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.