Combined effects of resveratrol and epigallocatechin-3-gallate on post thaw boar sperm and IVF parameters

Frozen-thawed boar semen suffer a fertility decrease that negatively affects its widespread use. In recent years supplementing frozen-thawed boar sperm with different antioxidants gave interesting and promising results; the aim of the present work was to study the effect of supplementing boar sperm thawing medium for 1 h with combination of epigallocatechin-3-gallate (EGCG, 50 μM) and Resveratrol (R, 2 mM), on boar sperm motility (assessed by CASA), viability, acrosome integrity, mitochondrial function, lipid peroxidation and DNA integrity (assessed by flow cytometry), protein tyrosine phosphorylation (assessed by immunofluorescence) and on in vitro fertilization (IVF). Our results demonstrate that sperm motility is negatively affected by R (alone or associated with EGCG, p < 0.05) in comparison to control and EGCG groups both at 1 h and 4 h; this effect is evident both in average motility parameters and in single cells kinematics, studied by cluster analysis, that showed the presence of a specific cell population with simil-hyperactivated features in R group (p < 0.01). Viability, acrosome integrity, mitochondrial functionality and lipid peroxidation are not influenced by the addition of the antioxidants; finally, DNA integrity is negatively influenced by R (both alone or associated with EGCG) both at 1 h and 4 h incubation (p < 0.05). Finally, tyrosine phosphorylated protein immunolocalization, used as capacitation parameter, is not affected by the different treatments. Penetration rate is strongly enhanced by R, both alone or associated with EGCG (p < 0.05); EGCG increases penetration rate as well but to a lower extent. Our findings demonstrate that the combination of R and EGCG could positively affect frozen-thawed boar sperm fertility in vitro; the effect is evident also in R groups, thus demonstrating that this antioxidant is predominant, and no synergic effect is present. Some insights are needed to understand if, in particular R (that showed the strongest effect) could be profitably used for artificial insemination in vivo, given the detrimental effect of this molecule on both sperm motility and DNA integrity.