The first success of glass eel production in the world: basic biology on fish reproduction advances new applied technology in aquaculture

The eel has long been esteemed as an important food fish in the world, especially in Japan, and has been used as an experimental fish for many fields of fish physiology. However, the decreases in eel resources have been a serious concern in recent years. The catches of glass eels as seedlings for aquaculture have shown a long-term decrease in both Europe and East Asia. To increase eel resources, the development of techniques for artificial induction of maturation and spawning and rearing their larvae have been eagerly desired. Recent progress of reproductive physiology of fish, especially mechanisms of oocyte maturation and ovulation in female and of spermatozoa maturation in male, facilitate to establish techniques for hormonal induction of maturation and spawning in sexually immature eels. With persistent effort to development of rearing techniques of larvae, we have first succeeded to produce glass eel. These applied techniques are may contribute to understand the basic reproductive physiology of the eel.     

鳗鲡繁殖生物学研究进展

鳗鲡是优质养殖鱼类,但鳗鲡苗种完全依靠天然捕捞。近年来鳗鲡苗种的短缺限制了鳗鲡养殖业的进一步发展,开展鳗鲡人工繁殖技术研究势在必行。文章从鳗鲡的催熟与催产、产后鳗鲡的某些繁殖生物学以及鳗鲡胚胎和仔鱼发育等方面简要回顾了鳗鲡繁殖生物学的研究概况,并着重介绍了日本的最新研究进展,日本已将人工苗培育至20多厘米的成鱼,实现了实验室内鳗鲡由卵到成鱼的全人工养殖。文章最后指出人工育苗中存在的问题,并提出今后应从内因和外因两方面研究,才能真正实现鳗鲡苗种规模化生产。     

Occurrence of spontaneous polyploids from the eggs obtained by artificial induction of maturation in the Japanese eel (Anguilla japonica)

The nuclear DNA contents of the Japanese eel larvae were measured to examine the genetic soundness of the eggs obtained by artificial induction of maturation and fertilization. Of the 12.4% abnormal polyploid larvae, 77.9% were triploid and the rest were haploid, tetraploid, pentaploid, or aneuploid.     

模仿自然繁殖条件下的黄鳝人工繁殖试验

黄鳝(Monopterus albus)属合鳃目,合鳃科,黄鳝属,是我国重要的经济鱼类之一,受环境污染及滥捕的影响,自然资源量不断下降。而人工养殖因受苗种繁殖的制约,尚难以形成规模。我国鱼类学家伍献文和刘建康等曾对黄鳝的生殖习性和幼体变态作过研究,国内外许多学者对黄鳝性逆转等方面亦作过许多报道,但有关黄鳝人工繁殖的报道却不多。由于黄鳝属吐泡营巢繁殖的鱼类,且怀卵量低,一般黄鳝在繁殖期间,黄鳝先在水表面吐泡沫,做成泡沫巢,然后雌鳝在水中产卵,与此同时,雄鳝即排精,精液将卵子托于泡沫巢中,受精卵在泡沫巢中     

Changes in serum vitellogenin levels and immunohistochemical localization of vitellogenin in hepatic cells during ovarian development in the Japanese eel

ABSTRACT: The present study was designed to quantify changes in serum vitellogenin (VTG) levels and to localize VTG in hepatic cells immunohistochemically during artificial maturation of the Japanese eel Anguilla japonica . Hormone treatment resulted in a gradual rise in serum VTG levels, in which the highest values were attained at the migratory nucleus stage. Hepatocytes responded to hormone treatment by increased immunoreactivity; both staining area and intensity were enhanced with ongoing gonadal development. Moreover, cytoplasmic vacuoles increased in number during artificial maturation, occupying large areas inside hepatocytes. The unusual appearance of hepatocytes may reflect the effect of artificial maturation.     

鳗鲡繁殖生物学研究进展

鳗鲡是优质养殖鱼类,但鳗鲡苗种完全依靠天然捕捞。近年来鳗鲡苗种的短缺限制了鳗鲡养殖业的进一步发展,开展鳗鲡人工繁殖技术研究势在必行。文章从鳗鲡的催熟与催产、产后鳗鲡的某些繁殖生物学以及鳗鲡胚胎和仔鱼发育等方面简要回顾了鳗鲡繁殖生物学的研究概况,并着重介绍了日本的最新研究进展,日本已将人工苗培育至20多厘米的成鱼,实现了实验室内鳗鲡由卵到成鱼的全人工养殖。文章最后指出人工育苗中存在的问题,并提出今后应从内因和外因两方面研究,才能真正实现鳗鲡苗种规模化生产。     

In vivo gonadotropic effects of recombinant Japanese eel follicle-stimulating hormone

Recombinant Japanese eel follicle-stimulating hormone (rjeFSH) produced by methylotrophic yeast was subjected to in vivo bioassay to assess its gonadotropic activities and availability to artificially induce maturation of this species. Intramuscular injections of rjeFSH into male eels at doses of 0.1 and 1.0 U/g body weight were repeated 3 times during 12 days. The rjeFSH significantly increased plasma 11-ketotestosterone levels, and induced both testicular growth and spermatogenesis in a dose-dependent manner. These results demonstrate that rjeFSH is effective in promoting male eel gamatogenesis in vivo. Instead of scarce native eel FSH, abundant recombinant eel FSH is now ready for future application to maturation induction of Japanese eel.     

Effect of aromatable androgen (17‐methyltestosterone) on induced maturation of silver European eels (Anguilla Anguilla): Oocyte performance and synchronization

The reproductive performances of silver European eel in term of gonad development and egg production, employing slow‐release implants with the androgen 17‐MT (1 mg) in combination with traditional weekly injection of carp pituitary extract (CPE) was evaluated. Wild female European eels (Anguilla anguilla) underwent a standard induction protocol with CPE and were randomly divided into three groups (N‐group, no implant; Y‐group, with implant; and control, C‐group, no treatment). The results showed that 17‐MT‐treated females (Y‐group) reproduced spontaneously about 6 weeks earlier than the N‐group females with a saving of almost 40% in CPE and time of induction. Concerning artificial induction of maturation in female silver eels, our study demonstrated that they positively respond to androgen exposure also in terms of eggs productivity. Indeed, Y‐group was more productive than N‐group: in Y‐group, 11 eels ensured an eggs production that exceeded 50% of initial body weight (BW), whereas in N‐group only three eels have exceeded this value. The results suggest that 17‐MT should be considered in future protocols for the improvement of the artificial reproduction of female silver European eels.     

The roles of bone and muscle as phosphorus reservoirs during the sexual maturation of female Japanese eels, Anguilla japonica Temminck and Schlegel (Anguilliformes)

We sought to determine the organs that function as phosphorus (P) reservoirs for supplying this mineral to the ovary during the sexual maturation of the Japanese eel, Anguilla japonica (Anguilliformes). The P content of the ovary increased markedly during sexual maturation despite the lack of an extrinsic P supply (e.g., diet). This phenomenon suggests that the P that accumulated in the ovary was derived from other parts of the eel. In addition, the P content of bone dramatically decreased during maturation, as did the total bone weight. This result suggests that resorption of bone occurred during the sexual maturation of the eel. Whereas the P content and mass of muscle markedly decreased during maturation, the Ca and P contents of the skin (which was scaled) did not. The total (whole-fish) P content was constant throughout maturation. Together, our results suggest that almost all of the P lost from bone and muscle is incorporated in the ovary and that bone and muscle function as P reservoirs during the sexual maturation of the Japanese eel. This revised version was published online in August 2006 with corrections to the Cover Date.     

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。     

Swimming physiology of European silver eels (<Emphasis Type="Italic">Anguilla anguilla</Emphasis> L.): energetic costs and effects on sexual maturation and reproduction

