A Multi-Platform Metabolomics Approach Identifies Urinary Metabolite Signatures That Differentiate Ketotic From Healthy Dairy Cows

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.