Determination of periods of sensitivity to low-salinity and low-temperature conditions during the early development of cultured Japanese eel Anguilla japonica larvae with respect to the rate of morphological deformity at completion of yolk resorption

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.     

Influence of temperature and feeding regimes on growth and notochord deformity in reared <Emphasis Type="Italic">Anguilla japonica</Emphasis> leptocephali

The high mortality rate of reared Japanese eel Anguilla japonica larvae is largely due to lower growth rate and the higher rate of deformed larvae. To establish an effective rearing protocol for this species, we examined the effects of water temperature and feeding regimes on their growth and notochord kyphosis. Larvae at 165 days post hatching were reared for 28 days at mean temperatures of 24, 25 and 27 °C, and were fed 4 or 6 times per day. Larval growth rate was significantly higher in larvae reared at 24–25 °C and fed 6 times per day. However, growth rate was significantly reduced at 27 °C, suggesting a shortage of metabolic energy due to an elevated cost of the higher basal metabolic rate at higher temperatures and low nutritional performance of currently used artificial diet. Notochord kyphosis was promoted by elevated water temperature, and two-way ANOVA showed that water temperature and feeding frequency had combined effects on the deformity. These findings suggest the importance of concurrently manipulating both environmental and nutritional factors to produce healthy eel larvae in captivity.     

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.     

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.     

The induced spawning of the grey mullet, Mugil macrolepis (Aguas) Smith and the large-scale rearing of its larvae

The grey mullet, Mugil macrolepis (Aguas) Smith could be induced to spawn by the administration of pituitary alone from the same species. The threshold dose was found to be three and four glands as the first and second injections after a 6-h interval. About 40% of the experiments were successful. The hand-stripped eggs were artificially fertilized with milt by the dry method.The embryonic development of the eggs are briefly described. The majority of the fertilized eggs hatched out about 23 h after fertilization. The salinity of the water ranged from 29 to 31‰ and temperature from 26 to 29°C.A method for the large-scale rearing of mullet larvae is outlined. “Seasoned greenwater” is produced by keeping slightly manured brackish water for about a fortnight in small tanks which can be exposed to sunlight in the afternoon. Blooms of Chlorella sp. developed and the larvae fed on the microplankton. The larvae at about 1 cm in length fed exclusively on copepods. Hence copepod cultures were maintained simultaneously in manured cisterns for feeding the mullet larvae until they accepted artificial feeds.     

Effect of salinity on occurrence of notochord deformities in Japanese eel <Emphasis Type="Italic">Anguilla japonica</Emphasis> larvae

One of the difficulties to rear Anguilla japonica larvae is the frequent occurrence of notochord deformities. We tested the effect of salinity on the occurrence of the deformities, because we have been using 50 % diluted seawater (50 % SW) for glass eel production, on the basis of the fact that intermediate salinity saves energy due to lower cost for osmoregulation and contributes higher survival and growth rates. We reared 6-day-old larvae in 50 and 100 % SW for 85 days and observed their morphology. The occurrence rate of deformed larvae, including kyphosis and scoliosis, was significantly higher in 50 % SW (35.8 %) than in 100 % SW (25.4 %), while survival rate was significantly higher in 50 % SW (69.8 %) than in 100 % SW (32.3 %) and growth in 50 % SW (mean body depth: 7.9 ± 5.3 mm) was better than in 100 % SW (6.8 ± 4.6 mm). We speculate that the most of severely deformed larvae could not survive in the tougher condition of 100 % SW, showing the lower occurrence of deformed larvae. Eventually, the yield of normal larvae after 85 days rearing was 1.9-fold higher in 50 % SW than in 100 % SW, implying that the advantage of 50 % SW for rearing eel larvae outweighs the risk of deformities.     

On the induced spawning and larval rearing of milkfish,Chanos chanos (Forskal)

A female milkfish, captured at sea, was injected with two hormonal injections of acetone-dried salmon pituitary powder and human chorionic gonadotropin, plus Vitamin B complex. It was stripped, and produced 128,000 ripe eggs with an average diameter of 1.15 mm. Fertilization rate was 38% following artificial fertilization with milt from an uninjected male. A total of 36,000 larvae hatched (74% of fertile eggs) after 26–32 h at 34 ‰ salinity and 27–32°C. The newly hatched larvae measured 3.4 mm in mean total length and possessed a large yolk sac. The mouth of the larvae opened about 54 h after hatching. The larvae were fed with fertilized oyster eggs, rotifers, copepods, brine shrimp, flour and prepared feed, together with Chlorella. A critical period was between the 4th and 6th days with mortality over 80%. The larvae started increasing in length by Day 8, and had the appearance of the wild fry by Day 11. On Day 13 a pigmentation pattern developed and the biggest larva measured 10.0 mm. By Day 18 the larvae measured 12.5 mm, and 14.5 mm by Day 21. A total of 2,859 fry was obtained; the highest larval survival rate obtained from different experimental groups was 46.8%.     

