Early weaning of winter flounder (Pseudopleuronectes americanus Walbaum) larvae on a commercial microencapsulated diet

Like most small marine fish larvae, the stomachs of winter flounder Pseudopleuronectes americanus are undeveloped at first feeding and have relatively reduced digestive capacity. This work was undertaken to test whether larvae at the onset of stomach differentiation (larval size about 5.5 mm) could be early weaned onto a commercial microencapsulated diet. We assessed the effect of early weaning by first comparing growth performance (standard length, total protein content and age at metamorphosis) of larvae fed enriched live prey from first feeding to a size of 5.5 mm and then reared on three different feeding regimes until metamorphosis: (1) live prey (LP) as a control group; (2) mixed feeding of live prey and microencapsulated diet (LP‐ME); (3) exclusively microencapsulated diet (ME) after fast weaning over 4 days (to a larval size of 6.2 mm). No differences were observed between larval development in the two first groups, which began metamorphosis at 40 days old. The larvae of the third group showed significantly slower growth that resulted in a delay of 4 days in the onset of metamorphosis. Differences in live prey availability between the treatments and the short transition period to allow the larvae to adapt to the new diet were identified as possible contributing factors to the slower growth and to the delay in metamorphosis of early weaned larvae. In a second experiment, the transitional weaning period was increased until the larvae were 6.6 mm in length. Weaning at that size resulted in no slowing of growth or delay in metamorphosis, suggesting that the feeding schedule was adequate.     

Growth of early juvenile winter flounder (Pseudopleuronectes americanus Walbaum)

Metamorphosis is a critical developmental stage in marine fish species, but mortalities are also elevated at the settling phase. Our objective was to evaluate the effects of different rearing systems and larval diets on survival, general condition and pigmentation of newly settled winter flounder (Pseudopleuronectes americanus) juveniles. At settlement, winter flounder juveniles were reared in two different types of tanks (plankton kreisel and rectangular raceway). The early juveniles were followed for 1 month and their condition was evaluated using biochemical indices. Juveniles did not show signs of fin rot and pigmentation was well developed, but they had different RNA/DNA ratios. This study highlights the fact that rearing conditions at the onset of the juvenile stage may be critical for successful juvenile production.     

Trichodina murmanica (Ciliophora) and Gyrodactylus pleuronecti (Monogenea) parasitizing hatchery-reared winter flounder, Pseudopleuronectes americanus (Walbaum): effects on host growth and assessment of parasite interaction

Winter flounder, Pseudopleuronectes americanus, caught in Passamaquoddy Bay, New Brunswick, Canada, and held captive under crowded conditions, developed mixed infections of Trichodina murmanica Polyanski, 1955 (Ciliophora) and Gyrodactylus pleuronecti Cone, 1981 (Monogenea). A protocol involving sequential sieving was used to separate the two species of parasites and produce viable experimental baths. Replicate groups of juvenile, hatchery-reared flounder received one of the following treatments: mixed bath of G. pleuronecti and T. murmanica , bath of G. pleuronecti, bath of T. murmanica or parasite-free (controls). The abundance of both parasites correlated negatively with condition factor of the flounder ( r =−0.354, P  < 0.001 for Trichodina ; r =–0.205, P  < 0.05 for Gyrodactylus ). During the periods of peak parasite abundance (1–2 weeks postinfection), the effect of the two parasite species was additive, as mean condition factor and the percentage change in weight were significantly lower (ANOVA, P  < 0.05) among fish with mixed infections compared to single infections or controls. The most common signs of tissue pathology were increased density of epidermal mucous cells on the fins and macrovesicular lipidosis of the hepatocytes. After a significant decline in parasite infrapopulations (3 weeks postinfection), infected fish resumed normal growth, indicating the observed effects were somewhat reversible.     

