Activity of the anti-oxidative enzymes glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and catalase (CAT), content of thiobarbituric acid reactive substances (TBARS) and SH-groups were determined in native stallion semen (n = 8 stallions). Semen was then diluted in Kenney extender, EquiPro((R)) extender either with or without addition of N-acetyl cysteine or phosphate-buffered saline (PBS) and stored for 72 h at 5 degrees C. Correlations between initial activity of enzymes and development of semen motility and membrane integrity were calculated. Activities of GSH-Px, SOD and CAT immediately after semen collections were 10.0 +/- 0.6 picokatals, 0.40 +/- 0.03 SOD units and 0.70 +/- 0.05 nanokatals/10(6) spermatozoa respectively. TBARS content was 0.06 +/- 0.01 nmol and SH-group content 1.7 +/- 0.5 mmol/10(6) spermatozoa. The loss of motile spermatozoa during storage did not differ between extenders. N-acetyl cysteine had no effect on semen motility and membrane integrity. The loss in membrane-intact spermatozoa was highest (P < 0.05) in semen diluted in PBS. Motility and membrane integrity after addition of extender were positively correlated with GSH-Px and CAT, indicating that anti-oxidative mechanisms contribute to the initial high percentage of motile and membrane-intact spermatozoa. However, in these samples the decrease in semen quality was most pronounced. No correlations existed between initial activity of anti-oxidative enzymes, peroxidation products and semen quality during storage. This indicates that once extender has been added, peroxidative damage to sperm membranes is not the predominant cause of losses in semen quality.
SummaryLosses in motility and fertilizing ability of stored semen can at least in part be attributed to lipid peroxidation of the sperm plasma membrane. Physiologically, mitochondrial respiration is the main source of reactive oxygen species (ROS). In processed semen, ROS originate from contaminating leucocytes and from spermatozoa with residual cytoplasm. In addition, normal spermatozoa produce ROS as a result of their flagellar activity. At low concentrations, ROS have positive biological effects and regulate physiological sperm functions. Mammalian sperm cell membranes have a specific lipid composition with a high content of polyunsaturated fatty acids, making them particularly susceptible to damage by ROS. Peroxidation increases membrane permeability and decreases metabolic activity of sperm cells. To control the effects of ROS, semen contains antioxidants. Enzymatic antioxidants are glutathione peroxidase, superoxide dismutase and catalase. Antioxidants have been substituted in semen through the diet or by adding antioxidants to semen extender. However, as the loss of sperm motility during cooled-storage is not only an effect of plasma membrane dysfunction but also of mitochondrial membrane dysfunction, addition of antioxidants to semen during cooled-storage may have only limited effects.
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