The effect of ultrafiltered fish protein hydrolysate level on growth performance,protein digestibility and mRNA expression of PepT1 in juvenile turbot (Scophthalmus maximus L.)

The intention of the study was to investigate the effect of ultrafiltered fish protein hydrolysate (UF) level on growth, feed utilization, apparent digestibility coefficients and proximal intestine peptide transporter 1 (PepT1) mRNA level for juvenile turbot (Scophthalmus maximus L.). Experimental diets (UF‐0, UF‐5, UF‐10, UF‐15 and UF‐20) were prepared containing about 68% plant protein, and fish meal protein was, respectively, replaced by 0%, 5%, 10%, 15% and 20% UF of dietary protein. Diet PP contained about 78% plant protein, and diet CAA contained about 10% crystalline amino acid mixture. All diets were fed to seven triplicate groups of turbot (initial weight 16.05 ± 0.03 g) for 68 days. Fish fed diet UF‐10 had an increasing tendency in growth compared with diets contained UF, while dietary UF level was not significantly correlated with specific growth rate and feed intake. Feed efficiency, protein efficiency ratio and protein productive value significantly correlated with dietary UF level, and fish fed diets contained low‐level UF had higher digestibility than that diets UF‐0, PP and CAA. There was a decreasing tendency in PepT1 expression level with dietary UF level. The results indicated that low‐level UF showed a positive effect on growth and feed utilization in juvenile turbot.     

1H NMR‐based metabolomics studies on the effect of size‐fractionated fish protein hydrolysate,fish meal and plant protein in diet for juvenile turbot (Scophthalmus maximus L.)

This study was designed to evaluate changes in the metabolic profile of liver and muscle of turbot (Scophthalmus maximus L.) fed fishmeal‐based diet, diets containing size‐fractionated fish protein hydrolysate and plant protein‐based diet using ¹H NMR‐based metabolomics approach combined with the growth. Fish protein hydrolysate (FPH) was obtained by enzymatic treatment, permeate fraction was obtained as UF by ultrafiltered step, and retentate fraction was retained as RF. FM diet contained fish meal used as a single protein source. Four other diets (PP, UF, FPH and RF) contained 180 g kg^?1 diet fish meal. 54, 55 and 55 g kg^?1 dry diet UF, FPH and RF were supplemented to UF, FPH and RF diets. All diets were formulated to be isolipidic and isonitrogenous fed to five triplicate groups of turbot (16.05 ± 0.03 g) for 68 days. O‐PLS‐DA in FM versus UF, FM versus FPH, FM versus RF and FM versus PP resulted in a reliable model for muscle and liver tissue, while O‐PLS‐DA in UF versus FPH and UF versus RF only showed metabolites changes in liver tissue. Results indicated that metabolite changes among the different treatments were consistent with the growth tendency.     

Effect of size‐fractionated fish protein hydrolysate on growth and feed utilization of turbot (Scophthalmus maximus L.)

Four experimental diets were fed to turbot to examine the effect of fish hydrolysate and ultra‐filtered fish hydrolysate on growth performance, feed utilization and non‐specific immune response. Fish hydrolysate was produced by enzymatic treatment and size fractionated using ultra‐filtration (UF). The permeate (molecular weight <1000 Da) after UF and the non‐ultra‐filtered fish hydrolysate (NUF) were tested as feed ingredients. Diets UF1, UF2 contained 3.7%, 1.2% ultra‐filtered fish hydrolysate to replace fish meal protein respectively. The diets UF1, NUF were identical in composition except that the molecular weight of fish hydrolysate in the diet. Fish meal was used in the control diet. All diets were made equal in protein, lipid and energy. Each experimental diet was fed to juvenile turbot (27.87 ± 0.04 g) in triplicate for 8 weeks. Results of this study indicate that the best overall growth and feed utilization of turbot juveniles were obtained with a diet containing higher dose of the small molecular weight compounds in fish hydrolysate. Acid phosphatase, alkaline phosphatase, lysozyme and superoxide dismutase activity in serum were not affected by diet. Total antioxidant capacity was improved with increasing level of low molecule weight fish hydrolysate (UF1).     

