Screening Of Blood Protein Markers And Its Establishment Of Warning System For Inactive Ovaries In Dairy Cows

In recent years,the negative energy balance(NEB)of high-yielding dairy cows in intensive cattle farms in China is relatively common,and the rate of anestrus caused by inactive ovaries(1O)is relatively high,which seriously affects the reproductive performance of dairy cows and reduces the production performance and economic value of dairy cows.However,the proteomic mechanism of the occurrence of IO in the NEB of postpartum energy in high-producing dairy cows is still unknown,and there is no corresponding early warning technology.Therefore,this study applied proteomics technology to carry out the screening of blood protein markers of IO cows and the signal pathways of key factors,in order to reveal the role of the obtained differential proteins in 10,and lay a foundation for establishing the risk early warning system of this disease.In this experiment,the energy positive balance(PEB)and negative energy balance(NEB)of Holstein cows were tracked from day 14-21 days to 60-90 days postpartum.After the estrus performance,rectal examination and B-ultrasound examination,the cows were tested according to the criteria.Divided into IO group(IO),normal estrus control group(CON),each group of 50 collected serum.In addition,follicular granulosa cells collected from slaughterhouses from the same breed,with similar background,no reproductive disease,and postpartum health,were divided into three groups based on follicle diameter,large follicles(L),middle follicles(M),and small follicles(S),10 samples per group.In addition,in the IO group and the CON group,15 cows were randomly selected from each group for proteomic analysis,and another 35 cows were used for ELISA.The test results were as follows:(1)In this experiment,iTRAQ/LC-MS/MS was used to study the NEB of dairy cow serum and 23 kinds of differentially expressed proteins were obtained,among which 16 were up-regulated in NEB group,and 7 were down-regulated in NEB group.These differential proteins were associated with lipid metabolism(APOA1,APOA5,APOF,TTR)and protease complex systems(PSMA2,PSMA4,PSMA7,and PSMB1)and also with glycolytic pathways(SORD and GAPDH).(2)In this experiment,the effect of NEB on postpartum reproductive hormone secretion in dairy cows was studied.Through the independent sample t-test of FSH,LH,E2,and P4 in serum of 10 group and CON group,it was found that dysfunction of reproductive hormones(decreased concentrations of FSH and E2)caused by NEB in dairy cows is the major cause of IO in cows.(3)This experiment applied ITRAQ/LC-MS/MS combination technology to study the IO serum proteomics of dairy cows and obtained 61 differentially expressed proteins,among which 14 differentially expressed proteins related to IO were upregulated by 11 types.Three kinds of expression were downregulated.ADIPOQ,IGFBP2 and RBP4 among 11 differential proteins verified by immunoblotting or ELISA methods affect follicular development through affecting reproductive hormone biological processes,and GPX3 affects follicular development through oxidative stress.(4)In this experiment,we used different sizes of follicles to study the role of glucose metabolism in follicular granulosa cells.We found that the key rate-limiting enzymes of glycolysis in granular cells such as HK,PEK and differential proteins such as ALDOB increased first and then decreased from small to large follicles.The key rate-limiting enzymes PKM2 and differential protein LDHB have been decreasing,suggesting that glycolysis plays a role in small to medium follicles,that is,it plays an early role in follicular development and is a potential mechanism for the occurrence of quiescence in dairy cows.(5)Three energy indexes,four reproductive hormones,and ten key differentially expressed proteins in the blood of IO group and CON group cows from the 60th to the 90th day postpartum were selected and analyzed by correlation analysis,binary logistic regression modeling and ROC analysis.An early warning technology based on single indicators of APOA4 and ITIH3 was established.The early warning values were APOA4>28.825 μg/L and ITIH3>195.07 ng/L.Potential biomarkers based on APOA4+ITIH3 and APOA4+ITIH3+E2 were established.The multi-indicator early warning system has APOA4>19.55 μg/L and ITIH3>191.14 ng/L;the latter has an early warning value of APOA4>47.56 μg/L,ITIH4>187.80 ng/L,and E2<69.63 ng/.L.(6)Based on the deep correlation analysis of the potential mechanism of differential protein in the occurrence of quiescence in dairy cows,we found that IO caused by NEB is associated with various metabolic abnormalities of the body,including lipid metabolism,oxidative stress,and vitamin metabolism,insulin and IGF systems,as well as the formation of COC,etc.,in order to build a metabolic network of disease mapping,suggesting the complexity of the mechanism of postpartum IO caused by NEB in dairy cows.Conclusion:This study used proteomics techniques to obtain negative energy balance in cows and differentially expressed proteins in IO.The relationship between differential protein and NEB and IO was clarified,and a network of signal transduction pathways for the occurrence of IO was constructed.Investigating the mechanism of glycolysis in the IO and establishing a single and multi-indicator risk early warning system for the occurrence of IO were conducted,which laid the foundation for further research on the mechanism and prevention of IO.