LC/MS-based Plasma And Follicular Fluid Metabolomics Analysis Of Dairy Cows Suffered From Inactive Ovaries

In the past few decades,due to the rapid development of the dairy industry and the selection of genetic genes,the global milk production of dairy cows has been greatly improved,but the fertility of dairy cows has shown a gradual decline.In most countries,low reproductive performance is the primary reason for the early elimination of dairy cows,which directly leads to serious damage to the economic benefits of farms.Inactive ovary is a pathological disorder of ovarian function,characterized by follicular diameter less than 8mm,no cyst and luteum on the ovary.The factors affecting ovarian quiescence are various,but the negative energy balance is considered as one of the main pathogenic factors.Therefore,in this study,the difference metabolites in serum and follicular fluid of cows with inactive ovary were obtained by UHPLC-QTOF-MS metabolomics,and their interaction in the occurrence of ovarian inactivity were clarified,which provided a basis for revealing the mechanism of postpartum ovarian inactivity in cows.In this study,the experimental cows were chosen in a large farm in heilongjiang province,which adopts a mixed diet(TMR)feeding mode with the free feeding in scattered column.After14 to 21 days postpartum,blood samples were collected from the tail vein of the experimental cows,which were divided into the energy negative balance group and the energy positive balance group,and followed to 50 to 55 days postpartum.Six cows were selected from the estrus group(group A)and the ovarian inactivity group(group B)after B-ultrasound detection,rectal examination and clinical estrus behavior.Through the blood clinicopathological examination of the two groups of cows,and based on the metabolomic analysis of serum and follicular fluid of UHPLC-QTOF-MS and bioinformatics,the differential metabolites and their metabolic pathways of serum and follicular fluid of cows with inactive ovary were obtained,which is to clarify the relationship and role between differential metabolites and inactive ovary of cows.The results showed that:(1)through blood clinicopathological analysis of the two groups of test cows,the serum GLU content decreased and the content of BHBA and NEFA increased,daily lactation increased(P<0.05)in the ovarian inactivity group compared with the estrus group,and ovarian inactivity group exhibit clinicopathological features of negative energy balance,fat mobilization,and hyperketolemia.(2)UHPLC-QTOF-MS analysis of serum in the two groups ofcows was performed to screen 14 different metabolites.Compared with the estrus group,the serum levels of L-glutamine,guanidyl acetic acid,DL-citrulline,lactose ceramide,L-arginine,phosphatidylcholine,glutamate,spatholipids,(R)-3-hydroxybutyric acid and3-methyl-2-oxybutyric acid were increased in the inactive ovary group;and L-ribulose,D-xylose,4-pyruvate,indoleacetic acid were decreased.they mainly involve in the enriched pathways of D-glutamine and D-glutamic acid metabolism,alanine,aspartic acid and glutamic acid metabolism,arginine and proline metabolism,pentose and glucuronate conversion glycerophospholipid metabolism.(3)UHPLC-QTOF-MS analysis of serum in the two groups of cows was performed to screen 14 different metabolites.Compared with the estrus group,the serum levels of choline,glutamine,lysophosphatidylcholine,phosphatidylcholine,phosphatidylethanolamine,uric acid,arachidonic acid,and maltose were increased in the inactive ovary group;and 6-hydroxy-5-methoxyindole glucuronic acid,2-methyl 2-valerate4-methyl,valine,Phenylpyruvate,2,5-dihydroxybenzoic acid,and 11,12-DHET were decreased.they mainly involve in the enriched pathways of valine,leucine and isoleucine biosynthesis,arachidonic acid metabolism,glycerophospholipid metabolism,starch and sucrose metabolism,phenylalanine metabolism,pentose and glucuronate interaction Transformation.(4)After integration analysis of the different metabolites of serum and follicular fluid in the two groups,only the change of phosphatidyl choline,glutamine was consistent,and other different metabolites changed differently,which involve sugar,amino acid and lipid metabolism,but different metabolites and their metabolic pathways exist obvious differencesin between serum and follicular fluid.Conclusion: in this study UHPLC-QTOF-MS technology was used to obtained a total of 28 different metabolites in serum and follicular fluid of cows with inactive ovary,build the mutual correlation diagram of different metabolites,discuss the difference of metabolites in glucose metabolism,lipid metabolism,amino acid metabolism and other metabolic changes,and its relationship with occurence of inactive ovary,and then lay a solid foundation for in-depth study of mechanism and new control strategy of partum inactive ovary in the future.