Urinary metabolite signatures as predictive biomarkers for estrus detection in water buffaloes: a proton-NMR based study

Developing a reliable, field ready estrus detection method is crucial for improving buffalo reproduction due to their subtle and poorly expressed estrus signs. This study investigated estrus phase-specific urinary metabolites in cyclic Murrah buffaloes (n=6) using proton nuclear magnetic resonance (¹H-NMR). A total of 90 urinary metabolites were identified, with 15 consistently detected across all animals during the estrus phases (proestrus, estrus and diestrus). PCA highlighted p-cresol, ornithine, phenol, chlorogenate, quinolinate, hippurate and 2-hydroxyisobutyrate as key metabolites differentiating the estrus phases. PLS-DA identified p-cresol and phenol for estrus; chlorogenate and o-acetylcholine for proestrus and ornithine in diestrus as the potential urinary markers for detection of estrus phases based on their VIP scores greater than 1.5. Metabolic pathway analysis revealed that the glycerophospholipid pathway, phenylalanine, tyrosine and tryptophan biosynthesis, aspartate and aldarate metabolism, and starch and sucrose metabolism were the major metabolic pathways involved in regulating and controlling estrus cycle. Notably, p-cresol and phenol exhibited significant abundance during estrus (over 9-fold and 5-fold, respectively), suggesting their potential as putative estrus detection biomarkers. However, given the limited sample size (n = 6), these findings should be considered preliminary, and independent validation in larger, well-characterized cohorts is needed to confirm diagnostic utility.