Pharmacokinetics of florfenicol in cod Gadus morhua and in vitro antibacterial activity against Vibrio anguillarum

The pharmacokinetic profile of the antibacterial agent florfenicol was studied in plasma after intravenous (i.v.) injection and in plasma, muscle and liver following oral (p.o.) administration to cod Gadus morhua, held in seawater at 8 degrees C and weighing 100 to 200 g. Following i.v. injection, the plasma drug concentration-time profile showed 2 distinct phases. The plasma distribution half-life (t1/2alpha) was estimated to be 1.6 h, the elimination half-life (t1/2beta) to be 43 h, the total body clearance (ClT) to be 0.015 1 kg(-1) h(-1) and mean residence time (MRT) to be 74 h. The volume of distribution at steady state, Vd(ss), was calculated to be 1.1 l kg(-1). Following p.o. administration, the bioavailability was estimated to be 91%, the peak plasma concentrations (Cmax) to be 10.8 microg ml(-1) and the time to peak plasma concentrations (Tmax) to be 7 h. Corresponding Cmax and Tmax values were 13.0 microg g(-1) and 9 h, respectively, in muscle and 12.1 microg g(-1) and 9 h, respectively, in liver. The in vitro minimum inhibitory concentration (MIC) values of florfenicol against 3 Vibrio anguillarum strains isolated from diseased cod (A-21, HI-610, HI-618) were 0.5 microg ml(-1) for all 3 strains.     

Minisatellite DNA fingerprints of Atlantic cod (Gadus morhua)

The human minisatellite probes 33.6 and 33.15 cross–hybridized to Hae III and Hinf I digested cod DNA, revealing complex fragment patterns in both Arctic and coastal cod. The DNA fingerprints were highly polymorphic, individual specific and stable in the germline. The potential applications of multi locus probes in cod research are discussed.     

Tissue distribution, inhibition and activation of gelatinolytic activities in Atlantic cod (Gadus morhua)

Gelatinolytic activities in fish tissues with properties like matrix metalloproteinases (MMPs) have been paid little attention. However, they have been proposed to participate in post mortem degradation during storage and the disintegration of pericellular connective tissue during spawning. In this paper the distribution of gelatinolytic activities in liver, heart, muscle, gill, and male and female gonad of Atlantic cod (Gadus morhua) was studied by using gelatin SDS-PAGE, proteinase inhibitors, gelatin and lentil lectin Sepharose affinity chromatography. The amount of gelatin degrading enzymes varied from tissue to tissue. Most of the gelatin binding enzymes were found to be matrix metalloproteinases by adding galardin, a broad range MMP inhibitor, to the incubation buffer. A 72 kDa form of cod gelatin degrading enzyme had properties similar to human proMMP-2, as it could be activated by p-aminophenylmercuric acetate and trypsin. Like the human MMP-2 it did not bind to lentil lectin. An 83 kDa cod gelatin degrading enzyme had properties similar to the 92 kDa progelatinase B (proMMP-9). These properties were also similar to that of the 72 kDa form, except that the 83 kDa cod gelatinase was bound to lentil lectin, showing that it is a glycoprotein like MMP-9.     

Ontogeny of redox regulation in Atlantic cod (Gadus morhua) larvae

The reduction potential of a cell is related to its fate. Proliferating cells are more reduced than those that are differentiating, whereas apoptotic cells are generally the most oxidized. Glutathione is considered the most important cellular redox buffer and the average reduction potential (E_h) of a cell or organism can be calculated from the concentrations of glutathione (GSH) and glutathione disulfide (GSSG). In this study, triplicate groups of cod larvae at various stages of development (3 to 63 days post-hatch; dph) were sampled for analyses of GSSG/2GSH concentrations, together with activities of antioxidant enzymes and expression of genes encoding proteins involved in redox metabolism. The concentration of total GSH (GSH+GSSG) increased from 610±100 to 1260±150 μmol/kg between 7 and 14 dph and was then constant until 49 dph, after which it decreased to 810±100 μmol/kg by 63 dph. The 14- to 49-dph period, when total GSH concentrations were stable, coincides with the proposed period of metamorphosis in cod larvae. The concentration of GSSG comprised approximately 1% of the total GSH concentration and was stable throughout the sampling series. This resulted in a decreasing E_h from −239±1 to −262±7 mV between 7 and 14 dph, after which it remained constant until 63 dph. The changes in GSH and E_h were accompanied by changes in the expression of several genes involved in redox balance and signaling, as well as changes in activities of antioxidant enzymes, with the most dynamic responses occurring in the early phase of cod larval development. It is hypothesized that metamorphosis in cod larvae starts with the onset of mosaic hyperplasia in the skeletal muscle at approximately 20 dph (6.8 mm standard length (SL)) and ends with differentiation of the stomach and disappearance of the larval finfold at 40 to 50 dph (10–15 mm SL). Thus, metamorphosis in cod larvae seems to coincide with high and stable total concentrations of GSH.     

