Study On Effects Of Dietary Lysine And Arginine On Growth Performance, And The Arginine/lysine Antagonism Mechanism In Juvenile Black Sea Bream, Acanthopagrus Schlegelii

The effects of dietary L-lysine and L-arginine levels on growth performance, feed utilization, body compositions, enzymes activity and serum biochemical parameters of black sea bream Acanthopagrus schlegelii juvenile were studied in the present research. The effects of dietary arginine on growth relating genes in tissues of black sea bream were studied to discuss the mechanism of dietary EAA controlling growth of fish. In this research, the effect of dietary imbalanced arginine/lysine was estimated and the potential mechanism of antagonism between these two EAAs in black sea bream was also discussed. The main contents and results in the current research present as follows.Trial 1. An 8-week feeding experiment was conducted to determine the quantitative L-lysine requirement of juvenile black sea bream (initial mean weight:9.13±0.09 g, mean±SD) in eighteen 300-L indoors flow-through circular fibreglass tanks by feeding diets containing six levels of L-lysine ranging from 2.08% to 4.05% dry diet at about 0.40% increments. The isonitrogenous and isoenergetic diets were formulated to simulate the amino acid profile of 38% whole fish body protein except lysine. Each diet was assigned to triplicate groups of 20 fish in a completely randomized design. The results showed that weight gain (WG) and specific growth rate (SGR) increased with increasing levels of dietary lysine up to 3.25% (P<0.05) and both showed a declining tendency thereafter.Dietary increasing lysine level have promoting effect on feed efficiency ratio (FER) and protein efficiency ratio (PER). The apparent digestibility coefficients (ADCs) of crude protein and gross energy, the protease and lipase activies were also influenced by dietary lysine level (P<0.05). The whole body crude protein and crude lipid contents were significantly affected (P<0.05) by dietary lysine level, while moisture and ash showed no significant differences. While for EAAs compositions, except for Val and His contents in dorsal muscle, Val, Phe, Met and Thr contents in liver were independent with dietary treatments (P>0.05),, other EAAs contents were significantly affected by dietary lysine level. The highest free lysine leve in serum was obtained in Diet 4 group. The activity of glutamic-oxalacetic transaminase (AST) and the glutamic-pyruvic transaminase (ALT) in serum decreased significantly when lysine level increasing (P<0.05), however, no significant difference (P>0.05) were found in contents of triacylglycerol and glucose nor the activities of catalyse and superoxide dismutase.Analysis of dose (lysine level)-response (SGR) with second order polynomial regression suggested a requirement of juvenile black sea bream to be 3.32% dry diet or 8.64% dietary protein.Trial 2. An 8-week feeding trial was conducted to determine the dietary arginine requirement of juvenile black sea bream in eighteen 350-L indoors flow-through circular fibreglass tanks. Six isonitrogenous and isoenergetic diets were formulated to contain graded levels of L-arginine (1.85,2.23,2.51,2.86,3.20 and 3.46% dry diet) from dietary ingredients and crystalline arginine. Each diet was randomly assigned to triplicate groups of 25 juvenile fish (10.51±0.15 g). Results showed that no significant difference in survival in experimental fish (P>0.05). SGR increased with increasing dietary arginine levels up to 2.51% and remained nearly the same thereafter. PER and protein productive value (PPV) showed increase tendency and then level off. FER was the poorest in Diet 1(P<0.05), the highest value of FER (91.32%) was observed in Diet 5, but no statistical difference was found when comparing with the Diet 3, Diet 4 or Diet 6 (P>0.05). ADCs of dry matter, crude protein and gross energy significantly improved up to 2.86% arginine diet and decreased at different extent thereafter, however, ADCs of crude lipid were unaffected. Fish fed 1.85% arginine diet had significant lower protein content in whole body and dorsal muscle than those fed diets supplemented with or more than 2.86% arginine. Lipid content decreased and lower value occurred at 3.46% dietary arginine (P<0.05). There were no significant differences in ash or moisture contents among dietary treatment (P>0.05). The dietary EAAs composition in whole body of black sea bream was significantly influenced by dietary arginine with exception of Thr. Arginine retention increased with dietary arginine level, then declined slightly at 3.46%arginine diet. Dietary increasing arginine level elevate arginase activity in liver up to 2.51% arginine diet (P<0.05), and the enzyme maintained at stable level thereafter (P>0.05). Almost all serum biochemical parameters were significantly affected by dietary arginine level except for cholesterol level.Broken-line regression based on SGR and second order polynomial regression based on PER indicated optimum dietary arginine requirement for juvenile