Compositions and functions of the hatching froth from ricefield eel (<Emphasis Type="Italic">Monopterus albus</Emphasis> Zuiew)

The ricefield eel (Monopterus albus) is an economic fish species in China. To improve its artificial reproduction, we studied the compositions and functions of the hatching froth secreted by ricefield eels. The froth showed a viscosity of 2.72 mPaS, composed of glycoproteins with a sugar content of 0.156 mg/ml and a protein content of 0.250 mg/ml at a ratio of 1.60. The froth proteins were identified by SDS-PAGE as four proteins with the molecular weights of 42.5, 58.6, 65.3, and 98.2 kDa respectively. Their total amino acid content was 201.88 mg/100 ml, consisting of 16 kinds of amino acids. Filed observation revealed three beneficial effects of the froth on the hatching of the eel eggs. First, the froth increased the hatching rates of fertilized eggs at a range of water temperatures. Second, the froth decreased the rates of infection of the fertilized eggs by the water mold. Finally, the froth accelerated velum breakage in the fertilized eggs. Of the different hatching methods, froth incubation achieved the highest hatching rate and larvae survival rate. These results demonstrate the indispensable function of the froth of ricefield eels, and offer practical reasons for protecting the froth during the breeding season.     

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.     

In vitro steroidogenesis by testicular fragments and ovarian follicles in a hybrid sturgeon, Bester

In this study, developmental changes in the steroidogenic capacity of testicular fragments and isolated ovarian follicles of a hybrid sturgeon, Bester, at a variety stage of developments were examined. Testicular fragments or isolated ovarian follicles were incubated in L-15 medium in the presence or absence of different concentrations of five preparations; forskolin, human chorionic gonadotropin (HCG), pregnenolone (P₅), 17-hydroxyprogesterone (17OHP) and testosterone (T) for 18 h at 15 °C. After incubation, concentrations of 11-ketotestosterone (11 KT) (testis) and, 17-estradiol (E₂) (ovarian follicles) and 17,20-dihydroxy-4-pregnen-3-one (DHP) (testis and ovarian follicles) were measured. 11KT was detected in the media following incubation with P₅, 17OHP and T. Its concentration was higher during late spermatogenesis and prespermiation and lower at the degeneration stage. Both P₅ and 17OHP were converted to DHP during the prespermiation stage. Forskolin had little stimulatory effect on the synthesis of 11KT and DHP and HCG did not induce the production of these steroids.E₂ was detected in the medium following incubation of follicles with P₅, 17OHP and T at all stages of oocyte development. The concentration of E₂ in the medium increased during vitellogenesis with the peak production occurring at the tertiary yolk stage. In contrast, the potencies of follicles to produce steroids shifted to the production of DHP during migratory nucleus stage. Forskolin and HCG had little effect on the synthesis of E₂ and DHP. These results demonstrated that the failure of spontaneous spermiation or ovulation is not due to the insufficient synthesis of DHP, but may due to the lack of availability of precursors.     

Effects of water temperature on early development of Japanese eel <Emphasis Type="Italic">Anguilla japonica</Emphasis>

ABSTRACT:   To determine an optimal temperature range for efficient production of healthy eel larvae Anguilla japonica , the effect of water temperature on hatching, survival, and deformity rates was examined. The early ontogeny of morphological features in this species by incubating eggs at five different temperatures (19, 22, 25, 28 and 31°C) was examined. Hatching occurred at 24, 28, 46 and 58 h after fertilization when incubated at 28, 25, 22 and 19°C, respectively; no eggs hatched at 31°C. The growth rate of prefeeding larvae increased as water temperature was elevated and acquisition of feeding ability was also accelerated at higher temperature. Significantly high hatching rates (76–86%, P  < 0.05) and survival rates (61–86%, P  < 0.05) were observed at relatively high temperatures (22–28°C). The deformities were 'open lower jaw', 'pericardial edema', and 'notochordal bending', in which the mouth was deformed with a downward projecting lower jaw, the pericardial cavity was swollen, and the notochord was bent or twisted to various degrees, respectively. Open lower jaw and pericardial edema were especially predominant at 19°C, with rates of 68 and 92%, respectively, compared with 31 and 10% at 25 and 28°C, respectively. The occurrence of notochordal bending was not affected by temperature. The optimal temperature for incubation and rearing A. japonica eggs and prefeeding larvae is approximately 25–28°C.     

