Ovum Pickup (OPU) and Intracytoplasmic Sperm Injection (ICSI) are advanced reproductive techniques used in equine breeding to overcome fertility challenges or increase productivity. These procedures involve retrieving oocytes (eggs) from a mare’s ovary and fertilizing them in a laboratory to produce embryos for future use.
OPU is the procedure whereby the oocytes are collected from the mare’s ovaries. The mare is withheld from food for approximately 6-12 hours. The procedure is performed following the preparation of the mare, including sedation, anti-inflammatories, antibiotics, muscle relaxants, and, in some cases, an epidural. A urinary catheter is placed, and her perineal region is cleaned.
A specialized ultrasound probe with an attached needle guide is inserted into the vagina (a process called Transvaginal Follicular Aspiration - TVA). The veterinarian then identifies the ovary rectally and positions it against the vaginal wall and ultrasound probe. Follicles 10mm and larger are identified on the ultrasound and punctured by inserting the needle through the vaginal wall into the follicle. The follicular fluid is aspirated, and the follicle wall is repeatedly scraped, flushed, and the fluid removed to collect the oocyte adhered to the follicular wall. This process is repeated for each
ovary and should not take longer than 60 minutes to complete.
The fluid collected from this procedure is taken into the laboratory at Westvets and filtered. Oocytes are identified, washed, and packaged for transport to another laboratory facility for maturation and ICSI procedures. The expected oocyte recovery rate from a follicle is, on average, 50-60%, although this can range from 0%-100%.
The oocytes collected from the ovary are not yet ready for fertilization and require maturation to reach a development phase where they can be fertilized. This maturation process requires specific conditions and takes an average of 30 hours. Not all oocytes will mature, and a current rate of 60% maturation is expected in normal oocytes.
Once the oocytes are matured, they are injected with a single sperm. This process requires a specialized microscope with high magnification, equipped with a micromanipulator and a sperm micro-injector. The oocyte is held still to allow the injection of an individual sperm into the cytoplasm. Once injected, these fertilized oocytes are cultured, or grown, under special laboratory conditions for 7-9 days, after which they will be developmentally equivalent to a day 5-6 embryo. Once developed, the embryos are frozen for future transfer. Being small, these embryos endure the freezing process well (unlike day 7-9 embryos in an ET program). The embryos are then stored indefinitely and only thawed for transfer into a recipient mare when convenient.
Fresh, chilled, or frozen semen can be used for the ICSI process. It is required to be present at the ICSI lab on the morning of the procedure, approximately 1-2 days after the OPU session. Only quality motile sperm are selected for the process. A very small volume of semen is needed. A single 0.5 ml semen straw can be cut into multiple sections (8-10), with only a single section being defrosted for use, allowing the remaining semen to stay frozen. This small amount of semen can be useful for multiple mares if OPU is conducted on the same day.
Not all semen is suitable for the ICSI procedure. Semen that contains DNA abnormalities may not be able to fertilize an oocyte or develop an embryo. It is crucial that a mare owner obtains permission from the semen owner to use the semen in the ICSI process. Some stallions have restrictions against the use of semen in this manner. Owners must also ensure they can register their foal with the relevant society before undertaking this process. Many societies require pre-approval prior to the OPU/ICSI process.
On average, an oocyte is collected from a follicle 60% of the time, although this can vary from 0% to 100%. Of these, a current rate of 60% maturation is expected. The overall chance of one or more frozen embryos from a single OPU session is 60%. To clarify, if five OPU-ICSI sessions are performed on a mare, 2 attempts may be unsuccessful, while the remaining 3 successful sessions should produce embryos.
Once an embryo is transferred into a suitable recipient mare, there is a 70-80% chance of establishing a pregnancy. Owners should be aware that there is a 15% pregnancy loss rate by 45 days gestation, which is slightly higher than a non-ICSI pregnancy.
The actual OPU procedure should not take longer than 1 hour. As with any veterinary procedure, there are risks to consider before performing the OPU technique. Although uncommon, possible complications include rectal tears, rectal, vaginal, or ovarian bleeding, rectal and ovarian abscesses, and peritonitis, which, in the worst case, can lead to the death of the mare. A small number of mares can develop a mild fever, slight discomfort, or a decreased appetite for a few days following OPU. It is recommended that the mare has a few days of rest following the procedure.
Despite cleaning the mare, the transvaginal probe can be exposed to bacteria as it passes through the entrance of the vagina. Thus, the needle transits through a potentially non-sterile environment as it is extended from the guide through the vaginal wall into the peritoneum and ovary. Mares with known pseudomonas infections of the reproductive tract are recommended to have a clear swab prior to the OPU procedure to decrease the risk of spreading this infection.
The donor mare will be treated with appropriate anti-inflammatories and antibiotics. The procedure is performed by our professionally trained staff and will be stopped if deemed too risky to continue. The occasional maiden mare may have tight ovarian ligaments that prevent the safe collection of oocytes from the ovary via the described technique. Any insurance against these risks is the responsibility of the owner.
As mentioned earlier, not all oocytes will mature, and of those that do, not all will develop into viable embryos. Having enough follicles at the start of the procedure will increase the chance of producing a viable embryo. The mare’s ovaries should be ultrasounded the week prior to the procedure and ideally have at least 15 follicles between 10mm and 35mm. If the mare has fewer follicles, postponing the procedure until she has further follicular growth may be worthwhile; however, this can be discussed with our veterinarians. It may be helpful to place a mare under lights for 6 weeks prior to her OPU session to help with follicular growth.
Depending on how many follicles a mare develops and how quickly she recovers from the procedure, an interval of at least 3 weeks between two OPU sessions on the same mare is regularly achieved.
Oocytes can be retrieved from mares of any age that are developing follicles on their ovaries, but age-related differences affect the efficiency of embryo production. The optimal age for mares to produce embryos through OPU and ICSI is between 5 and 15 years. Mares younger than 5 years old show a slightly lower rate of embryo production. In older mares, there is a reduction in the number of follicles and recovered oocytes, and the developmental potential of these oocytes to reach a usable embryo stage is lower. Nonetheless, embryos may still be successfully produced from older mares, possibly up to their mid-twenties.
A lot of research is directed at the impact of the health and diet of the mare on the quality of the oocytes and ultimately the production of viable embryos. As common sense would dictate, a mare in good body condition, with no chronic diseases, would potentially produce more and better-quality embryos than a poorer counterpart.
There are certain factors to consider when deciding on an advanced breeding technique for your mare. It is important to remember that no breeding technique can overcome inherent poor oocyte quality or DNA abnormalities of the oocyte or sperm.
Unfortunately, sudden death or a terminal illness or injury could impact your mare. In such cases, elective ovary harvest immediately after death or euthanasia for use in the ICSI program can occur. As mentioned above, mare health can play an important part in oocyte quality and embryo production and is an important consideration when deciding if this is a possibility for your mare. Coordination between ovary collection, semen availability, transport, and ICSI laboratory staff is required. For more information, please contact our WestVETS Reproduction team.
