星斑川鲽胚胎的玻璃化冷冻保存

为研究星斑川鲽胚胎玻璃化冷冻保存,首先,于室温条件下采用玻璃化液五步平衡法处理星斑川鲽的尾芽期胚胎30 min,比较分析分别含有葡萄糖、果糖、蔗糖和海藻糖的4种不同玻璃化液(PMG3G、PMG3F、PMG3S、PMG3T)的毒性,发现含有海藻糖的玻璃化液(PMG3T)对尾芽期胚胎的毒性最小,胚胎的成活率与孵化率相对较高,分别为75.00%±5.43%和50.00%±4.53%(P0.05)。其次,分析玻璃化液PMG3T对星斑川鲽囊胚期至出膜前期7个时期的胚胎的毒性,发现尾芽期胚胎和心跳期胚胎经处理后成活率较高,分别为69.33%±6.43%和72.67%±3.94%(P0.05),但心跳期胚胎的孵化率较高,为65.33%±3.91%(P0.05),尾芽期胚胎次之,说明心跳期胚胎对PMG3T的耐受能力相对于尾芽期胚胎更好,更适宜进行胚胎的玻璃化冷冻保存实验。最后,采用玻璃化液PMG3T对心跳期胚胎进行–196℃冷冻实验,发现所处理的60粒胚胎中有7粒成活,可漂浮水面,继续培养后可见进一步发育,但未孵化出膜。另外,将100 mL未受精卵(未经玻璃化液处理)置于–20℃,分别在冷冻5～70 min时取出10 mL复温并进行授精,结果显示,冷冻5 min的卵的受精率为89.83%±6.51%(P0.05),随着冷冻时间的延长,受精率越来越低,冷冻70min时受精率为3.71%±1.27%,畸形率越来越高,在冷冻70min时达到100%。本研究筛选出比较适宜的玻璃化液、低温耐受性较高的胚胎发育时期,为星斑川鲽胚胎低温冷冻保存技术的建立和种质的长期保存和应用提供了实验数据。     

牙鲆选育家系生长性状的因子分析

记录了2009年建立的12个牙鲆家系240日龄个体的体质量、全长、体长、头长、躯干长、吻长、尾柄长、尾长、体高,和尾柄高等10个生长性状,对这12个家系的生长性状进行了因子分析。结果表明：第一至第四公因子对变异的方差贡献率分别为81．36％、10．12％、5．3％,和1．31％,累积贡献率达98．1％,表明这4个公因子很好地反映了所控制的10个生长性状及其相互作用关系。贡献率最高的第一公因子包含了大多数生长性状,其因子得分排序在前两位的家系为F11和F4,与这2个家系生长性状表型值排序相一致,表明这两个家系具有结构较好的生长构成性状和较高的性状表型值,可作为培育快速生长型牙鲆新品种的候选家系,进行生产验证和推广养殖。     

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.     

Supplementation of Protein Hydrolysates to a Low‐fishmeal Diet Improves Growth and Health Status of Juvenile Olive Flounder,Paralichthys olivaceus

An 11‐wk feeding trial was conducted to evaluate three different protein hydrolysates as feed ingredients in high‐plant‐protein diets for juvenile olive flounder. Five experimental diets were fed to juvenile olive flounder to examine the effect of three different protein hydrolysates on growth performance, innate immunity, and disease resistance against bacterial infection. A basal fishmeal (FM)‐based diet was regarded as a high‐FM diet (HFM) and a diet containing soy protein concentrate (SPC) as a substitute for 50% FM protein was considered as a low‐FM diet (LFM). Three other diets contained three different sources of protein hydrolysates, including shrimp, tilapia, and krill hydrolysates (designated as SH, TH, and KH), replacing 12% of FM protein. All diets were formulated to be isonitrogenous and isocaloric. Triplicate groups of fish (15.1 ± 0.1 g) were handfed one of the diets to apparent satiation twice daily for 11 wk and subsequently challenged against Edwardsiella tarda. Growth performance and feed utilization of fish fed hydrolysate‐supplemented diets were significantly improved compared to those of fish fed the LFM diet. Dietary inclusion of the protein hydrolysates significantly enhanced apparent digestibility of dry matter and protein of the diets. In the proximal intestine, histological alterations were observed in the fish fed the LFM diet. The fish fed the hydrolysate diets showed significantly longer mucosal fold and enterocytes and greater number of goblet cells compared to fish fed the LFM diet. Respiratory burst activity was significantly higher in fish fed the TH and KH diets than fish fed the LFM diet. Significantly higher immunoglobulin levels were found in fish fed SH and KH diets compared to those of fish fed the LFM diet. Dietary inclusion of the protein hydrolysates in SPC‐based diets exhibited the highest lysozyme activity. Significantly higher superoxide activity was observed in groups of fish fed the KH diet. Fish offered the protein hydrolysates were more resistant to bacterial infection caused by E. tarda. The results of this study suggest that the tested protein hydrolysates can be used as potential dietary supplements to improve growth performance and health status of juvenile olive flounder when they were fed a LFM diet.     

