Fish protein hydrolysate affected amino acid absorption and related gene expressions of IGF‐1/AKT pathways in turbot (Scophthalmus maximus)

The effects of fish protein hydrolysate (FPH) on growth, peptide and amino acid (AA) transporters, postprandial free AA and related gene expression of IGF‐1/AKT pathway were evaluated in turbot (Scophthalmus maximus). Three diets were formulated to contain the same low level of fishmeal; meanwhile 0, 45 and 180 g/kg FPH were, respectively, supplemented to the FF (FPH‐free), FL (FPH‐Low) and FH (FPH‐High) diets. Fish fed the FH diet improved the growth compared with the other groups. For peptide and AA transporters, PepT1, B⁰AT1, CAT1 and PAT1 mRNA levels in proximal or distal intestine decreased in fish fed the FH diet. The concentration of free total essential AAs in serum was higher in the FH treatment than that in the FF treatment at 2 and 6 hr after feeding. For IGF‐1/AKT pathway in muscle, IGF‐1, 4E‐BP1 and FoxO1 mRNA levels were the highest in the FH group, whereas IGF‐1R mRNA levels were the highest expression level in the FF group. In conclusion, down‐regulated AAs transport, alleviated the delayed postprandial peak of serum‐free AAs and increased muscle protein synthesis were observed to improve the growth when turbot was fed high FPH level diets containing a high plant protein.     

Assessing feasibility of replacement of fishmeal with enzyme‐treated feather meal in the diet of juvenile turbot (Scophthalmus maximus L.)

A feeding trial was conducted to examine the potential of enzyme‐treated feather meal (EFM) as a fishmeal substitute in the diet of turbot. Fishmeal (650 g/kg) was incrementally replaced (0, 160, 320, 480, 640 and 800 g/kg) by EFM (0, 80, 160, 240, 320, 400 g/kg) to prepare six experimental diets (Control, EFM8, EFM16, EFM24, EFM32 and EFM40). Diet EFM24 was supplemented with lysine and methionine to form seventh diet (EFM24 + AA). The eighth diet was formulated with 240 g/kg steam‐processed feather meal to replace 480 g/kg fishmeal (SFM24). Eight diets were each fed to triplicate groups of juvenile turbot (37.47 g) in a recirculating aquaculture system for 7 weeks. The results showed that fish fed diet EFM24 grew better than those fed SFM24 (p < .05). The growth performance, body composition and serum biochemical indices of fish fed diet EFM8 were not statistically different compared to the control treatment (p > .05). However, further increase in fishmeal replacement (32%–80%) affected negatively growth, nutrients digestibility, serum biochemical indices and body composition (p < .05). Supplementation of lysine and methionine (EFM24 + AA) significantly improved growth performance and diet utilization compared to EFM24 (p < .05). Altogether results suggested that the EFM could comprise 80 g/kg in a diet for juvenile turbot by concurrent reduction of the fishmeal content by 160 g/kg without lysine and methionine supplementation.     

Fish protein hydrolysate in diets of turbot affects muscle fibre morphometry,and the expression of muscle growth‐related genes

The study was conducted to investigate the effects of fish protein hydrolysate (FPH) in diets for turbot on growth performance, muscle fibre morphometry, and the expression of muscle growth‐related genes. A control diet (FPH0) contained 0 g/kg FPH, and four experimental diets were formulated replacing fishmeal with FPH at levels of 45 (FPH4.5), 120 (FPH12), 180 (FPH18) and 300 (FPH30) g/kg. Fish fed the FPH12 and FPH18 diets had higher specific growth rate (SGR) than fish fed the FPH0 diet (p < .05), and a quadratic regression was found between SGR and dietary FPH level (p = .001, R² = .677). Cross‐section area (CSA) and the length of sarcomere in the FPH12 group increased compared with the control group (p < .05), and a quadratic regression was observed between CSA and dietary FPH level (p = .006, R² = .574) and between sarcomere length and dietary FPH level (p = .018, R² = .788). An appropriate level of FPH down‐regulated myostatin 2 gene expression and up‐regulated proliferating cell nuclear antigen gene expression, while the expression of myogenic regulatory factors was not affected by dietary treatments (p > .05). To conclude, an appropriate level of FPH may improve muscle growth by regulating the expression of muscle growth‐related genes, and muscle microstructure and ultrastructure.     

