Effect Of Dietary N-3 HUFA On Growth Performance And Lipid Metabolism In Juvenile Black Sea Bream, Sparus Macrocephalus

Four experiments were conducted to investigate the effect of dietary n-3 highly unsaturated fatty acids (HUFA, mainly EPA and DHA), on growth performance, body composition and plasma biochemistry in juvenile black sea bream, Sparus macrocephalus. The aim of this study was to deduce the optimum requirement of n-3 HUFA and DHA to EPA ratio for the best growth performance of juvenile black sea bream, Sparus macrocephalus, and then to explore the main mechanism of n-3 HUFA regulation on lipid metabolism in black sea bream. The present study include the following:(1) Effect of supplementation of dietary n-3 HUFA on growth and body composition of black sea bream juveniles, Sparus macrocephalus; (2) Effect of dietary DHA to EPA ratio on growth performance, body compositon and fatty acid profile of juvenile black sea bream, Sparus macrocephalus;(3) Cloning of fatty acid synthse (FAS) and hormone sensitive lipase (HSL) in black sea bream, Sparus macrocephalus;(4) Effect of dietary n-3 level and DHA to EPA ratio on lipogenic and lypolytic enzymes activities and relative gene expression in black sea bream, Sparus macrocephalus.(1) Three hundred and sixty black sea bream juveniles (average weight, 8.08±0.09) were randomly allocated into 18 net cages built in a big concrete pond. Six diets with a constant DHA to EPA ratio (2.8/1) were formulated to contain different levels (8.48,8.89,9.08,9.43,9.88,10.12%) of n-3 HUFA, by supplementing corn oil and fish oil to modulate the fatty aid content. All diets were assigned to six grous of fish with triplicate. Fish were fed twice every day (8:00 and 16:00) to apparent satiation. The experiment conditions were mentioned above. At the end of the feeding trial, growth performance, feed efficiency (FE) and protein efficiency ratio (PER) of black sea bream were calculated, and plasma biochemisty parameters, the proximate composition as well as fatty acid profiles in liver, dorsal muscle and white adipose tissue (WAT) were analysized. At the end of the experiment, hepotasomatic index (HSI) and intraperitoneal fat (IPF) ratio decreased with the increase of dietary n-3 HUFA and the values in group 5 and 6 were significantly lower than that in other four groups (P<0.05). Adipocyte diameter in IPF was decreased by dietary n-3 HUFA and the significance occurred between group 3 and 6. Proximate composition analysis showed that lipid content in muscle was affected by dietary n-3 HUFA significantly and reached the bottom at the level of 0.88%. No significance was found in whole body proximate composition of juvenile black sea bream. Concerning the fatty acid composition,ΣSFA and the main constituent 16:00 were negatively correlated with dietary n-3 HUFA; in contrast,Σn-3 HUFA content was significantly increased among all the treatment. DHA to EPA ratios in liver, dorsal muscle and WAT were unchanged with the supplementation of dietary n-3 HUFA. Plasma triglycerides (TG) and total cholesterol (T-CHO) were all significanlty decreased by dietary treatment. Quadratic analysis based on weight gain rate (WGR) indicated that dietary n-3 HUFA requirement for black sea bream was 0.87% DM, that is 8.7 g/kg in the diet.(2) Black sea bream juveniles (11.01±0.02 g, mean weight) were fed diets containing different DHA/EPA ratios (from 1.98 to 4.04) with constant dietary n-3 HUFA level (about 1.0%) for 8 weeks. Four hundred and twenty fish were allocated into seven groups with triplicate and were fed different experimental diets. Fish feeding and the experimental condition were all just the same with experiment 1. Growth performance, FE and PER were calculated, while tissue proximate composition, fatty acid composition and plasma biochemical parameters were all analyzed at the end of the feeding trial. IPF ratio in other treatments was significantly lower than that in group 1, and fish in group 3 showed the lowest fat deposit in viscera. Diameter of adipocyte in IPF was significantly reduced compared to the other groups. Proximate composition analysis showed that lipid content in muscle was decreased by 8.59% at large, and lipid deposit in whole body was decreased by 28.3%. Fatty acid profile analysis indicated that n-3 HUFA content in liver, dorsal muscle and WAT was independ of dietary DHA to EPA ratios, while DHA/EPA ratio in these tissues were positively correlated with dietary DHA/EPA ratio. Based on weigt gain rate (WGR), quadratic analysis (y=-9.9707x2+62.696x+10.309, R2=0.9258; x, DHA/EPA ratio; y, WGR) showed that at the present experiment condition, the DHA/EPA requiremet for juvenile black sea bream was 3.15.(3) FAS gene of 464 bp and HSL of 665 bp were cloned in this experiment. Blast analysis showed that the high similarity (76% and 69%) between black sea bream and zebrafish is a strong indication that these two sequences correspond to a FAS or HSL gene. Clone of partial FAS and HSL gene in the experiment made preparations for the next real-time PCR determination of FAS and HSL gene expression in the following experiment.(4) Postulated mechanism of fatty acid regulation in lipid metabolism in black sea bream was discussed according to the study of the effect of dietary n-3 HUFA and DHA to EPA ratio on FAS or HSL gene expression and enzyme activities. Determination of FAS and HSL activities showed that FAS activity was higher in liver than that in adipose tissue, which means the tissue specificity of FAS in black sea bream and liver was considered to be the main organ of fatty acid synthesis. In both experiment, FAS activity kept constant in group 1 to 4, and then decreased significantly with further increment of dietary n-3 HUFA or DHA/EPA ratio. HSL activity increased significantly with the elevation of dietary n-3 HUFA level or dietary DHA/EPA ratio, but reached a plateau when the ratio was higher than 2.88. Real-time PCR determination showed that FAS gene expression changed following the similar trend with FAS activity in liver; while liver HSL gene expression, just as in adipose tissue, was positively correlated with dietary n-3 HUFA level or DHA/EPA ratio. In summary, we can deduce that n-3 highly unsaturated fatty acid regulation in lipid metabolism was realized by affecting the two pathways, that is, lipogenesis and lipolysis at the same time.