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Four diets were formulated with or without fishmeal (FM) and essential amino acid (EAA) supplementation fed to the blunt snout bream throughout 8 weeks. The results showed that total cholesterol level (TC) was decreased significantly against FM reduction (P 0.05). Catalase (CAT) activity and glutathione (GSH) content were significantly augmented and diminished in the group fed diet 3 respectively (P 0.05). Glutathione peroxidase (GPx) activity was elevated significantly either by reducing FM or supplementing EAA (P 0.05), whereas the relative expression of copper-zinc superoxide dismutase (Cu/Zn-SOD) decreased against FM reduction (P < 0.05). The relative expressions of Interleukin 8 (IL-8), tumour necrosis factor-α (TNF-α), interleukin 10 (IL10), and nuclear factor-kappa B (NF-κB) were diminished significantly in FM-reduced groups (P < 0.05). The supplementation of EAA significantly increased the relative expression of signal transducer and activator of transcription 4 (STAT4) (P < 0.05). In conclusion, the results revealed that dietary plant proteins with EAA supplementation significantly affected the physiological status and immune responses in blunt snout bream.

The present study evaluated the mechanisms governing insulin signaling, glucose metabolism, and lipogenesis in juvenile fish fed with different dietary leucine levels. Fish were fed six practical diets with graded leucine levels ranging from 0.90 to 2.94% of dry basis for 8 weeks. The trial results showed that, compared to the control group (0.90%), optimal dietary leucine level (1.72%) resulted in the up-regulation of mRNA expression related to insulin signaling pathway, including target of rapamycin (TOR), insulin receptor substrate 1 (IRS-1), phosphoinositide 3-kinase (PI3K), and protein kinase B (Akt). However, an excessive leucine level (2.94%) led to protein S6 kinase 1 (S6K1) overexpression and inhibited TOR, IRS-1, PI3K, and Akt mRNA expressions. The protein level of TOR, S6K1, IRS-1, PI3K, and Akt showed a similar result with mRNA level of these genes. Optimal dietary leucine level (1.72%) significantly improved plasma insulin content, while high level of leucine showed an inhibiting phenomenon. Optimal dietary leucine level (1.72%) could reduce plasma glucose by enhancing the ability of glycometabolism including improving glucose transporter 2 (GLUT2), glucokinase (GK) expressions and down-regulating phosphoenolpyruvate carboxykinase (PEPCK) expression. While an excessive leucine level (2.94%) resulted in high plasma glucose by inhibiting the ability of glycometabolism including lowering GLUT2 and GK expressions, and improving glucose-6-phosphatase (G6Pase) and PEPCK expressions. The relative expressions of pyruvate kinase (PK) and glycogen synthase (GS) were not significantly affected by dietary leucine levels. Dietary leucine level of 1.33% could improve plasma triglyceride content (TG) by enhancing lipogenesis including improving sterol-response element-binding protein 1 (SREBP1), fatty acid synthase (FAS), acetyl CoA carboxylase (ACC), and glucose-6-phosphate dehydrogenase (G6PDH) expressions compared to the control group (0.90%). Total cholesterol (TC) was not significantly affected by dietary leucine levels. The present results indicate that optimal leucine level could improve glycolysis and fatty acid synthesis through improving insulin sensitivity in juvenile blunt snout bream. However, excessive dietary leucine level resulted in high plasma glucose, which led to insulin resistance by inhibiting the gene expressions of insulin signaling pathway and activating gluconeogenesis-related gene expression.

