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Hatchery produced leptocephalus larvae of the Japanese eel (Anguilla japonica) were reared on a research vessel and fed natural particulate organic matter (POM) collected in the Western North Pacific. A small net (36 cm diameter, 60 µm mesh) was vertically towed from 200 m depth to surface in 2013, and POM-enriched water (POMEW) filtered through 350 µm mesh was fed to the leptocephali for 11 days. Although the swollen gut corroborated the active ingestion of POM by the leptocephali, low survival rate and heavily melanized gut followed by necrosis in the mid-hindgut region of the leptocephali were observed. A large net (1.14 m diameter, 30 µm mesh) was used in 2014 and 2015, which was horizontally drifted subsurface (100–175 m). POMEWs filtered through 53 or 25 µm meshes were fed to the leptocephali for 5–18 days. Neither melanized gut nor necrosis occurred, but considerably low survival rate and little growth comparable with that in a starved condition were observed. No apparent shift in stable carbon and nitrogen isotope ratios was observed in the reared leptocephali. These indicated that the ingested POMs were not assimilated by the leptocephali and suggested that smaller particles may be important for the leptocephali.

Organogenesis of Japanese eels (Anguilla japonica) was investigated histologically from the late leptocephalus to the yellow eel stages. Early organogenesis, such as the formation of inner ears and the appearance of round blood cells that might be larval erythrocytes, had already begun at the late leptocephalus stage. During the first developmental phase (M1–M3 stages) of metamorphosing into early glass eels (G1 stage), the formation of gills and lateral muscles progressed conspicuously with a drastic body shape change from leaf-like to eel-like. In contrast, obvious regression in oesophageal muscle and pancreas occurred during metamorphosis. Formation of lateral line canals advanced continuously until the yellow eel stage. When the second developmental phase was initiated at the G1 stage, cone photoreceptor cells appeared, and the formation of oesophageal, stomach and intestinal muscles was initiated. Differentiation of gastric glands began at 1 week after metamorphosis. Erythrocytes increased continuously in density in glass eels and elvers (G1–E2 stages), and the morphological features of cone cells and olfactory epidermal cells became clearer with stage progression. In early elvers (E1 stage), the swimbladder initiated inflation, the stomach fully expanded and the rectal longitudinal fold changed to a circle. Swimbladder gas glands appeared in late elvers (E2 stage). In the yellow eels (juvenile stage), almost all organ structures were formed. These observations indicate that the organogenesis of A. japonica is ongoing after metamorphosis into glass eels, and the M1–E2 stages are considered to be a homologous phase to first metamorphosis, which is a transformation from the larval to the juvenile stages in other teleosts. In comparison to conger eels, the completion of the body shape change to eel-like occurs at the G1 stage, when organogenesis is still in progress, being followed by a prolonged duration of the G1–E2 stages before reaching the yellow eel juvenile stage, which may be a unique characteristic that is related to the early migratory life history of A. japonica. [ABSTRACT FROM AUTHOR]

During the induction of Japanese eel maturation, administering maturation-inducing steroids (MIS) or their precursors at an inappropriate maturational status is a major cause of poor egg quality. In this study, we investigated the feasibility of controlling oocyte maturation progress by rearing in cold and warm water to manipulate the timing of MIS administration. Mature females with oocytes at the migratory nucleus stage were reared for two terms (3 days to 1 day and 1 day to 0 days before MIS administration) at 20/20 °C, 20/15 °C, 15/20 °C, or 15/15 °C, and the maturational status was monitored based on their lipid droplet morphology and oocyte diameter. Oocytes matured faster at 20 °C than at 15 °C in either term. Next, the mature females were reared at 15 or 20 °C depending on the maturational status of each female 3 days and 1 day before MIS administration; the immature females were reared at 20 °C to accelerate their maturation. Consequently, the maturational status of most females was similar at MIS administration. After improvement, this method would lead most females to the optimum maturational status at MIS administration by properly rearing in cold and warm water. [ABSTRACT FROM AUTHOR]

The lipid droplet stage and oocyte diameter were determined using 10 oocytes from each female at each cannulation. a Frequency distribution of the lipid droplet stage from Monday to Thursday. b Changes in the lipid droplet stage. Values are the mean ± standard deviation of those for 3-7 females. d Rate of increase in oocyte diameter. [Extracted from the article]

