Evidence for modulation of the fecal microbiota profile by diet in lactating buffalo

Introduction The gastrointestinal microbiota of ruminants plays a crucial role in health, influencing immune responses, nutrient metabolism, and environmental impact. While the ruminal microbiota has been widely investigated, the hindgut microbiota, particularly the fecal microbiota, remains less explored. Diet strongly shapes microbial communities, thereby affecting digestion, metabolic pathways, and methane emissions. Next-generation sequencing enables detailed microbiota profiling; however, no studies have characterized the fecal microbiota of Italian Mediterranean buffaloes in relation to diet. This study aimed to evaluate the bacterial composition and dietary influences on the fecal microbiota of dairy buffaloes. Methods Over 6 months, monthly pooled fecal samples were collected from ~10 to 15% of lactating buffaloes across 10 farms. Concurrently, dietary data were recorded, and total mixed ration samples were analyzed for physicochemical properties and fatty acid profiles. DNA was extracted using the Quick-DNA™ kit, followed by 16S rRNA sequencing on an Illumina MiSeq System. Statistical analyses in R included alpha and beta diversity, differential abundance testing, and one-way ANOVA ( p < 0.05; trends at p < 0.10). Results In total, 10 phyla, 13 classes, 26 orders, 47 families, 86 genera, and 120 species were identified. Firmicutes was the most abundant phylum (55.8 ± 3.6%), followed by Bacteroidota (37.7 ± 3.4%). Among dietary variables, the forage-to-concentrate (FC) ratio and linseed (LS) inclusion exerted the greatest influence. Notably, the FC ratio affected beta diversity (community structure) but not alpha diversity (within-sample diversity), whereas LS inclusion influenced both alpha and beta diversity. A low FC ratio promoted phyla negatively associated with fiber digestibility, particularly families Lachnospiraceae and Succinivibrionaceae , consistent with cattle studies linking these taxa to high-concentrate diets rich in fine particles (