Effects Of Dietary Arginine On Growth,Nutrient Metabolism And Immune Capacity Involved In Fuctional Mechanism In Juvenile Blunt Snout Bream,Megalobrama Amblycephala

An 8-week feeding trial was conducted to investigate the effects of dietary arginine levels on the growth performance,protein metabolism,glycolipid metabolismin and immune response involved in fuctional mechanism in juvenile blunt snout bream.The juvenile blunt snout bream(20.0±0.03g)were randomly sorted into eighteen cages with 20 fish in each cage for farm pond culture.Dietary protein was supplied by fish meal,rapeseed meal and corn gluten,soybean oil and lecithin as lipid sources.Six isonitrogenous and isoenergetic practical diets were formulated to contain graded arginine levels(0.87(control),1.22,1.62,1.96,2.31 and 2.70%of dry diet),which replaced by the same proportions of glycine.This study includes the following five aspects:1 Dietary arginine requirement of juvenile blunt snout breamThis trial was conducted to estimate the dietary arginine requirement in juvenile blunt snout bream.Fish were cultured in farm pond for 8 weeks.At the end of the feeding trial,results showed that final weight(FW),weight gain(WG),specific growth rate(SGR)and protein efficiency ratio(PER)increased with increasing dietary arginine level up to 1.62%,and thereafter showed a decreasing trend,while feed conversion ratio(FCR)showed a converse trend.The hepatosomatic index(HSI)in the fish fed with 2.31 and 2.70%arginine diets were significantly higher than the other arginine diets.Viscerosomatic index(VSI)in the fish fed with 1.62 and 1.96%arginine diets were significantly lower than 0.87,2.31 and 2.70%arginine diets,but condition factor(CF)was not significantly affected by the graded dietary arginine levels(P>0.05).Significantly higher whole body protein contents and significantly lower moisture contents of fish fed with 1.96%dietary arginine compared to those fed with the other diets(P<0.05).Fish fed with 1.62%and 1.96%arginine diets showed significantly higher body lipid compared to control group(P<0.05).Whole body ash of juvenile blunt snout bream was not significantly affected by the graded dietary arginine levels(P>0.05).Plasma arginine content increased with increasing dietary arginine level,whereas plasma lysine decreased with increasing dietary arginine level(P<0.05).Plasma histidine,isoleucine,valine,tryptophan and methionine contents were significantly affected by dietary arginine levels(P<0.05).Plasma threonine and leucine content showed relatively stable level(P>0.05).Based on FCR and SGR,the optimal dietary arginine level was determined to be 1.85%of diet(5.60%of dietary protein)and 1.84%of diet(5.58%of dietary protein),respectively,using quadratic regression analysis.2 Different dietary arginine affects the biosynthesis of protein and lipid in juvenile blunt snout breamThis study evaluated the mechanisms governing protein synthesis and lipogenesis in juvenile fish fed with graded levels of dietary arginine.The core genes of protein and fat synthesis in liver and serum parameters were analyzed.At the end of the feeding trial,results showed that,compared to control group,the relative expression levels of target of rapamycin(TOR)mRNA in the liver of fish fed with 1.22%-2.70%arginine diets were significantly improved(P<0.05),the highest expression levels shown in 1.96%arginine diet;the relative expression levels of ribosomal protein S6 kinase 1(S6K1)shown an upward tendency with increasing arginine levels,and significantly higher relative expression levels of S6K1 mRNA in liver was observed in fish fed with 1.96%-2.70%arginine diets(P<0.05);significantly higher relative expression levels of insulin-like growth factor 1(IGF-1)mRNA in the liver was observed in fish fed with 1.62%and 1.96%arginine diets(P<0.05);dietary arginine level didn’t affect significantly to the relative expression levels of eukaryotic initiation factor 4E binding protein 2(4E-BP2)mRNA in liver(P>0.05);the relative expression of fatty acid synthase(FAS)was significantly increased in the group fed with a dietary arginine