Quantification of Cecal Short-Chain Fatty Acids

For each cecal content sample, 0.03 g of cecal content was weighed into a 2-mL centrifuge tube, to which 400 μL of ultrapure water was then added. The mixture was then homogenized and incubated for 30 min. The samples were then centrifuged at 10,000 rpm for 10 min at 4°C, and 300-μL volumes of supernatants were aspirated. The supernatants were mixed with 30 μL of metaphosphoric acid before being incubated at 4°C for 3 to 4 h. To remove contaminants from the protein samples, centrifugation was carried out at 13,500 rpm for 15 min at 4°C. An equal volume of transcrotonic acid was then added to the supernatants. A 0.22 μm water phase filter membrane was used to separate the cecal material from the supernatant after the mixture was incubated for 20 min. The supernatants were then stored in 2-mL screw-cap vials. An Agilent 7820A gas chromatograph (Agilent Technologies, Santa Clara, USA) was used to measure the SCFA concentrations in the samples (69 (link)).