PhD Candidate University of Michigan and Smithsonian's National Zoo and Conservation Biology Institute Ann Arbor, Michigan, United States
Abstract Authors: Katherine A. Dami 1,2,4; Sally L. Bornbusch 3; Carly R. Muletz-Wolz 5; Pierre Comizzoli 4; Jenny Santiestevan 4; Liliana Cortés-Ortiz 1; Thomas M. Schmidt 1; and Adrienne E. Crosier 2
1. Department of Ecology and Evolutionary Biology, University of Michigan, Ann Arbor, USA
2. Animal Care Sciences, Smithsonian’s National Zoo and Conservation Biology Institute, Washington D.C., USA
3. Center for Conservation Genomics, Smithsonian’s National Zoo and Conservation Biology Institute, Washington D.C., USA
4. Center for Species Survival, Smithsonian’s National Zoo and Conservation Biology Institute, Washington D.C., USA
5. National Institute of Standards and Technology, Gaithersburg, USA
Abstract Text: The vaginal microbiome is a dynamic and complex microbial ecosystem that plays an important role in maintaining female reproductive health and influencing reproductive outcomes. Recent advances in next-generation sequencing have led to increased investigation across species, revealing species-specific vaginal microbiomes and variation in microbial composition with estrous stage. Exploring these dynamics in felid species, as well as in other wildlife populations, is essential for understanding how host-associated microbiomes impact reproductive success across unique taxa.
The objectives of the study were to determine if vaginal microbial communities were (a) species-specific and (b) influenced by estrous stage in cheetahs (Acinonyx jubatus, N = 19 individuals, n = 59 samples), clouded leopards (Neofelis nebulosa, N = 11 individuals, n = 15 samples), and domestic cats (Felis catus, N =14 individuals, n = 14 samples). These samples were collected from 2019 – 2024 with some individuals, mainly cheetahs, opportunistically sampled multiple times. Vaginal cytology was used to determine the estrous stage of each individual at the time of sampling, and 16S rRNA gene sequencing was used to compare microbial communities across species and reproductive stages. Additionally, to address some common methodological variation in wildlife microbiome research, we assessed whether different storage methods, vaginal swabs frozen at −80°C with or without RNAlater, produced comparable microbial community profiles in cheetahs and clouded leopards.
Preliminary analyses across host species showed no significant differences in alpha diversity metrics (within-sample species richness and evenness) suggesting similar within-sample microbial diversity across felid species. However, beta diversity analyses (between-sample species composition) indicated distinct microbial community compositions across felid species (p < 0.05), highlighting species-specific vaginal microbiomes. Estrous stage significantly affected vaginal microbial communities, with alpha diversity varying during estrus (p < 0.05). In cheetahs, beta diversity also changed significantly during estrus (p < 0.05). Finally, the storage method (frozen with or without RNAlater) showed no significant impact on microbiome structure in both cheetahs and clouded leopards (p > 0.05), supporting the comparability of samples regardless of storage treatment.
By demonstrating that vaginal microbiomes are species-specific and vary with estrous cycle, our comparative research provides novel insights into microbial dynamics that can influence reproductive outcomes. This is particularly relevant for ex-situ wildlife populations managed for conservation, where improving reproductive success is a high priority. A better understanding of host–microbiome interactions can inform targeted, microbiome-based strategies to support reproductive success and overall population sustainability. Additionally, our results support the comparability of two commonly used storage methods, potentially enabling the integration of datasets that include samples collected using either approach.
