卵黄蛋白原在剑尾鱼体内不同组织的分布及雌激素作用对其表达的影响

为研究卵黄蛋白原(vitellogenin,vg)在剑尾鱼(Xiphophorus helleri)不同组织器官的表达情况,以及了解雌激素对不同组织中Vg表达的诱导作用,将成年雌、雄剑尾鱼及幼鱼暴露于50 μg/L的17β-雌二醇(E2)溶液中20 d,未暴露的剑尾鱼作对照,采用免疫组化法对各处理组剑尾鱼进行Vg的组织定位研究.结果表明,剑尾鱼Vg主要分布于成熟雌鱼的肝脏、脾脏和肠的血管、淋巴管,在正常幼鱼及雄鱼的以上各组织中均不表达.17β-雌二醇暴露下,剑尾鱼雌、雄成鱼和幼鱼的肝脏以及肾、脾、肠等组织的血管及淋巴管均呈Vg阳性.但Vg颗粒只在肝实质细胞中观察到,在其他组织中如脾脏、肠固有结缔组织等,Vg颗粒仅出现于血管,在细胞内未观察到.以上结果表明,剑尾鱼Vg在肝细胞中合成,通过血液循环到达其他组织;雌激素可诱导雌、雄剑尾鱼及幼鱼肝组织中Vg的表达.剑尾鱼幼鱼及雄鱼Vg的非正常表达可应用于环境雌激素效应评价.     

不同倍性雌性虹鳟(Oncorhynchus mykiss)性体指数与肝体指数的关系

文章研究了二倍体和三倍体雌性虹鳟（Oncorhynchus mykiss）在13月龄、17月龄、21月龄和35月龄时的性体指数（GSI）和肝体指数（HSI）及其相关性。二倍体雌性虹鳟的GSI在13月龄时最小（0．083％）,此后逐渐上升,至35月龄达到高峰（8．918％）,变化幅度极大;HSI在13-31月龄呈上升趋势,至31月龄时达最大值（1．444％）,然后下降。三倍体雌性虹鳟的GSI变化幅度很小,17月龄时达到最大值（0．103％）,35月龄时最小（0．032％）;HSI在17月龄达到高峰（1．179％）,而后下降,31月龄最低（1．067％）。试验结果表明,二倍体雌性虹鳟GSI与HSI的变化密切相关,可间接反映鱼类的生殖和生长状况;三倍体雌性虹鳟GSI与HSI无显著相关性,说明其肝脏与卵巢之间没有大规模的营养物质与能量的转移,有利于商品鱼的生产。     

三倍体虹鳟制种技术与倍性鉴定

虹鳟(Oncorhynchus mykiss)属鲑形目、鲑科,是重要的冷水性养殖鱼类,三倍体虹鳟以其不育或育性不良、肉质提升和生长周期缩短等特点得到养殖者的喜爱。尽管国内多地已经进行了三倍体虹鳟制种的研究,但效果并不理想,目前三倍体虹鳟苗种主要依靠国外进口。本文拟对三倍体人工诱导和倍性鉴定两个方面进行总结,以利于更有效地进行三倍体虹鳟规模制种。     

梭鱼卵黄蛋白原的化学发光免疫检测法

运用建立化学发光免疫(chemiluminescence immunoassay,CLIA)检测法,对梭鱼血清中主要卵黄蛋白原(Vg)类型(B类型;VgB)进行定量测定。梭鱼VgBCLIA法按两步法操作进行,使用纯化的梭鱼VgB制备特异型抗血清(a-VgB)。优化抗体浓度和培育时间等检验条件后,检验范围设定为3.91～500ng/mL。卵黄发生期的梭鱼雌鱼血清和雌激素处理后的梭鱼稚鱼血清稀释曲线与纯化的梭鱼Vg曲线平行,而梭鱼雄鱼稀释血清中几乎不发生相应的免疫学反应。对天津原种场养殖的10尾梭鱼血清VgB水平的定量检测结果表明,在雌鱼(9尾)血清中检测到VgB但个体间差异较大,变化范围为3.0～2700.1μg/mL,并表现出随着卵巢发育而增长的变化趋势。相对而言,成熟梭鱼雄鱼血清VgB水平则极低(2.7μg/mL),表明在其生长环境中不存在雌激素活性。另外,所有个体的组织学观察也未呈现出明显的性腺异常情况。研究为梭鱼主要雌激素诱导生物标志物(VgB)的量化提供了一种新的手段,较以往的检验方法具有更高的灵敏度,有助于建立多元化的水生环境中雌激素活性的检测体系。     

