Effects Of Dietary Phospholipid, Cholesterol And Their Interactions On Growth Performance, Digestive Enzymes And Expression Of Related Gene Of Tongue Sole (Cynoglossus Semilaevis Günther) Larvae

Tongue sole (Cynoglossus semilaevis Günther) is an important marine fishspecies. Two growth trials were conducted to investigate the effects of dietaryphospholipid, cholesterol and their interactions on growth performance and digestiveenzymes of tongue sole larvae. Based on above, we made a further research on thephospholipid synthesis mechanism caused by dietary phospholipid level. The resultsare summarized as follows:1. Effects of dietary phospholipid on survival, growth performance and activity ofdigestive enzymes in tongue sole (Cynoglossus semilaevis Günther) larvaeThe aim of the study was to determine the influence of dietary phospholipid onsurvival, growth performance and digestive enzymes in tongue sole (Cynoglossussemilaevis Günther) larvae (30days after hatchery, initial average weight:57±0.3mg,initial average body length:2.19±0.2cm), and also to determine the optimumrequirement of phospholipid. Larvae were fed from30d to60d with an isoproteic andisolipidic formulate diet which contained0.0%,2.5%,5.0%,7.5%and10.0%phospholipids separately(PL0, PL2.5, PL5, PL7.5and PL10), live prey (LP) wereused as control diet. The final dietary phospholipids concentrations were2.87%,4.59%,6.36%,8.75%and11.33%, respectively. The results showed that the finalbody length and SGR of larvae first significantly increased then decreased with theincrease of dietary phospholipid levels. The final body length and SGR in diet whichcontaining5%phospholipids significantly higher than diet PL0, PL2.5and LP group(P<0.05). The survival was significantly increased with the increase of dietaryphospholipid levels, wherase there was no significant difference among the dietscontaining phospholipid (P>0.05). Trypsin in digestive tract (pancreatic and intestinalsegments) and brush border membranes enzymes, alkaline phosphatase andaminopeptidase N showed higher activity in the treatment fed the diet containing5% phospholipids. Amylase and lipase in pancreatic and intestinal segment showed theopposite tendency. In this experiment, the results indicated that the optimal levelphospholipid in diet can significantly improve survival, growth performance anddigestive enzymes activities of tongue sole larvae. Base on SGR, the optimumphospholipid requirement was estimated to be around5.76%of diet.2. Effects of dietary phospholipid and cholesterol on survival, growthperformance and digestive enzymes of tongue sole (Cynoglossus semilaevisGünther) larvaeA3×3two-factorial experiment was designed to investigate the effects ofphospholipid levels (0%,5%and7.5%of diet), cholesterol levels (0%,1%and1.5%of diet) and their interaction on survival, growth performance and digestive enzymesof tongue sole (Cynoglossus semilaevis Günther) larvae(30days after hatchery, initialaverage weight:57±0.3mg, initial average body length:2.19±0.2cm). Each diet wasrandomly allocated to triplicate groups of fish in tanks (100×50×40cm), and eachtank was stocked with120fish. The results showed that there were significantinteraction between dietary phospholipid and cholesterol on growth performance oftongue sole larvae (P<0.001). The highest value of specific growth rate (SGR) andfinal body weight were found in the diet containing5%PL without supplementationof cholesterol. The SGR and final body weight significantly decreased with increasingdietary cholesterol (P<0.05). There were significant interaction between phospholipidand cholesterol on digestive enzymes activities of tongue sole larvae (P<0.001).Activities of trypsin in both pancreatic and intestinal segments and brush bordermembranes enzymes, alkaline phosphatase and leucine alanine peptidase activities indiets containing1%or1.5%cholesterol were significantly lower than those fed dietswithout supplementation of cholesterol(P<0.05). Furthermore, the enzymes activitiesof larvae fed diet that containing5%PL without adding cholesterol were significantlyhigher than other diets. Activities of amylase in both pancreatic and intestinal segmentshowed the opposite tendency. In this experiment, the results suggested that dietaryphospholipid played a critical role in saving dietary cholesterol, and the optimaldietary phospholipid (5%) could satisfy the nutrition requirements for tongue solelarvae without supplementation of cholesterol. 3. Molecular cloning, characterization and mRNA expression of CTP: phosphocholinecytidylyltransferase (CCT) from tongue sole (Cynoglossue semilaevis Günter)larvae in response to dietary phospholipidIn the present study, the cDNA of CTP: phosphocholine cytidylyltransferase(CCT) from tongue sole (Cynoglossus semilaevis Günther) was cloned by homologycloning with degenerate primer and RACE techniques. The full-length cDNA of CCTwas of2428bp, including an open reading frame (ORF) of1122bp encoding apolypeptide373amino acids with predicted molecular weight of42.81kDa andtheoretical isoelectric point of6.3. BLAST analysis revealed that CCT shared highsimilarity with other known CCT, and the area between the N-and C-terminus aminoacids portions are the most conserved, wherase the N-and C-terminus amino acidsportions appear to be quite variable. The expression levels of CCT in visceral masswere measured by real-time PCR after tongue sole were fed diets containing gradedlevels of phospholipid. The results showed that the expression of CCT wassignificantly up-regulated in fish fed diet PL-2.5%, and then down-regulated withdietary PL increasing from5%to7.5%. These results indicated that adequate dietaryphospholipid could inhibit the mRNA expression of CCT, speculating thatbiosynthesis of phospholipids is controlled by a feedback mechanism in tongue sole.