Abstract
Ketosis and subclinical ketosis are widespread among dairy cows especially after calving. Etiopathology of ketosis has been related to negative energy balance. The objective of this study was to investigate metabolite fingerprints in the urine of pre-ketotic, ketotic, and post-ketotic cows to identify potential metabolite alterations that can be used in the future to identify susceptible cows for ketosis and metabolic pathways involved in the development of disease. In this study, NMR, DI/LC-MS/MS, and GC-MS-based metabolomics were used to analyze urine samples from 6 cows diagnosed with ketosis and 20 healthy control (CON) cows at −8 and −4 weeks prepartum, the week (+1 to +3) of ketosis diagnosis, and at +4 and +8 weeks after parturition. Univariate and multivariate analyses were used to screen metabolite panels that can identify cows at their pre-ketotic stage. A total of 54, 42, 48, 16, and 31 differential metabolites between the ketotic and CON cows were identified at −8 and −4 weeks prepartum, ketosis week, and at +4, and +8 weeks postpartum, respectively. Variable importance in projection (VIP) plots ranked the most significant differential metabolites, which differentiated ketotic cows from the CON ones. Additionally, several metabolic pathways that are related to ketosis were identified. Moreover, two promising metabolite panels were identified which clearly separated ketotic from CON cows with excellent level of sensitivity and specificity. Overall, multiple urinary metabolite alterations were identified in pre-ketotic, ketotic, and post-ketotic cows. The metabolite panels identified need to be validated in the future in a larger cohort of animals.
Highlights
Ketosis is a prevalent metabolic disorder of dairy cattle characterized by increased levels of ketone bodies such as β-hydroxybutyric acid (BHBA), acetoacetate (AcAc), and acetone (Ac) in the blood, milk, and urine during the early lactation [1]
We found that several amino acids (AAs) [e.g., L-arginine (L-Arg), L-aspartic acid (L-Asp), L-glutamine (L-Gln), glycine (Gly), β-alanine (β-Ala), L-cysteine (L-Cys), L-isoleucine (L-Ile), L-lysine (L-Lys), L-phenylalanine (L-Phe), and L-tyrosine (L-Tyr)] and their metabolic products such as asymmetric dimethylarginine (ADMA), symmetric dimethylarginine (SDMA), carnosine, N-acetylglutamic acid, pantothenic acid, 1-methylhistidine, and 3-methylhistidine] were increased in the urine of pre-ketotic cows at one or more of the time points, especially at −8 and −4 weeks prior to parturition
Results of this study show that both nuclear magnetic resonance (NMR) and MS based metabolomics provides a powerful approach to discover predictive and diagnostic biomarkers of ketosis in dairy cows
Summary
Ketosis is a prevalent metabolic disorder of dairy cattle characterized by increased levels of ketone bodies such as β-hydroxybutyric acid (BHBA), acetoacetate (AcAc), and acetone (Ac) in the blood, milk, and urine during the early lactation [1]. Several recent metabolomics articles related to ketosis in dairy cows have been reporting multiple alterations in the blood and milk of the affected cows [2,3,4,5]. The studies are important contributions to the better understanding of the pathobiology of the Urinary Metabotypes of Ketotic Cows disease and its diagnosis. Using urine as an analytical biofluid would be more advantageous because it is non-invasively obtained and accessible in large volumes. Urine has a low content of proteins and lipids, comparable to plasma or serum, that interfere in the analytical process. To the best of our knowledge, no comprehensive urinary metabolomics and lipidomics profiling for the identification of predictive biomarkers of ketosis has been previously reported in dairy cows
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