Soybean protein concentrate as a protein source for turbot Scophthalmus maximus L.

In the first of two experiments, the effect of a gradual substitution of dietary fish meal with soybean protein concentrate (SPC) on growth, feed consumption and protein digestibility was examined in 13 g turbot Scophthalmus maximus. Five isonitrogenous and isoenergetic diets (50% protein and 22 kJ g^?1) containing SPC at protein replacement levels of 0, 25, 50, 75 and 100% were offered by hand twice daily. Growth rates of fish fed diets with zero and 25% replacement were not significantly different, with SGRs of 2.47 and 2.28, respectively. At higher replacement levels, growth rates decreased significantly with SGRs of 2.00, 1.33 and 0.68, respectively. Feed conversion ratios increased with soya replacement, with values of 0.68, 0.75, 0.89, 1.27 and 2.32, respectively, although there was no significant difference between the first two. Feed consumption rates remained constant up to 50% replacement, above which they decreased significantly, possibly because of reduced diet palatability. Apparent protein digestibility (APD) was not affected by the incorporation of SPC and ranged from 82.8 to 87.5%. Results suggest that protein catabolism increases in SPC‐rich diets, possibly because of rapid assimilation and utilization of the methionine supplement. In the second experiment, the importance of amino acid supplements and the beneficial effects of protecting these, either by coating them in protein or incorporating them in a protein–lipid emulsion, was investigated. Growth data provided some indication that the utilization of SPC may be improved by incorporating the methionine and lysine supplement in a protein–lipid emulsion prior to diet preparation, although this finding was not found to be statistically significant (0.1 < P < 0.2).     

Dietary protein requirement for young turbot (Scophthalmus maximus L.)

This study was conducted to determine the optimum dietary protein level for young (an initial weight of 89 g) turbot, Scophthalmus maximus L. Duplicate groups of the fish were fed the five isoenergetic diets containing the various protein levels ranging from 290 to 570 g kg^?1 diet for 45 days. Survival was not affected by dietary protein level. Weight gain and feed efficiency were improved with dietary protein level up to 490 g kg^?1 diet. Dietary protein requirement of young turbot using the broken‐line model was estimated to be 494 g kg^?1 diet based on weight gain response. Protein efficiency ratio was not influenced by dietary protein level. The highest protein retention was obtained from the fish fed the 490 g protein kg^?1 diet. Proximate composition of the fish was not significantly affected by dietary protein level. In considering these results, it was concluded that the 494 g protein kg^?1 diet with 100 g lipid kg^?1 diet (15 MJ kg^?1 diet) provided optimal growth of young turbot under these experimental conditions.     

Relationship between dietary phospholipid classes and neutral lipid absorption in newly-weaned turbot, shape Scophthalmus maximus

A 28-day feeding trial was conducted for comparing the effect of different dietary phospholipid (PL) classes on the growth of post-larval turbot and on the incorporation of dietary neutral lipid fatty acids into their body lipids. Prior to the experiment the turbot were weaned for one week on a PL-free diet. The nine experimental diets were isolipidic and contained an equal amount of highly unsaturated fatty acids in the form of ethyl esters. They differed by their PL content (0, 1 or 2%) and by the PL class composition of the added soybean PL fractions.Compared to the PL-free diet, diets enriched with phosphatidylcholine (PC) resulted in a better growth, a higher triglyceride content (% body dry matter) and increased levels of docosahexaenoic acid (% total fatty acids) in each of the examined body lipid classes (neutral lipid, phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol). The effects of the other soybean PL fractions were less explicit than those noted for soybean PC.The results support the idea that dietary PC plays a role in the intestinal absorption of neutral lipid fatty acids. This might, at least partially, explain the superiority of PC for enhancing growth. Abbreviations: DHA – docosahexaenoic acid (22:6n-3); EPA – eicosapentaenoic acid (20:5n-3); HUFA – highly unsaturated fatty acid; PA – phosphatidic acid; PC – phosphatidylcholine; PE – phosphatidylethanolamine; PI – phosphatidylinositol; PL – phospholipid; PS – phosphatidylserine; PUFA – polyunsaturated fatty acid.     

