外源性促性腺激素诱导日本鳗鲡卵子发生和卵巢发育成熟的机制

用兔抗促黄体素生成素受体(LHR)或称绒毛膜促性腺激素受体(CGR)、雌激素受体(ER)和孕激素受体(PR)的抗体对LHR,ER和PR进行免疫组织化学定位。目的在于揭示外源性促性腺激素(鲤鱼脑垂体提取物,CPE和人绒毛膜促性腺激素,hCG)诱发日本鳗鲡卵子发生和卵母细胞成熟的内分泌机制。结果表明,注射激素前后卵巢发育和卵子发生出现了十分显著的变化。卵巢组织学切片观察显示激素处理前鳗鲡卵巢发育处于卵黄发生早期,卵母细胞平均直径(220±0.01)μm。第一次注射这两种激素后10d,实验组卵母细胞中卵黄核分散在核的周围,核仁数量显著增加,多达18～20个左右,而对照组8～10个。第3和4次激素处理后,卵母细胞发育进入卵黄发生早-中期至中期,卵黄颗粒数量增加。第6和7次激素处理后,卵母细胞进入卵黄发生中后期到成熟期,卵母细胞胞质中充满卵黄颗粒,胞径和核径增加,分别为(570±1.39)μm和(128±1.19)μm,而对照组没有变化。其次,免疫组织化学染色结果显示LHR、ER和PR均定位在鳗鲡卵巢中卵母细胞胞质、核膜、核质、卵被膜和体细胞上。这里值得指出的是,从第3次和第4次激素处理后,这三种受体的定位各有特点,L...     

外源性促性腺激素诱导日本鳗鲡精子发生和成熟的作用机制

用兔抗血清对抗促黄体素生成素受体（LHR）或称绒毛膜促性腺激素受体（CGR）和雄激素受体（AR）进行LHR和AR免疫组织化学定位,以揭示外源性促性腺激素（鲤脑垂体激素和hCG）诱发日本鳗鲡精子发生及其内分泌机制。结果表明,经过注射激素处理后的实验组与注射前的对照组相比较,其精巢发育和精子发生出现十分显著的变化。组织学切片观察显示,激素处理前鳗鲡精巢处于精原细胞增殖期,而两种激素混合注射后第10天,实验组可见精小叶中精原细胞的有丝分裂和初级与次级精母细胞的数量显著的增加。注射后第35天,靠近生殖上皮除有少量精原细胞外,精小叶中有大量初级精母细胞和次级精母细胞和少数精子细胞以及管腔中存在少量精子。在注射后第83天,日本鳗鲡完成了精子发生和精巢发育成熟以及释精。免疫组织化学染色结果进一步揭示,激素处理前,LH受体免疫活性分布在生殖上皮,显示强的免疫阳性反应;激素处理后,LH受体定位在Sertoli细胞和间质细胞以及精原细胞和初级与次级精母细胞的胞膜上,均显示强的免疫阳性反应。激素处理前,雄激素受体定位在生殖上皮和早期生精细胞的胞膜上;激素处理后,AR则定位在这些生精细胞的核或胞质,而精子细胞和精子显示免疫阴性反应。这些结果首次证明了这两种激素诱导鳗鲡精子发生和成熟的作用机制是通过LH受体和雄激素受体的介导。     

盐度驯化对雌性日本鳗鲡形态特征及性腺发育的影响

为查明盐度驯化对5龄雌性日本鳗鲡(Anguilla japonica)形态特征及性腺发育的影响,通过150 d的模拟盐度驯化实验,分析雌鳗在不同盐度驯化下的形态特征和性腺发育状况。结果显示,从盐度0～35依次驯化至90d(在盐度15、25、35依次分别驯化30d)时,雌鳗的胸围(PC)、胸鳍指数(PI)、性体指数(GSI)和肝脏指数(LI)随盐度的升高均显著增加(P<0.05);而消化道指数(DTI)和眼径指数(EI)随盐度的升高均无明显变化(P>0.05)。通过对卵巢切片观察发现,雌鳗的卵母细胞直径(OD)随盐度的升高显著上升(P<0.05),但卵母细胞始终处于脂肪泡时期。在盐度35中继续驯化,发现雌鳗在第90、120和150天的上述7项指标在各盐度组间均不存在显著性差异(P>0.05)。为了解流水对雌鳗性腺发育的影响,设置了同步盐度驯化的流水组。结果显示,在整个盐度驯化过程中,流水组雌鳗7项指标的变化趋势与静水组一致,除卵母细胞直径(OD)外的其他6项指标与同一驯化盐度下的静水组均无显著性差异(P>0.05)。流水组的卵母细胞直径(OD)均显著高于同一...     

