虹鳟、山女鳟及其杂交子代(虹鳟♀×山女鳟♂)的微卫星分析

利用虹鳟(♀)和山女鳟(♂)进行种间杂交,获得了90.00%的受精率,80.52%的发眼率,90.68%的孵化率和30.68%的鱼苗成活率.运用13个微卫星分子标记对杂交亲本与杂交子代进行了分子遗传机制的研究,结果表明:(1) 在13个微卫星位点中,3个位点只在虹鳟中得到扩增产物,6个位点扩增出虹鳟和山女鳟清晰的差异条带,另外4个位点在双亲中没有扩增出显著差异条带;(2) 双亲遗传分化显著,虹鳟和山女鳟存在杂交现象,虹鳟和山女鳟杂交子代的遗传符合孟德尔遗传规律,属两性融合生殖,是真正意义上的杂交种. (3) 杂交后代与虹鳟和山女鳟的遗传相似性系数分别为0.461 7和0.596 5,遗传距离分别为0.772 9和0.516 8, 表明杂交F1与两亲本的遗传差异不是对等的, 而是偏向父本一方,UPGMA系统树也同样证明了这一点.     

哲罗鲑(♂)与细鳞鲑(♀)属间杂交不相容现象的SSR分析

利用哲罗鲑(Hucho taimen,♂)与细鳞鲑(Brachymystaxc lenok,♀)进行属间杂交,出现了仔鱼上浮率低和畸形率高等杂交不相容现象,为探究哲罗鲑与细鳞鲑属间杂交不相容现象产生的原因,本研究采用30对微卫星引物对杂交亲本及F1进行遗传分析.结果表明:(1)30对微卫星引物中,2对在细鳞鲑中未出现扩增带,3对在哲罗鲑中未出现扩增带,其余25对在双亲中均扩增出差异条带;(2)在双亲中具有差异的25个微卫星位点中,除Omi84TUF位点在1个F1出现非亲条带外,其余位点所有条带均来自于双亲,其中21个位点完全符合孟德尔遗传规律,表明哲罗鲑与细鳞鲑属间杂交属两性融合生殖,大部分遗传物质来源于父母本双方;(3)4个位点Omi105TUF、Omi156TUF、Omi165TUF及Omy16DIAS在部分子代中出现2条母本条带或父本条带缺失现象,可能是部分亲本染色体在配对或分离过程中产生了异常,出现染色体丢失或异源加倍现象,非整倍体的较早出现可能是导致杂交不相容的直接原因;4)F1与哲罗鲑和细鳞鲑的遗传相似系数分别为0.673 5和0.729 8,遗传距离分别为0.395 3和0.315 0,表明F1与细鳞鲑亲缘关系更近,UPGMA聚类分析也支持这一观点.     

日本金鳟(Oncorhyncus mykiss)和道氏虹鳟及其杂交后代生产性能的比较研究

本文对引进种日本金鳟和道氏虹鳟及其杂交后代的生存力及稚鱼的生长速度等进行了比较研究。结果表明,五项生存力指标由低到高的顺序均是:日本金鳟、杂交金鳟(F1 )、杂交金鳟自交后代(F2 )、野生型虹鳟(P <0 . 0 1 ) ,杂交日本金鳟对纯系日本金鳟体现出杂种优势,但是对于道氏虹鳟来说未体现优势,是由于道氏虹鳟是经过长期选育的良种,杂交后代遗传了它的部分优良性状。     

