在循环养殖系统中添加微生态制剂去除氨氮和亚硝酸氮的试验

利用活菌制剂,以闭路内循环方式净化水质,养殖点带石斑鱼。试验结果表明:3种活菌制剂对NH4 -N的降解率分别为:NMX菌39.0%,科恩菌30.5%,EM菌20.4%;对NO2--N的降解率分别为:NMX菌45.7%,EM菌40.4%,科恩菌13.6%。活菌制剂不仅能调节水质,而且还能促进养殖个体的生长,提高饵料转化率,NMX菌、科恩菌和EM菌试验组日增重分别为5.5 g/尾、5.1 g/尾、4.9 g/尾,而对照组为3.9 g/尾;相应饵料系数分别是1.00、1.02、1.02,对照组为1.06。     

循环水养殖密度和氨氮对斜带石斑鱼生长和免疫力的影响

选取平均体重为146.3 g/尾的斜带石斑鱼450尾分成3个密度组进行饲养,分别为低密度组D1(100尾/m³,14.6 kg/m³)、中密度组D2(150尾/m³,21.7 kg/m³)和高密度组D3(200 尾/m³,29.5 kg/m³),比较不同养殖密度引起的水质变化对斜带石斑鱼的生长性能、摄食率和免疫力的影响。结果表明：水体中氨氮含量随斜带石斑鱼养殖密度的增加而增加(P<0.05);斜带石斑鱼的摄食率随水体中氨氮浓度的增加而显著下降(P<0.05)。随着养殖密度的增加,斜带石斑鱼的生长速度呈现负相关性,各试验组的特定生长率分别为1.07、0.66、0.47;各实验组斜带石斑鱼体重也出现显著性差异(P<0.05),各实验组的尾均增重量分别为118.5 g、63.3 g和44.1 g;养殖密度对斜带石斑鱼的饵料系数和成活率无显著影响(P>0.05);斜带石斑鱼的免疫指标血清溶菌酶(LZM)、碱性磷酸酶(AKP)和超氧化物歧化酶(SOD)随养殖密度的增加而下降,随养殖时间的延长呈现出先上升后下降的趋势。阐明了养殖密度引起养殖水质状况的改变,继而影响斜带石斑鱼摄食率的变化,从而造成石斑鱼的生长性能出现显著性差异,可能是养殖密度对斜带石斑鱼生长性能影响的机制之一。     

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。     

盐度与pH对云纹石斑鱼胚胎发育和仔鱼活力的影响

为探讨盐度、pH对云纹石斑鱼Epinephelus moara胚胎发育和仔鱼活力的影响,采用单因子实验的方法,在水温22±0．2℃条件下,将受精卵在不同盐度（5、10、15、20、25、30、35、40和45）、不同pH（5．5、6．0、6．5、7．0、7．5、8．0、8．5、9．0和9．5）下孵化,观察比较其孵化率HR、畸形率DR以及对初孵仔鱼活力和仔鱼培育周期的影响。并在上述设定条件下,进行了初孵仔鱼耐受试验,测定其存活率SR及生存活力指数（SAI）。结果表明,云纹石斑鱼胚胎发育的最低和最高临界盐度分别为10和45,最低和最高临界PH分别为6．O和9．5;盐度、PH的适宜范围分别是20～40、6．5～9．0,最适范围分别是30～35、7．5～8．0。仔鱼生存盐度、pH的适宜范围分别是20～35、6．0～9．0,最适范围分别是25～30、7．5～8．0。     

