Effect of resveratrol treatment on apoptosis and apoptotic pathways during boar semen freezing

Resveratrol(3,5,4’-trihydroxystilbene,RSV) has been widely used in mammalian cells,but whether it can be used during freezing boar semen is still unknown.The effects of RSV treatment during boar semen freezing on its anti-freezing ability,apoptosis,and possible apoptotic pathways were observed in this study.Sperm motility,mitochondrial membrane potential(ΔΨm),adenosine triphosphate(ATP) content,terminal deoxynucleotidyl transferase(TdT)-mediated dUTP nick-end labeling(TUNEL)-positive apoptotic state,and messenger RNA(mRNA) expression levels of apoptotic genes involved in different apoptotic pathways after freezing with or without RSV treatment were tested.The results showed that:(1) Compared with fresh sperm,the motility,normal acrosome rate,and plasma membrane integrity rate of frozen boar sperm decreased significantly(P<0.05),and RSV did not significantly increase the sperm motility(0.44 vs.0.40,P>0.05),but it did significantly improve the normal acrosome rate(57.65% vs.47.00%,P<0.05) and plasma membrane integrity rate(46.67% vs.38.85%,P<0.05).(2) After freezing,most boar sperm showed low mitochondrial ΔΨm.RSV treatment could increase the rate of high mitochondrial ΔΨm of boar sperm.(3) RSV treatment significantly decreased reactive oxygen species(ROS) levels(58.65% vs.88.41%,P<0.05)and increased the ATP content(0.49 μmol/L vs.0.25 μmol/L,P<0.05) of boar sperm during freezing.(4) The apoptotic rate of the freezing group(80.41%) with TUNEL detection increased significantly compared to the fresh group(9.70%,P<0.05),and RSV treatment greatly decreased the apoptotic rate(68.32%,P<0.05).(5) Real-time polymerase chain reaction(RT-PCR) showed that not only the genes from the death receptor-mediated apoptotic pathway(tumor necrosis factor-α(TNF-α),Fas ligand(FasL),and Caspase-8),but also the genes from the mitochondria-mediated apoptotic pathway(manganese superoxide dismutase(MnSOD),B-cell lymphoma 2(Bcl-2),Bcl-2-associated X protein(Bax),and Caspase-9) were both significantly changed after freezing.RSV treatment during freezing greatly changed their expression levels.Although RSV treatment during boar semen freezing did not significantly increase motility after thawing,it still played an efficient antioxidant role,which could enhance the mitochondrial function and decrease the apoptotic level induced by both the death receptor-and mitochondria-mediated apoptotic pathways.