Effects Of CLC On Quality Parameters And Antioxidant And Anti-apoptotic Of Frozen-shawed Bovine Sperm

Cryopreservation can prolong the preservation time of sperm,but the process of freezing will cause a series of changes in the cell and physiological structure of sperm,which will be damaged to a certain extent,including the characteristics of sperm capacitation,sperm life,and interaction with oocytes.And fertilization ability,etc.The cryopreservation of semen is to use dry ice(-79 ?),liquid nitrogen(-196?)or other refrigeration equipment as a cold source.After special treatment,the semen is stored at very low temperature,completely inhibiting sperm.The metabolic activity causes the sperm to be dormant,resuscitate after warming up,and does not lose the ability to fertilize,thus allowing the sperm to reach a long-term preservation method.The purpose of this trial was to investigate the effect of adding cholesterol encapsulated methyl-?-cyclodextrin(CLC)on bovine frozen sperm quality(AR).The test was divided into control group(0 mg/ml)and different doses of CLC treatment group(0.5 mg/ml,1.0 mg/ml,1.5 mg/ml,2.0 mg/ml),respectively,to determine the biological characteristics of bovine sperm after freeze-thaw,tyrosine phosphorylation of proteins,antioxidant genes and apoptosis in sperm Gene expression and comparison.The test results are as follows:1.Different concentrations of CLC treatment groups(0 mg/ml,0.5 mg/ml,1.0 mg/ml,1.5 mg/ml,2.0 mg/ml)were added to the semen dilution.Stored in liquid nitrogen for storage for more than 5 hours,the sperm viability and plasma membrane integrity were detected,and the integrity of the acrosomal membrane was weakened to varying degrees.There were no significant differences between the different treatment groups.2.The control group(0 mg/ml)was subjected to freeze-thaw and treatment(0 mg/ml,0.5 mg/ml,1.0 mg/ml,1.5 mg/ml,2.0 mg/ml).Membrane protein separation,protein content determination,and analysis by SDS-PAGE and Western blot.The results showed that the acrosome protein showed that 0.5 mg/ml was closest to the capacitated sperm at 32 KDa.3.For different treatment groups(0 mg/ml,0.5 mg/ml,1.0 mg/ml,1.5 mg/ml,2.0 mg/ml),total RNA was extracted first,followed by reverse transcription and RNA concentration determination.The primer design and the reference sequence of the primer sequence were followed by PCR amplification of the target gene,followed by agarose gel electrophoresis,and finally the detection results of the antioxidant gene expression amount were analyzed.The results showed that the expression bands of CAT,GPx and SOD showed 0.5 mg/ml and 1.0mg/ml,which were significantly higher than the control group(P<0.05).The highest expression was shown at 1.0 mg/ml.4.For different treatment groups(0 mg/ml,0.5 mg/ml,1.0 mg/ml,1.5 mg/ml,2.0 mg/ml),total RNA was extracted first,followed by reverse transcription and RNA concentration determination.The primer design and the reference sequence of the primer sequence were followed by PCR amplification of the target gene,followed by agarose gel electrophoresis,and finally the detection result of the apoptosis gene was analyzed.The results showed that the expression bands of Caspase-3,Caspase-8 and BAX showed that 0.5 mg/ml and 1.0 mg/ml were significantly lower than the control group(P<0.05).The lowest at the 0.5 mg/ml treatment group.