| In the process of aquaculture,fish often suffer from starvation resulting in reduced growth,energy reserves,behavioral capacity and metabolic capacity due to various factors such as environmental changes,seasonal alternation,uneven food distribution,inter-species competition,and ecological damage.The effective way to solve this problem is to analyze the metabolic rules of fish organism under starvation conditions and formulate the best feeding strategy.Existing studies have demonstrated the effectiveness of probiotics in improving the metabolism of fish organisms,but their effects on the repair of fish organism functions after starvation still need to be studied in depth.Therefore,in this study,the irreversible time point of starvation damage in juvenile carp(Cyprinus carpio)was firstly determined by 28 days of starvation stress using Bacillus subtilis LSG2-1 as the test strain.The effect of Bacillus subtilis LSG2-1 on the re-feeding of juvenile carp was examined by feeding them diets with different concentrations of B.subtilis LSG2-1(NF:normal feeding group,Control:0 CFU/g,B6:1.0×10~6 CFU/g,B7:1.0×10~7 CFU/g and B8:1.0×10~8 CFU/g)for 4 weeks after 14 days of starvation stress.The effect of B.subtilis LSG2-1 on the growth performance,intestinal health,immune and antioxidant functions of juvenile carp after starvation and re-feeding was investigated.Finally,the mechanism of B.subtilis LSG2-1 in improving the metabolic functions of juvenile carp after starvation re-feeding was investigated by gut microbiomics and liver metabolomics analysis.This study aimed to provide theoretical reference and technical support for the optimization of starvation refeeding strategy in fish.The results of the related studies showed that:(1)Effects of starvation stress on the health of juvenile carpThe results showed that starvation treatment significantly(P<0.05)reduced the final body weight,weight gain rate,specific growth rate,liver-to-body ratio,organ-to-body ratio and fatness of juvenile carp and decreased survival rate at 21 and 28 days of starvation.Glucose,total protein,total cholesterol and triglyceride levels in the blood of juvenile carp were significantly reduced after starvation treatment(P<0.05).Intestinal and hepatic amylase activities were significantly increased(P<0.05)at 21 days of starvation and decreased(P<0.05)at 28 days of starvation;intestinal lipase and Na~+-K~+-ATPase activities were significantly increased(P<0.05)at 14 days of starvation,and hepatic lipase and Na~+-K~+-ATPase activities were significantly decreased(P<0.05).Acid phosphatase activity in blood,intestinal and hepatic malondialdehyde,glutathione peroxidase and peroxidase activities tended to increase significantly(P<0.05)during 14 days of starvation and decreased significantly(P<0.05)when starved for more than 14 days.Intestinal fold width,lamina propria width and muscle layer thickness were significantly higher(P<0.05)during 14 days of starvation and significantly lower(P<0.05)when starvation exceeded 14 days.(2)Effect of B.subtilis LSG2-1 on growth performance and intestinal health of juvenile starved re-feeding carpThe results of the study showed that at 14 d of re-feeding after starvation,there were no significant changes in any of the growth indicators in the B.subtilis LSG2-1 treated groups compared to the control group(P>0.05).Glucose,total cholesterol and triglyceride levels in blood were significantly lower in all treatment groups relative to the control group(P<0.05),while total protein levels did not change significantly(P>0.05).The muscle triglyceride content was significantly higher in the B7 and B8 groups(P<0.05),and myoglycogen,hepatic glycogen and triglyceride content were significantly lower in all treatment groups relative to the control group(P<0.05).The activities of digestive and absorptive enzymes(lipase,trypsin,γ-glutamyltransferase,Na~+-K~+-ATPase)in the carp intestine were significantly higher(P<0.05)after the addition of B.subtilis LSG2-1 compared to the control group.The intestinal tissue structure(fold height,fold width and muscle layer thickness)was significantly higher in each treatment group relative to the control group(P<0.05).While glucose and total cholesterol levels were elevated in the control group compared to the normal feeding group(NF group),there were no significant changes in total protein and total cholesterol levels in each treatment group.The intestinal tissue structure(fold height,fold width and muscle layer thickness)was decreased in the control group compared to the normal feeding group,but the addition of B.subtilis LSG2-1 to the feed increased the intestinal tissue structure in the treated group,even higher than the normal feeding group.At 28 d of re-feeding after starvation,the liver-to-body ratio was significantly lower in each treatment group relative to the control group(P<0.05)and returned to normal feeding levels.Blood species glucose,total cholesterol and triglyceride