Mycoplasma bovis is an important bovine pathogen. Artificial insemination (AI) using contaminated semen can introduce the agent into a naïve herd. Antibiotics, most often gentamycin, tylosin, lincomycin, spectinomycin (GTLS) combination are added to semen extender to prevent transmission of pathogenic bacteria and mycoplasmas. In a commercial AI straw production system with industrial scale procedures, we analyzed the mycoplasmacidal efficacy of GTLS and ofloxacin on M. bovis ATCC and wild type strain isolated from commercial AI straws. The strains were spiked at two concentrations (106 and 103 CFU/mL) into semen. Viable M. bovis in frozen semen straws was detected by enrichment culture and real-time PCR. We also compared different protocols to extract M. bovis DNA from spiked semen. None of the antibiotic protocols had any effect on the viability of either of the M. bovis strains at high spiking concentration. At low concentration, the wild type was inhibited by all other protocols, except low GTLS, whereas the ATCC strain was inhibited only by high GTLS. The InstaGene™ matrix was the most effective method to extract M. bovis DNA from semen. When there is a low M. bovis contamination level in semen, GTLS used at high concentrations, in accordance with Certified Semen Services requirements, is more efficient than GTLS used at concentrations stated in the OIE Terrestrial Code.
Keywords: DNA extraction; Mycoplasma bovis; antibiotics; bovine semen; prevention.