Equine embryo transfer

Embryo transfer is an established reproductive technology that involves the collection of an embryo (or embryos) from a donor female. The embryo (or embryos) is transferred into a recipient or surrogate mother where a pregnancy can be established and maintained, resulting in the birth of live offspring. Embryo transfer has been used to great commercial advantage in many species and has applications in equine reproduction.

Why use embryo transfer ?
A very attractive aspect to equine embryo transfer is that donor mares may remain in competition or training and still produce a foal without becoming pregnant themselves. In some cases, older mares that are able to become pregnant bonly to a resorb or abort may be used as embryo donor mares. For mares that foal late in a year, embryo collection may be performed, the embryo transferred into a recipient or frozen, and the donor mare bred earlier the following year. The international movement of equine genetics through the import and export of frozen embryos, the significantly lower cost of transporting embryos than live mares, and international concerns regarding disease control can all be addressed through embryo transfer.

How is an embryo produced ?
Through the breeding season, the mare's reproductive or estrous cycle has a predictable length of approximately 21 days. During her cyclic activity, fluid-filled follicles develop on the mare's ovaries. Towards the end of the 'heat' period or estrus during which time the mare is receptive to a stallion, a follicle (or occasionally two) grows, matures and releases an egg or ovum. The ovum is moved into the uterine tube where it can be fertilised by a sperm made available by natural or artificial breeding. The fertilised egg or zygote undergoes cell division to become an embryo which is then propelled along the uterine tube towards the uterus. By six days after ovulation the embryo is comprised of a mass of cells and is less than 0.2 mm in diameter when it enters the uterus. The mare is unique in that usually only viable embryos are transported into the uterus. An embryo which continues to develop moves freely in the uterus before attaching to the uterine lining about 25 days after ovulation.

What kind of mare makes a good embryo donor ?
The ideal embryo donor is a young healthy mare free of reproductive abnormalities. A potential donor mare should be in good physical condition and should be cycling normally before embryo recovery is attempted. A thorough reproductive examination must be performed and any abnormalities treated prior to embryo recovery being undertaken. While some clients wish to attempt embryo recovery from old, barren mares or mares with reproductive disorders, it should be understood that the probability of success in an infertile mare will be decreased. In those cases the decision to proceed is largely determined by the potential value of any foal that may result.

What kind of mare makes a good embryo recipient ?
Selection of healthy recipient mares is basic to the success of an embryo transfer program. Recipients should be young mares of proven fertility and normal reproductive health. Basically, recipients should undergo the same level of scrutiny as donor mares. Recipient mares must be at a similar stage of their estrous cycle as the donor at the time of embryo transfer. For example an embryo recovered from a donor on day 7 following ovulation should be transferred into the uterus of a recipient which ovulated from one day before (day 8) to one day after (day 6) the donor. To help ensure the availability of at least one suitable recipient, it is reasonable to synchronise two potential recipient mares for each donor.

When are embryos collected ?
Embryo donor mares may be bred by natural cover or by artificial insemination. In either case, it is important that the time of ovulation is known to within 12 hours. Embryo recovery is usually attempted on day 7, where day 0 is the day of ovulation. With natural breeding and assuming normal fertility of the stallion and mare, a fertilised embryo can be recovered on day 7 in 60-70% of attempts. If artificial insemination with frozen semen, or older infertile mares, are part of embryo program the rate of embryo recovery is lowered.

Embryo transfer in cattle often involves superovulation, or administering hormones to increase the number of developing follicles. Mares do not respond to super-ovulation so in the majority of embryo collection attempts only one embryo, and rarely two embryos in the event of double ovulations, is normally recovered.

How are embryos collected ?
In the early days of embryo transfer, all collections and transfers were performed surgically. The cost, risk to donor animals, and aftercare have been greatly reduced through the current techniques of non-surgical embryo collection and transfer.

