大菱鲆脾脏cDNA文库构建、EST序列分析与免疫抗病相关基因的筛选

以大菱鲆脾脏为材料,构建cDNA文库,通过对文库克隆的序列测定和初步生物信息学分析,共获得3 656个大菱鲆脾脏表达序列标签（EST）。经与GenBank数据库的序列比对后发现,1 891个EST代表了149个已知基因,其余1 765个EST为未知序列。这149个基因大致可分为6类：9个为细胞结构类（6.0%）;26个为代谢类（17.5%）;45个为细胞防御/免疫类（30.2%）;43个为基因/蛋白质表达类（28.9%）;16个为细胞信号转导/细胞交流类（10.7%）;10个无法明确分类（6.7%）。鉴定的与免疫抗病相关重要基因主要有：CC/CXC趋化因子;主要组织相容性复合体（MHC）class I α;C₃补体;天然杀伤细胞增强因子（NKEF）;胸腺素β-4/β-12;干扰素诱导蛋白Gig1;白介素8受体;STAT 4;热激蛋白70/90等。     

Colonization of the gut in first feeding turbot by bacterial strains added to the water or bioencapsulated in rotifers

Two bacterial strains, 4:44 and PB52, isolated from turbot(Scophthalmus maximus L.) were used during a first feedingexperiment with turbot larvae. Bacteria were either added directly tothe water on the day of hatching of the larvae (day 0), orbioencapsulated in rotifers (Brachionus plicatilis) distributedon day 2 after hatching. The two bacterial strains were found to bepresent in the water of the rearing tanks throughout the experiment. Theaddition of bacteria influenced the species-composition of themicroflora associated with intensively produced rotifers added to thetanks, and resulted in colonization of the gut of the larvae by theadded bacterial strains. The strain 4:44 showed a peak on day 9 afterhatching, and reached a mean(sem) of 2.5(1.4) × 10⁴bacteria per larva, when added in a mixture with PB52, and 0.4(0.1)× 10⁴ bacteria per larva, when added alone. The strainPB52, on day 12 after hatching, reached 5.2(1.5) × 10⁴bacteria per larva when added in a mixture with strain 4:44, and12.5(0.7) × 10⁴ bacteria per larva, when added alone.The added bacteria colonized the gut turbot larvae, whereas thebacterial land, the survival and growth of the larvae were in most casesnot influenced in a negative way by the addition of bacteria.     

Incorporation of fatty acids from dietary neutral lipid in eye, brain and muscle of postlarval turbot fed diets with different types of phosphatidylcholine

Previous results demonstrated the stimulating effect of soybean phosphatidylcholine (PC) on the utilization of dietary neutral lipid in larval and postlarval fish. The present study further investigated the effect of the degree of saturation of dietary PC on the enhancement of dietary fatty acid incorporation in lipids of turbot. Newly-weaned turbot were fed for 20 days on four isolipidic diets containing the same amount of highly unsaturated fatty acids (HUFA), presented either as neutral lipid, i.e. fish oil ethyl esters, or as polar lipid. Diet FO was a phospholipid-free control diet. Diets HPC, SPC and FPC were supplemented with 3% hydrogenated soybean PC, 3% native soybean PC and 3% marine fish roe PC, respectively.The three PC-supplemented diets resulted in better growth and higher muscle triacylglycerol levels than the PC-free diet FO. The fish fatty acids were determined in 3 lipid classes (neutral lipid, PC, phosphatidylethanolamine) of 3 organs or tissues (eye, brain and muscle). Despite the identical amounts of n-6 and n-3 fatty acids provided by the soybean oil and by the HUFA ethyl esters, the substitution of 3% hydrogenated coconut oil in diet FO by 3% hydrogenated PC in diet HPC caused, averaged over the various tissues and lipid classes, a 7 to 12% higher incorporation of 18:2n-6, 20:4n-6, 20:5n-3 and a 32% higher 22:6n-3 level in turbot lipid. Diet HPC appeared as efficient as diet SPC for enhancing the incorporation of the n-3 HUFA from the ethyl esters. Feeding diet FPC, in which the n-3 HUFA were provided through the marine PC source, resulted in slightly higher levels of these fatty acids in the fish than feeding the ethyl ester HUFA diets, even if supplemented with PC. Present results confirm the positive effect of PC, either hydrogenated or native, on the utilization of fatty acids provided in the diet as neutral lipid. The slightly higher incorporation of HUFA, when esterified on dietary PC instead of neutral lipid, raises the question regarding the form of intestinal absorption of PL in fish.p>     

