Personnel at the GVEH have been involved with equine embryo transfer (ET) since 1977 and have been performing the technique on a large number of mares both here in Australia and internationally for many years. Research carried out at the GVEH has been presented at international veterinary and scientific meetings and researchers have been responsible for the first foal born (worldwide) from the GIFT technique, the first successful transfer of a day 10 embryo, the first successful embryo transfer from a camel and the first foals conceived from sperm injection of matured mare eggs. Each year in Australia the demand for horse embryo transfer increases. It is anticipated that changing breed registry restrictions will only increase the demand for horse ET.
Embryo transfer in the horse involves collection and transfer of an embryo from one mare (donor) into another (recipient). The donor mare and stallion contribute their genetics to the foal, not the recipient. The recipient will help determine the size of the foal at birth but its subsequent growth is determined by its own genetics and nutrition.
The donor mare can be bred by fresh, cooled or frozen semen and breeding techniques are identical to breeding mares destined to carry their own foal. The day of donor ovulation (day of egg release) is termed day 0. Embryos may be removed from the mare by non-surgical flushing on day 6 or 7. Embryos collected on day 6 are significantly smaller compared to day 7 and more difficult to find When an embryo is obtained it is washed and graded and re-inserted into the recipient mare. The recipient should have ovulated (has not been bred) within one or two days of the donor. The embryo is re-inserted into the recipient mare either surgically or non-surgically. Pregnancy rates have improved from non-surgical transfer so that the pregnancy rates are almost identical, regardless of the method of transfer.
The first pregnancy test on the recipient can be determined as early as 5 to 6 days after embryo transfer (day 11 to 12). On occasion embryos can be cooled for transport to another facility or may be frozen for long term storage prior to being inserted into the recipient.
1) Obtaining pregnancies from mares that cannot maintain their own pregnancies, i.e. uterine disease or mare injury. This is the most common reason for using the technique We transferred an embryo in NZ from a mare that had damaged herself during foaling so badly that it was unlikely that she would carry a foal again.
2) Obtaining multiple pregnancies per season. A few years ago we were asked to collect embryos from three mares who had not had a foal for an average of five years. We obtained a total of six pregnancies in that one breeding season.
3) Obtaining pregnancies from 2 or 3 year old mares. This request is rare in Australia but quite common in the US.
4) Obtaining pregnancies from mares in competition. We are routinely asked to collect embryos from mares that are actively competing on the show circuits. They only have to visit our hospital for 15-25 minutes to collect the embryos every 19-21 days.
5) Obtaining pregnancies from mares foaling late in the breeding season and thus enabling them to conceive earlier next season. This is quite popular in the US but not in Australia.
1) Costs. The main cost with horse ET is the maintenance of the recipient mares. We frequently keep these mares for two or more years without using them. The mares are well fed and well cared for throughout the year and need to be carefully screened for reproductive soundness.
2) Considerable experience is necessary.
3) Embryo recovery from infertile mares is low. Normal mares will give about 70% embryo recovery per cycle. Infertile old mares may be as low as 20%.
4) Techniques to increase the ovulation rate (proportional to egg recovery) in the mare are generally unsuccessful. Mares usually only release one or sometimes two eggs for fertilisation per cycle. Much research has been aimed at increasing the ovulation rate, however this is expensive, mostly unsuccessful and usually only results in one extra embryo.
5) Mares and stallions are best located on the one premises. The rapid advancement in breeding technologies make it possible to breed mares with semen that has been cooled and transported or that has been frozen. This doesn’t help improve pregnancy rates and so most times it is recommended that the mare and stallion are kept on the same premise.
Embryo recovery is best from young, fertile mares and approximately 70- 80% of cycles may yield an embryo. An important influence on embryo recovery is the fertility of the stallion. Mares that consistently multiply ovulate (Warm-bloods, etc.) have a better embryo recovery rate. The worst embryo recovery rate is from old, barren mares that have persistent uterine infections (< 20%).
The quality of the recipient is the most important determinant of pregnancy rate after embryo transfer. Because of this, most embryo transfer facilities prefer to buy and manage recipient mares and do not accept offers from clients to supply their own mares. Non-surgical embryo transfer results in approximately 65-70% pregnancy rate per attempt, and surgical transfer improves the average by around 5-10%.
