Efficacy And Tolerance Evaluation Of N-Carbamylglutamate In Japanese Seabass(Lateolabrax Japonicus)Diet And The Related Nutrient Metabolism Regulation Mechanism

N-Carbamylglutamate(NCG),an analogue of N-acetylglutamate(NAG),can promote the synthesis of endogenous Arginine(Arg)in mammals,but not well studied in fish.Effects of NCG on growth performance,hematological parameters,antioxidant index,amino acid metabolism and histology of liver were detected to evaluate the efficacy and tolerance of NCG in the diet of Japanese seabass(Lateolabrax japonicus)diet.We explored the mechanism by which NCG improves liver health and reduces visceral adipose combined with relative mRNA levels of lipid metabolism and inflammatory cytokines,and immunofluorescence(Cleaved caspase-3),TUNEL test of liver in Japanese seabass.The results were summarized as follows:Part 1 Efficacy test on NCG requirement evaluation of Japanese seabassEfficacy test on NCG requirement evaluation of Japanese seabass.A 10-week growth trial was to determine the effects of NCG on growth performance,morphometric parameters and hematological parameters in the diet of Japanese seabass.Six experimental diets were designed with NCG supplement levels of 0(N0),120(N120),240(N240),360(N360),480(N480)and 720(N720)mg/kg.The results showed that N240 group had the highest PPV and the lowest VAI,and hepatic lipid was the lowest in the N360 group(P<0.05),indicating that optimal dietary NCG supplementation promoted the protein retention,and reduced visceral and lipid adipose deposition effectively.Appropriate dietary NCG supplementation decreased plasma BUN and TG levels in Japanese seabass(P<0.05),indicating that NCG improved protein and lipid metabolism of Japanese seabass.PPV and VAI were subjected to Broken-line analysis,and the results showed that at the dietary NCG requirement of Japanese seabass is 214.3-219.2mg/kg dry matter,and the highest recommended dose is 360mg/kg which authorized by MOA,China in fish feed..Part 2 Mechanism of NCG alleviates liver metabolic disease and tolerance test on NCG in Japanese seabass dietThis study was conducted to investigate the capacity of Arg endogenous synthesis by NCG,and the effects of various doses of dietary NCG on growth performance,hepatic health and underlying nutrient regulation metabolism on ERK1/2-mTOR-S6K1 signaling pathway in Japanese seabass.Four experimental diets were prepared with NCG supplement levels of 0(N0),360(N360),720(N720)and 3600(N3600)mg/kg,in which N3600 was 10-fold of N360 for tolerance test of an over-dose of dietary NCG.The results showed that the dietary NCG supplementation had no significant effects on the SGR and morphometric parameters of Japanese seabass,but 360-720 mg/kg NCG inclusion promoted PPV,while the 10-fold(3600 mg/kg)overdose of NCG reduced feed efficiency significantly.We found that Japanese seabass can utilize the 360-720mg/kg NCG to synthesis Arg to improve the amino acid metabolism by increasing plasma Arg level,up-regulating key Arg synthesis enzyme-ASL gene expression.Increased plasma GST and decreased MDA indicated the improved antioxidant response(P<0.05).Dietary NCG inclusion decreased plasma IgM and down-regulated the mRNA levels of inflammation(TNF-? and IL8),apoptosis(caspase family)and fibrosis(TGF-?1)related genes in the liver.An immunofluorescence examination revealed significantly decreased hepatic apoptosis and necrosis signals in the NCG groups.The ameliorated liver function and histological structure were closely related to the improved lipid metabolism parameters with decreased plasma VLDL and hepatic TG and NEFA accumulation,down-regulated fatty acid and cholesterol synthesis and simultaneously up-regulated lipolysis genes expression,which regulated by inhibiting phosphorylation of ERK1/2-mTOR-S6K1 signaling pathway(P<0.05).Given all that,it suggested that 360-3600 mg/kg NCG supplementation could improve hepatic health,and the N3600 group had no significant negative impact on Japanese seabass health.Therefore,10-fold of the maximum level of NCG(3600 mg/kg)is safe for the species.Part 3 Effects of NCG on lipid metabolism,inflammation and apoptosis of visceral adipose and adipocytes in Japanese seabassThis study aims to reveal the potential regulatory mechanism of NCG which reduced visceral adipose accumulation in Japanese seabass by in vivo and in vitro experiments.The results showed that dietary 720-3600 mg/kg NCG inclusion reduced visceral adipose with decreased the mRNA levels of fatty acid synthase(FASN),cholesterol synthase(HMGCR),adipocyte differentiation key factors(PPAR? and C/EBP?),LPL and Leptin in the adipose tissue(P<0.05),indicating that NCG reduced fat deposition in the Japanese seabass.Besides,there were no significant difference in ATGL and HSL mRNA levels.We isolated pre-adipocytes and induced them into adipocytes of Japanese seabass,and we found that during the differentiation process,lipid droplets accumulate in the cells,and small lipid droplets fuse into large lipid droplets,eventually forming into adipocytes.Moreovere,we found that the expression of FASN,HMGCR,LPL and Leptin in adipocytes of NCG treatment group was significantly inhibited(P<0.05).In addition,we found that NCG treatment had no significant effect on the lipolysis genes ATGL and HSL,and the key factors of adipocyte proliferation and differentiation(PPAR?,C/EBP? and C/EBP?).This indicated that NCG reduce fat accumulation by inhibiting adipogenesis.Furthermore,720-3600 mg/kg NCG inclusion significantly reduced the expression of inflammatory factors IL1?,IL8,caspase-3 and caspase-8 in visceral adipose of Japanese seabass(P<0.05).The pro-inflammatory factor-IL8 was significantly down-regulated and the anti-inflammatory factor-IL10 was significantly up-regulated of adipocyte in the NCG-treated group(P<0.05).In conclusion,NCG reduced visceral fat accumulation by inhibiting adipogenesis.