Comparison of stress responses in wild and captive winter flounder (Pseudopleuronectes americanus Walbaum) broodstock

Chronic stress is responsible for many problems occurring in fish holding facilities, such as increased susceptibility to disease, reduced growth rate and suppression of the immune response. The goal of this study was to verify if wild winter flounder (Pseudopleuronectes americanus Walbaum) suffer from chronic stress when kept several months in captivity. We hypothesized that winter flounder not subjected to chronic stress in captivity would have similar or higher condition indices and similar stress response compared to fish from the wild. Our results showed that the condition index of winter flounder kept in captivity was higher (1.50) than those from the wild (1.33) even after one year of captivity. The intensity of the acute stress response following short‐term exposure to air was similar between wild and captive fish (no significant interaction between the factors ‘stress’ and ‘origin of the fish’). Body water content (an indicator of energy reserves) was lower (76.1%) compared to wild fish (79.8%) after one year of captivity, suggesting that the winter fast had been less energetically demanding in captive fish. These results indicate that no chronic stress was present in captive fish. On the other hand, the mortality during captivity was around 30%, which is high for the aquaculture industry. Further work to address this problem is required.     

Sperm cryopreservation of silver barb (Barbodes gonionotus): cryoprotectants,cooling rate and storage time on sperm quality

Effectiveness and efficiency of frozen sperm on fertilization and hatching success of eggs from silver barb was examined in relation to cryoprotectants, freezing rate and storage period. Sperm was diluted in calcium‐free Hank's balanced salt solution, equilibrated with dimethylsulphoxide (DMSO), propylene glycol, sucrose or methanol at 5%, 10%, 15% or 20% final concentrations, and frozen in 250‐μL straws using a one‐step freezing procedure (1, 5 and 8°C min^?1 from 25 to ?40°C). Highest post‐thaw sperm motility was found from a treatment using 10% DMSO and 5°C min^?1 (82.2 ± 2.1%), similar to that of 10% DMSO and 8°C min^?1 (87.8 ± 3.2%). Post‐thaw motility of sperm frozen at 5 or 8°C min^?1 was significantly higher than 1°C min^?1. Relative sperm motility declined significantly after 10 months of cryostorage while viability did not change during a 12‐month cryostorage. Average fertilization rates of sperm after 1 and 4 months of storage were 64.5 ± 4.6% and 61.3 ± 3.4%, respectively, similar to those of fresh sperm (69.6–72.3%). Hatching rates of cryopreserved sperm (45.4–51.2%) were similar to those of fresh sperm (51.8–57.8%). This study developed suitable methods for cryopreservation of silver barb sperm that can be used to facilitate hatchery operation.     

Influence of temperature on growth and survival of juvenile winter flounder,Pseudopleuronectes americanus (Walbaum) reared under continuous light

Winter flounder, Pseudopleuronectes americanus, has emerged as a promising candidate flatfish for cold‐water aquaculture and restocking. Here, juveniles were reared for 8 weeks at three temperatures: 10, 15, and 20°C under 24‐hr light. All fish were imaged at stocking and at 2‐week intervals, where growth was measured as changes in standard length (S_L) and body area (B_A). By week 2, fish reared at 15 and 20°C were larger than those grown at 10°C. At weeks 4 to 6, fish at 15°C were larger than fish at 20°C. Linear regressions were used to model growth dynamics over time at each temperature. Highly significant linear growth trajectories were detected over time for S_L and B_A. S_L and B_A regressions also showed a significant difference among the slopes across temperatures, where comparing slopes showed the best temperature to rear the flounder was 15°C. Weights of fish held at 15°C and 20°C were greater than at 10°C at the termination of the experiment. Within each temperature, the growth rate of malpigmented fish was not different from that of the normally pigmented fish. Overall, growth of winter flounder was comparable to that of other commercially produced flatfish species, providing strong evidence for this flatfish species as a potential species for aquaculture.     

