A Comparison of Methods to Maintain the Equine Cecal Microbial Environment In Vitro Utilizing Cecal and Fecal Material

SIMPLE SUMMARY: In vitro systems for the fermentation of equine gastrointestinal (GI) content provide researchers with the ability to evaluate changes which may occur due to external influences but which cannot be accessed in vivo. The objective of this study was to evaluate three fermentation systems to replicate the equine cecal environment with regard to the microbiome and metabolite profile. The microbiome and metabolome of the fecal slurry used as inocula in this study were not representative of the cecal systems and care should be taken if feces are to be used to mimic proximal hindgut regions such as the cecum. However, the microbiome of the cecal inoculum maintained in either a chemostat batch fermenter or anaerobic chamber was fairly comparable. The metabolite concentrations, but not rate of production, were significantly different between the two cecal systems. These results provide a context to determine the most appropriate methods by which to create a fermentation system to reflect the equine cecal environment. They also highlight that caution must be exercised as many factors may influence the microbial and metabolic profiles within these systems; as such, they can best be used to demonstrate trends and gross reactions to environmental stimuli. ABSTRACT: The equine gastrointestinal (GI) microbiota is intimately related to the horse. The objective of the current study was to evaluate the microbiome and metabolome of cecal inoculum maintained in an anaerobic chamber or chemostat batch fermenter, as well as the fecal slurry maintained in an anaerobic chamber over 48 h. Cecal and fecal content were collected from healthy adult horses immediately upon death. Cecal fluid was used to inoculate chemostat vessels (chemostat cecal, n = 11) and vessels containing cecal fluid (anaerobic cecal, n = 15) or 5% fecal slurry (anaerobic fecal, n = 6) were maintained in an anaerobic chamber. Sampling for microbiome and metabolome analysis was performed at vessel establishment (0 h), and after 24 h and 48 h of fermentation. Illumina sequencing was performed, and metabolites were identified via nuclear magnetic resonance (NMR). Alpha and beta diversity indices, as well as individual metabolite concentrations and metabolite regression equations, were analyzed and compared between groups and over time. No differences were evident between alpha or beta diversity in cecal fluid maintained in either an anaerobic chamber or chemostat. The microbiome of the fecal inoculum maintained anaerobically shifted over 48 h and was not comparable to that of the cecal inoculum. Metabolite concentrations were consistently highest in chemostat vessels and lowest in anaerobic fecal vessels. Interestingly, the rate of metabolite change in anaerobic cecal and chemostat cecal vessels was comparable. In conclusion, maintaining an equine cecal inoculum in either an anaerobic chamber or chemostat vessel for 48 h is comparable in terms of the microbiome. However, the microbiome and metabolome of fecal material is not comparable with a cecal inoculum. Future research is required to better understand the factors that influence the level of microbial activity in vitro, particularly when microbiome data identify analogous communities.