Effects of dietary organic trace mineral mixture levels on survival,growth performance,body composition and antioxidant capacity of juvenile turbot (Scophthalmus maximus)

A 70‐day feeding trial was conducted to investigate effects of dietary organic trace mineral (OTM) mixture levels on survival, growth performance, body composition and antioxidant capacity of juvenile turbot (Scophthalmus maximus). The commercial diet with 0.03% inorganic trace mineral (ITM) premix was used as the control, and other four experimental isonitrogenous (50% crude protein) and isolipidic (12% crude lipid) diets were formulated to contain 0.0375%, 0.075%, 0.1125% and 0.15% OTM mixture respectively. Results showed that there were no significant differences in survival among dietary treatments. However, the growth was not significantly different between the control and OTM supplementation diets. Especially, turbot fed the diet with 0.075% OTM had significantly higher specific growth rate than 0.15%, 0.1125% and 0.0375% OTM treatments (p < .05). Moisture, crude protein, crude lipid and ash of the whole fish body showed no significant differences among dietary treatments. Activities of superoxide dismutase in serum of turbot fed diets with 0.1125% and 0.15% OTM were significantly higher than 0.0375% OTM treatment (p < .05). Activities of total antioxidant capacity and catalase in serum were the highest, while the malondialdehyde content was the lowest when the turbot were fed the diet with 0.075% OTM. In summary, results of the present study suggested that the supplementation of 0.0375% or more OTM in diets could obtain similar performance to the commercial diet with ITM premix, and the optimal amount of OTM supplementation in diets of turbot was approximately 0.075% on basis of growth performance and antioxidant capacity.     

饲料中赖氨酸和精氨酸含量对大菱鲆幼鱼生长、体成分和肌肉氨基酸含量的影响

为研究饲料中精氨酸(Arg)、赖氨酸(Lys)水平及其相互作用对大菱鲆生长、体成分和肌肉氨基酸含量的影响,本实验以初始体质量为(18.48±0.16)g的大菱鲆作为研究对象,采用3×3双因素设计,在基础饲料中分别添加Arg(0%、0.9%和2.0%)和Lys(0%、1.19%和2.39%),配制成9种等氮等能的实验饲料,每个处理设3个重复,每重复30尾鱼,养殖周期为8周。实验结果表明,当饲料中Lys添加量为1.19%时,大菱鲆增重率和特定生长率较其他两种添加量组显著升高(P < 0.05),但精氨酸的添加对其影响不显著且与赖氨酸之间不存在交互作用(P > 0.05)。饲料效率、蛋白质效率、蛋白质保留率和鱼体蛋白质含量受饲料中Lys和Arg添加量的交互影响(P < 0.05),在Arg和Lys添加量分别为0.9%和1.19%时,数值最高,显著高于赖氨酸未添加组和高添加组(P < 0.05)。全鱼粗脂肪、水分、灰分和形体指标不受Arg和Lys的交互作用影响(P > 0.05)。粗脂肪和水分随Lys的添加量升高而显著降低(P < 0.05);肝体比和脏体比均随饲料中Arg和Lys添加量的升高而显著降低(P < 0.05)。肌肉中大多数氨基酸含量受饲料Arg和Lys添加量的交互作用,显著性最低值均出现在Arg和Lys添加量分别为0.9%和2.39%组(P < 0.05)。以上结果表明,Arg和Lys的交互作用显著影响了大菱鲆幼鱼的饲料效率、鱼体蛋白质沉积和肌肉氨基酸含量;Arg和Lys添加量分别为0.9%和1.19%时,大菱鲆有最大生长和饲料利用效率;与Arg相比,Lys为主要影响因素,适量添加Lys可以促进生长,而添加量过高Lys会与Arg产生拮抗作用,抑制生长、饲料利用和肌肉氨基酸沉积。     

Characterization of nodavirus and viral encephalopathy and retinopathy in farmed turbot, Scophthalmus maximus (L.)

An outbreak of nodavirus infection in turbot larvae is described with respect to histopathology, immunohistochemistry, cell culture cultivation, RT-PCR amplification and sequence analysis of the capsid protein gene RNA2. Affected turbot developed classical signs of viral encephalopathy and retinopathy (VER) with abnormal swimming behaviour and high mortality levels. In the acute stage of infection, light microscopy revealed vacuolation of the central nervous system (CNS), with positive immunohistochemical staining for nodavirus. Later in the infection, CNS lesions appeared more chronic and contained clusters of cells immunopositive for nodavirus. Bacterial overgrowth in the intestines of the fish may have provoked or influenced the course of the nodavirus infection. We were unable to propagate the virus in cell culture. While RT-PCR using primers designed to detect Atlantic halibut nodavirus gave negative results, further testing with primers complementary to a more conserved region of RNA2 resulted in amplification of a product of the expected size. The entire RNA2 segment was cloned and sequenced. Sequence alignment showed that the turbot nodavirus (TNV) was different from previously described fish nodaviruses. In addition, phylogenetic analysis based on an 823 nt region of the sequence indicated that TNV clustered outside the four established fish nodavirus genotypes, suggesting a fifth genotype within the betanodaviruses.     

