The Physiology of Equine Reproduction and Embryo Transfer

Embryo transfer is one of many recently developed techniques to improve pregnancy rates in sub-fertile and older mares. Mares with normal reproductive capabilities are used as recipients for embryos from mares who are unable or unlikely to carry a foal to term, such as mares with pelvic fractures or uterine scaring as well as mares with problems associated with early embryonic death. Many factors affect the success rate of embryo transfer such as age and reproductive ability of the donor mare and synchronization of ovulation between the donor and recipient mare. The mare that will receive the transferred embryo must be in good reproductive health to carry the foal. There are two ways to transfer the oocyte, once collected, to the recipient mare, the flank incision technique and the nonsurgical, transvaginal transfer. Although embryo transfer is not as common in horses as it is in cattle due to higher costs, it is still an important new frontier in the physiology of equine reproduction.

Fertile mares have seasonal estrous cycles. They normally cycle between April and October. Estrus is the time in the mare’s reproductive cycle where she shows interest in mating. This usually lasts between four and eight days. The date when a mare comes into estrus after a winter of anoestrus is affected by the amount of daylight, temperature, nutrition and even climate.[1] During increasing periods of light the retina of a mare sends signals through the optic nerve to the pineal gland. This stimulates the pineal gland to produce less melatonin, a hormone that suppresses the Gonadatropin releasing hormone (GnRH). GnRH is produced in the hypothalamus and tells the pituitary gland which reproductive hormones to release. One of the hormones released from the pituitary in the presence of GnRH is the follicle-stimulating hormone (FSH), which begins the process of follicular enlargement. FSH stimulates many follicles to develop at the same time. These follicles produce an increased amount of estrogen, a hormone that prepares the mare for mating and fertilization. Once a follicle nears full development in a healthy cycling mare the amount of GnRH reaching the pituitary gland stimulates the release of luteinizing hormone (LH).[2] LH signals one or two follicles to mature and brings on ovulation, the release of the egg from the follicle.

Diestrus occurs about two to three days after estrus and lasts for around 16 days in healthy ovulating mares.[3] About eight hours after ovulation the follicle that ovulated fills with serum and blood. After another two to three days the corpus luteum (CL) forms on the ovary where the follicle was. The CL is a yellowish mass, which produces the hormone progesterone. Progesterone is not only responsible for the mare’s disinterest in mating but also prepares the uterus to receive, nurture and maintain the embryo. Progesterone levels must be maintained above two and half nanograms per milliliter to sustain the pregnancy.[4] Mares that are pregnant will maintain the CL with the help of hormones produced by the fetus and progesterone levels will remain high throughout the close to three hundred and forty days of gestation. If the mare is not pregnant the CL is only maintained for twelve to fourteen days before ceasing to function. This is called luteolysis. After this time the mare will return to estrus. If the mare is pregnant the embryo moves back and forth between the uterine horns and attaches to one of the horns at about fourteen to sixteen days after ovulation. This wandering is a signal to the mare that she is pregnant and the CL is not in luteolysis. Progesterone production is continued and the pregnancy is maintained in healthy mares.

Many factors can lead to sub fertile or infertile mares. Several of these factors can be over come by embryo transfer. Many mares cannot carry their own foal to term due to pelvic fractures or immature reproductive tracts. During parturition mares that have suffered a fracture in the pelvic region previously may further injure themselves. Embryo transfer offers a way for owners to pass on genetic characteristics without endangering these mares. Extensive uterine scaring may also make a mare a candidate for embryo transfer. Scaring can make it virtually impossible for the embryo to attach to the wall of the uterus, which may cause abortion of the embryo early in the pregnancy[5].

Embryo transfer techniques are also effective for mares with uterine tumors or disorders associated with early embryonic death. One of the most common causes of early embryonic death is a drop in the progesterone levels below two nanograms per milliliter, the concentration necessary to maintain a pregnancy. Low progesterone levels can be caused by various physiological disorders such as improper luteolysis of the CL, luteral insufficiency or failure to recognize the presence of the pregnancy. Luteolysis of the CL, which is caused by the release of Prostaglandin F2α (PGF2α), a naturally occurring long chain hydroxy fatty acid, from the endometrium during the early pregnancy would lead to the abortion or absorption of the embryo[6]. This can result from two different mechanisms, the first of which is the failure of the mare to recognize the pregnancy. If the period of movement within the uterine horns by the embryo is restricted or stopped, the mare’s body will not know there is a pregnancy and will release PGF2α to cause luteolysis of the CL. Without the proper amount of progesterone the mare will not maintain the pregnancy and come into estrus again shortly. The other mechanism that can lead to luteolysis of the CL during early pregnancy has to do with the invasion of pathogens into the uterus. These pathogens including yeast, secrete endotoxins that cause the endometrium to secrete PGF2α with the same results as above. These endotoxins may also result in embryonic death as a secondary cause of embryo loss from invading pathogens[7]. Luteral insufficiency is hard to classify outside of the presence of one of the previously mentioned reasons for low progesterone levels. Inconsistent progesterone levels can be treated with hormone therapy or embryos can be transferred into a mare that can maintain the pregnancy.

