Effects And Mechanisms Of Dietary Nucleotides On Growth Performance,Functional Organs Health And Flesh Quality Of Young Grass Carp (Ctenopharyngodon Idellus)

In this research,a total of 540 healthy young grass carp(Ctenopharyngodon idella)with initial body weight of 200.0 ±1.0 g were selected and divided into six treatments with 3 replicates,which were fed with graded nucleotides supplementation levels(0.0?200.0?400.0?600.0?800.0 and 1000.0 mg/kg)diets for 60 days.The study was carried out to investigate the effects and the potential mechanisms of dietary nucleotides on growth performance,intestinal development,intestinal bacteria and flesh quality of grass carp.After feeding trial,grass carp were challenged with Aeromonas hydrophila for 14 days and Flavobacterium columnare for 3 days to explore the effects of dietary nucleotides on fish functional organs(intestines,head kidney,spleen,skin and gill)structural integrity(antioxidant ability,anti-apoptosis ability and tight junctional protein)and immune function as well as the potential mechanisms in grass carp.Based on different parameters,optimal nucleotides supplementation levels on regulating functional organs health and flesh quality of young grass carp were also estimated.The results showed that the optimal nucleotides significantly increased the percentage weight gain(PWG),specific growth rate(SGR),feed intake(FI)and feed efficiency(FE)when compared with control group(P < 0.05).Dietary nucleotides also increased the intestinal weight and intestinal length,indicating that dietary nucleotides supplementation could promote growth performance of grass carp.Dietary nucleotides also increased the counting of Lactobacillus,Bacillus and Bifidobacterium,while reduced Escherichia coli and Aeromonas hydrophila in grass carp.Meanwhile,optimal dietary nucleotides levels decreased enteritis,skin lesion and gill rot morbidity of young grass carp,which were closely related to the maintenance of the intestinal structure integrity and the enhancement of immune function in the functional organs of grass carp.On the one hand,optimal dietary nucleotides levels down-regulated kelch-like-ECH-associated protein [Keap1a(not in PI,MI and DI)and Keap1a)] m RNA levels,up-regulated NF-E2-related factor 2(Nrf2)m RNA levels to promote the transcription of Cu Zn SOD,Mn SOD,CAT,GPx(1a,1b,4a and 4b),GST [GSTR,GSTO1,GSTO2,GSTP1 and GSTP2(not in head kidney,spleen and skin)] and glutathione reductase(GR),improved the activity of antioxidant enzymes and antioxidant ability,decreased the oxidative damage of functional organs caused by free radicals.Besides,optimal nucleotides supplementation prevented the DNA fragements in the intestines,head kidney,skin,gill and muscle,down-regulated c-Jun N-terminal protein kinase(JNK),p38 mitogen activated protein(p38MAPK)(not in PI,head kidney,spleen and skin),factor-related apoptosis ligand(Fas L),pro-apoptotic protein apoptotic protease activating factor 1(Apaf-1),B-cell lymphoma protein 2 associated X protein(Bax)(not in PI)and caspase-(2,3 7,8,9)m RNA levels but up-regulated B-cell lymphoma protein-2(Bcl-2),inhibitor of apoptosis proteins(IAP)and myeloid cell leukemia-1(Mcl-1)m RNA levels to inhibit apoptosis in the functional organs of grass carp.Optimal dietary nucleotides decreased molecule myosin light-chain kinase(MLCK)m RNA levels to up-regulate the m RNA levels of occludin(not in head kidney and gill),ZO-1,ZO-2,claudin-b(not in gill),claudin-c,claudin-f,claudin-3c(not in PI,MI and DI),claudin-7a,claudin-7b and claudin-11,and to down-regulate the m RNA levels of claudin-12,claudin-15 a,claudin-15 b,resulting in the enhancement of structural integrity of grass carp.On the other hand,optimal nucleotides supplementation increased immunologic substance contents including lysozyme(LZ),acid phosphatase(ACP),complement(C3 and C4)and Ig M,up-regulated ?