71 BOVINE SPERM DEATH KINETICS: CHANGES IN MOTILITY, ACROSOMES, AND PLASMA MEMBRANE

Extent and timing of alterations in structures and functions of sperm after its placement in the female reproductive tract are important for successful fertilization. To our knowledge, the few reports are available on the kinetics of alterations in bovine sperm structures and functions during pathway to their death. Therefore, the present study was conducted to determine the changes in motility, acrosome and plasma membrane asymmetry in fresh and frozen–thawed semen during incubation at 37°C over the period of 24 h. Semen was collected from 3 breeding beef bulls, pooled, and considered as one replicate (total replicates = 5). Each pooled semen sample was diluted in Tris-citric acid egg yolk glycerol extender (pH 6.8), cooled to +4°C over 90 min, and then cryopreserved by a programmable cell freezer. Fresh (pooled semen) and frozen–thawed semen were incubated at 37°C for 24 h. Each semen sample was evaluated for sperm motility with computer-assisted semen analysis and acrosomal integrity and plasma membrane asymmetry using fluorescein isothiocyanate-peanut agglutinin/propidium iodide and Annexin V/propidium iodide assays, respectively, at 0, 2, 4, 6, 12, and 24 h of incubation at 37°C, with a flow cytometer. Statistical analysis was conducted using PROC MIXED model in statistical analysis system as 2 (semen types) × 6 (times) factorial model, using time as repeated measure. Progressive motility was higher (P < 0.05) in fresh than in frozen–thawed semen until 6 h. Progressive motility declined (P < 0.05) below the threshold level (i.e. 30%) much later (12 h) in fresh as compared with frozen–thawed semen (2 h). However, acrosomal integrity and plasma membrane asymmetry deteriorated (P < 0.05) below threshold at the same time interval (2 h) in both fresh and frozen–thawed semen. Viable sperm (AN–/PI–) remained higher (P < 0.05) during the first 6 h in fresh than in frozen–thawed semen and declined (P < 0.05) below the threshold at 12 h in fresh and at 6 h in frozen–thawed semen. In fresh semen, the necrotic sperm (AN–/PI+) population increased (P < 0.05) over time and reached maximum (97%) at 24 h. In frozen–thawed semen, a mixed population of late apoptotic (53%) and necrotic (34%) sperm was found at 24 h. In conclusion, the alterations in sperm motility, acrosomes, plasma membrane integrity, and asymmetry were slower in fresh than in frozen–thawed semen. Fresh sperm followed necrosis and frozen–thawed sperm underwent necrosis and apoptosis-like pathways, respectively. This study was supported by the Canadian Commonwealth Scholarship Program by the Canadian Bureau for International Education (CBIE), and Agriculture and Agri-Food Canada.