Quantifying Short-Chain Fatty Acids

The short-chain fatty acid concentration was determined by using a gas chromatograph (Thermo Fisher Scientific, Milano, Italy) with a DB-FFAP capillary column (DB-FFAP, 30 m × 0.32 mm × 0.25 µm, Agilent Technologies Co., Ltd., Santa Clara, CA, USA) using the method described by Li et al. [13 (link)]. The feed samples were dried at 65 °C in a forced-air oven for 72 h and air-equilibrated for 12 h, then ground using a 1 mm screen. The feed samples were analyzed for DM (dried at 135 °C for 3 h in a forced-air oven); CP (AOAC 2000, Kjeldahl method 988.05); starch (using a commercial assay kit; Jiancheng Bioengineering Institute, Nanjing, China); and NDF and ADF using heat-stable alpha-amylase and sodium sulfite, following the methods of Van Soest et al. [14 (link)].