Dietary Protein Requirement of Juvenile Turbot(Scophthalmus maximus Linnaeus)

The dietary protein requirement of juvenile turbot(initial average weight, 38.2 g ± 0.1 g) reared indoor in aerated aquaria was determined in this study. Five energy equal experimental diets were formulated with fish meal as protein source, which contained different concentrations of protein(47.2%, 51.0%, 54.6%, 59.3% and 63.6% of dry diet). Three groups of fish with 18 individuals in each, were cultured in 300-L tanks and fed twice a day for 8 weeks. During culture, temperature was controlled between 15.0 and 18.0℃, salinity was controlled between 28.5 and 32.0, acidity was controlled between p H7.8 and p H8.5, and ammonia nitrogen was maintained below 0.03 mg L-1 and dissolved oxygen was maintained about 7 mg L-1. Results showed that the growth of fish was significantly affected by dietary protein content(P 0.05). Specific growth rate(SGR) of turbot increased when dietary protein content varied between 47.2% and 51.0%(P 0.05), and then kept stable when dietary protein content was higher than 51.0%. Fish which were fed the diet containing 63.6% protein showed the highest SGR while those fed the diet containing 59.3% protein showed the highest feed efficiency rate. No significant difference of feed intake and protein efficiency ratio was found among experimental diets(P 0.05). Broken-line regression analysis of SGR showed that the optimal dietary protein requirement of turbot was about 57.0%.     

Effects of Dietary Carbohydrate-to-Lipid Ratio on the Growth Performance and Feed Utilization of Juvenile Turbot(Scophthalmus maximus)

A 9-week feeding trial was conducted to investigate the effects of dietary carbohydrate to lipid ratio(CHO:LIP) on the growth performance and feed utilization of juvenile turbot Scophthalmus maximus(initial body weight 8.75 g ± 0.04 g). Four isonitrogenous and isoenergetic low protein level(39%) diets were formulated with increasing ratios of dietary carbohydrate to lipid(2:18, 6:18, 18:12 and 28:6). A high protein level(50%) diet with the 2:12 ratio of carbohydrate to lipid was used as the control. Results showed that the survival rate, contents of moisture, crude protein and ash in muscle were not significantly affected by dietary treatments. With the dietary CHO:LIP ratio increased from 2:18 to 18:12, weight gain rate significantly increased(P 0.05). Higher dietary CHO:LIP ratio(28:6) resulted in the significantly decreased weight gain rate(P 0.05). Meanwhile, this treatment also resulted in the highest daily feed intake and liver glycogen content, as well as the lowest feed efficiency(P 0.05). Muscle glycogen content in fish fed the diet with 2:12 or 2:18 CHO:LIP ratio was significantly lower than those fed with the other three diets(P 0.05). The present results confirmed that the juvenile turbot can utilize carbohydrate. Furthermore, the appropriate ratio of dietary carbohydrate to lipid was important to the growth and feed utilization of turbot. The proper CHO:LIP ratio based on the growth performance in the present study was determined to be 18:12 when the dietary protein level was 39%.     

Characterization of Turbot(Scophthalmus maximus) Skin and the Extracted Acid-Soluble Collagen

The biochemical composition of the turbot skin was investigated. The moisture level of the skin was found to be 51.4%. Based on dry matter content, there were relatively high protein(82.1%) and lipid(13.1%) concentrations in the turbot skin. Mineral element analysis revealed that the turbot skin had high Ca content(2069.0 mg kg^-1), and the concentrations of toxic heavy metals Hg and Pb were less than 0.005 mg kg^-1, which indicates that the turbot skin is a safe resource for collagen production. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) analysis showed that acid-soluble collagen(ASC) extracted from the turbot skin was type-I collagen. The imino acid content of the ASC was 241.6 per 1000 amino acids, which suggests a relatively high denaturation temperature. The Fourier transform infrared(FTIR) spectra of ASC reflected a highly stable structure, and the measured denaturation temperature of ASC was 29.5℃, which is higher than those from many temperate fishes. ASC was the most soluble at pH 4.0, and when the pH values were below or above 4.0, the solubility decreased rapidly. The ASC exhibited a relatively high solubility when NaCl concentration was lower than 2%. These results show that turbo skin can be employed as a source for producing collagen with high quality.     

