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.

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.

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.

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 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.