The first production of glass eel in captivity: fish reproductive physiology facilitates great progress in aquaculture

Weekly injections of salmon pituitary extracts (SPE) were administered to female Japanese eel, Anguilla japonica at a dose of 20 mg/fish. This induced vitellogenesis and caused oocytes to reach the migratory nucleus stage. Later, a majority of the females that received an injection of SPE at a priming dose, followed 24 h later by 17,20β-dihydroxy-4-pregnen-3-one (DHP), ovulated 15 to 18 h after the final injection. In cultivated males, repeated injections of human chorionic gonadotropin (hCG) at a dose of 1 IU/g BW/week induced spermatogenesis and spermiation. Since potassium ions were revealed to be an essential constituent for the maintenance of motility in the eel spermatozoa, artificial seminal plasma containing KCl was designed as a diluent of milt, and enabled the preservation of milt for several weeks in refrigeration. As a result, artificial fertilization performed immediately after ovulation with pre-diluted and stocked milt consistently resulted in the production of high-quality gametes. Recently, a slurry-type diet made from shark egg yolk has been found to be a suitable feed for captive-bred eel larvae. Although preleptocephalus larvae can be reared with this diet beyond the depletion of their yolk and oil droplet stores, it remains inadequate because larvae reared under this way cannot be raised to the following stage. Therefore, the diet was improved by supplements of krill hydrolysate, soybean peptide, vitamins and minerals. Larvae fed on this new diet have grown to 50 to 60 mm in total length (TL), and have begun to metamorphose into glass eels approximately 250 days after hatching.     

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.     

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.     

Biochemical composition of eggsin relation to egg quality in the Japanese eel, Anguilla japonica

This paper presents the relationship between egg quality and egg biochemical composition of cultured and wild Japanese eel, Anguilla japonica. Eggs were obtained by artificialinduction of maturation. Fertilization and hatching rates were used as characteristics of egg quality. Egg quality characteristics showed large variation; fertilization rate, 0–96; hatching rate, 0–84%. The biochemical composition also showed a large variation. There was no marked relationship between egg quality and fatty acid contents of eggs, except for n-6 highly unsaturated fatty acids (HUFA). Both the fertilization and hatching ratesincreased proportionally withincreases of the -tocopherol g(-Toc) contentin eggs. A more significant correlation was found between the amount of -Toc relative to the amount of HUFA and egg quality. The results of this study show that the egg quality of Japanese eel is affected by the –Toc level, andin particular, the ratio of -Toc to HUFAin the eggs. Abbreviations: BHT – butylhydroxytoluene; EFA – essential fatty acids; FAME – fatty acid methyl esters; HPLC – high performance liquid chromatography; HUFA – highly unsaturated fatty acids; NADH – nicotinamide adenine dinucleotide; NADPH - nicotinamide adenine dinucleotide phosphate; ROS – reactive oxygen species; -Toc –-tocopherol.     

大黄鱼（♀）与黄姑鱼（♂）人工杂交子代的胚胎发育

2008年10月,在福建宁德水产技术推广站进行了大黄鱼（Pseudosciaena crocea,♀）×黄姑鱼（Nibea alb-iflora,♂）杂交,观察了杂交子代胚胎发育。大黄鱼和黄姑鱼亲鱼平均体质量和体长分别为405 g、27.9 cm和390g、24.8cm,采用干法人工授精。受精卵浮性,为单油球端黄卵,卵径1060～1500μm,油球379.8μm左右。在水温25.8～26.2℃,盐度26的条件下,经18h55min半数受精卵破膜,胚胎发育分为卵裂期、囊胚期、原肠期、肌节出现期、心跳期、肌肉效应期、出膜前期和出膜期,初孵仔鱼全长2.75～2.98mm,受精率约为14.4%,孵化率约为31.9%,畸形率约为30%。共培育杂交初孵仔鱼5万尾。3日龄仔鱼开口摄食轮虫,7日龄开始摄食小型挠足类。     