The aim of this study was to develop sperm cryopreservation protocols for large-scale fertilization in Japanese eel ( Anguilla japonica ), by testing the feasibility of combining large-volume straws and a low sperm dilution ratio. Experiments were conducted using both 0.25-ml straws and 2.5- and 5-ml straws (macrotubes) for sperm cryopreservation. Cooling rates in macrotubes were measured at three internal positions and at different heights above the surface of liquid nitrogen (LN). There was no significant difference in the cooling rate among internal positions in the straws. The cooling rate could be adjusted to the appropriate range for Japanese eel sperm (6.3 °C–8.6 °C min −1 ) by positioning the 2.5- and 5.0-ml straw 0.5–5.0 cm above the surface of LN. We succeeded in scaling up the storage volume from 0.25 to 4.5 ml (18-fold) without decreasing the motility using a 5.0-ml straw. The number of storable motile spermatozoa was the highest at a range of dilution ratios (sperm to extender, v /v) from 1:1 to 1:5. Although frozen-thawed sperm showed a significant decrease in the proportion of motile sperm to egg compared with than fresh sperm, there was no significant difference in the fertilization rate, and fertilized eggs with cryopreserved sperm did not affect the hatching rate or survival rate at 7 days after hatching. Using a segmented linear analysis, we found that 10,180 ± 3077 motile spermatozoa per egg are required to maximize fertilization success. We estimated that spermatozoa stored in a 2.5- and 5.0-ml straw can fertilize approximately 184,185 and 331,532 eggs, respectively. Furthermore, our investigation of the effect of long-term storage in LN on the sperm quality showed that the storage period does not affect the post-thaw motility rate even after 2 years of storage. Our results demonstrate that cryopreservation techniques for Japanese eel sperm using 2.5- and 5.0-ml straws could be used for large-scale fertilization programs. [ABSTRACT FROM AUTHOR]

Japanese eel (Anguilla japonica) constitutes one of the most important food fish in Japan; accordingly, genome sequencing and linkage mapping have been conducted for the purpose of artificial cultivation. In the next stage, integration of genomic sequences within linkage groups (LG) is required to construct higher-resolution genetic markers for quantitative trait loci mapping and selective breeding of beneficial traits in farming. In order to identify LG1-linked scaffolds from the draft genome assembly (323,776 scaffolds) reported previously, we attempted to isolate chromosomes corresponding to LG1 by flow sorting and subsequent analyses. Initially, single chromosomes were randomly collected by chromosome sorting and subjected to whole-genome amplification (WGA). A total of 60 WGA samples were screened by PCR with primers for a known LG1-linked scaffold, and five positive WGA samples were sequenced by next-generation sequencing (NGS). Following reference mapping analysis of the NGS reads, four of the five WGA samples were found to be enriched by LG1-linked sequences. These samples were cytogenetically assigned to chromosome 5 by fluorescence in situ hybridization. Using blastn searches with 82,081 contigs constructed from the NGS reads of the four WGA samples as queries, 2323 scaffolds were identified as putative LG1-linked scaffolds from the draft genome assembly. The total length of the putative LG1-linked scaffolds was 99.0 Mb, comparable to the estimated DNA amounts of chromosome 5 (91.1 Mb). These results suggest that the methodology developed herein is applicable to isolate specific chromosome DNAs and integrate unanchored scaffold sequences onto a particular LG and chromosome even in teleost fishes, in which isolation of specific chromosomes by flow sorting is generally difficult owing to similar morphologies, sizes, and GC-contents among chromosomes in the genome. The putative LG1-linked scaffolds of Japanese eel contain a total of 6833 short tandem repeats which will be available for higher-resolution linkage mapping. [ABSTRACT FROM AUTHOR]

Bacteria with lethal effect on eel larvae were isolated from moribund captive-bred eel larvae and their 16S rRNA gene sequences were analysed. Nucleotide sequence of 10 isolates showed highest similarity with Lacinutrix algicola, Crocinitomix catalasitica and Pseudoalteromonas rubra. Age-dependent changes in the susceptibility of eel larvae were observed in response to challenge by a highly lethal isolate. Compared with 10 and 11 days after hatching (DAH), larval susceptibility to the highly lethal isolate was lower at 18 DAH. We found that the bacterial isolates have lethal effect on the captive-bred eel larvae, especially at the early developmental stage. These results will be useful to establish appropriate culture practices for eel larvae that will improve the success of mass production of glass eels for aquaculture. [ABSTRACT FROM AUTHOR]

