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CHAPTER 1: GENERAL

REPRODUCTIVE PHYSIOLOGY

Our knowledge of reproductive endo-crinology is derived from domestic animals, laboratory animals, pri-mates and humans, therefore all of

these animals receive some attention in this syllabus.

In view of the fact that this is a vet-erinary text, it is obvious that most emphasis is placed on the domestic species but some comparisons are drawn between domestic animals and man. This has been done for interest, and occasionally, because extrapolation between species is necessary. In addition, veterinarians are employed in significant numbers in primate centres therefore the syl-labus also contains occasional refer-

ences to the reproduction of primates or even humans.

Due to the fact that cattle have been studied in great detail with respect to some areas of reproductive physi-ology, the cow is often used as an extension of the general discussion on physiology in this syllabus and is also used as a model for comparison with other species.

THE HYPOTHALAMIC-PITUITARY AXIS & RELEASING HORMONES

The hypothalamus (Figure C1.1)produces various releasing hormones that control anterior pituitary func-tion.

A releasing hormone of cardinal importance in reproduction is gonad-otropin releasing hormone (GnRH or LHRH). This is a decapeptide mol-ecule that has been synthesized and

Fig C1.1 The base of the brain. This shows an equine brain, where the adenohypophysis actually wraps around the neurohypophysis. Note the location of the hypothalamus, just below the third ventricle. This is where many releasing hormones are produced. Abbreviations: Cer - cerebel-lum, Hypth - hypothalamus, Neu.H - neurohypophysis, Ade.H - adenohypophysis, 3rd V - third ventricle, Thal - thalamus.

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used experimentally and therapeuti-cally. It has the same structure in all mammals studied thus far, making it a unique and non-antigenic tool for manipulation of the reproductive system.

GnRHGnRH is released from the medial eminence of the hypothalamus in a pulsatile or episodic (in episodes)

fashion. GnRH causes the release of luteinizing hor-mone (LH) and follicle stim-ulating hormone (FSH) from the anterior pituitary gland. In animals that have breed-ing seasons, the pulses of GnRH secretion become more and more frequent

as the breeding season approaches. This in turn causes an increase in the pulse frequency of LH and FSH release, and ultimately results in the onset of cyclicity.

During LH or FSH surges in a cycling animal (for example, during a pre-ovulatory LH surge) the pulses of GnRH are so frequent that its secre-tion is almost continuous.

Some data shows that there may be a releasing factor/s for FSH other than GnRH, because FSH is sometimes elevated when LH is not elevated. For example, serum FSH levels can be quite high while there is a func-tional corpus luteum, or during the postpartum period, when serum LH levels are typically very low. In addi-tion, the FSH surge following bolus administration of GnRH is much less spectacular than the LH surge. How-ever, no other releasing factor for FSH has yet been identified.

GnRH is frequently used to release LH in clinical cases, e.g. for treat-ing cystic ovarian disease in cattle. It is also used to induce ovulation in cats, rabbits and camelids (all induced ovulators) and for various

treatments that synchronize estrous cycles in cattle. Unfortunately GnRH is not effective when used in attempts to convert anestrous (non-cycling) animals to a cycling animal. This is because anestrus is a complex, multi facto-rial problem. These problems are discussed in detail elsewhere.

It is very important to realize that there is another side to GnRH; sup-

Figure C1.2 The super active GnRH analog deslorelin in the form of an implant used to suppress fertility in dogs.

Figures C1 .3 The effect of a deslorelin implant on the semen volume in a dog. Semen volume is practically a bioassay for testoster-one production. Therefore, this graph shows how testosterone production was suppressed during the same time period.

frmutIiGm

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pression of gonadotrophins. This sounds incongruous in light of what we have just said about the release of LH and FSH. But, after GnRH has caused the release of LH and FSH, receptor sites on the cell membrane become refractory to GnRH and obligatory intermediary hormones are also depleted. What follows is knows as “down-regulation” of the pituitary. This causes profound sup-pression of LH and FSH in a condition known as “medical hypophysectomy”.

Medical hypophysectomy of practi-cal importance because it is used to suppress gonadal function in a myriad of clinical situations. These include prostate cancer, precocious puberty, androgen mediated acne and many others in human medicine. In veterinary medicine we use long term, potent GnRH analog treatment for population control in dogs (see figures C1.2 and C1.3) cats and wild animals, to suppress the function of ovarian remnants (after surgery), to treat prostatic hyperplasia and for numerous other reasons. We also see the effect of medical hypophy-sectomy when GnRH implants (used to induce ovulation in mares) are left in situ for too long; the interval to the next ovulation is abnormally prolonged.

Down regulation occurs after native GnRH treatments but is most pro-found when potent GnRH analogs (figure C1.2) are used. As mentioned elsewhere, there are many hundreds of these although only a handful are used clinically.

There are also some GnRH analogs that inhibit gonadotrophin release directly (without causing down regulation). They are competitive inhibitory analogs of GnRH that bind to the adenohyphysis, preventing native GnRH from binding there and functioning normally. Unlike GnRH

agonist, these antagonists do not cause an initial release of LH and FSH(which can cause prostate cancer to “flare” painfully) so they are some-times used in preference to super active agonists. Unfortunately these inhibitory analogs difficult to manu-facture and are very expensive to produce so they are seldom if ever used in veterinary medicine.

Now, back to the native form of GnRH i.e. the form produced by mother nature: Like all of the releasing hor-mones, GnRH has a very short half life and is rapidly metabolized in the liver. Therefore, the body has a clev-erly designed blood supply to deliver GnRH directly to the anterior pituitary gland in high concentrations, before it can be catabolized systemically. This is called the hypothalamic-pitu-itary portal system. Other releasing hormones (see below) also travel down this portal system to cause the release of other anterior pituitary hormones.

Since almost every hormone in the body has some sort of effect on reproductive function we must review many others too i.e.

Corticotrophin releasing hor-mone/factor

(CRH/F) causes the release of adre-nocorticotropic hormone (ACTH) by proteolytic cleavage of pro-opiomel-anocortin (POMC) into two parts, active ACTH and -lipotropin. Beta lipotropin then breaks up into two other molecules, Beta endorphins (which are much loved by exercise junkies for their opiate-like proper-ties) and melanocyte stimulating hormone (MSH, which promotes skin pigmentation).

Thyroid releasing hormone Thyroid releasing hormone (TRH) causes the release of thyroid stimu-lating hormone (TSH) and prolactin. Therefore TRH has been used experi-

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mentally to stimulate milk produc-tion in cows. Remember however (see below) that prolactin secretion is mostly under the negative control of dopamine.

Growth hormone releasing hor-mone

Growth hormone releasing hormone (GRH) causes the release of somato-tropic hormone (STH or growth hor-mone GH) while growth hormone inhibiting hormone (Somatostatin) causes suppression of growth hor-mone (GH).

Prolactin: a spe-cial caseProlactin inhibiting hormone (dopa-mine) suppresses the release of pro-lact in from the adenohypophysis. Therefore, unlike most other anterior pituitary hormones, prolactin has a control system that is primarily negative. Caber-

goline and other ergot alkaloids are dopamine-like substances and will cause significant sup-pression of serum pro-lactin levels.

In clinical veterinary medicine, ergot alkaloids (Figure C1.4) they are used to induce abortion in bitches and to suppress other prolactin depen-dent processes. On the other hand, substances l ike metoclopramide (Figure C1.5) reserpine and domperidone which block dopamine recep-tor sites, cause prolactin release. Domperidone is used for this purpose to treat agalactia in mares

which have been grazing on tall fescue pastures. This allows prolac-tin release, milk production and the mare is able to feed her foal.Others factors will also stimulate prolactin release, for example sero-tonin (5HT), histamine, and beta endorphins.

