Blood Metabolomics Study Of Heat Stress In Chinese Holstein Cows

Objectives:The objectives of this study were to detect changes in the blood metabolomes of dairy cows through the metabolomics platform,and analyze the changes in energy and glucose metabolism caused by heat stress.The discovery of the key metabolites in the metabolic pathway of dairy cows under heat stress,and the changes in the expression of genes related to the regulation of glucose.Furthermore,to screen the single nucleotide polymorphisms(SNPs)in the upstream,``downstream,and exon regions of the glucose facilitative transporters genes(GLUTs).These findings will provide a theoretical basis for improving animal management practices and selective breeding for heat tolerance in dairy cattle.Methods:A total of twenty animals with similar lactation days were selected from thermo-neutral and heat stress conditions to detect the changes in blood metabolomics and the same number of animals were selected for the glucose and m RNA expression of the GLUTs.Blood samples of 20 ml were collected from the tail vein in an anticoagulant tube.After centrifugation of 10 ml blood(half)at 3000 rpm for 15minutes,the upper plasma and middle white membrane were separated for metabolome detection and genes m RNA levels.The remaining 10 ml were used for the extraction of genomic DNA.The absolute concentration of metabolites in cow plasma was detected by the ~1HNMR absolute quantitative method.Changes in blood metabolites of cows as a result of heat stress were analyzed by PLS-DA analysis and t-test.The metabolic pathway was constructed by enrichment analysis and pathway analysis to study the metabolic pathway changes of cows under heat stress.Furthermore,the receiver operating characteristic(ROC)curve was used to analyze the accuracy of the above differential metabolites indicating individual heat stress response in lactating cows,and heat stress related key metabolites were obtained.The expression of related genes regulating the key metabolites was detected by real-time fluorescent quantitative PCR(RT-q PCR)to study the molecular regulation mechanism of the key metabolites under heat stress.Finally,genomic DNA was extracted from blood,and polymorphism in the 6 genes of the GLUTs family was screened by mixed pool sequencing method to obtain the genetic variation of related genes regulating important metabolites.Results:Based on the ~1HNMR platform,a total of 44 metabolites were detected in Holstein cow’s plasma,including 21 amino acids and their derivatives,13 organic acids,5 amines,3 alcohols,1 sugar,and 1 other.Through the statistical analysis,a total of 16 different metabolites were screened,among which the concentration of glucose,ethanol,glycine,hippuric acid,carnitine,citric acid,valine,leucine,methanol,and 2-oxoglutarate significantly down-regulated,while the concentration of acetone,citrate,butyric acid,2-hydroxy-3-methylvalerate,2-hydroxybutyrate and dimethylamine significantly upregulated under heat stress.Enrichment analysis and pathway analysis showed that these metabolites were involved in carbohydrates,amino acids,and lipid metabolism.The biomarker analysis using the ROC curve yielded a total of five substantial biomarkers for heat stress including glucose,acetone,ethanol,alanine,and butyrate having a cumulative AUC of 0.99.The AUC of glucose was the highest,reaching 0.98,which indicated that glucose had a significant effect on milk quality under heat stress and attracted our interest for further study among other metabolites.In order to further study the molecular regulation mechanism of glucose metabolism under heat stress,the m RNA expressions of SLC2A genes were detected.It was found that the m RNA expression of SLC2A1,SLC2A2,SLC2A3,SLC2A8,SLC2A10,and SLC2A13 significantly down-regulated while SLC2A6 significantly up-regulated(**P<0.01),(*P<0.05)during heat stress in comparison to thermo-neutral condition.These results indicate that heat stress substantially disturbed the energetic and metabolic pathways by changing the dynamics of blood metabolites,glucose metabolism,and the expression of glucose transporters(SLC2A)genes family.According to the gene sequences in the NCBI database,primers were designed for the upstream,downstream,and all exon regions of SLC2A1,SLC2A2,SLC2A3,SLC2A4,SLC2A6 and SLC2A8,and SNPs were screened by mixed pool sequencing.Total 21 SNPs were discovered in 6 genes,3,7,3,6 and 2 SNPs were identified in SLC2A1,SLC2A2,SLC2A3,SLC2A4 and SLC2A6 respectively,but no SNPs were identified in SLC2A8 gene.The above results provide effective genetic information for the molecular breeding of dairy cows.Conclusions:In conclusion,it is clear that HS substantially changed the thermo-physiological characteristics and metabolome profile of the dairy cows.Under heat stress,carbohydrates,amino acids,and lipid metabolism of dairy cows were changed.Among them,glucose was the most important energy substance,and its content changes most significantly.The AUC value was as high as 0.98,which indicates that glucose has good accuracy in judging the individual heat stress state of dairy cows,and can be used as a marker metabolite of heat stress in dairy cows.The m RNA expression of SLC2A genes indicated that heat stress might regulate the energy metabolism pathway of glucose by changing the expression of GLUTs.Besides,there were abundant genetic variations in the GLUTs gene,which may provide a theoretical basis for breeding heat-tolerant dairy cows.