Five pieces (3 cm × 3 cm per piece) of rumen wall were cut from the rumen of each goat. At the same time, microorganisms on the rumen epithelium were collected by scraping with glass slides. The rumen ISRIB in vitro contents were divided

TPX-0005 into rumen fluid and solid fractions by squeezing through two layers of cheesecloth and centrifugation at 800 × g for 15 min at 4°C. All samples were stored at −70°C. Establishment and maintenance of the mixed-cultures of anaerobic fungi and methanogens The mixed cultures of anaerobic fungi and methanogens were enriched from rumen content according to our previous study [29]. Rumen content was collected into pre-warmed thermos flasks from three rumen fistulated goats (Haimen goat) fed with Leymus Chinensis and immediately transported to the laboratory. The rumen content was homogenized prior to being squeezed through two layers of cheesecloth under anaerobic conditions. The resultant rumen liquid (5 ml) was placed into a CO2 gassed serum bottle with 45 ml of anaerobic diluting solution [30]. Three 10 ml aliquots were removed from the bottle and inoculated into three pre-warmed bottles (39°C) containing

90 ml of growth medium (Mixed-cultures). The mixed-cultures were incubated at 39°C in the incubator (PYX-DHS-50 × 65, Shanghai, China) without shaking and transferred every 3–4 days. OSI-744 In this study, the mixed cultures were transferred more than 62 times. A 7 ml portion of the culture supernatant from the 5th, 15th, 25th, 35th, 45th, 55th, and 62nd subcultures and 1.5 ml of the goat rumen content were collected for DNA extraction. Orpin’s medium [31] was prepared by boiling the mixture for 5 min prior to pumping with CO2 to remove O2. After 2–3 h gassing with CO2, the medium was then dispensed into 160 ml

serum bottles sealed with butyl rubber septa and aluminium crimp-seals (Bellco Glass Inc., Vineland, New Jersey, USA) in anaerobic condition. The growth medium composed of Orpin’s medium containing penicillin RANTES (1600 IU/ml) and streptomycin (2000 IU/ml) and 1% ground rice straw (1 mm) as the substrate. Throughout this study, the growth of methanogens relied on the anaerobic fungi in the co-cultures and no additional hydrogen was added. Methane produced by the mixed cultures was detected by GC during transfer, and the presence of methanogens in the mixed cultures was also monitored by PCR-DGGE. In our previous study, transfer frequency was conducted to investigate its effect on the diversity and activity of enriched ruminal cultures of anaerobic fungi and methanogens in the mixed cultures [18]. DNA samples extracted from our previous study [16] were further analyzed for the novel RCC survival in the present study. Briefly, the mixed cultures of anaerobic fungi and methanogens were subcultured with three transfer frequencies (three-day, five-day, seven-day), respectively, each with triplicates. A portion of 5 ml culture supernatants from each of the 2nd, 4th, and 9th subcultures was collected for DNA extraction.