Mortality of yellowtail flounder, Limanda ferruginea (Storer), eggs: effects of temperature and hormone-induced ovulation

Yellowtail flounder, Limanda ferruginea (Storer, 1839), is a highly marketable fish species that used to be a significant groundfish in the northwest Atlantic region. The potential of yellowtail flounder for mass rearing has recently been investigated, but studies into aspects of mortality during early ontogeny are lacking. We investigated the impact of spawning inducement on mortality of eggs from fertilisation until hatch, and the effects of rearing temperature on mortality and hatching time in this multiple batch spawning flounder. An ash-free dry mass (AFDM) technique for determining mortality was employed. This technique reduced rearing tank disturbances and provided quantitative mortality estimates. We found (1) that spontaneously ovulated (SO) mean egg mass (AFDM 24.84±0.73 μg) was significantly higher than mean egg mass from induced ovulation (IO) (AFDM 20.09±0.54 μg); (2) that mortality of SO eggs was lower than IO eggs (p=0.028), (3) that SO eggs reached a relatively constant (maximum) mortality by about day 3 and IO eggs by about day 5; (4) that there was no significant difference in mortality between eggs incubated at 10 °C and those incubated at 4 °C (p=0.320); and (5) that time to 100% hatch took significantly longer for eggs incubated at 4 °C (256.0±73.5 h) than those incubated at 10 °C (77.9±11.6 h) (p=0.003). There was considerable variation in egg mortality among batches and females; nevertheless, general trends indicate that GnRHa-induction of ovulation tends to decrease egg quality. In addition, the AFDM technique was an easily employed and accurate method of determining mortality. We recommend that unless coordination of spawning is important to a particular rearing protocol, induced ovulation should not be employed.     

The timing of puberty in cultured female yellowtail flounder, Limanda ferruginea (Storer): Oogenesis and sex steroid production in vivo and in vitro

This study examines puberty in female yellowtail flounder, correlating macroscopic, histological and endocrine parameters at the gonadal level. Short-term ovarian tissue incubations were used to study estradiol-17β production in relation to oogenic stage. Examining 2 year classes of young flounder demonstrated that cultured females retain phenotypic plasticity in reproductive age with pubertal onset occurring in one-year, two-year and three-year-olds. Immature ovaries were steroidogenic and capable of responding to gonadotropic stimulation. Endocrine puberty in females was detected by a peak in estradiol-17β production during the cortical alveolar oocyte stage prior to any ovarian evidence of vitellogenesis. Puberty, once initiated, proceeded to ovulation within 8 to 12 months; vitellogenic oocyte development followed the group synchronous pattern. Estradiol-17β was clearly the dominant of the two measured plasma hormones during pubertal onset and throughout vitellogenesis. Plasma testosterone was consistently detectable at low levels by mid-to late-vitellogenesis. Ovaries showed the highest sensitivity to gonadotropic stimulation in vitro during late-vitellogenesis. Variable plasma levels in both estradiol-17β and testosterone occurred in preovulatory and ovulating females during the captive spawning period. Together the results show that yellowtail flounder can mature at a young age and small size when culture conditions permit. In addition, the early sensitivity to gonadotropin by the immature ovaries may be an important physiological determinant for the timing of puberty in this species.     

Cytological abnormalities in the ovaries of triploid yellowtail flounder,Limanda ferruginea (Storer)

Abnormal oocytes were detected in the ovaries of triploid yellowtail flounder of three years of age. Triploidy in these females had been induced artificially by hydrostatic pressure shock. Binucleate oocytes were found in the ovaries of six different triploid females. These cells were always small and in a primary growth stage. Cytoplasmic vacuoles, presumed to be due to nucleoplasmic extrusion, was another abnormal feature observed in two primary growth oocytes, each seen in a separate female. The appearance of binucleate oocytes in triploid females, not previously noted in diploid yellowtail flounder, is proposed to be an ephemeral phenomenon associated with the triploid condition.     