Phenotypic flexibility of digestive system in Atlantic cod (Gadus morhua)

This study examined the restoration of the digestive capacity of Atlantic cod (Gadus morhua Linnaeus) following a long period of food deprivation. Fifty cod (48 cm, 1 kg) were food-deprived for 68 days and then fed in excess with capelin (Mallotus villosus Müller) on alternate days. Ten fish were sampled after 0, 2, 6, 14 and 28 days and the mass of the pyloric caeca, intestine and carcass determined. Two metabolic enzymes (cytochrome c oxidase and citrate synthase) were assayed in white muscle, pyloric caeca and intestine, and trypsin activity was measured in the pyloric caeca. A delay of 14 days was required before body mass started to increase markedly, whereas most of the increase in mass of both the pyloric caeca and intestine relative to fish length occurred earlier in the experiment. By day 14, the activities of trypsin and citrate synthase in the pyloric caeca as well as citrate synthase in the intestine had reached maxima. The growth of the digestive tissues and restoration of their metabolic capacities thus occur early upon refeeding and are likely required for recovery growth to take place. The phenotypic flexibility of the cod digestive system is therefore remarkable: increases in trypsin activity and size of pyloric caeca resulted in a combined 29-fold increase in digestive capacity of the fish during the refeeding period. Our study suggests that Atlantic cod are able to cope with marked fluctuations in food availability in their environment by making a rapid adjustment of their digestive capacity as soon as food availability increases.     

A reappraisal of activity metabolism in Atlantic cod (Gadus morhua)

Atlantic cod ( Gadus morhua ) were forced to swim in a swim tunnel respirometer until fatigued; oxygen consumption rate (O₂) was measured during swimming at incremental speeds until the fish was exhausted and during recovery from exhaustion. Maximal oxygen consumption (O_2max) occurred during maximal activity as has been found for other fish species, but at odds with the current paradigm for Atlantic cod. Earlier experiments had drawn the conclusion that O_2max in Atlantic cod occurs during recovery from exhaustive exercise. We found no support for this paradigm in our experiments and we propose that the respiratory physiology of Atlantic cod is not unlike that of other fishes.     

Feeding behaviour of cod (Gadus morhua) in relation to spawning

The food consumption and egg production of 26 adult (13 female and 13 male) Atlantic cod ( Gadus morhua ) were monitored during prespawning, spawning and postspawning periods. Females spawned from late January to mid-April. Feeding activity occurred from December to early January and ceased for females, on average, 36 days (15–54 days) before the onset of spawning. The duration of spawning by females was, on average, 42 days (10–61 days) and feeding was suppressed by both sexes during the first three-quarters of each female's spawning period. Mature females went, on average, 70 days or 19% of the year without eating. An abrupt increase in feeding activity, particularly by females, occurred during the last quarter of spawning or shortly after the release of the last egg batch (on average, feeding started again after 91% of a female's eggs had been released or 82% of egg batches). Females consumed greater quantities of food than males during both winter and postspawning feeding periods. During spawning, females lost, on average, 29% of their body weight and males 14%. Fecundity ranged from 0.75 to 3.97 million eggs per female. The volume of eggs produced by four individual females (range = 1285–5995 ml in four to 11 batches) ranged from 99 to 195% (mean 150%) of a female's postspawning body volume. Six immature cod fed throughout the experimental period and gained, on average, 8% of initial body weight. Laboratory results were supported by stomach fullness index values of Georges Bank cod exhibiting different maturity states.     

