Rumen sample collection and treatments before analysis During the 3-d feed challenge period, ruminal content samples (200 g)

were taken each day from the ruminal ventral sac 1 h before, and 3 h and 6 h after intraruminal feed dosing. Ruminal pH was immediately measured with a portable pH-meter (CG840, electrode Ag/AgCl, Schott Geräte, Hofheim, Germany). The samples were then treated for measurement of microbial and fermentation characteristics as follows: on d1 and d3 at −1 h and 3 h relative to intraruminal dosing, 30 g of ruminal content was immediately taken to the laboratory for enzyme extraction from the solid-adherent microorganisms (SAM) under anaerobic conditions. At the same time, 30 g of ruminal content was homogenized in ice using a Polytron grinding mill (Kinematica GmbH, Steinhofhalde HSP inhibitor Switzerland) at speed 5, for two 1 min cycles with 1 min Selonsertib manufacturer rest in ice between cycles. Two aliquots of 1.5 g were then stored at − 80°C until DNA extraction for bacterial qPCR and PCR-DGGE analysis. For each sampling time, an aliquot of ruminal contents was

dried at 103°C for 24 h for dry matter (DM) determination. At all sampling times, 100 g of ruminal content was strained through a polyester monofilament fabric (250 μm mesh aperture) and the filtrate was used for analysis of volatile fatty acids (VFAs), lactate, NH3-N and for protozoa counting. For VFAs, 0.8 mL of ruminal filtrate was mixed with 0.5 mL of a 0.5 N HCl solution containing 0.2% (w/v) metaphosphoric acid and 0.4% (w/v) crotonic acid. For NH3-N, 5 mL of ruminal

filtrate was mixed with 0.5 mL of 5% H3PO4. These samples were stored at − 20°C until analysis. For protozoa, 3 mL of the fresh filtrate was mixed Flavopiridol (Alvocidib) with 3 mL of methyl green, formalin and saline solution (MFS) and preserved from light until counting. Measurements Bacterial quantification by quantitative PCR Genomic DNA was extracted using the FastDNA® Spin Kit, and purified with the GeneClean® Turbo Kit (MP Biomedicals, Illkirch, France) according to the manufacturer’s instructions with minor modifications. Briefly, 250 mg of frozen milled ruminal contents was weighed into the tube provided containing silica beads and lysis buffer. Bacteria were lyzed using a beadbeater (Precellys 24, Bertin Technology, France). The yield and purity of the extracted DNA were assessed by optical density measurement with a Nanoquant Infinite M200 spectrophotometer (Tecan Austria GmbH, Grödig, Austria), using a dedicated quantification plate. Absorbance intensity at 260 nm was used to assay nucleic acids in 2 μL of sample. Absorbance mTOR activation ratios 260/280 and 260/230 were used to check sample purity. The quantitative PCR (qPCR) was carried out using the StepOnePlusTM real-time PCR system and software (Applied Biosystems, Courtaboeuf, France).