Effects Of Dietary Amino Acid Patterns And Arginine Levels For Hybrid Grouper (Epinephelus Fuscoguttatus ♀× Epinephelus Lanceolatus ♂) Juveniles

This thesis aimed to evaluate effects of dietary amino acid patterns and arginine levels on growth, feed utilization and mRNA expression levels of some related genes of hybrid grouper(Epinephelus fuscoguttatus ♀ x Epinephelus lanceolatus ♂) juveniles. The main contents and results of this thesis were presented as follows:1. Effects of dietary amino acid patterns on growth, feed utilization and hepatic IGF-I,TOR gene expression levels of hybrid grouper (Epinephelus fuscoguttatus ♀ × Epinephelus lanceolatus ♂) juveniles.A 7-week growth trial was conducted to evaluate effects of dietary amino acid patterns on growth,feed utilization and hepatic IGF-I,TOR genes expression levels of hybrid grouper(Epinephelus fuscoguttatus ♀ × Epinephelus lanceolatus (♂) juveniles. Four isoenergetic (350 kcal per 100 g dry matter), isoproteic (53.5% of dry matter) and isolipidic (7% of dry matter)experimental diets were formulated with crystalline-amino acids (AAs) replacing approximately 42% fishmeal protein-bound nitrogen to keep dietary AA patterns in line with the overall AA patterns of whole-egg protein (WEPAA), whole-body protein (WBPAA),muscle protein (MCPAA) of hybrid grouper and fishmeal (anchovy) protein (FMPAA),respectively. Each experimental diet was fed to triplicate groups of 15 hybrid grouper juveniles (average initial weight of 10.8 g/fish) which were stocked into small floating cages(L 120 cm xW70 cmx H 50 cm). Experimental cages were labeled and located in four connective 6m3 indoor concrete tanks (L 3 m ×W2 m × H 1 m) with 3 cages occurring in each tank.Weight gain (WG) of fish fed the FMPAA diet was significantly (P < 0.05) higher than that of fish fed the WEPAA diet or the WBPAA diet. Values of daily feed intake (DFI) of fish fed the WEPAA diet or the WBPAA diet was significantly higher than that of fish fed the MCPAA diet or the FMPAA diet. Fish fed the FMPAA diet had significantly lower feed conversion ratio (FCR), higher protein efficiency ratio (PER) and higher protein productive value (PPV) compared to fish fed other experimental diets. Whole-body protein content of fish fed the FMPAA diet was significantly higher than that of fish fed the WEPAA diet or the WBPAA diet. Muscle amino acid compositions were influenced by dietary amino acid pattern.Plasma total protein (TP) concentration of fish fed the FMPAA diet was significantly higher than that of fish fed the WEPAA diet or the WBPAA diet. The relative mRNA expression levels of insulin like growth factor-I (IGF-I) gene in liver of fish fed the FMPAA diet and the MCPAA diet were significantly higher than those of fish fed the WEPAA diet and the WBPAA diet. Fish fed the FMPAA diet showed significantly higher hepatic target of rapamycin (TOR) gene expression level than fish fed other experimental diets. Results of this study indicated that the amino acid pattern of fish meal (anchovy) protein was more suitable as a reference amino acid pattern in diet of juvenile hybrid grouper compared to the amino acid pattern of whole-egg protein,whole-body protein or muscle protein of this fish species.2. Effects of dietary arginine levels on growth, feed utilization and mRNA expression levels of related genes in liver of hybrid grouper (Epinephelus fuscoguttatus ♀ × Epinephelus lanceolatus ♂) juveniles.An 8-week growth trial was conducted to evaluate effects of dietary arginine requirement on growth, feed utilization and expressions of genes relative to target of rapamycin (TOR)signaling pathway in hybrid grouper (Epinephelus fuscoguttatus ♀ × Epinephelus lanceolatus♂) juveniles. Seven isoenergetic (350 kcal per 100 g dry matter), isoproteic (53.5% of dry matter) and isolipidic (7% of dry matter) were formulated to contain seven graded dietary arginine levels ranging from 1.9 to 4.7% (dry weight) at about 0.5% increments. Dietary amino acid nitrogen contents were adjusted to be constant by replacing arginine with aspartic acid and glutamate (1:1) mixture. Each diet was randomly assigned to triplicated groups of 16 juvenile fish (about 11.7 