Effects of docosahexaenoic, eicosapentaenoic, and arachidonic acids on the early growth, survival, lipid composition and pigmentation of yellowtail flounder (Limanda ferruginea): a live food enrichment experiment

The role of dietary ratios of docosahexaenoic acid (DHA, 22:6n−3), eicosapentaenoic acid (EPA, 20:5n−3) and arachidonic acid (AA, 20:4n−6) on early growth, survival, lipid composition, and pigmentation of yellowtail flounder was studied. Rotifers were enriched with lipid emulsions containing high DHA (43.3% of total fatty acids), DHA+EPA (37.4% and 14.2%, respectively), DHA+AA (36.0% and 8.9%), or a control emulsion containing only olive oil (no DHA, EPA, or AA). Larvae were fed differently enriched rotifers for 4 weeks post-hatch. At week 4, yellowtail larvae fed the high DHA diet were significantly larger (9.7±0.2 mm, P<0.05) and had higher survival (22.1±0.4%), while larvae fed the control diet were significantly smaller (7.3±0.2 mm, P<0.05) and showed lower survival (5.2±1.9%). Larval lipid class and fatty acid profiles differed significantly among treatments with larvae fed high polyunsaturated fatty acid (PUFA) diets having higher relative amounts of triacylglycerols (18–21% of total lipid) than larvae in the control diet (11%). Larval fatty acids reflected dietary levels of DHA, EPA and AA while larvae fed the control diet had reduced amounts of monounsaturated fatty acids (MUFA) and increased levels of PUFA relative to dietary levels. A strong relationship was observed between the DHA/EPA ratio in the diet and larval size (r²=0.75, P=0.005) and survival (r²=0.86, P=0.001). Following metamorphosis, the incidence of malpigmentation was higher in the DHA+AA diet (92%) than in all other treatments (50%). Results suggest that yellowtail larvae require a high level of dietary DHA for maximal growth and survival while diets containing elevated AA exert negative effects on larval pigmentation.     

Growth and survival of juvenile yellowtail flounder Pleuronectes ferrugineus (Storer) under different photoperiods

Yellowtail flounder Pleuronectes ferrugineus (Storer) have been identified as a potential species for coldwater aquaculture along the north‐east coast of Canada. It has been shown that increased photoperiods improve growth and survival during the larval stage of this species. We conducted two experiments to determine the effect of photoperiod on the growth and survival of juveniles. The first experiment compared growth and survival under 24‐, 18‐ and 12‐h lighting per day with a restricted food ration. The second experiment compared growth and survival rates under the above photoperiods and ambient lighting with fish being fed to satiation. No significant differences in growth or survival were found in juveniles under the different photoperiods in either experiment.     

Lipid composition of malpigmented and normally pigmented newly settled yellowtail flounder, Limanda ferruginea (Storer)

The lipid composition of malpigmented (MP) and normally pigmented (NP), newly settled yellowtail flounder (Limanda ferruginea, Storer) was compared in order to elucidate a possible connection between lipids and pigmentation development. Larvae were fed commercially enriched live food for 12 weeks post hatch and then differences in lipid composition and size were analysed. NP fish were found to be significantly larger (standard length 35 mm) than MP fish (32 mm) at 100% settlement. There were higher proportions of triacylglycerols in NP fish (P = 0.01), whereas MP fish had an increased percentage of phospholipids (P = 0.01). NP fish had a higher percentage of docosahexaenoic acid (DHA) in the polar lipids of their body (P = 0.03) and total lipids of their eyes (P = 0.04). These data support previously proposed theories for the importance of DHA in pigmentation development. Principal components analysis (PCA) described the majority of the variance (77%) within the data set using just two principal components axes. PCA demonstrated that differences between body zones were greater than those between NP and MP fish within a given zone.     

Rearing of flounder (Platichthys flesus) juveniles in semiextensive systems

A low-technology rearing system was implemented for rearing juvenile flounder for stock enhancement in a Danish fjord, the Limfjord. Each year during 1996–2002, between 13,000 and 153,000 juveniles were reared from the yolk-sac stage until metamorphosis in outdoor ponds relying on phyto- and zooplankton blooms as their main food source. In contrast to other similar systems, the blooms in this system are closely monitored and, to a certain extent, regulated. The zooplankton blooms consisted mainly of calanoid copepods, dominated by the species Temora longicornis and Centropages hamatus. Most juveniles produced (>99.5%) were normally pigmented with average yearly survival rates from hatch to metamorphosis varying from 7±9% to 48±18%, lowest in the first years of production.     

