Protective effects of aátocopherol on the activity and antioxidant profile of bovine spermatozoa subjected to ferrous ascorbateáinduced oxidative stress

Abstract

As spermatozoa are highly vulnerable to oxidative stress development, in vitro antioxidants offer an additional line of defense to the male reproductive system against oxidative insults. a-tocopherol (a-TOC) is the most abundant form of vitamin E identifed in the seminal plasma and spermatozoa membranes, being able to terminate numerous oxidative chain reactions causing substantial damage to biomolecules vital for sperm survival. This study was designed to shed more light on the in vitro effects of a-tocopherol with respect to the vitality and intracellular antioxidant profle of bovine spermatozoa subjected to ferrous ascorbate-induced oxidative stress. Spermatozoa were washed out from 50 bovine ejaculates, suspended in 2.9 % sodium citrate and subjected to a-TOC treatment (10, 50, 100 and 500 μmol/L) in the presence or absence of ferrous ascorbate (FeAA; 150 μmol/L FeSO4 and 750 μmol/L ascorbic acid) during a 6h in vitro culture. Spermatozoa motion parameters were assessed using the SpermVision™ computer-aided sperm analysis (CASA) system. Cell viability was examined with the metabolic activity (MTT) assay, and the nitroblue-tetrazolium (NBT) test was applied to quantify the intracellular superoxide formation. Cell lysates were prepared at the end of the experiments in order to assess the intracellular activity of superoxide dismutase (SOD), catalase (CAT), as well as glutathione (GSH) and malondialdehyde (MDA) concentrations. Treatment with FeAA reduced both spermatozoa motility parameters (P < 0.001) as well as viability (P < 0.05 with respect to Time 0 h; P < 0.01 in case of Time 2 h and P < 0.001 in relation to Time 6 h), decreased the antioxidant parameters of the samples (P < 0.001 in case of SOD; P < 0.01 with respect to CAT and GSH) but increased the superoxide production (P < 0.01 in case of Time 0h and P < 0.001 with respect to Times 2 h and 6 h) and lipid peroxidation (P < 0.001). a-TOC administration resulted in a preservation of the spermatozoa motility characteristics (P < 0.001 with respect to 500 μmol/L a-TOC), viability (P < 0.001 in case of 500 μmol/L a-TOC and P < 0.05 with respect to 100 μmol/L a-TOC) and antioxidant profle (P < 0.01 related to the impact of 500 μmol/L a-TOC on the SOD activity; P < 0.05 in relation to CAT; P < 0.01 with respect GSH; 100-500 μmol/L a-TOC), with 500 μmol/L a-TOC being the most effective. Our results suggest that a-tocopherol possesses signifcant antioxidant properties that may prevent the deleterious effects caused by free radicals to spermatozoa, and extend the fertilization potential of male reproductive cells.