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 × 10 7 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.
Bull semen, freezing, artificial insemination, antioxidant activities, lipid peroxidationSemen cryopreservation has allowed specific opportunities for the conservation and widespread dissemination of valuable genetic resources through sperm banks, the guarantee of a constant commercial supply of semen, and collaboration in breed improvement programs by means of artificial insemination (AI) (Holt et al. 1997). Some of the problems encountered using AI in pigs, sheep and exotic species include decreased motility, membrane integrity and fertilizing potential of spermatozoa during cryopreservation, premature activation in the female genital tract, and altered membrane responses to physiological stimuli of sperm that survive the freezing-thawing process (Viswanath and Shannon 2000). It is well known that phospholipids in the sperm plasma membrane undergo peroxidation, which results in the formation of reactive oxygen substances (ROS, namely, superoxide anion radical, hydrogen peroxide) and lipid hydroperoxides (Alvarez and Storey 2005). Excessive generation of ROS by immature and abnormal spermatozoa, and sperm processing (e.g., extending, freezingthawing process), accompanied by low scavenging and antioxidant concentrations in semina...