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In vitro fertility assessment using fluorescent technique is a better predictor of fertility status of bulls as compared to traditional semen quality assessment techniques, therefore, the study was planned to assess in vitro fertility status of bulls based on conventional and fluorescent techniques. Seventy-three ejaculates were collected from 12 Murrah buffalo bulls maintained at Artificial Breeding Research Centre, NDRI, Karnal, India for the experiment and subjected to statistical analysis using SYSTAT. The mean values of ejaculate volume (ml), mass activity, individual motility (%), sperm concentration (millions/ml), live sperm (%), total abnormalities (%), HOST (%) and acrosomal integrity (%) were 2.70 ± 0.28, 2.8 ± 0.14, 63.8 ± 2.16, 1749.7 ± 122.24, 77.3 ± 2.48, 6.2 ± 0.51, 75.1 ± 1.81 and 84.5 ± 2.26, respectively. The repeatability estimates were significant (P<0.05) for ejaculate volume (0.34 ± 0.137), acrosomal integrity (0.29 ± 0.134) and live percentage (0.28 ± 0.133), indicating sufficient bull to bull variation for the parameters. The mean values of seminal attributes of fluorescent based criteria of CMA3 (Chromomycin A3), SYBR-PI and FITC-PNA (fluorescent isothiocynate-conjugated peanut agglutinin) were 5.25 ± 0.41, 67.91 ± 1.24 and 82.00 ± 1.25 percent, respectively. Bulls were ranked on the basis of expected producing ability (EPA) for semen characteristics assessed by conventional and fluorescent criteria. Rank correlations were found to be significant for FITC with most of the parameters evaluated by conventional methods. In conclusion, among the conventional criteria, individual motility (%) revealed ranking of bulls almost similar to that of fluorescent criteria. [ABSTRACT FROM AUTHOR]