The effect of hatching time on the bioenergetics of northern pike (<Emphasis Type="Italic">Esox lucius</Emphasis>) larvae from a single egg batch during the endogenous feeding period

Size, caloric value and chemical composition were measured separately in the progeny of two northern pike (Esox lucius) females at 3-day intervals during the endogenous feeding period from hatching to final yolk resorption. Tissue, yolk and entire larvae were analysed separately in three groups of larvae that hatched at different times (between 88 and 106 degree-days post-fertilization). An integrated approach with the Gompertz model was used to compute the yolk conversion efficiency and time to maximum tissue size in early, mid and late hatched larvae. At hatching, unresorbed yolk of early hatched larvae contained more energy (39.20 J) and more protein (0.99 mg) compared to the yolk of larvae that hatched later (38.13 J and 0.92 mg protein for late hatched larvae, p < 0.05). In contrast, a significant reduction in tissue weight (?0.7 mg DW) and protein content (?0.5 mg) was found in early hatched larvae compared to those which hatched later (p < 0.05). Between days 9 and 12 post-hatching (108 and 144 degree-days post-hatching), close to the final yolk resorption, late hatched larvae stopped growing and their tissue began to be resorbed. This tissue resorption time was delayed in early hatched larvae which presented at the end of the experiment a greater tissue weight than late hatched ones. Yolk conversion efficiency in term of energy from hatching to complete yolk resorption stage was significantly higher for early and mid hatched larvae (51 %) compared to late hatched ones (44 %) (p = 0.004). Furthermore, the time to maximum tissue size was found to be negatively related to hatching time which implies that early hatched larvae take longer time to switch from one developmental stage to the next. The maximum tissue dry weight and energy content were found to be reached at approximately the same age post-fertilization for both early hatched and late hatched larvae, suggesting that the principal steps in a fish’s lifespan are better correlated with time of fertilization than hatching time.     

Observations on artificial fertilization of eggs and the embryonic and larval development of milkfish,Chanos chanos (Forskal)

Hydrated eggs obtained from a female milkfish, Chanos chanos, were artificially fertilized with the milt collected from a male injected with acetone-dried pituitaries of salmon. The fertilized eggs (1.1–1.25 mm in diameter) developed normally in seawater in basins and petri dishes at a salinity of 30–34‰, and successfully hatched in 25–28.5 hours at a temperature of 26.4–29.9°C. The yolk was completely absorbed in about 2.5 days and during this period many postlarvae died. A few larvae were reared up to 5 days but all died on the 6th day. Attempts were made to feed the postlarvae with freshly hatched trochophore larvae of oysters obtained from eggs artificially fertilized in the laboratory.     

Influence of temperature on yolk resorption in common snook Centropomus undecimalis (Bloch, 1792) larvae

To determine the optimal rearing temperature for Centropomus undecimalis larvae during the yolk resorption period, changes in larval development were measured at four different temperatures (23, 25, 28 and 31°C). Yolk and oil‐globule volume was recorded for 25 larvae at four different times. This involved an initial measurement at hatch and at 24, 48 and 72 h posthatch (hph). Additional morphological measurements included standard length, body height and eye diameter. On average, at the end of the three trials, larvae reared at 25°C had a longer mean standard length than larvae reared at 23, 28 and 31°C. Larvae reared at 25°C also had more yolk and oil globule reserves than larvae raised at 28 and 31°C. The body height:length residuals were also the highest at 25°C (i.e. larvae had deeper or stockier bodies). The yolk sac was present up to 72 hph at 23 and 25°C, while it was entirely consumed after 48 hph in larvae held at 28 and 31°C. Larvae showed the fastest growth during the first 24 hph in all temperature treatments; this period corresponded to the highest energy consumption as determined by the decrease in yolk sac and oil‐globule volume. Eye diameter did not vary significantly with time during yolk‐resorption. We conclude that a temperature near 25°C is optimal for raising snook larvae during the yolk‐resorption period.     

A study of the feeding of common carp larvae with artificial food

Common carp larvae were reared on artificial diets from hatching to 25 days old. The main components used in the diets were freeze-dried spleen, fish protein and chicken egg yolk. Free amino acids and bovine trypsin were added and the diet was made alkaline. All diets were gelatinized with agar to give a final dry matter content (15–20%) similar to that of zooplankton. Growth of larvae was negligible but survival was from 11 to 40%. Feeding on days 1, 2 and 3 with zooplankton and subsequently on an artificial diet (40% lyophilised egg yolk, 40% blood meal) showed some improvement both in growth and survival.     