The effect of ultrafiltered fish protein hydrolysate levels on the liver and muscle metabolic profile of juvenile turbot (Scophthalmus maximus L.) by 1H NMR‐based metabolomics studies

A ¹H NMR‐based metabolomics approach was to explore the effect of ultrafiltered fish protein hydrolysate (UF) levels on the liver and muscle metabolic profile of juvenile turbot (Scophthalmus maximus L.). Fish protein hydrolysate (FPH) was produced from by‐products by enzymatic treatment, and UF was obtained by diluting FPH followed filtration. Fish were fed diets containing fish meal protein, which was, respectively, replaced by UF protein 0, 50, 100, 150 and 200 g kg^?1 of dietary total protein (UF‐0, UF‐5, UF‐10, UF‐15 and UF‐20) for 68 days. OPLS‐DA of liver and muscle showed that high levels of UF in diets may lead to corresponding tissue metabolites changes. In liver tissue, changing metabolites included dimethylamine, N,N‐dimethylglycine, valine, isoleucine, leucine, arginine and alanine and were mainly involved in choline metabolism and amino acid metabolism. For muscle tissue, changing metabolites included lactate, alanine, proline, fumarate, tyrosine, histidine, cystathionine and taurine. And the metabolites were mainly involved in glycolysis and gluconeogenesis, proline metabolism and taurine metabolism. ¹H NMR‐based metabolomics is a useful approach to investigate different levels of UF on metabolic profile in liver and muscle tissues combined with the growth.     

Effect of fish meal replacement by plant protein blend on amino acid concentration,transportation and metabolism in juvenile turbot (Scophthalmus maximus L.)

The objective of the study was to evaluate the effects of incorporating plant protein blend in juvenile turbot (Scophthalmus maximus L.) diet on free amino acid (AA) concentration and the expression of genes related to peptide and AA transporters, key enzymes of AA metabolism and AA response (AAR) pathway. Fish were fed diets with fish meal (FM), or 400 g/kg FM replacement by plant protein blend for 9 weeks. Compared with the FM diet, PP40 diet did not affect plasma essential amino acid (EAA) concentration or AA metabolic enzymes gene in intestine, while it significantly upregulated all the detected peptide and neutral AA transporters gene. Results in muscle indicated that PP40 diet led to a great reduction of EAA concentrations and mRNA abundance of two kinds of AA transporters (SNAT2 and b^0,+AT), while it greatly increased the gene expression of L‐type and T‐type AA transporters (LAT2 and TAT1) and the enzymes of AA catabolism (BCKDH‐E2) and anabolism (asparagine synthetase). In addition, the expression of genes related to AAR pathway were all greatly stimulated by PP40 diet in muscle. Our results provide a molecular explanation for the change of tissues AA concentrations caused by plant protein in turbot, which maybe applicable for general carnivorous fish.     

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.     

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.     

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.     

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.     

Nutrient utilization in Atlantic salmon (Salmo salar L.) fed increased levels of fish protein hydrolysate during a period of fast growth

The present work was designed to study whether changes in dietary protein quality by means of partial inclusion of fish protein hydrolysate (FPH) would alter fish growth, feed utilization, protein retention and metabolism and fish health in general. FPH was produced after hydrolysing whole minced herring using the industrial enzyme Alcalase®. The dietary protein source, low‐temperature‐dried (LT) fishmeal nitrogen was exchanged with FPH nitrogen at six levels of inclusion ranging from 0 to 300 g kg^?1. The experimental diets were fed to post‐smolt (1+) Atlantic salmon (Salmo salar), with mean initial weight of 174 g for a period of 68 days. All diets were iso‐nitrogenous, iso‐energetic and contained the same amount of amino acids. Fish fed medium inclusion of FPH (180–240 g kg^?1) showed a tendency to have higher feed intake than fish fed lower and higher levels of FPH inclusions. Significant higher individual specific growth rates were present in fish fed diets with 180 and 240 g kg^?1 FPH when compared with those fed 300 g kg^?1. Feed conversion ratio increased significantly (R² = 0.61) and protein efficiency ratio decreased significantly (R² = 0.59) in fish fed increased levels of FPH. Further, apparent digestibility of crude protein and the amino acids arginine, lysine, methionine and phenylalanine increased significantly with increased dietary inclusion of FPH. Plasma free amino acids, ammonium and urea indicated that FPH amino acids was absorbed earlier and nonsynchronously, and may thus be more prone to be catabolized than in those fish fed the less solubilized protein. FPH inclusion did not have an impact on fish health, as evaluated by haematology and clinical parameters.     

