Effects Of Carnitine On Antioxidation,Anti-Apoptosis And Biology Character Of Boar Sperm After Frozen-Thawed

Since the invention of the frozen gamete technology,the frozen semen technology of mammals has developed rapidly with considerable economic benefit.For porcine sperm,because of the poor anti-freezing ability,the commercialization effect of frozen semen is not ideal.At present,the method of fresh artificial insemination is adopted.Carnitine has been used in various medicines for improving sperm quality since its discovery,and it has achieved certain effects in clinical practice.The aim of this study was to investigate the effects of carnitine on pig sperm quality and sperm binding ability after freezing and thawing,and the effects of sperm apoptotic factors:Bax,Bel-2,Caspase—3 and antioxidant enzyme SOD 2,CAT,detection The effect of GPxl mRNA expression levels.After adding different concentrations of carnitine to the frozen liquid,the sperm is frozen and thawed.The sperm biology character,the lipid peroxidation product malondialdehyde(MDA)and the total antioxidant are then tested to select the optimal concentration.The expression levels of apoptosis-related genes and antioxidant-related gene mRNAs in the fresh group were compared,and the freeze-thaw group was selected using the carnitine-free group and the optimal concentration of the freeze-thaw group selected in the first part.Fluorescence staining techniques were compared between the fresh group of sperm-oocyte combinations,the freeze-thaw and non-carnitine groups,and the freeze-thaw and optimal treatment groups.The results are as follows:1.The sperm activity rate,the plasma membrane integrity rate and the acrosome membrane integrity rate were significantly different between each group(P<0.05)except the acrosome membrane integrity rate of 0.025 mg/ml and 0.075 mg/ml groups,meanwhile the group with the concentration of 0.05 mg/ml were the highest.Compared with fresh semen,the concentrations of MDA in freeze-thaw groups significantly increased(P<0.05).The MDA concentration of the group with 0.05 mg/ml treatment was significant lower compared with other treatments(P<0.05),while the rest treatments had no significant difference(P<0.05).The total antioxidant capacity of each freeze-thaw group was significantly lower Compared with fresh sperm(P<0.05).The 0.05 mg/ml treatment group had significant higher total antioxidant capacity than other treatment groups which had no significant difference between each other.(p<0.05).The total antioxidant capacity of freeze-thaw groups was significantly lower than the fresh group,and the best concentration treatment group had the highest total antioxidant capacity and significant difference with other treatment groups,while the other treatment groups had no significant difference(P<0.05).It is proved that the quality of pig spermatozoa is decreased after freeze-thaw treatment,and carnitine has a positive effect on the cryopreservation quality of pig spermatozoa.2.Compared with fresh sperm,the expression levels of apoptotic-related genes in frozen-thawed sperm significantly increased(P<0.05),while the expression levels of anti-apoptotic genes and antioxidant enzyme genes significantly decreased(P<0.05).The expression levels of apoptotic-related genes in sperm of the optimal concentration group were significantly lower than the carnitine-free group(P<0.05),with the contrast of the expression levels of anti-apoptotic genes and antioxidant enzymes genes(P<0.05).3.The results of fluorescence staining after in vitro fertilization with the sperm form the three groups showed that the numbers of sperm adhesion were significant different between the three groups,the number of sperm adhesion in fresh sperm group was the highest and the number of sperm adhesion in the non-carnitine addition group was the lowest.In conclusion,the results showed that the addition of carnitine effectively reduce the adverse effects on sperm during freeze-thaw and improve the quality of cryopreservation sperm by affecting the expression of apoptosis-related genes and antioxidase-related genes during sperm cryopreservation.Carnitine can also improve sperm-oocyte binding ability of frozen sperm.