Estrogens can cause prolactin release too, explaining in part why we see such high serum concentrations of prolactin at parturition, prepar-ing the dam for milk production. In practice, many tranquilizers will also release prolactin because they are dopamine antagonists. That is why some patients in mental hospitals may show signs of milk production. It is also the logic behind the use of estrogen and phenothiazine tran-quilizers for induction of lactation in non-pregnant animals.

FSH and LH (gonadotropins)FSH and LH are produced by the basophilic cells of the anterior pitu-itary. In fact, both gonadotropins occur within the same cell. They are glycoproteins and huge molecules in comparison to the releasing hor-mones.

The large and complex struc-ture of the gonadotropins dic-tates that they are relatively unstable and must be refriger-ated for storage. This is why FSH, LH and similar hormones are usually sold and stored in the freeze-dried state and are only re-constituted just before use. FSH and LH consist of two subunits; and . The subunit is common to both hormones but the subunit is different, and dictates whether the molecule has FSH or LH activity.

It is important to remember that the structure of all the gly-coprotein hormones is slightly

Fig C1.4. Cabergoline (Galastop®). An ergot alkaloid (dopamine-like) used to abort bitches and to treat pseudopreg-nancy.

Figure C1.5 Metaclopromide This blocks dopamine receptors. It is often used as an anti-emetic.

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different from one species to another. Therefore laboratories need species specific assays to measure FSH and LH levels in most cases. Structural conservation may allow assays and the hormones themselves to work in other species but this is not always so. Occasionally antibodies may be formed against glycoprotein hor-mones that are derived for therapy from animals of another species.

With few exceptions, FSH and LH are secreted in an episodic fashion in response to the episodic pulses of GnRH; therefore single samples for assay of LH or FSH are not good reflections of true physiology. Also, due to their large size and complex nature, LH and FSH cannot be syn-thesized by ordinary methods and as a result, only pituitary extracts (Figure C1.4) are available for clinical use. Recently however, genetic engi-neering has been used to synthesize both LH and FSH. What is the function of FSH? From its name it is obvious that FSH can cause follicular development. But this is only true to a point i.e. when FSH has induced a primary follicle to develop into a larger secondary follicle (one that has both a theca externa and theca granulosa), LH is needed to assist in follicle maturation and LH is also needed to cause ovulation.

Essentially FSH “paves the way” for LH by inducing receptor sites for LH, its obligatory partner in this process. FSH also works in concert with LH to form the estrogens that dominate the steroid content of the follicle.

During steroid synthesis the outer (theca) cells of the follicle are stimu-lated to produce androgens by LH. Then these androgens diffuse into the inner (granulosa) cells off the fol-licle wall where they are aromatized to estrogens under the influence of FSH.

The follicle mature under the effect of LH then it ovulates and discharges its oocyte. The process of ovulation is poorly understood but prostaglandins F2 and E2, and collagenase as well as LH are involved.

Once ovulation has occurred, basal pulses of LH (as apposed to the LH surge) induce the theca granulosa cells to undergo hypertrophy ulti-mately forming the “large” cells of the corpus luteum (CL). There are also “small” luteal cells in the corpus luteum, arising via hyperplasia of the cells of the theca interna. The pri-mary function of the large luteal cells is to produce oxytocin and during pregnancy, relaxin. They also secrete progesterone. The small luteal cells mainly secrete progesterone.

The Basal LH pulsation is also respon-sible for main-tenance of the C L b e t w e e n estrous per i-ods a principle that is applied in the practise of estrous cycle control in sheep ( s e e l a t e r ) . In some ani-mals, LH is also responsible for the maintenance of the CL during pregnancy.

A p a r t f r o m GnRH , many other hormones regulate gonad-otropin secre-tion, but the pul-satile rhythms of FSH and LH secretion are mainly modulated by ovarian steroids. When a follicle approaches maturity, estrogen production by that follicle causes a positive feedback on the hypothalamic-pituitary system. This

Fig C1.4 Folltropin®-V is a highly purifi ed folltropin extract obtained from carefully selected porcine pituitary glands. Although this is used in another species from which it was harvested (it is used in cattle for superovulating prior to E.T.) it has enough of an effect on the target species to be of great value.

This product is a large and complex molecule, stored in a freeze dried state. Note the powder (L) and diluent (R). It is expensive too.

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causes the LH surge that is associ-ated with ovulation. Once ovula-tion has occurred, the CL produces progesterone which has a negative feedback effect on the hypothalamus. This is how progesterone suppresses the production of GnRH and gonado-tropin.

It is obvious then, that progesterone can potentially prevent the matura-tion and ovulation of follicles. This is basis of some forms of birth control. At the same time, it is important to remember that FSH secretion is not significantly suppressed by proges-terone (see the discussion on GnRH). This explains why progesterone alone cannot control the estrous cycle pre-cisely. It also explains why one can palpate large follicles on the ovaries of mares and cows in the middle of a luteal phase when there is a fully functional CL.

When the CL undergoes lysis towards the end of the estrous cycle, serum progesterone falls and the nega-tive feedback of progesterone on the gonadotropins (especially LH) diminishes. This allows a follicle to mature. Estrogen production by that follicle(s) causes an LH surge to occur and ovulation follows.

In contrast to progesterone, estro-gens do have a moderate negative feedback effect on FSH. In fact, this is of practical use when one wishes to suppress follicular development for estrous synchronization. For exam-ple, it is common to use estrogen in mares to suppress follicle devel-opment. This is used together with progesterone treatments to suppress the estrous cycle.

Domestic animals do not experience menopause but when women reach menopause, the negative feedback effect of ovarian steroids is with-drawn and FSH and LH rise. As a result, both of these gonadotropins

are voided in the urine and are col-lected for use as human menopausal gonadotropin (hMG). hMG is occa-sionally used in cows, women and other animals to stimulate follicle development for in-vitro fertilization and embryo transfer.

What are the functions of LH and FSH in the male? In male animals, LH causes the Leydig cells in the testicles to form testosterone. For this reason, it is sometimes called interstitial cell stimulating hormone (ICSH) but that name is seldom used. FSH causes the Sertoli cells to form androgen binding protein (ABP). ABP binds testosterone and makes it available in high concentrations in the seminif-erous tubules for spermatogenesis. FSH also controls estrogen synthesis by the Sertoli cells by stimulating the aromatization of testosterone that is produced by the Leydig cells. Most readers will be surprised to learn that the testicle produces a large amount of estrogen. In fact in the stallion, the testicle produces more estrogen than testosterone. That is why a total estrogen assay is so useful in diagnosing cryptorchidism (un-descended testicles) in stallions.

It is also known that FSH is an impor-tant stimulus for the early phases of spermatogenesis in mammals.

Prolactin Prolactin is secreted by the adeno-hypophysis.

We have already mentioned that pro-lactin is unique in terms of the fact that its secretion is controlled mostly by negative means via dopamine secretion, rather than by releasing hormones.

Prolactin is a long-chain peptide molecule that has a complex structure and is not synthesized for clinical use. Instead, endog-enous prolactin is stimulated through

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the use dopamine antagonists such as metaclopramide (Figure C1.4).

Prolactin plays an important role in maintenance of the corpus luteum, mustelids, pandas and numerous other wild and lab species. This is also true of the domestic bitch. If one suppresses prolactin produc-tion in bitches (ergot alkaloids can be used for this) abortion ensues. The role of prolactin in the queen is probably similar to that in bitches but this has not been well studied. The role of prolactin in the larger domes-tic species is also unclear but it is important in galactogenesis (the ini-tiation of lactation) in cows although it seems relatively unimportant in galactopoeisis (the maintenance of lactation). We know that it is also important in galactogenesis in mares because certain plant toxins that act like dopamine (e.g. tall fescue endo-phytic fungal poisoning) suppress mammary development in mares altogether.