The European eel migrates 5,000–6,000 km to the Sargasso Sea to reproduce. Because they venture into the ocean in a pre-pubertal state and reproduce after swimming for months, a strong interaction between swimming and sexual maturation is expected. Many swimming trials have been performed in 22 swim tunnels to elucidate their performance and the impact on maturation. European eels are able to swim long distances at a cost of 10–12 mg fat/km which is 4–6 times more efficient than salmonids. The total energy costs of reproduction correspond to 67% of the fat stores. During long distance swimming, the body composition stays the same showing that energy consumption calculations cannot be based on fat alone but need to be compensated for protein oxidation. The optimal swimming speed is 0.61–0.67 m s⁻¹, which is ~60% higher than the generally assumed cruise speed of 0.4 m s⁻¹ and implies that female eels may reach the Sargasso Sea within 3.5 months instead of the assumed 6 months. Swimming trials showed lipid deposition and oocyte growth, which are the first steps of sexual maturation. To investigate effects of oceanic migration on maturation, we simulated group-wise migration in a large swim-gutter with seawater. These trials showed suppressed gonadotropin expression and vitellogenesis in females, while in contrast continued sexual maturation was observed in silver males. The induction of lipid deposition in the oocytes and the inhibition of vitellogenesis by swimming in females suggest a natural sequence of events quite different from artificial maturation protocols.     

Positive feedback control by the gonads on gonadotropin (GTH) and gonadoliberin (GnRH) levels in experimentally matured female silver eels,Anguilla anguilla

Treatment of sham-operated female silver eels with carp pituitary extract stimulated ovarian development and induced increases in pituitary gonadotropin (GTH) and gonadoliberin (GnRH) contents. Both effects of carp pituitary extract were abolished in ovariectomized eels, indicating the involvement of the gonads. Endogenous sexual steroids, the secretion of which was increased during sexual maturation, should be responsible for the stimulation of GTH and GnRH levels. Ovariectomy itself had no significant effect on pituitary GTH and GnRH contents, reflecting the fact that, at the silver stage, sexual steroid levels are too low to exert any significant effect on pituitary GTH and GnRH. The positive feedback control exerted by the gonads on GTH and GnRH levels during sexual maturation, in the eel as well as in some other teleosts, would produce an amplification of the pubertal stimulation of the hypothalamo-pituitary-gonadal axis.     

激素缓释制剂诱导海水养殖鱼类性腺发育成熟与生殖行为的研究进展

综述促性腺素释放激素类似物（GnRH-a)缓释制剂（SRDS），人绒毛膜促性腺激素（HCG）SRDS，类固醇激素SRDS；雄烯二酮（ADSD）和17a-甲基睾丸酮（MT），以及鲤垂体匀浆（CPE）混合SRDS诱导海水养殖鱼类性腺发育成熟和生殖行为的效果，认为采用GnRH-a SRDS处理性腺发育良好的鲈形目鱼类和鲆鲽鱼类，可以显著提高血液促性腺激素（GtH)水平，且持续时间长，诱导排卵成功，HCG乳胶SRDS可诱导日本鳗鲡（Anguila japonicus)血液GtH持续 升高，性成熟系数明显升高并且性腺发育成熟；HCG和CPE水／油／水（W／O／W）复乳SRDS对日本鳗鲡的催熟效果显著，采用ADSD和MT SRDS SRDS可成功诱导日本鳗鲡性腺成熟与排卵。     

日本鳗鲡产卵后生存及其繁育

为证明日本鳗鲡(Anguilla japonica)生活史最后一步一产后鳗的命运,本研究模拟产后的日本鳗鲡继续在海水中养殖,观察其存活率及繁育情况.结果表明,产后鳗在海水中停食约18 d后,体能得到恢复,部分亲鱼开始出现摄食,1个月左右全部恢复摄食,经244 d养殖,雌、雄鳗体质量增加,存活率达94.6%.随后,给产后鳗注射外源性促性腺激素(鲤鱼脑垂体匀浆CPE和人绒毛膜促性腺激素HCG)后激发其退化的性腺(卵巢和精巢)重新发育(与当年银鳗作对照).通过性腺组织切片观察产后鳗和对照鳗性腺发育成熟的全过程及其差异,发现产后鳗起初性腺发育比当年银鳗差,但经多次注射激素后,产后鳗性腺成熟与当年银鳗同步,证明产后鳗生殖细胞对激素的敏感件高.应用17α,20β-双羟孕酮和促性腺激素释放激素类似物(GnRH-A3)使催熟的产后鳗和对照鳗均产卵和排精,并孵化出仔鱼,从而有力地证明,鳗鲡产后虽体质弱,但待体能恢复后能够继续生存和繁殖.本研究旨在探讨利用产后鳗作为今后鳗鲡人工繁殖亲鳗的可行性.     

