Fecal Samples Processing

At embryonic day 19, fecal samples were collected from each rat and stored in a −80°C refrigerator until metabolomic analysis. For fecal sample pretreatment, 50 mg of sample was weighed into an Eppendorf tube, and 1,000 μl of extract solution (acetonitrile:methanol:water = 2:2:1, with an isotopically labeled internal standard mixture) was added. After subjecting to a 30-s vortex, the samples were homogenized at 35 Hz for 4 min and sonicated for 5 min in an ice-water bath. The homogenization and sonication cycles were repeated three times. The samples were then incubated for 1 h at −40°C and centrifuged at 12,000 rpm for 15 min at 4°C. The resulting supernatant was transferred to a fresh glass vial for further analysis. The quality control sample was prepared by mixing an equal aliquot of the supernatant from all samples.