日本鳗鲡仔鱼的开口饵料和行为特征

在连续多年日本鳗鲡人工繁殖实验的基础上,研究了不同饵料对日本鳗鲡仔鱼存活率的影响,并记录了仔鱼的运动特征和摄食行为。结果显示,与对照组(不投饵)相比,投喂以鲨鱼卵、磷虾提取液为基础的饵料和以鲨鱼卵、海蜇匀浆液为基础的饵料以及微绿球藻液均提高了日本鳗鲡仔鱼的存活率,仔鱼发育至柳叶鳗前期阶段;而投喂以鲨鱼卵、卤虫匀浆液为基础的饵料和以卤虫匀浆液、磷虾提取液为基础的饵料,以及轮虫、海带+龙须菜匀浆液或海蜇以及发酵鲨鱼肉,均降低日本鳗鲡仔鱼的存活率。实验同时还研究了日本鳗鲡仔鱼的主动摄食行为,摄食时仔鱼先用吻端反复多次触碰食物,然后张开下颌咬食,证实了日本鳗鲡早期仔鱼的摄食方式是触碰后咬食。     

河鳗人工繁殖的初步研究

本文报道了自1973年以来河鳗人工繁殖研究的基本情况，包括亲鳗催熟、催产以及胚胎发育和培育等试验内容。试验结果表明，在人工催熟和水温逐渐升高的条件下，大部分亲鳗在4月上旬至5月中旬性腺成熟。从1974年至1978年总的产卵受精率为8．4％(其中流卵和产死卵者不计在内)。亲鳗自然产卵的时间一般在凌晨4时至6时20分之间，水温在18．5—24．5℃范围。产卵行为较特殊，产卵前发情时，雄鳗和雌鳗先后由池底阴暗处游至水表，沿池边环游追逐，最后在较小范围内急游产卵授精，产毕又回至池底。海水盐度在23—29．8‰范围，均能产卵受精，并孵出鳗苗。孵出后头两天的仔鳗悬于水表；第3天的仔鳗，卵黄囊大部分吸收，消化道全通，在肠内开始发现食物团，肠道内有强烈的纤毛运动，鱼体开始悬于水中，有极缓慢的下沉和上升；第4天眼球出现黑色素，晶体透明，上、下颌能启闭，有四对牙齿；第5天，肠道有蠕动波，出现肝脏原基，静止时侧卧水底；第14天，仔鳗在微流水中能不断游动，在静水中常处于水底。仔鳗养至第19天，存活434小时。     

Early development and allometric growth patterns in burbot Lota lota L.

This study analyzed the morphological development and allometric growth patterns of Lota lota L. (burbot) larvae reared under controlled laboratory conditions. From hatching to day 50, twenty larvae were sampled each [between 1 and 14 days post-hatch (DPH)] or every second day (between 14 and 50 DPH) and measured under a stereoscopic microscope using analytic software. Based on the external morphology, the different stages during early development of burbot were identified: yolk sac larva 0–8 DPH [3.92–4.37 mm total length (TL)]; preflexion larva 9–26 DPH (4.57–12.06 mm TL); flexion larva (between notochord degradation and its replacement with rays) 28–34 DPH (14.00–16.34 mm TL) and postflexion larva/juvenile 36–50 DPH (18.20–29.27 mm TL). Allometric growth patterns of some parameters (e.g., total length, head length, body length, tail length, head depth, body depth, eye diameter) were modeled by a power function and described by the growth coefficient. Organogenesis and changes of body proportions in burbot larvae were more rapid and complex during the yolk sac and preflexion phase of development as larvae developed most of their sensorial, feeding, respiratory and swimming systems and after notochord flexion, when most morphological changes were related to the progressive transformation from pelagic larva to demersal larva/juvenile.     

Early development,growth and survival of the yellow crab Cancer anthonyi Rathbun (Decapoda,Brachyura) in the laboratory

Larvae of the yellow crab, Cancer anthonyi Rathbun, were reared through five zoeal stages and one megalops stage in the laboratory. Total larval development times were 33 and 45 days at 22°C and 18°C, respectively. Survival rates to the first crab instar, for larvae reared in recirculating systems on a diet of Artemia nauplii, were 26% at 22°C and 17% at 18°C. Although no larvae reared in glass containers at 22°C survived past the first zoeal stage, 11% of those in similar containers at 18°C reached the first crab instar. Bacterial infections were associated with most observed mortalities. Antibiotics failed to increase the survival rates of larvae reared in glass containers.Juvenile crabs were reared in individual containers and in communal aquaria through 14 crab instars. Although instar durations were shorter at 22°C than at 18°C, mean carapace widths were significantly greater at the lower temperature. Crabs in communal aquaria at 22°C were larger than corresponding crabs at the same temperature in individual containers. Crabs reared in aquaria at 22°C reached the twelfth instar with a mean size of 90.3 mm, 195 days after hatching. Sexually mature thirteenth stage laboratory-reared crabs were mated and their offspring were reared through the ninth crab stage.     

