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

Abstract 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 (H 2 O 2 ) 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 6–100μmol/L, catalase 500IU+superoxide dismutase 9–150μmol/L, and glutathione peroxidase, 20IU) were investigated both separately and in combination. The amount of H 2 O 2 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. H 2 O 2 production in INRA-96- compared with RSD-1-diluted spermatozoa increased approximately 2–3-fold after 24h in storage at 5°C and then declined up to 72h, while that in RSD-1 showed no change over 72h; this had no effect on the sperm characteristics. Addition of antioxidants singly reduced H 2 O 2 production in INRA-96, regardless of concentration. Optimal concentrations were vitamin E phosphate 12.5μmol/L, catalase 500IU, superoxide dismutase 37μmol/L, and glutathione peroxidase, 20IU. 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.