Age is another factor affecting both conventional and artificial reproductive practices. Older mares are less likely to conceive naturally and carry a foal to term than younger mares, but their age also reduces the number of follicles ovulated and embryos produced. Oocytes from older mares are also less likely to accept fertilization and grow into healthy embryos. This affects all forms of assisted and normal reproduction from these mares. A study showed that while eleven out of twelve embryos from younger mares transferred to recipient mares were successful only eight out of twenty-six pregnancies occurred using embryos from mares over the age of twenty[8]. Embryo transfer reduces stress to an older mare by allowing a younger mare to care for the embryo and resulting foal.

One of the most important factors in embryo transfer is time. To collect an embryo for transfer a mare must be inseminated in the forty-eight hours prior to ovulation. The types of insemination, whether live cover or artificial, fresh or cooled, does not seem to affect the pregnancy rate for embryo transfer. The day of ovulation must be recorded for it will be considered day zero for all the time frames of embryo recovery and transfer. As stated earlier, in healthy mares fertilized embryos do not immediately attach to the wall of the uterus. This period of travel makes collecting the embryo and transferring it easier. Embryos are normally collected on the sixth day after ovulation if they are to be cryopreserved and at seven to eight days after ovulation if they are to be transferred fresh or cooled to five degrees Celsius. Seven to eight day embryos have little to no survival rate after cryopreservation. It is unclear why these larger embryos are less receptive to freezing than the six-day embryos but it is thought that it may have to do with the ability for the cryoprotectants, like 1,2 propanediol, to permeate into the equine blastocyst[9]. Even though six-day embryos survive the freezing and thawing process better than the larger embryos they still do not have a high success rate as many are aborted before sixty days of gestation. The type of cryoprotectants does not have a large effect on the survival rate of the embryos. To recover the embryos a catheter is inserted through the mare’s cervix and into the uterus. Then the uterine body is washed with three to four liters of Dulbecco’s phosphate-buffered saline with one percent fetal calf serum[10]. The fluid is recovered and examined for the presence of an embryo. If this procedure does not recover an embryo on the seventh day after ovulation it is repeated on the eighth. Once the embryo is recovered it can either be transferred fresh into the donor mare or be cooled or placed temporarily into a rabbit host to be shipped to the location of the recipient mare.

Embryos are graded on a four-grade scale, with one being excellent and four being poor. They are graded on size and morphological characteristics. Embryos that are smaller than normal or have morphological abnormalities receive a score of three or higher and usually result in decreased pregnancy rates. The highest pregnancy rate, seventy percent, is achieved with embryos with a score of one at the initial pregnancy exam.

One of the major limiting factors to embryo transfer is the fifty percent recovery of embryos from single-ovulating mares. Researchers are looking for a drug to induce multiple ovulations in any mare. Although in some breeds like thoroughbreds and draft breeds multiple ovulations are more common, most horses only ovulate one oocyte per estrous cycle. Carrying multiple healthy foals to term is nearly impossible in horses, so the multiple ovulations are not necessary in normal reproduction[11]. But for embryo transfer multiple ovulations would increase the number of embryos recovered per estrus and make embryo transfer less expensive. Although equine pituitary extract (EPE) has been shown to be effective in producing multiple ovulations it has not been approved for use outside the laboratory[12]. PGF2α is administered after the first dose of EPE and EPE treatment is continued for about a week. This stimulates two or more follicles to grow to about thirty-five millimeters in diameter. The follicles then grow to normal mature size of around forty-five millimeters then ovulate. Although unpractical in conventional forms of reproduction, in both embryo and oocyte transfer multiple ovulations would greatly increase recovery rates and thereby reduce the cost.

