橘色双冠丽鱼黑素皮质素受体1基因(MC1R)整胚原位杂交

为探究黑素皮质素受体1基因(MC1R)在橘色双冠丽鱼胚胎发育和体色形成过程中的表达定位及功能,本研究首先制备了MC1R基因的RNA正反义探针,T7方向转录的正义RNA探针质量浓度为447.529 ng/μL,SP6方向转录的反义RNA探针质量浓度为342.698 ng/μL。经10～20倍稀释后的探针用于原位杂交,MC1R基因探针表达定位显示,随橘色双冠丽鱼胚胎的发育杂交信号总体呈逐渐减弱趋势,原肠期和视泡期其卵黄和胚体侧卧部分有杂交信号分布;出膜期其脊柱、卵黄囊及其内容物和色素细胞内出现杂交信号;出膜期第1天,卵黄表面有信号分布。总体来看,杂交信号在脊索、卵黄囊、卵黄囊内容物质及色素细胞有特异表达,而以正义探针作为阴性对照组在5个胚胎阶段均无任何信号,杂交信号显现的MC1R表达定位说明其在橘色双冠丽鱼色素细胞的分化、迁移中起到重要调控作用,也与神经、营养、免疫功能相关,初步建立了鱼类体色相关基因功能和定位研究的整胚原位杂交方法。     

DNA芯片技术及其应用

本文介绍了DNA芯片技术的基本过程,主要包括DNA芯片的制备、样品的制备及与探针的杂交、杂交结果的检测与读出。还介绍了其应用前景及目前存在的一些问题。     

鳀科(Engraulidae)鱼类DNA条形码电子芯片研究

本研究将基于线粒体COⅠ部分序列的DNA条形码和DNA芯片技术相结合,以鳀科11属30种鱼类为研究对象,在比对分析其DNA条形码序列的基础上,利用软件Oligo Array 2.1筛选探针,经OligoCalc优化探针,去除易形成发夹(Hairpin)、茎环(Stem-loop)及自身二聚体结构(Homodimers)的探针,再利用Oligo heat map对探针与靶标序列进行虚拟杂交,共有14个物种的24条探针能与靶标序列特异性结合.利用DNA条形码芯片技术能将14个物种鉴定到种,虽然物种识别能力仅占总物种数的46.7％,但鉴定准确率可达100％.因此,基于COⅠ基因的DNA芯片技术对鳀科鱼类物种鉴定有一定的实用价值,但该技术对物种的识别能力尚有较大提升空间,通过筛选和优化得到高质量的分子探针则是突破该项技术的关键.     

杂交鲟源温和气单胞菌的分离鉴定及生物学特性的研究

为探明引起彭水县某鲟养殖场杂交鲟(Acipenser baerii♀×A.schrencki♂)患病的病原，本研究从患病杂交鲟的腹水中分离出一株细菌(A20)。通过菌落形态观察、革兰氏染色、溶血性实验、生理生化特征、16S rDNA基因和gyrB基因序列分析对菌株A20进行鉴定，同时探究菌株A20的耐受性、毒力基因、半致死浓度和药物敏感性等生物学特性。结果显示：菌株A20鉴定为气单胞菌属(Aeromonas spp.)短杆状革兰氏阴性菌温和气单胞菌(A.sobria);菌株A20生长的最适pH为7,对温度和盐度的耐受力较强；该菌携带hlyA、ompA、alt、aer 4种毒力基因；人工回归感染实验结果表明菌株A20为导致杂交鲟患病的病原菌，LD₅₀为1.37×10⁷ CFU/mL;药敏结果表明，该菌对氟苯尼考、恩诺沙星等15种抗菌药物敏感。     

鲍活体倍性检测技术的研究

在生物有机体中,一个细胞核中所含的DNA总量对于某种类有机体是一定的。利用流式细胞仪(FCM)测量细胞中的DNA含量,可以用来鉴别有机体的倍性。本研究分别取杂交鲍[即皱纹盘鲍(Haliotis discus hannai)×盘鲍(Haliotis.discus discus)]的一小块触手、足肌,设计不同的振荡时间、不同的DAPI染色时间、不同的取样部位,制备成不同的细胞悬液检查样品;用PA-Ⅱ型倍体分析仪测定其倍性,以探讨其对倍性检测效果的影响。试验结果表明:振荡的最适时间为1.5～2.0 min,染色的最适时间为4.0～5.0 min;取材部位的不同对检测结果无显著影响。     

