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The present study was designed to study the effect of traditional antibiotic combination (streptomycin and penicillin; SP) and relatively modern combination of antibiotics (gentamycin, tylosin, lincomycin and spectinomycin; GTLS) in extender on bacterial control and spermatozoal quality of liquid buffalo bull semen stored at 5°C. Semen collected from Nili-Ravi buffalo bulls (n = 10) was diluted with skim milk extender containing either SP (streptomycin 1000 μg/ml and penicillin 1000 IU/ml), GTLS (gentamycin 500 μg/ml, tylosin 100 μg/ml, lincomycin 300 μg/ml and spectinomycin 600 μg/ml) or negative control with no antibiotics (NA). Liquid semen was stored at 5°C for 5 days. Aerobic bacteria isolated from buffalo semen were Pseudomonas aeruginosa and Staphylococcus aureus. The only facultative anaerobic bacterium isolated was Klebsiella pneumoniae. In vitro antibiotic sensitivity test revealed that Ps. aeruginosa and Staph. aureus were susceptible to gentamycin. Staphylococcus aureus and K. pneumoniae were susceptible to tylosin and linco-spectinomycin. Total aerobic bacterial count was significantly lower in semen samples treated with GTLS than those of SP on third and fifth day of storage at 5°C. There was no difference (p > 0.05) in sperm motility, longevity and plasma membrane integrity (PMI) in extender containing SP or GTLS combination until the third day of storage at 5°C. On fifth day of storage sperm motility, longevity and PMI was significantly better in extender containing SP compared with GTLS and NA. Intact acrosomes, and sperm head, mid piece and tail abnormalities remained similar (p > 0.05) because of antibiotics up to 5 days of storage. In conclusion, GTLS is more capable than SP for bacterial control of buffalo bull semen. Moreover, GTLS and SP are equally efficient in preserving spermatozoal quality of extended buffalo bull semen for 3 days at 5°C.