In the continuing effort to increase the fertility of horses there comes a new weapon--superovulation. Inducing a mare to ovulate multiple oocytes can translate into producing more foals from selected females. Assisted reproductive technology can help solve the problem of how to increase the number of foals from a valuable mare, or how to obtain a single foal from a subfertile mare.

Fertility in the mare begins with ovulation. The mare naturally ovulates one ovum (egg) per 21-day cycle. In a typical breeding season, February through July, this presents six to eight opportunities for ova to be fertilized. Compared to the mare's spontaneous (unassisted) ovulation, superovulation maximizes the fertilization opportunities by inducing the development and ovulation of a greater number of follicles. This method can optimize fertility--ova mature in follicles, so a greater number of follicles means more oocytes and an increased likelihood of conceiving more foals.

With a higher probability of establishing a pregnancy, superovulation might result in the fertilization of multiple oocytes. This gives the equine practitioner a possible opportunity to collect more than one embryo for implanting into donor mares via embryo transfer.

Research at Colorado State University (CSU) has recorded multiple ovulation pregnancy rates per donor at approximately twice those of mares which ovulate a single follicle each cycle. Patrick McCue, DVM, PhD, Diplomate ACT, Department of Clinical Sciences, has reported on these efforts to breed more foals from selected females. "Development of a superovulation technique that is successful, safe, and commercially available would revolutionize the equine breeding industry," said McCue.

Follicular Development

In the ovary, a follicle contains an ovum. Ovulation, or the event of a follicle releasing an ovum, marks the beginning of the 21-day estrous cycle. Follicular dynamics include one or two waves of follicular development. During each wave, several follicles usually develop simultaneously.

Early in the estrous cycle, the first wave of follicles begins development. These might regress, or a follicle might ovulate in the middle of diestrus (the mare's sexually inactive period
of her estrous cycle).

The anterior pituitary gland secretes hormones that stimulate follicular development. Follicle stimulating hormone (FSH) surges to start the development of smaller follicles in the second wave. In this phase, a single follicle eventually emerges as the dominant one. The other follicles in the second wave disintegrate.

Although FSH regulates follicular development and the rate of ovulation, the hormone inhibin constrains FSH secretion and therefore suppresses smaller follicles. Inhibin is produced by the dominant follicle. Researchers surmise that inhibin's effect on FSH might be what reduces FSH concentrations and causes smaller follicles, which depend on FSH for growth, to degenerate and resorb before they mature.

"Follicle development is a dynamic process of growth and regression," said McCue. "Ovulation will occur only in the presence of proper gonadal stimulation."

Superovulation stimulates the entire follicular wave. Those follicles that might have degenerated--or not have been selected to ovulate--will go ahead and ovulate in addition to the dominant follicle.

Superovulation has been researched in other livestock species, with cattle achieving more success than horses. The ovulation rates in cattle are much higher than with horses. McCue noted physiological reasons in the mare, such as that the mare must ovulate through a very restricted area in the ovary.

Assisting Follicular Production

Treating the mare with hormones can produce more follicles. Research in the last 20 years has studied different approaches using five hormones, with one recently emerging as the most successful.

Equine pituitary extract (EPE) has succeeded in producing multiple ovulations. This preparation consists of gonadotropins from the horse's pituitary gland. At CSU, gonadotropins are extracted and processed to yield the EPE compound. McCue defined the resulting EPE as containing "approximately 6-10% LH (luteinizing hormone) and 2-4% FSH."

He described the use of EPE to superovulate mares at CSU.

"The exact day of ovulation is determined and the mares are then short-cycled with prostaglandin (PGF2alpha) five days later and administered 30 mg. of EPE as an intramuscular injection once daily." Prostaglandin destroys the corpus luteum, and it starts the estrous cycle just prior to the beginning of the next follicular wave. EPE treatment lasts approximately six to eight days.

"When multiple follicles greater than 35 mm in diameter are detected, the mares receive 2500 IU human chorionic gonadotropin (hCG) intravenously to promote synchronous ovulations," said McCue. A mare is bred prior to ovulation and also when she ovulates, immediately after researchers detect ovulation.

Recent research has shown that EPE provides better multiple ovulation results than other hormone treatments. For example, researchers have studied the effects of gonadotropin-releasing hormone (GnRH), which helped improve ovulation rates through multiple ovulations in seasonally non-cycling mares. However, this hormone showed less-promising results on cycling mares.

Another approach has been immunization against inhibin, the hormone that controls FSH secretion. McCue's research showed that immunization against inhibin led to greater levels of FSH. Studies showed a doubling of ovulation rates, with one mare ovulating eight follicles in a cycle so that researchers recovered four viable embryos. However, this treatment required multiple immunizations over several weeks and led to some negative reactions at the immunization sites on the mares.

Researchers also tried FSH to stimulate the mare's ovulation. This resulted in an increase of 1.6 to 1.7 follicles per cycle. Unfortunately, the lack of a commercial equine FSH product restricts administration to overly expensive products of porcine origin. Mares might require a much higher dose of this hormone (70 times more) than their porcine counterparts, with a lower response of ovulation.

Another hormone, equine chorionic gonadotropin (eCG), has shown to exert no effect on the development of follicles in the mare. Successful in ruminants, this hormone was formerly called pregnant mare serum gonadotropin.

CSU Findings

At CSU, the results of superovulating 55 mares in the embryo transfer program in the 1994 and 1995 breeding seasons were an overall 2.4 ovulations per cycle, with a range from one to five. Of mares treated with EPE, 74% had multiple ovulations. Researchers elsewhere had reported as many as 10 ovulations in one mare due to EPE treatment.

Mares were bred with either shipped cooled semen or frozen-thawed semen, and pregnancies monitored with ultrasound. Results were double the number of embryos collected per donor (from the superovulated mares as compared to non-treated mares).

Other studies at CSU have investigated whether superovulating normal mares can lead to increased pregnancy rates in mares bred to a subfertile stallion. McCue noted that mares ovulating multiple follicles had a 67% pregnancy rate, compared with 33% for the mares with a single follicle. This result was for mares inseminated with a limited dose of frozen semen.

Inducing a larger number of follicles can improve the breeding success of the subfertile stallion. In this case, superovulation in animals follows successes in human medicine. More available follicles in the human female can often counterbalance the reduced fertility of the male. In situations of reduced numbers of sperm, women have a greater chance of becoming pregnant if they increase the development of follicles and ovulation rate.

Also, EPE can prove more successful in inducing multiple ovulations in the older mare, even those older than 20. A CSU pilot study reported pregnancy rates more than doubled with superovulation in a group of mares averaging 17.2 years of age. Of mares in an EPE-treated cycle, 31% became pregnant following EPE treatment, compared to only 13% of the same mares during a non-treated cycle.

McCue noted, "These studies support the hypothesis that increasing ovulation rate, and therefore increasing the number of oocytes available for fertilization, results in enhanced pregnancy rates in older mares and in normal young mares bred with a limited number of motile spermatazoa."

About the Author

Charlene Strickland

Award-winning writer Charlene Strickland lives in Bosque Farms, N.M. She has published 8 books and over 600 magazine articles, and is a member of the International Alliance of Equestrian Journalists.

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