In embryo transfer, an embryo is taken from the donor mare, being the biological mother of the foal to come. A flush tube is inserted into the mare’s uterus.
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.
Many equine vets offer an Embryo Transfer service and likely each will have a different system but a basic transfer could be described as this:
More than one recipient mare would be selected for each attempted ET because they have to select at least two recipient mares from their band of broodmares whose cycle is close to that of your mare. If they chose one and things did not go right with the recipient mare, your embryo may be lost but you would still have a big vet bill because you have already done the AI and all the lead-up work for the ET.
If your recipient mare will stay for the term of the pregnancy and perhaps until weaning, you will be asked to pay an 'up-keep' or agistment fee whilst she is part of your breeding program. The vet will likely have bought and maintained these mares specifically for this type of work and they have to recoup some of these expenses. This fee is likely to start from the day the recipient mares are scheduled into the program.
An alternative and probably the most common option is to lease the mare to take home until foal is weaned, you may even have the option to buy she belongs to your vet so you must either lease her until the foal is weaned or buy her from the vet. Most vets would charge from $500-2000 for either the lease or a straight sale. Compared with the cost of you providing your own recipient mare and the risks associated with that as outlined earlier, this is not an expensive option.
Whether you leave your mare at the clinic for the whole process from insemination to transfer or whether you AI as you normally would then take the mare in for flushing in discussion with your vet is your choice.
Around 7 to 8 days after the AI, your mare is flushed and hopefully a viable embryo is captured.
Note - The embryo has to be retrieved from your mare at around 7 days of age so that it has a reasonable chance of survival. Embryos collected at day 6 or day 9 have a much lower viability so this is a critical date. Embryos are quite pliable at this age and become significantly more 'fragile' as they get older. This presents the first challenge for the ET breeder as it is virtually impossible to confirm the embryo even exists at 7 to 8 days of age and leaving it longer (which would enable confirmation by scanning) means that you may be able to confirm the AI has been successful but you have also virtually guaranteed that your ET will not be successful as an embryo beyond 10 days of age will almost always be too fragile to flush and transplant into the recipient. What all this means is that your vet will be flushing your mare and 'hoping' that they have a viable embryo and that they have captured it. The next step is for your vet to filter the flushing fluid and search for an embryo that will be almost impossible to see with the naked eye. At this age embryos are well under one millimetre in diameter. If the AI has been successful and the flush has recovered ALL material from the uterus and your vet has collected a healthy embryo you have passed a significant hurdle.
You must obtain approval from he AFHS/KFPS prior to breeding and there is a maximum of five breedings per mare per year allowed with each one requiring seperate approval.
One of the big pros of embryo transfer is that you can breed several foals a year with one good mare. Tom: “This way, a sport mare can produce foals without having tot take “maternity leave”. Also, when a mare has reproductive organs issues, embryo transfer may be a solution. Say a mare has a damaged cervix and is not able to carry the foals to full term. You might consider taking flushing the mare and transferring the embryos into recipient mares. Thanks to embryo transfer you can breed several foals with your best mares. If all goes well, you can try and flush the mare during the breeding season every two to three weeks. For most mares this is not a problem, so you could “create” a foal about seven times a year. Some mares aren’t particularly fond of the process though. Some riders notice their mares respond differently because of the hormone changes and witness behavioural changes. You could opt for a less frequent flushing in such cases.”
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%
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.
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 such as the cooled transport of embryos, freezing embryos, splitting embryos (for twins) Gamete Intra-Fallopian Transfer, In-Vitro Fertilisation, Sperm injection (ICSI) (for infertile mares and stallions) and sexing embryo's are now are progressing in ability and popularity through Equine Specialist Clinics such as the Goulburn Valley Equine Clinic.
UK-born Tom Stout moved to Utrecht to become Lecturer and, later, Head of the Clinic for Equine Reproduction at the Faculty of Veterinary Sciences and Professor at Utrecht University, he explains..
Are there any risks associated with flushing donor mares? Tom: “The flushing itself is safe. We do feel it’s good for a mare to have her own foal at least once. Flushing a mare over a period of years tightens the cervix, which might induce fertility problems. This goes for every mare, also mares that are never flushed. Like I said, some mares react strongly to the interference in their cycle, but that will settle if you give it a rest for a period of time. An embryo is very rarely damaged in the flushing process, but it happens. Once the foal is born, it has a different dam, but if the recipient mare is suitable, is does not pose any risks.
