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The bacterial load and degree of antibiotic resistance present in untreated and antibiotic-treated semen samples were investigated in five bulls standing at a cattle-breeding centre. Bacterial load was determined by colony counts from semen samples cultured on brain heart infusion and nutrient agar plates. Antibiotic resistance in these bacteria was assessed by measuring the diameter of bacterial growth inhibition zones around discs containing different concentrations of antibiotics. Representative antibiotic-resistant bacterial isolates were selected for identification. Untreated semen contained few culturable bacteria, and all were completely sensitive to gentamycin, spectinomycin and lincomycin: six of the isolates showed some resistance to tylosin. In semen to which antibiotics had been added as part of the routine production process, two isolates were sensitive to all of the antibiotics tested, and the remainder were resistant to all. Resistant Gram-negative isolates that were identified included Pseudomonas and Stenotrophomonas spp. both in the class Gammaproteobacteria and a Sphingomonas sp. which is in the class Alphaproteobacteria.