虹鳟pmela基因的克隆及其在不同发育阶段和组织中的表达分析

前黑素小体蛋白a (premelanosome protein, pmela)基因是黑色素合成通路中的关键基因之一，对动物的体色有着重要影响。为探讨pmela基因在虹鳟(Oncorhynchus mykiss)体色变异中的作用，本研究利用cDNA末端快速扩增(rapid amplification of cDNA ends, RACE)技术对pmela基因cDNA全长进行克隆并运用生物信息学方法分析该基因的序列特征，同时采用qRT-PCR比较pmela在野生型虹鳟(虹鳟)、黄色突变型虹鳟(金鳟)和杂交F1代受精期至孵化后3个月不同发育时期及成鱼不同组织中的相对表达量。研究获得pmela基因cDNA全长序列3 476 bp，包含2 532 bp开放阅读框，编码843个氨基酸。序列分析发现，Pmela为疏水性蛋白且存在PKD功能结构域。同源性比对发现，虹鳟Pemla氨基酸序列与红鲑(Oncorhynchus nerka)的同源性高达97.75%；进化分析结果显示，虹鳟与红鲑的亲缘关系最近，与人类(Homo sapiens)和小鼠(Mus musculus)的亲缘关系最远。qRT-PCR结果显示，pmela基因在虹鳟与金鳟胚胎及出膜后各时期均有表达，在受精期至16细胞期中的表达为虹鳟高于金鳟，出膜后该基因在金鳟背部皮肤中的表达均高于虹鳟，且在4细胞期、16细胞期、原肠期、神经期、体节期、心跳期、1 day post-hatching (1 dph)、3 dph、5 dph、7 dph、10 dph、1 month post-hatching (1 M)和2 M时期中，虹鳟与金鳟的表达存在显著差异。在各组织中，pmela基因在虹鳟与金鳟背部皮肤和眼睛中的表达显著高于其他组织。在杂交F1代中，pmela基因在不同发育时期和组织中的表达模式与双亲类似，且在大部分相同时期和组织中的表达与双亲存在显著差异。上述结果表明，pmela基因的表达量高低与虹鳟体色具有一定的相关性，且可能参与其体色的形成过程。本研究结果为进一步研究pmela基因在虹鳟体色变异中的作用提供基础资料。     

大西洋鲑、三倍体虹鳟和金鳟的肌肉营养成分与品质特性

为了解大西洋鲑(Salmo salar)、三倍体虹鳟(Oncorhynchus mykiss)、金鳟(Oncorhynchus mykiss)3种鱼肌肉营养成分和品质特性,利用生化分析、物性分析方法分析3种鱼肌肉的营养成分、氨基酸和脂肪酸组成、肉色、系水力和质构特性。结果表明,大西洋鲑、三倍体虹鳟、金鳟肌肉的水分质量分数分别为62.91%、67.15%、73.02%,粗蛋白质量分数分别为22.39%、21.03%、22.11%,粗脂肪质量分数分别为14.64%、17.16%、5.11%。3种鱼肌肉的滴水损失、黄色值(b~*)、羟脯氨酸含量、内聚性均显著不差异(P0.05)。3种鱼肌肉的硬度和咀嚼性由低到高依次为大西洋鲑、三倍体虹鳟、金鳟,而pH值的结果则与之相反(P0.05)。大西洋鲑和三倍体虹鳟肌肉的灰分、解冻损失、蒸煮损失、回复性、弹性和红色值(a~*)差异不显著(P0.05),但灰分、蒸煮损失、回复性均小于金鳟肌肉的对应指标(P0.05),弹性和红色值(a~*)则均大于金鳟肌肉的对应指标(P0.05)。大西洋鲑、三倍体虹鳟、金鳟肌肉中必需氨基酸含量占氨基酸总量分别为42.28%、41.84%、41.63%(质量分数),必需氨基酸/非必需氨基酸(EAA/NEAA)比值分别为73.25%、71.94%、71.32%,均符合联合国粮农组织/世界卫生组织(FAO/WHO)对优质蛋白质的评价标准;3种鱼肌肉中均检测到22种脂肪酸,组成丰富,其中不饱和脂肪酸含量较高。综上所述,3种鱼的肌肉都是符合人体营养需求的优质水产品,其中大西洋鲑和三倍体虹鳟肉质接近,且都优于金鳟的肉质。     

