Accumulation of immunomodulatory laminaran (β(1,3)-D-glucan) in the heart, spleen and kidney of Atlantic cod, Gadus morhua L.

The tissue distribution of ³H- and FITC-lammaran in Atlantic cod, Gadus morhua L., was investigated after intravenous administration. Liquid scintillation counting and whole-body autoradiography revealed a substantial accumulation of ³H-labelled laminaran in the heart throughout the experimental period (1 h to 12 days). High amounts of the immunomodulator were also present in the spleen, anterior kidney and posterior kidney during the study. Renal excretion and intestinal exsorption were the main elimination pathways. Fluorescence microscopic examination revealed accumulation of FITC-labelled laminaran in macrophages in the kidney and ellipsoid sheath cells (macrophages and reticular cells) in the spleen. Furthermore, endothelial entrapment in the heart was pronounced. Endothelial cells in the kidney also contained the FITC-labelled immunomodulator. Immunohistochemical analysis, using anti-β(l,3)-D-glucopyranose antibody, also showed endothelial uptake of laminaran in the heart. The present investigation shows that laminaran accumulates and is retained in immunologically relevant cells in the Atlantic cod.     

Tissue distribution and cellular uptake of Aeromonas salmonicida lipopolysaccharide (LPS) in some marine fish species

Τhe uptake and distribution of lipopolysaccharide (LPS), isolated from Aeromonas salmonicida, was investigated in Atlantic cod, Gadus morhua L., turbot, Scophthalmus maximus L., and Atlantic halibut, Hippoglossus hippoglossus L. LPS was radiolabelled by bromine oxidation and subsequent sodium borotritide reduction (³H-LPS), and fluorescence-labelled by introducing a fluorescein isothiocyanate derivative (FITC-LPS). After intravenous and intraperitoneal injections in cod, high amounts of radioactive LPS (³H-LPS) were present in heart, spleen and kidney throughout the experimental period (1–168 h). After peroral administration, a high amount of ³H-LPS was observed in intestinal tissues, whereas internal organs and tissues contained considerably lower amounts. Following intravenous administration of ³H-LPS in turbot, high contents of radioactivity were revealed in spleen, liver and kidney, whereas the content in heart was lower than in blood at the sampling times (1–24 h). The same pattern was observed after intraperitoneal administration. The spleen and liver contained high amounts of radioactivity when the turbots were intubated perorally with ³H-LPS. The spleen, kidney and heart were the main scavenging organs following intravenous administration of ³H-LPS in Atlantic halibut. A minor amount of radioactivity was present in the liver. The same pattern emerged after intraperitoneal injection in halibut. As observed for turbot, the spleen was the main accumulation site for ³H-LPS following peroral administration. Fluorescence microscopy of sections of organs and tissues from cod, intravenously and intraperitoneally injected with FITC-LPS, revealed that endocardial cells of both atrium and ventricle contained large amounts of the fluorochrome, whereas in turbot and halibut only atrial endothelial cells accumulated the substance. In all species, macrophages in kidney and spleen contained FITC-LPS and in the spleen the fluorochrome was trapped in the ellipsoidal walls. At later time points (e.g. 48 h) in the turbot spleen, FITC-LPS was located in cells adjacent to the ellipsoidal walls. Halibut endothelial cells that were located in the connective tissue of the intestine and gills also contained FITC-LPS. After peroral administration to the different fish species, specific fluorescence was found only in intestinal epithelial cells of halibut and in cells located in the lamina propria. Fluorescence was not detected in internal organs such as the kidney, spleen and liver after peroral administration of FITC-LPS. Gel chromatographic analysis of plasma samples from cod, turbot and halibut after intravenous and intraperitoneal injections showed that high molecular weight radioactivity was present. A minor amount of radioactivity that corresponded to low molecular weight substances was also observed. In conclusion, there is a high degree of variation with respect to the site of accumulation and some variation in the type of cells involved in the uptake of purified LPS in cod, turbot and halibut.     

