Molecular analysis of grass carp (<Emphasis Type="Italic">Ctenopharyngodon idella</Emphasis>) by SRAP and SCAR molecular markers

The sequence-related amplified polymorphism (SRAP) technique was used to analyze the gene differentiation between two cultured populations [Freshwater Fisheries Research Center (FFRC) and Qianzhou populations] and one wild population (Hanjian population) of grass carp (Ctenopharyngodon idella). Some loci showed quite different genetic frequencies, attributable to artificial selection, which imply that these fragments are putative markers of germplasm identification. We developed a simple and effective method to further characterize these SRAP fragments. Specific SRAP bands were cut directly from polyacrylamide gels, re-amplified, cloned, and sequenced. Twenty-one putative genetic markers were sequenced, ranging from 137 to 357 bp. The sequences were submitted to the database of the Genome Sequence Survey. A BLAST analysis showed that eight SRAP fragments were highly similar to functional genes, whereas the other 13 had no similarity, indicating that these markers are tightly linked to the germ identification trait although only eight are functional genes. Three primers were designed according to this sequence information and used for PCR amplification of the three populations. A sequence-characterized amplified region (SCAR1) was positively amplified in the artificially cultured populations but not in the wild population. The frequency of the SCAR3 marker in the cultured populations was 87% (26/174), whereas it was only 6% (6/100) in the wild population. A specific band was isolated from all individuals in the wild population with the SCAR3 primers, whereas the specific band was amplified from only seven individuals in the FFRC population and from none of the Qianzhou population. The frequency of SCAR2 in the artificially cultured populations was 96.5%. These results indicate that SCAR1 could be used as a specific molecular marker for population identification. The SCAR markers used in this study offer a powerful, easy, and rapid method for genetic analysis and the discrimination of different populations.     

Effect of waterborne zinc exposure on lipid deposition and metabolism in hepatopancreas and muscle of grass carp <Emphasis Type="Italic">Ctenopharyngodon idella</Emphasis>

The aim of the present study was to explore the effect of waterborne zinc (control, 0.85, 2.20, 3.10 mg/l, respectively) exposure on lipid deposition and metabolism in the hepatopancreas and muscle of grass carp Ctenopharyngodon idella. The lipid content, Zn accumulation, and the activities and expression levels of several enzymes involved in lipid metabolism were determined in hepatopancreas and muscle. Waterborne Zn exposure reduced growth performance and increased Zn accumulation in both tested tissues. In hepatopancreas, Zn exposure increased lipid content, the activities of lipogenic enzymes, such as 6PGD, G6PD, ME, ICDH and FAS, as well as the mRNA expression level of G6PD, 6PGD, ICDH, FAS and SREBP-1. But the activity of CPT I and the mRNA expression of HSL, CPT Iα1a, CPT Iα2a and PPARα were down-regulated by Zn exposure. In contrast, in muscle, waterborne Zn exposure decreased lipid deposition, activities of 6GPD, ICDH and ME, as well as the mRNA expression level of G6PD, ICDH, ME, FAS and SREBP-1. However, the activity of CPT I as well as the mRNA expression level of PPARα, HSL, CPT Iα2a, CPT Iα1b and CPT Iβ were up-regulated by Zn exposure. Our results indicate that waterborne Zn increases lipid content by up-regulating lipogenesis and down-regulating lipolysis in hepatopancreas. But, in muscle, waterborne Zn reduces lipid accumulation by up-regulating lipolysis and down-regulating lipogenesis. Differential patterns of lipid deposition, enzymatic activities and genes’ expression indicate the tissue-specific regulatory mechanism in fish.     

Cloning and characterization of <Emphasis Type="Italic">Bax1</Emphasis> and <Emphasis Type="Italic">Bax2</Emphasis> genes of <Emphasis Type="Italic">Ctenopharyngodon idellus</Emphasis> and evaluation of transcript expression in response to grass carp reovirus infection

