Antioxidant combinations are no more beneficial than individual components in combating ram sperm oxidative stress during storage at 5 °C

The unfrozen storage of ram semen for 2–4 days is an important goal for the acceptance of artificial insemination in sheep breeding programmes. The objective was to investigate the benefits of antioxidant supplementation on the production of hydrogen peroxide (H2O2) by ram spermatozoa stored at 5 °C over 3 days. Ejaculates from 9 rams were split between two defined diluents, INRA-96 and RSD-1, and cooled slowly to 5 °C for storage. Four different additives (vitamin E phosphate 6–100 μmol/L, catalase 500 IU + superoxide dismutase 9–150 μmol/L, and glutathione peroxidase, 20 IU) were investigated both separately and in combination. The amount of H2O2 generated was assessed by use of a 1-step fluorometric micro-plate assay. Sperm viability, acrosome integrity and membrane fluidity were assessed by flow cytometry. H2O2 production in INRA-96- compared with RSD-1-diluted spermatozoa increased approximately 2–3-fold after 24 h in storage at 5 °C and then declined up to 72 h, while that in RSD-1 showed no change over 72 h; this had no effect on the sperm characteristics. Addition of antioxidants singly reduced H2O2 production in INRA-96, regardless of concentration. Optimal concentrations were vitamin E phosphate 12.5 μmol/L, catalase 500 IU, superoxide dismutase 37 μmol/L, and glutathione peroxidase, 20 IU. In any combination, none was more effective than others. Viability was reduced but acrosomal integrity protected by the antioxidants in INRA-96 but not in RSD-1; membrane fluidity was not affected. Based on this study, there were no combinations more efficient at combating oxidative stress than any one alone.