Spawning of ocean pout (Macrozoarces americanus L.): evidence in favour of internal fertilization of eggs

Sperm physiology, in vivo artificial insemination and spawning of the ocean pout (Macrozoarces americanus L.), a marine bottom fish, were studied. Milt was collected from the reproductive tract of mature males by suction using a catheter. The uncontaminated milt, having a very low sperm concentration, contains highly motile spermatozoa and sperm motility was retained in vitro at 4 °C for at least 24 h in both seminal plasma and ovarian slime collected from the oviduct of pre-spawning females. Instead of activating sperm, dilution in sea water instantly immobilized the spermatozoa of ocean pout. Osmolarity and pH of ocean pout seminal plasma were in the ranges 365–406 mOsM and 7.2–7.5, respectively. A study of the ionic composition of ocean pout seminal plasma demonstrated the presence of various ions including Na⁺, K⁺, Ca²⁺, Mg²⁺, and Cl⁻, with a remarkably lower K⁺ concentration compared to that from other fish species. Since injections of milt containing motile sperm into the ovaries of pre-spawning females, which spawned in the absence of males, yielded fertilized ocean pout eggs, it is concluded that the ocean pout exhibits internal fertilization. The larvae hatched after 3 months of egg incubation in ambient sea water (9–10 °C). With proper timing of in vivo artificial insemination of mature females, fertilized ocean pout eggs can be obtained from fish reared in captivity.     

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.     

Changes in Serum Steroid Hormones and Vitellogenin Levels in Cultured Female European Eels Anguilla anguilla during Artificially Induced Ovarian Development

Changes in serum concentrations of estradiol-17β, testosterone, 17α,20βdihydroxy-4-pregnen-3-one, and vitellogenin were investigated during ovarian development induced by injection of a salmon pituitary extract in cultured European eel Anguilla anguilla . Vitellogenesis was induced with a weekly dose of 50 mg pituitary extract/kg body weight. In eels receiving that dose, gonadsomatic indices ranged from 20–43% after the 10th–11th weekly injection. Body weights were relatively stable during vitellogenesis, but increased dramatically during final maturation. Serum estradiol-17β levels increased slightly during vitellogenesis and peaked at an average of 6.95 ng/mL at final maturation. The profile of serum vitellogenin followed that of estradiol-17β which increased markedly from an average of 0.36 to 20.72 mg/mL. Control levels of vitellogenin were undetectable throughout the study. Average serum levels of testosterone increased to a peak of 17.74 ng/mL in the early stage of vitellogenesis, followed by a sharp drop to initial levels (3.86 ng/mL) in the late stage of vitellogenesis, and then increased again to an average of 8.84 ng/mL at the final maturation stage. Serum 17α,20β-dihydroxy-4-pregnen-3-one was not detected (<50 pg/mL) throughout the experiment. Profiles of serum estradiol-17β, testosterone, and vitellogenin observed during ovarian development appear different from those found in salmonids and other teleosts. This study indicates, however, that cultured European eels are a useful model for study of gonadal maturation in the eel.     

Histological study of the gastrointestinal tract in longfin yellowtail (<Emphasis Type="Italic">Seriola rivoliana</Emphasis>) larvae

This work contributes basic knowledge on larval development of Seriola rivoliana. A histological study describes the development of the digestive tract and accessory glands in S. rivoliana larvae reared under laboratory conditions at 24 °C from hatching to 30 days post-hatching (DPH). At hatching (2.6 ± 0.12 mm), larvae had an undifferentiated digestive tract with a closed straight tube and a large yolk sac with an oil globule. The liver and pancreas were observed at 1 and 2 days, and the mouth and anus opened at day 2. Enriched rotifers were visible in their digestive tract. At the beginning of the pre-flexion stage, a mixed nutritional period was observed. At day 3, exogenous feeding began; the digestive tract became differentiated into the buccopharynx, esophagus, an undifferentiated stomach, and the intestines. Zymogen granules were visible in the exocrine pancreas. At day 4, supranuclear vacuoles were present in the posterior intestine, indicating the beginning of intracellular digestion. At day 5, goblet cells were present in the esophagus and became functional at day 7 in the esophagus and intestine. The buccopharynx goblet cells developed at day 15. The presence of gastric glands and differentiation of the stomach in the fundic, cardiac, and pyloric regions during the post-flexion stage occurred at day 20. This was the onset of the juvenile period and the beginning of weaning; however, a long co-feeding phase is recommended. Pyloric caeca were observed at day 30 (13.6 ± 1.6 mm). These results provide valuable information on S. rivoliana larvae biology and digestive physiology, which should be useful to improve cultivation techniques and identify ecological features involved in ontogeny.