Other techniques associated with embryo transfer
1) Cooled Transported Embryos: The ability to cool embryos and transport them to remote facilities was developed by people at our clinic whilst working overseas. Subsequently we have demonstrated here in Australia that the technique has much application. We have achieved multiple pregnancies from this and are actively continuing to expand this technique. The advantage is that the embryo can be harvested from the mare by veterinarians on the farm where the mare normally lives and then the embryo can be transported to our facility where the recipients are housed. Maintenance of the recipients is expensive and having enough mares to schedule for a proposed embryo transfer is one of the most difficult aspects of horse ET. This program eliminates these difficulties for the veterinarian that only performs a few ET’s per year.
2) Freezing Embryos. We have obtained pregnancies from embryos frozen, stored and subsequently transferred. Currently only day 6 embryos are able to be successfully frozen and thawed with consistently good pregnancy rates. Because they are more difficult to obtain it is not a common technique Pictured here are the first two foals born from frozen embryos at Colorado State University (Frosty and Popsicle).
3) Splitting Embryos. Identical twins can be obtained by splitting an equine embryo and transferring the two identical halves into separate recipient mares. Horses do not usually form identical twins in the uterus (like cattle and humans), because of a major difference in the coating surrounding the embryo, thus micro-manipulation is necessary. We have produced identical equine twins by splitting a young embryo. However, only approximately 1 in every 6 embryos split result in identical twins and similar to freezing embryos, only day 6 embryos have been able to be successfully manipulated. Splitting of embryos will create genetically identical twins, however there are always some differences such as variation in exact coat colour markings. Identical twins have created enormous interest because of their potential as carriage horses or research animals. Any research that has a strongly inherited predisposition may be studied with much fewer animals if identical twins are used. To our knowledge there is no foals born from equine embryo splitting in Australia. The identical twins shown above were produced at Colorado State University. The slight differences in white markings are quite common and are thought to be related to different migration of pigment (and non pigment) cells according to in utero positioning and even temperature effects.
4) Gamete Intra-Fallopian Transfer (GIFT). This procedure involves aspirating mature eggs from the ovary prior to ovulation, placing them in the oviduct (site of fertilisation) of the recipient and breeding the recipient mare. We were first in the world to obtain a foal using this technique With this procedure great care must be taken to ensure that the recipients own egg does not get fertilised. Success rates with this technique are quite low (30%) but it does remain as an alternative to provide progeny from those mares that fail to produce embryos.
5) In-Vitro Fertilisation. Culture of immature or mature female eggs and co-incubation with sperm under laboratory conditions (in-vitro) is commonly accepted in humans and more recently cattle. This procedure has only been successful twice in the horse (in France) despite a large amount of time and finance invested by the research community. In the future we expect that this technique might become more commercially available for the horse.
6) Sperm injection (ICSI). Our most recent and exciting development was obtaining pregnancies from infertile mares using frozen semen from infertile stallions. This has huge (in our opinion) applications particularly for infertile mares, infertile stallions and in cases where there is limited access to sperm. The procedure is like GIFT in that the egg is removed from the donor mare about the time she would normally release it. It is then held with very delicate instruments and a single sperm injected through a tiny glass pipette into the middle of the egg. This step removes the barrier to the stallion sperm getting into the egg. It leaves the only job for the sperm as one of providing genes (DNA). Even non swimming sperm can get mares pregnant this way.
Pictured on the left is Art (an acronym for Assisted Reproductive Techniques). He was the first foal born in the world born from ICSI of In Vivo matured (from a live mare) female cells (oocyte). The Goulburn Valley Equine Hospital produced Art in conjunction with Monash University, Animal Research Group. A few weeks after Art was born the second foal arrived. He is pictured on the right. After an Australia wide foal naming competition the winning name chosen was Music (Method Using Sperm Injected Cell).
7) Sexing Embryos. There is quite an amount of interest in sexing embryos prior to transfer. Two current techniques have been used (neither are available commercially). a) HY antigen detection. This utilises an antibody/antigen reaction that combines a dye that fluoresces under ultraviolet light (male embryos). The success is reasonable, however the procedure is time consuming and not always accurate. b) Detection of the chromosomal sex is possible by taking a small sample of tissue from the embryo (biopsy). This procedure holds much promise, however to date it is apparent that any micro-manipulation to the embryo will reduce its viability. Embryos cannot be split and frozen or sexed then frozen. This technique may be commercially available in the future, but that depends on demand. Our attitude is that it is difficult enough to obtain a pregnancy from embryo transfer without stressing the embryo by sexing it. Ultrasonography techniques are now available that are accurate in determining foetal sex by approx. day 55-65 of pregnancy. Most people are utilising these techniques for foetal sex determination. A technique of the future is obtaining pregnancies from sex selected sperm. More on that later.