” enabling myome-
trial contractions to begin and 2) increased reproductive
tract secretions, particularly by the cervix.

The three stages of parturition are:
• I: initiation of myometrial

contractions (removal ofprogesterone
block)

• II: expulsion of the fetus

• III: expulsion of the fetal mebranes

Placental lactogen has both
lactogenic actions and soma-
totrophic actions. The lac-
togenic activity of placental
lactogen promotes mammary
function in the dam, while
the somatotropic activity
promotes fetal growth.

Removal of the “progesterone block” occurs
because fetal cortisol promotes the synthesis of three
enzymes that convert progesterone to estradiol. The
conversion pathway is illustrated in Figure 14-11.
Progesterone, that is high at the placental interface,
is converted to 17a-hydroxyprogesterone by the en-
zyme !?a-hydroxylase. Fetal cortisol also triggers
the enzyme 17-20 desmolase to convert 17a-hydroxy-
progesterone to androstenedione. Androstenedione is
converted to estrogen by activation of an aromatase
enzyme. This involves aromatization of the A ring of
the steroid and removal of the 19 carbon. The conver-
sion of progesterone to estradiol accounts, at least in
part, for the dramatic drop in progesterone and dramatic
elevation of estradiol. The relationship between pro-
gesterone and estradiol during gestation is presented
in Figure 14-12.

In addition to converting progesterone to es-
h·adiol, fetal corticoids also cause the placenta to syn-
thesize PGF2a.. The synthesis of PGF 2a helps abolish
the “progesterone block.” As both estradiol and prosta-
glandin become elevated, the myometrium becomes in-
creasingly more active and begins to display noticeable
contractions. Also, PGF 2a causes the CL of pregnancy
to regress, facilitating the decline in progesterone. The
drop in progesterone in some species is brought about
both by the conversion of progesterone into estradiol
and by the luteolytic process brought about by PGF2a·
Endocrine events associated with parhrrition are sum-
marized in Figures 14-13 and 14-14.

The fetus initiates Stage I of parturition.

Figure 14-11. Conversion of
Progesterone to Estradiol as

Parturition Nears
Corticoids from the fetus activate 17 a-hydroxylase,
17-20 desmolase and aromatase that convert
progesterone to estradio l. This conversion
removes the “progesterone block” to myometrial
activity.

17 a Hydroxyprogesterone

Androstenedione

CHJ
I

)

JJ-SD
CH1
I

· ‘ 1’
0

117: 20 I

l I Aromotase I
o)D'”

OH

. As the pressure inside the uterus continues to
mcrease, the feh1s in the cow, mare and ewe rotates so

the fi·ont feet and head are positioned to the poste-
of the dam (See Figure 14-15). Such a rotation is

tmportant to insure a proper delivery. If the fetus fails
to position itself correctly, dystocia (difficult birth)
may occur.

. As the levels of estradiol increase, coupled
With the e l_evation in levels of PGF2a , the contracting

begms to push the fetus toward the cervix, ap-
plymg pressure to the cervix. The endocrine events
that pro?1ote the firs t stage of parturition (dilation of
the cervtx and entry of the feh1s into the cervical canal)
are summarized in Figure 14-14.

Pressure ?n the cervix brought about by in-
myometnal contractions activates pressure-

sensttl_ve neurons located in the cervix that synapse in
the spmal cord and evenhmlly synapse with oxytocin

Ill

c
0

‘.P
1.’: …,
c
Q) v c

0 u

N w

“‘C c
Rl

Placentation, Gestation and Parturition 307

Figure 14-12. Estradiol and
Progesterone Profiles During
Gestation in the Mare, Cow,

Woman, Ewe and Sow
(P = Parturition)

Mare

I Woman I

p

I Sow I

t 10 20 30 40 so e Weel

Ve
tB
oo
ks
.ir

306 Placentation, Gestation and Parturition
Figure 14-10. Placental Lactogen in Blood Near
Termination of Gestation
(From Martal in Reproduction in Man and Mammals)
Woman
4000_1
‘5:b c – 600 c
CIJ
Q
Ewe
0 Somatotropic activity
0 Lactogenic activity
!)0
0 …. u
Ill
…J
iii …. c
CIJ u
Ill
0::
500
400
300
200
100 Cow
0
270 120 ISO 270
Day of Gestation
Parturition is a Complex Cascade of
Physiologic Events
Rat
12
The fetus triggers the onset of parh1rition by
initiating a cascade of complex endocrine/biochemical
events. The fetal hypothalamo-pihlitary-adrenal axis is
obligatory for the initiation of parturition. During the
conclusion of gestation, fetal mass approaches the in-
herent space limitations of the uterus. This space limita-
tion has been considered by some to be the stimulus that
causes adrenal corticotropin (ACTH) to be secreted
by the fetal pih1itary. The fetal pituitary then stimulates
secretion of adrenal corticoids from the fetal adrenal
cortex. The elevation of fetal corticoids initiates a
cascade of events that cause dramatic changes in the
endocrine condition of the dam. These endocrine
changes cause two major events to occur: 1) removal of
the myometrial “progesterone block,” enabling myome-
trial contractions to begin and 2) increased reproductive
tract secretions, particularly by the cervix.
The three stages of parturition are:
• I: initiation of myometrial
contractions (removal ofprogesterone
block)
• II: expulsion of the fetus
• III: expulsion of the fetal mebranes
Placental lactogen has both
lactogenic actions and soma-
totrophic actions. The lac-
togenic activity of placental
lactogen promotes mammary
function in the dam, while
the somatotropic activity
promotes fetal growth.
Removal of the “progesterone block” occurs
because fetal cortisol promotes the synthesis of three
enzymes that convert progesterone to estradiol. The
conversion pathway is illustrated in Figure 14-11.
Progesterone, that is high at the placental interface,
is converted to 17a-hydroxyprogesterone by the en-
zyme !?a-hydroxylase. Fetal cortisol also triggers
the enzyme 17-20 desmolase to convert 17a-hydroxy-
progesterone to androstenedione. Androstenedione is
converted to estrogen by activation of an aromatase
enzyme. This involves aromatization of the A ring of
the steroid and removal of the 19 carbon. The conver-
sion of progesterone to estradiol accounts, at least in
part, for the dramatic drop in progesterone and dramatic
elevation of estradiol. The relationship between pro-
gesterone and estradiol during gestation is presented
in Figure 14-12.
In addition to converting progesterone to es-
h·adiol, fetal corticoids also cause the placenta to syn-
thesize PGF2a.. The synthesis of PGF 2a helps abolish
the “progesterone block.” As both estradiol and prosta-
glandin become elevated, the myometrium becomes in-
creasingly more active and begins to display noticeable
contractions. Also, PGF 2a causes the CL of pregnancy
to regress, facilitating the decline in progesterone. The
drop in progesterone in some species is brought about
both by the conversion of progesterone into estradiol
and by the luteolytic process brought about by PGF2a·
Endocrine events associated with parhrrition are sum-
marized in Figures 14-13 and 14-14.
The fetus initiates Stage I of parturition.
Figure 14-11. Conversion of
Progesterone to Estradiol as
Parturition Nears
Corticoids from the fetus activate 17 a-hydroxylase,
17-20 desmolase and aromatase that convert
progesterone to estradio l. This conversion
removes the “progesterone block” to myometrial
activity.
17 a Hydroxyprogesterone
Androstenedione
CHJ
I
)
JJ-SD
CH1
I
· ‘ 1’
0
117: 20 I
l I Aromotase I
o)D'”
OH
. As the pressure inside the uterus continues to
mcrease, the feh1s in the cow, mare and ewe rotates so
the fi·ont feet and head are positioned to the poste-
of the dam (See Figure 14-15). Such a rotation is
tmportant to insure a proper delivery. If the fetus fails
to position itself correctly, dystocia (difficult birth)
may occur.
. As the levels of estradiol increase, coupled
With the e l_evation in levels of PGF2a , the contracting
begms to push the fetus toward the cervix, ap-
plymg pressure to the cervix. The endocrine events
that pro?1ote the firs t stage of parturition (dilation of
the cervtx and entry of the feh1s into the cervical canal)
are summarized in Figure 14-14.
Pressure ?n the cervix brought about by in-
myometnal contractions activates pressure-
sensttl_ve neurons located in the cervix that synapse in
the spmal cord and evenhmlly synapse with oxytocin
Ill c
0
‘.P
1.’: …,
c
Q) v c
0 u
N w
“‘C c
Rl
Placentation, Gestation and Parturition 307
Figure 14-12. Estradiol and
Progesterone Profiles During
Gestation in the Mare, Cow,
Woman, Ewe and Sow
(P = Parturition)
Mare
I Woman I
p
I Sow I
t 10 20 30 40 so e Weel308 Placentation, Gestation and Parturition

producing neurons in the hypothalamus (See Figure
14- I 5). Oxytocin, released into the systemic circula-
tion, acts to facilitate the myomeh·ial contractility
initiated by estradiol and by PGF2u· As the pressure
against the cervix continues to increase, so does the
oxytocin secretion, and thus the force of conh·action of
the myometrial smooth muscle begins to peak. When
this occurs, the fetus enters the cervical canal and the
first stage of parturition is complete.

Expulsion of fetus (Stage II) requires
strong myometrial and abdominal

muscle contractions.

Another important hormone involved in suc-
cessful parhrrition is relaxin. Relaxin is a glycopro-
tein that is produced by either the corpus luteum or the
placenta, depending upon the species. The synthesis
of relaxin is stimulated by PGF2a · Relaxin causes a
softening of the connective tissue in the cervix and
promotes elasticity of the pelvic ligaments. Thus,
this hormone prepares the birth canal by loosening
the supportive tissues so that passage of the fehts can
occur with relative ease.

One of the dramatic effects of estradiol
elevation prior to parturition is that it initiates secre-
tory activity of the reproductive tract in general and
particularly the cervix. As estradiol increases, the
cervix and vagina begin to produce mucus. This
mucus washes out the cervical seal of pregnancy
and thoroughly lubricates the cervical canal and the
vagina. Mucus reduces friction and enables the fetus
to exit the reproductive tract with relative ease. As
myometrial contractions continue to increase, the
feet and head of the fehts begin to put pressure on the
fetal membranes. When the pressure reaches a certain
level, the membranes rupture, with subsequent loss of
amniotic and allantoic fluid. This fluid also serves to
lubricate the birth canal. As the fetus enters the birth
canal, it becomes hypoxic (deprived of adequate levels
of oxygen). This hypoxia promotes fetal movement
that, in tum, promotes further myometrial contrac-
tion. This positive feedback system creates a set of
conditions where the time of parhtrition is reduced
because an increased strength of contraction follows
fetal movement. In a sense, the fehts is controlling
its exit from the uterus. The uterine contractions are
accompanied by abdominal muscle contractions of the
dam that further aid in expulsion of the fetus.

VI
1:
0

“” “‘”‘ 1:
Ql u
1:
0 u
Ql
1:
0
E
“” 0 :r:
Ql >

1i r:x:

Figure 14-13. Relative
Hormone Profiles in the Cow

During the Periparturient Period

Estrogens

I Prostaglandin

-I 0 -B -6 -4 -3 -2 -I 0 I 2 3 4 5
t Parturition

Days

Note that as fetal
cortisol levels rise,
P4 levels fall.

In most species, expulsion of the fetal mem-
branes quickly follows expulsion of the fetus. Expulsion
of the fetal membranes requires that the chorionic villi
become dislodged from the crypts of the matemal side
of the placenta. This release of the chorionic villi is
believed to be brought about by powerful vasoconstric-
tion of arteries in the villi. Vasoconstriction reduces
pressure and thus allows the villi to be released from
the crypts. Obviously in some fonns of placentation,
there must be some maternal vasoconsh·iction. For ex-
ample, in animals that have hemochorial placentation,
matemal blood is adjacent to the fetal placenta. Thus,
if vasoconstriction does not occur on the matemal side,
hemorrhage is likely.

The duration of parhlrition is variable among
species and this variation is summarized in Table 14-2.
Extension beyond what is considered to be the normal
upper-end duration of parturition constitutes a difficult
birth (dystocia). Such prolonged parturition can result
in serious complications to both the fetus and the dam.

Placentation, Gestation and Parturition 309

Figure 14-14. Cascade of Events Prompted by Fetal Cortisol

f t FETAL ACTH
f /I Fetal cortisol j \

Placental P4 Relaxin enzymes [!iJ I PGF2a I …,.I
.———–. t / t ….._____+ -l

I Luteolysis t Secretion by
<;;?tract

Lubrication

t Myometrial
contractions

I+ Pressure
f

t Cervical
stimulation

t Oxytocin

t
Maximum
pressure

Pelvic
ligament
stretching

Ve
tB
oo
ks
.ir

14
308 Placentation, Gestation and Parturition
producing neurons in the hypothalamus (See Figure
14- I 5). Oxytocin, released into the systemic circula-
tion, acts to facilitate the myomeh·ial contractility
initiated by estradiol and by PGF2u· As the pressure
against the cervix continues to increase, so does the
oxytocin secretion, and thus the force of conh·action of
the myometrial smooth muscle begins to peak. When
this occurs, the fetus enters the cervical canal and the
first stage of parturition is complete.
Expulsion of fetus (Stage II) requires
strong myometrial and abdominal
muscle contractions.
Another important hormone involved in suc-
cessful parhrrition is relaxin. Relaxin is a glycopro-
tein that is produced by either the corpus luteum or the
placenta, depending upon the species. The synthesis
of relaxin is stimulated by PGF2a · Relaxin causes a
softening of the connective tissue in the cervix and
promotes elasticity of the pelvic ligaments. Thus,
this hormone prepares the birth canal by loosening
the supportive tissues so that passage of the fehts can
occur with relative ease.
One of the dramatic effects of estradiol
elevation prior to parturition is that it initiates secre-
tory activity of the reproductive tract in general and
particularly the cervix. As estradiol increases, the
cervix and vagina begin to produce mucus. This
mucus washes out the cervical seal of pregnancy
and thoroughly lubricates the cervical canal and the
vagina. Mucus reduces friction and enables the fetus
to exit the reproductive tract with relative ease. As
myometrial contractions continue to increase, the
feet and head of the fehts begin to put pressure on the
fetal membranes. When the pressure reaches a certain
level, the membranes rupture, with subsequent loss of
amniotic and allantoic fluid. This fluid also serves to
lubricate the birth canal. As the fetus enters the birth
canal, it becomes hypoxic (deprived of adequate levels
of oxygen). This hypoxia promotes fetal movement
that, in tum, promotes further myometrial contrac-
tion. This positive feedback system creates a set of
conditions where the time of parhtrition is reduced
because an increased strength of contraction follows
fetal movement. In a sense, the fehts is controlling
its exit from the uterus. The uterine contractions are
accompanied by abdominal muscle contractions of the
dam that further aid in expulsion of the fetus.
VI
1:
0
“” “‘”‘ 1:
Ql u
1:
0 u
Ql
1:
0
E
“” 0 :r:
Ql >
1i r:x:
Figure 14-13. Relative
Hormone Profiles in the Cow
During the Periparturient Period
Estrogens
I Prostaglandin
-I 0 -B -6 -4 -3 -2 -I 0 I 2 3 4 5
t Parturition
Days
Note that as fetal
cortisol levels rise,
P4 levels fall.
In most species, expulsion of the fetal mem-
branes quickly follows expulsion of the fetus. Expulsion
of the fetal membranes requires that the chorionic villi
become dislodged from the crypts of the matemal side
of the placenta. This release of the chorionic villi is
believed to be brought about by powerful vasoconstric-
tion of arteries in the villi. Vasoconstriction reduces
pressure and thus allows the villi to be released from
the crypts. Obviously in some fonns of placentation,
there must be some maternal vasoconsh·iction. For ex-
ample, in animals that have hemochorial placentation,
matemal blood is adjacent to the fetal placenta. Thus,
if vasoconstriction does not occur on the matemal side,
hemorrhage is likely.
The duration of parhlrition is variable among
species and this variation is summarized in Table 14-2.
Extension beyond what is considered to be the normal
upper-end duration of parturition constitutes a difficult
birth (dystocia). Such prolonged parturition can result
in serious complications to both the fetus and the dam.
Placentation, Gestation and Parturition 309
Figure 14-14. Cascade of Events Prompted by Fetal Cortisol
f t FETAL ACTH
f /I Fetal cortisol j \
Placental P4 Relaxin enzymes [!iJ I PGF2a I …,.I
.———–. t / t ….._____+ -l
I Luteolysis t Secretion by
<;;?tract Lubrication t Myometrial contractions I+ Pressure f t Cervical stimulation t Oxytocin t Maximum pressure Pelvic ligament stretching Ve tB oo ks .ir

31 0 Placentation, Gestation and Parturition

Figure Pressure on the Cervix Causes Oxytocin Release and
Subsequent Myometrial Contractions

As the fetus moves through the
birth canal , elevated pressure
on the cervix stimulates sensory
neurons. A neural pathway ter-
minates in the paraventricular nu-
cleus (PVN) and causes
to be secreted from the postenor
pituitary lobe. Oxytocin
contraction of the myometnum.

Afferent
neurons

Hypothalamus

Difficulties in parturi tion usually occur in the second
stage (expulsion of the fetus). One cause of dystocia
is excessive size of the fetus. Fetal size is controlled
by both the dam and the sire. In primiparous dams, it
is always advisable to breed females to a male of small
body size so that fetal size does not exceed the ability
of the female to give birth successfully.

A second cause of dystocia is failure of proper
fetal rotation. About 5% of all births in cattle are char-
acterized by abnormal positioning of the fetus during
parturition. Such abnormal positioning results in dif-
ficult births and sometimes impossible presentations/
positions that require caesarean section.

A third cause of dystocia is multiple births in
monotocous species. Twins generally cause dystocia.
This is because: 1) both twins may be presented simul-
taneously, 2) the first fetus is positioned abnommlly and
therefore blocks the second or 3) the uterus becomes
fatigued by difficult and sustained contractions. A dis-
cussion of obstetrical procedures used to correct these
problems is beyond the scope of this book, but c …. atfbe
researched by consulting the appropriate references at
the conclusion of this chapter.

Placentation, Gestation and Parturition 311

Expulsion of fetal membranes
(Stage III) requires myometrial

contractions.

Myometrial contractions continue after expul-
sion of the fetus although they are not as strong. These
contractions are responsible for expelling the placenta.
The time required for expulsion of the placenta varies
significantly among species. This variation is presented
in Table 14-2. Retention of the fetal membranes (also
referred to as “retained placenta”), is not uncommon in
ruminants, especially dairy cows. This condition will
occur in 5-15% of parturitions in healthy dairy cows.
The underlying cause of retained placenta appears to
be that placental connective tissue is not enzymatically
degraded by cotyledonary proteolytic enzymes. Thus,
fetal cotyledons remain attached to matemal cotyledons.
Retained placenta is rare is mares, sows, bitches and
queens.

Table 14-2. Stages and Duration of Parturition Among Various

Species

Stage I Stage II Stage III
(Mllometrial Contractions/ (Fetal (Fetal Membrane

Cervical Dilation)

Alpaca 2 to 6h 5 to 90 min 45 to 180 min

Bitch 6 to 12h 6h (24h in large litters) most placentas pass with
neonate or within 15 min
of birth

Camel 3 to 48h 5 to 45 min 40 min

Cow 2 to 6h 30 to 60 min 6 to 12h

Ewe 2 to 6h 30 to 120 min 5 to 8h

Llama 2 to 6h 5 to 90 min 45 to 180 min

Mare 1 to 4h 12 to 30 min 1h

Sow 2 to 12h 150 to 180 min 1 to 4h

Queen 4 to 42h 4 kittens/litter, most placentas pass with
30-60 min/kitten neonate

Woman 8+h 2h 1h or less

14
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31 0 Placentation, Gestation and Parturition
Figure Pressure on the Cervix Causes Oxytocin Release and
Subsequent Myometrial Contractions
As the fetus moves through the
birth canal , elevated pressure
on the cervix stimulates sensory
neurons. A neural pathway ter-
minates in the paraventricular nu-
cleus (PVN) and causes
to be secreted from the postenor
pituitary lobe. Oxytocin
contraction of the myometnum.
Afferent
neurons
Hypothalamus
Difficulties in parturi tion usually occur in the second
stage (expulsion of the fetus). One cause of dystocia
is excessive size of the fetus. Fetal size is controlled
by both the dam and the sire. In primiparous dams, it
is always advisable to breed females to a male of small
body size so that fetal size does not exceed the ability
of the female to give birth successfully.
A second cause of dystocia is failure of proper
fetal rotation. About 5% of all births in cattle are char-
acterized by abnormal positioning of the fetus during
parturition. Such abnormal positioning results in dif-
ficult births and sometimes impossible presentations/
positions that require caesarean section.
A third cause of dystocia is multiple births in
monotocous species. Twins generally cause dystocia.
This is because: 1) both twins may be presented simul-
taneously, 2) the first fetus is positioned abnommlly and
therefore blocks the second or 3) the uterus becomes
fatigued by difficult and sustained contractions. A dis-
cussion of obstetrical procedures used to correct these
problems is beyond the scope of this book, but c …. atfbe
researched by consulting the appropriate references at
the conclusion of this chapter.
Placentation, Gestation and Parturition 311
Expulsion of fetal membranes
(Stage III) requires myometrial
contractions.
Myometrial contractions continue after expul-
sion of the fetus although they are not as strong. These
contractions are responsible for expelling the placenta.
The time required for expulsion of the placenta varies
significantly among species. This variation is presented
in Table 14-2. Retention of the fetal membranes (also
referred to as “retained placenta”), is not uncommon in
ruminants, especially dairy cows. This condition will
occur in 5-15% of parturitions in healthy dairy cows.
The underlying cause of retained placenta appears to
be that placental connective tissue is not enzymatically
degraded by cotyledonary proteolytic enzymes. Thus,
fetal cotyledons remain attached to matemal cotyledons.
Retained placenta is rare is mares, sows, bitches and
queens.
Table 14-2. Stages and Duration of Parturition Among Various Species
Stage I Stage II Stage III
(Mllometrial Contractions/ (Fetal (Fetal Membrane
Cervical Dilation)
Alpaca 2 to 6h 5 to 90 min 45 to 180 min
Bitch 6 to 12h 6h (24h in large litters) most placentas pass with
neonate or within 15 min
of birth
Camel 3 to 48h 5 to 45 min 40 min
Cow 2 to 6h 30 to 60 min 6 to 12h
Ewe 2 to 6h 30 to 120 min 5 to 8h
Llama 2 to 6h 5 to 90 min 45 to 180 min
Mare 1 to 4h 12 to 30 min 1h
Sow 2 to 12h 150 to 180 min 1 to 4h
Queen 4 to 42h 4 kittens/litter, most placentas pass with
30-60 min/kitten neonate
Woman 8+h 2h 1h or less
14
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14

312 Placentation, Gestation and Parturition

Further
PHENOMENA
for Fertility

The term “caesarean” was derived from the
false notion that Julius Caesar was born by
removing him from his mother through an
incision in the abdominal and uterine wall.
His family name, Caesar was derived from
the belief that Julius’ ancestors (centuries
before him) were hom in such a way. The
name Caesar is derived from the Latin word
“caesus” that means “to cut”. The name also
fits the way Julius died.

In a number of teleost fishes (fishes with a
more or less ossified skeleton) the female
incubates the eggs in her mouth and in some
species the male does the same. The term
“keep your mouth shut” has a special meaning
in this species.

In pipe fishes and sea horses the female lays
her eggs in a brood pouch of the male and he
is responsible for gestation. In fact, several
females may lay eggs in one male’s brood
pouch. The brood pouch offers a special
environment for developing offspring and is
under the control of prolactin.

Lampreys (a predatory eel) build nests in
sandy bottomed sh·eams. They assemble rock
walls to slow the water running over the nest.
At spawning, they stir up the sand that sticks
to the eggs. The sand weights the eggs and
prevents them from floating downstream. It
also reduces predation. This is mwtherform
of attachment that enables successful embryo-
genesis.

Infant kangaroos in their mother’s pouches
nurse from two nipples, and two babies of
different ages commonly nurse at the same
time. So, the mother kangaroo produces two
kinds of milk- on one side, fully rich for the
younger and 011 the other side, a sort of skim
for the elder.

The most prolific mammal in existence is the
tiny J’Odent known as the multimammate rat.
One female is capable of producing up to 120
offspring a year if conditions are favorable.
This is because she has 24 teats, the most of
any female mammal. It is rare that all of them
are used but when they are a multimammate
population explosion catt occur.

The female Egyptian spiny mouse acts as a
midwife to other females. She bites through the
umbilical cord and licks the neonates while the
mother continues to deliver the litter.

The female African elephant has a gestation
period of 1.8 years. The calf weighs about
300 pounds at birth and nurses for about three
years.

Durittg the 19th Century, adultery was so
feared that the chastity belt was invented. Such
belts were devices that were locked around the

genitalia to prevent copulation. It has
been recorded that afaitliful wife locked into a
chastity belt discovered that she was pregnant
some months after her husband had left 011 a
crusade. Her husband had the o11ly key. Her
pregnancy progressed and eventually the vil-
lage blacksmith had to be called in to remove
the chastity belt.

During the Middle Ages, prostitution was
considered to he an honest and essential pro-
fession. This was because prostitution was
considered as a means to prevellt adulte1y,
homosexual behavior and masturbation. The
Church actually condoned prostitution for tlzis
reason.

The Mayans believed in a maize god. Since
corn was a nutritional staple for these people,
they revered it and believed that corn was
symbolic of both the male am/female. From a
nutritional perspective they believed that corn
was nurturing like a breast and that
each individual kernel had powerful fertilizing
capabilities like spermatozoa. Once the seeds
were planted in the earth and the mature com
was produced, the cob represented the penis
and the husk represented the vagina. Thus, the
ear of com was also symbolic of copulation.

Kev References

Arthur, G.H., D.E. Noakes, H. Pearson and T.J. Parkin-
son. 1996. Veterinarv Reproduction and Obstetrics.
7th Edition. W.B. Saunders Co. Philadelphia. ISBN
0-7020-1 758-X.

Catchpole, H.R. 1991. “Hormonal mechanisms in
pregnancy and parturition” in Reproduction in Domestic
Animals. 4th Edition. P.T. Cupps, ed., Academic Press,
San Diego. ISBN 0-1 2-196575-9.

