Salivary TIMP1 and predicted mir-141, possible transcript biomarkers for estrus in the buffalo (Bubalus bubalis)

Successful reproductive management of buffaloes depends primarily upon timely estrus identification. However, 50% of the estrus events are undetected in buffaloes with the available estrus identification methods, leading to huge financial loss to buffalo farmers. Hence, there is an urgent need to develop an alternative and accurate estrus identification method, particularly on the basis of biomarkers in non-invasive fluids. Thus, the present study aimed to identify RNA based estrus biomarkers in cell free saliva in Bubalus bubalis, so that they can be used for future field applicable RT-LAMP colour reactions. RNA-Seq analysis of cell free salivary RNA showed 49 differentially abundant mRNAs between the estrus and diestrus stages. Among five mature miRNAs predicted from the RNA-Seq data, four were found differentially altered at the estrus stage than the diestrus stage. Validation study by direct salivary transcript analysis (DSTA) on 6 selected mRNAs (PPARGC1a, TIMP1, PEBP4, CSPG5, PRHR and ATOH7) and 5 miRNAs (bta-miR-92b, bta-miR-302d, bta-miR-141, bta-miR-27a and bta-let-7a-5p) showed significantly higher levels of TIMP1 (3.46 fold; P < 0.5) and bta-mir-141 (1.33 fold; P < 0.5) in cell-free saliva at the estrus stage compared to the diestrus stage. Hence, TIMP1 and miR-141 appear to be the possible transcript biomarkers for estrus in the cell free saliva of the buffalo. However, further validation studies are required in a large population of buffaloes to determine their estrus biomarker potential before considering them for RT-LAMP colour reaction.