Replacement of fish meal with cottonseed protein concentrate in Chinese mitten crab (Eriocheir sinensis): Nutrient digestibility, growth performance, free amino acid profile, and expression of genes related to nutrient metabolism.

This study aimed to investigate the application of cottonseed protein concentrate (CPC) in Chinese mitten crabs (Eriocheir sinensis). First, the apparent digestibility coefficient (ADC) of CPC, fish meal and soybean meal were compared in crabs (21.72 ± 0.33 g). The protein ADC of CPC was 90.42%, which was significantly higher than that of soybean meal (83.16%) (P < 0.05). The ADC of Phe, Cys and Glu of CPC were significantly higher than those of fish meal, while the ADC of Ile, Leu, Lys, Met, Thr and Ala of CPC were significantly lower (P < 0.05). Second, we investigated the effects of fish meal substitution by CPC on growth performance, free amino acid profile, and expression of genes related to nutrient metabolism in crabs. Six diets were formulated by replacing 0%, 15%, 30%, 45%, 60% and 75% fish meal with CPC, namely FM, CPC15, CPC30, CPC45, CPC60, and CPC75. A total of 630 crabs (1.68 ± 0.00 g) were randomly divided into 18 tanks (3 tanks per group) and fed 3 times daily for 9 weeks. Results showed that CPC75 group significantly reduced growth performance, feed conversion efficiency, and free Ile, Leu, Lys, Met, and Thr contents in muscle (P < 0.05). The contents of free amino acids (Arg, His, Ile, Leu, Lys, Met, Phe, Thr, Val, Ala, Cys, Glu, Gly, Ser and Tyr) in hepatopancreas decreased linearly with the increase of dietary CPC level (P < 0.05). The substitution of more than 45% fish meal with CPC significantly decreased the concentration of delicious amino acids (Ala, Glu and Gly) in hepatopancreas (P < 0.05), which might adversely affect crab flavor. The expression of genes related to antioxidant capacity, protein transport, TOR pathway and lipid metabolism was significantly downregulated by increasing dietary CPC level (P < 0.05). In conclusion, based on the quadratic regression analysis of FCR and PER, the optimal replacement levels of fish meal with CPC in crab diet containing 35% fish meal were 32.36% and 35.38%, respectively. It is recommended that Ile, Leu and Thr be supplemented in addition to Met and Lys in the application of CPC.

Comparision of nitrogen removal characteristic and microbial community in freshwater and marine recirculating aquaculture systems.

Recirculating aquaculture system (RAS) has a good prospect in aquaculture, but its nitrogen removal characteristics and microbial community changes in freshwater and marine water remain unclear. In this study, six RAS were designed and divided into freshwater group and marine water group with salinity of 0‰ and 32‰, respectively, and operated for 54 days to test changes in nitrogen (NH4+-N, NO2--N, NO3--N), extracellular polymeric substances and microbial communities. The results showed that ammonia nitrogen was rapidly reduced and almost converted to nitrate nitrogen in the freshwater RAS but to nitrite nitrogen in marine RAS. Compared with freshwater RAS, marine RAS had lower tightly bound extracellular polymeric substances and worse stability and settleability condition. 16S rRNA amplicon sequencing reflected significantly lower bacterial diversity and richness in marine RAS. Microbial community structure at phylum level showed lower relative abundance of Proteobacteria, Actinobacteria, Firmicutes, Nitrospirae, but higher abundance of Bacteroidetes under a salinity of 32‰. High salinity decreased the abundance of funtional genera (Nitrosospira, Nitrospira, Pseudomonas, Rhodococcus, Comamonas, Acidovorax, f_Comamonadaceae), which may account for nitrite accumulation and low nitrogen removal capacity in marine RAS. These findings could provide theoretical and practical basis for improving the start-up speed of high-salinity nitrification biofilm.

Effects of dietary dandelion extract on the growth performance, serum biochemical parameters, liver histology, and immune and apoptosis-related genes expression of hybrid grouper (Epinephelus lanceolatus♂ × Epinephelus fuscoguttatus♀) at different feeding period.