Development of an in vitro culture system for producing eel larvae from immature ovarian follicles in Japanese eel <Emphasis Type="Italic">Anguilla japonica</Emphasis>

To standardize conditions during the final maturation and ovulation of ovarian follicles from Japanese eel, we have developed a culture system for the production of fertilizable eggs from post-vitellogenic ovarian follicles in vitro. Post-vitellogenic ovarian follicles were incubated in culture medium supplemented with 17α,20β-dihydroxy-4-pregnen-3-one (DHP) with or without bovine serum albumin (BSA) to assess the effects of protein concentration. Eggs that ovulated during incubation were fertilized, and the remaining follicles were incubated in prostaglandin F_2α (PGF_2α) for a further 3 or 6 h before fertilization. Male eels were injected repeatedly with human chorionic gonadotropin. The quality of eggs obtained under the different culture conditions was evaluated after artificial fertilization in terms of hatching success. Hatching rates tended to decrease with increasing concentrations of BSA in the incubation medium in a dose-dependent manner. The addition of PGF_2α drastically increased the number of eggs that ovulated, but the rate of hatching was greatly decreased compared with eggs obtained earlier by DHP incubation alone. The larvae obtained from artificially fertilized eggs produced in vitro survived for 14 days without feeding. We conclude that in vitro culture systems thus have a great potential for the acquisition of good quality eggs under tightly controlled artificial conditions, culminating in the production of eel larvae.     

Some remarks on eel management in lakes

The paper presents basic factors determining eel production in Polish lakes. Analysis of data from 582 lakes over 29 years (1950–1978) showed that ell yield in lakes is basically determined by the artificial stocking of eels. The relationship between eel catch and eel stocking is very close and to a certain extent independent of the fishing effort. Prospects of further development of eel management in Europe are presented, as well as basic problems facing this management on a general scale. A need is identified for further studies in this field.     

人工条件下日本鳗鲡胚胎及早期仔鱼发育的生物学特征

在多年对日本鳗鲡人工育苗技术研究的基础上,通过采用对胚胎和早期仔鱼的活体观察、计算机图文分析以及组织切片等方法,系统研究了鳗鲡胚胎及孵化后19d前仔鱼的生物学特性。研究结果表明,在水温(23±0.5)℃条件下,日本鳗鲡从受精卵到仔鱼出膜需约38h30min,总积温为885.50℃·h。其特征与一般硬骨鱼类胚胎发育基本相同,鳗鲡胚胎发育可分为14期,为典型的盘状卵裂,原肠作用通过细胞的下包和内卷完成。但日本鳗鲡早期仔鱼与一般硬骨鱼类的仔鱼存在较大差异,表现为无色透明的血细胞和S形心管;开放型的口腔,发达且不能闭合的上下颌和3对颌齿;孵化后3～9d在身体两侧出现由神经细胞和支持细胞构成发达的感觉丘,其数目为6～9对不等;以及形态不断变化的尾垂体等生物学特性。分析认为这些生物学特性决定了日本鳗鲡仔鱼的孵化环境可能相对较稳定,以及仔鱼摄食方式可能为触碰后咬食的方式。     

鲤脑垂体匀浆液和人绒毛膜促性腺激_省略_性腺激素及性类固醇激素含量的影响

雄鳗注射５－６次、雌鳗注射９－１０次鲤脑体匀浆液（ＣＰＥ）＋人绒毛膜促性腺激素（ＨＣＧ）能分别诱导精巢和卵巢发育成熟。在雌雄鳗鲡,注射ＣＰＥ＋ＨＣＧ可显著增加端脑、间脑、中脑和下丘脑ｍＧｎＲＨ的含量,而对后脑和延髓ｍＧｎＲＨ的影响较小;注射ＣＰＥ＋ＨＣＧ增加雄鳗后脑和延髓ｃＧｎＲＨ－Ⅱ含量,对雌鳗脑区ｃＧｎＲＨ－Ⅱ则无显著影响。雌雄鳗鲡每次注射ＣＰＥ＋ＨＣＧ后１天,血清促性腺激素（ＧｔＨ）急剧上升