Probiotics Alleviate Metabolic Disturbance And Oxidative Stress Induced By High Carbohydrate Diet In Nile Tilapia

With the increasing demand of animal proteins,the sustainable and rapid development of aquaculture industry has been paid more and more attention.Carbohydrates,proteins,and fats are the main components of fish feeds,and carbohydrates are the most abundant energy sources in nature.Supplementing with an appropriate level of carbohydrates in the diets can promote the growth performance of fish and improve feed utilization efficiency which will contribute to the protein sparing effect in aquaculture.However,fish are naturally glucose intolerant,and they have lower utilization efficiency of carbohydrates than mammals.Long term high-carbohydrate diet(HCD)feeding usually impairs glucose homeostasis and leads to liver diseases and metabolic disorders in fish.Long term of feeding with HCD will also induce the inflammatory response and oxidative stress,which will damage the health of fish.Therefore,how to alleviate the metabolic disorders and oxidative stress caused by HCD should be paid more attention.Probiotics play important roles in regulation metabolism and maintenance health of host.However,it is unclear whether probiotics could alleviate metabolic disorders,inflammatory response and oxidative stress of fish induced by HCD.Probiotics isolated from the gut of host will be more safe,effective,and easier to be colonized in vivo.In this study,two strains were isoalted from the intestine of healthy Nile Tilapia.One of them was designated as Bacillus amyloliquefaciens SS1(B.amyloliquefaciens SS1)which belongs to Bacillus,and the other one was designated as Lactobacillus plantarum MR1(L.plantarum MR1)which belongs to Lactobacilli.Both strains could fement carbohydrate to produce SCFAs in vitro.In the present study,the diet containing 30%of corn starch was used as the normal control group(CON)and the diet containing 45%of corn starch was used as a high-carbohydrate model group(HCD).These two strains were added to the HCD to feed Nile tilapia(Oreochromis niloticus)for 10 weeks,respectively.The results showed that the two strains had different influences on fish.B.amyloliquefaciens SS1 allievated HCD-induced metabolic disorders in Nile tilapia,and L.plantarum MR1 obviously protected HCD-fed fish from oxidative stress.Therefore,we explored the possible mechanisms of these two strains.In this study,the addition of B.amyloliquefaciens SS1 to HCD can effectively promote the weight gain rate and increase the proportion of body protein accumulation.The increased production of amylase by B.amyloliquefaciens SS1 might account for the promotion of feed efficiency and growth performance of Nile tilapia.In addition,the fasting blood glucose level and lipid deposition were significantly reduced,and the glucose tolerance was markedly improved in B.amyloliquefaciens SS1 treatment group.To further explore the regulation effects of B.amyloliquefaciens SS1 administration on intestinal microbiota,the high-throughput sequencing analysis of 16S r RNA were performed.The results showed that B.amyloliquefaciens SS1 could alter the composition of intestinal microbiota and adjust it to be more similar with the control group.Further analysis showed that the abundance of SCFAs-producing bacteria increased significantly in B.amyloliquefaciens SS1-treated fish.Through the gas chromatography analysis,it was observed that the addition of B.amyloliquefaciens SS1could significantly increase the concentration of intestinal acetate which obviously upregulated the gene expression of free fatty acid receptor 2(ffar2)and increased the content of glucagon-like peptide-1(GLP-1)in the intestine and serum,thereby maintaining the metabolic homeostasis.In order to explore whether acetate was the effective molecule of B.amyloliquefaciens SS1 to exert the beneficial metabolic effects,different concentrations of sodium acetate were added to HCD,including 0.9 g/kg(HLA),1.8 g/kg(HMA)or 3.6 g/kg(HHA),to feed Nile tilapia for 8 weeks.The results showed that both HMA and HHA groups could mirror the effects of B.amyloliquefaciens SS1 in improving glucose tolerance and reducing liver lipid deposition.In conclusion,this part of study found that the addition of B.amyloliquefaciens SS1 can alleviate HCD-induced metabolic disorders by increasing the abundance of acetate-producing bacteria and the content of acetate in the intestine of Nile tilapia,suggesting that regulating intestinal microbiota and microbial metabolites may be a powerful strategy to balance fish nutritional metabolism and maintain fish health homeostasis in the future.In addition,excessive intake of HCD could induce the oxidative stress and fatty liver diseases.Regulating gut microbiota has the potential to alleviate oxidative stress and reduce lipid deposition caused by HCD,however,the potential mechanisms were not clear.In the present study,the addition of L.plantarum MR1 to HCD could significantly reduce lipid deposition,oxidative stress,and inflammatory response in the liver of Nile Tilapia.Further analysis showed that colonizing with L.plantarum MR1significantly changed the composition of intestinal microbiota and increased the concentration of intestinal acetate in the fish.The results of serum non-targeted metabolome analysis showed that the metabolic pathway with significant changes among groups was pyrimidine nucleotide metabolism,and the content of serum uridine in L.plantarum MR1 treatment group was significantly increased.It has been reported that acetyl coenzyme A(Acetyl-CoA)derived from fatty acid carbon promotes the de nove nucleotides synthesis.HPLC analysis showed that the content of Acetyl-CoA in the liver was obviously increased in L.plantarum MR1 treatment group,indicating that acetate derived from intestinal microbiota might be metabolized into Acetyl-CoA,which promoted the uridine synthesis.Sodium acetate was added to the primary hepatocytes of Nile tilapia and higher uridine content was detected.In order to verify that uridine is one of the effector molecules to reduce lipid deposition and attenuate oxidative stress in the liver of Nile Tilapia,uridine was added to the oleic acid(OA)or hydrogen peroxide(H₂O₂)induced-oxidative damage hepatocytes models.The results showed that uridine could reduce lipid droplets accumulation and directly alleviate oxidative stress in the hepatocytes.In summary,this part of study revealed that the administration of L.plantarum MR1 can decrease HCD-induced liver lipid deposition or oxidative stress by increasing the content of circulating uridine,indicating that intestinal microbiota can regulate nucleotides metabolism and maintain physiological homeostasis of the host.In this study,the effects of both probiotics are associated with the microbial metabolite acetate.B.amyloliquefaciens SS1 regulated intestinal microbiota to produce acetate,which acted as a signal molecule to maintain the metabolic homeostasis in HCD-fed Nile tilapia by promoting secretion of GLP-1.While,L.plantarum MR1derived acetate acted as a substrate for uridine synthesis to alleviate HCD-induced lipid deposition and oxidative stress in Nile tilapia.The present study illustrated that bacteria-derived acetate could act as a signal molecule or act as the substrate to synthesize other matabolites,but the underlying mechanism by which host regulted the destiny of aceate still need further investigation.