Development and evaluation of microparticulate diets for early weaning of Atlantic cod Gadus morhua larvae

Early weaning trials were conducted with cod larvae to investigate the effectiveness of microparticulate diets (microbound and microcoated) with and without lipid-walled capsules (LWCs). The microparticulate diets were evaluated by measuring physical parameters of the diet in the water column (leaching and settling rate), palatability (intestinal fullness), performance of the diet (survival and growth), and examination of the diet in the larval intestine (histological analysis). A feeding trial was conducted using four experimental diets (carrageenan microbound diet, carrageenan microbound diet with LWCs, zein microcoated diet and zein microcoated diet with LWC), one commercial diet (BioKyowa: A-250) and a live feed control (rotifers and Artemia ). Survival of cod larvae to 39 days post-hatch ranged from 5 to 10% with the experimental diets, 22.9% with the BioKyowa diet, and 36.5% with live prey. There was evidence of food absorption with all diets in the form of lipid vacuoles in the midgut and supranuclear vacuoles in the hindgut. Large vacuoles in the midgut were more abundant in the enterocytes of larvae fed the experimental diets compared with larvae on the BioKyowa diet and the live feed control. Based on observations of intestinal fullness, the experimental diets appeared to be less palatable than the BioKyowa diet. As a result, it took longer to wean the larvae and higher mortality was experienced during weaning. Once successfully weaned, the experimental diets yielded growth rates equivalent to larvae feeding on the commercial diet for the remainder of the experiment.     

Experimental mycobacteriosis in Atlantic cod, Gadus morhua L

Piscine mycobacteriosis causes losses in a number of fish species both in the wild and in aquaculture worldwide. Mycobacterium salmoniphilum infections have on several occasions been reported in farmed Atlantic salmon, Salmo salar L. The present study tested and confirmed the susceptibility of Atlantic cod, Gadus morhua L., an important yet relatively novel aquaculture species, to infection with M. salmoniphilum. Atlantic cod injected intraperitoneally with a suspension of this bacterium were maintained together with cohabitant (COH) fish in a flow-through marine water system at 10-11 °C. The fish were supervised daily and samples taken at 2, 7, 14, 23, 34 and 53 weeks post-infection and examined pathologically, bacteriologically and using molecular biology. Injected mycobacteria were re-isolated in high concentrations from both injected and COH fish groups. Death attributable to mycobacterial infection was observed in both injected (47%) and COH (28%) fish groups. Extensive development of granuloma in visceral organs, mainly the mesenteries, spleen, kidney and liver (lesser extent) and at later stages of the infection in heart tissues and gills, was observed in both injected and COH fish. Granulomas underwent a temporal progression of distinct morphological stages, culminating in well-circumscribed lesions surrounded by normal or healing tissue. Acid-fast bacilli were detected in both granulomas and non-granulomatous tissues. This study confirms that Atlantic cod is highly susceptible to M. salmoniphilum infection and that this bacterial species may be a threat to cod both in the wild and in the aquaculture.     

Stress response to anthropogenic noise in Atlantic cod Gadus morhua L.

The potential effects of anthropogenic noise on the physiology of Atlantic cod have not been well described. The aim of the present study was to investigate the impact of anthropogenic noise on Atlantic cod stress response using cortisol as a biomarker as well as on broodstock spawning performance. Results showed that artificial noise consisting of a linear sweep from 100 to 1000 Hz can induce a transient and mild cortisol elevation with a clear noise intensity dose response. In all cases plasma levels returned to baseline levels <1 h post sound exposure. Daily exposure to a similar intensity and frequency noise range applied habitually to a broodstock population during the spawning window resulted in a significant reduction in total egg production and fertilisation rates thus reducing the total production of viable embryos by over 50%. In addition, a significant negative correlation between egg cortisol content and fertilisation rate was observed. These results confirm that cod can perceive noise generated within a frequency range of 100–1000 Hz and display a heightened cortisol plasma level. In addition, anthropogenic noise can have negative impacts on cod spawning performances.     

