The sperm chromatin structure assay does not detect alterations in sperm chromatin structure induced by hydrogen...

The objective of this study was to investigate the effects of hydrogen peroxide (H₂O₂) on the chromatin structure of sperm. For this purpose, 44 cryopreserved bovine ejaculates were analyzed immediately post-thaw (control sperm, CON S), after 1 h of post-thaw incubation (non-oxidized sperm, NOX S), and after 1 h of post-thaw incubation in different concentrations of H₂O₂ (OX S; 50, 100 µM, 1000 µM H₂O₂). Sperm motility was determined using computer-assisted sperm analysis. Sperm plasma membrane and acrosome integrity were assessed by flow cytometry after staining with propidium iodide and fluorescein isothiocyanate-conjugated peanut agglutinin. Chromatin damage was assessed using the sperm chromatin structure assay (SCSA), and DNA damage was evaluated using the single cell gel electrophoresis (Comet) assay. Sperm motility and plasma membrane and acrosome integrity decreased while chromatin damage and DNA damage increased after 1 h of incubation (P < 0.05). The addition of H₂O₂ adversely affected all parameters (P < 0.05) except for chromatin structure. The role of H₂O₂ in sperm chromatin damage was investigated by supplementation of 10 IU catalase, which reversed the damage (P < 0.05). Interestingly, the chromatin decondensation induced by 10 mM dithioreitol, evidenced by an increase in chromatin damage in the SCSA (P < 0.05), was reversed by H₂O₂. In conclusion, H₂O₂ causes alterations in sperm functional status and DNA integrity. However, our results show that high concentrations of H₂O₂ can induce chromatin condensation, and thus the use of the SCSA for the assessment of H₂O₂-induced chromatin damage should be carefully considered.