Determining an optimal release site for juvenile winter flounder Pseudopleuronectes americanus (Walbaum) in the Great Bay Estuary, NH, USA

One of the main elements in developing an optimal release strategy for an enhancement effort is to evaluate and select release sites that will support growth and survival of newly released, cultured fish. Three potential release sites (New Castle (NC), Broad Cove (BC) and Oyster River (OR)) in the Great Bay Estuary, NH, USA were evaluated for pilot‐scale releases of winter flounder (Pseudopleuronectes americanus Walbaum). Cultured juvenile flounder were placed in cages at each of the three sites. Sites were evaluated based on growth and survival of the fish in relation to water temperature, prey availability and sediment composition. Fish grew faster in the two upper estuarine sites BC (0.54 mm day^?1) and OR (0.56 mm day^?1) than at the site at the mouth of the estuary (NC=0.37 mm day^?1). Fish survival (44–53%) and water temperature (17.8–19.7°C) did not vary between sites. Benthic samples showed that prey was available to, and eaten by, the flounder. Sediment composition was the main difference between the three sites, with one site (NC) characterized by gravel whereas the other two sites were sandy. These results corroborate other studies showing the importance of sediment quality for the distribution of flatfish populations. From these results, we can confidently eliminate NC as a potential release area and recommend that sandy sites are better for stocking cultured juvenile winter flounder than gravely sites.     

Evidence for the presence of a viral non-lymphocystis type disease in winter flounder, Pseudopleuronectes americanus (Walbaum), from the north-west Atlantic

Abstract. A non-lymphocystis type disease associated with virus particles has been documented in winter flounder, Pseudopleuronectes americanus (Walbaum), taken from a small estuary in Conception Bay, Newfoundland. The papillomatosis-type lesions found on the dorsal surface of the fish contained numerous electron-dense particles, approximately 30 nm in diameter. A number of lines of evidence, including cytochemical studies, indicated that the particles were not glycogen but virions. Sensitivity to deoxyribonuclease enzyme treatment and the presence of crystalline viral'factories' in the nuclei of infected cells further suggested that the particles were small DNA virions. Characteristics including nucleic acid composition, particle size, site of multiplication and the lack of an envelope indicate that this fish virus is similar to the Papovaviridae and Parvoviridae families of mammalian viruses. Lymphocystis is common in flatfish populations along the Newfoundland and Labrador coasts of the N.W. Atlantic, but this is the first record of flatfish infection with a small DNA virus.     

Feeding larvae of winter flounder Pseudopleuronectes americanus (Walbaum) with live prey or microencapsulated diet: linear growth and protein, RNA and DNA content

A commercial microencapsulated diet was used as a total or partial replacement of live prey for feeding larvae of winter flounder Pseudopleuronectes americanus (Walbaum), a potential alternative finfish species for coldwater marine aquaculture. Growth performance (morphometric measurements and biochemical composition) and nutritional condition (RNA/DNA ratios) of larvae fed live prey (Brachionus plicatilis Müller), a microencapsulated diet or a mixed diet of live prey and microcapsules were compared. Newly hatched larvae were unable to digest microencapsulated diet; live prey at initial feeding was required for their survival and growth. Larvae offered a mixed diet showed slower growth than larvae fed exclusively with live prey. However, at the onset of stomach differentiation, RNA/DNA ratios (indicators of protein synthesis potential) of the larvae fed both diets became similar. We suggest that, at that stage (size 5.5–6.3 mm), enzymatic activity had developed enough to allow digestion of inert food. As the RNA/DNA ratio is a good indicator of nutritional condition, it appears to be an interesting tool for the assessment of diet adequacy in marine larval feeding technology.     

