Trace 1300

For the effluent samples, DAN was determined using an auto discrete analyzer (AQ2, SEAL Analytical, UK). DOC was analyzed on a total organic carbon analyzer (TOC-V CPH, Shimadzu, Japan). VFAs (including acetate, propionate, butyrate, isobutyrate, valerate, and isovalerate) were determined using a gas chromatograph (TRACE 1300; Thermo Fisher Scientific, USA) equipped with a flame ionization detector. The FAN concentration was calculated using equation 1 (44 (link)):
$$\text{FAN}=(\text{TAN\hspace{0.17em}}\times {10}^{\text{pH}})/(\mathit{exp}\left\{\frac{6,334}{273+T}\right\}+{10}^{\text{pH}})$$ The volume of biogas collected was measured with a gas flowmeter (SQB-0.5; Yibin Mechatronics Research Institute, China), and the composition (CH₄, CO₂, and H₂) was analyzed using a gas chromatograph (Trace1300; Thermo Fisher Scientific, USA) equipped with a flame ionization detector and a thermal conductivity detector. The stable carbon isotope (¹³C) signatures of methane (δ¹³CH₄) and carbon dioxide (δ¹³CO₂) in biogas samples were analyzed using a gas chromatograph (Trace Ultra GC; Thermo Fisher Scientific, USA) coupled with a stable isotope ratio mass spectrometer (Delta V Advantage; Thermo Fisher Scientific, USA). α_C was calculated by using equation 2 (45 (link)):
$${\alpha }_{\text{C}}=({\delta }^{13}{\text{CO}}_2+1,000)/({\delta }^{13}{\text{CH}}_4+1,000)$$

The lambs were weighted on two consecutive days after they were reared in individual units for 50 days. Then, ruminal fluid was collected from each lamb by use of an orogastric tube 2–4 h after morning feeding. The ruminal fluid was mixed thoroughly and filtered through 4 layers of cheesecloth.
The ruminal fluid pH was immediately determined using a pH meter. An 8 mL aliquot of ruminal fluid was preserved with adding 1 mL of metaphosphoric acid (25% wt/vol) to determine volatile fatty acid (VFA) content. The rest of the samples were stored at -20°C for bacteria DNA extraction. For VFA determination, thawed samples of the rumen fluid were centrifuged for 15 min at 10,000 × g at 4°C. Two milliliters of the supernatant were then mixed with 200 μL crotonic acid (1% wt/vol), and the solution was filtered through a 0.45 μm filter. The ruminal VFAs were separated and quantified by using a gas chromatograph (Trace 1300, Thermo Fisher Scientific, United States) as described by Li et al. (2014) (link), using a 30 m × 0.32 mm × 0.33 μm fused silica column (DB-FFAP, Agilent Technologies, United States). Lactate concentrations in the ruminal fluid were determined using commercially available lactate assay kits (A019-2, Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

The VFA was analyzed as previously described [14 (link)]. Briefly, 200 mg of cecal content was weighed in the Eppendorf tube. Then the final volume of 1,500 μL was created by adding deionized water (1,100 μL), trimethyl acetic acid (100 μL), and metaphosphoric acid (100 μL). Following homogenization with a vortex, materials were centrifuged for 10 min at 15,000 r/min at 4 °C. A Gas Chromatography system (TRACE 1300; Thermo Scientific, Waltham, MA, USA) and supernatants (1,000 μL) were used to analyze VFA in the samples. Helium was used as a carrier gas at a 15.5 mL/min rate. Each sample's run duration was 17.5 min, with an injection volume of 0.5 μL. The initial temperature regimen was 120 °C for 4 min, followed by a 4 °C/min increase to 160 °C. 0.1, 0.5, 1, 2, 4, 6, 8, 10, 12, and 14 mmol/L of the standard stock solution mixture containing formic, acetic, propionic, isobutyric, butyric, isovaleric, valeric, isocaproic, hexanoic, and n-caproic acids were used. The software Chromeleon™ 7.2 (Thermo Scientific, Waltham, MA, USA) was used for data management and processing.