Multidomain proapoptotic Bcl-2-associated X (Bax) protein is an essential effector responsible for mitochondrial outer membrane permeabilization, resulting in cell death via apoptosis. In this study, two Bax genes of grass carp (Ctenopharyngodon idellus), designated as CiBax1 and CiBax2, were isolated and analyzed. The obtained CiBax1 cDNA is 2058 bp long, with a 579 bp open reading frame (ORF) coding a protein of 192 amino acid residues. The full-length cDNA of CiBax2 is 1161 bp, with a 618 bp ORF coding 205 amino acids. Both CiBax1 and CiBax2 are typical members of Bcl-2 family containing conserved Bcl and C-terminal domains, and they share conserved synteny with zebrafish Bax genes despite the grass carp Bax mapping to different linkage groups. Phylogenetic analysis showed that CiBax1 was clustered with Bax from most teleost fish, and CiBax2 was close to Bax2 from teleost fish but far separated from that of Salmo salar. Quantitative real-time PCR analysis revealed broad expression of CiBax1 and CiBax2 in tissues from healthy grass carp, but the relative expression level differed. The mRNA expression of CiBax1 and CiBax2 was both upregulated significantly and peaked in all examined tissues at days 5 or 6 post-infection with grass carp reovirus. Subcellular localization indicated that CiBax1 protein was localized in both nucleus and cytosol, while CiBax2 protein only in cytosol. Moreover, CiBax2, but not CiBax1 was colocalized with mitochondrion under normal condition. Taken together, the findings would be helpful for further understanding of the function of Bax in teleost fish.     

Molecular cloning,characterization and expression of FSH and LH beta subunits from grass carp (<Emphasis Type="Italic">Ctenopharyngodon idella</Emphasis>)

Follicle-stimulating hormone beta subunit (FSHβ) and luteinizing hormone beta subunit (LHβ) have been cloned and characterized from the pituitary of grass carp (Ctenopharyngodon idella). The full length of FSHβ and LHβ cDNA was 393 bp and 441 bp, with open reading frame encoding proteins of 130 and 146 amino acids, respectively. Phylogenetic analysis of the proteins of FSHβ and LHβ showed a high homology with other fishes. Homology analysis also indicated that LHβ has higher conservation than FSHβ. The expression analysis of grass carp FSHβ and LHβ by RT-PCR suggested that they were only expressed in the pituitary. Real-time quantitative PCR protocols were developed and validated to measure FSHβ and LHβ mRNAs during ovarian development. The FSHβ and LHβ mRNA level was very low in the pituitaries of early-pubertal fish and significantly increased during the ovulation period. These results suggested that in grass carp the gonadotropins synthesized synchronously in order for asynchronous oogenesis to take place.     

Respiratory response of grass carp <Emphasis Type="Italic">Ctenopharyngodon idellus</Emphasis> to dissolved oxygen changes at three acclimation temperatures

Respiratory parameters of grass carp were studied during dissolved oxygen (DO) changes from normal DO to hypoxia, then return to normal DO at 15, 25, and 30 °C acclimation, respectively. The results showed that with increases of acclimation temperature at normoxia the respiratory frequency (f_R), oxygen consumption rate (VO₂), respiratory stroke volume (V_S.R), gill ventilation (V_G), and V_G/VO₂ of grass carp increased significantly, but the oxygen extraction efficiency (EO₂) of fish decreased significantly (P < 0.05). With declines of DO levels, the f_R, V_S.R, V_G, and V_G/VO₂ of fish increased significantly at different acclimation temperatures (P < 0.05). A slight increase was found in VO₂, and the EO₂ of fish remained almost constant above DO levels of 3.09, 2.91, and 2.54 mg l^?1 at 15, 25, and 30 °C, while the VO₂ and EO₂ began to decrease significantly with further reductions in DO levels (P < 0.05). After 0.5 h of recovery to normoxia from hypoxia at three acclimation, the f_R, V_S.R, V_G, and V_G/VO₂ of the fish decreased sharply; meanwhile, the VO₂ and EO₂ increased sharply (P < 0.05). The respiratory parameters of fish gradually approached initial values with prolonged recovery time to normoxia, and reached their initial values in 2.5 h at 25 and 30 °C acclimation. The critical oxygen concentrations (C_c) of fish for VO₂ were 2.42 mg l^?1 at 15 °C, 2.02 mg l^?1 at 25 °C, and 1.84 mg l^?1 at 30 °C, respectively. The results suggest that grass carp are highly adapted to varied DO and short-term hypoxia environments.     