Highlights: [•] Japanese eel larvae provide a good model for early development of Anguilliformes. [•] Cloned TR genes encode proteins conserved in their active regions. [•] TRs showed TH-dependent activation of transcription from a TH-responsive promoter. [•] Expression of TRαs peaked during pre-metamorphic stage. [•] Expression patterns during metamorphosis resembled those in other Anguilliformes. [ABSTRACT FROM AUTHOR]

Abstract: Our objective was to evaluate the effects of hormonal treatments on the milt quality during the induction of spontaneous spawning in male eels. We measured the changes in milt characteristics (sperm motility, milt pH, seminal plasma ionic concentration) during the course of hormonal treatments intended to induce spontaneous spawning, and before and after spawning. Male eels received weekly injections of hCG (1IU/gBW/wk) to maintain spermiation. To induce spontaneous spawning, males received another priming injection of hCG (1IU/gBW) two days before spawning, followed 24h later by an injection of 17α-hydroxyprogesterone (OHP: 1μg/gBW). Three males, treated as described above, and one female, that had received hormonal injections to induce vitellogenesis and final maturation, were transferred to a tank to spawn. In Experiment 1, we measured the characteristics of milt (1) before administering the priming dose (1–2days after the weekly injection of hCG), (2) before injection with OHP, (3) after spawning (24h after the OHP injection), and (4) before weekly injections with hCG (4–5days after OHP injection). To evaluate the effects of hormonal treatment on the characteristics of milt we administered the same treatments but did not allow spawning (Experiment 2). In both experiments, the percent motility remained high prior to OHP injection, decreased significantly 24h after OHP injection, then recovered by the beginning of weekly hCG injection. The changes in potassium ion concentration were similar to those in sperm motility (%) but there was no change in pH and sodium ion concentrations. When the results were analyzed by individual in Experiment 1 (n=34), the relationship between sperm motility (%) and milt pH showed significant correlations without 24h after the OHP injection. The relationship between sperm motility (%) and potassium ion concentration showed a highly significant correlation 24h after the OHP injection. Thus, both pH and potassium ion concentration regulate motility in Japanese eel spermatozoa during hormonal treatment. Furthermore, administration of OHP into spermiating males reduced motility by decreasing the potassium ion concentration in the seminal plasma. [Copyright &y& Elsevier]

The Japanese eel (Anguilla japonica) is one of the most important aquaculture fish species in Eastern Asia. A major bottleneck in the mass production of glass eels is the long larval period that they undergo in captivity. In the present study, 1689 glass eels, originating from a partly factorial cross (43 sires × 32 dams), were used to investigate the potential value of genomic selection for 10 traits related to the timing of metamorphosis and the body size at that time. Genotyping by random amplicon sequencing-direct (GRAS-Di®) sequencing was used to identify and genotype single nucleotide polymorphism (SNP) markers, which were used to assign parentage and as a basis for genomic selection. The genomic heritabilities of the 10 metamorphic traits obtained by genomic best linear unbiased prediction (GBLUP) models were moderate (range 0.21–0.38), and the standard errors were smaller than those determined by the pedigree-based best linear unbiased prediction (ABLUP) models. The genomic prediction of breeding values outperformed the pedigree-based prediction, resulting in a 3%–19% improvement in the prediction accuracy. The impact of reduced SNP densities on the predictive ability of GBLUP was tested using a linkage disequilibrium-based pruning and random thinning method to reduce 17,017 SNPs to 2117 SNPs. An SNP set of 8000 was considered to be sufficient to achieve predictive ability comparable to that obtained using higher SNP densities for both methods. The selective genotyping of the top individuals in the training population led to a severe reduction in the predictive ability compared with the random genotyping of the same number of samples. These results will be useful for decision-making in establishing best-practice breeding programs when applied to genomic selection for metamorphic traits for shortening the larval period and improving seed quality in Japanese eels. • Heritabilities of the 10 metamorphic traits were moderate. • The GBLUP model with >8000 SNPs showed sufficient prediction accuracy. • Selective genotyping of top individuals significantly impacted prediction accuracy. • GRAS-Di genotyping is an effective tool for genomic selection in eels. • Genomic selection was shown to be valuable for shortening the larval period. [ABSTRACT FROM AUTHOR]

