The effect of fumarate used as a feed additive on the reduction of methanogenesis in the rumen was evaluated by in vitro experiments. The addition of fumarate to the culture of mixed ruminal microorganisms that were fermenting hay powder and concentrate reduced methane production. Most fumarate was metabolized to propionate, and a slight increase was noted in other volatile fatty acids. Fumarate was utilized by mixed bacteria but not by mixed protozoa. Fibrobacter succinogenes, Selenomonas ruminantium ssp. ruminantium, Selenomonas ruminantium ssp. lactilytica, Veillonella parvula, and Wollinella succinogenes oxidized H2 by using fumarate as a final electron acceptor, suggesting that these bacteria compete with methanogens for H2, which is the main substrate for methanogenesis in the rumen. However, the affinity of these bacteria to H2 was lower than their affinity to methanogens. These fumarate-utilizing bacteria metabolized malate to products that were similar to those from fumarate, suggesting the possession of fumarate dehydratase. Fibrobacter succinogenes, V. parvula, and W. succinogenes utilized formate, another substrate for methanogenesis, as an electron donor for fumarate reduction. The affinity of these bacteria to formate was higher than the affinity methanogenes have for formate. When methanogens were cocultured with an equal cellular amount of each of the fumarate-utilizing bacteria, methane production was markedly decreased, not only from formate, but also from H2. These results suggest that the addition of fumarate to ruminant feed reduces methanogenesis and enhances propionate production in the rumen.

The effects of the anti-methanogenic compound, bromochloromethane (BCM), on rumen microbial fermentation and ecology were examined in vivo. Japanese goats were fed a diet of 50 % Timothy grass and 50 % concentrate and then sequentially adapted to low, mid and high doses of BCM. The goats were placed into the respiration chambers for analysis of rumen microbial function and methane and H 2 production. The levels of methane production were reduced by 5, 71 and 91 %, and H 2 production was estimated at 545, 2941 and 3496 mmol/head per d, in response to low, mid and high doses of BCM, respectively, with no effect on maintenance feed intake and digestibility. Real-time PCR quantification of microbial groups showed a significant decrease relative to controls in abundance of methanogens and rumen fungi, whereas there were increases in Prevotella spp. and Fibrobacter succinogenes, a decrease in Ruminococcus albus and R. flavefaciens was unchanged. The numbers of protozoa were also unaffected. Denaturing gradient gel electrophoresis and quantitative PCR analysis revealed that several Prevotella spp. were the bacteria that increased most in response to BCM treatment. It is concluded that the methane-inhibited rumen adapts to high hydrogen levels by shifting fermentation to propionate via Prevotella spp., but the majority of metabolic hydrogen is expelled as H 2 gas.Key words: Rumen: Methane: Hydrogen: Bromochloromethane: Goats Enteric fermentation in livestock accounts for 19 % of anthropogenic sources of methane, a potent greenhouse gas (1) , for which rumen fermentation is the largest source of methane production. In rumen fermentation, several pathways involving both hydrogen-producing and -consuming steps are involved in the conversion of feedstuffs into various fermentation end products such as SCFA (2,3) . Although metabolic hydrogen in the rumen is incorporated in fermentation end products by bacteria, methanogenic archaea (methanogens) consume the greater majority of metabolic hydrogen to obtain energy for their metabolism and finally release methane, which accounts for 2 -12 % loss of the metabolic energy from feed (1,3,4) . Therefore, management of metabolic hydrogen and methane production in the rumen is an important factor to be considered, when developing strategies to reduce greenhouse gas emissions and improve efficiency of energy utilisation from feed.It is known that many chemical agents such as ionophores (e.g. monensin), unsaturated fatty acids, sulphate, nitrate, fumarate and halogenated methane analogues (e.g. bromochloromethane (BCM)) are able to reduce methane production from ruminants (1,4 -6) . BCM is one of the most effective inhibitors and apparently reduces methane production by interfering with the cobamide-dependent methyl transferase step of methanogenesis (7,8) . BCM complexed in cyclodextrin (CD; BCM-CD) results in the sustained inhibition of methane production when fed to ruminants (9 -11) . Moreover, an in vitro continuous fermentation system simulating rumen fermentation demon...

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Butyrivibrio fibrisolvens, a butyrate-producing ruminal bacterium, was evaluated for use as a probiotic to prevent colorectal cancer. Oral administration to Jcl:ICR mice of a new strain of B. fibrisolvens (MDT-1) that produces butyrate at a high rate (10(9) cfu/dose) increased the rate of butyrate production by fecal microbes, suggesting that MDT-1 can grow in the gut. The number of colorectal aberrant crypt foci (ACF), putative preneoplastic lesions induced by 1,2-dimethylhydrazine, was reduced after MDT-1 administration (10(9) cfu/dose, 3 times/wk for 4 wk). The number of aberrant crypts (ACs), number of foci having 3 or 4 ACs per focus, and the percentage of mice having 3 or 4 ACs per focus were also reduced, suggesting that the progress of lesions was suppressed by MDT-1. Interestingly, the MDT-1 cell homogenate did not have a similar beneficial effect. MDT-1 had low beta-glucuronidase activity, and administration of MDT-1 reduced the beta-glucuronidase activity in the colorectal contents. The numbers of natural killer (NK) and NKT cells in the spleen were markedly enhanced in response to MDT-1. Decreased beta-glucuronidase activity and increased numbers of NK and NKT cells and butyrate production may explain in part why MDT-1 administration suppressed ACF formation. These results suggest that colorectal cancer may be prevented or suppressed by the utilization of MDT-1 as a probiotic. Administration of MDT-1 had no harmful effect on the health of mice at least for 3 mo.

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