Screening of and Mechanistic Insights Into the Enteric Methane Mitigation Potential of European Native and Non-Native Forage Trees, Shrubs, and Herbs Using In Vitro Batch Culture

Enteric methane (CH₄) emissions from ruminant livestock must be mitigated to reduce their climate impact. Trees, shrubs, and herbs have gained attention for their nutritional value, climate resilience, and CH₄ reduction potential. This in vitro study evaluated 45 forage species harvested in Flanders, Belgium (July 2022), for their effects on enteric CH₄ production (μmol/g DM), total volatile fatty acid (VFA) production (μmol/g DM), and relative CH₄ production (CH₄:total VFA, mol/mol). Leaf traits from the TRY database were included. Twelve promising species were selected for a second experiment (July 2023) using fresh and ensiled substrate. Polyethylene glycol (PEG) was used to assess the activity of tannins in fresh substrates. Different headspace conditions (100% CO₂ vs. 50% CO₂/50% H₂) were used to assess the impact on methanogens. Reduced CH₄ production in the first experiment was mainly linked to reduced fermentability, and leaf traits are more closely related to fermentability than direct CH₄ mitigation. Alnus glutinosa, Castanea sativa, Catalpa bignonioides, Populus nigra, and Hedera helix emerged as the most effective CH₄ mitigators. Ensiling reduced the concentration of phenolic compounds. PEG assays confirmed the role of tannins in some species; however, PEG was ineffective against the hydrolysable tannins in C. sativa. C. sativa, A. glutinosa, H. helix, and C. bignonioides exhibited anti-methanogenic effects, likely due to plant secondary metabolites, some of which were only indirectly evaluated (tannins and total phenolic compounds). Further phytochemical and microbiological analyses, along with in vivo trials, are needed to confirm these forages' practical application in livestock diets.