Maternal stevioside supplementation improves intestinal immune function of chicken offspring potentially via modulating gut microbiota and down-regulating the promoter methylation level of suppressor of cytokine signaling 1 (SOCS1)

Abstract

The intestinal immune function of chickens is limited during the early growing stage. Maternal nutritional intervention has been suggested to affect the innate immunity of offspring. The present study aimed to investigate the effects of maternal stevioside supplementation on the intestinal immune function of chicken offspring. A total of 120 Jinmao yellow-feathered breeder hens were fed a basal diet or a diet supplemented with 250 mg/kg stevioside for 5 weeks. During the last week, 200 breeding eggs from each group were collected for incubation. After hatching, 80 male offspring (40 chickens from each group) were randomly selected and fed the same basal diet for 28 d. In addition, 90 well-shaped fertile eggs of non-treated breeder hens were incubated for the in ovo injection experiment. Steviol dissolved in 20% glycerol was injected at 7 d of incubation. The results showed that maternal stevioside supplementation could improve embryonic development, jejunal integrity and proliferation in the jejunal crypt (P < 0.05). Maternal stevioside supplementation could also increase the innate transcription levels of cytokines and endotoxin tolerance-related factors in the jejunum of chicken offspring (P < 0.05). At 28 d of age, the offspring following maternal stevioside supplementation exhibited higher jejunal secretory immunoglobulin A and serum interferons levels (P < 0.05). A higher abundance of Lactobacillales induced by maternal stevioside supplementation was positively correlated with intestinal immune-related factors (P < 0.05). The in ovo injection with steviol did not alter either embryonic development or intestinal immune function of hatching chickens (P > 0.05). Furthermore, maternal stevioside supplementation could induce hypo-methylation on the promoter region of suppressor of cytokine signaling 1 (SOCS1). In conclusion, maternal stevioside supplementation could improve the intestinal immune function of chicken offspring potentially via modulating the gut microbiota and down-regulating the promoter methylation level of SOCS1.

Keywords: Immune function; Jejunum; Maternal; Offspring; Stevioside; Transcriptome.

Fig. 1

Effects of maternal stevioside supplementation on the embryonic development and growth performance of chicken offspring. (A) Relative embryo weight during embryonic stage. (B) Relative embryo length during embryonic stage. (C) Length of tibia during embryonic stage. (D) Relative weight of breast muscle during embryonic stage. (E) Relative weight of liver during embryonic stage. (F) Hatching weight. (G) Relative weight of breast muscle on 1 d. (H) Length of tibia on 1 d. (I) Relative weight of liver on 1 d. (J) Average daily gain during 1–28 d. (K) Average daily feed intake during 1–28 d. (L) Feed conversion ratio during 1–28 d. Data are presented as mean values ± SEM (n = 8). Values with an asterisk represent statistically significant differences (P < 0.05). CON, the offspring of breeder hens fed a basal diet; STE, the offspring of breeder hens fed a basal diet supplemented with 250 mg/kg stevioside. Effect size and statistical power of parameters in Fig. 1 are shown in Appendix Table 5.

Fig. 2

Effects of maternal stevioside supplementation on the jejunal morphology and proliferation of chicken offspring. (A) The representative images of hematoxylin and eosin (H&E) staining on the jejunum. (B) The representative images of Alcian Blue-periodic acid Schiff (AB-PAS) staining on the jejunum. (C and D) Villus height, crypt depth, villus height-to-crypt depth ratio and the number of goblet cells per 100 μm jejunum in the chicken offspring on 1 and 28 d (n = 8). (E and H) The representative images of immunostaining of proliferating cell nuclear antigen (PCNA) on the jejunum of chicken offspring on 1 and 28 d. (F and I) Ratio of PCNA-positive cells in the jejunal crypt and villus on 1 and 28 d (n = 8). (G and J) Western blotting of PCNA in the jejunum on 1 and 28 d (n = 6). Data are presented as mean value ± SEM. Values with an asterisk represent statistically significant differences (P < 0.05). CON, the offspring of breeder hens fed a basal diet; STE, the offspring of breeder hens fed a basal diet supplemented with 250 mg/kg stevioside. Effect size and statistical power of parameters in Fig. 2 are shown in Appendix Table 6.

Fig. 3

Analysis of the jejunal transcriptome data in the chicken offspring. (A and E) Volcano plot of differentially expressed genes (DEGs) in the jejunum of chicken offspring on 1 and 28 d. (B and F) Number of up- and down-regulated DEGs on 1 and 28 d. (C and G) Heatmap plot of DEGs between the STE and CON groups on 1 and 28 d. (D and H) Plots of Principal Component Analysis (PCA) on 1 and 28 d. CON, the offspring of breeder hens fed a basal diet; STE, the offspring of breeder hens fed a basal diet supplemented with 250 mg/kg stevioside. The data of RNA-Seq were analyzed using randomly selected 4 samples from each group.

Fig. 4

Gene ontology (GO) and a Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis in the jejunum of chicken offspring. (A and D) The number of significantly enriched pathways based on GO and KEGG analysis in the jejunum of chicken offspring on 1 and 28 d. (B and E) The top 10 enriched items in the GO database on 1 and 28 d. (C and F) The top 20 enriched KEGG signal pathways on 1 and 28 d. The size of the dots indicates the numbers of differentially expressed genes (DEGs), and the color of the dots represents the adjusted P-value.