Phospholipids in formulated start-feeds – Effect on turbot (Scophthalmus maximus L.) larval growth and mitochondrial alteration in enterocytes

Turbot Scophthalmus maximus, larvae were start‐fed with formulated feeds containing marine phospholipids (MP) or soya phospholipids (SP). The experiment was performed with six experimental groups, four groups were given formulated feeds, one group was given live feed and one group was starved. Phospholipid (PL) contents of the formulated feeds were respectively 5% MP, 15% MP, 5% SP and 15% SP. Larvae were offered feed from day 3 post‐hatch. There was no significant size difference on day 5 between the group fed 15% MP and the group given rotifers. Electron microscopical examination of enterocytes was performed on larval intestine on day 5. Larvae fed 5% and 15% MP and larvae fed rotifers had normal looking enterocytes with numerous normal looking mitochondria. In the enterocytes of larvae fed 5% SP and 15% SP the mitochondria appeared swollen with a translucent matrix and fragmented cristae. Thus, MP and not SP seemed suitable as a lipid and PL source for start‐feeding turbot larvae.     

Glucose tolerance in turbot, Scophthalmus maximus (L.)

Eighty turbot, Scophthalmus maximus (L.), (average weight 61 g) were injected intraperitoneally with exactly 1 g glucose per kg body weight. There was a peak in plasma glucose 3 h post injection. Thereafter a gradual decrease to basal levels was seen within 24 h. Plasma triacylglycerol concentrations showed a rapid decline during the first 24 h, and thereafter stable values. Blood haematocrit values decreased from 20% before injection to 16% 72 h after injection. Liver glycogen concentrations showed an initial decrease from 8 to 5 g 100 g^?1 (w.w) during the first 12 h, and thereafter stable values, while muscle glycogen concentrations increased during the first 12 h, and thereafter showed a gradual decline until 72 h. This response was most probably caused by secondary changes upon handling in combination with the direct response to a glucose load. Thus turbot was able to restore alterations in carbohydrate metabolism efficiently within 24 h.     

Isolation of a highly pathogenic Vibrio pelagius strain associated with mass mortalities of turbot,Scophthalmus maximus (L.), larvae

A bacterial strain, characterized as Vibrio pelagius (Hq 222), was isolated from a turbot, Scophthalmus maximus (L.), larvae mass mortality in a commercial fish farm in Spain. Turbot larvae, post-larvae (0.2 g) and juveniles (5 and 15 g) were experimentally infected. The bacterium appeared to be very virulent for larvae and post-larvae, LD50 being < 5 bacteria mL(-1) for larvae 1 week post-infection and 3.9 x 10(5) bacteria mL(-1) in post-larvae at day 12 post-infection. The bacterial strain was recovered in pure culture from the internal organs of infected fish. Histological lesions in post-larvae exhibited swelling and necrosis of gill secondary lamellae, sloughing of intestinal mucosa and necrosis of haematopoietic tissue in the kidney. Vibrio pelagius (Hq 222) was able to grow in sterile sea water when incubated at room temperature or at 15 degrees C. Vibrio pelagius (Hq 222) was more adherent to the turbot cell lines TV-1 and TF than Escherichia coli. In both cell lines, the number of adhered bacteria increased with incubation time.     

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.     

Pathology of Edwardsiella tarda infection in turbot, Scophthalmus maximus (L.)

