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.     

Optimal dietary taurine level for growth of juvenile Japanese flounder Paralichthys olivaceus

The requirement for taurine in juvenile Japanese flounder Paralichthys olivaceus was determined by feeding diets containing various levels of taurine and cystine. Test diets supplemented with 0.5, 1.0 and 1.5% of taurine or with 0.5, 1.0 and 1.5% of L -cystine were prepared. The basal diet contained 55% protein from white fish meal. These diets were fed to juvenile Japanese flounder with an initial mean bodyweight of 0.9 g (total length (TL) 48 mm) for 5 weeks. Approximately 1.4% taurine content in the diet was required for optimum growth of juvenile flounder. A positive linear relationship was noted between the content of taurine accumulated in the muscle, liver and brain and the level of taurine in the diet. However, there was no increased taurine content in tissues of fish fed the cystine-supplemented diet. In contrast, the fish fed control and cystine-supplemented diets showed higher contents of cystathionine in the tissues. The concentration of cystathionine in tissues rapidly decreased with an increase of taurine in the diet. It was also observed that for each of the dietary groups, a trace amount of taurine was excreted. These results suggest that the taurine content in the diet affects the sulfur amino acid metabolism of juvenile Japanese flounder, and indicate that juvenile flounder are unable to biosynthesize taurine from cystine.     

Application of microparticle diets for Japanese flounder Paralichthys olivaceus larvae

ABSTRACT:   A feeding trial was conducted with two newly developed microparticle diets (MD-Q and MD-V), which differed in the mixing ratios of two types of casein hydrolysates (C700 and C800) as the protein source. The ratio of C700 and C800 were adjusted to 7:3 in MD-Q and 6:4 in MD-V, respectively. Japanese flounder larvae were fed from 7 days after hatching (d.a.h.) to 32 d.a.h. on live food (LF), MD solely, MD + 1/3 LF (either of the MD and one-third quantity of the live food) and 1/3 LF (one-third quantity of live food alone), respectively. They were then switched to Kyowa diet B from 33 d.a.h. until 40 d.a.h. The larvae fed on MD-Q exclusively had a higher survival rate (36.4%) than those fed on MD-V (24.2%) by 22 d.a.h. Unfed larvae could survive up to only 12 d.a.h. Larvae fed on MD-Q + 1/3 LF also had a significantly higher survival rate (34.2%) than those on MD-V + 1/3 LF (16.3%) and 1/3 LF solely (15.4%) at 32 d.a.h. These results suggest the potential of MD-Q as a part replacement for LF from the early developmental stage in the seed production of Japanese flounder.     

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.     

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.     

牙鲆亲本对子代贡献率的实验研究

利用18对高度多态性微卫星标记, 对13尾牙鲆(Paralichthys olivaceus)亲鱼的188尾后代进行亲子鉴定, 计算其非亲排除率、鉴定准确率以及亲本对子代的贡献率。使用4个微卫星标记进行亲子鉴定, 排除率≥0.999 9, 准确率为92.02%; 用6个标记的排除率≥0.999 999, 准确率为96.81%; 用8个标记的排除率≥0.999 999 999, 准确率为97.87%; 用10个标记的准确率为99.47%; 用12～18个标记的准确率为100%。亲子鉴定准确率随非亲排除率和微卫星标记数目的增多而升高。检验所有子代全部是实验亲本的后代, 即13个亲本对子代均有贡献, 但不同亲本的贡献率不同, 最高为47.34%, 最低为0.53%。实验表明, 基于多标记的非亲排除率检测方法可以用于牙鲆亲子鉴定, 其鉴定准确率可以达到100%; 不同亲本对子代贡献率不同, 差异很大, 产生差异的原因有待进一步研究。

     

Effects of dietary arginine and lysine levels on growth performance and biochemical parameters of juvenile Japanese flounder Paralichthys olivaceus

ABSTRACT: To investigate the effects of disproportionate levels of dietary arginine and lysine on juvenile Japanese flounder Paralichthys olivaceus , growth performance and biochemical parameters were evaluated by feeding five test diets, comprising different levels of arginine and lysine, to triplicate groups of juveniles (initial bodyweight 1.85 g) for 40 days. Crystalline amino acids were supplemented to test diets to correspond to the amino acid pattern found in the whole body protein of the Japanese flounder, except for arginine and lysine. After the feeding trials, plasma arginine, lysine and urea levels, excreted ammonia-N, and liver arginase activity were analysed. Survival, specific growth rate, feed conversion efficiency, and apparent protein retention were adversely affected ( P  <   0.05) by the deficiency in dietary arginine or lysine concentrations. An excess of either lysine or arginine in the diet did not depress growth when the diets contained adequate levels of either arginine or lysine, proving that there is no evidence for an arginine–lysine antagonism. Results for plasma arginine, lysine and urea levels, excreted ammonia, and liver arginase activity also demonstrated that Japanese flounder juveniles are not sensitive to excess dietary levels of lysine and arginine.     

