Effect of sand grain size on substrate preference and burial behaviour in cultured Japanese flounder juvenile,Paralichthys olivaceus

To improve release techniques of Japanese flounder, Paralichthys olivaceus, for the purpose of stock enhancement, the substrate preference and burial behaviour over a range of sand grain sizes were investigated under laboratory conditions. Ten 12‐hr trials (i.e. 10 experimental groups), which, respectively, contained two different levels of substrates, demonstrated that juvenile flounder (55–78 mm total length) selected substrate of grain size (GS) < 2.00 mm, and the degree (from high to low) of their preference could be listed as Level 3 (0.50–2.00 mm) > Level 4 (0.25–0.50 mm) > Level 5 (0.05–0.25 mm) > Level 2 (2.00–4.00 mm) > Level 1 (4.00–6.00 mm). The average burying of fish in substrates of GS < 2.00 mm was more than 93.1%. Additionally, along with food deprivation time increased from 12 to 24 hr, some flounder showed random off‐bottom swimming behaviour, and the appearance of off‐bottom swimming behaviour was <3.2%. These results suggest that releasing sites with appropriate substrate grain size (GS < 2.00 mm) are suitable for conducting mass releases of hatchery‐reared flounder juveniles, and starvation prior to release should be minimized. The results of the present study are essential to improve stocking strategies of Japanese flounder and reduce the juvenile mortality after release.     

Larval development and salinity tolerance of Japanese flounder (Paralichthys olivaceus) from hatching to juvenile settlement

Salinity tolerance and growth of Japanese flounder Paralichthys olivaceus at different developmental stages were evaluated, including newly hatched larvae (nhl), yolk sac larvae (ysl), oil droplet larvae (odl), post oil droplet larvae (podl), premetamorphic larvae (preml) and prometamorphic larvae (proml), at 11 salinities from 5 to 55 g L^?1 for 96 h. The ontogenesis during the early life of P. olivaceus was investigated under hatchery salinity 35 g L^?1. The results showed that suitable salinities for nhl, ysl, odl, podl, preml and proml larvae were 10 to 25 g L^?1, 10 to 30 g L^?1, 20 to 30 g L^?1, 30 g L^?1, 10 to 30 g L^?1, 15 g L^?1, respectively, demonstrating an ontogenetic variation of salinity tolerance. The salinity tolerance of nhl, ysl, preml was higher than that of odl, podl and proml. The ysl and preml larvae displayed wide salinity tolerances. The present findings demonstrate that the suitable salinity for larviculture of P. olivaceus is 20–25 g L^?1 before the depletion of oil droplet; after that, higher salinity (30 g L^?1) should be ensured for the post‐oil droplet larvae; the premetamorphic larvae can be cultured at a wide salinity range (10–30 g L^?1), and the metamorphosed larvae should be reared at salinity about 15 g L^?1.     

Dietary supplemental effects of red seaweed Eucheuma denticulatum on growth performance,carcass composition and blood chemistry of juvenile Japanese flounder,Paralichthys olivaceus

A 56‐day feeding trial was conducted to determine the effects of supplementing diets of juvenile Japanese flounder, Paralichthys olivaceus, with Eucheuma denticulatum (EDP). Isonitrogenous and isolipidic test diets were prepared using a fishmeal‐based positive control diet (PC) and a fishmeal‐soy protein concentrate negative control diet (NC) supplemented with 3 (D3%), 6 (D6%) and 9% (D9%) EDP. The test diets were assigned to tanks (12 fish tank^?1, initial mean body weight of 0.42 ± 0.01 g) in triplicates. Results of the feeding trial indicated that growth rates and feed efficiency significantly improved (P < 0.05) when 3% EDP was supplemented in the diets. However, there was a significant reduction (P < 0.05) of growth and feed efficiency in fish fed diets with higher supplementation levels. Protein, lipid, dry matter and ash contents of carcass and their corresponding nutrient retention values were not markedly altered in all dietary treatments. Fish fed diets supplemented with EDP exhibited higher (P < 0.05) omega‐3 polyunsaturated fatty acid accumulation in dorsal muscle when compared with those of fish fed NC. Blood chemical parameters showed significant differences (P < 0.05) in total cholesterol and triglyceride levels. These results suggest that EDP can be efficiently utilized by Japanese flounder and can promote best growth and feed utilization at a level of 3%.     