二倍体、三倍体虹鳟的血液指标分析

通过对二倍体和三倍体虹鳟红细胞大小的测量,发现两者之间存在着极其显著的差异.三倍体虹鳟红细胞长径和核长径分别是二倍体的1.34和1.33倍;红细胞面积和核面积分别为二倍体的1.43和1.41倍:核体积为二倍体的1.47倍.用MEK-6318血液自动分析仪检测二倍体和三倍体虹鳟血常规值,结果显示:在红细胞计数(RBC)和红细胞平均容量(MCV)上,二、三倍体之间都有着极其显著的差异.此外,三倍体虹鳟的淋巴细胞、中间细胞和中性粒细胞数量都显著超过了二倍体.     

卵黄蛋白原研究及其进展

甲壳动物和昆虫由于进化上亲缘关系近,被统称泛甲壳动物,大多数泛甲壳动物卵黄蛋白原为雌性特异性蛋白,是卵母细胞中储存的卵黄蛋白的前体,可为胚胎发育提供营养和能量,是决定繁殖性能的关键因素,在生殖发育中起着十分重要的作用。已有研究证明,调控泛甲壳动物卵黄蛋白原的主要激素有保幼激素、蜕皮激素、神经肽和胰岛素样肽等。昆虫保幼激素和蜕皮激素发挥促进作用,而神经肽和胰岛素样肽对卵黄蛋白原合成的调控作用因种类不同而存在不同。除了以上激素,甲壳动物特有的眼柄高血糖激素家族以及促雄腺激素(IAG)对卵黄蛋白原的合成起负调控作用。本文总结了近年来泛甲壳动物卵黄蛋白原合成调控机制的相关研究,概括并比较了调控昆虫与甲壳动物的卵黄蛋白原合成的主要激素及调控机制,为养殖虾蟹甲壳动物生殖调控相关研究提供参考。

     

水生动物卵黄蛋白原研究新进展

卵黄蛋白原（vitellogenin,VTG）是卵生动物卵黄中主要成分的前体。文章简述了水产动物如鱼类、两栖类及甲壳类动物中卵黄蛋白原基因的鉴定及其基因家族的分类及进化研究,介绍了VTG分子的生物学新功能,以及VTG作为标记分子在环境雌激素监测、反映雌性动物性腺成熟度、区分雌雄性别等方面的应用,以期为VTG在水产动物中的研究以及繁殖中的应用提供参考。     

虹鳟倍性鉴定SSR标记筛选与应用研究

虹鳟(Oncorhynchus mykiss)是我国冷水性鱼类的主要养殖品种。与二倍体相比,虹鳟三倍体具有生长速度快、肉质好和不育等优点。目前,鉴定虹鳟倍性主要是通过流式细胞术进行DNA含量分析,但这种方法需要采集鱼类血液或组织样本,会对其造成伤害。为了实现利用微创方法收集样本鉴定虹鳟倍性,本研究利用PCR扩增以及电泳分离技术对153个微卫星标记(SSR标记)进行分析,其中139个SSR标记能够在二倍体和三倍体中成功扩增,筛选出132个SSR标记具有多态性,最终鉴定出7个标记在三倍体中表现出较高的变异性。通过在52个已知倍性水平的参考样本和48个未知倍性的样本上进行验证,进一步从中筛选出3个SSR标记(SSR1054、SSR1056和SSR1468)足以区分倍性水平,并且根据3个标记序列重新设计了3对具有良好稳定性的引物序列进行倍性分析应用。本研究所筛选的SSR标记解决了现有虹鳟倍性鉴定中存在的技术流程复杂、耗材昂贵的问题,并有助于不同倍性虹鳟的遗传多样性研究。     

