Supplementary material- Sources of zinc

compounds) and inorganic sources (inorganic salts). Different studies have reported that dietary supplementation in diseased or stressed animals with organic Zn improves animal performance and immune response compared with supplementation with inorganic Zn. To identify potential mechanisms of differential function of organic and inorganic Zn supplements, RNA-seq analysis was performed to identify differentially expressed genes (DEGs) in the small intestine of pigs. Forty-five pigs (77.5 ± 2.5 kg body weight) were fed a corn-soybean meal basal diet without added Zn (ZnR), or ZnR diet supplemented with 50 ppm and 100 ppm of ZnCl2 (ZnCl50 and ZnCl100), and amino acid-bound Zn (LQ50 and LQ100) as inorganic and organic sources, respectively. Ileal and jejunal samples were collected from pigs fed experimental diets for 32 days. Supplementation with organic or inorganic forms of Zn did not result in different Zn serum levels. Among the DEGs identified by the RNAseq analysis, further analysis was focused on the proinflammatory cytokine interleukin 18 (<em>IL18)</em> that was downregulated and Toll-like receptor 2 (<em>TLR2)</em> that was upregulated in intestine of pigs fed diets supplemented by organic Zn compared with inorganic Zn. To determine if responses to Zn identified in tissues were mediated by the intestinal epithelial cells, mouse and swine enteroids were exposed luminally to ZnCl2 or LQ-Zn. Genes of interest did not change in mouse enteroids in response to Zn sources. Swine enteroids responded to Zn supplementation by decreasing the expression of <em>ZIP4 </em>compared with Zn restricted enteroids (<em>p = </em>0.006) but Zn sources did not change the expression of <em>IL18 </em>or <em>TLR2. </em>Taken together, our results from RNAseq analysis suggest that organic Zn potentially improves immune response through TLR2 signaling and decreasing production of proinflammatory cytokines. Further, although swine enteroids responded to Zn supplementation, changes in genes related to immune response were not observed in the epithelial cells. Overall, our findings provide information on the potential mechanisms by which Zn sources can affect animal performance and health.