Influence of various antioxidants on microscopic-oxidative stress indicators and fertilizing ability of frozen-thawed bull semen

Cryopreservation is associated with the production of reactive oxygen substances (ROS), which lead to lipid peroxidation of sperm membranes, resulting in a loss of sperm motility, viability and fertility. The aim of this study was to determine effects of the antioxidants of oxidized glutathione (GSSG), reduced glutathione (GSH) and bovine serum albumin (BSA) on standard semen indicators (motility, acrosome and total abnormalities, HOST), endogenous antioxidant enzyme activities and fertilizing ability of frozen-thawed bull semen. Eighteen ejaculates from each of 3 Holstein bulls were collected using an artificial vagina and 9 replicates of the ejaculates were diluted with a Bioxcell®-based extender supplemented with antioxidants, including BSA (5 mg/ml), GSH (2 mM), GGSG (2 mM), and an extender containing no antioxidants (control). Insemination doses (1.5 × 107 sperm/0.25 ml straw) were prepared for the insemination of cows at observed oestrus. Supplementation with antioxidants led to lower percentages of acrosome damage (4.0 ± 0.5%, 4.4 ± 0.5%, 4.0 ± 0.3%, respectively) and total abnormalities (10.3 ± 0.7%, 9.7 ± 0.8%, 10.4 ± 0.6%), compared to the controls (6.5 ± 0.6 and 14.9 ± 1.1% P < 0.01). Pregnancy rate after insemination was highest (72.2%) in the group which was given BSA (P < 0.05). There were no significant differences among groups in GSH and glutathione peroxidase (GSH-PX) enzyme activities. Superoxide dismutase (SOD) activities (0.05 ± 0.005, 0.03 ± 0.005, 0.05 ± 0.009 μkat/g protein, respectively) in all of the experimental groups with antioxidants were lower than the control group (0.11 ± 0.024 μkat/g protein, P < 0.001). Furthermore, BSA increased (P < 0.001) the activity of catalase (CAT, 304.23 ± 114.69 μkat/g protein), following the freezing-thawing process.