Flood, P.F. 199 I. “The development of the conceptus
and its relationship to the uterus” in Reproduction in
Domestic Animals. 4th Edition. P.T. Cupps, ed., Aca-
demic Press, San Diego. ISBN 0-1 2-196575-9.

Fuchs, A.R. and M.J. Fields. 1999. “Parturition, no.!Jhtl-
man mammals” in Encvclopedia o(Reproducilon: Vol.
3 p703-7 I 6. Knobil , E. and J.D . Neill, eds. Academic
Press, San Diego. ISBN 0- 12-227023- 1.

Ginther, OJ . 1992. Reproductive Biology o{the Mare.
2nd Edition. Equiservices, Cross Plains, WI. Library of
Congress Catalog No. 9 1-075595.

Johnston, S.D. M.V. Root, Kustritz and P.N.S. Olson.
200 I. Canine and Feline Theriogenologv. W.B. Saun-
ders, Philadelphia. ISBN 0-7216-5607-2.

Morrow, D.A. 1986. Current Therapy in Theriogenol-
2nd Edition. W.B. Saunders Co. Philadelphia.

ISBN 0-7216-6580-2.

Mossman, H.W. 1987. Vertebrate Fetal Membranes.
Rutgers University Press, New Brunsw ick. ISBN
0-8135-1132-1.

Thibault, C., M.C. Levasseur and R.H.F. Hunter.eds.
I 993. Reproduction in Man and Mammals. Ellipses,
Paris. ISBN 2-7298-9354-7.

Placentation, Gestation and Parturition 313

14
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14
312 Placentation, Gestation and Parturition
Further
PHENOMENA
for Fertility
The term “caesarean” was derived from the
false notion that Julius Caesar was born by
removing him from his mother through an
incision in the abdominal and uterine wall.
His family name, Caesar was derived from
the belief that Julius’ ancestors (centuries
before him) were hom in such a way. The
name Caesar is derived from the Latin word
“caesus” that means “to cut”. The name also
fits the way Julius died.
In a number of teleost fishes (fishes with a
more or less ossified skeleton) the female
incubates the eggs in her mouth and in some
species the male does the same. The term
“keep your mouth shut” has a special meaning
in this species.
In pipe fishes and sea horses the female lays
her eggs in a brood pouch of the male and he
is responsible for gestation. In fact, several
females may lay eggs in one male’s brood
pouch. The brood pouch offers a special
environment for developing offspring and is
under the control of prolactin.
Lampreys (a predatory eel) build nests in
sandy bottomed sh·eams. They assemble rock
walls to slow the water running over the nest.
At spawning, they stir up the sand that sticks
to the eggs. The sand weights the eggs and
prevents them from floating downstream. It
also reduces predation. This is mwtherform
of attachment that enables successful embryo-
genesis.
Infant kangaroos in their mother’s pouches
nurse from two nipples, and two babies of
different ages commonly nurse at the same
time. So, the mother kangaroo produces two
kinds of milk- on one side, fully rich for the
younger and 011 the other side, a sort of skim
for the elder.
The most prolific mammal in existence is the
tiny J’Odent known as the multimammate rat.
One female is capable of producing up to 120
offspring a year if conditions are favorable.
This is because she has 24 teats, the most of
any female mammal. It is rare that all of them
are used but when they are a multimammate
population explosion catt occur.
The female Egyptian spiny mouse acts as a
midwife to other females. She bites through the
umbilical cord and licks the neonates while the
mother continues to deliver the litter.
The female African elephant has a gestation
period of 1.8 years. The calf weighs about
300 pounds at birth and nurses for about three
years.
Durittg the 19th Century, adultery was so
feared that the chastity belt was invented. Such
belts were devices that were locked around the
genitalia to prevent copulation. It has
been recorded that afaitliful wife locked into a
chastity belt discovered that she was pregnant
some months after her husband had left 011 a
crusade. Her husband had the o11ly key. Her
pregnancy progressed and eventually the vil-
lage blacksmith had to be called in to remove
the chastity belt.
During the Middle Ages, prostitution was
considered to he an honest and essential pro-
fession. This was because prostitution was
considered as a means to prevellt adulte1y,
homosexual behavior and masturbation. The
Church actually condoned prostitution for tlzis
reason.
The Mayans believed in a maize god. Since
corn was a nutritional staple for these people,
they revered it and believed that corn was
symbolic of both the male am/female. From a
nutritional perspective they believed that corn
was nurturing like a breast and that
each individual kernel had powerful fertilizing
capabilities like spermatozoa. Once the seeds
were planted in the earth and the mature com
was produced, the cob represented the penis
and the husk represented the vagina. Thus, the
ear of com was also symbolic of copulation.
Kev References
Arthur, G.H., D.E. Noakes, H. Pearson and T.J. Parkin-
son. 1996. Veterinarv Reproduction and Obstetrics.
7th Edition. W.B. Saunders Co. Philadelphia. ISBN
0-7020-1 758-X.
Catchpole, H.R. 1991. “Hormonal mechanisms in
pregnancy and parturition” in Reproduction in Domestic
Animals. 4th Edition. P.T. Cupps, ed., Academic Press,
San Diego. ISBN 0-1 2-196575-9.
Flood, P.F. 199 I. “The development of the conceptus
and its relationship to the uterus” in Reproduction in
Domestic Animals. 4th Edition. P.T. Cupps, ed., Aca-
demic Press, San Diego. ISBN 0-1 2-196575-9.
Fuchs, A.R. and M.J. Fields. 1999. “Parturition, no.!Jhtl-
man mammals” in Encvclopedia o(Reproducilon: Vol.
3 p703-7 I 6. Knobil , E. and J.D . Neill, eds. Academic
Press, San Diego. ISBN 0- 12-227023- 1.
Ginther, OJ . 1992. Reproductive Biology o{the Mare.
2nd Edition. Equiservices, Cross Plains, WI. Library of
Congress Catalog No. 9 1-075595.
Johnston, S.D. M.V. Root, Kustritz and P.N.S. Olson.
200 I. Canine and Feline Theriogenologv. W.B. Saun-
ders, Philadelphia. ISBN 0-7216-5607-2.
Morrow, D.A. 1986. Current Therapy in Theriogenol-
2nd Edition. W.B. Saunders Co. Philadelphia.
ISBN 0-7216-6580-2.
Mossman, H.W. 1987. Vertebrate Fetal Membranes.
Rutgers University Press, New Brunsw ick. ISBN
0-8135-1132-1.
Thibault, C., M.C. Levasseur and R.H.F. Hunter.eds.
I 993. Reproduction in Man and Mammals. Ellipses,
Paris. ISBN 2-7298-9354-7.
Placentation, Gestation and Parturition 313
14
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Parturition

Fetal Attachment & Gestation

Early Embryogenesis &
Maternal Recognition of Pregnancy
Ovulation & Fertilization
Cyclicity
Regulation of
Reproduction
Tract Function
Puberty
Prenatal
Development
Spermatogenesis
Regulation of
Reproduction
Tract Function
Puberty
Prenatal
Development

Take Home Message
Immediately following parturition, the female hegins to lactate and enters a period

of reproductive repair called the pue1perium. For a period of time these two processes
overlap. During the puerperium uterine involution and return of ovarian function oc-
curs. Involution is the reduction in size and “remodeling” of the endometrium so that
the uterus can initiate and sustain another pregnancy.

Mammary gland development is initiated prenatally in the f emale fetus and con-
tinues through puberty and pregnancy. The anatomy and distribution of mammary
glands is diverse among mammals. Accumulation of secretions in the mammmy gland
hegins about two weeks before parturition. Lactation provides the neonate with the
opportunity to nurse and he nourished with minimal expenditure of energy. It also
provides immunoprotection for the neonate because initialmammmy secretions called
colostrum contain antibodies that provide passive immunity. Lactation continues until
the neonate is weaned. After weaning, the mammary glands undergo involution and
retum to a non-secretory state.

The puerperium and lactation are. init iated
immediately after parturition and for a period of time
these processes occur simultaneously. Lactation is
the synthesis, secretion and removal of milk from the
mammary gland. The puerperium is the period after
parturition when the reproductive tract retums to its
nonpregnant condition so that the female may become
pregnant again. This chapter will describe the basics
of these two important processes. Parturition results in
loss of placental function and deterioration of the mater-
nal tissue contributing to the placenta. Tissue damage
results . During the puerperium damaged reproductive
tissues are repaired and ovarian function returns.

The Puerperium

The puerperium begins immediately after par-
turition and lasts until reproductive function is restored
so that another pregnancy can occur. The time required
for complete uterine involution (repair) and ovarian
activity to resume in the postpartum female varies sig-
nificantly among species (See Table 15-1 ).

The four major events of the puerperium
are:
• myometrial contractions and expulsion
of lochia

• endometria/repair
• resumption of ovarian function
• elimination of bacterial contamination
of the reproductive tract

It must be emphasized that in many polyestrous
animals, the shortest possible puerperium is desirable
because eligibility for a subsequent pregnancy is of
high economic importance. For example, in dairy
cows frequent pregnancies are required for maximum
lifetime milk yield. In swine and beef cows, the shorter
the interval between pregnancies the more offspring are
produced and the more efficient the production of meat
becomes. Conversely, the longer the puerperium, the
longer the delay of a subsequent pregnancy and the less
efficient the production process becomes. Figure 15-1
summarizes the events that occur from parturition to the
subsequent pregnancy. These events will be described
in more detail below.

Reduction in Uterine Size and Volume is
Brought About by Myometrial Contractions

Immediately after parturition, the myome-
trium undergoes strong repeated contractions. The
purpose of these contractions is threefold. First, they
facil itate discharge of fluids and tissue debris from
the uterus. Secondly, the contractions compress the
uterine vasculature and help minimize the possibility
ofhemorrhage. Third, myometrial contractions reduce
the overall size of the uterus. Of the species presented
in this text, timely uterine involution is most important
in the postpartum dairy cow. In most species, frequent
postpartum suckling occms and oxytocin is secreted
(See Figure 15-13). In suckled animals, uterine contrac-
tions occur on a frequent basis. In the dairy cow how-
ever, the calf is usually removed within 24 hours after
parturition and milking takes place only two or three
times per day. Consequently, oxytocin episodes are

Ve
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Parturition
Fetal Attachment & Gestation
Early Embryogenesis &
Maternal Recognition of Pregnancy
Ovulation & Fertilization
Cyclicity
Regulation of
Reproduction
Tract Function
Puberty
Prenatal
Development
Spermatogenesis
Regulation of
Reproduction
Tract Function
Puberty
Prenatal
Development
Take Home Message
Immediately following parturition, the female hegins to lactate and enters a period
of reproductive repair called the pue1perium. For a period of time these two processes
overlap. During the puerperium uterine involution and return of ovarian function oc-
curs. Involution is the reduction in size and “remodeling” of the endometrium so that
the uterus can initiate and sustain another pregnancy.
Mammary gland development is initiated prenatally in the f emale fetus and con-
tinues through puberty and pregnancy. The anatomy and distribution of mammary
glands is diverse among mammals. Accumulation of secretions in the mammmy gland
hegins about two weeks before parturition. Lactation provides the neonate with the
opportunity to nurse and he nourished with minimal expenditure of energy. It also
provides immunoprotection for the neonate because initialmammmy secretions called
colostrum contain antibodies that provide passive immunity. Lactation continues until
the neonate is weaned. After weaning, the mammary glands undergo involution and
retum to a non-secretory state.
The puerperium and lactation are. init iated
immediately after parturition and for a period of time
these processes occur simultaneously. Lactation is
the synthesis, secretion and removal of milk from the
mammary gland. The puerperium is the period after
parturition when the reproductive tract retums to its
nonpregnant condition so that the female may become
pregnant again. This chapter will describe the basics
of these two important processes. Parturition results in
loss of placental function and deterioration of the mater-
nal tissue contributing to the placenta. Tissue damage
results . During the puerperium damaged reproductive
tissues are repaired and ovarian function returns.
The Puerperium
The puerperium begins immediately after par-
turition and lasts until reproductive function is restored
so that another pregnancy can occur. The time required
for complete uterine involution (repair) and ovarian
activity to resume in the postpartum female varies sig-
nificantly among species (See Table 15-1 ).
The four major events of the puerperium
are:
• myometrial contractions and expulsion
of lochia
• endometria/repair
• resumption of ovarian function
• elimination of bacterial contamination
of the reproductive tract
It must be emphasized that in many polyestrous
animals, the shortest possible puerperium is desirable
because eligibility for a subsequent pregnancy is of
high economic importance. For example, in dairy
cows frequent pregnancies are required for maximum
lifetime milk yield. In swine and beef cows, the shorter
the interval between pregnancies the more offspring are
produced and the more efficient the production of meat
becomes. Conversely, the longer the puerperium, the
longer the delay of a subsequent pregnancy and the less
efficient the production process becomes. Figure 15-1
summarizes the events that occur from parturition to the
subsequent pregnancy. These events will be described
in more detail below.
Reduction in Uterine Size and Volume is
Brought About by Myometrial Contractions
Immediately after parturition, the myome-
trium undergoes strong repeated contractions. The
purpose of these contractions is threefold. First, they
facil itate discharge of fluids and tissue debris from
the uterus. Secondly, the contractions compress the
uterine vasculature and help minimize the possibility
ofhemorrhage. Third, myometrial contractions reduce
the overall size of the uterus. Of the species presented
in this text, timely uterine involution is most important
in the postpartum dairy cow. In most species, frequent
postpartum suckling occms and oxytocin is secreted
(See Figure 15-13). In suckled animals, uterine contrac-
tions occur on a frequent basis. In the dairy cow how-
ever, the calf is usually removed within 24 hours after
parturition and milking takes place only two or three
times per day. Consequently, oxytocin episodes are
Ve
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316 The Puerperium and Lactation

Figure 15-1. Major Events From Parturition to Subsequent
Conception (Ruminant Model)

Conception

Uterine Involution
• .J. Uterine size (length and diameter)
• .J. Uterine volume
• Expulsion of lochia
• Endometrial repair

Table 15-1. Time Required for Uterine Involution and Resumption of Ovarian Activity in
Various Species

Species

Alpaca
Beef Cow
Bitch
Camel
Dairy Cow
Ewe
Llama
Mare
Queen
Sow
Woman

Time Required for
Complete Uterine Involution

20d
30d
90d
30-50d
45-50d
30d
20d
21-28d
30d
28-30d
40-45d

L = Lactation inhibits ovarian activity (See Chapter 7)

T ime Required for
Resumption of Ovarian Activity

5-10d
50-60d (L)
150d (A)
25-40d or up to 1 yr (L)
18-25d
180d (SOB)
5-1 0d
5-12d
30d
7d (L)
6-24mo (L) (See Chapter 7)

SOB = Short Day Breeder- ewes giving birth in the spring will not cycle until fall
A = Long natural postpartum anestrus (See Chapter 7)

reduced, myometrial contractions are not as frequent
and uterine involution can be delayed. In this light,
much of the material presented on uterine involution
will focus on the dairy cow since delayed uterine in-
volution is an important factor limiting fertility in this
animal.

Immediately after parturition the uterus un-
dergoes rapid but highly coordinated atTophy so that
in a relatively short period of time the uterine mass is
reduced to its nonpregnant size. In all species, marked
size reduction occurs during the first several days af-
ter parhrrition. In fact, in the dairy cow, myometrial
cell size decreases from 700pm on the firs t day after
parturition to a few days later. In most spe-
cies, myometrial contractions occur in three to four
minute intervals for the firs t several postpartum days.
These strong, high frequency myometrial contractions
subside within several days. The exact time that these
contractions stop depends on the species. The dramatic
postpartum size reduction of the uterus in the dairy cow
is illustrated in Figure 15-2. ‘

..-.
E

…
tl() c

J!

QJ

c ·;:::
QJ …

::J

@

Figure 15-2. Changes in
Uterine Length and Weight at

Various Postpartum Days

80
70
60
so
40
30
20
10

0 I s 10 IS 20

Days of postpar tum

The uterine length values here are used in Figures 15-4 through
15-8 to illuslrate approximate size changes. (From Gier, H.T.
and G.B. Marion, 1968. Amer. J. Vet. Res. 29: 83-96)

During and After Myometrial Contractions a
Bloody Fluid is Discharged fr·om the Tract

Shortly after parturition, a discharge called
lochia is expelled from the vulva. Lochia is typically
a blood-tinged fluid containing remnants of the fetal
placenta and endometrial tissue. L ochial discharge
occurs between 2 and 9 days in postpartum dairy cows.
An increase in blood and tissue debris in the lochia
is nonnal and occurs between 5 and 1 0 days. This is
due to the sloughing of caruncular surfaces that leaves
vascular “stubs” that leak blood. Lochial discharge is

The Puerperium and Lactation 317

physiologically nomml in all species. However, it is
often interpreted by observers to be the result of uterine
pathology (especially in the dairy cow). Therefore, the
first ” instinct” of the reproductive management team is
to treat the animal for nonexistent pathology. Unwar-
ranted treatment is financ ially wasteful, not effective
and often prolongs uterine involution especially if
the uterine lumen is invaded (infusion of antibiotics,
various solutions or to remove manually retained fetal
membranes).

Obviously, with s ignificant myometrial con-
tractions occmTing for the first 7 to I 0 days there will
be a reduction in the volume oflochia within the uterus.
In the dairy cow, up to 2000ml oflochia can be expelled
from the uterus during the first two to three days after
parturition. By 14 to 18 days, lochial discharge is al-
most nonexistent in most cows (See Figure 15-3).

..-.
I

QJ

E
::l
0 >
iii
‘ i

j

0

…1

@

Figure 15-3. Changes in
Lochial Volume at Various

Postpartum Days
1,500

1,000

soo

s 10 IS

Days of postpartum

20

(From Gier, H.T. and G.B. Marion, 1968. Amer. J. Vet. Res.
29: 83-96)

Caruncular Repair Requires Vasoconstriction,
Necrosis and Sloughing of Tissues Followed

by Growth of Surface Epithelium

After separation of the fetal cotyledons from
the maternal caruncle (within 8-12 hours after delivery
of the neonate) vasoconstriction takes place in the stalk
of the matemal caruncle. Necrosis of the caruncular
tissue follows. Necrosis is irreversible cell death that
leads to sloughing of the caruncular mass leaving ne-
crotic tissue in the lochial fluid inside the uterus. Some
blood is released from the canmcular stalk generating
a blood-tinged fluid. About 5 days after parturition,
the caruncles begin to lose their cellular organization
and integrity. This results in chunks of the caruncles
detaching from the surface of the caruncle leaving
remnants of blood vessels exposed to the surface. After
the decidual tissue of the caruncle has sloughed into

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316 The Puerperium and Lactation
Figure 15-1. Major Events From Parturition to Subsequent
Conception (Ruminant Model)
Conception
Uterine Involution
• .J. Uterine size (length and diameter)
• .J. Uterine volume
• Expulsion of lochia
• Endometrial repair
Table 15-1. Time Required for Uterine Involution and Resumption of Ovarian Activity in
Various Species
Species
Alpaca
Beef Cow
Bitch
Camel
Dairy Cow
Ewe
Llama
Mare
Queen
Sow
Woman
Time Required for
Complete Uterine Involution
20d
30d
90d
30-50d
45-50d
30d
20d
21-28d
30d
28-30d
40-45d
L = Lactation inhibits ovarian activity (See Chapter 7)
T ime Required for
Resumption of Ovarian Activity
5-10d
50-60d (L)
150d (A)
25-40d or up to 1 yr (L)
18-25d
180d (SOB)
5-1 0d
5-12d
30d
7d (L)
6-24mo (L) (See Chapter 7)
SOB = Short Day Breeder- ewes giving birth in the spring will not cycle until fall
A = Long natural postpartum anestrus (See Chapter 7)
reduced, myometrial contractions are not as frequent
and uterine involution can be delayed. In this light,
much of the material presented on uterine involution
will focus on the dairy cow since delayed uterine in-
volution is an important factor limiting fertility in this
animal.
Immediately after parturition the uterus un-
dergoes rapid but highly coordinated atTophy so that
in a relatively short period of time the uterine mass is
reduced to its nonpregnant size. In all species, marked
size reduction occurs during the first several days af-
ter parhrrition. In fact, in the dairy cow, myometrial
cell size decreases from 700pm on the firs t day after
parturition to a few days later. In most spe-
cies, myometrial contractions occur in three to four
minute intervals for the firs t several postpartum days.
These strong, high frequency myometrial contractions
subside within several days. The exact time that these
contractions stop depends on the species. The dramatic
postpartum size reduction of the uterus in the dairy cow
is illustrated in Figure 15-2. ‘
..-.
E
…
tl() c

J!
QJ
c ·;:::
QJ …

::J
@

Figure 15-2. Changes in
Uterine Length and Weight at
Various Postpartum Days
80
70
60
so
40
30
20
10
0 I s 10 IS 20
Days of postpar tum
The uterine length values here are used in Figures 15-4 through
15-8 to illuslrate approximate size changes. (From Gier, H.T.
and G.B. Marion, 1968. Amer. J. Vet. Res. 29: 83-96)
During and After Myometrial Contractions a
Bloody Fluid is Discharged fr·om the Tract
Shortly after parturition, a discharge called
lochia is expelled from the vulva. Lochia is typically
a blood-tinged fluid containing remnants of the fetal
placenta and endometrial tissue. L ochial discharge
occurs between 2 and 9 days in postpartum dairy cows.
An increase in blood and tissue debris in the lochia
is nonnal and occurs between 5 and 1 0 days. This is
due to the sloughing of caruncular surfaces that leaves
vascular “stubs” that leak blood. Lochial discharge is
The Puerperium and Lactation 317
physiologically nomml in all species. However, it is
often interpreted by observers to be the result of uterine
pathology (especially in the dairy cow). Therefore, the
first ” instinct” of the reproductive management team is
to treat the animal for nonexistent pathology. Unwar-
ranted treatment is financ ially wasteful, not effective
and often prolongs uterine involution especially if
the uterine lumen is invaded (infusion of antibiotics,
various solutions or to remove manually retained fetal
membranes).
Obviously, with s ignificant myometrial con-
tractions occmTing for the first 7 to I 0 days there will
be a reduction in the volume oflochia within the uterus.
In the dairy cow, up to 2000ml oflochia can be expelled
from the uterus during the first two to three days after
parturition. By 14 to 18 days, lochial discharge is al-
most nonexistent in most cows (See Figure 15-3).
..-.
I
QJ
E
::l
0 >
iii
‘ ij
0
…1
@
Figure 15-3. Changes in
Lochial Volume at Various
Postpartum Days
1,500
1,000
soo
s 10 IS
Days of postpartum
20
(From Gier, H.T. and G.B. Marion, 1968. Amer. J. Vet. Res.
29: 83-96)
Caruncular Repair Requires Vasoconstriction,
Necrosis and Sloughing of Tissues Followed
by Growth of Surface Epithelium
After separation of the fetal cotyledons from
the maternal caruncle (within 8-12 hours after delivery
of the neonate) vasoconstriction takes place in the stalk
of the matemal caruncle. Necrosis of the caruncular
tissue follows. Necrosis is irreversible cell death that
leads to sloughing of the caruncular mass leaving ne-
crotic tissue in the lochial fluid inside the uterus. Some
blood is released from the canmcular stalk generating
a blood-tinged fluid. About 5 days after parturition,
the caruncles begin to lose their cellular organization
and integrity. This results in chunks of the caruncles
detaching from the surface of the caruncle leaving
remnants of blood vessels exposed to the surface. After
the decidual tissue of the caruncle has sloughed into
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318 The Puerperium and Lactation

Fig’!re 15-4. Bovine Reproductive Organs- Day 1 Postpartum

Ovaries- There are no
functional structures on
the right ovary. The left
ovary contains two cor-
pora lutea (arrows 1 and
2) indicating a double
ovulation. Only one con-
ceptus developed. There
is no evidence of follicular
development on either
ovary.

Cervix (caudal view)- The
brownish mucus (M) is a
remnant of the cervical
seal of pregnancy. Mu-
cosal hemorrhaging (MH)
has resulted from abrasive
trauma to the cranial vagina
(CV) , fornix vagina (FV)
and portions of the cervix
(CX) during expulsion of
the fetus. A stainless steel
rod has been positioned in
the cervical canal to provide
spatial reference in Figures
15-4 through 15-8.

Uterus. in-situ- This photograph
and all subsequent in-situ pho-
tographs were taken from post-
partum dairy cows in which the
viscera was removed so that the
cranial surface of the reproduc-
tive tract can be viewed. Here,
the approximate overall length of
the uterus is 85cm. The right uter-
ine horn (RUH) is larger than the
left uterine horn (LUH) because
the right uterine horn housed
the fetus. The broad ligament
(BL) and rectum (R) are obvious.

Uterine Interior-The uterus contains many large caruncles (C) that consist
of intact tissue. Only a few caruncles have started to undergo necrosis (N)
as judged by the blackened regions. There is very little lochia (L) pres-
ent. The caruncular stalks (CS) are quite long and house the vasculature
that supplied the maternal cotyledon with blood during pregnancy. The
enlarged photograph illustrates a caruncular crown (CC) that has been
sliced open. The incision has extended into the center of the caruncular
stalk (CS). The entire layer of decidual tissue (DT) will soon slough into
the uterine lumen because of vasoconstriction of the caruncular arterioles.

CLP
\

The Puerperium and Lactation 319

Figure 15-5. Bovine Reproductive Organs- Day 4 Postpartum

RIGH

T

OVARY

….. -= …. – X
Cervix (caudal view)- Lo-
chia (L) has been expel led
through the cervix (CX) and
it has pooled in the ventral
reg ion of the cranial vagina
(CV) here. In the live cow,
lochia would be discharged
to the exterior.

LEFT
OVARY

Ovaries- A regressing CL from
the pregnancy (CLP) is present
on each of the right and left ova-
ries indicating a double ovulation.
Only one conceptus developed. A
regressing CL (RCL) from a cycle
prior to the pregnancy is present
on the right ovary. There is no
evidence of follicular development
in either ovary.

Uterus. in-situ- The most dramatic reduction in uterine size occurred
between day 1 and day 5. Uterine length is reduced from about 85cm
(day 1) to 58cm (day 4 ). The left uterine horn (LUH) housed the conceptus
during pregnancy and is larger than the right uterine horn (RUH). The
broad ligament (BL) and rectum (R) can be observed.
Uterine Interior- Much of the decidual tissue of the caruncles (C) has
sloughed into the uterine lumen along with blood and other fluids forming
lochia (L). This material is normally expelled from the uterus. The pres-
ence of lochia (L) in the uterus and its discharge from the vulva is normal.

