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.     

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.     

Surface disinfection of Atlantic halibut and turbot eggs with glutaraldehyde: evaluation of concentrations and contact times

To evaluate the effects of glutaraldehyde treatment at different disinfection temperatures, Atlantic halibut, Hippoglossus hippoglossus (L.), and turbot, Scophthalmus maximus (L.), eggs, incubated at 5 and 12°C, respectively, were disinfected with 400–1200 mg glutaraldehyde l-1 at three different contact times (2.5, 5 and 10 min). Egg batches of both poor and good quality were tested for halibut. Positive effects were more pronounced in poor than in good-quality batches at hatching. Egg disinfection had a highly positive effect on the viability of yolk-sac larvae in both types of batches. A level between 400 and 800 mg l-1 at a contact time of 5–10 min was optimal for halibut: at lower levels, the bactericidal effect was reduced, and at higher levels, there were indications of toxic effects. Halibut eggs disinfected with optimal doses of glutaraldehyde had furthermore a reduced hatching time and more synchronous hatching as compared with untreated eggs. Turbot was more sensitive to higher doses than halibut, and the best larval performance was obtained for 400–800 mg glutaraldehyde l-1 at a contact time of 2.5 min. A further evaluation should, however, be performed before recommendations are given for species incubated at temperatures higher than 5°C.     

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.     

Application of DNA markers to the management of Atlantic halibut (Hippoglossus hippoglossus) broodstock

For many aquaculture finfish species, the current broodstock have been collected from the wild or have undergone only a few generations of domestication. The Atlantic halibut (Hippoglossus hippoglossus) aquaculture industry in Atlantic Canada has retained F₁ juveniles (n=145) from the 1996 spawning of wild adults for candidate broodstock. Through the development and use of a five-microsatellite DNA marker multiplex, we determined the parentage of these 1996 F1 individuals, which are being reared at one government and two industry hatcheries, and evaluated the change in genetic variation between the wild and the 1996 F₁ stock. In the three groups of F₁ fish, single parental pairs were assigned to 98%, 96% and 100% of individuals. Large full- and half-sibling groups were identified within and across F₁ groups and, overall, only 36% of attempted crosses were represented in the retained fish. Effective population size in the parental group decreased from 27 to 13 when variance in family size was accounted for and to 12.5 when changes in gene diversity (compared to the combined F₁ stocks) were considered. Statistically significant differences in measures of genetic variation were not widely observed between groups (original wild sample, parental group, three F₁ groups and combined F₁). However, the F₁ population shows a 26% decrease in total allele numbers compared to the wild sample. These observations demonstrate the utility of genetic tools in the evaluation of genetic diversity and determination of pedigree during the establishment of new broodstock. They also emphasize the necessity for closely monitoring future matings among these fish and suggest the need to introduce additional genetic variation into this group of Atlantic halibut broodstock.     

Artemia enriched with high n-3 HUFA may give a large improvement in performance of Atlantic halibut (Hippoglossus hippoglossus L.) larvae

Atlantic halibut larvae were fed Artemia enriched with two different oil emulsions (cod liver oil and 2050TG) from first feeding to 70 days after first-feeding (dpff). Larvae fed 2050TG enriched Artemia had better growth, survival and eye migration than larvae fed the cod liver oil enriched Artemia, while pigmentation rate was similar in the two groups. In addition to the difference in fatty acids, the two emulsions differed in lipid class composition, since 2050TG is a synthetic oil and a mixture of mono-, di- and tri-acylglycerol, while cod liver oil is a tri-acylglycerol. Total lipid level, estimated as fatty acid methyl esters (FAME) was similar in the two Artemia types, but sum of n-6 and n-3 fatty acids, arachidonic acid (20:4n-6, ARA), docosahexaenoic acid (22:6n-3, DHA) and eicosapentaenoic acid (20:5n-3, EPA) were higher in Artemia enriched with 2050TG than in the cod liver oil enriched Artemia. However, the main difference in fatty acid composition in the larvae, was a higher DHA (% of total fatty acids) in 2050TG larvae than in cod liver oil larvae. The lipid level measured as FAME was up to four times higher in the 2050TG larvae than in the cod liver oil larvae, and the reason for this may have been a better bioavailability of the partly digested lipid in the 2050TG emulsion. The correlation between a high level of lipid in the larval tissues (e.g. high energy status) and improved eye migration in larvae fed the 2050TG enriched Artemia supports the hypothesis that energy limitation on the larval stage may be a cause of the impaired eye migration commonly observed in farmed Atlantic halibut juveniles.     

