Effect of supplementing Hermetia illucens larvae powder in feed on growth performance of weaned piglets
The animals were in good health since the initiation of the experiment. In this experiment, the piglets were growing well without death. The ADG, ADFI, F/G of each group of piglets are outlined in Table 4. From 28 days as a whole, no difference between ADG and F/G was evident. But in 7–14 days, the F/G of the HI4 group was found to be significantly lower than that of other groups (P < 0.05); a significant difference was also noted, in 21–28 days, between the F/G of the HI4 and HI8 groups from the C group (P < 0.05). This provides certain guiding significance for production and application: it is feasible for Hermetia illucens larval powder to completely replace a fish meal to feed weaned piglets with no effect on the growth performance of weaned piglets.
Effect of adding Hermetia illucens larvae powder in feed on serum biochemical parameters of weaned piglets
Before inoculating weaning piglets with bacteria, we collected blood, extracted serum, and adopted automatic biochemical analyzer BS-240VET (Mindray Bio, Shenzhen, China) to detect serum biochemical indicators. Table 5 demonstrates nonsignificant differences in GLB and AST between the three groups; however, the TP content of the HI4 group and HI8 group was significantly different from the C group (P < 0.05). Compared with the C group, Hermetia illucens larvae powder significantly increased P, Ca (P < 0.05), which may be related to the presence of calcium and phosphorus in the larval powder of Hermetia illucens. Hermetia illucens larvae powder significantly increased the ALB, ALT, TG, TC, and UREA content in the HI8 group as compared to the C group. These results indicated enhance digestion and absorption of protein and fat in the weaned piglets on supplementation of Hermetia illucens larvae powder to the feed.
Effects of Hermetia illucens powder on the composition and diversity of fecal microorganisms in weaned piglets
The bacterial community was confirmed by amplifying and sequencing the 16S rRNA gene (V3-plus-V4 region). 16S rRNA Illumina MiSeq sequencing revealed the microbial composition in the feces of the piglets treated by Hermetia illucens larvae. In this study, 65863 effective sequences from 24 samples were screened for subsequent analysis, with an average of 482 operation taxonomic units (OTUs) per sample in fecal. As illustrated in Fig. 1A, there was no significant difference in Shannon, Ace, Chao1, Simpson. However, the PCoA with the binary-Jaccard distance results validated that the HI4 group and HI8 group was separate from the C group (Fig. 1B). The outcome of the analysis between the groups using the PERMANOVA test revealed that R2 = 0.17, P = 0.001.
At the phylum level, Firmicutes and Bacteroidetes are two advantageous categories, the contents of Firmicutes in the C group, HI4 group, and HI8 group were 67.21%, 69.34%, and 70.92%, respectively. The contents of Bacteroidetes in the C group, HI4 group, and HI8 group were 26.55%, 23.94%, and 23.07%, respectively. The next two most dominant phyla, Proteobacteria and Actinobacteria, accounted for 2.41% and 2.63% in the C group, 1.84% and 3.52% in the HI4 group, and 1.68% and 2.48% in the HI8 group, respectively.
At the family level, the content of Lactobacillaceae in the HI4 group and HI8 group was higher than that in the C group; the most significant difference was reflected between HI8 and C groups (P < 0.05). On the other hand, significantly lower content of both Streptococcus (P < 0.05) and Staphylococcaceae (P < 0.01) was evident in the HI4 group and HI8 group than that in the C group (Fig. 2).
At the genus level, the ten most predominant genera were Lactobacillus, Megasphaera, Prevotella, uncultured_Muribaculaceae, Streptococcus, Agathobacter, Subdoligranulum, Prevotella_7, uncultured_Prevotellaceae, uncultured_Veillonellaceae. The levels of Lactobacillus in the HI4 group and HI8 group were higher than those in the C group, whereas the levels of Streptococcus were significantly lower than those in the C group (P < 0.05). The relative abundance of 16S rRNA gene sequence in pig feces was represented in the supplementary figure (Additional file 1:Figure S1), and the heat map portrayed the 20 species of bacteria most dominant at the genus level in the feces.
Effect of Hermetia illucens larvae powder in feed on diarrhea rate of weaned piglets after oral administration of ETEC K88
After inoculating piglets with ETEC K88, each group manifested different rates of diarrhea. As depicted in Table 6, the diarrhea rate was 50% in the C+K88 group, 37.5% in the HI4+K88 group, and 25% in the HI8+K88 group. This indicates that feed supplemented with Hermetia illucens larvae powder can improve the resistance of weaned piglets to ETEC K88 and diminish the diarrhea rate. The presence of antibacterial peptides and chitosan in Hermetia illucens larvae powder ameliorates the resistance of weaned piglets to disease.
