Dietary Linoleic Acid Nutrition Study In Oriental River Prawn,Macrobrachium Nipponense

Linolenic acid(18: 3n-3,LNA)is one of the important polyunsaturated fatty acids(PUFAs)for the regulation of growth,immunity,environmental stress resistance and lipid metabolism in oriental river prawn,Macrobrachium nipponense.LNA nutrition of the prawn was studied by the combination of nutrition,physiological,biochemical and molecular biology methods.The effects of dietary LNA levels on survival,growth and anti-ammonia-nitrite stress ability of the prawn were investigated.Effects of dietary linolenic acid levels on hepatopancreatic lipid deposition,tissue fatty acid profiles and lipid metabolism-related gene expression in prawn were also studied.Full-length cDNA of gene for ?6 desaturase fatty acyl(?6 FAD)was cloned,characterized and the relationship between dietary LNA and ?6 FAD expression in the hepatopancreas was also investigated.The results can be briefly summarized as follows:Linseed oil was added to the experimental diets to formulate six isonitrogenous and isolipidic diets containing 0.0%(L0),0.5%(L0.5),1.0%(L1.0),1.5%(L1.5),2.0%(L2.0)and 2.5%(L2.5)LNA of dry weight,respectively.Basal diet without LNA supplementation was considered as a control.Each LNA level had 5 tanks(replicates)with 50 prawns per tank.After 8 weeks of feeding,10 Prawns from each tank were exposed to ammonia nitrogen for 24 h,and then the growth,hepatopancreas malondialdehyde(MDA)contents,superoxide dismutase(SOD),glutathione peroxidase(GSH-Px)activity and total antioxidant capacity(T-AOC)were measured.The results showed as follows: with the LNA level increasing,the weight gain of prawns was increased firstly and then decreased,but the difference was not significant among all groups(P > 0.05).The SOD,GSH-Px and T-AOC activity in hepatopancreas increased with the increase of dietary LNA from 0.0 to 1.0% and then decreased as the dietary LNA increased to 2.50%.MDA content in LNA supplemental groups was significantly lower than the control group(P < 0.05).The prawn fed the diet with 1.0% and 1.5% LNA showed the highest acid phosphatase(ACP)and lysozyme(LYZ)activity(P < 0.05),respectively.After ammonia-nitrite stress,SOD reached highest activity at L1.5,in which had lowest MDA content.T-AOC and GSH-Px reached highest activities at L1.0.According to the bivariate regression analysis,the relationship between the SOD(y)and the LNA level(x)as was y =-0.4021x2 + 0.9539 x + 0.5125(R2=0.71848).As LNA level was 1.19%,juvenile prawn showed highest SOD activity.Our results indicated that moderate levels of dietary LNA(1.0 % ~ 1.5% of dry weight)could improve non-immune response and anti-ammonia-nitrite stress ability of oriental river prawn and the optimal dietary LNA level for juvenile prawn is 1.12%.The endogenous production of long-chain polyunsaturated fatty acids(LC-PUFAs)from the dietary C18 PUFA precursor,linolenic acid(LNA,18:3n-3),in omnivorous,freshwater crustaceans is poorly understood.Moreover,there is no information on the regulation of LC-PUFA biosynthesis by increasing dietary levels of LNA in prawns.In this study,the effects of different dietary LNA levels on lipid deposition,tissue fatty acid composition,and mRNA expression of genes related to lipid metabolism in the oriental river prawn(Macrobrachium nipponense)hepatopancreas were studied.Prawns(average weight 0.12±0.01 g)were fed six isonitrogenous and isolipid diets containing 0,0.5,1.0,1.5,2.0,and 2.5% LNA for 8 weeks.The hepatopancreas lipid content decreased significantly when dietary LNA content was > 2.0%.The lowest hepatopancreas lipid content was observed in prawns fed 2.5% LNA,which was significantly lower than that of prawns fed 0 ~ 2.0% LNA.Fatty acid analysis revealed that the percentage of 18:3n-3 and total n-3 PUFAs in the hepatopancreas and muscle significantly increased with increasing dietary LNA levels,while 20:5n-3,22:5n-3 and 22:6n-3 levels in the hepatopancreas and muscle decreased with increasing dietary LNA levels.Additionally,qRT-PCR results revealed that hepatopancreas mRNA expression of acetyl CoA carboxylase(ACC)decreased with increasing dietary LNA,and the lowest ACC mRNA expression was observed in prawns fed 2.0 and 2.5% LNA,while the greatest carnitine palmitoyl transterase-1(CPT1)mRNA expression was observed in the 2.0 and 2.5% LNA group.Hepatopancreas mRNA expression of acyl-CoA delta-9 desaturase(SCD)was lowest in the 2.5% group.Furthermore,fatty acyl elongase(elovl6)gene expression decreased with increasing dietary LNA content.These results indicate that dietary 18:3n-3 could decrease lipid deposition through increased fatty acid ?-oxidation and decreased fatty acid synthesis,and alter proximate fatty acid composition by regulating fatty acyl elongase and fatty acyl desaturase mRNA expression in the M.nipponense hepatopancreas.Moreover,the results of the present study suggest that M.nipponense might be capable of endogenous LC-PUFA production from the dietary C18 PUFA precursor,LNA,but increasing dietary LNA level has no effect on n-3 LC-PUFAs in either the muscle or the hepatopancreas.Full-length cDNA of gene for ?6-desaturase fatty acyl in M.nipponense was cloned by rapid amplification of cDNA ends(RACE),and the expressions in 9 tissues such as hepatopancreas,intestine and gill etc.were analyzed by real-time quantitative PCR(qRT-PCR).Analysis of the full-length cDNA of gene for ?6 desaturase fatty acyl revealed that it was 1906 bp,including 122 bp of 5' UTR and 394 bp of 3' UTR and encoded 453 amino acids.The presumed protein sequence had a typical desaturase structure: 3 histidine-rich motifs,a b5 domain of an N terminal cytochrome and a heme binding HPGG domain.qRT-PCR showed that gene for ?6 desaturase fatty acyl expressed in all 9 tissues while the highest in muscle and the least in the hepatopancreas.The mRNA levels of ?6 FAD in the hepatopancreas were significantly influenced by LNA in the diet,with peak expression in the L2.5 group.The results indicate that M.nipponense possesses ?6 desaturase fatty acyl and the high dietary LNA can induce ?6 FAD expression.