Phytogenic feed additives mitigate in vitro methanogenesis and alter microbial community and functional pathways in the dairy cow rumen

Using phytogenic feed additives (PFA) could be a promising strategy for mitigating enteric methane (CH 4 ) emissions from ruminants. This study aimed to evaluate the efficacy of specific phytogenic additives on rumen fermentation, methanogenesis, microbial community, and functional pathways. This 2 x 4 x 3 factorial study was conducted using an in vitro rumen fermentation system for a period of 72 h. Treatments included two silage-to-concentrate ratios (60:40 and 40:60), four PFA (great burnet leaves, GBL; oregano leaves, OL; cumin seeds, CS; and garlic bulbs, GB), and three inclusion levels (0, 10, and 20 g kg -1 DM) for each PFA. The GB addition proved the most potent anti-methanogenic additive, reducing CH 4 by up to 32.8% at 20 g kg -1 DM, followed by GBL with a 28.5% reduction at 10 g kg -1 DM, without impairing total volatile fatty acid production. Methane suppression was associated with a lower acetate-to-propionate ratio, decreased abundance of methanogenic archaea (particularly Methanobrevibacter ), and reduced expression of the key methanogenesis gene mcrA and fmdB . While GB exhibited a strong anti-protozoal effect, OL effectively reduced ruminal ammonia concentrations. Additionally, metagenomic analysis identified Porcincola was among the core and most abundant genera in our bovine rumen dataset. Optimising the inclusion of specific phytogenic additives can selectively manipulate the rumen microbiome, concurrently reduce methane production and influence nitrogen metabolism. Further research is warranted to evaluate potential synergistic interactions among these additives to enhance fermentation efficiency of ruminant diets. • Garlic bulb and great burnet reduced in vitro methane up to 32.8% with no VFA loss. • Methane drops linked to lower Methanobrevibacter abundance. • Methane mitigation correlated with reduced mcrA (methyl-coenzyme M reductase) gene. • Thymol-rich oregano lowered ammonia, indicating shifts in anaerobic N metabolism.