杂色鲍与盘鲍种间杂交受精率的影响因素

研究卵子排放后的时间、精子排放后的时间、授精精子浓度、胰蛋白酶、钙离子、pH和水温等因子对杂色鲍(Haiotisdiversicolor,以下简称S)与盘鲍(H.discus discu,以下简称J)种间杂交受精率的影响。结果表明,卵子排放后的时间和授精精子浓度是影响杂交受精率两个最重要的因素。与自繁组合相比,正反杂交受精率均较低且下降速度快得多。水温21.5℃时,两亲本自繁对照组中,卵子排放1 h对受精率均无显著影响。而SJ杂交,卵子排放时间2 min时,受精率为55.9%±8.1%,4.7 min时受精率已下降了50%;JS杂交,卵子排放时间为4 min时,受精率为31.5%±9.7%,10 min时受精率下降至4 min时的29.1%。相比之下,精子排放时间对杂交受精率的影响小得多,3 h内对正反交杂交受精率没有显著影响。杂交适宜精子浓度约为母本自繁的100倍,其中SJ杂交适宜授精精子浓度为6.0×106~2.4×107mL-1,JS杂交适宜授精精子浓度为3.8×107~7.5×107mL-1。水温对于杂交受精率也有影响,短时间内将水温控制在26~29℃可以得到较高受精率。其他实验因素,如添加胰蛋白酶、Ca2+或改变授精体系pH对杂交受精率均无正向作用。     

cDNA芯片结合抑制性差减杂交技术构建对虾白斑综合征病毒表达基因和螯虾免疫相关基因文库

以克氏原螯虾做为对虾白斑综合征病毒（WSSV）的增殖模型，试验组接种WSSV，对照组接种PBS。分别以试验组为检测组（tester）、对照组为驱逐组（driver），进行正向抑制性差减杂交；以对照组为tester、试验组为driver，进行反向SSH。将获得的正、反向差减杂交产物克隆入质粒载体PinPointTM Xa1 TVector，再转化大肠杆菌DH5α,共构建了2个分别含1620和400个克隆的正、反向差减文库。经PCR扩增，1514个差减克隆得到产物，其中正向差减克隆1221个，反向差减克隆293个。获得的PCR产物经浓缩、变性处理，用自动点样仪点制于尼龙膜载体上，制成cDNA芯片，用cDNA芯片对SSH获得的差减克隆进行鉴定，共获得差异表达克隆278个，其中攻毒组高表达克隆255个；对照组高表达克隆23个。     

丁香酚对罗非鱼的麻醉效果

对罗非鱼进行丁香酚药浴实验,研究丁香酚药浴浓度、药浴时间以及鱼体规格对麻醉效果的影响。结果表明:在10~120mg·L-1范围内,随着丁香酚药浴浓度的增加,罗非鱼麻醉时间逐渐缩短,苏醒时间逐渐增长,罗非鱼麻醉复苏率均为100%;丁香酚药浴浓度为45mg·L-1时,罗非鱼可以在3min内麻醉,5min内苏醒,为最适药浴浓度。在最适药浴浓度条件下,丁香酚对罗非鱼的麻醉效果与鱼的规格无相关性,且药浴时间超过120min,罗非鱼复苏率呈降低趋势,浸泡时间达到300min时,罗非鱼的死亡率为100%。     

螯虾免疫相关基因的检出及丝氨酸蛋白酶抑制物基因分析

通过对抑制性差减杂交和cDNA芯片技术分离的 2 0 1个阳性克隆的测序 ,得到螯虾 (Procambarusclarkii)免疫相关基因 4 8个 ,其中正向克隆中 4 2个 ,反向克隆 6个 ,除正向克隆中SNAP - 2 5 (synatosome-associatedproteinof2 5ku)已报道外 ,其余均为新基因 ,均在GenBank登录。正向克隆中的同源基因有微管蛋白、超氧化物歧化酶前体、丝氨酸蛋白酶抑制物Ⅰ、精氨酸激酶、70kD伴侣蛋白同类物等。进一步用DotNorthernblot对克隆号PCI188进行鉴定 ,结果攻毒组的杂交信号是对照组的 3.2 4倍 ,与cDNA芯片结果相符。用快速扩增cDNA末端技术扩增克隆号PCI188基因的 5’端片段和 3’端片段 ,全长共 112 8bp ,编码的蛋白质有 2 77个氨基酸 ,分子量为 30 .2 7kD。与GenBank序列号X795 12软尾太平剌蛄 (P .le niusculus)的蛋白酶抑制物Ⅰ (为丝氨酸蛋白酶抑制物 )的基因同源性为 5 8.7% ,氨基酸同源性为 6 9.7%。该蛋白质有 5个重复的结构域 ,与丝氨酸蛋白酶抑制物Kazal家族的结构域相似     