Influence of gonadotropic hormone-releasing hormone analog (GnRH-A) on plasma sex steroid profiles and milt production in male winter flounder,Pseudopleuronectes americanus (Walbaum)

The effects of gonadotropic hormone-releasing hormone analog (GnRH-A) treatment on the onset and duration of increases in plasma sex steroids and milt production (milt volume and number of spermatozoa) were investigated in prespawning male winter flounder. After treatment of maturing males during the winter with a single injection of either 20 or 200 g/kg [D-Ala⁶, Pro⁹-NHEt]LHRH (GnRH-A), plasma levels of testosterone and 11-ketotestosterone were increased within 12h and the steroid hormone levels remained elevated for long periods lasting several days. The androgenic steroid response of males was delayed after the administration of a lower dose of GnRH-A (2 g/kg). Although a single GnRH-A injection in December or January advanced the onset of spermiation in some males, only small amounts (<50 l) of milt could be collected. By March, all males were in spermiating condition following GnRH-A treatment; however, significant increases in sperm production, particularly increases in milt volume, occurred in fish twice treated with GnRH-A.Part of this work presented at the First Symposium of Fish Endocrinology, Edmonton, Alberta, Canada June 1988 and the Satellite Symposium on Applications of Comparative Endocrinology to Fish Culture, Almuñecar, Granada, Spain May 1989     

Effect of cryoprotectants, equilibration periods and freezing rates on cryopreservation of spermatozoa of mahseer, Tor khudree (Sykes) and T. putitora (Hamilton)

A study was conducted to standardize a protocol for cryopreservation of spermatozoa of the endangered mahseer, Tor khudree (Sykes) and T. putitora (Hamilton). The suitability of the cryoprotectants, dimethyl sulphoxide (DMSO) and glycerol, and the combination of the two were tested. Four equilibration periods and four freezing rates were also tested for their standardization. A combination of 9% DMSO and 11% glycerol gave significantly higher mean percentage of hatching in both T. khudree (45.59±1.86%) (control 71.08±0.59%) and T. putitora (45.00±1.25%) (control 73.48±1.19%) among the eight different treatments. Among the four different equilibration periods tested, the equilibration period of 30 min^?1 yielded the highest mean hatching percentage in T. khudree (39.46±1.94%) (control 71.70±0.61%) and T. putitora (38.28±1.06%) (control 73.11±0.82%). Freezing straws at a height of 8 cm above LN₂ surface for 10 min^?1 gave higher hatching percentages for both T. khudree (41.75±1.72%) (control 73.99±1.17%) and T. putitora (41.34±2.04%) (control 72.48±1.51%). The study reports the superior performance of the combination of DMSO and glycerol for the first time.     

Monoaminergic substances in the teleost brain: Catecholamine levels in male and female winter flounder,Pseudopleuronectes americanus Walbaum,associated with gonadal recrudescence

High performance liquid chromatography with electrochemical detection (HPLC-EC) was used to quantitate catecholamine (CA) levels in the winter flounder brain following perchloric acid extraction/alumina purification of CNS tissues. Greater concentrations of norepinephrine (NE) and dopamine (DA) were present in the hypothalamus compared with the CA levels in whole brain. A seasonal study of CA brain levels in reproductively active male and female flounder demonstrated that monoamine levels reach their maxima in October in association with the rapid increases in gonadosomatic index. When perchloric acid extracts of the teleost and rat hypothalamus were submitted to direct HPLC-EC analysis, without alumina purification of CA neurotransmitters, similar hypothalamic profiles were obtained indicating the presence of identifiable biogenic amine neurotransmitters substances including NE, DA and serotonin (5-HT).     

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.     

Growth and survival of winter flounder (Pseudopleuronectes americanus) larvae reared on different photoperiod regimes from hatch to metamorphosis

The aim of this study was to determine the effect of photoperiod on growth and survival of winter flounder, Pseudopleuronectes americanus, from hatch until metamorphosis. Sixteen 100 L upwelling tanks were stocked with larvae at an initial stocking density of 20 larvae/L. Larvae in eight tanks were provided with continuous light (24 hr light/0 hr dark), while those in the remaining eight tanks were grown under a photoperiod of 14 hr light/10 hr dark. Tanks were sampled for larval standard lengh from 6 to 28 days post‐hatch, and the number of live larvae, live metamorphosed and dead metamorphosed fish were recorded at the termination of the experiment (starting 46 dph). Results indicated that larvae provided with continuous light were larger than those sampled from 14 hr light/10 hr dark treatment. No effect of photoperiod on the number of non‐metamorphosed fish recovered at the end of the experiment was detected. On the contrary, over three times as many fish survived and reached metamorphosis under continuous light. An ANCOVA model showed a significant interaction between light and age. Both regressions were significant and provided the following growth equations: SL_{24 hr light/0 hr dark} = 0.101 × age + 2.55 (r² = 0.96) and SL_{14 hr light/10 hr dark} = 0.094 × age +2.45 (r² = 0.97). Using 6.36 ± 0.059 mm SL as the mean size at metamorphosis, larvae experiencing continuous and 14 hr light/10 hr dark photoperiods would reach metamorphosis in 38 and 42 days respectively.     

