Ai 3000

To determine the amount of unreacted 2-naphthol, the 2-naphthol remaining in the pretreatment liquor was extracted and analyzed by gas chromatography/mass spectrometry (GC/MS). 5 ml water was added to 10 ml pretreatment liquor and the mixture was extracted three times with 3 ml CHCl ${}_3$ . To determine remaining 2-naphthol in the biomass, 5 ml water was added to 10 g washed or filtered biomass and extracted three times with 10 ml CHCl ${}_3$ .
Two hundred and fifty microlitre of a syringaldehyde solution as internal standard was mixed with 750 μl of 2-naphthol extract. The concentration of the syringaldehyde solution was 0.5 g/l or 1 g/L for pretreament liquor extracts and biomass extracts, respectively. An autosampler (Thermo Scientific, AI 3000, Waltham, MA, USA) was used to inject the samples (5 μl for the extracts of the liquor and 1 μl for the extract from the biomass) into the GC/MS system (Thermo Scientific, Trace GC Ultra/Polaris ITQ ion trap, EI mode). The split ratio for the injections was 10:1. The GC system was equipped with a Supelco SLB 5 ms capillary column (30 m × 0.25 mm × 0.25 μm). Helium was used as the carrier gas with a flow of 1 ml/min. The following temperature program was used for the GC oven: 80 °C for 5 min, heating by 10 K/min to 280 °C, and 280 °C kept for 5 min.

The ruminal fluid pH was measured immediately after collection using a digital pH meter (PB21, Sartorius, Goettingen, Germany). Individual and total VFA in aliquots of strained ruminal fluid were quantified by gas chromatography54 (link). Samples were injected using an auto-sampler (AI 3000, Thermo Scientific, Waltham, MA, USA) into an AE-FFAP capillary column (30 m × 0.25 mm × 0.33 μm, ATEO, LanZhou, China) on a Varian GC (TRACE 1300, Thermo Scientific, MA, USA). Samples were run at a split-ratio of 20:1 with a column temperature of 45 °C to 150 °C with an increase of 10 °C/min followed by a 5-min hold. The injector and detector temperatures were 200 °C and 250 °C, respectively. Peak integration was performed using Chromeleon^® Software. All ruminal fluid samples were assayed in duplicate.

The concentration of ammonia nitrogen (NH₃-N) in the rumen fluid was determined using the method described by Feng & Gao (2010) (link). According to the method described by Erwin, Marco & Emery (1961) (link), the concentrations of volatile fatty acids (VFA), acetate, propionate, and butyrate in the rumen fluid were determined using a gas chromatography analyzer (AI 3000, Thermo Fisher Scientific GmbH, Dreieich, Germany). The length, width and muscular thickness of the rumen papilla were measured using a Digital microscope (BA210 Digital, Motic China Group Co., Ltd, China)