Protective effect of supplementation with water soluble β-carotene and α-tocopherol in boar sperm cooling-freezing extender, but not in the thawing extender

Cryopreservation of sperm is a crucial tool for the long-term preservation of male genetic material, causing significant issues in motility, membrane, and acrosome integrity, among other parameters. Antioxidants have been used to cope with these detrimental effects. We tested 1. the toxicity of a wide range of water soluble β-carotene (β: 250-4000 µM) and α-tocopherol (α: 31-496 µM) concentrations on boar ejaculated sperm (n = 6) in parameters as motility, viability, acrosome reaction, apoptosis, oxidation, mitochondrial activation and membrane potential; 2. the effect of various β-carotene (250-1000 µM) and α-tocopherol (31-124 µM) concentrations added to the cooling-freezing or thawing extenders (n = 30) before (0 min) and after 90 min incubation (37 °C). Toxicity results showed a decrease in the proportion of live spermatozoa with non-reacted acrosome from 75.1 ± 3.3 % using β250/α31 to 60.1 ± 5.7 % and 59.3 ± 5.4 % in samples with β2000/α248 and β4000/α496 respectively (p < 0.05), suggesting a detrimental effect of the highest concentrations. Antioxidant supplementation in the cooling-freezing extender decreased the apoptotic and oxidized spermatozoa in β500/α62 and β1000/α124, relative to the control. In contrast, antioxidants addition to the thawing extender induced some detrimental effects in several sperm parameters analyzed. In conclusion, water-soluble β-carotene and α-tocopherol prevent acrosome reaction and oxidation during cooling-freezing on boar sperm. High concentrations of these antioxidants negatively impacted motility and mitochondrial function, suggesting cytotoxic effects and potential capacitation-like changes. The β1000/α124 showed protective effects during cryopreservation, but post-thawing supplementation may stimulate oxidative stress rather than prevent it.