HOW TO INCORPORATE ASSISTED REPRODUCTIVE TECHNOLOGIES


fluid to enter prior to the oocyte to decrease the chance of it sticking to the sides. Immature oocytes collected in this manner will either be placed directly into mat- urational medium with hormones or a holding me- dium until being transferred into maturation medium the next day. The choice will be based upon collection timing along with the anticipated time for sperm injection.


Complications


Although the procedures described above are becom- ing more common as private practices implement them within their reproductive programs, it is im- portant to remember that they are invasive and should not be taken lightly. A few reports have described various complications including ovarian hemorrhage, peritonitis, colic, rectal hemorrhage, and ovarian abcesses19–22 following transvaginal as- piration procedures. As discussed previously, the peritoneal cavity is accessed during this procedure, therefore all equipment should be sterilized and the environment kept as clean as possible. Utilization of uterine or vaginal culture may be warranted in some cases as mares with an active bacterial endo- metritis, urine pooling problems and/or pneumova- gina are at particular risk for iatrogenic bacterial contamination during the TVA process. If a mare is encountered with a fibrotic cervix or cervical ad- hesions, and a small amount of anechoic uterine fluid, the author prefers to avoid endometrial cul- ture as to not introduce bacteria to a closed uterine environment. Originally, these procedures were devised for older problem mares without the inten- tion of continual embryo donation or carrying a foal to term. More research is needed to under- stand the longer-term effects on fertility, fibrotic changes to ovarian stroma, and overall effects of the primordial follicle pool in a younger population of mares with varied reproductive procedures intended in their future.23,24


Obtaining Oocytes from the Mare Postmortem


When amare dies of natural causes or is euthanized, her ovaries can be removed and oocytes collected from identifiable antral follicles. These oocytes can be in vitro matured and undergo sperm injection to create embryos, thus preserving valuable genetics. Postmortem ovary shipment has been available commercially since the early 2000s and pregnancies and subsequent foals have been successfully born from both oocyte transfer and ICSI produced em- bryos.25,26 The current practice is for referring vet- erinarians to remove the ovaries as soon as possible and ship them directly to a facility offering ovarian scraping and ICSI procedures. Occasionally, the euthanasia is coordinated, as with a chronic condi- tion such as laminitis so that the mare is within an ideal follicular phase. For these cases, it is recom- mended to provide prostaglandin administration and wait 4 or 5 days later for humane euthanasia in order to capitalize on the late diestrus/early estrus


356 2019  Vol. 65  AAEP PROCEEDINGS


follicular phase. More often than not, euthanasia occurs as an emergency and coordinating the last- minute logistics of immediate ovary removal, pack- aging, and shipping can be overwhelming and sometimes impossible. It has been suggested that practitioners adept with oocyte handling can effec- tively remove the ovaries and harvest oocytes at their facilities for shipment of just the oocytes to a lab offering ICSI services in a less emergent man- ner, achieving oocyte removal from the decaying tissue quicker than shipping the whole ovary. Pilot data collected recently has demonstrated signifi- cantly improved maturation rates in oocytes col- lected within 2 hours of ovary removal and “shipped” in holding media compared to a control ovary “shipped” in routine fashion prior to oocyte collection (72% vs. 22%; Hatzel JH, unpublished data, 2018). Therefore, the author does feel it will be beneficial for practitioners to learn the process of oocyte har- vesting at their location in order to ship just the collected oocytes for improved success. Regardless of the method chosen, early and continual commu- nication with the owner and ICSI team will help ease some of the stressors involved with ensuring not only the ovaries/oocytes are transported cor- rectly, but also that appropriate semen (either cooled or frozen) arrives no later than the day after the ovaries/oocytes. The process of ovary removal, packaging and ship-


ping has been previously described in detail27 and will not be described here. In general, ovaries be- ing shipped in close proximity to the receiving lab (within 2 hours) should be kept close to body tem- perature (37°C). More often than not, ovaries are shipped from a further location ( 2 hours) and it is recommended to ship them at a cool room tempera- ture (17–22°C). It is never recommended to ship them on ice. Any container that will consistently hold the temperature for the length of travel will suffice including: a Styrofoam box, a passive cool- ing device utilized for semen transport, or a thermo- regulating portable incubator. Approximately half the population of oocytes collected from ovaries will mature in vitro for ICSI. From that group, 30%– 50% will typically begin forming an early embryo and about half of the early embryos that begin form- ing will continue to develop to a transferrable blastocyst.27


Personnel


The most common method currently utilized by lab- oratories offering this service is scraping individual follicles to collect the oocytes by hand. In general, with a team of 3 people, it usually takes the author’s group about an hour to complete the entire proce- dure with an additional 30 minutes of cleanup. However, performing this technique can be achieved by a single person, which requires devotion of at least 2 hours of time. With a team, one or two members adept at scraping can process the ovaries while a third member with experience in identify-


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