Apoptotic-like changes in equine spermatozoa separated by density-gradient centrifugation or after cryopreservation

The objective was to evaluate apoptotic markers in ejaculated equine spermatozoa after separation by density-gradient centrifugation and after cryopreservation. Subpopulations of percoll-separated equine spermatozoa differed (P<0.05) in the percentage of live, caspase-activated spermatozoa (2.9+/-0.7% vs 14.2+/-6.4%; mean+/-S.E.M.), low mitochondrial membrane potential (MMP; 6.8+/-1.1 vs 23.8+/-3.7), altered plasma membrane permeability (1.3+/-0.2 vs 3.0+/-0.5), DNA fragmentation (2.0+/-1.3 vs 14.3+/-3.6), total motility (81.8+/-3.3 vs 35.1+/-5.4), and progressive motility (66.3+/-4.3 vs 24.1+/-4.5) for high-density versus low-density subpopulations, respectively. Phosphatidylserine externalization did not differ (P=0.67) between the high- and low-density subpopulations (2.6+/-0.7 vs 3.1+/-0.9). After cryopreservation, equine spermatozoa differed (P<0.01) in the percentage of active caspases (19.1+/-1.6 vs 52.1+/-2.8), low MMP (18.2+/-2.5 vs 48.7+/-2.6), altered plasma membrane permeability (6.8+/-1.7 vs 17.6+/-2.0), total motility (75.5+/-2.4 vs 45.2+/-5.6), and progressive motility (53.9+/-3.1 vs 28.3+/-4.5) for pre-freeze versus cryopreserved spermatozoa. There was no difference (P=0.21) in percentage of DNA fragmented cells before (5.5+/-1.2) versus after cryopreservation (6.6+/-1.1). We concluded that apoptotic-like changes were detectable in ejaculated equine spermatozoa and were more prevalent after cryopreservation.