３种虹鳟养殖群体的遗传结构及遗传多样性分析

从40个随机引物中筛选出20个,采用随机扩增多态DNA(RAPD)技术,对甘肃金鳟、道氏虹鳟和美国金鳟3个群体进行了遗传结构和遗传多样性分析.结果表明,甘肃金鳟、道氏虹鳟、美国金鳟群体多态位点百分比分别为82.79%、73.33 %、77.87%;平均等位基因数分别为1.8239、1.7429、1.7887,有效等位基因数分别为1 4823、1 4281、1.4527;基因多样度分别为0.2820、0.2528 、0.2675,Shannon遗传多样性指数分别为0.4219、0 3801、0 4022;以上参数表明,3个群体的遗传多样性均处于偏高的水平,且以甘肃金鳟的遗传多样性最为丰富.甘肃金鳟与美国金鳟之间的遗传距离最大,道氏虹鳟与美国金鳟之间的遗传距离最小.遗传分化指数表明,种内群体间的遗传分化程度很低,而群体内个体间的分化程度很高,变异主要来源于群体内个体间.综合分析认为,甘肃金鳟遗传多样性偏高,有进一步选育的遗传潜力;同时,群体内遗传分化程度较大,说明品种的纯度可能较低,经济性状不够稳定,需进一步选育.     

3种虹鳟养殖群体的遗传结构及遗传多样性分析

从40个随机引物中筛选出20个,采用随机扩增多态DNA(RAPD)技术,对甘肃金鳟、道氏虹鳟和美国金鳟3个群体进行了遗传结构和遗传多样性分析。结果表明,甘肃金鳟、道氏虹鳟、美国金鳟群体多态位点百分比分别为82.79%、73.33%、77.87%;平均等位基因数分别为1.8239、1.7429、1.7887,有效等位基因数分别为1.4823、1.4281、1.4527;基因多样度分别为0.2820、0.2528、0.2675,Shannon遗传多样性指数分别为0.4219、0.3801、0.4022;以上参数表明,3个群体的遗传多样性均处于偏高的水平,且以甘肃金鳟的遗传多样性最为丰富。甘肃金鳟与美国金鳟之间的遗传距离最大,道氏虹鳟与美国金鳟之间的遗传距离最小。遗传分化指数表明,种内群体间的遗传分化程度很低,而群体内个体间的分化程度很高,变异主要来源于群体内个体间。综合分析认为,甘肃金鳟遗传多样性偏高,有进一步选育的遗传潜力;同时,群体内遗传分化程度较大,说明品种的纯度可能较低,经济性状不够稳定,需进一步选育。     

虹鳟、金鳟苗小瓜虫病的中草药防治

小瓜虫病是对虹鳟、金鳟稚鱼危害较大的一种体表寄生性常见病和多发病，严重时常造成大批死亡。为实施无公害虹鳟、金鳟的养殖生产，我们在虹鳟、金鳟稚鱼培育期，试验总结出用辣椒 生姜和鱼虫克星(槟榔 苦楝)等中药浸洗防治小瓜虫病的有效方法，取得良好效果，现总结如下。     

大黄鱼（♀）与鮸（♂）杂交的遗传分析

用大黄鱼（♀）和鮸（♂）进行属间杂交，获得了56.25%的受精率，45.24%的孵化率和0.65%的鱼苗成活率。对杂交亲本与子代进行了微卫星和AFLP标记的比较分析，结果表明：4个微卫星位点（LYC0003，LYC0006，LYC0007，LYC0008）都可扩增出清晰的亲本差异条带，26个F1个体中均未出现雄鱼特有条带；4 对选择性扩增引物（E-ACC/M-CAG, E-AAC/M-CAT, E-AAC/M-CTA, E-AGC/M-CTC）共检出326个AFLP条带，143条母本特异性条带中有132 条出现在子代中，94 条父本特异性条带中只有18 条出现在子代中，子代中另出现了33 条非亲条带。F1个体之间的遗传相似系数为0.891，与母本、父本间的遗传相似系数分别为0.853、0.271；F1个体之间的遗传距离为0.116，与母本、父本间的遗传距离分别为0.159、1.307，表明杂交子代与母本大黄鱼之间具有极高的遗传同质性，属异精雌核发育个体。     

