A step forward in development of fish protein hydrolysate-based diets for larvae of Japanese eel Anguilla japonica

Eggs of spiny dogfish Squalus acanthias are an indispensable and effective component of the compound diets currently used for larvae of the Japanese eel Anguilla japonica. However, this fish species, a natural resource, is becoming more scarce, suggesting that suitable diets should be developed using more sustainable components. In the search for appropriate diets without shark eggs, we have designed test diets in which shark eggs were replaced with general feed materials, such as fish protein hydrolysate (FPH), with the notable improvement that the FPH had been pre-digested with integral enzymes from frozen krill. Feeding trials were conducted to assess the effects of the test diets on survival and growth of Japanese eel larvae at early developmental stages. Larvae fed on the test diets were found to be able to survive up to 65 days after hatching, the maximum total length and body depth were 14.1 and 1.5 mm, respectively. Although larvae fed on the test diets still exhibited a low survival rate and poor growth, the FPH-based diets had a nutritional value that promoted substantial larval growth based on the significant difference between the initial and final body sizes. Our results suggest that the development of FPH-based diets not based on shark eggs but rather on general feed ingredients is promising but that there is much room for improvement.     

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.     

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。     

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。     

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.     

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.     

日本鳗鲡早期幼苗趋光性及视觉发育特征

研究了出膜后第1～9 d的日本鳗鲡(Anguilla japonica)早期幼苗,在不同光强度下趋光性分布及视觉发育组织学特征。结果显示:在8900 lux的强光条件下,日本鳗鲡早期幼苗随着发育,表现出明显的负趋光性。到9 d时,占存活幼苗52.2%的幼苗都集中在最暗的110 lux区域;而在2300 lux的弱光条件下,表现出趋弱光性。到9 d时,占存活幼苗44.6%的幼苗都集中在25 lux区域。视网膜发育的组织学观察发现,日本鳗鲡幼苗的视网膜发育首先出现的是外界膜和内界膜,最后出现的是外丛层。幼苗的整个视网膜结构到9 d时,发育为9亚层结构。视网膜中最发达的是外核层,由3种细胞构成。发育最快的是内核层,由2种细胞构成。结果表明,与日本鳗鲡早期幼苗趋光性有关的因素包括视网膜分层、视网膜厚度、内核层细胞数等多种因素。另外,本研究还探讨了日本鳗鲡幼苗在自然界可能生存的水深和光环境。     

Characterization and ontogenetic development of digestive enzymes in Pacific bluefin tuna Thunnus orientalis larvae

The major digestive enzymes in Pacific bluefin tuna Thunnus orientalis larvae were characterized, and the physiological characteristics of the enzymes during early ontogeny were clarified using biochemical and molecular approaches. The maximum activity of trypsin (Try), chymotrypsin (Ct) and amylase (Amy) was observed at pH 6–11, 8–11 and 6–9, respectively. Maximum activity of Try, Ct and Amy occurred at 50 °C, that of lipase (Lip) was at 60 °C and that of pepsin (Pep) was at 40–50 °C. These pH and thermal profiles were similar to those for other fish species but differed from those previously reported for adult bluefin tuna. Enzyme activity for all enzymes assayed was found to decrease at high temperatures (Try, Ct, Amy and Pep: 50 °C; Lip: 40 °C), which is similar to findings for other fish species with one marked exception—increased Try activity was observed at 40 °C. Lip activity appeared to be dependent on bile salts under our assay conditions, resulting in a significant increase in activity in the presence of bile salts. Ontogenetic changes in pancreatic digestive enzymes showed similar gene expression patterns to those of other fish species, whereas marked temporal increases in enzyme activities were observed at 10–12 days post hatching (dph), coinciding with previously reported timing of the development of the pyloric caeca in bluefin tuna larvae. However, complete development of digestive function was indicated by the high pep gene expression from 19 dph, which contradicts the profile of Pep activity and previously reported development timing of the gastric gland. These findings contribute to the general knowledge of bluefin tuna larval digestive system development.     