Full-fat soybean meal in diets for Atlantic halibut: growth, metabolism and intestinal histology

Isonitrogenous and isocaloric diets containing 0, 18 or 36% toasted full-fat soybean meal (FFSM) were fed to Atlantic halibut. The diets were fed to five tanks of fish each for 34 days (period 1). Four tanks from each treatment were then retained in the growth experiment for a further 32 days (period 2), while the groups of fish from one tank from each of the 0 and 36% FFSM groups were split and transferred to two metabolism tanks each. The initial weight of the fish in the growth trial was 169 ± 1 g (mean ± SEM, n =12; weight range 89–253 g) and the final weight was 317 ± 5 g. There was no significant effect of dietary treatment on specific growth rate (range, 0.8–1.1% day⁻¹), feed consumption (0.5–0.7% body weight day⁻¹), feed efficiency (1.3–1.6 g wet gain g dry feed⁻¹), protein retention (48–55%) or energy retention (49–57%). The fat, protein and energy concentrations in the fish increased during the trial and were not affected by the diet. The hepatosomatic index in fish fed with 36% FFSM diet was significantly lower (1.7%) than in the other groups (2.2%) ( P  < 0.05). No differences in intestinal morphology were observed between dietary treatments and no pathological reactions were identified in any of the samples. In the metabolism trial, there were no significant differences in oxygen consumption or ammonia excretion between fish fed with 0 and 36% FFSM diets. In conclusion, up to 36% FFSM may be added to diets for Atlantic halibut without negative effects on growth, feed efficiency or intestinal morphology.     

Swimming behaviour as an indicator of low growth rate and impaired welfare in Atlantic halibut (Hippoglossus hippoglossus L.) reared at three stocking densities

The Atlantic halibut (Hippoglossus hippoglossus L.) is a new species in Norwegian aquaculture. However, in ongrowing trials, halibut shows variable and generally poor rates of growth. The halibut is a flatfish that spends most of its time resting on the bottom, and it was hypothesized that high stocking densities with frequent social interactions could lead to behaviour changes, reduced appetite and impaired welfare. Halibut were kept in six tanks at low, medium, and high densities (18%, 54% and 112% bottom coverage). All fish were individually tagged with Trovan™ passive implant transponder (PIT) tags, allowing an antenna to register fish swimming at the surface. Swimming and feeding behaviour was also recorded by underwater video cameras. Individual growth rates were highly variable, but food consumption and growth rates fell significantly with increasing stocking density, while individual swimming activity rose with increasing density. Frequent “surface swimmers” had a significantly lower growth rate than fish that were seldom recorded by the PIT antennae. Surface swimming may therefore be an indicator of suboptimal growth rates and impaired welfare in reared halibut.     

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.     

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.     

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 nutrient utilization in Atlantic halibut (Hippoglossus hippoglossus) fed diets containing a bacterial protein meal

Triplicate groups of Atlantic halibut were fed diets containing 0%, 9% or 18% of a bacterial protein meal (BPM) produced from natural gas in a 9‐week trial. Growth rates, relative feed intake, feed efficiency ratio and retention of all indispensable amino acids were significantly lower in fish fed the 18% BPM diets than in those fed the 0% and 9% BPM diets. There were no significant treatment effects on urea levels in plasma, liver or muscle, or in uric acid levels in plasma. The hepatosomatic index was lowest in fish fed the 18% BPM diet. Although the concentration of copper, an element abundant in BPM, increased in the liver as dietary BPM level increased, the total copper content in liver decreased. Fish fed the 0% and 9% BPM diets had a higher degree of supranuclear vacuolization of pyloric caeca and mid‐intestine epithelia compared with fish fed the 18% BPM diet. In conclusion, the halibut fed the 9% BPM diet performed equally well as the control group regarding growth, feed intake and feed efficiency ratio, whereas performance was reduced in the fish fed the 18% BPM diet.     