野生与人工养殖褐牙鲆亲鱼微量元素组成的差异比较

分析了野生与人工养殖褐牙鲆亲鱼肝脏、背肌及卵中硒、铁、锰、铜、铬、镉和铅等微量元素的含量。结果表明,野生褐牙鲆亲鱼肝脏中硒、铁含量均显著高于养殖亲鱼(P<0.05),铜含量则显著低于养殖亲鱼(P<0.05),其他各元素间均未见显著差异(P>0.05);野生褐牙鲆亲鱼背肌中除硒和锰的含量显著高于养殖亲鱼外(P<0.05),其他各元素含量均基本保持恒定(P>0.05);野生褐牙鲆亲鱼卵中硒、铁和锰的含量均显著高于养殖亲鱼(P<0.05),其中硒的含量约为养殖亲鱼的1.7倍,铁的含量约为养殖亲鱼的2.0倍,而锰含量则为养殖亲鱼的2.8倍。养殖褐牙鲆亲鱼卵中镉含量与野生亲鱼相比高出78%(P<0.05)。两种亲鱼卵中铜、镉、铅3种重金属含量均未见显著差异(P>0.05)。     

Dietary choline requirement of juvenile olive flounder (Paralichthys olivaceus)

An eight‐week study was conducted to determine the optimum dietary choline level in juvenile olive flounder, Paralichthys olivaceus. Seven diets were prepared to contain 0, 250, 500, 750, 1,000, 2,000 and 3,000 mg/kg diet. Juveniles (5.9 ± 0.03 g; 5.5 ± 0.4 cm; mean ± SD) were randomly distributed into 21 tanks (25 fish/tank) and fed one of the diets in triplicates. Survival rate of fish fed the diet containing the lowest choline level was significantly lower than those of fish fed the other diets (p < 0.05). Final body weight, weight gain, specific growth rate, feed efficiency and protein efficiency ratio significantly increased with increasing choline levels up to 1,000 mg/kg diet. Whole‐body protein and lipid contents increased in accordance with choline levels up to 750 mg/kg diet, beyond which they plateaued. Liver and muscle lipid contents elevated with increasing choline levels up to 2,000 mg/kg diet. Plasma cholesterol esters, triglycerides, cholesterol and total lipids were significantly influenced by the graded choline levels; however, responses of those indices were not identical. Broken‐line analyses of weight gain and liver choline concentrations responding to the graded choline levels revealed that choline requirements of the juvenile flounder could be between 847 and 1,047 mg/kg diet.     

Effects of dietary fish oil replacement by microalgae raw materials on growth performance,body composition and fatty acid profile of juvenile olive flounder,Paralichthys olivaceus

Replacing dietary fish oil with DHA‐rich microalgae Schizochytrium sp. and EPA‐rich microalgae Nannochloropsis sp. for olive flounder (Paralichthys olivaceus) was examined. Three experimental isonitrogenous and isolipidic diets with lipid source provided by 50% fish oil (F50S50), 50% (M50F25S25) and 100% microalgae raw material (M100) respectively were compared with a soybean oil (S100) diet as control. Triplicate groups of olive flounder juveniles (16.5 ± 0.91 g) were fed the experimental diets, and a group was fed the control diets for 8 weeks in a recirculation system. Results showed feed efficiency and growth performance were not significantly changed when fish oil (FO) was totally substituted by soybean oil (SO) or microalgae raw material (MRM). The whole‐body composition, lipid content of liver and muscle, and lipid composition of plasma were not significantly influenced by the total substitution of FO by MRM. The polyunsaturated fatty acids (PUFA) content of muscle and liver declined in fish fed S100 diet, whereas it was not significantly reduced in fish fed M50F25S25 and M100 diets. The total substitution of FO by MRM not only maintained the levels of arachidonic acid, EPA or DHA but also increased n‐3/n‐6 ratio. In conclusion, MRM as the sole lipid source is sufficient to obtain good feed efficiency, growth performance and human health value in olive flounder juveniles.     

指状拟舟虫诱导牙鲆抗血清免疫球蛋白分析

对引起牙鲆体表溃烂的原纤毛虫－指状舟虫诱导牙鲆免疫反应产生的免疫球蛋白ＩｇＭ进行分析,结果表明,病原纤毛虫免疫注射牙鲆 ,可诱导牙鲆发生特异性免疫反应,产生抗体型上鲆抗指状拟舟虫血清的凝集试验,发现纤毛虫停止游动并发虫体聚集;抗血清经与标准分子量和鼠ＩｇＭ单克隆抗体以及对照血清的ＳＤＳ－ＰＡＧＥ电泳比较分析证明,牙鲆抗指状拟舟虫血清免疫球蛋白ＩｇＭ重链分子量为７１０００,轻链分子量为２３０００;Ｉ     

