Effects Of Crystalline Amino Acid And Fish Hydrolysate On Growth Performance, Digestive Enzymes And PepT1Gene Expression In Tongue Sole (Cynoglossus Semilaeviss Günther) Larvae

Tongue sole (Cynoglossus semilaeviss Günther) is widely farmed in China. Studywas conducted to investigate the effects of fish meal replacement with crystallineamino acid and size fractionated fish hydrolysate on growth performance anddigestion, absorption in tongue sole larvae. The experiments content and results havebeen presented as follows:1. Effects of fish meal replacement with crystalline amino acid on growthperformance, activities of digestive and metabolic enzymes in tongue solelarvaeIn this experiment, five isoproteic and isolipidic diets were formulated to replace0%,25%,50%,75%,100%fish meal (0%,25%CAA,50%CAA,75%CAA and100%CAA) separately. At the replacement level of25%, another diet was formulatedwith amino acids coated with tripalmitin (C-25%CAA). The results showed SGRsignificantly decreased with increasing dietary crystalline amino acids (P<0.05).However, fish fed the diet with the C-25%CAA was significantly higher than fish fedthe diet without coating (25%CAA). Survival of larvae significantly decreased withincreasing dietary crystalline amino acids (P<0.05), but there was no significantdifferences between the C-25%CAA and0%CAA group (P>0.05). The crude proteincontent in larvae body significantly decreased with increasing dietary crystallineamino acids (P<0.05). Fish fed with fish meal was significantly higher than others(P<0.05), and there was no significant differences between C-25%CAA and25%CAAgroup (P>0.05). The activities of trypsin significantly decreased with increasingdietary crystalline amino acids (P<0.05), but there was no significant differencesbetween C-25%CAA and0%group (P>0.05). The activities of amylase significantlyincreased with increasing dietary crystalline amino acids (P<0.05). The activities of leucine-amino peptidase and alkaline-phosphatase in intestine and purified brushborder membrane of intestine had the same variation with trypsin. Glutamic-pyruvictransaminase and Glutamic-oxalacetic transaminease significantly increased withincreasing dietary crystaline amino acids. In conclusion, crystalline amino acids in themicro-diet significantly influenced the growth, digestive and metabolic enzymes oftongue sole larvae. Compared with other replacement groups, crystalline amino acidsreplace with25%fish meal protein in the micro-diet can enhance the growth andother physiological indexs in tongue sole larvae.2. Effects of fish meal replacement with size fractionated fish hydrolysate andcrystalline amino acid on growth performance and activities of digestiveenzymes in tongue sole larvaeThe study was conducted to investigate the effects of replacement of fish mealprotein with size fractionated fish hydrolysate and crystalline amino acid on growth,survival, activities of digestive enzymes in tongue sole larvae. Five isoproteic andisolipidic diets were formulated to replace30%fish meal, respectively. Whichincluded fish meal group, fish protein hydrolysate group, permeate after ultrafiltrationof fish hydrolysate group, retenate after ultrafiltration of fish hydrolysate group andcrystalline amino acids group. Each micro diet was randomly fed to triplicate groupsof150fish. The results showed that Larvae in fish meal group had the best SGR,which was significantly higher than other groups (P<0.05). No significant differencesin SGR were observed between permeate after ultrafiltration of fish hydrolysate groupand crystalline amino acids group. Results of survival rate showed the same trends toSGR. Whole body protein (%, wet weigh) in larvae fed permeate after ultrafiltrationof fish hydrolysate group was significantly higher than that in other treatments(P<0.05), except for the fish meal group. Activities of trypsin in pancreatic andintestinal segments showed a similar trend, and there were no significant differencesbetween fish meal group and permeate after ultrafiltration of fish hydrolysate group(P>0.05). The activities of amylase in pancreatic segments of larvae fed permeateafter ultrafiltration of fish hydrolysate group was significantly higher than othertreatments (P<0.05). No significant differences were observed between fish meal group and permeate after ultrafiltration of fish hydrolysate group (P>0.05). Thespecific activities of leucine-amino peptidase (LAP) and alkaline-phosphatase (AP) inintestinal segments and brush border membranes all showed the similar trend withtrypsin activities. Results of the present study showed that low molecular peptidesand crystalline amino acids could significantly improve growth performance oftongue sole larvae, suggesting that the low molecular protein hydrolysates cansignificantly enhance the larvae s pancreatic secretion capacity.3. Molecular cloning, characterization and the effects of fish meal replacementwith size fractionated fish hydrolysate and crystalline amino acid on geneexpression of PepT1in tongue sole larvaeIn the present study, the cDNA of oligopeptide transporter from tongue sole wascloned by homology cloning with degenerate primer and RACE techniques. Thefull-length cDNA of PepT1was of2684bp, including an open reading frame2190bp,5UTR98bp and3UTR396bp, encoding a polypeptide729amino acids. It has12potential membrane-spanning domains with a large extracellular loop betweentransmembrane domains9and10, and also predicts that both the amino and thecarboxyl termini are on the cytoplasmic side of the membrane. BLAST analysisrevealed that amino acids of membrane-spanning domains are highly conserved,whereas the large extracellular loop shows lower identity with PepT1from otherspecies. The expression levels of PepT1in visceral mass was measured by real-timePCR after larvae were fed diets fish meal, fish protein hydrolysate, permeate afterultrafiltration of fish hydrolysate, retenate after ultrafiltration of fish hydrolysate andcrystalline amino acids. The results showed that the gene expression of PepT1invisceral mass of fish larvae fed with fish protein hydrolysate group, permeate afterultrafiltration of fish hydrolysate group and retenate after ultrafiltration of fishhydrolysate group were significantly higher than fish meal group (P<0.05). Itindicated that different fractions of fish protein hydrolysate and crystalline amino acidin diets significantly affect the PepT1expression in tongue sole larvae, suggesting thatprotein hydrolysates can induce the expression of PepT1.