EquiNectar Leisure Horse Microbiome & Metabolome Study

Ten non-Thoroughbred leisure horses kept at a stable yard near Cambridge, Cambridgeshire, UK were maintained on their standard diets.

Baseline fecal samples were collected and frozen at −80°C.

The horses then received EquiNectar at 150 ml twice daily, approximately 0.7 ml/kg body weight, alongside their normal diet for eight weeks.

At the end of the supplementation period, fecal samples were collected again and analyzed using SIFT-MS metabolomics and 16S microbiome analysis.

The study used a before-and-after design and did not include a separate untreated control group.

Fecal samples were collected before supplementation and again after eight weeks.

The fecal metabolome was analyzed using specific ion flow tube mass spectrometry (SIFT-MS). Five grams of each fecal sample were incubated under standardized conditions before volatile compounds in the sample headspace were measured.

The resulting metabolomic data were analyzed using principal component analysis and machine-learning approaches including partial least squares discriminant analysis, random forest and Bayesian additive regression trees.

The researchers measured volatile metabolites including acetic, propionic and butyric acids, ammonia, methanol, ethanol, acetone and dimethyl disulphide.

For microbiome analysis, eight paired fecal sample sets underwent DNA isolation and 16S rRNA analysis using the Illumina platform. This allowed bacterial populations to be examined from phylum through to species level.