Hard and Easy Keeper Microbiomes Reveal Unique Microbial Responses to Carbohydrate and Protein Sources

Abstract This study reports differential responses of the equine gut microbiome to protein- and/or carbohydrate-based on keeper status [easy keeper (EK), medium keeper (MK), hard keeper (HK)]. Anaerobic microcosms were inoculated with equine feces from healthy animals on the same diets (n=12 total, n = 3 / EK, MK, HK of four breeds) with three dietary treatments [C = Carb (cornmeal), P = Protein (soybean meal), M = mix (50% C, 50% P), and F = fecal (no additive)]. Over 48 hours, fermentation products were measured using colorimetric assays and high performance liquid chromatography. Microbial populations were surveyed using 16S rRNA gene sequencing analyzed by QIIME2. Total SCFA production was not different between keeper status but the acetate: propionate ratio and isobutyrate production was greatest for HK and least for EK. Residual protein and carbohydrate at hour 48 was greatest in HK pointing to less efficient nutrient utilization. Diversity measures were not different between keeper statuses over time or within treatments (P > 0.05). Differential abundance (ANCOM and LEfSe), and Spearman correlations identified that while EK, MK and HK shared responses to treatment, groups had characteristic microbial responses to C and P. For example, Lactobacillales, a prominent lactate producer was differentially abundant for EK and MK across treatments but not a biologically significant marker in HK. Opportunistic Gamma proteobacteria bloomed in MK and HK by hour 48, but was only identified in EK at hour 8 indicating that EK communities can resist this growth. While the gut microbiome compositions of keeper groups were similar, they were functionally different in processing key nutrients. MK were more adaptive to dietary challenges and HK were less efficient at nutrient utilization. Analysis of the keeper status microbiomes may uncover novel functioning pathways and relationships that underlie the metabolic tendency of the host towards obesity or leanness.