青石斑鱼胃蛋白酶原的分离纯化及酶学性质

采用硫酸铵盐析、DEAE-Sephacel,DEAE-Sepharose阴离子交换柱层析、SP-Sepharose阳离子交换柱层析和Sephacryl S-200凝胶过滤柱层析,从青石斑鱼的胃中分离纯化到3种胃蛋白酶原(PG-1、PG-2和PG-3),SDS-PAGE结果显示,它们的分子量分别为35、36和37 ku.在pH 2.0下,3种胃蛋白酶原转化为有活性的胃蛋白酶P-1、P-2和P-3,分子量分别约为33、33和34 ku.3种酶的最适pH分别为3.0、2.5和2.5,最适温度均为40℃,且均能被典型的天冬氨酸蛋白酶抑制剂pepstatin A有效抑制.免疫交叉反应结果表明,3种胃蛋白酶原能与大鼠抗黄鳍鲷PG-Ⅰ,PG-Ⅱ,PG-3b,PG-3a,PG-4a及PG-4b多克隆抗体发生不同程度的免疫交叉反应.动力学参数测定结果表明,P-1、P-2和P-3对酸变性牛血红蛋白的KM/Kccat及Kcat/Km值分别为7.0×10-5 mol/L,17.6 S-1,2.5×105mol/(L· S);5.5×10-5 mol/L,22.8 S-1,4.1×105 mol/(L · S)和5.2 ×10-5 mol/L,18.7 S-1,3.6 ×105 mol/1(L· S).     

棕点石斑鱼的肌肉营养成分分析

试验结果表明,棕点石斑鱼的含肉率为77.3%,肌肉中粗蛋白、粗脂肪、粗灰分和水分的含量分别为18.8%、2.2%、1.16%和77.1%;18种氨基酸总量为18.22%(占鲜样),其中人体必需的8种氨基酸含量为6.88%,占氨基酸总量的37.36%,4种鲜味氨基酸含量为7.11%.其氨基酸构成比例符合FAO/WHO标准.必需氨基酸指数(EAAI)为514.DHA和EPA含量丰富,占脂肪酸总量的28.2%.     

Establishment of a novel fin cell line from Brown-marbled grouper, Epinephelus fuscoguttatus (Forsskål), and evaluation of its viral susceptibility

To lay a solid foundation of in vitro investigations of fish viral diseases, cytotechnology and cytotoxicology, a novel fin cell line from brown-marbled grouper, Epinephelus fuscoguttatus , was established and its viral susceptibility was evaluated. The fin tissues, digested with hyaluronidase and collagenase II, were used to initiate primary culture at 24 °C by using 20% foetal bovine serum-Dulbecco's modified Eagle medium/F12 medium, which was further supplemented with carboxymethyl–chitooligosaccharide, basic fibroblast growth factor and insulin-like growth factor-I. The fibroblastic fin cells grew at a steady rate during subsequent subculture and had a population doubling time of 50.6 h at passage 60. The modal diploid chromosome number was 48. A brown-marbled grouper fin cell line (bmGF-1) has been established and subcultured to passage 75 by now. Viral susceptibilities revealed that typical cytopathic effects of bmGF-1 cells emerged after being infected by turbot reddish-body iridovirus (TRBIV) or lymphocystis disease virus (LCDV). However, a large number of TRBIV and LCDV particles were also found in infected bmGF-1 cells. All these indicate that the bmGF-1 cell line has good susceptibility to TRBIV and LCDV, which may serve as a valuable tool for studies of cell–virus interactions and have potential applications in fish virus propagation and vaccine development.     

密度胁迫对点带石斑鱼幼鱼生长、代谢的影响

点带石斑鱼(Epinephelus malabaricus)幼鱼体质量(15±0.5)g,将其随机分为4组,每缸(直径60 cm×水深50cm)分别放养10尾、20尾、30尾、40尾幼鱼,密度分别相当于1.1 kg/m~3,2.1 kg/m~3,3.2 kg/m~3,4.2 kg/m~3,并分别标记记为G,O,G_20,G_30,G_40,每组3个重复,养殖6周后对幼鱼进行生长性能、饲料系数、血液指标及肝脏中相关酶活性进行分析以研究密度胁迫对点带石斑鱼幼鱼生长、代谢的影响.结果表明:1)G_10组幼鱼增重率显著低于其他各组(P<0.05),G_20组增重率最高且显著高于其他组(P<0.05).各组间特定生长率的变化与增重率的变化趋势类似.G_10和G2_20组的饲料系数显著低于G_30组(P<0.05),G_40组饲料系数最高且显著高于其他各组(P<0.05).2)G_30和G_40组间血浆皮质醇含量无显著差异(P>0.05),但均显著高于G_10和G_20组(P<0.05),G_10组皮质醇含量显著低于G_20组((P<0.05).血糖含量呈现与皮质醇含量相同的变化趋势.3)G_30和G_40组谷丙转氨酶活性均显著高于G_10和G_20组(P<0.05),各组谷草转氨酶活性随养殖密度的升高而升高;G_40组乳酸脱氢酶活性最高,与G_30组无明显差异(P>0.05),但G_40与2个低密度组(G_10和G_20)差异显著(P<0.05);乙酞胆碱酯酶活性随养殖密度的升高而降低.结果表明,养殖密度过高或过低都会对点带石斑鱼幼鱼的生长与代谢造成负面影响,20尾/缸(2.1 kg/m~3)是较为适宜的养殖密度.本研究旨在为工厂化养殖点带石斑鱼提供理论依据.     