The study explored on the effects of dietary 0.4% dandelion extract on the growth performance, serum biochemical parameters, liver histology and the expression levels of immune and apoptosis-related genes in the head kidney and spleen of hybrid grouper (Epinephelus lanceolatus♂ × Epinephelus fuscoguttatus♀) at different feeding period. The results showed that the weight gain rate (WGR) of the hybrid grouper were significantly increased at the second and fourth weeks (P < 0.05), but there was no significant difference in WGR at the eighth week (P > 0.05). Compared with the control group, dietary dandelion extracts supplementation improve lipid metabolism, reduce lipid accumulation in liver and maintain normal liver histology at the second and fourth weeks. At the end of the second week, the relative expression levels of antioxidant related genes (MnSOD, GPX and GR) in the head kidney of hybrid grouper fed with dandelion extract increased significantly; at the end of week 4 and week 8, the relative expression levels of antioxidant related genes other than MnSOD did not change significantly. However, in the spleen of hybrid grouper, the expression of these antioxidant genes showed the opposite trend. At the end of the eighth week, dietary dandelion extract supplementation significantly increased the expression of inflammatory response related genes in head kidney of hybrid grouper, but showed the opposite trend in spleen. In conclusion, the short-term (2 or 4 weeks) application of 0.4% dandelion extract in feed had the effects of growth improvement, liver protection and immune stimulation on hybrid grouper due to its antioxidant and anti-inflammatory activities. The beneficial effect of dandelion extract on hybrid grouper was time-dependent, and its action time on different immune organs of hybrid grouper was not synchronous.

The effects of dietary compound plant extracts on growth performance, liver and intestine health, and immune related genes expression in hybrid grouper (Epinephelus lanceolatus♂ × Epinephelus fuscoguttatus♀).

The study explored on the effect of dietary compound plant extract supplementation on the growth performance, serum biochemical indicators, liver and intestinal morphological and gene expression levels in the head kidney and spleen of the hybrid grouper (Epinephelus lanceolatusâ™‚× Epinephelus fuscoguttatus♀). The compound plant extracts (BDG) was a mixture of Bupleurum edulis extract, dandelion extract and Ginkgo biloba extract in a ratio of 1:4:1. Basal diets supplemented with BDG at 0, 0.75, 1.5, 3 and 6 g/kg were fed hybrid grouper for 8 weeks. The results showed that dietary 0.75 and 1.5 g/kg BDG supplementation could significantly increase the WGR and SGR of hybrid grouper (P < 0.05). And dietary 0.75 g/kg BDG could also significantly decrease serum aspartate aminotransferase, glucose and lactate dehydrogenase in hybrid grouper (P < 0.05). Dietary BGD supplementation protected the integrity of liver and intestinal morphological structure, reduced the accumulation of liver fat. Dietary BDG supplementation might enhance the immunity of hybrid grouper by regulating the expression of antioxidant and inflammation-related genes in head kidney and spleen of hybrid grouper. Our study demonstrated that the growth promoting effect of Bupleurum extract, dandelion extract and Ginkgo biloba extract in the ratio of 1:4:1 as a compound feed additive was better than any of them as a feed additive alone, and the dosage was less. The optimal additive dosage of BDG was 0.75 g/kg in hybrid grouper diets.

Dietary Ginkgo biloba leaf extracts supplementation improved immunity and intestinal morphology, antioxidant ability and tight junction proteins mRNA expression of hybrid groupers (Epinephelus lanceolatus ♂ × Epinephelus fuscoguttatus ♀) fed high lipid diets.

For many years, Ginkgo biloba has been used as a traditional Chinese medicine because of its antioxidant, anti-inflammatory and hepatoprotective effects. The present study aimed to investigate the effects of dietary Ginkgo biloba leaf extract (GBLE) supplementation on immune response, intestinal morphology, antioxidant ability and tight junction proteins mRNA expression of hybrid groupers fed high lipid diets. Basal diets supplemented with GBLE at 0, 0.50, 1.00, 2.00, 4.00 and 10.00 g/kg were fed to hybrid grouper for 8 weeks. The study showed that dietary GBLE supplementation significantly improved immune ability by increasing plasma complement 3, complement 4 and Immunoglobulin M content. Dietary supplementation of 0.50-2.00 g/kg GBLE improved intestinal morphology and increased the expression of zonula occludens 1, zonula occludens 2, zonula occludens 3, occludin and claudin 3a. Dietary supplementation of 0.50-2.00 g/kg GBLE improved antioxidant ability by increasing activities and expressions of glutathione peroxidase, catalase and glutathione reductase, suppressed inflammatory by increasing expression of interleukin 10, transforming growth factor ß1 and target of rapamycin, and decreased apoptotic responses by reducing the expression of caspase 3, caspase 8 and caspase 9 in the intestine of hybrid grouper fed high lipid diets. This study indicated that dietary GBLE supplementation was clearly beneficial for intestinal health and immunity in hybrid groupers fed high lipid diets and it could be used as a functional feed additive in aquaculture to promote the application of high lipid diets.

