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.     

Effect of diet composition on growth performance,hepatic metabolism and antioxidant activities in Siberian sturgeon (<Emphasis Type="Italic">Acipenser</Emphasis><Emphasis Type="Italic">baerii</Emphasis>, Brandt, 1869) submitted to starvation and refeeding

Many fish species undergo natural starvation periods. Adaptation to starvation is possible through the activation of behavioral, biochemical and physiological mechanisms. Knowledge of the effect of dietary nutrients on the intermediary metabolism during starvation and refeeding can be useful to improve fish health and optimize aquaculture production. To analyze the effect of dietary nutrients on liver metabolism of Siberian sturgeon (Acipenser baerii) submitted to starvation and refeeding, four isoenergetic diets differing in nutrient composition were designed: LP-St (38 % protein, 12 % lipid, 36 % carbohydrate), HP-St (44 % protein, 10 % lipid, 30 % carbohydrate), LP-L (38 % protein, 18 % lipid, 25 % carbohydrate) and HP-L (44 % protein, 16 % lipid, 22 % carbohydrate). Four groups of fish were fed 3 weeks to satiety with the corresponding diet, starved for 2 weeks and then refeed 5 weeks to satiety on the same diet. Starvation mobilized the hepatic lipid store to a greater extent than glycogen. Starvation increased superoxide dismutase activity irrespective of the diet, while low protein diets (LP-St and LP-L) increased catalase activity. The oxidative damage decreased after 5 weeks of refeeding. Refeeding the starved fish on the HP-St diet promoted the greatest growth performance. In addition to reporting for the first time the effect of diet composition on growth, liver composition and antioxidant activities in Siberian sturgeon submitted to starvation and refeeding, our findings suggest that refeeding on HP-St diet stimulated the use of dietary carbohydrates and allowed a protein sparing effect in Siberian sturgeon.     

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.     

Molecular characterization of cholecystokinin in grass carp (Ctenopharyngodon idellus): cloning, localization, developmental profile, and effect of fasting and refeeding on expression in the brain and intestine

Cholecystokinin (CCK) is a multi-functional brain–gut peptide in fish and mammals. To investigate the role of CCK in appetite regulation in fish, a 770-bp full-length cDNA sequence of CCK gene was obtained by RT-PCR and rapid amplification of cDNA ends methods in grass carp Ctenopharyngodon idellus. Homology analysis showed that the CCK cDNA sequence of grass carp had the highest similarity (90 %) to that of goldfish Carassius auratus and a higher similarity (>70 %) to those of other teleosts than to mammals. The PCR amplification using genomic DNA identified that the CCK gene of grass carp was comprised of three exons and two introns. Real-time quantitative PCR was used to detect CCK mRNA expression in adult tissues. High levels of gene expression were found in the hypothalamus and pituitary; moderate levels in the intestine, muscle and white adipose tissue; and low levels in other tissues. During early development (i.e., fertilized eggs to 35-day post-hatching larvae) the levels of CCK mRNA expression were higher during embryonic developmental stages than during post-hatch larval stages. Fasting decreased CCK mRNA expression levels in the brain and intestine, whereas refeeding resulted in an increase of expression. The results suggest that CCK mRNA expression has obvious tissue specificity and may have a role in feed intake regulation in grass carp.     

Immunogene expression in head kidney and spleen of common carp (<Emphasis Type="Italic">Cyprinus carpio</Emphasis> L.) following thermal stress and challenge with Gram-negative bacterium, <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis>

To evaluate the effect of thermal and microbial stress on the immune response of common carp (Cyprinus carpio L.), relative mRNA expression level of pro-inflammatory cytokines [tumor necrosis factor alpha (TNF-α) and interleukin (IL)-1β] and other genes related to immune or stress response [inducible nitric oxide synthase (iNOS), heat shock protein 70 (Hsp70), superoxide dismutase one (SOD1), and glucocorticoid receptor (GR)] was measured by quantitative PCR (qPCR). In addition, total protein and total immunoglobulin level in blood plasma of experimental common carp was also assayed. All the above parameters were estimated 24 h post-challenge with Gram-negative bacterium, Aeromonas hydrophila. Common carp (54.89?±?6.90 g) were initially exposed to 20 °C (control group) and 30 °C (thermal stress group) water temperature for 30 days, followed by experimental challenge with 2.29?×?10⁸ colony forming unit/mL (CFU/mL; LD₅₀ dose) of A. hydrophila. Exposure of fish to thermal stress and subsequently challenge with A. hydrophila significantly (P?<?0.05) increases the IL-1β mRNA expression in head kidney and spleen of common carp by ~?39.94 and ~?4.11-fold, respectively. However, TNF-α mRNA expression in spleen decreased ~?5.63-fold in control fish challenged with A. hydrophila. Thermal stress and challenge with bacterium suppresses the iNOS and GR mRNA expression in spleen of common carp. Moreover, significant (P?<?0.05) increase in total protein content of blood plasma (~?43 mg/g) was evident in fish exposed to thermal stress and challenged with A. hydrophila. In conclusion, our study highlights the importance of elevated temperature stress and microbial infection in differential regulation of expression of several immunogenes in common carp.     

