Changes in Diarrhea Score, Nutrient Digestibility, Zinc Utilization, Intestinal Immune Profiles, and Fecal Microbiome in Weaned Piglets by Different Forms of Zinc

Abstract

Twenty weaned piglets with initial body weight of 6.83 ± 0.33 kg (21 day of age, LYD) were randomly assigned to four treatments for a two-week feeding trial to determine the effects of different dietary zinc on nutrient digestibility, intestinal health, and microbiome of weaned piglets. The dietary treatments included a negative control (CON), standard ZnO (ZnO, 2500 ppm), zinc chelate with glycine (Chelate-ZnO, 200 ppm), and nanoparticle-sized ZnO (Nano-ZnO, 200 ppm). At 0 to 1 week, the diarrhea score was decreased in the CON group compared with the ZnO, Chelate-ZnO, and Nano-ZnO group. In overall period, the ZnO and Nano-ZnO groups exhibited improved diarrhea scores compared to the CON group. The apparent total tract digestibility of dry matter and gross energy was the lowest in the CON group after one week. Compared to the ZnO group, the chelate-ZnO group exhibited higher proportion of T-bet+ and FoxP3+ T cells and the nano-ZnO group had higher numbers of RORgt+ and GATA3+ T cells in the mesenteric lymph nodes. ZnO group increased IL-6 and IL-8 levels in the colon tissues and these positive effects were observed in both chelate ZnO and nano-ZnO groups with lower level. The 16S rRNA gene analysis showed that the relative abundance of Prevotella was higher in the ZnO-treated groups than in the CON group and that of Succinivibrio was the highest in the nano-ZnO group. The relative abundance of Lactobacillus increased in the ZnO group. In conclusion, low nano-ZnO levels have similar effects on nutrient digestibility, fecal microflora, and intestinal immune profiles in weaning pigs; thus, nano-ZnO could be used as a ZnO alternative for promoting ZnO utilization and intestinal immunity.

Keywords: ZnO alternative; digestibility; immune; microbiome; piglets.

Figure 1

Schematic diagram of experimental design.

Figure 2

Spleen weights in weaned piglets fed alternative form of zinc oxide. Weaned piglets were administered different form of zinc oxide for two weeks. The control piglets were fed diets with no zinc oxide. The figure displays gross pictures and average weights of the spleen from four dietary pig groups (control, CON; ZnO, positive control; chelate-ZnO, zinc chelate with glycerin; and nano-ZnO, nanoparticle-sized zinc oxide). Data are represented as mean ± SD. Values were statistically analyzed by one-way ANOVA with Tukey’s multiple comparison test.

Figure 3

Histological analysis of the intestinal morphology of weaned piglets fed alternative form of zinc oxide. Weaned piglets were given different type of zinc oxide for two weeks. Control piglets were fed without zinc oxide. Figures displays morphology of jejunum and colon tissue from pigs in four dietary treatments. Scale bar = 100 μM.

Figure 4

Population of CD4+ T cell subsets in mesenteric lymph node (MLN) of weaned piglets fed alternative form of zinc oxide. Forward scatter and side scatter were used to identify lymphocytes among live cells. Figures represent dot plots and graphs of CD4+ T cell (A,B) and its subsets (C,D) in piglets fed different forms of zinc oxide. CD4+ T cell subsets were identified by using the transcription factors T-bet, GATA3, RORγt, and FoxP3; T-bet, RORγt, and FoxP3 were gated simultaneously. Data are represented as mean ± SD. Values were statistically analyzed by unpaired t-test. * p < 0.05.

Figure 5

Major cytokine expression in the mesenteric lymph nodes (MLN) of weaned piglets fed different forms of zinc oxide. IL-8, IL-6, IL-22, IFNγ, IL-4, IL-10, and IL-17A gene expressions in the MLN were examined in piglets weaned on different forms of zinc oxide by using qRT-PCR. MLN cells were stimulated with 5 ug/mL of LPS for four hours on 37 °C and 5% CO₂ condition. mRNA expression levels of the cytokines in each group were calculated based on that of the negative control group (CON). All fold change values were normalized by GAPDH. Data are represented as mean ± SD. Values were statistically analyzed by one-way ANOVA with Tukey’s multiple comparison test.

Figure 6

Gene expression of T cell subsets in the colons of weaned pigs fed alternative forms of zinc. T cell subset changes in the colons of piglets exposed to different forms of zinc oxide over two weeks were examined by determining T cell transcription factor expression using qRT-PCR. mRNA expression levels of TBX21 (A), GATA3 (B), RORC (C), and FoxP3 (D) in each experimental group were calculated based on that of the negative control group (CON). All fold change values were normalized by GAPDH. Data are represented as mean ± SD. Values were statistically analyzed by one-way ANOVA with Tukey’s multiple comparison test.

Figure 7

Cytokine expression in the colon tissues of weaned piglets fed different forms of zinc oxide. IL-8, IL-2, IL-4, IFNγ, IL-17A, IL-10, IL1â, IL-22, and IL-12b gene expression was examined using qRT-PCR in the colon tissues of weaning piglets that were administered different forms of dietary zinc oxide. mRNA expression levels of the cytokines in each group were calculated based on that of the negative control group (CON). All fold change values were normalized by GAPDH. Data are represented as mean ± SD. Values were statistically analyzed by one-way ANOVA with Tukey’s multiple comparison test. * p < 0.05; ** p < 0.005; *** p < 0.001.

Figure 8

Barrier function in the colon tissues of weaned piglets fed different forms of zinc oxide. The gene expression of barrier functioning (A) and antimicrobial peptides (B) in colon tissues are shown. The mRNA expressions of the tight-junction related genes, ZO-1, OCLN, and CLDN4, were analyzed. The mRNA expressions of the antimicrobial peptide genes pBD1 and REGIIIγ were analyzed. Gene expression levels of each group were calculated based on that of the negative control group (CON). All fold change values were normalized by GAPDH. Data are represented as mean ± SD. Values were statistically analyzed by one-way ANOVA with Tukey’s multiple comparison test.

Figure 9

Principal coordinates analysis (PCoA) plots of ZnO treatment (CON; red. ZnO; blue, chelate-ZnO; yellow, nano-ZnO; green) in weeks. Week 0 (right) and week 2 (left) group clustered significantly based on unweighted UniFrac distance metrics (p < 0.05, R: 0.498346).

Figure 10

Taxonomic classification of the 16S rRNA gene sequences. Taxonomic analysis at the (A) phylum and (B) genus levels for the CON, ZnO, chelate-ZnO, and nano-ZnO groups were examined.

Figure 11

The bar plot identifying the different taxon between control and ZnO treatments. CON, ZnO, chelate-ZnO, and nano-ZnO groups at the genus (A–C) level were examined. The numbers on each bar indicates the normalized abundance of each strains. * p< 0.05; ** p < 0.005; *** p < 0.001; **** p < 0.0001.