Serum prolactin is invariably elevated in the suckled dam and suckling usu-ally causes the release of prolactin from the pituitary gland therefore suffice to say, prolactin must play an important general role in lactation.

Interestingly, prolactin is also asso-ciated with failure of ovulation. In stressed women (ballet dancers, marathoners etc.) and in lactating ewes in springtime, there are often high serum prolactin concentrations while the woman or ewe fails to have ovulatory cycles. Also intrigu-ing is the observation that serum endorphin concentrations are often elevated in parallel to prolactin con-centrations. Therefore there is link between prolactin and the opioids. In fact, endorphins and enkephalins cause an increase in serum prolactin concentrations through their ability to suppress the release of dopamine (dopamine comes into the picture

again!) and it is well known that endogenous opioids are elevated in these individuals. As mentioned earlier, opioids are associated with the so called “runners high” and may therefore be a root cause of the fail-ure of ovulation in these individuals. You will see later, that the opioids also have a direct suppressive effect on GnRH secretion.

Sometimes it is only necessary to suppress prolactin secretion to initiate ovulation, sometimes only endorphins. In many cases one may be suppressing one of these partners ovarian inactivity while unwittingly suppressing the other.

In summary, there is a complex inter-relationship between prolactin, the opioids and failure of ovulation.

Prolactin has a structure that is simi-lar to growth hormone and to pla-cental lactogen (a placental hormone with growth hormone and prolactin-like activity) therefore these hor-mones share similar immunological and physiological characteristics. Although important physiologically, prolactin is never used as a clinical hormone treatment.

OxytocinOxytocin is produced mainly in the hypothalamus and is transported into the posterior pituitary gland (neurohypophysis) via a neuronal transport system. To date, no releas-

Fig C1.5 Oxytocin; a simple and inexpensive molecule to manufacture. Note that its stability permits it to be stored in liquid form.

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ing hormone for oxytocin has been identified but it has recently been shown that its secretion is episodic like that of the releasing hormones. For example, in mares, secretion pulses occur about once every two hours.

Oxytocin is also produced by corpora lutea in the ovaries during the second half of the estrous cycle in most farm animals! You will see later that endometrial oxytocin is important for regulating the estrous cycle.

The name of the hormone reveals its principal function i.e. Oxy = fast, tocos = childbirth. Oxytocin hastens the process of birth via a direct effect on the myometrium and by releasing prostaglandins F2 and E2 from the endometrium as well. Oxytocin also causes contraction of the myoepi-thelial cells of the mammary gland, promoting milk letdown.

Human studies also suggest that oxytocin levels are elevated by mas-sage and intimacy and are implicated in maternal offspring relationships.Recent studies have shown that the corpus luteum of non-pregnant ruminants and horses contains oxy-tocin. As alluded to earlier, oxytocin plays an important part in luteolysis because it propagates the release of prostaglandins from the uterus and thereby ensures the completion of luteolysis. In this regard, when pus accumulates in the uterus (pyome-tra; a common find in the postpartum cow) and luteolysis fails to occur, it is interesting to note that the cor-pora lutea of the cow contains very little oxytocin. Along the same lines, the oxytocin content of the corpus luteum drops to low levels once preg-nancy has been established.

Oxytocin also causes contraction of the myoepithelial cells of the mam-mary gland, promoting milk letdown.For many years it was thought that

oxytocin could only have an effect on a uterus that had been primed with estrogen but this is not true. In ovariectomized bitches for example, estrogenic priming does not alter the effect of oxytocin on myometrial con-traction and women will also attest to the fact that there is significant and painful contraction of the uterus following oxytocin treatment, even weeks after childbirth when serum estrogen concentrations have fallen to basal levels. Oxytocin is also used every day to expel uterine fluid in old mares; from clinical experience we know that it works well, even when the uterus is not under an estrogenic influence.

Because of it has only nine amino acids, oxytocin has been artificially synthesized and oxytocin analogs are available. It is also quite stable and very inexpensive to make. A very useful hormone indeed!

HORMONES OF THE PINEAL GLAND

Melatonin Melatonin is the only hormone we will discuss under this heading. It is derived (via serotonin, also known as 5-hydroxytryptamine) from the amino acid tryptophan. Like other compounds that are derived from the breakdown of tryptophan melatonin is known as an indole (the smell of feces is due to indoles).

Although melatonin from cattle will cause skin blanching in frogs, the pineal gland in cattle and other domestic species do not have any known connection to skin color. How-ever, like its role in lower vertebrates, the pineal gland regulates seasonal cyclicity in farm animals. It does this via a rather remarkable nervous pathway that involves the retina, the inferior accessory optic tract and the

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cranial sympathetic ganglion in the neck.

Why is the pineal control pathway remarkable? See if you don’t agree after reading this: After passing along the inferior accessory optic tract (adjacent to the sight pathway) the signals travel down the neck of the animal and reach the sympa-thetic ganglion at the base of the neck. From here, impulses cross onto the sympathetic part of the vago-sympathetic trunk and returns to the cranium by piggy-backing on the blood vessels that eventually supply the pineal. Surely someone deserved a PhD for that work!

Seasonal day length variations affect the estrous cycles of the domestic animals via this cervical-cranial path-way. If the cranial cervical sympa-

thetic ganglion is destroyed or the pineal gland is removed in species that have breeding seasons, the animal will usually continue to cycle but will no longer occur in concert with the seasons. It is remarkable and indeed con-fusing, to find that the production of melatonin by the pineal gland increases when an animal is exposed

to darkness, irrespective of whether the animal has estrous cycles in the fall or the springtime! Therefore, in animals that breed when the day length is long (such as horses) mela-tonin has a suppressive effect on cyclicity but in animals that breed when the day length is short (such as sheep and goats) melatonin will stimulate cyclicity.

From a practical standpoint this knowledge is important because melatonin has become readily avail-able as a by-product of coffee de-caffeination and it is being used in commercial sheep production (Figure C1.6) It is also common to use vari-ous lighting treatments to control the reproduction of seasonal breeders, especially horses. These treatments are discussed in another course.

OVARIAN, PLACENTAL AND ADRENAL HORMONES

Inhibin There are usually many tertiary folli-cles (follicle with cavi-ties or antrums) on the ovaries so why is it that only one or two follicles ovu-late in most animals? The primary reason for this is that the mature follicle pro-duces a protein hor-mone called inhibin.

Inhibin has a profound and direct suppressive effect on FSH secretion. Therefore inhibin decreases the pos-sibility of multiple ovulations. This leads one to ask if dogs, cats and pigs produce less inhibin than cows, sheep and horses. The answer is unknown. However, it was recently demonstrated that the Baroola Merino (a sheep breed known for

OPAH

IITmctti

Fig C1.6 MELOVINE® (also marketed as REGULIN®) is a 4 mm-long subcutaneous implant inserted into the ear. It releases melatonin for a period of 70 to 90 days to simulate the shortening of the days. The implant is positioned at the base of the ear and does not need to be removed i.e. it is biodegradable. In ewes, It induces estrus and ovarian activity 50 to 70 days after it is implanted.

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its very high ovulation rate) does in fact have very low levels of inhibin! There is no doubt that inhibin could be vastly superior to estradiol for fol-licle control (see above) but this has not yet been demonstrated.

