Effects Of Dietary Cholesterol On Gowth Performance,Health Of Functional Organs And Fillet Quality And Its Potential Mechanisms In Young Grass Carp(Ctenopharyngodon Idella)

The present study explored the effects of cholesterol on the growth performance,the health of functional organs and fillet quality as well as its potential mechanisms in young grass carp(Ctenopharyngodon idella).A total of 540 of Grass carp(initial weight 225.37 ± 0.43g)were fed six experimental diets with graded levels of cholesterol(0.041-1.526%)for 60 days to investigate the effects of cholesterol on the growth performance and fillet quality of fish.After the feeding trial,the experimental fish were infected with Aeromonas hydrophila for 14 days and Flavobacterium columnare for 3 days respectively,to explore the effects of cholesterol on the fish functional organs immunity and structural integrity and its potential mechanisms by examining the antibacterial compounds,inflammation response,oxidation resistance,apoptosis,tight junctions and related signalling molecules in the intestine,head kidney,spleen,skin and gill of young grass carp.The results showed that optimal dietary cholesterol enhanced the feed intake(FI),feed efficiency(FE),percent weight gain(PWG)and specific growth rate(SGR)(P < 0.05),indicating that optimal dietary cholesterol could improve fish growth performance.Meanwhile,optimal dietary cholesterol increased the ability against enteritis,skin haemorrhage and lesion as well as gill rot morbidity of young grass carp(P < 0.05),this could be related to the functional organs(intestine,head kidney,spleen,skin and gill)immunity and structural integrity.(1)Optimal dietary cholesterol improved antibacterial compounds [lysozyme(LZ)and acid phosphatase(ACP)activities,complement 3(C3),C4 and Ig M] contents(P < 0.05),and up-regulated hepcidin,liver-expressed antimicrobial peptide(LEAP)-2A,Leap-2B and Mucin2 m RNA levels(P < 0.05),indicating that optimal dietary cholesterol could improve fish functional organs antibacterial ability;Meanwhile,optimal dietary cholesterol down-regulated the pro-inflammatory cytokines gene expression partly due to the suppression of nuclear factor kappa B(NF-κB)signalling,and up-regulated the gene expression of anti-inflammatory cytokines partly due to the activated target of rapamycin(TOR)signalling,thus,resulting in attenuating the functional organs inflammatory response of young grass carp.However,dietary cholesterol had no influence on the pro-inflammatory cytokines interleukin 1β(IL-1β)and IL-12p40 m RNA levels in the proximal intestine(PI),spleen and skin(P > 0.05),antimicrobial peptide β-defensin-1 m RNA levels in the skin(P > 0.05),anti-inflammatory cytokines transforming growth factor-β2(TGF-β2)and signaling molecule e IF4E-binding proteins 1(4E-BP1)m RNA levels in the PI,middle intestine(MI)and distal intestine(DI)(P > 0.05),anti-inflammatory cytokines IL-4/13 A m RNA levels in the PI,MI and DI(P > 0.05),as well as signaling molecule IκB kinase α(IKKα)m RNA levels in the PI,MI,DI,head kidney,spleen,skin and gill(P > 0.05).(2)Optimal dietary cholesterol decreased the reactive oxygen species(ROS),protein carbonyl(PC)and malondialdehyde(MDA)contents(P < 0.05),up-regulated the m RNA levels of antioxidant enzymes partly due to the activated NF-E2-related factor 2(Nrf2)signalling,thus,partly resulting in increasing the related antioxidant enzyme activities(except for CAT)(P < 0.05)and enhancing the ability of antioxidative damage in the functional organs of young grass carp;Optimal dietary cholesterol down-regulated the m RNA levels of pro-apoptotic proteins [factor-related apoptosis ligand(Fas L),apoptotic protease activating factor 1(Apaf-1),B-cell lymphoma protein 2 associated X protein(Bax),caspase-2,-3,-7,-8 and-9](P < 0.05)and up-regulated the m RNA levels of anti-apoptotic proteins [B-cell lymphoma 2(Bcl-2),inhibitor of apoptosis proteins(IAP)and myeloid cell leukemia 1(Mcl-1)](P < 0.05),partly due to the suppression of c-Jun N-terminal protein kinase(JNK)signalling,thus,resulting in inhibiting the functional organs apoptosis of young grass carp;Optimal dietary cholesterol up-regulated the m RNA levels of tight junction proteins partly due to the suppression of myosin light chain kinase(MLCK)signalling,thus,maintaining the intercellular structural integrity in functional organs of young grass carp,However,dietary cholesterol had no effect on the m RNA levels of antioxidant enzyme Mn SOD in the PI,MI and DI(P > 0.05),the antioxidant enzyme GSTP1 in the head kidney,spleen and skin(P > 0.05),the antioxidant enzyme Cu Zn SOD m RNA level in the gill,the signaling molecules Keap1 b and p38 mitogen-activated protein kinases(p38MAPK)m RNA levels in the PI,MI,DI,head kidney,spleen,skin and gill(P > 0.05).Dietary cholesterol had no effects on the m RNA levels of claudin-b and claudin-3c in the PI,MI,DI,and gill(P > 0.05),the claudin-11 and claudin-12 m RNA levels in the head kidney,spleen and skin(P > 0.05),as well as claudin-15 b in the gill(P > 0.05).In addition,optimal dietary cholesterol increased the protein and lipids contents in the muscle(P < 0.05),enhanced umami amino acids(aspartic acid and glutamic acid),sweet amino acids(serine,threonine and lysine)as well as sour amino acid(histidine)contents(P < 0.05),increased flavor substance inosinic acid(IMP)cotent(P < 0.05),increased C20:5n-3(EPA)and C22:6n-3(DHA)contents(P < 0.05),thus improving fillet quality.Optimal dietary cholesterol decreased the cooking loss,cathepsins B and L activities(P < 0.05),enhancing the water holding capacity of fish muscle.Optimal dietary cholesterol decreased the shear force and hydroxyproline(Hyp)content(P < 0.05),enhancing the tenderness of fish muscle.Optimal dietary cholesterol incrased the p H and decreased the lactic acid of fish muscle(P < 0.05),improving fillet quality.Furthermore,optimal dietary cholesterol decreased ROS,PC and MDA contents(P < 0.05),up-regulated the m RNA levels of antioxidant enzymes partly due to the activated casein kinase 2(CK2)/TOR/Nrf2 signalling,thus,partly resulting in increasing the related antioxidant enzyme activities(except for CAT)(P < 0.05)and enhancing fillet quality.As stated above,optimal dietary cholesterol increased the ability against enteritis,skin haemorrhage and lesion as well as gill rot,improved the growth performance of young grass carp,which were related to the improved functional organs(PI,MI,DI,head kidney,spleen,skin and gill)immunity and structural integrity.In addition,excess dietary cholesterol impaired functional organs immunity and structural integrity of young grass carp.In addition,optimal dietary cholesterol could enhance fillet quality by increasing nutrient and flavor substance,enhancing physicochemical quality and antioxidant ability.Finally,based on the percent weight gain(PWG)and the ability against enteritis,skin haemorrhage and lesion as well as gill rot,the optimal dietary cholesterol levels for young grass carp were estimated as 0.721%,0.853%,0.826% and 0.812%,respectively.