Macroscopic and histopathological changes in cultured turbot, Scophthalmus maximus (L.), in Spain caused by infection with Edwardsiella tarda are described. Eye tumefaction, inflammation, haemorrhages, ascites and the presence of a purulent fluid were the main macroscopic lesions observed. Histopathological lesions were found in the kidney, spleen and liver. In the kidney and spleen these were characterized by a severe apostematous inflammatory reaction, with a large number of abscesses. The liver was affected to a lesser degree and only some phagocytes loaded with bacteria were observed. Ultrastructural observations indicated that macrophages were the main cell type implicated in the inflammatory response. Most of the bacteria observed within the phagocyte cytoplasm showed no degenerative changes and some were dividing. Degenerative changes observed in macrophages indicate their failure in preventing the infection.     

Growth and survival of young turbot (Scophthalmus maximus L.) produced with cryopreserved sperm

This study assessed the growth and survival over a year of two groups of 4‐month‐old turbot Scophthalmus maximus (L.) derived from artificial fertilization with fresh (FG) and cryopreserved sperm (CG). Growth in both groups, measured monthly in terms of length and weight, were compared. Survival was also recorded. No significant differences were found when we compared weight and length data in both groups. Growth rates were similar between FG and CG young turbot during 1 year. Likewise, the same survival rate (92.2%) was found in both groups. Our results show the good survival and growth of young turbot obtained from cryopreserved sperm, and confirm the cryopreservation technique as a useful tool for raising turbot for commercial purposes.     

大菱鲆胚胎玻璃化方法研究

用大菱鲆(Scophthalmus maximus)神经期胚对6种抗冻剂的毒性进行检测,发现其毒性排列为1,2-丙二醇(PG)<甲醇(MeOH)<甘油(Gly)<二甲基甲酰胺(DMF)<乙二醇(EG)<二甲亚砜(DMSO).以DMF为主因子配制和筛选出11种玻璃化程度较好的玻璃化液,并用大菱鲆肌节期胚对其中5种玻璃化程度最好的玻璃化液进行检测,结果显示,A4(DMSO 20%+DMF 25%)、A7(DMSO 25%+DMF 20%)较适合于大菱鲆胚胎的平衡处理,利用A4和A7平衡处理神经期胚、肌节期胚、心跳期胚、出膜前期胚,结果显示大菱鲆神经胚和肌节胚对玻璃化液的适应能力较强.实验测试出不同时期胚胎在两种玻璃化液中的成活率,为大菱鲆各期胚胎在玻璃化液中的平衡处理提供了依据.利用大菱鲆肌节胚对不同的平衡步骤进行了筛选,发现五步法的平衡效果较好.     

Larval growth of turbot, Scophthalmus maximus (L.) produced with fresh and cryopreserved sperm

The present paper assesses the fertilization and hatching rates, as well as the growth, of larvae obtained from four artificial fertilizations (AF) using fresh and cryopreserved sperm of the turbot Scophthalmus maximus (L.). Larvae growth in both sperm groups, measured in terms of length and weight at culture days 0, 7, 14 and 31, are compared, as well as their growth rates. The two groups' fertilization and hatching rates were not significantly different. Likewise, no significant differences in length and wet weight of 7‐ and 14‐day‐old larvae were found using fresh and cryopreserved sperm; however, significant differences were found in 31‐day‐old larvae, which were more attributable to the variability inherent in larval turbot culture, and to variability in the reproductive specimens used in our study, than to the type of sperm employed. These results indicate that the type of sperm used in artificial fertilization, i.e. fresh or cryopreserved, is not a determining factor, either for fertilization and hatching, or for subsequent larval development. Our results also confirm once again the high quality of cryopreserved turbot sperm, and its usefulness in commercial hatcheries.     

The immune response of turbot, Scophthalmus maximus (L.), skin to high water temperature

The immune responses of mucus from the skin of turbot, Scophthalmus maximus, were studied in relation to changing water temperature. Groups of fish were exposed to a programmed increase of 3 °C per 48 h, until the experimental water temperatures of 16, 20, 23, 25, 27 or 28 °C were reached. After 48 h at the relevant temperature, the expressions of immune‐related factors were determined, including immunoglobulin M (IgM), IL‐1β, hepcidin, transferrin, lysozyme, acid/alkaline phosphatase and superoxide dismutase using RT–PCR and spectrophotometric methods. Significant changes in mucus immunity were observed, which paralleled with those previously reported for serum in other fish species. Hence, it is suggested that the serum and mucus immune system of turbot have a similar regulatory system. This information could be useful in better understanding the role of the mucus as a component of the innate immune system.     