Fig. 5

Effects of maternal stevioside supplementation on the gene expression in the jejunum of chicken offspring. (A and E) Relative mRNA expression of genes related with TLR4 signaling pathways on 1 and 28 d. (B and F) Relative mRNA expression of genes related with cytokine on 1 and 28 d. (C and G) Relative mRNA expression of genes related with endotoxin tolerance on 1 and 28 d. (D and H) Relative mRNA expression of genes related with tight junction on 1 and 28 d. Data are presented as mean values ± SEM (n = 8). Values with an asterisk represent statistically significant differences (P < 0.05). CON, the offspring of breeder hens fed a basal diet; STE, the offspring of breeder hens fed a basal diet supplemented with 250 mg/kg stevioside. TLR3 = toll like receptor 3; TLR4 = toll like receptor 4; TLR5 = toll like receptor 5; MYD88 = myeloid differentiation primary response gene 88; TRIF = toll like receptor adaptor molecule 1; IRF7 = interferon regulatory factor 7; TNF-α = tumor necrosis factor-alpha; IFN-α = interferon alpha; IFN-β = interferon beta; IFN-γ = interferon gamma; IL-4 = interleukin 4; IL-8 = interleukin 8; IL-18 = interleukin 18; PECAM1 = platelet and endothelial cell adhesion molecule 1; STAT1 = signal transducer and activator of transcription 1; SOCS1 = suppressor of cytokine signaling 1; SOCS2 = suppressor of cytokine signaling 2; SOCS3 = suppressor of cytokine signaling 3; OCLN = occludin; CLDN1 = claudin-1; CLDN2 = claudin-2; ZO-1 = tight junction protein 1; ZO-2 = tight junction protein 2. Effect size and statistical power of parameters in Fig. 5 are shown in Appendix Tables 7 and 8.

Fig. 6

Analysis of the gut microbiota in the chicken offspring. (A and F) Plots of Principal Co-ordinates Analysis (PCoA) on 1 and 28 d. (B and G) Venn diagram of operational taxonomic units (OTUs) on 1 and 28 d. (C and H) The results of unweighted pair-group method with arithmetic means (UPGMA) on 1 and 28 d. (D and J) Different structures of gut microbiota according to the linear discriminant analysis effect size (LEfSe) analysis on 1 and 28 d. (E and J) Cladogram plot of the biomarkers the CON and STE groups on 1 and 28 d. CON, the offspring of breeder hens fed a basal diet; STE, the offspring of breeder hens fed a basal diet supplemented with 250 mg/kg stevioside.

Fig. 7

Heatmap of the correlation analysis between gut microbiota (at order level) and intestinal immune-related factors on (A and B) 1 and 28 d. Positive and negative correlations are shown by the red and blue matrices, respectively. The color intensity shows the Pearson correlation coefficient (r) value in each matrix. The asterisk represents a significant correlation between the 2 parameters (P < 0.05). VH = villus height; CD = crypt depth; VCR = villus height-to-crypt depth ratio; SOCS1 = suppressor of cytokine signaling 1; IFN-γ = interferon gamma; IL-8 = interleukin 8; TLR4 = toll like receptor 4; IFN-α = interferon alpha; OCLN = occludin; IFN-β = interferon beta; STAT1 = signal transducer and activator of transcription 1; sIgA = secretory immunoglobulin A; IgA = immunoglobulin A; IgG = immunoglobulin G.

Fig. 8

Effects of in ovo injection with steviol on embryonic development and intestinal innate immune function. (A) Concentration of steviol deposited in the egg yolk after stevioside supplementation reveled by high-performance liquid chromatography (HPLC). The amount of steviol was shown to be 14.55 ± 0.49 μg steviol per g egg yolk. (B) The scheme of in ovo injection experiment. (C) The embryonic mortality after in ovo injection with steviol. (D) Hatching weight. (E) Relative weight of liver. (F) Length of tibia. (G) Relative weight of breast muscle. (H) Relative mRNA expression of genes related with TLR4 signaling pathways in the jejunum. (I) Relative mRNA expression of genes related with cytokine in the jejunum. (J) Relative mRNA expression of genes related with endotoxin tolerance in the jejunum. The data apart from the embryonic mortality are presented as mean value ± SEM (n = 8). TLR4 = toll like receptor 4; MYD88 = myeloid differentiation primary response gene 88; TRIF = toll like receptor adaptor molecule 1; IRF7 = interferon regulatory factor 7; IFN-α = interferon alpha; IFN-β = interferon beta; IFN-γ = interferon gamma; TNF-α = tumor necrosis factor-alpha; STAT1 = signal transducer and activator of transcription 1; SOCS1 = suppressor of cytokine signaling 1; SOCS2 = suppressor of cytokine signaling 2; SOCS3 = suppressor of cytokine signaling 3. Effect size and statistical power of parameters in Fig. 8 are shown in Appendix Table 9.

Fig. 9

Effects of maternal stevioside supplementation on the methylation levels of suppressor of cytokine signaling 1 (SOCS1) promoter cytosine-guanine (CpG) island. (A) Primer sequences of SOCS1 for methylation-specific PCR (MSP). (B) SOCS1 methylation revealed by MSP in the jejunum of chicken offspring on 1 and 28 d. M, methylated; U, unmethylated. (C) Map of SOCS1 promoter CpG island. Numbers above the line are nucleotides relative to the transcription start site. (D and G) Bisulphite sequencing of SOCS1 promotor in the jejunum on 1 and 28 d. A black circle represents a methylated cytosine, and a blank circle represents an unmethylated cytosine. (E and H) The overall methylation level of the SOCS1 promoter CpG island on 1 and 28 d. (F and I) The methylation level of each CpG site in the SOCS1 promoter CpG island on 1 and 28 d. U means unmethylation in the specific CpG site. Data are presented as mean value ± SEM (n = 3). Values with an asterisk represent statistically significant differences (P < 0.05).