Folate in eggs and developing larvae of Atlantic halibut, Hippoglossus hippoglossus L.

Folate mobilization from the yolk compartment during larval development was studied by analysing the folate concentration in whole body, embryo and yolk in a single batch of Atlantic halibut, Hippoglossus hippoglossus L., eggs and larvae that showed successful fertilization and development. There was a net loss of approx. 50% of folate from yolk during endogenous feeding. Further, only 23% of the decrease in yolk folate was retained in the larval body. The data suggest a need for folate for metabolic and growth purposes during embryogenesis of approximately 2 μg g^?1 weight gain. Relative to these data and published folate requirement for cold‐water species, batches of egg from 16 Atlantic halibut brood fish contained variable and, for some batches, critically low levels of folate. This may constitute a potential problem for larval development until start feeding.     

The influence of salinity on the morphological development of yolk sac larvae of Atlantic halibut, Hippoglossus hippoglossus (L.)

The effects of incubation salinity on survival and morphological development of yolk sac larvae of Atlantic halibut, Hippoglossus hippoglossus (L.), were studied at two salinities, 35.5%o and 32.3%o. Yolk sac larvae incubated at 35.5%o developed early tail deformities before 50 day-degrees (9 days post-hatch), and necrosis, oedema and calculus in the urinary bladder at about 150 day-degrees (28 days post-hatch). However, the incidence and severity of mouth deformity was not influenced by salinity. The cumulative mortality up to first feeding was 17% when the larvae were incubated at 3 5.5%o compared to 7% at 32.3%o. A broad variation between the different larval groups was noted in the extent of injuries induced by the higher salinity. Therefore, salinity must be considered as a critical environmental parameter for the yolk sac larvae of Atlantic halibut in aquaculture.     

Absorption of immunomodulating β(1,3)-glucan in yolk sac larvae of Atlantic halibut, Hippoglossus hippoglossus (L.)

A soluble immunomodulating β(1,3)-glucan, laminaran, was given in drinking water to yolk-sac larvae of Atlantic halibut, Hippoglossus hippoglossus L. The larvae absorbed laminaran when the concentration was 25 mg l^–1. After 5 days exposure to laminaran, each larva contained 2.3 ng and 46 ng of ³H- and ¹²⁵I-labelled laminaran, respectively. After 10 days exposure, each larva contained 4.5 ng and 47 ng of ³H- and ¹²⁵I-laminaran, respectively. The absorption was confirmed by fluorescence microscopy of larvae exposed to fluorescein-labelled laminaran (FITC-laminaran). The fluorescence was localized to cells in the intestinal epithelial layer and the skin epithelial layer.     

Large-scale rearing of Atlantic halibut, Hippoglossus hippoglossus L., yolk sac larvae: effects of flow rate on growth, survival and accumulation of bacteria

The role of flow rate in large-volume production units (2800 L silos) for Atlantic halibut, Hippoglossus hippoglossus L., larvae was studied. Correlations between flow rate, bacterial numbers (a measure of water quality), the larval growth and development rates, and mortality were assessed. The experiment included a total of six silos, two each at three different flow rates. Flow rate and mortality were positively correlated: the number of dead larvae on day 30 was highest (i.e. 2200 and 2000) in the silos with highest flow rate (8L min^?1) and lowest (i.e. 1300 and 1200) in the silos with the lowest flow rate (2L min^?l). Larval weight was negatively correlated with flow rate: on day 30, the mean dry weight was 968 μg in the silo with the lowest flow rate and 820 μg in the silo with highest flow rate. Yolk sac utilization efficiency was 92% in the silo with the lowest flow rate and 72% in the silo with the highest flow rate. The number of bacteria were highest (2.7-10⁶mL^?1) in the incubators with the lowest flow rate.     

Time of first feeding of Atlantic halibut, Hippoglossus hippoglossus L., larvae

Atlantic halibut, Hippoglossus hippoglossus (L.), eggs originating from one female were evenly distributed between four silos (4.8 m³) shortly prior to hatching. At days 30, 35, 40 and 44 after hatching [i.e. 200, 230, 260 and 290 day-degrees (days^o)], the larvae were successively collected and transferred to indoor start feeding tanks, and larvae were offered a diet of instar II Artemia nauplii which had been enriched short time (24 h). A significant correlation was found between the age of the larvae and onset of first feeding. The larvae transferred to start-feeding incubators at 290 days^o were able to capture Artemia only a few hours after transfer, whereas it took 6 days for the larvae transferred at 200 days^o to reach a corresponding ingestion level. Larval growth was also positively correlated to both larval age and prey consumption. However, there were no differences in survival between the larval groups.     