Effect of Dietary Vitamin E on the Sperm Quality of Turbot(Scophthalmus maximus)

A 3-month feeding experiment was conducted in an in-door seawater system to investigate the effect of dietary vitamin E(Ve) on the sperm quality of turbot(Scophthalmus maximus). D-α-tocopherol acetate was supplemented to the basal(control) diet(65.14 mg kg–1 Ve) to obtain low and high levels of dietary Ve(244.60 mg kg–1, LVe; 721.60 mg kg–1, HVe). Compared with the control, sperm concentration was significantly increased in Ve-supplemented groups(LVe and HVe); while relative sperm volume and testis-somatic index were significantly increased in group HVe only. Sperm motility duration was significantly longer in group HVe than in the control, but no significant difference was observed in percent motility among groups. Sperm size, the uniformity of mitochondrial size, and the integrity of mitochondria cristae and plasma membrane were improved by dietary Ve, especially in HVe. The content of Ve in testis and liver as well as polyunsaturated fatty acids in sperm increased with dietary Ve. These results suggested that dietary Ve, especially at the high level(721.60 mg kg–1), significantly improved sperm concentration and motility duration and maintained normal sperm morphology of turbot.     

Effects of Dietary Stachyose on Growth Performance,Digestive Enzyme Activities and Intestinal Morphology of Juvenile Turbot(Scophthalmus maximus L)

A 12-week feeding trial was conducted to evaluate the effects of dietary stachyose on the growth performance, digestive enzymes activities and intestinal structures of juvenile turbot(Scophthalmus maximus L). Five isonitrogenous(49.58% crude protein) and isolipidic(10.50% crude lipid) diets were formulated to contain 0(Control), 0.625%(S-0.625), 1.25%(S-1.25), 2.5%(S-2.5) and 5%(S-5) stachyose, respectively. With the increase of stachyose level, the growth performance and feed utilization of turbot, such as the specific growth rate, final mean body weight, weight gain rate and feed efficiency, increased significantly(P 0.05) and then stabilized. The feed intake of fish fed S-5 was significantly higher(P 0.05) than that of fish in other groups. The activities of trypsin, intestinal caseinolytic, stomach and intestinal amylase were significantly influenced by stachyose(P 0.05). The highest values of trypsin and intestinal caseinolytic activities were observed in group S-1.25, while the highest activity of stomach amylase and the lowest activity of intestine amylase were observed in group S-5. No lesion or damage was found on the distal intestine structures of fish from all treatments, while the height of simple folds in the distal intestine was significantly increased(P 0.05) when 1.25% or 2.5% stachyose was added in the diets. These results indicated that moderate level of stachyose(1.25%) improves the growth performance, feed utilization, digestive enzyme activities and the distal intestine structures of juvenile turbot.     

Expression Pattern of Peptide and Amino Acid Genes in Digestive Tract of Transporter Juvenile Turbot(Scophthalmus maximus L.)

Turbot(Scophthalmus maximus L.),a carnivorous fish species with high dietary protein requirement,was chosen to examine the expression pattern of peptide and amino acid transporter genes along its digestive tract which was divided into six segments including stomach,pyloric caeca,rectum,and three equal parts of the remainder of the intestine.The results showed that the expression of two peptide and eleven amino acid transporters genes exhibited distinct patterns.Peptide transporter 1(Pep T1) was rich in proximal intestine while peptide transporter 2(PepT2) was abundant in distal intestine.A number of neutral and cationic amino acid transporters expressed richly in whole intestine including B~0-type amino acid transporter 1(B~0AT1),L-type amino acid transporter 2(LAT2),T-type amino acid transporter 1(TAT1),proton-coupled amino acid transporter 1(PAT1),y~+L-type amino acid transporter 1(y~+LAT1),and cationic amino acid transporter 2(CAT2) while ASC amino acid transporter 2(ASCT2),sodium-coupled neutral amino acid transporter 2(SNAT2),and y~+L-type amino acid transporter 2(y~+LAT2) abundantly expressed in stomach.In addition,system b~(0,+) transporters(rBAT and b~(0,+)AT) existed richly in distal intestine.These findings comprehensively characterized the distribution of solute carrier family proteins,which revealed the relative importance of peptide and amino acid absorption through luminal membrane.Our findings are helpful to understand the mechanism of the utilization of dietary protein in fish with a short digestive tract.     