斑点叉尾■溃烂症的病原鉴定和致病特性

为探明斑点叉尾[鱼回](Ictalunes punctatus)溃烂症的病因,从4尾患鱼肝脾中分离纯化出4株优势菌株,并进行病原鉴定、毒力基因检测、动物回归感染和药敏试验。4株优势菌经鉴定并命名为杀鲑气单胞菌无色亚种(Aeromonas salmonicida subsp achromogenes)X-G1,杀鲑气单胞菌杀鲑亚种(A.s subsp salmonicida)X-P2、X-P3和嗜水气单胞菌(A.hydrophila)X-P4。15℃时,杀鲑气单胞菌X-G1、X-P2和X-P3的世代时间(约14 min)均小于嗜水气单胞菌X-P4(约20 min);25℃时,杀鲑气单胞菌X-G1、X-P2和X-P3株的世代时间(约20 min)均大于嗜水气单胞菌X-P4株(约16 min)。X-G1株可检到弹性蛋白酶、溶血素和甘油磷脂胆固醇酰基转移酶等3种毒力基因;X-P2株仅可检到弹性蛋白酶1种毒力基因;X-P3株可检测到弹性蛋白酶、溶血素、细胞毒性肠毒素、丝氨酸蛋白酶、酯酶、气溶素和甘油磷脂胆固醇酰基转移酶等7种毒力基因;X-P4株可检测到鞭毛、弹性蛋白酶、气溶素、细胞毒性肠毒素、热不稳定性肠毒素、丝氨酸蛋白酶和溶血素等7种毒力基因。分离株X-G1、X-P2、X-P3和X-P4在15~17℃水温下腹腔注射攻毒的半数致死浓度(LD 50)依次为0.49×10^4、0.78×10^4、0.53×10^4、3.84×10^4 CFU/g;而在23~26℃水温下测得的LD 50依次为1.48×10^4、1.80×10^4、0.82×10^4、0.68×10^4 CFU/g。分离株混合感染比单一株感染均表现出更强的致死能力。分离菌株对多西环素、恩诺沙星、氟苯尼考均敏感,但因患病鱼不能摄食药饵而导致治疗失败。     

母猪胎衣不下的病因与防治要点

猪的胎盘属于弥散型胎盘,这种胎盘的结构特点和饲养管理的不当,常常导致母猪胎衣不下发生,给生猪的生产繁殖带来极大损失。本文针对母猪胎衣不下发生病因、综合防治进行详细阐述,旨在对预防和治疗胎衣不下能有所帮助。     

Estimation of variation of virulence of Renibacterium salmoninarum by survival analysis of experimental infection of salmonid fish

The variation of virulence of Renibacterium salmoninarum , the causative agent of bacterial kidney disease (BKD) in salmonid fish, was studied by infecting rainbow trout, Oncorhynchus mykiss (Walbaum), with two isolates (strains 325 and 932) from diseased Atlantic salmon, Salmo salar L., and one isolate (strain 4366) from an apparently healthy Atlantic salmon. Coho salmon, Oncorhynchus kisutch (Walbaum), were injected with the strain 932 to estimate difference in fish species resistance. Fish were removed by random sampling for other study purposes, a study design possible with analysis of lifetime distributions incorporating both sampling-, death- and survival-times. At the end of the experiment, the rainbow trout infected with strains 325, 932 and 4366 had a survival probability of 33%, 51% and 72%, respectively. The coho salmon infected with strain 932 had a 26% survival probability. The strain differences were significant according to the log-rank test, and the risk ratio between the strains ranged from 1·8 to 5·4. The strain from the apparently healthy fish was least virulent. The survival of the fish species was different over time. Rainbow trout were more likely to die early in the time course, but high numbers of coho died later, resulting in an overall risk of mortality of 1·4 in favour of rainbow trout. Differences in virulence may reflect changed selective pressure on R. salmoninarum when introduced from feral stocks into the environment of fish farms.     