牙鲆养殖群体遗传变异的微卫星标记研究

应用10对微卫星引物对中国牙鲆一个养殖群体的30个个体进行了群体遗传结构分析。结果显示，微卫星标记比其他标记具有更高的多态性，10个微卫星座位的等位基因数在4～10之间，有效等位基因数在2．23～5．82之间，平均等位基因数为7．6，群体平均杂合度为0．6960，Hardy-weinberg遗传偏离指数的平均值为0．1774。     

野生与人工养殖牙鲆亲鱼不同组织脂肪酸的比较

为研究脂肪酸对牙鲆繁育性能的影响, 采用生物化学方法, 对野生和养殖牙鲆亲鱼肌肉、肝脏及卵中脂肪酸组成分别进行测定。结果表明: (1) 牙鲆肌肉、肝脏和卵中脂肪含量大小关系为肝脏>卵>肌肉。野生亲鱼肌肉和卵中脂肪含量显著低于养殖亲鱼(P<0.05), 肝脏脂肪含量与养殖亲鱼无显著差异; (2) 牙鲆亲鱼3种组织中均检测出21种脂肪酸。野生亲鱼肌肉中饱和脂肪酸(SFA)与养殖亲鱼无显著差异, 单不饱和脂肪酸(MUFA)显著低于养殖亲鱼(P<0.05)。肝脏和卵中SFA显著高于养殖亲鱼(P<0.05), MUFA与养殖亲鱼无显著差异; (3) 野生亲鱼肌肉、肝脏和卵中高不饱和脂肪酸(PUFA)的含量, 尤其是肝脏和卵中C20:5n-3(EPA)、C22:6n-3(DHA)的含量均显著低于养殖亲鱼(P<0.05), 但肌肉和卵中的C20:4n-6(ARA)含量明显高于养殖亲鱼(P<0.05); (4) 野生牙鲆亲鱼肌肉、肝脏和卵中n-3/n-6 PUFA及EPA/ARA显著低于养殖亲鱼(P<0.05), 肝脏中DHA/EPA显著高于养殖亲鱼(P<0.05), 但野生亲鱼肌肉和卵中的DHA/EPA与养殖亲鱼无显著差异。比较结果说明, DHA、EPA和ARA等PUFA是与牙鲆繁殖性能密切相关的重要脂肪酸。在牙鲆亲鱼养殖过程中, 除了提供牙鲆亲鱼足够的脂肪酸营养外, 也应注意各种脂肪酸, 尤其是PUFA中各种脂肪酸之间的添加比例, 从而保证亲鱼的繁殖性能及卵和仔鱼的质量。     

Methionine requirement of juvenile Japanese flounder Paralichthys olivaceus estimated by the oxidation of radioactive methionine

Growth and amino acid oxidation studies were conducted to estimate methionine requirement of juvenile Japanese flounder, Paralichthys olivaceus , by using the purified diets containing 500 g kg^–1 crude protein from casein, gelatine and crystalline amino acids (CAA). Diets with six graded levels of methionine (5.3, 8.3, 11.3, 14.3, 17.3 and 20.3 g kg^–1 diet) were fed to triplicate groups of the juvenile (initial weight 2.8 ± 0.05 g) twice a day for 40 days. To prevent leaching losses, CAA were precoated using carboxymethylcellulose (CMC), and further diets were bound by CMC and κ-carrageenan. Based on broken-line analysis of percentage weight gain and feed conversion efficiency, the methionine requirements of Japanese flounder in the presence of 0.6 g kg^–1 of cystine were 14.9 and 14.4 g kg^–1 dry diet, respectively. After the growth study was finished, a direct estimate of methionine requirement was made by examining the influence of dietary methionine level on ¹⁴C-methionine oxidation by determining radioactive carbon dioxide, protein and nonprotein fractions of the whole body. The dose–response curve between expired radioactive CO₂ and dietary methionine levels showed that the optimum methionine level for the flounder was estimated to be within the range of 14.3–17.3 g kg^–1 of diet in high agreement with values obtained from the growth study.     

牙鲆抗鳗弧菌病家系筛选及其分析

2007年以从养殖牙鲆(Paralichthys olivaceus)中筛选出的抗病选育群体、从日本引进的日本群体以及从黄海捕捞的野生群体为基础,进行各种组合方式的交配,建立了63个牙鲆家系;2008年又建立了30个家系。对2007年培育的59个家系和2008年培育的30个家系进行了鳗弧菌(Vibrio anguillarum)感染实验,结果表明,不同家系间的抗病力存在极显著的差异(P0.01)。从2007年培育的59个家系中筛选出4个抗病家系,鳗弧菌感染后存活率达到50%以上;2008年的30个家系中5个家系抗病力较强,鳗弧菌感染后的存活率达到60%以上。2008年培育的30个家系其4月龄和6月龄的感染后存活率的相关系数为0.403(P0.05),表明不同生长时期的感染结果具有一致性。230d的体长和体质量与对鳗弧菌的抗病力存在显著的相关关系(P0.05),皮尔森相关系数分别为0.282和0.237。研究发现,日本群体与抗病选育群体的杂交后代抗病性能表现优异,可以通过日本群体与中国群体间的杂交进行遗传改良,达到培育出高产、抗病牙鲆新品种的目的。     

