Assisted reproductive technologies (ARTs) are breeding techniques used to produce foals from subfertile mares and stallions. Some techniques, such as oocyte transfer, are primarily used for mares who are unable to carry a pregnancy to term or even produce embryos for embryo transfer.

Other techniques, such as intracytoplasmic sperm injection (ICSI), benefit subfertile and even infertile stallions as they only require one viable sperm to successfully achieve pregnancy.

Generally, assisted reproductive techniques used in horses require a donor mare to supply an egg, a recipient mare to carry the resulting foal, and sperm from a desired stallion. Harvesting oocytes from donor mares is an invasive and costly procedure, so typically ARTs are only performed if repeated breeding attempts between the desired pair have been unsuccessful.

The main ART used today with subfertile stallions is intracytoplasmic sperm injection, but it can be used for infertility in both mares and stallions. In this procedure, the oocyte is injected with a single sperm in a laboratory setting, and the resulting embryo is placed into a recipient mare or frozen for future embryo transfer.

Assisted Reproductive Technologies for Breeding Horses

Assisted reproductive technology (ART) typically refers to a group of reproductive procedures that use eggs from a donor mare to produce offspring. The main ARTs in equine reproductive are: [1]

  • Embryo transfer
  • Oocyte transfer
  • Intracytoplasmic sperm injection (ICSI)
  • Gamete intrafallopian transfer (GIFT)
  • In vitro fertilization (IVF)

Advantages and Disadvantages

The major benefit of ARTs is the ability to produce offspring from horses that could not reproduce through natural mating, artificial insemination, or even embryo transfer techniques.

Typically, the most advanced assisted reproductive techniques are used in one of two situations: [1]

  • Mares with fertility issues that prevent them from producing embryos for embryo transfer or who are unable to carry a foal to term
  • Using very poor-quality semen or semen in extremely limited supply, such as after the death of the stallion, to maximize pregnancy rates

The primary drawback of ARTs is that they require the invasive collection of oocytes (eggs) from the donor mare, demanding specialized expertise from the veterinarian. ART is also more expensive than more routine methods of breeding, because it requires specialized equipment for oocyte processing.

Mad About Horses
Join Dr. Chris Mortensen, PhD on an exciting adventure into the story of the horse and learn how we can make the world a better place for all equines.
Apple Podcasts Spotify Youtube
Mad Barn - Equine Nutrition Consultants | Mad Barn USA

Oocyte Recovery

The first step for all assisted reproductive technologies is recovery of oocytes from the donor mare. The two main methods of oocyte recovery are transvaginal oocyte pickup and flank puncture. [1][3][3]

Transvaginal oocyte pickup (OPU) of immature oocytes is becoming more common, as it is less invasive and more flexible regarding the timing of the procedure and handling of the oocytes. [2]

Mature vs. Immature Oocyte Collection

As part of the ART protocol, the veterinarian must decide whether to collect mature or immature oocytes from the donor mare. The veterinarian will select the best option based on their own skillset, equipment available, and other factors.

A mature oocyte is a fully developed egg cell that is ready for fertilization. Mature oocyte harvesting occurs in the period right before ovulation, requiring the veterinarian to track the mare’s estrus cycle closely so that harvesting occurs at the correct time point. [2][4]

Mature oocytes are also more sensitive to temperature changes, requiring immediate incubation after harvesting. [2][4] Benefits of collecting mature oocytes include: [2]

  • May improved oocyte quality, as the mare’s hormones stimulate maturation rather than artificial maturation after harvesting
  • Oocytes are easier to remove as they loosen from the follicle wall in the mature stage

Immature oocytes are undeveloped egg cells that have not yet completed the necessary stages of development to be fertilized. Immature oocytes must undergo a maturation procedure in the laboratory before they can be used for ICSI. [5]

The main disadvantage of using immature oocytes is that they can be more difficult to remove from the follicle because they tightly adhere to the follicle wall. [2][4] This can lead to ovarian tissue damage if the veterinarian has to scrape the follicle wall to extract the oocytes.