石斑鱼神经坏死病毒传播途径阻断的初步研究

本文报告了斜带石斑鱼（Epinephelus coioides）苗种培育中亲鱼、卵、饵料等几个关键环节的病毒携带检测以及几种药物对病毒的消除效果的试验。用RT—PCR法检测斜带石斑鱼亲鱼的粪便、卵、仔鱼、稚鱼,饵料轮虫、桡足类中都有检出神经坏死病毒（nervous necrosis virus,NNV）。聚维酮碘5—10mg／L浸泡卵20min、浸泡轮虫和桡足类30min,盐酸吗啉胍3～5mg／L浸泡卵30min、浸泡轮虫和桡足类40min,聚维酮碘5mg／L＋盐酸吗啉胍3mg／L浸泡卵20min、浸泡轮虫和桡足类30min可有效灭活所携带的神经坏死病毒活性。     

云纹石斑鱼精子冷冻保存

以云纹石斑鱼精液为实验材料,对精子稀释液、抗冻剂种类和适宜浓度、冷冻保存液进行了筛选。结果表明,利用9g/L NaCl、10g/L KHCO3和10%小牛血清配制而成的稀释液EM1-2适宜于云纹石斑鱼精子冷冻保存,以2ml冷冻管为精子容器,在60L液氮生物保存罐中冷冻保存精子,冷冻解冻精子活力可达56.67%±5.77%,要优于TS-2、ES1-3和其他EM系列稀释液冷冻保存精子活力。利用EM1-2为基础液对抗冻保护剂进行筛选,结果显示,10%~20%的二甲基亚砜(DMSO)和1-2-丙二醇(PG)冷冻保存后精子活力无显著差异(P0.05),其中15%的DMSO和10%PG冷冻保存精子效果最优,解冻后精子活力分别可达54.52%±7.81%和57.24%±3.69%。利用冷冻保存1年的精液与云纹石斑鱼卵进行受精,受精率和孵化率均达到80%以上,与新鲜精子无显著性差异(P0.05)。本研究表明,利用EM1-2配制15%的DMSO或10%的PG可用于冷冻保存云纹石斑鱼精液。在此基础上,建立了精子冷冻库,保存精子130ml,为人工繁育和杂交育种提供了丰富的精子源。     

云纹石斑鱼(♀)×鞍带石斑鱼(♂)杂交后代染色体核型分析

本研究对云纹石斑鱼(Epinephelus moara♀)×鞍带石斑鱼(Epinephelus lanceolatus♂)杂交后代利用植物血球凝集素(PHA)及秋水仙素通过活体注射法制作染色体标本。选用头肾和鳃组织制作细胞悬浊液,并对冷滴片和热滴片法进行比较,结合空气干燥法制作染色体分裂相玻片,经吉姆萨染色后,在显微镜下观察筛选清晰完整的分裂相。杂交子代(俗称云龙斑)选取75个分裂相,统计染色体数目并进行核型分析。结果显示,云龙斑二倍体染色体数为48,其中,19对染色体为端部着丝粒染色体,2对为亚中部着丝粒染色体,3对为亚端部着丝粒染色体,其染色体臂数(NF)为58,核型公式为2n=48,4sm+6st+38t。杂交后代云龙斑与父母本染色体数目相同,都为2n=48,但云龙斑染色体与其父母本组型不同,在遗传过程中发生了复杂的染色体重新配对和变异。本研究为杂交后代"云龙斑"的种质鉴定、亲缘关系分析、杂交育种等提供了重要的科学根据。     

