Effects of methane inhibitors on ruminal fermentation and microbial composition in vitro using inoculum from phenotypically high- and low-enteric-methane-emitting cows

The objective of this study was to investigate the interaction of ruminal inoculum type (obtained from high- or low-CH₄-emitting-phenotype cows) and potent antimethanogenic compounds (AMC), including bromoform (CHBr₃), 3-nitropropionic acid (3NPA), chloroform, and Rhodophyta macroalga (RM) on in vitro gas production, ruminal fermentation, and microbial composition parameters. Sixty-eight mid-lactation Holstein cows were initially screened for their enteric CH₄ emissions. Out of these 68 cows, 2 were designated as high- (HM, 24.8 ± 0.05 g CH₄/kg of DMI) and 2 as low- (LM, 14.8 ± 0.45 g CH₄/kg of DMI) CH₄ emitters. These cows were used as inoculum donors for the in vitro experiment. In vitro batch-culture incubations were carried out for 24 h. The experiment was a 2 × 5 factorial arrangement design with inoculum and inhibitor treatments as independent variables and their interaction included in the model. For gas production parameters, the AMC × inoculum type (INOC) interactions were significant for total gas production, CH₄ yield (g/mg of TMR), and CH₄ concentration in total gas. The low-CH₄ inoculum (LMI) resulted in similar CH₄ concentration in total gas as the high-CH₄ inoculum (HMI) for the negative control treatment (TMR + LMI or HMI inoculum with no AMC added). Total gas production was 21% lower for HMI when compared with LMI, regardless of the treatment. Acetate and propionate molar proportions and their ratio also had a significant AMC × INOC interaction in the current study. The inclusion of all AMC resulted in a decrease in acetate and an increase in propionate and butyrate molar proportions. Of the studied inhibitors, 3NPA tended to result in a greater CH₄ reduction, reduced acetate, and increased propionate concentrations when combined with LMI, rather than with HMI. A macroalgae inhibitor in combination with HMI, but not LMI, resulted in lower CH₄ and higher propionate concentration. Both LMI inoculum and inclusion of each AMC resulted in increased butyrate molar proportions. There was no significant INOC × AMC interaction for estimates of methanogenic activity of methanogenic archaea. Inoculum from low-CH₄-phenotype cows resulted in higher gene copy number of Methanobrevibacter ruminantium and Methanosphaera stadtmanae. In the bacterial community, 22 taxa had a significant interaction with INOC. Each AMC differentially affected methanogens and individual bacteria, altering H₂ fluxes. Regardless of the inoculum, CHBr₃ and 3NPA altered both methanogenic and bacterial communities to a greater extent than the rest of the AMC. The combination of LMI with 3NPA and HMI with RM resulted in a greater CH₄ reduction than HMI with 3NPA and LMI with RM, indicating that a different AMC may be more effective in reducing enteric CH₄ in HMI versus LMI. These results indicate that the synergistic effect between AMC and INOC are possible; however, due to the difficulty in classification of INOC as LMI or HMI, results of the study should be interpreted with caution.