PSIV-17 - Impacts of High Forage Crude Protein Concentrations on the Equine Fecal Microbiome

The objective of this study was to evaluate effects of forage crude protein (CP) concentrations on the equine fecal microbiome. Six mares were assigned to either a high-protein (HP; 24% CP) or normal-protein (NP; 15% CP) hay cube diet in a randomized cross-over design. Fecal samples were collected on the last day of each 35-d period. Branched-chain and short-chain fatty acids (SCFA) were measured by GC-MS; data were analyzed by mixed model ANOVA in R. 16S rRNA gene sequence analysis was conducted in Qiime2. Alpha and beta diversity were assessed by Kruskall-Wallis Tests and PERMANOVA. Taxonomy was assigned using SILVA. Metacyc pathways were predicted with PICRUSt2. Differential genus-level and pathway abundances were analyzed by LefSe (LDA >2.0). Significance was set at p≤0.05. Species richness did not differ by diet, but evenness was greater in NP vs. HP. Beta diversity metrics including Weighted UniFrac also differed by diet. Genera including Methanocorpusculum, Rikenellaceae RC9 gut group, and Eubacterium coprostanoligenes group were more abundant in HP. Treponema and Fibrobacters were more abundant in NP. Twenty pathways related to amino acid synthesis were identified as markers of NP, while 10 pathways related to amino acid degradation and SCFA synthesis were markers of HP. Pathway predictions were supported by metabolite analyses, with acetate (HP: 751; LP: 632 ± 49 ug/g), propionate (HP: 565; NP: 421 ± 43 ug/g), butyrate (HP: 341; LP: 210 ± 34 ug/g), and valerate (HP: 80; NP: 42 ± 11 ug/g) greater in HP than NP. Isobutyrate (HP: 108; NP: 94 ± 7 ug/g; P = 0.09), and isovalerate (HP: 91; NP: 48 ± 10 ug/g) were also greater in HP. These results demonstrate that consuming higher-CP forages impacts equine hindgut microbiota structure and function, leading to increased rates of protein fermentation. Further research is needed to determine physiological significance in forage-fed horses.