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.     

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.     

Comparative nutritional value of <Emphasis Type="Italic">Jatropha curcas</Emphasis> protein isolate and soy protein isolate in common carp

Jatropha seed cake (JSC) is an excellent source of protein but does contain some antinutritional factors (ANF) that can act as toxins and thus negatively affect the growth and health status of fish. While this can limit the use of JSC, detoxified Jatropha protein isolate (DJPI) may be a better option. An 8-week study was performed to evaluate dietary DJPI to common carp Cyprinus carpio. Five iso-nitrogenous diets (crude protein of 38%) were formulated that consisted of a C _ontrol (fish meal (FM) based protein), J ₅₀ or J ₇₅ (50 and 75% of FM protein replaced by DJPI), and S ₅₀ or S ₇₅ (50 and 75% of FM protein replaced by soy protein isolate, SPI) and fed to triplicate groups of 75 carp fingerlings (75; av. wt. ± SD; 11.4 ± 0.25 g). The growth, feeding efficiencies, digestibility, plasma biochemistry, and intestinal enzymes were measured. Results showed that growth performance of fish fed the S ₇₅- or DJPI-based diets were not significantly different from those fed the C _ontrol diet, while carp fed the S ₅₀ had significantly better growth than the J ₇₅ diet. Fish fed the J ₇₅ diet had significantly lower protein and lipid digestibility as well as significantly lower intestinal amylase and protease activities than all other groups. However, all plant protein-based diets led to significantly higher crude protein, crude lipid, and gross energy in the body of common carp compared to the control treatment. Plasma cholesterol and creatinine significantly decreased in the plant protein fed groups, although plasma triglyceride as well as the red blood cells count, hematocrit, albumin, globulin, total plasma protein, and lysozyme activity were higher in plant protein fed groups compared to FM fed group. White blood cells, hemoglobulin concentration, alkaline phosphatase and alanine transaminase activities, and glucose level in blood did not differ significantly among treatments. The results suggest that the DJPI is non-toxic to carp and can be used to replace FM in the diets of common carp up to 75%, but further research to potentially reduce some inherent ANF within this protein source, such as non-starch polysaccharides, may improve nutrient utilization.     

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.     

Effects of exogenous non-starch polysaccharide-degrading enzymes in diets containing <Emphasis Type="Italic">Gracilaria lemaneiformis</Emphasis> on white-spotted snapper <Emphasis Type="Italic">Lutjanus stellatus</Emphasis> Akazaki

A feeding experiment was conducted to investigate the effects of exogenous non-starch polysaccharide (NSP)-degrading enzymes in diets containing Gracilaria lemaneiformis (GL) on growth performance and digestive enzyme activities of white-spotted snapper Lutjanus stellatus Akazaki (initial mass 8.0 ± 0.1 g). A basal diet (D0) containing a mixed protein source (fish meal, soybean meal and GL meal) was used as the control. Two diets supplemented with 0.5 g (D1) and 1 g (D2) exogenous NSP-degrading enzymes per kilogram of diet were formulated. Each diet was assigned to triplicate groups of fish in a total of nine floating sea cages (270 fish, 30 fish per cage). After a 60-day feeding trail, significantly higher weight gain, specific growth ratio and feed efficiency ratio were observed in fish fed D2 diet compared to those of control (P < 0.05). Body lipid, moisture and ash contents were not significantly affected by NSP-degrading enzyme supplementation, but significantly higher protein content was noticed in fish fed D2 diet compared to that of control (P < 0.05). The amylase activity in the stomach and intestine was significantly higher in fish fed D2 diet (P < 0.05), but no significant differences were observed in pepsin and lipase activities in the stomach or trypsin and lipase activities in the intestine between all treatments. The results suggested that addition of 1 g kg^?1 NSP-degrading enzymes in diet could efficiently improve seaweed feed utilization and growth performance of white-spotted snapper fish.     

