Optimal Requirements And Plasma Glucose Regulation Mechanism Of Branch Chain Amino Acids In Juvenile Hybrid Grouper (Epinephelus Fuscoguttatus ♀ × Epinephelus Lanceolatus ♂)

Multiple feeding trials were conducted in this thesis,and the optimum requirements of branched amino acids and their nutritionally physiological function in hybrid grouper were further studied by using multiple methods and technologies of nutriology,molecular biology and biochemistry.The main results obtained were as followed:1.Studies on the optimum dietary requirement of leucine（Leu）and its nutritionally physiological function in hybrid grouper（Epinephelus fuscoguttatus♀×Epinephelus lanceolatus♂）.Seven experimental diets were formulated to contain graded Leu levels（1.83%to4.71%,dry matter basis）.A mixture containing equal proportions of aspartic acid and glycine（1:1）was used to substitute for Leu in the low-Leu diets.Hybrid grouper juveniles（average initial body weight:6.92±0.03g）was randomly distributed into 21 small floating cages at density of 12 fish/cage.Fish were fed twice daily（08:00 and 16:30）to apparent satiation for 9 weeks.Results showed that weight gain%（WG%）or protein productive value（PPV）was increased as dietary Leu level increased,reaching a peak value at the 3.28%dietary Leu level,and values of these two parameters started to reduce as dietary Leu level further rose.Fish fed 3.28%and 3.70%dietary Leu levels had higher growth hormone（GH）expressions in pituitary than fish fed other levels of Leu.For insulin like growth factor 1（IGF-1）,growth hormone receptor 1（GHR1）genes in hepatic,the fish fed 3.28%Leu diet had higher expressions than fish fed other dietary Leu levels.Expressions of rapamycin（TOR）and S6-kinase 1（S6K1）m RNA in fish fed 3.28%dietary Leu were significantly higher than those in fish fed diets with other Leu levels.Gut morphometric indices of experimental fish were improved when dietary Leu level was 3.28%or 3.70%.The analysis of a quadratic regression model demonstrated that optimum dietary Leu level for maximum growth of experimental fish was 3.25%（dry matter）.2.Studies on the optimum dietary requirement of isoleucine（Ile）and its nutritionally physiological function in hybrid grouper.Seven diets were formulated to contain graded Ile levels（0.73 to 3.08%,dry matter basis）.Alanine was used to substitute for Ile in the low-Ile diets.Each experimental diet was hand-fed to triplicate groups of 12 experimental fish for 6 weeks.After the sampling of the growth trial,the remaining fish in each group were fed their corresponding diets for2 d and then exposed to 4 mg Cu²⁺·L^-1 water for 24 h.Results showed that growth performance and feed utilization were obviously influenced by different Ile treatments（P<0.05）.WG%,PPV,PER,and FE were increased as dietary Ile level increased,reaching a peak value at 1.96%dietary Ile,and values of these indices started to reduce as dietary Ile level further rose.DFI showed an opposite tendency of variations as FE.Among all experimental treatments,fish fed 1.96%dietary Ile had the highest expression of growth and protein synthesis related genes,including GH in pituitary,IGF-1,GHR1,TOR and S6K1 in hepatic.Parameters（h F,w F,h E,h MV）about gut micromorphology of 1.96%Ile fed fish were better compared to those of fish fed other Ile levels.After the exposure to 4 mg Cu²⁺·L^-1 water for 24 hours,fish fed 1.96%dietary Ile had the highest survival and the best immunologic manifestation among all experimental treatments.Generally,optimum dietary Ile requirement of hybrid grouper was 1.98%.3.Studies on the optimum dietary requirement of valine（Val）and its nutritionally physiological function in hybrid grouper.Seven diets were formulated to contain graded Val levels（1.21 to 1.94%,dry matter basis）.Alanine was used to substitute for Val in the low-Val diets.Each experimental diet was hand-fed to