Evaluation of gross protein and lipid requirements in formulated feed for honey gourami,Trichogaster chuna (Hamilton 1822)

Gross protein and fat requirements in formulated feeds designed for the honey gourami, Trichogaster chuna (initial weight 1.34–1.38 g), were investigated. Feeds containing 30%, 40%, and 50% protein and 6% and 9% lipid at each protein level were tested. Protein and energy sources used were from fishmeal, shrimp meal, clam meal, soy flour, and wheat flour. An equal mixture of crude sardine oil and groundnut oil was used as the source of lipids. Comparison of the whole gourami amino acid profiles before and after the dietary treatments indicated a relative decline in all amino acids except methionine and lysine. Fatty acid profiles of whole individuals after dietary treatment showed a substantial increase in monounsaturated fatty acids relative to the initial fatty acid profiles. No significant differences were observed in fish growth between dietary treatments (p > .05). Feeds containing 30% protein and 6% lipid were found to be adequate for normal growth, while feeds with 40% protein and 6% lipid were seen to help accelerate growth and reproduction. In this study, protein and lipid levels required for regular maintenance, sexual maturation, and spawning of aquarium‐reared gourami were established.     

Susceptibility of a number of Australian freshwater fishes to dwarf gourami iridovirus (Infectious spleen and kidney necrosis virus)

Megalocytiviruses cause high mortality diseases that have seriously impacted aquaculture, with the most frequent outbreaks occurring in East and South‐East Asia. The international trade of juvenile fish for food and ornamental aquaculture has aided the spread of these viruses, which have spread to Europe and Australia and other regions. Australian freshwater fishes were examined for susceptibility to infection with the exotic megalocytivirus, dwarf gourami iridovirus (DGIV), which belongs to a group with the type species, Infectious spleen and kidney necrosis virus (ISKNV). Fish were held at 23 ± 1 °C and challenged by intraperitoneal (IP) injection or by cohabitation with Murray cod, Maccullochella peelii (Mitchell) infected with DGIV. A species was deemed to be susceptible to DGIV based on evidence of viral replication, as determined by qPCR, and megalocytic inclusion bodies observed histologically. Horizontal transmission occurred between infected Murray cod and golden perch, Macquaria ambigua (Richardson), Macquarie perch, Macquaria australasica (Cuvier) and Murray cod. This indicated that DGIV shed from infected fish held at 23 °C can survive in fresh water and subsequently infect these naïve fish. Further, DGIV administered IP was highly pathogenic to golden perch, Macquarie perch and Murray cod. Compared to these species, the susceptibility of southern pygmy perch, Nannoperca australis (Gunther) was lower. Freshwater catfish (dewfish), Tandanus tandanus (Mitchell), were not susceptible under the experimental conditions based on the absence of clinical disease, mortality and virus replication. This study showed the potential risks associated with naïve and DGIV‐infected fish sharing a common water source.     

Long-term culture of a cell population from Siberian sturgeon (Acipenser baerii) head kidney

In vitro cultures of native fish cell lines are of great importance, both for basic research and applied science. In particular, there is strong demand for long-term growable cell lines from breeding fish, like sturgeon. Here, we describe the culture of cells from Siberian sturgeon (Acipenser baerii) head kidney. The cells have so far been cultured over a period of 12 months (24 passages). Cytochemical and immunocytochemical examination suggests that, in vitro, the cells exhibit markers that are indicative for different cell types. In particular, fat storing cells (adipocytes) were observed, and the expression of cytokeratins and glial fibrilar acidic protein (GFAP) can be concluded on the basis of immuncytochemical analysis. The observation of different morphologies additionally underlines the heterogeneity of the cell population and matches the typical behaviour of in vitro cultures of stem/progenitor cells. Different applications can be imagined.     

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...     