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318 The Puerperium and Lactation
Fig’!re 15-4. Bovine Reproductive Organs- Day 1 Postpartum
Ovaries- There are no
functional structures on
the right ovary. The left
ovary contains two cor-
pora lutea (arrows 1 and
2) indicating a double
ovulation. Only one con-
ceptus developed. There
is no evidence of follicular
development on either
ovary.
Cervix (caudal view)- The
brownish mucus (M) is a
remnant of the cervical
seal of pregnancy. Mu-
cosal hemorrhaging (MH)
has resulted from abrasive
trauma to the cranial vagina
(CV) , fornix vagina (FV)
and portions of the cervix
(CX) during expulsion of
the fetus. A stainless steel
rod has been positioned in
the cervical canal to provide
spatial reference in Figures
15-4 through 15-8.
Uterus. in-situ- This photograph
and all subsequent in-situ pho-
tographs were taken from post-
partum dairy cows in which the
viscera was removed so that the
cranial surface of the reproduc-
tive tract can be viewed. Here,
the approximate overall length of
the uterus is 85cm. The right uter-
ine horn (RUH) is larger than the
left uterine horn (LUH) because
the right uterine horn housed
the fetus. The broad ligament
(BL) and rectum (R) are obvious.
Uterine Interior-The uterus contains many large caruncles (C) that consist
of intact tissue. Only a few caruncles have started to undergo necrosis (N)
as judged by the blackened regions. There is very little lochia (L) pres-
ent. The caruncular stalks (CS) are quite long and house the vasculature
that supplied the maternal cotyledon with blood during pregnancy. The
enlarged photograph illustrates a caruncular crown (CC) that has been
sliced open. The incision has extended into the center of the caruncular
stalk (CS). The entire layer of decidual tissue (DT) will soon slough into
the uterine lumen because of vasoconstriction of the caruncular arterioles.
CLP
\
The Puerperium and Lactation 319
Figure 15-5. Bovine Reproductive Organs- Day 4 Postpartum
RIGHT
OVARY
….. -= …. – X
Cervix (caudal view)- Lo-
chia (L) has been expel led
through the cervix (CX) and
it has pooled in the ventral
reg ion of the cranial vagina
(CV) here. In the live cow,
lochia would be discharged
to the exterior.
LEFT
OVARY
Ovaries- A regressing CL from
the pregnancy (CLP) is present
on each of the right and left ova-
ries indicating a double ovulation.
Only one conceptus developed. A
regressing CL (RCL) from a cycle
prior to the pregnancy is present
on the right ovary. There is no
evidence of follicular development
in either ovary.
Uterus. in-situ- The most dramatic reduction in uterine size occurred
between day 1 and day 5. Uterine length is reduced from about 85cm
(day 1) to 58cm (day 4 ). The left uterine horn (LUH) housed the conceptus
during pregnancy and is larger than the right uterine horn (RUH). The
broad ligament (BL) and rectum (R) can be observed.
Uterine Interior- Much of the decidual tissue of the caruncles (C) has
sloughed into the uterine lumen along with blood and other fluids forming
lochia (L). This material is normally expelled from the uterus. The pres-
ence of lochia (L) in the uterus and its discharge from the vulva is normal.
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320 The Puerperium and Lactation

Figure 1·5-6. Bovine Reproductive Organs- Day 1 0 Postpartum

Cervix (caudal view)-
Sites of mucosal hem-
orrhaging (MH) are still
apparent in the floor of the
cranial vagina (CV) in this
cow. The cervix (CX) has
decreased in diameter be-
cause its overall tone has
increased.

LEFT
– -oVARY ‘

LEFT
—.. OVARY

-·

Ovaries- The right ovary
contains several corpora
albicantia (CA) and a few
antral foll icles (AF). The left
ovary contains the regressing
corpus luteum of pregnancy
(CLP). It also contains an
antral follicle (AF) indicating
that a new follicular phase is
beginning.

Uterus. in-situ- The uterus continues to undergo a reduction in
size (41 em). The left uterine horn (LUH) remains larger than the
right uterine horn (RUH) because the left uterine horn housed the
conceptus. The rectum (R) and broad ligament (BL) are visib le.
Uterine Interior- The decidual tissue of each caruncle has been
sloughed into the uterine lumen. Some lochia (L) is still present but
it is more viscous and mucus-like. The endometrial and caruncular
epithelium is now beginning to cover the surface. The enlarged
photograph illustrates the marked reduction in size of the caruncle
(compare to days 1 and 4 ). The caruncular stalk is nonexistent.
This size reduction is a function of vasoconstriction of the caruncular
blood vessels (BV).

The Puerperium and Lactation 321

Figure 15-7. Bovine Reproductive Organs- Day 15 Postpartum
CERVIX

Cervix (caudal view)-
Strands of clear mucus
(M) secreted by the cervix
(CX) and cranial vagina
(CV) indicate that th is
cow is entering her first
foll icular phase after par-
tu rition (S ee ovaries).
FV = Fornix vagina.

Ovaries- The right ovary con-
tains the regressing corpus
luteum of pregnancy (CLP). It
also contains a developing antral
follicle (AF). The left ovary con-
tains a large antral follicle (AF)
indicative of the first postpartum
follicular phase. The follicles
present produce estradiol that
causes secretion of mucus by
the cervix and cranial vagina.

Uterus. in-situ- The right uterine horn (RUH) housed the
conceptus and is larger than the left uterine horn (LUH).
Continued reduction in size is evident. The broad ligament
(BL) and the rectum (R) can be observed. The dark coloration
at the tips of the uterine horns represents pooling of blood
following exsanguination of the cow.
Uterine Interior- The caruncles (C) have decreased further in
size and are almost completely covered in mucus. Lochia is
almost nonexistent and a puss-like material (P) is present in
localized areas. The presence of puss is normal and reflects
phagocytosis of deteriorating tissue by leukocytes. Carun-
cular blood vessels (arrows) can be seen as small knot-like
structures in the incised caruncles. M = Myometrium.

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320 The Puerperium and Lactation
Figure 1·5-6. Bovine Reproductive Organs- Day 1 0 Postpartum
Cervix (caudal view)-
Sites of mucosal hem-
orrhaging (MH) are still
apparent in the floor of the
cranial vagina (CV) in this
cow. The cervix (CX) has
decreased in diameter be-
cause its overall tone has
increased.
LEFT
– -oVARY ‘
LEFT
—.. OVARY -·
Ovaries- The right ovary
contains several corpora
albicantia (CA) and a few
antral foll icles (AF). The left
ovary contains the regressing
corpus luteum of pregnancy
(CLP). It also contains an
antral follicle (AF) indicating
that a new follicular phase is
beginning.
Uterus. in-situ- The uterus continues to undergo a reduction in
size (41 em). The left uterine horn (LUH) remains larger than the
right uterine horn (RUH) because the left uterine horn housed the
conceptus. The rectum (R) and broad ligament (BL) are visib le.
Uterine Interior- The decidual tissue of each caruncle has been
sloughed into the uterine lumen. Some lochia (L) is still present but
it is more viscous and mucus-like. The endometrial and caruncular
epithelium is now beginning to cover the surface. The enlarged
photograph illustrates the marked reduction in size of the caruncle
(compare to days 1 and 4 ). The caruncular stalk is nonexistent.
This size reduction is a function of vasoconstriction of the caruncular
blood vessels (BV).
The Puerperium and Lactation 321
Figure 15-7. Bovine Reproductive Organs- Day 15 Postpartum
CERVIX
Cervix (caudal view)-
Strands of clear mucus
(M) secreted by the cervix
(CX) and cranial vagina
(CV) indicate that th is
cow is entering her first
foll icular phase after par-
tu rition (S ee ovaries).
FV = Fornix vagina.
Ovaries- The right ovary con-
tains the regressing corpus
luteum of pregnancy (CLP). It
also contains a developing antral
follicle (AF). The left ovary con-
tains a large antral follicle (AF)
indicative of the first postpartum
follicular phase. The follicles
present produce estradiol that
causes secretion of mucus by
the cervix and cranial vagina.
Uterus. in-situ- The right uterine horn (RUH) housed the
conceptus and is larger than the left uterine horn (LUH).
Continued reduction in size is evident. The broad ligament
(BL) and the rectum (R) can be observed. The dark coloration
at the tips of the uterine horns represents pooling of blood
following exsanguination of the cow.
Uterine Interior- The caruncles (C) have decreased further in
size and are almost completely covered in mucus. Lochia is
almost nonexistent and a puss-like material (P) is present in
localized areas. The presence of puss is normal and reflects
phagocytosis of deteriorating tissue by leukocytes. Carun-
cular blood vessels (arrows) can be seen as small knot-like
structures in the incised caruncles. M = Myometrium.
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322 The Puerperium and Lactation

Figure 15-8. Bovine Reproductive Organs- Day 20 Postpartum

RIGHT
OVARY

Tlte photographs in Figures 15-4 through
15-8 were part of an Honors Thesis entitled
“A Full Color Photographic Description
of Postpartum Uterine Involution in the
Daily Cow” submitted to Washington State
University Honors College by Christina M.
Davis, Spring 2002. The Honors project was
sponsored by Current Conceptions, Inc.

Cervix (caudal view)-
The cranial vagina (CV}
and fornix vagina (FV)
are free of hemorrhagic
foci. The color, diameter
and tone of the cervix
(CX} are normal. Mucus
is present coating the
mucosal surfaces.

-..CA

Ovaries- The right ovary contains
the regressing corpus luteum of
pregnancy (CLP) and an antral
follicle (AF). The antral follicle is
not observed in the incised ovary
because it is out of the plane of sec-
tion. The left ovary contains several
antral follicles (AF} indicating this
cow is entering her first postpartum
follicular phase. A corpus albicans
(CA} represents a corpus luteum
from a cycle prior to the previous
pregnancy.

Uterus. in-situ- The uterine horns continue to decrease in size
and have almost returned to their normal nonpregnant size.
The right uterine horn (RUH) remains larger than the left uterine
horn (LUH} because the right uterine horn housed the concep-
tus. The broad ligament (BL} and the rectum (R} can be readily
observed.
Uterine Interior- Caruncles (C) are approaching the size of those
normally seen within the nonpregnant uterus. A cross-section of an
incised caruncle shows the mass of blood vessels (BV) between
the myometrium (M) and the epithelium (E) covering the caruncle.
The fluid within the uterine lumen is predominantly mucus.

the uterine lumen the caruncle begins to undergo re-
pair and is eventually covered again with endometrial
epithelium.

Figure 15-9. Changes in
Caruncular Height at Various

Postpartum Days
e s
u ._,
i: 4
00

‘ijj
J: 3 ,….
ns a 2 c
::l ,….
ns u

Days Postpartum

{From 1978)

At the same time caruncular repair is taking
place, the intercaruncular endometrial surfaces also
undergo repair. In general, the epithelium of the inter-
caruncular area of the endometrium repairs at a faster
rate than do the caruncles. The repair of the intercarun-
cular endometrium is generally complete by the eighth
postpartum day. The delay in caruncular repair, when
compared to the intercaruncular epithelium is associ-
ated with the large mass of the canmcular tissue that
must undergo necrosis and sloughing before surface
epithelial repair can take place.

Postpartum Bacterial Contamination of the
Uterus is Common in Most Domestic Animals

Generally, parturition in domestic animals oc-
curs in a non-sterile environment. As a result, bacte-
rial contamination of the reproductive tract, especially
the uterus is an inevitable sequela to parturition. The
postpartum reproductive tract (containing lochia) is
an ideal environment for the growth of bacteria. Even
though myometrial contractions tend to remove the
large volume of lochia produced in some species, bacte-
rial growth can continue. It must be emphasized that
bacterial contamination is not always associated with
pathology. Nom1al postpartum events tend to eliminate
the bacterial flora within a reasonable time. As you
recall, elevated estradiol promotes leukocytosis in the
uterus and elsewhere in the reproductive tract. Thus,
a high degree of phagocytosis can be observed in the
postpartum reproductive tract as a result of relatively
high postparhnn estradiol concentrations that exist for
a few days.

The Puerperium and Lactation 323

In some instances, high numbers of bacteria
can ove1whelm the natural defense mechanisms re-
sulting in postpartum uterine infection. Conditions
that predispose the uterus to infections are: retained
fetal membranes, dystocia and delay in lochial expul-
sion brought about by weak myometrial contractions.
Regardless of the cause, failure to eliminate bacterial
contamination will: 1) prolong uterine involution; 2)
prolong the puerperium and 3) delay subsequent preg-
nancies. Treatment of uterine infections is controversial.
There is little evidence that supports the effectiveness
of infusing the uterus with various pharmaceuticals in
dairy cows. The single most important nahtral factor
that aids in elimination of bacterial contamination is a
return to cyclicity (estrus) so that estradiol concentra-
tions will be elevated.

Photographic descriptions of the changes that
occur in the uterus, caruncles, cervix and ovaries of
the dairy cow during the first 20 days of the puerpe-
rium are presented in Figures 15-4 through 15-8. The
specimens were obtained from cows that were defined
as clinically normal as judged by palpation per rectum
by the veterinarian servicing the herd. All cows gave
birth to a single calf. To compare these various days
of involution to completely involuted organs, please
consult the figures in Chapter 2.

Lactation

Lactation ensures that the neonatal mammal
does not have to obtain food on its own. Instead, the
dam is responsible for consuming all of the nutritional
raw materials and transforming these into a highly
nutritious secretion called milk. The neonate benefits
from this synthetic and secretory process because its
only behavioral requirement in the early postnatal
period is suckling the dam. Some animals have been
domesticated and selected so they produce quanti-
ties of milk that far exceed that needed to nourish the
young. The dairy cow is the dominant producer of
milk for human consumption. However, goats, sheep,
water buffalo, camels and mares are also considered
important for their milk producing ability in some parts
of the world. The immense milk producing ability of
the modern dairy cow has provided a huge variety of
dairy products that contribute to a multi-billion dollar
industry in the western world. In this light, much of
the infom1ation provided in this section will be about
the dairy cow. However, the basic principles apply to
most mammals. The development of the mammary
gland (mammogenesis), anatomical diversity and milk
ejection from the gland will be the priority topics in the
remainder of this chapter.

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322 The Puerperium and Lactation
Figure 15-8. Bovine Reproductive Organs- Day 20 Postpartum
RIGHT
OVARY
Tlte photographs in Figures 15-4 through
15-8 were part of an Honors Thesis entitled
“A Full Color Photographic Description
of Postpartum Uterine Involution in the
Daily Cow” submitted to Washington State
University Honors College by Christina M.
Davis, Spring 2002. The Honors project was
sponsored by Current Conceptions, Inc.
Cervix (caudal view)-
The cranial vagina (CV}
and fornix vagina (FV)
are free of hemorrhagic
foci. The color, diameter
and tone of the cervix
(CX} are normal. Mucus
is present coating the
mucosal surfaces.
-..CA
Ovaries- The right ovary contains
the regressing corpus luteum of
pregnancy (CLP) and an antral
follicle (AF). The antral follicle is
not observed in the incised ovary
because it is out of the plane of sec-
tion. The left ovary contains several
antral follicles (AF} indicating this
cow is entering her first postpartum
follicular phase. A corpus albicans
(CA} represents a corpus luteum
from a cycle prior to the previous
pregnancy.
Uterus. in-situ- The uterine horns continue to decrease in size
and have almost returned to their normal nonpregnant size.
The right uterine horn (RUH) remains larger than the left uterine
horn (LUH} because the right uterine horn housed the concep-
tus. The broad ligament (BL} and the rectum (R} can be readily
observed.
Uterine Interior- Caruncles (C) are approaching the size of those
normally seen within the nonpregnant uterus. A cross-section of an
incised caruncle shows the mass of blood vessels (BV) between
the myometrium (M) and the epithelium (E) covering the caruncle.
The fluid within the uterine lumen is predominantly mucus.
the uterine lumen the caruncle begins to undergo re-
pair and is eventually covered again with endometrial
epithelium.
Figure 15-9. Changes in
Caruncular Height at Various
Postpartum Days
e s
u ._,
i: 4
00
‘ijj
J: 3 ,….
ns a 2 c
::l ,….
ns u
Days Postpartum
{From 1978)
At the same time caruncular repair is taking
place, the intercaruncular endometrial surfaces also
undergo repair. In general, the epithelium of the inter-
caruncular area of the endometrium repairs at a faster
rate than do the caruncles. The repair of the intercarun-
cular endometrium is generally complete by the eighth
postpartum day. The delay in caruncular repair, when
compared to the intercaruncular epithelium is associ-
ated with the large mass of the canmcular tissue that
must undergo necrosis and sloughing before surface
epithelial repair can take place.
Postpartum Bacterial Contamination of the
Uterus is Common in Most Domestic Animals
Generally, parturition in domestic animals oc-
curs in a non-sterile environment. As a result, bacte-
rial contamination of the reproductive tract, especially
the uterus is an inevitable sequela to parturition. The
postpartum reproductive tract (containing lochia) is
an ideal environment for the growth of bacteria. Even
though myometrial contractions tend to remove the
large volume of lochia produced in some species, bacte-
rial growth can continue. It must be emphasized that
bacterial contamination is not always associated with
pathology. Nom1al postpartum events tend to eliminate
the bacterial flora within a reasonable time. As you
recall, elevated estradiol promotes leukocytosis in the
uterus and elsewhere in the reproductive tract. Thus,
a high degree of phagocytosis can be observed in the
postpartum reproductive tract as a result of relatively
high postparhnn estradiol concentrations that exist for
a few days.
The Puerperium and Lactation 323
In some instances, high numbers of bacteria
can ove1whelm the natural defense mechanisms re-
sulting in postpartum uterine infection. Conditions
that predispose the uterus to infections are: retained
fetal membranes, dystocia and delay in lochial expul-
sion brought about by weak myometrial contractions.
Regardless of the cause, failure to eliminate bacterial
contamination will: 1) prolong uterine involution; 2)
prolong the puerperium and 3) delay subsequent preg-
nancies. Treatment of uterine infections is controversial.
There is little evidence that supports the effectiveness
of infusing the uterus with various pharmaceuticals in
dairy cows. The single most important nahtral factor
that aids in elimination of bacterial contamination is a
return to cyclicity (estrus) so that estradiol concentra-
tions will be elevated.
Photographic descriptions of the changes that
occur in the uterus, caruncles, cervix and ovaries of
the dairy cow during the first 20 days of the puerpe-
rium are presented in Figures 15-4 through 15-8. The
specimens were obtained from cows that were defined
as clinically normal as judged by palpation per rectum
by the veterinarian servicing the herd. All cows gave
birth to a single calf. To compare these various days
of involution to completely involuted organs, please
consult the figures in Chapter 2.
Lactation
Lactation ensures that the neonatal mammal
does not have to obtain food on its own. Instead, the
dam is responsible for consuming all of the nutritional
raw materials and transforming these into a highly
nutritious secretion called milk. The neonate benefits
from this synthetic and secretory process because its
only behavioral requirement in the early postnatal
period is suckling the dam. Some animals have been
domesticated and selected so they produce quanti-
ties of milk that far exceed that needed to nourish the
young. The dairy cow is the dominant producer of
milk for human consumption. However, goats, sheep,
water buffalo, camels and mares are also considered
important for their milk producing ability in some parts
of the world. The immense milk producing ability of
the modern dairy cow has provided a huge variety of
dairy products that contribute to a multi-billion dollar
industry in the western world. In this light, much of
the infom1ation provided in this section will be about
the dairy cow. However, the basic principles apply to
most mammals. The development of the mammary
gland (mammogenesis), anatomical diversity and milk
ejection from the gland will be the priority topics in the
remainder of this chapter.
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324 The Puerperium and Lactation

Mammary Glands are Sophisticated
Sweat Glands

Mammary glands arise in the developing
embryo along two lateral lines on the ventral surface
of the developing conceptus. These lines are slightly
thickened ridges of epidermis (skin) and are called
mammary ridges (See Figure 15-10). The mammary
ridges extend from the axillary region (armpit) of the
conceptus to the inguinal region. The number of mam-
mary glands that develop from the mammary ridges
depends on the species. For example, animals like
the pig, dog and cat have a series of individual glands
that develop at predictable positions along the entire
path of the mammary ridges. In contrast, animals like
the human and elephant have paired mammary glands
that develop from the thoracic portion of the mammary
ridges. Animals like the cow, mare and goat have mam-
mary glands that develop from the inguinal region of
the mammmy ridge. The diversity among mammals in
gland number, anatomic location and teat morphology
is presented in Figure 15-11.

The thickened epidermal epithelium creating
the mammary ridges gives rise to the primary mam-
mary bud (See Figure 15-1 0). The primmy mammary
bud pushes into the underlying dermis as it grows.
Continued growth results in secondary mammary
buds that fonn bud protrusions away from the primmy
bud. These seconda1y buds then lengthen and branch
throughout the remainder of embryonic development.
Finally, these branched buds begin to canalize form-
ing tiny ducts in the center of each bud. Each bud then
becomes a duct with a lumen. At birth, the mammary
glands consist oflactiferous ducts that open into larger
ducts and empty to the exterior of the mammary gland
through a teat or nipple (See Figure 15-1 0).

Postnatal changes in the mammary
gland occur:

• between birth and puberty
• between puberty and pregnancy
• during pregnancy
• during lactation
• during involution

Postnatal Growth of the Mammary Gland is
Endocrine Mediated

Complete anatomical development of the
mammary gland coupled with the ability to synthesize
and secrete milk does not occur until the female has
reached puberty, becomes pregnant and gives birth to
offspring.

Between Birth and Puberty, Mammary
Growth is Isometric

Between birth and puberty the mammary gland
experiences isometric gr·owth (at the same rate as the
other tissues). In other words, there is no noticeable
enlargement of the mammary glands when compared
to the rest of the body.

Mammary Glands Grow Significantly
Between Puberty and Pregnancy

After the onset of puberty, the mammary gland
begins to grow at a rate that is disproportionately
faster than normal body growth. This type of growth is
referred to as allometric growth. During repeated
estrous cycles, a duct and alveolar framework is con-
structed within the mammary gland. This framework
provides the cellular basis for future milk synthesis.
During the first several estrous (or menstrual) cycles,
the ducts begin to branch and their diameter increases
under the influence of estradiol. Under the influence
of progesterone (during the luteal phase), the terminal
portions of each branch begin to form the initial portions
ofthe alveoli. The alveoli fom1 the functional secretmy
elements of the mammaty gland (See Figure 15-13).
Estradiol alone will cause some duct development but
more complete and rapid duct development occurs
in the presence of prolactin and growth hormone
(somatotropin). Both of these hormones increase dur-
ing the onset of puberty. Repeated cyclic exposure of
the mammary cells to estrogen and progesterone can
stimulate mammogenesis to proceed only so far. The
mammary framework formed between puberty and
pregnancy needs fuh1re endocrine input during the
gestational period for complete development.

Final Mammary Development Occurs
During Pregnancy

Complete alveolar development in the dam
takes place during the last trimester of pregnancy. During
this time the terminal alveoli begin to grow into bunches
called lobules. A lobule would be analogous to a group
of grapes on a single stem among an entire bunch of
grapes (See Figure 15-13 ). A group oflobules that emp-

The Puerperium and Lactation 325

Figure 15-10. Prenatal Mammogenesis

Primary
mammary

bud

Secondary
mammary

bud

I Secondary mammary bud I

Canalization

Lactiferous
ducts

Myoepithelial —1
cells

Mammary Ridges
Mammary ridges are th ickened epider-
mal tissue that give rise to the mammary
gland.

Primary Mammary Bud
The thickened epidermal tissue begins
to develop inward and penetrate into the
mesenchyme (dermis).

Secondary Mammary Bud
The primary mammary bud begins to
send out branches that further penetrate
into the dermis.

Canalization
The fingerlike secondary buds begin to
lengthen and branch out. Finally they be-
gin to form canals or channels (canaliza-
tion) that will form the duct system of the
gland. Myoepithelial cells surround the
terminal portions of the developing gland.

