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.     

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.     

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.     

Feed intake, growth and feed conversion efficiency of Atlantic halibut, Hippoglossus hippoglossus (L.)

The individual food intake of each fish in each of four groups of Atlantic halibut, Hippoglossus hippoglossus (L.) (mean weight: 422 g) was monitored by direct observation over a period of 21 days. Gross feed conversion efficiency (= growth·feed intake^?1), net feed conversion efficiency and maintenance ration were estimated by regression analysis. Specific growth rates were found to be linearly related to weight-specific consumption at a temperature of 8–9°C: growth = 1.922· feed intake ? 0.242. Maintenance ration was 0.126% of body weight day^?1. The gross feed conversion efficiency increased asymptotically with increasing feed intakes and growth rates, and was found to approach 1.9 at high growth rates (0.5 on a dry weight basis). One feeding per day seemed to be sufficient for maximum food intake and growth rate.     

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.     

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.     

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.     

Successful early weaning of Atlantic halibut (Hippoglossus hippoglossus L.) in small shallow raceway systems

Small raceways were used in a weaning experiment with Atlantic halibut (Hippoglossus hippoglossus L.) larvae. The size of the tanks was 1.0 × 0.4 m with a 1‐ to 2‐cm water level. Duplicate larval groups were transferred to the raceways from circular first feeding tanks at 0.07, 0.10 and 0.16 g wet weight, while recommended weaning size of this species is 0.2–0.3 g. During the first 7 days of weaning, Artemia was used as a food supplement in combination with the formulated dry feed. Thereafter only dry feed was used. The dry feed used in this experiment was produced by a special heat technique. The 0.07, 0.10 and 0.16 g larval groups were evaluated after 31, 25 and 17 days respectively (same date) The corresponding average survival was 81.4%, 78.0% and 96.6% and the specific daily growth rate was 3.18%, 3.17% and 2.38% respectively. In the Artemia control group, a survival rate of 96.0% and a growth rate of 5.28% was achieved. To evaluate the weaning success, the groups were followed in a 22‐day post‐weaning period on a commercial dry diet. Higher growth rates, 5.8–6.9%, were then obtained in all experimental groups, except control. The survival here averaged approximately 80% in the three experimental groups, but showed some differences between replicates. One hundred per cent survival was achieved during weaning in the former Artemia group. From the start of weaning to the end of the post‐weaning period, the survival rates averaged 64% for the 0.07 and 0.10 g groups, approximately 80% for the 0.16 g group and 96% in the Artemia control group. Higher variance (CV) through the experiment and highest growth of the 75% quartiles of the fish groups compared with the 50% and 25%, indicated suppressed growth of the smaller fish. The successful weaning at these small sizes considerably reduced the live food period. Based on the present knowledge of the energetic demands of this species, it is calculated that weaning at 0.07 g compared with 0.25 g will reduce the amount of Artemia needed by at least 60%. No differences in pigmentation or degree of completed eye migration were detected between groups, indicating that this is determined at earlier developmental stages.     

Weaning of Atlantic halibut Hippoglossus hippoglossus L. using formulated diets with various levels of ascorbic acid

Atlantic halibut larvae (120 mg) were weaned to formulated diets with different supplementations of ascorbate- poly-phosphate, ApP (300, 2000 and 3000 mg ascorbic acid (AA) equivalents kg⁻¹ diet). The experiment lasted for 50 days with cofeeding of enriched Artemia and formulated diets during the first 30 days. During the last 20 days, only formulated diets were offered to the fish. One control group was fed only Artemia (770 mg AA kg⁻¹ dry weight) during the entire experimental period. The specific growth rate during the 50 days was ≈ 4.5% day⁻¹ and the mean weights in all dietary groups were ≈ 1 g when the experiment was terminated. No differences in mean weight and mortality were observed between the groups fed formulated diets and that fed Artemia during the experiment. The fish size in the groups fed formulated diets ranged between 0.10 and 3.05 g and this differed from the Artemia group where the size ranged between 0.35 and 1.35 g. Dietary levels of ApP had no positive effect on growth and survival. The retention of AA was significantly higher in the groups fed high dietary levels of ApP. Apparently, the bioavailability of high dietary levels of ApP appeared to be low for young halibut. After stressing the fish using a high-salinity challenge test, no significant difference in survival occurred among the dietary groups. Cortisol levels in plasma recorded 3 h post stress was significant lower in the Artemia group compared with the groups fed the formulated diets.     

Interaction of temperature and photoperiod on growth of Atlantic halibut Hippoglossus hippoglossus L.

