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Short-chain fatty acids (SCFAs) concentration in feces was measured by gas chromatography spectrometer (GCS) (7890B Plus GC System, Agilent, California). SCFAs were extracted from stool samples as follows: 100 mg of a dry fecal sample was put in a 10-ml centrifuge tube and gently suspended in 1.6-ml deionized water. About 0.4-ml 50% H₂SO₄ and 2-ml diethyl ether were then mixed with an orbital shaker for 45 min before centrifuging at 3,000 rpm for 5 min at room temperature. About 10-mg anhydrous CaCl2 was added to remove residual water for collecting a supernatant. Finally, a 2-μL supernatant was injected into the GCS. Gas chromatography analysis was carried out using Agilent 7890B GCS fitted with a flame ionization detector (FID). A GCS column (ZB-FFAP, Phenomenex, California) of 30 m × 0.32 mm × 0.25 μm was used. Nitrogen was supplied as the carrier gas at a flow rate of 1.69 ml/min in a non-split mode (injector temperature at 250°C). The initial oven temperature was 1006C for 2 min, and then rose at a rate of 8°C/min to 240°C before upholding there for 10 min. The temperatures of the FID and the injection port were 350°C. SCFAs were quantified by an external standard method using the mix standard solution of acetic, propionic, butyric, and valeric acids.

The pH values of GE and the control group were analyzed at 0, 6, 12, and 24 h during the fermentation process. The pH value of the supernatant of the fermentation broth after centrifugation (10,000× g, 10 min) was determined by a pH S-3B metera (Shanghai Leici Apparatus Corp., Shanghai, China).
The SCFA levels in the fermentation products were measured according to our previously described method [14 (link)]. The fermentation samples were centrifuged to obtain the supernatant fluid (0.8 mL). Then, 0.4 mL of 50% aqueous H₂SO₄ solution was added followed by vortex blending for 1 min. Subsequently, SCFA was extracted with 1.5 mL ethyl ether, shaken for 3 min, and centrifuged at 3000× g for 5 min. The residual water in the supernatant was removed with CaCl₂. After centrifugation, 2 μL of the supernatant were injected into a gas chromatography (GC) system (Agilent 5975 GC) equipped with a polar GC column (ZB-FFAP, Phenomenex, CA, USA; 30 m × 0.32 mm × 0.25 μm). The solvent delay time was 3.5 min, the initial temperature was 90 °C for 2 min, then increased to 220 °C at the rate of 15 °C/min, and maintained at this temperature for 5 min. The temperatures of flame ionization detector and injection were both 175 °C. Carrier gas He flow rate was 1.0 mL/min. SIM mode and Agilent Mass Hunter WorkStation software (Agilent Technologies, Santa Clara, CA, USA) were used for data acquisition.

Volatile fatty acid analysis followed the procedure by Khorasani et al. [28 (link)] and Lenahan et al. [29 (link)]. Briefly, digesta samples (ileum, cecum, and colon) were diluted with 25% metaphosphoric acid at a 2:1 ratio (w/v). Samples were centrifuged at 12,000×g for 10 min and the supernatant was collected into 2 mL centrifuge tubes and further centrifuged at 16,000×g for 10 min. Following that, the supernatant was collected and filtered through 0.45 μm PVDA filter (Fisher Scientific, Hampton, New Hampshire, USA) into 1.5 mL tubes. An internal standard (4.56 μmol/mL isocaproic acid in 0.15 mol/L oxalic acid) was added at 0.2 mL to 1 mL of the filtered sample supernatant and inverted to mix thoroughly. The VFA were determined on an Agilent 6890 gas chromatograph with a flame ionization detector (Agilent Technologies, Santa Clara, California, USA) and a capillary column ZB-FFAP (30 m length × 0.32 mm width × 0.25 μm film thickness; ZEBRON, Phenomenex, Torrance, California, USA). The initial oven temperature was set at 90 °C and a hold time of 0.1 min, then followed by the 1^st ramp; 10 °C per minute which increased until 170 °C with a hold time of 6 s. The 2^nd ramp was 20 °C per minute up to 230 °C with and a hold time of 2 min. Hydrogen gas was used for the FID and helium gas was used as a carrier.

For the analysis of the short-chain fatty acids (SCFAs), SCFAs were extracted from 60 mg of lyophilized stool samples. The samples were placed in a round-bottom flask and were gently suspended in 1.6 mL of distilled water. Subsequently, 0.4 mL of 50% aqueous H₂SO₄ and 2 ml of diethyl ether were added and mixed with an orbital shaker for 45 min, and then centrifuged at 3000 ×g for 5 min at room temperature. Anhydrous CaCl₂ was mixed with the collected supernatant to remove the residual water. Then, 2 μL of supernatant was analyzed by gas chromatography, using an Agilent 7890A gas chromatograph fitted with a flame ionization detector (FID) and a GC column (ZB-FFAP, Phenomenex, America) of 30 m×0.32 mm×0.25 μm. Nitrogen was supplied as the carrier gas at a flow rate of 1.69 mL/min in non-split mode (injector temperature: 250°C). The initial oven temperature was 100°C for 2 min, which was then increased at a rate of 8°C/min to 240°C, and kept there for 10 min. SCFAs were quantified by a standard external method using the standard mix solution of acetic, propionic, butyric, and valeric acids.

A Gas Chromatograph 6890 N with a flame ionisation detector (GC-FID) and Gas Chromatograph 7890A and Mass Spectrometer (GC–MS) 5975C MSD (Agilent Technologies Inc., USA), both with the capillary column ZB-FFAP 30 m × 0.25 mm × 0.25 µm (Phenomenex Inc., USA) were used for routine analysis and quantification of TCP and its metabolites or for verification of the presence of individual metabolites from the TCP pathway in selected samples, respectively. Samples (2 μl) were injected into the GC with an inlet temperature of 250 °C and split ratio 20:1. The operational conditions for the column were: helium carrier gas with an initial flow of 0.6 ml.min⁻¹ for 1 min, followed by a flow gradient from 0.6 to 1.8 ml.min⁻¹ (ramp 0.2 ml.min⁻¹), temperature program set to give an initial column temperature of 50 °C for 1 min, followed by a temperature gradient from 50 to 220 °C hold for 2 min (ramp 25 °C.min⁻¹). The temperature of the detector was 250 °C. MS scan speed was 6.9 s⁻¹. This method was used for all GC analyses. For that purpose, calibration curve of 0–5 mM of TCP, DCP, ECH, CPD and GDL with internal standard hexan-1-ol was prepared. Detection limits calculated using the software OriginPro v8 (OriginLab Corporation, USA) were 3, 5, 6, 186 and 22 μM for TCP, DCP, ECH, CPD and GDL, respectively.

Silage extracts (n = 3) were prepared by mixing 30 g of silage from each bag in 150 mL of deionized water. The pH of the filtrate was determined using a pH electrode (736 GP Titrino, Metrohm, Herisau, Switzerland) after shaking the solution at 4 °C overnight and filtering through Whatman No. 6 filter paper. Ammonia-N (NH₃-N) was determined using a calibrated ammonia ion-selective electrode (Orion Research, 1990). Volatile fatty zcids (VFA) and alcohols were measured following ethanol/water extraction using gas chromatography analysis (Varian Star 3400 CX GC, Varian Inc., Palo Alto, CA, USA) equipped with a Zebron ZB-FFAP (Phenomenex, CA, USA) Capillary column (30 m × 0.53 mm i.d., 1.0 micron film thickness), and nitrogen as the carrier gas. The instrument was coupled to a flame ionization detector.