Dietary Hermetia illucens Larvae Replacement Alleviates Diarrhea and Improves Intestinal Barrier Function in Weaned Piglets Challenged With Enterotoxigenic Escherichia coli K88

Abstract

A high-quality protein substitute, Hermetia illucens (black soldier fly) larvae powder, is rich in protein and often used in animal feed. This study aimed to investigate the feasibility and optimal ratio of replacing fish meal with H. illucens larvae in weaned piglets and to demonstrate the effects on piglets' growth performance, intestinal microflora and immune performance. Forty-eight female weaned piglets were randomly classified into three groups. Each group consisted of eight pens (replicates), with two piglets per pen. Three groups containing different proportions of H. illucens larvae (0, 4, and 8%) were referred to as C, HI4, and HI8. We first designed a 28-day feeding experiment to detect growth performance; after that, the piglets were induced with oral gavage of enterotoxigenic Escherichia coli K88 (ETEC K88) and recording diarrhea on day 29 of the experiment. Samples were taken on the 32nd day to detect the effect of H. illucens larvae on the immune performance of the weaned piglets. H. illucens larvae replacement did not cause any obvious change in the growth performance nether in HI4 nor in HI8 of weaned piglets with 28 d feeding stage. H. illucens larvae could improve the intestinal health of weaned piglets by increasing the content of Lactobacillus and reducing the content of Streptococcus. Compared with C+K88 group, the diarrhea rate was attenuated for the H. illucens supplemented group. The integrity of ileum villi in HI4+K88 and HI8+K88 groups was better than that in C+K88 group, and the villi in C+K88 group were severely damaged. The expression of IL-10, Occludin and Claudin-3 in the intestinal mucosa of the HI4+K88 group and HI8+K88 group were significantly increased (P < 0.05), and the expression of TNF-α was significantly decreased (P < 0.05) compared with the C+K88 group. The results of immunoblotting also validated that the same ETEC K88 treatment of weaned piglets enhanced the expression of tight junction protein in the intestinal mucosa of the H. illucens addition group. ETEC-induced diarrhea will be reduced by the diet of weaned piglets containing H. illucens larvae, ameliorating the immune performance of piglets. Our results indicates that the optimal dosage of H. illucens replacement in weaned piglets is 4%.

Keywords: Hermetia illucens larvae; growth performance; immune performance; intestinal health; weaned piglet.

Figure 1

Richness and diversity of fecal. (A) Chao1 index, Simpson index, Ace, Shannon. The values are means ± SEM (n = 8). (B) Principal coordinates analysis (PcoA) of bacterial communities in the fecal of pigs (based on the Binary-Jaccard). (n = 8). (C) Phylum-level and Family-level relative abundance of 16S rRNA gene sequences from the fecal of pigs (n = 8). The figure provided is an average of the three analyses.

Figure 2

Significantly changed bacterial family. The values were expressed as the mean ± SD (One-way ANOVA with a Tukey post-hoc test): *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, (n = 8). The figure provided is an average of three analyses.

Figure 3

Representative ileum morphology of different group piglets. (A) Morphology of ileum villi in C+K88 group. (B) Morphology of ileum villi in C+K88 group. (C) Morphology of ileum villi in C+K88 group. Representative optical micrographs of intestinal cross-sections. Scale bar = 200 μm. n = 3.

Figure 4

The challenge of ETEC affects the expression of ion transporter and aquaporin in the ileal mucosa of piglets, the content of antioxidant enzymes and immunoglobulins in serum, and the expression of inflammatory factors in the intestinal mucosa. (A) The relative mRNA expression of intestinal ion transporters and aquaporins (AQP) in ileum mucosa. AQP, aquaporins; CFTR, cystic fibrosis transmembrane conductance regulator; NHE3, Na+/H+ exchanger 3; NKCC1, Na-K-Cl cotransporter. Results are mean ± SD (n = 4). (B) Antioxidant enzyme activity and immunoglobulin concentration in serum of challenged weaned piglets (One-way ANOVA with a Tukey post-hoc test) (n = 4). (C) Effects of dietary supplementation of Hermetia illucens larvae on real-time mRNA expression of cytokines and barrier function genes in jejunal mucosa, ileum mucosa, colon mucosa (One-way ANOVA with a Tukey post-hoc test) (n = 4). *P < 0.05, **P < 0.01, ***P < 0.001, (n = 4). The presented data are the average of three independent experiments.

Figure 5

Effects of dietary supplementation of Hermetia illucens larvae on real-time mRNA expression and protein of barrier function genes in jejunal mucosa, ileum mucosa, colon mucosa. (A) Representative immunoblots and quantification of Claudin-3, and Occludin expression in the jejunum (n = 3). (B) Representative immunoblots and quantification of Claudin-3, and Occludin expression in the ileum. (C) Representative immunoblots and quantification of Claudin-3, and Occludin expression in the colon. *P < 0.05, **P < 0.01, ***P < 0.001, (n = 3). Data are based on an average of three independent experiments.

Figure 6

The overall picture shows the main results obtained in the current work. The up arrows (↑) indicate increasing effects, and the down arrow (↓) indicates decreasing effects.