Dietary plant protein utilization in Atlantic cod, Gadus morhua L.

The present trials aimed to investigate the effects of replacing fish meal with plant proteins in diets for cod, using a regression design where fish meal constituted the control. The plant protein diets were formulated to meet the amino acid requirements according to NRC (1993) and contained corn gluten meal, soybean meal, a mixture of these, or a mixture of wheat gluten meal and soybean concentrate. The plant protein fraction constituted up to 440 g kg⁻¹ of the extruded diet. Two feeding experiments were conducted, one at high (11 °C) and one at low (6.5 °C) temperature. High growth and feed utilization were obtained in all diet groups at both temperatures. However, only in Exp.1 (11 °C), growth and feed utilization were linearly reduced by increases in dietary soybean and corn gluten meal, while no such effect was detected in Exp.2 (6.5 °C). Reductions in protein retention were seen at both temperatures with use of all evaluated plant protein sources. Due to large amounts of plant protein ingredients in diet, differences in diet amino acid composition were seen. These were partly reflected in the muscle free amino acid pool. Dietary plant ingredients did not affect whole body, liver or muscle proximate compositions, or liver indices. In both experiments, blood parameters were within the range of earlier reported normal values and indicated, together with low mortality, good fish health status. The results show that there is high potential to use protein‐rich plant ingredients in diets for Atlantic cod.     

Immunostimulation of larvae and juveniles of cod, Gadus morhua L

Cod larval culture is currently hampered by high mortalities in the first 2-3 weeks after hatching, often due to infectious diseases. The immune system of cod is not fully competent until 2-3 months after hatching. Conventional vaccination is, therefore, not of value before this time, and the larvae are wholly reliant on non-specific parameters for their defence against infection. A range of substances, generally derived from bacterial, fungal or plant origin, can activate these non-specific parameters. During three hatching seasons, 2001-2003, at the Marine Institute's Experimental Station, Stadur, Grindavik, Iceland, the effects of several immunostimulants on survival and disease resistance of cod larvae and juveniles were examined. Both bathing treatments and administration in the feed were used. One of these substances, lipopolysaccharide (LPS), isolated from the bacterium Aeromonas salmonicida (ssp. salmonicida or achromogenes), appeared in some instances to improve survival and have a beneficial effect on disease resistance. Other substances tested had limited effects. The results emphasize the need for further work in this field.     

Growth and survival of Arcto-Norwegian and Norwegian coastal cod larvae (Gadus morhua L.) reared together in mesocosms under different light regimes

To evaluate genetic differences in growth and survival potential as a function of light regime, cod larvae from two stocks (CC: coastal cod and AC: Arcto-Norwegian cod, spawning at latitudes of 60°N and 69°N respectively) were co-reared in mesocosms. The experiment was carried out with two artificially illuminated light regimes, corresponding to the natural photoperiod at peak spawning of the respective stocks (CC: 7–8 h distinct dark period, AC: ‘mid-night sun’ as continuous light with 2–3 h reduced light intensity). One additional regime with ambient natural light analogous to 60°N was used. At termination on day 36 post-hatch, larvae of the co-reared stocks were distinguished from each other by a genetic marker in the CC stock. Stock-specific differences in growth rate and survival were indicated, with CC larvae growing and surviving better than AC larvae. This is in agreement with earlier comparisons of larval growth in these stocks. The 69°N light regime did not enhance growth for any of the two stocks. However, natural light greatly increased growth rate of both stocks, indicat ing important mechanisms in regulation of larval feeding relative to light quality or intensity. The results may be important for choice of stock and illumination in intensive aquaculture of this species.     