Abstract: Spontaneous polyploids and mosaics have often been observed in artificially propagated larvae of the Japanese eel, Anguilla japonica. However, the mechanisms responsible for such unusual cytotypes are unclear. In this study, we examined the relationship of such polyploidization and mosaicisms in larvae resulting from artificial propagation to egg quality (fertilization rate and hatching rate) and viability of larvae, and then clarified the inducing factors and the mechanism for occurrence of such phenomena. Eggs stripped from females after induced maturation were artificially inseminated with sperm pre-cultured with artificial seminal plasma. Ploidy was determined by measuring the relative DNA content of the nuclei with flow cytometry. Of 968 embryos from 32 full-sib families, 9.1% were determined to be abnormal, most of which were triploids (86.5% of abnormal embryos); others were haploids (1.1%), aneuploids (2.3%), and mosaics (10.1%). The percentage of normal diploids from each family varied between 56.3% and 100% (90.9±11.7%, n =32). A significant positive correlation was found between the fertilization rate (P <0.001) or the hatching rate (P <0.001) and the percentage of diploids. Survival rate of triploid eels was similar to diploid eels at 10days after fertilization whereas aneuploids were inviable. When eggs were left in the body cavity of the female for four hours after ovulation and subsequently fertilized, the percentage of diploids decreased. We tried to elucidate the cause for the occurrence of spontaneous triploids by genetic analysis using 26 microsatellite DNA markers, which have been developed and mapped in relation to the centromere. These results suggest that the occurrence of cytogenetically unusual progeny is associated with over-ripening or aging of ova caused by the lapse of time from ovulation until fertilization, and spontaneous triploid larvae are derived from the duplication of the maternal chromosome set by inhibition of the second polar body release after normal meiosis I (crossing over) in oocyte and fertilization with normal sperm. [Copyright &y& Elsevier]

The pancreatic digestive enzymes, trypsin, chymotrypsin, lipase and amylase were partially characterized, and changes in their activities were examined during the initial ontogeny of Japanese eel Anguilla japonica larvae from 5 to 34 days post-hatching (dph). The pH optima of the eel larval enzymes were narrower than those other fish species; trypsin activity was highest at pH 9, chymotrypsin and amylase activities were highest at pH 7 and 8, and lipase activity was highest at pH 8 and 9. In an analysis of thermal profiles, the larval pancreatic enzymes had a high optimal temperature and high thermal stability, which are typical of fish from the tropics. At 12 and 13 dph, lipase activity and gene expression levels of trypsin (- a and - b), lipase and amylase decreased markedly, suggesting a marked change in larval metabolism at that time. These data could be useful in the development of artificial larval diets in Japanese eel. [ABSTRACT FROM AUTHOR]

The present study analyzed the rate of occurrence of deformities at completion of yolk resorption after Japanese eel larvae were exposed to low-salinity (29 psu) or low-water-temperature (20 °C) conditions (after they had been reared under suitable conditions: 34 psu, 25 °C) at various points between 0 and 8 days post-fertilization (dpf) in order to determine the period of sensitivity of the larvae to low-salinity or low-temperature conditions. No significant differences were observed between the rate of deformed larvae obtained in the groups reared under suitable conditions and the corresponding rates in groups that were exposed to low-salinity or low-temperature conditions at 4 dpf or later, but the frequency of normal larvae showed a downward trend in experimental groups that were subjected to low-salinity or low-temperature conditions at 3 dpf or earlier. Occurrence rates of pericardial edema were significantly higher in experimental groups that were exposed to low-salinity or low-temperature conditions at 2 dpf or earlier. Therefore, it was concluded that the rearing conditions for Japanese eel larvae should be maintained at 34 psu salinity and 25 °C until at least 4 dpf in order to reduce the occurrence of larval deformity at completion of yolk resorption. [ABSTRACT FROM AUTHOR]

Primordial germ cells (PGCs) are segregated and specified from somatic cells during early development. These cells arise elsewhere and have to migrate across the embryo to reach developing gonadal precursors. Several molecules associated with PGC migration (i.e. dead-end, nanos1, and cxcr4) are highly conserved across phylum boundaries. However, since cell migration is a complicated process that is regulated spatially and temporally by multiple adaptors and signal effectors, the process is unlikely to be explained by these known genes only. Indeed, it has been shown that there are variations in PGC migration pattern during development among teleost species. However, it is still unclear whether the actual mechanism of PGC migration is conserved among species. In this study, we studied the migration of PGCs in Japanese eel (Anguilla japonica) embryos and tested the migration mechanism between Japanese eel and zebrafish (Danio rerio) for conservation, by transplanting eel PGCs into zebrafish embryos. The experiments showed that eel PGCs can migrate toward the gonadal region of zebrafish embryos along with endogenous PGCs, even though the migration patterns, behaviors, and settlements of PGCs are somewhat different between these species. Our results demonstrate that the migration mechanism of PGCs during embryonic development is highly conserved between these two distantly related species (belonging to different teleost orders). [ABSTRACT FROM AUTHOR]