The health and management of the recipient mare is extremely important for successful transfers. The recipient mare needs to be a healthy mare that is free of reproductive abnormalities, weighs between four hundred and five hundred and fifty kilograms and is between three and ten years of age[13]. Mares between these ranges show the highest rate of successful pregnancy and foaling. The recipient mare’s ovulation may also need to be synchronized with the donor mare so that the embryo can easily be transferred from one to the other. To induce ovulation and synchronize the mares, PGF2α is administered. This form of prostaglandin induces estrus and then GnRH can be used to synchronize ovulation. Another hormone that can be used when synchronizing ovulation is human chorionic gonadotropin(hCG), a hormone produced by the human fetus[14]. HCG is similar to the Equine chorionic gonadotropin (hCG), which is produced by the equine fetus. Both hCG and eCG are used to detect pregnancy by an antigen test performed on the urine. Recipient mares can ovulate anywhere from one day before to three days after the donor mare and still have a high pregnancy rate. To reduce the need for estrous cycle synchronization between donor and recipient mare deslorelin acetate,, can be administered in the form of an implant. Deslorelin acetate has been shown to suppress follicular activity in mares, so that recipient mares would not need synchronization because this hormone treatment can stop their follicular development and thereby prepare them for embryo transfer[15].

Success rates for surgical transfer of embryo via an incision in the flank are slightly higher than those of the non-surgical method. In the surgical method embryos are directly inserted into the exposed uterine horn through a puncture site. The non-surgical method uses an insemination rod that is passed through the cervix and deposited into the uterus. This is often used by facilities with fewer mares to breed because it is cheaper and relatively simpler. The success rates are similar for both methods but the surgical method, which is preferred by bigger breeding operations, is more sterile and there by has a slightly higher success rate[16]. Introduction of pathogens into the uterus via the insemination rod is a rare but possible risk to the non-surgical transfer method.

Embryo transfer is still a new frontier in equine assisted reproductive medicine and many aspects such as cryogenic preservation of embryos and a drug to induce multiple ovulations are still in the research stage while embryo recovery practices have remained mainly the same since their inception over thirty years ago. Embryo transfer is aiding in making possible offspring from infertile or sub fertile mares and in allowing mares to have more than one foal per season. While in some cases this may improve the gene pool and reproduction, it may in the long run reduce the gene pool by causing over breeding of supposed genetically advanced horses and allow for more abnormal recessive genes to become prominent in the gene pool. Transfer of embryos from mares with genetic reproductive problems may also lead to the necessity of embryo transfer and other assisted reproductive techniques to produce future generations of horses. But if used properly and researched further, embryo transfer could become a reasonable option for owners whose mare’s problems are not genetically related. For many mares both young and old, embryo transfer may be the only way to keep their genes from leaving the gene pool. For now the expense of embryo transfer and the lack of important technology makes it available to a relatively small number of horses. The techniques for embryo transfer have only moderate success rates, about sixty-six to seventy-five for the surgical flank incision technique and fifty to seventy-five for the non-surgical method. Until this rate increases and techniques for freezing embryos and inducing more than one ovulation per cycle, become more effective and reliable, embryo transfer will not decrease in cost or increase in usage. For now research to understand the physiology behind the intolerance of seven and eight day embryos to cryogenic preservation and the effects of equine pituitary extract on follicular development and other body systems is essential to further progress. Even with all of the unknowns associated with equine embryo transfer and reproduction, it is one of the few ways to secure offspring from infertile and sub-fertile mares.

[1] James M. Griffin, M.D.and Kjersten Darling, D.V. M., Veterinary Guide to Horse Breeding (New York: Howell Book House, 1999) 13.

[3] Dr. Clinton G. Depew, “Reproduction in the Mare,” Equine Veterinary Science 9 (1989): 48 and 49.

[4] Angus O. McKinnon and James L. Voss, Equine Reproduction (Philadelphia: Lea & Febiger, 1993) 526.

[5] Dr. Clinton G. Depew, “Infertility in mares,” Equine Veterinary Science 9 (1998): 47.

[8] E. L. Squires, P. M. McCue and D. Vanderwall, “The Current Status of Equine Embryo Transfer,” Theriogenology 51 (1999): 94

[10] M. F. Iuliano, E. L. Squires, and V. M. Cook, “Effect of Age of Equine Embryos and Method of Transfer on Pregnancy Rate,” Journal of Animal Science 60 (1985): 258.

[15] E. M. Carnevale, et al, “Use of Deslorelin Acetate to Suppress Follicular Activity in Mares Used as Recipients for Oocyte Transfer,” Theriogenology 55 (2001): 358.