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.     

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.     

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.     

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.     

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.     

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.     

Effect of feed restriction on the growth performance of turbot (Scophthalmus maximus L.) juveniles under commercial rearing conditions

Two trials were performed to study the effect of periodic feed deprivation (trial 1) or feed restriction, followed by satiation feeding (trial 2) on the growth performance of turbot juveniles under commercial rearing conditions. In trial 1, duplicate groups of 350 fish with an initial weight of 62 g were fed a commercial diet to apparent visual satiation for 7, 6, 5 or 4 days a week for 83 days. At the end of the trial, fish weight was directly related to the number of feeding days but feed efficiency and protein efficiency ratio were not affected by treatments. At the end of the trial, there were no differences in whole‐body composition among groups. In trial 2, duplicate groups of 500 fish with an initial weight of 33 g were fed a commercial diet to satiation (100%) or feed restricted to 90%, 80% and 70% of satiation for 90 days. Thereafter, all groups were fed to satiation for 34 days. During the feed restriction period, growth was directly related to feed intake, while during the satiation feeding period, it was inversely related to the previous feeding level. At the end of the trial, the final weight was not different among groups. At the end of the feed restriction period, whole‐body lipid content showed a trend to decrease with an increase in the feed restriction level. The results of this study indicate that under practical conditions, turbot juveniles should be fed daily as even cycles of short periods of feed deprivation negatively affect growth, while not improving feed efficiency. On the contrary, even after a relatively long feed restriction period, fish shows compensatory growth, and this may be used as a feed management strategy for controlling fish production in commercial farms.     

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.     

Finding suitable growth models for turbot (Scophthalmus maximus L.) in aquaculture 1 (length application)

Growth data of two different commercial turbot (Scophthalmus maximus) strains reared in recirculating aquaculture systems were analysed with the aim to determine the most suitable model for turbot. To assess the model performance three different criteria were used: (1) The mean percentage deviation between the estimated length and actual length; (2) the residual standard error with corresponding degrees of freedom and (3) the Akaike information criterion. The analyses were carried out for each strain separately, for sexes within strains and for a pooled data set containing both strains and sexes. We tested a pre‐selection of six models, containing three to four parameters. Models were of monomolecular shape or sigmoid shape with a flexible point of inflection including the special case of monomolecular shape in defined cases of their parameters. The 4‐parametric Schnute model achieved best fit in 62% of all cases and criteria tested, followed by the also 4‐parametric generalized Michaelis–Menten equation in 48% and the 4‐parametric Janoschek model (38%). The von Bertalanffy growth function achieved only 29%, Brody 24% and a new flexible function 19% best fit. In a 1–1000 day growth‐simulation sigmoid shaped curves were produced by the Schnute model in 71% of cases. The Janoschek and the Michaelis–Menten model each produced sigmoid curves in 57% of all cases. This indicates that a flexible 4‐parametric function reflects the growth curve of turbot the best and that this curve is rather sigmoid than monomolecular shaped.     

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 dietary water content on meal size, daily food intake, digestion and growth in turbot, Scophthalmus maximus (L.)

When fed once daily with wet squid, turbot (30–50 g) accustomed to dry pellets require many days to increase intake to meet their feed requirement (≈ 10 mg dry matter g⁻¹ bw meal⁻¹). Adaptation takes 1–2 days if several daily feedings are given. With dried squid, they ingest about 20% of the wet squid bulk because the stomach contents expand when moisturised. In contrast, turbot eat enough wet squid to fill most of the available stomach volume (≈ 7.6 mL 100 g⁻¹ bw). When presented in gelatine capsules, food water content is masked and does not affect the volume ingested. Moistening the contents shortens the delay before gastric emptying starts to one-third (0.6 h) compared with dry food (1.9 h). Daily dry-matter intake increased when dry contents were moistened but only if two or more meals were offered per day. Turbot adapt their digestion to supply water for dry diets but this may add extra metabolic costs. When offered 20 mg dry matter g bw⁻¹ day⁻¹, divided into four equal meals, turbot grew faster and more efficiently with moist than with dry squid. Protein, energy and dry-matter digestibilities were also enhanced. The increased daily protein absorption did not increase ammonia release, indicating that the extra protein was used for somatic growth.     

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.     

Effect of growth hormone transgenic Synechocystis on growth, feed efficiency, muscle composition, haematology and histology of turbot (Scophthalmus maximus L.)

The present study investigated the effect of supplementing feed with transgenic Synechocystis sp. PCC6803 containing the Paralichthys olivaceus growth hormone (GH) gene on growth, feed intake and feed efficiency ratio, muscle composition, haematology and histology of turbot (Scophthalmus maximus L.). At the end of the 40‐day feeding trial, the specific growth rate of fish fed the supplemented feed with 1.0% transgenic Synechocystis sp. PCC6803 was 21.67% higher (P<0.05) than that of control fish. Although body weight and feed efficiency ratio significantly increased (P<0.05) in fish fed the diet supplemented with transgenic alga, feed intake and condition factor of the experimental fish were unaffected. Muscle composition analysis showed that the protein content was positively influenced by the transgenic alga, whereas the lipid content was unaffected. Haematological parameters, including red blood cell, white blood cell, haemoglobin, and serum biochemical indices, such as enzyme activities of alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, lactate dehydrogenase, concentrations of total protein, glucose, blood urea nitrogen, creatinine, triglyceride and cholesterol and ion levels of K, Na, Cl, P were not influenced by supplementing the transgenic Synechocystis sp. PCC6803. Furthermore, no histopathological alterations were induced by transgenic alga treatment in the stomach, intestine, liver, spleen and kidney of the experimental fish. The results of the present study indicated that transgenic Synechocystis sp. PCC6803 containing P. olivaceus GH gene is an efficient growth promoter and a safe feed additive for fish.     