Anesthesia induces stress in Atlantic salmon (<Emphasis Type="Italic">Salmo salar</Emphasis>), Atlantic cod (<Emphasis Type="Italic">Gadus morhua</Emphasis>) and Atlantic halibut (<Emphasis Type="Italic">Hippoglossus hippoglossus</Emphasis>)

Stress in response to anesthesia with benzocaine, MS-222, metomidate and isoeugenol was studied in Atlantic salmon (Salmo salar), Atlantic halibut (Hippoglossus hippoglossus), and Atlantic cod (Gadus morhua) with no concomitant stress from handling or confinement in association with anesthesia or sampling. All of the anesthetics tested induced a stress response in all species, displayed by a release of cortisol to the water. MS-222 anesthesia elicited the highest cortisol release rates, reaching maximum levels 0.5 h post-exposure and returning to basal levels after 3–4 h. Benzocaine anesthesia caused a bimodal response where the initial peak in cortisol release rate was followed by a second increase lasting towards the end of the trial (6 h). This bimodality was more profound in Atlantic salmon than in Atlantic halibut and Atlantic cod. Metomidate anesthesia induced the lowest release of cortisol of the agents tested in both Atlantic halibut and Atlantic cod, but resulted in a bimodal response in Atlantic salmon where the initial increase in cortisol release was followed by a larger increase peaking at 2–2.5 h post exposure before returning to basal levels after 5 h. The stress induced in Atlantic salmon by isoeugenol anesthesia resembled that of MS-222, but did not reach the same elevated level. Overall, the cortisol release was most profound in Atlantic salmon followed by Atlantic halibut and Atlantic cod.     

Movements and spatiotemporal distribution of escaped farmed and local wild Atlantic cod (Gadus morhua L.)

Commercial farming of Atlantic cod (Gadus morhua L.) is now being developed in several countries. The ecological consequences of cod culture are poorly understood, but recent research suggests that Atlantic cod are more prone to escape from net pens than Atlantic salmon. Here, we describe the movements and the spatiotemporal distribution of farmed cod after escape relative to wild cod, both during and outside the natural spawning season. The experimental design included simulating escape incidents of farmed cod tagged with acoustic transmitters and using an array of automatic listening stations to monitor their dispersal and distribution. For comparison, local wild cod were monitored using the same array of receivers. The farmed cod dispersed rapidly after a simulated escape, they randomly distributed over large areas and their distribution overlapped with local wild cod. Moreover, escaped farmed fish were found at local cod spawning areas during the spawning season. The study also indicated that the recapture rate of escaped farmed cod was high compared with that of escaped farmed salmon. Thus, while our results showed that there is a considerable potential for ecosystem effects caused by escaped farmed cod, mitigating actions such as an efficient recapture fishery for escapees may be possible.     

Carnobacterium maltaromaticum vs. Vibrio (Listonella) anguillarum in the midgut of Atlantic cod (Gadus morhua L.): an ex vivo study

The present investigation addresses whether the midgut (MG) of Atlantic cod (Gadus morhua L.) is an infection route for Vibrio (Listonella) anguillarum serotype 02 β and if Carnobacterium maltaromaticum, a probiotic bacterium, can out‐compete the pathogen and modulate the autochthonous MG microbiota. This was investigated by using an ex vivo method the intestinal sac, utilized previously in studies on Atlantic cod and Atlantic salmon (Salmo salar L.). Exposure of the MG to V. (L.) anguillarum did not reveal any cell damage indicating that the MG does not appear to be an infection route for V. (L.) anguillarum in healthy Atlantic cod. This finding together with previous observations on Atlantic cod and Atlantic salmon indicate that intestine as an infection route might vary between these two species. When the MG was exposed to C. maltaromaticum, no cell damage or cellular disruptions were observed. As budding from the apices of microvilli was observed in all treatments exposed to bacteria, we suggest that budding might be involved in the primary barrier against bacterial infection. However, to clarify this hypothesis, further studies are needed. Exposure of the MG to the probiotics and pathogenic bacteria indicated that C. maltaromaticum, to some extent, is able to out‐compete V. (L.) anguillarum but the topic merits further investigation. Analysis of the MG microbiota after sterile saline solution and bacterial exposure indicates that bacteria related to Staphylococcus sciuri belong to the autochthonous gut microbiota in Atlantic cod.     

