Effects of thiol compounds on DNA-damage detection and <i>in vitro</i> fertilization outcomes in bovine sperm

Reduced glutathione (GSH), a thiol compound, plays an important role in protecting sperm from excessive levels of reactive oxygen species. Our previous study demonstrated that GSH supplementation was associated with improved in vitro fertilization (IVF) outcomes. However, GSH supplementation also increased the proportion of DNA-damaged sperm detected in the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, indicating involvement of its thiol components. In this study, we investigated the effects of several thiol compounds, including l-cysteine (Cys), N-acetyl-l-cysteine (NAC), and dithiothreitol (DTT), on bovine sperm DNA integrity. The addition of Cys or NAC (0.5, 1, or 5 mM) to the medium caused an increase in DNA damage, which was observed either throughout the sperm head or specifically in the post-acrosome (PA) region. DTT treatment increased DNA damage in both the PA and acrosomal regions, and it induced progressive sperm head lysis from the acrosome to the entire head. These treatments caused sperm DNA-damage rates that exceeded the normal range, suggesting that thiol groups enhance the detection of DNA damage. The effects of these thiols on IVF outcomes were further examined. Short-term exposure to 1 mM Cys before insemination improved cleavage rates, whereas prolonged exposure at concentrations ≥ 1 mM during insemination reduced cleavage rates. NAC supplementation had no significant effects. A mild negative correlation was observed between TUNEL-positive sperm rates and both cleavage and blastocyst formation rates following short-term treatment with 0.5 mM Cys. These findings suggest that thiol supplementation, when optimally dosed and timed, may enhance the sensitivity of DNA-damage detection rather than induce additional damage.