*2.5. Measurement of SCFAs*

The concentration of SCFAs in cecum contents was analyzed according to the method of Wu et al. [22] with some modifications. Briefly, 50 mg of cecum contents was suspended in 500 μL of saturated sodium chloride solution and vortexed uniformly for 30 min. Then, the solutions were acidified with 20 μL of 10% H2SO4 and extracted with 1 mL ethyl ether. After this, the mixtures were centrifuged at 12,000× *g* and 4 ◦C for 10 min, and the obtained organic layer of supernatant was mixed with 0.25 g of anhydrous sodium sulfate for 5 min to remove water. In the end, the supernatant was filtered with 0.22 μm organicbased filter membrane and the SCFAs in the organic layer were analyzed by a 7890A GC system (Agilent Technologies Inc., Santa Clara, CA, USA) equipped with a flame ionization detector; the carrier gas was N2, the shunt ratio was 20:1 and the flow rate was 1.5 mL/min. The chromatographic column was HP-INNOWAX (Agilent, 30 m × 0.25 mm × 0.25 μm), and the temperature procedure was as follows: temperature was increased from 60 ◦C to 190 ◦C at 20 ◦C/min and maintained for 4 min. The injection temperature was 200 ◦C, the ionization temperature was 250 ◦C and the injection volume was 5 μL. The standard curve was made by the external standard method and the concentrations of SCFAs were calculated according to the standard curve.

#### *2.6. DNA Extraction of Cecum Contents and High-Throughput Sequencing*

The cecum contents were sent to Majorbio Co., Ltd., China (Shanghai, China) for DNA extraction and sequencing of 16S rRNA gene. Briefly, the total microbial DNA was extracted from cecum contents (*n* = 10) with the DNA extraction kit and tested by agarose gel electrophoresis. The V3-V4 hypervariable region of 16S rRNA was amplified by polymerase chain reaction (PCR), and the primer sequences were 338F (5- - ACTCCTACGGGAGGCAGCAG-3- ) and 806R (5- -CTCCTACGGGAGG CAGCAG-3- ). Then, the PCR products of equimolar concentrations were sequenced using Illumina MiSeq platforms according to the operation manual.