大阪鲫卵黄蛋白原和钙离子关系的初步研究

注射苯甲酸雌二醇诱导雄性大阪鲫产生雌性特异血清蛋白(卵黄蛋白原),随着诱导天数的增加,血清中的钙离子浓度升高,同时血清中的卵黄蛋白原的含量也相应增加。在此过程中肝细胞的细胞质中存在大量的钙离子,并进行着旺盛的蛋白合成。雌鱼和被诱导的雄鱼一样在肝细胞内也存在大量的钙离子。正常雄鱼的肝细胞不合成卵黄蛋白原,只有很少的钙离子存在。腹腔注射氯化钙使卵母细胞的卵径增大(实验组为 1.08±0.14mm;对照组为 1.04±0.14mm),并使卵母细胞的蛋白含量升高,同时卵泡膜上毛细血管的分布更为稠密,这表明钙离子可促进卵母细胞的发育和蛋白的吸收。     

中华鲟血清卵黄蛋白原水平的初步观察

通过碱不稳定性蛋白结合磷法测定不同年龄中华鲟(Acipenser sinensis)血清中磷含量,以磷的含量变化反映卵黄蛋白原(VTG)变化情况。实验鱼为人工养殖0.5、1、2、3、4、5、6、8、9、10龄中华鲟和野生成熟中华鲟,共81尾,其中8尾野生雌鱼在人工繁殖前后各取样2~4次,共测97个血液样本。结果显示:低龄中华鲟VTG含量较低,最低值在5龄,含磷量为(4.6±1.6)mg/L,5龄后增加,成熟后达到最大,含磷量为(52.0±4.5)mg/L。在性腺不同发育时期,雌性个体在Ⅱ~Ⅴ期VTG的含量增加,Ⅱ~Ⅲ期增加速度较快,随后变缓,Ⅴ期达到最高水平,Ⅵ期降低;而雄性个体在Ⅱ~Ⅳ期VTG的增加量很少,Ⅳ期达到最高水平,含磷量为(28.7±12.2)mg/L。繁殖雌鱼产前VTG含量最高,含磷量为(62.5±8.7)mg/L,产后低于产前。此外,发现产后鲟鱼卵巢液中含量低于其血清中含量,但高于其他年龄组血清中的水平。本实验表明淡水养殖的中华鲟有可能达到性成熟。     

热休克诱导全雌虹鳟三倍体

以虹鳟二倍体的伪雄鱼为父本（遗传型为xx），探讨了采用热休克方法阻止第二极体排放诱导全雌虹鳟三倍体的适宜条件。结果表明：虹鳟三倍体诱导率明显受处理温度、起始时间以及持续时间等因子的影响。在孵化水温为6．5℃，卵子受精后20min经26℃热处理20min，孵化率为64．62％，三倍体诱导率为86．67％；卵子受精后lOmin经26℃热处理20min，三倍体诱导率为100％，但孵化率仅为3．87％。受精卵经26℃处理的诱导效果好于24℃和28℃的（P〈0．01）。     

