7 The use of in vitro fermentation methods to simulate an equine cecal environment

Abstract

The equine gastrointestinal (GI) microbiota is intimately related to the health and well-being of the horse. However, access to more proximal portions of the GIT, such as the cecum, are challenging in live horses. Surgical techniques which enable in vivo cecal sampling, such as cannulation, are difficult to perform and maintenance can be problematic. The objective of this study was to evaluate changes in the microbiome of cecal inoculum maintained in an anaerobe chamber or a chemostat, and of a fecal slurry maintained in an anaerobe chamber over 48h. Cecal and fecal samples were collected immediately upon death from an abattoir in QC CAN. Cecal and fecal material were transported on ice, anaerobically to the University of Guelph where 500mL of cecal fluid was used to inoculate a chemostat vessel (ch_c, n = 11) or 100mL was transferred to a vessel in an anaerobe chamber (an_c, n = 15). One hundred ml of 5% fecal slurry in sterile PBS was also maintained in an anaerobe chamber (an_f, n = 6). Sampling was performed at the time of vessel establishment (0h), after 24h and 48h of fermentation. Sequencing of the V4 region of the 16S rRNA gene was performed on an Illumina MiSeq. Bioinformatics analysis was run using mothur software. Measures of population richness (Chao1), evenness (Shannon Evenness), and diversity (Inverse Simpson) were analyzed between methods and over time using a repeated measures ANOVA in SAS 9.4. Measures of community membership (Jaccard) and structure (Bray-Curtis) were visualized using PCoA. No overall differences were noted between the systems with regards to measures of α diversity ( P > 0.05). However, richness changed in an_c over time (0h vs 24h and 48h, P < 0.05), and diversity as well as evenness changed in an_f over time (0h vs 24h and 48h, P < 0.05). Visual assessment of PCoA revealed a clustering of an_f samples. No clustering of an_c or ch_c samples was evident. The microbiome of cecal inoculum maintained in a chemostat vessel remains consistent over 48h based on measurements of α diversity. The microbiome of cecal inoculum maintained in an anaerobe chamber is also reasonably consistent over this time period. The microbiome of fecal inoculum maintained anaerobically shifts over 48h and fecal inoculum creates an inconsistent environment over time for in vitro use. It appears that the use of cecal inoculum in either a chemostat vessel, or if unavailable, an anaerobe chamber, will provide a stable community over 48h with respect to the composition of the microbiome for in vitro applications. Additional measures of microbe viability, as well as activity are required to accurately judge the stability of these environments.