This study investigated the combined effects of nitrate (NT) and medium-chain fatty acids (MCFA), including C8, C10, C12, and C14, on methane (CH4) production, rumen fermentation characteristics, and rumen bacteria using a 24 h batch incubation technique. Four types of treatments were used: control (no nitrate, no MCFA), NT (nitrate at 3.65 mM), NT + MCFA (nitrate at 3.65 mM + one of the four MCFA at 500 mg/L), and NT + MCFA/MCFA (nitrate at 3.65 mM + a binary combination of MCFA at 250 and 250 mg/L). All treatments decreased (P < 0.001) methanogenesis (mL/g dry matter incubated) compared with the control, but their efficiency was dependent on the MCFA type. The most efficient CH4 inhibitor was the NT + C10 treatment (- 40%). The combinations containing C10 and C12 had the greatest effect on bacterial alpha and beta diversity and relative microbial abundance (P < 0.001). Next-generation sequencing showed that the family Succinivibrionaceae was favored in treatments with the greatest CH4 inhibition at the expense of Prevotella and Ruminococcaceae. Furthermore, the relative abundance of Archaea decreased (P < 0.05) in the NT + C10 and NT + C10/C12 treatments. These results confirm that the combination of NT with MCFA (C10 and C12 in particular) may effectively reduce CH4 production.
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