人工诱导日本鳗鲡性腺发育组织学研究

从人工诱导日本鳗鲡(Anguill japonica)性腺成熟的角度,采用对亲鳗群体外形和解剖观察,以及性腺组织切片的方法,对日本鳗鲡从下海鳗到性腺发育成熟的全过程进行了组织学和细胞学研究。结果表明,人工诱导的日本鳗鲡精巢发育仍可分为5个时相,即精原细胞前增殖期(Ⅰ);精原细胞后增殖期(Ⅱ),精母细胞生长期(Ⅲ),精子出现期(Ⅳ)和精子成熟期(Ⅴ)。除日本鳗鲡下海鳗的精巢有73.3%的个体处在第Ⅰ时相外,其整个精巢的发育过程和细胞学特征与以前的研究无太大差异。在下海鳗群体中未观察到第Ⅰ时相的卵巢。因此,人工诱导日本鳗鲡的卵巢发育只有卵母细胞单层滤泡期(Ⅱ);脂肪泡出现期(Ⅲ);卵黄充满期(Ⅳ)和卵母细胞最后成熟期(Ⅴ)。本研究除对人工诱导下日本鳗鲡性腺发育各时相的组织学和细胞学特征进行了观察外,还探讨了人工诱导日本鳗鲡性腺发育的时间、下海鳗群体的组成、以及脂肪泡的形成和变化过程等问题,为以后的日本鳗鲡人工育苗技术提供了参考依据。     

雄烯二酮和甲基睾酮诱导雌性日本鳗鲡性发育的反馈调节作用

采用间隔１５ｄ多次埋植雄激素雄烯二酮（４－ａｎｄｒｏｓｔｅｎｅ－３,１７－ｄｉｎｅ,ＡＤＳＤ）或甲基睾酮（１７α－ｍｅｔｈｙｌ－ｔｅｓｔｏｓ－ｔｅｒｏｎｅ,ＭＴ）诱导雌性日本鳗鲡（Ａｎｇｕｉｌｌａ ｊａｐｏｎｉｃａ）性腺发育成熟;并着重研究埋植雄激素后,雌鳗血清中促性腺激素（ＧｔＨ）及脑和垂体中哺乳类促性腺激素释放激素（ｍＧｎＲＨ）含量的动态变化。一次或多次埋植ＡＤＳＤ后,１～５ｄ的血清ＧｔＨ含量上升,然后下降;并且,血清ＧｔＨ含量上升的幅度随埋植次数的增加而增加;对于ＭＴ处理组,只在埋植７次后第１５天测得血清ＧｔＨ水平显著高于对照组,但仍显著低于ＡＤＳＤ处理组。这表明埋植ＡＤＳＤ和ＭＴ可促进ＧｔＨ的分泌,但两者促进ＧｔＨ分泌的作用存在显著差异。埋植ＡＤＳＤ１ｄ后,脑和垂体中ｍＧｎＲＨ含明明显增加,第２天后和对     

日本鳗鲡人工繁育研究的进展

从日本鳗鲡亲本的选择、人工催熟催产技术、仔鳗的饵料开发、仔鳗的培育装置及培育方法等方面综述了目前在日本鳗鲡繁育方面的最新研究成果,以期为国内日本鳗鲡的人工繁殖研究提供参考资料.     

雄烯二酮和甲基睾酮诱导雌性日本鳗鲡性腺发育的反馈调节作用

采用间隔15d多次埋植雄激素雄烯二酮(4-androstene-3,17-dine,ADSD)或甲基睾酮(17α-methyl-testosterone,MT)诱导雌性日本鳗鲡(Anguilla japonica)性腺发育成熟;并着重研究埋植雄激素后,雌鳗血清中促性腺激素(GtH)及脑和垂体中哺乳类促性腺激素释放激素(mGnRH)含量的动态变化.一次或多次埋植ADSD后,1～5d的血清GtH含量上升,然后下降;并且,血清GtH含量上升的幅度随埋植次数的增加而增加;对于MT处理组,只在埋植7次后第15天测得血清GtH水平显著高于对照组,但仍显著低于ADSD处理组.这表明埋植ADSD和MT可促进GtH的分泌,但两者促进GtH分泌的作用存在显著差异.埋植ADSD 1d后,脑和垂体中mGnRH含量明显增加,第2天后和对照组无明显差别,表明ADSD促进了mGnRH的合成,并可能有一定程度的释放.研究表明雄激素对性腺未发育成熟的雌性日本鳗鲡在脑和垂体两个水平存在正反馈调节作用.     

