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 beta-carotene (beta: 250-4000 mu M) and alpha-tocopherol (alpha: 31-496 mu M) 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 beta-carotene (250-1000 mu M) and alpha-tocopherol (31-124 mu M) concentrations added to the cooling-freezing or thawing extenders (n = 30) before (0 min) and after 90 min incubation (37 C-degrees). Toxicity results showed a decrease in the proportion of live spermatozoa with non- reacted acrosome from 75.1 +/- 3.3 % using beta 250/alpha 31 to 60.1 +/- 5.7 % and 59.3 +/- 5.4 % in samples with beta 2000/alpha 248 and beta 4000/alpha 496 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 beta 500/alpha 62 and beta 1000/alpha 124, 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 beta-carotene and alpha-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 beta 1000/alpha 124 showed protective effects during cryopreservation, but post-thawing supplementation may stimulate oxidative stress rather than prevent it.