Embryo transfer (ET) is an advanced reproductive technology that allows breeders to produce multiple offspring from a single mare in a given year. This procedure involves taking an embryo from a donor mare, and depositing it in the uterus of a recipient mare who carries the pregnancy to term.

ET is becoming increasingly common in equine reproduction, and many equine veterinarians are able to perform the technique for horse breeders. However, embryo transfer requires extensive management of both the donor mare and the surrogate mare to have the greatest chance of pregnancy.

To perform embryo transfer, the donor mare is typically impregnated by artificial insemination to produce a fertilized embryo. Between Day 7 – 9 of gestation, the veterinarian flushes the embryo from the donor mare’s uterus, and prepares it for implantation into a synchronized recipient mare. The recipient mare’s ovulation must occur within 4 to 8 days of the donor mare’s ovulation.

Embryo transfer has a high success rate, with over 90% of embryo transfers successfully implanting in the recipient mare. [4] This article will discuss the process of retrieving and transferring embryos, as well as benefits and considerations of this reproductive technology.

Embryo Transfer in Horses

Embryo transfer is the process of taking an embryo from a donor mare, and transferring it into a recipient mare who will carry the pregnancy to term. It is essentially a form of surrogate pregnancy for horses.

The ability to have a different mare carry a pregnancy has many benefits, including: [1][2][3]

  • Producing foals from performance and show mares currently competing
  • Having multiple foals from the same mare in one year
  • Ability to produce foals from mares with health issues or musculoskeletal problems, which prevent them from carrying their own foals
  • Ability to produce foals from mares with reproductive health issues that prevent them from sustaining a pregnancy

However, not all breed organizations allow registration of embryo transfer foals, including the Jockey Club, which registers racing Thoroughbreds. [1] Horse breeders must investigate their breed’s specific regulations around assisted reproductive technologies before pursuing embryo transfer.

Stages of Embryo Transfer

There are three stages of embryo transfer that must be successful for pregnancy to occur. These stages are:

  • Pregnancy in the donor mare to produce the desired embryo
  • Flushing of the embryo from the donor mare
  • Implanting the embryo into a synchronized recipient mare at the same stage of estrus as the donor mare

Recent advances in embryo transfer include the ability to cool or freeze embryos for storage or transport. Embryo cooling is widely accessible, and can be performed by most equine reproductive veterinarians. Embryo freezing is more complex and may only be available from certain specialists.

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Donor Mare Management

Most donor mares are bred using artificial insemination, as this allows the veterinarian to precisely identify breeding dates and track the mare’s estrus cycle. [1]

Once fertilization occurs, the veterinarian continues to track the embryo’s development to identify the best time to perform a uterine flush, which removes the embryo from the donor mare. [1]

Embryos flushed around 7 days after ovulation are usually the ideal size for embryo transfer, however its size can vary depending on the mare’s age, stallion’s fertility, timing of breeding, and individual variation in the reproductive tract of the mare. [1]

Ultimately, the veterinarian must decide the best time to flush the embryo based on the results of ultrasounds of the reproductive tract.

Uterine Flushing

Historically, surgical techniques were the only available option for embryo recovery. These procedures were invasive, impractical and required general anesthesia of the donor mare. [4] Advances in embryo transfer techniques have led to the development of non-surgical procedures for embryo recovery, such as uterine flushing. [4]

To perform a uterine flush, the veterinarian introduces specialized tubing into the donor mare’s cervix, and connects the tubing to a bag of flush medium specially designed to support an embryo outside of the uterine environment. [5] Approximately 1 – 2 liters of flush medium enters the uterus, and then is allowed to flow back out of the uterus through a filter. [5]

The veterinarian will repeat this process 3 – 4 times to ensure that all embryos are flushed from the uterus, reducing the risk of embryos implanting in the donor mare and causing pregnancy. [5] Prostaglandins can also be administered after embryo collection to reduce the risk of pregnancy in the donor mare. [1][5]

After the uterine flush is complete, the contents of the embryo filter are examined to confirm collection of the embryo. After identifying an embryo, it is placed into a straw, which is a specialized, slender plastic tube that provides a sterile environment and is designed for holding and transferring embryos.