Palatability of Water-Soluble Extracts of Protein Sources and Replacement of Fishmeal by a Selected Mixture of Protein Sources for Juvenile Turbot(Scophthalmus maximus)

Poor palatability is a limiting factor for replacing fishmeal with other protein sources in aquaculture. The water-soluble molecules with low molecular weights are the major determinants of the palatability of diets. The present study was conducted to investigate the palatability of water-soluble extracts from single protein source(single extract pellets) and the mixture of these extracts with different proportions(blended extract pellets) in juvenile turbot(Scophthalmus maximus). Then according to the palatability of blended extract pellets, an optimal mixture proportion was selected, and a new protein source made from raw protein materials with the selected proportion was formulated to replace fishmeal. Summarily, the palatability of single extract pellets for turbot was descendent from fishmeal to pet-food grade poultry by-product meal, wheat gluten meal, soybean meal, peanut meal, meat and bone meal, and corn gluten meal. Subsequently, according to the palatability of single extract pellets, 52 kinds of blended extract pellets were designed to test their palatability. The results showed that the pellets presented remarkably different palatability, and the optimal one was diet 52(wheat gluten meal: pet-food grade poultry by-product meal: meat and bone meal: corn gluten meal = 1:6:1:2). The highest ingestion ratio(the number of pellets ingested/the number of pellets fed) was 0.73 ± 0.03, which was observed in Diet 52. Then five isonitrogenous(52% crude protein) and isocaloric(20 k J g~(-1) gross energy) diets were formulated by replacing 0(control), 35%, 50%, 65% and 80% of fishmeal with No.52 blending proportion. After a 10-weeks feeding trial, a consistent feed intake was found among all replacement treatments. Replacement level of fishmeal up to 35% did not significantly influence final body weight, specific growth rate, feed efficiency ratio, and protein efficiency ratio of turbot. Therefore, the water-soluble extracts of protein sources play an important role in improving the palatability of non-fishmeal protein sources in aquafeed.     

褐藻酸寡糖对大菱鲆幼鱼生长性能、血液学指标及非特异性免疫影响

用添加质量分数为0.00%(对照组)、0.02%、0.05%、0.20%、0.50%褐藻酸寡糖(alginate oligosaccharides,AOS)的基础饲料饲养初始体质量为(17.61±0.16)g的大菱鲆(Scophthalmus maximus)幼鱼10周,研究AOS不同添加量对大菱鲆幼鱼生长、血液学指标、非特异性免疫的影响。结果表明:与对照组相比,在4个不同添加量组中,增重率、特定生长率差异无统计学意义(P0.05);各组血液中白细胞含量均提高,差异有统计学意义(P0.05),血红蛋白、红细胞压积及红细胞数差异无统计学意义(P0.05);血清中谷丙转氨酶、谷草转氨酶、总胆固醇、甘油三酯、高密度脂蛋白含量降低,但差异无统计学意义(P0.05);0.02%、0.05%、0.20%组的血清中酸性磷酸酶活力高于对照组,差异有统计学意义(P0.05),但超氧化物歧化酶、溶菌酶活力与对照组差异无统计学意义(P0.05),其中0.05%、0.20%组碱性磷酸酶明显高于对照组(P0.05);添加AOS组肝脏中的酸性磷酸酶活力以及超氧物歧化酶SOD活力与对照组相比差异无统计学意义(P0.05);攻毒试验发现,大菱鲆幼鱼对腹水病的疾病抵抗力随添加量的增加而升高。在本实验条件下,饲料中添加AOS对大菱鲆幼鱼生长性能和血液学指标并无显著影响,但可提高其非特异性免疫功能。建议添加量为质量分数0.05%。     

Establishment,Characterization and Expression Pattern of a Spleen Cell Line,SMSP, Derived from Turbot(Scophthalmus maximus)

A turbot(Scophthalmus maximus) cell line named SMSP was obtained from the spleen. The origin of the cells was identified by morphology, chromosome number and COI gene. The optimal basic medium, serum concentration and growth temperature of the cells were detected. SMSP cell line is mainly composed of fibroblast-like cells. Most of the SMSP cells contained 44 chromosomes, and the sequence of COI gene confirmed that the cells were originated from turbot. The optimal culture conditions were 24℃,DME...     

