外源人绒毛膜促性腺激素诱导黑边石斑鱼排卵的研究

【目的】了解外源人绒毛膜促性腺激素(human chorionic gonadotropin,HCG)诱导黑边石斑鱼(Epinephelus fasciatus)排卵的影响。【方法】以成熟的黑边石斑鱼为研究对象,测量其体长、体质量和肥满度后,在2018年7—8月,使用HCG激素(剂量为200 IU/kg或500 IU/kg)注射分别对6尾雌鱼进行成熟诱导。【结果】在200 IU/kg HCG处理中,注射24 h前有5尾雌鱼的卵母细胞处于第三次卵黄球前期阶段;注射48 h后2尾雌鱼出现核移动卵母细胞,1尾雌鱼的卵母细胞为第三次卵黄球后期阶段;注射60 h后1尾雌鱼出现排卵现象,其余5尾雌鱼均为第三次卵黄球后期阶段。在200 IU/kg HCG处理的排卵个体中,注射时其卵母细胞直径为477.0 μm,注射后60 h卵母细胞的直径增加至624.4 μm。此外,经催产、排卵、受精和孵化后共获得总卵数16 906粒,受精率为68.7%,孵化率为43.0%。而500 IU/kg HCG处理的黑边石斑鱼排卵失败。【结论】使用剂量为200 IU/kg的外源HCG激素可诱导黑边石斑鱼成熟且排卵,但为了提高排卵率、受精率和孵化率,其注射剂量和效应时间还需要进一步调节。     

用微卫星标记研究南海四种石斑鱼的遗传变异与种间关系(英文)

[目的]从DNA分子水平揭示我国南海的四种重要经济海水鱼类点带石斑鱼(Epinephelusmalabaricus)、斜带石斑鱼(E.coioides)、鲑点石斑鱼(E.fario)、蜂巢石斑鱼(E.merra)的遗传群体变异水平与种间关系。[方法]本研究利用从GenBank中搜索到的21对微卫星引物,对点带石斑鱼、斜带石斑鱼、鲑点石斑鱼及蜂巢石斑鱼的等位基因数A、有效等位基因数Ne、多态信息含量PIC、观察遗传杂合度Ho、期望遗传杂合度He等遗传参数进行检测,并采用UPGMA类聚分析法探讨这四种石斑鱼的种间关系。[结果]点带石斑鱼群体的A、Ne、PIC、Ho和He等遗传参数的平均值分别为4.38±1.60、3.69±0.86、0.69±0.08、0.67±0.08、0.72±0.06,斜带石斑鱼群体的为3.88±1.13、3.55±1.04、0.66±0.10、0.68±0.21、0.70±0.08,鲑点石斑鱼群体的为6.00±1.07、4.68±0.65、0.78±0.03、0.73±0.25、0.79±0.03,蜂巢石斑鱼群体的为5.50±1.07、4.58±0.80、0.76±0.05、0.75±0.18、0.78±0.04。[结论]综合比较上述数值可知,四个石斑鱼群体的遗传变异程度依次为鲑点石斑鱼群体>蜂巢石斑鱼群体>点带石斑鱼>斜带石斑鱼群体;与其他海水鱼类的遗传变异水平比较,四种石斑鱼的群体遗传变异水平都较高,说明我国这些石斑鱼种类的遗传种质资源尚好。另外,遗传距离与类聚分析表明,点带石斑鱼与斜带石斑鱼遗传距离最近,而斜带石斑鱼与蜂巢石斑鱼的亲缘关系最远。     

赤点石斑鱼神经坏死病毒MCP基因原核表达条件优化(英文)

[Objective] To optimize the prokaryotic expression of MCP gene of red-spotted grouper nervous necrosis virus. [Method] The MCP gene was amplified from red-spotted grouper nervous necrosis viral genome by RT-PCR. The recombinant expression vector pRSET A-MCP was constructed and transformed into BL21(DE3)plysS to express proteins with induction in different media, at different pH, or at different temperatures. [Result] The expression level of recombinant bacteria reached a peak with induction under the following condition: SOB or LB medium, pH 7.0, 37 ℃ while the fusion protein was about 44.5 kD in molecular weight. [Conclusion] This study provided a basis for the development of RGNNV-MCP vaccine.     