Effects of dietary monocalcium phosphate supplementation on the anti-oxidative capacity, anti-bacteria function in immune organs of obscure puffer (Takifugu obscurus) after infection with Aeromonas hydrophila.

The purpose of the present study was to explore the impaired anti-bacteria ability in immune organs and immune systems of obscure puffer induced by chronic dietary phosphorus (P) deficiency. Fish were fed diets supplemented with 6 g/kg P (P6) and 0 g/kg P (P0) respectively for 15 weeks, and lower final body weight, feed intake, weight gain, whole body P content and bone P content were observed in fish fed P0 diet (P < 0.05). Then the fish were continued to feed for 3 weeks and intraperitoneal injection with PBS (P6+PBS) and Aeromonas hydrophila (A.hydrophila) (P6 + A.hydrophila and P0 + A.hydrophila), and sampled at 3, 6, 12 and 24 h. The results showed that dietary P deficiency lowered survival rate, total hemocyte count, whereas enhanced ROS production and apoptosis rate of obscure puffer compared to the 6 g/kg P supplemented group after infection. Moreover, compared to the P sufficient group, puffer fish fed P deficient diet decreased the expressions of antioxidant genes catalase (cat) and glutathione reductase (gr), immune-related genes toll-like receptor 2 (tlr-2) and anti-inflammatory factors transforming growth factor ß1 (tgf-ß1) and interleukin 11 (il-11) while increased pro-inflammatory cytokines tumor necrosis factor α (tnf-α), interleukin 1ß (il-1ß) and interleukin 8 (il-8) in head kidney post-infection. In addition, dietary P deficiency decreased the hepatic gene expressions of anti-apoptotic factor B-cell lymphoma 2 (bcl-2) and bax-inhibitor 1 (bi-1), accompanied by increasing the mRNA expressions of pro-apoptotic factor caspase 3, caspase 8 and caspase 9 compared to the P sufficient group after A.hydrophila infection. In conclusion, dietary P deficiency impaired the anti-bacteria function of the immune system as well as immune organs by increasing oxidative stress and aggravating the inflammatory response and apoptosis in obscure puffer under the A.hydrophila challenge.

Effects of dietary Senecio scandens buch-ham extracts on growth performance, plasma biochemical, histology and the expression of immune-related genes in hybrid grouper (Epinephelus lanceolatus♂ × Epinephelus fuscoguttatus♀).

The study mainly aimed at the effects of dietary Senecio scandens buch-ham extract (SSBE) on the growth performance, body composition, plasma biochemical index, intestinal and liver histology and the expression of antioxidant, apoptosis and inflammatory related genes in hybrid grouper (Epinephelus lanceolatus♂ × Epinephelus fuscoguttatus♀). Basal diets supplemented with SSBE (10:1) 0%, 0.05%, 0.1%, 0.2% and 0.4% were fed hybrid grouper for 8 weeks. The results showed that WGR and SGR were significantly increased in the week 2 and week 4 in Diet 0.05% group (P < 0.05). The total protein, globulin and albumin significantly increased whereas alanine aminotransferase, triglyceride and alkaline phosphate in the plasma were significantly decreased in Diet 0.1% group (P < 0.05). The villi length, width, muscle thickness and the cross-sectional area of intestine were improved in Diet 0.05% and Diet 0.1% group. The expression levels of PPAR-α and CPT-1 in the liver of hybrid grouper were significantly increased following the supplementation of SSBE (P < 0.05). The expression levels of antioxidant related genes (CAT, GPX, GR and Keap1) and anti-inflammatory factor (IL-10) in liver, head kidney and spleen of hybrid grouper decreased significantly (P < 0.05). In addition, diets supplemented with 0.05%-0.1% SSBE had a good liver-protecting effect, but it would have a detrimental effect on hepatocytes when the content exceeds 0.2%. The above results indicated that the suitable additive amount of SSBE in hybrid grouper feed was 0.05%-0.1%.

Xiaochaihu Decoction reduces hepatic steatosis and improves D-GalN/LPS-induced liver injury in hybrid grouper (Epinephelus lanceolatus♂ × Epinephelus fuscoguttatus♀).

