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

Abstract

The aim of this study was to assess the dose- and time-dependent in vitro effects of ferrous sulphate (FeSO4.7H2O) 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 μM) of FeSO4.7H2O using the SpermVisionTM 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 FeSO4.7H2O). After 2 h, FeSO4.7H2O stimulated the overall percentage of spermatozoa motility at the concentrations of ≤ 125 μM. However, experimental administration of 250 μM of FeSO4.7H2O 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 ≥ 500 μM of FeSO4.7H2O (P < 0.001). The concentrations of ≤ 62.50 μM of FeSO4.7H2O markedly stimulated (P < 0.001) spermatozoa activity after 24 h of exposure, while at high concentrations of ≥ 500 μM of FeSO4.7H2O 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 (≥ 500 μM) of FeSO4.7H2O 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 μM of FeSO4.7H2O; Time 6 h) induced even necrotic alterations to spermatozoa. These results suggest that high concentrations of FeSO4.7H2O 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.

Fig 1. Spermatozoa motility (MOT; %) exposed to FeSO₄.7H₂O in different time periods.

The control group (Ctrl) received a culture medium without FeSO₄.7H₂O administration; Group A– 3.90 μM; B– 7.80 μM; C– 15.60 μM; D– 31.20 μM; E– 62.50 μM; F– 125 μM; G– 250 μM; H– 500 μM; I– 1000 μM of FeSO₄.7H₂O. Results of the analysis were obtained of 10 repeated experiments at each concentration. The statistical difference between the values of Ctrl and treated spermatozoa was indicated by asterisks ***P < 0.001; **P < 0.01; *P < 0.05 (One-way ANOVA with Dunnett’s multiple comparison test). CASA system.

Fig 2. The effect of FeSO₄.7H₂O on the viability of spermatozoa in different time periods.

Each bar represents the arithmetic mean (± SD) absorbance in % of (untreated) control group (Ctrl), which represented 100%. Results of the analysis were obtained of 7 independent experiments at each concentration. A decline in absorbance reflects a decline in the cell viability. The control group (Ctrl) received a culture medium without FeSO₄.7H₂O administration; Group A– 3.90 μM; B– 7.80 μM; C– 15.60 μM; D– 31.20 μM; E– 62.50 μM; F– 125 μM; G– 250 μM; H– 500 μM; I– 1000 μM of FeSO₄.7H₂O. The statistical difference between the values of Ctrl and treated spermatozoa was indicated by asterisks ***P < 0.001; **P < 0.01; *P < 0.05 (One-way ANOVA with Dunnett’s multiple comparison test). MTT assay.

Fig 3. Fluorescent staining of bovine spermatozoa in the control group without FeSO₄.7H₂O administration after Time 2 h of culture.

Blue-stained (DAPI positive) chromatin of spermatozoa heads (A). The cells are Annexin V-negative (B), without apoptotic changes (400x magnification).

Fig 4. In vitro culture of spermatozoa with 500 μM of FeSO₄.7H₂O after Time 6 h.

Blue-stained (DAPI positive) chromatin of spermatozoa heads (A, C). Detection of apoptosis in spermatozoa (green staining) (B). Annexin V fluorescence reaction was detected in the mitochondrial segment and head (acrosomal part) of bovine spermatozoa (D; arrow) (400x magnification).

Fig 5. In vitro culture of spermatozoa with the highest concentration (1000 μM) of FeSO₄.7H₂O after Time 6 h.

Blue-stained (DAPI positive) chromatin of spermatozoa heads (A). Typical apoptotic (B) as well as necrotic spermatozoa alterations (C), fluorescently detected by propidium iodide (red staining) (400x magnification).

Fig 6. More detailed image of altered spermatozoa in the group with the highest concentration (1000 μM) of FeSO₄.7H₂O and the longest time of exposure (6 h).

Blue-stained (DAPI positive) chromatin of spermatozoa heads (A). Annexin-positive regions of apoptotic changes (B) were found not only in the mitochondrial portion, but also on the spermatozoa head (acrosomal part). Necrosis positivity by propidium iodide at the excitation 488 nm was detected (C) (400x magnification).