Effects of Methanogenic Inhibitors on Methane Production and Abundances of Methanogens and Cellulolytic Bacteria in in Vitro Ruminal Cultures

The objective of this study was to systematically evaluate and compare the effects of select antimethanogen compounds on methane production, feed digestion and fermentation, and populations of ruminal bacteria and methanogens using in vitro cultures. Seven compounds, including 2-bromoethanesulphonate (BES), propynoic acid (PA), nitroethane (NE), ethyl trans-2-butenoate (ETB), 2-nitroethanol (2NEOH), sodium nitrate (SN), and ethyl-2-butynote (EB), were tested at a final concentration of 12 mM. Ground alfalfa hay was included as the only substrate to simulate daily forage intake. Compared to no-inhibitor controls, PA, 2NEOH, and SN greatly reduced the production of methane (70 to 99%), volatile fatty acids (VFAs; 46 to 66%), acetate (30 to 60%), and propionate (79 to 82%), with 2NEOH reducing the most. EB reduced methane production by 23% without a significant effect on total VFAs, acetate, or propionate. BES significantly reduced the propionate concentration but not the production of methane, total VFAs, or acetate. ETB or NE had no significant effect on any of the above-mentioned measurements. Specific quantitative-PCR (qPCR) assays showed that none of the inhibitors significantly affected total bacterial populations but that they did reduce the Fibrobacter succinogenes population. SN reduced the Ruminococcus albus population, while PA and 2NEOH increased the populations of both R. albus and Ruminococcus flavefaciens. Archaeon-specific PCR-denaturing gradient gel electrophoresis (DGGE) showed that all the inhibitors affected the methanogen population structure, while archaeon-specific qPCR revealed a significant decrease in methanogen population in all treatments. These results showed that EB, ETB, NE, and BES can effectively reduce the total population of methanogens but that they reduce methane production to a lesser extent. The results may guide future in vivo studies to develop effective mitigation of methane emission from ruminants.