Effects of methane inhibitors on ruminal fermentation and microbial composition in vitro using inoculum from...

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.