Proteome profile comparison of two differently fed groups of Atlantic cod (Gadus morhua) larvae

Proteome analysis was used to study the effects of feeding early Atlantic cod (Gadus morhua) larvae with a saithe (Pollachius virens) protein hydrolysate (SPH). Protein hydrolysates have previously been shown to beneficially affect fish larval development. Feeding was initiated on day 2 post hatch (ph) or as soon as the larvae opened their mouth and the protein expression was monitored 4 days later or in 6‐dph cod larvae. The results demonstrated changes in the abundance of 13 protein spots in the cod larvae fed SPH. Of these, seven protein spots were up‐regulated and six protein spots showed down‐regulation. Five of the up‐regulated proteins in cod larvae are known to be involved in energy metabolism. A few early larval specific proteins were down‐regulated in the SPH‐fed cod larvae possibly because of an enhanced development in this group relative to the control group. Two trypsin isoforms were detected within the cod larval proteome. The detection of the trypsin spots was made possible by co‐electrophoresis of known cod trypsins with the cod larval protein extract. Surprisingly, no difference in trypsin content was observed between the SPH‐fed and the control larval groups.     

Application of prospective probionts at early stages of Atlantic cod (Gadus morhua L.) rearing

This work aimed at validating the use of two prospective probionts (Arthrobacter sp. and Enterococcus sp.) at early stages of cod (Gadus morhua L.) rearing. Ova at late post‐fertilized stage and larvae during their first 4 weeks of life were bathed with both probionts, isolated previously from the cod‐rearing environment. This treatment was compared with groups fed rotifers supplemented with a commercial probiotic (Remus^®) and those untreated. Microbiological analyses (total viable counts, presumptive Vibrio and lactic acid bacteria) were performed in rearing systems and larval survival, growth and development were assessed. Larval development was evaluated by proteolytic activity of larval lysates and immunological analysis of important proteins: apolipoprotein A‐I, haemoglobin, C‐reactive protein, C3 and cod serum proteins. Bacterial bathing led to a significantly higher larval weight, length and culturable microbial load in larval gastrointestinal (GI) tract when compared with the control and Remus groups. Development occurred earlier in bathed larvae. However, their survival was negatively affected compared with the control group, but was significantly higher than for the Remus group. The non‐pathogenicity of both probionts was demonstrated by intraperitoneal injection of 13 g cod juveniles. The results suggest that Arthrobacter and Enterococcus probionts affected the larval GI microbiota and contributed to growth, development and digestion, either directly or indirectly.     

Heritability of economically important traits in the Atlantic cod Gadus morhua L

During the development of breeding programme for Atlantic cod Gadus morhua L., in Iceland, genetic parameters were estimated for 1402 individuals, which were assigned with DNA profiling to 140 dams and 70 sires. The cod was reared in cages on the eastern and western coasts of Iceland from 2004 to 2005. At the average body weight of 1.8 kg, the estimated heritability (h² ± SE) for body weight, gutted weight and the condition factor (CF) were 0.31 ± 0.06, 0.34 ± 0.04 and 0.24 ± 0.06 respectively. Genetic correlation (r_G) in body weight between the two rearing locations was estimated as 0.95, which reflects a low G × E interaction. The estimated heritability for hepatosomatic index (HSI) and fillet yields was 0.061 ± 0.04 and 0.04 ± 0.04 respectively. The HSI and fillet yields were highly genetically correlated with body weight or 0.67 and 0.82 respectively. The genetic correlation between the CF and body weight was estimated as 0.31. There appears to be substantial amount of additive genetic variation for body weight suggesting that selection is likely to be successful. Low heritability for fillet yields and the HSI indicates less promise of genetic improvement. Assigning of parentage to individuals with DNA profiling was 80% successful.     

