Effects Of L-cysteine On Boar Sperm During Liquid Preservation And Its Mechanism

The maintenance of boar sperm quality during liquid storage at 17℃ is crucial to the conception rate and litter size of sows after artificial insemination.Therefore,improving the preservation effect of boar sperm during liquid preservation is the focus of current semen preservation research.However,boar sperm produces reactive oxygen species during the storage at 17℃.With the extension of storage,antioxidant substances in boar semen and sperm are gradually consumed and boar sperm suffers oxidative stress,leading to the decline of sperm quality and the effect of artificial insemination.According to studies,adding certain antioxidants to boar sperm extender could help increase the antioxidant level in sperm,reduce the degradation of semen quality caused by the accumulation of active oxygen radicals,and prolong the storage time of semen.Cysteine is a common thiol-containing amino acid in organisms.It has good antioxidant capacity and is a prerequisite for the biosynthesis of glutathione.However,there is no conclusion of the optimal concentration of cysteine during liquid preservation of boar sperm and whether the antioxidant effect of cysteine is related to the synthesis of glutathione.In this study,different concentrations of cysteine were added during the preservation of pig semen at17℃.Quality parameters,antioxidant indicators of boar sperm were tested to investigate the antioxidant effect of cysteine and the optimal concentration.Moreover,we also established oxidative stress model of boar sperm,detected sperm quality parameters and glutathione synthesis pathway to further explore the antioxidative mechanism of cysteine.The main results are as follows:1.During liquid storage at 17℃,boar sperm quality was improved by the addition of cysteine,and the optimal concentration is 1.25 m M.On the 13 th day of storage,sperm motility in cysteine treatment group(1.25 m M)was significantly higher than that in the control group(P<0.05).Sperm membrane integrity and acrosome integrity were also significantly higher than those in the control group(P<0.05).During the middle and late period(5-13d)of preservation,mitochondrial membrane potentials and ATP content of boar sperm in cysteine group(1.25 m M)were significantly higher than those in the control group(P<0.05).2.Addition of cysteine protects boar sperm by its antioxidant capacity during liquid preservation.Addition of cysteine in the diluent could decrease the oxidative stress and improve the glutathione(GSH)contents of liquid preserved sperm.On the 5th day of storage,ROS contents and the level of sperm lipid peroxidation(LPO)were significantly lower in the group treated with 1.25 m M cysteine than that in the control(P<0.05),yet the glutathione(GSH)contents of sperm in 1.25 m M cysteine treatment group were significantly higher when compare with the control(P<0.05).During the late period(9-13d)of preservation,ROS contents,LPO level of sperm in the cysteine group(1.25 m M)were extremely significantly lower than that of the control group(P<0.01),yet the glutathione(GSH)contents of sperm in 1.25 m M cysteine treatment group were extremely significantly higher when compare with the control(P<0.05).3.Cysteine protects boar sperm not only by its antioxidant capacity but also the biosynthesis of GSH during liquid preservation.γ-glutamylcysteine synthetase(γ-GCS)and glutathione synthetase(GSS)required for cysteine biosynthesis of GSH were found in boar sperm.During incubation at 37 ° C for 3h,menadione was added to boar semen in all groups except the control.We found the ROS content of sperm in the menaquinone-added group was significantly higher than that in the control group(P<0.05).However,the ROS content of sperm pretreated with 1.25 m M cysteine was significantly lower than the menadione addition group(P<0.05).Sperm supplemented with γ-GCS-specific inhibitor methionine-sulfoxide imine(L-BSO)had a significantly higher content of ROS than sperm in the 1.25 m M cysteine-added group(P<0.05).Sperm motility,plasma membrane integrity,mitochondrial membrane potentials,ATP content,GSH content and γ-GCS activity in the menadione addition group were significantly reduced when compared with control(P<0.05).Sperm motility,membrane integrity,mitochondrial membrane potentials,ATP content,GSH content and γ-GCS activity in the 1.25 m M cysteine-pretreated group were significantly higher than those in the menadione-added group(P<0.05).Sperm motility,membrane integrity,mitochondrial membrane potentials,ATP content,GSH content and γ-GCS activity of sperm in the L-BSO treated group sperm were significantly lower than that in the 1.25 m M cysteine-added group(P<0.05).In summary,the addition of 1.25 m M cysteine to the extender could effectively improve the quality of boar sperm during liquid preservation.Cysteine could protect sperm by its own antioxidant capacities,and may promote the synthesis of GSH in boar sperm.