Effect of Hermetia illucens meal on ileum Morphology in Piglets Challenged with ETEC K88
The morphology of ileum is sketched in Fig. 3 and Table 7. We made paraffin sections of the ileum and found that the intestinal morphological integrity of the HI4+K88 and HI8+K88 groups was superior to that of the C group under the same concentration of ETEC K88. As observed in Fig. 3, the ileum villi in the C+K88 group witnessed the most severe damage with shortened and blunt, deep crypts. However, the integrity of ileum villi in the HI4+K88 and HI8+K88 groups was significantly different from that in the C+K88 group. As described in Table 7, comparing the Vh (villus height) and Cd (Crypt depth) of the ileum in the C+K88 group, the Vh was found to be significantly increased in HI4+K88 and HI8+K88 groups (P < 0.05), whereas there was no significant difference in the Cd. Moreover, the villus height to crypt depth ratios in the ileum in the HI4+K88 and HI8+K88 groups were significantly higher than that in the C+K88 group. These findings signify that improved resistance to pathogenic bacteria and protection to intestinal health may be attributed to the early feeding of Hermetia illucens larvae powder.
The challenge with ETEC affects the expression of ion transporters and aquaporins in the ileum mucosa of piglets
NHE3 is mainly distributed in villous epithelial cells. As substantiated in Fig. 4A, the mRNA expression of NHE3 and CFTR was increased significantly in the ileum mucosa of the ETEC K88 challenged piglets belonging to the HI4+K88 and HI8+K88 groups (P < 0.05) as compared to the C+K88 group. Among these, the difference in the HI4+K88 group was the most significant.
Abundantly expression of AQP1 and AQP3 are known in piglet gastrointestinal tract and epithelial cells. The results of AQP1 and AQP3 are demonstrated in Fig. 4A. Compared with the C+K88 group, the expression of AQP1 and AQP3 in the HI4+K88 group was significantly different (P < 0.05); however, the HI8+K88 group was insignificant. Nevertheless, there is no difference between the three groups of AQP7, AQP9, and NKCCL.
The challenge of ETEC affects Antioxidant enzyme activity and immunoglobulin content
We estimated the activities of peroxidase and catalase in serum on day 32 of the experiment. As shown in Fig. 4B, the CAT activity of the HI4+K88 and HI8+K88 groups significantly differed from the C+K88 group (P < 0.05). The result of POD activity was parallel to that of CAT; both the Hermetia illucens larvae addition group was significantly higher than the control group (P < 0.05). Compared with the C group, the IgG and IgA concentrations of the HI4+K88 group exhibited significant differences (P < 0.05). Though there was no significant difference in the HI8+K88 group, there was a tendency to increase.
Gene expression of inflammatory factors in jejunum mucosa, ileum mucosa, and colon mucosa
To evaluate the impact of feeding Hermetia illucens larvae on the intestinal immune function, we elucidated the mRNA expression of several inflammatory factors and tight junction proteins. After inoculation with ETEC K88, significant down-regulation of the mRNA expression of the pro-inflammatory cytokine TNF-α in the jejunal mucosa, whereas significant up-regulation of the expression of the anti-inflammatory cytokine IL-10 in the jejunal mucosa (P < 0.05) was reported for the HI4+K88 group compared to the C+K88 group (Fig. 4C). However, only the expression level of IL-10 in the HI8+K88 group witnessed a significant increase than that in the C+K88 group (P < 0.05), and there was no difference in the others in the jejunal mucosa. In the ileum mucosa, the Hermetia illucens supplemented group experienced increased expression of inflammatory cytokine IL-10 (P < 0.05), especially in the 8% addition group. There is no difference in the expression of IL-8, TNF-α, and IFN-γ among the three groups in the ileum. In the colon mucosa, the HI4+K88 group demonstrated better advantages, such as significantly increased expression of anti-inflammatory cytokine IL-10 (P < 0.01), whereas significantly decreased pro-inflammatory cytokine TNF-α expression (P < 0.05). On the other hand, the expression levels of IL-10 and TNF-α in the HI8+K88 group were significantly different from those in the C+K88 group (P < 0.05). No significant difference in the expression of IL-8 and IFN-γ among the three groups was noted.
The effect of a diet supplemented with Hermetia illucens larvae on the mRNA expression and protein of barrier function genes in jejunum mucosa, ileum mucosa, and colon mucosa
As evident in Fig. 5A, a significant increase in the expression of Occludin and Claudin-3 in the jejunum mucosa (P < 0.05) of the HI4+K88 group compared with the C+K88 group was observed. However, in the HI8+K88 group, the expression level of only Occludin was found to be significantly higher than that in the C+K88 group (P < 0.05). The expression levels of tight junction proteins Occludin, ZO-1, and Claudin-3 in the ileum mucosa of the HI4+K88 group manifested a significant increase than those in the C+K88 group (P < 0.05) (Fig. 5B). The Occludin and Claudin-3 expressions were significantly increased in the HI8+K88 group when compared with that in the C+K88 group (P < 0.05) (Fig. 5B). Results from the colon mucosa revealed a significant increase (P < 0.05) in the tight junction proteins Occludin, Claudin-3, and ZO-1 in the HI4+K88 group, whereas, a significant increase (P < 0.05) in only Occludin in the HI8+K88 group, in comparison to the C+K88 group (Fig 5C).
The results of western blotting substantiated the results of fluorescence quantification. There were significant differences in Claudin-3 and Occludin in jejunum mucosa, ileum mucosa, and colon mucosa in the HI4+K88 group and HI8+K88 group compared with the C+K88 group (P < 0.05). This indicates that the early feeding of Hermetia illucens larvae powder feed may protect intestinal health by stimulating the expression of tight junction proteins in the mucosa when invaded by pathogenic bacteria.