Conditional Antioxidant Protection of Stallion Spermatozoa by Butylated Hydroxytoluene Under Induced Oxidative Stress

Despite advances in the method, equine semen cryopreservation still leads to a marked decline in sperm quality, partly attributed to oxidative stress, which motivates the search for lipophilic antioxidants capable of stabilizing the plasma membrane during cooling and freezing. This study performed three experiments to evaluate butylated hydroxytoluene (BHT) as an additive to commercial skim milk-based (Botu-Sêmen) and egg yolk-based (Botu-Crio) extenders. In Experiment 1, a dose-response curve (0.5, 1.0, and 1.5 mM) was performed using stallion semen to assess toxicity and establish a safe concentration by analysing sperm kinetics (CASA) and membrane integrity immediately after dilution and after 24 h at 5°C. In Experiment 2, BHT at 0.5 mM was tested in standardized cooling (24 h at 5°C) and freezing protocols. Stallions were classified as Good/Bad Coolers and Good/Bad Freezers, and sperm kinetics (CASA), plasma membrane integrity assessed by epifluorescence microscopy (CFDA/PI), and plasma/acrosomal integrity and oxidative stress markers assessed by flow cytometry were evaluated. In Experiment 3, spermatozoa were exposed to an oxidative challenge with arachidonic acid (50 μM for 15 min at 37°C) to elucidate the mechanism of action of BHT. BHT at 0.5 mM was pharmacologically safe; however, it did not improve cryotolerance or reduce basal oxidative stress in commercial cooling and freezing protocols (p > 0.05). Under oxidative challenge, BHT markedly reduced lipid peroxidation and intracellular reactive oxygen species production (p < 0.05), indicating a conditional protective effect dependent on oxidative imbalance and impaired antioxidant defences. These findings suggest that BHT acts as an antioxidant primarily under critically increased ROS generation, but does not enhance the performance of commercial cryopreservation when procedures already operate within optimal conditions.