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

Zuzana Knazicka,Hana Duranova,Peter Massanyi,Anton Kovacik,Michal Miskeje,Tomas Jambor,Shubhadeep Roychoudhury,Norbert Lukac,Veronika Fialkova,Alexander V Makarevich,Joël R Drevet

doi:10.1371/journal.pone.0257766

Zuzana Knazicka, Hana Duranova + Show 9 more

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https://doi.org/10.1371/journal.pone.0257766

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Journal: PloS one	Publication Date: Sep 23, 2021
Citations: 2	License type: CC BY 4.0

Affiliation: Slovak University of Agriculture, Assam University

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.

Highlights

The natural environmental factors and differentiated anthropogenic pollutants, as well as many other sources strongly influence the male reproductive system, both in animals and humans [1, 2]
Fe participates in various physiological, regulatory, and biochemical processes such as oxidation/reduction reactions, electron transport associated with cellular respiration, deoxyribonucleic acid (DNA) synthesis/ repair, cell division and proliferation, all of which are closely related to spermatozoa production and metabolism [7,8,9,10,11,12,13,14,15,16,17]
The quantified Fe in seminal plasma was 0.025 μg/measurements were performed–volume (mL) and spermatozoa Fe concentration measured by the flame atomic absorption spectrophotometry (FAAS) method was 0.049 μg/mL

Summary

Introduction

The natural environmental factors and differentiated anthropogenic pollutants, as well as many other sources strongly influence the male reproductive system, both in animals and humans [1, 2]. Fe has a crucial role as part of metalloproteins such as hemoglobin or myoglobin, as well as enzymes that are associated with energetic reactions [6] It is a biologically essential element of every living organism because Fe cofactors activate enzymes involved in major metabolic processes in the cell. Disturbances in the regulative absorption mechanism can appear due to pathological conditions or prolonged intake of high Fe doses In these cases, since the capacity for storage of Fe in ferritin is exceeded, the metal is complexed to phosphate or hydroxide to form hemosiderin (or it is bound into proteins), and in this form it is present in the liver [3]. Fe can induce cell death by generating free radicals as it interconverts between ferrous (Fe2+) and ferric (Fe3+) forms [24]

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