Untargeted metabolomics of buffalo urine reveals hydracyrlic acid, 3-bromo-1-propanol and benzyl serine as potential estrus biomarkers.

Buffalo is a silent heat animal and doesn't show prominent signs of estrous like cattle so it becomes difficult for farmers to determine the receptivity of the animal based purely on the animal behaviour. India, having a huge population size, needs to produce more milk for the population. Successful artificial insemination greatly depends on the receptivity of the animal. Hence the present study aimed to identify the changes in the metabolome of the buffalo. GC-MS based mass spectrometric analysis was deployed for the determination of estrous by differential expression of metabolites. It was found that hydracrylic acid, 3-bromo-1-propanol and benzyl serine were significantly upregulated in the estrous phase of buffalo (p.value ≤0.05, FC ≥ 2). The pathway enrichment analysis also supported the same as pathways related to amino acid metabolism and fatty acid metabolism were up regulated along with the Warburg effect which is linked to the rapid cell proliferation which might help prepare animals to meet the energy requirement during the estrous. Further analysis of the metabolic biomarkers using ROC analysis also supported these three metabolites as probable biomarkers as they were identified with AUC values of 0.7 or greater. SIGNIFICANCE: The present study focuses on the untargeted metabolomics studies of buffalo urine with special reference to the estrous phase of reproductive cycle. The estrous signals are more prominent in cattle, where animals show clear estrous signals such as mounting and discharge along with vocal signals. Buffalo is a silent heat animal and it becomes difficult for farmers to detect the estrous based on the physical and behavioral signals. Hence the present study focuses on GC-MS based untargeted metabolomics to identify differentially expressed urine metabolites. In this study, hydracrylic acid, 3-bromo-1-propanol and benzyl serine were found to be significantly upregulated in the estrous phase of buffalo (p-value ≤0.05, FC ≥ 2). Further confirmation of the metabolic biomarkers was done using Receiver operating characteristics (ROC) analysis which also supported these three metabolites as probable biomarkers as they had AUC values of 0.7 or greater. Hence, this study will be of prime importance for the people working in the area of animal metabolomics.