公豬稀釋精液添加 Trolox 對冷凍保存精液品質之影響

Abstract

Liquid and frozen methods are usually used to apply in boar semen preservation. The principal semen preservation is liquid method in pig breeding farm. However the number of semen dosages of collected and diluted the boar semen is usually greater than the number of mated sows. It wastes the excellent boar semen. If we could cryopreserve the liquid boar semen, we would increase the application of the excellent boars. The objective of this study was to evaluate the effect of Trolox in diluent boar semen for 2 and 3 days storage on the sperm quality and in vitro fertility of cryopreserved boar diluent semen. In experiment 1, the fresh boar semen was mixed with SafeCell diluent containing 200 μM Trolox in a 50% ratio, and stored at 16 to 18 ˚C in a refrigerator for 2 and 3 days. Before and after cryopreservation, the sperm motility, acrosomal integrity, viability and mitochondrial activity of the stored semen were analyzed using phase contrast microscopy and flow cytometry. The thawed semen was examined during 4 hours (0, 2 and 4 hours) post-thawed. The results indicated that the sperm quality parameters including motility, acrosomal integrity, viability and mitochondrial activity of liquid stored semen with Trolox after 2 d and 3 d storage were no significant difference compared with the control. The acrosomal integrity of cryopreserved diluent semen with Trolox for 2 d storage was 37.8% that was significantly better than the control (28.1%) (P < 0.05). At post-thawed 2 hours, the motility of sperm in diluent semen with Trolox for 2 d storage was 46% that was significantly higher than the same treatment for 3 d storage (40%) (P < 0.05). At post-thawed 4 hour, all quality parameters of the 4 groups were not different significantly.In experiment 2, fresh boar semen was mixed with SafeCell or BTS diluents containing 200 μM Trolox in a 50% ratio, and stored at 16 to 18 ˚C in a refrigerator for 3 d. Before cryopreservation, the free radical scavenging of diluent semen sored at 0 and 3 days was analyzed by DPPH antioxidant assay. Before and after cryopreservation, the sperm motility, acrosomal integrity, viability and mitochondrial activity of the cryopreserved semen were analyzed by phase contrast microscopy and flow cytometry. Finally, the fertility of the frozen semen was evaluated by in vitro fertilization of porcine oocytes. The results showed that the free radical scavenging among all groups was not significantly different before semen cryopreserved. The quality parameters of thawed semen, including motility, acrosomal integrity, viability and mitochondrial activity did not differ significantly between SafeCell and BTS diluent semen with or without Trolox. However, the incubated time (0, 2 and 4 hours) of post thawed semen affected all quality parameters. The longer time of incubation decreased the sperm quality. The diluent semen supplemented with Trolox did not improve the sperm quality of cryopreserved semen. Finally, there was no difference of penetration rate and polyspermy rate in IVF experiments among the 4 groups.In conclusion, the Trolox in diluent semen could improve the integrity of sperm acrosome, and extension the time of liquid stored semen decreased the sperm motility of frozen-thawed semen. All parameters and fertility of BTS and SafeCell diluent semen with or without Trolox that stored 3 days and cryopreserved did not differ significantly after been thawed. We also suggested that the shorter time diluent (BTS) could be used to dilute boar semen for cryopreservation, but the longer time diluent (SafeCell) maintained the better sperm quality of post-thaw semen.