Effect of fecal preservation method on captive southern white rhinoceros gut microbiome

The southern white rhinoceros (Ceratotherium simum simum) faces an uncertain future in the wild due to increased poaching pressure and habitat fragmentation, thus the management of reproductively successful populations is of critical importance. Successful reproductive outcomes in rhinoceros may be mediated by diet and gut microbial diversity; therefore, understanding gut microbial dynamics within and between captive and wild populations may help improve conservation efforts. Accordingly, gut microbiome preservation methods are needed that are practical for in situ field sampling of wild populations. We evaluated the efficacy of 3 different preservation methods over 2 timepoints for stabilizing microbial communities in feces from southern white rhinoceros (n = 10) at the North Carolina Zoo in Asheboro, North Carolina, USA, during July-September 2020 and January-March 2021. Samples were immediately frozen at -80 degrees C, stored in PERFORMAbiome (TM)center dot GUT (PB) tubes or stored in 95% ethanol at ambient temperatures (to simulate field conditions), and processed after 14 or 230 days post-collection. We quantitatively compared alpha and beta diversity across microbial communities and identified taxa that were enriched in each treatment group. Samples preserved in 95% ethanol consistently harbored lower Shannon diversity index (SHDI) and Simpson's diversity (SDI) values compared to Frozen and PB samples. This trend was apparent in both Ethanol day-14 samples (SHDI 4.94; SDI 0.98) versus Frozen day-14 (SHDI 5.19; W = 518, P < 0.001; SDI 0.99; W = 476, P < 0.001) and PB day-14 (SHDI 5.15; W = 430, P < 0.01; SDI 0.99; W = 1075, P = 1) samples, and in Ethanol day-230 samples (SHDI 4.48; SDI 0.97) versus Frozen day-230 (SHDI 5.18; W = 0, P < 0.05; SDI 0.99; W = 0, P = 0.032) and PB day-230 (SHDI 5.23; W = 0, P < 0.05; SDI 0.99; W = 0, P = 0.032) samples. Ethanol day 230 samples differed (P < 0.05) from all other treatments in both alpha and beta diversity indices. Notably, frozen and PB preservation methods maintained compositionally similar microbial communities across both time points. Our results indicate that PB tubes stored at ambient temperatures perform similarly to freezing at -80 degrees C, highlighting their utility for microbiome fieldwork applications. Identifying optimal and versatile microbiome preservation techniques will enable future studies of the gut microbiome in reproductively-successful wild populations, an effort central to conservation efforts in the southern white rhinoceros and other threatened species.