Inhibin consists of two major sub-units joined together (rather like insulin like two rulers {Sub unit and sub unit } overlapping one another. The glue between the two “rulers” is a series of disulfide bonds. There are several forms of inhibin

(Figure C1.7) so it is important to request an assay for the form of inhibin that you are interested in. In veterinary medicine, we commonly use inhibin assays to diagnose the presence of granulosa cell tumors in mares (Figure C1.7) and because

the subunit seems to be produced most consistently by these tumors, we request that inhibin assay. Interestingly, if one combines two sub units of inhibin, another hormone called “activin” is formed; unlike inhibin, it actually stimulates the secretion of FSH! Relatively little is known about activin.

Because of its size and complexity, inhibin is not available for clinical use. The closest we can come to the activity of inhibin in clinical use, is

through GnRH down-regulation (discussed elsewhere).

The sex steroids All steroids are small molecules, stable and easy to manufacture. Because of this, there are often hundreds of analogs of each type of the native steroids. They are discussed under the headings: Androgens, Progestagens and Estrogens.

● AndrogensMany of the functions of tes-tosterone (an androgen) are well known and include mascu-linization, anabolic effects and increased libido. Less obvious effects include the maintenance of spermatogenesis, stimulation of the growth and function of the accessory sex glands, and break-down of the penile frenulum at puberty.

At many peripheral sites, tes-tosterone is converted to dihy-droxytestosterone (DHT), a highly active androgenic metab-olite. This occurs under the action of the enzyme 5- reduc-

tase. Many tissues only have receptor sites for DHT and not testosterone itself. This is significant because a lack of 5-reductase will make the tissues unable to respond to circu-lating testosterone making the indi-vidual appear phenotypically female

t(

TTAsBhodAE

●MtwlieooaFig C1.7 This image shows that there are at least two forms of inhibin and

three of activin. We are most interested in inhibin therefor activin is greyed-out here. It turns out that the alpha sub-unit of inhibin is most consistently produced by granulosa cell tumors in mares; more than the alpha,betaA. That is why alpha,betaA is not colored orange here. In this illustrations, the color orange indicates (the arrow and rings) marks the principle effect and forms that we are interested in. Note how inhibin production by the tumor has inhibited FSH and caused the contralateral ovary to shrink. This is easily felt on rectal palpation.

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even though they have testicles and an X and Y chromosome! This “male feminizing syndrome” is seen in both humans and domestic animals.

Other androgens have similar activi-ties to testosterone.

Some androgens are aromatized to estrogens in the placenta (and peripheral fat tissue which is rich in aromatase enzymes). Androgen aromatization in the placenta is a hallmark of pregnancy and in wild ruminants and horses, it is often used to detect pregnancy. These estrogens may also be important in mammary gland development. Androgens that are aromatized to estrogens may also be important in female sexual behav-ior. In fact, serum androgen levels have been linked to libido in women. This may also be the case in some domestic animals because a mare will often show estrous behavior soon after an injection of testosterone!

Testosterone and other androgens are also produced by the adrenals under the control of ACTH.

● ProgestagensProgesterone (< pro = for, gest = gestation) is a progestagen.This hormone is of importance in the maintenance of pregnancy, hence its name. It stimulates endometrial glands to hypertrophy and produce embryotroph. Progesterone is pro-duced by the corpus luteum and the placenta. The adrenal gland also produces some progesterone but its contribution is a relatively insignifi-cant by-product of cortisol synthesis.

Progesterone from the corpus luteum is responsible for suppressing GnRH secretion, and hence, LH secretion (it has little effect on FSH secretion) by the pituitary gland. It also primes the behavioral centres in the brain so that estrous behavior is properly elicited. It is ironic that progesterone

is essential for priming sexual behav-ior in some species, yet it can block sexual receptivity at other times. For example, an elevation of serum progesterone a few days prior to ovu-lation is essential for normal sexual behavior in cows but if that same animal is treated with an appropriate dose of progesterone during estrous behavior, estrous behavior would be suppressed!

Because of the ability of progester-one to suppress estrous behavior & the LH surge and ovulation, proges-terone and various other progesta-gens (hormones with progesterone-like activity) are used for estrous synchronization and contraception in many mammals.

In concert with many other hor-mones, progesterone is also impor-tant in lactogenesis. This is why progesterone can be used for induc-ing lactation, together with estrogens and other hormones that release prolactin.

● EstrogensEstrogens such as estradiol 17, estrone and estriol are important for maintaining the female pheno-type and stimulating female sexual behavior. Estrogens are generally mitogenic and cause tissue with the appropriate receptor sites to undergo hyperplasia. This effect is obvious in the mammary gland, uterus, vaginal mucosa and vulva. The mitogenic effect of estrogens is striking during an examination of vaginal cytology from a bitch in estrus.

Together with prostaglandins E1 and E2 (see later) estrogens also cause cervical relaxation and increase the secretion of cervical and vaginal glands in preparation for copulation. The string of mucus seen hanging from the vulva of a cow during estrus is an effect of estrogens.

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Together with progesterone, estro-gens are also responsible for the remarkable uterine tone that is pal-pable in cows during estrus and in mares during early pregnancy.

The important role of estrogens and prolactin release in preparation for lactation has already been men-tioned.

The principal site of estrogen produc-tion in the non-pregnant female is the ovarian follicle. The most plenti-ful estrogen in the follicle is estra-diol 17. In pregnant mammals, the placenta produces estrone, estradiol 17 estriol and in mares, the unique estrogens equilin and equilenin. Most of the placental estrogen is the prod-uct of conversion from androgens supplied by the fetal adrenal.

Estrogens are excreted in such high levels in the urine of pregnant mares that they are reaped commercially and used as replacement therapy for post-menopausal women to restore mental acuity, skin texture, feminine vitality and to prevent osteoporosis (estrogens stimulate the production of thyrocalcitonin).

● Adrenal sex steroidsThe adrenal gland produces an array of steroid hormones. Some of these steroids are regarded as “sex hor-mones” i.e. androgens, estrogens and progestagens while others are not regarded as “sex hormones”, such as cortisol and other glucocorticoids. Yet all of these hormones act on the reproductive process to a greater or lesser degree. Therefore they are all really “sex hormones”. Interestingly, many adrenal hormones differ from non-adrenal hormones by only one or two double bonds, or an OH or methyl group. Therefore, it is not surprising to find that many of these hormones have profound effects on reproduction. For example, cortisol can block ovulation in cows, it is an

essential trigger in the birth process, and like corticosteroids, estrogens can cause Na+ and water retention. Therefore, the adrenal gland cannot be excluded from discussions on reproductive function.

PLACENTAL GONADOTRO-PINS

hCG In humans and primates, a placental hormone called chorionic gonadotro-pin (hCG in the case of humans) is produced during the first trimester of pregnancy. It is similar to LH in its activity but bears structural resem-blance to FSH and TSH as well.

Because of its LH-like activity, hCG is used in both human and veterinary medicine to induce ovulation.

In pregnant women and in primates, the role of hCG is not clear but it appears to stimulate gonadal and placental steroidogenesis and car-bohydrate metabolism as well. hCG is excreted in the urine of humans and primates and forms the basis of a convenient early pregnancy test in both of these species.

eCGEquine chorionic gonadotropin (eCG) (Figure C1.8) was once known as pregnant mare serum gonadotropin (PMSG) but is more correctly called eCG because it is specifically a chori-onic product. It is formed by the cells of the chorionic girdle between about 30 and 120 days of gestation (some-time quite a bit later) and is also the basis of a pregnancy test in mares. Unlike tests for hCG, most tests for eCG require the use of serum rather than urine because eCG does not appear in the urine in an unchanged form, recognized by common assays.

