Hp6890n

The concentrations of ruminal SCFAs were determined by using a gas chromatograph (HP6890N, Agilent Technologies, Wilmington, DE, USA) according to the description of Yang et al. [37 (link)]. A two-tailed t-test was used in the analysis of rumen SCFA concentrations and rumen pH. Differences were considered significant when p < 0.05. These analyses were performed by using SPSS software package (SPSS Inc., Chicago, IL, USA).

On day 29, all goats were slaughtered at 6 h after receiving the morning feed. Ruminal content (30 mL) were strained through a 4-layer cheesecloth and immediately subjected to pH measurement by using a pH meter (Mettler-Toledo Delta 320, Halstead, United Kingdom). A 5% HgCl₂ solution was added to the fluid samples, which were subsequently stored at −20°C for the determination of SCFA concentration and osmolarity. Rumen tissue from the ventral blind sac was quickly excised and washed in ice-cold phosphate-buffered saline (PBS; pH 7.4). The epithelium was subsequently separated from the muscle layers and cut into 1–2 cm² pieces. For each animal, five pieces of rumen epithelium were immediately fixed in 4% paraformaldehyde (PFA) (Sigma, St. Louis, MO, 123 United States) for morphometric analyses. Ten pieces were stored at −20°C for the later extraction of microbial DNA. Ten pieces were stored at −80°C for the later extraction of epithelial RNA.
Ruminal SCFAs concentrations were measured by using a gas chromatograph (HP6890N, Agilent Technologies, Wilmington, DE) as described by Yang et al. (2012) (link). 10 mL of rumen fluid was centrifuged at 18,000 g for 20 min at 4°C (Eppendorf Centrifuge 5424 R, Eppendorf AG, Hamburg, Germany). The supernatant was collected and the osmolarity was measured by using an osmometer (Osmomat 030-D, GONOTEC Berlin, Germany).

Determination of ammonia nitrogen by spectrophotometer colorimetric method [16 (link)]. Instantly measured rumen pH with a portable pH meter (Model PHB-4, Shanghai Leica Scientific Instrument Co., Ltd., Shanghai, China). Volatile fatty acid (VFA) concentrations were determined as described previously [17 (link)]. In brief, 2 mL of thawed rumen content was centrifuged at 5400 rpm at 4 °C for 10 min, and then 0.2 mL of 25% metaphosphoric acid was added to 1 mL of the supernatant for homogenization. The mixed solution was centrifuged at 10,000 rpm for 10 min at 4 °C, and VFAs (acetate, propionate and butyrate) were determined by a gas chromatograph (HP6890N, Agilent Technologies, Wilmington, DE, USA) equipped with an AT-FFAP capillary column (50 m × 0.32 mm × 0.25 µm). The column temperature was held at 60 °C for 1 min, then ramped unrestricted at 5 °C/min to 115 °C, then to 180 °C at 15 °C/min.

THMs and nine HAAs (HAA9) were analyzed using a gas chromatograph (Agilent HP 6890N) outfitted with a ⁶³Ni electron capture detector (ECD), an auto-sampler and a Supelco Equity^TM-5 column (30 m × 0.25 mm ID). The instrument operating conditions were set according to our previous work [9 (link)]. Three replications of THM and HAA9 measurements were performed for each sample.
Four THM compounds, namely chloroform (CHCl₃), bromodichloromethane (CHBrCl₂), dibromochloromethane (CHBr₂Cl) and bromoform (CHBr₃), were monitored in this study. The THMs were extracted with hexane in accordance with Standard Method 6232B [24 ].
The HAA9 compounds, namely monochloroacetic acid (MCAA), mono bromoacetic acid (MBAA), dichloroacetic acid (DCAA), bromochloroacetic acid (BCAA), trichloroacetic acid (TCAA), bromodichloroacetic acid (BDCAA), dibromoacetic acid (DBAA), chlorodibromoacetic acid (CDBAA) and tribromoacetic acid (TBAA), were quantified according to the United States Environmental Protection Agency (USPEA) Method 552.2 [25 ].

The biogas production of the CSTR reactor was monitored and recorded daily using the water displacement method. The biogas will then be collected in a Tedlar bag, and the biogas composition was analyzed using a gas chromatograph (HP 6890 N) (Agilent, Santa Clara, CA 95051, United States) equipped with a thermal conductivity detector (TCD) (Agilent, Santa Clara, CA 95051, United States). The column used was HP Molesieve (Agilent Technologies, Santa Clara, CA, United States) of 30 m length × 0.5 mm ID × 40 μm film thickness capillary column. The splitless inlet, oven, and TCD detector temperatures will be kept at 60°C, 70°C, and 200°C. Argon was used as the carrier gas, while nitrogen was used as the makeup gas.