无公害工厂化养殖牙鲆技术研究

按照水产品无公害养殖行业标准，在工厂化条件下进行牙鲆养殖研究，经1年试验，平均规格401g／尾，成活率为51．4％。饵料系数1．93，饵料效率0．52。     

Advance in the Culture Research of Small-Eye Flounder,Paralichthys microps,and Chilean Flounder,P. adspersus,in Chile

ABSTRACT

Over 71% of the aquaculture production of Chile is based on salmonids, and given the recents problems in the world salmon industry, it is necessary to investigate the culture of new species as a vital priority to maintain an industry of great social and economic impact for Chile. Small-eye flounder, Paralichthys microps, and chilean flounder P. adspersus, are native, high-value flatfish present off the coasts of Perú and Chile, that are commonly exploted by artisanal fishermen. Since 1986 both species have been objects of research studies for culture based on techniques developed for Japanese flounder. This paper is a review of current knowledge of this species and to identify research needs for the near future. Natural spawning of small-eye flounder and chilean flounder broodstock in captivity is already possible, but the manipulation of spawning using photoperiod and hormonal inducement, as well as the role of broodstock nutrition in egg and larval quality, needs to be explored. Successful incubation and larval culture protocol for both species have been developed, with larval survival over 24% from certain egg batches. Nutritional characterization and requirements for chilean flounder eggs and larvae during the culture period, optimal temperature, density, light intensity and green-water culture, are all subjects of ongoing research. Efforts to improve early culture protocol continues with emphasis on causes of larval mortality, early use of dry feed, microbial ecology of culture tanks and water quality. Weaning from live to formulated diets has been investigated, demonstrating survival of 30-50%. Presently only about 1-5% of hatchery raised flounder show a pigment disorder. Research on formulated diets and the weaning process are under way to ameliorate these results. Juveniles are being grown experimental in tanks and net pens systems and their performance (growth, survival, health) in both systems is being evaluated. Chilean flounder require approximately 2.8 years to reach 1 kg. Effort is also focusing on improving juvenile growing protocol, including recirculation systems, culture environment and diet formulation. Much future research will be required to attain optimal production of the species, such as species-specific nutritional requirements, optimum stocking density, disease and health management and growing protocols with new systems (shallow raceway). At present there is enough known about chilean flounder to begin a pilot-stage that will be the key for future development of its culture.     

Effects of dietary supplementation of citrus by‐products fermented with a probiotic microbe on growth performance,innate immunity and disease resistance against Edwardsiella tarda in juvenile olive flounder,Paralichthys olivaceus (Temminck & Schlegel)

Two consecutive studies were conducted to evaluate the dietary supplementation of citrus by‐products (CB) fermented with probiotic bacteria on growth performance, feed utilization, innate immune responses and disease resistance of juvenile olive flounder. In Experiment I, five diets were formulated to contain 0% (control) or 3% four different CB fermented with Bacillus subtilis (BS), Enterococcus faecium (EF), Lactobacillus rhamnosus (LR) and L. plantarum (LP) (designated as CON, CBF‐BS, CBF‐EF, CBF‐LR and CBF‐LP, respectively). During 10 weeks of a feeding trial, growth performance and feed efficiency were not significantly different among all the fish groups. However, fish fed CBF containing diets had significantly higher survivals than the CON group. Disease resistance of fish against Edwardsiella tarda was increased by the fermentation of CB. In Experiment II, we chose the BS as a promising probiotic and formulated five diets to contain 0%, 2%, 4%, 6% and 8% CBF‐BS. Growth performance was not significantly affected by the CBF‐BS supplementation during 6 weeks of a feeding trial. Innate immunity of fish was significantly enhanced by CBF‐BS supplementation. Myeloperoxidase and lysozyme activities were increased in a dose‐dependent manner by dietary CBF‐BS inclusions. In a consecutive challenge test against E. tarda, an increased disease resistance was found by CBF‐BS supplementation. These studies indicate that the fermentation process of CB with probiotic has beneficial effects on innate immunity and thereby increases disease resistance of olive flounder against E. tarda. Bacillus subtilis can be used as a promising probiotic microbe for by‐product fermentation in fish feeds.     