The tolerance and safety assessment of taurine as additive in a marine carnivorous fish,Scophthalmus maximus L.

The effects of dietary taurine on growth performance, liver and intestine morphology, serum physiological and antioxidant parameters, serum thyroid hormone level, muscle taurine content and fatty acid composition of turbot were first evaluated, for the safe utilization in marine fish feed and for human food safety. Four experimental diets were formulated to contain 0, 10, 50 and 100 g/kg taurine. Each diet was randomly assigned to six replicates of 30 juvenile turbot (initial mean weight of 7.46 g). The feeding trial lasted for 10 weeks. The growth performance of fish was significantly enhanced by 10 g/kg dietary taurine. The integrity of the distal intestine was impaired and the absorptive surface was found to be significantly reduced by 100 g/kg dietary taurine. The obvious pathological changes in liver were observed in fish fed 100 g/kg taurine. Dietary taurine with 10 and 50 g/kg significantly increased the activities of serum superoxide dismutase, lysozyme and thyroid hormone. The taurine content in muscle was found to be significantly increased by dietary taurine; however, no significant differences were observed among taurine‐supplemented treatments. This study suggested that 10 g/kg taurine was safe in turbot feed, and fivefold of safety margin was obtained.     

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.     

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.     

Fishmeal replacement by mixed plant proteins and maggot meal on growth performance,target of rapamycin signalling and metabolism in juvenile turbot (Scophthalmus maximus L.)

Fishmeal could only be replaced by plant proteins at limited levels in aquafeeds, especially for carnivorous fish. In this study, an experiment was designed to evaluate the possibility of improving the utilization of plant proteins by maggot meal supplementation in turbot diet. Five diets were formulated: a reference diet (FM) containing 63% fishmeal and four experimental diets (35(0%), 35(3%), 40(0%), 40(3%)) in which fishmeal was substituted at different levels by plant proteins with 0–3% maggot meal. Turbot (4.90 ± 0.03 g) was fed with these diets for 9 weeks. Fishmeal was successfully replaced by plant proteins in turbot diet without growth reduction at 35% but not 40%. However, maggot supplementation (3%) at 40% plant protein replacement level achieved comparable growth performance with that of fishmeal. Maggot meal supplementation improved apparent digestibility coefficients, plasma hydroxyproline levels, intestine trypsin activities and activated target of rapamycin (TOR) signalling, all of which were decreased or down‐regulated after high plant protein replacement. Therefore, this study demonstrated maggot meal, a potential valuable protein source for turbot.     

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.     

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 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.     

Effects of replacing fishmeal with different cottonseed meals on growth,feed utilization,haematological indexes,intestinal and liver morphology of juvenile turbot (Scophthalmus maximus L.)

A ten‐week feeding trial was conducted to evaluate the effect of replacing fishmeal with two differently processed cottonseed meals (CSM), namely XC and SC, separately in turbot (5.28 ± 0.02 g). Nine isonitrogenous and isoenergetic diets were formulated without fishmeal replacement (FM), 150 g/kg (XC15, SC15), 250 g/kg (XC25, SC25), 350 g/kg (XC35, SC35) and 450 g/kg (XC45, SC45) of fishmeal replaced by CSM. Fishmeal was successfully replaced by XC in turbot diets without growth reduction at 350 g/kg, but not by SC even at 150 g/kg. The apparent digestibility coefficients of SC‐included diets were significantly lower than XC‐included diets at same replacement level. The activities of aspartate aminotransferase and superoxide dismutase were significantly affected in XC45 and SC45 group. The XC45 and SC‐included diets caused shortened distal intestine villi height and structural damage of liver compared with FM diet. This study indicated that different processing methods could affect the nutritional value of CSM and effect as a protein source for turbot. CSM with high quality could be an important alternative dietary protein source for juvenile turbot.