39 FERTILITY POTENTIAL OF FROZEN-THAWED WOOD BISON SEMEN USING EXTENDER WITHOUT EXOGENOUS PROTEIN

Abstract

Cryopreservation of semen free of infectious agents is critical for the recovery and preservation of genetic diversity in Canada’s threatened wood bison populations. Egg yolk is a common constituent of conventional semen extenders, but it raises biosafety concerns related to transmission of infectious agents. The purpose of this experiment was to determine the fertility potential, both in vitro and in vivo, of semen frozen without the use of exogenous protein but with addition of cholesterol loaded cyclodextrin (CLC). Semen was collected by electro ejaculation from 4 wood bison bulls. Fresh sperm total motility and concentration were analysed using a computer-assisted sperm analyzer. Ejaculates with total sperm motility of >60% and a concentration of >200 × 10⁶ sperm mL^–1 were selected and pooled among bulls. For the control group, pooled semen was diluted to 50 × 10⁶ sperm mL^–1 with conventional extender containing 20% egg yolk and 7% glycerol. For the test group, pooled semen was diluted to 100 × 10⁶ sperm mL^–1 in Tris-citric acid buffer and incubated with 2 mg mL^–1 CLC for 15 min before 1 : 1 dilution with 14% glycerol extender. Extended semen was placed in 0.5-mL straws, cooled, and frozen. Post-thaw motility analysis was conducted using a computer-assisted sperm analyzer, and the 2 collections with highest post-thaw motility were selected for fertility testing. Heterologous IVF was conducted using bovine oocytes obtained from an abattoir. After 20 to 22 h of in vitro maturation, the cumulus-oocyte complexes were placed into drops with either control or test semen. After 18 h, potential zygotes were denuded and moved to culture media. Cleavage and blastocyst rates were assessed on Day 4 and 8, respectively, from oocyte collection. The fertility potential of the semen was also tested in vivo using synchronized wood bison cows. At 24 h after hCG treatment, bison cows were assigned randomly to 2 groups and inseminated twice, 12 h apart, with frozen-thawed control semen (n = 23) or test semen (n = 23). Pregnancy was assessed 34 to 36 days after insemination by transrectal ultrasonography. Total sperm motility (mean ± standard error of the mean) of fresh semen was 80.2 ± 3.3%. The post-thaw motility was 41.7 ± 2.9% and 44.6 ± 3.3% for control and test semen, respectively. The cleavage rates of bovine oocytes fertilized in vitro with bison semen from the control group (n = 278) and test group (n = 299) were 49.9 ± 2.1% and 41.3 ± 4.5%, respectively, and the blastocyst rates were 17.7 ± 1.7% and 17.3 ± 2.5%, respectively. Pregnancy rates of wood bison artificially inseminated with control and test semen were 39 and 0%, respectively. In conclusion, wood bison semen frozen with CLC (without exogenous protein) resulted in post-thaw motility and in vitro fertility potential comparable to that of conventional egg yolk extender. However, CLC semen failed to fertilize in vivo, perhaps because of disruption of the processes of capacitation. The dichotomy between in vivo and in vitro results was surprising. It may be necessary to exercise caution when using IVF as a tool to assess fertility in vivo.