Molecular adaptations of glucose and lipid metabolism to different levels of dietary carbohydrates in juvenile Japanese flounder Paralichthys olivaceus

Japanese flounder juveniles (initial body weight: 7.12 ± 0.02 g) were fed three diets containing 0, 120 and 200 g/kg of carbohydrates (C0, C120 and C200, respectively) for 10 weeks. Results showed that higher dietary carbohydrate intake enabled further deposition of glycogen and lipids in liver and muscle. The mRNA levels of glucokinase (gck), phosphofructokinase (pfkl) and hexokinase (HK) activity involved in glycolysis were significantly up‐regulated in C120 (p < .05) but showed no further up‐regulation except for gck in C200. Besides, the gluconeogenic phosphoenolpyruvate carboxykinase 1 (pck1) mRNA levels were down‐regulated significantly in fish fed the C120 (p < .05). However, further increase in dietary carbohydrate levels increased the mRNA levels and activities of enzymes involved in gluconeogenesis and lipolysis (p < .05). Additionally, plasma glucose remained unchanged in C120 (p > .05) but significantly increased in C200 group (p < .05). In conclusion, Japanese flounder was able to use carbohydrates efficiently through regulation of glucose and lipid metabolism when dietary carbohydrate was not higher than 120 g/kg, while 200 g/kg of dietary carbohydrate caused the deregulation of glucose homoeostasis.     

牙鲆5个养殖群体的遗传多样性分析

利用16对微卫星分子标记对牙鲆（Paralichthys olivaceus）的5个养殖群体进行遗传多样性分析。结果表明,等位基因数为2-9个,平均等位基因数为6．0625;有效等位基因数1．2596-5．5161,平均有效等位基因数3．692;各位点的杂合度观测值（Ho）0．2200-0．8000;杂合度期望值（He）0．2061-0．8187。各群体之间无偏倚杂合度期望值从小到大依次为丹东、北戴河、威海、青岛、荣成。Kruskal-Wallis检验结果（H=0．672,df=4,P=0．955）则说明,5个群体遗传多样性差异不显著。群体间基因分化系数（Gs,）为0．0991,各群体之间存在中度遗传分化。采用UPGMA法对5个群体进行聚类,可分3类：丹东和北戴河各为一类,威海、荣成、青岛为一类,其中威海与荣成群体的分化最小。结合Hardy-Weinberg平衡,拟合度检验和遗传偏离指数（动的分析结果表明,各群体的遗传平衡状况存在很大差别。遗传变异的分析结果说明5群体遗传多样性比较丰富。     

Distribution of immunoglobulin-positive cells in the spleen and kidney of Japanese flounder Paralichthys olivaceus

SUMMARY: The distribution of surface immunoglobulin-positive (sIg⁺) cells in the spleen, the head-kidney, and the trunk-kidney of adult Japanese flounder Paralichthys olivaceus was investigated by a flow cytometric analysis using a monoclonal antibody FB17 against flounder immunoglobulin M. Although the percentage of sIg⁺ cells varied among organs, a good correlation was revealed between the percentage in the head-kidney and that in the trunk-kidney ( r = 0.996, P < 0.001). In contrast, no correlation was obtained between the percentage of sIg⁺ cells in the spleen and the head-kidney or trunk-kidney. The head-kidney and the trunk-kidney contained cells bearing fewer sIg that were stained dimly by FB17 in addition to brightly stained sIg⁺ cells regarded as mature B cells, while the spleen contained only sIg⁺ cells stained brightly. Moreover, the immunocytochemical analyses showed that the head-kidney contains plasma cells with strongly stained cytoplasm in addition to B cells with stained cell surface, which suggests that the cells with fewer sIg detected by flow cytometric analysis are presumed to be at a stage during the processes of differentiation to plasma cells. These results suggest that there are some differences between the spleen and kidney in the role of the immune system and it is considered that differentiation of B cells to plasma cells occurs in the kidney.     

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。