Effect of different fermentation substrates on rumen microorganisms and microbe-derived extracellular vesicles (EVs)

Diet composition and microbiota play a crucial role in animal health and productivity. The study aimed to explore the effects of different fermentation substrates on rumen microbiota and their extracellular vesicles (EVs). Straw (fiber), corn starch (starch), and casein (protein) were used as substrates for in vitro fermentation. After 24 h of fermentation, samples were collected and subjected to 16 S rRNA gene sequencing to analyze rumen microbiota. Microbe-derived EVs were extracted and their morphology and particle size were determined. Results showed that fiber increased the diversity of rumen microorganisms, protein increased richness, and starch decreased both diversity and richness of the microbes. Rumen microbiota was dominated by Firmicutes in the protein group, Bacteroidota in the fiber group and Prevotella in the starch group. Principal co-ordinates analysis (PCoA) revealed significant differences in microbial community structure among the three groups. LEfSe analysis at the genus level identified that Prevotella, Succinivibrio, Clostridia_UCG_014 were enriched in the starch group, whereas Acidaminococcus, Muribaculaceae, Pyramidobacter were enriched in the protein group. For the fiber group, the enriched genera included F082 and Rikenellaceae_RC9_gut_group. Phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt2) analysis showed that the top ten microbial functions were mainly involved in signaling and cellular processes (K06142, K03310, K02030, K06147, K01990, K02004, K01992, K02014) and genetic information processing (K06180, K03088), with the fiber group showing better performance in these processes compared to other two groups. Additionally, the particle sizes of extracellular vesicles ranged from 20 to 400 nm, with an average particle distribution coefficients (PDI) close to 0.3 in each group, indicating uniform particle size. Overall, different fermentation substrates significantly affected the diversity of rumen microbes, without affecting the morphology and particle size of microbial EVs.