Ontogenetic development of digestive enzymes in yellowtail kingfish Seriola lalandi larvae

The development of digestive enzymes was examined in laboratory-reared yellowtail kingfish larvae from hatching to 36 days after hatching (DAH). The specific activities of amylase, lipase, and alkaline phosphatase showed three distinct phases: a sharp increase in enzyme activity from hatching to the onset of exogenous feeding on 3 DAH, followed by a fluctuation and a general decline toward 18 DAH, and then a period of low activity from 18 to 36 DAH. The total activities of these three enzymes showed a gradual increase from hatching to 18 DAH, followed by a sharp increase toward 36 DAH. In contrast to other enzymes, the specific and total activities of trypsin reached the maximum on 15 DAH and 24 DAH, respectively, and then both activities declined to low levels toward 36 DAH. The dynamics of digestive enzymes corresponded to the anatomical development of the digestive system. The enzyme activities tend to be stable after the formation of gastric glands in the stomach on 15 DAH. The composition of digestive enzymes indicates that yellowtail kingfish is able to digest protein, lipid and carbohydrates at an early stage. However, due to the low level of amylase specific activity after 18 DAH, the carbohydrate component should remain at a low level in formulated diets for fish larvae.     

Effects of body weight, water temperature and ration size on ammonia excretion by the areolated grouper (Epinephelus areolatus) and mangrove snapper (Lutjanus argentimaculatus)

The effects of body weight, water temperature and ration size on ammonia excretion rates of the areolated grouper Epinephelus areolatus and the mangrove snapper Lutjanus argentimaculatus were investigated. Under given experimental conditions, L. argentimaculatus had a higher weight-specific ammonia excretion rate than E. areolatus. Weight-specific ammonia excretion rates of fasted individuals of both species showed an inverse relationship with body weight (W, g wet wt.), but a positive relationship with water temperature (t, °C). The relationships for total ammonia nitrogen (TAN) were: E. areolatus: TAN (mg N kg⁻¹ d⁻¹)=21.4·exp^0.11t·W^−0.43 (r²=0.919, n=60); L. argentimaculatus: TAN (mg N kg⁻¹ d⁻¹)=121.5·exp^0.12t·W^−0.55 (r²=0.931, n=60). Following feeding, the weight-specific ammonia excretion rate of E. areolatus increased, peaked at 2 to 12 h (depending on temperature), and returned to pre-feeding levels within 24 h. A similar pattern was observed for L. argentimaculatus, with a peak of TAN excretion being found 6 to 12 h after feeding. Stepwise multiple regression analysis indicated that weight-specific TAN excretion rates of both species increased with increasing temperature and ration (R, percent body wt. d⁻¹): E. areolatus: TAN (mg N kg⁻¹ d⁻¹)=22.8·t−28.8·R−378.2 (r²=0.832, n=24); L. argentimaculatus: TAN (mg N kg⁻¹ d⁻¹)=22.9·t−25.4·R−216.4 (r²=0.611, n=24). The effect of body weight on weight-specific postprandial TAN excretion was not significant in either species (p>0.05). This study provides empirical data for estimating ammonia excretion of these two species under varying conditions. This has application for culture management.     

Effect of dietary taurine levels on growth and feeding behavior of juvenile Japanese flounder Paralichthys olivaceus

This study was conducted to investigate the effect of dietary taurine levels on growth and feeding behavior of juvenile Japanese flounder. Three different taurine level diets were prepared by supplementation of taurine (T-0%, 0.5% and 1.5%) to the basal diet. Fish meal washed with 70% ethanol to remove taurine was used as the sole protein source. Feeding experiments were carried out twice at 20 °C by using different size of fish (average body weight: 0.3 g in Experiment I and 3.7 g in Experiment II). The feeding behavior of fish was observed throughout the experimental period. At the end of experiments, fish were killed for amino acids analysis.

The final average body weight and feed efficiency of juvenile Japanese flounder fed the T-1.5% diet was significantly higher than those of fish fed the T-0% diet in Experiments I and II. Abnormal feeding behavior such as multiple feeding while swimming in the water column was observed in the T-0% group in Experiment I. These findings indicate that taurine is essential for normal growth and development of normal feeding behavior of juvenile Japanese flounder.     

