Different energy sources are often used in horse diets to enhance health and performance. Understanding how diet impacts the cecal and fecal microbiota is crucial for meeting the nutritional needs of horses. High-throughput sequencing and qPCR were used to compare the fecal and cecal microbiota of five healthy horses receiving three different diets: hay diet (HAY), hay + starch and sugar (SS), and hay + fiber and oil ingredients (FO). Assessment of short-chain fatty acids, pH, and buffer capacity was also performed. The HAY diet was associated with the highest values of fecal pH; the FO and SS diets were associated with higher values of BC6 in the cecum, and the SS diet had higher BC5 values in feces ( < 0.05). HAY was associated with a lower alpha diversity in feces and with a higher abundance of Treponema, Fibrobacter, Lachnospiraceae AC2044, and Prevotellaceae UCG-003 in feces. SS was associated with a higher abundance of Desulfovibrio, the Lachnospiraceae AC2044 group, and Streptococcus in the cecum, and Streptococcus and Prevotellaceae UCG-001 in feces, while FO was associated with higher Prevotella, Prevotellaceae UCG-003, and Akkermansia in the cecum, and the Rikenellaceae RC9 gut group and Ruminococcus in feces. This study indicated that different energy sources can influence cecal and fecal microbiota composition and fecal diversity without significantly affecting fermentation processes under experimental conditions. These findings suggest that the diets studied may not pose immediate health risks; however, further research is needed to generalize these effects on gastrointestinal microbiota in broader equine populations.
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