Excessive lipid accumulation and chemical abuse can induce fatty liver diseases in fish, but the underlying mechanism and therapies are unknown. The present study aims to evaluate the effects of Xiaochaihu Decoction (XCHD) on the growth performance, lipid metabolism and antioxidant function of hybrid grouper in vitro and in vivo, and provide evidence as to whether it can be potentially used as a medicine for liver diseases in aquaculture. In vitro, steatosis model of hybrid grouper primary hepatocytes were incubated for 48 h in control or lipid emulsion (LE)-containing medium with or without 24 h post-treatment with XCHD. XCHD treatment reversed the LE-induced intracellular lipid accumulation, cell viability and hepatocytes morphological structure. In vivo, a total of 300 hybrid grouper with an average initial weight of 25.43 ±â€¯0.18 g were fed diets containing five graded levels of XCHD at 150-1200 mg/kg diet for 8 weeks. After that, a challenge trial was conducted by injection of D-GalN/LPS to induce liver injury. As a result, dietary supplementation with 150-300 mg/kg XCHD diets can significant improve growth performance and feed utilization (P < 0.05). Dietary XCHD down-regulated the expression of lipogenic-related genes (G6PD, DGAT2 and ME1) and up-regulated lipolysis-related genes (ATGL, PPARα and LPL) expression in the liver of hybrid grouper. Livers challenged with D-GalN/LPS exhibited extensive areas of vacuolization with the disappearance of nuclei and the loss of hepatic architecture. These pathological alterations were ameliorated by XCHD treatment. XCHD significantly down-regulated the D-GalN/LPS induced apoptosis-related genes caspase-3, caspase-9 and p53 mRNA expression and up-regulated the antioxidant-related genes CAT and MnSOD mRNA expression in dose dependent manner, respectively. XCHD potently reduced hepatic lipid accumulation and enhanced antioxidant capability in hybrid grouper and may be a potential fish-feed additive to prevent fatty liver diseases onset and progression.

Dietary Radix Bupleuri extracts improves hepatic lipid accumulation and immune response of hybrid grouper (Epinephelus lanceolatus♂ × Epinephelus fuscoguttatus♀).

In this study, two experiments were performed to explore the effect of Radix Bupleuri extracts (RBE) on growth, lipid deposition and metabolism and immune response of hybrid grouper (Epinephelus lanceolatus♂ × Epinephelus fuscoguttatus♀) using in vitro and in vivo models. In vitro, we used 2 ml/L 20% lipid emulsion (LE)-induced steatosis in hybrid grouper primary hepatocytes, then RBE (200, 400 and 800 µg/ml) was added to the hepatocytes after (post-treatment) the incubation with 20% LE (2 ml/L) in the culture medium. We found that RBE markedly increased cell viability, which were consistent with hepatocytes morphological structure examination and lipid metabolism and immune related genes study. The above result suggested that RBE has a protective effect on this model of hepatocytes damage. In vivo, five graded levels of RBE at 0, 200, 400, 800 and 1600 mg/kg diet were supplemented to a basal diet with 15% lipid levels (high lipid), and fed to a total of 300 hybrid grouper with an average initial weight of 25.58 ±â€¯0.05 g for 8 weeks. Growth performance, liver histology, plasma biochemical parameters, and expression of genes involved in lipid metabolism and immune-related were measured. The study indicated that dietary RBE significantly improved growth performance and feed utilization and reduced hepatosomatic index. Dietary supplementation with 200-800 mg/kg RBE diets effectively decreased serum ALP, ALT, AST and LDH contents in fish. Furthermore, adipogenesis relative mRNA levels of DGAT2, G6PD, ME1 and DGKα in fish fed 200-400 mg/kg RBE diets were lower (P < 0.05) than in those fed RBE0 diets, while dietary supplementation with 200-800 mg/kg RBE diets up-regulated lipolysis-related genes (CPT1, LPL and PPARα) expression in the liver of hybrid grouper. Moreover, dietary RBE down-regulated the expression of apoptosis-related genes (caspase-9), up-regulated the expression of antioxidant genes (CAT) and immune-related genes (MHC2, IKKα and TGF-ß1). Thus, our data suggest that RBE suppressed lipid accumulation and enhanced immune capability in hybrid grouper both in vitro and in vivo. These results offer new insight into RBE as a hepatoprotective in fish.

The effects of dietary Lycium barbarum extract on growth performance, liver health and immune related genes expression in hybrid grouper (Epinephelus lanceolatus♂ × E. fuscoguttatus♀) fed high lipid diets.