level of 1.96%compared with that of the other groups(P<0.05);1.62%dietary arginine level significantly increased the levels of acetyl CoA carboxylase(ACC)and glucose-6-phosphate dehydrogenase(G6PDH)mRNA levels(P<0.05).Additionally,plasma triglyceride content(TG)was significantly increased in the groups fed with a dietary arginine level of 1.62%(P<0.05).Total cholesterol(TC)was not significantly affected by the graded levels of dietary arginine(P>0.05).3 Different dietary arginine affects glucose metabolism in juvenile blunt snout breamThis study evaluated the mechanisms governing insulin resistance and glucose metabolism in juvenile fish fed with graded levels of dietary arginine.The results showed that,compared with the control group(0.87%),2.31%dietary arginine level resulted in the upregulation of the relative gene expression of insulin receptor substrate 1(IRS-1),phosphoinositide 3-kinase(PI3K)and protein kinase B(Akt)in the insulin signaling pathway(P<0.05),while 2.70%dietary arginine level led to inhibition of these genes.1.62%dietary arginine level upregulated glycolysis by increasing glucokinase(GK)mRNA level(P<0.05);2.70%dietary arginine level upregulated gluconeogenesis and resulted in high plasma glucose content by increasing phosphoenolpyruvate carboxykinase(PEPCK)and glucose-6-phosphate(G6P)mRNA level(P<0.05).Furthermore,2.70%dietary arginine level significantly lowered glucose transporter 2(GLUT2)and insulin level of serum and increased pyruvate kinase(PK)mRNA levels(P<0.05).These results indicate that 1.62%dietary arginine level improves glycolysis in juvenile blunt snout bream.However,2.70%dietary arginine level results in high plasma glucose,which could lead to negative feedback of insulin resistance,including inhibition of IRS-1 mRNA levels and activation of gluconeogenesis-related gene expression.This mechanism seems to be different from mammals at the molecular level.4 Effects of different dietary arginine on NO synthesis in juvenile blunt snout breamThe present study assessed the effects of dietary arginine on antioxidant status and immunity involved in AMPK-NO signaling pathway in juvenile blunt snout bream.The results showed that,compared with the control group(0.87%),significantly higher mRNA levels of adenosine monophosphate activated protein kinase(AMPK)and nitric oxide synthetase(NOS),activities of total nitric oxide synthetase(T-NOS)and nitric oxide synthetase(iNOS),and plasma nitric oxide(NO)contents were observed in fish fed with 1.62%-2.70%dietary arginine levels(P<0.05).Significantly higher levels of NOS and iNOS were observed in fish fed with 1.62%-2.70%dietary arginine levels in enzyme-linked immune sorbent assay(P<0.05).At dietary arginine levels of 1.22%-2.70%,the mRNA levels of iNOS were significantly improved.Dietary arginine also significantly influenced plasma interleukin 8(IL-8)and tumour necrosis factor-α(TNF-α)contents(P<0.05).Furthermore,dietary arginine significantly affected the activity and mRNA level of glutathione peroxidase(GPx),the mRNA levels of pro-inflammatory factor including IL-8 and TNF-α and plasma malondialdehyde(MDA)content(P<0.05).However,total superoxide dismutase(T-SOD)activity,plasma complement component 3(C3)content,plasma immunoglobulin M(IgM)content,plasma interleukin 1β(IL-1β)content and the mRNA levels of copperzinc superoxide dismutase(Cu/Zn-SOD),manganese superoxide dismutase(Mn-SOD)and IL-1β were not significantly affected by dietary arginine(P>0.05).After Aeromonas hydrophila challenge,the death rate was significantly lowered in fish fed with 1.62%-1.96%dietary arginine levels(P<0.05).Furthermore,the mRNA levels of AMPK,NOS and iNOS,plasma NO content and the activities of T-NOS and iNOS showed an upward trend with increasing dietary arginine levels(P<0.05).Significantly higher levels of NOS and iNOS were observed in fish fed with 1.62%-2.70%dietary arginine levels in enzyme-linked immune sorbent assay(P<0.05).At dietary arginine levels of 1.96%-2.31%,T-SOD activities were significantly improved(P<0.05).Significantly higher GPx activities were observed in fish fed with 