Dietary fermented mixed ingredient product enhances growth performance and intestinal stem cell-mediated epithelial regeneration through Wnt/β-catenin pathway in layer chicks

Abstract

This study aimed to investigate the effects of dietary supplements of fermented mixed ingredient product (FMIP) on the growth performance, intestinal health, and immune performance of layer hens during the brooding period. Four hundred eighty healthy one-day-old layer chicks were randomly divided into four groups (six replicates/group, twenty hens/replicate) and were fed with different experimental diets for eight weeks (from day 1 to day 56): (1) Corn-soybean-base diet (CON); (2) Chlortetracycline group (CTC; CON diet supplemented with 0.5g/kg chlortetracycline); (3) 4 % fermented mixed ingredient product (4 % FMIP); (4) 8 % fermented mixed ingredient product (8 % FMIP). The results showed that, compared with the CON group, feeding with CTC, 4 % or 8 % FMIP increased the average daily feed intake (ADFI), average daily gain (ADG), immune organs index, serum IgA, IgM, and IgG levels, as well as the apparent metabolic rates of dry matter, crude protein, crude fiber, and crude ash (P < 0.05). Meanwhile, FMIP supplementation improved jejunal morphology and barrier function, as reflected by increased villus height and transepithelial electrical resistance, decreased DAO activity in serum, and up-regulated Occludin protein expression (P < 0.05). Additionally, FMIP supplementation significantly increased protein expression of the stem cell markers (SOX9 and Lgr5), proliferative cell marker (PCNA), and differentiated absorptive cell marker (Villin) (P < 0.05). The immunofluorescence results were consistent with the above results, and FMIP groups have the same effects as the CTC group. Furthermore, the CTC or 4 % FMIP treatment group resulted in a remarkable increase in Wnt/β-catenin signaling proteins (including β-catenin, TCF4, c-Myc, and Cyclin-D1) compared with the CON group (P < 0.05). In conclusion, dietary supplementation of 4 % FMIP improves growth and immune performance, and promotes the intestinal stem cell expansion of layer chicks through Wnt/β-catenin pathway activation.

Keywords: Fermented mixed ingredient product; Growth performance; Immune performance; Intestinal health; Layer chicks.

Fig. 1

Dietary supplementation with fermented mixed ingredient product promotes growth performance and decreases diarrhea scores in layer chicks. (A) Average daily feed intake (ADFI). (B) Average daily gain (ADG). (C) Feed to gain ratio (F/G). (D) 1-4 week diarrhea score. (E) 5-8 week diarrhea score. (F) 1-8 week diarrhea score. Significant differences are indicated between groups by different lowercase letters (P < 0.05); the values are the means ± SEM. CON, control (n= 6); CTC, chlortetracycline (n= 6); 4 % FMIP, 4 % fermented mixed ingredient product (n= 6); 8 % FMIP, 8 % fermented mixed ingredient product (n= 6).

Fig. 2

Dietary supplementation with fermented mixed ingredient product enhances immune capability in layer chicks. (A) Spleen index. (B) Bursal index. (C) Thymus index. (D) The expression of IgA in serum. (E) The expression of IgG in serum. (F) The expression of IgM in serum. (G) The expression of LPS in serum. (H) The expression of sIgA in the jejunum. Significant differences are indicated between groups by different lowercase letters (P < 0.05); the values are the means ± SEM. CON, control (n= 6); CTC, chlortetracycline (n= 6); 4 % FMIP, 4 % fermented mixed ingredient product (n= 6); 8 % FMIP, 8 % fermented mixed ingredient product (n= 6).

Fig. 3

Dietary supplementation with fermented mixed ingredient product promotes the morphological and structural integrity of the jejunum in layer chicks. (A) Duodenal weight per unit length. (B) Jejunum weight per unit length. (C) Ileum weight per unit length. (D) Images of hematoxylin and eosin staining (100× magnification, scale bar = 500 μm). (E) Villus height. (F) Crypt depth. (G) Villus height/crypt depth. (H) Structure of the jejunum under scanning electron microscopy (250× magnification). Significant differences are indicated between groups by different lowercase letters (P < 0.05); the values are the means ± SEM. CON, control (n= 6); CTC, chlortetracycline (n= 6); 4 % FMIP, 4 % fermented mixed ingredient product (n= 6); 8 % FMIP, 8 % fermented mixed ingredient product (n= 6).

Fig. 4

Dietary supplementation with fermented mixed ingredient product enhances intestinal barrier function in layer chicks. (A) Diamine oxidase (DAO) activity of the Serum. (B) DAO activity of the jejunum. (C) Jejunal transepithelial electrical resistance (TEER). (D) Jejunal protein levels of occludin. Significant differences are indicated between groups by different lowercase letters (P < 0.05); the values are the means ± SEM. CON, control (n= 6); CTC, chlortetracycline (n= 6); 4 % FMIP, 4 % fermented mixed ingredient product (n= 6); 8 % FMIP, 8 % fermented mixed ingredient product (n= 6).

Fig. 5

Dietary supplementation with fermented mixed ingredient product promotes ISC proliferation and differentiation in layer chicks. (A-B) Jejunal protein levels of Lgr5, PCNA and villin. (C,F) Jejunal IF staining of SOX9 (100× magnification, scale bar = 200 μm). (D,G) Jejunal IF staining of PCNA (100× magnification, scale bar = 100 μm). (E,H) Jejunal IF staining of villin (100× magnification, scale bar = 200 μm). Significant differences are indicated between groups by different lowercase letters (P < 0.05); the values are the means ± SEM. CON, control; CTC, chlortetracycline; 4 % FMIP, 4 % fermented mixed ingredient product (n= 6); 8 % FMIP, 8 % fermented mixed ingredient product (n= 6).

Fig. 6

Dietary supplementation with fermented mixed ingredient product potentiates the canonical Wnt/β-Catenin signaling pathway in the intestinal of layer chicks. (A-B) Jejunal protein levels of β-catenin, TCF4, c-Myc, and Cyclin-D1. Significant differences are indicated between groups by different lowercase letters (P < 0.05); the values are the means ± SEM. CON, control (n= 6); CTC, chlortetracycline (n= 6); 4 % FMIP, 4 % fermented mixed ingredient product (n= 6); 8 % FMIP, 8 % fermented mixed ingredient product (n= 6).