Growth performance, hepatic morphology and antioxidant ability, and expressions of antioxidant, inflammatory and apoptosis related genes were investigated in hybrid grouper fed high lipid diets containing 0, 0.5, 1, 2 and 10 g kg-1Lycium barbarum extract (LBE) for 8-week feeding. The study showed that dietary LBE significantly increased weight gain rate (WGR) and specific growth rate (SGR) of fish (P < 0.05), the highest WGR and SGR were observed in fish fed 10.00 g kg-1 LBE diet. Dietary LBE improved liver morphology by decreasing hepatocyte necrosis and inflammatory cell infiltration induced by high lipid diets. Meanwhile, high lipid diets supplemented with 0.5-2 g kg-1 LBE improved hepatic antioxidant ability by increasing the expression of antioxidant genes (GPx and CAT) and decreasing Keap1 mRNA levels. Moreover, dietary supplementation with 0.50-2.00 g kg -1 LBE significantly decreased IL-8, caspase-3, caspase-8 and caspase-9 mRNA levels and significantly increased IL-10 and TGF-ß1 mRNA levels in the liver of fish fed high lipid diets. In conclusion, high lipid diets supplemented with LBE improved growth performance, feed utilization and liver health in hybrid groupers by increasing hepatic antioxidant enzymes activity and its genes expression, as well as inhibition of hepatic inflammatory response and apoptosis.

Effects of dietary dandelion extracts on growth performance, liver histology, immune-related gene expression and CCl4 resistance of hybrid grouper (Epinephelus lanceolatus♂ × Epinephelus fuscoguttatus♀).

The study investigated the effects of dietary supplementation with dandelion extracts (DE) on growth performance, feed utilization, body composition, serum biochemical, liver histology, immune-related gene expression and CCl4 resistance of hybrid grouper (Epinephelus lanceolatus♂ × Epinephelus fuscoguttatus♀). A basal diet supplemented with DE at 0% (diet 0%), 0.1% (diet 0.1%), 0.2% (diet 0.2%), 0.4% (diet 0.4%) and 0.8% (diet 0.8%) were fed to hybrid grouper for 8 weeks. The results revealed that dietary DE had not a significant impact on growth performance and feed utilization (P > 0.05), but it could decrease the percent of crude lipids in whole body and increase the percent of crude protein in muscle (P < 0.05). Dietary DE increased the mRNA levels of antioxidant enzymes (catalase, glutathione peroxidase and glutathione reductase) and reduced inflammatory factor in the spleen and head-kidney of fish (P < 0.05), but reduced the expression of the liver antioxidant gene except for glutathione reductase (P < 0.05). Dietary supplementation with 0.2%-0.4% DE could effectively improve liver health. After injection of CCL4 by 72 h, fish fed Diet0.2% and Diet0.4% showed regular hepatocyte morphology while fish fed Diet 0%, Diet 0.1% and Diet 0.8% showed hepatocyte damage. Higher survival rate and total blood cell count was observed in fish fed 0.1%-0.4% dietary DE (P < 0.05). In conclusion, DE could be used as a functional feed additive to enhance liver function of farmed fish. The best level of it should be between 0.2% and 0.4%.

Liver transcriptome analysis and de novo annotation of the orange-spotted groupers (Epinephelus coioides) under cold stress.

Cold stress has caused great economic loss in fish culture worldwide. Orange-spotted grouper (Epinephelus coioides) is one of the most serious lost aquatic animals in 2016 cold fronts in South China. However, the molecular mechanism of grouper's cold resistance has remained largely unknown. In the present study, HiSeq™2000 (Illumina) was used to analyze the transcriptomic profiles of the liver from grouper under control temperature (CT, 28 °C) and low temperature (LT, 13 °C). Two normalized liver cDNA libraries of CT and LT groups were created. We obtained 51,944,970 and 51,905,036 clean reads from CT and LT groups, respectively. Comparing the LT group to the CT group, a total of 5905 significantly differentially expressed genes (DEGs) were identified, including 2093 up-regulated unigenes and 3812 down-regulated unigenes. GO annotation and functional enrichment analysis indicated that all of the DEGs were classified into three categories: biological process (23 subclasses), cellular component (18 subclasses) and molecular function (13 subclasses). KEGG analysis of the DEGs showed that 2732 DEGs were annotated to 253 signaling pathways. The most highly enriched pathways were cell adhesion molecules, Staphylococcus aureus infection, PPAR signaling pathway, Vibrio cholerae infection, primary immunodeficiency, fatty acid elongation, and we found cold stress mainly affects immunity, metabolic and signal transduction. Thirteen of the DEGs were further validated by qRT-PCR. Our results provide valuable information for further analysis of the mechanisms of groupers response under cold stress.

Effects of replacing fish meal with soybean meal on growth performance, feed utilization and physiological status of juvenile obscure puffer, Takifugu obscurus.

