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

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 60 s. 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 3 h 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 0 h, respectively. The percent change in post-thaw sperm total and progressive motilities, VAP, VCL, VSL, IPM-high ΔΨm and IPM-IACR during 3 h 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 0 h, and total motility, progressive motility, VCL, IPM and IPM-IACR at 3 h 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 3 h 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 0 h to 3 h, 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 0 h whereas sperm survival was greater in TCA extender during 3 h post-thaw incubation. Bison sperm had greater survival (P < 0.05) when frozen at −40 °C/min freeze rate.