大菱鲆营养成分与食用价值研究概述

根据国内外研究成果,对大菱鲆Scophthalmus maximus L.(多宝鱼)的主要营养成分、食用价值以及养殖管理方式进行综述.结果表明,大菱鲆不仅因含有丰富的鲜味氨基酸而味道鲜美,而且含有丰富的人体所必须的营养物质,如必须氨基酸和高度不饱和脂肪酸(DHA和EPA)等,是一种有益于人类心脏保健、能延缓衰老和促进儿童生长发育的优质海水鱼类.大菱鲆在我国主要采用"温室大棚 深井海水"工厂化养殖模式流水养殖,养殖水源洁净无污染,严格遵循现有的养殖和管理技术规范,保证养殖产品安全和无公害,可以放心食用.     

Stress responses of larval turbot, Scophthalmus maximus L., exposed to sub-lethal concentrations of petroleum hydrocarbons

Turbot larvae were exposed to dilutions of the water soluble fraction (WSF) of crude oil (1–25%), in a laboratory flow-through system. Whole body immunoreactive cortisol (IRC) content of premetamorphic 345 degree C day and early metamorphosing 450 degree C day larvae (23 and 30 days post hatch, respectively) was elevated when exposed to 25% WSF for 6 h, but the lower WSF concentrations did not induce IRC elevations. Larvae of 450 degree C days exposed to WSF for 6 h and then left in clean sea water for 24 h showed recovery of IRC, whilst the IRC content of 450 degree C day larvae exposed to WSF for 30 h remained elevated. Whole body thyroxine content of 345 degree C day larvae exposed to 25% WSF for 6 h was significantly elevated, which may have implications for larval development/metamorphosis. However, whole body triiodothyronine content of these larvae was not altered by WSF-exposure. Furthermore, larvae of 450 degree C days, WSF-exposed for 6 h or 30 h showed no thyroidal disturbance. Larvae of 345 degree C days exhibited significantly elevated whole body adrenaline and noradrenaline content after exposure for 6 h to 25% WSF; however, more diluted WSF did not influence adrenaline or noradrenaline content. These studies have demonstrated that larvae of 345 and 450 degree C days can successfully mount endocrine stress responses but that environmentally realistic concentrations of petroleum hydrocarbons do not induce these responses.     

Estimation of genetic parameters for early growth trait of turbot (Scophthalmus maximus L.) using molecular relatedness

In this study, a data set of 1140 progenies from 19 families that were composed of 13 half‐sib and six full‐sib families was used in the estimation of the genetic parameters of weight for juvenile turbot at 100 days post hatch. Sixty progenies were randomly selected from each family, and 20 of these individuals were genotyped at 12 microsatellite loci. Two sorts of relatedness, including pedigree relatedness (PR) from a complete pedigree and average molecular relatedness (AMR) from molecular markers, were compared to explore the feasibility of AMR in selective breeding when only parents were known. Two different animal models, Model 1 and Model 2 which were without and with the addition of maternal and common environmental effects, respectively, were used to estimate the variance component and breeding values for weight using restricted maximum likelihood (REML) method. Thereafter, cross‐validation was applied to investigate predictive ability of model and accuracy of breeding values. Pearson correlation analysis showed that AMR was highly correlated (0.91) with PR. According to conditional Akaike Information Criterion, the best models for AMR and PR methods were Model 2 and Model 1 respectively. Heritability estimates from AMR and PR methods based on their best models were 0.19 (±0.06) and 0.66 (±0.17). Cross‐validation showed that AMR was comparable to PR in terms of predictive abilities and accuracies of breeding values based on the same model. This study therefore suggests that the AMR can be applied as alternative of PR when only parents were known.     