达氏鲟精巢细胞消化分离和超低温冷冻保存

通过研究两种酶对精巢细胞的消化效果,探究抗冻剂、降温程序、糖类和卵磷脂对达氏鲟(Acipenser dabryanus)精巢细胞冻存效果的影响,获得消化冻存后高存活率的细胞。实验使用0.25%胰蛋白酶和2 mg/m L胶原酶H+500 U/m L中性酶II的组合酶对达氏鲟精巢细胞进行消化,获得不同消化时间内的活细胞数量。另外,还研究了冷冻稀释液中分别添加10%二甲基亚砜(DMSO)、乙二醇(EG)、甲醇(MET)作为抗冻剂,采用-1℃/min慢速降温以及直接投入液氮中的快速降温方法冻存,冷冻稀释液采用D-海藻糖或同浓度D-蔗糖,以及添加5%、8%、11%卵磷脂对冻存效果的影响。结果显示:两种消化酶在同一时间消化所得的活细胞数和活细胞率无显著差异,并且都在3 h获得最多活细胞。慢速降温的冻存效果极显著地好于快速降温(P=0.01)。不同抗冻剂的保存效果差异显著,复苏后细胞相对存活率EG(51.70%±5.24%)MET(45.09%±3.15%)DMSO(40.18%±3.90%)。不同糖对达氏鲟精巢细胞冻存效果无显著影响;不同浓度的卵磷脂冻存效果有极显著差异。含8%卵磷脂的冻存液对细胞的冻存效果最好,细胞存活率可达(93.55±2.56)%,培养10 d后细胞数目为0 d时的3.19倍。     

Effect of cryoprotectants, extenders and freezing rates on the fertilization rate of frozen striped catfish, Pangasius hypophthalmus (Sauvage), sperm

The effects of four cryoprotectants (methanol, MeOH; dimethyl sulphoxide, DMSO; dimethyl acetamide, DMA; and ethylene glycol, EG), three extenders (calcium‐free Hanks' balanced salt solution, C‐F HBSS, Hanks' balanced salt solution, HBSS and sodium chloride, NaCl) and two different freezing procedures (one‐ and two‐step) on the cryopreservation of striped catfish (Pangasius hypophthalmus (Sauvage)) sperm were investigated. Sperm were frozen using a controlled‐rate freezer in 250 μL straws and stored for 2 weeks in a liquid nitrogen (LN₂) container. They were then airthawed at room temperature, and fertilization, motility and viability were assessed. The highest fertilization rate of 41% (81% of control) was achieved with the combination of 12% DMSO and 0.9% NaCl using a one‐step freezing procedure (10°C min^?1). Also, DMA resulted in a higher fertilization rate (30% or 51% of the control) than MeOH (18% or 38% of the control) or EG (8% or 12% of the control). In addition, the three extenders used did not affect fertilization rates after cryopreservation with each cryoprotectant. There were no significant differences among the three cryoprotectant concentrations and between the one‐ and two‐step freezing procedures. However, fertilization rates of cryopreserved sperm were significantly lower than the controls (P<0.05). The results of this study indicate that high fertilization rates of striped catfish eggs can be achieved using cryopreserved sperm when frozen at 10°C min^?1 in DMSO or DMA with either 0.9% NaCl or C‐F HBSS.     

