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.     

The potential impact of summer flounder (Paralichthys dentatus) aquaculture on regional flounder price

Abstract

Increasing consumer demand for flounder and dramatic reductions in ocean flounder harvests have raised interest in summer flounder (Paralichthys dentatus) aquaculture. Flounder farmers have several potential markets, including regional seafood dealers and high‐end niche (e.g., sashimi and ike‐jime) buyers. Prices paid by buyers are exogenous to typical aquacultural budgeting models, yet they are a key determinant of potential profitability. We develop a simple economic model to assess the impacts of aquaculture industry supply on prices paid by regional seafood dealers. The model is parameterized for a region with established flounder dealers and active field research in flounder production methods. We derive the price elasticity of regional seafood dealer demand for farmed flounder, and we examine the impacts of changes in production cost and ocean harvest on regional price and the level of aquacultural production that maximizes returns to the aquaculture industry.     

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.     

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.     

Arachidonic acid requirements in larval summer flounder, Paralichthys dentatus

The arachidonic acid (20:4n-6,AA) requirements of larval summer flounder weredetermined for the rotifer- and Artemia-feeding stages. Experimental emulsionscontained adequate n-3 highly unsaturated fattyacid (HUFA) ratios and emulsion levels of AAwere set at 0, 3, 6, 9, and 12% (AA0, AA3,AA6, AA9, and AA12). Examination of fatty acidlevels in live feeds and larval tissuesconfirmed the physiological incorporation offatty acids relative to dietary levels. In thefirst experiment, survival, growth, andsalinity tolerance (2-h in 70) were measuredat 18 days after hatch (dah) after feeding thelarvae the various levels of AA. Larvae fedAA6-enriched rotifers were better able tosurvive the salinity tolerance test. AAenrichment up to 12% had no effect on growthand survival. In the second experiment, larvaewere fed AA0- or AA6-enriched rotifers until 23dah, followed by unenriched 24- and 48-h Artemia nauplii until 32 dah. These larvaethen were subdivided and fed AA-enriched Artemia from 33-45 dah. At the end of thisexperiment, larvae fed AA6-enriched rotifershad higher survival, increased growth, andsurvived better in the salinity tolerance test(2-h in 80). The enrichment of Artemiadid not have any effect on these variables.Thus, the provision of AA6-enriched rotifers(10 mg AA g^–1 DW) early in larvaldevelopment may serve to enhance larval stresstolerance at the rotifer stage while alsoincreasing larval survival, growth, and stresstolerance later in the Artemia stage.     

Effects of clioquinol on the scuticociliatosis‐causing protozoan Miamiensis avidus in olive flounder Paralichthys olivaceus

Scuticociliatosis is a devastating and intractable protozoal disease in olive flounder, leading to a significant loss throughout the year. This study aimed to investigate a systemically effective antiscuticociliatosis agent for olive flounder for better absorption into the infected internal organs. The in vitro and in vivo antiscuticociliatosis effects of clioquinol (CQ) were examined after screening 30 biocidal agents against the highly pathogenic scuticociliate Miamiensis avidus. CQ was the most potent in vitro drug of those tested against cultured M. avidus. CQ was the least toxic in healthy olive flounder among the drugs that exhibit high potencies. In olive flounder, a single intramuscular injection of 40 mg/kg CQ significantly reduced mortality caused by artificial infection with M. avidus, and 10–20 mg/kg CQ increased fish survival times. CQ was also effective in naturally infected scuticociliatosis. Ciliate cell numbers were lower when CQ was injected in most organs, including the brain. CQ was well absorbed by the internal organs after intramuscular injection. This study suggests that CQ can be considered as a potential antiscuticociliatosis agent for systemic administration in olive flounder.     

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。     

Nutritional comparison in muscle of wild,pond and factory cultured Japanese flounder (Paralichthys olivaceus) adults

Muscle proximate composition, amino acid profiles, fatty acid compositions and selected minerals of wild, pond and factory cultured Japanese flounder adults were compared in this study to elucidate their nutritive values and dietary nutrition requirements. Wild and pond cultured Japanese flounder flesh had higher crude proteins, but crude lipid contents of factory cultured fish were 3.8~4.0 fold greater. Major amino acids in Japanese flounder were aspartic acid, glutamic acid and lysine. Wild fish had higher levels of total amino acids, essential amino acids, nonessential amino acids and half‐essential amino acids and the contents in pond cultured samples were similar. Arachidonic acids (ARA), eicosapentaenoic acids (EPA), docosahexaenoic acids (DHA), polyunsaturated fatty acids (PUFA), DHA + EPA and n–3 PUFA in wild Japanese flounder were significantly higher than that in factory cultured fish, whereas fatty acid levels were close between pond cultured and wild fish. Japanese flounder were rich in zinc, iron and selenium, but poor in copper, chromium and nickel. Results indicated wild Japanese flounder had higher nutritional value and better meat quality, but the nutritional compositions of pond cultured fish were close to wild Japanese flounder, which suggested that pond culture of Japanese flounder offers broad application prospects.     