Severe oxidative stress is thought to be a principal cause of jaundice of yellowtail Seriola quinqueradiata

The jaundiced yellowtail Seriola quinqueradiata had yellow discolored skin and hyperbilirubinemia. Histology revealed that hemosiderin and ceroid occurred in the spleen. In the vascular system, there were vasculitis and sinusoidal dilatation. Hepatic cells showed degenerative figures with pyknotic or karyolytic nuclei, basophilic cytoplasm, cytoplasmic vacuoles in various sizes, and ceroidosis. In the bile duct system, however, there was no noticeable change. Hemoglobin contents of jaundiced fish were similar to those of control fish. Plasma taurine levels of jaundiced fish were significantly lower than those of control (p0.05). Judging from 2-thiobarbituric acid reactive substance values, in vivo lipid peroxidation progressed in the liver of jaundiced fish. In the plasma of jaundiced fish, contents of unconjugated bilirubin were significantly higher than those of conjugated bilirubin. A large amount of substance Xs, breakdown products of bilirubin scavenging active oxygen species, existed in the plasma of jaundiced fish. Therefore, jaundiced fish may suffer from severe oxidative stress, which might be a principal cause of jaundice. Liver superoxide dismutase and glutathione peroxidase activities of jaundiced yellowtail were significantly lower than those of controls.     

Immobilization antigen-independent protection of olive flounder (Paralichthys olivaceus) against Philasterides dicentrarchi (Ciliophora: Scuticociliatia) infection

To determine whether olive flounder (Paralichthys olivaceus) immunized with lysate of Philasterides dicentrarchi could be protected immobilization antigen (i-antigen) type independently, fish were immunized with lysate of ciliates obtained from in vitro cultures or ciliates obtained from infected fish. Fish immunized with ciliates obtained from infected fish were completely protected against artificial infection with in vitro cultured P. dicentrarchi, in spite of a weak or no serum agglutination activity against in vitro cultured ciliates. The present results indicate that i-antigen-independent protection was elicited by immunization of fish with the ciliates lysate, and the cultured ciliates would provide a good source for preparation of vaccines, which are cross-protective against various i-antigen types of P. dicentrarchi.     

Genetic characterization, morphometrics and gonad development of induced interspecific hybrids between yellowtail flounder, Pleuronectes ferrugineus (Storer) and winter flounder, Pleuronectes americanus (Walbaum)

Viable interspecific hybrids between yellowtail flounder (Pleuronectes ferrugineus, Storer) and winter flounder (Pleuronectes americanus, Walbaum) were produced by artificial insemination of yellowtail flounder eggs with winter flounder sperm. However, mean fertilization rate, hatching success and early survival up to 3 weeks post hatch were significantly lower than those of parental pure cross controls (P < 0.01). Overall, cytogenetic traits (karyological analysis and estimation of cellular DNA contents using flow cytometry) of hybrid flounder were intermediate between the two parental species. Microsatellite assay was used to distinguish the parental genomes in the hybrids; in most cases, one allele was specific to each of the parents. Morphometrics assessed by body proportions indicated that hybrids generally displayed a morphology intermediate between the maternal and paternal species. Interspecific hybrids exhibited abnormal and retarded gonad development in both sexes based on histological analysis of gonads from adult fish. The sterility of the hybrids presents a significant advantage for their use in aquaculture, as potential escapees would not be capable of reproducing in the wild and contaminating natural stocks.     

