Multi Omics Analysis Reveals That Compound Radix Pulsatillae and Lactic Acid Bacteria Reprogram the Microbiome...

Oat (Avena sativa L.) silage fermentation often fails due to insufficient lactic acid bacteria (LAB) and low water-soluble carbohydrate content. We investigated the effects of Compound Radix Pulsatillae (CRP; 40 g/kg FM) alone or combined with a commercial LAB inoculant (containing L. plantarum, L. buchneri, and Enterococcus faecium, CRP_LA) on oat silage after 60 days. Compared to control (CK), both CRP and CRP_LA increased dry matter and water-soluble carbohydrate retention while reducing fiber components and ammonia nitrogen (p < 0.05). CRP_LA exhibited superior fermentation quality (lowest pH 4.82, highest lactic acid 47.83 g/kg DM). Using 16S rRNA sequencing and UPLC-MS/MS metabolomics integrated with weighted gene co-expression network analysis (WGCNA), we identified a brown module strongly associated with CRP_LA treatment. Six hub metabolites, belonging to flavonoids, terpenoids, alkaloids, phenolic acids, and nucleotide derivatives, were significantly elevated in CRP_LA silage and showed strong correlations with Lactobacillus abundance and fermentation quality parameters. Correlation-based network analysis revealed that these hub metabolites positively correlated with Lactobacillus abundance, lactic acid, and water-soluble carbohydrate retention, while negatively correlating with spoilage microorganisms (Enterobacter, Acinetobacter, Leuconostoc) and ammonia nitrogen. This multi-omics study provides a metabolite-centric molecular map of the silage microecosystem reshaped by CRP and LAB co-fermentation. The identified hub metabolites-with predicted antimicrobial, antioxidant, and plant-protective functions-represent potential quality markers for functional silage additive development. Mechanistic validation via targeted metabolite supplementation or pathway-specific gene expression analysis is warranted in future studies.