The embryo is then washed several times in flush medium to remove any contaminants or cellular debris. The embryo is then transferred into a petri dish for temporary storage while the recipient mare is prepared. [5]

Embryo Evaluation

After flushing, some veterinarians choose to evaluate the embryo to ensure it is viable and has a good chance of producing a successful pregnancy. Features that are assessed during an embryo examine include: [2][5]

  • Size of the embryo
  • Shape and symmetry
  • Developmental stage of the embryo
  • Whether the embryo wall is intact

Most embryos are good or excellent quality, likely because poor quality embryos cannot move through the oviduct properly and therefore are not able to be flushed. [3][5]

Embryo Biopsy

Embryo biopsy is another method of evaluating the embryo, testing for specific genetic traits that the embryo carries. Most embryo biopsies focus on serious hereditary diseases and are performed when one or both parents are known to be a carrier of the disease.

Example of genetic diseases that are tested for include: [5]

Embryo biopsy can also determine the sex of the embryo, for breeders who desire to produce a colt or filly specifically. [5] Embryo biopsy does not affect the success rate of embryo transfer. [6]

Recipient Mare Management

Recipient mares require special management both before and after the embryo transfer occurs. Prior to the transfer, they must be synchronized with medication, or have their estrus cycle tracked closely to ensure they will be a suitable recipient for the donor mare.

After the transfer, they require specific management to ensure successful implantation of the embryo and subsequent pregnancy.

Synchronizing a Recipient Mare

Recipient mare management is very complex, as the recipient must be “synchronized” such that their estrus cycle matches the estrus cycle of the donor mare. The two main methods of synchronization are: [1][3]

  • Selecting specific recipient mare(s) for the donor, and using medication to adjust their estrus cycle to match the donor mare
  • Maintaining a large herd of recipient mares, so that there is likely to be an appropriately synchronized recipient mare available for the donor mare at any given time

The ideal level of synchronization is having the two mares ovulating within 4 – 8 days of each other. [1] Most veterinarians recommend having at least three potential recipient mares available, so that the transfer is successful even if one or more of the mares is out of sync or develops a reproductive problem. [1]

To synchronize the mares, progesterone is administered daily to the donor mare and recipient mares starting on the same day. This medication maintains their reproductive cycle in diestrus, the phase immediately after ovulation. [1]

After 8 – 10 days of progesterone, the medication is stopped and a dose of prostaglandin is given, which triggers the mares to begin their cycle again. [1]

The veterinarian then monitors the developing follicles for synchronicity, and gives ovulation-stimulating medications to the mares once their follicles have developed sufficiently. [1][3] The hope is that at least one of the three potential recipients will be a suitable candidate for embryo transfer.

Recipient Mare Herds

Many veterinary practices who specialize in equine reproduction will maintain their own herd of recipient mares for the benefit of their clients. Large breeding farms may also maintain their own recipient mare herd.

In these situations, each recipient mare’s estrus cycle is tracked closely using ultrasound so that the managing veterinarian knows which day each recipient will ovulate.

With a large enough recipient mare herd, a recipient mare should be available at all times without having to specifically synchronize the recipient to the desired donor mare. [1][3]

Studies show that embryos implanted into naturally ovulating mares have similar success rates to medically induced ovulation. [7]

Embryo Transfer Procedure

Similar to embryo recovery, embryo transfer was once an exclusively surgical procedure. [4] Many veterinarians thought that non-surgical procedures would not be successful, as manipulating the cervix would release prostaglandins that would terminate the pregnancy. [1]

Further research into this specific question identified that manipulating the cervix had no effect on the ability of recipient mares to become pregnant, allowing for development of non-surgical techniques. [1]

Transcervical Embryo Transfer

The main technique used today is transcervical embryo transfer. [5] To transfer the embryo, the veterinarian uses a specialized pipette or delivery device which is inserted through the cervix of the recipient mare.

Some veterinarians may use cervical forceps to bring the cervix into a more accessible position prior to inserting the embryo transfer device. [8] The goal of embryo transfer is to deposit the embryo into the uterine body or into a uterine horn. [2][5]

After placement, the veterinarian removes the embryo transfer device and rinses it in flush medium for examination under the microscope. In some cases, the embryo becomes trapped in the delivery device, so examination of the rinsing fluid confirms placement of the embryo. [5]

Management after Transfer

Management of recipient mares after the embryo transfer is somewhat controversial, and no one protocol is firmly established. [1] The main goals of management are to make the recipient’s reproductive tract hospitable for the embryo, and to encourage the recipient’s body to recognize the pregnancy.