Isolation and characterization of collagens from the skin of giant grouper (Epinephelus lanceolatus)

ABSTRACT

Acid-solubilized collagen (ASC) and pepsin-solubilized collagen (PSC) were extracted from the skin of giant groupers (Epinephelus lanceolatus) with yields of 39.51 and 19.12%, respectively. ASC and PSC consisted of two different α chains (α1 and α2) and were characterized to be type I collagen with no disulfide bond. The imino acid contents of the ASC and PSC from giant grouper skin were 189 and 181 per 1,000 residues, respectively. The maximum endothermic temperatures (T_max) of ASC and PSC measured by differential scanning calorimetry (DSC) were 31.71 and 31.33°C, respectively. The denaturation temperatures of ASC and PSC measured by viscometry were 29.84 and 29.05°C, respectively. The maximum solubility in 0.5 M acetic acid was observed at pH 5 and pH 6 for ASC and PSC, respectively. A sharp decrease in solubility was observed for both ASC and PSC in the presence of NaCl above 3% (w/v).     

River pen culture of giant freshwater prawn Macrobrachium rosenbergii (De Man) in southern Vietnam

A technical and socio‐economic survey conducted in the Dong Thap province of Vietnam to assess the current status of river pen culture of giant freshwater prawn Macrobrachium rosenbergii (De Man) showed that pen culture, which has been developed and operated by farmers' indigenous knowledge, requires more study in order to optimize stocking density and to assess its environmental impacts. In this study, the prawn pens were of rectangular shape, with an average size of 209 m². Prawns were stocked in June at an average density of 62 pieces m^?2 and fed on farm‐made feed and were harvested 2–4 times starting from 4 months after stocking until late December or the following January. The average prawn yield was 0.52 kg m^?2 year^?1, ranging from 0.14 to 1.6 kg m^?2 year^?1. The average net return was US$0.71 m^?2 year^?1, ranging from US$1.24 to 4.37 m^?2 year^?1. About 73% of the farmers achieved positive net returns. The top five constraints for prawn pen culture were lack of knowledge of culture technologies, poor water quality, disease problems, poor quality of wild seed and lack of capital. Almost all farmers have no environmental awareness, and environmental regulations for prawn pen culture have not been established. Field measurements showed that all measured water quality parameters were within the range for good growth of giant freshwater prawns, and indicated that there was no significant accumulation of nutrients and organic matter at the bottoms of rivers or canals.     

Development of two cell culture systems from Asian seabass Lates calcarifer (Bloch)

Two new cell culture systems namely epitheloid cells of Lates (LCE) and fibroblastic cells of Lates (LCF) have been developed from fry and fingerling of the economically important brackishwater fish Lates calcarifer. Primary cultures were initiated by explant technique using caudal fin of fingerling and whole body tissue of the fry. The nutritional requirements and the growth pattern in response to different culture environment were similar for the two cell cultures. The culture medium used was Leibovitz‐15 supplemented with 20% fetal bovine serum (FBS) and 1% fish serum. The LCE comprised of epithelioid cells and LCF cells were fibroblastic. With a split ratio of 1:2, the confluency of cells was attained in 8–10 days at an incubation temperature of 28°C. The cells were found to grow well in a wide range of temperature (24–32°C) and stable at 20 and 36°C. The growth rate of LCF and LCE cells increased proportionately with the concentration of FBS from 5% to 20%. A decrease of serum level to 10% after eight subcultures produced no apparent change in cell morphology and growth rate. The viability of cells was found to be 70% when revived after a month of storage in liquid nitrogen (?196°C).     

红螯螯虾XO-SG神经细胞的原代培养

本研究采用酶解的方法获得红螯螯虾眼柄XO-SG复合体的单个神经细胞,并依据显微观察对XO-SG神经细胞进行分类,同时利用Leibovitz's L-15等作为基础培养基离体培养解离的神经细胞。目的是建立虾类XO-SG神经细胞的分类标准并确定合适的培养条件,便于在体内外开展神经内分泌系统的调控研究。结果显示,根据神经细胞形态特点,红螯螯虾XO-SG神经内分泌细胞分为6种类型,不同类型的细胞在细胞大小以及显微结构上存在明显差异;建立了红螯螯虾眼柄XO-SG神经元的体外原代培养方法,细胞在改良的L-15培养基中存活状态良好,原代培养的神经细胞可以在体外存活14 d。离体培养过程中,不同类型神经细胞的再生速率存在差异,部分细胞在第2天就出现再生的轴突或树突。再生过程基本可以持续7 d,随后细胞开始萎缩凋亡。本研究为红螯螯虾眼柄神经细胞的分类以及利用神经细胞在体外开展虾类的内分泌代谢和调节机制研究提供了基础依据和研究平台。     

