Intestinal Immune Cell Populations, Barrier Function, and Microbiomes in Broilers Fed a Diet Supplemented with Chlorella vulgaris

Abstract

This study aimed to evaluate the effects of dietary Chlorella vulgaris (CV) on the distribution of immune cells, intestinal morphology, intestinal barrier function, antioxidant markers, and the cecal microbiome in 10-day-old broiler chickens. A total of 120 day-old Ross 308 male broiler chicks were assigned to two dietary treatments using a randomized complete block design, with body weight as the blocking factor. Birds fed a diet containing CV showed an increase in CD4⁺ T cells (p < 0.05) compared to those fed the control diet. The relative mRNA expression of intestinal epithelial barrier function-related markers (occludin and avian β-defensin 5) was elevated (p < 0.05) in the CV-supplemented group compared to the control group. The alpha diversity indices (Chao1 and observed features) of the cecal microbiome in 10-day-old birds increased (p < 0.05), indicating higher richness within the cecal bacterial community. In the microbiome analysis, enriched genera abundance of Clostridium ASF356 and Coriobacteriaceae CHKCI002 was observed in birds fed the diet containing CV compared to those fed the control diet. Taken together, dietary CV supplementation might alter intestinal barrier function, immunity, and microbiomes in 10-day-old broiler chickens.

Keywords: broilers; gut health; immune cells; microalgae; microbiome.

Figure 1

Villus height (VH), crypt depth (CD), and VH:CD of 10-day-old broilers fed a control diet or a Chlorella vulgaris (CV)-containing diet. Each data point represents six replicates and is presented as mean ± standard deviation. Significant differences between the treatments were analyzed using the 1-sample 2-tailed t-test.

Figure 2

(A) Relative mRNA expression of antioxidant markers in jejunal tissues and (B) concentrations of antioxidant enzymes in the serum of 10-day-old broilers fed a control diet or a Chlorella vulgaris (CV)-containing diet. Each data point represents six replicates and is presented as mean ± standard deviation. Significant differences between the treatments were analyzed using the 1-sample 2-tailed t-test.

Figure 3

Effects of dietary Chlorella vulgaris (CV) supplementation on the relative mRNA expression of occludin (OCLN), mucin 2 (MUC2), and avian β-defensin 5 (AvBD5) in the jejunal tissue of 10-day-old broiler chickens. Each data point represents six replicates and is presented as mean ± standard deviation. Significant differences between the treatments were analyzed using the 1-sample 2-tailed t-test. * p < 0.05.

Figure 4

Changes in the populations of immune cells in the jejunal lamina propria of 10-day-old broilers fed a control diet or a Chlorella vulgaris (CV)-containing diet. (A) The distribution of monocyte/macrophages (Mono/Macro) and B cells in MHCII cells; (B) populations of Mono/Macro and B cells in MHCII cells; (C) the distribution of CD8 and CD4 cells in CD3 cells; (D) populations of CD4 and CD8 T cells in CD3 cells; (E) the distribution of TCRγδ and CD8 cells in CD3 cells; (F) populations of TCRγδ T cells in CD3 cells. In figure (A,C,E), colors represent the density of cells in that location and indicate the density of cells in the order of red, yellow, green, and blue. Each data point represents six replicates and is presented as mean ± standard deviation. Significant differences between the treatments were analyzed using the 1-sample 2-tailed t-test. * p < 0.05.

Figure 4

Changes in the populations of immune cells in the jejunal lamina propria of 10-day-old broilers fed a control diet or a Chlorella vulgaris (CV)-containing diet. (A) The distribution of monocyte/macrophages (Mono/Macro) and B cells in MHCII cells; (B) populations of Mono/Macro and B cells in MHCII cells; (C) the distribution of CD8 and CD4 cells in CD3 cells; (D) populations of CD4 and CD8 T cells in CD3 cells; (E) the distribution of TCRγδ and CD8 cells in CD3 cells; (F) populations of TCRγδ T cells in CD3 cells. In figure (A,C,E), colors represent the density of cells in that location and indicate the density of cells in the order of red, yellow, green, and blue. Each data point represents six replicates and is presented as mean ± standard deviation. Significant differences between the treatments were analyzed using the 1-sample 2-tailed t-test. * p < 0.05.

Figure 5

Effects of Chlorella vulgaris (CV) supplementation on the phylum, class, order, family, and genus-level compositions of cecal microbial communities in 10-day-old broilers. All levels that accounted for less than 1% of the bacterial taxonomic abundance were classified into the “Others” category. Each color represents a different taxonomic unit (n = 6).

Figure 6

Effects of dietary Chlorella vulgaris (CV) supplementation on the alpha diversity indices of the cecal microbial communities in 10-day-old broilers. Each data point represents six replicates. Significant differences between the treatments were analyzed using the 1-sample 2-tailed t-test. * p < 0.05.

Figure 7

Linear discriminant analysis effect size (LEfSe) and linear discriminant analysis (LDA) based on operational taxonomic units were used to differentiate the cecal microbial communities of birds fed a control diet or a Chlorella vulgaris (CV)-containing diet. (A) Cladogram generated using LEfSe indicating the phylogenetic distribution of the cecal microbiome in 10-day-old broilers fed a diet containing CV. (B) Histogram of LDA scores indicating differences in the cecal microbiome of 10-day-old broilers fed a control diet or a CV-containing diet.

Figure 8

Concentrations of short-chain fatty acids in the cecal digesta of 10-day-old broilers fed a control or a diet containing Chlorella vulgaris (CV). Each data point represents six replicates and is presented as mean ± standard deviation. Significant differences between the treatments were analyzed using the 1-sample 2-tailed t-test.