Shifts in the vaginal microbiota of nulliparous ewe lambs following initial reproductive handling and oestrus synchronization

Current knowledge of the ovine vaginal microbiota is limited to adult ewes, leaving the bacterial community of nulliparous ewes and its dynamics during their first reproductive handling undefined. This study aimed to characterize the vaginal microbiota of ewe lambs prior to their first breeding and to evaluate the changes caused by routine handling and oestrus synchronization. A total of 66 vaginal samples were collected from 22 nulliparous Latxa ewe lambs at three time points: baseline, post-handling, and post-oestrus synchronization using intravaginal devices containing oxytetracycline. Bacterial composition was characterized by sequencing the V3-V4 hypervariable regions of the 16S rRNA gene. Data were processed using DADA2 to infer Amplicon Sequence Variants, and ecological shifts were evaluated through beta and alpha diversity metrics, alongside differential abundance analysis with ANCOM-BC2. A baseline community was revealed, with high diversity and inter-individual variability. This initial state was dominated by the phylum Firmicutes and showed a strong connection to the gut microbiota, including enteric genera such as Fibrobacter, Rikenellaceae RC9 gut group, and Lachnospiraceae NK3A20 group. The first physical examination caused a significant shift in community structure (beta diversity p = 0.001), marked by a rapid drop in these gut-associated bacteria and an increase in opportunistic genera such as Trueperella (log2FC = 2.18; padj < 0.05). The subsequent synchronization treatment further reduced both alpha and beta diversity, creating a uniform environment where genera such as Aerococcus, Corynebacterium, Finegoldia, and Trueperella increased, while Alistipes, Acinetobacter, and Jeotgalicoccus decreased (Padj < 0.05). Despite these changes, the cohort achieved an 89% pregnancy rate via natural mating. Routine handling and synchronization protocols induce temporary shifts in the vaginal microbiota, decreasing diversity and promoting some opportunistic genera. Crucially, the high fertility rate achieved suggests that these ecological alterations are compatible with successful conception under natural mating conditions.