Effects of silymarin on growth performance,antioxidant capacity and immune response in turbot (Scophthalmus maximus L.)

A 9‐week feeding trial was conducted using triplicate groups of turbot (6.50 ± 0.01 g) to explore the potential effects of silymarin. Three concentrations of silymarin (100, 200, and 400 mg/kg) were added to the plant protein‐based diet. Fish were randomly distributed into fiberglass tanks (30 fish per tank). The results showed that adding 100 mg/kg silymarin significantly improved the growth performance, with no effects on feed utilization. The antioxidant capacity in the liver was significantly improved in the 100 mg/kg and 200 mg/kg silymarin groups by not only inducing the activities of superoxide dismutase (SOD) and catalase but also increasing the messager RNA (mRNA) expression levels of SOD, glutathione peroxidase, and peroxiredoxin 6. Meanwhile, supplying 100 and 200 mg/kg of silymarin enhanced the heights of villi and enterocytes. Silymarin supplementation reduced the mRNA expression of interleukin‐8 and tumor necrosis factor‐α but induced the expression of transforming growth factor‐β (TGF‐β) in the intestine. These results indicated that silymarin was a potential nutraceutical that could enhance the growth performance and health status of turbot fed in a high plant protein diet. Adding 100 mg/kg silymarin to the plant protein diet achieved optimal performance in turbot.     

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.     

Statistical properties and performance of pairwise relatedness estimators using turbot (Scophthalmus maximus L.) family data

The statistical properties and performance of four estimators of pairwise relatedness were evaluated in several scenarios using the microsatellite genotype data from a set of large known full‐sibships of turbot. All estimators showed a significant negative bias for the four kinships commonly used in these studies (unrelated: UR, half‐sibs, full‐sibs and parent–offspring), when allele frequencies of the reference population were estimated from the individuals analysed. When these frequencies were obtained from the base population from which all families proceeded, the bias was mostly corrected. The Wang (W) and Li (L) estimators were the least sensitive to this factor, while the Lynch and Ritland (L&R estimator) was the highest one. The error (mean around 0.130) was very similar in all scenarios for W, L and Queller and Goodnight (QG) estimators, while L&R was the highest error‐prone estimator. Parent–offspring kinship resulted in the lowest error, when using W, L and QG estimators, while UR resulted in the lowest error with the L&R estimator. Globally, W was the best‐performing estimator, although L&R could perform better in specific sampling scenarios. In summary, pairwise estimators represent useful tools for kinship classification in aquaculture broodstock management by applying appropriate thresholds depending on the goals of the analysis.     

Influence of several non‐nutrient additives on nonspecific immunity and growth of juvenile turbot,Scophthalmus maximus L.

The effects of three non‐nutrient additives on nonspecific immunity and growth of juvenile turbot (Scophthalmus maximus L.) were studied in this feeding experiment. The five treatments are basal diet alone, basal diets containing three different additives [0.4 g kg^?1 of xylo‐oligosaccharides (XOS), 1.3 g kg ^?1 of yeast cell wall and 0.8 g kg ^?1 of bile acids] individually or in combination. Two hundred and twenty‐five turbots (average initial weight 151.3 ± 11.3 g) were randomly allotted in five treatments with three replicates within each treatment in a 72‐day period. Comparing with basal diet group, activities of C3, C4, phagocyte, lysozyme, specific growth rate and feed conversion rate in yeast cell wall, XOS and the combined groups was enhanced significantly (P < 0.05); however, these parameters in bile acid groups were increased slightly (P > 0.05) except for phagocyte (P < 0.05); superoxide dismutase activity in additive groups was not significantly increased (P > 0.05) except for the combined group (P < 0.05). In conclusion, supplementation of yeast cell wall and XOS enhanced the nonspecific immunity of juvenile turbot. Synergistic or additive effect of the three additives was not observed.