New methods for the primary culture of gill epithelia from freshwater rainbow trout

Gill epithelia from freshwater rainbow trout can be grown in primary culture in Leibovitz's L-15 medium, by seeding freshly isolated gill cells on two successive days, from two different fish, directly onto permeable filter supports (DSI technique). This preparation allows the measurement of transepithelial resistance (TER) and exposure of the apical surface to freshwater, as in vivo. New culture methods were developed and evaluated, using TER as an indicator of epithelial integrity, in an effort to improve the utility of the preparation for proteomic and toxicological research. TER was not related to cell density or protein content in DSI epithelia. To eliminate bovine proteins, the 5% foetal bovine serum (FBS) normally required for epithelial development was replaced with trout plasma. While previously frozen trout plasma proved toxic, freshly collected heparinized plasma, provided by chronically cannulated adult trout, was not. The use of 5% fresh trout plasma supported a TER development curve identical to that with 5% FBS, a useful advance for proteomic research because foreign (bovine) proteins are eliminated. However, 10% plasma reduced TER development, and 100% plasma abolished it. The inhibitory effect on TER of high plasma levels was seen only early in epithelial development, and was exerted from the apical side, likely an effect on tight junction formation. Mature plasma-supplemented preparations mounted a TER rise in response to apical freshwater exposure comparable to that of FBS-supplemented epithelia. Yolk-sac fry extract was inhibitory to TER development, even in the presence of 5% FBS. Transfer of mature epithelia from 18 °C to 4 °C maintained stable TER and extended the useable lifespan by at least ten days, thereby facilitating storage of preparations for toxicity testing. A new method of growing epithelia, involving only a single seeding of cells from a single fish, directly onto filter inserts (SDSI technique), provided mature epithelia with much lower TER, a smaller TER response to apical freshwater, and lower cell density and protein content than DSI epithelia. These SDSI epithelia offer the advantage of multiple preparations grown directly from unique individuals for in vitro toxicity testing. This revised version was published online in July 2006 with corrections to the Cover Date.     

刀额新对虾原代淋巴细胞培养及其感染白斑综合征病毒（WSSV）的病理特征

为深入了解对虾白斑综合征病毒(WSSV)的致病机理和体外培养的对虾细胞对WSSV的敏感性,实验以1.5×L-15培养基培养刀额新对虾原代淋巴细胞,待细胞形成单层后接种WSSV,通过倒置显微镜、荧光显微镜、透射电子显微镜观察接种病毒后细胞的病理变化。结果显示,使用1.5×L-15培养基培养对虾淋巴组织,3 h后可观察到有细胞迁出,并能迅速形成单层,36 h后细胞的迁出汇合率可达80%,且能存活20 d以上。接种WSSV 24 h后,出现病变的细胞变圆、漂浮,细胞之间的网状结构消失,最后细胞破碎、溶解;接种WSSV 48 h后Hoechst 33342染色结果显示,感染的细胞核深染,且变形、膨大;电镜下,细胞核内含大量成簇分布的杆状病毒,细胞器被挤向细胞边缘,细胞膜轮廓模糊。研究表明,纯化的病毒粒子接种体外培养的淋巴细胞,能够使其产生明显病理变化,证明了WSSV对体外培养的淋巴细胞具有感染力,并且可在淋巴细胞中增殖。     