Role of cyclooxygenase-mediated metabolites in lipid metabolism and expression of some immune-related genes in juvenile grass carp (<Emphasis Type="Italic">Ctenopharyngodon idellus</Emphasis>) fed arachidonic acid

Cyclooxygenase (COX) catalyzes the conversion of arachidonic acid (ARA) to prostaglandins, and COX-mediated metabolites play important roles in the regulation of lipid metabolism and immunity in mammals. However, such roles of COX in fish remain largely unknown. In this study, we designed three semi-purified diets, namely ARA-free (control), ARA, and ARA + acetylsalicylic acid (ASA; a COX inhibitor), and used them to feed grass carp (27.65 ± 3.05 g) for 8 weeks. The results showed that dietary ARA significantly increased the amount of ARA in the hepatopancreas, muscle, and kidney (P < 0.05), whereas this increase was reduced by dietary ASA. The hepatopancreatic prostaglandin E₂ content increased in the ARA group, and this increase was inhibited by ASA (P < 0.05). ARA decreased the lipid content in the hepatopancreas, whereas ASA recovered lipid content to a significant level (P < 0.05). ARA significantly decreased the messenger RNA (mRNA) expression levels of fatty acid synthase and stearoyl-CoA desaturase in the hepatopancreas (P < 0.05). However, ASA did not rescue the mRNA expression of these genes (P > 0.05). Interestingly, ARA significantly enhanced the level of peroxisome proliferator-activated receptor α gene expression, and this increase was attenuated by ASA (P < 0.05). Finally, ARA significantly enhanced the mRNA expression of myeloid differentiation factor 88 (MyD88) in the kidney, and ASA attenuated the expression of toll-like receptor 22 and MyD88 (P < 0.05). In conclusion, our findings suggest that COX metabolites play important roles in the inhibition of lipid accumulation in the hepatopancreas of grass carp fed with ARA and that regulation of gene expression promotes lipid catabolism rather than lipogenic activities. Additionally, these eicosanoids might participate in the upregulation of immunity-related genes in the kidney.     

Stimulatory effect of dietary taurine on growth performance,digestive enzymes activity,antioxidant capacity,and tolerance of common carp, <Emphasis Type="Italic">Cyprinus carpio</Emphasis> L., fry to salinity stress

The present study was carried out to evaluate the effect of dietary taurine (Tau) on performance, digestive enzymes, antioxidant activity, and resistance of common carp, Cyprinus carpio L., fry to salinity stress. Fish (0.97?±?0.033 g) were fed on different taurine levels of 0.0 (control), 5, 10, 15, or 20 g/kg diet up to satiation twice daily for 8 weeks. At the end of the feeding trial, fish were stressed by exposure to 10 ppt salinity for 3 days during which fish mortality was observed. Fish performance was significantly (P?<?0.05) improved by dietary taurine up to 15 g Tau/kg diet after which fish growth and feed intake were almost the same. Also, taurine supplementation significantly (P?<?0.05) elevated activities of intestinal amylase, lipase, and protease resulting in an improving in feed intake giving better performance. Furthermore, Tau-stimulated antioxidant activity of common carp was observed in a dose-related manner, where activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) were significantly (P?<?0.05) higher, but malondialdehyde (MDA) value was significantly (P?<?0.05) lower in Tau-fed fish groups than those fed the control diet. In salinity stress experiment, highest survival rate was observed at fish fed Tau-supplemented diets without significant (P?>?0.05) differences over fish fed the control diet. It appears that taurine could be used as a feed supplement to confer better growth and health of common carp fry with optimal level of 15 g/kg diet.     

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.     

Modulation of antioxidant defense and immune response in zebra fish (<Emphasis Type="Italic">Danio rerio</Emphasis>) using dietary sodium propionate

The present study explores the effect of dietary sodium propionate on mucosal immune response and expression of antioxidant enzyme genes in zebra fish (Danio rerio). Six hundred healthy zebra fish (0.42 ± 0.06 g) supplied, randomly stocked in 12 aquariums and fed on basal diets supplemented with different levels of sodium propionate [0 (control), 5, 10 and 20 g kg^?1] for 8 weeks. At the end of the feeding trial, mucosal immune parameters (TNF-α, IL-1β, Lyz), antioxidant enzyme (SOD, CAT) as well as heat shock protein 70 (HSP70) gene expression were measured. The results revealed feeding on sodium propionate significantly up-regulated inflammatory response genes (TNF-α, IL-1β, Lyz) in a dose-dependent manner (P < 0.05). However, antioxidant enzyme genes significantly down-regulated in the treated group compared with control (P < 0.05). Also, HSP70 gene expression was higher in the liver of fish fed the basal diet and deceased with elevation of sodium propionate levels in the diet. These results showed beneficial effects of dietary sodium propionate on mucosal immune response as well as the antioxidant defense of zebra fish.     

