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Cryopreservation of bison semen



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ABSTRACT The cryopreservation of bison semen is not as successful as for dairy and beef bull semen. The overall objective of this research was to develop a cryopreservation procedure to improve the post-thaw survival rate of bison sperm. For this purpose two different studies were conducted. The first study consisted of two experiments. In Experiment 1, damage to bison sperm motility characteristics at different stages of cryopreservation was quantified. In Experiment 2, the effects of extenders and freeze rates on post-thaw motility and structural characteristics were investigated. In Experiment 1, semen was diluted in Triladyl extender and frozen with a freeze rate –10°C/min. Sperm motility characteristics, i.e., total motility, progressive motility, curvilinear velocity (VCL), average path velocity (VAP) and straight-line velocity (VSL) were recorded in fresh, diluted, chilled (4°C) and frozen-thawed semen using computer-assisted sperm analysis (CASA). There was a significant decline (P < 0.001) in total and progressive sperm motilities (35% and 42%, respectively) after the freeze-thaw stage compared to pre-freeze stages. All sperm velocities declined (P < 0.05) approximately 32% at dilution, and in post-thaw semen, VCL was reduced more (P < 0.05) than VAP and VSL. In Experiment 2, semen was diluted in Triladyl or TCA extender and frozen at three different freeze rates: –10, –25 and –40°C/min. Post-thaw sperm motility characteristics were assessed using CASA, and sperm structural characteristics, i.e., intact plasma membrane (IPM), mitochondrial membrane potential (∆ΨM) and intact acrosomes (IACR) were evaluated using flow cytometry, at 0 and 3 h post-thaw after incubating at 37°C. Triladyl yielded better (P < 0.05) post-thaw sperm total and progressive motilities (41% and 34%, respectively) than TCA (36% and 29%, respectively) at 0 h. However, post-thaw percent of decline in sperm motilities and structural characteristics after 3 h of incubation at 37°C, was less (P < 0.05) in TCA than in Triladyl extender. At post-thaw 0 h freeze rate did not affect any sperm characteristics, while a freeze rate of –40°C/min revealed less (P < 0.05) percent decline in total and progressive motilities (38% and 45%), than other freeze rates (average 50% and 60%, respectively), after 3 h of post-thaw incubation. In the second study, three experiments were conducted to determine the effects of zwitterion extender and temperature of glycerol addition (Experiment 1), the addition of reduced glutathione (GSH; Experiment 2) and cholesterol-loaded cyclodextrin (CLC; Experiment 3) on post-thaw quality of bison sperm. In Experiment 1, bison semen was diluted in Triladyl (control) or zwitterion-based (ZI) extenders, i.e. HepesT (Hepes-Tris) and TesT (Tes-Tris). In addition, glycerol in ZI extenders was added either at 37 or 4°C, before cryopreservation. Extenders had no significant effect (P > 0.05) on immediate (0 h) post-thaw total and progressive sperm motilities. However, sperm velocities (VCL, VAP, VSL) were better (P < 0.05) in ZI extenders than Triladyl. After 3 h of post-thaw incubation at 37°C, sperm motilities were the best (P < 0.05) in semen diluted in Triladyl. The percentage of sperm with IPM and IPM-IACR were higher (P < 0.05) in Triladyl compared to ZI extenders, while IPM-∆ΨM did not differ due to extenders, at 0 and 3 h post-thaw incubation. Temperature of glycerol addition did not affect sperm motility, or structural characteristics, but sperm VCL and IPM-High ∆ΨM at 0 h were higher (P < 0.05), when glycerol was added at 4°C than at 37°C. The interaction between ZI extender and temperature of glycerol addition showed significant effect on VCL only. At 0 h, VCL was better (P < 0.05) in TesT extender with glycerol addition at 4°C than 37°C and than HepesT with glycerol addition at either 37 or 4°C (P < 0.05). Generally, Triladyl was superior than the ZI extenders and addition of glycerol in semen extender at 4°C was not significantly effective than at 37°C. In Experiment 2, GSH was used as an antioxidant to protect bison sperm against the possible production of reactive oxygen species (ROS) to improve their post-thaw quality. Semen was diluted at 37°C in Triladyl extender containing GSH (0, 0.5, 1.0, 2.0 mM/50 x 106 sperm). Post-thaw analyses at 0 and 3 h did not reveal any significant effect of GSH treatment on sperm motility or structural characteristics (P > 0.05). In Experiment 3 fresh bison sperm (100 x 106/ml in Tris-citric acid [TCA] buffer) were pre-treated with different concentrations of CLC (0, 1, 2 or 3 mg) and then diluted in TCA extender for cryopreservation. The CLC-treated sperm showed better (P < 0.05) post-thaw (0 h) total and progressive motilities, IPM and IPM-IACR, and also less (P < 0.05) percent of decline in these characteristics after 3 h of post-thaw incubation at 37°C compared to control (0 mg CLC) samples. The overall results of the study showed that freeze-thaw is the most damaging stage to bison sperm. Post-thaw bison sperm quality at 0 h was better in Triladyl than TCA and zwitterion extenders; however, after 3 h sperm survival was better (P < 0.05) in TCA extender and in semen frozen at a freeze rate of –40°C/min. The addition of reduced glutathione (GSH) in extender did not improve post-thaw bison sperm quality. The pre-freeze treatment of bison semen with cholesterol-loaded cyclodextrin (CLC) improved (P < 0.05) post-thaw bison sperm quality at 0 and 3 h. In conclusion, Triladyl or TCA extender and higher freeze rate –40°C/min can be used successfully for cryopreservation of bison semen, and cholesterol-loaded cyclodextrin should be included in semen cryopreservation procedure for better post-thaw quality of bison sperm.



bison, cryopreservation, semen, extender, freeze rate, glycerol, post-thaw, motility.



Master of Science (M.Sc.)


Veterinary Biomedical Sciences


Veterinary Biomedical Sciences


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