Butyric acid

The concentrations of short‐chain fatty acids (SCFAs; acetate, propionate and butyrate) in the cecal contents were measured using gas chromatography. Acetic acid, propionic acid, and butyric acid (Macklin, Shanghai, China) were added to the fecal samples as standards. The cecal contents were diluted to 3 mL with 10 mmol/L NaOH. The fecal samples were extracted by vortexing and sonication at 0°C for 5 minutes. The pooled extract was then centrifuged (15 minutes, 10 000 rpm, 4°C). The supernatant (1 mL) was mixed with chloroform at a ratio of 1:1 to extract liposoluble components, and the aqueous phase was obtained after vortexing and sonication. Then, 0.7 mL of the aqueous phase was reacted with 1.2 µL of HCl for 10 minutes. N‐hexane (1.4 mL) was used to extract SCFAs in the aqueous phase. The extract was stored at 4°C and analyzed with an Agilent 7890A Series gas chromatograph. The capillary column was 0.25 mm × 0.25 µm × 30 m (HP‐INNOWAX). The injector temperature was maintained at 220°C, and the carrier gas was N₂ at a flow rate of 1 mL min⁻¹ in the capillary column. The column temperature was maintained at 70°C for 2 minutes. The temperature was first increased at a rate of 10°C min⁻¹ to 150°C for 10 minutes. Then, the temperature was increased to 230°C at a rate of 15°C min⁻¹ for 5 minutes. The running time was 30.333 minutes.

The concentrations of VFAs were determined using headspace sampler gas chromatography (Agilent Technologies, Wilmington, DE, USA) according to previous studies (Thanh et al., 2009 (link); Yang et al., 2019 (link)). The external standards of VFAs (acetic acid, propionic acid, butyric acid, isobutyric acid, valerate acid, and isovaleric acid) and metaphosphoric acid were purchased from Macklin Biochemical Co., Ltd. (Shanghai, China). One gram of cecal content was blended with 6% phosphorous acid (m/v, 1:4) and injected into Agilent Technologies GC7890 Network System, equipped with a 30 m × 0.25 mm × 0.25 μm column with a flame ionization detector.