Growth,hematological and biochemical indices of common carp <Emphasis Type="Italic">Cyprinus carpio</Emphasis> fed diets containing corn gluten meal

The effects of replacing fish meal (FM) with corn gluten meal (CGM) on growth and physiological performance were evaluated in common carp (Cyprinus carpio). Six experimental diets were formulated by substituting 0 (control), 20, 40, 60, 80, and 100% CGM protein for FM protein. The concentrations of dietary protein in the experimental diets were 27.8–29% and the P:E ratios were 14.7–15.46 mg/kJ. Eighteen fish with an initial weight of 13.5?±?0.1 g were allocated to each of 18 circular tanks (450 L) to give triplicate groups for each dietary treatment. The fish were fed to satiation for 8 weeks. At the end of the feeding trial, growth indices, body proximate composition, and hematological and biochemical parameters were measured. Blood samples were taken from six fish in each tank. Final weight and total length were significantly higher in fish fed 100% CGM (27.8?±?1.2 g and 11.9?±?0.3 cm) than for those fed the control (22.7?±?1.4 g and 10.9?±?0.5 cm) or 20% CGM (22.3?±?1.2 g and 11?±?0.4 cm) diets. No effect of FM replacement by CGM was observed for condition factor or hepatosomatic index (P?>?0.05). The highest value of protein productive value (14.31?±?0.65) was observed in fish fed 20% CGM (P?<?0.05). There were no significant differences in percentage body moisture and fat, but percentages of protein and ash were significantly different among experimental groups; the highest values of protein (15.6?±?0.24%) and ash (3.01?±?0.26%) were recorded in fish fed 40% CGM. For hematological parameters, the highest number of white blood cells (4.1?±?0.1?×?10³ mm^?3) was observed in fish fed 100% CGM (P?<?0.05). In addition, the highest hematocrit (42.1?±?0.7%) and triglyceride (294.11?±?23.82 mg dl^?1) were seen in fish fed the diet containing 40% CGM, while 80% CGM gave the highest cholesterol level (204.44?±?9.0 mg dl^?1; P?<?0.05). Replacement of FM with CGM had no negative effects on growth and physiological parameters of common carp fingerlings in this short (8 weeks) trial, suggesting that it may be feasible to replace FM with CGM in diets formulated for juvenile common carp.     

Effects of nutritional status and diet composition on fatty acid transporters expression in zebrafish (Danio rerio)

This study investigated the expression of zebrafish (Danio rerio) fatty acid transporters, such as scavenger receptor CD36, plasma membrane fatty acid‐binding protein (FABPpm), and fatty acid transport protein family (FATPs), in responses to nutritional status and diet composition. For diet composition study, three isonitrogenous (43% crude protein) purified diets were formulated to contain 6%, 12% and 18% crude lipid levels. Meanwhile five isonitrogenous and isoenergetic purified diets were formulated to contain different lipid sources including fish oil (FO; rich in n‐3 HUFA), palmitic acid (PA; rich in C16:0), olive oil (OO; rich in C18:1n‐9), sunflower oil (SO; rich in C18:2n‐6) or perilla oil (PO; rich in C18:3n‐3) as the sole lipid source. Results showed that liver CD36, FABPpm‐a, FABPpm‐b, FATP1‐a and FATP6, intestine CD36, FABPpm‐a and FABPpm‐b, and muscle CD36 and FATP1‐b were significantly increased during postprandial period. During starvation, liver CD36, FABPpm‐a, FABPpm‐b and FATP1‐a, intestine FABPpm‐a, FABPpm‐b, FATP4 and FATP6, and muscle FATP1‐b, FATP4 and FATP6 were significantly increased. The expression of some fatty acid transporters (including liver CD36 and FABPpm‐b, intestine FABPpm‐a, and muscle CD36, FABPpm‐a, FATP1‐a, FATP2 and FATP4) was up‐regulated by refeeding, while the expression of other fatty acid transporters (liver FABPpm‐a and intestine FATP1‐a, FATP4 and FATP6) was down‐regulated by refeeding. The expression of several fatty acid transporters (liver FATP2 and FATP4, and intestine CD36, FATP2 and FATP4) was significantly increased by high‐lipid diet, whereas CD36, FATP1‐b, FATP2 and FATP4 expression in the muscle were reduced by increasing dietary lipid level. Compared with FO group, the muscle of fish fed the diet with OO had a higher CD36 mRNA abundance, while the muscle of fish fed the diet with OO and PO had a higher FATP1‐b expression. Compared with the FO group, the hepatic FATP2 mRNA was significantly increased in the PA group, while the intestine FATP2 mRNA was significantly increased in the SO and PO groups. In addition, intestine FATP4 expression in the PA and OO groups was lower compared with the FO group. The results indicate that zebrafish fatty acid transporters play a central role in coordinating the mobilization of fuel substrates in responses to nutritional status and diet composition.     