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.     

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.     

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。     

Survival of larval summer flounder Paralichthys dentatus on formulated diets and failure of thyroid hormone treatment to improve performance

Summer flounder is a new aquaculture species, the culture of which could be made simpler by the replacement, to the greatest extent possible, of live feed with formulated diets. Previous work has shown that development of the summer flounder stomach can be accelerated by treatment of the larvae with thyroid hormone. Two series of experiments were conducted. In the first experiment, 2-week feeding trials were conducted beginning with fish (which were not treated with thyroid hormone) 14, 21, 28, 35, or 42 days after hatch (DAH) to determine when they could begin to utilize commercially available formulated diets. In the second experiment, two groups of sibling larvae, one treated with thyroid hormone, the other not, were placed in similar 2-week feeding trials at 25, 30 or 35 DAH to test whether survival of thyroid hormone-treated larvae would surpass those of untreated larvae. The first series of experiments demonstrated that the larvae could not effectively utilize the formulated diets until 35 DAH. The second series of experiments indicated that thyroid hormone-treated larvae do not exhibit significantly greater survival than untreated larvae.     

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%.     

Quantitative investigation of Enteromyxum leei (Myxozoa: Myxosporea) infection and relative condition factor in cultured olive flounder Paralichthys olivaceus (Temminck and Schlegel)

Enteromyxum leei has been reported to cause emaciation disease in various fish species. To determine the effect of parasite intensity on cultured olive flounder Paralichthys olivaceus, we investigated the relationship between the relative condition factor (rCF = CF/standard CF × 100) and parasite load with quantitative polymerase chain reaction (qPCR) and the challenge test. A total of 57 cultured olive flounders were obtained from 11 fish farms and divided into five groups based on their rCF. We investigated the parasite intensity in the posterior intestine of the fish. The parasite load was closely matched to severe loss of body weight. In addition, olive flounders were inoculated either orally or anally with intestinal scrapings of infected fish or phosphate‐buffered saline. The fish were reared at natural water temperature and transferred to different tanks, and the water temperature was adjusted to 20°C after 6 weeks of inoculation. When the water temperature was increased to 20°C, the rCF decreased in the experimentally infected group. The results demonstrated that qPCR can be utilized to determine the relative abundance of E. leei in olive flounders and water temperature is an important factor to track the progress of the emaciation disease.     

Evaluation of Antarctic krill (Euphausia superba) meal supplementation in diets for olive flounder (Paralichthys olivaceus)

The supplemental effect of Antarctic krill meal (KM) into a low fish meal (FM) diet was evaluated for olive flounder (Paralichthys olivaceus). A 56% FM‐based diet was regarded as a high FM inclusion diet (HFM), and a low‐FM diet (LFM) was prepared by replacing 50% FM from the HFM. Four other diets were prepared by supplementing 3%, 6%, 9% and 12% KM into the LFM diet gradually replacing soy protein concentrate and tankage meal (designated as KM3, KM6, KM9 and KM12 respectively). Quadruplicate groups of fish were fed one of the diets for 12 weeks. The growth performance and feed utilization efficiency were improved by the dietary KM supplementation. Digestibility of dietary protein and dry matter was increased by the KM3‐9 diets. Haematocrit and haemoglobin were increased by KM supplementation. The innate immunity and antioxidant capacity assessed by Ig, antiprotease, lysozyme, GPx and SOD and the condition factor of fish were significantly increased by KM3‐9 diets. Moreover, goblet cell counts, villi length and fillet yield of fish were significantly improved by all the KM‐containing diets (KM3‐12). A 25‐day‐long challenge test with the Edwardsiella tarda pathogen showed that the cumulative mortality was higher in fish fed the LFM diet than in fish fed the HFM or KM‐supplemented diets. The results indicate that dietary KM supplementation in a LFM diet can increase growth performance and feed utilization efficiency, diet digestibility, intestinal development and functions, innate immunity and disease resistance of olive flounder. The recommended level of KM inclusion in a LFM diet seems to be 6.6% according to quadratic regression analysis.     

