| With the development and promotion of artificial compound feed,the breeding area and scale of largemouth bass Micropterus salmoides(LMB)have expanded rapidly in recent years,which contribute to an rapid increase of its annual production.In 2020,the total production of LMB in China has exceeded 600,000 tons.Although the application of feed for the entire culture cycle of LMB is almost successful,it is very common that liver lesions of LMB are induced with the ingestion of compound feed in the middle and late stages of the culture cycle.Dietary starch level is the most important factor affecting the liver health of LMB.Liver injury will be induced in LMB when dietary starch level exceeds 10%,but the underlying mechanism is still unknown.Metabolic liver injury of LMB promoted the popularity of low starch diets in practice.However,the high protein level in low-starch diets not only increases the cost,but also might exacerbate the metabolic burden of the liver in LMB.Therefore,this thesis aimed at investigating the effects of dietary protein and starch levels on the growth,feed utilization,plasma(serum)biochemistry and liver health of LMB.Moreover,the potential mechanism of liver injury that was induced by high starch diet in LMB was ascertained by transcriptome analysis.The main results of this thesis are shown as follows:1.Effects of feeding low protein and high starch diet on growth,feed utilization,plasma biochemistry,hepatocyte inflammation and apoptosis of LMBThe present study was conducted to investigate the regulatory mechanism of liver injury in LMB fed low protein high starch diets.Two isolipidic and isoenergetic diets were formulated with different protein and starch ratios,being named as diets P49S9(48.8 % protein and 9.06 % starch)and P42S18(42.4 % protein and 18.2 % starch).Juvenile LMB with an initial weight of 4.65 g were randomly divided into 6 tanks containing a volume of 250 L with continuous aeration and filtration,and 3 replicates(25fish/tank)were set up for each treatment group.Fish in each tank were fed to apparent satiation for 8 weeks.The results indicated that though the P42S18 fish up-regulated the feeding ratio to meet their protein requirements,feeding efficiency ratio and growth performance were impaired in treatment P42S18 as compared to treatment P49S9.Periodic acid-Schiff(PAS)staining showed glycogen accumulated in the liver of LMB fed low protein high starch diets,and the reason should be attributed to down-regulated expression of the glycogenolytic glycogen debranching enzyme.Lower liver lipid level was associated with feeding low protein high starch diets in LMB,which should be resulted from the changes in hepatic glycerolipid metabolism regulated by lipoprotein lipase(representative of triglyceride synthesis,up-regulated)and diacylglycerol acyltransferase(representative of triglyceride breakdown,down-regulated).Though fasting plasma glucose level was comparable,treatment P42S18 performed inferior glucose tolerance to treatment P49S9.Hematoxylin-eosin(H&E)and Td T-mediated d UTP Nick-End Labeling(TUNEL)staining suggested that feeding low protein high starch diets induced disruption of structural integrity,inflammation and apoptosis in the hepatocytes of LMB.As expected,KEGG pathways analysis indicated that many of the up-regulated differentially expressed genes were enriched in AGE(advanced glycation end product)/RAGE(receptor for AGE),Toll-like receptor and apoptosis signaling pathways.Our transcriptome data revealed that feeding low protein high starch diets might promote the accumulation of AGEs in LMB,which bound to RAGE and subsequently induced PI3K/Akt signal pathway.The activation of Akt induced NF-κB translocation into the nucleus thus releasing proinflammatory factors including tumor necrosis factor-α(tnf-α)and interleukin-8(il-8).The release of these inflammatory factors concomitantly induced T cell stimulation and natural killer cells chemotactic effects through Toll-like receptor signaling pathway.Besides mediating inflammation and immune response,TNF-α signal transduction participated in mediating apoptosis through the receptor of TNF(TNF-R1)pathway by up-regulating the expression of cysteinyl aspartate specific proteinase 8(caspase 8)and cytochrome c(cytc).In conclusion,our results demonstrated that feeding low protein and high starch diets induced hepatocytes inflammation and apoptosis in LMB through the PI3K/Akt/NF-κB signaling pathway.2.Effects of dietary starch levels on growth,feed utilization,serum biochemistry and liver health of LMBThree diets containing different starch levels were formulated in this experiment,being designated as diets LC(low starch,8.13% starch),MC(medium starch,14.1%starch)and HC(high starch,20.1% starch).LMB juveniles with an initial body weight of6.37±0.07 g were randomly divided into 9 tanks(250 L each)with continuous aeration and filtration,and 3 replicates(20 fish/tank)were set up for each treatment.Fish were fed to apparent satiation for 8 weeks.The results showed that although the feed efficiency of treatment HC was improved,feeding ratio and growth performance were suppressed in treatment HC compared to treatment LC.With the increase of dietary starch level,glycogen concentration in the liver of LMB gradually increased,which might be the reason for the increase of hepatosomatic index in treatment HC.H&E,Masson and TUNEL staining showed that feeding high starch diets induced disruption of structural integrity,inflammation,apoptosis and fibrosis in the liver of LMB.KEGG enrichment analysis showed that DEGs were prominently enriched in the Apoptosis,Toll-like receptor,NOD-like receptor and Hedgehog signaling pathways.Transcriptome analysis showed that liver inflammation was mediated by the release of pro-inflammatory chemokine factor(interferon-inducible protein,ip-10)and pro-inflammatory factor(interleukin-8,il-8)through the PI3K/Akt/NF-κB signaling pathway.When apoptotic signals were produced in the organism,the Hedgehog signaling pathway was induced to activate the Gli A/Gli signaling axis,leading to an increase of extracellular matrix(ECM)(type I collagen,col1a1)proteins,which activated hepatic stellate cells and induced liver fibrosis.The apoptosis of hepatocytes in largemouth bass was mediated by caspase 8through the Fas-L/Fas/FADD signaling pathway.Moreover,the survival factor in the Apoptosis signaling pathway would reduce the release of Direct IAP binding protein with low p I(diablo)from the mitochondria through the activation of PI3K/Akt signaling pathway,thus reducing its inhibitory effects on the X-linked apoptosis inhibitory protein(xiap),which finally inhibited the apoptotic program of hepatocytes.This might be an autonomously apoptosis inhibitory program in the liver of LMB that were fed high starch diets,which deserves further investigations.3.Effects of dietary protein levels on growth,feed utilization,serum biochemistry and liver health of LMBThree diets containing different protein levels were formulated in this experiment,being designated as diets LP(low protein,44% protein),MP(medium protein,49%protein)and HP(high protein,54% protein),respectively.LMB juveniles with an initial body weight of 6.42±0.03 g were randomly divided into 12 tanks(250 L each)with continuous aeration and filtration,and 4 replicates(20 fish/tank)were set up for each treatment.Fish were fed to apparent satiation for 8 weeks.The results showed that dietary protein levels did not affect the growth performance and feed utilization of LMB.Liver protein levels gradually increased with an increase of dietary protein.The activities of aspartate transaminase(AST)and alanine aminotransferase(ALT)in the serum of LMB were lowest in treatment HP as compared with the other treatments.H&E and TUNEL staining showed that the ingestion of diet HP had a restorative effect on the integrity and apoptosis of the hepatocytes in LMB.At the molecular level,the expression of the proinflammatory interleukin-1β(il-1β)along with the pro-apoptotic bcl-2-Associated X protein(bax)and cysteinyl aspartate specific proteinase 7(caspase 7)in the liver of LMB decreased with an increase of dietary protein levels,and significant differences were found between treatments LP and HP.Taken together,the results in this study showed that although dietary protein levels did not affect the growth of LMB,the increase of dietary protein was beneficial for the liver health status of LMB. |
