Oxidative and nitrosative stress in frozen-thawed pig spermatozoa. I: Protective effect of melatonin and butylhydroxytoluene on sperm function

Abstract

The addition of antioxidants to the cryopreservation medium has been shown to exert a positive effect on the quality of frozen-thawed sperm in different species. The objective of the present study was to evaluate the effects of supplementing the freezing medium with butylhydroxytoluene (BHT) and melatonin (MEL) in frozen-thawed pig spermatozoa. With this purpose, six ejaculates coming from six separate boars were cryopreserved in traditional freezing medium (i.e. lactose/egg-yolk/glycerol; Control) supplemented with 1.0 mM BHT (BHT-1), 2.0 mM BHT (BHT-2), 0.01 μM MEL (MEL-1) and 1.0 μM MEL (MEL-2). We evaluated sperm viability, membrane lipid disorder, acrosome integrity, mitochondrial membrane potential, lipid peroxidation, oxidation of thiol groups, and levels of total reactive oxygen species (ROS), peroxynitrite and superoxide anion (·O2−). We also analysed total (TM) and progressive sperm motilities (PM), and kinetic parameters at post-thaw (T0, T30 and T60). The BHT-2 and MEL-2 groups presented higher viability and acrosome integrity, and lower levels of peroxynitrite, ·O2− and lipid peroxidation than the control (P < 0.05), whereas MEL-2 diminished the levels of total ROS (P < 0.05). TM and PM were not affected by the treatment, while, LIN and STR shows differences between experimental groups. In conclusion, the addition of BHT and MEL to cryopreservation medium diminishes oxidative and nitrosative stress markers, which has repercussions for the integrity of plasma and acrosomal membranes of frozen-thawed spermatozoa.