Because of its predominant FSH-like activity, eCG is used to stimulate fol-

Notes

13

licle development in super-ovulation programs for embryo transfer. In practise however, more FSH is used than eCG nowadays, because the eCG molecule contains a great deal of sialic acid. This lengthens its cir-culating half life and makes its fol-

licle stimulating activity difficult to control.oCG

An ovine chorionic gonadotropin (oCG) has also been isolated from the placenta of sheep and it appeared after only two weeks of gestation. It had LH-like properties like hCG but did not cross react with LH or hCG. When more is known about this hor-mone, it may become the basis of a pregnancy test for sheep. A similar chorionic gonadotropin (bCG) may exist in cows.

OTHER PLACENTAL HORMONES

Placental lactogens These are named PL or oPL, hPL, bPL,

etc. according to species in which it is found) These are called are poly-peptide hormones that have proper-ties in common with both growth hormone and prolactin i.e. placental lactogen has been shown to stimulate weight gain and epiphyseal growth (like growth hormone) and also to increase casein synthesis in mam-mary tissue (like prolactin) Although placental lactogens are found in higher concentrations in the serum of high producing cows than in low producing cows their function in domestic animals is largely unknown.

Placental lactogen forms the basis of an experimental pregnancy test in ewes but laboratories do not run this test on a commercial basis so it has no practical significance at this time.

Relaxin Relaxin is a polypeptide hormone similar in structure to insulin. It is produced by both the placenta and the corpus luteum. The corpus luteum is the major source of the hormone in pigs and is a valuable source of relaxin for investigational purposes. In other animals, relaxin is formed mostly by the placenta.

Relaxin causes relaxation of the cervix and pelvic ligaments in the last trimester of gestation by activating several enzymes, including collage-nase and proteoglycanase. Therefore it is not surprising then, to find that if the ovaries of a sow are removed, parturition is protracted and the inci-dence of stillbirth increases. In other domestic species that have been ovariectomized, this does not occur because the main source of relaxin in those species is the placenta.

Serum relaxin concentrations are elevated throughout pregnancy in the sow and are especially high at parturition but in most other animals,

Fig C1.8 Equinex, also sold as PREGNECOL® (Bioniche animal health) To help stimulate follicle development in functional ovaries of cows, ewes and sows. Strangely eCG is still referred to by its old name, pregnant mare’s serum gonadotropin (PMSG). This product, obtained from the serum of pregnant mares, has both LH- and FSH-like effects but is generally used for its strong FSH-like activity.

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14

relaxin is only elevated in the last few days of gestation. In the bitch however, relaxin concentrations start to rise quite early in gestation and after 30 days, form the basis for a pregnancy test (Figure C1.9).

During parturition, relaxin is released under the effects of both prostaglan-din F2 and oxytocin.

Like insulin, relaxin has not been synthesized for clinical use because it is a complex molecule that consists of 2 polypeptide chains linked by

disulfide bridges.

PROSTAGLANDINS

Prostaglandins (Figure C1.10) are often abbreviated as PG.

Prostaglandins occur throughout the body in many different forms but those which are most important in reproduction belong to the E and F series.

Prostaglandins are formed from the unsaturated fatty acid arachidonic acid. Arachidonic acid is released from intracellular phospholipid stores via the action of the enzyme phos-pholipase A2. This is worthy of men-tion because corticosteroids block

the action of phospholipase A2 and can therefore prevent prostaglandin synthesis and prostaglandin medi-ated inflammation.

Once arachidonic acid is liberated from the membrane phospholipid stores, it is converted to prostaglan-dins G, H, E and F and to throm-boxanes and prostacyclines as well. The conversion of arachidonic acid is mediated by the action of several enzymes, including cyclooxygenase. (COX). Aspirin and indomethacin block the activity of cyclooxygenase and as a result, can also block pros-taglandin synthesis.

Prostaglandins are important in many reproductive functions, including the release of GnRH, the processes of ovulation, early recognition of preg-nancy, luteolysis and parturition. Therefore it is not surprising to find that corticosteroids and indometha-cin can block ovulation and that aspirin and indomethacin can inhibit luteolysis and even delay parturition!

During early pregnancy, it is important that prostaglandin mediated luteolysis is blocked so that the CL can continue to function and support pregnancy.

When an animal falls pregnant, the fetoplacental unit produces “preg-nancy specific substances”, including steroids, sialic acid, -feto protein, glycoprotein hormones and many more. These substances act on the endometrium and prevent luteolysis by suppressing prostaglandin pro-duction. Alternatively, pregnancy specific hormones can cause pros-taglandins to be secreted into the uterine lumen rather than into the bloodstream, thereby preventing luteolysis. This is known to occur in pigs. The direction of prostaglandin synthesis may also change during pregnancy, so that the E series are produced instead of the F series.

Fig C1.9 The Witness test for canine pregnancy; based on the production of relaxin after 30 days in bitches and queens.

Notes

15

Prostaglandin E is luteotropic under some conditions while prostaglandin F is almost always luteolytic.

Prostaglandins play an important role in the “recognition” and main-tenance of pregnancy. In fact, in horses, it the production of PGE2 that signals to the uterine tube to allow the embryo to enter the uterus. Non fertilized oocytes don’t produce this prostaglandin so, in horses unfertil-ized oocytes don’t enter the uterus. This is very helpful when one is doing embryo transfer because there is not need to determine if the embryo one flushes from the uterus is a live embryo of just an unfertilized oocytes (one has to do this in cattle). very handy indeed!

In several mam-mals, it is currently believed that the one of the principal embryonic signalling proteins is inter-feron tau, which supp resses the maternal immune response locally and prevents rejection of the embryo. Other pregnancy specific proteins have also been discovered. One of these is used in the commercial and successful prod-uct “Biopryn™” By 30 days of preg-nancy, this protein is produced in suffi-cient amounts to be a reliable pregnancy test for cattle

In sheep, anti-luteolytic prostaglan-dins (PGE1, PGE2) are secreted into uterine venous blood from day eight through day 13 post breeding and chronic intrauterine infusions of PGE1

or PGE2 delay spontaneous luteoly-sis in un-mated ewes. In mares and pigs, the early embryo also produces estrogens and these also seem pre-vent luteolysis. (Interestingly, in other animals estrogens are most often luteolytic!)

Fig C1.10 Two forms of prostaglandin used in veterinary medicine. On top is the native hormone, prostaglandin F2 and below, a potent analog, fenprostalene. Note the package warning pertaining to pregnant women and asthmatics! This applies to all forms of prostaglandin used in veterinary medicine. The therapeutic index of Fenprostalene is low in dogs and cats. Prostaglandin F2 is the prostaglandin of choice in those species

Notes

16

Embryos that are delayed in their development or which secrete insuf-ficient signalling proteins, will fail be recognized by the mother. The CL will regress and consequently, the embryos will be lost when and the female returns to estrus.

From a clinical standpoint, prosta-glandins have become tremendously important in both human and veteri-nary medicine. In veterinary medi-cine, the F series and its analogs are used to cause luteolysis in cycling and pregnant animals (figure C1.10) and the E series (figure C1.11) are used for cervical relaxation prior to mechanical abortion or the removal of dead fetuses.

The exact role of prostaglandin F2 in luteolysis is still unknown but it appears to involve: 1) Direct inhibi-tion of steroidogenesis, 2) Starvation of the CL by constriction of luteal capillaries or 3) Inhibition of LH receptor sites.

The mechanism of luteolysis in pri-mates and women is poorly under-stood but is an entirely intra-ovarian event, not requiring the presence of the uterus. Unlike most domestic species, in the event of hysterec-

tomy, the ovarian cycle is not altered in women.

The supporting role of oxytocin in luteolysis has already been men-tioned.

THE ENDOCRINOLOGY OF PUBERTY

We still have a very poor understand-ing of puberty.

