Garlic oil and rubber seed oil mixtures modulate rumen fermentation and microbiome for methane mitigation in vitro

Ruminant methane emissions account for approximately 14.5% of anthropogenic greenhouse gas emissions, underscoring the need for microbiome-targeted mitigation strategies that preserve fermentation efficiency. This <italic>in vitro</italic> rumen fermentation study evaluated garlic oil (GO; 0.03% v/v), rubber seed oil (RO; 2–6% substrate v/v), and their combination (GORO) on fermentation, methanogenesis, and microbial ecology using 16S rRNA gene sequencing and PICRUSt2 functional predictions (n=5 replicates/treatment). The GO and GORO treatments reduced methane yield by ~50% (3.21–3.66 mL vs. 6.54–7.11 mL in control (CON)/RO; <italic>p </italic>< 0.001) and decreased the dry matter digestibility (64.1–66.8% vs. 68.5–70.7%; <italic>p </italic>< 0.001). Volatile fatty acid (VFA) profiles shifted toward propionate (21.2–21.6 vs. 19.6–19.9 mol/100 mol; <italic>p </italic>< 0.001), and reduced the acetate to propionate ratios (2.72–2.77 vs. 3.12–3.19; <italic>p </italic>< 0.001), while total VFA remained unchanged (<italic>p </italic>= 0.08). Microbial analyses revealed that GO induced alpha diversity loss (Chao1: 865 vs. 1,257 in CON; <italic>p </italic>< 0.05), but GORO restored richness (Chao1:1,256) and enriched cellulolytics (<italic>Ruminococcus</italic> 8.2% vs. 2.1%; <italic>Fibrobacter</italic> 1.9% vs. 0.45%). For GO, methanogenesis suppression was associated with a 76% decline in <italic>Methanobrevibacter</italic> (<italic>p </italic>< 0.001) and a 5.6–fold rise in <italic>Selenomonas</italic> (<italic>p </italic>< 0.001). Functional predictions showed GO upregulated methylmalonyl-CoA decarboxylase, and GORO restored endo-1,4-β-xylanase activity. Random Forest classification identified <italic>Selenomonas</italic> as the top biomarker, and network analysis linked <italic>Clostridium</italic> to methanogenesis (ρ = 0.85 with CH<sub>4</sub>; <italic>p </italic>< 0.001). These findings support the use of phytogenic oils as biotechnological tools to redirect hydrogen toward sustainable pathways while preserving microbial ecosystem stability.