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).

妊娠107天时，于母猪排便后5分钟内采集新鲜粪便样本。采用FastDNA土壤基因组DNA提取试剂盒（FastDNA SPIN Kit for Soil，MP Biomedicals，美国），严格按照制造商提供的操作手册提取总细菌DNA。以引物338F（5'-ACTCCTACGGGAGGCAGCAG-3'）和806R（5'-GGACTACHVGGGTWTCTAAT-3'）为扩增引物，在GeneAmp 9700型PCR仪（ABI，美国）上对细菌16S rRNA基因的V3-V4高变区进行扩增。20 μL的PCR反应体系组成如下：4 μL 5×FastPfu缓冲液、2 μL 2.5 mM dNTP混合液、0.8 μL（5 μM）各引物、0.4 μL FastPfu DNA聚合酶、10 ng模板DNA，最后以无核酸酶水补至反应总体积。PCR反应程序设置为：95℃预变性3分钟；27个循环，每个循环包含95℃变性30秒、55℃退火30秒、72℃延伸45秒；最终于72℃延伸10分钟。PCR扩增产物经2%琼脂糖凝胶电泳分离后，采用AxyPrep DNA凝胶回收试剂盒（Axygen Biosciences，美国）进行纯化，并通过QuantiFluor-ST荧光计（Promega，美国）完成定量。将等摩尔浓度的纯化扩增子混合后，在Illumina MiSeq测序平台（上海美吉生物医药科技有限公司，中国上海）上进行双端测序。