高温—最佳温度循环对褐牙鲆生长、能量分配和身体成分的影响

环境温度短周期波动对鱼类的生长和能量代谢有重要影响,实验在室内可控条件下,对褐牙鲆幼鱼在经历高温—最佳温度循环的生长与能量分配进行了研究。在为期40 d的实验中,驯养于20 ℃的褐牙鲆幼鱼在每个为期10 d的循环中分别经历0 d(H₀)、2 d(H₂)、4 d(H₄)、6 d(H₆)和8 d(H₈)的26.5 ℃高温胁迫后,剩余的天数恢复到最佳温度(20 ℃)下养殖。实验结束时,经历不同天数高温胁迫的各处理组的褐牙鲆幼鱼以干重、湿重、蛋白质和能量表示的鱼体重均显著小于对照组(H₀)。实验期间摄食量少和分配于生长的能量减少是导致经历高温胁迫的处理生长减慢的主要原因。经历高温胁迫的处理组的摄食量都显著低于对照组,但摄食率、消化率以及饲料转化效率都与对照组无明显差异。经受高温处理的H₂、H₄和H₆组分配于生长的能量与对照组没有显著差异,但H₄组的能量损失于粪便最多,H₆组的能量损失于排泄的比例最高。H₈组褐牙鲆幼鱼个体分配于生长的能量比例显著低于其他处理,代谢消耗的能量比例显著高于其他处理。经历不同温度循环处理的褐牙鲆幼鱼单位体重日摄食能与对照组相比没有显著差异,但分配于生长的能量却显著低于对照组。结果表明,褐牙鲆幼鱼对高温—最佳温度循环的适应能力较差,生长受到温度周期波动的抑制。     

Evaluation of optimum dietary protein-to-energy ratio in juvenile olive flounder Paralichthys olivaceus (Temminck et Schlegel)

An 8‐week feeding trial was conducted to estimate the optimum dietary protein to energy (P/E) ratio in juvenile olive flounder Paralichthys olivaceus. Eight experimental diets were formulated with two energy levels and four protein levels at each energy level. Two energy levels of 12.5 and 16.7 kJ g^?1 diets were included at crude protein (CP) levels of 25%, 30%, 35% and 45% with 12.5 kJ g^?1, and CP levels of 35%, 45%, 50% and 60% with 16.7 kJ g^?1. After 1 week of the conditioning period, fish initially averaging 8.1±0.08 g (mean±SD) were randomly distributed into the aquarium as groups of 15 fish. Each diet was fed on a dry‐matter basis to fish in three randomly selected aquariums at a rate of 3–5% of total wet body weight per day for 8 weeks. After 8 weeks of the feeding trial, weight gain (WG), feed efficiency ratio and specific growth rate of fish fed 45% CP with 16.7 kJ g^?1 energy diet were significantly higher than those from the other dietary treatments (P<0.05). WG of fish fed 12.5 kJ g^?1 energy diets increased with the increase of dietary protein levels. However, WG of fish fed 16.7 kJ g^?1 energy diets increased with the increase of dietary protein levels up to 45% CP and then decreased when fish fed 50% and 60% CP diets. Both dietary protein and energy affected protein retention efficiency and energy retention efficiency. Haemoglobin (Hb) of fish fed 35% and 45% CP diets with 12.5 kJ g^?1 energy were significantly high and not different from Hb of fish fed 45% and 50% CP diets with 16.7 kJ g^?1 energy. Haematocrit of fish fed 45% CP diet with 16.7 kJ g^?1 energy was significantly higher than those from fish fed 25% and 30% CP diets with 12.5 kJ g^?1 energy (P< 0.05). Based on the results of this experiment, we concluded that the optimum dietary P/E ratio was 27.5 mg protein kJ^?1 with diet containing 45% CP and 16.7 kJ g^?1 energy in juvenile olive flounder.     

花鲈冷冻精子诱导漠斑牙鲆雌核发育

采用紫外线(UV)对冻存的花鲈精子进行照射使其遗传物质失活,作为异源精子诱导源,与漠斑牙鲆卵子进行“授精”,可以诱导漠斑牙鲆卵进行雌核发育。结合静水压处理,抑制第二极体排出,成功获得了漠斑牙鲆雌核发育二倍体鱼苗。实验表明,同源精子和异源精子均可在紫外线照射遗传失活后诱导漠斑牙鲆卵雌核发育,经实验筛选出异源精子诱导漠斑牙鲆雌核发育的最佳条件为花鲈冻存精子采用80 mJ/cm²的紫外线照射,然后与漠斑牙鲆卵子进行授精,培育水温18 ℃条件下,受精后4~5 min,施以65 MPa的静水压休克处理6 min,可有效诱导漠斑牙鲆卵的雌核发育。采用形态学、流式细胞仪DNA含量分析和微卫星标记技术对雌核发育鱼苗进行了分析,证明了雌核发育鱼苗为雌核发育二倍体。本文首次报道了采用异源冷冻精子诱导漠斑牙鲆鱼卵进行雌核发育的技术方法,为漠斑牙鲆性别控制和遗传改良提供了技术手段。     