Cryopreservation of summer flounder,Paralichthys dentatus L., sperm

The summer flounder, Paralichthys dentatus L., is a high‐value species and considerable research has been conducted to determine practices conducive for its culture. As milt can be limited in this species, experiments were conducted to develop a practical sperm cryopreservation protocol for hatchery use. Two dilution ratios (1:2 and 1:4; sperm:extender), 2 diluents (saline and sucrose‐based), 2 cryoprotectants (10% DMSO and 12% glycerol) and 3 freezing rates (?5, ?10 and ?15°C min^?1) were evaluated using differential staining to assess post‐thaw sperm survival. Seven combinations of the factors examined reduced post‐thaw viability by less than 30%. The average viability of sperm from fresh, pooled flounder milt (67.2 ± 2.9%) was not different from that of thawed milt diluted 1:4 with sucrose diluent (10% DMSO) frozen at ?5°C min^?1 (38.4 ± 7.7%) and fertilization and hatch success were not different in trials using fresh or thawed, cryopreserved sperm. From these experiments a practical sperm cryopreservation method was developed, but further refinement of the freezing protocol is necessary to optimize results.     

牙鲆微卫星标记的开发及多态性分析

将牙鲆基因组DNA经限制性内切酶RsaⅠ和BstUI双酶切后采用FIASCO法构建酶切片段基因组文库。共挑取269个克隆,177个为阳性克隆,阳性克隆率为65.79%。经测序后获得191条微卫星序列,其中完美型占74.35%;非完美型占14.66%;混合型占10.99%。用引物设计软件Primer Premier 5.0设计引物153对,挑选其中的50对合成并在32个野生牙鲆个体中进行扩增,共31个位点具有多态性,统计结果后使用POPGENE软件进行分析,平均等位基因个数为3.939 4,平均有效等位基因数为3.052 2,平均观测杂合度为0.650 1,平均期望杂合度为0.586 6,各引物的Hardy-Weinberg平衡指数在0.012 587~0.984 917变动。这些筛选出的多态性微卫星标记可应用于进一步的牙鲆遗传多样性分析、家系分析及遗传图谱的构建等工作中。     

抗迟缓爱德华氏菌病牙鲆家系的筛选与分析

牙鲆(Paralichthys olivaceus)养殖病害日益严重,迟缓爱德华氏菌(Edwardsiella tarda)是牙鲆养殖中的主要致病菌,带来了极大的经济损失,为了培育出对该病抵抗能力强的牙鲆新品种,2014年4–6月间,利用我们长期以来建立和筛选出来的牙鲆抗病群体和家系的亲鱼,通过杂交,自交,雌核发育建立47个家系,9–10月间,对其中39个家系进行感染实验,先用少量鱼进行预实验摸索出半致死浓度,然后再进行正式感染实验,感染数量为每个家系80尾。各家系的存活率范围为1.19%~51.19%,最终平均存活率为20.29%,认为存活率高于30%的7个家系为抗病力强家系,存活率在20.29%~30%的9个家系为抗病力一般家系,平均存活率以下的23个家系为抗病力弱家系。1406#家系抗病力最强,它为1005#家系的自交后代,而其他抗病力强家系也大多是1005#、09104#、0915#的后代。而09104#为0768#的后代,并且0768#抗鳗弧菌病能力强。1005#为韩国群体自交后代,0915#为韩国群体和日本群体杂交后代。这些抗病力强的牙鲆可以作为新品系进行推广养殖,可望减少牙鲆腹水病发生。     

牙鲆体质量与形态性状的异速生长分析

为分析牙鲆（Paralichthys olivaceus）体质量与形态性状之间异速生长的遗传规律,通过人工受精的方法建立牙鲆19个全同胞家系,测量了牙鲆不同日龄的体质量和形态性状。采用逐步回归方法建立最优联合异速生长模型,将此模型镶嵌到体质量动物模型的每个遗传和环境效应中,进一步分析对多个形态性状异速生长遗传规律。静态异速生长分析表明：体质量与全长之间存在最大异速生长指数（1.415 5）,表现为正异速生长,剩余形态性状与体质量之间的异速生长指数为0.061 5~0.718 0,皆表现为负异速生长。全长与尾柄高之间异速生长指数的正遗传相关最大（0.907 8）;全长与尾柄长之间的负遗传相关最大（0.946 8）。不同模型通过统计标准比较,确定模型Ⅰ为进行牙鲆动态异速生长分析的最优随机回归模型。     