Facilitative glucose transporters (GLUT) are transmembrane transporters involved in glucose transport across the plasma membrane. In this study, blunt snout bream GLUT2 gene was cloned, and its expression in various tissues and in liver in response to diets with different carbohydrate levels (17.1; 21.8; 26.4; 32.0; 36.3; and 41.9% of dry matter). Blunt snout bream GLUT2 was also characterized. A full-length cDNA fragment of 2577 bp was cloned, which contains a 5'-untranslated region (UTR) of 73 bp, a 3'-UTR of 992 bp, and an open reading frame of 1512 bp that encodes a polypeptide of 503 amino acids with predicted molecular mass of 55.046 kDa and theoretical isoelectric point was 7.52. The predicted GLUT2 protein has 12 transmembrane domains between amino acid residues at 7-29; 71-93; 106-123; 133-155; 168-190; 195-217; 282-301; 316-338; 345-367; 377-399; 412-434; and 438-460. Besides, the conservative structure domains located at 12-477 amino acids belong to the sugar porter family which is the major facilitator superfamily (MFS) of transporters. Blunt snout bream GLUT2 had the high degree of sequence identity to four GLUT2s from zebrafish, chicken, human, and mouse, with 91, 63, 57, and 54% identity, respectively. Quantitative real-time (qRT) PCR assays revealed that GLUT2 expression was high in the liver, intestine, and kidney; highest in the liver and was regulated by carbohydrate intake. Compared with the control group (17.1%), fed by 3 h with higher starch levels (32.0; 36.3; and 41.9%), increased plasma glucose levels and glycemic level went back to basal by 24 h after treatment. Furthermore, higher dietary starch levels significantly increase GLUT2, glucokinase (GK), and pyruvate kinase (PK) expression and concurrently decrease phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6P) mRNA levels (P 0.05), and these changes were also back to basal levels after 24 h of any dietary treatment. These results indicate that the blunt snout bream is able to regulate their ability to metabolize glucose by improving GLUT2, GK, and PK expression levels and decreasing PEPCK and G6P expression levels.

An 11-week feeding trial was carried out to investigate the effects of supplemented chromium picolinate (Cr-Pic) on the growth, whole-body composition, and relative mRNA expression related to lipogenesis and glucose metabolism in juvenile blunt snout bream. Seven isonitrogenous and isoenergetic diets with graded Cr supplementation levels were fed to triplicate groups. The final weight (FW), feed conversion ratio (FCR), and specific growth rate (SGR) were improved with increasing dietary Cr supplementation levels up to 0.4 mg/kg, and thereafter showed relatively constant. However, 12.0 mg/kg dietary Cr supplementation decreased growth and feed utilization. Based on SGR and FCR, the optimal dietary Cr supplementation level for the juvenile was estimated to be 0.28 mg/kg. Significantly higher plasma insulin levels were found in juvenile fed diets with 0.4 and 0.8 mg/kg Cr supplementation compared to those fed diet sans supplemented Cr. Plasma glucose levels decreased with increasing dietary Cr supplementation, and the lowest value was remarked in the group added 3.2 mg/kg of Cr. Adding 0.4-0.8 mg/kg Cr enhanced insulin receptor substrate 1 (IRS-1), phosphoinositide-3-kinase (PI3K), and pyruvate kinase (PK) and inhibited expression of phosphoenolpyruvate carboxykinase (PEPCK), glucose-6-phosphatase (G6Pase), and glycogen synthase (GS) mRNA levels. High dietary Cr (12.0 mg/kg) supplementation resulted in high G6Pase and PEPCK expression. The highest content of whole-body lipid was remarked in fish fed with 0.4 mg/kg dietary Cr, which related to the enhanced gene expression related to lipogenesis; thereafter, mRNA levels showed a diminishing trend. These findings indicate that optimum dietary Cr-Pic supplementation has a positive effect on growth and blood glucose homeostasis by modifying the mRNA levels related to glucose metabolism and lipogenesis in juvenile blunt snout bream.

