Investigating dose-dependent effects of chemical compounds targeting rumen fermentation pathways using an in-vitro...

Ruminal fermentation leads to the formation of methane (CH₄) as a byproduct, which is one of the major greenhouse gases. Despite extensive research efforts involving the use of various anti-methanogenic and hydrogen sink compounds, the current understanding of the dose-response effects of these compounds on the rumen microbiome and fermentation profile is limited. In this study, potential methanogenesis inhibitors or electron acceptors were evaluated for their effects on methane production, fermentation, and prokaryotic community composition. Dose-response effects of sodium 2-bromoethanesulfonate (BES: 0, 2.5, 5, 10 mmol/L), p-hydrocinnamic acid (HoC: 0, 5, 10 mmol/L), and sodium fumarate dibasic (DFS: 0, 5, 10, 20 mmol/L) on dry matter degradation, total gas production, methane concentration and yield, composition and yield of volatile fatty acids, and prokaryote composition were studied during 48 h rumen fermentations.

The BES decreased the yield (ml/ g DM) and concentration (%) of CH₄, acetic, isobutyric, and total VFA (t-VFA) concentrations (mmol/g DM), and increased propionic and butyric acid concentrations (mmol/g DM) without affecting dry matter degradability (dDM) as the dose increased. The HoC decreased dDM, total gas production (TGP), CH₄ yield (ml/ g DM) and increased tVFA concentration (mmol/g DM) as the dose increased. The increasing dose of DFS increased the pH, propionic acid and tVFA concentrations (mmol/g DM) and decreased the yield (ml/ g DM) and concentration (%) of CH₄ without affecting dDM. Sodium 2-bromoethanesulfonate, HoC, and DFS doses did not significantly change the alpha-diversity and beta-diversity indices of the prokaryotic communities at the amplicon sequence variant level, although the relative abundances of specific phyla were affected by the treatments. The major bacterial phyla across all samples were Bacteroidetes, Proteobacteria, Firmicutes, Spirochaetota, Verrucomicrobiota, and Patescibacteria.

This study demonstrated that (i) all the evaluated compounds affected the targeted metabolic pathways without influencing the structure of the rumen microbial community, (ii) BES inhibited methanogenesis without affecting dry matter degradability, and (iii) HoC and DFS shifted hydrogen utilization towards acetate and propionate production. The recommended doses, to reduce methane during in-vitro rumen fermentation for BES, HoC, and DFS were determined to be 2.5 mmol/L, 5 mmol/L, and 10 mmol/L, respectively. Further research is suggested to understand the interactive effects of methane inhibition compounds, such as BES, in conjunction with H₂ sink compounds such as HoC and DFS. However, caution is advised when using halogenated compounds like BES, as some methanogens have developed resistance and BES is not approved for use as a feed additive for live animals.