A formulated diet for Atlantic halibut (Hippoglossus hippoglossus, L.) larvae

A diet for Atlantic halibut-larvae was formulated taking into account the fact that marine-fish larvae have a limited ability to assimilate protein and lipid. Dietary protein consisted of a free amino-acid premix (7.2% of crude protein), predigested-squid mantle (7.2%), squid mantle (8.6%) and cod-muscle mince (77.0%). Lipid sources were soyabean lecithin (33% of crude lipids), crude phospholipids extracted from cod roe (10%) and sardine oil (57%). Larvae were weaned onto the experimental diet at wet-body weights of 0.07, 0.10 or 0.16 g, respectively. The experimental diet was fed for 31, 25 or 17 days, respectively, and the experiment was terminated on the same calendar day for all groups. A control group was fed with Artemia nauplii enriched with DHA Selco™ from 0.07 g. Survivals ranged from 78% in larvae transferred at 0.10 g to 96% in those transferred at 0.16 g and in the control group. Daily specific-growth rates (SGR) were 3.1 ± 0.07, 3.3 ± 0.11 and 2.2 ± 0.01% day⁻¹ in larvae transferred at 0.07, 0.10 and 0.16 g, respectively, while growth in the control group was 5.1% day⁻¹. It was concluded that weaning of Atlantic-halibut larvae is feasible from 0.7 g (approximately 20 days post first-feeding) when the formulated diet contains predigested protein and ample amounts of phospholipids.     

Atlantic halibut, Hippoglossus hippoglossus L., larvae cultivation literature, including a bibliography

The first attempts to rear Atlantic halibut, Hippoglossus hippoglossus L., larvae were carried out in Norway in the period from 1974 to 1980, when ripe adult specimens of Atlantic halibut were net-caught, and stripped for eggs and milt. Both incubation of yolk-sac larvae and first-feeding were carried out in large submerged plastic bags and the larval food consisted of natural zooplankton collected from surrounding lagoon water. This semi-extensive production method was further developed and led to the establishment of several commercial production trials by the end of the 1980s. During recent years, research has been focused on intensive methods for first-feeding and the combined effort of several research institutes has resulted in a reliable production method. During the late 1980s and early 1990s, the main research activity was focused on the biology of and rearing techniques for eggs and yolk-sac larvae. These techniques provided satisfactory yields for several years. However, during the past few years, a certain decrease in survival through the yolk-sac stage has been experienced at several hatcheries. Since the early 1990s, the first-feeding period has represented the bottleneck in the development of a reliable rearing method. The main effort has been concentrated on system configuration and on improving live prey quality. In the future, new feeding strategies including further improvement of live prey (i.e. Artemia), the use of copepods and early weaning onto a formulated diets should be emphasized. Further research on hygiene and technological improvements is needed to increase growth and survival through metamorphosis.     

A tank design for first feeding of Atlantic halibut, Hippoglossus hippoglossus L., larvae

An intensive method of juvenile Atlantic halibut, Hippoglossus hippoglossus (L.), production has been under development over the last decade because of the problems associated with the extensive method. The lack of initiation of feeding behaviour has been the main obstacle for successful indoor rearing under artificial light and feeding conditions. In the present paper, an intensive method for the first feeding of halibut larvae is described and verified by practical feeding trials. The method involves circular 1.5-m³ indoor tanks with a peripheral ring-shaped cover, the use of continuous light, UV-A radiation during the first 24 h, central up-welling of water made by aeration and the use of microalgae in the rearing water. The single most important factor in such systems is to maintain a current pattern which allows the larvae to orientate and position themselves to face the water current for easy capture of prey.     

Lipids in eggs from first-time and repeat spawning Atlantic halibut, Hippoglossus hippoglossus (L.)

Abstract The production of viable larvae is critical to any aquaculture venture. Since lipids are important components of fish eggs, the amount and nature of the lipid may be crucial to the survival of the larvae. In this study the lipid content of unfertilized eggs from first-time and repeat spawning Atlantic halibut females was compared. The egg lipid content for the first-time spawner ranged from 0·18 to 0·41mg/egg; the range for the repeat spawner was 0·27 to 0·38mg/egg. However, the mean total lipid per egg values were similar for both fish at ca. 0·31mg/egg. The repeat spawner prodticed more eggs than the first-time spawner, thus allocating more lipid to egg production. In the eggs from the two fish 62–84% of the lipids was phospholipid.     

