III. ESTROUS CYCLE

1. OVERVIEW

   The estrous cycle of the horse is complex and variable. Individual horses generally differ from the "text book horse". The equine estrous cycle is most easily viewed as a multilayered process, with variation occurring at each level. We shall consider behavior as the outermost layer, then work inwards, describing in sequence follicular dynamics, and hormonal changes. We will also describe luteolysis, and finally the maternal recognition of pregnancy.

2. BEHAVIOR

   1. Teasing- Estrous behavior in the mare is a display to a stallion. Without a stallion, assessing behavioral estrus is difficult, if not impossible. Regular exposure allows the mare to familiarize herself with the stallion, and the observer to better read the individual mare. Monday, Wednesday, and Friday provides a convenient teasing schedule for most situations. Sometimes, it may be necessary to tease every day. In the absence of a stallion, mares may rarely show estrus to geldings or exceptionally to people. In all cases, regular observation and recording is essential.

   2. In heat- The textbook estrus mare tolerates and frankly encourages rude advances by the stallion. She squats, raises her tail, urinates, everts the clitoris (clitoral wink) and stands still, as the stallion eagerly calls, nibbles, licks, even bites or threatens her. As the stallion nibbles the stifles and hocks the mare may further tilt her pelvis. The posture of the estrous mare, with the rounded back (kyphosis) is unlike the estrous posture of other animals (eg cats, dogs, cattle, rodents) which arch their backs in the opposite direction (lordosis).

   3. Out of heat-The textbook out of heat mare (diestrus mare) is agitated by the approach of the stallion. She moves restlessly, pins her ears back, strikes, kicks, voices her objection, clamps her tail down, and will not urinate or evert the clitoris. She signals to the stallion her utter disgust at his approach.

   4. Cyclic variation- Cycles of estrous behavior are manifestations of the hormonal shifts deep in the mare. Followed closely they, more than any other parameter, reveal a summary of her endocrinologic state. During the ovulatory season, alternating periods of estrus and diestrus can be detected by conscientious teasing. Because follicular development is driven faster during extended daylight, horses display longer estrus during the spring and fall (7-10 days), and shorter estrus during the midsummer (4-5 days). Unlike the developing follicle, the corpus luteum is insensitive to daylength, and diestrus behavior is a consistent 14-15 days throughout the ovulatory season. Some studies have demonstrated slightly longer diestrus during midsummer (16 days) than spring or late fall days (13 days) but others have detected no differences.

   5. Mare variation- The individual nature of a mare pervades her sexual behavior. Some mares will shamelessly court the stallion, while others may accept him grudgingly, or passively, and yet others will persistently refuse him even in deep physiologic estrus. More importantly, some mares will display estrus for the expected 5-7 days, depending on the time of year, while others will only show estrous behavior for a day at most. "One day" mares may be missed in a Monday, Wednesday, Friday teasing program. As mares are coming into heat or going out of heat they may provide useful "mixed signals" of what to expect in the coming days. A twitch or lip chain may be necessary to induce an estrous display in some cases. Teasing mares regularly, and recording their responses diligently day-by-day, allows one to develop a feel for each individual mare. Many times this may make the difference between successful or unsuccessful breeding.

3. FOLLICULAR DYNAMICS

   The individuality of a mare is also reflected in follicular growth patterns which are quite unruly. As rules of thumb in a clinical setting, a large follicle can be present at any stage of the estrous cycle; ovulations usually occur 24-48 hours before the end of estrus, but may sometimes occur in diestrus. However, underlying this apparently haphazard follicular growth, researchers have discerned the following patterns.

   1. Follicular waves- Like the cow, follicular waves drive folliculogenesis in the horse. A follicular wave emerges and after about 3 days of growth the wave "dissociates" into the single growing dominant follicle which goes on to ovulate, and the smaller follicles which regress. According to this pattern, at about day 10 of diestrus a wave emerges that dissociates at the beginning of estrus, leading to ovulation about 5 days later of the dominant follicle 24-48 hours before the end of estrus. This wave is known as the primary wave and occurs in all normal equine estrous cycles. However, in addition to the primary wave, a secondary wave may emerge at the end of estrus, yielding a large dominant follicle during mid diestrus. This diestrous follicle may ovulate (diestrous ovulation). Diestrous ovulations occur in approximately 20% of all estrous cycles. Thus the horse presents at least 3 possible scenarios.

