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A total of 48 semen ejaculates from 6 Murrah bulls were collected during winter period (15 deg C to 25 deg C) and heat stress (42 deg C to 48 deg C) through artificial vagina method to study the effect of heat stress on seminal characteristics of Murrah buffalo bull semen. Overall mean values of ejaculated volume (3.95±0.33 Vs 3.43±0.21 ml), sperm concentration (1324.80±67.84 Vs 1133.11±74.27 millions/ml), total number of sperm per ejaculate (6575.74±1306.37 Vs 4785.31±619.18), mass motility (3.63±0.09 Vs 3.33±0.10), initial progressive motility (72.50±0.89 Vs 73.33±0.87%), live sperm (85.82±0.77 Vs 84.12±0.94%), abnormal sperm (8.71±0.47 Vs 12.25±0.72%), intact acrosome (83.54±0.30 Vs 82.92±0.32%), cervical mucus penetration (21.10±0.32 Vs 24.31±0.48 mm) and hypo osmotic swelling reactive sperm (55.88±2.65 Vs 52.08±0.34%) were in fresh semen during winter period (15 C to 25 C) and heat stress (42 deg C to 48 deg C), respectively. The overall mass activity (P LT 0.05) and percent HOS positive sperm (P LT 0.05) were found significantly lower during heat stress whereas percent abnormal sperm and the mean distance travelled by vanguard spermatozoa (CMPT) was found significantly higher (P LT 0.01) during heat stress. There is variation in sensitivity among individual bulls to heat stress for sperm concentration, initial progressive motility, abnormal sperm, intact acrosome, cervical mucus penetration and hypo osmotic swelling reactive sperm.