响应曲面法分析低盐条件下温度与pH对斜带石斑鱼幼鱼能量代谢的联合效应

采用二因素五水平的中心复合设计和响应曲面分析方法,以温度(21~33℃)和pH(6.5~8.5)为因子,以耗氧率和排氨率为响应指标,探讨了在低盐条件下温度和pH对斜带石斑鱼(Epinephelus coioides)幼鱼能量代谢的联合影响。结果表明,本实验条件下,随着温度的上升,耗氧率(MO_2)和排氨率(MTAN)呈上升的变化;随着pH的上升,MO_2和MTAN呈先上升后下降的变化。温度一次效应对幼鱼的MO_2影响极显著(P0.01),二次效应对幼鱼的MO_2影响显著(P0.05);pH一次效应对幼鱼MO_2影响显著(P0.05),二次效应影响极显著(P0.01);温度与pH互作效应对MO_2影响显著(P0.05)。温度一次效应对幼鱼的MTAN影响极显著(P0.01),二次效应对幼鱼的MTAN无显著影响(P0.05);pH一次效应和二次效应对幼鱼MTAN影响显著(P0.05);温度与pH互作效应对MTAN无显著影响(P0.05)。所考察因子与幼鱼MO_2、MTAN间二次多项回归过程的决定系数分别为0.981 1、0.954 6(P0.01),可用于分析与预测。通过对所建立的回归方程同时优化,得到温度与pH的最佳组合为:温度31.2℃和pH 7.6。在此组合条件下,幼鱼MO_2、MTAN最大值分别为188.44 mg O_2·(kg·hr)-1;7.01 mg TAN·(kg·hr)-1,可靠度为0.902。实验结果提示,低盐条件下斜带石斑鱼养殖可在本实验中得到的最优因子组合下进行,以提高生产效益。     

Growth and feed performance,digestibility and acute stress response of juvenile grouper (Epinephelus fuscoguttatus) fed diets with hydrolysate from milkfish offal

Nutritional qualities of fish processing by‐products can further be improved through enzymatic hydrolysis. The objective of this study was to elucidate the efficacy of hydrolysed milkfish offal at different inclusion levels when fed to juvenile grouper, Epinephelus fuscoguttatus, with an initial body weight of 2.88 ± 0.06 g. The animals were fed for 56 days with seven diets supplemented with 0 (control), 5%, 15% and 25% of milkfish offal (MO) and milkfish offal hydrolysate (MOH). The diets were formulated to be isonitrogenous (45%) and isolipidic (11%). The diets were assigned to 21 tanks (15 fish per tank) with each diet having three replicates. Results from the experimental trials indicated that feed conversion efficiency, feed intake and weight gain of fish significantly (P < 0.05) improved when fed diets with MOH. No significant differences within the rest of the dietary treatments were observed. Survival rate (>90%) did not differ in all the dietary treatments. Proximate composition (crude protein, crude fat and ash) indicated no significant difference among fish fed from all the dietary treatments. Apparent digestibility of MOH indicated a 95% and 66% digestibility for protein and dry matter respectively. Plasma stress parameters (cortisol and glucose) were not influenced by the dietary treatment when fish were subjected to an acute stressor (5‐min chasing). Liver morphology indicated normal hepatocyte shape and the presence of lipid droplets in fish fed from all the dietary treatments. The results indicated that milkfish offal processed as hydrolysate can be utilized in grouper diets and can promote growth and feed efficiency when supplied at 10–15%.     

Quantitative trait locus mapping of growth‐related traits in inter‐specific F1 hybrid grouper (Epinephelus fuscoguttatus × E. lanceolatus) in a tropical climate

Growth‐related traits are the main target of genetic breeding programmes in grouper aquaculture. We constructed genetic linkage maps for tiger grouper (Epinephelus fuscoguttatus) and giant grouper (E. lanceolatus) using 399 simple sequence repeat markers and performed a quantitative trait locus (QTL) analysis to identify the genomic regions responsible for growth‐related traits in F₁ hybrid grouper (E. fuscoguttatus × E. lanceolatus). The tiger grouper (female) linkage map contained 330 markers assigned to 24 linkage groups (LGs) and spanned 1,202.0 cM. The giant grouper (male) linkage map contained 231 markers distributed in 24 LGs and spanned 953.7 cM. Six QTLs affecting growth‐related traits with 5% genome‐wide significance were detected on different LGs. Four QTLs were identified for total length and body weight on Efu_LG8, 10, 13 and 19 on the tiger grouper map, which explained 6.6%–12.0% of the phenotypic variance. An epistatic QTL with a reciprocal association was observed between Efu_LG8 and 10. Two QTLs were identified for body weight on Ela_LG3 and 10 on the giant grouper map, which explained 6.9% of the phenotypic variance. Two‐way analysis of variance indicated that the QTL on Efu_LG13 interacts with the QTLs on Ela_LG3 and 10 with large effects on body weight. Furthermore, these six QTLs showed different features among the winter, summer and rainy seasons, suggesting that environmental factors and fish age affected these QTLs. These findings will be useful to understand the genetic structure of growth and conduct genetic breeding in grouper species.