|Other Abstract||With the increase of breeding density, the cultivation of sea cucumber faces the trouble of bacterial infectious diseases. It is necessary to search for a green and safe way to improve the intestinal ecological health and promote the healthy cultivation of sea cucumber Apostichopus japonicus. Therefore, in this dissertation, the Jerusalem artichoke powder (JAP) was added to the microbial solid-state fermentation feed. JAP is rich in inulin and can be used as a prebiotic ingredient. In this study, the fermented feed with JAP was applied to the artificial culture of Apostichopus japonicus. The purpose of this test is to investigate the effects of Jerusalem artichoke powder and fermented feed on the gut microbiota, intestinal short-chain fatty acid content, intestinal morphology and structure, digestive enzymes, non-specific immunity, growth and body nutritional composition of Apostichopus japonicus, and to study the synergistic effects of them. The main research contents and conclusions of the dissertation are as follows:
1. The Saccharomyces cerevisiae, Bacillus subtilis, and Lactobacillus Bulgaricus were cultured respectively and mixed in proportion to obtain compound probiotics (8.75×109 CFU/mL). The crude powder of basal diet was cultured by solid-state fermentation with probiotics, and the nutritional components were measured before and after fermentation. The results showed that the crude protein content was increased, the crude fiber content was reduced, and the crude fat content did not change significantly. The nutrient composition ratio of fermented feed was better than that of basal diet, which was beneficial to the growth of Apostichopus japonicus.
2. In this work, 240 sea cucumbers were randomly divided into 4 groups, each group had 6 replicates, and each replicate had 10 sea cucumbers. The groups were as follows: Control group (CT group, basal diet), Fermented Feed group (FF group), JAP Basic Feed group (IBF group, 1.5% JAP), and JAP Fermented Feed group (IFF group, 1.5% JAP). After feeding for 60 days, the diversity of intestinal flora, the content of short-chain fatty acids (SCFAs) in intestinal contents, the morphology of intestinal tissues, the activities of digestive enzymes, immunity, growth performance, and body wall nutritional composition of Apostichopus japonicus were measured.
(1) The effect of fermented feed with JAP on the gut microbiota and intestinal short-chain fatty acid content: Compared with the control group, the diversity and richness of intestinal microflora in the experimental groups were significantly increased (P<0.05), and the intestinal microorganisms of the IFF group with the control group were more homologous. At the phylum level, compared with the control group, the abundance of Proteobacteria was reduced significantly and the abundance of Firmicutes was increased significantly in the FF group and IFF group (P<0.05). Meanwhile, the abundance of Proteobacteria and Verrucomicrobiota was significantly reduced in the IBF group (P<0.05). At the order level, the abundance of Rhodobacterales was significantly reduced in the IBF group and IFF group (P<0.05) but the abundance of Flavobacteriales was increased, and the abundance of Staphylococcales and Bacillales was significantly increased in the IFF group (P<0.05). In addition, compared with the control group, the total SCFAs contents were increased in all the experimental groups. The content of acetic acid was significantly increased in the IBF group (P<0.05), the contents of butyric acid were increased in all the experimental groups, but there was no significant difference (P>0.05).
(2) The effect of fermented feed with JAP on the morphology of intestinal tissues and the activities of digestive enzymes: Compared with the control group, the intestinal villi height and microvilli number in the FF group were increased. In the IBF group, the width of foregut villi was reduced, the number of midgut microvilli was increased, and the thickness of intestinal muscle layer was increased. The height and number of intestinal villi were increased in the IFF group. The feed of experimental groups all contributed to varying degrees to promoting the development of intestinal villi and the improvement of muscle layer thickness. In addition, the activities of protease, amylase, lipase, and cellulase in the FF group were significantly increased (P<0.05). The feed of IBF group had no significant effect on the improvement of digestive enzyme activity (P>0.05). In the IFF group, the activities of protease, cellulase, and lipase were significantly increased (P<0.05).
(3) The effect of fermented feed with JAP on immunity and the correlation analysis between immune enzyme activities and intestinal flora: Compared with the control group, the feed of FF and IFF groups significantly increased the activity of phenol oxidase (PO), but significantly reduced the activity of nitric oxide synthase (NOS) (P<0.05). The total number of coelomic cells (TCC) and phagocytic activity were significantly increased in the IBF and IFF groups (P<0.05). The activities of acid phosphatase (ACP) and alkaline phosphatase (AKP) in the IFF group were also significantly increased (P<0.05). The feed of experimental groups had no significant effect on superoxide dismutase (SOD) activity (P>0.05). The correlation analysis between immune enzymes activities and antioxidant enzymes activities with intestinal flora showed that NOS activity was negatively correlated with the abundance of Pseudoruegeria, Bacillaceae, Actibacter, and Staphylococcus and positively correlated with the abundance of Ruegeria, Halocynthiibacter, and Vermiphilaceae (P<0.05). The activities of SOD and PO were positively correlated with the abundance of Tropicibacter and Pseudoruegeria, respectively (P<0.05). However, ACP and AKP activities were negatively correlated with Halocynthiibacter (P<0.05).
(4) The effect of fermented feed with JAP on growth performance and body wall nutritional composition: In terms of growth performance, compared with the control group, the feed of experimental groups all significantly increased the weight gain rate (WGR) and special growth rate (SGR), reduced the feed coefficient (FC) (P<0.05), and improved the feed utilization rate of Apostichopus japonicus. In the large-scale feeding trial, the fermented feed with JAP increased WGR significantly (P<0.05). In terms of body nutritional composition, the feed of experimental groups had no significant effect on moisture and ash content (P>0.05) but increased the level of crude protein to a certain extent, and significantly reduced the content of crude lipid (P<0.05).
The results of this study showed that the fermented feed with JAP can improve the intestinal microflora, promote the proliferation of probiotics such as Bacillus and increase the intestinal short-chain fatty acid content. At the same time, it can promote the development of intestinal morphology and digestive enzyme activities, improve immunity and growth performance and improve the quality of Apostichopus japonicus. Moreover, the combination uses of 1.5% JAP and fermented feed showed better synergistic effects. This study provides a data support for the application of the fermented feed with JAP in the cultivation of sea cucumbers.|