饲料中大豆卵磷脂/鱼油比例对斜带石斑鱼稚鱼生长发育及肠道组织的影响

为探讨饲料中不同大豆卵磷脂/鱼油比例对斜带石斑鱼稚鱼成活率、生长行为、饲料利用以及肠道组织学的影响,本实验设计了4种添加不同大豆卵磷脂/鱼油比率的微颗粒饲料：0%/22%（饲料1）,5%/14%（饲料2）,10%/8%（饲料3）以及15%/7%（饲料4）,饲喂斜带石斑鱼稚鱼。每种实验饲料均设3个平行。试验持续40d。结果表明,饲料中不同大豆卵磷脂/鱼油比例对斜带石斑鱼稚鱼生长以及成活率有显著影响（P≤0.05）。饲料3和饲料4的增重分别为：（449.6±11.4）g和（588.3±61.7）g,显著高于饲料1的数值（185.0±2.5）（P≤0.05）。饲料1鱼体脂肪含量为：（154.7±12.8）g·kg-1,显著低于其他饲料组（P≤0.05）,但其鱼体蛋白含量为（728.3±25.5）g·kg-1,显著高于其他饲料组。饲料效率、肥满度、特定生长率、摄食量和蛋白质效率以及终末体重中,4种饲料之间无显著性差异。随饲料卵磷脂/鱼油比例升高,石斑鱼稚鱼肠道脂滴生成数量出现下降,且在饲料4中未观察到肠道脂滴。研究表明斜带石斑鱼稚鱼生长发育阶段需要较高的饲料卵磷脂/鱼油比例（如：15%/7%）。     

七带石斑鱼精子的超微结构

利用透射电子显微镜技术研究了七带石斑鱼（Epinephelus septemfasciatus）精子的超微结构,结果发现：七带石斑鱼精子由头部和尾部（鞭毛）两部分组成,没有明显的中片,头部前段无顶体,主要有高电子密度的球形细胞核,核中主要是致密的染色质,还分布有一些核泡,核外由核膜包围,将核与细胞质分隔开,核膜最前端紧贴质膜,核后端有植入窝,近端中心粒和中心粒间体位于其中。头部后端为袖套结构,由质膜包围的袖套结构延伸到后部,使整个头部呈椭球形,质膜后端沿鞭毛形成一个袖套腔,袖套结构中分布有中心粒复合体、线粒体以及囊泡等。精子细胞的细胞膜不光滑,呈现小的皱折。七带石斑鱼精子的尾部细长,紧接于头部细胞核,穿过袖套腔,通过基体与核紧密结合,中心粒复合体也位于此,且近端中心粒和远端中心粒呈钝角,鞭毛的轴丝由此形成,基体与鞭毛间的过渡区轴丝无中央微管,呈“9+0”型。尾部的主要结构为轴丝,其轴丝为典型的“9+2”结构,其起始端开始于前端基体,轴丝外仅有少许细胞质。     

中国南部养殖石斑鱼“肿胀病”的研究

1998年5月至7月间,在中国南部养殖石斑鱼暴发"肿胀病",引起石斑鱼大量发病、死亡.从体表和鳍条的溃疡病灶处分离出2个菌株.形态学观察和生化试验结果表明这2个菌株均为鳗弧菌.将肝、脾、心和肾的上清液接种到CO、FHM、CAB、CK、PG和PC细胞上,没有观察到细胞病变.患病鱼的腹腔中有大量黑色的球状物和腹水,使患病鱼呈现明显的腹部肿胀的症状.组织病理学观察结果表明,这些黑色的球状物由多核细胞组成的增生性组织.     

