Effects Of Glucose,Pyruvate And Lactic Acid On Metabolism And Related Genes Of Liquid Stored Boar Sperm

Artificial insemination（AI）has been widely used in modern pork industry.About 99%of AIs are performed with liquid stored boar semen for 1-5 days at 15-25℃.Moreover,85%semen is preserved for 1-2 days at room temperature.Generally,different components（Antibiotics,sugars,antioxidants and sperm stimulants etc.）are added to extenders to inhibit sperm metabolism,slow down sperm movement,reduce energy consumption and achieve the purpose of long-term preservation.The effects of different diluents on the metabolism and life span of pig sperm were also different.However,the domestic pig farms often use the self-made diluent,which has short preservation time and poor effect,which seriously affects the conception rate and litter size of sows.The metabolic activity of sperm is the basis for sperm to maintain its life span,motile and fertility.How to long-term storage of boar sperm and maintain its normal motility,viability and fertilization ability become a key problem in modern pork production.However,the molecular mechanism of sperm metabolism on sperm is still unknown.Furthermore,the way of sperm metabolism in boar is still controversial.Therefore,in this study,different concentrations of glucose,lactic acid,pyruvate and some metabolic inhibitors,electron transport chain inhibitors（Rotenone,CCCP）and glycolysis pathway inhibitors（iodoacetic acid Sodium）,were added to determine the effects on sperm metabolism,kinetic parameters and metabolism-related m RNA and miRNA during liquid storage.The results will help to reveal the metabolic pathways of pig sperm during liquid storage.The results are as follows:（1）To evaluate the effects of metabolic substances on boar sperm quality parameters,different concentrations of glucose,lactic acid and pyruvic acid were added during liquid storage.The results showed that the plasma membrane integrity（PMI）,mitochondrial membrane potential（MMP）,ATP content,and progressive forward speed of sperm with 5 m M glucose,3 m M lactic acid and 5 m M pyruvate were significantly higher than those of the control group（p<0.05）.The optimal concentration of glucose,lactic acid and pyruvate added to basic solution of PBS without Ca²⁺and Mg²⁺were 5 m M,3 m M and 5 m M,respectively.（2）The results of dual luciferase reporter system indicated that PGAM1 is the direct target gene of miR-1343.In boar sperm,miR-1343 targets PGAM1 to regulate phosphoglycerate mutase to catalyze the conversion between 3-phosphoglycerate and 2-phosphoglycerate,thereby participating in glucose metabolism.（3）After treating boar sperm with oxidative phosphorylation inhibitor（Rotenone,CCCP）,ATP content was significantly lower than that of glycolysis inhibitor（sodium iodoacetate）（p<0.05）,and progressive forward speed of sperm was significantly reduced（p<0.05）.The results show that the boar spermatozoa mainly rely on the oxidative phosphorylation pathway to provide energy to maintain the sperm’s movement and metabolism during liquid storage at 17℃in vitro.（4）To detect the effect of different metabolic substances on expression level of pig sperm miR-26a/PDHX and miR-1343/PGAM1,5 m M glucoseand was added at room temperature during liquid storage.The results showed that the relative expression levels of PDHX and PGAM1 in 5 m M glucose group wassignificantly higher than those in the control group（p<0.05）.While,the relative expressions of miR-26a and mir-1343 were significantly lower than those in the control group（P<0.05）.After incubation with metabolic inhibitors for 24 hours,the expression levels of PDHX and PGAM1 in the rotenone and CCCP group were significantly higher than those in the control group（p<0.05）by RT-qPCR,and the relative expression levels of miR-26a and miR-1343 were significantly lower than those in control group（p<0.05）.The expression levels of PDHX and PGAM1 in the sodium iodoacetate group were significantly lower than those in the control group（p<0.05）,while the relative expression levels of miR-26a and miR-1343 were significantly higher than those in the control group（p<0.05）.The results suggested that boar sperm metabolism was regulated by the miR-26a/PDHX and miR-1343/PGAM1 pathways during liquid storage.In conclusions,the optimal concentration of glucose,lactic acid and pyruvate added to basic solution of PBS without Ca²⁺and Mg²⁺were 5 m M,3 m M and 5 m M,respectively.Except for glycolysis,oxidative phosphorylation is the main metabolic pathway of boar sperm to maintain motility and life span during liquid storage.