Some of the benefits of harvesting immature oocytes include: [2][4]

  • The oocytes are less affected by temperature variation after harvesting
  • This method does not require the veterinarian to track the mare’s estrus cycle

Flank Puncture Procedure

The flank puncture procedure is a surgical technique in which the ovary is accessed through the flank of the mare to retrieve oocytes.

Prior to oocyte recovery, mares receive a dose of gonadotropin, a hormone that stimulates the ovaries to produce 1 – 2 mature follicles that are ready for ovulation. [1] These follicles contain the oocytes harvested during the recovery procedure.

In horses, inducing “superovulation” (the maturation of multiple follicles) is less effective than in other species. The unique anatomy of the mare ovary usually only allows for the aspiration of only one or two mature follicles at a time per ovary per cycle. [1][5]

To perform a flank puncture, the veterinarian places a cannula in the flank of the mare. They then would manipulate the ovary into position via rectal palpation and a needle is passed through the cannula into the mature follicle. [1]

Once the follicle is targeted, its contents including the oocyte and surrounding fluid are drawn into a syringe through a process called aspiration. The collected oocyte then undergoes further examination for use in assisted reproductive techniques.

Transvaginal Oocyte Pickup (OPU) Procedure

Transvaginal Oocyte Pickup (OPU) is another type of recovery procedure where eggs are collected from the ovaries of a mare using a needle inserted through the vaginal wall. Typically, this procedure is used for gathering immature oocytes, but can also be used for mature oocytes[2][5]

OPU does not require stimulation of the mare’s ovaries, and can be performed at any point in the mare’s estrus cycle. [2] Some large operations perform OPU procedures every 14 days on their mares, to maximize the oocyte recovery. [2][4][5]

In OPU, the veterinarian inserts a specialized ultrasound probe into the mare’s vaginal canal. The probe has a channel for a needle attached to a vacuum pump. [1] After placing the probe, the veterinarian manipulates the ovary into place through rectal palpation, until the ovary is visible on the ultrasound screen.

The veterinarian then insert the needle into a follicle and activates the vacuum pump to drain it. [2] The veterinarian may also choose to fill and then drain the follicle with sterile fluid several times to try and loosen the oocytes from the follicle wall. [1][2][5][6]

The procedure is repeated for each follicle that is accessible to the veterinarian.

Oocyte Transfer

Oocyte transfer is a reproductive technique where an oocyte (egg) recovered from a donor mare is transferred to a recipient mare’s oviduct for fertilization and subsequent development.

The oviduct, also known as the fallopian tube in humans, is the tube that connects the ovary to the uterus in female mammals. This is where fertilization of an egg by a sperm typically occurs in natural mating.

In this procedure, the recipient mare’s oocytes are first removed and replaced with the oocyte from the donor mare to ensure that any offspring produced have the donor’s genetic material.

Overall, oocyte transfer has the highest success rate of the ARTs, with around 40% of oocyte transfer procedures successfully achieving pregnancy. [1][5]

Benefits of Oocyte Transfer

The main reason for using oocyte transfer is to produce foals from mares that cannot produce their own embryos. Common conditions in the donor mare that may require oocyte transfer include: [1]

Drawbacks of Oocyte Transfer

Disadvantages of oocyte transfer procedures include: [1][3][5]

  • Requires good semen quality to maximize the chance of pregnancy
  • There is a risk that not all of the recipient mare’s oocytes are removed, resulting in an embryo with the recipient mare’s genetics
  • Only able to implant as many oocytes as there are recipient mares synchronized to the donor mare
  • High risk of post-breeding endometritis in the recipient mare because the smooth muscle relaxants required for ovary manipulation reduce semen clearance after breeding

 

