CHAPTER 10 Control and Synchronization of the Estrous Cycle and Ovulation
Accurate and predictable synchronization of estrus and ovulation is vital to equine breeding. The objectives of controlling the reproductive cycle of mares during the breeding season center on the ability to induce a fertile estrus and ovulation.
Synchronization of estrus has been used successfully as a management tool in the cattle industry. In cows, estrus and ovulation are simultaneously synchronized by hormonal therapies because ovulation predictably occurs shortly after the end of estrus (see Chapter 38). In contrast, synchronization of the mare’s estrus and ovulation is more difficult.
The mare’s estrus is relatively long, averaging 5 to 7 days; the maturation of the preovulatory follicle until ovulation takes 36 to 48 hours to complete. In addition, ovulation cannot be predicted from the first day of estrus as in cows. Actually, in the mare, behavioral signs of estrus typically cease within 2 days after ovulation; thus, the presumptive day of ovulation can only be determined retrospectively. Even though many effective methods have been developed for inducing estrus with a reasonable degree of synchronization, hormonal therapies that predictably induce estrus and ovulation are limited. In the United States, very few drugs have been approved for control of the equine estrous cycle. Because many horse breed associations are now accepting the use of frozen and shipped cooled semen in artificial insemination programs, the development of synchronization protocols that would allow a single breeding at a predetermined time remains a challenge for veterinarians and a great expectation for horse breeders.
The luteal phase of the estrous cycle can be shortened by treatment with PGF2α and its analogues.1 PGF2α was shown to be luteolytic in mares in 1972 and since then has been widely used in breeding farms that require intensive management of broodmares and stallions and mares.2 The corpus luteum is not responsive to one single injection of PGF2α until day 5 after ovulation, although daily administration of PGF2α for 2 or 3 days starting on day 3 after ovulation may cause luteolysis. In horse mares, 9 μg/kg of the PGF2α free acid equivalent to 1.2 μg of dinoprost tromethamine prostaglandin (Lutalyse, the only PGF2α approved for horses by the Food and Drug Administration [FDA]) has been shown to be luteolytic, but the manufacturer recommends the administration of 1 mg/100 lb (∼22 μg/kg). Recently, a dose as low as 0.5 mg of Lutalyse per horse mare (∼1.2 μg/kg) administered twice 24 hours apart caused luteolysis in 100% (21/21) of mares in the study without inducing well-known side effects (sweating, colic) associated with recommended dose.3
Other indications for administration of PGF2α include termination of unwanted pregnancies before and after formation of endometrial cups, termination of abnormal pregnancies such as trophoblastic vesicles without an embryo proper, and therapy for mares susceptible to persistent postmating endometritis. Induction of parturition with PGF2α is possible but not recommended because of an increased incidence of premature placental separation and decreased foal viability associated with its use.
Without a sound understanding of endocrinology and knowledge of the follicular and luteal structures present in the ovaries at the time of PGF2α administration, some of the clinical responses observed (such as the interval from treatment to display of estrus and time of ovulation) may be confusing to veterinarians not familiar with the mares’ reproductive physiology.
Prostaglandin F2α only has a significant effect when a mature CL is present.5 Maturity of a CL and hence its responsiveness to the action of PGF2α may vary considerably among individual mares. Although the CL of most mares will respond to PGF2α from the fourth day after ovulation, some will respond as early as the first day after ovulation, and others appear to be refractory.6 Studies in cows have shown that stage of diestrus7 and follicular status at the time of PGF2α administration8 have dramatic effects on the interval between PGF2α treatment and display of behavioral estrus. Large and small luteal cells have been shown to differ in their response to luteotrophic and luteolytic agents through regulation of PGF2α receptors.9 The mare’s CL has two distinct populations of luteal cells but it has been reported that the relative proportion of small to large luteal cells increases with the age of the CL during diestrus.10 The effect of this phenomenon on clinical response to treatment with PGF2α is currently unknown.
Follicular status also has an effect on the interval from treatment with PGF2α to the onset of estrus and ovulation.4 When a large follicle (30 to 40 mm or greater) is present at the time of PGF2α administration, a number of different clinical responses have been demonstrated.5 In most cases, mares come into estrus and ovulate within 6 days after PGF2α treatment,11 but in a few instances ovulation occurs within 24 to 72 hours and the mare shows few or no signs of estrus.12 In the latter situation, PGF2α may play a role in accelerating ovulation, because analogues of PGF2α have been shown to speed the ovulatory process.13 On occasion, the large diestrual follicles present at the time of PGF2α treatment may be atretic. Ovulation at the subsequent estrus is the result of growth and maturation of smaller follicles following regression of the larger one. In this situation, an increased amount of time is required for the mare to display signs of estrus and to ovulate.
Spontaneous diestrual ovulations occur in approximately 5% of estrous cycles and may account for apparent failure of luteolysis following treatment with PGF2α.4 If PGF2α is administered shortly before spontaneous ovulation of a large (greater than 35 mm) diestrual follicle, luteolysis of the primary CL may occur, but the CL that develops following spontaneous ovulation of the diestrous follicle (and increased progesterone concentrations) may mask the effects of lysis of the primary CL and the mare may not return to estrus as anticipated.
