Serum Metabonomics Analysis Of Dairy Cow With Subclinical Vitamin E Deficiency Based On GC-MS

Dairy cows are prone to metabolic stress in the early stages of lactation,and it is very important to meet the nutritional needs of dairy cows at this stage.Vitamin E(VE)has important antioxidant and immune-enhancing effects.however.Due to the lack of VE subclinical clinical symptoms and rapid detection methods,it is difficult to early monitor blood VE level.The incidence of high-yield dairy cows in large-scale cattle farms can be as high as 20%,which is becoming a metabolic disease endangering the health of dairy cows.To this end,this study used non-targeted gas chromatography-mass spectrometry(GC-MS)technology to screen the differential metabolites in the serum of postpartum VE-deficient dairy cows and healthy dairy cows,and used mass spectrometry multiple reaction monitoring(MRM)target technology to verify the selected differential metabolites,and discuss the changes in the serum metabolic profile and differential metabolites of dairy cows with subclinical deficiency of VE and their role in the occurrence and development of the disease.It is of great significance for the prevention and treatment of VE deficiency in the future.This study was conducted on an intensive cattle farm in Heilongjiang Province.According to the requirements of experimental animal ethics,Holstein cows with similar age,parity,body condition and other information were randomly selected as experimental animals.Serum VE content is divided into subclinical VE deficiency group(VE content 2～3 μg/mL)and normal group(VE content above 4 μg/mL)40 heads each.According to serum biochemical indicators and clinical manifestations,13 cows with subclinical VE deficiency and 14 healthy cows were selected for non-targeted metabolomics analysis(GC-MS).Subsequently,the differential metabolites related to the subclinical deficiency of VE were screened out through multivariate statistical methods and bioinformatics analysis.In addition,10 serum samples from each group of dairy cows were assigned to verify change of the selected differential metabolites using MRM targeting analysis,and their relationship and effects with VE subclinical deficiency in cows.Results:(1)The content of malondialdehyde(MDA)and β-hydroxybutyric acid(BHBA)in serum of dairy cows in the subclinical VE deficiency group was significantly increased,of VE,total antioxidant capacity(T-AOC)and glutathione(GSH)was significantly reduced(P < 0.05),indicating that the postpartum subclinical VE deficiency of dairy cows is related to metabolic stress,ketosis,and oxidative stress caused by negative energy balance.(2)Thirty-one differential metabolites in serum of cows with VE subclinical deficiency were obtained by GC-MS,and 20 of them were elevated,namely glycine,1,5-anhydrous glucose,glutamic acid,trans fatty acids,phenylalanine,methionine,L-cysteine,glycerin ether,6-fructose phosphate,citrate,norepinephrine,biuret,decanoic acid,salicylic acid,5-methyluracil,prostaglandin E2,glutamine,hexanediamide,n-acetyl-beta-alanine,lyxose.And 11 of them were decreased,namelyα-tocopherol,methyl phosphate,cholesterol,D-glycerol 1-phosphate,fructose 2,6-diphosphate,4-methylcatechol,benzoic acid,6-sucrose phosphate,mannitol,allylmalonic acid,hesperidin.Their main metabolic enrichment pathways refer to nitrogen metabolism,glycine,serine and threonine metabolism,primary bile acid biosynthesis,cysteine and methionine metabolism,glycerol phospholipid metabolism,GSH metabolism,fructose and mannose,etc.And the increase of lipid metabolism is related to negative energy balance,decreased VE,decreased antioxidant activity and lipid peroxidation.(3)The amino acid results verified by MRM target technology were consistent with the results of GC-MS non-target metabolomics.The validation results of fatty acids showed that the fatty acids of dairy cows with subclinical VE deficiency was generally higher than that in the healthy cows,which was consistent with the decrease of fatty acid synthesis pathway and the increase of glycerol ester metabolism and arachidonic acid metabolism pathway in the untargeted metabolic results.It suggested that VE deficiency was related to the decrease of antioxidant and the increase of lipid peroxidation in dairy cows.Conclusion: In this study,serum differential metabolite profiles of postpartum subclinical VE deficiency in dairy cows were obtained,and an interaction map of serum differential metabolite profiles of postpartum subclinical VE deficiency in dairy cows was constructed to identify the main metabolic enrichment pathways for metabolites of carbohydrate,lipid and amino acid in dairy cows with subclinical VE deficiency.The relationship between negative energy balance,excessive lipid mobilization,oxidative stress and subclinical VE deficiency in dairy cows was elucidated,which laid a foundation for further revealing the mechanism of subclinical VE deficiency in early lactation cows and providing new prevention strategies.