42 Influence of oocyte grade on bovine in vitro embryo production and post-thaw viability

In vitro production (IVP) of cattle embryos has increased significantly over the last 10 years. The IVP embryos are sensitive to cryopreservation, and the survival rate of IVP embryos after thawing is lower than in vivo-derived embryos. While improved methods for freezing IVP embryos are needed, understanding which embryos are most likely to survive the freezing process will also help reduce the waste of embryos and costs associated with transferring embryos that do not result in pregnancies. Our objective was to determine how oocyte quality influences the number of IVP embryos produced and the ability of those embryos to survive slow freeze cryopreservation. Abattoir-derived oocytes were divided into four grades according to IETS guidelines (grade 1, n = 415; grade 2, n = 438; grade 3, n = 436; grade 4, n = 377) and embryos generated using a standard IVP protocol (4 replicates). Embryo cleavage rates were assessed on Day 3 and blastocyst rates evaluated on Day 7 postfertilization. On Day 7, embryos were divided according to stage of development (early blastocysts, blastocysts, and expanded blastocysts) and slow frozen in 1.5 M ethylene glycol and 0.5 M sucrose in a 0.25-mL straw using a standard freeze curve for cattle embryos. Embryos were frozen for at least 24 h before being thawed and cultured for 24 h in a chemically defined culture medium containing 5% fetal calf serum. Viability of embryos was assessed 24 h after thawing. Cleavage, blastocyst, and post-thaw viability rates of embryos according to oocyte grade were evaluated using a Pearson’s chi-squared test. Differences between groups were identified using an equality of proportions test without continuity correction. Grade 1 and 2 oocytes resulted in significantly higher cleavage and blastocyst rates (83 ± 3% and 83 ± 4% cleavage and 23 ± 2% and 21 ± 3% blastocysts, respectively) than grades 3 and 4 (67 ± 6% and 75 ± 5% cleavage (P < 0.05) and 11 ± 6 and 16 ± 7% blastocysts (P < 0.001), respectively). Post-thaw viabilities indicated a trend for embryos from grade 3 oocytes to have poorer viability (4/17, 25%) compared to grades 1, 2, and 4 (20/37, 54%; 24/47, 51%; and 15/25, 60%, respectively), though there was no significant difference between groups likely owing to the small sample size. There was also no significant difference when post-thaw viability was evaluated according to stage of embryo, likely for the same reason (early blast 7/25, 28%; blastocyst 15/27, 56%; expanded blastocyst 41/74, 55%). Further replication of the experiment will improve statistical robustness, and transfer of embryos to generate pregnancies will need to be done to determine whether 24-h post-thaw viabilities will be predictive of pregnancy for embryos generated from oocytes of different qualities. Collectively, these data could be used to make decisions on which embryos to transfer fresh or freeze to maximize pregnancy rates with IVP bovine embryos.