日本鳗鲡幼体的耳石微化学分析及其环境指示元素筛选

在长江口日本鳗鲡鳗苗捕捞汛期,于长江靖江段采集日本鳗鲡幼体,采用同步辐射X射线荧光(XRF)定量分析方法测定了幼体矢耳石中14种元素的含量,分析了在海洋与淡水早期生活史阶段的微化学特征。结果显示,耳石中Ca和Sr为常量元素,Ba、Fe、Mn、Zn的含量也在1-10μg/g之间,Cr、Ni、Se、Co、Cu、Au则在1 mg/g以下。Ni、Cu、Mn、Cr、Co、Zn和Sr含量的稳定性较好,变异系数在30%以下;Se、Au的含量低且极不稳定,变异系数达40.8-75.0%;Fe在不同个体间存在显著的含量差异(p=0.007<0.05),但其他元素不存在个体差异。在海洋生活史阶段,Mn、Se、Co、Ba为强富集元素(BCFa-e >1000),Sr、Fe、Zn、Ni、Cu为中等富集元素(BCFa-e在100~1000)。除Sr、Ba和Se外,卵黄囊期和柳叶鳗期的耳石富集系数并无显著差异(p>0.05)。在淡水生活史阶段,Sr和Co为强富集元素(BCFa-e >1000),Se、Zn、Cu、Ba、Mn为中等富集元素(BCFa-e在100~1000),Ni、Fe为低富集元素(BCFa-e在10~100)。与海水阶段相比,耳石在淡水阶段的Fe、Ba、Mn、Se、Co、Ni富集系数均大幅减小,而对Sr、Zn、Cu的富集能力有所增大。分析表明,耳石内的Sr、Ba、Ni、Co为环境强响应元素,Fe和Mn为环境弱响应元素,Zn、Cu和Se为环境负响应元素,幼鳗自海洋至淡水的迁徙过程中,前者存在明显的时滞效应,但后两者的日间含量波动较大,缺乏响应的规律性。本文认为,用作鱼类迁移行为或栖息地环境变化的指示元素,需要具备耳石富集效应强、时滞效应小、不同环境间含量差异大、稳定性好、且为非必需元素等特点。从本研究结果看,只有Sr和Ba两种元素符合这些条件 。     

循环水养殖系统培育日本鳗鲡和美洲鳗鲡苗种的研究

采用具有气泡反冲洗型珠子过滤器的循环水养殖系统对日本鳗鲡和美洲鳗鲡进行了苗种培育的研究。2种鳗苗的放养密度约为传统模式的8～10倍,试验期间换水量约为20%～45%。结果显示:日本鳗苗和美洲鳗苗成活率均≥96.74%,高于传统模式。投喂水丝蚓阶段,日本鳗苗特定生长率和饲料系数分别为(7.08±0.08)%/d和4.92±0.03,美洲鳗苗分别为(4.83±0.06)%/d和4.74±0.19。投喂配合饲料阶段,日本鳗苗分别为(2.30±0.19)%/d和1.30±0.02,美洲鳗苗分别为(1.69±0.05)%/d和1.76±0.06。研究表明,该循环水养殖系统可适用于日本鳗苗和美洲鳗苗的培育,并具有节能、减排和无药残等优点,为开发资源节约型和环境友好型的鳗鲡育苗模式提供参考。     

日本鳗鲡人工繁殖研究现状及存在问题

鳗鲡属(Anguilla)鱼类共有19个种和亚种,其中以日本鳗鲡(A.japonica)和欧洲鳗鲡(A.anguilla)的人工养殖最为广泛。鳗鲡主要生长于热带及温带地区水域,除了欧洲鳗鲡及美洲鳗鲡(A.     

First capture of post-spawning female of the Japanese eel Anguilla japonica at the southern West Mariana Ridge

Two adult male freshwater eels, Anguilla japonica, were captured in June 2008 in the West Mariana Ridge (13°N, 142°E) in the North Pacific, but collections of females have yet to be reported. In September 2008, we successfully caught two adult female A. japonica, 55.5 and 66.2 cm in total length, in the adjacent but northern area (14°N, 143°E). Six newly hatched eel larvae (pre-leptocephali) were also collected by subsequent plankton sampling conducted near the female catch area. Female adults appeared to be in the post-spawning state, probably a considerable time after spawning, since a small number of remarkably regressed oocytes (50–250 μm in diameter) were observed in the ovaries. Capture of post-spawning female eels and newly hatched larvae near the Suruga Seamount (14°N) together with the previous collection of mature males in the southern area (13°N) corroborates that the area along the West Mariana Ridge is the spawning area of this species, but suggests that the eel spawning may occur over a wider area than previously expected.     

