INTRODUCTION
Methane production in the rumen is a nutritionally wasteful process which represents 2 to 15% feed energy loss (Moss, 1993). In addition, methane production by animals, principally ruminants, is estimated to account 15 to 20% of the global production of methane (Crutzen et al., 1986). Therefore, extensive research effort has been made to find ways to reduce methane production in the rumen. Various compounds have been used to decrease ruminal methane production by various methods (Bhatta et al., 2007), and some of examples are: ionophores (Van Nevel and Demeyer, 1988), organic acids (Martin, 1998), fatty acids (Czerkawski et al., 1966; Dohme et al., 2001), plant extracts (Busquet et al., 2005) and halogenated compounds (Martin and Macy, 1985).
Halogenated compounds such as bromoethanesulfonic acid (BES) are known to be the most effective inhibitors due to their direct inhibitory effects to methanogenic bacteria. BES is known to inhibit the action of methyl coenzyme M reductase in the last step of methanogenesis (Balch and Wolfe, 1979). Martin and Macy (1985) observed that 30 [micro]M BES reduced methane production by 76% in mixed cultures of rumen fluid. However, Immig et al. (1996) observed that methane production was recovered after 4 days of BES infusion into the rumen of sheep. Ungerfeld et al. (2004) observed different sensitivity to methane inhibitors including BES by pure culture of ruminal methanogens. Methanobrevibacter ruminantium was the most sensitive to BES, Methanosarcina mazei was the least sensitive and Methanomicrobium was intermediate.
Traditional culture-based techniques have allowed for the isolation and identification of only a limited number of species of ruminal methanogens due to their fastidious growth requirement (Joblin, 2005). However, non-cultured techniques developed in recent years made identification and enumeration of methanogens easier. Some of molecular techniques used for this purpose include DNA hybridization, the development of clone libraries of 16S rRNA gene sequences, quantitative real-time PCR, denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) (Lin et al., 1997; Tajima et al., 2001; Skillman et al., 2006; McSweeney et al., 2007; Nicholson et al., 2007). Yu et al. (2005) developed group-specific primer to detect four orders of methanogens (Methanobacteriales, Methanomicrobiales, Methanosarcinales, and Methanococcales) using quantitative real-time polymerase chain reaction (qPCR).
Although previous experiments clearly show that BES reduces methane production, which can be related to the number and composition of methanogens (Sparling and Daniels, 1987; Van Nevel and Demeyer, 1995), direct evidence on the relationship between BES and methanogen population has not been clearly presented in the literature. Therefore, current in vitro study was conducted to find relationship between methane production and the population of methanogen and to determine effects of BES on ruminal fermentation characteristics with two different substrates.
MATERIALS AND METHODS
In vitro incubation
Rumen fluid was obtained from rumen-cannulated Holstein steers before morning feeding. Steers were fed twice a day with a ration consisting of 60% timothy and 40% commercial concentrate mixture (12% crude protein and 75% TDN). The collected rumen content was filtered through four layers of cheesecloth and then mixed with two volume of buffer (Menke and Steingass, 1988) under [O.sub.2]-free C[O.sub.2] gas. The 50 ml of rumen fluid-buffer mixture was dispensed anaerobically into serum bottles containing 0.5 g of substrates which had two different ratios of timothy and concentrate (100% timothy vs. 40% timothy-60% concentrate). The solution of BES sodium salt (Aldrich, WI, USA) was added to have the final concentration of 0, 1 and 5 mM in the bottle, which then was filled with [O.sub.2]-free C[O.sub.2] gas and capped with a rubber stopper. The bottles were incubated for 0, 24, 48 and 72 h in a 39[degrees]C incubator.
Analyses
Total gas production was measured after 24, 48 and 72 h incubation by the method of Theodorou et al. (1994) and then headspace gas in the bottle was collected for the analysis of methane and hydrogen. Methane and hydrogen were measured with a gas chromatography (Varian 3800, USA) equipped with Carbosieve S 8100 mesh column (Supelco, USA). The culture fluid was subsampled for the determination of pH, volatile fatty acid (VFA) concentration and total DNA extraction. VFA analysis was performed with a gas chromatography (HP 6890) as described by Erwin et al. (1961).
DNA extraction and real-time PCR
DNA extraction : Total DNA was extracted according to Lee et al. (2007). The culture fluid was mixed with TE buffer (10 mM Tris HCl, 1 mM EDTA, pH 8.0), Tris-buffered phenol, sterilized glass beads (0.5 mm) and 10% sodium lauryl sulfate solution in the 2 ml tube. The mixture was shaken for 2 min two times using Mini-beadbeater (BioSpec. Product Inc., USA). The mixture was cooled down on ice for 2 min after each bead-beating. The tubes were centrifuged at 14,000xg for 5 min and supernatant was collected. The RNA was removed by incubation with DNase-free RNase (iNtRON Biotechnology, Korea). The extracted DNA solution was filtrated using MicroSpin S-200 HR Columns (GE Healthcare, UK) and then DNA concentration was determined using spectrophotometer (UV-1601PC, Shimadzu, Japan).
PCR primers : The PCR primer sets used in this study for amplification of total bacteria, total methanogens and different groups of methanogen (the order Methanobacterials, the order …
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