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.     

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.     

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。     

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。     

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。     

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.     

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.     

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。     

牙鲜幼鱼能量代谢的初步研究

对个体重２．５～８７．６ｇ的牙鲆幼鱼的代谢进行测试。结果表明：温度和体重对牙鲆幼鱼的耗氧率及二氧化碳排出率有明显的影响;牙鲆幼鱼的呼吸商为０．８２±０．０８,氧氮比为２３．８±６．６,其能源物质以蛋白质和脂肪为主;牙鲆在盐度为３０、２５、２０和１５的水中,平均代谢率分别为３．０８、３．０７、３．８７和３．２８Ｊ／ｇ·ｈ,说明牙鲆幼鱼适盐较广,在本实验盐度阈值内,对鱼体代谢基本没影响。     

牙鲆头肾巨噬细胞的分离培养与鉴定

本研究使用密度梯度离心法从牙鲆(Paralichthys olivaceus)头肾中分离得到了巨噬细胞,通过差速贴壁法对获得的细胞进行纯化,后续经过优化培养条件和培养过程,采用L-15培养基(Gibco)、5％胎牛血清、1％青–链霉素、1％非必需氨基酸、30％L929细胞培养基在24℃、无CO2的条件下进行培养.显微镜...     

Effect of artemia nauplii enriched with vitamin A palmitate on hypermelanosis on the blind side in juvenile Japanese flounder Paralichthys olivaceus

ABSTRACT:   The effect of Artemia nauplii enriched with different level of vitamin A (VA) palmitate (1 µg = 1 IU) on the occurrence of hypermelanosis on the blind side of Japanese flounder Paralichthys olivaceus was determined. Artemia were enriched with 0, 1, 2, 5 or 10 mg VA palmitate/L (control group, and 1-, 2-, 5-, and 10-mg groups). The enriched Artemia were fed to the larvae from 27 to 31 days post hatching (dph) corresponding to the F–G stage. VA palmitate, retinol and retinoic acid (RA) contents of Artemia were correlatively elevated with increasing VA palmitate in the culture medium. RA was detected in Artemia enriched with 5 mg and 10 mg, and a significantly high frequency of hypermelanosis on the blind side was observed in these groups at 65 dph ( P  < 0.05). These results suggest that RA synthesized from VA palmitate in Artemia could induce hypermelanosis on blind side of flounder when Artemia are enriched with more than 5 mg VA palmitate/L.     

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.     

Optimum dietary protein level for maximum growth of juvenile olive flounder Paralichthys olivaceus (Temminck et Schlegel)

This experiment was conducted to determine the optimum dietary protein level for juvenile olive flounder Paralichthys olivaceus (Temminck et Schlegel) fed a white fish meal and casein‐based diets for 8 weeks. Olive flounder with an initial body weight of 4.1 ± 0.02 g (mean ± SD) were fed one of the six isocaloric diets containing 35%, 45%, 50%, 55% and 65% crude protein (CP) at a feeding rate of 4–5% of wet body weight on a dry‐matter basis to triplicate groups of 20 fish per aquarium. After 8 weeks of feeding, per cent weight gain (WG) and feed efficiency ratios of fish fed the 55% CP diet were not significantly higher than those from fish fed the 50% and 65% CP diets, but significantly higher than those from fish fed the 35% and 45% CP diets. Fish fed the 50%, 55% and 65% CP diets had significant higher specific growth rates than did fish fed the 35% and 45% CP diets; however, there was no significant difference among fish fed the 50%, 55% and 65% CP diets. The protein efficiency ratio was inversely related to the dietary protein level; that is, maximum efficiency occurred at the lowest dietary protein level. Broken‐line model analysis indicated that the optimum dietary protein level was 51.2 ± 1.8% for maximum weight gain in juvenile olive flounder. The second‐order polynomial regression analysis showed that the maximum WG occurred at 57.7% and it revealed that the minimum range of protein requirement was between 44.2% and 46.4%. These findings suggest that the optimum dietary protein level for maximum growth could be greater than 46.4%, but less than 51.2% CP in fish meal and casein‐based diets containing 17.0 kJ g^?1 energy for juvenile olive flounder.     

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。     

牙鲆主要生长性状与体质量的灰色关联度分析

采用灰色关联分析方法对牙鲆(Paralichthys olivaceus)10个选育家系的9个生长性状与体质量的关联度进行分析和评价。首先对体质量与生长性况进行数列无量纲化处理,计算出每个生长性状与体质量的关联系数和关联度,根据灰色关联度构建两两比较判断矩阵定量分析各生长性状对体质量的影响度。结果显示,每个生长性状与体质量的关联度由大到小依次为:全长(0.875 8)、体长(0.859 3)、尾柄高(0.830 7)、躯干长(0.827 7)、头长(0.803 5)、尾长(0.781 2)、体高(0.740 2)、吻长(0.665 7)和尾柄长(0.654 0)。体质量的主控因素为:全长、体长、尾柄高和躯干长4个性状,影响度分别为:28.51%、21.62%、16.41%和11.69%,四者的影响度总和达到78.23%。分析表明,与体质量关联度最大的分别是全长、体长、尾柄高和躯干长,且它们对体质量的影响度最高,可以将其作为培育高产牙鲆的重要评价指标,以提高选择效率。本研究旨在为制定理想的牙鲆选育目标、提高育种效率提供理论依据,同时拓展灰色关联度方法在水产动物育种研究中的应用。     

A new device for monitoring the activity of freely swimming flatfish, Japanese flounder Paralichthys olivaceus

ABSTRACT:   The tail beat and activity behavior of four captive Japanese flounder Paralichthys olivaceus , were monitored with acceleration data-loggers while the fish swam in an aquarium. Depth, swimming speeds and two-axis acceleration data were collected continuously for approximately 20 h per fish. Simultaneously, the swimming behaviors of the fish were filmed at different angles. Using the specific characteristic of the acceleration profiles, in tandem with other types of data (e.g. speed and depth), four behavioral patterns could be distinguished: (i) 'active' swimming; (ii) burying patterns; (iii) 'inactive' gliding; and (iv) lying on the bottom. Tail beat frequency ranged from 1.65 ± 0.47 to 2.04 ± 0.25 Hz (mean ± SD; n  = 4). Using the relationship between tail beat frequency and swimming speed, the 'preferred' swimming speed of the fish was estimated to be between 0.6 and 1.2 body lengths (BL)/s. Additionally, fish rarely swam faster than 1.2 BL/s. This study shows that the acceleration data-loggers represent a useful and reliable system for accurately recording the tail beat of free-ranging fish and estimating flatfish behavior.