Enzymes from the gut bacteria of Atlantic cod,Gadus morhua and their influence on intestinal enzyme activity

Gut‐associated bacteria of fish are known to produce enzymes which aid in digestion. The presence and activities of these bacteria in cold‐water fishes like Atlantic cod are less known. Therefore, we have characterized the activities of extracellular enzymes of GP21 (Pseudomonas sp.) and GP12 (Psychrobacter sp.), two bacteria isolated from the gastrointestinal tract of Atlantic cod. Additionally, we examined if these bacteria when delivered through feeds could influence the activity of selected intestinal enzymes. GP21 was able to produce amylase, chitinase, cellulase and protease, whereas GP12 could produce only chitinase and protease. These enzymes were produced extracellularly and they were found to be catalytically active at acidic conditions (pH 2–5) and at temperatures ranging from 15 to 30 °C. Orally delivered bacteria could possibly influence the activity of intestinal enzymes after 40 days, rather than after 20 days of feeding. Thus GP21 and GP12, the potential probiotic organisms, could support digestion in Atlantic cod.     

Optimum temperature and food-limited growth of larval Atlantic cod (Gadus morhua) and haddock (Melanogrammus aeglefinus) on Georges Bank

We estimated recent growth of Atlantic cod (Gadus morhua) and haddock (Melanogrammus aeglefinus) larvae collected on the southern flank of Georges Bank in May 1992–94 from the ratio of RNA to DNA (R/D) and water temperature. Growth of both species increased with water temperature to about 7°C and then decreased. The highest growth rates were observed in May 1993 at water temperatures around 7°C. These data confirm an earlier observation of comparable temperature optima for growth of Atlantic cod and haddock larvae in the north‐west Atlantic. Comparisons of field growth rates and temperature optima with data for larvae cultured at high temperatures and prey densities in the laboratory suggest that growth may have been food‐limited at higher temperatures on Georges Bank. Given that 7°C is the long‐term mean water temperature on the southern flank in May and that climate models predict a possible 2–4°C rise in water temperatures for the western North Atlantic, our findings point to a possible adverse effect of global warming on Atlantic cod and haddock.     

Effects of Long-Term Feed Deprivation on the Development of Rigor Mortis and Aspects of Muscle Quality in Live-Stored Mature Atlantic Cod (Gadus Morhua L.)

Fresh Atlantic cod is available in large amounts in Norway during the first 5 months of the year. Live-storage of cod may extend the marketing period of fresh cod products throughout the year. In addition, this concept makes pre-rigor processing possible. The main problem of keeping wild cod in captivity is that it does not easily accept formulated dry feed. The purpose of this study was, therefore, to investigate how long-term live-storage of mature Atlantic cod in the absence of feed (79 days) affects the onset and development of rigor mortis, as well as fillet quality by assessing hardness and water-holding capacity of the muscle, pH, protein, and water content. The results showed that starvation of Atlantic cod for 23 days reduces the pre-rigor time from 29 to 17 h. Further starvation did not decrease this period significantly, suggesting that live-stored cod deprived of feed for 79 days may still be industrially processed before the onset of rigor mortis. More than 51 days of starvation reduced protein concentration and increased water content of the muscle. After 51 days, the muscle texture was softer than in fish starved for a shorter period.     

A synthesis of large-scale patterns in the planktonic prey of larval and juvenile cod (Gadus morhua)

Data from 40 published studies of the diet composition of larval and juvenile cod (Gadus morhua) from around the northern North Atlantic were summarized to assess generic patterns in ontogenetic and regional variability in the key prey. The results showed that larvae at the northern edge of the latitudinal range of cod depend primarily on development stages of the copepod Calanus finmarchicus, whilst those at the southern edge depend on Para‐ and Pseudocalanus species. Juvenile cod preyed on a wider range of taxa than larvae, but euphausiids were the main target prey. Analysis of regional variations in the relative abundances of C. finmarchicus and Para/Pseudocalanus spp. in the plankton, as estimated by the continuous plankton recorder (CPR) surveys, showed a similar geographical pattern to the larval cod stomach contents. Comparison of CPR data from the 1960s and 70s with data from the 1990s showed that the boundary between C. finmarchicus and Para/Pseudocalanus spp. dominance has shifted northwards on both sides of the Atlantic, whilst the abundance of euphausiids in the southern cod stock regions has declined. The results are discussed in relation to regional differences in the response of cod stocks to climate variability.     

