Apoptotic - like changes of boar spermatozoa in freezing media supplemented with different antioxidants

This study evaluated the effect of supplementing the freezing extender with exogenous antioxidants on apoptotic-like changes in post-thaw boar spermatozoa. A total of 36 ejaculates were resuspended in standard lactose-egg yolk-glycerol extender supplemented with antioxidant to final concentrations of 0 (as control), 2.5mM GSH (group I), 5.0 mM GSH (group II), 150 IU/mL SOD (group III), 300 IU/mL SOD (group IV), 200 IU/mL CAT (group V), 400 IU/mL CAT (group VI), 150 IU/mL SOD+200 IU/mL CAT (group VII), 300 IU/mL SOD+400 IU/mL CAT (group VIII). Sperm motility and apoptotic-like changes were determined before and after freeze-thawing. The various markers of apoptotic-like changes were measured: plasma membrane permeability by YO-PRO-1/PI assay, phosphatidylserine (PS) translocation across the plasma membrane using fluorescein-labeled Annexin-V, mitochondrial transmembrane potential detected by JC-1, and DNA fragmentation evaluated by TUNEL assay. The highest percentage of progressive motile sperm was noticed in group II (PM% 64.2±15.4) compared with control (PM% 36.8±5.5). The supplementation of 400 IU/mL CAT (group VI) revealed significant (P<0.01) reduction of apoptotic-like changes (YO-PRO-1⁺/PI⁻: 13.1±7.5%, AnV⁺/PI⁻: 9.9±4.1%) in frozen-thawed spermatozoa compared with extendersupplemented with200 IU/mL CAT (groupV). Irrespectiveof the concentrationused, SOD and CAT in combination (group VII and group VIII) significantly (P<0.01) improved post-thaw sperm survival compared with the control. Evaluation by TUNEL assay revealed that cryopreservation and thawing did not induce DNA fragmentation in boar spermatozoa.