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

Chiara Evangelista^1,2*Daniele Pietrucci²Marco Milanesi²Federica Gabbianelli²Loredana Basiricò¹Sebastiana Failla³Giovanni Chillemi⁴Umberto Bernabucci¹

• ¹Department of Agricultural and Forestry Sciences, University of Tuscia, Viterbo, Italy
• ²Department for Innovation in Biological Agro-Food and Forest Systems, Viterbo, Italy
• ³CREA Centro di ricerca Zootecnia e Acquacoltura Sede di Monterotondo, Monterotondo, Italy
• ⁴Department of Experimental Medicine, University of Rome “Tor Vergata”, roma, Italy

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 faecal 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 faecal microbiota of Italian Mediterranean buffaloes in relation to diet. This study aimed to evaluate the bacterial composition and dietary influences on the faecal microbiota of dairy buffaloes. Over six months, monthly pooled faecal samples were collected from ~10–15% of lactating buffaloes across 10 farms. Concurrently, dietary data were recorded, and total mixed ration samples were analysed 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). 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 fibre digestibility, particularly families Lachnospiraceae and Succinivibrionaceae, consistent with cattle studies linking these taxa to high-concentrate diets rich in fine particles (<4 mm). Linseed inclusion reduced species richness and increased Firmicutes, Spirochaetota, and Proteobacteria, the latter including potential pathogens implicated in ruminal dysbiosis. Conversely, LS inclusion decreased Verrucomicrobiota, a phylum important for gut health and mucus layer maintenance. This study provides the first characterization of the faecal microbiota of Italian Mediterranean dairy buffaloes and highlights its responsiveness to diet. Findings underscore the potential of faecal microbiota as a non-invasive biomarker for evaluating dietary effects, with implications for animal health, productivity, and environmental sustainability.