The study was conducted to test the feasibility of protocols for field collection of cumulus–oocyte complexes (COC) for invitro embryo production (IVP) in wild bison. The study was done with captive wood bison during the late anovulatory season (July). In Experiment 1, bison were assigned randomly to 2 groups (n=8/group) in which transvaginal ultrasound-guided COC collection was done in a chute without sedation or in lateral recumbency after chemical immobilization using a dart gun to compare collection efficiencies. In Experiment 2, a 2×2 design was used to examine the effects of superstimulation treatments [single-dose equine chorionic gonadotrophin (eCG) vs. multiple-dose FSH] and methods of drug administration (manual injection vs. field darting) on COC collection and IVP. Initial COC collection was done to serve as a non-superstimulated random start and to synchronize follicular wave emergence on the following day (Day 0; n=16). Half of the bison were given a single dose of 5000IU of eCG intramuscularly (IM) on Day −1. The other half were given 200mg of FSH IM on Day 0 and Day 2, followed by 2000IU human chorionic gonadotrophin IM on Day 4. Superstimulatory treatments were given by manual injection (chute restrained) to half of the bison in each group and by field darting in the other half. The COC were collected on Day 4 in the eCG group and Day 5 in the FSH group. Recovered COC were matured invitro for 25 to 28h at 38.8°C, fertilized (2×106 sperm mL−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. A commercially available IVF media set was utilised (IVF Bioscience). Data were compared by ANOVA and GLIMMIX. In Experiment 1, no difference was detected between chute-restrained vs. chemically immobilized groups in the time required to complete COC collection (6.9±1.0 vs. 8.9±1.0 min; P=0.2), the number of follicles aspirated (11.5±1.9 vs. 9.3±1.8; P=0.4), or the COC recovery rate [COC recovered/follicle aspirated; 58/92 (63%) vs. 44/69 (64%); P=0.9]. In Experiment 2, no differences were detected between superstimulation treatments (eCG vs. FSH) or method of drug administration (manual injection vs. field darting) for any endpoint. The number of medium (4.5–7.5 mm; 9.5±1.0 vs. 2.5±0.6; P<0.0001) and large (>8 mm; 8.6±0.8 vs. 3.4±1.5; P=0.004) follicles available was greater after superstimulation (groups combined) than without superstimulation (random start). Although there was no difference in the number of recovered compact COC between the superstimulated and non-superstimulated bison (8.9±1.3 vs. 6.2±1.7; P=0.2), the embryo production rate (number of embryos produced/number of COC matured) was greater after superstimulation than without superstimulation [54/189 (39%) vs. 19/108 (18%); P<0.05]. We conclude that COC collection in a field setting is feasible and minimum-handling superstimulation with a single dose of eCG is as effective as a multiple-dose FSH protocol for the invitro production of embryos in bison.
This research was supported by NSERC and Vetoquinol.
Fas and Fas ligand protein and mRNA in normal and atretic mouse ovarian follicles