Expression of pancreatic enzyme genes during the early larval stage of Japanese eel Anguilla japonica

ABSTRACT: To reveal the ontogeny of pancreatic exocrine function in the early larval stage of eel, cDNAs encoding major pancreatic enzymes, trypsinogen, amylase and lipase were identified from the Japanese eel Anguilla japonica and their expression pattern in larvae was analyzed. The cloned eel trypsinogen precursor consisted of 224 amino acids and showed 82.2% identity to trypsinogen-2 of winter flounder Pleuronectes americanus . The eel amylase precursor consisted of 512 amino acids and showed 77% identity to winter flounder amylase. Eel pancreatic lipase was composed of 470 amino acids and had 58.3% of identity to human pancreatic lipase. In the eel larvae, mRNA expression of trypsinogen and amylase was first detected at 6 days post-hatching (d.p.h.), and the expression level increased between 7 and 8 d.p.h. In contrast, mRNA expression of lipase was first detected at 8 d.p.h. Eel larvae start to feed actively at 8 d.p.h. Thus, it was indicated that eel pancreas starts to synthesize digestive enzymes at 6 d.p.h. and acquires full function by the onset of exogenous feeding at 8 d.p.h.     

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.     

Vitamin contents of eggs that produce larvae showing a high survival rate in the Japanese eel Anguilla japonica

This paper describes the relationship between the egg vitamin concentrations and the egg quality in the Japanese eel Anguilla japonica . No notable relation was found between any vitamin and the fertilization rate. Hatching and survival rates of larvae, however, significantly increased with an elevated level of egg vitamin C (VC). In contrast to VC, the relation between vitamins E (VE) and A (VA) concentrations and survival rate showed a clear peak, with a reduced survival rate at both higher and lower vitamin concentrations. The ratio of VE to lipid or highly unsaturated fatty acid (HUFA) in eggs positively correlated with hatching and survival rates of larvae. High-quality (HQ) eggs were determined as eggs that produced larvae having a survival rate higher than 80% at 8 days post hatch, and low-quality (LQ) eggs were determined as eggs that did not hatch. The level of VC of HQ was significantly higher than LQ. The results of this study suggest that HQ eggs, which produce larvae having a high survival rate, must have high levels of VC and VE/HUFA ratio and contain optimum levels of VA and VE in 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.     

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.     

日本鳗鲡人工育苗及仔鱼饲料研究

日本通过40年对鳗鲡成熟控制技术的研究,特别是近几年来采用17,20β-dihydroxy-4-pregnen-3-one(DHP)的鳗鲡雌鱼成熟诱导技术,使高质量鳗鲡受精卵的取得具有相当高的把握性。日本水产厅养殖研究所采用以鲨鱼卵冷冻干燥粉末为主要成份并含有磷虾或其它鲜鱼虾抽提液的糊状饲料,喂食人工孵化的日本鳗鲡仔鱼取得举世嘱目的突破性进展,但柳叶状仔鱼全长达到30mm以后几乎不再摄食,出现生长停滞,并已变得十分衰弱。全面攻克日本鳗鲡人工育苗难题尚待时日,特别是研制能有效促进后期柳叶状仔鱼生长并变态的全价育苗饲料已成为目前关注的焦点。     

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.     

Decreasing dietary lipids improves larval survival and growth of Japanese eel Anguilla japonica

Shark eggs-based diet is the only diet by which eel larvae can grow to glass eels in captivity. However, the high level of lipids in the diet is suggested to negatively affect eel larvae. This paper examines the effect of defatted shark eggs (DSE) and hen egg yolk (HY) on growth and survival of larvae of Japanese eel Anguilla japonica. Lyophilized shark egg and commercial HY were defatted with n-hexane, and four experimental diets were prepared using both defatted and untreated shark eggs and HY. Larvae were reared for 3 weeks by feeding the experimental diets. The highest survival rate was observed in the larvae fed DSE, and larvae fed HY showed the lowest survival rate. The best growth was found in larvae fed DSE, followed by shark eggs and defatted HY, and the worst growth was in HY-fed larvae. These results show that decreasing dietary lipids improves the nutritional value of both shark eggs and HY for eel larvae. Regulation of the dietary lipid level may positively affect the larval performance of eels by combination of ingredients with a low lipid content.