A correlation between phototactic response and first-feeding of Atlantic halibut (Hippoglossus hippoglossus L.) larvae

The correlation between positive phototaxis and feeding incidence at first‐feeding for groups of Atlantic halibut larvae ranging in age from 210 to 240 degreedays, post hatch, was examined. Phototactic response was measured as the fraction of larvae that responded by horizontal swimming towards a light source, and the median travel distance for the responding fraction of larvae. Within the ranges investigated, larval age, size or proportion of deformed larvae had no significant effect on the phototactic response, or on the feeding incidence (deformed larvae excluded from analysis). The fraction of larvae responding phototactically and the median travelling distance for responding larvae were significantly correlated. Feeding incidences after 24 and 48 h were also significantly correlated with both measures of phototactic response. The possibility of using phototactic response in quality assessment is discussed.     

The effect of light on activity and growth of Atlantic halibut, Hippoglossus hippoglossus L., yolk-sac larvae

The growth and activity of Atlantic halibut, Hippoglossus hippoglossus L., yolk-sac larvae exposed to light of differing intensities and wavelengths were investigated every fifth day. The experiments were conducted at 6 ^oC from day 1 until day 34 post hatch. Four intensities of constant white light (2.0, 0.3, 0.03 and 0.005 μ Em^?2 s^?1, λ_max 590 nm), and constant coloured light of equal intensities (0.03μ Em^?2s^?1) in the blue, green and red spectrums (λ_max 450, 560 and 670 nm, respectively) were used. In addition to a control treatment in constant darkness, one treatment was incubated in a 12:12 h light: dark photoperiod. The light treatments did not have any discernible effect on the total length, myotome height, dry weights or yolk conversion efficiencies. The most intense white light resulted in an increased activity on days 24 and 30 post hatch, resulting in a temporarily reduced length and myotome height for the larvae in these groups compared to the other treatments. Larvae from all treatments were of the same size 34 days post hatch. The dry weights of the larvae and yolk-sacs were unaffected by light treatment. The activity increased independently of light treatment until 120 degree-days, and thereafter, the strongest white light resulted in an temporarily increased activity. The distribution of activity changed independently of light regime in the beakers during development.     

Effect of recovery salinity on survival of acutely stressed halibut (Hippoglossus hippoglossus L) larvae

First‐feeding halibut larvae (245‐day degrees; 40 days post hatch), reared at 34 g L^?1 salinity and 7°C, were subjected to handling and allowed to recover in a range of salinities (0–34 g L^?1) and at 10°C. Survival of the unfed larvae was determined daily for 18 days. Mortality rates approached 0 after 4 days in all treatments and presumed starvation‐induced mortality started at about 11 days post handling. By 20 days post treatments, all larvae had died. Salinities in the range of 10–20 g L^?1 produced significantly (anova , P<0.01) higher initial survival (71–95%) than salinities above 20 g L^?1 (24–48%) or below 10 g L^?1 (0–19%) and this survival pattern changed little in unfed larvae for the first 10 days following the stressor. For example, 24 hour post handling, survival of halibut was improved from 28.7±16.5% (mean±standard error, n=3) at 34.0 g L^?1 to 95.2±4.8% at 13 g L^?1. A second‐order polynomial regression of 4‐day post‐handling survival data (y=?0.002x ²+0.0603x+0.0699, r²=0.3936) predicted a maximum survival at 15.1 g L^?1 salinity. These results have important implications for halibut aquaculture and research when handling of larvae is unavoidable. For practical applications, we recommend reducing salinity of receiving waters to 15–20 g L^?1 with a slow (3–4 days) reacclimation to ambient conditions.     

Egg yolk nutritional constituents as indicators of egg quality in Atlantic halibut (Hippoglossus hippoglossus L.)

Variation in oocyte quality affects sustainability of finfish aquaculture products including Atlantic halibut (Hippoglossus hippoglossus L.). To have an insight into role of major egg yolk constituents in the oocyte quality, egg fatty acid (FA), amino acid (AA) and folate contents were related to normal blastomere symmetry, and fertilization and hatching success. Significant correlations were found between dihomo‐γ‐linolenic acid (DGLA, 20:3n6), palmitic acid (PA, 16:00) and docosapentaenoic acid (DPA, 22:5n3), and normal blastomere symmetry and survival success. Egg concentrations of myristic acid (MA, 14:0), oleic acid (OA, 18:1n9), stearidonic acid (SA, 18:4n3) and eicosadienoic acid (EDA, 20:2n6) explained 56 % variation in fertilization success. OA and EDA explained 70 % of variation in blastomere symmetry. Palmitoleic acid (POA, 16:1n7) and linolenic acid (LNA, 18:3n3) concentrations explained 57% of variation in hatching success. Egg valine concentrations correlated with fertilization rates, and aspartic acid and leucine correlated with normal blastomere symmetry. Alanine and valine concentrations explained together 45% of variation in fertilization. Glutamic acid and tyrosine concentrations explained 68% of variation in normal blastomere symmetry while serine, arginine and valine explained 36% of variation in hatching. These FAs and AAa may be potential indicators of oocyte quality in Atlantic halibut.     