水硬度对七彩神仙鱼幼鱼发育的影响

采用不同硬度的水对七彩神仙鱼幼鱼进行饲养。6周龄幼鱼在硬度为7.94°dH±0.30°dH时饲养84d后,比在硬度为14.71°dH±0.23°dH水中的幼鱼个体大,生长速度快。表明较高硬度的水有利于七彩神仙鱼幼鱼的生长发育。     

虎斑乌贼受精卵卵黄营养成分分析

本实验对虎斑乌贼受精卵卵黄的营养成分进行分析,旨在探讨其幼体的营养需求量,为其幼体配合饲料研制提供参考数据。随机选取大约800个虎斑乌贼受精卵的卵黄,采用国家标准方法测定其水分、灰分、粗蛋白质、粗脂肪、氨基酸、脂肪酸和矿物元素含量。结果表明:1)虎斑乌贼受精卵卵黄中粗蛋白质含量为76.33%(干重基础);总氨基酸(TAA)和必需氨基酸(EAA)含量分别为71.22%和32.38%(干重基础),EAA/TAA为45.46%,氨基酸中以谷氨酸(Glu)含量最高(9.97%),必需氨基酸中亮氨酸(Leu)含量最高(7.58%)。2)其粗脂肪含量12.71%(干重基础);共检出17种脂肪酸,包括8种饱和脂肪酸(SFA)、5种单不饱和脂肪酸(MUFA)和4种多不饱和脂肪酸(PUFA),SFA、MUFA和PUFA分别占脂肪酸总量的43.47%、7.54%和49.25%,其中以DHA含量最高,达32.80%,EPA含量为7.70%,DHA/EPA为4.26。3)检测出Na、K、Ca、Mg、Sr、Mn、Fe、Cu、Zn、Al和As 矿物元素,微量元素中富含Zn、Al和Fe,含量分别为 0.77、0.71和0.43 mg/kg(鲜重基础)。由此可见,卵黄具有高蛋白、低脂肪,富含n-3PUFA的特点;虎斑乌贼幼体饲料中蛋白质需求量参考值为76.33%;氨基酸需求量参考值,如赖氨酸(Lys)为5.49%,蛋氨酸(Met)为2.63%;脂肪的需求量参考值为12.71%,DHA为4.17%,EPA为0.98%;微量元素需求量参考值,如Zn为2.77 mg/kg,Cu为0.19 mg/kg(干重基础)。     

斑节对虾虾苗活力检测方法

该研究通过肉眼观察、镜检，进行干露、饥饿、盐度突降、福尔马林等抗性试验，并采用病毒检测等方法，以期建立评估斑节对虾（Penaeus monodon）虾苗活力和质量标准。结果表明，斑节对虾健康虾苗具有趋光性、集群性，体表光洁，肌肉透亮，肠胃食物充盈等特性。测试虾苗干露时间以15min为宜，健康虾苗干露后能立即恢复活力，而病弱虾苗多出现死亡、昏迷现象；虾苗的成活率随饥饿时间的延长而降低，随福尔马林浓度升高和时间延长而降低，随盐度突降幅度增加而降低。健康虾苗能忍受100～200μL·L^-1福尔马林溶液30min，成活率近100％；在盐度20～30下虾苗的成活情况较好，而其在淡水中仅能存活1h。对虾苗进行病毒检测，可以避免养殖中因虾苗携带病毒而可能导致的病毒性疾病的暴发。     

金鳟、虹鳟苗种长途运输实例

鳟鱼发眼卵和旨种运输是养鳟生产的重要环节。发眼卵多在冬季和早春运输，苗种多在晚春和夏季运输。苗种运输大多以鱼篓充氧进行短距离汽车运输，长途运输冷水性鱼苗种的实例很少。本文总结了金鳟和虹鳟苗种长途运输方法，以期与养鳟业界共同交流提高。一、包装材料和方法内包装材料是苗种运输专用塑料袋，规格54×104cm，外包装是泡沫塑料箱，规格63.5×45.5×30．5cm，纸箱规格64×46×32．5cm。塑料袋采取双层式，内装苗种和水共12.5kg，其中苗种0.5-1．3kg，100－2000尾。起运前苗种停食1－2天。运鱼用水水温调至6℃，鱼、水入袋后袋内…     

