Antimicrobial therapy is a mainstay for treating reproductive diseases, including endometritis. Ceftiofur, a third-generation cephalosporin, is a common antibiotic used to treat equine bacterial endometritis. It is also routinely given empirically as an intra-uterine (IU) infusion in broodmare practice. We hypothesized that ceftiofur IU would disrupt the resident microbial community within the healthy uterus of mares. To test our hypothesis, eight university-owned mares were selected for characterization of the estrual uterine microbiome before and after IU ceftiofur. Double-guarded swabs of the estrual endometrium were taken before and 3 days after both IU saline and ceftiofur in a crossover design. Isolation of DNA from endometrial swabs was performed, followed by amplification of the V4 region of the 16S rRNA gene by Illumina Miseq sequencing to examine core bacterial communities present before and after ceftiofur. The uterine microbial composition of sham and ceftiofur-treated mares was not significantly different as measured by beta diversity. The only notable difference was a lower abundance of Christensenellaceae_R-7_group after ceftiofur (0.14 ± 1.05% vs. 2.89 ± 1.07% control; p = 0.0428). In conclusion, three-day treatment of ceftiofur did not change the microbial composition acutely within the mare uterus when sampled directly after treatment. Ceftiofur may have a long-term effect on the uterine microbiome, which may require sampling several weeks post treatment. In conclusion, ceftiofur does not change the healthy uterine microbiome acutely during estrus and but should still be used judiciously.

Dysbiosis of the lower reproductive tract (LRT) in mares may play a role in clinical diseases, including endometritis and placentitis. Metagenomic/metagenetic analysis of bacterial DNA can identify organisms that are not readily cultured and, thus, may go undetected. In this study, we tested the following hypotheses: (1) the clitoris of estrual mares harbors a unique resident microbiome, (2) topical Lactobacillus genus complex (LGC)-containing probiotic will alter the equine clitoral microbiome, and (3) early pregnancy rates following clitoral LGC application will not differ significantly from industry standards. Mares (n = 12) in estrus had sterile clitoral swabs collected (0) prior to daily topical LGC for 4 days. Second (12 h) and third clitoral swabs (48 h) were collected following final LGC application. During the next estrus, the mares were bred by artificial insemination. Genomic DNA was extracted and used for 16S rRNA sequencing via the Illumina Miseq platform. Abundance was evaluated via Friedman test with pairwise Dunn’s post hoc comparisons. Statistical significance was set at p < 0.05. Compared to time 0, Desulfobacterota decreased and Corynebacterium spp. increased at 12 h and 48 h compared to 0, while Actinobacillus and Fusobacterium spp. increased in a time-dependent manner. Furthermore, Mobiluncus spp. and Christensenellacea_R-7_group decreased at 12 h and 48 h compared to 0. LGC changed the beta but not alpha diversity at both 12 h and 48 h. Mares with LGC application achieved an 85% pregnancy rate in the subsequent estrus. Future investigations are needed to understand the role of the LRT microbiome and probiotics in equine breeding.