Evaluating the most suitable nonlinear growth model for turbot (Scophthalmus maximus) in aquaculture 2 (weight application): Multi‐criteria model selection and growth prediction

Seeking the most suitable model to describe the growth of turbot, we analysed growth data of two different turbot (Scophthalmus maximus) strains reared communally in a recirculating aquaculture system. We fitted 10 different nonlinear growth models to individual weight gain data (n = 2,010) during the grow‐out phase. Analyses were carried out for each strain, for sexes within strains and for a pooled data set containing both strains and sexes. To assess the model performance, three different criteria are used. Further, a growth‐simulation was performed to evaluate the shape of the generated curve. This way we could assess the capability of the models to predict future growth. The 3‐parametric Gompertz model achieved the best fit in 42.9% of all cases tested and the lowest Bayesian information criterion in 100% of cases. The model produced realistically shaped curves and asymptotic values matching the biological attributes of the species. In contrast, 5‐parametric functions projected unrealistically shaped curves and predicted improbable mature sizes. Our results show that increasing number of parameters do not lead to increasing goodness of fit, but tend to result in overfitting, and demonstrate the advantages of the 3‐parametric Gompertz model for describing the growth of turbot.     

拟微绿球藻粉替代鱼粉对大菱鲆幼鱼生长性能、体组成和血清生化指标的影响

为探讨拟微绿球藻(Nannochloropsis sp.)粉替代鱼粉对大菱鲆(Scophthalmus maximusL.)幼鱼生长性能、体组成和血清生化指标的影响,用拟微绿球藻粉替代基础饲料中0%、3.88%、7.76%、11.64%和15.52%的鱼粉,配制成5种等氮等能的饲料(N_0、N_(3.88)、N_(7.76)、N_(11.64)、N_(15.52))。选取初始体重为(24.60±0.02) g的大菱鲆幼鱼600尾,随机分成5组,每组3个重复,每个重复40尾鱼,养殖周期70d。结果显示:1)各实验组大菱鲆幼鱼的增重率(WGR)、特定生长率(SGR)、蛋白质效率(PER)、饲料系数(FCR)、日摄食率(DFI)、肥满度(CF)和成活率(SR)均无显著差异(P0.05);2)随着藻粉添加量的增加,全鱼及肌肉中粗脂肪含量显著降低(P0.05),粗蛋白、粗灰分和水分含量无显著差异(P0.05);3)血清溶菌酶(LZM)、补体蛋白C3、补体蛋白C4及酸性磷酸酶(ACP)活力均呈先上升后下降的趋势,分别在N_(7.76)、N_(7.76)、N_(11.64)、N_(7.76)组达到最大值,且显著高于N_0组(P0.05),N_(15.52)组碱性磷酸酶(ALP)显著低于其他组(P0.05),其他组之间无显著差异(P0.05);4)藻粉组血清总超氧化物歧化酶(T-SOD)、总抗氧化能力(T-AOC)和谷胱甘肽过氧化物酶(GSH-PX)活力,均呈先上升后下降的趋势,在N_(7.76)组达到最大值,且显著高于N_0组(P0.05);5) N_(7.76)组血清甘油三酯(TG)含量显著低于其他组(P0.05),其他组之间无显著差异(P0.05),藻粉组血清总胆固醇(TCHO)显著低于N0组(P0.05),各藻粉组之间差异不显著(P0.05);6)藻粉组血清谷草转氨酶(AST)活力呈先下降后上升的趋势,N_(11.64)组达到最小值,显著低于N_0组(P0.05),藻粉组谷丙转氨酶(ALT)活力显著低于N_0组(P0.05)。研究表明,本实验条件下,拟微绿球藻粉替代大菱鲆幼鱼饲料中15.52%的鱼粉对其生长无显著影响,替代7.76%可显著提高其非特异性免疫力,降低血脂水平。     