不同抗冻剂对牙鲆胚胎毒性研究以及玻璃化颗粒冷冻保存方法的应用

以牙鲆(Paralichthys olivaceus)不同发育期胚胎为实验材料,研究了7种渗透性和5种非渗透性抗冻剂对牙鲆尾芽期和心跳期胚胎的毒性,同时对玻璃化液在不同胚胎发育期的毒性作用,以及玻璃化颗粒冷冻过程中,冷冻颗粒降温和解冻的时间进行了研究。结果表明,单一渗透性抗冻剂对牙鲆胚胎的毒性随着抗冻剂浓度的升高、平衡时间的延长而提高,其毒性由大到小依次为乙二醇(EG)、酒精(EtOH)、甘油(Gly)、二甲基甲酰胺(DMF)、二甲基亚砜(DMSO)、甲醇(MeOH)、1,2-丙二醇(PG)。非渗透性抗冻剂中聚乙烯吡咯酮(PVP)对牙鲆尾芽期胚胎的毒性最强,其次是蔗糖和D果糖,葡聚糖和葡萄糖毒性最弱。在总体积分数一定的情况下,PG、MeOH与DMSO体积比为9∶6∶5的混合抗冻剂对牙鲆尾芽期胚胎毒性最低;各发育期胚胎经过玻璃化液平衡后,尾芽期以前胚胎的成活率随胚胎发育期逐渐升高,心跳期以后逐渐降低,尾芽期和心跳期成活率最高。含胚胎的玻璃化颗粒冷冻降温时间最短为15.09 s,解冻时间最短为6.22 s;而不含胚胎的玻璃化颗粒冷冻降温和解冻时间分别为(13.83±1.86)s和(7.20±0.90)s。将PG、MeOH与DMSO按体积比9∶6∶5配成总体积分数为35%的混合溶液,再添加5%的蔗糖配制成玻璃化液,采用此玻璃化颗粒冷冻方法对173粒牙鲆尾芽期至心跳期胚胎进行超低温冷冻保存,解冻后共获得4粒成活胚胎。     

Short-term storage and cryopreservation of Urechis unicinctus (Echiura: Urechidae) sperm

In the present study, attempts were made to preserve Urechis unicinctus sperm at 4°C. Cryopreservation procedures were optimized for various cryoprotectants and freezing rates, equilibration times and dilution ratios. During short‐term storage, the motility of undiluted sperm was extended for 6 days of cold storage,and in 70% and 100% artificial seawater only persisted for 2 and 4 days respectively. The survival rate of undiluted sperm was maintained at a high level accordingly. After cryopreservation, the highest motility and survival rate (41.5±2.2%) were obtained in 15% dimethyl sulphoxide (Me₂SO) using a freezing rate of 30°C min^?1. After thawing the sperm cryopreserved in glycerol lost almost all motility. The motility and survival rate of post‐thawing sperm did not show significant differences after 8 and 15 min equilibration using 15% Me₂SO as cryoprotectant; the values were significantly higher than those of 2 min equilibration. Comparisons of motility and survival rate between treatments pooled by dilution ratio showed that the effect of 1:1 ratio (sperm volume to cryoprotectant volume) was best. There was no difference between 1:3 and 1:5, and other ratioswere significantly worse.     

中华乌塘鳢精子的生物学特性及其超低温保存

对中华乌塘鳢精子激活的生理生态学进行了研究,探讨了中华乌塘鳢精子超低温保存方法,中华乌塘鳢精子激活的适宜盐度为５－１５;其精子的激活不仅与激活溶溶盐度有关,而且与激活溶液的离子成份有关,激活溶液中Ｋ＾＋的存在一定程度上对精子的活动有抑帛和;中华乌塘鳢精子对ＰＨ的适应性强,在ＰＨ５．５－９．５范围内,激活率均在７０９％以上,尤以中性及弱酸性条件下的激活率最高,在ＰＨ６．０时精子活力最好。使用筛选出的