16s
The bacterial DNA from the cecal contents (8 samples/group) was extracted as previously described. 1% agarose gel was used to determine the quality of DNA samples. After measuring the concentration, approximately 2030 ng of qualified DNA was used to prepare the library and subjected to Illumina sequencing performed by Meiji Biotechnology Co., Ltd (Shanghai, China). The V3 and V4 hypervariable microbial 16S rDNA regions were amplified by PCR using the following primers: 38F (5-ACTCCTACGGGAGGCAGCAG-3); 806R (5-GGACTACHVGGGTWTCTAAT-3). The PCR products were recycled using 2% agarose gel and purified using AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA). The library construction quality was checked using QuantiFluor-ST (Promega, USA). The amplicon was sequenced using an Illumina MiSeq instrument (Illumina, San Diego, CA, USA), and the raw data were processed with QIIME (1.70) software. All raw data were filtered and trimmed by the Trimmomatic method. UPARSE software (version 7.1, http://drive5.com/uparse/) was used to cluster OTUs according to 97% similarity against the Silva_138 16SrRNA database (http://www.arbsilva.de/). ANOSIM and unweighted principal component analysis (PCoA) were used to compare the bacterial diversity between the control and treatment groups. The above 16S rRNA sequencing analysis was performed by Meiji Biotechnology Co., Ltd (Shanghai, China).