Acute ammonia toxicity and gill morphological changes of Japanese flounder Paralichthys olivaceus in normal versus supersaturated oxygen

Ammonia toxicity and morphological changes in gills of juvenile Japanese flounder Paralichthys olivaceus (5.76 ± 0.12 g) were investigated when fish were separately exposed to normal dissolved oxygen (DO) at 6.5 ± 0.5 mg L^?1 and supersaturated oxygen at 16.0 ± 2.0 mg L^?1 at different ammonia concentrations. Under normal oxygen, ammonia concentrations were tested from 0.04 (control) to 93.3 mg L^?1 total ammonia nitrogen (TAN), whereas under oxygen supersaturation, ammonia concentrations ranged from 0.04 (control) to 226.7 mg L^?1 TAN in the trial. After exposure to ammonia for 96 h, the ammonia LC₅₀ for fish was 62.48 mg L^?1 TAN (0.50 mg L^?1 NH₃–N) at normal oxygen and 160.71 mg L^?1 TAN (0.65 mg L^?1 NH₃–N) at oxygen supersaturation. Light microscopic observations confirmed that gill damage in normal oxygen was more profound than in oxygen supersaturation when fish were exposed to the same level of TAN (93.3 mg L^?1). Furthermore, electron microscopic scanning also showed more crimple, retraction and fibrosis on the secondary lamella surface in fish exposed to normal oxygen than those in fish exposed to supersaturated oxygen at the same TAN (93.3 mg L^?1). This study suggests that supersaturated oxygen can increase ammonia tolerance in Japanese flounder through reducing gill damage by ammonia, which partially explains the merit of using pure oxygen injection in intensive fish farming.     

Pathogenicity of Edwardsiella tarda to olive flounder, Paralichthys olivaceus (Temminck & Schlegel)

The LD50 and cytotoxic and enzymatic activities of both cells and extracellular products (ECPs) of eight Edwardsiella tarda strains were determined and their bacterial superoxide dismutase gene (sodB) and catalase gene (katB) were sequenced. Strains were also examined for their ability to resist the immune responses of olive flounder, Paralichthys olivaceus. LD50 values of strains (FSW910410, KE1, 2, 3, 4, 5 and 6) in olive flounder ranged between 10(2.5) and 10(5.3) cfu (colony forming units) per fish. Unlike the avirulent strain SU100 (LD50>or=10(7)), all pathogenic strains were able to survive in flounder serum and head kidney leucocytes (except for KE2). The virulent strains possessed type I sodB and katB, whereas SU100 had type II sodB but not katB. However, there was no difference between avirulent and virulent strains in haemolytic and cytotoxic activities. The results of this study demonstrated that the ability of E. tarda to resist complement activity and phagocytosis is conferred by its superoxide dismutase and catalase, which thus play an essential role in the pathogenicity of this bacterium. In addition genotyping of sodB and kat B proved to be a very useful tool to distinguish virulent from avirulent strains.     

Susceptibility of marine fish species to a megalocytivirus, turbot iridovirus, isolated from turbot, Psetta maximus (L.)

Turbot iridovirus (TBIV), a member of the genus Megalocytivirus in the family Iridoviridae, was isolated from diseased turbot, Psetta maximus (L.), in Korea in 2003. In this study, experimental infection of turbot, Japanese flounder, Paralichthys olivaceus (Temminck & Schlegel), and rock bream, Oplegnathus fasciatus (Temminck & Schlegel), with TBIV was performed to evaluate the viral susceptibility of these fish species. After virus exposure, the mortalities of turbot reared at 22 and 25 degrees C were 60% and 100%, respectively, suggesting that TBIV is the causative agent of the mass mortality of turbot that occurred in Korea in 2003. Moreover, TBIV was detected in Japanese flounder and rock bream by polymerase chain reaction after experimental infection (26 days post-inoculation) despite no viral pathogenicity in these fish, suggesting that these two fish species are also susceptible to the virus. It is possible that horizontal transmission of TBIV occurs among these three fish species because turbot is routinely cultured with Japanese flounder and rock bream in Korea.