The normalized assay values were analyzed statistically by single-factor anova at a level of significance of 0.05. DGGE was used to examine the relationship Temsirolimus chemical structure between diet and the

rumen Treponema community. The analysis was carried out in a Bio-Rad DCode Universal Mutation Detection System (Bio-Rad Laboratories, Hercules, CA). The g-TrepoF and BAC926R primers used for real-time PCR were used to amplify the V3–V5 regions of the 16S rRNA gene of Treponema in the sheep rumen samples. Genomic DNA from T. bryantii ATCC 33254 was also included in the analysis. An amplicon of c. 575 bp for DGGE analysis was obtained by modifying the reverse primer by addition of a 40-bp GC clamp (5′CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGCACGGGGGG-3′). PCR was performed with a Veriti 96-well thermal cycler (Applied Biosystems, Singapore). NVP-AUY922 supplier A reaction mixture containing 0.4 μM of each primer, 5 μL of 10 × ExTaq buffer, 0.2 μM of

each dNTP, 1.25 U ExTaq polymerase (Takara, Otsu, Japan) and 10 ng of template DNA in a total volume of 50 μL was prepared. The temperature program for cycling consisted of an initial denaturation at 94 °C for 5 min, followed by 30 cycles of 94 °C for 30 s, annealing at 64 °C for 30 s and extension at 72 °C for 30 s with a final extension at 72 °C for 5 min. PCR-amplified 16S rRNA gene fragments were separated using an 8% polyacrylamide gel with 0.5 × TAE buffer (20 mM Tris-acetate, 10 mM sodium acetate, 0.5 mM EDTA, pH 8.0) and a 35–60% linear gradient of denaturant [100% denaturant corresponded to 40% (v/v) deionized formamide and 7 M urea]. Each gel was run at 60 °C, 80 V for 16 h, and then placed in fixing

solution (10% ethanol and next 0.5% acetic acid) for 2 h, stained in 0.1% (w/v) silver nitrate solution for 20 min and developed in 1.5% sodium hydroxide (w/v), 0.1% sodium borohydride (w/v) and 0.4% formaldehyde (v/v) for 8 min. Thereafter, the gel was rinsed and kept in distilled water until the image was scanned. Gel images were analyzed by bionumerics software version 4.5 (Applied Maths, Kortrijk, Belgium). Normalized banding patterns were used to generate dendrograms by calculating Dice similarity coefficients and by an unweighted pair group method with an arithmetic average clustering algorithm. For statistical analysis, the DGGE banding patterns were converted into binary data as the presence or the absence of bands using bionumerics software, and principal component analysis (PCA) was conducted using the primer 5 data analysis software system (PRIMER-E Ltd, Plymouth, UK). Three clone libraries were constructed for the respective feeding conditions. Mixed DNA samples obtained from the rumen content DNA from three animals under the same dietary conditions were used for library construction.