An 8-week feeding trial was conducted to evaluate the growth performance, feed utilization and physiological status of obscure puffer, Takifugu obscurus (13.03 ±â€¯0.14 g) fed diets in which fish meal (FM) was replaced with various levels of dehulled and defatted soybean meal (SBM): 0% (SBM0), 15% (SBM15), 30% (SBM30), 45% (SBM45), 60% (SBM60) and 75% (SBM75). No significant differences were observed in weight gain and specific growth rate (SGR) of fish when FM replacement level was lower than 30%, and the broken-line model of SGR showed the maximum replacement level was 40%. Fish fed the SBM-containing diets had a lower red blood cell value compared to the control. The hemoglobin and methemoglobin values showed a declining tendency as dietary SBM level increased. Plasma triacylglycerol, cholesterol and low-density lipoprotein cholesterol levels also showed a decreasing trend that was associated with the reduced crude lipid content of whole body as dietary SBM level increased. The activities of alanine aminotransferase and aspartate aminotransferase in fish fed the SBM-containing diets were all higher than those fed the control diet while glutathione peroxidase and catalase activities were lower than the control group. Results indicated that up to 40% FM protein, based on the broken-line analysis of SGR, can be replaced with SBM in diet for obscure puffer juveniles with supplemental lysine, methionine and taurine.

The hepatoprotective effects of Radix Bupleuri extracts against D-galactosamine/lipopolysaccharide induced liver injury in hybrid grouper (Epinephelus lanceolatus♂ × Epinephelus fuscoguttatus♀).

The present study is aiming at evaluating the hepatoprotective of Radix Bupleuri extracts (RBE) on the d-galactosamine/lipopolysaccharide (D-GalN/LPS) induced liver injury of hybrid grouper in vitro and in vivo. In vitro, RBE (0, 200, 400 and 800 µg/ml) was added to the hybrid grouper primary hepatocytes before (pretreatment) the incubation of the hepatocytes with D-GalN (20 mM) plus LPS (1 µg/ml) in the culture medium. RBE at concentrations of 200, 400 and 800 µg/ml significantly improved cell viability and inhibited the elevation of TNF-α, IL-1ß and IL-6 and significantly down-regulated the caspase-3, caspase-9 and P53 mRNA levels. In vivo administration of RBE at the doses of 0, 200, 400, 800 and 1600 mg/kg in the diet for 8 weeks prior to D-GalN (500 mg/kg) and LPS (20 µg/kg) intoxication. The study indicated that the RBE not only ameliorated liver injury, as evidenced by well-preserved liver architecture, but also significantly increased hepatic antioxidant enzymes activities in the D-GalN/LPS-induced liver injury animal model. Further demonstrating the protective effects of the RBE, we found that pretreatment with the RBE up-regulated the expression of antioxidant genes (GPx and MnSOD), while down-regulated apoptosis-related genes (caspase-3, caspase-9 and P53), immune related genes (MHC2 and TLR3) and pro-inflammatory cytokines (TOR and IKKα) mRNA expression in the liver of hybrid grouper. In brief, the present study showed that RBE can protect hepatocyte injury induced by D-GalN/LPS through elevating antioxidant enzyme activity and suppressing apoptosis and immune inflammatory responses. The results support the use of RBE as a hepatoprotective in fish.

The protective effects of vitamin C on apoptosis, DNA damage and proteome of pufferfish (Takifugu obscurus) under low temperature stress.

The aim of this study was to investigate the protective effects of vitamin C on apoptosis, DNA damage and proteome of pufferfish under low temperature stress. Six diets were formulated to contain 2.60, 48.90, 95.50, 189.83, 382.40, 779.53mg/kg vitamin C. After 8-week feeding trial, fish were exposed to low temperature challenge. The results showed that pufferfish receiving vitamin C diet exhibited a significant decrease in ROS production (48.9-189.83mg/kg vitamin C diet groups), cytoplasmic free-Ca2+ concentration (48.9-779.53mg/kg vitamin C diet groups), apoptotic cell ratio (95.5-779.53mg/kg vitamin C diet groups) and DNA damage (189.83-779.53mg/kg vitamin C diet groups) under low temperature stress in comparison with those of control. We also investigated the alteration in protein expression under low temperature stress by a comparative proteomic analysis. The results demonstrated that 24 protein spots showed significantly differential expression in the cold-stress-treated group compared with those of the control group, and 5 protein spots were successfully identified. Furthermore, comparative proteomic analysis revealed that vitamin C could increase expressed proteins related to energy metabolism, immune responses and cytoskeleton. These findings would be helpful to understand the protective effects of vitamin C against cold stress.

Effect of dietary astaxanthin on the growth performance, non-specific immunity, and antioxidant capacity of pufferfish (Takifugu obscurus) under high temperature stress.

