HOW TO TREAT THE SUB-FERTILE MARE


ity for some practices. It is a huge undertaking to attempt to set up an equine ICSI laboratory, and even when set up, new laboratories are often not successful. If an effective laboratory is available, my strong suggestion is to ship oocytes to that lab- oratory; if no ICSI laboratory is available, consider shipping oocytes internationally, performing OT, or recommending cloning. International shipment of immature oocytes is a relatively simple procedure. Oocytes are placed in a suitable passive insulated devicea, set up at room temperature,1 and shipped via a standard overnight delivery service. Typically, international shipment by a delivery service will take 2 days. At Texas A&M, we have had good blastocyst rates with re- search oocytes shipped from France, with a ship- ment time of approximately 50 hours. However, if resulting embryos are to be transferred for preg- nancy, the health regulations can be complex. In- ternational oocyte shipment requires meeting the same regulations set up for import of semen and embryos into the country in which the laboratory is established, and thus often quarantine and testing of the donor mare is necessary for each oocyte har- vest. For the United States, a new import permit is needed for each shipment. When choosing an ICSI laboratory to which to


send oocytes, the practitioner should ask what the laboratory’s success rate is in terms of percentage of blastocysts achieved per injected oocyte and the on- going pregnancy rate (with heartbeat) after trans- fer. It would also be good to know the numbers of ICSI cases performed (one case referring to all the oocytes collected from one mare in one session). Currently, there are only a few laboratories in the world that have published effective equine ICSI re- sults (i.e., 25% blastocysts per injected oocyte when using research mares and stallions); others report good rates anecdotally. The efficiency de- creases when clinical work is performed, due to mare age, cause of subfertility, and variation in sperm quality—the rate of blastocyst development per injected oocyte can be much lower depending on type of clinical caseload, and still be clinically useful. Now that ICSI is becoming more accepted as a technique for equine clinical reproduction, many larger equine reproduction centers are attempting to establish this technique. Remember that anyone with the capability of injecting a sperm into an egg with a micromanipulator can say that they are doing ICSI, but the important thing is whether a viable embryo results from the procedure. For some rea- son, production of embryos for equine ICSI seems to be more difficult, or at least technically different, than that for other species. Technicians experi- enced in human ICSI may produce no blastocysts when performing equine ICSI.


Oocyte Recovery and Shipment for ICSI


ICSI is more efficient when immature oocytes are recovered from all immature follicles present on the


mares’ ovaries, followed by in vitro maturation of the recovered oocytes. The practitioner should strive to attain a 50% or greater oocyte recovery rate on transvaginal aspiration of immature follicles, as- pirating all follicles greater than 5 mm in diameter. This typically results (in Quarter-type mares) in aspiration of approximately 12 follicles and recovery of approximately six oocytes per aspiration session, of which four oocytes undergo successful maturation in vitro with subsequent ICSI. If only the domi- nant stimulated preovulatory follicle is aspirated, recovery rates are approximately 80%, with only one mature oocyte to undergo ICSI. Some practitioners do both: follow the preovulatory follicle and stim- ulate it, then during the TVA session recover oocytes from both the preovulatory follicle and the subordi- nate follicles present on the ovary. Interestingly, there seems to be no effect of this timing, either positive or negative, on the developmental compe- tence of immature oocytes from subordinate follicles when compared with aspirating immature follicles during diestrus. Although the above “combined” approach yields


both quantity (the immature oocytes) and quality (the preovulatory oocyte), the two kinds of oocytes are ready for fertilization at different times. This requires ICSI to be performed at two separate times, and thus the expense to the client is increased and the return from ICSI of the one preovulatory oocyte is low. In clinical mares at Texas A&M, our blas- tocyst rate for in vivo–matured oocytes from the dominant stimulated follicle is only slightly higher than that for in vitro–matured oocytes (38 vs 23%, respectively per injected oocyte), thus in our hands, the increase in viability of the preovulatory oocyte does not make up for the fact that only one oocyte is available. Immature oocytes can be shipped at room temper-


ature by overnight courier, as noted above. In con- trast, the maturing oocyte from the stimulated preovulatory follicle must be shipped in culture me- dium in a portable incubator; we try to keep the temperature between 37 and 38.2oC. Some airlines do not want to accept active devices and so such incubators may have to be transported by car. Recovery of oocytes from immature follicles can be performed on a set schedule (i.e., once every 14 d), without checking the mare in between.2 At Texas A&M, because each recovered oocyte involves 12 days of monitoring and handling as it progresses through maturation, ICSI, and embryo develop- ment, we perform TVA on Mondays, Tuesdays, and Wednesdays so that ICSI is performed on Wednes- days, Thursdays and Fridays. The weekends in- volve embryo evaluation, handling, and shipment only. In contrast, recovery of the maturing oocyte from the dominant stimulated follicle requires ex- amining mares repeatedly to monitor follicle activity and evaluate the size and appearance of the preovu- latory follicle, timing gonadotropin stimulation of the dominant follicle, and aspirating the follicle at a


AAEP PROCEEDINGS  Vol. 62  2016 155


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