40 Rapid-freeze of bison semen in cholesterol-based extender

Conventional semen cryopreservation protocols necessitate an equilibration time of 90 to 120 min at 4–5°C before freezing to allow glycerol and egg yolk to interact with the sperm plasma membrane. However, prolonged processing time increases risks for glycerol toxicity and may not be feasible when cryopreserving wild bison semen under field conditions. Alternatively, cholesterol is rapidly delivered to the sperm membrane by cyclodextrin and does not require a long equilibration time. Therefore, the present study was conducted to test the hypothesis that bison semen can be frozen rapidly by minimising equilibration time and freezing at a high rate, using a novel cholesterol-based semen extender rather than egg yolk. A 2 × 2 experiment was conducted comparing two extenders (Tris-egg yolk-glycerol (TEYG, control) vs. cholesterol-cyclodextrin+Tris-glycerol (CC+TG)) and two cryopreservation protocols (routine- vs. rapid-freeze). Semen was collected from three wood bison bulls by electroejaculation and pooled (5 collection sessions). Semen was then diluted in either TEYG or CC+TG extender and subjected to either a routine or a rapid-freeze protocol. For routine freezing, the diluted semen was cooled to and held at 4°C for 120 min. After equilibration, semen was frozen in a programmable freezer with the 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, semen was diluted in extender at 25°C and immediately frozen in a programmable freezer with the freezing curve of −1°C min⁻¹ from 25°C to 10°C, −40°C min⁻¹ from 10°C to −80°C, and then plunged into liquid nitrogen. Post-thaw sperm motion parameters were evaluated with computer-assisted sperm analysis, and plasma membrane and acrosome integrity were determined by flow cytometry at 0 and 2 h. Values were compared by ANOVA for repeated measures. Data were presented as mean ± s.e.m. Post-thaw total motility was the greatest with CC+TG routine freeze (51.5 ± 4.08%), and there was no difference between TEYG routine freeze (44.7 ± 1.0%) and CC-TG rapid freeze (38.6 ± 4.8%), while TEYG rapid freeze resulted in the lowest total motility (9.8 ± 1.21%; P < 0.05) at 0 h. At 2 h, routine freeze resulted in greater total motility than rapid freeze (TEYG routine (31.9 ± 2.8%) and CC+TG routine (31.4 ± 2.1%) vs. TEYG rapid (3.5 ± 0.1%) and CC+TG rapid (3.1 ± 0.4%); P < 0.05). Routine freeze resulted in greater sperm population with intact plasma membrane and acrosome integrity compared with rapid freeze (TEYG routine (47.4 ± 1.5%) and CC+TG routine (51.8 ± 6.1%) vs. TEYG rapid (17.0 ± 1.9%) and CC+TG rapid (29.6 ± 4.6%); P < 0.05) at 0 h. Results demonstrated that TEYG extender failed to protect bison sperm during cryopreservation in a rapid-freeze protocol (without the equilibration time). Equilibration time may be minimised using a rapid-freeze protocol with egg yolk-free CC+TG extender; however, sperm longevity in vitro was short. Further studies are required to improve sperm longevity in the rapid-freeze group and to determine the sperm fertility potential.

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