The settlement and reproductive success of Lepeophtheirus salmonis (Krøyer 1837; Copepoda: Caligidae) on atypical hosts

The reproductive success of Lepeophtheirus salmonis settled on host and non‐host fish has been compared. Triplicate single species tanks of Atlantic salmon, marine three‐spined sticklebacks, saithe and Atlantic cod were exposed to 10 adult female L. salmonis per tank (n=30 lice per species). Adult female L. salmonis settlement and egg string production occurred only on salmon and cod, with no egg production occurring on saithe and three‐spined sticklebacks. The number of eggs in egg strings, hatching success of eggs and the survival of all larval stages to the copepodid stage were severely affected by the species of fish on which female L. salmonis had settled. L. salmonis settled on cod produced significantly fewer eggs, lower hatching rates and lower survival rates of larvae than females on Atlantic salmon. The production of egg strings by L. salmonis females infecting cod, which successfully hatch and moult through to the infective copepodid stage, albeit in small numbers, is discussed in terms of the implications to aquaculture and salmon and cod farming scenarios.     

The effect of dietary chitin on growth and nutrient digestibility in farmed Atlantic cod,Atlantic salmon and Atlantic halibut

The effect of adding 0%, 1%, 2% and 5% chitin from prawn shells in the diets for Atlantic cod, Atlantic halibut and Atlantic salmon on growth was investigated. Nutrient digestibility and feed utilization was investigated in salmon and cod. Atlantic cod grew from 186 ± 29 to 383 ± 78 g (N = 960) over 13 weeks. Dietary chitin had no effect on length, weight, condition, liver size or specific growth rate (SGR). The apparent digestibility (ADC) for protein ranged from 84.7% to 86.5%, lipid between 88.8% and 93.1% and dry matter from 96.1% to 96.6%. Feed utilization varied between 1.08 and 1.11 and was not correlated with dietary chitin content. Atlantic salmon tripled their weight from 199 ± 9 to 615 ± 75 g (N = 480) during the 13 weeks. High inclusions of chitin (>1%) reduced both growth rate and condition. Protein and lipid ADC was negatively correlated with dietary chitin. Feed utilization ranged between 0.86 and 0.90 and was not significantly affected by dietary chitin. Faecal protein increased significantly with increasing dietary chitin, while faecal dry matter and lipid did not. Individually tagged Atlantic halibut grew from 1300 ± 470 to 2061 ± 714 g (N = 70) during 6 months. Individual growth rates varied within each group from being slightly negative to 0.81%·day^?1. Diet had no significant effect on growth rates. Atlantic cod and Atlantic halibut seems unaffected by up to 5% chitin additions in the diet, while chitin >1% of diet negatively affects growth and nutrient utilization in Atlantic salmon.     

Pre-anaesthetic metomidate sedation delays the stress response after caudal artery cannulation in Atlantic cod (Gadus morhua)

Recovery from caudal artery cannulation with and without pre-anaesthesia metomidate sedation was assessed in Atlantic cod (Gadus morhua). The levels of plasma cortisol, glucose, electrolytes and acid–base parameters were compared between sedated and unsedated cod and to those in uncannulated individuals, where the samples were obtained by sacrificial sampling (reference level). Metomidate sedation delayed the stress response, causing sedated cod plasma cortisol to return to the reference level more slowly [day 4 post surgery (PS)] than in unsedated cod (day 2 PS). Plasma glucose was elevated in both sedated and unsedated cod up to and including day 5 PS. Plasma K⁺ was lower and pH was higher in cannulated cod than in the reference from 24 h PS until the end of experimentation, indicating a stress effect of sacrificial sampling on plasma K⁺ and pH that was likely caused by an acute stress response. Metomidate sedation delayed the stress response following CA cannulation and should therefore not be used as a pre-anaesthetic sedation in Atlantic cod. The caudal artery cannulation can be a useful tool in obtaining repeated blood samples from Atlantic cod given an adequate recovery time, which was determined to be 6 days irrespective of pre-anaesthesia sedation status.     