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324 The Puerperium and Lactation
Mammary Glands are Sophisticated
Sweat Glands
Mammary glands arise in the developing
embryo along two lateral lines on the ventral surface
of the developing conceptus. These lines are slightly
thickened ridges of epidermis (skin) and are called
mammary ridges (See Figure 15-10). The mammary
ridges extend from the axillary region (armpit) of the
conceptus to the inguinal region. The number of mam-
mary glands that develop from the mammary ridges
depends on the species. For example, animals like
the pig, dog and cat have a series of individual glands
that develop at predictable positions along the entire
path of the mammary ridges. In contrast, animals like
the human and elephant have paired mammary glands
that develop from the thoracic portion of the mammary
ridges. Animals like the cow, mare and goat have mam-
mary glands that develop from the inguinal region of
the mammmy ridge. The diversity among mammals in
gland number, anatomic location and teat morphology
is presented in Figure 15-11.
The thickened epidermal epithelium creating
the mammary ridges gives rise to the primary mam-
mary bud (See Figure 15-1 0). The primmy mammary
bud pushes into the underlying dermis as it grows.
Continued growth results in secondary mammary
buds that fonn bud protrusions away from the primmy
bud. These seconda1y buds then lengthen and branch
throughout the remainder of embryonic development.
Finally, these branched buds begin to canalize form-
ing tiny ducts in the center of each bud. Each bud then
becomes a duct with a lumen. At birth, the mammary
glands consist oflactiferous ducts that open into larger
ducts and empty to the exterior of the mammary gland
through a teat or nipple (See Figure 15-1 0).
Postnatal changes in the mammary
gland occur:
• between birth and puberty
• between puberty and pregnancy
• during pregnancy
• during lactation
• during involution
Postnatal Growth of the Mammary Gland is
Endocrine Mediated
Complete anatomical development of the
mammary gland coupled with the ability to synthesize
and secrete milk does not occur until the female has
reached puberty, becomes pregnant and gives birth to
offspring.
Between Birth and Puberty, Mammary
Growth is Isometric
Between birth and puberty the mammary gland
experiences isometric gr·owth (at the same rate as the
other tissues). In other words, there is no noticeable
enlargement of the mammary glands when compared
to the rest of the body.
Mammary Glands Grow Significantly
Between Puberty and Pregnancy
After the onset of puberty, the mammary gland
begins to grow at a rate that is disproportionately
faster than normal body growth. This type of growth is
referred to as allometric growth. During repeated
estrous cycles, a duct and alveolar framework is con-
structed within the mammary gland. This framework
provides the cellular basis for future milk synthesis.
During the first several estrous (or menstrual) cycles,
the ducts begin to branch and their diameter increases
under the influence of estradiol. Under the influence
of progesterone (during the luteal phase), the terminal
portions of each branch begin to form the initial portions
ofthe alveoli. The alveoli fom1 the functional secretmy
elements of the mammaty gland (See Figure 15-13).
Estradiol alone will cause some duct development but
more complete and rapid duct development occurs
in the presence of prolactin and growth hormone
(somatotropin). Both of these hormones increase dur-
ing the onset of puberty. Repeated cyclic exposure of
the mammary cells to estrogen and progesterone can
stimulate mammogenesis to proceed only so far. The
mammary framework formed between puberty and
pregnancy needs fuh1re endocrine input during the
gestational period for complete development.
Final Mammary Development Occurs
During Pregnancy
Complete alveolar development in the dam
takes place during the last trimester of pregnancy. During
this time the terminal alveoli begin to grow into bunches
called lobules. A lobule would be analogous to a group
of grapes on a single stem among an entire bunch of
grapes (See Figure 15-13 ). A group oflobules that emp-
The Puerperium and Lactation 325
Figure 15-10. Prenatal Mammogenesis
Primary
mammary
bud
Secondary
mammary
bud
I Secondary mammary bud I
Canalization
Lactiferous
ducts
Myoepithelial —1
cells
Mammary Ridges
Mammary ridges are th ickened epider-
mal tissue that give rise to the mammary
gland.
Primary Mammary Bud
The thickened epidermal tissue begins
to develop inward and penetrate into the
mesenchyme (dermis).
Secondary Mammary Bud
The primary mammary bud begins to
send out branches that further penetrate
into the dermis.
Canalization
The fingerlike secondary buds begin to
lengthen and branch out. Finally they be-
gin to form canals or channels (canaliza-
tion) that will form the duct system of the
gland. Myoepithelial cells surround the
terminal portions of the developing gland.
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326 The Puerperium and Lactation

• Cow
• Camel

• Ewe
• Goat
• Mare

• Sow

Figure 15-11. Diversity in Anatomical Position, Number and Teat
Morphology Among Mammals

Inguinal I
J

Inguinal

Camel
2 canals per teat

(camel)

Cow

I canal I cistern
per teat

(cow, ewe, goat)

2 ducts
per teat

(mare, sow)

The Puerperium and Lactation 327

Figure 15-11. Diversity in Anatomical Position, Number and Teat
Morphology Among Mammals

• Rat
• Mouse
• Rabbit
• Bitch
• Queen

• Primates
• Elephant

I Thoracic I

/
( \ Primate

ties into a common duct is called a lobe. During the final
trimester of pregnancy, the lobulo-alveolar structur·es
develop to the point where they represent nearly 90%
of the cellular mass of the mammary gland at parturi-
tion. Prolactin, adrenal cortical hormones and placen-
tal lactogen are important in allowing the mammmy
epithelium to synthes ize milk. As seen in Chapter 14,
all ofthese bonn ones increase dramatically just before
the time of parturition . The induction of parturition is
carefully timed with the onset of the mammary gland’s
abil ity to secrete copious quantities of milk so that the
neonate has immediate access to mi lk.

Elephant
(Elephant photograph courtesy
of Dr. Janine L. Brown, Con-
servation & Reasearch Center,
National Zoological Park)

5 – 6 ducts
per teat

(bitch, queen)

8- 10 ducts
per nipple/ teat

(primate, elephant)

Lactation Provides Immunoprotection and
Nutrition for the Neonate

The first secretions from the mammary gland
(called colostrum) are critical to neonatal survival be-
cause the milk from the dam contains immunoglobulins
(antibodies). These immunoglobul ins are ingested by
the neonate and are h·ansported unaltered by the cells
of the gut mucosa to provide passive immunity. In
ruminants (and other animals) with an epitheliocho-
rial placenta, maternal immunoglobulins cannot be
transferred in-utero because the placenta is a baiTier.
Thus, ingestion of colosh·um soon after birth provides
necessary immunologic protection for the newborn. In

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326 The Puerperium and Lactation
• Cow
• Camel
• Ewe
• Goat
• Mare
• Sow
Figure 15-11. Diversity in Anatomical Position, Number and Teat
Morphology Among Mammals
Inguinal I
J
Inguinal
Camel
2 canals per teat
(camel)
Cow
I canal I cistern
per teat
(cow, ewe, goat)
2 ducts
per teat
(mare, sow)
The Puerperium and Lactation 327
Figure 15-11. Diversity in Anatomical Position, Number and Teat
Morphology Among Mammals
• Rat
• Mouse
• Rabbit
• Bitch
• Queen
• Primates
• Elephant
I Thoracic I
/
( \ Primate
ties into a common duct is called a lobe. During the final
trimester of pregnancy, the lobulo-alveolar structur·es
develop to the point where they represent nearly 90%
of the cellular mass of the mammary gland at parturi-
tion. Prolactin, adrenal cortical hormones and placen-
tal lactogen are important in allowing the mammmy
epithelium to synthes ize milk. As seen in Chapter 14,
all ofthese bonn ones increase dramatically just before
the time of parturition . The induction of parturition is
carefully timed with the onset of the mammary gland’s
abil ity to secrete copious quantities of milk so that the
neonate has immediate access to mi lk.
Elephant
(Elephant photograph courtesy
of Dr. Janine L. Brown, Con-
servation & Reasearch Center,
National Zoological Park)
5 – 6 ducts
per teat
(bitch, queen)
8- 10 ducts
per nipple/ teat
(primate, elephant)
Lactation Provides Immunoprotection and
Nutrition for the Neonate
The first secretions from the mammary gland
(called colostrum) are critical to neonatal survival be-
cause the milk from the dam contains immunoglobulins
(antibodies). These immunoglobul ins are ingested by
the neonate and are h·ansported unaltered by the cells
of the gut mucosa to provide passive immunity. In
ruminants (and other animals) with an epitheliocho-
rial placenta, maternal immunoglobulins cannot be
transferred in-utero because the placenta is a baiTier.
Thus, ingestion of colosh·um soon after birth provides
necessary immunologic protection for the newborn. In
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328 The Puerperium and Lactation

conh·ast, humans and other animals with hemochorial
placentation have placental transfer of immunoglobulins
from the dam to the fehts. The feh1s is thus born with
at least partial passive immunity. Immunoglobulins in
milk are still important to neonatal immunoprotection
in primates.

Breastfed infants suffer fewer ear infections,
respiratory infections and gastrointestinal disorders
compared to infants who are formula fed. Early feed-
ing in the neonatal period can have lifelong impact.
Women who breastfeed their babies have a lower risk
of breast cancer. Adults who were breastfed as infants
have a lower incidence of obesity, cardiac disease and
Type I diabetes compared to those who received for-
mula. Calves fed milk have higher growth rates and
lower morbidity and mortality compared to calves fed
milk-replacer. In addition, calves that grow faster during
the milk-feeding period can produce more milk during
their lifetimes.

Colostrum is provided for a brief period (2 to
3 days) and then milk composition remains relatively
constant for the remainder of the lactation. During the
course of lactation, milk synthesis increases and then
peaks shortly after parh1rition. After the secretory peak,
the synthetic rate decreases and this generally coincides
with the time of weaning. It is important to recognize
that growth of the neonate is directly proportional to
milk protein production by the dam. In some instances,
failure of the neonate to grow can be due to mastitis
(inflammation of the mammary gland) or agalactia
(failure to synthesize milk). It should be emphasized
that in some breeds of sheep and goats the reproductive
goal is to produce triplets and quadruplets. This goal
conflicts with the anatomy/lactation ability of the dam
since these species have only two teats. Nutrition of
the neonate may thus be compromised since there may
not be enough milk to serve the nutritional demands of
the offspring.

Involution is the Return to a
Nonsecretory State

As the need for milk as the sole nutritional
source begins to decrease, the neonate begins to suckle
less frequently. Consequently, there is a buildup of pres-
sure within the manunmy gland causing the secretory
cells to become less and less functional. This phenom-
enon is called pressure atrophy. Pressure atrophy is
such a powerful force that milk synthesis can be stopped
in just a few days if the intramammaty pressure is al-
lowed to buildup suddenly. The milk synthesis occur-
ring in the alveolar epithelium decreases to the point
that the cells undergo almost complete atrophy. Secre-
tory cells will remain nonfunctional until a subsequent
pregnancy. With a subsequent pregnancy, prolactin,

adrenal cortical hormones and placental lactogen will
restimulate alveolar cells to produce milk for another
lactation.

During involution, immune cells such as lym-
phocytes and macrophages invade the mammary tissue.
Mammary involution is a critical process because it
allows the mammaty gland to recover and develop new
secretory tissue for a subsequent lactation. Changes in
the tissue mass of the mammary gland as a function of
reproductive stage are presented in Figure 15-1 2.

Milk Contains Hormones
and Growth Factors

Many substances that are found in blood can
also be found in milk. Thus, milk naturally contains
hormones and growth factors that are derived from the
blood of the lactating female. Also, exogenous materi-
als such as alcohol, drugs, antibiotics, etc. can be found
in milk if they are consumed by the dam. Before the
advent of controlled nutrition in dairy cattle, it was not
uncommon for milk to have an onion-like flavor in the
spring because cows grazed in pastures and consumed
wild onions growing there. Chemicals causing the
onion flavor pass directly into the milk because they
are lipid soluble.

Protein hormones such as prolactin, GnRH,
growth hom1one (somatotropin), thyroid hormone (thy-
roxine) and their releasing factors have been identified
in milk. It should be emphasized that protein hormones
have little or no physiological effect on the neonate (or
the consumer) because they are hydrolyzed into amino
acids in the gash·ointestinal tract and therefore lose their
biologic activity.

All steroid hormones can be found in milk.
The concentration of estrogen and progesterone in milk
reflects cyclic hormone production by the ovaries and
is highly conelated with blood concentrations. Such a
phenomenon enables progesterone to be easily assayed
in milk to detem1ine the reproductive status of the fe-
male. Cowside ELISA teclmology enables progesterone
concentrations in mille to be determined. Procedures to
assay progesterone at each milking through the use of
“in-line” assay technology in the milking parlor is a
worthy research and development pursuit. The concept
would utilize a small sensor in each milking machine
that can transduce the progesterone concentration into
an electrical signal that could be transferred to the com-
puter. The development of such technology would en-
able the producer to determine whether a cow is cycling,
the stage of the estrous cycle, the pregnancy stah1s and
some fom1s of ovarian pathology (e.g. cystic ovarian
disease) for each cow on a daily basis. The availability
of such technology would revolutionize reproductive
management of dairy cows.

The Puerperium and Lactation 329

Figure 15-12. Changes in the Mammary Gland as a Function of
Reproductive Stage

(Modified from Mepham. 1987. Phvsio/oqy of Lactation)

‘ Stromal Tissue

1st
Pregnancy

2nd
Pregnancy

2nd
Lacation

The mammary gland undergoes continuous change from prenatal life through subsequent lactations. During
pubertal onset the ductal and secretory tissue of the mammary gland increases. During the first pregnancy
these tissues continue to increase but at a faster rate. At the time of parturition, the secretory tissue mass is
high and continues to increase until it peaks shortly after parturition during the fi rst lactation. At the conclu-
sion of the fi rst lactation (either weaning or drying-off in the dairy cow) the secretory tissue mass decreases
significantly (mammary involution, INV). During the second pregnancy and lactation secretory tissue and
ductal tissue increases significantly. Following lactation a second involution (INV) takes place.

Growth Factors in Milk May Provide New
Insights to Neonate Health

It is known that a number of growth factors
are present at high levels in colostrum. Colostrum is
the fi rst milk produced after parturition and contains
antibodies to provide the neonate with passive immu-
nity. These growth factors mirror the profile of immu-
noglobulins secreted into the colostrum. Researchers
have hypothesized that the accumulation of growth
factors in colostrum evolved to promote neonatal
growth and development. Examples of growth factors
found in colostrum are Insulin Like Growth Factors I
& 2 (IGF1&2), Epidermal Growth Factor (EGF) and
Transforming Growth Factor a and b (TGF-a, TGF-b).
Most of the discoveries related to the presence of these
growth factors in milk are relatively recent. Since
growth factors are present in milk and have significant
biologic activity, two outcomes could be important.
First, the discovery that these growth facto rs exist
opens a new avenue of study implicating mammary
secretions in neonatal health and development that go

beyond simply meeting nutritional needs. Secondly,
there must be some molecular protection mechanism
for these growth factors that prevents digestion by the
gastTointestinal tract. Better understanding in both areas
could open doors regarding neonatal health and growth
and protection mechanisms for various proteins.

Peptides are Physiologically Derived from
Milk Proteins

Over 15 physiologically active peptides are
derived from milk proteins. These peptides have been
implicated in controlling blood pressure (antihyper-
tensive}, prevention of blood clots (antithrombotic)
and activating the immune system (immunostimula-
tion). Opioid peptides from milk proteins (caseins and
lactalbumin) have morphine-like activity. Some of these
“casomorphins” are believed to prolong gastrointesti-
nal transit time by inhibiting gut moti lity. Such an effect
is antidianheal. Further, the dynamics of amino acid
transpori and induction of insulin and somatostatin
production may be a function of these casomorphins.

[li]
I

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328 The Puerperium and Lactation
conh·ast, humans and other animals with hemochorial
placentation have placental transfer of immunoglobulins
from the dam to the fehts. The feh1s is thus born with
at least partial passive immunity. Immunoglobulins in
milk are still important to neonatal immunoprotection
in primates.
Breastfed infants suffer fewer ear infections,
respiratory infections and gastrointestinal disorders
compared to infants who are formula fed. Early feed-
ing in the neonatal period can have lifelong impact.
Women who breastfeed their babies have a lower risk
of breast cancer. Adults who were breastfed as infants
have a lower incidence of obesity, cardiac disease and
Type I diabetes compared to those who received for-
mula. Calves fed milk have higher growth rates and
lower morbidity and mortality compared to calves fed
milk-replacer. In addition, calves that grow faster during
the milk-feeding period can produce more milk during
their lifetimes.
Colostrum is provided for a brief period (2 to
3 days) and then milk composition remains relatively
constant for the remainder of the lactation. During the
course of lactation, milk synthesis increases and then
peaks shortly after parh1rition. After the secretory peak,
the synthetic rate decreases and this generally coincides
with the time of weaning. It is important to recognize
that growth of the neonate is directly proportional to
milk protein production by the dam. In some instances,
failure of the neonate to grow can be due to mastitis
(inflammation of the mammary gland) or agalactia
(failure to synthesize milk). It should be emphasized
that in some breeds of sheep and goats the reproductive
goal is to produce triplets and quadruplets. This goal
conflicts with the anatomy/lactation ability of the dam
since these species have only two teats. Nutrition of
the neonate may thus be compromised since there may
not be enough milk to serve the nutritional demands of
the offspring.
Involution is the Return to a
Nonsecretory State
As the need for milk as the sole nutritional
source begins to decrease, the neonate begins to suckle
less frequently. Consequently, there is a buildup of pres-
sure within the manunmy gland causing the secretory
cells to become less and less functional. This phenom-
enon is called pressure atrophy. Pressure atrophy is
such a powerful force that milk synthesis can be stopped
in just a few days if the intramammaty pressure is al-
lowed to buildup suddenly. The milk synthesis occur-
ring in the alveolar epithelium decreases to the point
that the cells undergo almost complete atrophy. Secre-
tory cells will remain nonfunctional until a subsequent
pregnancy. With a subsequent pregnancy, prolactin,
adrenal cortical hormones and placental lactogen will
restimulate alveolar cells to produce milk for another
lactation.
During involution, immune cells such as lym-
phocytes and macrophages invade the mammary tissue.
Mammary involution is a critical process because it
allows the mammaty gland to recover and develop new
secretory tissue for a subsequent lactation. Changes in
the tissue mass of the mammary gland as a function of
reproductive stage are presented in Figure 15-1 2.
Milk Contains Hormones
and Growth Factors
Many substances that are found in blood can
also be found in milk. Thus, milk naturally contains
hormones and growth factors that are derived from the
blood of the lactating female. Also, exogenous materi-
als such as alcohol, drugs, antibiotics, etc. can be found
in milk if they are consumed by the dam. Before the
advent of controlled nutrition in dairy cattle, it was not
uncommon for milk to have an onion-like flavor in the
spring because cows grazed in pastures and consumed
wild onions growing there. Chemicals causing the
onion flavor pass directly into the milk because they
are lipid soluble.
Protein hormones such as prolactin, GnRH,
growth hom1one (somatotropin), thyroid hormone (thy-
roxine) and their releasing factors have been identified
in milk. It should be emphasized that protein hormones
have little or no physiological effect on the neonate (or
the consumer) because they are hydrolyzed into amino
acids in the gash·ointestinal tract and therefore lose their
biologic activity.
All steroid hormones can be found in milk.
The concentration of estrogen and progesterone in milk
reflects cyclic hormone production by the ovaries and
is highly conelated with blood concentrations. Such a
phenomenon enables progesterone to be easily assayed
in milk to detem1ine the reproductive status of the fe-
male. Cowside ELISA teclmology enables progesterone
concentrations in mille to be determined. Procedures to
assay progesterone at each milking through the use of
“in-line” assay technology in the milking parlor is a
worthy research and development pursuit. The concept
would utilize a small sensor in each milking machine
that can transduce the progesterone concentration into
an electrical signal that could be transferred to the com-
puter. The development of such technology would en-
able the producer to determine whether a cow is cycling,
the stage of the estrous cycle, the pregnancy stah1s and
some fom1s of ovarian pathology (e.g. cystic ovarian
disease) for each cow on a daily basis. The availability
of such technology would revolutionize reproductive
management of dairy cows.
The Puerperium and Lactation 329
Figure 15-12. Changes in the Mammary Gland as a Function of
Reproductive Stage
(Modified from Mepham. 1987. Phvsio/oqy of Lactation)
‘ Stromal Tissue
1st
Pregnancy
2nd
Pregnancy
2nd
Lacation
The mammary gland undergoes continuous change from prenatal life through subsequent lactations. During
pubertal onset the ductal and secretory tissue of the mammary gland increases. During the first pregnancy
these tissues continue to increase but at a faster rate. At the time of parturition, the secretory tissue mass is
high and continues to increase until it peaks shortly after parturition during the fi rst lactation. At the conclu-
sion of the fi rst lactation (either weaning or drying-off in the dairy cow) the secretory tissue mass decreases
significantly (mammary involution, INV). During the second pregnancy and lactation secretory tissue and
ductal tissue increases significantly. Following lactation a second involution (INV) takes place.
Growth Factors in Milk May Provide New
Insights to Neonate Health
It is known that a number of growth factors
are present at high levels in colostrum. Colostrum is
the fi rst milk produced after parturition and contains
antibodies to provide the neonate with passive immu-
nity. These growth factors mirror the profile of immu-
noglobulins secreted into the colostrum. Researchers
have hypothesized that the accumulation of growth
factors in colostrum evolved to promote neonatal
growth and development. Examples of growth factors
found in colostrum are Insulin Like Growth Factors I
& 2 (IGF1&2), Epidermal Growth Factor (EGF) and
Transforming Growth Factor a and b (TGF-a, TGF-b).
Most of the discoveries related to the presence of these
growth factors in milk are relatively recent. Since
growth factors are present in milk and have significant
biologic activity, two outcomes could be important.
First, the discovery that these growth facto rs exist
opens a new avenue of study implicating mammary
secretions in neonatal health and development that go
beyond simply meeting nutritional needs. Secondly,
there must be some molecular protection mechanism
for these growth factors that prevents digestion by the
gastTointestinal tract. Better understanding in both areas
could open doors regarding neonatal health and growth
and protection mechanisms for various proteins.
Peptides are Physiologically Derived from
Milk Proteins
Over 15 physiologically active peptides are
derived from milk proteins. These peptides have been
implicated in controlling blood pressure (antihyper-
tensive}, prevention of blood clots (antithrombotic)
and activating the immune system (immunostimula-
tion). Opioid peptides from milk proteins (caseins and
lactalbumin) have morphine-like activity. Some of these
“casomorphins” are believed to prolong gastrointesti-
nal transit time by inhibiting gut moti lity. Such an effect
is antidianheal. Further, the dynamics of amino acid
transpori and induction of insulin and somatostatin
production may be a function of these casomorphins.
[li]
I
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330 The Puerperium and Lactation

One additional proposed function of casomorphins is
that they produce an analgesic effect causing drowsi-
ness and sleep in infants. While little is known about
the physiologic activity of these milk protein derived
peptides, the fact that many of these materials have
distinct phannacological effects opens new doors for
the potential use of milk in a therapeutic sense.

Pharmaceutical Proteins are Secreted in Milk
of Transgenic Animals

By employing genetic engineering techniques,
it is now possible to “genetically engineer” a mam-
mary gland that would secrete materials that can have
significant therapeutic effects on the consumer beyond
the known nutritional effects of milk. For example,
recombinant human antithrombin purified from the
milk of transgenic goats has been approved for human
use by the U.S. Food and Drug Administration and the
European Medicines Agency to treat a rare clotting
disorder. The amount of this dmg obtained from one
goat in a year is equivalent to that from 90,000 human
blood donations. A second product, recombinant human
C I inhibitor, purified from rabbit milk and used to treat
hereditaty angioedema, has been approved for use in
the European Union. Other phammceutical proteins se-
creted in milk from transgenic animals and cunently in
FDA clinical trials include fibrinogen, malaria antigen,
albumin and several other clotting factors. Exploiting
the mammary gland of transgenic animals as an organ
to synthesize and secrete pharmaceutical proteins holds
great promise as health promotion interventions.

Milk Ejection Transfers Milk from
the Mammary Alveoli into the Ducts

Milk ejection is the active transferofmilk from
the alveoli and alveolar ducts into the larger mammary
ducts, the cisterns and into the teat or nipples where it
can be removed by the suckling neonate. Milk ejec-
tion should not be confused with milk secretion. Milk
is synthesized and secreted by the alveolar cell into
alveolar lumina. Prior to suckling (or milking) milk is
predominately located in alveolar lumina and the fine
ducts draining the alveoli. Milk stays in these ana-
tomical regions because there is a strong resistance to
milk flow in such a small diameter network (a fonn of
capillary action causing retention of the milk). Between
sucklings (ormilkings) 70% to 80% of all secreted milk
is located within the lumina of the alveoli and small
ducts of the mammary gland. Therefore, an active
mechanism for removal of this large quantity of milk
is necessaty so that the neonate can have access to it
through suckling.

Milk ejection requires:

• sensory activation (auditory, tactile
and visual)

• neural activation of the hypothalamus

• o:A.ytocin release into the blood

• contraction oftlze myoepithelial cells

• mechanical transfer of milk from
alveoli into ducts and finally into the
teat/nipple

Milk ejection is an active neuroendocrine reflex
involving sensmy neurons in the teat or nipple, release
of oxytocin from the neurohypophysis and contraction
of myoepithelial cells that surround each alveolus and
some of the ducts . The ejection process results in a rapid
transfer of milk from the alveolus and smaller ducts into
the larger ducts and cisterns of the mammary gland.
Myoepithelial cells are spindle shaped contractile cells
that surround each alveolus in a mesh-like fashion (See
Figure 15-13). Myoepithelial cells are very similar in
structure to smooth muscle cells. The process of milk
ejection is also referred to as ” milk letdown.” Efficient
and timely removal of milk from the mammary gland is
important not only for extraction of milk by the neonate,
but also is an important part of the milk harvest to pre-
vent pressure atrophy. In general, the more frequently
milk is removed, the less the pressure atrophy and
greater the quantity of milk that can be secreted.

Tactile stimulation of the teat or nipple is the
primary sensory “driver” for milk ejection. In addition
to direct tactile stimulation of the teat or nipple, sounds
of the neonate (or the milking parlor), visual sight of
the newbom or a milking faci lity can stimulate release
of oxytocin from the neurohypophysis. Release of
oxytocin is brought about by afferent nerve fibers that
synapse with oxytocin synthesizing neurons in the para-
ventricular and the supraoptic nuclei. When sufficient
frequency of stimulation has been accomplished, nerves
in the two nuclei begin to fire and release oxytocin
from their tern1inals located in the neurohypophysis.
Oxytocin is then secreted into the blood and enters the
systemic circulation of the dam. The physiology of
milk ejection is presented in Figure 15- .13.

The myoepithelial cells within the mammary
gland have receptors for oxytocin and contract imme-
diately upon exposure to it. When myoepithelial cells
contract they cause the diameter of the alveolus to be
greatly reduced. Thus, milk is ejected into larger ducts
and is transferred into the larger spaces and finally into
the teat or nipple.

The Puerperium and Lactation 331

Figure 15-13. The Anatomy and Physiology of Milk Ejection

The milk ejection mechanism is initiated by suckling (1 ). The teat contains sensory neurons and
impulses from these neurons travel through afferent nerves (2) to the hypothalamus. Nerves in
the paraventricular nuclei are stimulated by these afferent neurons and the terminals in the pos-
terior lobe of the pituitary (3) release oxytocin. Oxytocin then enters the blood and is delivered
to the mammary gland (4 ). The target cells for oxytocin are the myoepithelial cells that surround
the alveolus. Contraction of the myoepithelial cells (5) causes milk to be “squeezed” out of each
individual alveolus into small ducts and then into larger ducts. The net effect of simultaneous
contraction of the myoepithelial cells throughout the entire mammary gland is to deliver milk to the
large ducts and the gland cistern so that it is available for removal by the neonate.

