Effects Of Docosahexaenoic Acid On Growth,Muscle Fiber Development And Cell Cycle Of Blunt Snout Bream (Megalobrama Amblycephala)

DHA,as an important polyunsaturated fatty acid,has been used as a nutritional and health supplement in mammalian feeds,marine fish farming and even human food.It provides animals with a large amount of highly unsaturated fatty acids,which not only promotes growth and enhances immunity,but also promotes the development of animal muscle fibers,which in turn affects the quality and taste of muscles.However,for freshwater fish,traditional studies generally believe that it can synthesize highly unsaturated fatty acids such as DHA on its own,and does not need to be added to the feed.In order to study the optimum dietary amount of DHA for blunt snout bream,and to determine the effect of DHA on the growth,muscle fiber development and cell cycle of freshwater fish,thereby promoting the growth of freshwater fish,muscle quality and health,this study is divided into three parts:Experiment 1 Effects of different dietary DHA levels on growth performance,fatty acid profile and lipogenesis of blunt snout bream (Megalobrama amblycephala)Six diets were designed to investigate the effects of dietary docosahexaenoic acid(22:6n-3;DHA) levels (0.05,0.13,0.23,0.42,0.81 and 1.59 % diets) on growth performance,fatty acid profile and expression of some lipogenesis related genes of blunt snout bream (Megalobrama amblycephala).Fish (average weight 26.40±0.11 g)were randomly fed one of six diets for 8 weeks.Results indicated that the final body weight (FBW) and specific growth rate (SGR) of fish fed 0.13% DHA was significantly higher than other groups except for the 0.23% DHA (P<0.05).The optimum amount of dietary DHA for blunt snout bream was 0.11% based on the two slope broken-line regression analysis of SGR.The number of lipid droplet clusters of the liver stained with oil red O in the 0.23% DHA group was significantly higher than that of other groups (P<0.05),which was consistent with the lipid contents of whole-body and liver.The DHA proportion in liver and muscle significantly increased with the increasing dietary DHA levels (P<0.05),which reflected fatty acid profiles of diets.The mRNA expressions of ACCa and FAS in the 0.13% DHA group and 0.23%DHA group were significantly higher than that of other groups (P<0.05).In summary,the optimum amount of dietary DHA for blunt snout bream was 0.11%,0.13-0.23%dietary DHA could improve growth performance and lipogenesis,and improve DHA and PUFA proportion in liver and muscle.Experiment 2 Effect of DHA on muscle fiber development and muscle quality of blunt snout bream (Megalobrama amblycephala)A feeding trial and a primary hepatocyte culture were designed to study the effect of DHA on muscle development mechanism of blunt snout bream.Fish (average weight 26.40±0.11 g) were randomly fed three levels of dietary DHA:0.05% DHA,0.13% DHA and 0.81% DHA for 8 weeks.Results were indicated that the weight gain rate (WGR) of fish feed with 0.13% DHA was significantly higher than 0.05% DHA and 0.81% DHA (P<0.05).The texture (hardness,gumminess and chewiness),fibers density,the proportion of small-sized fiber (<20μm) and sarcomere lengths in the white muscle were significantly increased in the 0.81% DHA group than 0.05% DHA and 0.13% DHA (P<0.05).The gene expression of Myog and Mrf4 were significantly increased (P<0.05),and the gene expression of MSTNa and MSTNb were significantly decreased in muscle of fish fed 0.81% DHA than 0.05% DHA and 0.13% DHA(P<0.05).These results suggested that 0.81% DHA increased the flesh quality and promoted the white muscle hyperplasia and muscle fiber development.In addition,the gene expression of AMPKα2 and Sirt1 in the 0.13% DHA and 0.81% DHA group was significantly higher than the 0.05% DHA group (P<0.05).The protein level of AMPK,phosphor-AMPK and AMPK/phosphor-AMPK were significantly increased in DC and DH group (P<0.05).In vitro,the isolated primary hepatocytes from blunt snout bream were incubated with 0,100 and 400 μM DHA for 24h.The results showed that 400 μM DHA was significantly up-regulate the gene expression of AMPKa2 and Sirt1,and increased the protein level of AMPK,phosphor-AMPK,AMPK/phosphor-AMPK and Sirtl (P<0.05).In summary,0.81% dietary DHA promoted the white muscle hyperplasia and muscle fiber development,which may be in association with the activation of AMPK/Sirtl pathway.Experiment 3 High level of DHA affects the growth by inducing cell cycle arrest in blunt snout bream (Megalobrama amblycephala)Pervious study showed that the high levels of docosahexaenoic acid (DHA) diets caused growth retardation in Megalobrama amblycephala,in order to explore the mechanism,a feeding trial and primary hepatocytes culture were designed.Fish(average weight 26.40±0.11 g) were randomly divided into three groups (three diets:0.05% DHA,0.23% DHA and 1.59% DHA),which lasted for 8 weeks.Results indicated that the final body weight and weight gain of fish fed 1.59% DHA were significantly decreased than 0.23% DHA (P<0.05).The gene and protein expression of P53 were significantly increased (P<0.05),and the expressions of downstream Cyclin D1 and Cyclin El were significantly inhibited in 1.59% DHA group than 0.05% DHA and 0.23% DHA (P<0.05).In vitro,the isolated primary hepatocytes from were incubated with 500 μM DHA,which showed that cell cycle arrest and apoptosis were significantly increased (P<0.05),the gene and protein levels of P53 were significantly increased (P<0.05),whereas Cyclin D1 and Cyclin E1 were significantly decreased (P<0.05) than other groups.When primary hepatocytes were incubated with DHA and the P53 inhibitor (Pifithrin-α),the P53 expression and P53-mediated signaling were inhibited,cell cycle was recovered andapoptosis was decreased.In summary,DHA activated the P53/Cycling pathway to induce cell cycle arrest.The inhibition of P53 activity may be a potential target for reducing the side effects of DHA on the growth of Megalobrama amblycephala.In a word,the optimum amount of dietary DHA for blunt snout bream was 0.11%.0.13%-0.23% dietary DHA could promote the growth,increase the content of DHA and PUFA in the liver and muscles;0.81% dietary DHA promoted the white muscle hyperplasia,muscle fiber development and fish quality by activating the AMPK/Sirtl pathway;However,excessive DHA induced cell cycle arrest and inhibited the growth of bream bream by activating the P53/Cycling pathway.