Quantification of Caecal SCFA Content

The SCFA (acetate, propionate, and butyrate) content of 40 caecal samples (10 from R− CTR, 10 from R− LE, 11 from R+ CTR, and 9 from R+ LE) was determined by gas chromatography following the protocol previously described by Bedu-Ferrari et al.^{63 (link)}. Between 100 and 250 mg of caecal content were diluted in two volumes of deionised water. Samples were homogenised and mixed for 2 h at 4 °C before centrifugation at 12,000 × g for 15 min at 4 °C. The supernatant was then collected and weighed, and 10% (vol/vol) of phosphotungstic acid saturated solution (Sigma-Aldrich) was added for protein precipitation overnight at 4 °C. As an internal standard, 10 µL of 2-ethylbutyrate (Sigma-Aldrich) were added to 40 µL of acidified supernatant, and the solution was analysed using a gas–liquid chromatograph (GC-FID Agilent 7890B). All samples were analysed in duplicate. Data were collected and peaks were integrated using Agilent OpenLab Chemstation software. Prior to modelling, the relative concentrations of the three SCFAs were computed as the SCFA concentration (in µmol/g) divided by the total concentration of the three main SCFAs (acetate, butyrate, and propionate).