Early introduction of an inert diet and unenriched Artemia enhances growth and quality of Atlantic cod (Gadus morhua) larvae

The effects of two weaning diets and different weaning protocols on growth, survival, skeletal deformity and gut morphology of Atlantic cod larvae were studied in four groups from 16 to 45 days posthatch (dph). Cod larvae in groups 1 (early weaning with control diet) and 2 (early weaning with experimental diet) were used to evaluate the effects of different polar lipid content of weaning diets on larval and juvenile performance. Cod larvae in groups 2, 3 (early weaning with experimental diet + cofeeding with Artemia) and 4 (earlier weaning with experimental diet and earlier cofeeding with Artemia) were used to evaluate the effects of early introduction of dry diet and Artemia. From 45 to 170 dph, cod juveniles from all four groups were reared using a standard feeding protocol. No significant differences in growth, survival, deformities and gut morphology were found between cod larvae and juveniles from groups 1 and 2. Cod larvae fed on cofeeding regime with Artemia nauplii (groups 3 and 4) were bigger and had lower frequencies of jaw and neck deformities and higher foregut microvillus circumference than cod larvae from group 2. Our results demonstrate the importance of proper weaning protocols in producing better quality cod juveniles.     

Efficacy of egg surface disinfectants in captive spawning Atlantic cod Gadus morhua L. and haddock Melanogrammus aeglefinus L.

In an effort to optimize the efficiency of high‐density incubation of Atlantic cod Gadus morhua and haddock Melanogrammus aeglefinus eggs, the per cent hatch of eggs treated with four disinfectants (3% hydrogen peroxide, 1% polyvinylpyrrolidone iodine, 0.1% sodium hypochlorite and a 0.005% antibiotic solution – penicillin/streptomycin) was compared in both species. The per cent hatch of eggs of each species was greatest after a 24 h treatment with the antibiotic solution. The hatching success of eggs treated within the different disinfectant treatments depended upon the embryonic developmental stage in both species. The sodium hypochlorite treatment had the lowest % coverage of colony growth after disinfected haddock eggs were plated onto sterile agar media, highest survival rates to the end of the embryonic period, but the lowest per cent hatch.     

Spawning‐related mortality in captive Atlantic cod (Gadus morhua L.)

Two year‐classes of Atlantic cod were reared in indoor tanks from November 2002 to November 2004. The average annual mortality was 21% and 36% for males and females respectively. The highest monthly mortality rates in females were observed around and during the spawning periods from March to July, maximally 7% and 20% for year‐class 2001 and 18% and 34% for year‐class 2000. The mortality in males was less dependent on season, reaching a maximum of 7% and 5% month^?1 for year‐class 2001 and 2000 respectively. The maximum mortality during spawning increased with age from: 7%, 19% and 34% per month for 2‐, 3‐ and 4‐year‐old females respectively. The average gonadosomatic indices in 3‐ and 4‐year‐old mature females that died during the spawning periods were 22% and 26% respectively and most of them were spawning when they died. The results suggest that egg‐retention may have been the main cause of death in females during spawning.     

Changes in Atlantic cod (Gadus morhua L.) sperm quality during the spawning season

The biology of cod reproduction is well described in the scientific literature. However, sperm biology and spermatozoa management are poorly studied in this species. Because of its recent farming expansion, a better knowledge of cod gametes is becoming especially useful. This work aimed at establishing tools to study sperm biology in cod, and also investigated the existence of changes in cod sperm quality during the spawning period. We showed that sperm concentration could be assessed using spectrophotometry at 260 nm. Sperm motility significantly decreased after a 168‐h storage at 4 °C. A 1:9 dilution of sperm in a non‐activating medium (1/3 seawater and 2/3 freshwater, osmotic pressure: 360 mOsm kg^?1) improved sperm storage. Sperm concentration, sperm velocity and storage capacity at 4 °C peaked during the medium period of the spawning season and then decreased to values close to those observed at the beginning of the reproductive period. The measured values of osmotic pressure, pH, protein, Na⁺, Cl^? and Ca²⁺ concentrations of the seminal fluid were modified along the spawning period. Cell damage was noted at the end of the spawning period: local blebs were observed on the flagellum but also loops at its distal part. On the other hand, spermatocrit did not vary with the sampling date. In conclusion, cod sperm quality is modified during the spawning period, the highest‐quality samples being collected during the medium part of this season.     

