Biochemical Index And Marker Protein Screening Of High And Low Motility Sperm Of Yak After Freezing And Thawing

Yak is a precious species resource in the plateau area of China,providing herdsmen with important means of production and living.Semen freezing technology plays an important role in the development of modern biotechnology,providing support for the protection and utilization of germplasm resources and the efficient development of yak breeding.However,sperm is very sensitive to temperature changes,freezing and thawing process will damage the structure and function of sperm and other aspects,resulting in the phenomenon of freezing damage is still common,and there are relatively few studies on the viability of frozen yak sperm,so the cryopreservation of semen is still facing a huge challenge.In this study,freeze semen of three healthy yaks were selected,45% Percoll and90% Percoll density gradient centrifugation were used to divide frozen and thawed yak sperm into high motility sperm group and low motility sperm group.In order to explore the structural and functional differences of yak sperm after freezing and thawing,the quality of the two separated and purified sperm groups was tested.TMT labeling and liquid chromatography-tandem mass spectrometry were used to compare the proteomics differences between high motility sperm and low motility sperm after freezing and thawing of yaks.Bioinformatics analysis was carried out to screen sperm proteins that might be related to sperm motility of yaks.Western blot and immunofluorescence methods were used to detect the expression distribution of the screened proteins.The correlation between the screening proteins and the motility and quality of yak sperm was analyzed in combination with the quality differences of high and low motility sperm.The results showed that:(1)After percoll separation and purification,CASA detected the motility of yak sperm with high motility and low motility was 95.75±0.71 and 18.82±4.58 respectively.The membrane integrity,mitochondrial activity and ATP content of high motility sperm were significantly higher than those of low motility sperm(p<0.01).Lactate dehydrogenase(LDH)enzyme and Acid phosphatase(ACP)enzyme activities in high motility sperm were significantly higher than those in low motility sperm(p<0.05),the activities of Superoxide dismutase(SOD)enzyme and Alkaline phosphatase(AKP)enzyme in high motility sperm were significantly higher than those in low motility sperm(p < 0.01).Ros levels in high motility sperm were significantly lower than those in low motility sperm(p < 0.01).(2)TMT labeling,combined with HPLC-MS/MS,was used to quantify and analyze proteins extracted from high and low motility sperm of thawed yaks.A total of 3280 proteins were detected,and 303 proteins with fold change ≥1.2 were identified(p<0.05),including 262 significantly up-regulated differential proteins in high motility sperm.Differential proteins were significantly down-regulated in 41 highly active sperm.Bioinformatics analysis of differential proteins showed that subcellular localization showed that differential proteins were mainly located in cytoplasm.GO analysis showed that most of the differential proteins were involved in cellular processes,metabolic processes and biological regulation.In cell composition,most of them are located in organelles and membranes.In terms of molecular function,differential proteins are mainly involved in binding function,followed by catalytic activity.KEGG signal pathway enrichment analysis showed that different proteins were involved in metabolic pathway,amino acid synthesis,glycolysis and gluconeogenesis,TCA cycle,PPAR signal pathway,etc.(3)Combined with GO annotation,KEGG pathway and PPI analysis,three potential marker proteins including Fatty acid-binding protein 5(FABP5),Cystathionine gamma-lyase(CTH)and Phosphoglycerate kinase 1(PGK1)that may be related to sperm motility were screened out from the differential proteins.The relative expression levels of FABP5,CTH and PGK1 in yak sperm with high motility were detected using GAPDH as internal reference protein.The results showed that FABP5,CTH and PGK1 proteins were mainly located in the posterior end of sperm head in yak sperm with high motility,and the expression levels were significantly higher than those in low motility sperm group(p <0.01).In this study,protein markers related to semen motility of yaks after freezing and thawing were screened to provide theoretical support for improving semen quality after thawing and improving semen freezing technology of yaks.