舟山褐牙鲆（Paralichthys olivaceus）仔鱼摄食和生长的研究

试验研究了褐牙鲆Paralichthys olivaceus仔鱼的摄食和生长。结果表明,仔鱼初次开口摄食时间在6日龄（16～18℃）,初次摄食率为30％,8日龄饥饿仔鱼最大初次摄食率为65％,孵出后第9天达到不可逆点（PNR）,混合营养期为3d左右。初次摄食强度和初次摄食率开始时较低,在卵黄囊耗尽时达最高,然后下降。随着饥饿时间的延长,仔鱼出现“负增长”,孵化第11天存活率仅为10％,12日龄全部死亡。     

Development of microparticle diets for Japanese flounder Paralichthys olivaceus larvae

ABSTRACT:   Two newly designed microparticle diets (MD), with two kinds of peptide (C700 and C800) as a protein source were developed. Microparticle diet Q (MD-Q) contained C700 (molecular weight ∼30 000 Da) and C800 (molecular weight 1000–2000 Da); in contrast, microparticle diet T (MD-T) contained C700 only. Two separate trials, representing larvae from different hatches, were conducted. Japanese flounder Paralichthys olivaceus larvae were fed newly designed MD or a combination of them with live food (LF) between 3 and 10 days after hatching (d.a.h.) in experiment I, or between 11 and 20 d.a.h. in experiment II, and compared them to LF and a commercial diet (CD) feeding groups. The growth and survival rates in both experiments were the highest in the LF treatment. But in the microparticle treatment larvae fed MD-Q had a higher survival rate and better growth than those fed MD-T, either alone or in combination with LF. First-feeding larvae fed on MD-Q had a 20.5% survival rate and 1.12 mm gain by 10 d.a.h. Unfed larvae died within 6 d.a.h. These results indicate that using a mixture of different molecular weight peptides is good protein sources and also this type of microparticle diet can be applied to flounder from larval to juvenile stages.     

牙鲆消化道发育的组织学观察

牙鲆（Paralichthys olivaceus）肉质鲜美、营养价值高,是我国重要的海水养殖鱼类之一,同时,牙鲆二次变态显著,是研究硬骨鱼类变态发育很好的模式生物。在其变态过程前后,外型上最大的变化是其右眼的移位,内部器官如脑颅骨、骨骼肌等都发生结构和功能上的改变,而消化道的发育和分化,特别是其胃在变态前后有显著的变化。消化系统的发育关系到饵料的选择,与仔鱼成活率有直接关系,有较为重要的研究价值。[第一段]     

Effects of high dose of vitamin A on reproduction and egg quality of Japanese flounder Paralichthys olivaceus

ABSTRACT: The present study was conducted to investigate the effects of dietary vitamin A on reproduction and egg quality in Japanese flounder Paralichthys olivaceus . Broodstock were fed experimental pellets containing two levels of vitamin A [11 × 10³ IU/100 g (control diet; CD), 337 × 10³ IU/100 g (experimental diet; ED)] for approximately 2 months before spawning and during the spawning period. Two groups of five females (average weight 1.4 kg) and 10 males (average weight 0.7 kg) were randomly allocated to two 30 m³ indoor tanks. Total egg production of the CD group was slightly higher than the ED group. Percentage of buoyant eggs and hatching rate of the ED group were significantly higher than the CD. In other egg quality parameters, such as percentage abnormal larvae and starvation tolerance of larvae, no notable difference was found between these two groups. At the end of the experiment, the skin color of broodstock in the ED group was darker than that of the CD group. Vitamin A content in eggs of the ED group was significantly higher than that of the CD group. However, the difference in vitamin A content in eggs between the ED and CD groups was much smaller than that in the liver of the females between the two groups. These results indicate that feeding broodstock a higher level of vitamin A increases the vitamin A content in eggs but does not affect egg quality in Japanese flounder because excess dietary vitamin A was stored mainly in the broodstocks' liver.     

