49 Exploring relationships between forage nutrients and the equine fecal microbiome

Abstract

Dietary nutrients have been identified as drivers of gut microbial composition across animal species. The aim of this study was to apply a biologically relevant computational approach to explore relationships between forage nutrients and the equine fecal microbiome. Fecal samples were collected from 8 mares grazing cool-season (CSG) and warm-season grass (WSG) rotational pastures at 5 time points: following initial 3-wk adaptation to CSG hay; after 2–3 weeks grazing CSG (spring), WSG (summer), and CSG (fall); and after transition back to hay in the fall. Forage samples were collected on 3 random days per period (0800–1000), and nutrient content was analyzed by NIRS. Sequence analysis of the 16S rRNA gene was conducted in QIIME 2, and taxonomy was assigned using SILVA. From 3482 amplicon sequence variants (ASV) generated in QIIME 2, 354 bacterial co-abundance groups (BCG) were then identified using Sparse Cooccurrence Network Investigation for Compositional data. Random forest regression showed that mean Crude Protein (CP), Acid Detergent Fiber (ADF), and Water-Soluble Carbohydrate (WSC) concentration could be predicted based on microbial composition (R > 0.5; P < 0.0001). Spearman correlations between these nutrients and BCG abundances were subsequently evaluated in R (v.4.0.2), with threshold criteria of R > 0.5 and P < 0.05. Only 2 BCG were correlated (positively) with ADF and included ASV from Prevotellaceae and Oscillispiraceae families and Streptococcus and Lachnospiraceae_XPB1014_group genera. Six BCG were correlated with WSC (5 pos: 1 neg). Positively correlated BCG included ASV from the Lachnospiraceae family and ASV assigned to Rikenellaceae RC9 gut group, Colidextribacter, Christensenellaceae R-7 group, Fibrobacter, Bacteroidales BS11 gut group, Treponema , and Ruminococcus genera; however, negatively correlated BCG included another distinct Christensenellaceae R-7 group ASV. Nine BCG were correlated with CP (1 pos: 8 neg). The BCG positively correlated with CP included ASV from Muribaculaceae and Lachnospiracea e. Negatively correlated BCG members were also from Lachnospiraceae and Oscillispiraceae as well as genera including Treponema, Christensenellaceae R-7 group, Papillobacter, and Ruminococcus, and Clostridium butyricum. These results suggest that CP, ADF, and WSC were forage nutrients with the greatest influence on microbial composition and through this approach key bacteria positively and negatively responding to these nutrients were identified. However, these finding also underscore limitations of taxon-based analysis by demonstrating differential responses of distinct ASV within a single taxonomic classification to forage nutrients.