大黄鱼(♀)与(鱼免)( ♂)杂交的遗传分析

用大黄鱼(♀)和(鱼免)( ♂)进行属间杂交,获得了56.25%的受精率,45.24%的孵化率和0.65%的鱼苗成活率.对杂交亲本与子代进行了微卫星和AFLP标记的比较分析,结果表明:4个微卫星位点(LYC0003,LYC0006,LYC0007,LYC0008)都可扩增出清晰的亲本差异条带,26个F1个体中均未出现雄鱼特有条带;4 对选择性扩增引物(E-ACC/M-CAG,E-AAC/M-CAT,E-AAC/M-CTA,E- AGC/M-CTC)共检出326个AFLP条带,143条母本特异性条带中有132 条出现在子代中,94 条父本特异性条带中只有18 条出现在子代中,子代中另出现了33 条非亲条带.F1个体之间的遗传相似系数为0.891,与母本、父本间的遗传相似系数分别为0.853、0.271;F1个体之间的遗传距离为0.116,与母本、父本间的遗传距离分别为0.159、1.307,表明杂交子代与母本大黄鱼之间具有极高的遗传同质性,属异精雌核发育个体.     

山女鳟的生物学特性及繁殖力

本文就山女鳟的生物学特性及繁殖力进行了测定 ,结果表明 :山女鳟可量比例性状为体长 /体高 4 2 0± 0 2 7,体长 /头长 3 99± 0 35 ,体长 /尾柄长 1 0 60± 1 5 5 ,体长 /尾柄高 1 2 1 4± 0 79,头长 /吻长 2 5 3± 0 63,头长 /眼径 6 84± 1 1 6,头长 /眼间距 3 0 9±0 33;可数性状为背鳍鳍式 :D 1 0～ 1 6,胸鳍鳍式 :A 8～ 1 3,腹鳍鳍式 :8～ 1 1 ,臀鳍鳍式 :1 1～ 1 5 ,侧线鳞 :1 1 2～ 1 40 ,侧线上鳞 :2 6～ 2 9,侧线下鳞 :2 5～ 35 ,第一鳃弓鳃耙数 :1 3～ 1 5 ,脊椎骨 63～ 65 ,幽门垂 2 0～ 5 7。绝对繁殖力 61 7 83± 2 5 2 70 ,相对繁殖力 2 0 9± 0 67。     

山女鳟同工酶的研究

采用淀粉凝胶电泳方法，对山女鳟的肝脏、肌肉等组织的LDH、MDH、IDH、ADH、EST、SOD等同工酶进行电泳，并对其表型进行生化遗传分析。结果表明，在检测到6种同工酶是由13个基因座位编码；其中m-MDH基因座位呈多态，其多态座位比例为7.6%，平均杂和度为0.059。在生化遗传水平上山女鳟表现出较低的遗传变异。     

QTL for IHNV resistance and growth identified in a rainbow (Oncorhynchus mykiss) × Yellowstone cutthroat (Oncorhynchus clarki bouvieri) trout cross

Infectious hematopoietic necrosis virus (IHNV) is a major constraint to rainbow trout culture. Yellowstone cutthroat trout (Oncorhynchus clarki bouvieri) have greater resistance to this virus than do rainbow trout (O. mykiss), but the genetic mechanism of this resistance is not understood. We conducted a genome scan using a backcross of cutthroat trout into a rainbow trout background to estimate the number and locations of quantitative trait loci (QTL) associated with IHNV resistance and growth in trout. IHNV resistance was considered in terms of both survival (binary trait) and days to death (quantitative trait). The genetic map was scanned using interval mapping via two different approaches: one model considered survival alone and a second two-part model combined both survival and days to death. Three QTL were significantly (P ≤ 0.05) associated with virus resistance genome-wide, explaining 32.5% of the phenotypic variation. Cutthroat alleles at two of these QTL resulted in increased resistance to the pathogen, as expected. No growth QTL were detected in this cross. We suggest that these traits are genetically independent.     

