Embryo transfer — For best results, donor, recipients and stallions should be at same location and managed by same veterinarian

Equine embryo transfer has become a widely accepted technique in which a donor mare is mated and the resulting conceptus (embryo) is removed from her uterus and transferred to a suitable recipient mare, where it will undergo suitable maturation and development. The first successful embryo transfer in horses was performed in 1972, and since that time, the procedure has gained widespread acceptance.

• Embryo transfer is used so:
• Multiple offspring from one donor mare can be used.
• Donor mares can continue in athletic competition without having to carry a foal.
• Offspring can be produced from mares at an earlier age than usual (i.e. 2 years old).
• Mares that have experienced various reproductive problems can breed or carry.
• Mares with physical disabilities that make it difficult for them to carry a foal to term can still breed.
• Researchers can learn more about various aspects of equine reproduction.
• Rare or endangered equines have a better chance prospering.

Most breed registries allow this technique, and some even allow the registration of multiple foals in any given year, such as the Quarter Horse. The breed registry guidelines should be consulted. Using reproductively normal donor mares and stallions of known, good fertility, an embryo can be recovered approximately 70 percent of the time a recovery attempt is made. In contrast, mares with poor reproductive histories or aged mares (greater than 18 years of age) have poor recovery rates (less than 30 percent). Aged mares also have fewer normal oocytes, which contribute to poorer embryo recovery. Remember, embryo transfer is not a cure for infertility!

In recent years, a purified eFSH product has come available to potentially increase the number of ovulations and recovery of more than one embryo per attempt. The selection of the stallion for use in an embryo transfer program is also extremely important. The use of fresh or fresh-cooled semen from a stallion of known good fertility can increase your embryo recovery rate. In contrast, the use of poor-quality semen or frozen semen in general will lower your embryo collection rates. The worst-case scenario is to breed older subfertile mares with poor-quality semen or frozen semen.

Initial mare exams

Each donor mare should undergo a complete reproductive examination prior to enrolling in an embryo transfer program to help ascertain a realistic probability of harvesting an embryo successfully.

Donor mares should be cycling normally and in good physical condition. Any uterine pathology or physical abnormalities should be corrected prior to the breeding season. For best results, donor, recipients and stallions should be on the same premises and managed by the veterinarian in charge of the embryo transfer program to maximize embryo recovery.

Recipient mares ideally should be young, reproductively normal mares that have undergone a thorough physical and reproductive examination. The proper selection and management of recipient mares may be the most important factors affecting a successful embryo transfer program.

Both donors and recipients must be reproductively managed to be in synchrony with each others' reproductive cycles. Ideally, two to three recipients should be available for each donor mare. Cycles usually are synchronized using a combination of progesterone-estrogen (P and E) and prostaglandin. Ovulation usually is induced by the use of HCG or GnRH analog. The recipient should ovulate from two days before the donor or up to three days after the donor. Ideally, the recipient should ovulate one day after the donor mare. Alternatively, ovariectomized mares that are suitably prepared with hormones also can be used as recipients.

Embryo transfer procedure

The most common method of embryo recovery is the non-surgical transcervical uterine lavage technique. This procedure usually is carried out on day seven post ovulation (where detected ovulation is day zero). Donor mares that are older (18 years of age) generally are flushed on day eight because the embryo seems to be smaller and less developed in these older mares. Because the equine embryo doesn't enter the uterus until about day six post ovulation, earlier attempts to recover the embryo may result in not recovering an embryo. Recent work has indicated that the non-surgical transfer of day-nine embryos can be as successful as day seven or eight post-ovulation embryos.

The actual recovery of an embryo involves the passage of a suitable embryo-recovery catheter (usually 80cm in length with a 80-100ml inflatable cuff) through the cervix into the uterus. The cuff is inflated with either air or flush media to a sufficient size to seal up against the internal os of the cervix to prevent any leakage of fluid from the uterus. The uterus is lavaged usually with 2 liters of warmed flush media. After the media is in the uterus, it is allowed to exit via the catheter through an embryo recovery filter. This is repeated three to four times (for a total of 6-8 liters of media). The most common media used is Dulbecco's phosphate buffered saline containing 1 percent (v/v) fetal or newborn calf serum, penicillin (100 IU/ml) and streptomycin (100 micrograms/ml). More recently, other suitable flush media have become available. The majority of fluid infused into the uterus should be recovered (95 percent). This is aided my massaging and manipulating the uterus per rectum.

Once the flush is completed, the catheter is removed from the donor mare, and the mare is administered prostaglandin to return her to estrus.

The filter containing recovered fluid then is examined with a stereo-microscope at about 15x magnification, or alternatively the fluid is poured into a search dish for embryo identification. Larger embryos (day eight) generally are visible with the naked eye. Once the embryo is located, it is washed by sequential transfer through four to eight drops of holding media in preparation for transfer.

Embryo transfer

Embryos can be transferred to same premise recipient mares either surgically or non-surgically. Alternatively, embryos can be packaged in a suitable storage media, placed in an Equitainer (Hamilton Research) for cooling and shipped to another facility where suitable recipient mares are located. Embryos handled this way can be stored for 12-24 hours.

Longer-term storage would involve the freezing of embryos at -196°C in liquid nitrogen. Short-term storage is the more practical of the two alternative storage techniques. Freezing of embryos is a much more difficult procedure and requires, at this time, embryos that are very small (<300 micrometers).

Surgical transfer involves making a flank incision in a standing recipient mare and transferring the embryo via a special pipette directly into an exposed uterine horn.

Non-surgical transfer involves depositing the embryo into the uterus via a transvaginal procedure identical to the insemination technique. The embryo is loaded into an insemination pipette with media or into a .25-.5ml semen straw, which is used with an insemination "gun". Non-surgical transfer generally has replaced the surgical technique, and the success rate can be equal to or greater than those obtained with surgical transfer (70-75 percent).

After transfer, recipient mares generally are given supplemental progesterone and are examined for pregnancy approximately seven to eight days after the transfer. If determined to be not pregnant, progesterone supplementation is stopped so that she will return to cyclicity. Pregnant mares then are managed normally.

Dr. John V. Steiner is a diplomate and president-elect of the American College of Theriogenologists. He works for the Equine Reproductive Practice at Hagyard Davidson McGee Associates PLLC in Lexington, Ky. He is a 1968 graduate of Cornell University.