      1. Scenario 1 : Primary wave with ovulation in estrus.

      2. Scenario 2 : Additional secondary wave without ovulation in diestrus.

      3. Scenario 3 : Additional secondary wave with ovulation in diestrus.

      As an additional complication, late emerging secondary waves may yield dominant follicles that are still present early in estrus, either regressing or ovulating in estrus independently of the primary wave.

      Incidence of secondary waves and diestrous ovulations are relatively high in Thoroughbreds and Standardbreds, and relatively low in Quarter horses and ponies.

   2. Follicular changes prior to ovulation- In the pre-ovulatory period the dominant follicle undergoes characteristic changes. Diameter increases at about 3 mm/day and the wall appears to thicken slightly. As ovulation becomes imminent (within 24 hours) 85% of follicles develop a tear drop shape pointing towards the ovulation fossa. In general, follicles do not ovulate until at least 35mm in diameter. Average size of an ovulatory follicle is between 40-45mm. Arabians and double follicles may ovulate at smaller diameters (30 mm or greater). The follicle may palpably soften close to ovulation, being softer at 12 hours pre ovulation than at 72 hours pre ovulation. As the follicle becomes flatter and softer, it's point migrating towards the ovulation fossa, adjacent follicles may become more spherical. Evacuation of the follicular antrum takes about 60 seconds. Follicles ovulate at a smaller size in midsummer than at the beginning of the breeding season.

   3. Multiple ovulations- Mares sometimes ovulate 2 or more follicles during the same estrus. Synchronous ovulations occur on the same day, asynchronous ovulations are at least one day apart. Both follicles may originate from the primary wave, or one may originate from the secondary wave. Breed differences exist, with approximate incidence of multiple ovulations as follows: draft horses, 25%; Thoroughbreds, 20%; Standardbreds, 15%; Quarter Horses, 10%; Arabians, 2% and ponies less than 2%. Certain mares and mare families have increased rates of double ovulations. Incidence is 50% lower in foaling mares compared to barren or maiden mares. There may be an increased incidence during midsummer but the data is controversial. Incidence of triple ovulations is about 1%, and quadruple ovulation 0.05%. Double ovulations from the same ovary have a follicular diameter almost 10 mm smaller than a single ovulation. The interval between double ovulations can be as long as 6 days.

4. GONADOTROPIN RELEASING HORMONE (GNRH)

   1. Measurement- Levels of GnRH are too low to detect in peripheral blood. However, cannulation of the pituitary vein enables release of GnRH to be measured before it is diluted out in the general circulation.

   2. Pulsatile release- GnRH is released in pulses throughout the estrous cycle, each pulse being followed by a corresponding pulse of gonadotropins. At the time of onset of the LH surge, pulse frequency increases to about 1 pulse per 2 hours. At the time of ovulation, pulse frequency increases to 2 pulses every hour.

5. FSH AND LH

   1. LH- LH levels are low throughout most of diestrus, begin to increase a few days before the onset of estrus, reach a peak about one day after ovulation, then subside over the next 4-6 days. Thus LH profiles in the horse are distinctive in at least two ways:

      1. Rise in LH is gradual, not a "spike" as in other species

      2. LH peaks after ovulation.

      LH is responsible for maturation of the follicle and drives ovulation.

   2. FSH- FSH levels appear to be reciprocally related to LH levels. Overall, FSH is low through most of estrus, starts to rise about the time of ovulation, and is high during diestrus, and falls as estrus begins. There is some evidence for two FSH surges, one shortly after ovulation and another at about day 10 of diestrus. These may be responsible for the two waves of follicles sometimes seen. Overall, the details of equine FSH profiles in horses are complex, variable, and yet to be fully elucidated. FSH is believed to stimulate emergence of follicular waves.