The combined effect of temperature and photoperiod on the growth of juvenile Atlantic halibut Hippoglossus hippoglossus (initial weight 11.6 g) was investigated under different temperature (6 °C and 12 °C) and photoperiod (continuous light and simulated natural photoperiod) combinations by studying the effects on fish weight, specific growth rate (SGR), feed consumption (F%), feed conversion (FCE) and oxygen consumption and ammonia excretion under routine and fasting conditions. SGR was greater at the higher temperature, and growth was faster under continuous light than under natural photoperiod, irrespective of temperature, SGRs being 0.83% and 0.98%, and 1.61% and 1.71% at 6 °C and 12 °C respectively. The weight data revealed a significant interaction between temperature and light, and provided evidence of a stronger growth‐enhancing effect of continuous light at the lower temperature. F% increased with temperature but was independent of photoperiod, whereas FCE was higher among fish exposed to continuous light, irrespective of temperature. Independently of photoperiod, oxygen consumption and ammonia excretion increased with temperature, but the differences in these rates in relation to photoperiod became more pronounced at the lower temperature, where continuous light gave the highest rates. Higher oxygen consumption during darkness suggested that light may suppress activity in halibut. It was proposed that reduced activity and anabolic effects of photoperiod contribute to explain the increased growth and growth efficiency in fish subjected to continuous light.     

Change in flesh quality associated with early maturation of Atlantic halibut (Hippoglossus hippoglossus)

To determine the effect of maturation on flesh quality, 20 Atlantic halibut (Hippoglossus hippoglossus) consisting of mature and immature fish of both sexes were killed on site, exsanguinated and stored on ice. After 6 days of storage, the fish was gutted and filleted before colour, texture hardness and shear force were evaluated to determine the effect of maturation. Results show that mature males excreted black mucus from the skin during ice storage, while a greyish mucus was observed from immature fish. Mature fish had approximately 2% lower slaughter yield and 6% lower fillet yield compared with immature fish. The fillets from mature males were significantly harder compared with fillets from immature fish and the muscle structure proved to be stronger as an increased fracturability was observed in mature fish. In colour, fillets from mature fish proved to have a whiter appearance (L^*) than immature fish, while no difference was seen in a^* and b^* values. We conclude that the physiological changes associated with maturation affects the end quality, and may be related to slower growth.     

Growth, feed conversion efficiency and growth heterogeneity in Atlantic halibut (Hippoglossus hippoglossus) reared at three different photoperiods

Juvenile Atlantic halibut, Hippoglossus hippoglossus (initial weight (SD) 191.3 (±44.7)) g, were reared for 99 days at a constant temperature of 11°C and subjected to three different light regimes from 13 September to 21 December: continuous light (LD24:0), simulated natural photoperiod of Bergen (60°25′N, LDN) and constant 20 h light:4 h dark (LD20:4). The fish reared on the different photoperiod regimes differed in their growth patterns as juveniles exposed to long days, i.e. LD20:4 and LD24:0, exhibited faster growth than those experiencing a natural photoperiod. The LD20:4 group showed the highest average specific growth rate (0.72% body weight day^?1), whereas fish on LDN displayed the lowest average specific growth rate (0.60% body weight day^?1). The final mean weights of the LD20:4 and the LD24:0 groups were 15% and 12% higher than those of the LDN group. Dividing the duration of the experiment into three time periods shows that the LD20:4 and LD24:0 had a higher feed conversion efficiency (FCE) as compared with the LDN group during the first and the last period, while a reversed situation was observed in the second period. Our data indicate a larger variation in growth rates among individuals in the best‐performing groups (here LD20:4 and LD24:0). This may indicate that formation of size hierarchies is more pronounced in groups with more homogenous growth (here LDN). Overall, our findings indicate that extended light regimes result in faster growth and better feed conversion in juvenile Atlantic halibut. In line with findings on other flatfish species, this supports the concept that constant long day:short night or continuous light regimes should be used by the farmer in order to maximize growth and improve feed conversion in Atlantic halibut.     

Evaluation of Calanus finmarchicus copepod meal in practical diets for juvenile Atlantic halibut (Hippoglossus hippoglossus)

The copepod, Calanus finmarchicus, has potential as a new ingredient in practical feeds for marine fish. Two experiments were conducted to evaluate the nutritional value of C. finmarchicus meals when fed to juvenile Atlantic halibut. The first study determined protein, lipid and energy digestibility coefficients of four C. finmarchicus meals prepared under different processing conditions. The second study evaluated growth and nutrient utilization efficiency of juveniles fed diets containing varying proportions of fish meal, plant meals and C. finmarchicus meal. Moisture, ash, protein, lipid and gross energy contents of the C. finmarchicus meals were 28–93, 74–138, 505–648, 123–269 g kg⁻¹, and 21–26 MJ kg⁻¹, respectively. Protein, lipid and energy digestibilities of C. finmarchicus meals were 91–99%, 90–95% and 90–99%. The digestibilities significantly decreased for the Calanus meals processed at higher temperatures. During the growth study, halibut fed a diet containing 240 g kg⁻¹ C. finmarchicus meal had significantly higher weight gain and growth rate than all other groups. Nitrogen and energy retention efficiencies ranged between 35–45% and 33–43%, and were significantly higher for fish fed 160–240 g kg⁻¹ C. finmarchicus meal than for fish fed plant protein and control diets. The results indicate that growth and nutrient utilization efficiency are improved in Atlantic halibut fed diets supplemented with C. finmarchicus meal.     