饲料中添加褐藻酸寡糖对大菱鲆肠道结构、消化酶活性及表观消化率的影响

在基础饲料中添加质量分数0、0.02%、0.05%、0.20%、0.50%的褐藻酸寡糖,饲喂体质量(17.61±0.16)g的大菱鲆(Scophthalmus maximus L)70 d,研究褐藻酸寡糖对大菱鲆肠道结构、消化酶活性及表观消化率的影响。结果表明:1)褐藻酸寡糖处理组的肠道微绒毛长度与对照组差异无统计学意义(P0.05),肠道皱襞高度与对照组差异无统计学意义(P0.05),杯状细胞大于对照组(P0.05);2)饲料中添加褐藻酸寡糖对大菱鲆肠道内蛋白酶和淀粉酶活性影响无统计学意义(P0.05),0.02%和0.05%褐藻酸寡糖实验组中的脂肪酶活性与对照组差异有统计学意义(P0.05);3)褐藻酸寡糖对饲料干物质消化率、蛋白质消化率以及脂肪消化率影响无统计学意义(P0.05)。在大菱鲆饲料中添加褐藻酸寡糖可增大肠道杯状细胞,促进肠道脂肪酶活性,但对肠绒毛长度,皱襞高度,淀粉酶、蛋白酶活性,表观消化率无影响。     

Administration of Yeast Glucan on Immunity of Offspring in Turbot Scophthalmus maximus: A Trans-Generational Immune-Enhancing Effect

β-glucan has been shown to increase immunity and survival in various juvenile and adult fish both in freshwater and marine aquaculture species as well as in marine fish larvae. However, information about the trans-generational immune-enhancing effects of β-glucan remains rather limited. Here we clearly show that dietary intake of β-glucan enhanced the levels of C3, Bf and lysozyme in the serum of turbot as well as in their eggs released. We also show that yeast glucan induced a significant increase in lysozyme activity in both the serum and eggs. Moreover, the embryos derived from yeast glucan-treated turbot were more resistant to bacterial challenge than control embryos. By contrast, the administration of yeast glucan on female turbots had little influence on the egg development and embryonic development. Collectively, these data indicate that yeast glucan can be safely used to promote the non-specific trans-generational immunity in offspring of turbot.     

Heritability and genetic correlation of survival in turbot (Scophthalmus maximus)

We analyzed the survival data of the offspring from 21 sires and 42 dams of turbot. The results show that the cumulative survival rates for turbot from 2 to 18 months range from 17.5% to 28.5%; main mortality occurred during months 2–5; and the highest survival rates of families were 97.9%, 98.8%, 99.4%, 99.7% during months 2–5, 5–6, 6–8, 8–11, and 11–18, respectively, and 99.5%, being 53.5%, 23.8%, 19.5%, 14.9%, and 13.2% higher, respectively, than the mean values in each period. In all periods, the estimated heritabilities for survival were very low without significant difference from zero (P≫0.05) (values ranged from 0.06 to 0.12), indicating low additive genetic effects. The genetic correlations of survival among families in different periods were all positive, but low in magnitude (values range from 0.03 to 0.31). Genetic correlations between long-term survival and other periods’ survival had negative values (−0.06 and −0.15) and three positive values (0.16, 0.12 and 0.14). Genetic correlations between survival and weight were all positive, except for survival at months 2–5 and weight at 18 months, which was not significantly negative (−0.18).     

Heritability and genetic correlation of survival in turbot(Scophthalmus maximus)

We analyzed the survival data of the offspring from 21 sires and 42 dams of turbot.The results show that the cumulative survival rates for turbot from 2 to 18 months range from 17.5% to 28.5%;main mortality occurred during months 2-5;and the highest survival rates of families were 97.9%,98.8%,99.4%,99.7% during months 2-5,5-6,6-8,8-11,and 11-18,respectively,and 99.5%,being 53.5%,23.8%,19.5%,14.9%,and 13.2% higher,respectively,than the mean values in each period.In all periods,the estimated heritabilities for survival were very low without significant difference from zero(P0.05)(values ranged from 0.06 to 0.12),indicating low additive genetic effects.The genetic correlations of survival among families in different periods were all positive,but low in magnitude(values range from 0.03 to 0.31).Genetic correlations between long-term survival and other periods' survival had negative values(-0.06 and-0.15) and three positive values(0.16,0.12 and 0.14).Genetic correlations between survival and weight were all positive,except for survival at months 2-5 and weight at 18 months,which was not significantly negative(-0.18).     