Experimental susceptibility of Atlantic cod, Gadus morhua (L.), and Atlantic halibut, Hippoglossus hippoglossus (L.), to different genotypes of viral haemorrhagic septicaemia virus

Viral haemorrhagic septicaemia (VHS) is a well-characterized disease of rainbow trout, Oncorhynchus mykiss, which has also caused economic losses in marine turbot farms in the British Isles. We have previously demonstrated that turbot, Scophthalmus maximus, are susceptible to isolates of viral haemorrhagic septicaemia virus (VHSV) that are endemic in the marine environment, highlighting a potential risk to marine aquaculture. Given the increasing interest in the intensive rearing of additional aquaculture species such as Atlantic cod, Gadus morhua, and Atlantic halibut, Hippoglossus hippoglossus, this study aimed at investigating the susceptibility of these species to VHSV. Both species were found to be largely resistant to VHS following immersion challenge with a selection of 18 isolates, representing the known marine VHSV genotypes. Only one and two VHSV-associated mortalities occurred out of a total of 1710 and 1254 halibut and cod, respectively. These findings suggest that there is a low direct risk to the development of commercial cod and halibut aquaculture from the existing endemic reservoir of VHSV. This study, coupled to field observations has, however, highlighted the fact that both species can become infected with VHSV. The known adaptability of RNA viruses, together with the selection pressures associated with intensive aquaculture would thus advocate a cautious approach to VHSV surveillance within these emerging industries.     

Distribution of α-tocopherol in fillets of turbot (Scophthalmus maximus) and Atlantic halibut (Hippoglossus hippoglossus), following dietary α-tocopheryl acetate supplementation

The present study investigated the distribution of α‐tocopherol (vitamin E) in fillets of turbot (Scophthalmus maximus) and Atlantic halibut (Hippoglossus hippoglossus). Turbot and Atlantic halibut were fed commercial diets, supplemented with different levels of α‐tocopheryl acetate at the dietary target levels of 100, 500 and 1000 mg α‐tocopheryl acetate kg^?1 diet. The actual levels were 72, 547 and 969 for turbot, while halibut received 189, 613 and 875 mg α‐tocopheryl acetate kg^?1 diet. Turbot were fed the diets for 24 weeks, while Atlantic halibut were fed for 20 weeks prior to slaughter. At the end of the feeding periods fish had reached a final weight of around 1 kg. Fish were slaughtered and filleted. From the four fillets obtained per fish, 22 samples were taken from designated areas and analysed for their α‐tocopherol content. The average concentrations of α‐tocopherol incorporated in turbot and Atlantic halibut increased with increasing levels of α‐tocopheryl acetate in the diet. Atlantic halibut had significantly (P < 0.05) more α‐tocopherol in positions 2/II and 1/I than in position 9/IX. Turbot had significantly (P < 0.05) more α‐tocopherol in position 2/II than in positions 1/I, 4/IV and 11/XI. By mapping α‐tocopherol concentrations in fish fillets, a high degree of quality prediction may be established. Moreover, this study may help scientists in their choice of sampling position, when investigating if α‐tocopheryl acetate supplementation resulted in successful α‐tocopherol incorporation.     

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.     

The influence of water temperature on spawning patterns and egg quality in the Atlantic halibut (Hippoglossus hippoglossus L.)

A three-year study was conducted to investigate the effects of water temperature on Atlantic halibut broodstock reproductive performance. Two groups of fish held under ambient photoperiod were established onto contrasting temperature regimes. The ‘chilled’ group were held at below 9 °C from late October and at approximately 6 °C from December until the end of the spawning period whilst the ‘ambient’ group received no temperature control. The temperature profiles for the ‘ambient’ group changed over the 3 years but were generally characterised by warmer conditions prior to spawning and an earlier temperature rise in the spring. Total egg production was higher in the ‘chilled’ group each year. Absolute fecundity was significantly reduced in the ‘ambient’ group compared to the ‘chilled’ group every year of the study (0.6 million eggs/female for ‘chilled’ group vs. 0.3 to 0.4 million eggs/female for ‘ambient’ group) and egg viability, in terms of fertilisation and hatch rate, was significantly impaired in the ‘ambient’ group in years 2 and 3 (mean fertilisation rate in the ‘ambient’ group was between 27.0% and 54.8%; vs. 63.4% to 77.4% in the ‘chilled’ group, mean hatch rate in the ‘ambient’ group was between 3.1% and 25.6% vs. 60.7% to 71.7% in the ‘chilled’ group). Eggs spawned at high temperatures, later in the season were generally of low viability. In the ‘ambient’ group the spawning season became progressively delayed during the study and average duration of spawning season over 3 years was shorter (between 23.5 to 26.3 days for the ‘ambient’ group vs. between 30.5 and 41.2 days for ‘chilled’ group). It is hypothesised that high temperature during the vitellogenesis period caused a delay in spawning and a reduction in quantity and quality of eggs and that this effect was exacerbated by high temperature during spawning.     

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.     

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.