Molecular sieve 13x

Gas and SCFA analyses were carried out essentially as described by Oh et al. [14 (link)]. Head space gas was analyzed using gas chromatography (GC-8A; Shimadzu, Kyoto, Japan) with attached parallel columns, Porapak Q (Waters, Milford, MA) and Molecular Sieve 13X (Restek, Bellefonte, PA), and a thermal conductivity detector. Flame ionization detector-attached gas chromatography (GC-14B; Shimadzu, Kyoto, Japan) was used for SCFA analysis, using a fused silica capillary column (ULBON HR-20 M, 0.53 mm i.d. × 30 m length, 3.0 μm film; Shinwa, Kyoto, Japan).

Proximate chemical composition, NDF and acid detergent fiber of the experimental feeds was analyzed according to the method of the Association of Official Analytic Chemists [10 ]. Culture pH was measured using an electrode (pH METER F-51; HORIBA, Kyoto, Japan). Gases (H₂, CH₄, and CO₂) in batch cultures were analyzed using a GC-8A gas chromatograph (Shimadzu, Kyoto, Japan) equipped with parallel columns of Porapak Q (Waters, Milford, MA, USA) and Molecular Sieve 13X (Restek, Bellefonte, PA, USA) and a thermal conductivity detector. SCFA was analyzed using a GC-14B gas chromatograph (Shimadzu, Japan) equipped with an ULBON HR-20M fused silica capillary column (0.53 mm i.d.× 30 m length, 3.0-μm film; Shinwa, Kyoto, Japan) and a flame-ionization detector. Injected samples were prepared as follows. Culture fluid or rumen fluid was mixed with 25% meta-phosphoric acid at a 5:1 ratio, incubated overnight at 4°C, and centrifuged at 10,000×g at 4°C for 10 min to obtain the supernatant, to which crotonic acid was added as an internal standard. The operational details of gas chromatography for gases and SCFA were as described by Watanabe et al [11 (link)]. Ammonia nitrogen was measured by the phenol-hypochlorite reaction method [12 (link)] using a microplate reader at 660 nm (ARVO MX, Perkin Elmer, Yokohama, Japan).