微米纯氧气泡增氧技术养殖大菱鲆效果初探

试验旨在分析新型微米纯氧气泡增氧养殖大菱鲆的效果。试验采用微米纯氧气泡增氧和机械增氧2种方式,设置机械增氧组(溶解氧6~9 mg/L)、微米纯氧增氧Ⅰ组(溶解氧6~9 mg/L)和微米纯氧增氧Ⅱ组(溶解氧15~20 mg/L)3个试验组。结果表明,微米纯氧Ⅰ组大菱鲆的体重增长、成活率、肥满度及饵料转化率高于机械增氧组;微米纯氧Ⅱ组各指标低于机械增氧组。7个月的大规模生产试验(800 m2水面,溶解氧6~9 mg/L)表明,采用微米纯氧气泡增氧养殖大菱鲆,各测定指标均显著高于机械增氧,可以加快大菱鲆生长,提高成活率和饵料转化率。     

Application of amplified fragment length polymorphism markers to assess molecular polymorphisms in gynogenetic haploid embryos of turbot (Scophthalmus maximus)

Among the variety of cultured marine species, the turbot Scophthalmus maximus is a fish of growing importance in European aquaculture. In this paper, an advanced application of AFLPs to estimate the genetic diversity of haploid gynogenetic families with the aim of obtaining a preliminary genetic map is presented. Ten EcoRI/TaqI primer combinations were tested in four families comprising diploid mothers and their haploid progenies. The amplified fragment length polymorphism (AFLP) analysis revealed an average of 6.8 polymorphic bands per primer combination and a total number of 88 polymorphisms out of 579 fragments. Among various primer pairs, seven combinations were selected in relation to the quality of profiles and number of polymorphic fragments, to be used in the determination of genetic linkage relationship between AFLP markers within the largest haploid family. Co‐migration of non‐homologous fragments was also investigated in one primer combination adding a fourth selective nucleotide to the three used in the classic TaqI AFLP protocol. Surprisingly, a rate of 38.7% of non‐homologous fragments co‐migrating with monomorphic bands was identified, due to the combined effect of homoplasy and the protocol used. Additional polymorphic markers discovered by this protocol were included in the linkage map. The turbot AFLP linkage map comprises 52 AFLP markers distributed in 12 linkage groups. On the basis of this map, turbot expected total genome length sums up to 1225.6 cM. The results confirm the usefulness of AFLPs in revealing genome segregation in haploid turbot progeny.     

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.     

低氧和高氧对大菱鲆幼鱼红细胞核异常及氧化抗氧化平衡的影响

研究了极低氧(0.2、1.0、2.0 mg/L)、低氧(3.0、5.0 mg/L)和高氧(11.0、14.0 mg/L)处理28 d期间,大菱鲆幼鱼外周血红细胞核异常以及血清超氧化物岐化酶(SOD)、过氧化氢酶(CAT)活力和肝脏丙二醛(MDA)含量等氧化抗氧化平衡中参数的变化。结果显示,极低氧条件下,0.2、1.0和2.0 mg/L组的实验鱼分别在处理2 h、1 d和2 d内全部死亡,其余各组则没有出现死亡现象。溶解氧(DO)对大菱鲆核异常包括微核及总核异常率的影响表现出明显的时间和剂量累积效应,到28 d时才在5.0、14.0 mg/L组和对照组中(正常充气,约7.0 mg/L)观察到微核现象。恢复正常充氧14 d后,高氧组和对照组中仍有微核现象出现。2 h时极低氧组SOD活力较高。其他各处理时间时,SOD活力在各组之间差异显著。各组CAT活力在2 h时未发生明显变化,在7、28和42 d时差异显著(〖WTBX〗P〖WTBZ〗<0.05)。极低氧组大菱鲆肝脏MDA含量较低,低氧组和高氧组MDA含量均随着处理时间的延长逐渐降低。恢复后低氧组MDA含量显著升高(〖WTBX〗P〖WTBZ〗<0.05)。研究还发现,对照组大菱鲆也会受到过氧化的胁迫作用,而5.0 mg/L组的总核异常率和所测氧化还原指标在整个实验期间均未发生明显变化。