HOW TO INCORPORATE ASSISTED REPRODUCTIVE TECHNOLOGIES


88.8% (mean, 56.3%) for stallions compared to 0% to 100% for mares (mean, 66.7%). Overall, mares that yielded an embryo at the first OPU had a 70% likelihood of yielding embryos at subsequent at- tempts, whereas those that did not yield an embryo at the first treatment had only a 30.5% likelihood of producing embryos at subsequent attempts. In short, mare identity was a more important pre- dictor of blastocyst production than stallion. Sur- prisingly, the mare effect was not primarily an effect of age. Indeed, although mares older than 20 years yielded fewer oocytes and blastocysts (mean 0.8 per OPU), the percentage of blastocysts per injected oocyte (13.7%) was no different to that in younger mares. Of the first 419 embryos transferred, 270 resulted


in a pregnancy 7 to 10 days later (64.4%). Thirty- five of these pregnancies (13%) were lost in the pe- riod up to day 37 after transfer (equivalent to day 41–42 of gestation since, based on vesicle size 7 days after transfer, the in vitro produced (IVP) embryos behaved like day 5 or 6 in vivo embryos).4 The likelihoods of pregnancy and pregnancy loss were independent of donor mare age. On the other hand, there were significant effects of year of production/ transfer on likelihood of pregnancy and pregnancy loss, with results improving over time. This was at least in part due to identifying the ideal stage of the recipient mare’s cycle to transfer a frozen-thawed ICSI embryo. In this respect, the likelihood of pregnancy was higher after transfer to recipients on days 3 and 4 after ovulation (72% and 73%, re- spectively) than on days 5 or 6 (57% and 42%, respectively).


4. Discussion


Setting up a commercial OPU and/or ICSI program involves a considerable learning curve, with results improving over time, sometimes due to subtle experience-related changes and sometimes because of planned changes in protocol. For example, while blastocyst production rates in our program have im- proved over time (from approximately 0.9 in 2014 to around 1.4 per OPU in 2018), this period has also included changes in oocyte transport systems (from Equitainer to programmable incubator to organ transport boxes) and follicle flushing solutions (from embryo flushing medium supplemented with hepa- rin sodium, to a commercial oocyte recovery medium including reagent grade heparin). However, since these changes were not randomized, it is difficult to prove the suspicion that, for example, the preserva- tives in the heparin solution were compromising results. In addition, the realization that there is a strong mare effect, and communicating this to cli- ents, has led to an increasing bias to “reliably pro- ducing” mares, which will in itself have contributed to an improvement in overall results. The authors also advise clients to avoid using refrozen semen since, although there are reports of satisfactory re- sults in other programs, they have produced only 1


372 2019  Vol. 65  AAEP PROCEEDINGS


blastocyst and saw very low cleavage rates (10% compared to 68.5%) when using refrozen semen from 4 different stallions on the oocytes from 9 mares. In terms of expectation, with the right equipment


and an experienced team, average oocyte recovery from immature follicles should be around 50%; how- ever, low recovery is not necessarily a predictor of poor blastocyst production. Importantly, oocytes can be shipped overnight at 22°C to a dedicated equine ICSI lab, with little or no loss of fertility3 as long as the temperature is maintained. Moreover, embryo production (1 blastocyst per treatment cy- cle) and pregnancy after transfer of ICSI embryos (70%) are, respectively, better than or only a little worse than for conventional ET. This is particu- larly true for aged mares (20 years) that, although they yield fewer oocytes, still achieve blastocyst pro- duction per injected oocyte and likelihoods of preg- nancy and pregnancy maintenance after transfer of those embryos similar to young mares (i.e., they do not suffer from the age-related losses seen for other breeding techniques [natural mating, artificial in- semination (AI), ET]). In addition, OPU-ICSI of- fers a route to producing embryos from some poorly fertile stallions, and for making efficient use of lim- ited semen. On the other hand, ICSI embryos seem to be more exacting in terms of the synchrony of the recipient mare (day 3 and 4 after ovulation are bet- ter than days 5 and 6), while pregnancy losses after transfer of ICSI embryos are currently higher (8– 15%) than for other breeding techniques, such that approximately 50% to 60% of transferred ICSI em- bryos can be expected to result in the birth of a live foal.2 Finally, while major complications of OPU are uncommon, around 20% of mares suffer from post-procedure abdominal discomfort, fever or distal limb edema mostly for only 1 to 2 days but occasion- ally for longer. Although the symptoms are usually mild and can be countered by administration of NSAIDs and moderate exercise, this can be a reason for owners of actively competing sport horses to de- cide not to use OPU.


Acknowledgments


Oocyte searching, preparation for transport, and embryo thawing was performed by Mabel Beitsma, Claudia Deelen, and Marilena Rizzo. IVM, ICSI, and IVP and embryo cryopreservation were carried out by staff at Avantea (Cremona, Italy).


Declaration of Ethics


The Authors have adhered to the Principles of Vet- erinary Medical Ethics of the AVMA.


Conflict of Interest The Authors have no conflicts of interest.


References and Footnotes 1. Galli C, Colleoni S, Duchi R, et al. Developmental compe- tence of equine oocytes and embryos obtained by in vitro pro- cedures ranging from in vitro maturation and ICSI to embryo


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