Abstract: The first genetic linkage map of the Japanese eel (Anguilla japonica) was constructed based upon Amplified Fragment Length Polymorphism (AFLP) and microsatellite (STR) markers with an F1 pseudo-testcross strategy. The segregation of 106 microsatellite loci, which included 74 newly developed, and 463 polymorphic loci identified from 56 AFLP primer combinations was studied in 46 F1 individuals derived from a single female and a male. A total of 319 markers (99 STRs+220 AFLP) are placed on the female map and 314 markers (91 STRs+223 AFLP) on the male map. The female map spans 1732.4cM (Kosambi) in 22 linkage groups with an average spacing of 7.2cM; the male map spans 964.9cM in 22 linkage groups with an average spacing of 6.3cM. The average ratio of male:female recombination rates was 1:2.11. The estimated coverage of the genome was 82.4% for the female map and 74.0% for the male map. This map is the first linkage map in the order Anguilliformes and provides a basis for mapping quantitative trait loci (QTL) and for breeding applications. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

The occurrence of morphological deformities under different rearing water temperatures (18, 20, 22, 24, 26, 28, and 30 C) was examined in Japanese eel larvae. The rates of hatching and survival until yolk resorption at 22–26 C were higher than those at other water temperatures. Fertilized eggs never hatched at 18 and 30 C. The rates of occurrence of abnormal larvae reared at the water temperatures 24–28 C were lower than those at 20 or 22 C. Pericardial edema and lower jaw deformities occurred most frequently at lower temperatures (20 and 22 C). In contrast, the incubation temperature did not significantly affect the relative frequency of some neurocranial deformities and of spinal curvature. These results imply that the optimal temperatures for rearing Japanese eel eggs and embryos are 24–26 C from the viewpoints of survival and deformity. [ABSTRACT FROM AUTHOR]

Abstract: Genetic improvement of the Japanese eel (Anguilla japonica) can be achieved by artificially controlling its life cycle using recent advances in reproductive biology. In this study, we developed 43 microsatellite loci to confirm Mendelian inheritance at 10 of them as well at 16 previously reported in two full-sib families produced by artificial insemination. In order to establish a base for aquaculture genetics of this species in the near future, these microsatellite loci were mapped in relation to the centromere by half-tetrad analysis using four artificially induced triploid families. The second division segregation frequency (y) of the microsatellite loci ranged from 0.008 to 0.968 (mean±SD=0.645±0.298). These results suggest the presence of strong chiasma interference in the eel. Significant differences were observed for the map distances of microsatellite loci between the two isolation procedures. Microsatellites isolated using the enrichment procedure were mapped to various sites starting from the centromere to the telomere, whereas those from the conventional size-selected library showed a tendency to be distributed in the telomeric region. [Copyright &y& Elsevier]

The Japanese eel (Anguilla japonica) spends a long period as the leptocephalus larval form under current rearing conditions. The duration of the larval stage until metamorphosis is influenced by body size and growth; however, little knowledge exists of the regulatory mechanism of growth in eel larvae. The present study focused on growth hormone (GH), insulin-like growth factors (IGFs), and IGF binding protein (IGFBP) as the central regulators of growth in teleost fishes and transforming growth factor-beta 3 (TGF-β3) as a possible key modulator of muscle growth and body component synthesis. Japanese eel IGFBP-1a and TGF-β3, comprising 264 and 411 amino acids, respectively, were cloned. Short-term (5-day) fasting and refeeding experiments were performed to understand changes in growth-related genes affected by nutritional status. The relative expression of gh increased with fasting and subsequently decreased with refeeding to the basal levels of the fed control. Relative igf-1 and igf-2 expression levels were high in the fasted group. Relative igf-1 was reduced after 2-day refeeding, whereas igf-2 decreased to the basal level after 1-day refeeding, suggesting that IGF-1 and IGF-2 might be regulated independently and contribute to postnatal growth in eel larvae. Relative igfbp-1a expression was sharply increased by fasting, whereas tgf-β3 showed high and low values in the fed and fasted groups, respectively. Relative igfbp-1a and tgf-β3 levels were negatively and positively correlated with body size, respectively. These results suggest that igfbp-1a and tgf-β3 are potential indices of growth for exploring optimal rearing conditions to shorten the larval stage in Japanese eels. [Display omitted] • Fasting increased the expressions of gh , igfs , and igfbp-1a in early larvae. • Expression of IGFBP-1a is a useful indicator of growth retardation and stress. • TGF-β3 gene would involve in growth in the early larvae of Japanese eel. [ABSTRACT FROM AUTHOR]

