2 ethylbutyrate

Short-chain fatty acid concentrations in the cecal contents were quantified after carrying out water extraction (2 vol/wt) and protein precipitation [10% vol/vol phosphotungstic acid (Sigma-Aldrich)] as described elsewhere (48 (link)). Briefly, the short-chain fatty acids (SCFAs) in the acidified supernatant (0.3 μl) were separated using a 7890 Gas Chromatography System (Agilent, Les Ulis, France) equipped with a split/splitless injector (ALS7650), a flame-ionization detector, and a Nukol-SP-1000-capillary GC column (15 m × 0.53 nm, 0.5 μm; FSCAP Nukol; Supelco, Saint-Quentin-Fallavier, France). Hydrogen was the carrier gas (flow rate = 10 ml/min). The inlet, column, and detector temperatures were 200, 100, and 240°C, respectively. We used 2-ethylbutyrate (Sigma-Aldrich) as the internal standard. Samples were analyzed in duplicate. The data were collected and the peaks were characterized using OpenLab ChemStation C.01.06 software (Agilent, Les Ulis, France).

Acetate, butyrate, and propionate were quantified in the bacterial supernatant using gas chromatography (Agilent Technologies, Les Ulis, France) [28 (link)]. 2-Ethylbutyrate (Sigma, Angers, France, 2-ethylbutyric acid, 99%) at a concentration of 20 mM was used as internal standard in a 1:4 ratio to the bacterial supernatant for normalization of the data per run. An external standard (Sigma, Angers, France, volatile free acid mix) was used for identification and quantification of acetate, butyrate, and propionate in the samples in each run. Analyses were made using the OpenLab Chemstation software (Agilent, Les Ulis, France). Concentrations are given in mM.

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).