Cryopreservation of turbot Scophthalmus maximus (L.) sperm: fertilization and hatching rates

The present paper assesses the fertilization and hatching rates of an artificial fertilization series (n=1153) using fresh and cryopreserved sperm from 49 specimens of turbot Scophthalmus maximus (L.), carried out to confirm the results of a previous study, with the ultimate aim of transferring these cryo‐preservation techniques to commercial hatcheries. No significant differences were found between the fertilization rates of the two groups (fresh and cryopreserved sperm) when their respective fertility rates were >69.2%, which was the case in 75% of all fertilizations. Likewise, no significant differences in hatching rates were found. In order to use the cryopreserved sperm more efficiently, a key concern for commercial use of this technique, we also experimented with a lower sperm:diluent ratio (1:1) than used previously at our centre. We also compared the traditional 0.5‐mL straws with 2‐mL cryotubes able to contain a higher volume of sperm, finding no significant differences in the resulting fertilization and hatching rates. In conclusion, the use of cryopreservation for turbot sperm presents major advantages for broodstock management in commercial hatcheries.     

饲料中添加外源酶对大菱鲆幼鱼生长和饲料利用率的影响

研究了饵料中添加植酸酶、非淀粉多糖酶及二者的组合酶对大菱鲆幼鱼生长、体成分以及饵料利用率的影响。在水温14．0～18．5℃将144尾初始平均体质量为（20．53±0．10） g的大菱鲆幼鱼随机分成4组,每组3个重复,每个重复放养12尾鱼,分别饲喂基础饵料（对照组）和在每千克基础饵料中添加200 mg植酸酶、100 mg非淀粉多糖酶、200 mg植酸酶＋100 mg非淀粉多糖酶的组合酶（试验组）。计42 d。试验结果表明,与对照组相比,添加外源酶显著提高了大菱鲆幼鱼的特定生长率（ P＜0．05）,且添加组合酶的特定生长率最高,较对照组提高了16．30％;但摄食率、饵料系数、蛋白质效率和成活率均无显著影响（P＞0．05）。饵料中添加外源酶对大菱鲆幼鱼的鱼体水分、粗脂肪、灰分和能量无显著影响（P＞0．05）;但鱼体粗蛋白含量均有增加（P＞0．05）,且添加非淀粉多糖酶鱼体粗蛋白含量最高（P＜0．05）。与对照组比较,添加非淀粉多糖酶和组合酶显著提高大菱鲆幼鱼的干物质、粗蛋白、磷和能量的表观消化率（P＜0．05）;添加植酸酶显著提高粗蛋白表观消化率（P＜0．05）,干物质、磷和能量表观消化率较对照组高,但差异不显著（P＞0．05）。饵料中添加外源酶显著提高大菱鲆幼鱼氮、磷贮积率,显著降低氮排放率（P＜0．05）;添加组合酶显著降低磷排放率（P＜0．05）;饵料中分别添加植酸酶和非淀粉多糖酶未显著降低磷排放率（P＞0．05）,但有下降趋势。     

Vaccination of turbot, Psetta maxima (L.), against the protozoan parasite Philasterides dicentrarchi: effects on antibody production and protection

The efficacy of a vaccine against the fish pathogen Philasterides dicentrarchi was evaluated in turbot by measuring the production of specific antibodies and duration of protection. Four groups of turbot were vaccinated twice, on days 0 and 30, with phosphate-buffered saline, mineral oil adjuvant, antigen or antigen plus adjuvant. Specific serum antibodies were determined on day 0 and 1 month after the first and the second vaccinations. Protection was evaluated 1 month after the first vaccination and 1 and 5 months after the second vaccination. Serum antibody titres, measured by enzyme-linked immunosorbent assay, and protection, assessed by challenges, increased significantly 1 month after the second vaccination in the group injected with antigen plus adjuvant and the protection lasted for at least a further 5 months in this group. The relative protection was 77% and 66% 1 and 5 months after the second vaccination, respectively. Administration of antigen or adjuvant separately had no effect on antibody response or protection. The results indicate that emulsion containing antigen plus adjuvant induced durable protection against P. dicentrarchi after the administration of the two vaccinations, and that this preparation can be used as a vaccine against the pathogen.     

Effects of hypoxia on respiratory physiology of turbot, Scophthalmus maximus

Variations in respiratory and acid-base status were studied in turbot (Scophthalmus maximus) during progressive severe hypoxia followed by recovery under normoxic conditions. The first behavioural strategy of turbot under hypoxia was an increase in amplitude and frequency of ventilation. Consequently, standard O₂ consumption remained unchanged over a broad range of O₂ tensions, until a low critical level of 30 mmHg. The hyperventilation induced a moderate blood alkalosis, compensated by a lactic acidosis. The fact that blood pH did not decrease below control values could be explained by the retention in white muscle of most of the lactate produced and by a high capacity for H⁺ excretion. During the recovery period, the marked increase in O₂ uptake corresponding to an oxygen debt repayment, was partly related to the lactate elimination. When total energy contributions of aerobic and anaerobic processes were assessed in terms of ATP, the anaerobic contribution, estimated at the deepest hypoxia level, was higher than 20% of the total energy budget and appeared to totally compensate for the decline in aerobic metabolism. Moreover, the high value of O₂ tension in arterial blood in normoxia and during recovery from hypoxia showed high diffusing capacity of gills in turbot. Our results explain the high tolerance of turbot for O₂ deficient waters.