Effect of dietary chromium picolinate on growth performance and blood parameters in grass carp fingerling, <Emphasis Type="Italic">Ctenopharyngodon idellus</Emphasis>

An experiment was conducted to investigate the effect of dietary chromium picolinate supplement on growth and haematology parameters of grass carp, Ctenopharyngodon idellus. Six diets with increasing dietary chromium picolinate levels 0, 0.2, 0.4, 0.8, 1.6 and 3.2 mg kg⁻¹ were fed to triplicate groups of 20 fish (initial weight of 12.78 ± 1.16 g, mean ± SD) in a flow water system for 10 weeks. Fish fed the diet supplemented with 0.8 mg Cr kg⁻¹ had significantly improved weight gain (WG), feed efficiency ratio (FER), protein efficiency ratio (PER) and protein retention (PR). Fish fed high-chromium diets exhibited lower whole-body crude lipid contents than fish fed low-chromium diets. Liver glycogen concentrations for fish fed the diet with 0.2 mg Cr kg⁻¹ was the highest (77.67 mg g⁻¹). Fish fed the diet supplemented with 1.6 and 3.2 mg Cr kg⁻¹ had significantly lower liver glycogen concentrations than other groups (P < 0.05). The highest serum insulin concentrations were observed in fish fed the diet supplemented with 0.8 mg Cr kg⁻¹, but serum insulin concentrations decreased (P < 0.05) when dietary supplementation of chromium was higher than 0.8 mg Cr kg⁻¹. Cholesterol concentrations decreased in direct proportion to dietary chromium level and achieved the lowest level when the fish were fed the 0.8 mg Cr kg⁻¹ diet, but increased when the fish were fed the diet with more than 0.8 mg Cr kg⁻¹ (P < 0.05). Fish fed the diet supplemented with 0.8 mg Cr kg⁻¹ had higher triglyceride and high-density lipoprotein cholesterol (HDL-C) concentrations compared to other treatments. The results of the present study suggested that chromium picolinate could modify serum carbohydrate and lipid metabolism profile, and that the optimal dietary chromium level was 0.8 mg kg⁻¹ for grass carp according to growth.     

Effect of N-acetyl cysteine and glycine supplementation on growth performance,glutathione synthesis,and antioxidative ability of grass carp, <Emphasis Type="Italic">Ctenopharyngodon idella</Emphasis>

An 8-week feeding trial was conducted to evaluate the effect of N-acetyl cysteine (NAC) and glycine supplementation on growth performance, glutathione (GSH) synthesis, and antioxidative ability of grass carp, Ctenopharyngodon idella. Four practical diets were formulated: control, control + 0.2% NAC, control + 0.5% glycine, and control + 0.2% NAC + 0.5% glycine. Each diet was randomly assigned to quadruplicate groups of 30 fish (approximately 8.8 g). Weight gain and specific growth rate were significantly increased with the supplementation of NAC. Supplementation of NAC plus glycine significantly increased the feed efficiency. Glutathione peroxidase (GPx) and γ-glutamine cysteine synthase (γ-GCS) in plasma were significantly increased with the supplementation of NAC plus glycine. GSH in plasma increased and malondialdehyde (MDA) decreased in fish fed diets supplemented with NAC. Respiratory burst, superoxide dismutase (SOD), and catalase (CAT) activity were not affected by NAC or glycine. These results clearly indicated that NAC improved the growth performance and restored GSH of grass carp, supplemented NAC together with glycine enhanced GSH synthesis, and improved the antioxidative ability of grass carp.     

A study of the damage of the intestinal mucosa barrier structure and function of <Emphasis Type="Italic">Ctenopharyngodon idella</Emphasis> with <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis>

The aim of this study is to explore the effect of Aeromonas hydrophila on the intestinal mucosal barrier structure and intestinal permeability in grass carp (Ctenopharyngodon idella). Histopathological examinations showed that A. hydrophila induced severe intestinal lesions, including inflammatory cell infiltration and intestinal villus fusion and swelling. Messenger RNA (mRNA) expression of the inflammatory cytokines TNF-α, IL-1β, IL-8, IL-10 and MyD88 was significantly increased after infection with A. hydrophila. The permeability of intestinal mucosa was determined using Evans blue (EB) and d-lactic acid. The results indicated that the levels of EB and serum d-lactic acid were significantly increased after infection with A. hydrophila (p < 0.05). Our results also indicated that the intestinal mucosal barrier injury induced by A. hydrophila infection was closely associated with the expression of the tight junction (TJ) protein zonula occludens-1 (ZO-1), occludin, claudin b and claudin c as well as the activity of Na⁺, K⁺-ATPase and Ca²⁺, Mg²⁺-ATPase. Lower mRNA levels of occludin and lower Na⁺, K⁺-ATPase and Ca²⁺, Mg²⁺-ATPase activity in the intestines were observed after challenge. ZO-1 and claudin c were significantly increased 24 h after infection with A. hydrophila. The most interesting finding was that claudin b also significantly increased 24 h after challenge and then decreased to lower levels at 72, 120 and 168 h post-infection compared to the PBS-treated control group. The results demonstrated that grass carp infection with A. hydrophila induced intestinal inflammation and impaired the structure and function of the intestinal mucosal barrier.     

