Embryo Transfer in Mares

Embryo transfer is an assisted reproduction technique by which an embryo is transferred from the uterus of a donor mare to the uterus of a recipient mare that will carry the pregnancy to term. The embryo is usually recovered from the donor mare by uterine lavage (wash) at around 6 to 8 days after ovulation. The embryo is then placed in the uterus of a synchronized recipient mare.

Advantages

• A valuable mare can produce more than one foal per season.
• Mares can be in competition while producing foals.
• Young, genetically valuable mares can start producing foals before reaching physical maturity or while in training.
• Aged mares incapable of carrying a foal themselves due to pathology based on uterine biopsy can continue to produce foals.
• Mares with physical disabilities preventing pregnancy or foaling such as tearing of abdominal muscles or old pelvic fractures can still produce offspring.

Disadvantages

• The procedure is costly.
• Restrictions or prohibition are enforced by some breed associations.
• Performing embryo transfer in mares requires specialized training, and therefore not all equine facilities or practitioners are prepared to offer this service

The success of this procedure depends on the ability to control any of the following factors:

• The inherent fertility of the donor mare, which cannot be controlled. Furthermore, in many cases, subfertility is the reason why some mares become embryo transfer candidates.
• The inherent fertility of the recipient mares. These mares should be chosen based on young age and a normal reproductive tract.
• Breeding management or timing should be appropriate when performing embryo transfer.
• The embryo recovery and transfer technique. Experience of the veterinarian plays an important role in the success of the procedure.
• Degree of synchronization of donor and recipient. The donor and recipient mares are synchronized with the use of hormones; tight synchronization is important to ensure that the embryo is transferred into an appropriate uterine environment.

Equipment for Embryo Transfer

Starting an embryo transfer program requires setting-up a specialized laboratory. Embryo transfer equipment is commercially available from most companies selling equine breeding supplies. Apart from the usual equipment used for routine assisted breeding, such as an ultrasound machine, the following is required:

• Foley catheters to flush the donor's uterus. This is a special type of catheter used to flush the uterus of the donor mare for embryo recovery. A balloon at its end allows lodging of the catheter in the cervix of the mare, preventing loss of fluid during the flushing procedure.
• Extra tubing and connectors that are attached to the end of the Foley catheter to introduce and flush out fluid from the mare's uterus.
• Special filters to recover the embryo from the flushing fluid.
• A dissecting microscope to search for the embryo.
• Sterile culture dishes, capillary glass pipettes, and miscellaneous laboratory utensils.

The flushing medium most commonly used for embryo transfer is Dulbecco's phosphate buffered saline (DPBS) supplemented with 10 percent fetal calf serum and antibiotics. This medium is also used to wash and transfer the embryo into the recipient's uterus. The medium may be purchased in liquid or in powder form and reconstituted in the laboratory. As an alternative to DPBS, which is expensive, some practitioners use Lactated Ringers Solution instead.

Prior to attempting embryo recovery, all utensils and media should be warmed to body temperature in an incubator to avoid cold shock to the embryo. All equipment should be either disposable or washed properly and sterilized prior to use. No detergents or disinfectants should be used for washing embryo transfer equipment, since they may leave residues that might compromise viability of the embryo. Equipment is washed with warm tap water only and is then thoroughly rinsed with distilled water. Reusable catheters, tubing and filters should be gas sterilized prior to use.

Separate Foley catheters should be reserved for embryo recovery procedures and not mixed with those used to lavage the uterus of mares with infections.

Choosing Recipients for Embryo Transfer

It is well worthwhile to invest time and money in choosing optimal recipients, as it would be wasteful to invest resources and expertise in breeding a donor mare and recovering an embryo and then place it in the uterus of a mare that could not carry a foal to term. Therefore all mares purchased with the purpose of becoming recipients for an embryo transfer program should be submitted to a complete breeding soundness exam. All mares that make it into the recipient program should pass all aspects of the BSE and have a healthy endometrium (lining of the uterus) when evaluated in a biopsy sample.