Evaluation of different procedures for fertilization and larvae production in Hediste diversicolor (O.F. Müller, 1776) (Nereididae,Polychaeta)

Different experimental trials were performed to clarify some aspects of the biology of the polychaete Hediste diversicolor (O.F. Müller, 1776) as a further step towards the development of appropriate breeding protocols for indoor farming systems. In particular, the trials were addressed to evaluate the effectiveness of two fertilization conditions (in vitro and “natural‐like”); induce gamete spawning by exposing mature individuals to thermal shock or to tissue homogenates; estimate the density effects on larval growth and survival; and evaluate the most suitable parameters to be used as proxy for biomass assessment. The highest percentages of fertilized eggs and larvae were obtained by the in vitro fertilization condition. Mature organisms were induced to spawn by exposure to thermal shock although the spawned eggs revealed low rates of fertilization and hatching. The treatment with male tissue homogenates induced females to successful spawning, and the resulting eggs showed high fertilization and hatching rates. The density of larvae in the rearing phase had no effect on growth or on survival rates of juveniles. Finally, allometric evaluations showed that fresh weight and L3 length are the most reliable parameters to be used as proxy for biomass assessment of this species.     

大黄鱼（♀）与黄姑鱼（♂）人工杂交子代的胚胎发育

2008年10月,在福建宁德水产技术推广站进行了大黄鱼（Pseudosciaena crocea,♀）×黄姑鱼（Nibea alb-iflora,♂）杂交,观察了杂交子代胚胎发育。大黄鱼和黄姑鱼亲鱼平均体质量和体长分别为405 g、27.9 cm和390g、24.8cm,采用干法人工授精。受精卵浮性,为单油球端黄卵,卵径1060～1500μm,油球379.8μm左右。在水温25.8～26.2℃,盐度26的条件下,经18h55min半数受精卵破膜,胚胎发育分为卵裂期、囊胚期、原肠期、肌节出现期、心跳期、肌肉效应期、出膜前期和出膜期,初孵仔鱼全长2.75～2.98mm,受精率约为14.4%,孵化率约为31.9%,畸形率约为30%。共培育杂交初孵仔鱼5万尾。3日龄仔鱼开口摄食轮虫,7日龄开始摄食小型挠足类。     

Changes in fertility of rainbow trout eggs retained in coelom

ABSTRACT:    Effects of prolonged retention time of ovulated eggs in the parental coelom on fertilization success were studied in rainbow trout Oncorhynchus mykiss using cryopreserved sperm with a uniform fertilizing ability. Proportions of successful fertilization, eyed eggs and hatched alevins were examined at different time periods up to a retention time of 14 days beyond the ordinary stripping time, and were compared with eggs incubated in artificial coelomic fluids (ACF). Eggs that were retained longer in the coelom showed gradual decreases in all the proportions of successful fertilization, eyed eggs and hatched alevins. The progress of cleavage after fertilization slowed with prolonged retention times. Eggs incubated in ACF lost their fertilizing ability much sooner than those retained in the coelom. The hatching rate of eggs retained for 2 weeks in coelom was 36%, while it was 1% in those eggs incubated for 4 days in ACF. Thus, eggs retained in the coelom showed higher fertilization success than those incubated in ACF.     

Larval Development and Rearing of Longtooth Grouper Epinephelus bruneus in Jeju Island, Korea

Eggs and sperm were obtained from a female (6.3 kg/BW) and a male (8.4 kg/BW) longtooth grouper Epinephelus bruneus following HCG injection in July 2003. The eggs were fertilized artMcially with the sperm and incubated in one of two 50-m³ tanks after washing the fertilized eggs. The fertilized eggs were 830–950 pn (average 900 ± 2 μm) in diameter and the respective fertilization and hatching rates were 97.7 ± 0.6% and 96.8 ± 0.5% at a water temperature of 25.0 ± 0.5 C. With this regime, the survival rate by day 93 was 7.5% in the 50-m³ tank. The elapsed time from hatching to opening the mouth was 3 d at 25 C. The initial mouth size (z) of the larvae was 0.22–0.23 mm. The newly hatched larvae were 2.02 ± 0.02 mm TL; this increased to 4.12 ± 0.09 mm TL by day 11. By day 54, the larvae had metamorphosed into juveniles and reached 41.12 ± 1.20 mm TL, and by day 93 the juveniles reached 93.78 ± 1.98 mm TL. In all, 49.5% of the larvae were malformed and the type of malformation was diverse.     