斜带石斑鱼肝细胞分离及原代培养方法的建立

本研究以斜带石斑鱼肝细胞为实验对象,在不同培养条件下进行原代培养,旨在探讨稳定可靠的斜带石斑鱼肝细胞分离及原代培养方法。采用组织块分离法和胰蛋白酶(含EDTA)消化法分离肝细胞,并通过密度梯度离心法分离纯化肝细胞,细胞悬液于DMEM/F-12、M199和L-15培养液中培养;细胞活力及数量采用血球计数板计数,并通过MTT法测定细胞增殖率;同时,测定不同时间培养上清液中乳酸脱氢酶(LDH)活性、白蛋白(ALB)和尿素氮(BUN)的含量,以分析肝细胞生长状态。结果表明,组织块方法不适于斜带石斑鱼肝细胞的培养,未见细胞从组织块中迁出,而胰蛋白酶消化法获得良好稳定的培养效果,细胞产量达到1.6×108个/g肝重,活细胞数达到95%;L-15培养基细胞生长明显优于DMEM/F-12和M199培养基;启动原代培养的48~72 h阶段肝细胞生长代谢旺盛,培养上清液中LDH活性显著降低,ALB和BUN含量显著升高。结果显示,0.25%的胰蛋白酶常温消化法适合斜带石斑鱼肝细胞的分离,斜带石斑鱼肝细胞原代培养的最适培养基为L-15培养基,肝细胞在启动原代培养的48~72 h生长代谢旺盛。     

Economic analysis of monosex culture of giant freshwater prawn (Macrobrachium rosenbergii De Man): a case study

All‐male monosex culture of Macrobrachium rosenbergii (De Man) has emerged as a popular practice in India, especially in the state of Andhra Pradesh. A study was conducted to compare the economics of all‐male, mixed and all‐female culture in 15 adjacent, rectangular ponds of 4000 m² each by stocking juveniles previously reared in a nursery for 60 days. The experiment was conducted using a completely randomized design with three treatments; T₁ (all male), T₂ (mixed) and T₃ (all female), and five replicates for a period of 5 months after the nursery phase. Statistical analysis showed highly significant (P<0.01) differences among the three types of culture. The cost of production was estimated and the economic feasibility of the culture methods was evaluated by cost‐return and partial budgeting analysis. The average weight, productivity and specific growth rate were the highest for all male culture, being 80.92±2.41 g, 1532 kg ha^?1 and 1.97±0.02 respectively. All‐female culture registered significantly higher survival (89.16±0.77%) and the best apparent feed conversion ratio of 1.26±0.02. The economic analysis revealed that all‐male monosex culture of M. rosenbergii was 63.13% and 60.20% more profitable than mixed and all‐female cultures respectively.     

水温对幼鲵生长影响的初步研究

研究了不同时间段泉水和河水对幼鲵(Andrias davidanus)生长的影响。结果显示:幼鲵在不同时间和不同地点增重存在差异。第二阶段幼鲵平均日增重极显著高于第一阶段(P<0.01)。1号点(泉水)幼鲵平均增重和日平均增重极显著高于2号点(河水)幼鲵(P<0.01);第一阶段1号点日平均水温极显著高于2号点(P<0.01),最低温度较河水高6.95℃,且1号点水温变化较小。结果表明,水温是影响幼鲵生长的重要因素之一。     

哲罗鲑的集约化流水养殖

本文对利用泉水集约化养殖哲罗鲑的生长情况、饵料系数、养殖成活率、病害防治进行了总结,并进行了养殖密度和饲料对比试验.经过323d的养殖,哲罗鲑从初始时的1.59g/尾增长到1020g/尾,达到了上市标准,养殖成活率84.84%,饵料系数0.86;密度和饲料实验表明放养800尾/池的哲罗鲑能够获得较好的效果,且投喂进口饲料的哲罗鲑饵料系数0.92,每Kg鱼饲料成本9.05元,投喂国产饲料的哲罗鲑饵料系数1.05,每Kg鱼饲料成本7.66元;影响哲罗鲑的主要疾病是小瓜虫病、烂鳃病和出血病,要提早预防.     