Effect of hydrogen peroxide exposures on mucous cells and lysozymes of gill tissues of olive flounder Paralichthys olivaeceus

We investigated the effects of hydrogen peroxide on mucous cells and lysozymes in gill tissue of olive flounder. Morphological analysis revealed significant changes in the number of mucous cells after exposure to 300 and 500 mg L ^{? 1} hydrogen peroxide; however, there was no detectable difference in the 100 mg L ^{? 1} group. Lysozyme activities declined gradually in the gill mucus, but in contrast activities in serum increased significantly following the completion of treatment. The hepatic c‐ and g‐type lysozyme levels showed significant responses, indicating that the liver is a major lysozyme producer in response to stress condition; however, in the head kidney, there were no significant changes in the g‐type lysozyme level in all the groups, while the only detectable difference with the c‐type gene in the 300 mg L ^{? 1}. While the treatments showed no significant changes compared with the control for either branchial lysozyme, there were significant differences in c‐type lysozyme between the 3rd treatment and 5 day. Taken together, our data indicate that hydrogen peroxide treatment may lead to modulation of the innate immune response of olive flounder; therefore, the application of hydrogen peroxide in a field system will require more attention to dose and timing effects.     

Pharmacodynamics of amoxicillin against field isolates of Streptococcus parauberis from olive flounder (Paralichthys olivaceus)

Olive flounder is the most important species for the Northeast Asian fish farming industry. However, this species is substantially affected by multiple infectious agents, including Streptococcus parauberis. Evaluation of antibiotics before their application is critical to treat infections and prevent drug resistance. Therefore, in this study, the pharmacodynamics of amoxicillin (AMX) and other antimicrobials against the planktonic‐ and biofilm‐forming bacteria were assessed. The minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and Time–kill curve assay were analysed using micro‐dilution method. The minimum biofilm eradicating concentration (MBEC) was determined using the Calgary Biofilm device. The effects of temperature, pH, hardness and salinity were detected for both planktonic‐ and biofilm‐forming bacteria. The MIC of AMX ranged from 0.015 to 2 μg/ml, whereas that of cephalexin (CEP), enrofloxacin (ENR) and oxytetracycline (OTC) ranged from 0.125 to 256, 0.125 to >512 and 0.25 to >512 μg/ml respectively. No bacteria were resistant against AMX, while the percentage of resistance to CEP, OTC and ENR were 68.7%, 52.6% and 11.1% respectively. The IC₅₀ of AMX, CEP, ENR and OTC was 0.03, 0.091, 0.015 and 0.213 μg/ml respectively. The MBEC of amoxicillin against S. parauberis ranged from 0.5 to 16 μg/ml. Higher rates of bacterial growth were obtained at 30°C, pH = 8 and salinity of 7.5–10 ppt. The hardness of the media suppressed the bacterial growth. In conclusion, AMX was found to be effective against both the planktonic and the biofilm forms of the prominent fish pathogen, S. parauberis.     

漠斑牙鲆胚胎细胞系的建立与鉴定

漠斑牙鲆(Paralichthys lethostigma)是中国近年新开发的重要海水养殖鱼类,本研究以漠斑牙鲆囊胚期胚胎为材料,探索了鱼类胚胎细胞分离和培养的条件,建立了漠斑牙鲆胚胎细胞培养技术。建立的漠斑牙鲆胚胎细胞系(SFEC)至今已经培养了240多天,传至80多代。SFEC的完全培养基采用添加抗生素、胎牛血清(FBS)、花鲈血清(SPS)、成纤维生长因子(bFGF)的DMEM。SFEC形态小而呈圆形、多边形,在培养基中生长迅速。本实验检测了温度、胎牛血清浓度、成纤维生长因子对SFEC生长的影响。在24~30℃之间SFEC生长良好,但当温度低于18℃时细胞生长速度明显减慢。在含15?S的培养基中,SFEC生长速度明显比在含7.5?S的培养基中快,在培养基中添加bFGF可以使细胞生长速度显著提高。SFEC的二倍体核型为2n=6st 42t。将GFP报告基因通过脂质体介导的方法转入SFEC中并成功地获得了表达,转化率为20%。