Cytochrome P450 2AA molecular clone,expression pattern,and different regulation by fish oil and lard oil in diets of grass carp (<Emphasis Type="Italic">Ctenopharyngodon idella</Emphasis>)

Cytochrome P450 enzymes (CYP enzymes) catalyze important metabolic reactions of exogenous and endogenous substrates, including fatty acid. In this study, we cloned the complete CDS of the cytochrome P450 2AA (CYP2AA) gene from the grass carp (Ctenopharyngodon idella) for the first time. CYP2AA consisted of 1500 bp, which encoded a predicted protein of 499 amino acids. The identities of CYP2AA between C. idella and zebrafish were 86%. It consists of the conserved heme-binding motif FXXGXXXCXG. Quantitative real-time PCR analysis indicated that CYP2AA mRNA in C. idella was highly expressed in liver and adipose tissue. The effects of fish oil and lard oil in diets on expression of CYP2AA mRNA in vivo were also investigated. The fish oil (FO) group exhibited significantly higher CYP2AA expression in adipose tissue than the lard oil (LO) group (P?<?0.01), whereas the mRNA expression of CYP2AA was not notably different in liver. It suggested that the high abundance of CYP2AA mRNA expression in adipose tissue could be induced by fish oil. Our findings provided molecular characterization and expression profile of CYP2AA, and enhanced our understanding of CYP2AA in fish lipid metabolism.     

Nitrogen removal characteristics of <Emphasis Type="Italic">Pseudomonas stutzeri</Emphasis> F11 and its application in grass carp culture

This study was designed to investigate the denitrification characteristics of Pseudomonas stutzeri F11 under different environmental conditions and to evaluate the effect of these characteristics on nitrogen removal and the water microbiome in an experimental grass carp aquaculture system. The results showed that the optimal growth conditions of strain F11 were (1) ammonia-nitrogen (N), nitrite-N, or nitrate-N as sole N source; (2) initial ammonia-N concentration of 10–30 mg N/l; (3), initial nitrite-N concentration of 200 mg N/l; (4) sodium citrate as carbon source; (5) rotation (r) speed of 200 r/min; a C:N ratio of between 2 and 10; (6) culture at 32–37 °C. The addition of P. stutzeri F11 to the experimental grass carp aquaculture system reduced the levels of ammonia-N, nitrite-N, and total N in the water over an extended range, but had no effect on nitrate-N level. Results of the 454 pyrosequencing analysis indicated that the structure of the microbial community in the aquaculture water changed significantly after the addition of P. stutzeri F11 preparations. The addition of P. stutzeri F11 to the aquaculture system also altered the microbiome metabolism in the water, especially the bacteria involved in nitrogen metabolism. These results suggest that the addition of P. stutzeri F11 to an experimental grass carp aquaculture system decrease nitrogen levels and alter the microbial community structure of the water; as such, this bacterial strain could be a potential candidate for the regulation of water quality in aquaculture systems.     

Effects of low-voltage constant direct current on plasma biochemical profiles and gene expression levels in crucian carp <Emphasis Type="Italic">Carassius carassius</Emphasis>

This study investigated the plasma biochemical profiles and gene expression levels of crucian carp Carassius carassius subjected to anesthetization with low-voltage constant direct current (DC) and tricaine methanesulfonate (MS222). Fish in the MS222 treatments and the DC treatments were exposed to either two concentrations of MS222 (100 and 200 mg/l) or constant DC with a voltage of 26 V, at a voltage gradient of 0.68 V/cm, respectively. The results showed that low-voltage constant DC immobilized fish more quickly, resulting in significant elevations of plasma cortisol levels compared to exposure of high or low doses of MS222. Electronarcosis led to an increased expression of heat shock proteins 70 (HSP70) as well as HSP90 compared to the treatment group with MS222. Also, electronarcosis enhanced mitochondrial respiratory chain gene (atp6, cox1 and nd5) and antioxidant enzyme gene (CAT, SOD and GSH-px1) expression levels. In conclusion, low-voltage constant DC can disturb fish plasma biochemical profiles and gene expression levels.