云纹石斑鱼(♀)×七带石斑鱼(♂)杂交子一代胚胎发育及仔稚幼鱼形态学观察

以云纹石斑鱼(Epinephelus moara)♀为母本,七带石斑鱼(E.septemfasciatus)♂为父本进行种间远缘杂交,系统观察了杂交子一代胚胎及仔稚幼鱼的生长发育及形态特征。对杂交子一代的胚胎发育按卵裂期、囊胚期、原肠胚期、神经胚期和器官形成期各阶段进行观察,在水温21～22.5℃、盐度29、pH 8～8.1的海水中,整个胚胎发育过程经35 h 20min完成。根据在卵黄囊期、第二背鳍棘和腹鳍棘生长消退过程、鳞片生长、体表色素变化的不同,观察比较了胚后发育的仔鱼期、稚鱼期、幼鱼期形态特征。鱼苗培育至58 d后完成了稚鱼至幼鱼的变态:初孵仔鱼4 d以前为内源性营养(卵黄囊仔鱼);4 d后开口摄食,为外源性营养;(42±2)d基本完成变态进入稚鱼阶段,(58±4)d全身被鳞,进入幼鱼期。同时,用同一母本(云纹石斑鱼)同批卵与同种雄鱼采集的精子授精后获得的受精卵作为对照组,进行孵化和仔稚幼鱼培育,与杂交子一代进行比较,结果发现杂交子一代在仔稚幼鱼阶段生长速度快于云纹石斑鱼,与七带石斑鱼相近。     

温度变化对七带石斑鱼早期发育及开口摄食的影响

观察比较了9个温度梯度(13、15、17、19、21、23、25、27、29℃)对七带石斑鱼(Epinephelus septemfasciatus)受精卵孵化时间、孵化率和畸形率的影响。用在(21±0.5)℃条件下孵出的健康仔鱼进行耐饥饿和摄食实验,实验温度处理设两种方式:一、处理温度始终保持不变;二、将温度在处理48 h后统一调节至21℃。实验期间,每天统计仔鱼死亡数,测定不投饵条件下的存活指数(SAI),并观察仔鱼投饵后的开口摄食情况及其形态发育状况。结果表明:1)受精卵孵化的最适温度范围为17~23℃,当温度低于13℃或高于27℃时,受精卵不能孵化。21℃时,受精卵孵化率最高,为(93.67±1.52)%;而畸形率最低,为(1.06±1.06)%。当温度低于或高于21℃时其孵化率降低,而畸形率升高;2)在13~28℃范围内,初孵仔鱼的SAI值随着温度的上升先升高后逐渐降低,21℃时的SAI值最高,为(20.26±0.44)%。各组温度保持不变时,13℃和27℃的SAI值分别为(2.18±0.01)%、(8.47±0.28)%;调整一致后分别为(6.90±0.44)%、(13.30±0.31)%。温度调节组与不调节组相比,其SAI值明显提高。温度调节后,每个梯度组的畸形率也出现了不同程度的降低;3)不同温度处理的仔鱼开口摄食存在差异。21℃时,仔鱼的初始摄食率和饱食率分别可达80.0%和40.0%。随着温度偏离21℃,摄食率和饱食率均逐渐降低。温度调节恢复组的仔鱼摄食情况改善明显,其中17℃不变和恢复组对比变化最为明显:当保持17℃不变时仔鱼的摄食率和饱食率分别为20.0%、13.3%,而处理48 h后恢复到21℃后,两者分别提高到66.7%和33.3%,分别提高了2.3和1.5倍。     

Vibrio harveyi (VirB11) recombinant vaccine development against vibriosis in orange‐spotted grouper (Epinephelus coioides)

Despite significant improvements in aquaculture to compensate wild catch, disease organisms have thrived in limiting its national and global potential. Using antibiotics, in a bid to remedy the havoc, has given rise to complications, attracting attention to disease prevention by immune enhancement against diseases. Grouper production has been inhibited for the threats of bacterial infection, particularly of Vibrio origin. Considering the rise in vibriosis cases, improved vaccines are necessary; moreover, recombinant vaccines, the choice for trial in the present experiment have been effective and more specific in improving immunity. The current work deals with grouper immune system enhancement with a recombinant vaccine developed from VirB11 gene in Vibrio harveyi. VirB11 was cloned in V. harveyi for recombinant vaccine development against vibriosis in orange‐spotted grouper (Epinephelus coioides). As indicated by the results, recombinant VirB11 protein showed effectiveness in conferring protection against vibriosis with observable specific antibody response in enzyme‐linked immunosorbent assay (ELISA) analysis; a significant increase (p < 0.05) in antibody levels was observed after a week and after 8 weeks post‐vaccination. From the weeks post‐vaccination, log2 (antibody titres) in the sera of vaccinated groups reached a peak of 14.2 at week 5 in the vaccinated group in comparison with a peak of approximately 5 and 2 in adjuvant and PBS controls. As indicated by the challenge results, 90% relative survival was observed in vaccinated group and 13% relative survival in control group I (adjuvant control). The cumulative performance of protein concludes VirB11 commendable for recombinant vaccine development.     