Photoperiod influences the growth, food intake, feed efficiency and digestibility of red sea bream (Pagrus major)

Two consecutive trials were conducted to investigate the effects of photoperiod manipulation on growth rate, food intake and feed conversion efficiency (Trial 1), and the digestibility of nutrients and energy (Trial 2) in red sea bream, Pagrus major (body weight 19–120 g). Fish were exposed to four photoperiods (6L:6D, 12L:12D, 16L:8D and 24L:0D) with light intensity 1500 lx on the water surface. The fish were fed with a commercial diet to apparent satiation. In Trial 2, 0.5% chromic oxide (Cr₂O₃) was used as an inert marker. Significantly higher weight gain and specific growth rates were observed in fish exposed to a 24L:0D photoperiod followed by 16L:8D, 6L:6D and 12L:12D photoperiods (P < 0.05). Food intake and feed conversion efficiency (FCE) were also significantly higher in fish exposed to 24L:0D followed by 16L:8D, 6L:6D and 12L:12D photoperiods (P < 0.05). Fish exposed to 24L:0D and 16L:8D photoperiods showed significantly higher lipid and energy digestibility than those exposed to a 12L:12D photoperiod (P < 0.05). The results demonstrated that the enhancement of growth performances under 16L:8D and 24L:0D photoperiods were attributed to improved appetite, greater food intake and higher feed conversion efficiency as well as higher digestibility.     

Swimming behaviour of winter flounder (Pleuronectes americanus) on natural fishing grounds as observed by an underwater video camera

Swimming behaviour of winter flounder (Pleuronectes americanus) was recorded near baited hooks on natural fishing grounds using an underwater video camera. Winter flounder were observed to stay on or very close to the seabed, never rising to more than 0.6 m off bottom during 1 month of observation. Winter flounder were recorded to take bait actively at temperatures as low as −1.2 °C. Movement of winter flounder was characterised by a period of swimming off seabed followed by a period of resting on the seabed. The proportion of time swimming off seabed as opposed to resting on the seabed was positively related to water temperature. Flounder spent an average of 32% of time in swimming when at −1.2 °C compared with 67% when at 4.4 °C. Voluntary swimming speed of the flounder during the period of swimming was lower at lower temperatures. An average swimming speed of 0.52 body lengths per second (L s⁻¹) at −1.2 °C was recorded compared with 0.95 L s⁻¹ at 4.4 °C. Overall rate of movement was reduced by three-fold when water temperature fell from 4.4 to −1.2 °C. The reduced rate of movement at lower temperatures is discussed in relation to potential fishing area of fixed fishing gears such as gillnets.     

Optimum dietary protein to lipid ratio for Starry flounder (Platichthys stellatus)

An 8‐week feeding trial was conducted to evaluate the optimum dietary protein to lipid ratio (P/L) for Starry flounder Platichthys stellatus (30.7 ± 0.7 g initial weight). Nine fishmeal‐based diets with three protein levels (40%, 45% and 50%) and three lipid levels (8%, 11% and 14%) were fed to 30 fish in triplicate. Results showed that the growth performance was affected by dietary P/L ratio significantly, and the best weight gain rate, specific growth rate and feed conversion ratio were appeared at fish fed P45L14 and P50L11 diets. Apparent digestibility coefficient of protein was increased with increasing dietary lipid, but lipid digestibility was decreased by dietary lipid. Lipid deposition in tissues was increased with increasing dietary lipid at each protein level and was higher for the lower protein diets. In liver, the per cent of both eicosapentaenoic acid and docosahexaenoic acid were increased by dietary protein. High dietary protein level (50%) at high lipid level (14%) increased arachidonic acid significantly. Aspartate aminotransferase and alanine aminotransferase were affected significantly by dietary P/L ratios. High density lipoprotein cholesterol and total cholesterol of 45% protein groups were significantly higher than others. The study revealed that the optimum dietary protein to lipid ratio for starry flounder was P45L14 or P50L11, or P/E ratios were 25.49 and 27.70 mg protein kJ^?1 gross energy respectively.