• Recombinant Japanese eel growth hormones with CTP were produced. • Recombinant Gh was found to promote the growth of eel larvae. • Addition of CTP to recombinant Gh enhanced growth promoting effect. • Recombinant Gh may have negative effects on the Gh receptors and Igf-I. Growth hormone (Gh) regulates somatic growth in fishes, particularly through the Gh - insulin-like growth factor-I (Igf-I) axis. In this study, recombinant Japanese eel Ghs with or without C-terminal peptides of human chorionic gonadotropin (CTP), which are known to prolong the half-life, were produced using the HEK 293 and CHO expression system. The effect of recombinant Gh administration to eel larvae on their somatic growth was investigated in short-term feeding experiments, and it was found that three types of recombinant Ghs with CTP (CTP-reGh, reGh-CTP and reGh-CTP × 2) were more effective in promoting somatic growth in eel larvae than recombinant Ghs without CTP. Among the three recombinant Ghs with CTP, reGh-CTP × 2 had the highest growth-promoting effects, however only when provided in the short term. After long-term administration of reGh-CTP × 2, there was no difference in growth between the Gh administrated group and the control group. The survival rate of eel larvae were not affected by recombinant Ghs. In addition, the mRNA expression of gh , Gh receptors, Igf-I and IGF-II were measured by quantitative real-time PCR, and significant reductions in the expression of gh, Gh receptors and Igf-I were observed. These findings provide useful tools to study the mechanisms of somatic growth and increase understanding of Gh regulation in anguillid eel larvae. [ABSTRACT FROM AUTHOR]

Abstract: To elucidate the utilization of major yolk nutrients in eggs and yolk-sac larvae of Japanese eel Anguilla japonica, contents of free amino acids (FAA), 380 kDa lipovitellin (oLv; the major yolk protein in ovulated eggs), and lipids were measured. All larvae hatched by the 2nd day after fertilization at a water temperature of 23 °C, and the hatched larvae absorbed almost all of the yolk mass by the 10th day. The FAA composition in ovulated eggs is unique in that glutamine (Gln) is markedly high (32% of total FAA) in comparison to other pelagic eggs of marine teleosts suggesting the importance of Gln for early development of the Japanese eel. Total FAA content decreased to 45% of the initial level by the 4th day after fertilization. Gln content showed a rapid decrease to 5% of the initial level within 2 days after fertilization. oLv content, measured by enzyme-linked immunosorbent assay using an antiserum against lipovitellin, decreased until the 4th day and was, thereafter, undetected. Total content of triacylglycerol (TG) was stable until the 2nd day, and then decreased until the 8th day. On the other hand, the content of phospholipids (PL) gradually decreased until the end of yolk sac absorption. From these data, we divided the pattern of nutrient utilization in embryos and larvae of Japanese eel into two phases: first, utilization of FAA (especially Gln) and oLv from the 1st day, followed by TG utilization from the 2nd to 4th day by which time more than 30% of the FAA and the oLv stock have been utilized. In addition, PL was utilized as a subsidiary energy source throughout development. Almost all the yolk nutrient were absorbed by the 8th day after fertilization, and it is considered that the larvae need to start feeding before this period. [Copyright &y& Elsevier]

In the present study, methods for the cryopreservation of Anguilla japonica spermatozoa were examined. Spermatozoa were collected from artificially matured males and incubated in Japanese eel K30 artificial seminal plasma (K30 ASP) before experiments. Sperm motility was investigated using computer-assisted sperm analysis (CASA). 10% and 15% MeOH as cryoprotectant was the most successful cryoprotectant with percentage of the initial motility of 59.7 ± 12.1%; the combination of 5% MeOH and 5% DMA was also viable. DMSO was unsuitable as a cryoprotectant with K30 ASP as it showed no cryopreservation properties and was toxic to sperm, causing sperm to be immotile immediately after dilution. Japanese eel spermatozoa had a narrow range of optimal cooling rates (6.3–28.6 °C min − 1 ) and immersion temperatures of − 40 to − 70 °C were effective. The different fetal bovine serum (FBS) concentration in extender, temperature of milt before cooling, dilution rates (3–100 times) and equilibrium time showed no significant difference. The results indicate that the type of extender media greatly influenced the suitability of cryoprotectant. The establishment of a protocol to cryopreserved Japanese eel sperm will benefit the artificial seed production of Japanese eel and provide an important tool for genetic and breeding studies. Statement of relevance The development of the sperm cryopreservation protocol for Japanese eel will ensure a stable supply of high quality seed for experimental and commercial purposes. [ABSTRACT FROM AUTHOR]