Inter‐individual variation in the dominant intestinal microbiota of reared Atlantic cod (Gadus morhua L.) larvae

The diversity of the microbiota in 15 Atlantic cod (Gadus morhua L.) larvae from three different hatcheries with variable food type and water treatment was characterized with culture dependent and independent approaches. The culture‐dependent approach was phenotypic characterization of bacterial isolates based on 16 tests, and the culture‐independent approach was terminal restriction fragment length polymorphism (T‐RFLP) of PCR amplified 16S rRNA. Both approaches revealed large inter‐individual differences in the microbiota of cod larvae. Data from real time PCR indicate substantially lower variation in bacterial number larva^?1 both within and between hatcheries than the data on colony forming units, resulting in an estimated four orders of magnitude variation in culturability between larvae. Considerably higher richness and diversity were observed with T‐RFLP than with phenotypic characterization, and no correlation between the two approaches was found for diversity indexes calculated at the individual level. Dissimilarity in the composition of the microbiota between individuals from the same rearing environment was comparable to the dissimilarities between hatcheries. The large inter‐individual variation is important to consider when designing experiments, as pooling of individuals will completely mask this variability. We conclude that colonization of individual cod larvae by bacteria seems to be a stochastic and not a deterministic process, which is not well understood.     

Behavioural responses in wild cod (Gadus morhua L.) exposed to fish holding water

Local fishermen in several areas of Norway assert that salmon farms have caused the wild migrating cod to change their migratory behaviour, so that they no longer enter their natural spawning grounds in the fjords. If the asserted changes in behaviour of wild cod populations can be linked to establishment of salmon farms, water-soluble odorants are then possible candidates to explain such a connection. Chemical stimulants are important to the individual fish's conception of the surrounding environment. High density stocks of fish in a farm are expected to release large amounts of waterborne information. The present laboratory experiments were conducted to test behavioural responses in Atlantic cod exposed to water containing metabolites and waste from farmed salmon. The trials were conducted on single fish in a multiple chamber preference system. The results show that migrating wild Atlantic cod chose to spend more time in chambers without addition of water from the salmon tank, regardless of their maturation status, and even at very low concentrations (0.2%). The avoidance is probably due to presence of chemical compounds with olfactory properties from salmon, since anosmic cod did not elicit such response. Farmed cod, on the other hand, does not avoid water from the salmon tank, and stationary wild cod caught nearby a fish farm had a less pronounced response as compared to wild migrating cod. The response seen is not species specific, as wild migrating cod responded similar to water from a tank holding farmed cod as to water from salmon. These results do not preclude fishermen's observations that cod change their behaviour in areas with fish farming activity. Such a change in behaviour could be a response to water-soluble odorants, but needs to be validated and detailed in further laboratory experiments as well as in nature before any conclusions can be made.     

Effects of pre-rigour filleting on quality aspects of Atlantic cod (Gadus morhua L.)

Aquaculture is promising in increasing the amount of fresh Atlantic cod available all year around. The aim of this study was to investigate the effects of pre‐rigour filleting on fillet quality of wild and farmed cod. Pre‐rigour filleting resulted in significantly less fillet gaping than in fillets produced post rigour. The pre‐rigour fillets were, however, 12–13% shorter post rigour. In spite of the significantly lower water content of the muscle 6 days post mortem, a significantly higher liquid loss was seen in the pre‐rigour group. Differences in gaping score and muscle pH were dependent on whether the fish were wild or fed. Fillet gaping increased with storage time and was particularly pronounced for well‐fed cod. Fed cod had a significantly higher hydroxyproline (Hyp) content and significantly lower amount of glycosaminoglycans (GAGs) than wild cod. The ratio between GAGs and Hyp was thus significantly lower in fed cod. A connection between connective tissue components and fillet gaping may exist. However, further investigations are required.