Effects Of Trehalose On Protamine Deficiency,Lipid Peroxidation,and Tyrosine Phosphorylation After Capacitation

In the process of cryopreservation,glycerol as an osmotic protective agent prevents the ice crystals from damaging the sperm membrane,so that the quality parameters such as sperm vitality,acrosomal membrane integrity,and plasma membrane integrity are improved.However,the toxicity of glycerin will cause the loss of sperm protamine,resulting in a reduction in fertilization rate.The main purpose of this experiment was to investigate the effect of replacing some glycerol with trehalose after glycerol concentration was lowered on the protein tyrosine phosphorylation of sperm of Yanbian yellow cattle,the lack of protamine and the peroxidation of membrane lipid.The test was divided into a control group(6%glycerol+0 mM trehalose)and different concentrations of trehalose treatment group(3%glycerol+0,50,100 and 150 mM).The quality parameters of frozen and thawed bovine sperm,tyrosine phosphorylation level after capacitation in vitro,mitochondrial membrane potential and viability,protamine deficiency and membrane lipid peroxidation were detected.The test results are as follows:1.Compared with fresh sperm,sperm motility,kinetic parameters,plasma membrane integrity and acrosomal integrity of each freeze-thaw treatment group were significantly decreased(P<0.05).The sperm motility,kinetic parameters,plasma membrane integrity and acrosomal membrane integrity of the treatment group(3%glycerol+100 mM trehalose)were significantly higher than those of the control group and other treatment groups(P<0.05).2.After the sperm of Yanbian Yellow Cattle after lyophilization was processed,the acrosomal membrane protein was separated and the protein content was measured.Finally,SDS-PAGE and Western blot analysis.Compared with the control group,the sperm protein tyrosine phosphorylation level of the 3%glycerol treatment group(3%glycerol+100 and 150 mM trehalose)was significantly higher than that of the control group(P<0.05).3.Hoechst 33342,PI and JC-1 were used to simultaneously assess the semen viability(head)and mitochondrial membrane potential level(middle).Compared with fresh sperm,sperm viability and mitochondrial membrane potential of each freeze-thaw treatment group decreased significantly(P<0.05).The sperm viability and mitochondrial membrane potential of the treatment group(3%glycerol+100 mM trehalose)were significantly higher than those of the control group(P<0.05).4.CMA3 staining was used to detect the protamine deficiency of freeze-thaw sperm.Compared with fresh sperm,the levels of sperm protamine deficiency in each freeze-thaw treatment group were significantly increased(P<0.05).The protamine-deficient sperm in the treatment group(3%glycerol+100 mM trehalose)was significantly lower than that in the control group(P<0.05).5.In order to detect the level of sperm membrane lipid disorders,sperm samples were stained with anthocyanin 540(M540)and Yo-Pro-1 and detected by flow cytometry.Compared with fresh sperm,the sperm membrane lipid disorder of each freeze-thaw treatment group decreased significantly(P<0.05).The rate of live sperm in the treatment group(3%glycerol+100 mM trehalose)was significantly higher than that in the control group(P<0.05).The conclusion of this study is:1.Adding 100mM trehalose to replace part of glycerol can significantly improve sperm motility and plasma membrane integrity;2.Adding 100 and 150mM trehalose to replace part of glycerol can significantly increase the tyrosine phosphorylation level of sperm protein after energy acquisition;3.Adding 100mM trehalose to replace part of glycerol can significantly improve the sperm viability and mitochondrial membrane potential;4.Adding 100mM trehalose to replace part of glycerol can significantly improve the protamine deficiency level of sperm;5.Adding 100mM trehalose to replace part of glycerol can significantly improve the membrane lipid disorder of sperm.