Supplementary material- Sources of zinc

Animals that receive Zinc (Zn) dietary supplementation with organic sources respond better to challenges than those receiving Zn from inorganic Zn sources. The objective of this study was to identify intestinal epithelial responses to different Zn sources. Forty-five pigs (nine per treatment) were fed a corn-soybean meal diet without supplemented Zn (ZnR) or supplemented with 50 and 100 ppm of inorganic ZnCl2 (Zn50 and Zn100), and amino acid-bound organic Zn sources (LQ50 and LQ100). After 32 days on diets, RNA-seq analysis of ileum and jejunum showed expected changes on genes related to Zn insufficiency when comparing ZnR to other diets. When comparing organic with inorganic Zn sources, pro-inflammatory cytokine interleukin 18 (<em>IL18)</em> was downregulated and Toll-like receptor 2 (<em>TLR2)</em> upregulated. To determine the role of epithelial cells in response to dietary Zn, swine intestinal organoids (enteroids) were exposed to ZnCl2 or LQ-Zn. In enteroids, <em>ZIP4</em> expression decreased with added Zn compared with Zn-restriction but Zn sources did not change the expression of <em>IL18 </em>or <em>TLR2. </em>These observations suggest that organic Zn may improve immune response through TLR2 signaling, while decreasing expression of proinflammatory cytokines in non-epithelial cells of the intestinal mucosa.

经有机源膳食锌（Zinc, Zn）补充的动物，相较于从无机锌源获取锌的个体，对应激刺激的应答效果更优。本研究旨在明确肠道上皮细胞对不同锌源的应答反应。
将45头仔猪（每组9头）分为5组，饲喂基础玉米-豆粕日粮：不添加锌的对照组（ZnR）、添加50 ppm与100 ppm无机氯化锌（ZnCl₂）的两组（分别记为Zn50与Zn100），以及添加50 ppm与100 ppm氨基酸结合型有机锌源的两组（分别记为LQ50与LQ100）。饲喂32天后，对回肠与空肠组织进行RNA测序（RNA-seq）分析，结果显示：相较于ZnR组，其余各组与锌缺乏相关的基因表达出现预期变化。对比有机锌源组与无机锌源组，可见促炎细胞因子白细胞介素18（interleukin 18, IL18）表达下调，而Toll样受体2（Toll-like receptor 2, TLR2）表达上调。
为明确上皮细胞在膳食锌应答中的作用，本研究将猪肠道类器官（肠类器官）分别暴露于氯化锌（ZnCl₂）与LQ-Zn环境中。在肠类器官中，与锌限制组相比，添加锌后ZIP4的表达水平降低，但不同锌源并未改变IL18与TLR2的表达。
上述结果表明，有机锌可能通过TLR2信号通路改善免疫应答，同时降低肠黏膜非上皮细胞中促炎细胞因子的表达水平。