The interrelation between temperature regimes and fish size in juvenile Atlantic cod (<Emphasis Type="Italic">Gadus morhua</Emphasis>): effects on growth and feed conversion efficiency

The present paper describes the growth properties of juvenile Atlantic cod (Gadus morhua) reared at 7, 10, 13 and 16 °C, and a group reared under “temperature steps” i.e. with temperature reduced successively from 16 to 13 and 10 °C. Growth rate and feed conversion efficiency of juvenile Atlantic cod were significantly influenced by the interaction of temperature and fish size. Overall growth was highest in the 13 °C and the T-step groups but for different reasons, as the fish at 13 °C had 10% higher overall feeding intake compared to the T-step group, whereas the T-step had 8% higher feeding efficiency. After termination of the laboratory study the fish were reared in sea pens at ambient conditions for 17 months. The groups performed differently when reared at ambient conditions in the sea as the T-step group was 11.6, 11.5, 5.3 and 7.5% larger than 7, 10, 13 and 16 °C, respectively in June 2005. Optimal temperature for growth and feed conversion efficiency decreased with size, indicating an ontogenetic reduction in optimum temperature for growth with increasing size. The results suggest an optimum temperature for growth of juvenile Atlantic cod in the size range 5–50 g dropping from 14.7 °C for 5–10 g juvenile to 12.4 °C for 40–50 g juvenile. Moreover, a broader parabolic regression curve between growth, feed conversion efficiency and temperature as size increases, indicate increased temperature tolerance with size. The study confirms that juvenile cod exhibits ontogenetic variation in temperature optimum, which might partly explain different spatial distribution of juvenile and adult cod in ocean waters. Our study also indicates a physiological mechanism that might be linked to cod migrations as cod may maximize their feeding efficiency by active thermoregulation.     

Quality and Shelf Life of Liver of Farmed Cod (Gadus morhua)

ABSTRACT

Livers of Atlantic cod (Gadus morhua) are traditionally used in cod liver oil production or consumed cooked or canned. The farming of cod is a relatively new industry in Norway. The aim of this study was to determine quality and shelf life of fresh liver from farmed cod during chilled storage on ice by hydrolysis and oxidation state and sensory quality and the influence on canned liver. In two experiments, livers from farmed cod were stored chilled and sampled from Days 0 to 13, respectively. Quality, measured as hydrolytic and oxidation degradation, was reduced after 7 days of storage, while sensory quality was reduced after 4 days. Free fatty acids increased from Day 7 in both experiments, while peroxide value and anisidine value showed no change when the livers were single wrapped. Rancid odor was the first sign of oxidation and was registered after three to four days of storage. Canning within 2 days of storage prevented leakage of oil from the canned livers. Sensory analyses of oxidation are recommended as a sensitive and rapid method to detect oxidation of chilled cod liver.     

Isolation, cultivation and characterization of head kidney macrophages from Atlantic cod, Gadus morhua L.

Head kidney macrophages from Atlantic cod, Gadus morhua L., were isolated by density sedimentation and maintained under serum-free conditions for up to one week. The cells adhered and spread well on glass and plastic, were highly phagocytic, and had typical macrophage morphology as shown by phase contrast and scanning and transmission electron microscopy. Histochemical studies with the light microscope showed that the macrophages were acid phosphatase and non-specific esterase positive, and alkaline phosphatase and peroxidase negative. Compared with unstimulated control cells, LPS-treated cells showed enhanced superoxide anion formation, as measured by the reduction of nitroblue tetrazolium, and increased levels of acid phosphatase activity.     

Infectious pancreatic necrosis virus establishes an asymptomatic carrier state in kidney leucocytes of juvenile Atlantic cod, Gadus morhua L

Juvenile Atlantic cod (10 g) were infected with infectious pancreatic necrosis virus (IPNV) by intraperitoneal injection and cohabitation. Fish showed no signs of disease but IPNV could be re-isolated from kidney tissue for up to 12 weeks. On weeks 2, 5, 8, 10, 11 and 12 following infection, kidney leucocytes were fractionated on Percoll gradients, and cells separated into plastic adherent and non-adherent cell populations after overnight incubation. IPNV was detectable in lysates of both cell populations and in supernatants by culture in CHSE-214 cells. Wells containing 10(5)-10(6) macrophages had an IPNV TCID(50) of about 10(3)/well and in serially diluted macrophages the minimum number of cells required to detect virus ranged from 10(1) to 10(4). These data indicate that about one in 10(4) macrophages were infected and the mean number of virus/infected cell was about 10. Replication of IPNV in the macrophages was low as the titre of the virus in macrophage lysates did not increase between days 1 and 3 of culturing the macrophages, but virus was released into the supernatant over this time.     