三疣梭子蟹受精卵离体孵化技术

为了探究三疣梭子蟹受精卵的离体孵化技术及效果,本研究先后开展了受精卵块最适分离液种类及作用条件的筛选、分离液处理不同发育期受精卵离体孵化的差异、分离液处理对受精卵卵膜的结构影响,及分离液处理受精卵对孵化后幼体活力的影响实验。结果显示,木瓜蛋白酶是一种较为理想的分离液,在浓度为0.09 g/mL,分离时间30 min时,分离率可达到99%以上;经过分离液处理后的各期受精卵均能孵化出幼体,卵内溞状幼体期离体胚胎孵化率最高,为89.0%±3.3%,未经处理的对照组为70.0%±4.8%;卵裂期离体胚胎孵化率最低,为58.0%±3.9%,对照组为31.0%±2.3%,与对照组相比,处理组的孵化率明显提高。透射电镜结果显示,经过分离液处理的受精卵卵膜结构疏松,且厚度降低,符合处理组孵化率增加这一现象,干露、福尔马林溶液胁迫和行为学测试对不同处理组的幼体进行质量评价的结果显示,处理组和对照组幼体活力无显著差异。研究表明,实验所获得的分离液可以有效提高三疣梭子蟹受精卵的分离率和孵化率,且不影响幼体质量,可为三疣梭子蟹及其他甲壳动物受精卵的离体孵化提供参考。本研究可为三疣梭子蟹的苗种繁育提供新的技术手段,也将为基因编辑辅助育种等技术的实施奠定基础。     

Melatonin, but not somatostatin-14 influences in vitro steroidogenesis by ovarian follicles of rainbow trout

Ovarian follicles taken from sexually maturing rainbow trout at the mid-vitellogenic stage of ovarian development were incubated in vitro in the presence or absence of melatonin or somatostatin-14 (SRIF-14) to determine whether there is evidence of a direct action of these factors on gonadal steroidogenesis in fishes. The steroidogenic capacity of the ovarian follicles was assessed by measuring testosterone (T) and 17-estradiol (E₂) release into the incubation medium, and by examining the steroid metabolites produced following incubation of follicles with radiolabelled steroid precursors.Melatonin appears to elicit a biphasic effect on steroidogenesis by in vitro rainbow trout ovarian follicles; at a concentration of 1 × 10^–3 M, melatonin stimulated basal T and E₂ production, but at a concentration of 1 × 10^–2 M there was an inhibition of basal and sGtH-stimulated T and E₂ Melatonin may act to reduce the activity of specific steroidogenic enzymes, since there was evidence of melatonin at 1 × 10^–2 M enhancing the accumulation of [³H]17-hydroxyprogesterone in the medium following incubation with [³H]pregnenolone, possibly suggesting the inhibition of C_17,20-lyase activity. In contrast, SRIF-14, used at concentrations of 1 × 10^–8 M and 1 × 10^–6 M, had no effect on basal or sGtH-stimulated E₂ or T production by ovarian follicles, incubated in vitro.     

Fertilization strategies for Sea Bass Dicentrarchus labrax (Linnaeus, 1758): effects of pre‐incubation and duration of egg receptivity in seawater

Studying gamete biology can provide important information about a species fertilization strategy as well as their reproductive ecology. Currently, there is a lack of knowledge about how long sea bass Dicentrarchus labrax eggs can remain viable after being activated in seawater. The objectives of this study were to understand the effects of pre‐incubation of fresh and overripe sea bass eggs in seawater and to determine the duration of egg receptivity. Pooled eggs (fresh and overripe) from four females were pre‐incubated in seawater for 0 min (control), 0.5 min, 1 min, 3 min, 10 min and 30 min and then fertilized by pooled sperm from four males. The fresh eggs had a higher fertilization success than overripe eggs. Our results revealed a significant effect of pre‐incubation time for both the fresh (P < 0.01) and overripe eggs (P < 0.01). Fertilization success of eggs significantly declined for both these treatments after 3 min of pre‐incubation, which clearly indicates that sea bass eggs are able to be fertilized by sperm for up to 3 min after release into seawater. This study has particular importance for understanding fertilization strategies, reproductive potential, as well as reproductive ecology of sea bass.     

