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The gut microbiome of mammals engages in a dynamic relationship with the body and contributes to numerous physiological processes integral to overall health. Understanding the factors shaping animal-associated bacterial communities is therefore paramount to the maintenance and management in ex situ wildlife populations. Here, we characterized the gut microbiome of 48 endangered black-footed ferrets (Mustela nigripes) housed at Smithsonian's National Zoo and Conservation Biology Institute (Front Royal, Virginia, USA). We collected longitudinal fecal samples from males and females across two distinct reproductive seasons to consider the role of host sex and reproductive physiology in shaping bacterial communities, as measured using 16S rRNA amplicon sequencing. Within each sex, gut microbial composition differed between breeding and non-breeding seasons, with five bacterial taxa emerging as differentially abundant. Between sexes, female and male microbiomes were similar during non-breeding season but significantly different during breeding season, which may result from sex-specific physiological changes associated with breeding. Finally, we found low overall diversity consistent with other mammalian carnivores alongside high relative abundances of potentially pathogenic microbes such as Clostridium, Escherichia, Paeniclostridium, and (to a lesser degree) Enterococcus-all of which have been associated with gastrointestinal or reproductive distress in mammalian hosts, including black-footed ferrets. We recommend further study of these microbes and possible therapeutic interventions to promote more balanced microbial communities. These results have important implications for ex situ management practices that can improve the gut microbial health and long-term viability of black-footed ferrets., (© The Author(s) 2023. Published by Oxford University Press on behalf of The American Genetic Association. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Reproductive microbiomes contribute to reproductive health and success in humans. Yet data on reproductive microbiomes, and links to fertility, are absent for most animal species. Characterizing these links is pertinent to endangered species, such as black-footed ferrets (Mustela nigripes), whose populations show reproductive dysfunction and rely on ex-situ conservation husbandry. To understand microbial contributions to animal reproductive success, we used 16S rRNA amplicon sequencing to characterize male (prepuce) and female (vaginal) microbiomes of 59 black-footed ferrets at two ex-situ facilities and in the wild. We analyzed variation in microbiome structure according to markers of fertility such as numbers of viable and non-viable offspring (females) and sperm concentration (males). Ferret vaginal microbiomes showed lower inter-individual variation compared to prepuce microbiomes. In both sexes, wild ferrets harbored potential soil bacteria, perhaps reflecting their fossorial behavior and exposure to natural soil microbiomes. Vaginal microbiomes of ex-situ females that produced non-viable litters had greater phylogenetic diversity and distinct composition compared to other females. In males, sperm concentration correlated with varying abundances of bacterial taxa (e.g., Lactobacillus), mirroring results in humans and highlighting intriguing dynamics. Characterizing reproductive microbiomes across host species is foundational for understanding microbial biomarkers of reproductive success and for augmenting conservation husbandry., (© 2024. The Author(s).)