Integrative analysis of fermentation characteristics, bacterial community, metabolome, and in vitro rumen fermentation of alfalfa, paper mulberry, and whole-plant quinoa silages

Ensiling changes nutrient availability and fermentation products that subsequently enter the rumen. This study compared alfalfa ( Medicago sativa L., AL), paper mulberry ( Broussonetia papyrifera , PM), and whole-plant quinoa ( Chenopodium quinoa Willd., QU), harvested and prepared under the conditions appropriate to each material. Fermentation characteristics were measured after 7, 14, 30, and 60 d of ensiling, and chemical composition was determined before ensiling and at 60 d. Full-length 16 S rRNA gene sequencing, untargeted metabolomics, and 48-h in vitro rumen incubation were used to characterize bacterial succession, metabolite profiles, and fermentation responses. The three materials showed different fermentation trajectories. At 60 d, QU had lower pH and butyric acid concentration than AL and PM, whereas nitrogen fractions differed among silages. Terminal communities of AL and QU were dominated by Lactiplantibacillus and Weissella , while PM retained higher relative abundances of Enterobacter and Aerococcus . Metabolomic profiles differed among fresh materials and terminal silages. In vitro, QU had lower absolute methane output and higher total volatile fatty acid (VFA) concentration than AL and PM, although its in vitro dry matter digestibility (IVDMD) was lower than that of AL. Under the ensiling conditions used, QU showed lower terminal pH and butyric acid concentration and a distinct nitrogen-fraction profile. It also showed lower absolute methane output and higher total VFA concentration during in vitro fermentation, but did not have the highest IVDMD. Because forage identity was confounded with harvest maturity, initial composition, and wilting, these differences apply to the prepared materials and require validation in animal-feeding studies.