Characterization of apoptosis induced by grouper iridovirus in two newly established cell lines from barramundi,Lates calcarifer (Bloch)

Two new cell lines have been established from the muscle and swim bladder tissues of barramundi, Lates calcarifer, and designated as BM (barramundi muscle) and BSB (barramundi swimbladder), respectively. The cells multiplied well at 28 °C in Leibovitz’s L‐15 medium supplemented with 10% foetal bovine serum, and have been continuously subcultured more than 100 times to date. Morphologically, BM cells were mostly fibroblastic, whereas BSB were mostly epithelial. Both cell lines were susceptible to grouper iridovirus (GIV) and displayed characteristics of apoptosis after viral infection. The induction of apoptosis was further assayed in GIV‐infected BM and BSB cells by various methods. The inhibition of cell growth by GIV was demonstrated by MTT [3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide] assay. Morphological observations revealed typical apoptotic features in the infected cells, including cell shrinkage and rounding, chromosome condensation and formation of apoptotic body‐like vesicles. Chromosome fragmentation was detected by DNA laddering and TUNEL assays. Finally, the appearance of phosphotidylserine on the outer leaflet of apoptotic cell membranes was confirmed by annexin V staining. This is the first report of apoptosis induced by GIV in fish cells.     

黑斑侧褶蛙脑微血管内皮细胞的原代培养及鉴定

为了建立蛙脑微血管内皮细胞原代培养方法,实验以黑斑侧褶蛙脑组织为实验材料,在无菌条件下分离出大脑灰质,利用0.1%Ⅱ型胶原酶和胶原酶-分散酶进行消化,经过Percoll密度梯度离心后,将获取的脑微血管段接种于鼠尾胶包被的培养瓶中,于28°C、5%CO₂条件下原代培养蛙脑微血管内皮细胞,并通过细胞形态学观察和Ⅷ因子相关抗原免疫荧光对细胞进行鉴定,最后用四唑蓝比色法(MTT)测定细胞的生长状态。结果显示,微血管段在体外培养1 d后开始贴壁生长,并逐渐扩大成团簇状,培养至7 d时,细胞呈“铺石路样”镶嵌式排列,表现为区域性单层生长;Ⅷ因子免疫荧光检测发现胞质呈红色,表达为阳性,阳性细胞率达97%以上;MTT实验显示,细胞在第3～5天时生长速率最快。本研究首次建立了蛙脑微血管内皮细胞的原代培养与鉴定方法,为体外研究蛙类神经性疾病的发病机理奠定基础,也为相关药物的研发和筛选提供了细胞模型。     

Lymphoid organ cell culture system from Penaeus monodon (Fabricius) as a platform for white spot syndrome virus and shrimp immune-related gene expression

Shrimp cell lines are yet to be reported and this restricts the prospects of investigating the associated viral pathogens, especially white spot syndrome virus (WSSV). In this context, development of primary cell cultures from lymphoid organs was standardized. Poly-l-lysine-coated culture vessels enhanced growth of lymphoid cells, while the application of vertebrate growth factors did not, except insulin-like growth factor-1 (IGF-1). Susceptibility of the lymphoid cells to WSSV was confirmed by immunofluoresence assay using monoclonal antibody against the 28 kDa envelope protein of WSSV. Expression of viral and immune-related genes in WSSV-infected lymphoid cultures could be demonstrated by RT-PCR. This emphasizes the utility of lymphoid primary cell culture as a platform for research in virus-cell interaction, virus morphogenesis, up and downregulation of shrimp immune-related genes, and also for the discovery of novel drugs to combat WSSV in shrimp culture.     

Comparative chemical,taste, and functional components in different tissues of giant grouper (Epinephelus lanceolatus)

The chemical composition, nutrition, and flavor components of various giant grouper tissues were determined. The muscle protein content was about 20%, and the muscle fat content was in the range of 3.9–6.4%, which is higher than that of most other groupers. The bone had the highest fat value (12.5–14.2%). The major nucleotide-related compound in the tissues was inosine monophosphate (IMP); whereas in the skin, adenosine monophosphate (AMP) was predominant. The predominant free amino acid (FAA) in the tissues was taurine (Tau), followed by arginine (Arg), lysine (Lys), and glycine (Gly). Tau content was higher in chin meat, and lower in bone. The total peptide content was in the range of 660–1,390 mg/100 g, which was much higher than that observed in other fish species. Muscle was rich in C16:0, C18:0, C18:1, and omega-3 highly unsaturated fatty acids, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA).