星斑川鲽雌核发育二倍体、单倍体与普通二倍体及杂交胚胎发育的比较

通过紫外线(UV)对冷冻的鲈(Lateolabrax japonicus)精子进行灭活,利用冷休克和压力休克方法诱导星斑川鲽(Platichthys stellatus)雌核发育二倍体,同时利用灭活鲈精子制备单倍体胚胎,未灭活鲈精子受精制备杂交胚胎,星斑川鲽精子受精制备正常发育胚胎。对以上几种胚胎发育时序、发育生物学特征进行了观察比较。结果表明,卵裂期单倍体、杂交二倍体和雌核发育二倍体胚胎发育速度与普通二倍体胚胎没有明显差异,从低囊胚期开始各实验组胚胎发育速度均慢于普通二倍体胚胎;杂交胚胎在胚体形成期基本死亡,单倍体胚胎在尾芽期停止发育死亡,均不能正常孵出。雌核发育二倍体与普通二倍体具有相似的发育时序,普通二倍体100 h 10 min孵化出膜,而雌核发育二倍体104 h 50 min孵化出膜。雌核发育胚体畸形率(53.59±0.36)%,孵化率(0.11±0.01)%;普通二倍体胚体畸形率(35.11±6.19)%,孵化率(58.01±5.30)%;与普通二倍体相比,雌核发育二倍体胚体畸形率高,孵化率低,但孵化鱼苗能够正常发育,获得了雌核发育群体。本研究为星斑川鲽雌核发育提供了技术方法,同时为单倍体、杂交和雌核发育胚胎的发育生物学研究提供了细胞生物学证据。     

星斑川鲽(♀)与石鲽(♂)杂交子代胚胎发育研究

采用人工催产和干法受精技术,以星斑川鲽为母本,石鲽为父本进行属间杂交,受精卵在13~14℃海水中培养。利用DCM130-2.0显微电子成像系统观察受精卵胚胎发育,借助XTZ-D体视显微镜拍摄发育过程,分别在卵裂期、孵化前期统计杂交胚胎受精率、孵化率和畸形率。试验结果表明,杂交胚胎发育可分为卵裂、囊胚、原肠胚、器官发生、脱膜孵化5个阶段,在水温13~14℃条件下,受精卵历时98h完成孵化,该杂交组合胚胎发育时间介于双亲之间,杂交胚胎受精率为15.6%,孵化率为42.1%。杂交胚胎发育在卵裂、囊胚、原肠、器官形成和孵出期均有畸形胚胎出现,畸形率为57.1%。本研究结果为星斑川鲽和石鲽杂交育种提供了直接的发育生物学依据。     

异源精子诱导条斑星鲽雌核发育

用冷冻保存的花鲈精子作为异源精子，采用紫外线（UV）对精子进行照射使其遗传物质失活，然后与卵子进行授精，可以刺激条斑星鲽鱼卵进行雌核发育，在受精后一定时间采用冷休克处理“受精”卵，抑制第二极体排出成功获得了条斑星鲽雌核发育二倍体鱼苗。实验表明，花鲈精子只有经过紫外线照射遗传失活后，才能诱导产生条斑星鲽雌核发育二倍体鱼苗。经过大量实验筛选出诱导条斑星鲽雌核发育的最佳条件为花鲈冷冻精子采用80 MJ/cm² 的紫外线照射，然后与条斑星鲽卵子进行授精，在受精后7~9 min，将受精卵放在-1.0~1.5 ℃海水中进行冷休克处理，持续时间为60~90 min，在此条件下，获得的雌核发育二倍体的相对诱导率达40.68%±7.24%。由于不失活精子与条斑星鲽卵形成的杂交胚只能存活到原肠期，而染色体未被成功加倍的胚胎会由于单倍体的致死效应在孵化前后死亡，所以存活的仔鱼全部为条斑星鲽雌核发育二倍体。采用微卫星标记技术对雌核发育鱼苗进行了分析，证明了雌核发育鱼苗为雌核发育二倍体。首次报道了采用异源冷冻精子诱导条斑星鲽鱼卵进行雌核发育的技术方法，为条斑星鲽性别控制和遗传改良提供了技术手段。     

线纹尖塘鳢胚胎和前期仔鱼发育的初步研究

连续观察了线纹尖塘鳢(Oxyeleotrislineolatus)自然产卵受精的胚胎和前期仔鱼各发育阶段的形态特征。在室温28～29℃下,线纹尖塘鳢的胚胎发育历时70h完成,仔鱼孵化出膜。胚胎发育可分为受精卵、卵裂期、囊胚期、原肠期、神经胚期、器官形成期、肌肉效应期和孵化出膜期8个连续的典型发育期。胚后发育以卵黄囊的消失为依据,划分为前期仔鱼。     

江鳕胚胎发育的研究

观察了人工繁殖江鳕(Lota lota)的胚胎发育的全过程.根据对江鳕胚胎发育的观察与分析,将其胚胎发育分为受精卵、卵裂期、囊胚期、原肠期、神经胚期、器官形成期6个阶段,共20个发育时期,同时描述了各个发育时期的特征.在0～3℃下,胚胎历时998 h后孵出仔鱼,从受精卵至孵化出膜需要积温887～1023 h·℃.     

星斑川鲽远缘杂交初步研究

为了探索川鲽(Platichthys stellatus)远缘杂交的可行性,2007年进行了星斑川鲽♀×条斑星鲽♂、星斑川鲽♀×圆斑星鲽♂、大菱鲆♀×星斑川鲽♂杂交试验.结果表明:星斑川鲽♀×条斑星鲽♂、星斑川鲽♀×圆斑星鲽♂杂交不存在配子不亲和,受精卵可以正常发育,杂交子代存活和生长正常;而大菱鲆♀×星斑川鲽♂杂交胚胎能够发育,但孵化出仔鱼为畸形,24 h后死亡.     

紫外灭活的异源和同源精子诱导的半滑舌鳎单倍体胚胎发育过程比较

为探讨在雌核发育研究中紫外灭活的异源精子的细胞质等是否对“受精”卵的发育产生影响，本文对紫外灭活的异源和同源精子诱导的半滑舌鳎单倍体胚胎发育过程进行了比较研究。结果显示，在胚盘形成期、二细胞期、多细胞期和囊胚期，异源和同源精子诱导的半滑舌鳎单倍体胚胎发育时序及形态无显著差异。之后，灭活的异源精子诱导的单倍体胚胎发育速度比同源精子诱导的单倍体胚胎要慢，至受精后18h 48min已慢1个发育时期，说明在发育前期，异源精子诱导的单倍体胚胎发育阻力更大。肌节期后，同源精子诱导的半滑舌鳎单倍体胚胎发育也严重受阻，与异源精子诱导的单倍体胚胎发育速度无显著差异；且单倍体胚胎陆续大量死亡，但异源精子诱导的单倍体胚胎死亡率明显要大于同源精子诱导的单倍体胚胎。异源和同源精子诱导的单倍体胚胎孵出的仔鱼均表现有单倍体综合症，外形基本一致。为确保紫外线处理将精子完全灭活，当紫外灭活的异源和同源精子诱导的半滑舌鳎单倍体胚胎发育至原肠后期时，各取30粒用倍性分析仪检测其倍性，结果显示全为单倍体。     

细鳞裂腹鱼胚胎和卵黄囊仔鱼的发育

对人工繁殖的细鳞裂腹鱼(Schizothorax chongi)胚胎和卵黄囊仔鱼的发育过程进行了连续观察。在水温(17±1)℃下,胚胎发育历时124h后孵出仔鱼,孵出9d后仔鱼鳔充气并开始平游;从受精卵到孵化出膜所需要的平均积温为2108.00h℃。根据对细鳞裂腹鱼胚胎发育外部形态及典型特征的观察与分析,将其胚胎发育分为受精卵期、卵裂期、囊胚期、原肠胚期、神经胚期和器官形成期6个阶段,共25个发育时期,描述了各发育时期的特征。分析发现,在裂腹鱼亚科中,细鳞裂腹胚胎和仔鱼发育速度较快,特别是在肌肉效应期以后更加明显;心跳频率并不是简单的由慢到快的线性增长,而是一个快慢相间的曲线变化的过程;胚胎发育的各个时期对环境的敏感性不同,原肠期对外界环境变化最为敏感。     

圆斑星鲽胚胎及仔鱼发育的观察

对圆斑星鲽(VeraspervariegatusTemmincketSchlegel)胚胎及胚后发育进行观察,描述了各发育时期的形态特征和发育速度。在水温为(11±0.5)℃时,受精卵历时153h脱膜孵出,孵化后仔鱼在水温为(15±0.5)℃时经过6d开口摄食,50d完成变态而成幼鱼。圆斑星鲽的卵子为半浮性卵,其卵黄量很多。卵裂后期,卵裂球的大小和卵裂速度均出现差异。眼泡在体节形成之前出现。刚孵化仔鱼消化道细而直,孵化5d肛门打通,6d开口。随着卵黄囊的消失,消化道开始弯曲。色素细胞首先出现在86h的胚体中,至140h在卵黄囊上零星分布,且随着胚体发育,色素细胞增多,至变态完成后全身密布黑色色斑。胚体色素细胞的形状由孵化前的雪花状逐渐变为圆形,但卵黄囊上的色素细胞始终为雪花状。     

星斑川鲽与其他鲆鲽类杂交研究简报

以星斑川鲽为母本,分别以条斑星鲽、圆斑星鲽为父本进行了属间杂交,成功获得了星斑川鲽(♀)×条斑旱鲽(♂)、星斑川鲽(♀)×圆斑星鲽(♂)的杂交后代,并且性状优良,生长良好.以大菱鲆为母本、星斑川鲽为父本进行了属问杂交,获得的大菱鲆(♀)×星斑川鲽(♂)杂交胚胎发育顺利,但仔鱼1日龄全部死亡,不能进一步发育.同时对杂交胚胎的发育情况进行了详细观察和记录,并确定了胚胎发育的时序.     

Aquaculture performance of triploid barfin flounder Verasper moseri

ABSTRACT:   To evaluate the aquaculture performance of triploid barfin flounder Verasper moseri , the sex ratio, maturation, growth and the relative proportion of body parts were examined. The sex ratio of triploids was similar to diploids under communal rearing conditions, but the proportion of female diploids was higher than that of triploids under separate rearing conditions. The gonadosomatic index of triploid females was very low even during the spawning season, and the ovaries were rudimentary. These results suggest that triploid barfin flounder females were sterile. In addition, triploid males produced a small quantity of milt containing very few spermatozoa with abnormal shapes. Spermatozoa obtained from triploids were aneuploidies. When normal eggs were fertilized with sperm from triploid males, no fry developed. These results suggest that triploid barfin flounder males were functionally sterile. Triploid males grew more slowly than diploid males, and triploid females showed similar or slower growth than diploid females, whether reared separately (23 months) or communally (35 months). The ratios of visceral weight to the edible parts for triploid males were similar to those for diploid males, but ratios for triploid females were higher than for diploid females during the spawning period. In conclusion, a significant improvement of growth was not found in triploid barfin flounders.     

条斑星鲽人工繁育技术研究

对条斑星鲽人工繁育技术,孵化适温、适盐范围及亲鱼、仔稚鱼营养强化进行了研究,探讨了温度、盐度、营养强化与性腺、胚胎、胚后发育的关系。结果表明:采用先降温再升温+营养强化方法促熟亲鱼可获得优质卵子。最适孵化温度(10.5±0.5)℃,最适孵化盐度33.6。以小球藻强化轮虫有助于提高仔鱼开口率、生长率及成活率。     

Molecular cloning, characterization expression of P450c17-I and P450c17-II and their functions analysis during the reproductive cycle in males of barfin flounder (Verasper moseri)

P450c17, a key steroidogenic enzyme, plays important roles in the production of sex steroid and cortisol. In teleost, there are two types of P450c17, P450c17-I possessing 17α-hydroxylase and 17, 20-lyase activities, and P450c17-II only possessing 17α-hydroxylase activity. This work describes the molecular cloning of the cDNA encoding the barfin flounder (Verasper moseri) P450c17-I and P450c17-II by means of RT-PCR and 5' and 3' rapid amplification of cDNA ends (RACE) analyses and mRNA expression profiles analyzing by semiquantitative RT-PCR. Respectively, P450c17-I and P450c17-II mRNA levels in the testes correlated with serum testosterone (T) level, as well as gonadosomatic index (GSI) of males during specific stages of spermatogenesis. P450c17-I and P450c17-II mRNA were expressed in the testis and ovary, suggesting that both of them participate in the production of sex steroid in barfin flounder gonads. P450c17-I mRNA was undetectable; in contrast, P450c17-II mRNA was detected at the highest level in the head kidney, meaning that only P450c17-II is involved in the production of cortisol in barfin flounder. The results demonstrated that both of P450c17-I and P450c17-II participate in the production of sex steroid in male barfin flounder gonads.     

条斑星鲽和圆斑星鲽线粒体12S rRNA与16S rRNA基因序列及其系统分析

采用PCR产物直接测序法分别测定了条斑星鲽和圆斑星鲽线粒体基因组序列,并对12SrRNA和16S rRNA基因核苷酸全序列进行分析,条斑星鲽和圆斑星鲽线粒体12S rRNA的核苷酸序列长度分别为948 bp和949 bp,16S rRNA均为1716 bp。试验结果表明,由12S rRNA和16S rRNA基因所构建的两个系统树的结构基本一致,21种鱼主要分为3个大的分支:鲤形目、鲶形目聚为1支,鲑形目独立聚为1支,鲈形目和鲽形目聚为1大支。鲆鲽类与鲈形目的鲹科、鲷科鱼类聚类在一支,且与鲹科的日本竹荚鱼、大西洋竹荚鱼构成姊妹群,支持鲆、鲽类是从鲈形目分化出来的观点,而同属于鲽形目的鳎科鱼类塞内加尔鳎在12S rRNA树中成为一个独立的分支,在16S rRNA树中聚到鲈形目和鲽形目的分支中,并且与鲈形目关系更近些,鳎类是1个特殊类群,其分类地位有待进一步探讨。线粒体基因组序列已提交到GenBank中,序列号为:DQ403797和EF025506。     

Interaction of orexin/hypocretin-like immunoreactive neurons with melanin-concentrating hormone and α-melanocyte-stimulating hormone neurons in brain of a pleuronectiform fish, barfin flounder

Immunohistochemical localization of orexin/hypocretin in the brain of a pleuronectiform fish, the barfin flounder Verasper moseri was examined as the first step in unraveling the possible function of the hormone in the brain. Orexin-A-like immunoreactive (ir) cell bodies were found to be located in the nucleus posterioris periventricularis (NPPv) of the hypothalamus, and orexin-A-like-ir fibers were detected not only in the hypothalamus but also extensively throughout the brain. The orexin-A-like-ir cell bodies did not project their fibers to the pituitary gland. Since melaninconcentrating hormone (MCH) and α-melanocyte-stimulating hormone (α-MSH) are suggested to regulate food intake in addition to orexin/hypocretin in the teleost fish, it was examined whether neural connections exist between orexin neurons and the MCH and α-MSH neurons in the barfin flounder brain by using double-staining immunohistochemistry. Some orexin-A-like-ir fibers were in close contact with the MCH-ir and α-MSH-ir cell bodies in the hypothalamus. Moreover, a few MCH-ir and α-MSH-ir fibers were in close contact with the orexin-A-like-ir cell bodies in the hypothalamus. These results suggest that reciprocal connections exist between the orexin and MCH neurons and between the orexin and α-MSH neurons in the brain of the barfin flounder.     

Involvement of Gonadotropin-Releasing Hormone in Thyroxine Release in Three different forms of Teleost Fish: Barfin Founder, Masu Salmon and Goldfish

Effects of gonadotropin-releasing hormone (GnRH) on thyroxine (T₄) release in vivo and in vitro were studied in barfin flounder Verasper moseri, masu salmon Oncorhynchus masou and goldfish Carassius auratus. Seabream GnRH (sbGnRH) at a dose of 200 ng/50 g body weight (BW) significantly increased plasma T₄ levels 1 h after the in vivo injection in the barfin flounder, but thereafter the levels normalized. Salmon GnRH (sGnRH) significantly increased plasma T₄ levels l h after the injection with a significant return to initial levels in male masu salmon and male goldfish. In contrast, sGnRH and cGnRH-II in barfin flounder, and cGnRH-II in male masu salmon and male goldfish were not effective in stimulating T₄ release. To clarify direct involvement of GnRH in T₄ release, dissected lower jaw including scattered thyroid follicles was incubated with sbGnRH (1 μg/well) in barfin flounder, and with two doses (0.1 and 1 μg/well) of sGnRH in masu salmon and goldfish in vitro. T₄ concentrations of control were stable during 24 h. Incubation of lower jaw with high dose (1 μg/well) of GnRH significantly (P<0.05) increased T₄ concentrations of incubation medium at 1 h in all experimental fishes. These results indicate that direct stimulation of T₄ secretion by GnRH occurs widely in teleost fish.     

Fishery characteristics of barfin flounder Verasper moseri in southern Tohoku,the major spawning ground,after the start of large-scale stock enhancement in Hokkaido,Japan

Commercial landings of a rare pleuronectid flatfish, barfin flounder Verasper moseri, recovered drastically after large-scale stock enhancement in Hokkaido conducted since 2006. This study investigated commercial landings, fishing grounds, size distributions, and sex and age compositions of barfin flounder during 2007–2011 in southern Tohoku, their major spawning ground, which is over 700 km south of Hokkaido. Landings, mostly comprising stocked fish, increased drastically in southern Tohoku: from 2.0 tons in 2007 to 20.8 tons in 2010. Over 98 % of them were landed during January–April, with the peak period during February–March. Fishing logbook data of offshore bottom-trawl vessels during 2007–2010 revealed the upper continental slope off southernmost Tohoku as the main fishing ground (35°40′–36°50′N, annual weighted mean depth 267–299 m). Two size modes in the landings consisted of males (ca. 40 cm) and females (ca. 60 cm). The main age at fishery recruitment in southern Tohoku was younger for males (age 2+) than for females (ages 3+ and 4+), the result of which was that the female percentage was lowest at 1.5 % in 2009 and highest at 7.9 % in 2011. Our results showed clearly how the effectiveness of large-scale stock enhancement in Hokkaido extended to southern Tohoku in association with spawning migration of stocked barfin flounder.     

Effects of photoperiod on gonadotropin-releasing hormone levels in the brain and pituitary of underyearling male barfin flounder

ABSTRACT:   A pleuronectiform fish, the barfin flounder Verasper moseri , expresses three gonadotropin-releasing hormone (GnRH) forms in the brain: salmon GnRH (sGnRH), chicken GnRH-II (cGnRH-II) and seabream GnRH (sbGnRH). To clarify the effects of photoperiod on GnRH systems, changes in brain and pituitary GnRH peptide levels were examined using time-resolved fluoroimmunoassays. In experiment 1, 5-month-old male barfin flounder (mean total length 9.0 cm, body weight 11.0 g) were divided into short (8:16 h light : dark [L:D] cycle; lights on 08.00–16.00 hours) and long photoperiod (16:8 h L:D cycle; lights on 04.00–20.00 hours) groups in mid September and maintained until November under natural water temperature (19.3–15.2°C). Brain sGnRH concentrations were significantly higher in the 16:8 h L:D group than in the 8:16 h L:D group, whereas no significant differences were observed in total length, body weight, plasma testosterone concentration, brain cGnRH-II concentration and pituitary sbGnRH content. In experiment 2, 7-month-old male barfin flounder (mean total length 16.5 cm, body weight 76.8 g) were divided into short and long photoperiod groups in mid December and maintained until February under natural water temperature (12.5–6.6°C). Total length, body weight and condition factor were significantly greater in the 16:8 h L:D group than in the 8:16 h L:D group, whereas no significant differences were observed in plasma testosterone concentration and GnRH levels in the brain and pituitary. These results indicate that levels of sGnRH in barfin flounder are influenced by photoperiodic treatment dependent on water temperature and/or body size.     

Self-feeding activity of a pleuronectiform fish, the barfin flounder

The self-feeding activity of the barfin flounder (Verasper moseri) was examined under natural photoperiod and temperature. The experiment, carried out over 12 weeks from September to December, involved tagged fish (mean body weight: 371 g) reared in three 1000-l tanks (3 or 5 fish per tank) with a self-feeder device. The sensor comprised a switch and trigger string with a bead. Self-feeding activity was recorded in all tanks from day 1. The fish fed mostly at night. Strong positive correlations were observed between the number of daily feeder activations and water temperature/photoperiod, and all fish grew during the experiment. These results demonstrate that barfin flounder can operate self-feeding systems and it is suggested that self-feeding activity is influenced by photoperiod and water temperature.