2.4. Total DNA Isolation and 16S rDNA Sequencing Analysis

Total fecal DNA was extracted and purified with the PowerSoil^® DNA Isolation Kit (MO BIO, Carlsbad, CA, USA). The samples were quantified with the Solexa PCR system and microbial 16S rDNA was sequenced on the Illumina Hiseq 2500 platform (Norcross, GA, USA) with paired-end reads. The flash software (version 1.2.11, Johns Hopkins University, USA), Trimmomatic software (version 0.33), and UCHIME software (version 8.1) were utilized to merge paired-end reads into clean and effective tags. The operational taxonomic unit (OTU) number of each sample was obtained by clustering sequences with 97% similarity using Usearch software (version 10.0). Then, we further performed α-diversity, β-diversity, and correlation analysis to explore the species richness and diversity, and microbiome composition change in terms of phylum, genus, and species levels.