Dietary docosahexaenoic acid decreased lipid accumulation via inducing adipocytes apoptosis of grass carp, <Emphasis Type="Italic">Ctenopharygodon idella</Emphasis>

The purpose of this study was to explore the mechanism of by which docosahexaenoic acid (DHA) inhibit the accumulation of adipose tissue lipid in grass carp (Ctenopharyngodon idella). We therefore designed two semi-purified diets, namely DHA-free (control) and DHA-supplemented, and fed them to grass carp (22.19 ± 1.76 g) for 3 and 6 weeks. DHA supplementation led to a significantly lower intraperitoneal fat index (IPFI) than that in the control group by reducing the number of adipocytes but significantly higher adipocyte size (P < 0.05). In the intraperitoneal adipose tissue, the DHA-fed group showed significantly higher peroxisome proliferator-activated receptor (PPAR)γ, CCAAT enhancer-binding protein (C/EBP)α, and sterol regulatory element-binding protein (SREBP)1c mRNA expression levels at both 3 and 6 weeks (P < 0.05). However, the ratio of the expression levels of B cell leukemia 2 (Bcl-2) and Bcl-2-associated X protein (Bax) was significantly lower in the DHA-fed group than in the control group (P < 0.05), and the protein expression levels of the apoptosis-related proteins caspase 3, caspase 8, and caspase 9 were also significantly higher (P < 0.05). Overall, although DHA promotes lipid synthesis, it is more likely that DHA could suppress the lipid accumulation in adipocytes of grass carp by inducing adipocyte apoptosis.     

Fat deposition pattern and mechanism in response to dietary lipid levels in grass carp, <Emphasis Type="Italic">Ctenopharyngodon idellus</Emphasis>

This study aimed to evaluate the fat deposition pattern and lipid metabolic strategies of grass carp in response to dietary lipid levels. Five isonitrogenous diets (260 g kg^?1 crude protein) containing five dietary lipid levels (0, 20, 40, 60, 80 g kg^?1) were fed to quadruplicate groups of 15 fish with initial weight 200 g, for 8 weeks. The best growth performance and feed utilization was observed in fish fed with lipid level at 40 g kg^?1. MFI and adipose tissue lipid content increased with increasing dietary lipid level up to 40 g kg^?1, and higher lipid level in diet made no sense. Fish adapted to high lipid intake through integrated regulating mechanisms in several related tissues to maintain lipid homeostasis. In the present study, grass carp firstly increased PPARγ and CPT1 expressions in adipose tissue to elevate adipocyte differentiation and lipolysis to adapt to high lipid intake above 40 g kg^?1. In liver, fish elevated hepatic lipid uptake but depressed biosynthesis of hepatic FAs, resulted in no difference in HSI and liver lipid content among the groups. Only in muscle, fish showed a significant fat deposition when the lipid intake above 40 g kg^?1. The excess lipid, derived from enhanced serum TC and TG contents, was more likely to induce deposition in muscle rather than lipid uptake by adipose tissue in grass carp fed with high dietary lipid, indicating the muscle of grass carp might be the main responding organ to high lipid intake.     

Identification of an aminopeptidase from the skeletal muscle of grass carp (<Emphasis Type="Italic">Ctenopharyngodon idellus</Emphasis>)

