With recent poaching of southern white rhinoceros (SWR [Ceratotherium simum simum]) reaching record levels, the need for a robust assurance population is urgent. However, the global captive SWR population is not currently self-sustaining due to the reproductive failure of captive-born females. Dietary phytoestrogens have been proposed to play a role in this phenomenon, and recent work has demonstrated a negative relationship between diet estrogenicity and fertility of captive-born female SWR. To further examine this relationship, we compared gut microbial communities, fecal phytoestrogens, and fertility of SWR to those of another rhinoceros species—the greater one-horned rhinoceros (GOHR [Rhinoceros unicornis]), which consumes a similar diet but exhibits high levels of fertility in captivity. Using 16S rRNA amplicon sequencing and mass spectrometry, we identified a species-specific fecal microbiota and three dominant fecal phytoestrogen profiles. These profiles exhibited various levels of estrogenicity when tested in an in vitro estrogen receptor activation assay for both rhinoceros species, with profiles dominated by the microbial metabolite equol stimulating the highest levels of receptor activation. Finally, we found that SWR fertility varies significantly not only with respect to phytoestrogen profile, but also with respect to the abundance of several bacterial taxa and microbially derived phytoestrogen metabolites. Taken together, these data suggest that in addition to species differences in estrogen receptor sensitivity to phytoestrogens, reproductive outcomes may be driven by the gut microbiota’s transformation of dietary phytoestrogens in captive SWR females. IMPORTANCE Southern white rhinoceros (SWR) poaching has reached record levels, and captive infertility has rendered SWR assurance populations no longer self-sustaining. Previous work has identified dietary phytoestrogens as a likely cause of this problem. Here, we investigate the role of gut microbiota in this phenomenon by comparing two rhinoceros species to provide the first characterizations of gut microbiomes for any rhinoceros species. To our knowledge, our approach, combining parallel sequencing, mass spectrometry, and estrogen receptor activation assays, provides insight into the relationship between microbially mediated phytoestrogen metabolism and fertility that is novel for any vertebrate species. With this information, we plan to direct future work aimed at developing strategies to improve captive reproduction in the hope of alleviating their threat of extinction.

29With recent poaching of southern white rhinoceros (Ceratotherium simum simum; SWR) 30 reaching record levels, the need for a robust assurance population is urgent. However, the global 31 captive SWR population is not currently self-sustaining due to the reproductive failure of 32 captive-born females. Dietary phytoestrogens have been proposed to play a role in this 33 phenomenon, and recent work has demonstrated a negative relationship between diet 34 estrogenicity and fertility of captive-born female SWR. To further examine this relationship, we 35 compared gut microbial communities, fecal phytoestrogens, and fertility of SWR to another 36 rhinoceros species-the greater one-horned rhinoceros (Rhinoceros unicornis; GOHR), which 37 consumes a similar diet but exhibits high levels of fertility in captivity. Using 16S rRNA 38 amplicon sequencing and mass spectrometry, we identified a species-specific fecal microbiota 39 and three dominant fecal phytoestrogen profiles. These profiles exhibited varying levels of 40 estrogenicity when tested in an in vitro estrogen receptor activation assay for both rhinoceros 41 species, with profiles dominated by the microbial metabolite, equol, stimulating the highest 42 levels of receptor activation. Finally, we found that SWR fertility varies significantly with 43 respect to phytoestrogen profile, but also with the abundance of several bacterial taxa and 44 microbially-derived phytoestrogen metabolites. Taken together, these data suggest that in 45 addition to species differences in estrogen receptor sensitivity to phytoestrogens, reproductive 46 outcomes may be driven by gut microbiota's transformation of dietary phytoestrogens in captive 47