Oocyte Transfer Procedure

There are two main types of oocyte transfer procedures, depending on the type of recipient mare used. Veterinarians and owners have the option of using either an actively cycling recipient mare, or a non-cycling mare. [1][5][7]

Actively Cycling Mare

If the recipient mare is actively cycling, then the recipient is synchronized to the donor mare, so that their estrus cycles occur at approximately the same time. After synchronization, the mares receive a dose of gonadotropin on the same day. [1]

Once the mature follicles are ready in both mares, they undergo an oocyte recovery procedure. For the donor mare, the oocyte recovery procedure provides the oocyte for implantation in the recipient.

The recipient mare’s recovery procedure removes any oocytes in her mature follicles, preventing her oocytes from entering her reproductive tract after ovulation. [1]

Non-Cycling Mare

Recipient mares that are not actively cycling receive estrogen treatment prior to the transfer and progesterone afterwards. These hormones prime the reproductive tract to support an embryo, even if the mare’s ovaries are not active. [1]

The major benefits of using non-cycling recipient mares include: [1]

  • Eliminates the need to synchronize the donor and recipient mare
  • Recipient mare does not need an oocyte recovery procedure as part of the preparation for oocyte transfer

Oocyte Implantation

Once the recipient mare is prepared and the donor oocyte is acquired, the recipient mare undergoes surgical implantation of the donor oocyte. For cycling mares, the surgical procedure occurs at around the time the mare ovulates, so that her hormonal balance can support the developing embryo. [1]

To implant the oocyte, the veterinarian performs a standing abdominal surgery by making an incision in the donor mare’s flank. The veterinarian then locates the mare’s oviduct and passes the donor oocyte into the oviduct through a pipette or catheter. [1][5][7]

The veterinarian will then inseminate the recipient mare with the desired semen through a typical artificial insemination procedure. [1][5]

In Vitro Fertilization

In vitro fertilization (IVF) is the process of fertilizing a harvested oocyte with sperm within a laboratory setting. [1]

In this procedure, the harvested oocyte is placed in a Petri dish with sperm, allowing the sperm to fertilize the oocyte. Compared to other species, in vitro fertilization has been very unsuccessful in horses.

Equine sperm has a poor ability to penetrate through the oocyte wall on its own, necessitating more invasive procedures such as intracytoplasmic sperm injection (ICSI). [1][8]

Intracytoplasmic Sperm Injection

Intracytoplasmic sperm injection (ICSI) is the main alternative to in vitro fertilization available for horses. ICSI is becoming increasingly popular as a treatment for subfertility, particularly in sport horses. [6]

In this procedure, veterinarians inject a harvested oocyte with a single sperm under a microscope, bypassing the requirement for the sperm to penetrate the oocyte wall on its own. [1] The fertilized oocyte is then incubated allowing development, with ultimately the embryo being transferred to a recipient.

Benefits of ICSI

ICSI can benefit both mares and stallions, allowing them to produce foals that may not have been produced under traditional circumstances. Mares that benefit from ICSI include: [2][6]

  • Mares with chronic infertility from endometritis or damage to the reproductive tract
  • Competition mares, since ICSI-derived embryos can be frozen for later use
  • Older mares experiencing age-related fertility issues

Additional benefits of ICSI for mare owners include: [1][6][8]

  • Ability to achieve multiple pregnancies from a single donor mare oocyte recovery procedure, if multiple oocytes are harvested
  • Do not need to surgically implant the oocyte into the recipient mare
  • More flexibility in synchronizing of donor and recipient mares

For stallion owners, benefits of ICSI include: [2][3]

  • Maximizing the use of semen in limited supply due to death or old age of a high-value stallion
  • Ability to use frozen semen, which can be transported and stored for long periods
  • Ability to split a single semen dose into multiple doses, potentially producing multiple pregnancies
  • Maximizes the chance of pregnancy in stallions with poor sperm quality, because the procedure only requires one viable sperm

Drawbacks of ICSI

The main disadvantages of ICSI are related to the cost of the procedure, and the requirement for the reproduction veterinarian to have specialized training and equipment available. [2]

Success rates are highly variable, and range from 21 – 55% in different clinical studies. [1]

Intracytoplasmic Sperm Injection Procedure

The veterinarian first examines the harvested oocytes to ensure they are at the correct stage of development for the ICSI procedure. After confirming oocyte maturity, they perform a “swim-up” procedure to select the sperm that will be implanted in the oocyte.