A current consensus of opinion sug-gests that the hypothalamus in the prepubertal animal is very sensitive to the negative feedback effects of gonadal steroids. As the reproductive system matures, increasing concen-

trations of gonadotropins stimulate the gonads to produce more steroids. These steroids then feed back on the hypothalamus which appears to become less and less sensitive to their suppressive effects. The hypo-thalamus also begins to respond pos-itively to the effects of estrogen. In the female it is one of the LH surges generated by this positive feedback that results in the first ovulation.

The first luteal phase after puberty is usually abbreviated, probably

Fig C1.11. Prostaglandin E 2. Prepidil® Upjohn. In veterinary medicine, this is occasionally used to dilate the cervix to assist one in the removal of mummifi ed fetuses in cattle. Although it works well to induce cervical dilation in women, its effect in cattle is less predictable.

Notes

17

because of a relative deficiency of LH, but the luteal phases soon become normal in duration.

From a practical standpoint, we nor-mally describe puberty as the first time that estrus and ovulation occur together, or in the male, the first time that a fertile ejaculate can be produced.

“THE BASIC CYCLE”

NomenclatureIn humans and non-human primates, reference is made to the menstrual cycle (< L. menses = month) but in dealing with domestic species, we refer to estrous cycles (oistros < Gr. meaning frenzy).

It should be noted that estrus is a noun and estrous is an adjective or adverb. In the same way, one refers to the menstrual cycle not the men-struation cycle, similarly, one uses the term estrous cycle not estrus cycle.

General information on the estrous cycle.

The pig and the cow are polyestrous which means that they cycle con-tinuously, irrespective of the season, but the ewe, doe, queen and mare are seasonally polyestrous which means that they only cycle during specific breeding seasons. Bitches are sometimes called monestrous animals because they only show estrus and ovulate once every 4 to 12 months. In wild canids estrus only occurs during the spring, so they are referred to as seasonally mones-trous animals.

Estrus is the period of time when the female animal is receptive to the sexual advances of the male animal. The duration of estrus can vary from several hours to several days, depending on the species. All of the domestic species ovulate while they

are in estrus except for the cow. She ovulates about 12 hours after estrus has ended.

Quite logically, the period just before estrus is called proestrus and the period just after estrus is called met-estrus (<meta < Gr. meaning after or beyond).

There is only one domestic species that has a distinct and protracted proestral period and that is the bitch. In the bitch, proestrus can last for 10 days or more. In other animals, behavioral changes only occur sev-eral hours before estrus. Usually therefore, the signs of proestrus are indistinct.

Estrous behavior can be quite differ-ent from one species to another but two behavioral characteristics are common to all domestic and wild spe-cies that are in estrus: 1. The female tends to seek out the male and 2. The female always remains immobile for mounting. In fact, immobility is one of the greatest stimuli for any male animal to mount and copulate. This is why phantoms (dummy females) or even immobilized males can be used as mounts when semen is to be collected.

Homosexual behavior is seen in all the domestic species but is generally rare in animals other that cattle and pigs. In those two species, it is more common and consistent in cattle than in pigs. Homosexual behavior can mislead those who are trying to detect estrus. Generally speaking, the females who are mounting other females are sometimes in proestrus but more often the stage of the cycle is uncertain. By contrast, the females allowing mounting are almost always in estrus. In some cases, mounting can be a display of dominance rather than something that is specifically sexual. This may occurs between two males or two females. In wild stal-

Notes

18

lions for example, mounting behavior is common as the herd sires establish a hierarchy of dominance.

After ovulation, the new CL starts to produce progesterone and this brings behavioral estrus to a halt. In the dog and cat, the corpora lutea take approximately two to three weeks to become fully functional but in most other species, maximum progester-one production is reached as early as 6 or 7 days after ovulation.

Although metestrus is loosely defined as “the period shortly after estrus” in behavioral terms, it is sometimes also defined as “the time during which the CL is still forming” or “the time during which the forming CL is not yet susceptible to therapeutic luteolysis caused by prostaglandins”. As a result of the last definition, metestrus is taken to be about 4 or 5 days long in most of the farm spe-cies because prostaglandin induced luteolysis starts to become effec-tive by the fourth or fifth day after ovulation. In actual fact, corpora lutea only becomes fully functional and predictably susceptible to pros-taglandins several days later than that. In dogs and cats metestrus is very long by this definition because exogenous prostaglandins do not cause luteolysis until at least 25 days after ovulation. Just for variety we find that in pigs, the corpora lutea are not susceptible to prostaglandins until shortly before the next estrous period. Therefore pigs have a “met-estral” period almost as long as the luteal phase!

From the discussion in the previous paragraph, it should be apparent that the limits of metestrus are different for different species and metestrus can not be defined both accurately and conveniently using a single state-ment for all domestic animals.

If an animal does not fall pregnant,

recognition of pregnancy does not occur. Therefore prostaglandin pro-duction by the endometrium is not blocked or modified (see recognition of pregnancy above). As a result, prostaglandin F2 destroys the corpus luteum and the animal returns to estrus.

Prostaglandin production by the uterus reaches the ovary via a local vascular pathway in ruminants, a systemic pathway in horses and by both routes in pigs. In ruminants and pigs, branches of the uterine vein that are very convoluted are closely apposed to the ovarian arter-ies. This special anatomy provides a route for the diffusion of prosta-glandin from the vein to the artery and is a therefore direct and quick link between the endometrium and the ovary in those species. This link does not exist in the horse therefore any prostaglandin that is produced by the endometrium is diluted in the systemic blood pool before it returns to the ovary. Nevertheless, the luteo-lytic mechanism still works perfectly in the horse, possibly because the CL of the horse is extremely sensitive to prostaglandins. This is born out by the clinical finding that much lower doses of prostaglandin are required for luteolysis in the horse than in the cow.

The day of ovulation is referred to as day 1 in domestic animals . It is not day “0” as if often seen in publications because there is of course, no such thing as day 0, only time 0. This is different from the nomenclature used for women where day 1 is the day that men-struation begins. In this regard some primates have light but overt menstruation (such as the chim-panzee) while others (such as the gorilla and orangutan) do not show visible menstruation. Therefore it is common in all non-human primates

Notes

19

to use genital swelling to detect the approximate time of ovulation.

The day of ovulation is the end of the estrous cycle in the domestic animals. After ovulation, another cycle begins.

In domestic species the term “follicu-lar phase” is sometimes used instead of proestrus and estrus but this is neither wise nor correct because large follicles can be found on the ovaries throughout the entire estrous cycle in all domestic species.

The term “follicular phase” has actu-ally been borrowed from human and primate physiology. After menstrua-tion rapid follicle growth occurs, this being referred to as the follicular phase. However recent data shows that women actually have two or more follicle waves during their men-strual cycles (like cattle and sheep) therefore the term may not be valid for humans either!

FERTILIZATION AND IMPLANTATION

The fertile life span of oocytes is about 12 to 24 hours after ovulation, depending on the species. However, spermatozoa have much longer life spans than this, once they are in the female tract and have become bound to membrane receptors or are free in parts of the uterine tube (fallopian tube). Sperm can remain in situ for many days, just waiting for a newly follicle to pass by. For example, it is known that mares and bitches will often conceive to a single insemination as long as 7 days before ovulation.

In fruit bats, poultry (and doubt-less, many other species) copula-tion and ovulation can be weeks or even months apart, still resulting in conception!

Despite these statements about sperm longevity, one should always strive to ensure that ovulation and insemination (natural or artificial) should occur as synchronously as possible. This is especially impor-tant in ruminants where asynchrony of greater than 12 hours between ovulation and insemination results in poor conception rates.