fish in triplicate（12 fish/tank）for 6 weeks.Results showed that WG%,PPV,PER,FE were increased as dietary Val level increased,reaching a peak value at1.58%dietary Val,and values of these indices started to reduce as dietary Val level further rose.Fish fed 1.21%dietary Val had significantly higher DFI than fish fed other dietary Val levels.Fish fed 1.58%Val had better gut micromorphology than fish fed other dietary Val levels.Val had higher Expression of GH in pituitary,IGF-1,TOR and S6K1 in liver of1.58%Val fed fish was higher than that of fish fed other levels of Val.The SOD,CAT,LZM activities and Ig M concentrations of serum of fish fed 1.58%dietary Val were higher than those of fish fed other dietary Val levels.Fish fed 1.58%dietary Val had higher expression of Nrf2 in head kiney,and Kelch-like-ECH-associated protein 1（Keap1）showed an opposite tendency of variations as Nrf2.In conclusion,optimum dietary Val requirement based on the maximum growth of hybrid grouper was 1.56%.4.Study on the regulation mechanism of Leu in plasma glucose of hybrid grouperThree experimental diets were formulated to contain graded Leu levels（1.78,3.18%and 4.66%,dry matter basis）.The Leu regulation in plasma glucose and expression of TOR,Gluts and IGF-1 of hybrid grouper was studied.In the long effects trial,each experimental diet was hand-fed to sextuplicate groups of 21 hybrid grouper juveniles（average initial body weight:7.90±0.01）which were randomly distributed into 21 small floating cages glass tanks.In the short effects trial,18 cages of fish were fed commercial feeds.Fish were fed twice daily（08:00 and 16:30）to apparent satiation for 8 weeks.and then fish in both the long effects trial and the short effects trial were injected DMSO+saline and rapamycin inhibitors（Rapamycin,Rapa）,and then fed 1 meal of different Leu levels diets.The sampling was carried out 2 hours after the last meal.Results showed that after the 8-week feeding,WG%,PPV,PER,FE,whole-body and muscle protein content of fish fed 3.18%dietary Leu was significantly higher than that of fish fed 1.78%or 4.66%dietary Leu.Resluts from the long effects trial showed that prior of post the Rapa blocking,plasma glucose concentrations in the fish fed 3.18%dietary Leu were significantly lower than those in the fish fed 1.78%or 4.66%dietary Leu,and the expression levels of hepatic TOR,S6K1 genes and protein,IGF-1,GLUT1 and GLUT2and muscle GLUT1 and GLUT4 genes,plasma IGF-1 and insulin concentrations showed an opposite trend to plasma glucose.After the TOR blocking,plasma glucose levels of fish fed any of the experimental diets elevated,and the m RNA levels of GLUTs and IGF-1were reduced.Results from the short effects trial showed that prior the TOR blocking,plasma glucose concentrations in fish fed 3.18%dietary Leu were significantly lower than those in fish fed 1.78%or 4.66%dietary Leu,and the expression levels of hepatic TOR,S6K1 genes and protein,IGF-1,GLUT1 and GLUT2 genes,plasma IGF-1 and insulin concentrations showed an opposite trend to plasm glucose,but no significant difference in muscle GLUT1 and GLUT4 gene expression was observed.Post the TOR blocking,there were no significant differences in plasma glucose concentrations and expression of those genes among fish fed different dietary Leu levels,but for fish fed any level of dietary Leu,plasma glucose concentration increased and expression of those genes decreased.In conclusion,the optimum dietary Leu inculsion could reduce plasma glucose concentration,activate the expression of hepatic TOR,S6K1 genes and protein,GLUTs and IGF-1 genes,plasma IGF-1 and insulin concentrations;under the effects of Leu,TOR expression had a negative correlation with plasma glucose concentration but a positive correlation with GLUTs,IGF-1,insulin concentrations;Leu could regulate plasma glucose level through the TOR/GLUTs/IGF-1/insulin axis.