Cecal Short-Chain Fatty Acid Analysis

As was previously described [28 (link)], cecal samples were homogenized in HCl (2 mL, 3%, 1 M), centrifuged and combined with ethyl acetate (100 µL) and acetic acid-d4 (1 µg/mL) before collecting the organic phase to determine short-chain fatty acid (SCFA) composition. Samples were quantified via GC-MS using a TRACE™ 1310 gas chromatograph (Thermo Fisher Scientific, Waltham, MA, USA) and a TraceGOLD™ TG-WaxMS A column (Thermo Fisher Scientific, Waltham, MA, USA). The pH of cecum content was determined using an Oakton^® model 700 digital pH meter (Oakton Instruments, Vermon Hills, IL, USA). Before testing, the potentiometer was calibrated with pH buffers at 1.68, 4.01, 7.00, 10.01, and 12.45 according to the manufacturer’s recommendations.