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.     

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.     

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.     

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。     

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.     

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.     

Demonstration of infectious pancreatic necrosis virus strain VR-299 in Japanese eel, Anguilla japonica Temminck & Schlegel

Abstract. Six isolates of infectious pancreatic necrosis virus (IPNV) were obtained from trout or eel farms in Taiwan in 1987. By using the electrophoretic analysis of ³⁵S-methionine-labelled early viral polypeptide patterns and silver-stained ds RNA gel patterns, a comparison of these isolates with the three archetypal IPNV serotypes (AB, SP and VR-299) was made. They were all identical with VR-299, both in the RNA and early viral polypeptide patterns. From the results obtained, it was apparent that the VR-299 serotype of IPNV was just as widespread in eel as in trout in Taiwan. This is the first case of VR-299 isolation from eel.     

Assessing sexual maturity of feminized Japanese eel Anguilla japonica by measuring eye size

For efficient production of Japanese eel Anguilla japonica eggs, knowledge of the status of the sexual maturity of potential broodstock females is important because this status directly influences the time required to produce mature eggs by successive hormone doses. Here we apply an eye index (relative eye size) to evaluate the gonadal status of feminized A. japonica, which were induced by administration of estradiol-17β. Examination of gonad somatic and eye indexes of 267 feminized eels, cultivated for 12–56 months, revealed a significant correlation between these two indexes. Artificial maturation of 35 feminized eels showed that the number of hormone injections administered before ovulation was significantly lower as the eye index score increased, indicating availability of this noninvasive method of predicting sexual maturity of feminized eel. There was no correlation between eye index and fertilization rate, hatching rate, or larval survival rate, suggesting that sexual maturity before hormonal treatment does not affect egg quality.     

Fine structure and differentiation of the alimentary canal in captive-bred Japanese eel Anguilla japonica preleptocephali

ABSTRACT:   The fine structure of the alimentary canal in preleptocephali produced by artificially matured Japanese eel was examined. At 1 day posthatch (dph), the alimentary canal was found only above the dorsal side of the yolk mass, and the epithelium was composed of a single layer of epithelial cells. By 5 dph, the alimentary canal was divided into three segments based on the structure of the epithelial cells: foregut, midgut and hindgut, corresponding to the future esophagus, intestine and rectum, respectively. After 7 dph, the epithelium in the foregut was surrounded by a circular muscle layer, suggesting a role in the transportation of food materials. The epithelial cells of the midgut exhibited well-developed membranous structures, which are deduced to be invaginations of the cytoplasmic membrane. Pinocytotic invaginations and vacuoles were observed in the epithelial cells of the hindgut; this observation suggests that this region is involved in the uptake of food. Significant changes in morphological features of the epithelial cells in each segment were observed until 7 dph; however, these were not evident between 7 dph and 13 dph. Consequently, the differentiation of the alimentary canal was completed by 7 dph, and preleptocephalus had developed the ability to absorb food by 7 dph.     

Androgen secretion activity of recombinant follicle-stimulating hormone of Japanese eel, Anguilla japonica in immature and maturing eel testes

The androgen secretion activities of recombinant Japanese eel follicle-stimulating hormone (rjeFSH) were investigated in immature and maturing eel testes. The rjeFSH stimulated testosterone (T) and 11-ketotestosterone (11-KT) secretion in immature testis but not in maturing testis. This result suggests that eel FSH plays an important role through the sex steroid secretion in immature testis rather than in maturing testis.