Fertilization actually occurs in the uterine tubes (probably in the region of the ampullary-isthmic junction) not the uterus.

Via a combination of peristalsis and cilial action in the uterine tube, the spermatozoon and oocyte are brought together for fertilization. The motility of the spermatozoon itself is probably more important in its final approach to the oocyte than in its transport from the cervix to the uter-ine tube. Travel through the uterus is mainly due to uterine contractions, usually getting sperm to the site of fertilization within minutes or even seconds after copulation or A.I.

Once fertilization has occurred, the zygote is transported to the uterus within 2 to 8 days, depending on the species. Pig embryos can be found in the uterus after only 2 to 2 ½ days

The fertile life span of oocytes is about 12 to 24 hours after ovulation, depend-ing on the species. However, spermatozoa have much longer life spans than this

Notes

20

and those of cats and ruminants somewhat later, after about 5 days. The embryo of a mare reaches her uterus after 5 to 6 days but in a bitch this process takes even longer, with estimates ranging from 6 to 8 days. In new world camelids, embryos can only be found in the uterus 8 days after the LH surge!

All of this information is clinically important because one must know when to flush the uterus or uterine tubes to collect embryos for embryo transfer. For example most bovine embryos are collected from the uterus, 6 to 7 days after ovulation.

In most of the domestic species, the embryos arrive in the uterus as mor-ulas with 4 to 16 cells but often they have become expanded blastocyst.

Most embryos reach the uterus, still surrounded by the thick, translucent zona pellucida, the “egg shell” around the oocyte. The embryos “hatch” several days later, the time being dependant on the species. However, in camelids, the embryos hatch while

they are still inside the uterine (fal-lopian tubes). Therefore one should not be concerned if llama or alpaca embryos look like flabby spheres or ovoid structures without zona pel-lucida!

As a general rule, recognition of pregnancy occurs 3 to 5 days before the end of the luteal phase, just in time to block endometrial prostaglan-din production. In horses, however it appears that pregnancy is recognized earlier than this because pregnant mares often maintain functional corpora lutea if embryos are flushed from their uteri 7 or 8 days after conception.

In dogs and cats, it is not known when pregnancy is recognized but recognition of pregnancy is not nec-essary for prolongation of the lifes-pan of the CL. Their luteal phases are very long even if pregnancy is not recognized.

Ruminants and pigs have elongated fetal membranes (figure C1.12) and pigs have multiple conceptuses, therefore it is easy to visualize how the signal for recognition of preg-nancy can reach most of the endo-metrial surface so that luteolysis can be prevented and pregnancy can continue. In the horse however (figure C1.12) the feto-placental unit resembles a marble and its fetal membranes cannot possibly make contact with all of the endometrium simultaneously. As a result, the equine embryo must migrate through the uterus, so that all of the endo-metrium can eventually receive the signal for recognition of pregnancy.

Embryo migration is evident on ultrasound examinations in mares showing the embryo is still migrat-ing around the uterus 14 to 15 days after conception. The embryo may be in one uterine horn when an exami-nation is first performed but several

Fig C1.12. Equine and bovine embryos of the same age; about 13 to 14 days. The equine embryo (above) is spherical and can easily be seen on ultrasonography at this age. However, the bovine embryo fl attens easily between the folds of the endometrium and therefore its cross section is diffi cult to detect. It is only visible on ultrasound at about 22 to 23 days of age. As discussed in the text, an equine embryo of this age will still be moving within the lumen for another day or two.

Notes

21

minutes later, it may be in the uterine body or at the tip of the other horn! We see this every day in equine studs because pregnancy examinations are usually done 14 to 16 days after ovu-lation. By day 16 or 17, the equine embryo stops moving.

It has been shown that canine and feline embryos also migrate around the uterus before implantation but this does not seem to be an essen-tial part of recognition of pregnancy. Instead, intra-uterine migration in dogs and cats simply appears to be a method of distributing embryos evenly between both uterine horns. The same phenomenon has been shown to occur in pigs and is proba-bly common to all polytocous species.

Implantation is a nebulous concept because it is gradual and has no obvi-ous beginning or end. Implantation begins earliest in those species that have the most intimate placenta-tion. For example, in primates and humans, species that have hemo-endothelial placentation, the embryo starts to invade the endometrium within a week of ovulation. Note that “hemo-endothelial” refers to a his-tological classification of placentation in which the intimacy of placenta-tion is described by mentioning the tissues of the dam (first) and the fetus (second) that are in contact with one another. In this case for example, the blood of the dam is in contact with the endothelium of the blood vessels of the chorion. In the dog (endotheliochorial placentation) implantation is well under way by 13 or 14 days while pig and ruminant embryos (epitheliochorial placen-tation) only implant by about one month after conception.

Placental attachment is still not intimate enough in either the horse or the cow to result in retained pla-centa (after abortions) until after the fourth month of gestation. After that,

retained placenta is fairly common, especially in the cow.

Horse embryos (also epitheliocho-rial placentation) take a long time to implant. Even at 2 months of age, they are still not firmly attached to the endometrium; the fetoplacental unit can easily be removed from the uterus. This is possible in spite of the advanced development of diffuse micro-cotyledonous placentation that typifies the horse. It is only at 70 to 100 days that the placenta becomes firmly attached to the endometrium.

A quick reminder that the ruminants have a multiple complex (multiples) type of placenta, dogs and cats a zonary form of placenta and cam-elids, pigs and horses diffuse form of placentation (figure 1.13)

THE ENDOCRINOLOGY OF PREGNANCY

In bitches, the lifespan of the corpus luteum is similar whether the bitch is pregnant or not but in other species (even the cat) the life of the corpus luteum is normally extended only when pregnancy is recognized.

In some animals such as pigs, goats, bitches and probably the cat, corpora lutea are required throughout preg-nancy, but in this is not so in cows where the ovaries can be removed at 150 days. The same is possible in mares at 100 days and in ewes at 55 days of gestation, all without disrupting pregnancy. Therefore, the placenta can maintain pregnancy in some animals but not in others. These data are important because many clinical procedures and tests are based on this knowledge.

In women and primates, ovariec-tomy can be performed very early in pregnancy.

Notes

22

Fig C1.13. Gross placentation of domestic species. Placenta multiplex of ruminants in a sheep (A), bovine conceptus (B) and the uterine wall of a neonatal calf (C) showing how the placentation anatomy has already been formed at birth. D and E show the zonary placentation of dogs and cats respectively while F, G and H are the diffuse placentas of pigs, horses and camelids respectively.

Notes

23

It is well known that LH and FSH control ovarian steroidogenesis but what controls placental steroidogen-esis? hCG performs this function in humans and primates but the main-tenance of placental steroidogenesis in the farm species is less certain.

In species that need corpora lutea to maintain pregnancy (pigs, goats, bitches etc.) the pituitary gland appears to maintain pregnancy by producing LH, albeit in low concen-trations during pregnancy. For exam-ple, if the pituitary gland is removed in the pig, pregnancy will fail but this is not so in the ewe, where the CL is not required during late pregnancy. In other words, the placentas of the ewe, cow and mare appear to have their own mechanisms for control-ling steroid synthesis and do not depend on pituitary gonadotropin in advanced pregnancy!

In all the farm species, the placenta starts to produce estrogens during the first trimester of gestation. The principal form of estrogen in these species is estrone and without excep-tion, tests for estrone or its conju-gates, estrone sulfate or estrone glucuronide in serum, milk or urine can be used to confirm pregnancy and to determine the viability of the conceptus. For the sake of reference, tests for estrone will be positive and reliable as early as 25 days in pigs, 50 days in the small ruminants and 90 days in the horse and cow.