Dietary vitamin A requirements of juvenile Japanese flounder Paralichthys olivaceus

A 70‐day feeding experiment was conducted to assess the dietary vitamin A (VA) requirements of juvenile Japanese flounder (Paralichthys olivaceus). Six semi‐purified diets with VA supplementations of 0, 5000, 10 000, 15 000, 20 000 and 25 000 IU kg^?1 were fed twice a day to triplicate groups of 20 juveniles per tank with an initial weight of 1.59 ± 0.01 g (mean ± SE). Weight gain (WG) and specific growth rate (SGR) increased as dietary VA increased up to 10 000 IU kg^?1. Significantly lower WG and SGR were observed for the 0 IU kg^?1 treatment than for treatments of 5000, 10 000 and 15 000 IU kg^?1. Highest WG and SGR were observed in fish fed 10 000 IU kg^?1; slightly lower values were recorded in fish fed 15 000, 20 000 or 25 000 IU kg¹. No significant difference was observed in survival rate among treatments. Whole body total lipid was significantly higher in fish fed 0 and 5000 IU kg^?1 than for other levels. Reduced growth and small livers were observed as signs of VA deficiency in fish fed 0 IU kg^?1. Slightly reduced growth and pale fragile livers were observed as effects of VA excess in fish fed 25 000 IU kg^?1. Total retinol contents in liver and eyes increased with increasing levels of dietary VA. No retinol was detected in livers, and significantly lower total retinol content was observed in eyes, of fish fed 0 IU kg^?1. WG analysed by the broken line method indicated that an optimum dietary VA requirement of 9000 IU kg^?1.     

双列杂交分析比较两个养殖群体杂交牙鲆抗病力

采用双列杂交设计对牙鲆Paralichthys olivaceus两个养殖群体：日本群体（J）和中国抗病选育群体（C）进行群体间杂交和群体内自繁,获得C（♀）×C（♂）、J（♀）×J（♂）、J（♀）×C（♂）和C（♀）×J（♂）4个交配组合的子一代。针对受精率、孵化率、白化率和存活率4个性状进行了组合间的比较,计算了各项性状的杂种优势率,对组间各性状进行了方差分析,并进行了相关分析。结果表明,杂交组在受精率、孵化率及存活率上存在着不同程度的杂种优势,其中C（♀）×J（♂）后代在孵化率和存活率两个性状上的杂种优势率分别为33.99%和31.37%。对4个性状的表型相关分析表明,受精率和孵化率的相关系数为0.657,受精率和存活率相关系数为0.432,分别达到了极显著水平（P〈0.01）。分析结果认为,两个不同群体间的种内杂交是牙鲆优良品种培育的有效途径。     

mtDNA 和微卫星标记在放流牙鲆和非放流牙鲆鉴定中的应用

用mtDNA控制区特异性引物对牙鲆(Paralichthys olivaceus)亲鱼、放流牙鲆和回捕牙鲆的DNA进行扩增,获得了3个群体的mtDNA控制区第一高变区部分序列。分析结果表明:母本55尾具有26种单倍型,作为其子代的预备放流牙鲆129尾具有5种单倍型,与亲本的单倍型全部一致,验证了mtDNA方法鉴定子代的准确性。回捕牙鲆435尾具有70种单倍型,其中330尾具有17种单倍型,与母本的单倍型相一致,为疑似放流牙鲆;另外105尾具有53种单倍型,与母本的单倍型不一致,为野生牙鲆或非北戴河站母本后代放流牙鲆。利用4个高多态性微卫星标记对330尾疑似放流牙鲆做进一步鉴定。结果表明,330尾检测个体中有310尾等位基因与候选亲本等位基因全部对应,可确认其为放流牙鲆;另外20尾等位基因与候选亲本不对应,可确认其为非放流牙鲆。结合mtDNA和微卫星标记鉴定的结果,可以确认2013年的435尾回捕牙鲆中有310尾放流牙鲆,占71.26%;另外125尾为非放流牙鲆,占28.74%。本研究结果表明,利用mt DNA标记可快速排除回捕鱼中的非放流牙鲆,利用微卫星标记作进一步鉴定,可以排除疑似放流牙鲆中的非放流牙鲆,是区分放流牙鲆与非放流牙鲆,准确评价放流效果的好方法。     

