Ruminal fluid and fecal samples of 5 cattle that were close to the group average BW in each group were detected by 16S rRNA gene sequencing. The main methods of ruminal and fecal bacterial DNA extraction and PCR amplification were followed the procedure described by our previous study (Ma et al. 2020c (link)). Briefly, the ruminal fluid and fecal samples were used for total genomic DNA extraction via the TIANamp Stool DNA Kit (TIANGEN, Beijing, China). The 0.8% Agarose Gel Electrophoresis and a NanoDrop 2000 Spectrophotometer (Thermo Scientific, Waltham, MA, United States) were used to evaluate the concentration and purity of DNA. The universal primers 341F (5′-CCTACGGGRSGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) with 12 nt unique barcodes were used to amplify the V3–V4 variable region of the 16S rRNA gene from all DNA samples (Metzler-Zebeli et al. 2015 (link)). PCR products from all samples were pooled with equal molar amount for subsequent sequencing analysis. Sequencing libraries were generated using TruSeq DNA PCR-Free Sample Prep Kit (Illumina, San Diego, CA, USA) reference to the manufacturer’s instructions and index codes were added. The library were sequenced on an Illumina HiSeq 2500 platform (Illumina, San Diego, CA, USA) by 2 × 250 bp paired-end sequencing.
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