Partial replacement of fish oil by flaxseed oil in Atlantic halibut (Hippoglossus hippoglossus L.) diets: effects on growth,nutritional and sensory quality

Three isonitrogenous (520 g protein kg^?1 DM) and isoenergetic (25 MJ kg^?1 DM) diets containing increasing levels of flaxseed oil (FxO; 0%, 40% and 70% of total added oil) at the expense of fish oil (FO) were tested for 33 weeks in groups of 61 individually PIT‐tagged halibut (initial weight, 849 ± 99 g). Effects on fish growth performance, fillet nutritional and sensory quality were determined. Specific growth rate (0.2% day^?1), feed conversion ratio (1.2–1.3) and nitrogen and energy retention were not affected by dietary treatments. Dietary fatty acid composition was reflected in fatty acid profiles of halibut muscle, liver and heart. Muscle of fish fed FxO diets contained higher 18:2n‐6 and 18:3n‐3 concentrations whereas 20:5n‐3 and 22:6n‐3 levels were significantly reduced. However, increasing FO replacement induced preferential retention of 22:6n‐3 especially in heart, and a trend for 20:5n‐3 conservation in heart and muscle was observed. FO replacement did not affect colour, texture and the characteristic fish odour and flavour of cooked fillets. By selectively retaining long‐chain polyunsaturated fatty acids halibut can adapt to a lower dietary supply without adverse effects on growth, feed conversion ratio, survival, and fillet nutritional and sensory quality.     

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.     

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.     

Atlantic halibut (Hippoglossus hippoglossus) vitellogenin: induction,isolation and partial characterization

Vitellogenin (VTG) synthesis was induced by repeated injections of estradiol-17 in juvenile Atlantic halibut (Hippoglossus hippoglossus). VTG eluted as a large, phosphoprotein containing peak on DEAE-Sephacel chromatography of plasma from estradiol-17 treated juvenile and mature female, but not mature male halibut. A purification procedure for Atlantic halibut VTG was developed, where VTG was precipitated with MgCl₂, EDTA and distilled water, and the precipitated protein submitted to anion exchange chromatography on DEAE-Sephacel. Precipitated VTG eluted as a broad, partly dissociated peak on DEAE-Sephacel, when chromatography was run at 4°C, but the protein appeared intact when analysed both by SDS PAGE and native PAGE. DEAE-Sephacel chromatography at room temperature resulted in an irregular elution pattern and a dissociated protein fraction, as analysed by SDS PAGE. Biochemical characterization of VTG showed that the molecular mass of the monomer was ca 160 kDa, as estimated by SDS-PAGE. The total lipid content was 19.8% w/w, with 64%, or 12.7% of the total weight, as phospholipid. Protein bound phosphorus constituted 0.62% w/w of halibut VTG. Plasma dilution curves from mature and maturing female halibut were parallel with a dilution curve from halibut egg yolk homogenate in an homologous RIA. Plasma from mature male, but not juvenile halibut crossreacted with the VTG antiserum.     

Histopathology of culture‐associated skin erosions and papillary hyperplasia of Atlantic halibut,Hippoglossus hippoglossus (L.)

Atlantic halibut, Hippoglossus hippoglossus (L.), that were cultured in tanks with a smooth bottom (gel‐coated fibreglass) substrate developed papillary hyperplasia and skin erosions on the blind (ventral) side. No viruses, bacteria or parasites were observed in any sections of affected skin. Comparison of microscopic pathology with that of skin from normal, wild‐caught halibut showed severe epidermal proliferation with foci of severe mucous cell hyperplasia. Both epidermal thickness and mucous cell density were significantly greater in fish held on a smooth substrate compared to skin of healthy fish. Spongiosis was present, especially at the base of the papillary, hyperplastic epidermis, and there was a chronic inflammatory infiltrate in the scale pockets composed of lymphocytes, histiocytes and erythrocytes. Skin erosions had various degrees of epidermal loss, in some cases to the basement membrane. A predisposing factor for the epidermal sloughing may have been related to the spongiosis, and the vacuolated, degenerated basal cells. When affected halibut were cultured for an additional 28, 62 or 97 days on a sand substrate, which is conducive to skin lesion healing, there was no apparent change in epidermal thickness over time. However, mucous cell density significantly increased from day 0 to 97 during the healing process.