The present study assessed the effects of dietary arginine on intestinal antioxidant status and immunity involved in Nrf2 and NF-κB signaling pathway in juvenile blunt snout bream. Fish were fed three practical diets with graded arginine levels (0.87%, 1.62% and 2.70%) for 8 weeks. Compared with the control group (0.87%), the counts of white blood cell (WBC), red blood cell (RBC) and hemoglobin (HGB) content were significantly improved at dietary arginine levels of 1.62% (P0.05). Plasma albumin (ALB) levels and alkaline phosphatase (ALP) activities were significantly improved at dietary arginine levels of 1.62% and 2.70% (P 0.05). Alanine transaminase (ALT) activity was decreased in fish fed with 1.62% dietary arginine level (P0.05). Plasma glutathione peroxidase (GPx) activities, copper-zinc superoxide dismutase (Cu/Zn-SOD) activities, total antioxidant capacity (T-AOC) activities and glutathione (GSH) levels were significantly increased at dietary arginine levels of 1.62% and 2.70% (P0.05). Plasma total superoxide dismutase (T-SOD) activities and catalase (CAT) activities were significantly improved in fish fed with 1.62% dietary arginine level. Significantly higher manganese superoxide dismutase (Mn-SOD) activity was observed in fish fed with 1.62% dietary arginine level compared with 2.70% dietary arginine level (P0.05). 1.62% and 2.70% dietary arginine levels significantly lowered malondialdehyde (MDA) levels. The relative expression of nuclear factor erythroid 2-related factor 2 (Nrf2) was significantly increased in fish fed with 1.62% dietary arginine level, inversely, the relative expression of Kelch-like ECH-associated protein 1 (Keap1) showed a converse trend. 1.62% and 2.70% dietary arginine levels significantly improved the relative expressions of Cu/Zn-SOD, GPx and CAT. Furthermore, 2.70% dietary arginine level significantly lowered the relative expression of Mn-SOD compared with the control group and 1.62% dietary arginine levels. The relative expressions of Interleukin 1β (IL-1β), tumour necrosis factor-α (TNF-α) and nuclear factor-kappa B (NF-κB) were lowered in fish fed with 1.62% dietary arginine level. 1.62% and 2.70% dietary arginine levels significantly improved the relative expressions of transforming growth factor-β (TGF-β). Hematocrit (HCT), aspartate aminotransferase (AST) activities, interleukin 8 (IL-8) and interleukin 10 (IL-10) expressions were not significantly affected by the graded dietary arginine levels. These results suggest that the optimal dietary arginine level plays an important role in enhancing antioxidant and immune status to maintain the intestinal health of juvenile blunt snout bream.

The present study assessed the effects of dietary leucine on growth performance, antioxidant status and immunity in juvenile blunt snout bream. Fish were fed six practical diets of graded leucine levels ranging from 0.90% to 2.94% of dry basis for 8 weeks. Trail results showed that compared to control group (0.90%), 1.72% dietary leucine level significantly improved final weight (FW), weight gain rate (WG) and specific growth rate (SGR), and significantly lowered feed conversion ratio (FCR). Based on WG and SGR, the optimal dietary leucine level was obtained at 1.40% and 1.56%, respectively. Whole body crude lipid and protein contents were improved with increasing dietary leucine up to 2.14% and thereafter showed a downward trend, while whole body moisture content showed a converse trend. No significant change was found in whole body ash content. 1.72% dietary leucine level significantly improved the antioxidant capacity of fish by regulating the plasma superoxide dismutase (SOD) activity, glutathione peroxidase (GPx) activity, total antioxidant capacity (T-AOC) activity, catalase (CAT) activity, aspartate aminotransferase (AST) activities and malondialdehyde (MDA) content, furthermore, 1.72% dietary leucine level also significantly improved the antioxidant genes expressions of associated with Nrf2 signaling pathway by regulating heme oxygenase-1 (HO-1), GPx, copperezinc superoxide dismutase (Cu/Zn-SOD), manganese superoxide dismutase (Mn-SOD), 2.14% dietary leucine levels also significantly improved glutathione transferase (GST) mRNA level. Dietary leucine levels significantly affected plasma immunity parameters such as the contents of plasma complement component 3 (C3), immunoglobulin M (IgM) and lowered the hepatopancreas genes expressions of pro-inflammatory factor by regulating interleukin 1β (IL-1β), interleukin 8 (IL-8) and tumour necrosis factor-α (TNF-α) mRNA levels. The present study indicated that optimal dietary leucine level plays an important role in improving growth, enhancing antioxidant and immune status to maintain the health in juvenile blunt snout bream.