一株虹鳟源嗜冷黄杆菌CH06的基因组进化及毒力相关基因分析

为了深入揭示我国鱼类病原菌嗜冷黄杆菌的基因组进化及其致病机制,本实验对嗜冷黄杆菌毒力菌株CH06进行全基因组测序,比较基因组分析并挖掘其毒力相关基因。基因组测序结果显示,CH06的基因组大小为2 836 981 bp,GC含量为32.56%,注释出2 437个编码基因。通过平均核苷酸一致性(ANI)分析结果显示,CH06与12株嗜冷黄杆菌的ANI值均高于99%,菌株CH06应属于嗜冷黄杆菌分支。基因组序列分析发现,CH06含有5个插入序列、4个基因岛和3个前噬菌体,揭示这些可移动遗传元件推动了CH06的基因组进化。系统进化树和基因组共线性分析发现,CH06与菌株CSF259-93和FPG101亲缘关系较近。分泌系统分析显示,CH06中存在Ⅰ型分泌系统(type Ⅰ secretion system,T1SS)和Ⅸ型分泌系统(T9SS)。通过蛋白结构域特征分析发现,CH06包含56个T9SS效应蛋白和29个双组分系统元件,表明嗜冷黄杆菌可能存在复杂的致病机制和调控机制。本研究对CH06的基因组进化以及毒力相关基因进行分析,为我国鱼类病原嗜冷黄杆菌的进化、多样性及致病机制研究提供参考。     

Loma salmonae (Protozoa: Microspora) infections in seawater reared coho salmon Oncorhynchus kisutch

Loma salmonae (Putz et al., 1965) infections were observed in five groups of coho salmon, Oncorhynchus kisutch, reared in seawater net-pens in Washington State, U.S.A. in 1984–1986. Ultrastructural characteristics, size of spores, tissues and host infected, and geographical location identified the microsporidium as Loma salmonae. Preserved spores measured 4.4×2.3 (4–5.6×2–2.4) μm and exhibited 14–17 turns of the polar filament. Infections were evident in the gills of some fish before seawater entry, but few parasites were observed and they caused little tissue damage. Infections observed in fish after transfer to seawater were associated with significant pathological changes in the gills. A mixed inflammatory infiltrate was associated with ruptured microsporidian xenomas within the vessels and interstitium of the primary lamellae. Microsporidian spores were dispersed throughout the lesions and were often seen inside phagocytes. The parasite was also observed in the heart, spleen, kidney and pseudobranchs; however, the inflammatory lesions were common only in the heart.

Monthly examination of fish after transfer to seawater showed peak prevalences (33–65%) of gill infections during the summer. Although moribund fish were often infected with other pathogens, the high prevalence of L. salmonae infections and the severity of the lesions it caused, suggested that this parasite significantly contributed to the recurrent summer mortalities observed at this net-pen site.     

Oral pharmacological treatments for ichthyophthiriosis of rainbow trout (Oncorhynchus mykiss)

The aim of this work was to explore the efficacy of a veterinary drug, Triclabendazole (5-chloro-6-(2, 3-dichlorophenoxy)-2-methylthio-1H-benzimidazole), in an inclusion complex with β-cyclodextrin as a suitable treatment for parasitic diseases caused by Ichthyophthirius multifiliis in rainbow trout. The efficacy was determined by the reduction in the infection intensity. The complexes were prepared by the kneading method and were characterized by DSC and X-ray diffractometry. The selected stoichoimetry was 1:3 because of the higher percentage of Triclabendazole complexed with cyclodextrin. Administration of Triclabendazole and complex was carried out by including appropriate doses in animal feed. Our studies suggest that the Triclabendazole–cyclodextrin complex results in a reduction in the infection degree and trophont size is decreased in the animals treated. The oral treatment of Triclabendazole in inclusion complexes may be an alternative to bath treatments in trout farming.     