Fast development of genetically uniform strains by gynogenesis in Japanese flounder (Paralichthys olivaceus)

The Japanese flounder is a major species in both aquaculture and research. Inbred strains of Japanese flounder were developed efficiently in our laboratory by meiotic and mitotic gynogenetic reproduction techniques. To determine the induction efficiency of gynogenesis and the rate at which full homozygosity is produced, six meiotic gynogenetic females (G1–G6) that had experienced meiotic gynogenesis once and three common females (C1–C3) were selected for production of mitotic gynogenesis in our experiments. Of the nine adult females, all six gynogenetic fish successfully produced viable offspring. However, only one of the three common fish did. Using microsatellite markers, we estimated the homozygosity of gynogenetic fish induced by mitotic gynogenesis. We found that the homozygosity of seven lines (C1, G1–G6) increased quickly, individually from 43%, 72%, 69%, 71%, 78%, 69% and 58% to 100%, 100%, 100%, 90%, 100%, 100% and 90% offspring complete homozygosity. Under mitotic gynogenesis, individuals with higher homozygosity had a higher induction rate. The G4 line showed the highest induction rate, achieving 44.59% convert hatching rate and 29.28% convert normality rate. We conclude that meiotic gynogenesis may be a feasible method to produce DH genetic material in Japanese flounder.     

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。     

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.     

牙鲆幼鱼消化能力与肠道蛋白酶活性的研究

对体重7.5~20.0 g的牙鲆幼鱼的摄食比率、食物的消化时间、消化道不同部位蛋白酶的最适pH值、蛋白酶和脂肪酶的分布和摄食后不同时间蛋白酶的变化规律进行了研究。结果表明:牙鲆幼鱼的平均摄食量为净体重的5.89%。摄食配合饲料后约5 h,胃内的食物基本排空。pH值为2.6~3.0时胃蛋白酶的活性最高,pH值为8.2时幽门盲囊和肠道的蛋白酶活性最高。消化道中蛋白酶活性的变化趋势为由前向后逐渐降低,以胃蛋白酶活性最高;脂肪酶活性变化趋势为由前向后逐渐增高,以肠道脂肪酶活性最高。摄食后不同时间牙鲆消化道各部位的蛋白酶活性有显著变化,胃蛋白酶活性在摄食后3 h达到最高值,摄食后7 h降至极低水平;肠道蛋白酶活性在摄食后呈现逐渐增高趋势;幽门盲囊中蛋白酶活性变化与胃和肠道密切相关。在下一次摄食前消化道各部位蛋白酶活性均处于较高水平。     

褐牙鲆水中阻力的初步研究

鱼类在水中游泳时,需要克服水的阻力从而需要消耗一定的能量.鱼类依鱼种、大小以及形态特征的差异,在同样的水流条件或游泳速度下,其水中阻力也不相同.本研究通过对褐牙鲆(Paralichthys olivaceus)在垂直循环水槽中进行游泳阻力测试实验,分别用3种特征面积(横截面积、湿表面积和鱼体体积的2/3次方),获得褐牙鲆3种不同的阻力系数.经比较分析发现,在雷诺数Re为2.7×104～1.91×105时,采用湿表面积作为褐牙鲆的特征面积获得的阻力系数的平均值为0.020,此时标准差最小,仅为0.000 76.而分别用横截面积和鱼体体积的2/3次方时获得的阻力系数的标准差相对较大.因此,用褐牙鲆的湿表面积计算得到的阻力系数更适合于该种鱼类个体水中阻力的研究.     

