GEA effects of dietary supplementation with gallnut ellagic acid on the gut microbiota of sows
At 107 days of gestation, fresh fecal samples were collected from sows within 5 minutes after defecation. Total bacterial DNA was extracted using the FastDNA SPIN Kit for Soil (MP Biomedicals, USA) according to manufacturer protocols. The V3-V4 hypervariable regions of bacterial 16S rRNA gene were amplified with primers 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3') on a GeneAmp 9700 PCR system (ABI, USA). The 20 uL PCR reaction system contained: 4 uL 5x FastPfu buffer, 2 uL 2.5 mM dNTPs, 0.8 uL of each primer (5 uM), 0.4 uL FastPfu polymerase, 10 ng template DNA, and nuclease-free water to volume. PCR conditions were: 95 degrees C for 3 min; 27 cycles of 95 degrees C for 30 s, 55 degrees C for 30 s, 72 degrees C for 45 s; final extension at 72 degrees C for 10 min. PCR products were separated by 2% agarose gel electrophoresis, purified with AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, USA), and quantified using QuantiFluor-ST fluorometer (Promega, USA). Equimolar purified amplicons were pooled for paired-end sequencing on Illumina MiSeq platform (Majorbio Bio-Pharm Technology Co., Ltd., Shanghai, China).