不同维生素E水平饲料对云纹石斑鱼幼鱼低盐胁迫前后抗氧化和渗透压调节功能的比较

维生素E(VE)在鱼类繁殖、生长代谢、抗氧化能力和免疫机能等方面有至关重要的作用。为探究饲料VE添加量对云纹石斑鱼低盐度胁迫下抗氧化和渗透压调节功能的作用,本实验设计0、20、40、80和160 mg/kg等5个VE水平饵料添加量,依次为A、B、C、D、E组,饲喂云纹石斑鱼幼鱼56 d。然后进行6 h低盐度(12)胁迫实验,测试皮质醇(COR)、葡萄糖(GLU)、乳酸(LD)等血清生化指标,超氧化物岐化酶(SOD)、过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GSH-Px)、总抗氧化能力(T-AOC)和丙二醛(MDA)等抗氧化指标,以及鳃Na⁺/K⁺-ATP酶(NKA)、Ca²⁺-ATP酶活力等。结果显示,血清COR含量最高为盐度胁迫后A(32.74±1.53) ng/mL,最低为胁迫前E(19.06±3.88) ng/mL;GLU含量最高为盐度胁迫后A(16.46±0.99) mmol/L,最低为养殖前O(7.90±0.34) mmol/L;LD含量最高为低盐度胁迫前C(1.94±0.15) mmol/L,最低为胁迫后E组(1.30±0.06) mmol/L。说明VE的添加可以增强云纹石斑鱼抗应激的能力,且在47.524~134.566 mg/kg(B~D)的添加范围,效果较好。血清SOD、CAT、GSH-Px活力、T-AOC和MDA含量与投喂饲料中VE水平密切相关,SOD最高为胁迫前C(105.29±9.07) U/mL,最低为胁迫后A(11.23±2.30) U/mL;CAT最高为养殖前O(4.09±0.17) U/mL,最低为胁迫后A(0.35±0.10) U/mL;GSH-Px最高为胁迫前E(972.58±55.35) U,最低为胁迫后A(47.90±10.64) U;MDA最低为胁迫前D(33.48±2.34) nmol/mL,最高为胁迫后A(101.79±7.79) nmol/mL。饲料中添加91.378~178.924 mg/kg(C~E)水平VE对其抗氧化能力有增强作用。VE可增强NKA活力,当VE添加量为91.378 mg/kg(C)时,低盐度胁迫对NKA影响较小。而添加91.378~178.924 mg/kg(C~E)水平VE可增强云纹石斑鱼Ca²⁺-ATP酶活力。研究表明,饲料中添加91.378~134.566 mg/kg范围的维生素E,可以增强云纹石斑鱼的机体活力、抗应激及环境胁迫能力。     

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.     

Nucleotide sequence and mRNA expression analysis of <Emphasis Type="Italic">β</Emphasis>-actin gene in the orange-spotted grouper <Emphasis Type="Italic">Epinephelus coioides</Emphasis>

The full-length cDNA, encoding the orange-spotted grouper β-actin and spanning 1920 bp including a poly (A) tail, was cloned from its brain cDNA library. The open reading frame encodes a protein of 375 amino acids. Sequence analysis indicated that it contained the typical structural features of cytoplasmic actins, and showed higher homology with other vertebrate β-actin than any other members of the actin family. The partial genomic sequence indicated that the organization of the β-actin gene in the orange-spotted grouper might also be conserved. Northern blot analysis indicated that it was expressed at high levels in the brain, spleen, adipose tissue, ovary, and liver, but at low levels in the gill filament and heart, and at a very low level in the kidney. The expression of β-actin gene in the skeletal muscle was barely detectable. These results indicated that the expression of the orange-spotted grouper β-actin gene showed significant variation in different tissues. Therefore, caution should be taken when using β-actin gene as an internal control in the normalization of gene expression among tissues. Whereas, semi-quantitative RT-PCR analysis indicated that treatment with 17α–methyltestosterone (MT) had little effect on the mRNA expression of β-actin gene in the in vitro incubated hypothalamus, pituitary, and ovary fragments of the orange-spotted grouper, suggesting β-actin can be used as an internal control for RT-PCR analysis of MT effects on gene expression in these tissues.