The age of the recipients is also important. It is more likely that a mature but relatively young mare (5 to 8 yr old) will be able to carry a foal to term. Often, mares purchased as recipients are acquired through public auctions and the age is unknown. One should be confident about assessing age by teeth evaluation.

Ideally, mares in a recipient program should be chosen in accordance to a body size similar or slightly larger than the breed size of the donor mare. It is known that size of the mare will influence size of the foal, regardless of the genetic input by the donor.

Finally, it is well worthwhile to choose mares with a good predisposition, not only because they will be much easier to handle throughout the process, but also because they may make better mothers. In this regard, knowing the background of a mare and choosing a mare that has foaled uneventfully in the past should be ideal. Not only would you know for sure that the mare is capable of carrying a foal to term but also the motherhood of the mare. Unfortunately, unless provided by the client, the past of recipient mares is often unknown.

Synchronization Protocol for Donor and Recipients

Many synchronization protocols for donor and recipients are used, depending on personal preferences and experience. Hormones are given to the donor and recipients to manipulate their estrus cycle with the objective that they all come into heat at the same time. These treatments can vary from giving mares a single shot of prostaglandin, to placing the mares in a full treatment schedule of progesterone and estrogen shots.

Although cumbersome, a series of progesterone/estrogen shots provide the best synchrony between donor and recipients. Briefly, the protocol consists of a daily injection of a combination of progesterone and estrogen for 8 to 10 days (150 mg of progesterone plus 10 mg Estradiol-17b per shot). This suppresses the mare's own hormones, and when the treatment is discontinued most mares should come into heat in 3 to 5 days. Because some mares may ovulate while in treatment, a prostaglandin shot is usually given at the end of the progesterone-estrogen series for better synchrony.

Oral progestagens marketed to keep mares out of heat do not work as well for synchronization protocols.

Once donor and recipients are in heat and the donor mare is bred to the appropriate stallion, ovulation may be induced in all mares by giving them a shot of human chorionic gonadotropin. This will ensure a tighter ovulation synchrony between donor and recipient. A recipient is ideal if she ovulated on the same day or one day after the donor. Less ideal recipients are those ovulating one day before or farther from the ovulation date of the donor.

Unfortunately, even the best synchronization protocols can fail and donor and recipients may not always come into synchronized heat and ovulate as expected. This is a good reason to have always more than one recipient available for each donor mare. At least two recipients per donor is recommended, three would be ideal.

Breeding Management of the Donor Mare

The first limiting factor for the success of the embryo transfer program is that the donor mare must become pregnant. It is therefore imperative to practice good breeding management of the donor mare.

The donor mare may be bred with fresh or transported-cooled semen. It is important that the semen is of good quality and that the stallion has good established pregnancy rates. Obviously, the ideal stallion for a mare is not always chosen in regards to his fertility potential. However, old and subfertile stallions should be avoided to prevent introducing another factor that may preclude success of the embryo transfer program. For this same reason, the use of frozen-thawed semen should be avoided.

Optimal breeding management consists of breeding a mare during estrus as close to ovulation as possible. Ideally, the reproductive tract of donor and recipients should be examined daily by palpation and ultrasonography per rectum to ascertain the optimal time for breeding the donor and to assess synchrony with the recipients.

Around breeding, the donor must be given an injection of human chorionic gonadotropin to induce ovulation, and the recipients are treated in a similar manner (but not bred) for tighter ovulation synchrony.

Ovulation should always be confirmed by ultrasound examination. The day of ovulation is considered 'day 0' and the donor mare's uterus is usually flushed on day 7 after ovulation.

Embryo Collection Procedure

Embryo recovery from the donor mare is usually performed on day 7 postovulation. First, the mare is checked by palpation and ultrasonography per rectum to ascertain tone and appearance of the uterus. A mare that is pregnant will most likely have a very toned, homogeneous uterus, a closed cervix, and there will be no fluid in the uterus as visualized with ultrasound. At this time, the embryo is too small to be seen on ultrasound.

