50 BOVINE SPERM DEATH KINETICS: TEMPORAL CHANGES IN PRODUCTION OF REACTIVE OXYGEN SPECIES AND PLASMA MEMBRANE INJURY OF DAIRY AND BEEF FROZEN–THAWED SEMEN

The extent of changes in sperm structure and function affect the success of fertilization ultimately during the pathway to ovum in the female reproductive tract. The success of AI with frozen-thawed semen varies in dairy and beef breeds of bovine because of differed alterations in sperm during transport in female tract after insemination. To our knowledge, no report is available comparing the changes in dairy and beef sperm leading to death in female tract. Therefore, this study was aimed to investigate the changes in motility, generation of reactive oxygen species (superoxide and hydrogen peroxide), and their relation to sperm death [asymmetry (apoptosis) and rupture of plasma membrane] of dairy and beef frozen-thawed semen during incubation at 37°C for 24 h. This incubation was aimed to mimic the environment of female reproductive tract. Frozen dairy semen (n = 4 bulls) was procured from a Canadian breeding station, whereas beef semen was collected from breeding beef bulls (n = 3; 5 replicates), diluted with Tris-based extender (composition was same as used in dairy semen), cooled to +4°C over 90 min, and cryopreserved by programmable freezer using standard rate as used in dairy semen. Two straws per replicate were thawed at 37°C from both types of semen, pooled separately, and incubated at 37°C for 24 h in capped tubes. Each pooled semen sample was evaluated for motility with CASA, superoxide (O2–, and hydrogen peroxide (H2O2) radical using HE/YoPRO and H2DCFDA/PI assay, respectively, and asymmetry of plasma membrane using YoPRO/PI assay through flow cytometric analysis at 0, 2, 4, 6, 12, and 24 h of incubation. The MIXED procedure of SAS (SAS Institute Inc., Cary, NC, USA) was used to analyse the data as 2 × 6 factorial model for 2 types of semen (dairy and beef) and 6 time points using time as repeated measure. A threshold limit of 30% was considered for motility and live sperm to get optimum fertility. Sperm motility remained higher (P < 0.05) than threshold limit till 6 h in dairy (50.95 ± 2.62%) and 2 h in beef semen (30.28 ± 6.95%). Dairy semen possessed more (P < 0.05) nonapoptotic sperm without O2– (HE–/YoPRO–) till 6 h of incubation than beef semen. The increase in apoptotic sperm containing superoxide radical (HE+/YoPRO+) over time was more (P < 0.05) in beef semen till 6 h of incubation. The rise in dead sperm containing H2O2 (H2DCFDA+/PI+) was recorded more in beef than in dairy semen until 6 h of incubation. Live sperm without apoptosis (YoPRO–/PI–) were higher until 24 h in dairy (49.36 ± 4.56%) compared with beef semen (24.89 ± 3.85%), whereas viable sperm with apoptosis (YoPRO+/PI–) were found similar in both types of semen over time. In conclusion, dairy frozen-thawed semen possessed more live sperm without reactive oxygen species (superoxide and hydrogen peroxide) until 6 h of incubation than did beef semen. The decrease in superoxide radical was more in dairy than in beef semen. Dead and apoptotic sperm increased more in beef frozen-thawed semen over time during incubation. This inference suggests performing the insemination late near ovulation with beef frozen-thawed semen because of less viable life than dairy semen.