Design and operation of Atlantic halibut, Hippoglossus hippoglossus L., egg incubators

An egg incubator system specially designed to meet the requirements of Atlantic halibut, Hippoglossus hippoglossus L., eggs is described. Compared to other pelagic marine fish eggs, halibut eggs are frequently found to be heavy and lacking in buoyancy in seawater incubators. The biological features of halibut eggs which explain the choice of system design are discussed. In addition to the biological requirements, the system described is designed to meet the demands of commercial hatcheries where handling of large egg masses with a minimum of labour is needed. The incubation method has proved by experience to give high survival rates throughout egg development.     

Suitable methods for cryopreservation of semen from Atlantic halibut, Hippoglossus hippoglossus L.

Decrease in the quality and quantity of Atlantic halibut, Hippoglossus hippoglossus L., semen towards the end of the reproductive season hampers production of good-quality embryos. Therefore, cryopreservation of spermatozoa is a method showing potential to facilitate controlled reproduction in Atlantic halibut. The present study aimed at establishing the appropriate cryopreservation procedure. We tested 20 extenders composed of four various diluents and five cryoprotectants (DMSO, DMA, methanol, propylene glycol, and glycerol) to determine the best extender. Then, we examined cryopreservation quality using various methods of loading and various volumes of cryopreserved samples. In most of the tested variants, sperm diluted with an extender showed high motility after 24-h incubation despite the high osmotic pressure of the extender. Modified turbot extender (MTE) was the best of the tested diluents, securing the highest post-thaw motility (P < 0.05), and DMSO, DMA, and methanol were the best cryoprotectants (P < 0.05). There was no significant effect of 15-min equilibration of semen in MTE-based extenders prior to freezing on post-thaw motility (P > 0.05). MTE-based extender was chosen as the most suitable. Semen cryopreserved in straws, Eppendorfs or Ziploc bags in volumes ranging from 0.25 to 20 ml showed similar high fertilization ability. Survival of larvae produced with the cryopreserved sperm did not differ from controls produced with freshly collected sperm. Motility 3 h after thawing was high but depended on the type of cryoprotectant and the volume of cryopreserved sperm (P < 0.05). The developed cryopreservation procedure has been applied at our Atlantic halibut breeding station for seed production.     

Effect of predigested protein on growth and survival of Atlantic halibut larvae (Hippoglossus hippoglossus L.)

In this study Atlantic halibut (Hippoglossus hippoglossus L.) larvae (0.12 ± 0.04 g) were, from day 40 post first feeding, offered six diets in which 10% or 30% of the dietary protein was hydrolysed with (a) pepsin (P), (b) pepsin + trypsin (PT) or (c) pepsin + trypsin + chymotrypsin (PTC). In addition, a diet without hydrolysed protein was offered, and enriched Artemia was fed as control. The amount of soluble protein increased progressively with the enzyme treatments P, PT and PTC and with higher inclusion levels of hydrolysed protein. Survival was highest among the larvae offered Artemia (83 ± 0%) or the diet 10P (10% pepsin hydrolysed protein; 67 ± 4%). The diet 10P supported survival significantly better than the more hydrolysed diets 10PTC, 30P, 30PT and 30PTC, but not significantly better than the non‐hydrolysed diet and 10PT. Specific growth rate (SGR) was 1.76 ± 0.20 in average for all groups of larvae and was not significantly affected by the diets. Still, the larvae offered pepsin hydrolysed diets tended to have better growth (2.10 ± 0.05 SGR; P < 0.06) than the larvae offered the other hydrolysed diets. The larvae offered the formulated diets did not differ in chemical composition.     

Effects of light administration and algae on first feeding of Atlantic halibut larvae, Hippoglossus hippoglossus (L.)

The aim of the study was to investigate the effects of two light regimes that independently had shown positive effects on feeding and growth in cultures of Atlantic halibut, Hippoglossus hippoglossus (L.), larvae. The regimes were low-intensity overhead light and submerged light at intermediate light intensities. Secondly, an alleged beneficial effect of algae was investigated. An experiment was designed to include four different regimes in the larval cultures: low-intensity overhead light with and without algae (Tetraselmis sp.), and submerged light with and without algae. The results showed that submerged light was superior to overhead light with respect to larval growth, survival and feeding incidence. It was further indicated that algae improved larval growth and survival, but no effect was shown on feeding incidence. There was, however, no interaction between the effects of algae and those of the light regime. The causal effect of the algae may be ascribed to indirect factors, such as light attenuation as well as a direct and indirect nutritional effect.