175 Use of equine chorionic gonadotrophin in a minimum-handling protocol for oocyte collection in bison

Abstract

Wood bison (Bison bison athabascae) and plains bison (Bison bison bison) are threatened subspecies native to North America. The creation of a germplasm biobank will connect valuable and inaccessible genetics from geographically distant herds in a biosecure manner. Protocols that are feasible in field conditions are required for cumulus-oocyte complex (COC) collection for the purpose of invitro embryo production (IVP). The efficacy of a single dose of equine chorionic gonadotrophin (ECG) was tested in an effort to develop a minimum-handling ovarian superstimulation protocol for bison. The experimental design enabled comparison between ECG-treated and non-superstimulated bison. Transvaginal ultrasound-guided follicle ablation was performed in mature wood bison (n=24) during May (anovulatory season) to induce follicular wave emergence the following day. Immediately after ablation, the bison were assigned to one of three groups (n=8 per group) and treated intramuscularly with 5000IU of ECG (Folligon, Merck), 2500IU of ECG, or saline (control). Transvaginal COC collection was performed 5 days later. Follicular and COC data were recorded, and only grade 1 and 2 COC were used for IVP. The COC were matured invitro for 25-28h at 38.8°C, fertilised (2×106 spermmL−1), and co-incubated at 38.8°C in 5% O2, 5% CO2, and 90% N2 for 18h. Presumptive zygotes were denuded and cultured at 38.8°C in 5% O2, 5% CO2, and 90% N2. Nominal data were compared among groups using analysis of variance, and proportional data were compared using GLIMMIX. The total number of follicles ≥3mm on the day of COC collection was greater in the 5000-IU ECG group than in the 2500-IU ECG and control groups (37.5±6.9, 17.5±2.0, and 16.9±2.0, respectively; P<0.005). The number of follicles 5-8mm was also greater in the 5000-IU ECG group than in the 2500-IU ECG and control groups (12.5±2.1, 7.6±1.0, and 5.8±0.9, respectively; P<0.01), as was the number of follicles >8mm (21.1±5.4, 3.3±1.2, and 0.9±0.2, respectively; P<0.0005). The proportion of grade 1 and 2 COC/total COC recovered was greater in the 5000-IU ECG group than in the 2500-IU ECG and control groups (84/124 (68%), 39/76 (51%), and 37/75 (49%), respectively; P<0.05). The proportion of cleaved zygotes/number of COC matured, assessed at 56h after fertilisation, was similar among the 5000-IU ECG, 2500-IU ECG, and control groups (42/84 (50%), 27/39 (69%), and 21/37 (57%), respectively; P=0.20). The proportion of embryos of IETS grades 1-3/number of COC matured was also similar among the 5000-IU ECG, 2500-IU ECG, and control groups (15/84 (17.9%), 8/39 (20.5%), and 7/37 (18.9%), respectively; P=0.94), but the bison in the 5000-IU ECG group produced twice as many embryos than those in the other groups. In summary, a single dose of 5000IU of ECG increased the number and size of follicles available for COC collection, more than doubled the number of COC collected for IVP, and resulted in the production of more embryos than the other groups. A single dose of 5000IU of ECG is effective in a minimum-handling protocol for ovarian superstimulation and IVP in bison. This research was supported by NSERC.