g) twice daily (08:00h and 16:00h).Results indicated that the weight gain (WG) of fish were significantly improved with dietary Arg increasing. Fish fed diets with 3.8%, 4.1% Arg level had higher WG than fish fed diets with 1.9%, 2.2% or 4.7% Arg levels. The lowest WG of fish was found in the 1.9% Arg group. Feed conversion ratio (FCR) showed a decline trend with the increasing of dietary arginine level. Protein efficiency ratio (PER) and protein productive value (PPV) were increased with dietary arginine level up to 3.1% and thereafter decreased. Fish fed the 3.8%arginine diet had higher whole-body and muscle protein contents than fish fed diets with other arginine levels. Whole-body protein content was increased as dietary arginine increasing up to 3.8% and then decreased. Similar trends were observed in muscle protein content. Relative mRNA expression levels of insulin like growth factor-I (IGF-I), TOR and ribosomal protein S6 kinase-polypeptide 1 (S6K1) mRNA in fish fed with 3.8% dietary arginine were significantly higher than those in fish fed diets with other arginine levels. Based on the quadratic regression analysis of weight gain against dietary arginine levels, the optimal dietary arginine requirement for hybrid grouper was estimated to be 3.66% of the diet(corresponding to 6.8% of dietary protein on a dry weight basis).3. Effects of dietary arginine levels on immune response and mRNA expression levels of some immunity-related genes in head kidney of hybrid grouper (Epinephelus fuscoguttatus ♀× Epinephelus lanceolatus ♂) juveniles.An 8-week feeding trial (trial 1) and consecutively a 3 6-h environmental copper exposure trial (trial 2) were conducted to investigate the effects of graded dietary arginine(Arg) levels (1.9%-4.7% of dry matter) on the hepatic antioxidant enzymes activities,hematological parameters, hepatic histology index and mRNA expression of hepatic genes of antioxidant-immune-cytokine-related signaling molecules in juvenile hybrid grouper(Epinephelus fuscoguttatus ♀ x Epinephelus lanceolatus ♂). In trial 1, seven isoenergetic(350 kcal per 100 g dry matter), isoproteic (53.5% of dry matter) and isolipidic (7% of dry matter) were formulated to contain seven graded dietary arginine levels ranging from 1.9 to 4.7% (dry weight) at about 0.5% increments. Results of trial 1 indicated that survival rates of fish showed no significant differences among all experimental treatments. The plasma immunoglobulin M (IgM) contents were significantly improved by the increasing of dietary Arg and reached a peak value at 3.1% -3.8% Arg level of dry matter. Similar trend was found in activities of hepatic catalase (CAT). With the increasing of dietary Arg level, the values of malondialdehyde (MDA) were decreased. In the team of immunity-related genes, the head kidney kelch-like-ECH associated protein 1 (Keapl) gene expression of fish fed with 3.1%-4.1% Arg level diets were significantly lower than those in other groups. The gene expression of interleukin 1 beta (IL-1β) were increased with the dietary arginine level up to 3.8% and thereafter decreased.After exposed to waterborne Cu for 36 hours, fish fed diets with the Arg level being lower than 2.7% had low survival rates. With dietary Arg levels above 4.1%, plasma IgM contents and lysozyme activities as well as the hepatic CAT activities of fish were significantly enhanced (P < 0.05), and thereafter tended to be decreased. Values of MDA in liver showed an opposite variation trend as IgM. The results of liver histology showed that exposure of grouper specimens to Cu induced produced progressive deleterious morphological alterations in liver. Fish fed diets with 1.9% arid 2.2% Arg levels were shown severe damaged. Fish fed the diet with the 3.8% Arg level were slightly damaged compared to other treatments. Supplementations of dietary Arg significantly up-regulated the gene expressions of Nrf2 and Hsp70 of head kidney. The mRNA level of Keapl, IL-1β, IL-8 of head kidney were significantly down-regulated by dietary Arg. Results of this study indicated that the supplementation of dietary 3.1%-4.1% Arg levels improved the antioxidant capacity and immunity of hybrid grouper.