58 Fertility potential of bull semen cryopreserved without equilibration time

Equilibration time of 90–120 min at 4–5°C is necessary for sperm to maintain post-thaw viability in conventional semen cryopreservation procedures. During this period, conventional semen extenders containing egg yolk and glycerol interact with the sperm to stabilise and protect the plasma membrane against cryodamage. Alternatively, cholesterol-based extenders rapidly interact with the sperm membrane and may not require equilibration time. The objective of the present study was to determine the effect of equilibration time removal on fertility potential using bull sperm cryopreserved in cholesterol-based extenders. Semen was collected from Black Angus bulls (n = 5 bulls, 4 replicates), transported to the laboratory within 1.5 h at 32°C, and screened for motility and concentration. Ejaculates with >60% total motility and >200 × 10⁶ sperm/mL were pooled to minimise bull-to-bull effect. Pooled samples were diluted in either egg yolk (control) or cholesterol-based extender and subdivided for either routine or rapid freezing (without equilibration time). For routine freezing, semen was cooled and equilibrated at 4°C for 120 min and underwent programmed freezing with a freezing curve of −3°C min⁻¹ from 4°C to −10°C, −40°C min⁻¹ from −10°C to −80°C, and then plunged into liquid nitrogen. For rapid freezing, equilibration time was replaced with programmed cooling of −1°C min⁻¹ from 22°C to 4°C followed by programmed freezing as described in routine freeze. Post-thaw sperm motility, and plasma membrane and acrosome integrity at 0, 2, 4, and 6 h were determined with computer-assisted sperm analysis and flow cytometer, respectively. Routine-freezing egg yolk and rapid-freezing cholesterol-based extended semen were then used in a fixed-time artificial insemination trial in lactating beef cows (n = 203) synchronised with a five-day progesterone protocol. Pregnancy was diagnosed by ultrasonography at 31 to 32 days after insemination. Sperm motility, and plasma membrane and acrosome integrity were compared among groups using ANOVA. Pregnancy rates between groups were compared using binomial generalised mixed model ANOVA. Within freezing protocols, sperm parameters did not differ between egg yolk and cholesterol-based extenders. Progressive motility was greater with routine freezing vs rapid freezing in egg yolk (44 ± 4.4% vs 31 ± 0.3%) and cholesterol-based (38 ± 3.0% vs 28 ± 6.5%) extenders at 0 h post-thaw, respectively (P < 0.05). Pregnancy rate was greater in routine freezing egg yolk extender (78/103; 77%) compared to rapid freezing cholesterol-based extender (59/100; 59%, P < 0.05). Although conventional egg yolk extender with equilibration time was superior, cholesterol-based extender with no equilibration yielded pregnancy rates comparable to industry standards.

Research was supported by NSERC Canada, the Government of Saskatchewan, and Agriculture and Agri-Food Canada.