日本鳗鲡人工催产后亲鱼恢复培养与再催产效果

日本鳗鲡(Anguillajaponica)产后亲鱼的恢复培养全过程包括:产后亲鱼的海水淡化、诱导开口以及亲鱼培育。对2种不同淡化方式的结果比较发现,日本鳗鲡亲鱼的海水淡化方式以缓慢连续的方式较好,即每天换水1次,每次淡化量为总盐度的3%～4%,总淡化时间为1个月。淡化后的亲鱼存活率分别为:自然产卵的产后亲鱼达到100%,人工授精的产后亲鱼为86.3%,而难产的产后亲鱼则为81.5%。在各种开口驯养方式中,以水蚯蚓为开口饵料的驯养效果较好。诱导亲鱼开始摄食所需时间最短,为18d;摄食量也最大,达到平均体重的2.5%。从水蚯蚓逐步转为全人工饵料后,进行日常亲鱼培育。经18个月的恢复培养后,使产后亲鱼的平均体重雌鳗330g、雄鳗150g分别恢复到765g和470g。但2次繁殖的各项指标中,催熟率、成熟系数和性腺发育情况远远低于野生鳗,而催产率和幼苗存活率则略高于野生鳗,其原因尚有待进一步研究。     

日本鳗鲡排卵的人工诱导

对日本鳗鲡(Anguillajaponica)卵巢发育的时间与催产率的关系进行研究,对卵母细胞在体内胚泡破裂(GVBD)的发生过程、以及用17α-OHP和17α,20β-DHP诱导日本鳗鲡排卵的方式、时机和催产效果进行观察,结果表明,卵巢发育时间越长,催产率越低。造成催产率低的原因主要是由于日本鳗鲡在卵母细胞成熟后期,雌鳗个体对催产药物敏感性差异较大,催产时机难以掌握。通过采用适合日本鳗鲡卵母细胞的透明液观察GVBD的发生过程,并根据每个个体GVBD的发生速度,制订适宜的催产方案基本解决了上述难题。本实验采用CP、HCG、17α-OHP或17α,20β-DHP混合催产,使其平均催产率由35 4%分别提高到91 9%和93 0%。17α-OHP和17α,20β-DHP的催产剂量为5mg/(500g)和10mg/(500g)。在(21±0 5)℃水温条件下,催产效应时间分别为15～18h和13～16h。     

Serum steroid profiles in artificially maturing female Japanese eel,Anguilla japonica

To investigate whether steroid profiles in salmon pituitary homogenate (SPH)-induced artificially maturing Japanese eel, Anguilla japonica, resemble those in other, naturally maturing fishes, the daily changes in 11 steroids were analyzed for a 70-day period (average time needed to reach the maturational phase). Concentrations of most steroids were low and changed on a weekly basis, with maximum values 2–5 days after an SPH injection. Thus, pregnenolone, 17α-hydroxypregnenolone, dehydroepiandrosterone, progesterone, 17α-hydroxyprogesterone, 17α,20 β-dihydroxy-4-pregnen-3-one, androstenedione and estrone levels were barely or not detectable in serum throughout the experimental period, which is largely in keeping with what is known about oogenesis-related steroids in other fishes. In contrast, serum testosterone (T) levels were high, but fluctuated considerably with each SPH injection (about 0.3–8.3 ng/ml). The serum estradiol-17β (E₂) levels increased after SPH injections and gradually rose throughout the experiment, peaking at the end of the experimental period (about 0.2–7.8 ng/ml). Serum levels of 11-ketotestosterone (11-KT) before SPH treatment were higher (approximately 2 ng/ml) than those of the other steroid hormones (less than 0.5 ng/ml). 11-KT levels increased gradually over the experimental period, and, like E₂, levels peaked towards the end of the experimental period (about 15 ng/ml). The observed patterns for T, E₂ and 11-KT are unlike those in other fishes. Furthermore, the consistent elevations in levels of 11-KT, both before and after SPH treatment, are suggestive of an important role for this steroid in controlling oocyte growth.     