虾青素在虹鳟体内的沉积与降解研究

为研究着色虹鳟在摄食状态和饥饿状态下的虾青素降解规律,实验选用平均体质量为(101.9±1.3)g的未着色虹鳟和已着色虹鳟,对未着色虹鳟饲喂含虾青素(100 mg/kg)饲料以研究虾青素在机体的沉积规律(虾青素沉积组);对着色虹鳟,饲喂含虾青素(100 mg/kg)饲料(正对照)、不含虾青素饲料(摄食降解组)或予以饥饿处理(饥饿降解组),实验共设4个处理组,每个处理组3个重复.实验共持续4周,每周采样一次,测定肌肉、肝脏和全鱼虾青素含量,血清类胡萝卜素含量,肌肉色差值(L*,a*,b*)和肌肉罗氏比色卡得分.结果表明,虾青素沉积组的虹鳟肌肉、肝脏虾青素含量、肌肉红度值和罗氏比色卡得分随饲养时间延长而增加(P＜0.05),在第3周时达到基本稳定,与第4周测定值无显著差异(P＞0.05);在4周养殖期内,全鱼虾青素含量呈增加趋势,而全鱼虾青素沉积率则呈下降趋势,第1周、第4周时的全鱼虾青素含量和全鱼虾青素沉积率分别为3.71 mg/kg、8.59 mg/kg和21.31％、15.26％;饥饿降解组和摄食降解组虹鳟的全鱼、肌肉、肝脏虾青素含量、肌肉红度值和罗氏比色卡得分随养殖时间延长逐渐降低(P＜0.05),其中饥饿降解组的下降趋势更为明显,第3周后,这种降低趋势减缓.上述研究表明,在虹鳟饲料中添加100 mg/kg虾青素,经过3周饲养即可使肌肉达到着色要求,虾青素的沉积率随时间延长而下降;饥饿和投喂不含虾青素的饲料均可使已着色虹鳟肌肉虾青素含量和红色度显著下降,饥饿使虾青素的减少更加迅速,虾青素在鱼体内的减少量随时间的延长而降低.     

Apparent amino acid availability from feedstuffs in extruded diets for rainbow trout Oncorhynchus mykiss

Apparent amino acid availability coefficients of several typical and novel feed ingredients were determined in rainbow trout using extruded diets and the faecal stripping technique. The ingredients were tested included five fish meals, three terrestrial animal by‐products, five plant protein concentrates, four plant meals, and seven low‐protein plant ingredients. Amino acid availability from the fish meals was relatively high ranging from 90 to 101%. Lower coefficients overall were observed for Menhaden fish meal FAQ when compared to the other fish meals. No differences in apparent amino acid availability were detected among the animal by‐products. Within the plant concentrate group, rice protein concentrate and barley protein concentrate exhibited generally lower amino acid availabilities compared to other concentrates tested. Among the plant meals, only the availabilities of histidine, valine, isoleucine and lysine in flaxseed meal were lower than those of soybean meal. Apparent amino acid availabilities among the low‐protein plant products were variable and significantly different.     

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。...     

Effects of dietary chromium picolinate supplementation on serum glucose, cholesterol and minerals of rainbow trout (Oncorhynchus mykiss)

The effect of chromium (chromium picolinate, CrPic) supplementation at various levels (0, 400, 800 or 1600 μg Cr/kg diet) on serum concentrations of glucose and cholesterol and on serum and liver concentrations of Cr, Zn, Cu and Fe of rainbow trout (Oncorhynchus mykiss) was evaluated. One-year-old rainbow trout (n=120) were randomly assigned to four treatment groups, each with two replicates of 15 fish. The fish were fed either a control diet or the control diet supplemented with either 400, 800 or 1600 μg Cr/kg diet. Serum glucose and cholesterol concentrations decreased (p=0.001) with higher dietary chromium supplementation. Increasing dietary chromium supplementation linearly increased serum chromium and zinc concentrations (p=0.001). Serum copper and iron concentrations did not change (p>0.10) as dietary chromium supplementation increased. Liver chromium and zinc concentrations linearly increased (p=0.001). Also, liver Cu and Fe concentrations did not change (p>0.10) as dietary chromium supplementation increased. The results of present study conclude that supplemental chromium to the diet of rainbow trout influences serum and liver Cr and Zn concentrations, thus most probably functions of the organs as well as the welfare of raibow trout.     