Characterization of protease inhibitors of seminal plasma of cyprinids

Anti-proteinase activity was demonstrated in the seminal plasma of cyprinid fish species (bream, chub, ide, dace, asp, goldfish, roach, common carp) using electrophoretic techniques combined with a detection method based on inhibition of bovine trypsin. We found species-specific protease inhibitors in the seminal plasma of cyprinids. At least three bands of protease inhibitors with different migration rates could be identified by native PAGE. Higher variability was characterized for bands with slower migration rates. Visualization of inhibitors after SDS-PAGE under non-reducing conditions allowed estimation of their molecular weights. Apparent molecular weights were within the range of 51–59 and 47–54 kDa for the bands with slower and moderate migration rates, respectively. The molecular weight of fast migration bands for roach and common carp were estimated to 23 and 30 kDa, respectively. Inhibitors of common carp seminal plasma differed in their affinity toward serine proteases. Three inhibitors in common carp seminal plasma could be visualized using cod and bovine trypsin, but only two inhibitors (of high molecular weight) were recognized with chymotrypsin. There were differences in anti-proteinase activity and seminal plasma protein concentration in relation to the origin of common carp seminal plasma (breeding lines) and time of milt collection (spawning vs. post-spawning season).     

鱼类热忍耐温度研究进展

The paper is a review of the studies on the temperature of the thermal tolerance in fishes. It was described from four aspect: (1) The key parameters of temperature of thermal tolerance in fishes; (2) The influence factors of temperature of thermal tolerance in fishes; (3) The research methods of thermal effect of fishes; (4) The evaluation of effects of thermal pollution on fishes, especially, with the coastal power station will be‘built in China recent years, the effects of thermal discharge water on the marine fisheries resource wered iscussed.     

Histopathology of respiratory organs of certain air-breathing fishes of India

Air-breathing fishes have evolved bimodal respiratory mechanisms for exploitation of water (through gills and highly vascularized skin) as well as atmospheric air (through aerial respiratory organs, ABO). Mucous cells in these respiratory organs of variously stressed fishes exhibit periodic fluctuations in their density and staining properties. The main types of damage in the gills include congestion of blood capillaries (BLCs), periodic lifting and sloughing of respiratory epithelia of the secondary lamellae causing haemorrhage, extensive fusion of secondary lamellae and hyperplasia of the respiratory epithelia due to uncontrolled regeneration leading to asphyxiation, altered excretion, and death of the fish. Haemolysis has also been observed following lead exposure. The damage in the ABO of Heteropneustes fossilis includes sloughing of the epithelial cells, leading to haemorrhage causing decreased red blood corpuscles density and degeneration of the secondary gill lamellae with reduced respiratory area. Subsequent hyperplasia of the respiratory epithelia and fusion of gill lamellae increase the respiratory barrier distance. The BLCs often bulge out and protrude into the lumen, bringing blood nearer to air. The ladder-like pillar cell (PLC)-BLC components of the gill lamellae frequently collapse. Damage to the ABO of Channa striata is less severe. Often haemorrhaging due to bursting of extensively stretched BLCs causes aerial respiratory failure. Chloride cells of the ABOs also show hyperplasia. While the highly mucogenic epidermis of C. striata shows less damage, the epidermis of Clarias batrachus and H. fossilis shows severe wear and tear, sloughing, and haemorrhage. Side-by-side regeneration continues, causing altered histomorphology of the epidermis. The different gland cells also show periodic fluctuations in their density and staining. The dermis also shows severe damage with loosening of their connective tissue fibres. These fibres give stronger reactions for sulfated mucin that not only retain additional water molecules for continuance of skin breathing, but also bind the toxic ambient pollutants.     

不同年龄细鳞鱼的繁育效果

研究了不同年龄组(3～8龄)细鳞鱼(Bachymystax lenok Pallas)亲鱼的繁育效果,分析其繁育主要技术,包括亲鱼培育、繁育技术、受精卵孵化、鱼苗培育等,结果发现,5～6龄和7～8龄细鳞鱼产卵量没有明显差异,产卵量、孵化率、出苗率均较高,繁殖效果明显好于3～4龄亲鱼。