The cecal SCFA concentration was determined using ion‐exclusion HPLC according to the method of Tsukahara et al. (2014 ). The collected cecal contents (0.05 g) were mixed with distilled water (0.1 ml) and 12% perchloric acid (v/v; 15 µl). The mixture was then centrifuged for 10 min at 4°C, 13,000 g. The supernatants were collected and filtered using a 0.45 µm cellulose acetate membrane filter (Cosmonice Filter W; Nacalai Tesque). The samples were injected into a SIL‐30AC autosampler (Shimadzu). Two serial organic columns (Shim‐pack SCR‐102H, Shimadzu) with a guard column (SCR‐ 102HG; Shimadzu) were used to separate the SCFAs, (acetic acid, butyrate acid, propionic acid, and isobutyric acid). The column conditions were set at 50°C with an isocratic elution (0.8 ml/min) of 5 mmol/L p‐toluene sulfonic acid aqueous solution using a solvent delivery pump (LC‐30AD; Shimadzu). SCFAs were detected using an electronic conductivity detector (CDD‐10Avp, Shimadzu) following postcolumn dissociation (0.8 ml/min) with 5 mmol/L p‐toluene sulfonic acid, 20 mmol/L bis‐Tris, and 100 µmol/L EDTA. SCFAs were quantified with a system controller (CBM‐20A; Shimadzu), and the concentrations of acetate, butyrate, propionate, and isobutyrate were expressed in nmol/mg wet matter.
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