Effects of dietary live or heat‐inactivated autochthonous Bacillus pumilus SE5 on growth performance,immune responses and immune gene expression in grouper Epinephelus coioides

To evaluate the possible dietary application of live and heat‐inactivated probiotic Bacillus pumilus SE5 in grouper Epinephelus coioides, juveniles (14.6 ± 0.2 g) were fed either a basal control diet (without probiotic) or the basal diet supplemented with 1.0 × 10⁸ CFU g^?1 live (T1) and heat‐inactivated B. pumilus SE5 (T2). The heat‐inactivated probiotic significantly improved the final weight, weight gain (WG) and specific growth rate (SGR) at day 60 and significantly decreased the feed conversion ratio (FCR) at day 30 and 60, while the viable probiotic significantly decreased the FCR at day 60 (P < 0.05). Phagocytic activity, serum complement C3 and IgM levels as well as SOD activity elevated significantly in fish fed the heat‐inactivated probiotic for 60 days (P < 0.05). Furthermore, the heat‐inactivated probiotic remarkably up‐regulated expression of TLR2 and pro‐inflammatory cytokines (IL‐8 and IL‐1β) in head kidney (P < 0.05), but the viable probiotic failed to do so. These results indicated that heat‐inactivated B. pumilus SE5 can effectively improve the growth performance and immune responses of E. coioides.     

温度和盐度对鞍带石斑鱼受精卵发育及仔鱼成活率的影响

研究了在8个盐度梯度(18.3‰、19.7‰、21.2‰、23.5‰、25.6‰、27.1‰、29.5‰和31.7‰)下鞍带石斑鱼Epinephelus lanceol受精卵和仔鱼在水中的分布状态、出膜率和仔鱼存活率;在9个温度梯度(18℃、20℃、22℃、24℃、26℃、28℃、30℃、33℃和35℃)下,其受精卵孵化率、畸形率和仔鱼成活率。结果表明:(1)在水温为25~26℃,当盐度≤25.6‰时,受精卵表现为沉性;当盐度≥27.1‰时,受精卵表现为浮性。仔鱼的分布与受精卵有相似的特点,当盐度≤21.2‰时仔鱼分布在底层水体;当盐度≥27.1‰时,仔鱼集中分布在上层水体。(2)以孵化率和成活率为指标,鞍带石斑鱼孵化的适宜盐度范围为21.2‰~31.7‰,最适盐度范围为25.6‰~31.7‰。在盐度为31.7‰时仔鱼成活率最高达到75.9%。(3)鞍带石斑鱼胚胎发育的适温范围是26~30℃。28℃时受精卵孵化率、仔鱼成活率均最高,畸形率最低。     

Immunization of grouper, Epinephelus coioides, confers protection against a protozoan parasite, Cryptocaryon irritans

The present study aimed to determine whether protection is conferred by immunization of grouper, Epinephelus coioides, against a protozoan parasite, Cryptocaryon irritans. The immunization of E. coioides was carried out by a low level exposure of fish to live C. irritans theronts from predetermined number of tomonts and by an intraperitoneal injection of a vaccine consisting of formalin-killed C. irritans theronts.

Mucus titers detected by ELISA were significantly higher in fingerling and adult grouper subjected to the low level of exposure to C. irritans theronts at 3-week post-exposure compared to fish that had no previous exposure. In addition, significantly smaller tomonts were produced from adult grouper after three successive exposures than the tomonts produced after a single exposure to the parasite.

In the vaccine-immunization experiment, no mortality was monitored in fish that received high dose vaccine (100 μg/fish), while 40% cumulative mortality and 100% cumulative mortality were recorded in low dose group (10 μg/fish) and control group (PBS-injected), respectively. In the succeeding replicate, the vaccine-immunized group (high dose) had 37.5% cumulative mortality and 100% cumulative mortality for the control. In addition, a total of 1830 tomonts were collected at 5-day post-challenge from the control group while none from the vaccine-immunized group. Significantly fewer trophonts and tomonts were enumerated at 5-day and 7-day post-challenge, respectively, in the vaccine-immunized group than the control.

Results suggest that a protective immunity has been conferred on the immunized grouper as indicated by high antibody titers in the mucus of C. irritans-exposed fish and higher survival and fewer parasites in vaccine-immunized fish than the control groups. The conferred immunity played a major role in preventing or limiting the adhesion, invasion, and development of C. irritans theronts on the skin of the immunized grouper.