The present study was conducted to investigate the effects of astaxanthin on growth performance, biochemical parameters, ROS production, and immune-related gene expressions of the pufferfish (Takifugu obscurus) under high temperature stress. The experimental basal diets supplemented with astaxanthin at the rates of 0 (control), 20, 40, 80, 160, and 320 mg kg-1 were fed to fish for 8 weeks. The results showed that the fish fed diet with 80, 160, and 320 mg kg-1 astaxanthin significantly improved weight gain and specific growth rate. Furthermore, fish fed the moderate dietary astaxanthin increased plasma alkaline phosphatase activities, and decrease plasma aspartate aminotransferase and alanine aminotransferase activities. After the feeding trial, the fish were exposed to high temperature stress for 48 h. The results shown that astaxanthin could suppress ROS production induced by high temperature stress. Meanwhile, compared with the control group, the astaxanthin groups increased SOD, CAT, and HSP70 mRNA levels under high temperature stress. These results showed that the basal diet supplemented with 80-320 mg kg-1 astaxanthin could enhance growth, nonspecific immune responses, and antioxidant defense system and improve resistance against high temperature stress in pufferfish.

Effects of dietary Panax notoginseng extract on growth performance, fish composition, immune responses, intestinal histology and immune related genes expression of hybrid grouper (Epinephelus lanceolatus ♂ × Epinephelus fuscoguttatus ♀) fed high lipid diets.

Growth, plasma biochemical parameters, fish composition, immune parameters, intestinal histology, and expressions of immune-related genes were examined in hybrid grouper (Epinephelus lanceolatus ♂ × Epinephelus fuscoguttatus ♀) that fed respectively six diets containing Panax notoginseng extract (PNE) at 0, 0.5, 1, 2, 4, and 10 g kg-1 after 8 weeks. Results indicated that dietary PNE significantly improved growth, feed efficiency ratio, protein efficiency ratio, and protein deposit rate, and significantly increased crude protein and crude lipid levels of whole body and crude protein level of muscle. Dietary PNE significantly increased plasma total protein, alkaline phosphatase, immunoglobulin, complement 3 and complement 4 contents, but significantly decreased cholesterol, triglyceride, glucose, and low density lipoprotein cholesterol contents. Furthermore, dietary PNE increased villus length and muscle thickness in foregut, midgut, and hindgut, activities of hepatic superoxide dismutase and total antioxidant capacity, and increased the expression levels of immune related genes (IL-10, TGF-ß1, TOR, MHC2 and TLR3) in the head kidney and the expression levels of antioxidant genes (CAT and GR) in fish that fed PNE at 0.5-4 g kg-1. In conclusion, grouper fed high lipid diets supplemented with PNE at 0.5-10 g kg-1 improved growth, feed utilization, blood immune parameters, hepatic antioxidant status, intestine morphology and expression levels of immune related genes in the head kidney.

Effects of dietary dandelion extract on intestinal morphology, antioxidant status, immune function and physical barrier function of juvenile golden pompano Trachinotus ovatus.

Intestinal morphology, antioxidant status, immune function and tight junction proteins mRNA expression were examined in golden pompano (Trachinotus ovatus) that fed respectively six diets containing dandelion extracts (DE) at 0, 0.5, 1, 2, 4 and 10 g kg-1 after 8 weeks feeding. The study indicated that dietary DE significantly improved intestinal antioxidant abilities by increasing SOD, CAT, T-AOC activities and up-regulating intestinal cat, gpx mRNA levels, but by decreasing MDA content and down-regulating intestinal keap1 mRNA levels in golden pompano. Meanwhile, dietary DE improved intestinal morphology, suggesting that enhances intestinal digestion and absorption, by increasing muscle thickness, villus length, villus width and villus number in the foregut and hindgut; as well as villus number, villus width and muscle thickness in the midgut (P < .05). Dietary DE enhanced intestinal barrier function by increasing intestinal zo-1 and occludin mRNA levels, but by decreasing the mRNA levels of claudin-12 and claudin-15. Furthermore, dietary DE improved intestinal immunity via increasing goblet cells numbers and regulating expression of immune-related genes. In conclusion, dietary DE supplementation promoted intestine health by improving intestine morphology, immunity, antioxidant abilities and intestinal barrier in golden pompano.

Effects of dietary ginkgo biloba leaf extract on growth performance, plasma biochemical parameters, fish composition, immune responses, liver histology, and immune and apoptosis-related genes expression of hybrid grouper (Epinephelus lanceolatus♂ × Epinephelus fuscoguttatus♀) fed high lipid diets.