Aminopeptidases play important roles in turnover of proteins, metabolism of hormones and neurotransmission, cell maturation and immunological regulations. In the present study, an aminopeptidase was purified to homogeneity from the skeletal muscle of grass carp by ammonium sulfate fractionation and sequential chromatographic steps, including DEAE-Sephacel, Sephacryl S-200, hydroxyapatite and Phenyl-Sepharose. The purified enzyme revealed a molecular mass of approximately 105 kDa both on SDS–PAGE and on gel filtration of Superdex 200. The enzymatic activity toward synthetic substrates was optimal at 40°C and pH 7.0–7.5. Metal-chelating agents such as EDTA and EGTA effectively inhibited the enzyme activity while inhibitors to serine, asparatic and cysteine proteinases did not show much effect, suggesting its belonging to metalloproteinase family. A specific aminopeptidase inhibitor bestatin was most effective in suppressing the enzymatic activity and performed in a competitive fashion. The enzymatic activity was slightly enhanced by metal ions of Mg²⁺ and Mn²⁺ while inhibited to different extents by Co²⁺, Cu²⁺, Zn²⁺ and Ca²⁺. Sulfhydryl reagent was necessary to maintain its activity. Purified enzyme demonstrated amidolytic activity most effectively against synthetic aminopeptidase substrate Leu-methylcoumarylamide (MCA) while N-terminal-blocked substrates and myofibrillar proteins were not hydrolyzed. The enzyme purified in the present study was quite possibly a leucine aminopeptidase (LAP) and functions during muscular protein metabolism.     

Effects of <Emphasis Type="SmallCaps">l</Emphasis>-carnitine against H<Subscript>2</Subscript>O<Subscript>2</Subscript>-induced oxidative stress in grass carp ovary cells (<Emphasis Type="Italic">Ctenopharyngodon idellus</Emphasis>)

This study was designed in vitro to investigate the effects of l-carnitine against H₂O₂-induced oxidative stress in a grass carp (Ctenopharyngodon idellus) ovary cell line (GCO). GCO cells were pre-treated with different concentrations of l-carnitine, followed by incubation with 2.5 mM H₂O₂ for 1 h to induce oxidative damage. The results indicated that adding l-carnitine at concentrations of 0.01–1 mM into the medium for 12 h significantly increased cell viability. Pre-treatment with l-carnitine at concentrations of 0.1–5 mM for 12 h significantly inhibited 2.5 mM H₂O₂-induced cell viability loss. The significant decreases in the level of reactive oxygen species and cell apoptosis were observed in 0.5 mM l-carnitine group compared to the H₂O₂ group. Malondialdehyde values of all of the l-carnitine groups were significantly lower than those of the H₂O₂ group, while total glutathione levels of all of the l-carnitine groups were significantly higher than of the H₂O₂ group. The activity of antioxidant enzymes, such as total superoxide dismutase (0.1 and 0.5 mM l-carnitine), catalase (0.5 mM l-carnitine) and γ-glutamyl cysteine synthetase (0.5 and 1 mM l-carnitine), was significantly increased. In addition, pre-treatment of l-carnitine in GCO cells exposed to 2.5 mM H₂O₂ significantly increased the mRNA expression of copper, zinc superoxide dismutase, catalase (0.5 mM l-carnitine), glutamate cysteine ligase catalytic subunit (0.1–1 mM) and glutathione peroxidase (0.1 mM l-carnitine). In conclusion, l-carnitine promotes GCO cell growth and improves antioxidant function, it plays a protective role against oxidative stress induced by H₂O₂ in GCO cells, and the appropriate supplemental amount of l-carnitine is 0.1–1 mM.     

Effect of <Emphasis Type="Italic">Cratoxylum formosum</Emphasis> on innate immune response and disease resistance against <Emphasis Type="Italic">Streptococcus agalactiae</Emphasis> in tilapia <Emphasis Type="Italic">Oreochromis niloticus</Emphasis>

The effect of aqueous extract of Cratoxylum formosum on innate immune response and disease resistance in tilapia Oreochromis niloticus was investigated. The fish were fed diets containing 0% (control), 0.5% (diet 1), 1% (diet 2), and 1.5% (diet 3) of C. formosum aqueous extract for 30 days. At the end of the experimental period, parameters of innate immune response including phagocytosis of blood leukocytes, lysozyme activity in plasma, and respiratory bust activity were examined. Feeding the fish with diet 2 and diet 3 for 20 days enhanced phagocytic activity and for 30 days stimulated lysozyme and respiratory burst activities. Diet 1 increased the phagocytic activity at 30 days, but did not affect the other measured parameters. All parameters were not significantly changed (P > 0.05) in the control group throughout the experiment. Following 30 days of feeding, fish were infected with S. agalactiae. The cumulative mortalities of bacterial-infected tilapia that were fed diet 1, diet 2, and diet 3 were 56, 12, and 10%, respectively, compared with 85% in the control group. These results indicate that the aqueous extract of C. formosum may elevate the innate immune response and enhance disease resistance in tilapia.     