black sea bream were 2.79 and 3.09% diet, corresponding to 7.74 and 8.13% of dietary protein, respectively.Trial 3. An 8-week feeding trial was conducted to determine the effects of imbalanced arginine/lysine levels in the diets of black sea bream juvenile. Eight isonitrogenous diets were formulated using a combination of intact protein and crystalline amino acids to contain different arginine/lysine levels (2.83/3.25,3.46/4.06,2.27/4.09,3.51/2.70,2.31/2.74,2.87/4.03, 2.91/4.68 or 2.85/5.24,% dry diet). The control diet contained 2.83% and 3.25% of arginine and lysine, respectively, which is known to satisfy the requirements of this fish. Each diet was fed to triplicate groups of 25 fish (10.0±0.1g).The results showed that dietary imbalanced levels of arginine and lysine had serious influences on growth performance and feed utilization of black sea bream, these corresponding indicators exhibited decreasing tendency in different extent. Based on growth performance, feed utilization efficiency, ADCs of dietary lysine and arginine, lysine and arginine retention ratio in body as well as liver arginase activity obtained in the present study suggest that arginine/lysine antagonism may exist in black sea bream. Adding arginine level to diets which containing superfluous lysine could alleviate this antagonism in this marine fish species.Trial 4. In the present study, total RNA extracted from black sea bream liver and dorsal muscle were reverse transcribed as described for TaqMan assays. Cloning primers were designed based on conserved regions of the fish genes'sequences from NCBI website to amplify IGF-I, IGF-IR and Arginase genes of black sea bream, and the sequence length were 890 bp,570bp and 430 bp, respectively. Blast analysis showed that the high similarity among black sea bream and other fish species, which indicated that these three sequences correspond to their corresponding gene family.Trial 5. The liver and dorsal muscle samples obtained from the arginine requirement study were used for genes expression analyzing. The results showed that the lowest level of IGF-I gene expression in liver was found in fish fed diet without arginine supplement (Diet 1) (P<0.05). IGF-I mRNA expression level increased with increasing arginine level to 3.25% (P<0.05), and then level off (P>0.05). The IGF-I mRNA expression in dorsal muscle presented a similar variation tendency with it was in liver. The relative lower and higher IGF-IR mRNA expression in dorsal muscle were observed in fish fed Diet 1 and Diet 4 (P<0.05), respectively. GHR gene mRNA expression tended to be enhanced by dietary arginine level, however, no significant differeces were found among the later four arginine level diets (P>0.05). Higher arginase gene expression levels in liver were detected in experimental fish fed Diet 5 and Diet 6, while lower values were seen in Diet 1 and Diet 2 (P<0.05).The liver samples obtained from the arginine/lysine ratios study were also used for genes expression analyzing. The results showed that fish fed diet with appropriate level of arginine and lysine simultaneously could inducing relative higher IGF-I mRNA expression in liver (P<0.05). Also, adding arginine into diet with excessive lysine had comparable gene expression level with control group. Black sea bream fed diet with inadequate lysine and arginine (Diet 5), or fish fed diets with constant arginine level but increasing lysine content, hepatic IGF-I mRNA expression decline significantly (P>0.05). Similarly, when comparing with the control group, dietary superfluous lysine content (Diet 7 and Diet 8) depressed hepatic Arginase gene mRNA expression significantly. In Diet 3 and Diet 5, which containing insufficient arginine level, Arginase mRNA expression levels also showed down regulation. These was no significant difference on regulation effect by dietary treatment on hepatic Arginase gene mRNA level among the control diet, Diet 2, Diet 4 and Diet 6 group (P>0.05).Results of this trial indicated that the effect of growth-promoted of dietary arginine was by regulated growth axis genes transcriptional control in black sea bream. Elevations in liver arginase gene mRNA expression have been observed under the dietary arginine level, and lysine level had influence on this gene expression.The following conclusions could be drew based on the present research.1, Lysine and arginine are necessary for black sea bream juvenile in diet for growth.2, Based on the growth performance of black sea bream juvenile, the optimal lysine and arginine levels in diet are 3.32% dry diet, corresponding to 8.64% dietary protein; and 2.79% diet, corresponding to 7.74% of dietary protein, respectively.3, Judged by growth performance, feed utilization efficiency and metabolism of amino acid, antagonism between arginine and lysine exists in black sea bream.4, The growth of black sea bream could be influenced by dietary arginine level.5, Dietary arginine level influencing the arginase gene expression in black sea bream. Meanwhile, lysine level also could affect this gene expression.