Ve
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330 The Puerperium and Lactation
One additional proposed function of casomorphins is
that they produce an analgesic effect causing drowsi-
ness and sleep in infants. While little is known about
the physiologic activity of these milk protein derived
peptides, the fact that many of these materials have
distinct phannacological effects opens new doors for
the potential use of milk in a therapeutic sense.
Pharmaceutical Proteins are Secreted in Milk
of Transgenic Animals
By employing genetic engineering techniques,
it is now possible to “genetically engineer” a mam-
mary gland that would secrete materials that can have
significant therapeutic effects on the consumer beyond
the known nutritional effects of milk. For example,
recombinant human antithrombin purified from the
milk of transgenic goats has been approved for human
use by the U.S. Food and Drug Administration and the
European Medicines Agency to treat a rare clotting
disorder. The amount of this dmg obtained from one
goat in a year is equivalent to that from 90,000 human
blood donations. A second product, recombinant human
C I inhibitor, purified from rabbit milk and used to treat
hereditaty angioedema, has been approved for use in
the European Union. Other phammceutical proteins se-
creted in milk from transgenic animals and cunently in
FDA clinical trials include fibrinogen, malaria antigen,
albumin and several other clotting factors. Exploiting
the mammary gland of transgenic animals as an organ
to synthesize and secrete pharmaceutical proteins holds
great promise as health promotion interventions.
Milk Ejection Transfers Milk from
the Mammary Alveoli into the Ducts
Milk ejection is the active transferofmilk from
the alveoli and alveolar ducts into the larger mammary
ducts, the cisterns and into the teat or nipples where it
can be removed by the suckling neonate. Milk ejec-
tion should not be confused with milk secretion. Milk
is synthesized and secreted by the alveolar cell into
alveolar lumina. Prior to suckling (or milking) milk is
predominately located in alveolar lumina and the fine
ducts draining the alveoli. Milk stays in these ana-
tomical regions because there is a strong resistance to
milk flow in such a small diameter network (a fonn of
capillary action causing retention of the milk). Between
sucklings (ormilkings) 70% to 80% of all secreted milk
is located within the lumina of the alveoli and small
ducts of the mammary gland. Therefore, an active
mechanism for removal of this large quantity of milk
is necessaty so that the neonate can have access to it
through suckling.
Milk ejection requires:
• sensory activation (auditory, tactile
and visual)
• neural activation of the hypothalamus
• o:A.ytocin release into the blood
• contraction oftlze myoepithelial cells
• mechanical transfer of milk from
alveoli into ducts and finally into the
teat/nipple
Milk ejection is an active neuroendocrine reflex
involving sensmy neurons in the teat or nipple, release
of oxytocin from the neurohypophysis and contraction
of myoepithelial cells that surround each alveolus and
some of the ducts . The ejection process results in a rapid
transfer of milk from the alveolus and smaller ducts into
the larger ducts and cisterns of the mammary gland.
Myoepithelial cells are spindle shaped contractile cells
that surround each alveolus in a mesh-like fashion (See
Figure 15-13). Myoepithelial cells are very similar in
structure to smooth muscle cells. The process of milk
ejection is also referred to as ” milk letdown.” Efficient
and timely removal of milk from the mammary gland is
important not only for extraction of milk by the neonate,
but also is an important part of the milk harvest to pre-
vent pressure atrophy. In general, the more frequently
milk is removed, the less the pressure atrophy and
greater the quantity of milk that can be secreted.
Tactile stimulation of the teat or nipple is the
primary sensory “driver” for milk ejection. In addition
to direct tactile stimulation of the teat or nipple, sounds
of the neonate (or the milking parlor), visual sight of
the newbom or a milking faci lity can stimulate release
of oxytocin from the neurohypophysis. Release of
oxytocin is brought about by afferent nerve fibers that
synapse with oxytocin synthesizing neurons in the para-
ventricular and the supraoptic nuclei. When sufficient
frequency of stimulation has been accomplished, nerves
in the two nuclei begin to fire and release oxytocin
from their tern1inals located in the neurohypophysis.
Oxytocin is then secreted into the blood and enters the
systemic circulation of the dam. The physiology of
milk ejection is presented in Figure 15- .13.
The myoepithelial cells within the mammary
gland have receptors for oxytocin and contract imme-
diately upon exposure to it. When myoepithelial cells
contract they cause the diameter of the alveolus to be
greatly reduced. Thus, milk is ejected into larger ducts
and is transferred into the larger spaces and finally into
the teat or nipple.
The Puerperium and Lactation 331
Figure 15-13. The Anatomy and Physiology of Milk Ejection
The milk ejection mechanism is initiated by suckling (1 ). The teat contains sensory neurons and
impulses from these neurons travel through afferent nerves (2) to the hypothalamus. Nerves in
the paraventricular nuclei are stimulated by these afferent neurons and the terminals in the pos-
terior lobe of the pituitary (3) release oxytocin. Oxytocin then enters the blood and is delivered
to the mammary gland (4 ). The target cells for oxytocin are the myoepithelial cells that surround
the alveolus. Contraction of the myoepithelial cells (5) causes milk to be “squeezed” out of each
individual alveolus into small ducts and then into larger ducts. The net effect of simultaneous
contraction of the myoepithelial cells throughout the entire mammary gland is to deliver milk to the
large ducts and the gland cistern so that it is available for removal by the neonate.
Ve
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332 The Puerperium and Lactation

Further
PHENOMENA
for Fertility

“Bedroom Talk:
R eproductive Physiology Style”

by Ruth Loomis

Hey honey, wake-up and quit your snoring
I think I can fee/my E1 /eve/s soaring.
My ovary is primed for the LH surge,
Come 011, wake-up, I’ve got tlte urge!

I’m certain this egg is ripe for fertilization,
But, in case you ‘•’e forgottell, that does require
i11semination!
Ami I recall, it’s bee11 nearly a week
Your epididymal reserves must be at peak!

· 0/r, I see you need a bit more stimulation.
I could continue with some more plwnation?
No, don ‘t close your eyes. Wake-up and take
notice.
I’m displaying some absolutely fabulous lordo-
sis!

What’s that you say, you want me to look
On page- of my reproductio11 book?
So your telling me this passage It as led you to
reflect
That what would really work for you is the
Coolidge Effect?!

Is that so? Well tlo as you wislt, my darling, my
sweets
But /mow this, you won’t be sleeping between
these two sheets!

Ruth Loomis was a student inA11imal Reproduc-
tive Physiology at Washington State University in
the spring of 2002 a11d based the poem above Oil
the nomenclature she /eamed i11 the reproduction
course. Size graduated from Was!lington State
University with a BA in English Literature. She
is 11ow in the College of Veteri11ary Medicine at
WSU (Class of2006).

The 19th Century British explorer, Sir
Richard Burton, developed a recipe that he
believed enhanced sperm production am/
viability. Such a recipe would result in an
increased probability of conception. He
used a mixture of honey, opium, spices and
a small lizard. The pmpose of the lizard has
not been disclosed. The author beliel’es that
Sir Burton knew something about sperm
motility and related the rapid crawling mo-
tion of a lizard to that of spermatozoa.

Oysters can change from one gender to
another and back again.

The tale of the mini ball pregnancy gives
new meaning to the term target tissue. A
surgeon in the Civil War treated two patients
that had been shot near one anothe1: One
patient was a soldier who was protecting the
treatment ward and suffered a gunshot that
passed through his scrotum and took off the
left testicle completely on its way out. The
other patient was a nurse who received a
serious shot to the left side of her abdomen,
the bullet lost somewhere inside. Miracu-
lously the woman sun,ived the wound but
months later she began to notice abnormal
swelling of her abdomen. The surgeon
was sure the woman was pregnant, but the
patient and the villagers all swore to her
absolute virginity. Upon examination it was
found that the woman was indeed pregnant,
with hymen still intact. The child was born
without difficulty but soon it was noticed
that the young boy had a large, hard mass
contained within his right testicle. The doc-
tor operated on the young child to remove
the lump and was astounded to discover
the contents of the testicle was none other
than the missing mini ball that wounded
the mother nine months befoJ·e. The solu-
tion to this mysterious conception? The
surgeon thought that this mini ball must
have been the same one to have mutilated
the soldier’s testicle, canying sperm with it
into the uterus of the nurse after the bullet
left the first victim, where it remained and
functioned to fertilize one of her eggs! How
could the ball get into the scrotum of the
neonate? What parts of this narrative are
absolutely false and which could be tme?
A great final exam q uestionl

Key Refer ences

Akers, R.M. 2002. Lactation and the Mammarv Gland.
Iowa State Press, Ames ISBN 0-8138-2992-5.

Arthur, G.H. D.E. Noakes, H . Pearson, and T.J. Par-
kinson. 1996. Veterinarv Reproduction and Obstetrics,
7th Edition. W.B. Saunders, Co. Philadelphia. ISBN
0-7020-1 785-X.

Gier, H.T. and G.B. Marion. 1968. “Uterus of the cow
after parturition: involutional changes.” Am. J. Vet. Res.
29:83-96.

Larson, B.L. ed. 1985. Lactation. Iowa State Press,
Ames. ISBN 0-8138- 1063-9.

McEntee, K. 1990. Reproductive Patholof!Y of’Don!estic
Animals. Academic Press, Inc. San Diego.)SJ?N. 012-
483375-6.

Mepham, T.B. 1987. Phvsiologv o(Lactation. Open
University Press, Philadelphia. ISBN 0-335-1 5152-3.

Morrow, D.A. 1969. “Postpartum ovarian activity and
involution of the uterus and cervix in dairy cattle.” Vet-
erinm )i Scope. Vol1 4.

Salamonsen, L.A. 2003. “Tissue injury and repair in the
female human reproductive h·act.” Reprod. 125:30

I.

Salisbury, G.W. , N.L. VanDemark and J.R. Lodge. 1978.
Phvsiology o[Reproduction and Artificial Insemination
in Cattle. 2nd Edition. W.H. Freeman and Co., San
Francisco. ISBN 0-7167-0025-5.

Schmidt, G.H. 1971. Biology o(Lactation. W.H. Free-
man, San Francisco. ISBN 07-1670821 -3.

The Puerperium and Lactation 333
Ve
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332 The Puerperium and Lactation
Further
PHENOMENA
for Fertility
“Bedroom Talk:
R eproductive Physiology Style”
by Ruth Loomis
Hey honey, wake-up and quit your snoring
I think I can fee/my E1 /eve/s soaring.
My ovary is primed for the LH surge,
Come 011, wake-up, I’ve got tlte urge!
I’m certain this egg is ripe for fertilization,
But, in case you ‘•’e forgottell, that does require
i11semination!
Ami I recall, it’s bee11 nearly a week
Your epididymal reserves must be at peak!
· 0/r, I see you need a bit more stimulation.
I could continue with some more plwnation?
No, don ‘t close your eyes. Wake-up and take
notice.
I’m displaying some absolutely fabulous lordo-
sis!
What’s that you say, you want me to look
On page- of my reproductio11 book?
So your telling me this passage It as led you to
reflect
That what would really work for you is the
Coolidge Effect?!
Is that so? Well tlo as you wislt, my darling, my
sweets
But /mow this, you won’t be sleeping between
these two sheets!
Ruth Loomis was a student inA11imal Reproduc-
tive Physiology at Washington State University in
the spring of 2002 a11d based the poem above Oil
the nomenclature she /eamed i11 the reproduction
course. Size graduated from Was!lington State
University with a BA in English Literature. She
is 11ow in the College of Veteri11ary Medicine at
WSU (Class of2006).
The 19th Century British explorer, Sir
Richard Burton, developed a recipe that he
believed enhanced sperm production am/
viability. Such a recipe would result in an
increased probability of conception. He
used a mixture of honey, opium, spices and
a small lizard. The pmpose of the lizard has
not been disclosed. The author beliel’es that
Sir Burton knew something about sperm
motility and related the rapid crawling mo-
tion of a lizard to that of spermatozoa.
Oysters can change from one gender to
another and back again.
The tale of the mini ball pregnancy gives
new meaning to the term target tissue. A
surgeon in the Civil War treated two patients
that had been shot near one anothe1: One
patient was a soldier who was protecting the
treatment ward and suffered a gunshot that
passed through his scrotum and took off the
left testicle completely on its way out. The
other patient was a nurse who received a
serious shot to the left side of her abdomen,
the bullet lost somewhere inside. Miracu-
lously the woman sun,ived the wound but
months later she began to notice abnormal
swelling of her abdomen. The surgeon
was sure the woman was pregnant, but the
patient and the villagers all swore to her
absolute virginity. Upon examination it was
found that the woman was indeed pregnant,
with hymen still intact. The child was born
without difficulty but soon it was noticed
that the young boy had a large, hard mass
contained within his right testicle. The doc-
tor operated on the young child to remove
the lump and was astounded to discover
the contents of the testicle was none other
than the missing mini ball that wounded
the mother nine months befoJ·e. The solu-
tion to this mysterious conception? The
surgeon thought that this mini ball must
have been the same one to have mutilated
the soldier’s testicle, canying sperm with it
into the uterus of the nurse after the bullet
left the first victim, where it remained and
functioned to fertilize one of her eggs! How
could the ball get into the scrotum of the
neonate? What parts of this narrative are
absolutely false and which could be tme?
A great final exam q uestionl
Key Refer ences
Akers, R.M. 2002. Lactation and the Mammarv Gland.
Iowa State Press, Ames ISBN 0-8138-2992-5.
Arthur, G.H. D.E. Noakes, H . Pearson, and T.J. Par-
kinson. 1996. Veterinarv Reproduction and Obstetrics,
7th Edition. W.B. Saunders, Co. Philadelphia. ISBN
0-7020-1 785-X.
Gier, H.T. and G.B. Marion. 1968. “Uterus of the cow
after parturition: involutional changes.” Am. J. Vet. Res.
29:83-96.
Larson, B.L. ed. 1985. Lactation. Iowa State Press,
Ames. ISBN 0-8138- 1063-9.
McEntee, K. 1990. Reproductive Patholof!Y of’Don!estic
Animals. Academic Press, Inc. San Diego.)SJ?N. 012-
483375-6.
Mepham, T.B. 1987. Phvsiologv o(Lactation. Open
University Press, Philadelphia. ISBN 0-335-1 5152-3.
Morrow, D.A. 1969. “Postpartum ovarian activity and
involution of the uterus and cervix in dairy cattle.” Vet-
erinm )i Scope. Vol1 4.
Salamonsen, L.A. 2003. “Tissue injury and repair in the
female human reproductive h·act.” Reprod. 125:30 I.
Salisbury, G.W. , N.L. VanDemark and J.R. Lodge. 1978.
Phvsiology o[Reproduction and Artificial Insemination
in Cattle. 2nd Edition. W.H. Freeman and Co., San
Francisco. ISBN 0-7167-0025-5.
Schmidt, G.H. 1971. Biology o(Lactation. W.H. Free-
man, San Francisco. ISBN 07-1670821 -3.
The Puerperium and Lactation 333
Ve
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oo
ks
.ir

The Puerperium
& Lactation

Parturition
Fetal Attachment & Gestation
Early Embryogenesis &
Maternal Recognition of Pregnancy
Ovulation & Fertilization
Cyclicity
Regulation of
Reproduction
Tract Function
Puberty
Prenatal
Development

Menopause

,
I

-.. I’ Andropause .. I’-
\

Spermatogenesis
Regulation of
Reproduction
Tract Function
Puberty
Prenatal
Development

Take Home Message
Previous chapters have described the physiology of the 15 events along the pathway

of the reproductive process. In this chapte1; we will address human reproduction by
describing four factors that distinguish reproduction in humans from other mammals.

These are:
• The menstmal cycle that involves monthly endometrial sloughing
• Hormonal contraception that is used as a pregnancy management intervention
• Assisted reproductive technologies (ART) that are used to overcome infertility
• Menopause and andropause that are natural consequences of extended lifespans

A ve1y high percentage of the population is directly experiencing one or more aspects
of the above. Understanding the physiologic basis for these factors is important for high
compliance with inten,ention use, good reproductive health and a high quality of life.

In animals, reproductive processes can :l)e –discussed openly, manipulated without reservation
and pregnancies are viewed as an essential expectation
because high reproductive rates are obligatory for ef-
ficient food animal production and maintenance of wild
populations of animals. Unfortunately, reprod uctive
physiology in humans is often confused with sex and
this confusion results in controversies focusing around
ethical, religious, political and personal values that
detract from the fimdamental value of understanding
how the reproductive system works. Consequently,
there is a significant degree of misunderstanding about
reproductive fimction. These misunderstandings result
in untrue hearsays, myths, unfounded opinions and poor
Imowledge of reproductive scie nce in general. This
chapter will focus on the physiologic principles ofhow
the reproductive system works in humans especially as
it relates to contemporary interventions that directly
impact reproductive function.

It is predicted that the world population will
approach 10 billion by the year 2050. This will create
frightening pressures on the production and allocation
of food resources especially the production of animal
based protein (meat, milk and eggs). The field of re-
productive physiology likewise is under similar pres-
sures because on one hand the goal is to improve and
maximize reproductive performance in food-producing
animals, while on the other hand to restrict and manage
reproductive rate in the human population. An addi-
tional challenge is to find ways that knowledge about
reproductive science can be objectively presented to
different culh1res, religions and political stmch1res with
the ultimate goal of improving reproductive health and
the quality of life.

The Physiology of the Menstrual Cycle
has a Different Starting Point

Than the Estrous Cycle

Understanding the physiology of the menstrual
cycle is an important prerequisite for good reproductive
health, pregnancy prevention and family-planning. It
should be understood by both women and men. Under-
standing the menstrual cycle requires basic knowledge
about: l) the female reproductive organs and their fimc-
tions; 2) the major hormones and their secretory patterns
during the cycle; 3) how the major hom10nes influence
the fimction of the reproductive organs; 4) how the
major organs impact behavioral/emotional status of the
woman and 5) the major ovarian and uterine changes
that occur during the cycle.

A recent study indicated that almost 40% of
survey participants incorrectly identified or didn’t know
the menstrual cycle length and 22% did not lmow if their
own menstrual cycles were normal or abnormal. In ad-
dition, only 2% of adolescent girls reported receiving
infom1ation regarding menstruation from their health
care providers. The majority of the infonnation was
obtained from their mothers (85%), friends or sisters
(6.5%) or no one (6%). These data suggest the girls
are not receiving scientifically accurate information
about their menstrual cycles (See Houston in Key
References).

An earl ier study involving female university
students found that: 1) 59% of participants could not
properly describe the sequence of menstrual cycle
events; 2) 30% of the women could not provide a basic
defini tion of menstruation; 3) approximately 33% of
the participants did not know how hormones fluctuated

16

Ve
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oo
ks
.ir

The Puerperium
& Lactation
Parturition
Fetal Attachment & Gestation
Early Embryogenesis &
Maternal Recognition of Pregnancy
Ovulation & Fertilization
Cyclicity
Regulation of
Reproduction
Tract Function
Puberty
Prenatal
Development
Menopause
,
I
-.. I’ Andropause .. I’-
\
Spermatogenesis
Regulation of
Reproduction
Tract Function
Puberty
Prenatal
Development
Take Home Message
Previous chapters have described the physiology of the 15 events along the pathway
of the reproductive process. In this chapte1; we will address human reproduction by
describing four factors that distinguish reproduction in humans from other mammals.
These are:
• The menstmal cycle that involves monthly endometrial sloughing
• Hormonal contraception that is used as a pregnancy management intervention
• Assisted reproductive technologies (ART) that are used to overcome infertility
• Menopause and andropause that are natural consequences of extended lifespans
A ve1y high percentage of the population is directly experiencing one or more aspects
of the above. Understanding the physiologic basis for these factors is important for high
compliance with inten,ention use, good reproductive health and a high quality of life.
In animals, reproductive processes can :l)e –discussed openly, manipulated without reservation
and pregnancies are viewed as an essential expectation
because high reproductive rates are obligatory for ef-
ficient food animal production and maintenance of wild
populations of animals. Unfortunately, reprod uctive
physiology in humans is often confused with sex and
this confusion results in controversies focusing around
ethical, religious, political and personal values that
detract from the fimdamental value of understanding
how the reproductive system works. Consequently,
there is a significant degree of misunderstanding about
reproductive fimction. These misunderstandings result
in untrue hearsays, myths, unfounded opinions and poor
Imowledge of reproductive scie nce in general. This
chapter will focus on the physiologic principles ofhow
the reproductive system works in humans especially as
it relates to contemporary interventions that directly
impact reproductive function.
It is predicted that the world population will
approach 10 billion by the year 2050. This will create
frightening pressures on the production and allocation
of food resources especially the production of animal
based protein (meat, milk and eggs). The field of re-
productive physiology likewise is under similar pres-
sures because on one hand the goal is to improve and
maximize reproductive performance in food-producing
animals, while on the other hand to restrict and manage
reproductive rate in the human population. An addi-
tional challenge is to find ways that knowledge about
reproductive science can be objectively presented to
different culh1res, religions and political stmch1res with
the ultimate goal of improving reproductive health and
the quality of life.
The Physiology of the Menstrual Cycle
has a Different Starting Point
Than the Estrous Cycle
Understanding the physiology of the menstrual
cycle is an important prerequisite for good reproductive
health, pregnancy prevention and family-planning. It
should be understood by both women and men. Under-
standing the menstrual cycle requires basic knowledge
about: l) the female reproductive organs and their fimc-
tions; 2) the major hormones and their secretory patterns
during the cycle; 3) how the major hom10nes influence
the fimction of the reproductive organs; 4) how the
major organs impact behavioral/emotional status of the
woman and 5) the major ovarian and uterine changes
that occur during the cycle.
A recent study indicated that almost 40% of
survey participants incorrectly identified or didn’t know
the menstrual cycle length and 22% did not lmow if their
own menstrual cycles were normal or abnormal. In ad-
dition, only 2% of adolescent girls reported receiving
infom1ation regarding menstruation from their health
care providers. The majority of the infonnation was
obtained from their mothers (85%), friends or sisters
(6.5%) or no one (6%). These data suggest the girls
are not receiving scientifically accurate information
about their menstrual cycles (See Houston in Key
References).
An earl ier study involving female university
students found that: 1) 59% of participants could not
properly describe the sequence of menstrual cycle
events; 2) 30% of the women could not provide a basic
defini tion of menstruation; 3) approximately 33% of
the participants did not know how hormones fluctuated
16
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336 The Human Factor

during the cycle and 4) 54% could name only one of the
hom1ones involved, but could not describe the f1mction
of the hormone (See Koffin Key References).

The data above indicates a compelling Jack of
knowledge about the menstrual cycle among Alneri-
can women. This lack of knowledge undoubtedly
translates into the fact that approximately 50% of the
six million annual pregnancies in the United States are
unintended.

The menstrual cycle consists of the
following six events:

• menstruation
• follicular growth
• ovulation
• corpus luteum formation and growth
• endometrial growth and secretion
• luteo/ysis

In Chapter 7, we compared the menstrual cycle
with the estrous cycle. The menstrual cycle differs
fi·om the estrous cycle in two fundamental ways. First,
endometrial sloughing (menses) occurs in a predictable
manner dming eve1y cycle ifthe woman is not pregnant.
Second, there is no defined period of sexual receptivity.
In this chapter, we want to discuss the menstrual cycle
as a series of six distinctly different events (See Figure

16-1 ). Remember, the menstrual cycle starts at the first
day of the menstrual period (menses). This timing con-
vention originated because the menstrual period was an
observable event and marked the start of each menstrual
cycle. Typically, the length of the menstrual cycle is
28 days, but can range from 25 to 34 days. This is the
period of time from the start of one menstrual period
to the start of the next menstrual period.

The onset of menstruat ion signals the start
of the cycle and is it designated as day 1. Following
menstruation, marked follicular growth takes place in
response to FSH and LH from the anterior pituitary. At
about day 14, ovulation occurs. The newly ovu lated
follicle then develops into a corpus luteum (CL). The
CL secretes progesterone and some estrogens. These
hormones promote endometrial growth. Near the end
of the cycle, if a woman is not pregnant, the corpus
luteum undergoes luteolysis and loses its ability to se-
crete progesterone (See Figure 9-14). The rapid drop in
progesterone stimulates the onset of the next menstrual
period. Figure 16-1 describes the events of the menstrual
cycle in a c ircular and linear fashion.

The menstrual cycle is usually described as
having t\vo components. One component is the ovarian
cycle that describes changes that occur in the ovary dur-
ing the cycle. The uterine cycle describes the changes
that take place in the endometrium of the uterus during
the cycle.

The menstrual cycle also consists of the fol-
licular phase and the luteal phase (See Figure 16-2).
Follicular growth during and fo llowing menstruation is
referred to as the follicular phase because the dominant

Figure 16-1. Menstrual Cycle Sequence

•
t

Doy I

Lutcolysis

CL&
C!ndome:trlo:d

growth

Follicle growth

•
28 days

(Ronge = doys}

Doy 14

Follicle
growth

The six major events of the men-
strua l cycle are shown by the
circled numerals. Their relation-
ship to the timing of the cycle is
illustrated in circular form in the
top portion of the graphic and in
linear form in the bottom portion
of the graphic.

ovarian structures are foll icles. The dominant ovarian
hormone is estradiol. After ovulation, the luteal phase
begins. The dominant ovarian structure is the corpus
luteum and the dominant ovar ian hormone is proges-
terone. lt should be emphasized that while both the
menstrual cycle and estrous cycle are characterized by
the follicular and luteal phases, fo llicles are constantly
developing and regressing in both phases of the cycle.
In other words, even during the luteal p hase fo llicles
develop and regress.

During the follicular p hase, the anterior pitu-
itary secretes FSH and LH (See Figure 16-2) . These
hormones promote growth of ovarian fo llicles and
the growing follicles secrete increasing quantities of
estrogens. A threshold concentration of estmdiol trig-
gers the LH surge that causes ovulation. After ovulation
the luteal phase begins . The follicle that just ovulated
becomes the coqnrs luteum and secretes high concen-
trations of progesterone and some estradiol. The high
concentrations of progesterone inhib it GnRH secretism
from the hypothalamus and FSH and LH secretioq.fiom
the anterior pihlitary. Therefore, follicles do not develop
to the preovulatory stage during the luteal phase. At

The Human Factor 337

about day 23-24, progesterone drops rapidly because
luteolysis has occurred. This sudden drop in proges-
terone is thought to cause symptoms of premenstrual
syndrome (PMS) in many women. Further, the drop
in progesterone initiates endometrial sloughing and the
next menstrual period.

The uterine cycle is subdivided into the prolif-
erative and sec•·eto•·y phases. The proliferative phase
is the increase in endometrial thickness in response to
estradiol secreted by growing follicles. This increased
thickness is referred to as the proliferative phase be-
cause the cells of the endometr ium divide by mitosis
(proliferate). After oV1ilation and formation of the CL,
progesterone promotes further increased thickness in
the endometrium and it develops secretory capacity.
This is important because if conception takes place at
around day 14, the embryo will enter an environment
about three days later that is ideal for sustaining emb1yo
development prior to implantation. If pregnancy does
not occur, luteolysis is initiated, progesterone drops pre-
cip itously and a new menstrual period (and menstrual
cycle) begins (See Figure 16-3.).

