200 Effect of duration of in vitro maturation and cumulus–oocyte complex morphology on in vitro embryo production in wood bison

North American wood bison are currently designated a threatened species in Canada and there is impetus for developing in vitro culture methods to produce disease-free bison embryos as a conservation strategy. In vitro maturation is one of the main rate-limiting steps of in vitro embryo production. The objective of the study was to evaluate the influence of the duration of IVM in relation to the morphologic grade of the cumulus–oocyte complex (COC) on in vitro production of bison embryos. Ultrasound-guided COC collection was done at random stages of the follicular wave in adult female wood bison (n = 35 animals/10 replicates) during the anovulatory season. A 2 × 2 factorial experiment was conducted to compare two IVM durations (24 vs 30 h) and two morphologic grades of COC (good vs poor). For this experiment, Grade 1 and 2 COC were considered good quality (52%, 104/201), and Grade 3 and 4 COC were considered poor quality (48%, 97/201). After collection, the COC were subjected to IVM at 38.5°C in commercial buffered medium. Frozen-thawed semen was processed through 80/40% gradient of BoviPure. Matured COC (n = 2 to 9) were co-incubated with sperm in a 100-µL volume of fertilization medium for 18 h (2.0 × 10⁶ sperm/mL). Presumptive zygotes were incubated in two-step embryo culture medium (ART Laboratory Solutions Inc.). In vitro fertilization and embryo culture were performed in 5% CO₂, 5% O₂, and 90% N₂ at 38.5°C. Cleavage and blastocyst rates were evaluated on Day 4 and 8, respectively (Day 0 = start of IVF). Proportional data were arcsine transformed, compared among groups by 2-way analysis of variance, and presented as mean ± s.e.m. The good morphologic quality COC group had a higher rate of cleavage than the poor quality COC group (73.4 ± 2.9% vs 36.9 ± 3.8%; P < 0.01) and greater blastocyst formation (29.9 ± 2.5% vs 7.5 ± 3.1%; P < 0.01). The 24 h IVM group had a higher rate of cleavage than the 30 h IVM group (62.8 ± 4.3% vs 47.5 ± 5.7%; P < 0.01) and greater blastocyst formation (22 ± 3.5% vs 15.3 ± 4.0%; P = 0.05). There was no interaction between two main effects analysed in this study for either cleavage rate (P = 0.28) or blastocyst rate (P = 0.83). In conclusion, a duration of 24 h IVM appears optimal, and the COC morphological grade at the beginning of IVM is positively related to in vitro developmental competence of bison embryos and may be used as an important indicator in conservation strategies involving germplasm collection from free-roaming bison herds.

This research was supported by grants from Genome Canada and MITACS.