A flushed embryo is transferred into the recipient mare. This mare has the responsible task of carrying, bringing forth and nurturing a foal. Naturally, the demands are high. Tom explains: “At our clinic there are fifteen recipient mares. They are healthy and in good shape and are of ample height. We prefer them having had a foal before. Of course the recipient mare’s height should match the donor mare’s height. It’s not a good idea to transfer a Friesian horse embryo in a pony mare. We know that a maiden mare’s first foal is often small. An experienced dam knows how to deal with foals. We are really happy with harness horse mares, in this respect, especially when combined with Friesian horses. Harness horse mares are relatively inexpensive, big and they usually have broad bellies. We avoid mares with behavioural issues. We want them to be friendly and easy to handle. Before they’re introduced into the group, they go into quarantine for a while. If all goes well, the chance of gestation after the transfer is 80%.
Not just the recipient mares, the embryos also are carefully monitored. Tom: “Luckily horses score a 90% average on good-quality embryos. We usually flush on the eighth day. The embryo is then between a quarter of a millimetre and half a millimetre in size. A normal embryo looks like a light-coloured, tight, transparent little ball. It’s sometimes surrounded by darker dead cells, which are often bigger than healthy cells. It goes without saying that many dead cells and delayed development of the embryo are not good signs.”
The average costs for producing an embryo transfer foal are approx $5,000 - $7,000 depending on the semen used (fresh, chilled, frozen) and usually includes 3 AI cycles, flushing and transfer fees and lease of the recip mare) excluding service fee.
Frozen versus fresh semen
Research shows there is a much bigger chance of an embryo developing with fresh semen than there is with cooled or frozen semen, a 70% versus a 35 to 50% chance. Tom: “It’s not like I advise against frozen semen, but it’s important as a breeder to be aware of the difference in success rates. It also varies enormously from stallion to stallion. Some stallions show very low frozen semen conception rates, others have a 70% success rate. In the Netherlands frozen Friesian horse semen is hardly used yet, but colleagues abroad have lots of difficulty inseminating mares with frozen semen from Friesian stallions. This might be the stallion, or the accuracy with which the semen is frozen. It’s very important to choose a diluent that fits the stallion. All stallions are different and sometimes the diluent does not match the stallion’s semen. It’s essential to try different diluents beforehand. Not everyone does, so unfortunately a lot of low-quality semen is being sold. It’s bad for a country’s reputation and for a breed’s reputation and that’s a real shame.
Like semen embryos can also be frozen. “But it is much harder to freeze an embryo than it is semen,” Tom explains. For freezing, an embryo is best flushed the sixth day. As soon as it gets any bigger, the chance of success decreases rapidly. You should check on the mare several times a day to make sure she’s flushed at exactly the right moment. Good results call for lots of experience. There are many advantages, especially for Friesian horses. You could flush the embryos of a two-year-old mare that is subsequently started and prepared for competitions. If she turns out to be successful, the embryo is there to use. There are no clear rules for bringing embryos outside Europe yet, so it’s not easy. But who knows, perhaps we can sell embryos to far away foreign countries in the future.”
Tom Stout reflects on an option in the near future: performing a biopsy on a flushed embryo without compromising its viability and having it tested for genetic disorders. Such a procedure could be of use for the Friesian horse. It will also be possible to determine the embryo’s sex. Tom: “We’re working on mapping out genetic anomalies, common also in Friesian horses. We do not know exactly which gene causes what problem nor where it is located. We have however pinpointed several genetic disorders in Quarter Horses as well as Arabians. Once you know which genes are responsible for the disorders, you can have the embryos tested for their presence. This way, you can rule out breed-related anomalies prenatally by embryo selection. Utrecht and Wageningen Universities, among others, are working on this closely together with universities outside the Netherlands. We know now in which chromosome area we’re supposed to look, but I reckon it’ll take another three to four years before we’ve located the genes and their exact location.”
Embryo at 7 days
Flushing taking place
Interesting article by thehorse.com CLICK PHOTO TO VIEW
Embryo Transfer foal Poppe fan Regal from Regal Park Stud VIC first foal of Zaretta TK by Omer R S fan Top en Twel out of surrogate Standardbred mare 'Penny' from Westvets QLD
Armani of Bonadelca with her recip mare at the Victoria Keuring 2017
Embryo transfer foal 8.5 hours old from Terarossa Friesian stud VIC