To achieve this, management strategies may include: [1][5]

  • Anti-inflammatory medications such as a banamine, to reduce inflammation caused by the embryo transfer procedure
  • Antibiotics to treat any potential bacterial contamination of the reproductive tract
  • Supplemental progesterone to adjust the hormonal balance to a pregnancy-maintaining state

Embryo Management

Current advancements in cooling and freezing embryos allow them to be transported long distances prior to implanting in a recipient mare. The ability to transport embryos has several benefits for horse breeders: [1][5]

  • Recipient mares can be maintained at specialty centers, rather than each breeder maintaining their own recipient mare herd
  • The cost of shipping embryos overseas or between countries is significantly less than shipping a mare
  • Increased access to unique genetics or combinations of genetics not available in their current geographical location

Cooled Embryos

For shipping cooled embryos, the veterinarian places the embryo into a vial of embryo holding medium. The embryo is then slowly cooled using a specialized cooling shipment container, similar to the ones used for fresh-cooled semen. [1][5]

Once the embryo arrives at its destination, it is removed from the embryo holding medium and prepared for implantation. Studies show that implantation success rates for immediately transferred embryos and cooled transported embryos are similar, averaging between 75 – 80%. [1]

Frozen Embryos

Frozen embryos are becoming more common, but is not currently a standard procedure offered by all reproductive veterinarians. [1] Major benefits of embryo freezing include: [5][9]

  • The recipient mare does not have to be synchronized to the donor mare
  • Ability to import or export embryos
  • Long-term storage of embryos, including after death of the mare
  • Collection of embryos from competition mares during the off-season, for implantation during the following breeding season

Liquid nitrogen is the most common coolant for freezing embryos, as it does not require specialized equipment and most reproductive veterinarians already have liquid nitrogen on hand for freezing semen. [5]

To use a frozen embryo, the veterinarian removes it from the liquid nitrogen storage and thaws it in a warm water bath, before implanting it in the recipient mare. [5] Success rates of frozen embryo implantation range from around 65 – 85% depending on the study. [1][10]

Summary

  • Embryo transfer involves flushing an embryo out of a donor mare, and placing it in a recipient mare who will carry the pregnancy
  • Both the donor and recipient mares require extensive management to ensure their estrus cycles synchronize
  • Embryo cooling and freezing is available for storage or transport of embryos

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References

  1. McKinnon, A. O. et al. Equine Reproduction. Wiley-Blackwell, 2010.
  2. Brinsko. S. P. and Blanchard. T. L., Eds.Manual of Equine Reproduction, 3rd edition. St. Louis, Mo: Mosby/Elsevier. 2011.
  3. Dordas-Perpinya. M. and Bruyas. J. F. Practical Aspects of Equine Embryo Transfer. Translational Research in Veterinary Science. 2019.
  4. Allen. W. R. (Twink) and Wilsher. S. Historical Aspects of Equine Embryo Transfer. Journal of Equine Veterinary Science. 2020. View Summary
  5. Dascanio. J. J. et al., Eds. Equine Reproductive Procedures, 2nd edition. Wiley-Blackwell. 2021.
  6. Guignot. F. et al. Establishment of Pregnancies after Transfer of Biopsied Equine Embryos. Journal of Equine Veterinary Science. 2012.
  7. Vicioso. R. et al. Influence of Hormonal Induction of Estrus and/or Ovulation on Embryo Transfer Success. Journal of Equine Veterinary Science. 2023.
  8. Ramirez. H. et al. Embryo Transfer Technique Affects Pregnancy Rates in Recipient Mares. Journal of Equine Veterinary Science. 2023.
  9. Squires. E. L. and McCue. P. M. Cryopreservation of Equine Embryos. Journal of Equine Veterinary Science. 2016.
  10. Araujo. G. et al. Pregnancy Rates after Vitrification, Warming and Transfer of Equine Embryos. Animal Reproduction Science. 2010.