For thousands of years, leaves from the Ginkgo biloba tree have been a common treatment in Chinese medicine. The present study was conducted to investigate the effects of dietary ginkgo biloba leaf extract (GBE) supplementation on growth performance, plasma biochemical parameters, fish composition, immune responses, liver histology, and immune and apoptosis-related genes expression of hybrid grouper (Epinephelus lanceolatus♂ × Epinephelus fuscoguttatus♀) fed high lipid diets. A basal diet supplemented with GBE at 0, 0.50, 1.00, 2.00, 4.00 and 10.00 g kg-1 was fed to hybrid grouper for 8 weeks. The study indicated that dietary GBE did not improve growth performance and feed utilization but it reduced intraperitoneal fat rate. There were no significant differences in condition factor, viscerosomatic index, hepatosomatic index, spleen index, relative gut length, food intake, protein deposit rate and survival among all groups (P > 0.05). Dietary supplementation with 0.50-4.00 g GBE kg-1 diets effectively increased plasma HDL content and decreased plasma GLU, LDL and TG content in fish. Furthermore, dietary GBE had a significant effect on moisture, crude protein and lipid in the liver, and protein in the whole body of fish (P < 0.05). Dietary supplementation with 0.50-1.00 g GBE kg-1 diets effectively decreased occurrence rates of the hepatocyte swelling, hepatocyte vacuolization, and nuclei shifting to the cellular periphery cytoplasmic vacuolization, meanwhile hepatic antioxidant enzymes (SOD, CAT and T-AOC) activities significantly increased whereas MDA content significantly decreased in fish fed diets supplemented with GBE (P < 0.05). Moreover, dietary GBE up-regulated the expression of antioxidant genes (CAT, GPx and GR), immune-related genes (MHC2 and TLR3) and anti-inflammatory cytokines (IL-10 and TGF-ß1), while dietary supplementation with 0.50-4.00 g GBE kg-1 diets down-regulated apoptosis-related genes (p53, caspase-9, caspase-8 and caspase-3) expression in the head kidney of hybrid grouper. These results indicated that hybrid grouper fed diets supplemented with GBE did not improve growth performance and feed utilization but it had hypolipidemic effects, improved hepatic antioxidant status, maintained normal liver histology and preserved liver function, increased immune-related genes expression and decreased apoptosis-related genes expression in the head kidney of hybrid grouper.

Effects of dietary dandelion extracts on growth performance, body composition, plasma biochemical parameters, immune responses and disease resistance of juvenile golden pompano Trachinotus ovatus.

The present study was conducted to investigate the effects of dietary dandelion extracts (DE) supplementation on growth performance, feed utilization, body composition, plasma biochemical indices, immune responses, hepatic antioxidant abilities, and resistance to the pathogen Vibrio harveyi in Trachinotus ovatus. A basal diet supplemented with DE at 0, 0.50, 1.00, 2.00, 4.00 and 10.00 g kg-1 were fed to golden pompano for 8 weeks. The study indicated that dietary supplementation with DE could significantly improve final body weight (FBW), weight gain rate (WGR), specific growth rate (SGR), feed efficiency ratio (FER), feed intake (FI), protein efficiency ratio (PER) and protein deposit rate (PDR) (P < 0.05). The highest FBW, WGR, SGR, FI and PDR were observed in fish fed 1.00 g kg-1 dietary DE (P < 0.05). The highest FER and PER were recorded at 0.50 g kg-1 dietary DE (P < 0.05). Condition factor, viscerosomatic index, hepatosomatic index and survival were not significantly different among all groups. Fish fed 1.00 g kg-1 dietary DE showed significant increase in plasma total protein, complement 4 content and alkaline phosphatase, lysozyme, glutathione reductase (GSR) activity, but significant decrease in triglyceride, low density lipoprotein cholesterol, malondialdehyde (MDA) content and aspartate aminotransferase activities compared to the control group (P < 0.05). Hepatic antioxidant enzymes (SOD, T-AOC, CAT, GSH-Px, GSR) significantly increased whereas MDA content significantly decreased in fish fed 1.00 g kg-1 DE supplement (P < 0.05). After challenge with Vibrio harveyi, significant higher post-challenge survival was observed in fish fed DE supplement (P < 0.05). These results indicated that golden pompano fed a diet supplemented with DE (especially at 1.00 g kg-1 of fed supplement) could significantly promote its growth performance, feed utilization, body protein deposit, immune ability, hepatic and plasma antioxidative enzyme activities and improve its resistance to infection by Vibrio harveyi.