250 Effect of overnight cooling on post-thaw bull sperm characteristics

Conservation strategies for bison include collection and cryopreservation of semen under wild conditions with limited equipment. While vapour freezing can be accomplished in the field using minimal supplies, post-thaw sperm characteristics may be reduced compared to programmable freezers. To overcome this issue, semen may be shipped overnight at 4°C after dilution in semen extenders and frozen in a laboratory with a programmable freezer. The current study involves beef bulls as an animal model for bison to evaluate the efficacy of cryopreservation protocols for field-collected semen. A 2 × 3 experiment was conducted to examine the effect of semen extender (egg yolk [TEYG] vs cholesterol-based [CC-TG, animal-product-free]) and freezing protocol (vapour vs programmed vs next-day programmed) on post-thaw sperm quality. Semen was collected from Simmental bulls (n = 4 bulls, six replicates) by electroejaculation. Ejaculates with >60% total motility and >200 × 10⁶ sperm/mL were pooled for further processing. Semen was diluted in either TEYG or CC-TG extender at 22°C. For vapour freezing, semen was equilibrated at 4°C for 3 h, placed 5 cm above the level of liquid nitrogen for 10 min, and then plunged into liquid nitrogen. For programmed freezing, semen was equilibrated at 4°C for 3 h, and frozen at a rate 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 next-day programmed freezing, the equilibration period was extended to 24 h before programmed freezing. Sperm motility and the integrity of the sperm plasma membrane and acrosome were determined using computer-assisted sperm analysis and flow cytometry, respectively, at 0 and 2 h post-thaw. Values were compared among groups using ANOVA in R statistical software. Sperm motility was not influenced by treatments (no main effects or interactions). The overall motility at 0 h was 47 ± 3.1% versus 41 ± 3.2% for TEYG versus CC-TG, and 41 ± 3.4% versus 42 ± 3.5% versus 50 ± 4.5% for vapour versus programmed versus next-day programmed freezing, respectively. The greatest live sperm population 2 h post-thaw (intact plasma membrane and acrosome) was in the next-day programmed group (programmed: 44 ± 2%, vapour: 43 ± 2.6%, next-day programmed: 52 ± 2.8%; P < 0.05). Using a Bos taurus model, results demonstrated that semen may be held overnight at 4°C and frozen the next day without compromising post-thaw sperm quality.

This research was supported by funding from the Saskatchewan Agriculture Development Fund and the Natural Sciences and Engineering Research Council of Canada.