Cadmium detoxification effect of Chinese parsley Coriandrum sativum in liver and kidney of rainbow trout Oncorhynchus mykiss

Although fish feed supplemented with Chinese parsley (CP), Coriandrum sativum, does not affect the health of rainbow trout Oncorhynchus mykiss, it has the remarkable effect of reducing the uptake of Cd (12–17% compared with controls without CP). Here, we measured the change in the amount of metallothionein (MT) in the liver and kidney using gel permeation chromatography-high-performance liquid chromatography (TSK GEL SW 3000 column). The increase in MT content over time in the Cd-fed groups corresponded to the change in Cd accumulation. Gel permeation was performed with soluble fractions from the fish liver and kidney, and changes in the Cd, Cu, and Zn concentrations over time for each fraction, and the relationship between the form of Cd that accumulated in tissues and its toxicity were measured. A nontoxic MT fraction of 7,000 Da and a high-molecular-weight fraction of approximately 60,000 Da were isolated from the soluble fractions. The induction of nontoxic MT–Cd was higher in the CP-supplemented groups than in the control groups. The synergetic actions of several compounds in CP may contribute to bind MT, thereby preventing accumulation of harmful heavy metals and essential metals in fish.     

温度、流速对恩诺沙星在虹鳟体内吸收、分布的影响

环境因素会对药物在动物机体内的代谢产生不同的影响,为研究温度和水流速度对药物在虹鳟体内药物代谢的影响,以虹鳟为实验对象,设置3个温度(5、10、15°C)和流速(8、16、24 cm/s),水温由自动循环水族缸的控温系统调控,流速由鱼类生态测量仪调控,高效液相色谱法(HPLC)检测组织中恩诺沙星含量。结果发现,在5、10、15°C时,血浆T_(max)分别是8.67、4.78、2.39 h;T_(1/2α)分别是0.86、0.80、0.77 h;T_(1/2β)分别是49.18、45.81、38.35 h;AUC分别是140.49、130.40、112.78μg/(L·h)。实验温度下,表现为温度升高会加快药物的吸收和分布,即吸收分布速率增大,达峰时间缩短。在8、16、24 m/s 3个实验流速下,血浆T_(max)分别是8.57、6.03、4.04 h;T_(1/2α)分别是5.47、2.16、0.27 h;T_(1/2β)分别是26.54、6.93、2.13 h;T_(1/2ka)分别是7.68、5.00、2.01 h。实验流速下,表现为流速增大会加快药物的吸收和分布,即吸收分布速率增大,达峰时间缩短。研究表明,在一定范围内温度的升高及水流速度的增加会加速恩诺沙星在虹鳟体内的吸收与分布。     

Ovarian fluid pH enhances motility parameters of rainbow trout (Oncorhynchus mykiss) spermatozoa

All evidence to date suggest that sperm motility is the primary determinant of fertilization success in externally fertilizing fish species. Ovarian fluid, which comprises 10–30% of the total egg volume in salmonids, enhances sperm motility with respect to swimming speed, trajectory and the duration of movement. It was recently demonstrated that there is individual variability in sperm motility enhancing potential of ovarian fluid of particular females. In the present study we examined the effect of particular ovarian fluids collected from 31 females on the sperm motility parameters of one male of rainbow trout (Oncorhynchus mykiss) using computer-assisted sperm analysis (CASA). During our experiment we also monitored the pH of ovarian fluid. We found that particular fluids differed in the ability to activate spermatozoa; sperm remained immotile in four fluids and exhibited 50–100% motility in 27 samples. The percentage of motile sperm, velocity and duration of movement positively correlated with ovarian fluid pH (r² = 0.34–0.62). These data strongly suggest that the pH of the ovarian fluid is the primary determinant of sperm motility in rainbow trout under natural conditions of fertilization.     

Survival and growth of vaccinated, medicated and untreated coho salmon (Oncorhynchus kisutch) exposed to Vibrio anguillarum

Vaccination and medication with terramycin resulted in increased survival of pen-reared coho salmon (Oncorhynchus kisutch) exposed to vibriosis in a Maine estuary. Mortality was significantly lower (P < 0.001) in vaccinated fish than in fish receiving terramycin in their food. An unexpected increase in growth also occurred in vaccinated and medicated fish. Six months after the outbreak began, 25% of vaccinated, 12% of the medicated, and 6% of the untreated group reached minimum market size (200 g).