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.     

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.     

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.     

Nutrient composition and metamorphosis success of Atlantic halibut (Hippoglossus hippoglossus, L.) larvae fed natural zooplankton or Artemia

Atlantic halibut larvae were fed docosohexanoic acid- (DHA) selco enriched Artemia (RH-cysts) or wild zooplankton in duplicate tanks from first-feeding and 60 days onward. The zooplankton were collected from a fertilized sea water pond and consisted mainly of different stages of Eurytemora affinis and Centropages hamatus . There were no differences in survival, or in growth during the first 45 days of feeding, between larvae fed the two prey items, but the larvae fed Artemia showed much higher incidence of malpigmentation and impaired eye migration than larvae fed zooplankton. The prey organisms contained similar amounts of dry matter and protein, but Artemia was higher in lipid and glycogen than the zooplankton. Larvae fed Artemia were higher in both glycogen and lipid than the zooplankton-fed larvae towards the end of the feeding period. There were large differences between the prey organisms in the concentrations of essential fatty acids (% of total fatty acids) which was reflected in the fatty acid composition of the larval body. It is concluded that the macronutrient composition of Artemia in the present study was probably within the optimal range for promotion of growth and survival in young Atlantic halibut. The concentration of n-3 HUFA, and especially DHA, is however, very much lower in enriched Artemia than in copepods, and may be one of the factors triggering developmental errors in Atlantic halibut.     

Pigmentation and eye migration in Atlantic halibut (Hippoglossus hippoglossus L.) larvae: new findings and hypotheses

Atlantic halibut juveniles, which have been fed Artemia during larval development, frequently demonstrate malpigmentation and impaired eye migration. This is in contrast to the high percentage of normally developed larvae fed copepods, reared under similar conditions. Nutrition is therefore an important component influencing larval development. Analyses of the nutrient composition of Artemia and copepods show that Atlantic halibut larvae fed Artemia probably receive sufficient amounts of vitamin A by converting canthaxanthin, while iodine may be deficient, possibly leading to interrupted thyroid hormone synthesis. An unbalanced fatty acid composition, such as high levels of arachidonic acid and low levels of docosahexaenoic acid, can be another limiting factor in Artemia. Vitamin A, fatty acids and thyroid hormones have all been shown to affect pigmentation in flatfish. They are ligands to nuclear receptors, thyroid hormone receptors, retinoic acid receptors, retinoic X receptors and peroxisomal proliferator‐activated receptors, which are members of the superfamily of steroid hormone receptors. The receptors interact with each other to promote gene expression that modulates proliferation and differentiation of cells. Our hypothesis is that these interactions are important for development during flatfish metamorphosis. Very little data exist on the topic of impaired eye migration. However, energy limitation, iodine deficiency and an unbalanced fatty acid composition have been proposed as possible explanations. Here, we review the literature on development of pigment cells and the possible mechanisms behind the effects of vitamin A, fatty acids and thyroid hormone on pigmentation and eye migration during development of Atlantic halibut larvae.     

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.     

The problem of meeting dietary protein requirements in intensive aquaculture of marine fish larvae, with emphasis on Atlantic halibut (Hippoglossus hippoglossus L.)

Atlantic halibut (Hippoglossus hippoglossus) achieve a mature gastrointestinal tract approximately 2 months after first feeding (12 °C). The immature digestion may be the reason that compound diets fail to sustain growth and survival in first feeding halibut larvae and in larvae of other marine fish species. On the other hand, larvae fed with live feeds are capable of extraction of sufficient quantities of nutrients to sustain high growth rates. A lower availability of the protein in formulated diets compared with live prey is considered to be an important reason for the low performance of formulated diets. One approach to increase dietary protein availability is supplementation of pre‐digested proteins. Experiments using tube fed individual larvae show that halibut larvae are able to utilize hydrolysed protein more efficiently than intact protein. However, Atlantic halibut in culture did not respond well to dietary supplementation of hydrolysed protein, in contrast to some other species. One reason may be extensive leaching of pre‐hydrolysed proteins from the microparticulate feed. Atlantic halibut are slow feeders and may thus suffer more from nutrient leaching than species eating more rapidly. Feed formulation techniques affect dietary protein leaching, and in this paper, different techniques and their impact on feed properties are described. Microbound diets are most widely used in larval rearing, but show high rates of nutrient leaching. Lipid‐based capsules seem to have the best potential to prevent leaching, however, they are not able to deliver a complete diet. The high need for improvements in larval feed formulation techniques are clearly stated, and some suggestions are given. Among these are production of complex particles, where small lipid‐based capsules or liposomes containing the low molecular weight water‐soluble nutrients are embedded. In such feed particles the water‐soluble molecules are protected from leaching. Techniques for delivery of water‐soluble nutrients that are needed in large quantities, i.e. free amino acids or hydrolysed and water‐soluble protein, remain to be developed.     

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.     

The effects of feeding frequency on growth of juvenile Atlantic halibut, Hippoglossus hippoglossus L.

Four experiments were conducted to investigate the effects of feeding frequency on growth of juvenile Atlantic halibut, Hippoglossus hippoglossus L. Fish (22–75 g) fed three (3 ×) or five times per day (5 × day^?1) under constant light and temperature (13±1°C) consumed significantly more feed than fish fed 1 × day^?1 but by the end of the experiment only fish fed 5 × day^?1 were heavier and had greater specific growth rates (SGR). Under simulated winter conditions (9L:15D, 5±1°C), halibut (～300 g) fed every other day consumed more feed, had a greater SGR and final weight compared with fish fed every third day. Feed conversion ratios were not different among treatment groups in any of the experiments. These results suggest that growth rates may be improved by feeding juvenile halibut more than 1 × day^?1.     