Identification of a novel gill-specific calpain from rainbow trout (Oncorhynchus mykiss)

Calpains are calcium-dependent neutral proteases responsible for many cellular functions. The two forms of calpain ubiquitously expressed in mammalian tissues are known as μ-calpain and m-calpain. We report here the identification of a novel calpain that is similar to but distinct from the μ- and m-calpains in rainbow trout. The cDNA of the novel gene is 2623 bp in length with a single open reading frame. The predicted protein (676 amino acids) contains the conserved calpain characteristic domains that include: domain I (pro peptide), II (cysteine catalytic site), III (electrostatic switch), and IV (calmodulin-like) with five Ca²⁺-binding EF hands. Northern blot and RT-PCR analyses demonstrated that the novel calpain gene is predominantly expressed in rainbow trout gills. Comparison of the novel protein with the ubiquitously expressed calpains and several mammalian tissue-specific calpains revealed that the novel calpain is an orthologue of the mammalian digestive tract specific calpain (calpain 9).     

虹鳟鱼核型及DNA含量分析

采用植物血球凝素（ＰＨＡ）体内注射、肾组织细胞短期培养、常规空气干燥法制备了虹鳟的染色体，并进行了核型分析。虹鳟二倍体的染色体为６０条，其核型为２２ｍ＋１ｓｔ＋７ｔ，ＮＦ＝１０４。以外周血红血球为样本，鸡红血细胞为对照，用美国产的ＦＡＣＳｔａｒｐｌｕｓ流式细胞仪测定了虹鳟的二倍体细胞核的ＤＮＡ含量，其ＤＮＡ含量为鸡血对照的１．５１倍，绝对含量为３．４７±０．２１ｐｇ／Ｎ。     

Growth, digestibility and nutrient utilization of rainbow trout (Oncorhynchus mykiss) and European sea bass (Dicentrarchus labrax) juveniles fed different dietary soybean oil levels

A shortage of marine raw ingredients, such as fish oil, is predicted in the near future. The use of suitable alternative lipid sources, such as vegetable oils, is crucial for sustainable growth of the aquaculture sector. Three isonitrogenous (50% Crude Protein) and isoenergetic (19 kJ g⁻¹) diets, in which fish oil was gradually replaced by soybean oil (0, 25 and 50% of the oil fraction) were tested for 12 weeks, using European sea bass (Dicentrarchus labrax) and rainbow trout (Oncorhynchus mykiss) juveniles. No dietary effects (p>0.05) on growth performance, feed conversion, digestibility or body composition were observed with either species. The results obtained suggest the inclusion of soybean oil up to 50% of the dietary lipid is possible, in diets for sea bass and/or rainbow trout juveniles.     

Effects of dietary nucleotides on the antioxidant status and serum lipids of rainbow trout (Oncorhynchus mykiss)

An 8‐week feeding experiment was conducted to evaluate the effects of dietary nucleotides (0, 0.5, 1, 1.5 and 2 g kg^?1) on serum antioxidative biomarkers and lipid contents of rainbow trout fingerlings. The serum triglycerides and low‐density lipoprotein (LDL)‐C levels were significantly decreased in fish fed diets containing 1.5 and 2 g kg^?1, whereas serum high‐density lipoprotein (HDL)‐C was higher in trout receiving diets with those levels of nucleotides. A significant decrease in thiobarbituric acid reactive substances concentration was observed in groups receiving 1.5 or 2 g kg^?1 of dietary nucleotides in comparison with control group (?32% and ?27%, respectively). A significant decrease was observed in catalase and serum glutathione peroxidase activity in trout fed 1.5 and 2 g kg^?1 dietary nucleotides compare with other groups. There were no significant changes in serum superoxide dismutase activity among the experimental groups. In conclusion, these results suggest that dietary nucleotides may reduce lipid peroxidation by increasing the levels of antioxidant lipoprotein, HDL‐C, and decreasing the LDL‐C, the main substrate of lipid peroxidation.