Distribution of the novel RCC species in the rumen The distribution of the novel RCC species in the rumen

epithelium, in the liquid and solid fractions of goats fed with diets of different 17-AAG research buy concentrate levels is shown in Table 2. The16S rRNA gene copy numbers of the novel RCC species in the rumen epithelium, the liquid and solid fraction ranged from 0.50 to 2.56, 14.44 to 93.45 and 50.30 to76.09 (×106per cm2, ml or g), respectively. The total archaea ranged from16.34 to 36.68, 162.69 to 248.93 and 1385.19 to 2079.26 (×106 per cm2, ml or g), respectively. The abundance of the novel RCC species in the rumen of goats fed low concentrate diet was numerically higher than that of goats fed high concentrate diet. But, the abundance of the total archaea was not affected by the high concentrate feeding. The relative abundance of the novel RCC species within total archaea (12.01 ± 6.35% to 56.47 ± 30.84%) in the liquid fraction was numerically higher than in the other two fractions (1.56 ± 0.49% to 29.10 ± 35.99% and 2.68 ± 2.08% to 5.71 ± 2.07%) in each diet group. Table 2 The 16S rRNA copy numbers of the total Archaea and the novel RCC species in the rumen as quantified by real-time PCR Level of concentrate inclusion* Archaea The novel RCC species The novel RCC species/Archaea   Epithelium × 106 cm2 Liquid × 106 ml Solid × 106 g Epithelium × 106 cm2 Liquid × 106 ml Solid × 106 g Epithelium

% Liquid % Solid % High (65%) 33.25 133.94 2079.26 0.50a 14.44 50.30 1.56 12.01 2.85 Medium (40%) 36.68 248.93 1857.66 0.66a 30.97 38.46 12.90 check details 19.06 5.71 Low (0%) 16.34 162.69 1385.19 2.56b 93.45 SPTLC1 76.04 29.10 56.47 2.68 SEM 6.22 35.73 285.15 0.40 16.56 10.73 7.98 9.23 0.78 P-value 0.413 0.450 0.661 0.034 0.106 0.393 0.421 0.086 0.219 a, b, c, means with different letters in the same column are different P < 0.05; n = 3. *, The pH value of rumen content, 5.60 ± 0.11 (High); 5.79 ± 0.15 (Medium); 6.17 ± 0.25 (Low). Purification of the novel RCC species with anaerobic fungus One fungal culture containing the novel RCC species

was obtained after purification with trimethylamine to support the novel RCC and with Lumazine to inhibit the growth of Methanobrevibacter sp. The anaerobic fungus was identified as belonging to Piromyces sp. as revealed by morphological examination (monocentricthallus; spherical or oval sporangium with filamentous rhizoids; uniflagellate zoospores). The sequencing results showed only one 16S rRNA gene sequence from the total DNA extracted from the supernatant of the fungal culture, and this sequence was 100% identical to LGM-AF04 (DQ985540) and 99% to the clone from Jinnan cattle rumen (EF055552). Further confirmation was also performed by sequencing the mcrA gene coding the alpha subunit of the methyl-coenzyme M reductase that plays a crucial role in the methanogenesis, and the results showed that only one mcrA gene sequence (GenBank: KC859622) was present.