1.22%-2.70%dietary arginine levels(P<0.05).At dietary arginine levels of 1.22%-2.31%,the plasma TNF-α and IL-8 contents were significantly decreased(P<0.05).Significantly lower plasma IL-1βcontents were observed in fish fed 1.62%-1.96%dietary arginine levels(P<0.05).Dietary arginine significantly influenced the mRNA levels of antioxidant and pro-inflammatory genes including Cu/Zn-SOD,Mn-SOD,GPx,IL-8,TNF-α and IL-1β(P<0.05).Significantly higher plasma C3 contents and significantly lower plasma MDA contents were observed in fish fed with 1.62%-1.96%arginine levels(P<0.05).Furthermore,plasma IgM contents were significantly improved at dietary arginine levels of 1.62%-2.31%(P<0.05).However,high dietary arginine group(2.70%)significantly improved the mRNA levels of pro-inflammatory genes including IL-8,TNF-α and IL-1β and plasma MDA,IL-8,TNF-α and IL-1β contents as compared with optimal dietary arginine levels(1.62%and 1.96%)(P<0.05).The present results indicate that optimal arginine level(1.62%and 1.96%)could improve antioxidant capacity,immune response and weaken tissues inflammatory involved in arginine-AMPK-NO signaling pathway,while high arginine level resulted in excessive NO production,leading to increase oxidative stress damage and inflammatory response in juvenile blunt snout bream.5 Effects of different dietary arginine on intestinal antioxidant status and immunity in juvenile blunt snout breamThe present study assessed the effects of dietary arginine on intestinal antioxidant status and immunity.The results showed that,compared with the control group(0.87%).The results of hematological parameters were shown in Table 3.Compared with the control group,at dietary arginine level of 1.62%,the counts of white blood cell(WBC)and red blood cell(RBC)were significantly improved(P<0.05),likewise,hemoglobin(HGB)content was also significantly increased in fish fed with 1.62%of dietary arginine(P<0.05).Hematocrit(HCT)level showed a downward trend,but the results were not significantly affected by dietary arginine level(P>0.05).Plasma albumin(ALB)levels and alkaline phosphatase(ALP)activities were significantly improved at dietary arginine levels of 1.62%and 2.70%(P<0.05).Alanine transaminase(ALT)activity was decreased in fish fed with 1.62%dietary arginine level(P<0.05).Aspartate aminotransferase(AST)activity was not significantly affected by dietary arginine levels(P>0.05).Plasma GPx activities,Cu/Zn-SOD activities,total antioxidant capacity(T-AOC)activities and glutathione(GSH)levels were significantly increased at dietary arginine levels of 1.62%and 2.70%(P<0.05).Plasma total superoxide dismutase(T-SOD)activities and catalase(CAT)activities were significantly improved in fish fed with 1.62%dietary arginine level(P<0.05).Significantly higher Mn-SOD activity was observed in fish fed with 1.62%dietary arginine level compared with 2.70%dietary arginine level(P<0.05).1.62%and 2.70%dietary arginine levels significantly lowered MDA levels(P<0.05).The relative expression of nuclear factor erythroid 2-related factor 2(Nrf2)was significantly increased in fish fed with 1.62%dietary arginine level(P<0.05),inversely,the relative expression of Kelch-like ECH-associated protein 1(Keapl)showed a converse trend(P<0.05).1.62%and 2.70%dietary arginine levels significantly improved the relative expressions of Cu/Zn-SOD,GPx and CAT(P<0.05).Furthermore,2.70%dietary arginine level significantly lowered the relative expression of Mn-SOD compared with the control group and 1.62%dietary arginine levels(P<0.05).The relative expressions of IL-1β,TNF-α and nuclear factor-kappa B(NF-κB)were lowered in fish fed with 1.62%dietary arginine level(P<0.05).1.62%and 2.70%dietary arginine levels significantly improved the relative expressions of transforming growth factor-β(TGF-β).IL-8 and interleukin 10(IL-10)expressions were not significantly affected by the graded dietary arginine levels(P>0.05).These results suggest that the optimal dietary arginine level plays an important role in enhancing antioxidant and immune status to maintain the intestinal health of juvenile blunt snout bream.