Effect of β-Alanine Metabolite on Gut Integrity and Immunity in Commercial Broiler Chickens Infected with Eimeria maxima

Abstract

(1) Background: In a metabolomics analysis conducted to investigate the mechanisms behind the growth-promoting effects of probiotics in broilers, β-alanine was found to be significantly elevated. This led to the hypothesis that β-alanine could also contribute to growth-promoting effects in infected broilers. (2) Methods: An in vitro culture system was developed to assess β-alanine's impact on proinflammatory cytokine response in chicken macrophage cells, gut integrity in chicken intestinal epithelial cells, and muscle differentiation in quail muscle cells and primary chicken embryonic muscle cells. In vivo animal feeding studies were then conducted to investigate the effects of dietary β-alanine on various disease parameters in Eimeria maxima-infected broiler chickens. (3) Results: In vitro, β-alanine treatment significantly decreased the gene expression of cytokines in chicken macrophage cells and increased occuldin expression in chicken intestinal epithelial cells. Dietary β-alanine increased the body weight of chickens following Eimeria maxima infection in the H-ALA group. Dietary β-alanine also suppressed cytokines and increased JAM-2 and occludin expression in the H-ALA group compared to the infected group without β-alanine supplementation. (4) Conclusions: These results strongly support the positive effects of dietary β-alanine on intestinal immune responses and gut barrier function in broiler chickens infected with Eimeria maxima.

Keywords: antibiotic alternative feed additives; gut health; gut metabolite; in vitro test; β-alanine.

Figure 1

Experimental design outline for experiment 2. dpi: day post infection.

Figure 2

Anticoccidial effect of β-alanine on Eimeria maxima sporozoites. Each bar represents the mean ± SEM (n = 3). The sporozoite concentration in the control (CON) group was 2.5 × 10⁵ sporozoites/mL. Significance was determined using a PROC GLM in SAS. A p-value less than 0.05 (*) was considered significant.

Figure 3

mRNA expression level of tight junction proteins and mucin in chicken epithelial cells treated with β-alanine. Each bar represents the mean ± SEM (n = 3). Transcript levels were measured using quantitative RT-PCR and normalized to GAPDH transcript levels. Significance was determined using PROC GLM in SAS, with a p-value less than 0.05 (*) considered significant.

Figure 4

Secretion of proinflammatory cytokines in chicken macrophages treated with LPS and β-alanine. Each bar represents the mean ± SEM (n = 3). Cytokine transcript levels were measured using quantitative RT-PCR and normalized to GAPDH levels. Significant results are indicated by: * = p < 0.05.

Figure 5

Proliferation and differentiation of quail muscle cells (a,b) and chicken embryo muscle cells (c,d) influenced by FBS concentration and β-alanine. Each bar represents the mean ± SEM (n = 3). Transcript levels were measured using quantitative RT-PCR and normalized to GAPDH transcript levels. Significant results are indicated by: * = p < 0.05.

Figure 6

Lesion score and oocyst shedding in chickens fed a β-alanine supplemented diet during infection with Eimeria maxima. NC: basal diet, PC: basal diet for infected chickens, H-ALA: β-alanine at 10.0 mg/kg feed, L-ALA: β-alanine at 1.0 mg/kg feed, n.d.: not detected. All chickens, except NC, were infected by oral gavage at day 14 with 1.0 × 10⁴ oocysts/chicken of E. maxima. ^a~c Bars with different letters indicate significant differences (p < 0.05). Each bar represents the mean ± SEM (n = 6). Lesion score was collected from distal jejunal tissue on day 20 (6 dpi: days post infection), and fecal samples were collected from 6 to 8 dpi to calculate oocyst shedding.

Figure 7

Transcripts of proinflammatory cytokines in the jejunum of chickens fed a β-alanine supplemented diet during Eimeria maxima infection in experiment 2. NC: basal diet, PC: basal diet for infected chickens, H-ALA: β-alanine at 10.0 mg/kg feed, L-ALA: β-alanine at 1.0 mg/kg feed, all chickens, except NC, were infected by oral gavage at day 14 with 1.0 × 10⁴ oocysts/chicken of E. maxima. ^a~d Bars with different letters indicate significant differences (p < 0.05). Each bar represents the mean ± SEM (n = 6). Data were collected on day 20 (6 dpi: days post infection). Cytokine transcript levels were measured using quantitative RT-PCR and normalized to GAPDH levels.

Figure 8

Transcripts of Th1 cytokines in the jejunum of chickens fed a β-alanine supplemented diet during Eimeria maxima infection in experiment 2. NC: basal diet, PC: basal diet for infected chickens, H-ALA: β-alanine at 10.0 mg/kg feed, L-ALA: β-alanine at 1.0 mg/kg feed, all chickens, except NC, were infected by oral gavage at day 14 with 1.0 × 10⁴ oocysts/chicken of E. maxima. ^a~b Bars with different letters indicate significant differences (p < 0.05). Each bar represents the mean ± SEM (n = 6). The data were collected on day 20 (6 dpi: days post infection). Cytokine transcript levels were measured using quantitative RT-PCR and normalized to GAPDH levels.

Figure 9

Transcripts of tight junction proteins in the jejunum of chickens fed a β-alanine supplemented diet during Eimeria maxima infection in experiment 2. NC: basal diet, PC: basal diet for infected chickens, H-ALA: β-alanine at 10.0 mg/kg feed, L-ALA: β-alanine at 1.0 mg/kg feed, all chickens, except NC, were infected by oral gavage at day 14 with 1.0 × 10⁴ oocysts/chicken of E. maxima. ^a~b Bars with different letters indicate significant differences (p < 0.05). Each bar represents the mean ± SEM (n = 6). The data were collected on day 20 (6 dpi: days post infection). Transcript levels were measured using quantitative RT-PCR and normalized to GAPDH levels.