Is there a genetic basis to growth in Atlantic cod?

There is still much disagreement and debate about whether or not genetically based growth differences occur in Atlantic cod, and there is evidence on both sides. In this review, data on genetically based growth differences in cod will be presented to shed light on this hypothesis. Motivated by the hypothesis that growth patterns may reflect specific genotype adaptations, we review stock‐specific responses on growth. An example of genetically based differences between the population units at two spawning localities off south Iceland is discussed. Here, significant differences in growth performance of the different Syp‐I genotypes were found. Also, the cod sampled at Loftstaðahraun displayed higher mean weight and length compared to the cod from Kantur indicating that these population units may display different life histories. Other studies have shown conflicting results depending on which side of the Atlantic the problem has been investigated. We propose that a common‐garden meta‐analysis with several cod stocks from both sides of the Atlantic is needed to give any reasonable answer to the question of genetically based growth differences. Until such studies have been conducted, it is premature to conclude one way or the other. In this review, we have not tried to quantify how large the environmental part of growth regulation versus the genetic part is, as this information is not available in the published literature on cod. Based on recent research on two flatfish species (turbot and Atlantic halibut), approximately 30% of growth variation is caused by genetical factors, but it remains to be seen if this is similar in cod. A fruitful way to continue this research might be to conduct controlled experiments, where performance (growth, food intake, feed conversion efficiency, feeding behaviour, etc.) and environmental factors (e.g. temperature, oxygen, photoperiod, predation risk, food availability) are studied simultaneously for different genotypes and different stocks.     

Inclusion of camelina meal as a protein source in diets for farmed Atlantic cod Gadus morhua

Camelina meal Camelina sativa (CM) is a potential protein source in aquaculture feeds, because of its crude protein level (39%) and essential amino acids. Two feeding experiments were conducted with Atlantic cod Gadus morhua. Cod in Experiment I (19.4 g fish^?1) were fed diets with 0%, 12% or 24% CM for 9.5 weeks at 10°C; and cod in Experiment II (14.4 g fish^?1) were fed diets with 0%, 15%, 30% or 40% CM for 13 weeks at 10°C. Growth, lipid and amino acid tissue composition were compared amongst cod fed varying levels of CM. In Experiment I, cod could tolerate the highest level of CM inclusion (24%) without affecting growth compared to cod fed the control diet. In Experiment II, growth performance was significantly affected at 30% CM inclusion compared to the control treatment, and cod fed 15% CM displayed some signs of depressed growth (reduced feed intake and weight gain). Both treatment and duration were interacting factors (P = 0.015) that determined growth performance when comparing both experiments. Muscle tissue composition was relatively unaltered with less than 30% CM inclusion; however, multivariate statistics revealed significant differences in muscle tissue fatty acid composition between cod fed 40% CM and the control diet. The tissue amino acid profile was generally unaltered because the dietary amino acid profile was consistent after CM inclusion. A few antinutritive compounds in CM may have affected palatability in diets with greater than 30% CM inclusion, which may have resulted in reduced growth performance.     

Trypsin Activity Measurement in Fish and Mammals

Abstract

Trypsin activity is usually measured using synthetic substrates, principally amide and ester derivatives of the amino acids lysine or arginine. The aim of this study was to compare trypsin activity measurements done with two assays using amide substrates and two assays using ester substrates. The activity of purified commercial enzyme extracts (bovine and Atlantic cod, Gadus morhua) were measured as was the activity in animal tissues (mice and Atlantic cod) at 5, 15, 25, and 35°C. The results clearly showed the potential impact of the substrate choice on the results when comparing different taxonomic groups at different temperatures. The sensitivity, the measurement reproducibility and even the thermal sensitivity of the enzyme varies according to the assay used.