3 methylbutyric acid

Stool SCFAs were measured using a gas chromatography-mass spectrometry (GC-MS; Gas chromatograph (type 7890A, Agilent, USA) equipped with a mass selective detector (type 5975C, Agilent, USA). The measurement details were described in our previous study (30 (link)) and Supplementary File 1. The standards, including acetate-d3, propionate-d5, butyrate-d7, valerate-d9, isobutyric acid, 2-methylbutyric acid, and 3-methylbutyric acid (isovaleric acid), were purchased from Sigma (St. Louis, MO, USA). A set of diluted standards was prepared (1,000 μg/mL, 100 μg/mL, 10 μg/mL, 1 μg/mL, 0.1 μg/mL, and 0.01 μg/mL) with ethyl acetate before analysis. Data analysis was performed with ChemStation (Agilent, CA, USA, v E.02.02.1432) and Chroma TOF software (LECO, St. Joseph, MI, USA, v 4.34). SCFA concentrations in fecal samples were reported in milligrams per gram (mg/g) of feces.

Hexanal, heptanal, hexanoic acid, octanal, nonanal, decanal and undecanal were obtained from (Sigma-Aldrich Chemie (GmbH, Germany) while propionic acid, 3-methylbutyric acid, and 6,10-dimethyl-5,9-undecadien-2-one (geranyl acetone) were sourced from Sigma-Aldrich Corporation (3050 Spruce Street, St. Louis, Missouri 63103 USA). Purities of the compounds ranged between 95% and 99%. The BG lure used in this study was purchased from Biogent, with an expiry date of December 2015. It mainly contains lactic acid, hexanoic acid and ammonia [13 ].

Valeric acid and isovaleric acid were extracted from the homogenized samples with Milli‐Q water according to our developed method for the analysis of animal feeds. The water extracts were acidified with sulfuric acid (pH <2) to obtain free acids (R‐COOH), which were extracted with diethyl ether (Merck) and analyzed with a gas chromatograph using a flame ionization detector. The acids were separated on a 10 m × 0.53 mm × 1.00 μm FFAP column using a temperature gradient in splitless mode. The injector was maintained at 220°C and the detector at 300°C, and the nitrogen flow (carrier gas) was 8 ml min⁻¹. The injected volume into the column was 1 μl, which was maintained at 80°C for 5 min. Then, the temperature was increased by 5°C min⁻¹ up to 130°C, and by 30°C min⁻¹ up to 200°C. The final temperature was maintained for 8 min. The retention times of valeric acid and isovaleric acid were 9.4 min and 11.0 min, respectively. The analytical standards for valeric acid (n‐valeric acid and pentanoic acid, CAS 109‐52‐4) and isovaleric acid (3‐methylbutanoic acid and 3‐methylbutyric acid, CAS 503‐74‐2) were from Sigma‐Aldrich (USA). All valeric acid was in the form of isovaleric acid. Isovaleric acid content results were expressed in g kg⁻¹ on a dry weight basis.