From this discussion it is clear that the use of PGF2α in mares may not be straightforward. Administration of PGF2α on days 6 to 9 after ovulation will result in the onset of estrus and subsequent ovulation within 3 to 4 days and 9 to 10 days, respectively, following treatment in most mares.5 It should be emphasized that the behavioral signs of estrus may not be observed and the interval between treatment and ovulation may range from 2 to 15 days.14
Appropriate adjustments in the management of individual nonpregnant mares may be required when treatment with PGF2α is anticipated. For example, careful examination of the ovaries by palpation per rectum and ultrasonography, noting the exact size, location, and texture of follicular structures prior to PGF2α treatment may alert the practitioner to the need to examine the mare more frequently so that short intervals between treatment and estrus and ovulation do not result in missed breeding opportunities.
During the physiologic breeding season when mares cycle regularly, the primary indication for extending the luteal phase is synchronization of estrus and ovulation for embryo transfer and for timed insemination. The administration of progesterone (or synthetic progestin) alone or in combination with estradiol-17β suppresses estrus behavior, thus mimicking an extended luteal phase for as long as the progestins are administered.14 Progesterone is also used alone to assist in the maintenance of pregnancy and to delay the onset of estrus (e.g., at foal heat). Because progesterone has an inhibitory effect on the release of LH from the anterior pituitary, the rationale is to artificially maintain an elevated concentration of progesterone until the corpora lutea (CL) in all treated animals have regressed. Estrus is delayed by progesterone administration until the onset is desired. Following withdrawal of progesterone, estrus and ovulation should occur at a predictable time. This method is generally successful for estrus synchronization in mares; however, progesterone, as with any other single hormone used to synchronize estrus and ovulation in mares, does not exert complete control over the physiologic events of the mare’s estrous cycle. The limiting factor is the degree to which spontaneous follicular growth and ovulation are suppressed.
Treatment with progesterone in oil (50 mg/day IM) initiated before estrus prevents the behavioral signs of estrus but does not prevent ovulation, whereas a dose of 100 mg/day blocks both estrus and ovulation.15 Neither dose is very effective if started after the first day of estrus. A dose of 200 mg/day is needed to suppress behavioral signs if a mare is in estrus at the beginning of treatment. Also, the dose required to inhibit ovulation once estrus has begun is higher than that required by mares in diestrus. Even the high endogenous concentrations of progesterone that are present during the midluteal phase of the estrous cycle are sometimes not sufficient to suppress gonadotropin release, follicular development, and ovulation. Administration of progesterone in oil (100 to 200 mg/day IM) for 7 to 10 days is followed by estrus 2 to 7 days after treatment ends. Daily injections of progesterone in oil may be painful, are not well tolerated by mares, and have the potential to cause seromas, abscesses, and fibrosis at the injection site.
In attempts to overcome the inconvenience of daily injections, other preparations and routes of administration have been tested; however, none are currently approved for use in horses and are commercially available only through veterinary compounding pharmacies. Intravaginal sponges impregnated with progesterone and synthetic progestins may have some degree of success in synchronizing estrus in mares.16 Unfortunately, sponges have the potential to adhere to the vaginal wall and induce necrotic vaginitis. Intravaginal devices that release progesterone slowly have been investigated in Europe and Australia and hold promise for future use. Microspheres containing progesterone and progesterone plus estradiol-17β have been injected to control or synchronize the estrous cycles of mares. Some long-acting preparations of progesterone plus estradiol were available by prescription from a pharmacy in Kentucky but the FDA recently seized compounded equine drugs. The FDA understood the pharmacy was illegally manufacturing drug products from bulk ingredients without FDA approval and selling them outside a valid veterinarian-client-patient relationship.
The only progestin approved by the FDA for use in horses is altrenogest (allyl-trenbolone*). Consequently, altrenogest (Regu-Mate) is the most extensively studied synthetic progestin. Altrenogest is widely used because of the convenience of oral administration, safety, and a common belief in its efficacy. Examples of efficacy include the following:
Lofstedt and Patel18 questioned the ability of altrenogest to control the equine estrous cycle. At the recommended dose and duration of treatment, altrenogest failed to suppress follicular development. Ovulation occurred in three of four mares when treatment was initiated during the first 3 days of estrus. Apparently the binding affinity of altrenogest for receptors in the hypothalamus is 60% that of progesterone. Also, LH is only slightly or not at all suppressed by altrenogest.16 Of the four mares that were treated during estrus in one study, the mean duration of estrus was 4.5 days. Nine of the 12 mares treated with altrenogest ovulated and formed CL during treatment. In four of the mares, the CL persisted between 2 and 10 days beyond the 15-day altrenogest treatment. To ensure closer synchrony, PGF2α should be administered at the end of the progestin therapy. Altrenogest or progesterone in oil given for 9 days in addition to an injection of PGF2α on day 9 or 10 of treatment with progestin has been shown to be an effective method of synchronizing estrus. Oral administration of altrenogest for 8 to 12 days with an injection of PGF2α on the last day of altrenogest treatment will result in most mares exhibiting estrus in 2 to 5 days after withdrawal. If any large follicles exist at the end of treatment, they can ovulate without the mare exhibiting signs of estrus, and these ovulations may be missed. However, not all mares with preovulatory follicles present at the end of treatment ovulate within 3 to 4 days. In one study, 4 of 12 preovulatory follicles remained until ovulation occurred 6 to 9 days later.17 Examination of the ovaries with transrectal ultrasonography at the end of the treatment period and administration of human chorionic gonadotropin (hCG) when a preovulatory follicle has reached 35 mm in diameter will help alleviate this problem and provide tighter synchrony of ovulation. During routine breeding management of the majority of mares at breeding operations, there are few indications for use of progestins to control the reproductive cycle.