Objective:To characterize HBP of seminal plasma, fresh- and frozen-thawed sperm extracts and determine their relationship with post-thaw sperm function tests and bull fertility.Methods:Both fresh (1-2 mL) and frozen semen (50 straws per bull) were collected from thirty breeding Murrah buffalo bulls and subjected to immunoblotting. Further, frozen-thawed semen was evaluated for first service conception rate (FSCR), percent acrosome reaction, hypoosmotic swelling test (HOST), viability, DNA integrity and total motility and linked to HBP.Results: Fourteen immunoreactive bands in seminal plasma (135, 75, 70, 65, 60, 55, 45, 37, 33, 31, 28, 24, 18 and 16 kDa), twelve in fresh sperm extracts (75, 70, 65, 55, 48, 37, 31, 28, 24, 20, 16 and 11 kDa) and thirteen in frozen-thawed spermatozoa (135, 100, 75, 70, 65, 55, 48, 45, 37, 31, 28, 24 and 20 kDa) were detected in west blots. In seminal plasma, fresh- and frozen-sperm extracts, bulls positive for 70 and 18 kDa; 55 kDa and 135, 75, 55, 45, 28 and 24 kDa, respectively, had higher (P<0.05) FSCR as compared to their negative counterparts and had also higher (P<0.05) percentages of most seminal parameters in positive ones. The antibody binding was most prevalent in acrosomal and postacrosomal regions of head in majority of spermatozoa.Conclusion: We have identified buffalo bull seminal HBP that influence semen quality and subsequent fertility of bulls.