-defensin-1,hepcidin,liver expressed antimicrobial peptide 2 [LEAP-2A(no in gill)and LEAP-2B] and Mucin2(not in PI and MI)m RNA levels,downregulated pro-inflammatory cytokine tumor necrosis factor ?(TNF-?)(no in head kidney and gill),interferon ?2(IFN-?2)(no in gill),interleukin-1?(IL-1?)(not in PI,MI and DI),IL-6,IL-8(not in PI,MI and DI),IL-15,IL-17 D,IL-12p35(not in gill)and IL-12p40(not in head kidney,spleen and skin)m RNA levels,up-regulated anti-inflammatory cytokines IL-10(not in PI,MI and DI),IL-11,TGF-?1,TGF-?2(not in PI,MI and DI),IL-4/13A(not in head kidney,spleen and skin)and IL-4/13B(not in gill)m RNA levels,down-regulated pro-inflammatory signaling molecules I?B kinase ?(IKK?)and IKK?(excpet IKK?),nuclear factor-?B p52(NF-?Bp52)(not in PI,MI,DI and gill),nuclear factor-?B p65 and cRel m RNA levels,up-regulated inhibitor of ?B?(I?B?),TOR and S6K1 m RNA levels and down-regulated 4E-BP1 and 4E-BP2(not in PI,MI and DI)m RNA levels,thus resulting in the improved immune function of grass carp.Furthermore,optimal nucleotides also improved the nutritive quality,flavor quality,health quality and physicochemical quality of grass carp.The nutritive quality was improved by dietary nucleotides though increasing the content of protein and fat and decreasing the content of water in muscle.Optimal nucleotides enhanced the flavor quality via increasing the content of umami amino acids(aspartic acid and glutamic acid),sweet amino acids(serine,glycine and alanine)and flavor inosinic acid in muscle.Optimal nucleotides also improved the health quality of young grass carp by increasing the content of n-3 series unsaturated fatty acids especially EPA and DHA,while decreasing the unhealthy saturated fatty acid in muscle(P < 0.05).Optimal nucleotides enhanced the physicochemical quality by increasing water holding capacity,tenderness and p H.Furthermore,optimal nucleotides enhanced apoptosis related water holding capacity with down-regulating the gene expression of p38 MAPK,JNK,Fas L,Apaf-1,Bax and caspase-(2,3,7,8 and 9)m RNA levels,while up-regulating anti-apoptotic protein Bcl-2 and IAP m RNA levels.The increased tenderness was associated with decreased collagen content and increased cathepsins B and L activities,while the increased p H was related to the decreased lactate by dietary nucleotides in the muscle of grass carp.Optimal dietary nucleotides up-regulated the transcription of antioxidant enzymes such as Cu Zn SOD with the regulation of nuclear transcription factor(Nrf2),which resulted in the increased activities of antioxidant enzymes,enhanced antioxidant ability and reduced the oxidation of protein and lipids caused by free radicals.Therefore,the results showed that optimal nucleotides could improve flesh quality by inhibition apoptosis.In summary,optimal nucleotides supplementation could enhance growth performance,intestinal development,the capacity of against enteritis,skin lesion and gill rot morbidity and flesh quality of young grass carp.Dietary nucleotides maintained the structural integrity of cells by enhancing antioxidant,anti-apoptotic and tight junction properties of functional organs,improved the immune function of functional organs with increasing the content of antimicrobial substances,antimicrobial peptide gene expression and regulating inflammatory cytokines.In addition,dietary nucloetides enhanced the flesh quality by improving nutritive quality,flavor quality,health quality and physicochemical quality.Finally,based on the regression analysis of PWG,enteritis,skin lesion and gill rot resistance,EPA,DHA,shear force and p H,the optimal nucleotides supplementation levels for grass carp(200-734 g)were 517.97,580.35,626.71,526.38,604.53,594.28,540.61 and 615.76 mg/kg,respectively.