By comparing 16S rRNA gene sequences from sheep fed different diets, we tested the hypothesis that distinct members of Treponema may relate to the digestion of either hay or concentrate diet. All procedures with live animals were approved by the Animal Care and Welfare
Committee of Hokkaido University, Japan (Protocol number 09-0046). Three rumen fistulated sheep (average body weight, 90.7 ± 6.9 kg) were used in three consecutive periods PF-02341066 datasheet corresponding to three dietary regimens. In the first period, each animal was given an alfalfa hay diet (1.2 kg day−1), and in the second period an orchardgrass hay diet (1.2 kg day−1). The orchardgrass hay diet was supplemented with soybean meal. In the third period, each animal was fed a concentrate-diet containing 1.0 kg of a commercial formula feed (Ram 76ME, Mercian, Tokyo, Japan) and 0.5 kg of the
orchardgrass hay. The three diets were formulated to be isonitrogenous (18.2% crude protein). Each diet was fed for 3 weeks and rumen contents were sampled from individual animals before feeding on the last day of the experimental period. The samples were stored at −30 °C until DNA was extracted. Throughout the experimental 3-deazaneplanocin A mw period, animals were kept in individual pens and fed once daily at 09:00 hours. Water and a mineral block was available ad libitum. Total DNA was extracted from 0.25 g wet rumen content samples following the RBB+C method according to Yu & Morrison (2004). Briefly, cells were lysed by repeated beating with glass beads (Mini Bead Beater, BioSpec Products, Bartlesville, OK) in the presence of 4% (w/v) sodium dodecyl Amobarbital sulfate, 500 mM NaCl, 50 mM Tris-HCl (pH 8.0) and 50 mM EDTA. Two different sized (0.1 and 0.5 mm) glass beads were used for
disrupting the cells. After incubation of the lysate at 70 °C for 15 min, nucleic acids were recovered by isopropanol precipitation. DNA was treated with DNase-free RNase and proteinase K, and purified by a QIAamp DNA Stool Mini Kit (Qiagen, Hilden, Germany). Purified DNA was quantified by a NanoDrop 2000 spectrophotometer (Thermo Scientific) and the final concentration of DNA extracts was adjusted to 10 ng μL−1 for use in all downstream applications. In order to design a PCR primer targeting rumen Treponema, 63 currently available 16S rRNA gene sequences of rumen Treponema were obtained from the GenBank database as well as from our clone library sequence collections. Sequences for the three known Treponema species were also included in the analysis. In addition, 10 mammalian and 14 termite Treponema sequences were included in the in silico analysis. The sequences were aligned with clustal x v.1.81 multiple sequence alignment software (Thompson et al., 1997). The Treponema group-specific forward primer was designed based on a region conserved among all rumen Treponema, while the universal primer 926R (Watanabe et al.