The study involved 58 Thoroughbred racehorses, all bred and trained under uniform conditions for over four months, to evaluate their fitness based on mental state, veterinary assessments, and athletic performance. The horses followed a structured training regimen and were fed a controlled diet, with no antimicrobial treatments administered in the two months prior to sampling. To compare the exercise-associated microbiota, four non-racehorse breeds were selected as controls, all raised under different conditions and not subjected to racing or specialized training.

Faecal samples were collected from both racehorses and non-racehorses for DNA and RNA extraction, followed by metagenome and metatranscriptome sequencing. The methodology included a detailed process for DNA extraction, library preparation, and sequencing using the Illumina NovaSeq 6000 platform. The analysis aimed to identify microbial communities and their functional potential in the gut microbiome of the horses, as well as to assess the impact of exercise on these microbial populations.

In addition to the horse study, a butyrate treatment experiment was conducted on mice to explore the effects of short-chain fatty acids on athletic performance and muscle function. Mice were divided into control and butyrate groups, undergoing treadmill tests and grip strength assessments. The study also included histological analyses of muscle tissues and quantification of serum SCFAs, aiming to understand the relationship between gut microbiota, exercise, and overall health in both horses and mice. Statistical analyses were performed to compare microbial diversity and functional pathways between the groups.