| Summary: | Antimicrobials are added to semen extenders to inhibit the growth of bacteria that are transferred to the semen during collection. However, this non-therapeutic use of antimicrobials could contribute to the development of antimicrobial resistance. The objective of this study was to determine changes in the antibiotic susceptibility of vaginal microbiota after artificial insemination. Swabs were taken from the vagina of 26 mares immediately before artificial insemination and again 3 days later. Bacteria isolated from the vagina at both time points were subjected to antibiotic susceptibility testing and whole-genome sequencing. In total, 32 bacterial species were identified. There were increases in the resistance of <i>Escherichia coli</i> to trimethoprim (<i>p</i> = 0.0006), chloramphenicol and (<i>p</i> = 0.012) tetracycline (<i>p</i> = 0.03) between day 0 and day 3. However, there was no significant effect of exposure to antibiotics in semen extenders with respect to the resistance of <i>Staphylococcus simulans</i> and <i>Streptococcus equisimilis</i> (<i>p</i> > 0.05). Whole-genome sequencing indicated that most phenotypic resistance was associated with genes for resistance. These results indicate that the resistance patterns of vaginal bacteria may be affected by exposure to antibiotics; therefore, it would be prudent to minimize, or preferably, avoid using antibiotics in semen extenders.
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