Effects of temperature on vitellogenesis in Japanese eel Anguilla japonica

ABSTRACT:   Cultured immature female Japanese eels acclimated to sea water at either 10 or 20°C were treated weekly with salmon gonadotropin fraction (sGTH) in order to investigate the effects of water temperature on artificial induction of ovarian maturation. In eels maintained at 20°C, ovulation was induced in 11 of 18 fish during the experimental period of 13 weeks, whereas at 10°C all fish showed a low gonadosomatic index (GSI) at the end of the experiment. Plasma vitellogenin levels were higher in eels kept at 20°C than in eels at 10°C throughout the experiment. However, no significant differences were observed in the plasma testosterone and estradiol-17β levels between groups. Eels pretreated with sGTHs at 10°C for 13 weeks were separated to two different temperature (10 and 20°C) groups, and received the same weekly sGTH injections. In eels transferred to 20°C, plasma vitellogenin levels, GSI and oocyte diameter were increased, but these values were maintained at low levels in eels that remained at 10°C. These results clearly indicate that water temperature is an important factor regulating vitellogenesis in the Japanese eel.     

Artificial induction of maturation and fertilization in the Japanese eel, Anguilla japonica

Repeated injections of salmon pituitary extract (20 mg per fish per week) induced vitellogenesis in feminized, cultivated Japanese eels (Anguilla japonica). Oocytes were attained at the migratory nucleus stage after 11 or 12 injections. Addition of 17,20-dihydroxy-4-pregnen-3-one (DHP) into the incubation medium induced germinal vesicle breakdown (GVBD) in the oocytes at the migratory nucleus stage. An injection of DHP (2 µg g^-1 BW), given 24h after an injection of salmon pituitary extract (20 mg fish^-1), succeeded in inducing maturation and ovulation in females which contained occytes at the migratory nucleus stage. Most fish ovulated 15–18h following the DHP injection. Eggs that were ovulated within 15h after the DHP injection showed high fertility and hatchability, but eggs ovulated 18 or 21h after the DHP injection, showed considerably lower fertility and hatchability. A delay between ovulation and stripping of the eggs rapidly decreased both the fertility and hatchability within 6–9h after ovulation, indicating that artificial fertilization must be carried out immediately after ovulation. Repeated injections of human chorionic gonadotropin (hCG) at a concentration of 1 IU g^-1 BW week^-1 induced spermatogenesis, spermiation, and the acquisition of potential for sperm motility in cultivated males. Most males spermiated after the fifth or sixth injection of hCG, and the milt weight gradually increased and remained constant (1–2 g) from the 11th to 31th injection. Sperm motility peaked 24h after each weekly injection, and decreased from the 3^rd day after the injection. Potassium ions are an essential constituent for the maintenance of motility in the eel spermatozoa. Artificial seminal plasma containing 15.2 mM KCl is applicable as a milt diluent. Using these techniques developed for female and male eels, we have succeeded in obtaining many fertilized eggs from cultivated eels.     

Maturational factors as indicators of egg quality in Japanese eel, Anguilla japonica

In order to identify maturational factors that can serve as indicators of egg quality in artificially matured Japanese eel, Anguilla japonica, maturation-promoting factor (MPF; consists of cdc2 and cyclin B) related proteins and chromosomes were examined. Mitogen-activated protein kinase (MAPK; 44 kDa band) and cdc2 (35 kDa and 34 kDa band) were detected. Chromosomes aligned on a vertical spindle appeared normal; however, chromosomes on a horizontal spindle and dispersed chromosomes seemed unusual.     

Demographic survey of the yellow-phase Japanese eel Anguilla japonica in Japan

Catch statistics and biological data of the Japanese eel Anguilla japonica from 12 river systems in nine prefectures of Japan (36°N, 140°E–31°N, 130°E) during 1999–2004 were analyzed in order to evaluate the demography of A. japonica in Japan. Significant differences in biological characteristics of 6388 eels were found among the river systems. Fishery catches of eels in all locations have declined, but the magnitudes or patterns of decline seem to be different. Of sex-identified eels, mean total lengths ± SD (mm) of females (n = 3776) and males (n = 962) were 495.6 ± 104.3 and 412.9 ± 80.7, respectively, and overall sex ratio (% female) was 79.6 %. Mean age (years) and growth rate (mm year^?1) were 5.0 ± 1.9 and 96.7 ± 38.6 for females (n = 3643) and 3.6 ± 1.7 and 120.5 ± 65.4 for males (n = 907), respectively. This study highlights the demographic heterogeneity of the A. japonica among the studied river systems, which suggests that it is important to conserve a variety of habitats in multiple river systems as an inclusive management target in addition to restoration of the diversity of habitats for eels in a single river system.