In vitro effect of ferrous sulphate on bovine spermatozoa motility parameters, viability and Annexin V-labeled membrane changes

The aim of this study was to assess the dose- and time-dependent in vitro effects of ferrous sulphate (FeSO.sub.4 .7H.sub.2 O) on the motility parameters, viability, structural and functional activity of bovine spermatozoa. Spermatozoa motility parameters were determined after exposure to concentrations (3.90, 7.80, 15.60, 31.20, 62.50, 125, 250, 500 and 1000 [mu]M) of FeSO.sub.4 .7H.sub.2 O using the SpermVision.sup.TM CASA (Computer Assisted Semen Analyzer) system in different time periods. Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay, and the Annexin V-Fluos was applied to detect the membrane integrity of spermatozoa. The initial spermatozoa motility showed increased average values at all experimental concentrations compared to the control group (culture medium without FeSO.sub.4 .7H.sub.2 O). After 2 h, FeSO.sub.4 .7H.sub.2 O stimulated the overall percentage of spermatozoa motility at the concentrations of [less than or equal to] 125 [mu]M. However, experimental administration of 250 [mu]M of FeSO.sub.4 .7H.sub.2 O significantly (P < 0.001) decreased the spermatozoa motility but had no negative effect on the cell viability (P < 0.05) (Time 2 h). The lowest viability was noted after the addition of [greater than or equal to] 500 [mu]M of FeSO.sub.4 .7H.sub.2 O (P < 0.001). The concentrations of [less than or equal to] 62.50 [mu]M of FeSO.sub.4 .7H.sub.2 O markedly stimulated (P < 0.001) spermatozoa activity after 24 h of exposure, while at high concentrations of [greater than or equal to] 500 [mu]M of FeSO.sub.4 .7H.sub.2 O the overall percentage of spermatozoa motility was significantly inhibited (P < 0.001) and it elicited cytotoxic action. Fluorescence analysis confirmed that spermatozoa incubated with higher concentrations ([greater than or equal to] 500 [mu]M) of FeSO.sub.4 .7H.sub.2 O displayed apoptotic changes, as detected in head membrane (acrosomal part) and mitochondrial portion of spermatozoa. Moreover, the highest concentration and the longest time of exposure (1000 [mu]M of FeSO.sub.4 .7H.sub.2 O; Time 6 h) induced even necrotic alterations to spermatozoa. These results suggest that high concentrations of FeSO.sub.4 .7H.sub.2 O are able to induce toxic effects on the structure and function of spermatozoa, while low concentrations may have the positive effect on the fertilization potential of spermatozoa.