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Not AvailableEstrus detection in buffaloes has been a major concern for decades, and lack of reliable methods affects their effective reproductive management. Luteinizing hormone (LH) detection in urine is in practice for several mammals for timed insemination, whereas very few reports are available on buffalo urinary LH. The focus of this study is to detect the presence of LH in buffalo urine, quantitate variation in urinary LH during different estrous cycle phases and examine the duration of mid-cycle LH window. Nearly hundred buffaloes were examined, longitudinal urine samples (n = 42) were collected from seventeen animals and classified into respective phases based on several estrus detection parameters. The urinary LH was detected using bovine LH ELISA kit validated for serum/plasma/tissue homogenate. Detection of buffalo LH in the neat urine convincingly proved the competence of the bovine LH kit. Variation in the LH range was observed between different phases of estrous cycle and significant fold variation (P < 0.05) was noticed during estrus phase (1.01 ± 0.23) with average baseline value of 46.73 ± 3.36 mIU/mL. Interestingly, an extended window (A1–A3) of mid-cycle LH surge was observed due to its lingering excretion in urine. The results, altogether, revealed that LH can be detected in buffalo urine with noticeable fold variation during estrus phase and the extended LH window intensifies the chance of ovulation prediction for timed insemination.Not Availabl