The mare is prepared for embryo recovery by cleaning the perineal area (vulva, anus and buttocks) with povidone iodine (Betadine) scrub three times, and rinsing thoroughly with water after each scrub. After generous rinsing, the area is dried with clean paper towels.

The person performing embryo transfer recovery should wear clean palpation sleeves and sterile gloves, and a sterile, water based jelly should be used for lubrication. One hand is introduced in the mare's vagina to pass the Foley catheter through the cervix and into the body of the uterus. Then, the catheter's balloon is inflated with air, using a small syringe, and thus is securely lodged within the cervix to avoid losing fluid during the uterine flush.

It takes at least two people to perform the embryo collection procedure, one holding the catheter within the mare's tract, and an assistant helping with fluid insertion and recovery. One liter of fluid is run into the mare's uterus by gravity, then recovered into a large glass cylinder, or directly passed through a filter to trap the embryo. This is repeated three times.

Some fluid should be kept within the recovery filter, to avoid drying out of the embryo. Once the recovery procedure is finished, fluid within the filter is decanted into a Petri dish. Searching for the embryo is performed under magnification with a dissecting microscope.

Embryo Transfer Procedure

If recovery is done on day 7 postovulation, the embryo should be at the blastocyst stage and should be easy to visualize under a dissecting microscope by an experienced operator.

Once found, the embryo is washed in sterile DPBS with 10 percent fetal calf serum. Embryos can be handled with 0.25 ml French semen freezing straws or glass pipettes connected to a rubber bulb or to a small tuberculin syringe.

The embryo is commonly transferred transcervically, that is, through the cervix of the mare as if performing artificial insemination. For this purpose:

• The chosen recipient is prepared for transfer by wrapping and tying her tail. Her uterus must be checked for tone and evidence of fluid. The embryo should never be transferred to a mare that has fluid in the uterus.
• Then, the perineal area of the recipient mare is thoroughly scrubbed as explained for the donor.
• The embryo can be loaded into an insemination pipette connected to a 5-ml Air-Tite syringe. About 1-ml of air is loaded into the syringe first, then the pipette is loaded with three small pockets of medium separated by pockets of air, with the fluid pocket in the middle containing the embryo.
• The person performing the transfer procedure should wear a surgical sterile glove over a clean shoulder length plastic sleeve. The pipette is inserted in the vagina and carefully passed through the cervix. Once advanced into the uterine body the syringe plunger is pushed forward and the embryo injected into the uterus.
• The recipient mare should be checked for pregnancy around 7 days after the transfer.

Another possibility is to perform surgical embryo transfer. This is done with the sedated recipient in the standing position through a flank incision that allows exteriorization of the uterine horn. There is no obvious advantage in transferring embryos surgically, and the procedure is more traumatic for the recipient mare.

In some cases, owners or practitioners performing embryo transfer choose not to have embryo recipients for a given donor mare. This avoids the hassles of purchasing, checking and maintaining recipients, as well as the expense and work involved with synchronization. In this case, the recovered embryo can be shipped to a referral facility that holds numerous recipients available for transfer at any given time. The drawbacks of this alternative are the risks involved with shipping an embryo and also that pregnancy rates may be slightly reduced when an embryo is not transferred immediately after collection.

For shipment, embryos are packaged into a sterile vial containing a commercialized medium, Hams F-10, that is buffered by diffusing it with a mixture of 90 percent nitrogen, 5 percent oxygen and 5 percent carbon dioxide. Prepared medium can be ordered from the transfer facility just prior to performing the embryo recovery procedure. This medium is also supplemented with fetal calf serum and antibiotics.

Once collected and washed, the embryo is placed into a 3-ml sterile plastic vial containing equilibrated warm medium, and this is then placed inside a 50-ml medium containing centrifuge tube, as a precaution.

The 50-ml tube is packaged into an Equitainer for cooled shipment.

Packaged embryos should be shipped by airline or overnight courier service, and transferred as soon as they reach the transfer facility, optimally before 24 hr postrecovery.