Effect Of Bile Acid,Mulberry Leaf And Perilla Oil On Improving Liver Damage Induced By High-starch Diet In Largemouth Bass (Micropterus Salmoides)

Starch is widely used in aquatic feed because of its cheapness,feed binding and protein economizing.However,fish have lower ability to make use of the dietary starch.In the practice of aquaculture production,excessive intake of carbohydrates will cause liver damage,hyperglycemia,and decreased disease resistance in fish,especially carnivorous fish,which will affect the growth of fish.How to maintain fish liver health is the key to solving the problem of aquaculture.Therefore,the study designed three experiments,taking largemouth bass（Micropterus salmoides）as the research object to explore the effects of adding bile acid,mulberry leaf and perilla oil respectively on the growth performance,plasma biochemistry and liver tissue morphology,liver antioxidant capacity and immunity in largemouth bass fed high-starch diet.To evaluate the effect of functional additives（bile acids,mulberry leaves and perilla oil）on the liver injury of largemouth bass induced by high starch feed,also to provide valuable reference for the screening and development of therapeutic drugs for liver disease.1.Effect of supplemental bile acid on liver damage induced by high-starch diet in largemouth bass（Micropterus salmoides）Corn starch as the starch source,the experiment set three isonitrogenous and isoenergetisemi-purified diets,low-starch diet（5%starch level）,high-starch diet（15%starch level）and high-starch diet with bile acids（300 mg/kg diet）.Largemouth bass with an average initial weight 10.14±0.07g was selected and reared for 60 days.The experimental results showed that growth performance of largemouth bass in the high starch group was inhibited.Its specific of growth rate（SGR）and protein in efficiency（PER）were significantly declined than the bile acid treatment and the low starch treatment（P<0.05）.Compared with the high starch treatment,the addition of bile acid reduced the liver fat content and the liver glycogen content,and the hepatopancreas somatic index（HSI）was significantly reduced（P<0.05）.At the same time,the activities of alanine aminotransferase（ALT）,aspartate aminotransferase（AST）and alkaline phosphatase（ALP）and the glucose content in the plasma of largemouth bass in the bile acid treatment were significantly lower than the high starch treatment（P<0.05）.The results of liver tissue morphology showed that the morphology of hepatocytes in the high starch group was severely damaged and the cell structure was blurred.The vacuolization symptom of liver tissue was significantly reduced in bile acid treatment.The supplemental bile acids can significantly enhance the relative expression and enzyme activity of catalase（CAT）in the liver,while also significantly reducing the concentration of malondialdehyde（MDA）（P<0.05）.The relative expression of liver pro-inflammatory cytokines（NF-κB,TNF-α,IL-8）in the bile acid group was significantly lower than the high starch group（P<0.05）,and the relative expression of the anti-inflammatory cytokine interleukin 10（IL-10）was significantly higher than the high starch group（P<0.05）.In addition,the supplemental bile acids significantly up-regulated the expression of heterodimer chaperone（SHP）,and significantly down-regulated hepatocyte nuclear factor 4α（HNF4α）（P<0.05）,and the key genes of gluconeogenesis,phosphoenolpyruvate carboxykinase（PEPCK）and glucose-6-phosphatase（G6Pase）expression level（P<0.05）.In summary,the supplemental bile acids in high starch diet can ameliorate liver health,and inhibit the expression of hepatocyte nuclear factor 4α（HNF4α）through heterodimer chaperone（SHP）,thereby inhibiting gluconeogenesis to alleviate stress response and liver damage induced by high-starch diet,accordingly improving its growth performance.2.Effect of supplemental mulberry leaf（Morus alba L.）on liver damage induced by high-starch diet in largemouth bass（Micropterus salmoides）Corn starch as the starch source,the experiment set three isonitrogenous and isoenergetisemi-purified diets,low-starch diet（5%starch level）,high-starch diet（15%starch level）and high-starch diet with mulberry leaf（50g/kg diet）.Largemouth bass with an average initial weight 10.12±0.06g was selected and reared for 60 days.The experimental results showed that the low starch group had the highest SGR,followed by the mulberry leaf group,and the high starch group had the lowest SGR（P<0.05）.The PER of the mulberry of leaf group was significantly higher of than the high starch group,while the HSI,liver fat and liver glycogen content were significantly lower than the high starch group（P<0.05）.Simultaneously,the suppressed growth accompanied by liver damage was found in fish fed the high starch diet compared to the low starch group.However,supplementation with mulberry leaf diet significantly reduced plasma glucose,TG and TC content,and weakened the activity of ALT,AST and ALP（P<0.05）.The liver histological investigation showed the hepatocyte nucleus had migrated and severely vacuolated in the high starch group,while an improvement of liver histostructure in the mulberry leaf group.Furthermore,hepatic SOD and CAT were significantly up-regulated by mulberry leaf supplementation（P<0.05）.Meanwhile,the superoxide dismutase（SOD）and catalase（CAT）activity of mulberry leaf group were significantly increased,while significantly reduced malondialdehyde（MDA）concentration in liver compared with the high starch group（P<0.05）.In addition,mulberry leaf supplementation significantly down-regulated the expression in levels of N_iF-κ_nB,TN_nF-αand I_nL-8 in the liver（P<0.05）.It can be seen that mulberry leaves can alleviate hepatic injuries induced by regulating inflammation and oxidative stress,supplying a theoretical foundation for the evolution of mulberry leaf as a therapeutic drug for liver disease.3.Effect of supplemental perilla oil on liver damage induced by high-starch diet in largemouth bass（Micropterus salmoides）Corn starch as the starch source,the experiment set three isonitrogenous and isoenergetisemi-purified diets,low-starch diet（5%starch level）,high-starch diet（15%starch level）and high-starch diet with perilla oil（0.1%）.Largemouth bass with an average in initial weight of 10.11±0.08g was selected and reared for 60 days.The experimental results indicated that the specific growth rate of the perilla oil group was of significantly high_er than the high starch group（P<0.05）.The HSI,_inliver fat and liver glycogen content of the high starch treatment and the perilla oil treatment were significantly increased than the low starch treatment（P<0.05）.However,the supplemental perilla oil could significantly reduce the activity of ALT and AST in plasma（P<0.05）.while the activity of ALP and the content of glucose and TC in the plasma were not significantly different from the high starch group（P>0.05）.It can be observed from the liver tissue slices that the degree of hepatocyte vacuolation and the number of ballooning changes in the perilla oil group was significantly reduced compared with the high starch group.In addition,supplementation with perilla oil could significantly increase the relative expression and the enzyme activity of SOD and CAT in the liver,and significantly reduced the MDA content（P<0.05）.Moreover,compared with the low starch group,the relative expression of IL-8 was significantly up-regulated and the relative expression of IL-10 and TGF-βin the liver were significantly reduced in the high starch treatment（P<0.05）,while the addition of perilla oil could inhibited the in expression of IL-8 in liver.In summary,perilla oil can relieve the high starch-induced liver damage by enhancing the antioxidant capacity of the liver and reducing inflammation in largemouth bass.