Iodine enrichment of Artemia and enhanced levels of iodine in Atlantic halibut larvae (Hippoglossus hippoglossus L.) fed the enriched Artemia

Flatfish metamorphosis is initiated by the actions of thyroid hormones (TH) and iodine is an essential part of these hormones. Hence, an iodine deficiency may lead to insufficient levels of TH and incomplete metamorphosis. In this study, different iodine sources for enrichment of Artemia were evaluated and the levels of iodine obtained in Artemia were within the range of 60–350 μg g^?1 found in copepods. Larval Atlantic halibut was fed Artemia enriched with either normal DC‐DHA Selco or DC‐DHA Selco (commercial enrichments) supplemented with iodine from days 9 to 60 postfirst feeding. There was no significant difference in growth, mortality or metamorphic development between the groups. The analyses showed that we were able to enrich Artemia with iodine. Further, the larvae‐fed iodine‐enriched Artemia had higher whole body iodine concentration compared to larvae‐fed Artemia without iodine enrichment.     

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.     

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 comparison of retinol, retinal and retinyl ester concentrations in larvae of Atlantic halibut (Hippoglossus hippoglossus L.) fed Artemia or zooplankton

Atlantic halibut (Hippoglossus hippoglossus L.) larvae were fed enriched Artemia or zooplankton in duplicate tanks from 0 to 60 days after first‐feeding. Both diets and the larvae were analysed for vitamin A (VA) in order to confirm earlier findings, in which Artemia fed larvae had lower levels of VA compared with larvae fed zooplankton. Furthermore, we wanted to investigate the composition of the retinoids in the larvae. The results showed that Artemia and zooplankton contains low levels of VA, probably too low to sustain the assumed requirement. Nevertheless, larvae fed Artemia had the same level of retinal and retinol as larvae fed zooplankton. We found a significant lower level of retinyl esters in larvae fed Artemia. The total VA level was lower in larvae fed Artemia only at the end of the feeding trial after the onset of metamorphosis. Our conclusion is that feeding Artemia to Atlantic halibut larvae is not likely to cause VA deficiency.     

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.     

Growth, feed conversion and chemical composition of Atlantic salmon (Salmo salar L.) and Atlantic halibut (Hippoglossus hippoglossus L.) fed diets supplemented with krill or amphipods

The effects of partial replacement of fish meal (FM) with meal made from northern krill (Thysanoessa inermis), Antarctic krill (Euphausia superba) or Arctic amphipod (Themsto libellula) as protein source in the diets for Atlantic salmon (Salmo salar L.) and Atlantic halibut (Hippoglossus hippoglossus L.) on growth, feed conversion, macro‐nutrient utilization, muscle chemical composition and fish welfare were studied. Six experimental diets were prepared using a low‐temperature FM diet as control. The other diets included northern krill where 20, 40 or 60% of the dietary FM protein was replaced with protein from northern krill, and two diets where the FM protein was replaced with protein from Antarctic krill or Arctic amphipod at 40% protein replacement level. All diets were iso‐nitrogenous and iso‐caloric. Atlantic salmon grew from 410 g to approximately 1500 g during the 160 day experiment, and Atlantic halibut grew from 345 g to 500–600 g during the 150 day experiment. Inclusion of krill in the diets enhanced specific growth rate in salmon, especially during the first 100 days (P < 0.01), and in a dose–response manner in halibut for over the 150 day feeding period (P < 0.05). Feed conversion ratio did not differ between dietary treatments, and no difference was found in dry matter digestibility, protein digestibility and fish muscle composition. Good growth rates, blood parameters within normal ranges and low mortalities in all experimental treatments indicted that fish health was not affected either Atlantic salmon or Atlantic halibut fed the various zooplankton diets.     

Experimental infection of eggs and yolk sac larvae of halibut, Hippoglossus hippoglossus L.

Abstract Scanning electron micrographs of halibut eggs with an epiflora dominated by Flexibacter sp., showed ulcerations colonized by large numbers of bacteria. The chorion was dissolved in most ulcerations and the zona radiata was severely damaged. Infection experiments showed that exposure to these bacteria caused high mortalities at the late egg stage, hatching and the early yolk sac stage. Eggs exposed to three different Vibrio spp. showed different mortality patterns, with low mortality at hatching, followed by a continuous high mortality throughout the yolk sac stage. Mortality in the uninfected control group was low throughout the experiment. Transmission electron microscopy of the Vibrio -infected larvae showed bacteria present in the gill, heart and frontal yolk sac regions.     

A study of the susceptibility of Atlantic halibut,Hippoglossus hippoglossus (L.), to viral haemorrhagic septicaemia virus isolated from turbot,Scophthalmus maximus (L.)

The susceptibility of Atlantic halibut, Hippoglossus hippoglossus (L.), to viral haemorrhagic septicaemia virus (VHSV) was tested. Juvenile halibut of approximately 5 g weight were subjected to challenge by intraperitoneal injection, cohabitation and immersion to a VHSV isolate from an outbreak of the disease in turbot, Scophthalmus maximus (L.). The intraperitoneal injection gave the highest mortality rate of 28% after 50 days. The cohabitee group suffered 19% mortality rate and the immersion group only 2%. Control groups included turbot exposed either by intraperitoneal injection or immersion which suffered mortality rates of 93 and 50%, respectively. The results suggest that halibut are markedly less susceptible to VHSV than turbot.     

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.     

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.