Exploration of urinary metabolite dynamicity for early detection of pregnancy in water buffaloes

Early and precise pregnancy diagnosis can reduce the calving interval by minimizing postpartum period. The present study explored the differential urinary metabolites between pregnant and non-pregnant Murrah buffaloes ( Bubalus bubalis ) during early gestation to identify potential pregnancy detection biomarkers. Urine samples were collected on day 0, 10, 18, 35 and 42 of gestation from the pregnant ( n = 6) and on day 0, 10 and 18 post-insemination from the non-pregnant ( n = 6) animals. 1 H-NMR-based untargeted metabolomics followed by multivariate analysis initially identified twenty-four differentially expressed metabolites, among them 3-Hydroxykynurenine, Anthranilate, Tyrosine and 5-Hydroxytryptophan depicted consistent trends and matched the selection criteria of potential biomarkers. Predictive ability of these individual biomarkers through ROC curve analyses yielded AUC values of 0.6–0.8. Subsequently, a logistic regression model was constructed using the most suitable metabolite combination to improve diagnostic accuracy. The combination of Anthranilate, 3-Hydroxykynurenine, and Tyrosine yielded the best AUC value of 0.804. Aromatic amino acid biosynthesis, Tryptophan metabolism, Phenylalanine and Tyrosine metabolism were identified as potential pathway modulations during early gestation. The identified biomarkers were either precursors or products of these metabolic pathways, thus justifying their relevance. The study facilitates precise non-invassive urinary metabolite-based pen-side early pregnancy diagnostics in buffaloes, eminently before 21 days post-insemination.