levels were significantly lower in the treated groups relative to the control group(P<0.05),while total protein levels did not change significantly(P>0.05).Myoglycogen and liver triglyceride contents were significantly lower in the treated group compared to the control group(P<0.05).The activities of digestive and absorptive enzymes(lipase,trypsin,γ-glutamyl transferase,Na~+-K~+-ATPase)in the intestine of juvenile carp in the treated group were significantly higher(P<0.05)compared to the control group.The intestinal tissue structure(fold height,fold width and muscle layer thickness)was significantly higher in the treated group relative to the control group(P<0.05),and the length of foregut microvilli was significantly higher in the B6 group than in the control group(P<0.05).The relative expressions of brain NPY and AGRI-1 genes,liver AMPK gene,Sirt1 gene and PGC-1 gene in B6,B7 and B8 groups were significantly higher than those in the control group.were significantly higher relative to the control group(P<0.05).The relative expression of intestinal ZO-1,Occludin and Claudin-1 genes was significantly higher in each treatment group compared to the control group(P<0.05).Relative expression of NPY and AGRI-1 genes of brain tissue species and intestinal ZO-1,Occludin and Claudin-1 genes were decreased in the control group relative to the normal feeding group,whereas the expression of B.subtilis LSG2-1-treated group was increased to the normal feeding level or even higher than the normal feeding level.(3)Effect of B.subtilis LSG2-1 on immunity and antioxidant capacity of juvenile starved re-feeding carpThe results of the study showed that malondialdehyde(MDA)activity was significantly reduced in the liver and intestine of the B.subtilis LSG2-1-treated groups at 14 d of re-feeding after starvation relative to the control group(P<0.05).The addition of B.subtilis LSG2-1 did not cause significant changes in glutathione peroxidase(GSH-PX)and peroxidase(SOD)activities(P>0.05),while trace reduced glutathione(GSH)activities in the liver,foregut and midgut were significantly increased relative to the control group(P<0.05).The trace reduced glutathione(GSH)activity in the liver,foregut and midgut of the control group was decreased relative to the normal feeding group,whereas the trace reduced glutathione(GSH)activity in the B.subtilis LSG2-1 treated group was increased and higher than that of the normal feeding group.Complement C4,acid phosphatase(ACP)and alkaline phosphatase(AKP)activities in the blood of juvenile carp were significantly higher in the B.subtilis LSG2-1-treated group compared to the control group(P<0.05),with alkaline phosphatase(AKP)being higher than in the normal feeding group.Malondialdehyde(MDA)activity in liver and intestine was significantly lower in the B.subtilis LSG2-1-treated groups at 28 d of starvation compared to the control group(P<0.05).glutathione peroxidase(GSH-PX)activity in liver was significantly higher in the B6 group compared to the control group(P<0.05),and glutathione peroxidase(GSH-PX)activity in the foregut was significantly higher in the B7 and B8 groups compared to the control group(P<0.05).Peroxidase(SOD)and trace reduced glutathione(GSH)activities in the liver and intestine of juvenile carp in the three treatment groups were significantly higher(P<0.05)relative to the control group.Complement C4,acid phosphatase(ACP)and alkaline phosphatase(AKP)activities in the blood of juvenile carp in the treated group were significantly higher than those in the control group(P<0.05),and AKP activities in the B7 group were higher than those in the normal feeding group.Relative to the control group,the relative expression of IL-8 gene in liver,intestine and muscle was significantly lower in the treated group(P<0.05);the relative expression of IL-10 and Keap-1 gene in liver,intestine and muscle,and the relative expression of Nrf2 gene in intestine and muscle were significantly higher(P<0.05).The relative expressions of IL-8 genes in liver,intestine and muscle were elevated in the control group relative to the NF group,and the relative expressions of IL-8 genes in the foregut,midgut and hindgut of the B7 group and in the muscle of the B7 and B8 groups were lower than those of the NF group.The relative expression of IL-10 genes,Keap-1 and Nrf2 genes in liver,intestine and muscle were decreased in the control group relative to the NF group.The relative expression of IL-10 gene in liver,foregut and hindgut of B.subtilis LSG2-1 treated group was higher than that of normal feeding group.the relative expression of Keap-1 gene in foregut,midgut and hindgut of B7 and B8 groups was increased compared with normal feeding group.the relative expression of Nrf2 gene in foregut of B6 and B7 groups and in muscle of B7 group was significantly higher than that of NF group.