J. G. Brom-de-Luna

Vitrification of germinal-vesicle stage equine oocytes: Effect of cryoprotectant exposure time on in-vitro embryo production

Previous studies have found low rates of blastocyst development (0-11%) after vitrification of germinal vesicle (GV)-stage equine oocytes. In this study, we systematically evaluated a short (non-equilibrating) system for GV-stage oocyte vitrification. In Exp. 1, we assessed oocyte volume in cumulus-oocyte complexes (COCs) exposed to components of a short protocol, using 2% each of ethylene glycol and propylene glycol in the first solution (VS1); 17.5% of each plus 0.3 M trehalose in the second solution (VS2); and fetal bovine serum as the base medium. Based on the time to oocyte minimum volume, we selected a 40-sec exposure to VS1. In Exp. 2, we evaluated exposure times to VS2 and, based on rates of subsequent maturation in vitro, we selected 65 s. In Exp. 3, we used the optimized vitrification system (40-VS1; 65-VS2) and evaluated three warming procedures. Blastocyst development after ICSI was equivalent (15%) for COCs warmed in either standard (trehalose stepwise dilution) or isotonic (base medium) solutions, but was reduced (0%) for COCs warmed in a highly hypertonic (1.5 M trehalose) solution. Exposure to the vitrification and warming solutions, without actual vitrification, was associated with reduced blastocyst development (0-5%; Exp. 4). We conclude that this optimized short protocol supports moderate blastocyst production after vitrification of GV-stage equine COCs. Oocytes can be warmed in isotonic medium, which simplifies the procedure. The systems used still showed a high level of toxicity and further work is needed on both vitrification and warming methods to increase the efficiency of this technique.

Culture of somatic cells isolated from frozen-thawed equine semen using fluorescence-assisted cell sorting

Nuclear transfer using somatic cells from frozen semen (FzSC) would allow cloning of animals for which no other genetic material is available. Horses are one of the few species for which cloning is commercially feasible; despite this, there is no information available on the culture of equine FzSC. After preliminary trials on equine FzSC, recovered by density-gradient centrifugation, resulted in no growth, we hypothesized that sperm in the culture system negatively affected cell proliferation. Therefore, we evaluated culture of FzSC isolated using fluorescence-assisted cell sorting. In Exp. 1, sperm were labeled using antibodies to a sperm-specific antigen, SP17, and unlabeled cells were collected. This resulted in high sperm contamination. In Exp. 2, FzSC were labeled using an anti-MHC class I antibody. This resulted in an essentially pure population of FzSC, 13-25% of which were nucleated. Culture yielded no proliferation in any of nine replicates. In Exp. 3, 5 × 103 viable fresh, cultured horse fibroblasts were added to the frozen-thawed, washed semen, then this suspension was labeled and sorted as for Exp. 2. The enriched population had a mean of five sperm per recovered somatic cell; culture yielded formation of monolayers. In conclusion, an essentially pure population of equine FzSC could be obtained using sorting for presence of MHC class I antigens. No equine FzSC grew in culture; however, the proliferation of fibroblasts subjected to the same processing demonstrated that the labeling and sorting methods, and the presence of few sperm in culture, were compatible with cell viability.

88 EFFECT OF BICARBONATE/CO2 LEVEL DURING EMBRYO CULTURE ON EQUINE BLASTOCYST RATE AFTER INTRACYTOPLASMIC SPERM INJECTION

Equine embryos appear to require a high glucose concentration for development to the blastocyst stage. The complete cell-culture medium, DMEM/F-12 (DM), which contains 17mM glucose, has been widely used for equine embryo culture; however, in other species, high glucose during the early stages of embryo development is detrimental. To avoid this, we initiated a 2-step system using a low-glucose human embryo culture medium (Global) from Days 0 to 5 [Day 0=day of intracytoplasmic sperm injection (ICSI)], with glucose (20mM) added to the medium in the second step (Days 5 to 10; Choi et al. 2015 Reproduction 150, 31-41). We noted a high pregnancy loss rate (20%) in our clinical ICSI program (Hinrichs et al. 2014 J. Equine Vet. Sci. 34, 176), which used this 2-step Global system. Limited data are available on pregnancy with DM-produced embryos, but in one study, the loss rate was 1/13 (7.7%; Choi et al. 2011 Reproduction 142, 529-538). It is possible that use of DM in the second step of culture would better support normal blastocyst development than does Global with added glucose. However, DM is typically used at 5% CO2, and Global at 6% CO2, so use of both media would necessitate 2 sets of incubators. In the present study, we explored the use of DM in the second step of a two-step equine embryo culture system, under different CO2 environments. Oocytes were collected from research mares via follicle aspiration and were held overnight before being matured in vitro for 30h. All media included 10% fetal bovine serum. On Days 0 to 5 after ICSI, all embryos were cultured in Global under 6% CO2 in mixed gas (5% O2 and remainder N2) at 38.2°C. In Experiment 1, on Day 5, embryos were transferred to DM prepared according to our standard method, with 14.3mM NaHCO3 and 5mM NaOH, and were cultured in mixed gas at either 5% CO2 or 6% CO2. Five replicates were performed. In Experiment 2, DM was prepared by our standard method, or with 24.2mM bicarbonate and no NaOH. When pH was measured using a pH meter after media were equilibrated overnight, this higher bicarbonate provided the same pH at 6% CO2 (pH 7.3), as was achieved with the standard DM preparation at 5% CO2. Six replicates were performed. In both experiments, blastocyst development was assessed on Days 7 to 10, and blastocyst rates were compared between treatments by Fishers exact test. In Experiment 1, blastocyst rates were 43%, 13/30 and 27%, 8/30 for the standard DM preparation in 5% and 6% CO2, respectively (P>0.05). In Experiment 2, the blastocyst rates were 34%, 14/44 for the standard DM preparation at 5% CO2 and 43%, 19/44 for the high-bicarbonate DM at 6% CO2 (P>0.05). We conclude that a 2-step Global-DM system can support equine blastocyst production under a consistent CO2 environment (6%) if DM bicarbonate levels are adjusted to balance the increased CO2.