Metabonomics Analysis Of Ovarian Cortical Tissue And Follicular Fluid In Inactive Ovary Dairy Cows Based On LC/MS

Background and purpose: In the past ten years,with the rapid economic development and the country’s policy support for the dairy industry,large-scale dairy farming has continued to expand.While the annual yield of dairy cows is steadily increasing,the incidence of reproductive disorders is on the rise,causing serious economic losses to the cattle farm.Inactive ovary(IO)caused by negative energy balance(NEB)in dairy cows is a kind of pathological anestrus caused by temporary ovarian function disorders.It not only affects the recovery of ovarian function in dairy cows after calving,hinders normal estrus and breeding,but also reduces the reproductive efficiency of dairy cows,has become an important issue that restricts the healthy development of large-scale cattle farms.In view of the fact that there is no report on the metabonomics of the ovarian tissue of postpartum IO caused by NEB in dairy cows,this study used non-targeted liquid chromatography/mass spectrometry(LC/MS)technology to analyze the ovarian cortex and follicles of postpartum IO cows.Metabolomics analysis of the liquid was carried out to clarify the changes and effects of different metabolites on the occurrence of IO in dairy cows,and lay the foundation for in-depth exploration of the mechanism and prevention strategies of IO in dairy cows in the future.Methods: In this study,lactating cows with similar age,parity and condition were randomly selected as experimental animals in an intensive cattle farm in Heilongjiang Province(n=100),with similar ages,parities,and conditions.14-21 days after calving,according to the levels of serum β-hydroxybutyric acid(BHBA)and glucose(GLU),they were divided into energy balance(PEB)group and energy negative balance(NEB)group,30 heads in each group.In50～55 days postpartum,estrus identification was carried out,divided into PEB normal estrus group(A group)and NEB ovarian quiescent group(B group),each group selected 6 cattle for slaughter,and collected blood,ovarian cortex tissue and follicular fluid.Subsequently,the blood clinical pathology was analyzed in the A and B groups of cows.The LC/MS technology was used to detect the metabolic profile of the ovarian cortex and follicular fluid of the A and B groups of cows,and combined with multivariate statistics and bioinformatics analysis to obtain differential metabolites and their metabolic pathways,and then explore the relationship between differential metabolites and IO.Results:(1)The blood clinical pathological analysis of experimental cow showed tha tcompared with PEB group,NEB cows had higher body condition loss,lower estrus rate,more number of sperm transfusion and longer calving interval during early lactation.Compared with group A,the blood glucose concentration of cows in group B decreased,and contents of NEFA and BHBA increased,indicating that NEB not only caused the decrease of reproductive performance,but also the occurrence of IO was related to the enhancement of fat mobilization and hyperketonemia.(2)Through LC/MS detection,multivariate statistical analysis and univariate analysis,29 different metabolites of ovarian cortex between group A and group B were screene d out.Compared with group A,the 8 metabolites of 5-HETr E,Elaidamide,ethyl oleate,Histamine 8 metabolites including amine and phosphatidylcholine in the ovarian cortex tissue of cows were increased in group B,while the 20 metabolites of 13-Hp ODE,Lyso PE(0:0/20:2(11Z,14Z)),8,9-Di HETr E,L-Glutamic acid,L-proline,8-methylnonanoic acid,L-valine,2-oxopentanedioic acid were decreased.Their main enrichment pathways were D-glutamine and D-glutamate metabolism,Pentose phosphate pathway,Linoleic acid metabolism,Histidine metabolism,and other biological metabolic processes.(3)45 different metabolites of follicular fluid between group A and group B were screened through LC/MS detection,multivariate statistical analysis and univariate analysis.Compared with group A,the 28 metabolites of 12-keto-leukotriene B4,sphingomyelin,20-carboxyarachidonic acid,phosphatidylcholine,oleamide,16-hydroxypalmacid ester,pyrogl utamic acid,gluconolactone,lactulose,xanthine,cytosine,guanosine,uric acid in follicular fluid of cows were increased in group B,while the 17 metabolites of propiolactone,11,12-dihydroxyeicosatrienoic acid,L-valine,indole acetaldehyde,4-aminobutyraldehyde,phenylpyruvate,L-lysine,D-mannose,sucrose were decreased.Their mainenrichment pathways are arachidonic acid metabolism,purine metabolism,phenylalanine metabolism,glyceroph ospholipid metabolism,and histidine metabolism.(4)The common different metabolites in the ovarian cortex tissue and follicular fluid of cows in the A and B groups were phosphatidylcholine,lysophosphatidylcholine,arachidonic acid metabolites and glutamine with same changes,but other difference metabolit eswere different.Although the enrichment pathways of different metabolites between two tissues involve in sugar,amino acid and lipid metabolism,they were obvious differences between ovarian cortical tissue and follicular fluid.At the same time,according to the differential metabolites and main enrichment pathways of ovarian cortex and follicular fluid,the interaction maps of differential metabolites were constructed respectively,and the relationship between differential metabolites and ovarian quiescence was explored.Conclusion: In this study,differential metabolite profiles of follicular fluid and ovarian cortical tissue of postpartum IO in dairy cows were obtained,and the interaction map of differential metabolites was constructed to identify the main metabolic pathways of carbohydrate,lipid and amino acid differential metabolites in dairy cows with postpartum IO induced by NEB and their interrelationships.It provides a direction for further research on the pathogenesis and prevention of postpartum IO in dairy cows in the future.