GC/MS And LC/MS Based Plasma Metabolomic Analysis Of Dairy Cows Suffered From Inactive Ovaries

At present,with the continuous increase of the number and milk yield of cows in the intensive dairy farms in China,the anestrus of dairy cows is also increaseing,which leads to low reproductive efficiency of dairy cows and brings great economic losses to the dairy industry.It has become an important reproductive problem for high-yield dairy cows.Inactive ovaries is a disease of postpartum anestrus in cows.It refers to the absence of luteum and follicle on the ovary or the fact that the diameter of the follicle is less than 8 mm and there is no clinical signs of estrus.In fact,it is the stop or obstruction of follicle development.Negative energy balance(NEB)is the main cause of inactive ovaries in cow.Therefore,this study used the GC-TOF-MS and UHPLC-QTOF-MS techniques of metabolomics to obtain the plasma differential metabolites of inactive ovaries cows caused by NEB and estrus cows,to explore the role of differential metabolites in postpartum inactive ovaries in dairy cows,and to provide a basis for further research on the mechanism of postpartum inactive ovaries in dairy cows.To this end,this study used metabolomics GC-TOF-MS and UHPLC-QTOF-MS techniques to obtain plasma differential metabolites of ovarian resting cows and normal estrus cows caused by negative energy balance,and explored the occurrence of ovarian quiescence in dairy cows after delivery.The role played by it provides a basis for further research on the mechanism of postpartum ovarian rest in dairy cows.This study was carried out in an intensive dairy farm fed free tatal mixed ration in Heilongjiang Province,eighty healthy cows with similar age,body condition and parity were randomly selected from 14-21 days postpartum,which were assinged into energy positive balance group(BHBA < 1.00 mmol/L,Glc > 3.00 mmol/L)and energy negative balance group(BHBA > 1.20 mmol/L,Glc < 2.80 mmol/L)according to the plasma levels of BHBA and Glc.Subsequently,the two groups of cows were followed up to 45-60 days postpartum,and the ovaries were examined by rectal examination and B-ultrasound.According to cows estrus signs and follicle size,the fifteen anestrus cows from the negative energy balance group were identifed as an inactive ovarian group(n=15),and the fifteen estrus cows from the positive energy balancegroup were were identifed as estrus cows(n=15).On the basis of blood clinicopathological examination of cows in inactive ovarian group and estrus groups,GC-TOF-MS and UHPLCQTOF-MS techniques were used to obtained the plasma metabolic profiles of the two groups of cows,and to screen the plasma differential metabolites of the two groups of cows by multivariate statistical analysis and univariate analysis,then to explore the relationship and functions of differential metabolites with cows inactive ovaries through metabolic pathway analysis.Results:(1)The clinicopathological changes in the two groups of dairy cows showed that the postpartum milk yield in the inactive ovaries cows was significantly higher than that in the estrus cows(P<0.05),and plasma E2 level was significantly lower than that in the estrus cows(P<0.01),other plasma biochemical parameters,such as ALT,AST,TP,Ca,Mg,P,Glc,BHBA,SOD,GSH-Px,P4,etc.were not significantly different from that in the estrus cows(P>0.05).(2)Sixteen plasma differential metabolites from two groups of cows were screened by GC-TOF-MS analysis.Among them,nine differential metabolites were significantly increased in the inactive ovaries cows compared with in the estrus cows,including Glycose 2,6-phospho glyconic acid,D-Fructose-2,6-diphosphate,D-Galactonic acid,1-Methylhydantoin,oxamic acid,24,25-dihydrolanosterol,Elaidic acid,and alpha-Tocopherol,respectively.Seven differential metabolites were significantly decreased in the inactive ovaries cows,including Glytathione,5-oxoproline,thymidine,3-Hydroxypropionic acid,Monostearin,Glyceric acid and D-Talose,respectively.(3)Fourteen plasma differential metabolites from two groups of cows were screened by UHPLC-QTOF-MS analysis.Among them,eleven differential metabolites were significantly increased in the inactive ovaries cows compared with in the estrus cows,including D-tagatose,L-Leucyl-L-proline,L-Anserine,Pro-Val,Lys-Leu,9-cis-Retinal,?-Tocopherol,bete-Cryptoxanthin,Lathosterol and Phenylpropionylglycine,Sphingomyelin(d18:1/18:0),respectively.Three differential metabolites were significantly decreased in the inactive ovaries cows,including Cytosine,1-Palmitoyl lysophosphatidylcholine and1-Stearoyl-2-hydroxy-sn-glycero-3-phosphocholine,respectively.(4)The metabolic pathway analysis found that the plasma differential metabolites of dairy cow with inactive ovaries obtained by two metabolomics techniques were involved in ?-alanine metabolism and steroidbiosynthesis metabolism,and other metabolic pathways including Glyoxylate and dicarboxylate metabolism,Fructose and mannose metabolism,Glutathione metabolism,Glycerolipid metabolism,pyrimidine metabolism,Glycine,serine and threonine metabolism and Retinol metabolism,indicated that the abnormalities in postpartum Glycose metabolism,lipid metabolism,amino acid metabolism,and Glytathione metabolism in dairy cows will impede the development of follicles,which can lead to postpartum inactive ovaries in dairy cows.Conclusion: This study used GC/MS and LC/MS to screen the thirty differential metabolites in plasma of inactive ovaries cows suffered from negative energy balance,construct the metabolic network paths diagram related to differences metabolites,explores the changes and its effect of differences metabolites on glycose metabolism,lipid metabolism and amino acid metabolism and other metabolism in the occurrence of postpartum inactive ovary cows caused by NEB.It provides a new direction for further research on the mechanism and controll strategies of postpartum inactive ovary in dairy cows.