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...

The effects of cashew nut shell liquid (CNSL) feeding on methane production and rumen fermentation were investigated by repeatedly using 3 Holstein nonlactating cows with rumen fistulas. The cows were fed a concentrate and hay diet (6:4 ratio) for 4 wk (control period) followed by the same diet with a CNSL-containing pellet for the next 3 wk (CNSL period). Two trials were conducted using CNSL pellets blended with only silica (trial 1) or with several other ingredients (trial 2). Each pellet type was fed to cows to allow CNSL intake at 4 g/100 kg of body weight per day. Methane production was measured in a respiration chamber system, and energy balance, nutrient digestibility, and rumen microbial changes were monitored. Methane production per unit of dry matter intake decreased by 38.3 and 19.3% in CNSL feeding trials 1 and 2, respectively. Energy loss as methane emission decreased from 9.7 to 6.1% (trial 1) and from 8.4 to 7.0% (trial 2) with CNSL feeding, whereas the loss to feces (trial 1) and heat production (trial 2) increased. Retained energy did not differ between the control and CNSL periods. Digestibility of dry matter and gross energy decreased with CNSL feeding in trial 1, but did not differ in trial 2. Feeding CNSL caused a decrease in acetate and total short-chain fatty acid levels and an increase in propionate proportion in both trials. Relative copy number of methyl coenzyme-M reductase subunit A gene and its expression decreased with CNSL feeding. The relative abundance of fibrolytic or formate-producing species such as Ruminococcus flavefaciens, Butyrivibrio fibrisolvens, and Treponema bryantii decreased, but species related to propionate production, including Prevotella ruminicolla, Selenomonas ruminantium, Anaerovibrio lipolytica, and Succinivibrio dextrinosolvens, increased. If used in a suitable formulation, CNSL acts as a potent methane-inhibiting and propionate-enhancing agent through the alteration of rumen microbiota without adversely affecting feed digestibility.

Summary Given the growing concerns over human and animal health issues related to confined animal feeding operations, an in‐depth examination is required to monitor for airborne bacteria and associated antibiotic resistance genes. Our 16S rRNA‐based pyrosequencing revealed that the airborne microbial community skewed towards a higher abundance of Firmicutes (> 59.2%) and Bacteroidetes (4.2–31.4%) within the confinement buildings, while the office environment was predominated by Proteobacteria (55.2%). Furthermore, bioaerosols in the confinement buildings were sporadically associated with genera of potential pathogens, and these genera were more frequently observed in the bioaerosols of pig and layer hen confinement than the turkey confinement buildings and office environment. High abundances of tetracycline resistance genes (9.55 × 102 to 1.69 × 106 copies ng−1 DNA) were also detected in the bioaerosols sampled from confinement buildings. Bacterial lineages present in the poultry bioaerosols clustered apart from those present in the pig bioaerosols and among the different phases of pig production, suggesting that different livestock as well as production phase were associated with a distinct airborne microbial community. By understanding the diversity of biotic contaminants associated with the different confinement buildings, this study facilitates the implementation of better management strategies to minimize potential health impacts on both livestock and humans working in this environment.

Japanese goats fed a diet of 50% Timothy grass and 50% concentrate with increasing levels of the anti-methanogenic compound, bromochloromethane (BCM) were investigated with respect to the microbial population and functional shifts in the rumen. Microbial ecology methods identified species that exhibited positive and negative responses to the increasing levels of BCM. The methane-inhibited rumen appeared to adapt to the higher H2 levels by shifting fermentation to propionate which was mediated by an increase in the population of H2-consuming Prevotella and Selenomonas spp. Metagenomic analysis of propionate production pathways was dominated by genomic content from these species. Reductive acetogenic marker gene libraries and metagenomics analysis indicate that reductive acetogenic species do not play a major role in the BCM treated rumen.

To visualize and localize specific bacteria associated with plant materials, a new fluorescence in situ hybridization (FISH) protocol was established. By using this protocol, we successfully minimized the autofluorescence of orchard grass hay and detected rumen bacteria attached to the hay under a fluorescence microscope. Real-time PCR assays were also employed to quantitatively monitor the representative fibrolytic species Fibrobacter succinogenes and Ruminococcus flavefaciens and also total bacteria attached to the hay. F. succinogenes was found firmly attached to not only the cut edges but also undamaged inner surfaces of the hay. Cells of phylogenetic group 1 of F. succinogenes were detected on many stem and leaf sheath fragments of the hay, even on fragments on which few other bacteria were seen. Cells of phylogenetic group 2 of F. succinogenes were often detected on hay fragments coexisting with many other bacteria. On the basis of 16S rRNA gene copy number analysis, the numbers of bacteria attached to the leaf sheaths were higher than those attached to the stems (P < 0.05). In addition, R. flavefaciens had a greater tendency than F. succinogenes to be found on the leaf sheath (P < 0.01) with formation of many pits. F. succinogenes, particularly phylogenetic group 1, is suggested to possibly play an important role in fiber digestion, because it is clearly detectable by FISH and is the bacterium with the largest population size in the less easily degradable hay stem.Fibrobacter succinogenes and Ruminococcus flavefaciens are considered to be the predominant cellulolytic bacteria present in the rumens of ruminant animals (16,17,18,27,28). Transmission electron microscopy observations of the fibrous materials digested by rumen microbes have shown that F. succinogenes-or R. flavefaciens-like bacteria are distributed over materials such as fescue and orchard grass and that sometimes these bacteria account for more than 70% of fiber-attaching bacteria (1, 12). In contrast, when species-specific quantification was carried out, F. succinogenes and R. flavefaciens accounted for 0.1 to 6.6% and 1.3 to 2.9% of total bacteria, respectively (8,17,20,26). Furthermore, in an analysis of fiber-associated rumen bacteria based on a 16S rRNA gene clone library, only a few clones belonging to F. succinogenes or R. flavefaciens were obtained, although other species and uncultured bacteria were frequently detected. Thus, the approaches used so far have been inconclusive with respect to clarifying the significance of these cellulolytic species. A new approach allowing both specific visualization and quantification of bacteria, especially fiber-attaching bacteria, might provide more useful information to allow elucidation of their ecology.It is generally accepted that F. succinogenes makes a large contribution to fiber digestion, given that this species has a potent ability to solubilize crystalline cellulose and is found in relatively large numbers or a relatively large biomass in the rumen (13,26). Although F. succinogenes can b...