Abstract: To obtain fertilized eggs of the Japanese eel Anguilla japonica, mature females are injected with maturation-inducing steroid (MIS) to induce final oocyte maturation and ovulation and then paired with mature males for spawning (induced spawning method) or stripped to collect eggs for insemination (stripping and insemination method). Progress of final oocyte maturation and ovulation and consequent egg quality are affected by the environmental water temperature. To detect the optimum temperature of the rearing water during artificial induction of ovulation by MIS injection, we investigated the effects of water temperature on ovulation, egg quality, and its deterioration due to over-ripening using the stripping and insemination method. Mature females injected with 17α-hydroxyprogesterone were stocked in tanks maintained at 17.5°C, 20°C, 22.5°C or 25°C. Earlier ovulation occurred at the higher water temperatures. Viability of the eggs that were collected from females immediately after observation of ovulation was similar at 20°C and 22.5°C but inferior at 17.5°C and 25°C. After eggs are retained in the body cavity for an hour, their viability was increasingly reduced due to over-ripening at the higher temperatures between 20°C and 25°C. Moreover, hatchability of unfertilized eggs placed in tubes and maintained at 15°C, 17.5°C, 20°C, 22.5°C or 25°C for 3h showed a time-dependent decrease due to over-ripening, which progressed slower at the lower temperature band between 20°C and 25°C. However, 15°C and 17.5°C did not delay the progress of over-ripening compared to 20°C. We conclude that 17.5°C and 25°C are not appropriate for ovulation induction, because egg quality just after ovulation is lower than at 20°C and 22.5°C. In the stripping and insemination method, 22.5°C is disadvantageous compared to 20°C because of the faster progress of over-ripening, though this does not matter in the induced spawning method as spawning behavior and consequent fertilization are expected to occur at an appropriate timing after ovulation. [Copyright &y& Elsevier]

Abstract: The quality of eggs obtained from maturation-induced Japanese eel, Anguilla japonica, is unstable. One of the causes for low quality eggs is that females are artificially induced to ovulate at an inappropriate maturational status. Here we investigated the feasibility to use the morphological changes of lipid droplets in oocytes as a biomarker to show the optimum timing for injections of salmon pituitary extract (SPE) for priming and maturation-inducing steroid (MIS) for final oocyte maturation and ovulation. Various sizes of oocytes (700 to 1000μm) were collected by cannulation from maturation-induced female eels during five days until ovulation. Morphology of the lipid droplets in these oocytes were classified into 10 stages mainly on the basis of their diameter, which increased with the progress of maturation as the droplets fused together. To assess the maturational status of each female, the lipid droplet stage was determined for 10 oocytes randomly chosen among the most advanced mode of developing oocytes and the median value was defined as the stage for the individual. Relationship between the lipid droplet stage at induction of ovulation and hatchability of the ovulated eggs was examined in 23 females, in which the hatching rate markedly fluctuated. Stages of females from which good quality eggs were obtained converged around stage 4 (five largest droplets, 55 to 70μm) at priming with SPE, stage 6 (90 to 110μm) at injection with MIS, and stage 7 (110 to 130μm) at ovulation, while those of females from which low quality eggs were obtained showed distributions over broader ranges. Furthermore, 466 females were induced to mature and the lipid droplet stage at induction of ovulation was examined in 13 females from which high quality eggs (hatching rate >80%) were obtained. Most of the 13 females were at stage 3 (40 to 55μm) or 4 when primed with SPE, at stage 6 when injected with MIS, and at stage 7 when they ovulated. These results suggest that the optimum stages for SPE priming, MIS injection, and ovulation are stages 3 to 4, 6, and 7, respectively, though the optimum stage for priming will be variable if the temperature and/or the time between priming and MIS injection is varied. We conclude that the lipid droplet stage can be used as a sensitive biomarker to show the optimum timing for inducing ovulation by hormonal treatments. [Copyright &y& Elsevier]

