title: Quantification of damage at different stages of cryopreservation of endangered North American bison (Bison bison) semen and the effects of extender and freeze rate on post-thaw sperm quality.
abstract: Semen cryopreservation is an important technique for the banking of animal germplasm from endangered species and exploitation of genetically superior sires through artificial insemination. Being a member of bovidae family, bison semen has poor freezing ability as compared to dairy and beef bulls' semen. This study was designed to quantify the damage to bison sperm at different stages of cryopreservation, and to determine the effects of extender (commercial Triladyl(®) vs. custom made tris-citric acid [TCA]) and freeze rate (-10, -25 and -40°C/min) on post-thaw quality of bison semen. Semen was collected from five bison bulls (three woods and two plains) via electroejaculation. In Experiment 1, semen was diluted in Triladyl® extender and frozen with freeze rate -10°C/min. Sperm motility characteristics were recorded in fresh, diluted, cooled (4°C) and freeze-thawed semen using computer-assisted sperm analyzer (CASA). In Experiment 2, semen was diluted in Triladyl® or TCA extender, and frozen with three different freeze rates, i.e. -10, -25 or -40°C/min. Thawing was performed at 37°C for 60s. Post-thaw sperm motility characteristics were assessed using CASA, and sperm structural characteristics (plasma membrane, mitochondrial membrane potential and acrosomes) were evaluated using flow cytometer, at 0 and 3h while incubating semen at 37°C. In Experiment 1, total and progressive motilities did not differ among pre-freeze stages of cryopreservation (P>0.05). However, sperm total and progressive motilities declined (P<0.001) in freeze-thawed semen by 35% and 42%, respectively, compared to after cooling (pre-freeze) semen. In Experiment 2, Triladyl®, as compared to TCA, yielded greater (P<0.05) post-thaw sperm total motility (41% compared to 36%) and progressive motility (34% compared to 29%) at 0h, respectively. The percent change in post-thaw sperm total and progressive motilities, VAP, VCL, VSL, IPM-high ΔΨm and IPM-IACR during 3h incubation at 37°C, was less (P<0.05) in TCA than in Triladyl®. There was an effect of freeze rate on post-thaw sperm average path velocity at 0h, and total motility, progressive motility, VCL, IPM and IPM-IACR at 3h were the greatest (P<0.05) when bison semen was frozen at -40°C/min. Likewise, the percent change in post-thaw sperm total and progressive motilities, during 3h incubation at 37°C, was less (P<0.05) in bison semen frozen at -40°C/min. All post-thaw bison sperm characteristics decreased (P<0.05) from 0h to 3h, during incubation at 37°C. In conclusion, the maximum damage to bison sperm occurred during freeze-thaw processes. Post-thaw total and progressive motilities of bison sperm were greater in Triladyl® at 0h whereas sperm survival was greater in TCA extender during 3h post-thaw incubation. Bison sperm had greater survival (P<0.05) when frozen at -40°C/min freeze rate.
authors.affiliation: Agriculture and Agri-Food Canada, Saskatoon Research Center, Saskatoon, Saskatchewan, Canada.
authors.forename: S A
authors.initials: SA
authors.lastname: Hussain
authors.forename: C
authors.initials: C
authors.lastname: Lessard
authors.forename: M
authors.initials: M
authors.lastname: Anzar
mesh_terms: D000177:Acrosome / Q000187:drug effects; D000818:Animals; D016164:Bison / Q000502:physiology*; D015925:Cryopreservation / Q000379:methods / Q000662:veterinary*; D003451:Cryoprotective Agents / Q000494:pharmacology; D005615:Freezing; D007315:Insemination, Artificial / Q000379:methods; D008297:Male; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D055101:Semen Analysis / Q000662:veterinary*; D012662:Semen Preservation / Q000379:methods / Q000662:veterinary*; D013094:Spermatozoa / Q000187:drug effects
chemical_list: D003451:Cryoprotective Agents