Taurine alone or in combination with fish protein hydrolysate affects growth performance,taurine transport and metabolism in juvenile turbot (Scophthalmus maximus L.)

The study was conducted to investigate the effects of taurine (Tau) alone or in combination with fish protein hydrolysate (FPH) on growth performance, the expression of Tau transporter (TauT) and metabolic profile in juvenile turbot. FM, FPH0, FPH0+T, FPH10 and FPH10+T diets, respectively, contained 300, 150, 150, 80, and 80 g/kg fishmeal. FPH10 and FPH10+T diets contained 62 g/kg FPH. FPH0+T and FPH10+T diets were, respectively, prepared by supplementing the FPH0 and FPH10 diet formulations with 8 g/kg Tau. Specific growth rate was the highest in FM group and the lowest in FPH10 group. TauT mRNA levels in fish fed Tau supplemented diets were significantly lower than that in Tau unsupplemented diets. NMR‐based metabolomics analysis showed that Tau contents in liver of FPH0+T and FPH10+T were significantly higher than that of FM, FPH0 and FPH10. In muscle, Tau contents were significantly decreased in the FPH10+T versus FPH0 and the FPH10+T versus FPH10 comparisons. In conclusion, 62 g/kg FPH to replace fishmeal may not affect Tau synthesis, transport and metabolism. However, Tau supplemented alone or in combination with a certain level of FPH could reduce the requirement for Tau synthesis and transport and increased Tau levels in muscle and liver.     

Dietary nucleotides improve the growth performance,antioxidative capacity and intestinal morphology of turbot (Scophthalmus maximus)

A growth trial was conducted to evaluate the effects and safety of nucleotides in low fish meal diets on the growth performance, antioxidative capacity and intestinal morphology of turbot (Scophthalmus maximus). High fish meal control diet was formulated with 500 g kg^?1 fish meal. Seven levels (0.075, 0.15, 0.225, 0.300, 1.5 and 3.0 g kg^?1, respectively) of nucleotides were added to a low fish meal basal diet, which was formulated with 400 g kg^?1 fish meal. The eight experimental diets were fed to groups of juvenile turbot (initial weight: 6.0 ± 0.03 g) for 60 days. Results showed that compared with high fish meal control diet, low fish meal basal diet treatment had lower total antioxidative capacity (T‐AOC), glutathione peroxidase activity, fold height of proximal and distal intestine, enterocyte height of all evaluated enteric section and microvillus height of mid‐intestine and distal intestine (P < 0.05). However, supplemented nucleotides in diets could significantly improve growth (specific growth rate, SGR), feed utilization, antioxidative capacity and intestinal morphology of turbot (P < 0.05). Broken‐line regression analysis of SGR and T‐AOC showed that the optimal supplemental levels of dietary nucleotide for juvenile turbot were 0.366 and 0.188 g kg^?1, respectively. In summary, 0.300 g kg^?1 of dietary nucleotides was helpful in improving growth, feed utilization, antioxidative capacity and intestinal morphology of turbot fed with low fish meal diet. Excessive dietary nucleotides (3.0 g kg^?1) might cause oxidative stress and morphological damage in intestine and then reduce the growth of turbot.     

Inbreeding evaluation using microsatellite and its effect on growth traits in turbot,Scophthalmus maximus

Turbot is an economically relevant fish, and simultaneously at the risk of inbreeding depression in China. Microsatellite DNA markers were used to evaluate inbreeding level of a random turbot breeding population and further investigate its effects on growth traits. DNA based inbreeding coefficients (F_DNA) were ranged from 0 to 0.4124. Average inbreeding coefficient was 0.0621 with a standard deviation of 0.0658. Regression analysis results showed inbreeding had a very significant (p < .01) effect on harvest body weight (BW) and significant (p < .05) effect on harvest body long (BL). Inbreeding depression, expressed as the percent change in phenotype per 10% increase in F, was ?4.00% for BW and ?1.14% for BL. The inbreeding depression on high inbreeding coefficient (more than 10%) individuals was further analyzed. A set of significant (p < .05) and larger inbreeding depression was obtained, ?7.15% and ?2.49% for BW and BL respectively. This study demonstrates that inbreeding has a negative effect on growth traits and highlights the need of DNA markers to control inbreeding and maximize the genetic diversity in selection breeding programs.     

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.     

Dietary krill hydrolysates affect the expression of growth‐related genes in juvenile turbot (Scophthalmus maximus L.)

A 10‐week feeding experiment in indoor flow‐through seawater system was conducted to investigate the effects of dietary krill hydrolysate on the expression of growth‐related genes in juvenile turbot (Scophthalmus maximus L.; initial body weight 9.45 ± 0.01 g). Three isonitrogenous and isolipidic experimental diets containing high plant protein were formulated to contain 0 (control), 50 g/kg (LKH) and 100 g/kg (HKH) krill protein hydrolysate (KH) to replace fishmeal, respectively. Triplicate groups of 30 fish were fed for 10 weeks to apparent satiation twice daily. At the end of the feeding trial, the mRNA expressions of insulin‐like growth factor (IGF‐1) gene in liver, peptide transporters (PepT1) gene in pyloric caeca and proximal intestine and neuropeptide Y (NPY) gene in brain in all groups were determined. IGF‐1, PepT1 and NPY expression levels in HKH group were significantly increased compared with those of LKH and control (p < 0.05), which was consistent with the SGR, feed efficiency, PER and PPV. These results indicated that dietary 100 g/kg krill hydrolysate could improve growth performance and upregulate the mRNA expression of IGF‐1, PepT1 and NPY genes in juvenile turbot.