   3. Synchrony and asynchrony- FSH and LH share the same releasing hormone (GnRH) so one would expect their levels to be more synchronous. Although clearly asynchronous during estrus, small LH pulses occur during diestrus which are synchronous with FSH pulses. These small LH pulses are necessary for maintenance of the corpus luteum, but may also contribute to diestrus ovulations.

6. ESTRADIOL, PROGESTERONE AND INHIBIN

   1. Estradiol- The mare synthesizes a variety of estrogens during the estrous cycle, estradiol 17b being the one most commonly measured. Estradiol begins to rise about 6 days prior to ovulation, close to the beginning of estrus. It peaks approximately 2 days before ovulation and then subsides. Throughout most of diestrus estradiol levels remain low. However, surges of estradiol in diestrus, at about day 6, have been suggested, linked to the emergence of secondary follicular waves.

   2. Progesterone- Levels of progesterone are low throughout estrus (< 1 ng/ml), but start rising between 12 and 24 hours post ovulation. Maximal levels are reached by days 5 to 7 post ovulation (4 - 22 ng/ml). Levels remained high until days 13-14 post ovulation. Thereafter they decline rapidly to the low values of estrus.

   3. Inhibin- Inhibin produced by the dominant follicle of estrous, suppresses FSH, preventing growth of additional follicles. Inhibin levels peak at the time of ovulation.

7. INTERRELATIONSHIPS

   1. Progesterone and FSH- Progesterone does not inhibit FSH release.

   2. Progesterone and LH- Progesterone inhibits release of LH.

   3. Estradiol and FSH- Estradiol inhibits release of FSH.

   4. Estradiol and LH - Estradiol stimulates release of LH.

   5. Inhibin and FSH- Inhibin inhibits release of FSH.

8. PROSTAGLANDINS AND LUTEOLYSIS

   Prostaglandin (PGF2a ) is released from the uterus at about day 14 post ovulation. Because of its short half life, studies commonly measure its metabolite, PGFM.

   1. Systemic pathway- The horse is unusual in that PGF released by the uterus reaches the ovary via the systemic circulation; a utero-ovarian counter current system as exists in the cow does not exist in the mare. Thus the equine corpus luteum is more sensitive to systemic administered PGF than other species.

   2. Action of PGF2a- PGF2a induces demise of the luteal cells and a precipitous drop in progesterone levels. The corpus luteum resists systemically administered prostaglandins until day 5 post ovulation.

   3. Mechanism of PGF2a release- PGF2a release is believed to require prolonged exposure to progesterone which may stimulate a prostaglandin synthetase enzyme, raising uterine prostaglandin levels. Estrogen stimulates release of PGF from the uterus directly, and possibly by increasing the uterine response to oxytocin. Oxytocin, released by the CL in response to PGF, is an important component of the luteolytic cascade in ruminants; oxytocin causes PGF release from the uterus and exogenous oxytocin is luteolytic in cattle. In horses, levels of circulating oxytocin are highest during luteolysis. However, it is not yet clear whether the CL is the source for oxytocin. In the mare, exogenous oxytocin is not luteolytic.

9. MATERNAL RECOGNITION OF PREGNANCY

   The early embryo blocks the luteolytic cascade of PGF release. Exactly how this is achieved is still unclear but the following facts appear to be pieces in the puzzle.

   1. Fetal mobility- The ability of the embryo to migrate throughout the uterus from days 10-14 is necessary to prevent PGF release.

   2. Estrogen release- The early equine embryo vigorously secretes estrogen. However, this does not appear to be the signal that prevents PGF release; in normal luteolysis estrogen promotes PGF release.

   3. EPSI molecule?- It has been hypothesized that the equine embryo secretes an endometrial prostaglandin synthetase inhibitor (EPSI).

   4. Glycoprotein capsule- The equine embryo is surrounded by a glycoprotein capsule whose function is still unclear. It has been suggested that its slippery surface promotes uterine mobility. The embryo ceases migration when the capsule is shed (approximately 16 days post ovulation).

10. SENESCENCE

    Most mares greater than 20 years will continue to cycle normally throughout the ovulatory season. However, a proportion (about 30%) will cease cycling during the ovulatory season. The mechanism underlying this senescence is unclear.