HOW TO TREAT THE SUB-FERTILE MARE


Providing a consistent environment can be achieved by using a shipping container intended for semen. A passive cooling devicea,b with the coolant cans removed, a cooler insulated container, or a sim- ple Styrofoam box with added layers of insulation are viable options. A couple of 1-L bags of room temperature saline can be added to provide protec- tion and help to stabilize the ambient temperature. Ideally, a thermoregulating portable incubatorc could be set to an ultra-precise temperature and maintain a reliable environment for shipping post- mortem ovaries. Newer units of this incubator of- fer the convenience of setting the two separate chambers within the device at different tempera- tures. This device would be very useful in a prac- tice offering postmortem oocyte extraction or additional assisted reproductive procedures.


Stallion Choice


Finally, a key step to consider from the very begin- ning of this process is stallion selection and semen shipment. Although equine oocytes have been suc- cessfully cryopreserved and foals have been pro- duced, the efficiency is not high. Therefore, we recommend fertilization of the collected oocytes by ICSI, even if the embryos are to be cryopreserved. Consequently, a stallion or stallions must be se- lected for sperm injection. If the collected oocytes successfully mature, sperm will be needed for ICSI by approximately 24 hours after oocyte harvesting. If possible, frozen semen should be organized to arrive either with the ovaries or the following day. We often recommend two straws, to allow one for a backup, but this may need to be adjusted based on the specific straws. Cooled/shipped semen may of- fer better results and can be collected the day of ovary arrival and shipped to arrive the next day, prior to the sperm injection procedure. A limited amount of cooled semen is needed. One advantage for ICSI is that a mare owner may choose more than one stallion to inject individual oocytes; however, we generally do not recommend picking more than two stallions. Although the ICSI procedure uses mini- mal amounts of sperm, good-quality semen from which to select sperm for oocyte injections is still essential for optimal results.


Available Facilities


As of today, there are two main facilities within the United States and a facility in Europe with pub- lished results, offering commercial postmortem ovary processing with an ICSI procedure. Colorado State University7 and Texas A&M University4 have had the longest running programs and have been paramount in conducting research that has led the way to successful pregnancies and foals born from oocytes collected from postmortem ovaries, and a similar program is available for the European com- munityd. More facilities are offering the oocyte harvesting aspect of excised postmortem ovaries and will subsequently ship the oocytes to a facility offer-


184 2016  Vol. 62  AAEP PROCEEDINGS


Fig. 3. A bone curette is utilized to scrape the interior surface of visible follicles.


ing ICSI services. Location within the country should dictate which facility is chosen, given that the race against time is a big factor toward success.


3. Results


What to Expect Although beyond the scope of this article, below is a brief overview of the overall process as previously described.3 Upon arrival, the temperature is im- mediately checked within the fluid surrounding the ovarian surface and recorded. The ovaries are then rinsed with warmed saline or a complete embryo flush medium to gently remove potential organic debris. The surface of the ovary is examined and loose parenchyma and remaining oviductal tissue is removed to facilitate easier access to palpable folli- cles. Palpable follicles are incised with a scalpel blade and the follicular fluid collected within a petri dish. The interior of each follicle is carefully scraped with a bone curette of a size proportional to the follicular dimensions (Fig. 3) to physically de- tach the cumulus oocyte complex, which has unique anatomical features and a tight attachment to the follicular wall.10 After each visible follicle has been processed, the ovary is sliced into sections to expose any additional follicles for scraping. Although time consuming, this procedure has provided the best method to collect complete cumulus oocyte com- plexes that result in competent oocytes for ICSI.11 The most optimal scenario for success is when a practitioner is comfortable with the process of har- vesting oocytes from postmortem equine ovaries prior to shipping. Once obtained by the practitio- ner, immature oocytes can be placed within a suit- able holding medium. One such published medium is 40% M199 containing Hank’s salts and HEPES, 40% M199 containing Earle’s salts and 20% fetal bovine serum, and 25 ugmL-1 gentamycin (EH me- dium), which can be maintained at room tempera-


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