Effect of Antibiotics in Extender on Bacterial and Spermatozoal Quality of Cooled Buffalo (Bubalus bubalis) Bull Semen S Akhter 1 , MS Ansari 1 , SMH Andrabi 2 , N Ullah 2 and M Qayyum 1 1 Department of Zoology, University of Arid Agriculture, Rawalpindi, Pakistan; 2 Animal Reproduction Laboratory, Animal Sciences Institute, National Agricultural Research Centre, Islamabad, Pakistan Contents The present study was designed to study the effect of traditional antibiotic combination (streptomycin and penicil- lin; 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 ¼ 10) was diluted with skim milk extender containing either SP (streptomycin 1000 lg/ml and penicillin 1000 IU/ml), GTLS (gentamycin 500 lg/ml, tylosin 100 lg/ml, lincomycin 300 lg/ml and spectinomycin 600 lg/ 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 bac- terium 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-spectino- mycin. 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 mem- brane 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. Introduction It is well known that bacteriospermia in bovine is detrimental to spermatozoal quality (Aleem et al. 1990; Dela Pena et al. 1995). Bacteria can transmit through natural mating or artificial insemination (AI) in bovine, and can impair the reproductive function of female, resulting in lowered fertility rates (Andrabi et al. 2001). There are few microbiological studies available on buffalo semen. The bacteria isolated from buffalo semen are Bacillus cereus, Bacillus subtilis, Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas testosteroni, Corynebacterium equi, Staphylococcus aureus and Streptococcus pyo- genes (Naidu et al. 1982; Aleem et al. 1990; Ahmed et al. 2001). Antibiotics are added to bovine semen extender to control bacteria over the last 50 years. Streptomycin and penicillin (SP) is the traditional combination added to buffalo semen extender. However, some of the micro- organisms, which were previously sensitive to SP, may have become resistant to these antibiotics (Andrabi et al. 2001; Hasan et al. 2001). An alternative combina- tion of antibiotics comprising of gentamycin, tylosin, lincomycin and spectinomycin (GTLS) added to extender was tested for bull semen for the first time by Shin et al. (1988). This combination (GTLS) proved to be more efficient in eliminating bacteria like Mycoplas- ma bovis, Mycoplasma bovigenitalium, Ureaplasmas, Haemophilus somonus, Campylobacter fetus subsp. venerealis in bull semen (Shin et al. 1988). Moreover, GTLS was not detrimental to post-thaw semen quality (Lorton et al. 1988a; Shin et al. 1988; Hasan et al. 2001) or fertility in bovine (Lorton et al. 1988b; Andrabi et al. 2001). Buffalo is the main dairy animal in most of the developing countries of the world particularly in Asia, where facilities for artificially breeding the animals are limited. These countries produce buffalo bull semen mainly for their own AI use. There is consequently no need to store nationally produced semen for a long period, or to make use of highly diluted semen. Thus, the use of liquid semen stored at refrigerated temperature is the most appropriate for AI in these countries. Chilled semen also makes AI programme independent from the use of liquid nitrogen, which is expensive and difficult to keep regular supplies in most of the buffalo rearing countries. To our knowledge, there is no single study available on the use of GTLS in milk based extenders for liquid storage of buffalo semen. Hence, the present study was designed to study the effect of traditional antibiotic combination (SP) and relatively modern combination of antibiotics (GTLS) in extender on bacterial control and spermatozoal quality of liquid buffalo bull semen stored at 5°C. Materials and Methods Microbial analysis Culture media preparation Culture media was prepared by adding 20 g tryptose soya agar (Oxoid, Hampshire, England), 5 g bacterio- logical agar (Oxoid) and 0.5 g yeast extract (Oxoid) to 500 ml distilled water. The culture media was auto- claved and than poured in sterilized Petri plates. The media in Petri plates was allowed to solidify before incubation at 37°C for 24 h. After incubation, plates were checked for any contamination for further use. Reprod Dom Anim 43, 272–278 (2008); doi: 10.1111/j.1439-0531.2007.00890.x ISSN 0936-6768 Ó 2007 The Authors. Journal compilation Ó 2007 Blackwell Verlag