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 (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 6-100 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 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 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 H(2)O(2) 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.

Survival of ram spermatozoa at high dilution: protective effect of simple constituents of culture media as compared with seminal plasma.

During incubation of ram spermatozoa at 1 x 10(7) cells mL-1 or less in a simple HEPES-buffered saline medium, high levels of cell death were detected using propidium iodide as a probe of viability (membrane integrity): some 70% of the cells died during 3 h incubation at 37 degrees C. Because the conditions of incubation were similar to those encountered during manipulations for in vitro fertilization, this phenomenon was investigated further. If ram spermatozoa were diluted in an equivalent sucrose-based medium, or if the saline medium was supplemented with 10% seminal plasma, survival was greatly improved (only 5-15% died during a 3-h incubation at 37 degrees C); the protective effect of seminal plasma resided in a 5-10 kDa fraction. Sperm death in the basal saline medium was strongly dependent on cell concentration below 5 x 10(7) spermatozoa mL-1 whereas little effect of concentration was seen in the sucrose medium or in the presence of seminal plasma. The presence of Ca2+ (2 mM), EGTA (1 mM) or mercaptoethanol (1 mM) enhanced sperm survival in saline medium, but no effect was gained by replacing NaCl with KCl, and neither BSA nor fetal calf serum were beneficial. However, when a combination of pyruvate (1 mM), lactate (21.7 mM), Mg2+ (0.4 mM), phosphate (0.3 mM) and Ca2+ (2 mM) was included in the saline medium (to render it similar to Tyrode's medium), cell survival was greatly improved (12% died during the 3-h incubation).(ABSTRACT TRUNCATED AT 250 WORDS)