LC-MS And 1~H NMR-based Metabolomics Study On Heat-stressed Lactating Dairy Cows

Present study took the advantage of fast, sensitive, specific, high-throughput analytical technique of ultra-fast liquid chromatography (UFLC) combined with triple quadrupole mass spectrometer (MS), and integrated the advantage of non-discriminative detection and powerful identification of 1H NMR to perform metabolomics analysis on plasma and milk of heat-stressed (HS) lactating dairy cows, discover diagnostic biomarkers, and explore its physiological mechanism. Research contents include the following three parts:1. LC-MS-based metabolomic analysis on plasma of HS lactating dairy cowsThe integration of LC-MS metabolomics and lipidomics study in combination with multivariate statistical analysis was deployed to find discriminating metabolites in plasma between HS and HS-free dairy cows. And 25 potential biomarkers were obtained, including glucose, lactate, ornithine, citrulline, arginine, proline, creatine, urea, arachidonic acid, oleic acid, linoleic acid, phytosphingosine, C16 Sphinganine, choline, lysoPC(0:0/18:0), lysoPE(18:0), lysoPC(16:0), lysoPC(18:0), lysoPC(18:2), PC(16:0/14:0), PC(14:1/18:3), PC(12:0/22:2), PC(15:1/18:2), PC(20:2/12:0), PC(18:1/18:3), suggesting that HS disturbed carbohydrate, amino acids, and lipid metabolism of dairy cows.2.1H NMR-based metabolomic analysis on plasma of HS lactating dairy cows1H NMR-based metabolomic study in combination with multivariate statistical analysis was deployed to find discriminating metabolites in the plasma between HS and HS-free dairy cows. And 21 potential biomarkers were identified, including lactate, pyruvate, glucose, proline, lysine, creatinine, glycine, threonine, isoleucine, leucine, creatine, acetoacetate, acetone, ?-Hydroxybutyrate, choline, HDL, VLDL/LDL, isobutyrate, trimethylamine, trimethylamine N-Oxide, betaine. Consistent with results of present first part, discovered potential biomarkers also indicate HS-induced disturbances of carbohydrate, amino acids, and lipids metabolism. In addition, gut microbiome-derived metabolism was proved to be perturbed by HS.3. LC-MS and 1H NMR-based metabolomics analysis on milk of HS lactating dairy cowsA metabolomics study using LC-MS and 1H NMR spectroscopy was performed to analyze the metabolomic differences in the milk between HS-free and HS dairy cows, and discover diagnostic biomarkers and changes in the metabolic pathway. A total of 53 discriminating metabolites were significantly up- or down-regulated in the HS group compared with the HS-free group (P< 0.05). These biomarkers were involved in pathways of carbohydrate, amino acid, lipid, and gut microbiome-derived metabolism. Comparing these potential biomarkers with previously identified HS candidate biomarkers in plasma, significant correlations between the levels of lactate, pyruvate, creatine, acetone, P-hydroxybutyrate, trimethylamine, oleic acid, linoleic acid, lysophosphatidylcholine 16:0, and phosphatidylcholine 42:2 in milk and plasma were found, indicating that the blood-milk barrier became leaky and the levels of these 10 biomarkers in milk can reflect HS-induced metabolomic alterations in blood.