Figure 16-2. Relative Blood Concentrations of FSH, LH, Estradiol and
Progesterone During the Follicular and Luteal Phases of

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the Menstrual Cycle
Ov ulation • .———·– -,.

Menses ;_ __ j

day 1 day 14

day 28

0

Estradiol

LH

•

FSH

• Progesterone

During the menstrual period, all hormones are low. During the follicular phase, FSH promotes foll icular de-
velopment. Developing follicles secrete increasing amounts of estradiol. The estradiol peak during the late
follicular phase stimulates the preovulatory surge of LH and ovulation takes place shortly thereafter. After
ovulation the corpus luteum develops and secretes progesterone and estradiol. If the woman is not preg-
nant, luteolysis is initiated during the late luteal phase and progesterone drops rapidly and a new menstrual
period begins. Note that FSH and LH are low during the luteal phase because of the negative feedback by
progesterone on the hypothalamus that inhibits GnRH and thus FSH and LH.

Ve
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I
I’
336 The Human Factor
during the cycle and 4) 54% could name only one of the
hom1ones involved, but could not describe the f1mction
of the hormone (See Koffin Key References).
The data above indicates a compelling Jack of
knowledge about the menstrual cycle among Alneri-
can women. This lack of knowledge undoubtedly
translates into the fact that approximately 50% of the
six million annual pregnancies in the United States are
unintended.
The menstrual cycle consists of the
following six events:
• menstruation
• follicular growth
• ovulation
• corpus luteum formation and growth
• endometrial growth and secretion
• luteo/ysis
In Chapter 7, we compared the menstrual cycle
with the estrous cycle. The menstrual cycle differs
fi·om the estrous cycle in two fundamental ways. First,
endometrial sloughing (menses) occurs in a predictable
manner dming eve1y cycle ifthe woman is not pregnant.
Second, there is no defined period of sexual receptivity.
In this chapter, we want to discuss the menstrual cycle
as a series of six distinctly different events (See Figure
16-1 ). Remember, the menstrual cycle starts at the first
day of the menstrual period (menses). This timing con-
vention originated because the menstrual period was an
observable event and marked the start of each menstrual
cycle. Typically, the length of the menstrual cycle is
28 days, but can range from 25 to 34 days. This is the
period of time from the start of one menstrual period
to the start of the next menstrual period.
The onset of menstruat ion signals the start
of the cycle and is it designated as day 1. Following
menstruation, marked follicular growth takes place in
response to FSH and LH from the anterior pituitary. At
about day 14, ovulation occurs. The newly ovu lated
follicle then develops into a corpus luteum (CL). The
CL secretes progesterone and some estrogens. These
hormones promote endometrial growth. Near the end
of the cycle, if a woman is not pregnant, the corpus
luteum undergoes luteolysis and loses its ability to se-
crete progesterone (See Figure 9-14). The rapid drop in
progesterone stimulates the onset of the next menstrual
period. Figure 16-1 describes the events of the menstrual
cycle in a c ircular and linear fashion.
The menstrual cycle is usually described as
having t\vo components. One component is the ovarian
cycle that describes changes that occur in the ovary dur-
ing the cycle. The uterine cycle describes the changes
that take place in the endometrium of the uterus during
the cycle.
The menstrual cycle also consists of the fol-
licular phase and the luteal phase (See Figure 16-2).
Follicular growth during and fo llowing menstruation is
referred to as the follicular phase because the dominant
Figure 16-1. Menstrual Cycle Sequence
•
t
Doy I
Lutcolysis
CL&
C!ndome:trlo:d
growth
Follicle growth
•
28 days
(Ronge = doys}
Doy 14
Follicle
growth
The six major events of the men-
strua l cycle are shown by the
circled numerals. Their relation-
ship to the timing of the cycle is
illustrated in circular form in the
top portion of the graphic and in
linear form in the bottom portion
of the graphic.
ovarian structures are foll icles. The dominant ovarian
hormone is estradiol. After ovulation, the luteal phase
begins. The dominant ovarian structure is the corpus
luteum and the dominant ovar ian hormone is proges-
terone. lt should be emphasized that while both the
menstrual cycle and estrous cycle are characterized by
the follicular and luteal phases, fo llicles are constantly
developing and regressing in both phases of the cycle.
In other words, even during the luteal p hase fo llicles
develop and regress.
During the follicular p hase, the anterior pitu-
itary secretes FSH and LH (See Figure 16-2) . These
hormones promote growth of ovarian fo llicles and
the growing follicles secrete increasing quantities of
estrogens. A threshold concentration of estmdiol trig-
gers the LH surge that causes ovulation. After ovulation
the luteal phase begins . The follicle that just ovulated
becomes the coqnrs luteum and secretes high concen-
trations of progesterone and some estradiol. The high
concentrations of progesterone inhib it GnRH secretism
from the hypothalamus and FSH and LH secretioq.fiom
the anterior pihlitary. Therefore, follicles do not develop
to the preovulatory stage during the luteal phase. At
The Human Factor 337
about day 23-24, progesterone drops rapidly because
luteolysis has occurred. This sudden drop in proges-
terone is thought to cause symptoms of premenstrual
syndrome (PMS) in many women. Further, the drop
in progesterone initiates endometrial sloughing and the
next menstrual period.
The uterine cycle is subdivided into the prolif-
erative and sec•·eto•·y phases. The proliferative phase
is the increase in endometrial thickness in response to
estradiol secreted by growing follicles. This increased
thickness is referred to as the proliferative phase be-
cause the cells of the endometr ium divide by mitosis
(proliferate). After oV1ilation and formation of the CL,
progesterone promotes further increased thickness in
the endometrium and it develops secretory capacity.
This is important because if conception takes place at
around day 14, the embryo will enter an environment
about three days later that is ideal for sustaining emb1yo
development prior to implantation. If pregnancy does
not occur, luteolysis is initiated, progesterone drops pre-
cip itously and a new menstrual period (and menstrual
cycle) begins (See Figure 16-3.).
Figure 16-2. Relative Blood Concentrations of FSH, LH, Estradiol and
Progesterone During the Follicular and Luteal Phases of
c
0
‘,P
c
QJ
u c
0 u
QJ
c
0
E ……
0 :r:
QJ
> ·.p
(lJ
QJ
0:::
the Menstrual Cycle
Ov ulation • .———·– -,.
Menses ;_ __ j
day 1 day 14 day 28
0 Estradiol
LH
• FSH
• Progesterone
During the menstrual period, all hormones are low. During the follicular phase, FSH promotes foll icular de-
velopment. Developing follicles secrete increasing amounts of estradiol. The estradiol peak during the late
follicular phase stimulates the preovulatory surge of LH and ovulation takes place shortly thereafter. After
ovulation the corpus luteum develops and secretes progesterone and estradiol. If the woman is not preg-
nant, luteolysis is initiated during the late luteal phase and progesterone drops rapidly and a new menstrual
period begins. Note that FSH and LH are low during the luteal phase because of the negative feedback by
progesterone on the hypothalamus that inhibits GnRH and thus FSH and LH.
Ve
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338 The Human Factor

Figure 16-3. Major Endometrial Changes During the Menstrual Cycle

-·

The spiral arteries deliver blood to the uterine
glands during the proliferative and secretory
phases before luteolysis. A high blood flow to
the endometrium facilitates secretion.

The endometrium begins to proliferate immediately after
menstruation (about day 7) and continues to grow during
the proliferative phase until the time of ovulation. After
ovulation, a CL is formed and progesterone causes contin-
ued proliferation of the endometrium during the secretory
phase. Luteolysis, caused by intraovarian PGF2u causes
progesterone and estrad io l to drop dramatically. Please
review Figure 9-14 for the mechanism of luteolysis mecha-
nism and see Figure 16-3 for the hormonal profile.

.·

After luteolysis, the dramatic drop in P 4 promotes PGF2″ synthesis by the
endometrium that causes sustained vasoconstriction in the spiral arter-
ies. Sustained vasoconstriction causes ischemia and the endometrium
undergoes necrosis and sloughs into the uterine lumen. Endometrial
sloughing (menstruation) lasts from 2 to 6 days.

The Human Factor 339

Figure 16-4. Relationships Between Emotional/Mood Status and
Estradiol and Progesterone During the Menstrual Cycle

Preovulatory Component

High self esteem
1′ Memory
t Social interaction
t Intimacy
t Acuity & cognit ion
t Olfaction

Premenstrual Component

t Anxiety & tension
t Distress
t Anger & Irritability
t Interpersonal conflicts

Depression 12

10
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8
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6 0 ,_
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11’1

4
e

2 a..

2 4 6 8 ”S” 20 22 24 26 Day of Cycle 28
During the 5-6 days prior to ovulation (“preo-
vulatory component”), estradiol increases and
decreases dramatically. The elevated estradiol
has been associated with emotional changes
that reflect confidence and agressiveness.

Progesterone and estradiol undergo huge
fluctuations during the menstrual cycle:

• estradiol increases about 5X during the
5-6 days before ovulation

• progesterone decreases by about 1 OX
during the 2-3 days preceding menses

It should be emphasized that estradiol and pro-
gesterone undergo dramatic changes in concentration
during the course of one menstrual cycle. For example,
during the mid-follicular phase, estradiol concentrations
are about 30 pg/mL of blood. In the 5- 6 days that fol –
low, estradiol increases to about 140 pg/mL of blood. In
other words, the concentration of estradiol increases by
about 5X during this 5 or 6 day period. Dming the luteal
phase, progesterone increases from about 1-2 ng/mL of
blood to 9-1 0 ng/mL ofblood. This represents a 5-9 fold
increase in progesterone during a 4-6 day period. After
luteolysis, progesterone drops from a peak of 9-1 0 ng/
mL to l ng/mL during a 2-3 day period, another l OX
change in progesterone . No other honnone in the body
changes this dramatically in such a short period of time
(See Figure 16-4).

In contrast, the 3-5 days during precipitous pro-
gesterone decline that precedes the menstrual
period (“premenstrual component”) is character-
ized by emotions reflecting tension, anger and
anxiety in many women.

Throughout the course o f histo ry it has
been !mown that profound emotional and behavioral
changes occur during the menstrual cycle. However,
only recently have we begun to understand how the
hormonal fluctuations in estradiol and progesterone
during the menstrual cycle influence brain function,
cognition, emotions, sensory processing, appetite and
probably many more as yet unidentified functions.
Research involving the stages of the menstrual cycle
on emotional status and other central nervous system
functions has given validity to the concept that PMS
is a set of physiologic-driven responses to rapid and
dramatic concentration changes in estradiol and pro-
gesterone during the cycle. The fact that hormonal
changes influence behavioral and emotional changes
in the female should be recognized by everyone. It is
particularly important that men understand the relation-
ship between stage of the cycle and behavioral changes.
This is because most women intuitively understand the
emotional changes that are occmTing, but men need to
understand the magnih1de of the honnonal “swings”
and the behavioral/emotional changes that accompany
them. Such an understanding would enable empathetic
responses that would undoubtedly foster more positive
relationships during the premenstrual component of
the cycle.

Ve
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.ir

338 The Human Factor
Figure 16-3. Major Endometrial Changes During the Menstrual Cycle
-·
The spiral arteries deliver blood to the uterine
glands during the proliferative and secretory
phases before luteolysis. A high blood flow to
the endometrium facilitates secretion.
The endometrium begins to proliferate immediately after
menstruation (about day 7) and continues to grow during
the proliferative phase until the time of ovulation. After
ovulation, a CL is formed and progesterone causes contin-
ued proliferation of the endometrium during the secretory
phase. Luteolysis, caused by intraovarian PGF2u causes
progesterone and estrad io l to drop dramatically. Please
review Figure 9-14 for the mechanism of luteolysis mecha-
nism and see Figure 16-3 for the hormonal profile.
.·
After luteolysis, the dramatic drop in P 4 promotes PGF2″ synthesis by the
endometrium that causes sustained vasoconstriction in the spiral arter-
ies. Sustained vasoconstriction causes ischemia and the endometrium
undergoes necrosis and sloughs into the uterine lumen. Endometrial
sloughing (menstruation) lasts from 2 to 6 days.
The Human Factor 339
Figure 16-4. Relationships Between Emotional/Mood Status and
Estradiol and Progesterone During the Menstrual Cycle
Preovulatory Component
High self esteem
1′ Memory
t Social interaction
t Intimacy
t Acuity & cognit ion
t Olfaction
Premenstrual Component
t Anxiety & tension
t Distress
t Anger & Irritability
t Interpersonal conflicts
Depression 12
10
E ……..
8
QJ
t:
6 0 ,_
QJ ……
11’1
4
e
2 a..
2 4 6 8 ”S” 20 22 24 26 Day of Cycle 28
During the 5-6 days prior to ovulation (“preo-
vulatory component”), estradiol increases and
decreases dramatically. The elevated estradiol
has been associated with emotional changes
that reflect confidence and agressiveness.
Progesterone and estradiol undergo huge
fluctuations during the menstrual cycle:
• estradiol increases about 5X during the
5-6 days before ovulation
• progesterone decreases by about 1 OX
during the 2-3 days preceding menses
It should be emphasized that estradiol and pro-
gesterone undergo dramatic changes in concentration
during the course of one menstrual cycle. For example,
during the mid-follicular phase, estradiol concentrations
are about 30 pg/mL of blood. In the 5- 6 days that fol –
low, estradiol increases to about 140 pg/mL of blood. In
other words, the concentration of estradiol increases by
about 5X during this 5 or 6 day period. Dming the luteal
phase, progesterone increases from about 1-2 ng/mL of
blood to 9-1 0 ng/mL ofblood. This represents a 5-9 fold
increase in progesterone during a 4-6 day period. After
luteolysis, progesterone drops from a peak of 9-1 0 ng/
mL to l ng/mL during a 2-3 day period, another l OX
change in progesterone . No other honnone in the body
changes this dramatically in such a short period of time
(See Figure 16-4).
In contrast, the 3-5 days during precipitous pro-
gesterone decline that precedes the menstrual
period (“premenstrual component”) is character-
ized by emotions reflecting tension, anger and
anxiety in many women.
Throughout the course o f histo ry it has
been !mown that profound emotional and behavioral
changes occur during the menstrual cycle. However,
only recently have we begun to understand how the
hormonal fluctuations in estradiol and progesterone
during the menstrual cycle influence brain function,
cognition, emotions, sensory processing, appetite and
probably many more as yet unidentified functions.
Research involving the stages of the menstrual cycle
on emotional status and other central nervous system
functions has given validity to the concept that PMS
is a set of physiologic-driven responses to rapid and
dramatic concentration changes in estradiol and pro-
gesterone during the cycle. The fact that hormonal
changes influence behavioral and emotional changes
in the female should be recognized by everyone. It is
particularly important that men understand the relation-
ship between stage of the cycle and behavioral changes.
This is because most women intuitively understand the
emotional changes that are occmTing, but men need to
understand the magnih1de of the honnonal “swings”
and the behavioral/emotional changes that accompany
them. Such an understanding would enable empathetic
responses that would undoubtedly foster more positive
relationships during the premenstrual component of
the cycle.
Ve
tB
oo
ks
.ir

I.
”

340 The Human Factor

Figure 16-4 describes some of the emotional
differences that occur during the late follicular phase
and late luteal phase. During the late follicular phase,
estradiol promotes an overall feeling of well being,
desire for intimacy, confidence and increased cognitive
ability. There is evidence that during the late follicular
phase, there is a significant increase in the number of
synaptic junctions in the hippocampus (a region of the
cerebral cortex that is thought to play a role in learn-
ing and memory). In contrast, during the late luteal
phase (about 5 days prior to the onset of menstruation)
significant temporary mood changes occur in a high
percentage of women. These changes have been labeled
as premenstrual syndrome (PMS). A syndrome is
a group of symptoms that occur together. The emo-
tional symptoms associated with PMS vary significantly
among women and can be characterized by feelings of
anxiety or tension, sadness, irritability, anger, changes
in appetite and feelings of being overwhelmed or out of
control. Physical symptoms include cramps, backaches,
muscle spasms, nausea, dizziness, breast tenderness
and unpleasant tingling or swelling of the hands and
feet. There are no precise or predictable symptoms of
PMS and the degree of severity is quite variable among
women. Between 70 and 90% of women experience
some physical and emotional difficulties before men-
struation begins. While most women experience one
or more of these symptoms, only 5-l 0% of women
experience severe and debilitating symptoms.

It is important to recognize that there is a sig-
nificant amount of variation in the expression of the
symptoms ofPMS both within and among women. In
other words, the symptomatic expressions may vary
from cycle-to-cycle and from woman-to-woman. Re-
gardless, it is clear that physical and emotional changes
occur during the menstrual cycle and these are linked
to the dramatic changes in estradiol and progesterone
concentrations that occur during the menstrual cycle.
It is important for both men and women to understand
that these physical and emotional changes have a strong
physiologic basis and should not be considered a “black
box” of unexplained behavior.

Steroidal Birth Control is a Method to
Control Ovulation

As pointed out earlier, there is no defined pe-
riod of sexual receptivity associated with the menstrual
cycle. Therefore, sexual intercourse can take place
at any time during the cycle. Thus, frequent sexual
intercourse can occur and increases the probability
of pregnancy. In this context, contraception method-
ologies have been an important component of human
reproduction throughout history, especially during the
last century. Here we will address steroidal contracep-

tion because, unlike barrier methods, understanding the
reproductive physiology underlying its use increases
the chances of success.

Contraception means opposing concep-
tion. It is defined as the prevention of pregnancy as
a consequence of sexual intercourse. There are many
contraceptive methods that can be used to minimize the
probability of pregnancy. Steroidal contraception is a
physiologic intervention that utilizes progestins to pre-
vent ovulation and thus prevent pregnancy. Preventing
fertilization (conception) is a contraceptive approach to
birth contt·ol. Birth control means managing or pre-
venting birth. Fundamentally, there are three forms of
bi1th control. These are: a) contraception or prevention
of conception (preventing the union of sperm and the
oocyte); b) interception (preventing implantation) and
c) abortion (disruption of a pregnancy after implanta-
tion). An ethical/moral consideration should be realized
by all women who use steroidal contraception. In some
cases, conception can occur, but the steroidal interven-
tion prevents optimal uterine conditions for embryo
survival and implantation. The woman has no way of
knowing if pregnancy was prevented by preventing
ovulation (contraception) or minimizing the chance of
implantation (interception). This is not to be confused
with an abortive intervention. Abortion refers to the
termination or loss of an embryo after implantation. It
is important to recognize that even when couples are
tlying to conceive, 30-50% of embryos fai l to implant
under norn1al conditions. Please refer to Figure 12-13
and adjacent text for pregnancy probability relative to
time of ovulation. The discussion in this chapter will
focus entirely on steroidal contraception because it
is a physiologic intervention that involves honnonal
manipulation that prevents ovulation.

From a physiologic perspective, steroidal
contraception can be used as a method of reproductive
management for family-planning. It is well known that
about 50% of all pregnancies in the United States are
unintended. Furthennore, about 78% of all pregnancies
among American teenagers are unintended. Therefore,
the mechanisms responsible for the effectiveness of
steroidal contraception should be understood by both
women and men in order to maximize the effectiveness
of this important intervention.

Regardless of the delivery method, the net ef-
fect is a sustained luteal phase. Figure 16-5 compares
the estradiol and progesterone profile in a unaltered
cycle with the progesterone profile of the cycle in which
exogenous progesterone is administered. Notice, that
women using progestin contraception have no follicular
phase. Therefore, follicles do not develop to maturity
and will not ovulate. Like in the normal cycle, when
progestin concentrations drop, the woman will menstru-
ate. In summary, regardless of the type of hormonal

“‘ <=c: Co IE Q)QJ >U ·- c: ;oo
QjU
cr:

Figure 16-5. Estradiol and
Progesterone Profiles During a

Normal Menstrual Cycle and With
Steroidal Contraception

Menses
+

Ovulation
+

Menses
+

CJ Estradiol _ _j • Progesterone

day 1

Menses
+
day 1

d ay 14

day 14

day 28
Menses
+
day 28

Steroidal contraception results in a sustained luteal
phase when compared to a normal menstrual cycle.
Shortly after administration of progesterone, blood
levels increase and remain high for the remainder of
the cycle until progesterone is withdrawn (placebo
pill, removal of patch or vaginal ring, or metabolism
of the injected progestin).

contraception used, ovulation is usually prevented
because progestin and estrogens inhibit GnRH and
therefore FSH and LH is inhibited. Follicles don’t grow
and ovulate. If ovulation does not occur, pregnancy is
not possible.

The various steroidal contraception
delive1y methods are:

• pill (daily)

• transdermal patch (weekly)

• intravaginal ring (monthly)

• injection (every 90 days)

The Human Factor 341

The primary active ingredient in steroidal
contraception is progestin. Here, we use the term
progestin to refer to any natural or synthetic material
that has progesterone-like actions. Progestins can be
administered orally, by injection, by release from a
transdennal patch or release from an intTa-vaginal ring.
They can also be released fi·om some intrauterine de-
vices (IUDs) or from implants. Each method delivers
progestins at different frequencies.

Many interventions contain an estrogen. The
purpose of estradiol is two-fold. First, estrogens pro-
mote normal reproductive tract f·unction. Second, low
concentrations of estrogens cause negative feedback
on GnRH neurons and thus have a negative effect on
FSH and LH secretion.

Oral contl·aception applications are character-
ized by a 28-day hormonal regimen and these are sum-
marized in Figure 16-6. The woman takes a progestin
or progestin/estradiol pill for 2 1 consecutive days.
On the fo llowing 7 days, a placebo pill containing no
hormone is taken and this mimics luteolysis because
progestin drops rapidly and a new menstrual period is
initiated. The key to the success of this method is dili-
gence in taking the pill every day and approximately
the same time eve1y day. This ensures that progesterone
concentrations wi ll remain high and stable. It should
be emphasized that failure to take one or more pills
in succession will result in decreased progestin levels
in the blood and the probability of e levated FSI-1 and
LH increases, particularly if several pills are missed in
succession.

The transdermal patch contains progestin that
diffuses tlu-ough the skin and enters the blood. Patches
are replaced every week and during the patch-free
week progestin concentrations drop and a new men-
stmal period begins. Patches can be placed at various
locations in the body including the upper arm, the
abdominal region, the buttocks and the shoulder blade.
In order to be effective, a patch that is removed must
be replaced by a new patch every week except during
the patch-free week.

The vaginal ring is inse1t ed into the vagina and
steadily releases unifonn concentrations of progestin
that are absorbed through the vaginal tissues and enter
the blood. One vaginal ring releases progestin for three
weeks. After the ring is removed, blood progesterone
drops and a new menstrual period is initiated.

Progestin injections provide a continual 90-
day hormonal absorption from the injection site. The
progestin injection is not reversible. Therefore, for a
period of 90 days there will be neither ovulation nor
menstrual periods. After approximately 90 days, the
progesterone source is depleted and menses will occur
and so will ovulation in about 2 weeks if progestin is
not administered during or after the menstrual period.