For non-surgical embryo collection, the mare is restrained, her tail is wrapped and held out of the way, while her rectum is emptied of feces. The vulva and perineum is cleansed with a mild soap and a sterile, large bore embryo collection catheter is passed through the vagina, the cervix and into the uterus. The inflatable cuff of the catheter is filled to ensure a snug seal against the cervix. One end of a sterile tubing set is attached to the catheter, and the other connected to a container of warmed sterile solution containing electrolytes, antibiotics and serum. A volume of about 1 litre of fluid is allowed to flow by gravity into the uterus. By gentle massage of the uterus, embryos present within the uterus become suspended in the flush fluid which is then drained out of the uterus and passed into a fine filter dish which retains the embryo(s) and a small volume of flush fluid.

This process is repeated with an additional 2-3 litres of fluid to ensure complete irrigation of the uterus. Finally, the fluid remaining in the filter dish is poured into a gridded dish and examined under a dissecting microscope to locate the recovered embryo or embryos. Identifying and manipulating embryos under the microscope (without losing any !) requires considerable practice and some manual dexterity. Once an embryo is identified, it is picked up in a fine catheter or pipette and transferred into a small volume of embryo holding medium. The embryo can be assessed with respect to it's developmental stage, according to a system used by the International Embryo Transfer Society. Equine embryos can be held at room temperature for 2-3 hours but generally they should be transferred into a recipient mare or frozen as soon as possible.

How often can embryo recoveries be performed in a season ?
Embryo recovery attempts can be repeated on consecutive heats. To shorten the interval between collection attempts, or to concentrate collections within a particular time frame, mares can be 'short cycled' using prostaglandins (Estrumate or Lutalyse). Careful monitoring of follicle development and ovulation, is important. After embryo recovery is attempted, and regardless if other collection attempts are planned, an injection of prostaglandin is given following a flush to remove residual fluid from the mare's uterus. This will should stimulate a return to normal estrus within a few days although the interval between estrus periods may be slightly longer than normal.

Embryo collections should not normally increase the risk of uterine infections or reduce mare fertility. In fact, embryo collection attempts by uterine flushing may be a useful diagnostic test and can form part of the treatment of subtle uterine infections. The fluid recovered from an infected uterus is usually cloudy and may contain flecks of pus, indicating the need for additional investigation or treatment.

How are embryos transferred ?
Once an embryo has been recovered, it will only result in a pregnancy if it is returned to another equine uterus. The uterine environment of the recipient uterus must be very similar to that of the donor, and the levels of hormones involved in pregnancy must be similar. It is for these reasons that the synchrony of donor and recipient cycles is very important An embryo may be transferred into the uterus of a recipient in one of two ways - either through a small flank incision - a surgical transfer - or through the cervix - non-surgical transfer. Surgical transfers may be performed under heavy sedation and local anesthesia. While surgical transfers can increase pregnancy rates, this must be offset by higher costs, more aftercare and possible complications. Non-surgical transfers are faster, less invasive and are virtually free of complications. In either case, attention to detail, minimising trauma and donor-recipient synchrony are essential to maximising pregnancy rates.

Can equine embryos be stored ?
Short term (a few hours) storage of equine embryos requires careful regulation of temperatures and holding in a special solution. Longer term storage storage, generally anything in excess of a day requires that embryos be frozen or cryo-preserved under controlled programs and stored in liquid nitrogen. Once frozen, embryos remain stable for many years. This means that embryos may be collected at times when a pregnancy is not normally desired and frozen for transfer at a later date. This can add considerable flexibility to many equine embryo transfer programs, and allows the long distance movement (trans-continental and international) of equine genetics. However, in addition to the concerns that apply to the transfer of fresh embryos, frozen embryos must be handled with care and thawed under appropriate conditions prior to their transfer into recipients at the appropriate stage of their cycle. It is unusual for pregnancy rates with frozen embryos to be as high as those obtained with fresh transferred embryos.