牙鲆血清免疫球蛋白的分离纯化及部分特性分析

运用Sephacryl S-200凝胶层析和HiTrap rProtein ASepharose亲和层析2种方法对牙鲆(Paralichthys olivaceus)血清免疫球蛋白进行分离纯化,结果表明,牙鲆免疫球蛋白分布于33%~50%的硫酸铵饱和溶液中,其中45%的分离效果最好。凝胶层析和亲和层析样品均出现2个蛋白峰,用还原SDS-PAGE检测确定牙鲆免疫球蛋白存在于第2个蛋白峰中。牙鲆免疫球蛋白重链分子量约为75.4 kD,轻链分子量约为29.9 kD和28.2 kD,推测牙鲆血清免疫球蛋白的分子量为836 kD。制备了兔抗牙鲆免疫球蛋白多克隆抗体,免疫双扩散法检测多克隆抗体效价为1∶32,免疫斑点法检测多克隆抗体效价至少为1∶1 600。运用免疫印迹法(Western-bloting)检测了兔抗牙鲆免疫球蛋白多克隆抗体的特异性,实验证明该抗体与牙鲆全血清中免疫球蛋白重链、轻链反应均成阳性。     

Cytological studies on induced meiogynogenesis in Japanese flounder Paralichthys olivaceus (Temminck et Schlegel)

The cytological process of induced gynogenetic development and subsequent chromosome duplication by a cold shock treatment was observed in Japanese flounder Paralichthys olivaceus (Temminck et Schlegel). Mature eggs were at the metaphase of the second meiosis when inseminated with ultraviolet (UV)-irradiated sperm of red sea bream Pagrus major . After the beginning of cold shock treatment, the previously visible spindle became invisible, probably due to the side effect caused by cold shock treatment. The chromosomes at the centre of the metaphase plate were condensed. This condition continued during the duration of the cold shock treatment and several minutes after it. The release of the second polar body was blocked and it developed into a female-like pronucleus. Then, it fused with the female pronucleus to generate a diploid zygotic nucleus, and the egg exhibited the first mitosis. Consequently, the haploid female chromosome set of the egg was doubled by the inhibition of the second polar body release. There was a significant delay in developmental time in the gynogenetic eggs when compared with that in the normal eggs. From the time of insemination to early cleavage, the UV-irradiated heterospecific sperm nucleus remained condensed.     

牙鲆家系4 个选育性状的测定与分析

为了研究牙鲆(Paralichthys olivaceus)不同选育性状之间的相关性,2009年建立了38个牙鲆实验家系和1个养殖场对照组。选取其中33个家系和对照组进行鳗弧菌感染实验并对存活个体进行荧光标记后混合养殖在2个水泥池中,其中一个混养池在平均400日龄时暴发了淋巴囊肿病(Lymphocystis Disease,LD),LD发病率为62.4%,包括16个家系和对照组。从这些家系中筛选出4个发病率低于40.0%的抗病家系(F0939,F09104,F0915,F0908)。另外,在4~5月龄时,从每个家系中平均选取100尾鱼进行荧光标记后混合养殖,580日龄时测定这些家系的养殖存活率和体重,平均养殖存活率为32.0%,将7个养殖存活率高于55.0%的家系定义为高养殖存活率家系(F0908,F0975,F0990,F09125,F0927,F09119,F0905)。对牙鲆LD的抗病个体和易感个体的体重和全长进行卡方检验,发现抗病个体的体重和全长均显著高于易感个体(P0.01),同时分析牙鲆各个家系的LD发病率与发病时的体重和全长的相关性,结果显示牙鲆各个家系LD发病率与发病时体重和全长均呈强负相关(r–0.6)。对牙鲆家系鳗弧菌感染存活率、LD发病率、580日龄的养殖存活率和体重这4个性状两两之间进行相关性分析,发现LD发病率与580日龄的体重呈强负相关(r=–0.790);鳗弧菌感染存活率与养殖存活率之间为弱正相关(r=0.371);其他性状两两之间不存在显著相关性(–0.092≤r≤0.185)。本研究共筛选到了4个牙鲆LD抗病家系和7个高养殖存活率家系,并揭示了4个牙鲆选育性状之间的相关性,为培育速生多抗的牙鲆新品种提供了遗传材料和理论依据。