26 Influence of reproductive status on oocyte collection and in vitro embryo production in bison

Novel approaches are being explored to improve in vitro embryo production in wild, free-roaming bison herds for the creation of a germplasm biobank. The objectives of the study were to determine the effects of reproductive status (nonpregnant, pregnant, and prepubertal) and advancing gestation (pregnant bison, 90 vs 120 days) on ovarian follicles, cumulus-oocyte complex (COC) collection, and in vitro embryo production. A secondary objective was to examine the effect of follicular wave status (random vs synchronised) on in vitro embryo production. Transvaginal ultrasound-guided aspiration of follicles ≥ 3 mm was done in nonpregnant, pregnant, and prepubertal bison (n = 4/group) at a random stage of the follicular wave and again five days later (synchronised) in two replicates (i.e. ∼90 days and 120 days of gestation). The COC were matured in vitro for 24–28 h at 38.8°C, fertilised (2 × 10⁶ sperm/mL) and co-incubated at 38.8°C in 5% O₂, 5% CO₂, and 90% N₂ for 18 h. Presumptive zygotes were denuded and cultured at 38.8°C in 5% O₂, 5% CO₂, and 90% N₂. A commercially available IVF media set was used (IVF Bioscience). Nominal data were compared among groups by ANOVA, and proportional data by GLIMMIX. Prepubertal bison had a greater number of follicles ≥ 3 mm than nonpregnant and pregnant bison (42.0 ± 3.3 vs 23.7 ± 3.1 vs 21.8 ± 2.3; P = 0.0001) and more COC recovered (23.0 ± 2.1 vs 12.9 ± 2.4 vs 5.7 ± 1.0; P = 0.0001). The total number of follicles was not different between random and synchronised collections (29.8 ± 3.4 vs 28.8 ± 2.7, respectively; P = 0.7), but as expected, fewer large follicles were present on Day 4 in the synchronised group (1.8 ± 0.3 vs 2.6 ± 0.4; P = 0.04). Collections on pregnant bison were more difficult with advancing gestation; COC collection efficiency (number of follicles ≥ 3 mm/number of follicles aspirated) were lower at 120 vs 90 days (35/66 [53.0%] vs 50/63 [79.4%]; P = 0.0001). The embryo development rate (COC submitted to in vitro maturation ÷ morulae or blastocysts) tended to be lowest in the prepubertal group, intermediate in the pregnant group, and highest in the nonpregnant group (17/332 [5.1%] vs 5/64 [7.8%] vs 19/182 [10.4%], respectively; P = 0.08). The number of embryos produced per bison tended to be lower in the pregnant group than the nonpregnant and prepubertal groups (5/16 [0.3] vs 19/16 [1.2] vs 17/16 [1.1]; P = 0.06, data combined between random and synchronised waves). In conclusion, both pregnant bison and prepubertal bison are valid options for COC collection in field conditions. Results document successful COC collection and embryo production from pregnant bison, but production was limited by ovary inaccessibility, fewer follicles, and lower COC collection efficiency. Yearling prepubertal bison provide the most promise as COC donors in the field, as they produced a similar number of embryos (∼1 freezable embryo per collection) to nonpregnant mature bison, and are available for collection throughout the winter season without the limitations imposed by advanced stages of pregnancy or unknown pregnancy status.