The Regulation Of Lipid Metabolism And Immunity Of Zebrafish By Dietary Supplemented Nucleotides And The Associated Mechanisms

Nucleotides(NT)are important low molecular compounds in organisms,and nucleotides and related metabolites play key roles in many biological processes.Nowadays aquaculture develops rapidly,but the problem of antibiotic abuse is increasingly prominent,which seriously threatens the safety of aquaculture.In the process of aquaculture,we should pay attention to not only the nutritional needs of aquatic animals,but also the effect of feed on the health of aquaculture animals.In recent years,the addition of nutrients with growth-promoting and immuno-regulating functions to feed has gradually become an important strategy to replace antibiotics.By adding exogenous nucleotides into the feed,the growth performance,immunity and disease resistance of aquatic animals have been reported to be improved.In this paper,we investigated the regulation of exogenous nucleotides on the lipid metabolism and innate immunity of zebrafish,which may provide more evidence for the application of nucleotides in animal production.In the first part of this experiment,we first studied the effect of adding 0.1% mixed nucleotides on the growth and lipid metabolism of zebrafish.The 0.1% nucleotides improved growth performance of zebrafish comprared with control group.Moreover,addition of 0.1% mixed nucleotides could reduce liver fat and increase muscle fat,as indicated by both TAG content test and tissue HE analysis.We also tested the effect of nucleotides on zebrafish larvae.After feeding 0.1% complex nucleotide,the fish larvae were weighed and stained with oil red.The results showed that the liver fat of the fish larvae was significantly reduced in the 0.1% nucleotides group versus control,while the final body weight was significantly increased,which was consistent with the results in adult zebrafish.We further investigated the functions of mono-nucleotides by adding 0.1% AMP,CMP,GMP,UMP or IMP,we found that AMP was more beneficial compared with other mononucleotide in terms of growth and liver TAG content.Therefore,we further explored the mechanism of the effect of AMP on the lipid metabolism of zebrafish.We firstly investigated the dose effect of AMP and observed that 0.02%AMP was the optimal dose.Therefore,we conducted further mechanistic study at this dose.By HE analysis,expression analysis of lipid metabolism related genes and AMP content detection,and through the Western blot detection of AMPK phosphorylation,we propose that the AMP activates AMPK phosphorylation,which stimulates the oxidation of fatty acid and therefore reduces the liver fat content.In the second part,we studied the effect and exogenous nucleotide addition on the disease resistance and innate immunity of zebrafish.Firstly we challenged nucleotides-fed and control zebrafish with A.hydrophila NJ-1,and observed that 0.1% NT enhanced disease resistance of zebrafish.Further study indicated that 0.1% NT supplementation increased the intestinal expression of mucin,tight junction proteins,antibacterial peptides,and Mpo and Saa(P < 0.05),suggesting that the nucleotides enhanced the physical barrier and intestinal mucosal immunity of zebrafish.Meanwhile,ROS levels in the head kidney were significantly increased by NT supplementation,suggesting enhancement of the systematic immunity.In order to study the role of intestinal microbiota in the immuno-regulation effect of nucleotides,germ-free(GF)zebrafish and microbiota transfer technique were used.GF zebrafish colonized with microbiotas from control and NT-fed zebrafish showed no significant difference in the relative expression of Mpo,Saa and ROS activities,and no difference in disease resistance was observed between the two groups.Moreover,the relative expression of mpo,saa and ROS activity in GF zebrafish directly fed with 0.1% NT were significantly increased,and the disease resistance of GF zebrafish fed NT was increased compared with control.Therefore,NT can directly stimulate the innate immune response of fish independent of the intestinal microbiota.Also,the protective effect of NT against pathogen infection in fish was also mediated by direct effect.In summary,we investigated the regulation of dietary nucleotides on lipid metabolism of zebrafish.We found the role of AMP in regulating lipid metabolism,and investigated the associated mechanisms.In addition,we investigated the immuno-regulatory effect of dietary nucleotides in zebrafish,and found that the immuno-stimulatory effect of nucleotides is mediated by direct effect of nucleotides independent of the intestinal microbiota.