The “swim-up” procedure involves placing a thawed semen straw in an incubator for around 15 minutes. The veterinarian then removes the very top sample of the semen straw, because this portion should contain the most actively motile sperm. [2][5]

From this sperm population, the veterinarian selects a single sperm and draws it into a specialized pipette. They insert the pipette into the oocyte under the microscope and inject the sperm. The fertilized oocyte is further incubated for approximately 7 – 9 days, during which time it matures into an embryo.

Once the embryo is sufficiently developed, the veterinarian transfers the embryo into a recipient mare using a transcervical procedure similar to artificial insemination. [2] Alternatively, the fertilized oocytes can be transferred surgically into a recipient mare as soon as the ICSI procedure is completed. [1]

Gamete Intrafallopian Transfer

Gamete intrafallopian transfer (GIFT) is another possible alternative to IVF for horses. In this procedure, both the oocytes and sperm are surgically transferred into the oviduct of a recipient mare, in a procedure similar to oocyte transfer. [1]

GIFT addresses some of the potential disadvantages of oocyte transfer, as lower quality semen can be used, and there is a reduced risk of post-breeding endometritis in the recipient mare. [1][8][9]

However, GIFT has a low success rate with frozen and fresh-cooled semen. Fresh semen is generally required for the procedure, which limits its clinical application. [1][5][8][9]

Currently, GIFT is uncommon for breeding horses due to the relative availability and success of ICSI. [8]

Summary

  • Assisted reproductive technologies used for breeding horses can result in foals from otherwise subfertile and even infertile mares and stallions
  • Most ARTs require a donor mare to supply an oocyte (egg) and a recipient mare to carry the embryo
  • ICSI is increasing in popularity as it benefits both infertile mares and stallions, and has few disadvantages

Is Your Horse's Diet Missing Anything?

Identify gaps in your horse's nutrition program to optimize their well-being.

References

  1. Brinsko. S. P. and Blanchard. T. L., Eds.Manual of Equine Reproduction, 3rd edition. St. Louis, Mo: Mosby/Elsevier. 2011.
  2. Dascanio. J. J. et al., Eds. Equine Reproductive Procedures, 2nd edition. Wiley-Blackwell. 2021.
  3. Squires, E. L. Perspectives on the Development and Incorporation of Assisted Reproduction in the Equine Industry. Reprod. Fertil. Dev. 2019. View Summary
  4. Carnevale, E. M. Advances in Collection, Transport and Maturation of Equine Oocytes for Assisted Reproductive Techniques. Veterinary Clinics of North America: Equine Practice. 2016. View Summary
  5. McKinnon, A. O. et al. Equine Reproduction. Wiley-Blackwell, 2010.
  6. Stout, T. a. E. Clinical Insights: Assisted Reproductive Technologies. Equine Veterinary Journal. 2019. View Summary
  7. Carnevale, E. M. & Maclellan, L. J. Collection, Evaluation, and Use of Oocytes in Equine Assisted Reproduction. Veterinary Clinics of North America: Equine Practice. 2006. View Summary
  8. Hinrichs, K. Assisted Reproduction Techniques in the Horse. Reprod. Fertil. Dev. 2013. View Summary
  9. Carnevale, E. M. Clinical Considerations Regarding Assisted Reproductive Procedures in Horses. Journal of Equine Veterinary Science. 2008.