16
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I.
”
340 The Human Factor
Figure 16-4 describes some of the emotional
differences that occur during the late follicular phase
and late luteal phase. During the late follicular phase,
estradiol promotes an overall feeling of well being,
desire for intimacy, confidence and increased cognitive
ability. There is evidence that during the late follicular
phase, there is a significant increase in the number of
synaptic junctions in the hippocampus (a region of the
cerebral cortex that is thought to play a role in learn-
ing and memory). In contrast, during the late luteal
phase (about 5 days prior to the onset of menstruation)
significant temporary mood changes occur in a high
percentage of women. These changes have been labeled
as premenstrual syndrome (PMS). A syndrome is
a group of symptoms that occur together. The emo-
tional symptoms associated with PMS vary significantly
among women and can be characterized by feelings of
anxiety or tension, sadness, irritability, anger, changes
in appetite and feelings of being overwhelmed or out of
control. Physical symptoms include cramps, backaches,
muscle spasms, nausea, dizziness, breast tenderness
and unpleasant tingling or swelling of the hands and
feet. There are no precise or predictable symptoms of
PMS and the degree of severity is quite variable among
women. Between 70 and 90% of women experience
some physical and emotional difficulties before men-
struation begins. While most women experience one
or more of these symptoms, only 5-l 0% of women
experience severe and debilitating symptoms.
It is important to recognize that there is a sig-
nificant amount of variation in the expression of the
symptoms ofPMS both within and among women. In
other words, the symptomatic expressions may vary
from cycle-to-cycle and from woman-to-woman. Re-
gardless, it is clear that physical and emotional changes
occur during the menstrual cycle and these are linked
to the dramatic changes in estradiol and progesterone
concentrations that occur during the menstrual cycle.
It is important for both men and women to understand
that these physical and emotional changes have a strong
physiologic basis and should not be considered a “black
box” of unexplained behavior.
Steroidal Birth Control is a Method to
Control Ovulation
As pointed out earlier, there is no defined pe-
riod of sexual receptivity associated with the menstrual
cycle. Therefore, sexual intercourse can take place
at any time during the cycle. Thus, frequent sexual
intercourse can occur and increases the probability
of pregnancy. In this context, contraception method-
ologies have been an important component of human
reproduction throughout history, especially during the
last century. Here we will address steroidal contracep-
tion because, unlike barrier methods, understanding the
reproductive physiology underlying its use increases
the chances of success.
Contraception means opposing concep-
tion. It is defined as the prevention of pregnancy as
a consequence of sexual intercourse. There are many
contraceptive methods that can be used to minimize the
probability of pregnancy. Steroidal contraception is a
physiologic intervention that utilizes progestins to pre-
vent ovulation and thus prevent pregnancy. Preventing
fertilization (conception) is a contraceptive approach to
birth contt·ol. Birth control means managing or pre-
venting birth. Fundamentally, there are three forms of
bi1th control. These are: a) contraception or prevention
of conception (preventing the union of sperm and the
oocyte); b) interception (preventing implantation) and
c) abortion (disruption of a pregnancy after implanta-
tion). An ethical/moral consideration should be realized
by all women who use steroidal contraception. In some
cases, conception can occur, but the steroidal interven-
tion prevents optimal uterine conditions for embryo
survival and implantation. The woman has no way of
knowing if pregnancy was prevented by preventing
ovulation (contraception) or minimizing the chance of
implantation (interception). This is not to be confused
with an abortive intervention. Abortion refers to the
termination or loss of an embryo after implantation. It
is important to recognize that even when couples are
tlying to conceive, 30-50% of embryos fai l to implant
under norn1al conditions. Please refer to Figure 12-13
and adjacent text for pregnancy probability relative to
time of ovulation. The discussion in this chapter will
focus entirely on steroidal contraception because it
is a physiologic intervention that involves honnonal
manipulation that prevents ovulation.
From a physiologic perspective, steroidal
contraception can be used as a method of reproductive
management for family-planning. It is well known that
about 50% of all pregnancies in the United States are
unintended. Furthennore, about 78% of all pregnancies
among American teenagers are unintended. Therefore,
the mechanisms responsible for the effectiveness of
steroidal contraception should be understood by both
women and men in order to maximize the effectiveness
of this important intervention.
Regardless of the delivery method, the net ef-
fect is a sustained luteal phase. Figure 16-5 compares
the estradiol and progesterone profile in a unaltered
cycle with the progesterone profile of the cycle in which
exogenous progesterone is administered. Notice, that
women using progestin contraception have no follicular
phase. Therefore, follicles do not develop to maturity
and will not ovulate. Like in the normal cycle, when
progestin concentrations drop, the woman will menstru-
ate. In summary, regardless of the type of hormonal
“‘ <=c: Co IE Q)QJ >U ·- c: ;oo
QjU
cr:
Figure 16-5. Estradiol and
Progesterone Profiles During a
Normal Menstrual Cycle and With
Steroidal Contraception
Menses
+
Ovulation
+
Menses
+

CJ Estradiol _ _j • Progesterone
day 1

Menses
+
day 1
d ay 14
day 14
day 28
Menses
+
day 28
Steroidal contraception results in a sustained luteal
phase when compared to a normal menstrual cycle.
Shortly after administration of progesterone, blood
levels increase and remain high for the remainder of
the cycle until progesterone is withdrawn (placebo
pill, removal of patch or vaginal ring, or metabolism
of the injected progestin).
contraception used, ovulation is usually prevented
because progestin and estrogens inhibit GnRH and
therefore FSH and LH is inhibited. Follicles don’t grow
and ovulate. If ovulation does not occur, pregnancy is
not possible.
The various steroidal contraception
delive1y methods are:
• pill (daily)
• transdermal patch (weekly)
• intravaginal ring (monthly)
• injection (every 90 days)
The Human Factor 341
The primary active ingredient in steroidal
contraception is progestin. Here, we use the term
progestin to refer to any natural or synthetic material
that has progesterone-like actions. Progestins can be
administered orally, by injection, by release from a
transdennal patch or release from an intTa-vaginal ring.
They can also be released fi·om some intrauterine de-
vices (IUDs) or from implants. Each method delivers
progestins at different frequencies.
Many interventions contain an estrogen. The
purpose of estradiol is two-fold. First, estrogens pro-
mote normal reproductive tract f·unction. Second, low
concentrations of estrogens cause negative feedback
on GnRH neurons and thus have a negative effect on
FSH and LH secretion.
Oral contl·aception applications are character-
ized by a 28-day hormonal regimen and these are sum-
marized in Figure 16-6. The woman takes a progestin
or progestin/estradiol pill for 2 1 consecutive days.
On the fo llowing 7 days, a placebo pill containing no
hormone is taken and this mimics luteolysis because
progestin drops rapidly and a new menstrual period is
initiated. The key to the success of this method is dili-
gence in taking the pill every day and approximately
the same time eve1y day. This ensures that progesterone
concentrations wi ll remain high and stable. It should
be emphasized that failure to take one or more pills
in succession will result in decreased progestin levels
in the blood and the probability of e levated FSI-1 and
LH increases, particularly if several pills are missed in
succession.
The transdermal patch contains progestin that
diffuses tlu-ough the skin and enters the blood. Patches
are replaced every week and during the patch-free
week progestin concentrations drop and a new men-
stmal period begins. Patches can be placed at various
locations in the body including the upper arm, the
abdominal region, the buttocks and the shoulder blade.
In order to be effective, a patch that is removed must
be replaced by a new patch every week except during
the patch-free week.
The vaginal ring is inse1t ed into the vagina and
steadily releases unifonn concentrations of progestin
that are absorbed through the vaginal tissues and enter
the blood. One vaginal ring releases progestin for three
weeks. After the ring is removed, blood progesterone
drops and a new menstrual period is initiated.
Progestin injections provide a continual 90-
day hormonal absorption from the injection site. The
progestin injection is not reversible. Therefore, for a
period of 90 days there will be neither ovulation nor
menstrual periods. After approximately 90 days, the
progesterone source is depleted and menses will occur
and so will ovulation in about 2 weeks if progestin is
not administered during or after the menstrual period.
16
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342 The Human Factor

Figure 16-6. Influence of the Pill , Patch, Vaginal Ring and Injection
Upon Progestin Profile

Progestin pills (21)
000000000000000000000
+ + + + + + + + + + + + + + + + + • + + + Placebo pills (7) No progestin
t t t 0000000 ++++++ Patch 1 Pa tch 2 Patch 3

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

t t

Ring inserted Day of Cycle Ring removed

No ovulation } 90 da s No menses y

t 3D d ays 60 days
Progestin injection

The pill, patch and vaginal ring all release progestins. In all three cases, the menstrual period is initiated
after progestin administration stops. The difference between these three applications is the frequency at
which they are administered. The pill is taken orally for a period of 21 days followed by 7 days of placebo
pills. The patch is applied and then replaced with a new patch each week for three weeks. The vaginal ring
is inserted and is removed after 21 days. Progestin injection resu lts in elevated blood progestin for 90 days
during which there will be no ovulation and no menstrual periods.

Assisted Reproductive Technology (ART)
Provides Conception Opportunities

For Infertile Couples

Assisted reproductive technology (ART) de-
scribes any procedure in which the sperm and oocytes
are united outside the body that result in a viable zygote
and embryo. One or more embryos are then transferred
back into the woman’s uterus to generate a pregnancy.
These techniques are performed by a physician in con-
junction with a reproductive biologist who is trained in
embryology and andrology. The most commonly used
ART method is in-vitro fertilization (IVF) in which
oocytes are fertilized by one of two methods. First,
spem1 may fertilize oocytes in-vitro under “their own
power”. In other words, motile sperm penetrate the cu-

mulus cells, zona pellucida and the oocyte plasma mem-
brane to fonn a zygote. This is called conventional IVF
in which the man provides adequate numbers of viable
spenn. In cases where the man cannot provide adequate
numbers of viable sperm, a single sperm is injected into
the oocyte. This technique is called intracytoplasmic
sperm injection or ICSI.

In-vitro fertilization requires:
• semen collection
• semen evalution and prepamtion
• ovarian stimulation
• oocyte retrieval and preparation

IVF is intended to generate pregnancies in
women with blocked or missing oviducts, women with
endometriosis, women who fail to ovulate, men with in-
adequate sperm function and couples with unexplained
infertility. ART procedures are conducted in ferti lity
clinics that specialize in IVF procedures, early emb1yo
culture and development and transfer procedures.

Now, Jet’s look at the sequence of events (See
Figure 16-7) that take place for the woman and the
man during typical IVF procedures. In the woman,
the ovaries are hormonally stimulated so that a higher
than nomml number of foll icles develop. After ovarian
stimulation, oocytes are retrieved from each preovula-
tory follicle transferred to a culture environment. In
the male, semen is typically collected by masturbation
and processed for ferti lization. A semen analysis is
pe1fonned in advance of ovarian stimulation and used
to detenn ine the method of ferti I izati on (either conven-
tional IVF or ICSI). If there are adequate numbers of
nom1al sperm, the specimen can be used for conven-
tional IVF. If there are inadequate numbers of sperm
then ICSI is used. A successful ferti lizatim).resfllts in
the development of the emb1yo that progresses from
the pronuclear stage to the 2, 4, 8 cell, morula and then
to a blastocyst.

The Human Factor 343

Semen Evaluation is Performed Prior to a
Couple Beginning IVF Procedures

The first step in male fertil ity evaluation is
collection of semen. Typically, a semen evaluation is
conducted prior to initiation of the IVF procedure. This
evaluation could be considered as a screening test to
detem1ine whether the man is producing sufficient quan-
tities of viable sperm for conventional IVF. The three
most important characteristics of the spermatozoa are:
concentration ofspenn in the ejaculate, adequate num-
bers of viable spenn (motile spenn) and low numbers of
abnormal sperm. While each ART clinic has its own set
of criteria, guidelines are provided by the World Health
Organization (WHO). These guidelines indicate that a
fertile ejaculate should contain more than 20 million
spenn per millil iter, w ith greater than 50% motility. An
ejaculate that meets these criteria is eligible for conven-
tional in-vitro fertilization where spennatozoa fer tilize
oocytes under their “own power”. If the ejaculate does
not meet these criteria then plans are made to perform
intracytoplasmic sperm injection (ICSI).

Figure 16-7. Sequence of IVF Events and Preimplantation
Embryo Development

!ovarian stimulation I
For ART to be effective, successfu l ovarian stimulation and oocyte
retrieval are required. Oocytes retrieved (day 0) are then eligible
for either conventional IVF or ICSI.

4 8 Morula Blastocyst

Viable embryos will
be cu ltu red in-vitro
and then transferred
into the uterus.

Sperm quality determines wh ich fertilization technique will
be used. After semen collection and preparation, if there are
sufficient viable sperm conventional IVF will be performed. If
insufficient sperm are present, ICSI is used.

16
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342 The Human Factor
Figure 16-6. Influence of the Pill , Patch, Vaginal Ring and Injection
Upon Progestin Profile
Progestin pills (21)
000000000000000000000
+ + + + + + + + + + + + + + + + + • + + + Placebo pills (7) No progestin
t t t 0000000 ++++++ Patch 1 Pa tch 2 Patch 3
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28
t t
Ring inserted Day of Cycle Ring removed
No ovulation } 90 da s No menses y
t 3D d ays 60 days
Progestin injection
The pill, patch and vaginal ring all release progestins. In all three cases, the menstrual period is initiated
after progestin administration stops. The difference between these three applications is the frequency at
which they are administered. The pill is taken orally for a period of 21 days followed by 7 days of placebo
pills. The patch is applied and then replaced with a new patch each week for three weeks. The vaginal ring
is inserted and is removed after 21 days. Progestin injection resu lts in elevated blood progestin for 90 days
during which there will be no ovulation and no menstrual periods.
Assisted Reproductive Technology (ART)
Provides Conception Opportunities
For Infertile Couples
Assisted reproductive technology (ART) de-
scribes any procedure in which the sperm and oocytes
are united outside the body that result in a viable zygote
and embryo. One or more embryos are then transferred
back into the woman’s uterus to generate a pregnancy.
These techniques are performed by a physician in con-
junction with a reproductive biologist who is trained in
embryology and andrology. The most commonly used
ART method is in-vitro fertilization (IVF) in which
oocytes are fertilized by one of two methods. First,
spem1 may fertilize oocytes in-vitro under “their own
power”. In other words, motile sperm penetrate the cu-
mulus cells, zona pellucida and the oocyte plasma mem-
brane to fonn a zygote. This is called conventional IVF
in which the man provides adequate numbers of viable
spenn. In cases where the man cannot provide adequate
numbers of viable sperm, a single sperm is injected into
the oocyte. This technique is called intracytoplasmic
sperm injection or ICSI.
In-vitro fertilization requires:
• semen collection
• semen evalution and prepamtion
• ovarian stimulation
• oocyte retrieval and preparation
IVF is intended to generate pregnancies in
women with blocked or missing oviducts, women with
endometriosis, women who fail to ovulate, men with in-
adequate sperm function and couples with unexplained
infertility. ART procedures are conducted in ferti lity
clinics that specialize in IVF procedures, early emb1yo
culture and development and transfer procedures.
Now, Jet’s look at the sequence of events (See
Figure 16-7) that take place for the woman and the
man during typical IVF procedures. In the woman,
the ovaries are hormonally stimulated so that a higher
than nomml number of foll icles develop. After ovarian
stimulation, oocytes are retrieved from each preovula-
tory follicle transferred to a culture environment. In
the male, semen is typically collected by masturbation
and processed for ferti lization. A semen analysis is
pe1fonned in advance of ovarian stimulation and used
to detenn ine the method of ferti I izati on (either conven-
tional IVF or ICSI). If there are adequate numbers of
nom1al sperm, the specimen can be used for conven-
tional IVF. If there are inadequate numbers of sperm
then ICSI is used. A successful ferti lizatim).resfllts in
the development of the emb1yo that progresses from
the pronuclear stage to the 2, 4, 8 cell, morula and then
to a blastocyst.
The Human Factor 343
Semen Evaluation is Performed Prior to a
Couple Beginning IVF Procedures
The first step in male fertil ity evaluation is
collection of semen. Typically, a semen evaluation is
conducted prior to initiation of the IVF procedure. This
evaluation could be considered as a screening test to
detem1ine whether the man is producing sufficient quan-
tities of viable sperm for conventional IVF. The three
most important characteristics of the spermatozoa are:
concentration ofspenn in the ejaculate, adequate num-
bers of viable spenn (motile spenn) and low numbers of
abnormal sperm. While each ART clinic has its own set
of criteria, guidelines are provided by the World Health
Organization (WHO). These guidelines indicate that a
fertile ejaculate should contain more than 20 million
spenn per millil iter, w ith greater than 50% motility. An
ejaculate that meets these criteria is eligible for conven-
tional in-vitro fertilization where spennatozoa fer tilize
oocytes under their “own power”. If the ejaculate does
not meet these criteria then plans are made to perform
intracytoplasmic sperm injection (ICSI).
Figure 16-7. Sequence of IVF Events and Preimplantation
Embryo Development
!ovarian stimulation I
For ART to be effective, successfu l ovarian stimulation and oocyte
retrieval are required. Oocytes retrieved (day 0) are then eligible
for either conventional IVF or ICSI.
4 8 Morula Blastocyst
Viable embryos will
be cu ltu red in-vitro
and then transferred
into the uterus.
Sperm quality determines wh ich fertilization technique will
be used. After semen collection and preparation, if there are
sufficient viable sperm conventional IVF will be performed. If
insufficient sperm are present, ICSI is used.
16
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344 The Human Factor

Ovarian Stimulation Promotes
Development of Multiple Follicles

Having multiple oocytes provides increased
probability for successful fertilization and embryo
development. Ovarian stimulation begins with an injec-
tion ofFSH, or and FSI-1-like honnone and concludes
with human chorionic gonadotropin (hCG). It should
be emphasized that there are many options for ovarian
stimulation. These vary depending on the reproduc-
tive status of the woman and from clinic-to-clinic. As
shown in Figure 16-8, exogenous FSH or and FSH-like
hormone stimulates growth of more than one follicle.
In this example, four follicles within one ovary respond
to FSH and these follicles secrete estradiol like in the
normal cycle. The four follicles continue to grow larger

Figure 16-8. Ovarian Stimulation
With FSH and hCG

Early Development

Eura.d iol

Mid-Development

Euradiol

Estradiol

Ovarian stimulation causes development of
higher than normal numbers offollicles. In this
example, four follicles develop simultaneously
after stimulation with FSH.

Aspiration

Estradio l Estradiol

At the appropriate time, the woman is treated
with human chorionic gonadotropin (hCG) to
stimulate final development and maturation of the
follicles. Oocytes are retreived from the follicles
using transvaginal ultrasound aspiration .

into mid-development and secrete more estradiol. Final
ovarian stimulation continues using hCG. Like LH,
hCG promotes final follicular mah1ration and growth
coupled with elevated estradiol secretion. When ready,
oocytes are aspirated from these large follicles.

Oocyte Retrieval is Accomplished by
Transvaginal Ultrasound Aspiration

Transvaginal aspiration is a technique that
combines ultrasound imaging with the mechanical
process of inserting a needle into each mature follicle
and applying slight suction to dislodge the oocyte and
remove it from the follicle. This procedure is typically
conducted in the physician’s office or in an outpatient
clinic. Some fonn of mild general analgesia is generally
administered. The first step in the procedure is to insert
an ultrasound probe into the vagina that enables it to be
positioned in close proximity to the ovary. Preovulatory
follicles that are eligible for aspiration are identified.
In the ultrasound images, they appear as dark circles
within the ovaty. Upon identification of eligible fol-
licles, a small needle is guided along the wall of the
ultrasound probe, through the wall of the vagina and
into each follicle (See Figme 16-9). Once inside the fol-
licle, slight suction is applied to the needle. This suction
dislodges the oocyte and can be aspirated. In general,
all eligible follicles in both ovaries can be aspirated
within 30 minutes. While this procedme is minimally
invasive, some women may experience cramping. This

Figure 16-9. Oocyte Retrieval Using
Transvaginal Ultrasound Aspiration

An ultrasound probe is
inserted into the vagina
and positioned in close

mity to the ovary.

A small needle is guided along the ultrasound probe
and inserted through the wall of the vagina and
into preovulatory follicles. Slight suction is applied
to dislodge the oocyte and it is aspi rated into a
collection vessel.

The Human Factor 345

Figure 16-10. Retrieved Oocyte, ICSI and Male and Female Pronuclei

Spenn

I ,- ‘ lnjectlon Mala
pi polio

ZP • ZP

A recently retrieved oocyte
with cumulus ce lls (CC)
su rrounding the zona pel-
lucida (ZP). In conventional
IVF, sperm penetrate the
cumulus cells and zona pel-
lucida before fertilizing the
oocyte. Micrograph courtesy of
West Virginia University Center for
Reproductive Medicine.

With ICS I, the cumulus cells a re re-
moved by enzymatic digestion and then
the denuded oocyte is inseminated .
The oocyte is held in position by a
pipette that applies s lig ht suction to
the zona pellucida (ZP). The sperm is
injected with a glass pipette. Micrograph
courtesy of West Vj rginia University Center for
Reproductive Afedicine.

The presence of a male
and female pronuclei in the
oocyte cytoplasm indicates
that ferti lization has taken
place. Each pronucleus
contains the genetic mate-
rial from the wo man and
the man. Micrograph courtesy
of West Virginia University Center
for Reproductive Medicine.

is not serious and generally subsides within one hour.
Immediately after aspiration, the oocytes are placed in
a culture medium that supports their viability.

Retrieved Oocytes are Surrounded by a
Layer of Cumulus Cells

Cumulus cells are remnants of granulosa! cells
that surround the freshly retrieved oocyte (See Figure
16-l 0). In the case of conventional IVF, sperm are
capacitated in-vitro and added to the culrure medium
containing the oocyte where they penetrate the cumulus
cells and eventually the zona pellucida. Typically, spenn
are allowed to interact with the oocytes overnight. In
cases where ICSI is used, the cumulus cells are removed
by enzymatic digestion so that the zona pellucida is de-
nuded of cells thus enabling the procedure to be more
efficient.

Figure 16-10 is a composit of photomicrographs
illustrating the ICSI procedure. After the cumulus cells
are removed, the oocyte is held in place using a pipette
that applies slight suction to the zona pellucida. At
the opposite pole of the oocyte, a small glass pipette
containing a single spenn is injected through the zona
pellucida, the plasma membrane of the oocyte and into
the cytoplasm. The ICSI procedure generally takes less
than one minute per oocyte.

Iffertilization is successfitl, the oocyte is char-
acterized as having a male and female pronucleus (See
Figure 16-1 0). Typically it takes about 18 hrs for the pro-
nuclei to fom1 after addition/injection of the spenn.

Embryo Transfer is a Non-Surgical Procedure

The transfer procedure is illustTated in Figure
16-l l. A small flexible catheter is inserted into the va-
gina and tlueaded through the cervix into the uterus.
The highest quality embryos are transferred into the
uterus. The goal ofiVF is one healthy baby. Guidelines
for the number of embryos transferred as a function of

Figure 16-11. Embryo Transfer

A small, flexib le catheter is inserted through the
vagina, cervix and into the lumen of the ute rus. The
catheter usually contains two e mbryos. It is attached
to a syringe and the flu id containing the embryos is
deposited. The catheter is then removed.

16
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344 The Human Factor
Ovarian Stimulation Promotes
Development of Multiple Follicles
Having multiple oocytes provides increased
probability for successful fertilization and embryo
development. Ovarian stimulation begins with an injec-
tion ofFSH, or and FSI-1-like honnone and concludes
with human chorionic gonadotropin (hCG). It should
be emphasized that there are many options for ovarian
stimulation. These vary depending on the reproduc-
tive status of the woman and from clinic-to-clinic. As
shown in Figure 16-8, exogenous FSH or and FSH-like
hormone stimulates growth of more than one follicle.
In this example, four follicles within one ovary respond
to FSH and these follicles secrete estradiol like in the
normal cycle. The four follicles continue to grow larger
Figure 16-8. Ovarian Stimulation
With FSH and hCG
Early Development
Eura.d iol
Mid-Development
Euradiol
Estradiol
Ovarian stimulation causes development of
higher than normal numbers offollicles. In this
example, four follicles develop simultaneously
after stimulation with FSH.
Aspiration
Estradio l Estradiol
At the appropriate time, the woman is treated
with human chorionic gonadotropin (hCG) to
stimulate final development and maturation of the
follicles. Oocytes are retreived from the follicles
using transvaginal ultrasound aspiration .
into mid-development and secrete more estradiol. Final
ovarian stimulation continues using hCG. Like LH,
hCG promotes final follicular mah1ration and growth
coupled with elevated estradiol secretion. When ready,
oocytes are aspirated from these large follicles.
Oocyte Retrieval is Accomplished by
Transvaginal Ultrasound Aspiration
Transvaginal aspiration is a technique that
combines ultrasound imaging with the mechanical
process of inserting a needle into each mature follicle
and applying slight suction to dislodge the oocyte and
remove it from the follicle. This procedure is typically
conducted in the physician’s office or in an outpatient
clinic. Some fonn of mild general analgesia is generally
administered. The first step in the procedure is to insert
an ultrasound probe into the vagina that enables it to be
positioned in close proximity to the ovary. Preovulatory
follicles that are eligible for aspiration are identified.
In the ultrasound images, they appear as dark circles
within the ovaty. Upon identification of eligible fol-
licles, a small needle is guided along the wall of the
ultrasound probe, through the wall of the vagina and
into each follicle (See Figme 16-9). Once inside the fol-
licle, slight suction is applied to the needle. This suction
dislodges the oocyte and can be aspirated. In general,
all eligible follicles in both ovaries can be aspirated
within 30 minutes. While this procedme is minimally
invasive, some women may experience cramping. This
Figure 16-9. Oocyte Retrieval Using
Transvaginal Ultrasound Aspiration
An ultrasound probe is
inserted into the vagina
and positioned in close
mity to the ovary.
A small needle is guided along the ultrasound probe
and inserted through the wall of the vagina and
into preovulatory follicles. Slight suction is applied
to dislodge the oocyte and it is aspi rated into a
collection vessel.
The Human Factor 345
Figure 16-10. Retrieved Oocyte, ICSI and Male and Female Pronuclei
Spenn
I ,- ‘ lnjectlon Mala
pi polio
ZP • ZP
A recently retrieved oocyte
with cumulus ce lls (CC)
su rrounding the zona pel-
lucida (ZP). In conventional
IVF, sperm penetrate the
cumulus cells and zona pel-
lucida before fertilizing the
oocyte. Micrograph courtesy of
West Virginia University Center for
Reproductive Medicine.
With ICS I, the cumulus cells a re re-
moved by enzymatic digestion and then
the denuded oocyte is inseminated .
The oocyte is held in position by a
pipette that applies s lig ht suction to
the zona pellucida (ZP). The sperm is
injected with a glass pipette. Micrograph
courtesy of West Vj rginia University Center for
Reproductive Afedicine.
The presence of a male
and female pronuclei in the
oocyte cytoplasm indicates
that ferti lization has taken
place. Each pronucleus
contains the genetic mate-
rial from the wo man and
the man. Micrograph courtesy
of West Virginia University Center
for Reproductive Medicine.
is not serious and generally subsides within one hour.
Immediately after aspiration, the oocytes are placed in
a culture medium that supports their viability.
Retrieved Oocytes are Surrounded by a
Layer of Cumulus Cells
Cumulus cells are remnants of granulosa! cells
that surround the freshly retrieved oocyte (See Figure
16-l 0). In the case of conventional IVF, sperm are
capacitated in-vitro and added to the culrure medium
containing the oocyte where they penetrate the cumulus
cells and eventually the zona pellucida. Typically, spenn
are allowed to interact with the oocytes overnight. In
cases where ICSI is used, the cumulus cells are removed
by enzymatic digestion so that the zona pellucida is de-
nuded of cells thus enabling the procedure to be more
efficient.
Figure 16-10 is a composit of photomicrographs
illustrating the ICSI procedure. After the cumulus cells
are removed, the oocyte is held in place using a pipette
that applies slight suction to the zona pellucida. At
the opposite pole of the oocyte, a small glass pipette
containing a single spenn is injected through the zona
pellucida, the plasma membrane of the oocyte and into
the cytoplasm. The ICSI procedure generally takes less
than one minute per oocyte.
Iffertilization is successfitl, the oocyte is char-
acterized as having a male and female pronucleus (See
Figure 16-1 0). Typically it takes about 18 hrs for the pro-
nuclei to fom1 after addition/injection of the spenn.
Embryo Transfer is a Non-Surgical Procedure
The transfer procedure is illustTated in Figure
16-l l. A small flexible catheter is inserted into the va-
gina and tlueaded through the cervix into the uterus.
The highest quality embryos are transferred into the
uterus. The goal ofiVF is one healthy baby. Guidelines
for the number of embryos transferred as a function of
Figure 16-11. Embryo Transfer
A small, flexib le catheter is inserted through the
vagina, cervix and into the lumen of the ute rus. The
catheter usually contains two e mbryos. It is attached
to a syringe and the flu id containing the embryos is
deposited. The catheter is then removed.
16
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346 The Human Factor

patient age, stage of embryo development and embryo
a quality are available through the Society for Assisted
Reproductive Technology (SART). Often, two and
sometimes three embryos are transferred to increase
the probability of a single birth even though twins and
triplets are possible.

Reproductive Aging in Women (Menopause)
and Men (Andropause)

Menopause is part of the natural aging process
and is defined as lack of menstrual periods. Meno-
pause is defined as the age of the last menstrual period.

Perimenopause refers to the timeframe (e.g., few years
before and after) around the last menstrual period. The
average age of menopause in western countries is 51
years. However, age of menopausal onset is heavily
influenced by genetics, ethnicity and general health
status. Some factors associated with early menopause
include: having relatives who reached menopause early,
being African or Hispanic, have a history of smoking,
have lived at high altitudes for most of one’s life, or
being a vegetarian. Women in these categories can
reach menopause up to two years sooner than women
without these factors.