Silymarin inhibits adipogenesis in the adipocytes in grass carp <Emphasis Type="Italic">Ctenopharyngodon idellus</Emphasis> in vitro and in vivo

In this study, two experiments were performed to explore the function of silymarin in adipogenesis in grass carp (Ctenopharyngodon idellus) using in vitro and in vivo models. In experiment 1, differentiated grass carp pre-adipocytes were treated with silymarin for 6 days. Treatment with 100 μg mL^?1silymarin (SM100 group) significantly reduced triglyceride accumulation at day 6. The adipogenic gene expression levels of PPARγ, C/EBPα, SREBP1c, FAS, SCD1, and LPL, and the protein expression level of PPARγ were significantly down-regulated in the SM100 group. Additionally, the SM100 group had significantly lower reactive oxygen species production and reduced glutathione contents compared with the control in vitro. In experiment 2, the juvenile grass carp (mean body weight= 27.4 ± 0.17 g) were fed six isonitrogenous and isocaloric diets in a factorial design containing 0, 100, or 200 mg kg^?1 silymarin (SM0, SM100, SM200) associated with either 4 or 8% lipid levels (low lipid, LL, and high lipid, HL, respectively) for 82 days. The results demonstrated that dietary silymarin supplementation significantly reduced the elevated intraperitoneal fat index in grass carp fed with high-lipid diets, and the gene expression of adipogenesis (PPARγ, FAS) when supplemented with dietary silymarin was notably lower than when no silymarin was supplemented under the high-lipid diets. Thus, our data suggest that silymarin suppressed lipid accumulation in grass carp both in vitro and in vivo, and the effect might be due to an influence on the expression of adipogenesis factors and ROS production partly associated with effects on antioxidant capability.     

Spatial learning-induced <Emphasis Type="Italic">egr</Emphasis>-<Emphasis Type="Italic">1</Emphasis> expression in telencephalon of gold fish <Emphasis Type="Italic">Carassius auratus</Emphasis>

The immediate-early gene (egr-1) expression was used to examine the neuron’s response in telencephalon of goldfish during spatial learning in small space. Fishes were pre-exposed in the experimental apparatus and trained to pick food from the tray in a rectangular-shaped arena. The apparatus was divided into identical compartments comprising three gates to provide different spatial tasks. After the fish learned to pass through the gate one, two more gates were introduced one by one. Fish made more number of attempts and took longer time (P < 0.05) to pass through the first gate than the gate two or three. This active learning induces the expression of egr-1 in telencephalon as established by western blot analysis. Subsequently, the fish learn quickly to cross the similar type of second and third gate and make fewer errors with a corresponding decline in the level of egr-1 expression. As the fish learned to pass through all the three gates, third gate was replaced by modified gate three. Interestingly, the level of egr-1 expression increased again, when the fish exhibit a high exploratory behavior to cross the modified gate three. The present study shows that egr-1 expression is induced in the telencephalon of goldfish while intensively acquiring geometric spatial information to pass through the gates.     

Modulation of appetite,lipid and glucose metabolism of juvenile grass carp (<Emphasis Type="Italic">Ctenopharyngodon idellus</Emphasis>) by different dietary protein levels

This study was undertaken to explore the systemic metabolic strategies of juvenile grass carp (Ctenopharyngodon idellus) to maintain growth when fed with different dietary protein levels. The optimal growth group and two growing discomfort groups were selected through the basic data, to explain the growth difference from appetite regulation and lipid and glucose metabolism perspective. Three experimental diets were formulated with three dietary protein levels at 200.3, 296.1 and 442.9 g kg^?1, named P1, P2 and P3, respectively. Juvenile grass carp (initial body weight 12.28 ± 0.14 g) were fed with three diets with 3 replications per dietary treatment in an indoor recirculation system for an 8-week feeding trial. Fish fed with diet P2 dietary group showed significantly higher WG, SGR, FI and PER than other groups. Compared with other groups, mRNA expressions of NPY, Y8a and Y8b in fish fed with P2 significantly down-regulated, while the expressions of CCK and CART in fish fed with P3 significantly down-regulated (P < 0.05). With increasing dietary protein levels, G6Pase, GK, PK and PEPCK were all significantly inhibited (P < 0.05). For lipid metabolism, the mRNA expression of ACC in P1 dietary group was significantly higher than P3 dietary group; besides, LPL expression in P3 group was significantly higher than other two groups (P < 0.05). PPARα expression in P2 was significantly lower than other groups (P < 0.05). These results suggested that grass carp fed with P2 (296.1 g kg^?1 protein level) showed highest weight gain, contributed to more balanced nutrient metabolism and appetite regulation. Too high dietary protein (442.9 g kg^?1) should be avoided because it induced lowest PER, body lipid and liver lipid, and inhibited glucose and lipid metabolism in juvenile grass carp.     