赤潮异弯藻对牙鲆早期发育的影响

就赤潮异弯藻(Heterosigma akashiwo)藻液及各组分对牙鲆(Paralichthys olivaceus)早期发育阶段的影响及敏感性时期作了探讨。实验结果表明，原肠期前的发育卵和初孵仔鱼对赤潮异弯藻较其他发育阶段更为敏感。在同等条件下藻液对牙鲆早期发育各阶段的毒性最大，其次是藻细胞碎片和藻细胞内容物，去藻过滤液的毒性最小。暴露在高浓度80000cells／ml藻液中，96h后仔鱼的存活率为47．6％；而在同浓度的去藻过滤液中，96h后仔鱼的存活率为90％。藻液对牙鲆仔鱼96h LC50为35042cells／ml。     

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。     

Effects of protein and lipid sources on the growth and survival of red sea bream Pagrus major and Japanese flounder Paralichthys olivaceus receiving micro-bound diets during larval and early juvenile stage

The effects of dietary protein and lipid sources on growth and survival of the red sea bream Pagrus major (30‐days old) and Japanese flounder Paralichthys olivaceus (15‐days old) were examined using four zein‐microbound diets (MBD) containing the following nitrogen and lipid sources: MBD‐1, SBP‐1000 (soybean peptides with molecular weights of about 1000) + fish oil calcium soap (FOCS); MBD‐2, SBP‐3000 (molecular weights of about 3000) + FOCS; MBD‐3, soybean protein isolate (SPI) + FOCS; MBD‐4, SBP‐1000 + stearoyl calcium lactate (SCL). Six groups of the test fish were maintained on the following diets for 30 days; group 1, live food; group 2, 1/2 live food + 1/2 MBD‐1; group 3, MBD‐1; group 4, MBD‐2; group 5, MBD‐3; group 6, MBD‐4. In the red sea bream, high performance in terms of total length (TL) and body weight gain (BWG) was obtained in groups 1, 2, 3 and 4. However, the fish receiving MBD‐3 (group 5) and MBD‐4 (group 6) had shorter TL and lower BWG than those receiving the other diets. Regarding the Japanese flounder, high performance in terms of TL, BWG, and survival rate (SR) was obtained in groups 1 and 2. Group 3 receiving MBD‐1 gave the BWG and SR close to those of group 2 but lower than group 1. In contrast, groups 5 and 6 receiving MBD‐3 or MBD‐4 alone had significantly (P < 0.05) shorter TL and lower BWG than groups 1 and 2. The present study thus indicated that soybean peptides with molecular weights of 1000–3000 and FOCS are superior to SPI and SCL as nitrogen and lipid sources of MBD for the red sea bream and Japanese flounder at larval and early juvenile stage.     

Pharmacokinetics of Eugenol in Japanese Flounder,Paralichthys olivaceus

Eugenol, which has been used as an anesthetic for fish, was administered to Japanese flounder, Paralichthys olivaceus (127 ± 50.8 g), by bath treatment at concentrations of 0.025, 0.125, and 0.25 mL/L seawater for 10 min and intramuscular injection at 40 μL/fish to investigate the pharmacokinetic characteristics of eugenol in the plasma of Japanese flounder. In the bath treatment test, plasma eugenol concentration increased with increasing eugenol concentration up to 0.125 mL/L and reached steady state within 5 min. After a 10‐min bath treatment in 0.25 mL/L eugenol, plasma eugenol concentration was about 58.4 µg/mL. After transfer into running seawater, plasma eugenol concentration decreased biphasically with half‐lives of 0.0296 h (α‐phase) and 0.289 h (β‐phase). The AUC_0000→0800 was about 16.5 µg h/mL. In administration by intramuscular injection, plasma eugenol concentration increased rapidly after administration and decreased biphasically with half‐lives of 0.0329 h (α‐phase) and 8.08 h (β‐phase). The AUC_0000→0800 was about 52.5 µg h/mL. In both methods of administration, Japanese flounder with average weight of 127 g were effectively anesthetized when plasma eugenol concentrations were between 2.19 and 4.88 µg/mL.     

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。