Abstract: Pepsinogen is the precursor form of the gastric-specific digestive enzyme, pepsin. Ghrelin is a representative gastric hormone with multiple functions in vertebrates, including the regulation of growth hormone release, stimulation of food intake and gastrointestinal motility function. We investigated chronological changes in the distribution of pepsinogen-expressing cells by in situ hybridization and ghrelin-immunoreactive cells by immunohistochemistry in the Japanese eel (Anguilla japonica) during metamorphosis from the leptocephalus sage to the elver stage. The ghrelin-producing cells first appeared in the gastric cecum and pyloric portion of the stomach in the late phase of metamorphosing leptocephali, whereas the pepsinogen-producing cells were first detected in the early phase of the glass-eel stage. These suggest that endocrine cells differentiated earlier than exocrine cells in the eel stomach. Accompanying eel development, the distribution of ghrelin-producing cells spread to the esophagus and other regions of the stomach, but not to the intestine. These results may be related to the changes in dietary habits during metamorphosis in the Japanese eel. [Copyright &y& Elsevier]

Abstract: The occurrence of morphological deformities under different incubation salinities (24, 30, 33, 36 and 42 practical salinity units [psu]) was examined in Japanese eel larvae. Fertilized eggs hatched at all salinity treatments. The survival rate until yolk resorption decreased at higher than 36 psu. The proportions of deformed larvae reared at 36 psu were significantly lower than at other treatments. Pericardial edema and abnormal lower jaw increased at lower than 33 psu and spinal curvature occurred at high salinity (42 psu). In contrast, the incubation salinity did not significantly affect the relative frequency of abnormal neurocranium. These results imply that the optimal salinities for rearing Japanese eel eggs and embryos are 34–35 psu from the viewpoints of survival and deformity. Taken together with previous study [Kurokawa, T., Okamoto, T., Gen, K., Uji, S., Murashita, K., Unuma, T., Nomura, K., Matsubara, H., Kim, H.S., Ohta, H., Tanaka, H., in press. Influence of Water Temperature on Morphological Deformities in Cultured Larvae of Japanese Eel, Anguilla japonica, at Completion of Yolk Resorption. J. World Aqua. Soc.], the rearing of eel eggs and embryos at 25 °C and 34–35 psu probably decreases the mortality and deformity rate of eel larvae at yolk resorption stage. [Copyright &y& Elsevier]

Abstract: Eggs from maturation-induced Japanese eels, Anguilla japonica, often sink in seawater immediately after artificial insemination and do not hatch. In the present study, the specific gravity of unfertilized eggs from individual females was measured in isotonic (about 310 mOsm/kg) and hypertonic (about 875 mOsm/kg) saline solutions and the relation of specific gravity to egg quality was examined. Egg specific gravity under isotonic conditions showed a significant negative correlation with egg fertility, hatchability and water content, suggesting that inadequate hydration of oocytes during final maturation, which leads to insufficient egg buoyancy, is one of the causes of poor egg quality. Some of the eggs that showed lower specific gravity than seawater under isotonic conditions exhibited higher specific gravity than seawater under hypertonic conditions, indicating that the buoyancy acquired by the eggs is sometimes lost after their transfer to seawater. Only eggs that retained low specific gravity under hypertonic as well as isotonic conditions exhibited high fertility and hatchability. Taken together with data from other studies, these results suggest that the poor quality of eggs that sink immediately after artificial insemination is attributable to at least two causes: the failure of oocytes to acquire sufficient buoyancy during maturation and the loss of buoyancy in seawater because of the inability of egg osmoregulation. [Copyright &y& Elsevier]

A method was developed for assessing fertility, hatchability and larval survival in the Japanese eel, Anguilla japonica. Fertilized eggs were stocked in 24-, 48-, and 96-well plates at one egg to one well and maintained at 23 °C without removing dead larvae, changing the water or feeding. Upon hatching, the larvae were often trapped on the surface of the water where they died (surface death). The remaining larvae then showed rather stable survival in the three types of plates beyond the yolk-sac stage, which was completed by day 8 after hatching (DAH). Polyethylene glycol 6000 (PEG 6000) added to the rearing water at 1 or 10 μg/ml markedly suppressed surface death without apparent effect on hatchability, the survival profile or abnormality. These results indicated that the survival rate can be determined easily by stocking the fertilized eggs individually in the wells of microplates with water containing PEG 6000 and by rearing the hatched larvae until the completion of yolk absorption, with little management. By this method, a series of parameters for evaluating the egg quality, rates of fertilization, hatching, survival and abnormality were determined in the artificially maturation-induced Japanese eel: unstable fertility and frequent abnormal development were revealed to be impediments to constant production of larvae at first feeding. [Copyright &y& Elsevier]