In Vitro screening of methanogenesis inhibitors and dihydrogen sinks for mitigating methane emissions in ruminants

Introduction: Enteric methane production represents an inefficiency in ruminant systems and contributes to greenhouse gas emissions.Methods: To mitigate ruminal methanogenesis, this study performed a systematic comparative valuation of the dose-dependent effects of two distinct additive categories: methanogenesis inhibitors (MI) such as 3-nitroxipropanol (3NOP), bromoform (BRO) and iodoform (IOD), as well as alternative dihydrogen sinks (dHS) including fumaric acid (FA), encapsulated slow-release fumaric acid (EFA) and phloroglucinol (PHG). The experimental arrangement followed a dose-response design where each additive was incubated at three concentrations (low, medium, and high) for 24 hours with buffered rumen fluid and a total mixed ration as substrate. Control (CON) and blank (BLK) samples were included. IOD and BRO were dissolved in ethanol as they are not soluble in water. To take into account the ethanol effect on fermentation parameters, ethanol-amended CON and BLK were added to correct for BRO and IOD values. Total gas production (TGP) was calculated from pressure readings using the ideal gas law. The volatile fatty acid (VFA) profile and the methane (CH4) and hydrogen (H2) concentrations in the headspace were evaluated using gas chromatography. Statistical analysis included dosage as a fixed effect and run as a random effect.Results: The addition of 3-NOP caused a quadratic decrease in CH4 production and a corresponding linear increase in H2. However, increasing doses of 3-NOP reduced both TGP and total VFA (TVFA) while shifting fermentation toward propionate production. Among the MI, only IOD maintained TVFA production, linearly reducing methanogenesis and TGP, while increasing H2 availability. BRO strongly inhibited CH4 at all doses and increased H2 production, while significantly reducing TGP and TVFA. Regarding dHS, no treatment affected H2 accumulation. Both FA and EFA linearly decreased acetate and increased propionate proportions without influencing TVFA. Notably, EFA, unlike FA, linearly increased TGP and reduced CH4 proportion. In contrast, PHG increased both TGP and TVFA, favoring acetate formation.Conclusion: The study identified specific dose-response behavior of MI and dHS, highlighting the modulation of different fermentative products.