How Studying Poop May Help Us Boost White Rhino Populations

Researchers at North Carolina State University have identified significant differences in the gut microbiome of female southern white rhinos who are reproducing successfully in captivity, as compared to females who have not reproduced successfully in captivity. The work raises questions about the role that a particular genus of gut microbes may be playing in limiting captive breeding of this rhinoceros species.

“Our work focuses on the southern white rhinoceros (Ceratotherium simum simum), because while it is not yet endangered, species numbers are declining in the wild due to poaching,” says Christina Burnham, first author of a paper on the work and a former graduate student at NC State.

“There is a significant population of southern white rhinos under human care in the United States, but there have been challenges in getting many of these animals to reproduce successfully. It is critical we understand why, as the managed rhinos serve as important assurance populations in case wild rhino numbers continue to fall. We wanted to know how the gut microbiome may influence the reproductive ability of these rhinos.”

To that end, the researchers collected multiple fecal samples from eight female southern white rhinoceroses over a six-month period. The study population consisted of two juveniles; two “subadults” who are no longer nursing but are not yet of reproductive age; two adults who have reproduced successfully; and two adults who have not reproduced successfully.

“We wanted to have a robust sample size that would allow us to assess the gut microbiome of females in this species while accounting for age, the time of year and reproductive status,” Burnham says.

The researchers extracted and sequenced DNA from the fecal samples, which allowed them to identify the diversity and abundance of bacteria in the gut of the study animals.

“We found differences between rhinos in each age group,” says Erin McKenney, co-author of the study and an assistant professor of applied ecology at NC State. “In other words, microbial communities in the gut microbiome change predictably as animals age from juveniles to subadults to adults. This likely reflects, among other things, changes in the diet and behavior of the animals. This sort of microbiome ‘succession’ is fairly common in many animal species. And it’s important here because it casts the differences we found between reproductively successful adults and reproductively unsuccessful females in stark relief.”

The researchers found that reproductively successful females had less diversity in the types of microbial species present in their gut microbiome, when compared to the microbiome of reproductively unsuccessful females. The researchers also found that each group of adults was playing host to microbial species that have previously been associated with reproductive health.

“One of the microbial families we found at significant levels in reproductively successful adults was Rikenellaceae,” Burnham says. “This is of interest because Rikenellaceae has previously been theorized to play a role in helping southern white rhinos break down dietary plant compounds – including phytoestrogens that are associated with limiting reproductive success.

“On the other hand, we only saw significant enrichment of Mobiluncus microbes in reproductively unsuccessful adults,” Burnham says. “Previous work has found that Mobiluncus is associated with a range of reproductive health problems in a variety of non-rhinoceros species.

“However, in those previous studies, Mobiluncus was detected in cervical and vaginal microbiomes. We looked only at the gut microbiome. We hypothesize that the gut may serve as a reservoir for Mobilincus, but we need to collect cervical or vaginal swabs from the adult females we studied to determine whether Mobiluncus may be present in those microbiomes.”

“Because this was a longitudinal study, we collected multiple samples from each animal over the course of half a year,” McKenney says. “And the differences we saw in the gut microbiomes of adult females were consistent over time, which suggests that these differences in microbial communities may be playing an important role in the reproductive health of these animals. That said, we will need to do additional research to determine what that role may be, if any.”

The paper, “Effects of Age, Seasonality, and Reproductive Status on the Gut Microbiome of Southern White Rhinoceros (Ceratotherium simum simum) at the North Carolina Zoo,” is published open access in the journal Animal Microbiome. Corresponding author of the paper is Shweta Trivedi, a teaching professor of animal science at NC State and director of NC State’s Veterinary Professions Advising Center. The paper was co-authored by Kimberly Ange-van Heugten, a teaching associate professor of animal science at NC State; and by Jb Minter, director of animal health at the North Carolina Zoo and adjunct faculty member at NC State.

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Note to Editors: The study abstract follows.

“Effects of Age, Seasonality, and Reproductive Status on the Gut Microbiome of Southern White Rhinoceros (Ceratotherium simum simum) at the North Carolina Zoo”

Authors: Christina M. Burnham, Erin A. McKenney, Kimberly Ange-van Heugten and Shweta Trivedi North Carolina State University; Larry J. Minter, North Carolina State University and North Carolina Zoo

Published: May 5, Animal Microbiome

DOI: 10.1186/s42523-023-00249-5

Abstract:
Background:
Managed southern white rhinoceros (Ceratotherium simum simum) serve as assurance populations for wild conspecifics threatened by poaching and other anthropocentric effects, though many managed populations experience subfertility and reproductive failure. Gut microbiome and host health are inextricably linked, and reproductive outcomes in managed southern white rhinoceros may be mediated in part by their diet and gut microbial diversity. Thus, understanding microbial dynamics within managed populations may help improve conservation efforts. We characterized the taxonomic composition of the gut microbiome in the managed population of female southern white rhinoceros (n=8) at the North Carolina Zoo and investigated the effects of seasonality (summer vs. winter) and age classes (juveniles (n=2; 0–2 years), subadults (n=2; 3–7 years), and adults (n=4; >7 years)) on microbial richness and community structure. Collection of a fecal sample was attempted for each individual once per month from July-September 2020 and January-March 2021 resulting in a total of 41 samples analyzed. Microbial DNA was extracted and sequenced using the V3-V4 region of the 16S rRNA bacterial gene. Total operational taxonomic units (OTUs), alpha diversity (species richness, Shannon diversity), and beta diversity (Bray-Curtis dissimilarity, linear discriminant analysis effect size) indices were examined, and differentially enriched taxa were identified.

Results:
There were differences (p<0.05) in alpha and beta diversity indices across individuals, age groups, and sampling months. Subadult females had higher levels of Shannon diversity (Wilcoxon, p<0.05) compared to adult females and harbored a community cluster distinct from both juveniles and adults. Samples collected during winter months (January-March 2021) possessed higher species richness and statistically distinct communities compared to summer months (July-September 2020) (PERMANOVA, p<0.05). Reproductively active (n=2) and currently nonreproductive adult females (n=2) harbored differentially enriched taxa, with the gut microbiome of nonreproductive females significantly enriched (p=0.001) in unclassified members of Mobiluncus, a genus which possesses species associated with poor reproductive outcomes in other animal species when identified in the cervicovaginal microbiome.

Conclusion:
Together, our results increase the understanding of age and season related microbial variation in southern white rhinoceros at the North Carolina Zoo and have identified a potential microbial biomarker for reproductive concern within managed female southern white rhinoceros.

This post was originally published in NC State News.