(4)Effect of B.subtilis LSG2-1 on the gut microbiome and liver metabolome of juvenile starved refeeding carpThe results of the study showed that there was no significant difference between groups in the Alpha diversity index of carp gut microorganisms(P>0.05).At the phylum level,the dominant flora consisted mainly of Proteobacteria,Bacteroidota,Firmicutes,Cyanobacteria,and Fusobacteriota.The relative abundance of Proteobacteria and Cyanobacteria was increased and the relative abundance of Bacteroidota,Firmicutes and Fusobacteriota was decreased in the control group compared to the NF group with normal feeding.In contrast,Bacteroidota and Firmicutes increased and Cyanobacteria decreased in each treatment group of B.subtilis LSG2-1 relative to the control group.The relative abundance of Proteobacteria was higher in group B8 than in the control group,and the relative abundance of Fusobacteriota was higher in group B6 than in the control group.The genus level groups were mainly composed of Herminiimonas,Pseudomonas,Hafnia-Obesumbacterium,Gemmobacter,Aeromonas,and Chloroplast,and Cetobacterium.Compared to the NF group,the relative abundance of Gemmobacter and Chloroplast increased in the control group,while Aeromonas,Flavobacterium and Cetobacterium decreased.Compared with the control group,the relative abundance of Pseudomonas,Aeromonas and Flavobacterium increased in the B.subtilis LSG2-1-treated group,and the relative abundance of Chloroplast decreased in the B6 group.The relative abundance of Cetobacterium increased in group B6.The liver metabolomics results showed that the control group had 48 differential metabolites relative to the normal feeding group(NF group),of which 44 differential metabolites were upregulated and 4 differential metabolites were downregulated.After feeding B.subtilis LSG2-1 supplemented diets,there were 56 differential metabolites in the B6 group relative to the control group,of which 19 differential metabolites were up-regulated and 37differential metabolites were down-regulated;63 differential metabolites in the B7 group relative to the control group,of which 3 differential metabolites were up-regulated and 60differential metabolites were down-regulated;66 differential metabolites in the B8 group relative to the control group,of which 8 differential metabolites were up-regulated.There were66 differential metabolites in the B8 group relative to the control group,of which 8 differential metabolites were up-regulated and 58 differential metabolites were down-regulated.The differential metabolites mainly included lipids,nucleotides and organic acids.KEGG enrichment analysis of the detected differential metabolites showed that the differential metabolic pathways among the groups were mainly concentrated in lipid metabolism,amino acid metabolism and nucleotide metabolism.After starvation and re-feeding,juvenile carp in the control group promoted lipid metabolism by up-regulating lipid metabolites,causing lipid deposition in the liver,while juvenile carp in the treatment group improved lipid metabolism levels in the liver mainly by intervening in amino acid metabolism and nucleotide metabolism to maintain liver function and down-regulating lipid metabolites to inhibit lipid metabolism.After correlation analysis of the differential secondary metabolites obtained from metabolomic screening and significant differential genus-level flora obtained from 16S sequencing analysis,it was found that the metabolites that were significantly correlated with the flora in the intestine were mainly lipids.The control group was involved in glycerophospholipid metabolism through Lactobacillus and Coriobacteriaceae＿UCG-002 upregulated lipid metabolites Lyso PE 20:1 and Lyso PE 16:1,respectively,relative to the normal feeding group(NF group).In contrast,after the addition of B.subtilis LSG2-1 to the feed,the differential intestinal flora Streptococcus,Escherichia-Shigella,Pseudoalteromonas,Hydrophilus and Sphingomonas.in the B6 group intervened in hepatic lipid by upregulating the metabolites oleic acid,aspartic acid and 5-hydroxyindole-3-acetic acid and downregulating the metabolites inosine-5’-monophosphate adenosine and ethoxyquinoline compared to the control group Differential microorganisms in the gut of groups B7 and B8 regulated lipid metabolism mainly through down-regulation of glycerophospholipid metabolites.In summary,under the conditions of this experiment,re-feeding after starvation of juvenile carp can affect the metabolic level of fish,damage the intestinal health,reduce the immunity and antioxidant function of the body,and also promote liver lipid metabolism causing lipid deposition in the liver.In contrast,feeding B.subtilis LSG2-1 supplemented with 1.0×10~6～1.0×10~8 CFU/g of B.subtilis could regulate liver metabolism and enhance liver function by optimizing the structure of intestinal flora in juvenile carp after starvation re-feeding,thus promoting intestinal health,improving body immunity and antioxidant capacity,and improving growth performance. |
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