温度对奥尼罗非鱼受精卵孵化和仔鱼活力的影响

研究了温度对奥尼罗非鱼(Oreochromis niloticus×O.aureus)受精卵孵化和仔鱼活力的影响。在盐度为5不同温度(22、24、26、28、30、32和34℃)下,比较观察奥尼罗非鱼受精卵的培育周期、孵化周期、孵化率和畸形率。同时在以上不同温度条件下对初孵的奥尼罗非鱼仔鱼进行耐饥饿试验,测定其不投饵存活系数(Survivalactivity index,SAI)。结果表明:在温度为24~32℃,受精卵孵化率较高(84.3%~91%),温度高于34℃和低于22℃时,孵化率较低。温度与培育周期呈负相关关系,函数关系为y=0.3607x2-26.836x+553.5,R2=0.973。仔鱼生存适宜温度是24~30℃,最适的温度是24~26℃。温度为22℃时,SAI值分别为19.9,但是仔鱼发育的缓慢,部分仔鱼的身体色素一直没有出现,不适合仔鱼的培育。     

Minimally invasive surgical technique for egg collection from the Persian sturgeon, Acipenser persicus

A minimally invasive surgical technique (MIST) for the removal of ovulated eggs from Persian sturgeon Acipenser persicus was tested on broodstocks caught from the wild to determine whether it affected fecundity or hatching rates compared with the traditional stripping method of killing and removing the eggs (commonly used in hatchery). Morphological parameters of females, germinal vesicle (GV) position, weight of obtained egg, number of eggs/gram, fertilization rate, and percentage of hatching during incubation were not significantly different between the MIST and traditional stripping methods. Obtained ova were 4.8 ± 0.4 kg female⁻¹ in the MIST and 4.6 ± 0.5 kg female⁻¹ in traditional stripped groups, respectively; the fertilization rate was 83.4 ± 11.2% and 80.0 ± 7.2% in groups, respectively. The results of this study showed that the minimally invasive surgical technique approach is efficient, practical, and less stressful to Persian sturgeon broodstocks during artificial propagation than other reported egg collection procedures.     

The Influence of Ovarian Fluid pH of Stripped Unfertilized Channel Catfish,Ictalurus punctatus,Eggs on the Hatching Success of Channel Catfish ♀ x Blue Catfish,Ictalurus furcatus♂, Hybrid Catfish Eggs

This study was designed to determine the effect of ovarian fluid pH of stripped unfertilized channel catfish, Ictalurus punctatus, eggs on fertilization and hatch rate of channel catfish ♀ x blue catfish, Ictalurus furcatus♂, hybrid catfish eggs. A significant correlation was established between ovarian fluid pH of stripped channel catfish eggs and hybrid embryo hatch rate (R² = 0.75, P = 0.01), suggesting ovarian fluid pH of stripped catfish eggs prior to fertilization can be predictive of the hatching success of hybrid catfish embryos. These data were used to categorize pH of stripped eggs: pH <7.0 as “low pH eggs,” pH 7.0–7.4 as “medium pH eggs,” or pH >7.4 as “high pH eggs” quality eggs. The range in percent hatch rate for these pH categories was <15%, 15–30%, and >30%, respectively. Higher calcium concentrations during incubation do not appear to improve hatching success of “low pH eggs.” The predictive model presented herein describes a quick method for evaluating stripped catfish eggs for hybrid fry production in catfish hatcheries.     

青蛤受精卵和幼虫密度对孵化和生长的影响

本文报道了青蛤受精卵密度对孵化的影响和青蛤幼虫培养密度对生长的影响。受精卵密度在26个/ml、30个/ml、38个/ml、44个/ml、53个/ml时,孵化率在87.8～89.9％之间;受精卵密度为53个/ml时,幼虫畸形率较高。实验发现,幼虫的培养密度应控制在6～11个/ml,幼虫变态后,减少培养密度,可以提高幼虫成活率。