Analysis of gut microbiota in Sows Supplemented with Gallnut tannic acid during Early Pregnancy
Fresh fecal samples were collected from each sow within 15 min of defecation on D30 of gestation, and transferred to a 5-ml sterile fecal collection tube, then  immediately snap-frozen in liquid nitrogen and stored at 80 C until DNA extraction. Microbial DNA was extracted from fecal samples using FastDNA SPIN Kit for soil (MP Biomedicals, CA,USA) following kit instructions. The concentration and purity of DNA were determined using a NanoDrop 2000 spectrophotometer (Thermo Fisher, Wilmington, USA) and 1 % agarose gel electrophoresis. The specific Primers 338F (5-ACTCCTACGGGAGGCAGCAG-3) and 806R (5-GGACTACHVGGGTWTCTAAT-3) was used to amplify the V3V4 hypervariable regions of the bacterial 16S rRNA gene in a thermocycler PCR system (GeneAmp 9700, ABI, USA). The PCR reaction mixture including 4 L 5  Fast Pfu buffer, 2 L 2.5 mM dNTPs, 0.8 L each primer (5 M), 0.4 L Fast Pfu polymerase, 10 ng of template DNA, and ddH2O to a final volume of 20 L. PCR amplification cycling conditions were as follows: initial denaturation at 95  for 3 min, followed by 27 cycles of denaturing at 95  for 30 s, annealing at 55  for 30 s and extension at 72  for 45 s, and single extension at 72  for 10 min, and end at 4 . The resulting PCR products were extracted from a 2% agarose gel and further purified using an AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, CA, USA) and quantified using a QuantiFluorTM-ST fluorometer (Promega, USA) according to the manufacturers protocol. Purified amplicons were pooled in equimolar amounts, and paired-end sequencing was performed on an Illumina MiSeq platform at Majorbio Bio-Pharm Technology Co. Ltd (Shanghai, China).