Effect of stocking density on performances of juvenile turbot (Scophthalmus maximus) in recirculating aquaculture systems

Limited information has been available about the influence of loading density on the performances of Scophthalmus maximus, especially in recirculating aquaculture systems (RAS). In this study, turbot (13.84±2.74 g; average weight±SD) were reared at four different initial densities (low 0.66, medium 1.26, sub-high 2.56, high 4.00 kg/m²) for 10 weeks in RAS at 23±1°C. Final densities were 4.67, 7.25, 14.16, and 17.47 kg/m², respectively, which translate to 82, 108, 214, and 282 percent coverage of the tank bottom. Density had both negative and independent impacts on growth. The final mean weight, specific growth rate (SGR), and voluntary feed intake significantly decreased and the coefficient of variation (CV) of final body weight increased with increase in stocking density. The medium and sub-high density groups did not differ significantly in SGR, mean weight, CV, food conversion rate (FCR), feed intake, blood parameters, and digestive enzymes. The protease activities of the digestive tract at pH 7, 8.5, 9, and 10 were significantly higher for the highest density group, but tended to be lower (not significantly) at pH 4 and 8.5 for the lowest density group. The intensity of protease activity was inversely related to feed intake at the different densities. Catalase activity was higher (but not significantly) at the highest density, perhaps because high density started to induce an oxidative effect in turbot. In conclusion, turbot can be cultured in RAS at a density of less than 17.47 kg/m². With good water quality and no feed limitation, initial density between 1.26 and 2.56 kg/m² (final: 7.25 and 14.16 kg/m²) would not negatively affect the turbot cultured in RAS. For culture at higher density, multi-level feeding devices are suggested to ease feeding competition.     

Isolation and characterization of 45 Polymorphie microsatellite loci of turbot (Scophthalmus maximus) and cross-species amplification

Turbot(Scophthalmus maximus) is a flatfish species commercially important for aquaculture.In this study,we generated a microsatellite-enriched genomic DNA library for Scophthalmus maximus,and then isolated and characterized 45 microsatellite loci by genotyping 30 individuals.The observed number of alleles ranged from 2 to 19 with an average of 6.24,while the effective number of alleles ranged from 1.30 to 11.11 with an average of 3.66.The expected heterozygosities varied from 0.235 to 0.925 4 and polymorphic information content ranged from 0.204 4 to 0.903 3,with an average of 0.622.Twelve loci deviated significantly from Hardy-Weinberg equilibrium,and no significant linkage disequilibrium was observed between any pair of loci after Bonferroni correction.In cross-species amplification,five flatfish species(Paralichthys lethostigma,Verasper moseri,platichthys stellatus,Hippoglossoides dubius and Cynoglossus semilaevis) showed at least one polymorphic locus.These polymorphic microsatellite loci should prove useful for population analysis of turbot and other related species.     

Molecular characterization and expression analysis of Lily-type lectin (SmLTL) in turbot Scophthalmus maximus,and its response to Vibrio anguillarum

A full-length lily-type lectin(Sm LTL) was identified from turbot(Scophthalmus maximus) in this study. By searching database for protein identification and function prediction, Sm LTL were confirmed. The full-length c DNA of Sm LTL is composed of 569 bp and contains a 339 bp ORF that encodes 112 amino acid residues. The Sm LTL peptide is characterized by a specific β-prism architecture and contains three mannose binding sites in a three-fold internal repeat between amino acids 30–99; two of the repeats share the classical mannose binding domain(Qx Dx Nx Vx Y) while the third binding site was similar to other fish-specific binding motifs(Tx Tx Gx Rx V). The primary, secondary, and tertiary structures of Sm LTL were predicted and analyzed, indicating that the S m LTL protein was hydrophilic, contained 5.36% α-helices, 39.29% extended strands, 16.07% β-folds, and 39.29% random coils, and three β-folds. Quantitative realtime polymerase chain reaction(qPCR) analysis revealed that the Sm LTL mRNA was abundantly expressed in skin, gill, and intestine. Low levels of Sm LTL expression were observed in other tissues. The expression of Sm LTL in gill, skin and intestine increased at m RNA level after stimulation of V ibrio anguillarum, our results suggest that Sm LTL serve as the first line of defence against microbial infections and play a pivotal role in the innate mucosal immune system. The current study indicates that Sm LTL is a member of the lilytype lectin family and the information reported here will provide an important foundation for future research on the role of this protein.