In all domestic species (except the mare) serum estrogen concentra-tions continue to rise, and increase remarkably towards the end of pregnancy. This stimulates mam-mary development and as discussed below, appears to prepare the dam and conceptus for parturition.

Generally, serum progesterone con-centrations fall just before birth. In mares however, there is a short-live

rise in serum progesterone con-centrations that occurs in the last few days before foaling. However, progesterone does actually fall to baseline concentration by the time foaling occurs. The source of the pro-gesterone is not certain but it is prob-ably of adrenal origin, perhaps even mediated as a fetal stress response to impending parturition.

THE PHYSIOLOGY OF PARTURITION

Many studies on parturition have been performed on sheep (figure C1.14) and lab animals and the find-ings have been extrapolated to the other species. It is not known what triggers parturition, except that the fetus itself initiates the process. Experimentally, fetal starvation or hypoxia can trigger birth.

In fetal lambs there is a dramatic increase in the production of fetal corticotropic hormone (CRH) and hence, ACTH, during the last two weeks of gestation. This causes an equally remarkable enlargement of the fetal adrenal cortex. (ACTH is a large molecule and cannot cross the placenta.) These changes cause an increase in fetal serum cortisol and androgen concentrations. The rise in fetal cortisol production changes pla-cental steroidogenesis by stimulating the activity of the enzyme system responsible for estrogen production and steroidogenesis turns towards estrogen synthesis and away from progesterone synthesis. Interest-ingly, the androgens from the fetal adrenal provide a substrate for estro-gen synthesis, are rapidly aromatized into estrogens by the placenta and contribute to the rising estrogen concentrations.

Increasing estrogen concentrations stimulate the release of cotyledonary and inter-cotyledonary prostaglan-

Notes

24

din F2 and E2 and this stimulates myometrial contraction. Increased myometrial activity may start several weeks before parturition but when it first becomes remarkable and cervi-cal dilation begins, the first stage of parturition has begun.

Another function that the adrenal performs during birth is the produc-tion of corticosteroids for lung matu-ration. Corticosteroids are important in the maturation of the fetal lung because they stimulate the produc-tion of lethicin, an alveolar surfactant which is essential for the neonatal pulmonary function.

The action of prostaglandins on the myometrium is potentiated by

declining serum progesterone con-centrations but in addition to causing uterine contraction, prostaglandins also release oxytocin from the neuro-hypophysis and this helps to advance the fetus toward the cervix. The combined effects of prostaglandin E2, E1 and relaxin (all coming from the placenta) cause initial dilation of the cervix but fetal pressure on the cervix also causes it to dilate.

Relaxin and serotonin (found in high concentrations in fetal serum at this time) also cause the release of collagenase from sources such as fibroblasts, leukocytes and myome-trial cells themselves. Collagenase changes the collagen structure of the cervix and facilitates cervical dilation.

Fig.C1.14 The cascade of events leading to parturition in sheep. This model is sometimes used as a model for other species as well (not mares). Briefl y, the origin of the cascade is in the FETAL hypothalamus, causing the release of fetal ACTH, and in turn, cortisol. Cortisol alters placental steroidogenesis stimulating estrogen production and causing progestagen production to decline. Estrogen mediates prostaglandin production by the placenta and this effects on the myometrium contraction (PGF) and cervical relaxation (PGE 1 and PGE2). Relaxin from the placenta relaxes the reproductive tract adnexa and also assists in cervical relaxation. The fi nal expulsion of the fetus is mediated by oxytocin, released from the neurohypophysis. Note that the production of cortisol is also essential to activate surfactants in the fetal lings

Notes

25

This collagenase is also important in allowing the release of the placenta from the endometrium

On the day of parturition when pas-sive cervical dilation is complete, the fetus starts to expand the cervix and the cranial vagina. When the cervix is fully dilated and the cranial vagina begins to stretch, a massive amount of oxytocin is released from the posterior pituitary. This is known as Ferguson’s reflex and it makes the uterus push the fetus into the cranial vagina. The dam senses the expansion of her vagina and experi-ences an almost uncontrollable urge to expel the fetus by contracting her abdominal muscles. This is called an “abdominal press” and it marks the onset of the expulsive or second stage of parturition.

The expulsion of the fetal membranes is called the third stage of parturition. As noted earlier, fetal serum collage-nase activity increases towards par-turition. In the placenta, collagenase loosens the connections between the superficial epithelium of the placenta and the submucosa on both the maternal and fetal side and probably at that level at least, facilitates pla-cental separation. Other proteolytic enzymes dissolve the proteinaceous interface between the tissue of the fetus and dam.

Optimal maturation of the placental epithelium is obviously dependant on good nutrition. Therefore, when factors such as vitamin E, vitamin A and selenium are lacking in the diet, it is not surprising to find that the placenta is not expelled normally (retained placenta). When myome-trial contraction is compromised by dystocia or large fetuses, failure of placental expulsion is also quite common.

In all mammals, the placenta is expelled within 12 hours of birth. The

uterus contracts strongly even after the placenta has been expelled and it continues to do so, although with diminishing strength and frequency, for the next 7 to 21 days. During this time, placental debris called lochia are expelled from the uterus and the uterus returns to its normal non-gravid state in a process called involution. The cow is also used as a model for a detailed discussion of uterine involution.

Note that the mare is an “odd man out” in terms of our under-standing of parturition. This is because there is no apparent rise in estrogens or corticosteroids shortly before foaling. It is no wonder then that corticosteroids work well to induce parturition in ruminants but seem to be clinically useless in mares! Add to this the finding that progesterone concentrations RISE shortly before foaling and you have another fact to baffle you about this species!

Autonomic effects The autonomic nervous system has some control over parturition.

Beta-2 sympathetic stimulation causes uterine relaxation and passive cervical dilation. This effect manifests itself in nervous and wild animals that are in the process of birth. When the female is disturbed, sympathetic stimulation follows and parturition is inhibited. See figure C1.15.

In obstetrics, it is common to use beta 2 agonists to prevent premature delivery or to inhibit uterine contrac-tions.

By contrast, parasympathetic stimu-lation causes uterine contraction and propagates birth. That is why neostigmine is occasionally used stimulate parturition in pigs.

From the fore going, one can see that

Notes

26

variations in sympathetic or para-sympathetic tone can affect the rate of parturition. However, it is not clear how the autonomic nervous system interacts with hormones to control of parturition.

In summary: Knowledge on the physiology of parturition and the role of estrogens, corticosteroids, prosta-glandins and autonomic agents leads us to use all these factors to induce or control parturition in various domes-tic species. These procedures are discussed in detail in the course on theriogenology.

THE RETURN TO CYCLICITY AND FERTILITY AFTER PARTURITION

The return to cyclicity occurs at dif-ferent times during the postpartum period in different species and is mainly dependant on whether or not the neonate is suckled and whether or not the species in question nor-mally cycles during the season in which it has just given birth. These factors are discussed in detail in the

sections that are devoted to specific animals.

It is remarkable that mares and cats, despite being suckled return to cyclicity soon after parturition.

The return to fertility (not cyclicity) is largely dependant on the intimacy and complexity of placentation in a particular species, with those species that have complex and intimate pla-centation usually taking the longest time to return to fertility. A remark-able exception to this statement is the cat. It has very intimate placentation yet it can return to fertility within 7 to 10 days of having kittens! Once again, these facts receive adequate discussion in other sections.

Fig. C1.15. A wild moose giving birth in Alaska. Ask yourself: “Would this moose cow give birth if a wolf was standing by?” The answer of course is NO. What physiological mechanism inhibits birth in such cases? Image by permission of Mark McDermott 2010. Anchorage, Alaska.

Notes

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