248 IN VITRO EMBRYO PRODUCTION USING IN VIVO-MATURED OOCYTES COLLECTED TRANSVAGINALLY FROM WOOD BISON (BISON BISON ATHABASCAE)

Abstract

Reproductive technologies are being developed to help conserve the genetic diversity of wood bison, a threatened species. To date, the efficiency of in vitro embryo production in bison is very low and appears to be related to inadequate in vitro conditions for oocyte maturation. Recently, we have attempted to circumvent the problem by inducing oocyte maturation in vivo and found that more than one-third of superstimulated oocytes collected 30 h after administration of hCG were at metaphase II (Cervantes et al. 2013 Reprod. Fertil. Dev. 25, 283; Cervantes et al. 2014 Reprod. Fertil. Dev. 26, 199). We hypothesise that additional maturation time in vitro, after in vivo maturation, will allow the remaining oocytes to reach the MII stage, and thus improve in vitro embryo production in wood bison. The objective of this study was to determine the effect of an additional 4 h of in vitro maturation on the developmental competence of oocytes collected 30 h after hCG treatment. Wood bison cows (n = 24) were superstimulated by the administration of 300 mg of FSH (Folltropin-V) diluted in 0.05% hyaluronan on the day of follicular wave emergence and 100 mg of FSH in hyaluronan 2 days later. Bison were administered 2500 IU of hCG (Chorulon) IM 2 days after the last dose of FSH. Transvaginal ultrasound-guided follicle aspiration was performed 30 h after hCG treatment to collect cumulus-oocyte complexes (COC). Expanded COC (with no evidence of degeneration) were selected and assigned randomly to 2 groups (n = 38 COC/group) in which IVF was done immediately, or after 4 h of in vitro maturation in TCM 199 with 5% calf serum, 5 μg mL–1 pLH, 0.5 μg mL–1 pFSH, and 0.05 μg mL–1 gentamicin, at 38.5°C, 5% CO2 and high humidity. In vitro fertilization (Day 0) was done with frozen-thawed wood bison semen (dose 5 × 106 sperm mL–1) in Brackett-Oliphant medium at 38.5°C, 5% CO2, and high humidity. Presumptive zygotes were cultured in CR1aa plus 5% calf serum, at 38.5°C and in 5% CO2, 5% O2, and 90% N2 and high humidity. Cleavage was recorded on Day 3, and blastocyst formation was recorded on Days 7 and 8. Cleavage and blastocyst rates (calculated from the total number of oocytes submitted to IVF) were compared between groups by chi-square analysis. No difference was detected between groups (immediate fertilization v. after an additional 4 h in vitro) in cleavage rate on Day 3 (55.3 v. 60.5%, respectively, P = 0.82), or blastocyst rate on Day 7 (13.2 v. 23.7%, respectively, P = 0.37). However, the blastocyst rate on Day 8 was higher in the COC group exposed to an additional 4 h of in vitro maturation (18.4 v. 44.7%, respectively, P = 0.03). Results support the hypothesis that an additional short period of in vitro maturation improves the developmental competence of oocytes collected after 30 h of in vivo maturation.We thank Bioniche Animal Health for providing FSH (Folltropin-V) and hyaluronan (MAP-5), and Merck Animal Health for hCG (Chorulon).