| Half-smooth tongue sole(Cynoglossus semilaevis) is an important commercial flatfish in China. Two feeding experiments were carried out in an indoorculture system to investigate the effects of dietary lipid and arachidonic acid level on growth, activities ofdigestive enzymes, fatty acid composition and genes expression of metabolism related enzymes of larval tongue sole. The main results are summarized as follows:1. Effects of dietary lipid levels on survival, growth, activities of digestive enzymes and fatty acid composition of larval half-smooth tongue sole (Cynoglossus semilaevis)A 30-day feeding trial was conducted to investigate the effects of dietary lipid levels on survival, growth, activities of digestive enzymes and fatty acid composition of half-smooth tongue sole (Cynoglossus semilaevis). Fish oil and phospholipids were used as the lipid sources. Five isoproteic diets were formulated with graded levels of lipid (6.68%,9.84%,13.47%,17.89% and 21.88% dry weight). Each diet was randomly allocated to triplicate groups of 150 larval tongue sole (35 DAH,54mg), and live prey (LP) were used as the control diet. During the feeding experiment, water temperature was kept at 23±1℃, salinity was 30±3‰ and light intensity was about 7.9WM-2 during daytime at the water surface. Results showed that, the survival rate (SR) of larvae increased significantly with increasing dietary lipid from 6.68% to 13.47% and then decreased significantly, but SR in all treatments were much lower than the control. The specific growth rates (SGR) of larvae fed the diet with 13.47% lipid were significantly higher than other treatments and the control. Larvae fed 13.47% or 17.89% dietary lipid showed higher trypsin, lipase, leucine aminopeptidase (LAP) and alkaline phosphatase (AP) activities than other treatments and the control, while amylase activity nearly showed reverse trend with them. The fatty acid composition of the tongue sole larvae was well correlated with dietary fatty acid profile. However, the contents of saturated fatty acid (SFA) (% total fatty acid) and n-6 poly unsaturated fatty acid (PUFA) in whole body were lower than in diets, and whole-body DHA and EPA contents of larvae fed 13.47% and 17.89% dietary lipid were higher than other treatments, but no significant difference (P>0.05) was found in all treatments.2. Molecular cloning and nucleotide sequence analysis for lipogenic and lipolytic key enzymes, and effects of dietary lipid on mRNA expression of these genes in larval half-smooth tongue sole (Cynoglossus semilaevis)The lipogenic key enzymes (Acetyl-CoA carboxylase, ACC and Fatty acid synthase, FAS)and the lipolysis related key enzymes (Hormone-sensitive lipase, HSLand Adipose triglyceride lipase, ATGL), were cloned and the effects of dietary lipid level on ACC, FAS, HSL and ATGL genes expression were investigated in half-smooth tongue sole(Cynoglossus semilaevis) larvae. The obtained partial cDNA ofACC1, ACC2, FAS, HSL and ATGL were 661bp,732bp,505bp,589bp and 581bp. respectively, and they shared high identities with many other fish species. A 30-day feeding trial was conducted to investigate the effects of dietary lipid level on ACC1, ACC2, FAS, HSL and ATGL genes expression. Five isoproteic diets were formulated with graded levels of lipid ranging from 6.68% to 21.88% dry weight. Each diet was randomly allocated to triplicate groups of 150 larval tongue sole (35 DAH,54±1 mg) and fish were fed five times daily to apparent satiation during the feeding experiment. The relative mRNA expression level was decided by real-time quantitative polymerase chain reaction (qPCR). Results showed that, no significant differences of both ACC1 and ACC2 genes expression were observed among different lipid treatments (P>0.05). High-lipid diet (21.88%) depressed FAS expression andpromoted HSL expression of larval visceral mass significantly (P<0.05), while dietary lipid level did not affect the ATGL mRNA expression significantly (P>0.05).3. Effects of dietary arachidonic acid(ARA) levels on survival, growth, activities of digestive enzymes and fatty acid composition of larval half-smooth tongue sole (Cynoglossus semilaevis)A 30-day feeding trial was conducted to investigate the effects of dietary arachidonic acid (ARA) on survival, growth, activities of digestive enzymes and fatty acid compositionof larval half-smooth tongue sole, Cynoglossus semilaevis. Defatted fish meal, krill meal, squid meal and hydrolyzed fish meal, together with casein were chosen as primary protein sources.Six isoproteic and isolipidic diets were formulated with graded levels of ARA (0.01%,0.39%,0.70%,1.07%,1.42% and 2.86% dry weight) and the ratio of dietary docosahexaenoic to eicosapentaenoic acid (DHA/EPA) was approximately fixed at 2.0. Each diet was randomly allocated to triplicate groups of 180 larvae (35 DAH,54±0.2 mg), and larvae were fed five times daily to apparent satiation for 30 days. During the feeding experiment, water temperature was kept at 23±1℃, salinity was 30±3‰ and light intensity was about 7.9WM-2 during daytime at the water surface.Results showed that survival ratio and specific growth rate in treatment with 1.42% ARA were significantly higher than other treatments. As dietary ARA increased, activities of trypsin, leucine aminopeptidase (LAP) and alkaline phosphatase (AP) increased at first and then decreased significantly, and the highest activity showed in larvae fed the diet with 1.07% or 1.42% ARA. However, activities of amylase had an opposite trend with trypsin. The whole-body fatty acid composition of tongue sole larvae was well correlated with dietary fatty acid profile, and EPA content of larvae decreased with increasing dietary ARA. These results suggested that higher dietary ARA level (approximately 1.42% dry weight) significantly enhanced survival, growth and activities of digestive enzymes, and affected the fatty acid composition of larval tongue sole.4. Molecular cloning for ARA metabolism related key enzymes, and effects of dietary ARA on mRNA expression of these genes in larval half-smooth tongue sole (Cynoglossus semilaevis)Cyclooxygenase (COX), Lipoxygenase (LOX) and Cytochrome P450 (CYP450)are the primary three metabolic pathways of ARA found in mammal. The present study cloned and obtainedthe partial sequences of COX-la (cyclooxygenase la), COX-1b (cyclooxygenase 1b), COX-2 (cyclooxygenase 2),5-LOX (5-lipoxygenase) and CYP2J6-like (cytochrome P450 2J6-like)in half-smooth tongue sole (Cynoglossus semilaevis). The lengthes of COX-la, COX-1b, COX-2,5-LOX and CYP2J6-like were 1021bp,721bp,1021bp,961bp and 901bp, respectively. They shared high identities with many other fish species, and their Genebank accession number were KF533723, KF533724, KF533725, KF533726 and KJ578726, respectively. The effects of dietary ARA level onCOX-1a, COX-1b, COX-2,5-LOX and CYP2J6-like genes expression were also investigated intongue solelarvae.Six isoproteicand isolipidic diets were formulated with graded levels of ARA (0.01%, 0.39%,0.70%,1.07%,1.42% and 2.86% dry weight) and the ratio of dietary docosahexaenoic to eicosapentaenoic acid (DHA/EPA) approximately fixed at 2.0. Each diet was randomly allocated to triplicate groups of 1801arvae (35 DAH, 54±0.2mg), and larvae were fed five times daily to apparent satiation for 30 days. The relative mRNA expression level was decided by real-time quantitative polymerase chain reaction (qPCR). Results showed that, increase in dietary ARA up to 1.07% or 1.42% significantly raised COX-2 and 5-LOX genesexpression(P<0.05), whileno significant differences were found in genes expression of COX-1a, COX-1b and CYP2J6-like(P>0.05). |
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