Immune response of sea bass <Emphasis Type="Italic">Dicentrarchus labrax</Emphasis> to <Emphasis Type="Italic">Tenacibaculum maritimum</Emphasis> antigens

ABSTRACT:   Seawater fishes are affected by a pathology commonly called 'myxobacteriosis', caused by Tenacibaculum maritimum (formerly Flexibacter maritimus ). The disease is characterized by fin erosion and necrotic ulcers of skin and muscle, and by low but constant mortality in cultured marine fish; in Italy is one of the most important and widely spread diseases affecting sea bass Dicentrarchus labrax , gilthead seabream Sparus aurata , sharp-snouted bream Diplodus puntazzo , white bream Diplodus sargus , and six-tooted bream Dentex dentex . In order to obtain an effective vaccine against the disease, formalin killed cells (FKC), extracellular products (ECPs) and crude lipopolysaccharide (LPS) preparations were obtained from the T. maritimum strain SPVId and injected intraperitoneally twice into the sea bass. The fish immune response to the preparations was studied: agglutinating antibody titer and in vitro phagocytosis were determined after the first and second injection in order to evaluate whether the preparations are immunogenic or not and if the booster effect took place. The results show that FKC and LPS preparations increased the antibody titer after the first injection when compared to the control sea bass. Moreover, all the preparations stimulated a secondary (booster) response. In vitro phagocytosis of the total blood was significantly higher for all the preparations when compared to the controls, but the crude LPS immunized sea bass showed the highest activity.     

Dietary supplementation of curcumin augments heat stress tolerance through upregulation of <Emphasis Type="Italic">nrf-2</Emphasis>-mediated antioxidative enzymes and <Emphasis Type="Italic">hsps</Emphasis> in <Emphasis Type="Italic">Puntius sophore</Emphasis>

Heat stress is one of the major environmental concerns in global warming regime and rising temperature has resulted in mass mortalities of animals including fishes. Therefore, strategies for high temperature stress tolerance and ameliorating the effects of heat stress are being looked for. In an earlier study, we reported that Nrf-2 (nuclear factor E2-related factor 2) mediated upregulation of antioxidative enzymes and heat shock proteins (Hsps) provide survivability to fish under heat stress. In this study, we have evaluated the ameliorative potential of dietary curcumin, a potential Nrf-2 inducer in heat stressed cyprinid Puntius sophore. Fishes were fed with diet supplemented with 0.5, 1.0, and 1.5% curcumin at the rate 2% of body weight daily in three separate groups (n = 40 in each group) for 60 days. Fishes fed with basal diet (without curcumin) served as the control (n = 40). Critical thermal maxima (CTmax) was determined for all the groups (n = 10, in duplicates) after the feeding trial. Significant increase in the CTmax was observed in the group fed with 1.5% curcumin- supplemented fishes whereas it remained similar in groups fed with 0.5%, and 1% curcumin-supplemented diet, as compared to control. To understand the molecular mechanism of elevated thermotolerance in the 1.5% curcumin supplemented group, fishes were given a sub-lethal heat shock treatment (36 °C) for 6 h and expression analysis of nrf-2, keap-1, sod, catalase, gpx, and hsp27, hsp60, hsp70, hsp90, and hsp110 was carried out using RT-PCR. In the gill, expression of nrf-2, sod, catalase, gpx, and hsp60, hsp70, hsp90, and hsp110 was found to be elevated in the 1.5% curcumin-fed heat-shocked group compared to control and the basal diet-fed, heat-shocked fishes. Similarly, in the liver, upregulation in expression of nrf-2, sod, catalase, and hsp70 and hsp110 was observed in 1.5% curcumin supplemented and heat shocked group. Thus, this study showed that supplementation of curcumin augments tolerance to high temperature stress in P. sophore that could be attributed to nrf-2-induced upregulation of antioxidative enzymes sod, catalase, gpx, and the hsps.