Figure 16-12. Changes in Numbers Throughout the Life-span
of the Human Female

Ill
C1l …
0
0 -0 :r..

C1l .c
E
::::1 z

(Modified from Palter and Olive in Novak’s Gynecology, 11th Ed.)

Prenatal

About midway through
gestation, the number
of oocytes peaks within
the fetal ovary. About
25% of oocytes (poten-
tial follicles) degenerate
before birth.

6-7 X 106

600,000

1,000
t t

Oocyte numbers de-
cline from between
1 and 2 million to
about 300,000.

The number of oocytes is reduced
from about 300,000 to near zero at
menopause. About 25,000 primorid-
al follicles are available at age 37.

After 37, the rate of recrui tment
increases and therefore the rate of
atresia increases and the follicular
pool decreases rapidly until meno-
pause.

Fertilization Birth Puberty
( 10-13 yr)

Menopause
(45-50 yr)

There are profound physiological and psy-
chological changes that accompany menopause. Some
changes that occur during the menopausal transition
include: decreased cognitive function, genital atrophy,
vasomotor fluctuations (“hot flashes”), bone Joss, higher
risk of cardiovascular disease and collagen loss. These
changes are mainly due to marked decreased secretion
of estradiol that will be described later in this chapter.

Depletion of Follicles is the Cause of
Menopause in Women

The decline in ovarian follicle numbers over the
lifetime of the female is summarized in Figure 16- I 2.
At birth, the ovaries contain 1-2 million primordial fol-
licles . These follicles undergo a steady rate of decline
until about the age of 3 7, when approximately 25,000
primordial follicles remain. After age 37, the rate of
atresia increases until the woman enters menopause.
At this time, approximately 1000 follicles remain. It,is
likely that these 1000 fo llicles never get ·be-
cause they are probably not sensitive to gonadotropins.
Thus, further follicular development cannot occur.

Follicular Depletion Changes Many
Hormone Profiles

During menopausal onset, at least 7 hormones
undergo dramatic changes. These are: antiMi.illerian
hormone (AMI-I), inhibin, estradiol, testosterone, pro-
gesterone, FSH and LH (See Figure 16-13). All of the
hormones are directly related to follicular depletion and
will be discussed below.

AntiMiillerian Hormone

As follicles are depleted, antiMi.illerian hor-
mone (secreted by the granulosa! cells of preantral
follicles and early antral fo llicles) also decl ines. It
is important to understand that AMH is responsible
for controlling recruitment of primary follicles. AMH
also inhibits the FSH sensitiv ity in antral follicles.
Therefore, the net effect of AMH is to promote atresia
in developing antral follicles. In other words, sup-
pression of AMH also inhibits the FSH sensitivity in
antral follicles . AMH begins to slowly decline with
age. However, when AMI-I declines faster after age 3 7,
this allows more follicles to be recruited and undergo
atresia. As a result, the follicular pool is depleted at
a faster rate and the number of antral follicles in each
cohort decreases with age.

The Human Factor 34 7

Figure 16-13. Hormone Profile
Changes During Menopause

(Modified from F.J. Broekmans , et al. , 2009)

c
0

OJ·;:;
>ro
·..-:;; l:;
roc – OJ OJ u a::c

0 u
c
0

OJ·;:;
> ro ·- …. …… …. ro c -OJ OJ u a::c

0 u
c
0

OJ·;:;
>ro ·- …. ….. …… ro c – OJ OJu a::c

0 u
c
0

OJ ·.;::; >ro ·- …. ………… roc – OJ OJu O::c
0 u

c
0

OJ ·.;:;
> ro ·- …. …… …… ro c – OJ OJ u O::c

0 u
c
0

OJ·;:;
>ro ·- …. ……….. me – OJ OJu a::c

0 u
c
0

OJ ·;:; >ro ·;::; ,::;
roc – OJ OJu a::c

0 u

E2

lnhibin

T

p4

FSH
LH

Reproductive Post-
years reproductive

I
years

I
Menopause
Ve
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oo
ks
.ir

346 The Human Factor
patient age, stage of embryo development and embryo
a quality are available through the Society for Assisted
Reproductive Technology (SART). Often, two and
sometimes three embryos are transferred to increase
the probability of a single birth even though twins and
triplets are possible.
Reproductive Aging in Women (Menopause)
and Men (Andropause)
Menopause is part of the natural aging process
and is defined as lack of menstrual periods. Meno-
pause is defined as the age of the last menstrual period.
Perimenopause refers to the timeframe (e.g., few years
before and after) around the last menstrual period. The
average age of menopause in western countries is 51
years. However, age of menopausal onset is heavily
influenced by genetics, ethnicity and general health
status. Some factors associated with early menopause
include: having relatives who reached menopause early,
being African or Hispanic, have a history of smoking,
have lived at high altitudes for most of one’s life, or
being a vegetarian. Women in these categories can
reach menopause up to two years sooner than women
without these factors.
Figure 16-12. Changes in Numbers Throughout the Life-span
of the Human Female
Ill
C1l …
0
0 -0 :r..
C1l .c
E
::::1 z
(Modified from Palter and Olive in Novak’s Gynecology, 11th Ed.)
Prenatal
About midway through
gestation, the number
of oocytes peaks within
the fetal ovary. About
25% of oocytes (poten-
tial follicles) degenerate
before birth.
6-7 X 106
600,000
1,000
t t
Oocyte numbers de-
cline from between
1 and 2 million to
about 300,000.
The number of oocytes is reduced
from about 300,000 to near zero at
menopause. About 25,000 primorid-
al follicles are available at age 37.
After 37, the rate of recrui tment
increases and therefore the rate of
atresia increases and the follicular
pool decreases rapidly until meno-
pause.
Fertilization Birth Puberty
( 10-13 yr)
Menopause
(45-50 yr)
There are profound physiological and psy-
chological changes that accompany menopause. Some
changes that occur during the menopausal transition
include: decreased cognitive function, genital atrophy,
vasomotor fluctuations (“hot flashes”), bone Joss, higher
risk of cardiovascular disease and collagen loss. These
changes are mainly due to marked decreased secretion
of estradiol that will be described later in this chapter.
Depletion of Follicles is the Cause of
Menopause in Women
The decline in ovarian follicle numbers over the
lifetime of the female is summarized in Figure 16- I 2.
At birth, the ovaries contain 1-2 million primordial fol-
licles . These follicles undergo a steady rate of decline
until about the age of 3 7, when approximately 25,000
primordial follicles remain. After age 37, the rate of
atresia increases until the woman enters menopause.
At this time, approximately 1000 follicles remain. It,is
likely that these 1000 fo llicles never get ·be-
cause they are probably not sensitive to gonadotropins.
Thus, further follicular development cannot occur.
Follicular Depletion Changes Many
Hormone Profiles
During menopausal onset, at least 7 hormones
undergo dramatic changes. These are: antiMi.illerian
hormone (AMI-I), inhibin, estradiol, testosterone, pro-
gesterone, FSH and LH (See Figure 16-13). All of the
hormones are directly related to follicular depletion and
will be discussed below.
AntiMiillerian Hormone
As follicles are depleted, antiMi.illerian hor-
mone (secreted by the granulosa! cells of preantral
follicles and early antral fo llicles) also decl ines. It
is important to understand that AMH is responsible
for controlling recruitment of primary follicles. AMH
also inhibits the FSH sensitiv ity in antral follicles.
Therefore, the net effect of AMH is to promote atresia
in developing antral follicles. In other words, sup-
pression of AMH also inhibits the FSH sensitivity in
antral follicles . AMH begins to slowly decline with
age. However, when AMI-I declines faster after age 3 7,
this allows more follicles to be recruited and undergo
atresia. As a result, the follicular pool is depleted at
a faster rate and the number of antral follicles in each
cohort decreases with age.
The Human Factor 34 7
Figure 16-13. Hormone Profile
Changes During Menopause
(Modified from F.J. Broekmans , et al. , 2009)
c
0
OJ·;:;
>ro
·..-:;; l:;
roc – OJ OJ u a::c
0 u
c
0
OJ·;:;
> ro ·- …. …… …. ro c -OJ OJ u a::c
0 u
c
0
OJ·;:;
>ro ·- …. ….. …… ro c – OJ OJu a::c
0 u
c
0
OJ ·.;::; >ro ·- …. ………… roc – OJ OJu O::c
0 u
c
0
OJ ·.;:;
> ro ·- …. …… …… ro c – OJ OJ u O::c
0 u
c
0
OJ·;:;
>ro ·- …. ……….. me – OJ OJu a::c
0 u
c
0
OJ ·;:; >ro ·;::; ,::;
roc – OJ OJu a::c
0 u
E2
lnhibin
T
p4
FSH
LH
Reproductive Post-
years reproductive
I
years
I
Menopause
Ve
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oo
ks
.ir

348 The Human Factor

Testosterone, Estradiol and Inlzibin

In cycling women, testosterone, estradiol and
inhibin are important secretory products of antral fol-
licles. Without antral follicles, testosterone, estradiol
and inhibin drop dramatically (See Figure 16-13). Re-
call fi:om Chapter 8 (Figure 8-9) that estradiol secretion
takes place in a “2 cell-2 gonadotropin” model where
testosterone is secreted by the theca intema cells and
converted to estradiol by the granulosa) cells. Without
these cells neither hormone can be synthesized and
secreted.

Progesterone

Progesterone significantly declines when the
corpus luteum from the last cycle is lysed. Without
future antral follicles and ovulations, corpora lutea
cannot be formed. As you recall from Chapter 9, the
human corpus luteum secretes estradiol in addition
to progesterone (See Figure 9-14). Without estradiol
secretions from antral follicles or the corpus lutem,
circulating estradiol concentrations in the blood drop
dramatically.

FSHandLH

Without AMH, testosterone, estradiol, inhibin
and progesterone, negative feedback on the hypothala-
mus and pituitary does not exist. As a result, FSH and
LH concentrations increase dramatically. Post meno-
pausal FSH concentrations are six times greater than
FSH concentrations in the normal reproductive years.
Concurrently, LH concentrations are four times greater
than LH concentrations in the normal reproductive
years.

Estrogen deficiency results in:
• genital atrophy
• decreased seCI’etion by the

reproductive tract
• modification of lipid metabolism

and ofthe vascular walls
• increase in the physiological loss

of bone (osteoporosis)
• vasomotor symptoms

(“hot flashes’?
• decreased cognitive function
• increased fat mass

Regardless of the number of overall honnonal
changes, the single most important hormonal change
is the decrease of estradiol. Almost all negative effects
associated with menopause are due to lack of estradiol.
The primary physiological and psychological effects of
menopause relate to estradiol deficiency.

The majority of the symptoms of menopause
can be reversed with estradiol. Unforhmately, hormone
replacement therapy is smTounded by controversial
issues relating to the possible carcinogenic effects of
estradiol. More recently, AMH has been suggested
as a possible alternative to the conventional hormone
replacement therapy. AMI-I could be used to slow the
rate of follicular recruitment and ah·esia. In this way,
the onset of menopause would be delayed. The negative
health effects associated with estradiol absence such
as osteoporosis, increased cardiovascular disease and
decreased cognitive ability would be minimized. For
more details about the risks and benefits of hormone
replacement therapy, consult the references at the end
of this chapter.

Reproductive Aging in Men (Andropause)

Andropause is a decline in reproductive fl.mc-
tion as it relates to advancing age. However, andropause
is not a defined, finite cessation of reproductive capac-
ity. The changes are significantly slower than in the
woman. Andropause is characterized by a decline in
libido, an increased incidence in erectile dysfunction,
loss of muscle and bone mass, physical fi.mction, and an
increase in fat mass. The biochemical causes of erectile
dysfunction are presented in Figure 11-9.

Andropause results in:
• decreased libido
• decreased muscle mass
• decreased bone density
• increased fat mass

Men in the seventh and eighth decade of life
have about 70% of daily sperm production when com-
pared to men in their early 30s. Circulating testosterone
concentrations decrease approximately I %-3% per year
beginning at the age of35-40, thus men aged 70-80 have
about 50% of circulating testosterone concentrations
when compared to younger men. Although hormones
decrease with age in males, these changes are minimal
compared to the hormonal changes in women (See
Figure 16-14).

c
0

“P ro …..
-1-‘ c
QJ
u c
0 u
QJ
c
0
E …..
0

I
QJ
> ·p
ro
QJ

0::::

The Human Factor 349

Figure FSH and LH Profiles Associated With Gender and Age

Women
FSH LH

FSH concentrations in older women (post-
menopausal years) are approximately six
times greater than FSH concentrations
when compared to women in their re-
productive years. LH concentrations are
approximately four times higher in older
women when compared to women in their
reproductive years.

Men
FSH LH

FSH and LH concentrations increase with
age in men as well. However, FSH and
LH concentrations in older men are not as
dramatic when compared to the hormone
changes during and after menopause.
Despite higher FSH and LH concentra-
tions, sperm production is still possible.

16
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348 The Human Factor
Testosterone, Estradiol and Inlzibin
In cycling women, testosterone, estradiol and
inhibin are important secretory products of antral fol-
licles. Without antral follicles, testosterone, estradiol
and inhibin drop dramatically (See Figure 16-13). Re-
call fi:om Chapter 8 (Figure 8-9) that estradiol secretion
takes place in a “2 cell-2 gonadotropin” model where
testosterone is secreted by the theca intema cells and
converted to estradiol by the granulosa) cells. Without
these cells neither hormone can be synthesized and
secreted.
Progesterone
Progesterone significantly declines when the
corpus luteum from the last cycle is lysed. Without
future antral follicles and ovulations, corpora lutea
cannot be formed. As you recall from Chapter 9, the
human corpus luteum secretes estradiol in addition
to progesterone (See Figure 9-14). Without estradiol
secretions from antral follicles or the corpus lutem,
circulating estradiol concentrations in the blood drop
dramatically.
FSHandLH
Without AMH, testosterone, estradiol, inhibin
and progesterone, negative feedback on the hypothala-
mus and pituitary does not exist. As a result, FSH and
LH concentrations increase dramatically. Post meno-
pausal FSH concentrations are six times greater than
FSH concentrations in the normal reproductive years.
Concurrently, LH concentrations are four times greater
than LH concentrations in the normal reproductive
years.
Estrogen deficiency results in:
• genital atrophy
• decreased seCI’etion by the
reproductive tract
• modification of lipid metabolism
and ofthe vascular walls
• increase in the physiological loss
of bone (osteoporosis)
• vasomotor symptoms
(“hot flashes’?
• decreased cognitive function
• increased fat mass
Regardless of the number of overall honnonal
changes, the single most important hormonal change
is the decrease of estradiol. Almost all negative effects
associated with menopause are due to lack of estradiol.
The primary physiological and psychological effects of
menopause relate to estradiol deficiency.
The majority of the symptoms of menopause
can be reversed with estradiol. Unforhmately, hormone
replacement therapy is smTounded by controversial
issues relating to the possible carcinogenic effects of
estradiol. More recently, AMH has been suggested
as a possible alternative to the conventional hormone
replacement therapy. AMI-I could be used to slow the
rate of follicular recruitment and ah·esia. In this way,
the onset of menopause would be delayed. The negative
health effects associated with estradiol absence such
as osteoporosis, increased cardiovascular disease and
decreased cognitive ability would be minimized. For
more details about the risks and benefits of hormone
replacement therapy, consult the references at the end
of this chapter.
Reproductive Aging in Men (Andropause)
Andropause is a decline in reproductive fl.mc-
tion as it relates to advancing age. However, andropause
is not a defined, finite cessation of reproductive capac-
ity. The changes are significantly slower than in the
woman. Andropause is characterized by a decline in
libido, an increased incidence in erectile dysfunction,
loss of muscle and bone mass, physical fi.mction, and an
increase in fat mass. The biochemical causes of erectile
dysfunction are presented in Figure 11-9.
Andropause results in:
• decreased libido
• decreased muscle mass
• decreased bone density
• increased fat mass
Men in the seventh and eighth decade of life
have about 70% of daily sperm production when com-
pared to men in their early 30s. Circulating testosterone
concentrations decrease approximately I %-3% per year
beginning at the age of35-40, thus men aged 70-80 have
about 50% of circulating testosterone concentrations
when compared to younger men. Although hormones
decrease with age in males, these changes are minimal
compared to the hormonal changes in women (See
Figure 16-14).
c
0
“P ro …..
-1-‘ c
QJ
u c
0 u
QJ
c
0
E …..
0
I
QJ
> ·p
ro
QJ
0::::
The Human Factor 349
Figure FSH and LH Profiles Associated With Gender and Age
Women
FSH LH
FSH concentrations in older women (post-
menopausal years) are approximately six
times greater than FSH concentrations
when compared to women in their re-
productive years. LH concentrations are
approximately four times higher in older
women when compared to women in their
reproductive years.
Men
FSH LH
FSH and LH concentrations increase with
age in men as well. However, FSH and
LH concentrations in older men are not as
dramatic when compared to the hormone
changes during and after menopause.
Despite higher FSH and LH concentra-
tions, sperm production is still possible.
16
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350 The Human Factor

Further
PHENOMENA
for Fertility
Some African tribes believed that menstrual
blood kept in a covered pot for nine months
had the power to tum itself into a baby.

The oldest woman to conceive naturally is
Dawn Brooke. She gave birth to her son at
59 years of age.

Guiness Book of World Records reported
that Jacilyn Dalenberg gave birth to her
three gmnddaughters at age 56. She was a
surrogate for her daughter.

Female pilot whales as old as 51 years of
age have been observed to be lactating. One
female was recorded to have lactated for
approximately 11 years after the last ovula-
tion and parturition. The last calf may be
suckled until puberty (8 years for females
and 11 years for males).

In the 1700s, it was reported that a peasant
wife from Russia holds the record for the
greatest number of children born to one
mother. 27 pregnancies resulted in 16 sets
of hVins, 7 sets of triplets and four sets of
quadruplets, for a total of 69 children. It
was also reported that only hVo children died
in their infancy. What is the probability of
this story?

40 species of lizards are known to reproduce
by parthenogeneis (natural cloning). These
species consist of all females. Who needs a
male around?

In 2007, Nanu Jogi is reported to have been
the oldest known father in the world. He was
90 years old when his 21st child was bom.

The typical person spends about 600 hours
having sex behveen the ages of 20 and 70.

Shaking hands is one way to say hello to a
friend. However, Walibri tribesmen from
Central Australia greet each other by shak-
ing each other’s penises.

Besides the eyelid, the scrotal skin is the
only part of the body with little or no sub-
cutaneous fat.

The” nesting behavior of the Silvery-Cheeked
Hornbill adds new meaning to the term
“cabin fever”. When the time comes to in-
cubate the eggs, the female-finds a suitable
hole in a tree and goes inside. The male then
brings mud to his spouse who “plasters”
herself inside for over three months. She
leaves a narrow opening so that the male can
deliver food for her and the chicks.

Two separate British courts in the 1980s
reduced the sentences of women who killed
their husbands on the grounds that severe
PMS (premenstrual syndrome) was respon-
sible for transforming the normally sane
women into maniacs.

It has been caluculated that the average man
will ejaculate approximately 18 quarts of
semen containing over half a trillion sperm
over his lifetime.

The voice of a male frog deepens and gets
louder with age.

The average speed of the ejaculate during
a male orgasm is 28 mph, according to the
Kinsey Institute.

A dragonfly’s penis has a shovel on the end
that scoops out a rival male’s sperm.

Key References

Berek, J. ed. 1996. Novak’s 13th Edition. Wil-
liams and Williams. Baltimore. ISBN 0-7817-3262-X.

Broekmans, F.J., M.R. Soules and B.C. Fauser. 2009.
Ovarian aging: Mechanisms and Clinical Consequences.
Endo Rev. 30(5) 465-493.

Driancourt, M.A., A Gougeon, A. Roy ere and C. Thibault.
1993. “Ovarian function” in Reproduction in Mammals and
Man. p281-306. C. Thibault, M.C. Levasseur and R.H.F.
Hunter, eds, Ellipses, Paris. ISBN 2-7298-9354-7.

Houston, A, Abraham, A, Zhi huan Huang, Z. , and
D’Angelo, L. (2006). Knowledge, attitudes, and conse-
quences of menstrual health in urban adolescent females.
J Pediatr Ado/esc Gynecol. 19:271 -275.

Horstman, A.M. , Dill ion, E., Urban, R. and M. Sheffield-
Moore. 2012. The role of androgens and estrogens on
healthy aging and longevity. J Gerontal A Bioi Sci Med
Sci, doi: 10.1 093/gerona/gls068.

Koff, E., Rierdan, J. , and Shtbbs, M. (1990). Conceptions
and misconceptions of the menstrual cycle. Women &
Health, 16(3/4): 119-136.

Lobo, R. 2004. “Menopause and Aging” in Yen and Jaffe s
Reproductive Endocrinology-5th Edition, Strauss and Bar-
bieri, eds. Elsevier, Philadelphia. ISBN 0-72 16-9546-9.

Netter, A. 1993. “The menopause” in Reproduction in
Mammals and Man. p627-642. C. Thibault, M.C. Le-
vasseur and R.H.F. Hunter, eds., Ellipses, Paris. ISBN
2-7298-9354-7.

Synder, P. 2004. “Male Reproductive Aging” in Yen and
Jaffe’s Reproductive Endocrinology-5th Edition, Strauss
and Barbieri, eds. Elsevier, Philadelphia. ISBN 0-7216-
9546-9.

Udolff, L.C. and E. Y. Adashi 1998. “Menopause” in
Encvclopedia o(Reproduction, Vol. 3 p 183- 188. Knobil
and Neill, eds. Academic Press, San Diego. ISBN 0-1 2-
227023-1.

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350 The Human Factor
Further
PHENOMENA
for Fertility
Some African tribes believed that menstrual
blood kept in a covered pot for nine months
had the power to tum itself into a baby.
The oldest woman to conceive naturally is
Dawn Brooke. She gave birth to her son at
59 years of age.
Guiness Book of World Records reported
that Jacilyn Dalenberg gave birth to her
three gmnddaughters at age 56. She was a
surrogate for her daughter.
Female pilot whales as old as 51 years of
age have been observed to be lactating. One
female was recorded to have lactated for
approximately 11 years after the last ovula-
tion and parturition. The last calf may be
suckled until puberty (8 years for females
and 11 years for males).
In the 1700s, it was reported that a peasant
wife from Russia holds the record for the
greatest number of children born to one
mother. 27 pregnancies resulted in 16 sets
of hVins, 7 sets of triplets and four sets of
quadruplets, for a total of 69 children. It
was also reported that only hVo children died
in their infancy. What is the probability of
this story?
40 species of lizards are known to reproduce
by parthenogeneis (natural cloning). These
species consist of all females. Who needs a
male around?
In 2007, Nanu Jogi is reported to have been
the oldest known father in the world. He was
90 years old when his 21st child was bom.
The typical person spends about 600 hours
having sex behveen the ages of 20 and 70.
Shaking hands is one way to say hello to a
friend. However, Walibri tribesmen from
Central Australia greet each other by shak-
ing each other’s penises.
Besides the eyelid, the scrotal skin is the
only part of the body with little or no sub-
cutaneous fat.
The” nesting behavior of the Silvery-Cheeked
Hornbill adds new meaning to the term
“cabin fever”. When the time comes to in-
cubate the eggs, the female-finds a suitable
hole in a tree and goes inside. The male then
brings mud to his spouse who “plasters”
herself inside for over three months. She
leaves a narrow opening so that the male can
deliver food for her and the chicks.
Two separate British courts in the 1980s
reduced the sentences of women who killed
their husbands on the grounds that severe
PMS (premenstrual syndrome) was respon-
sible for transforming the normally sane
women into maniacs.
It has been caluculated that the average man
will ejaculate approximately 18 quarts of
semen containing over half a trillion sperm
over his lifetime.
The voice of a male frog deepens and gets
louder with age.
The average speed of the ejaculate during
a male orgasm is 28 mph, according to the
Kinsey Institute.
A dragonfly’s penis has a shovel on the end
that scoops out a rival male’s sperm.
Key References
Berek, J. ed. 1996. Novak’s 13th Edition. Wil-
liams and Williams. Baltimore. ISBN 0-7817-3262-X.
Broekmans, F.J., M.R. Soules and B.C. Fauser. 2009.
Ovarian aging: Mechanisms and Clinical Consequences.
Endo Rev. 30(5) 465-493.
Driancourt, M.A., A Gougeon, A. Roy ere and C. Thibault.
1993. “Ovarian function” in Reproduction in Mammals and
Man. p281-306. C. Thibault, M.C. Levasseur and R.H.F.
Hunter, eds, Ellipses, Paris. ISBN 2-7298-9354-7.
Houston, A, Abraham, A, Zhi huan Huang, Z. , and
D’Angelo, L. (2006). Knowledge, attitudes, and conse-
quences of menstrual health in urban adolescent females.
J Pediatr Ado/esc Gynecol. 19:271 -275.
Horstman, A.M. , Dill ion, E., Urban, R. and M. Sheffield-
Moore. 2012. The role of androgens and estrogens on
healthy aging and longevity. J Gerontal A Bioi Sci Med
Sci, doi: 10.1 093/gerona/gls068.
Koff, E., Rierdan, J. , and Shtbbs, M. (1990). Conceptions
and misconceptions of the menstrual cycle. Women &
Health, 16(3/4): 119-136.
Lobo, R. 2004. “Menopause and Aging” in Yen and Jaffe s
Reproductive Endocrinology-5th Edition, Strauss and Bar-
bieri, eds. Elsevier, Philadelphia. ISBN 0-72 16-9546-9.
Netter, A. 1993. “The menopause” in Reproduction in
Mammals and Man. p627-642. C. Thibault, M.C. Le-
vasseur and R.H.F. Hunter, eds., Ellipses, Paris. ISBN
2-7298-9354-7.
Synder, P. 2004. “Male Reproductive Aging” in Yen and
Jaffe’s Reproductive Endocrinology-5th Edition, Strauss
and Barbieri, eds. Elsevier, Philadelphia. ISBN 0-7216-
9546-9.
Udolff, L.C. and E. Y. Adashi 1998. “Menopause” in
Encvclopedia o(Reproduction, Vol. 3 p 183- 188. Knobil
and Neill, eds. Academic Press, San Diego. ISBN 0-1 2-
227023-1.
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