Evaluation of microsomes isolated from fresh and frozen myotomal tissue of Atlantic cod (Gadus morhua)

Microsomes were isolated from fresh and frozen myotomal tissue of Atlantic cod by two procedures. Electron microscopy revealed one method to yield microsomes containing greater quantities of myofibrillar debris than the other and this was reflected as reduced 5'-nucleotidase, acid phosphatase and succinate dehydrogenase marker enzyme activity. Overnight freezing of myotomal tissue did not affect the marker enzyme activity of microsomes isolated by either procedure. Morphological changes were observed among microsomes prepared from myotomal tissue retained for 8 weeks at -30 degrees C. Accordingly, 5'-nucleotidase was marginally elevated and acid phosphatase and succinate dehydrogenase activity reduced in comparison to fresh microsome preparations.     

The carotenoids of eggs of wild and farmed cod (Gadus morhua)

• 1.1. Eggs of wild cod, and of farmed cod fed (a) a diet supplemented with astaxanthin and (b) a diet supplemented with both astaxanthin and canthaxanthin, were analysed with respect to carotenoids.
• 2.2. The total carotenoid contents in eggs were 0.7 ppm for wild cod and 0.5 ppm for farmed cod.
• 3.3. Cod, having white flesh, deposit ketocarotenoids in the eggs, preferably astaxanthin.
• 4.4. Canthaxanthin can replace astaxanthin in the eggs, but astaxanthin appears to be deposited preferentially when both carotenoids are present in the diet.
• 5.5. The isomer distribution of (3S, 3′S):(3R, 3′S, meso):(3R, 3′R) astaxanthin in the eggs reflected the isomer composition of the diet.
• 6.6. Echinenone, 4′-hydroxyechinenone, adonixanthin and zeaxanthin encountered in cod eggs may represent reductive metabolites of canthaxanthin and astaxanthin.

     

Purification and characterization of pancreatic elastase from Atlantic cod (Gadus morhua)

1. A pancreatic elastase from Atlantic cod (Gadus morhua) has been purified and characterized. 2. The enzyme is a very basic protein with an approximate mol. wt of 28,000. 3. The cod elastase has higher elastin specificity than porcine elastase, and it is inhibited by soybean trypsin inhibitor, which has no effect on porcine elastase. 4. The cod elastase expresses a higher turnover number (kcat) and catalytic efficiency (kcat/Km) than porcine elastase, but it is less thermostable.     

Neutrophils and B-cells in Atlantic cod (Gadus morhua L.)

Proportions of leucocytes from head kidney, blood and spleen were identified as B-cells and neutrophils using a polyclonal antibody to cod IgM and a monoclonal antibody which previously has been shown to bind specifically to salmon and trout neutrophils. The cell specific binding of the antibodies was supported by double immunostaining. The morphology of isolated leucocytes was examined on Diff Quick stained slide preparations, and myeloperoxidase positive neutrophils were identified by diaminobenzidine staining. The antibodies clearly identified distinct cell populations. Using flow cytometry, high proportions of neutrophils were observed in peripheral blood leucocytes and high proportions of B-cells were found in head kidney leucocytes when compared to proportions of these cells in Atlantic salmon (Salmo salar L.). The spleen contained the highest proportion of B-cells. Cytoplasmic staining of immunoglobulin positive cells in slide preparations indicated that plasma cells were present, but not strikingly abundant, in head kidney, spleen and peripheral blood. Staining for myeloperoxidase identified, in accordance with the flow cytometry results, a large number of neutrophils, especially in peripheral blood leucocytes. The neutrophil nucleus was not clearly segmented, but appeared more irregular than rounded. The findings of high proportions of neutrophils in peripheral blood suggest that these cells of the innate immune system might have a central role in defence and protection against infections in cod.     

The distribution of Cryptocotyle spp. metacercariae in the skin of caged Atlantic cod (Gadus morhua)

In caged Atlantic cod (>40cm), Cryptocotyle spp. metacereariae were concentrated on the dorsal surface, but relatively scarce on the fins. Behaviour and mechanical forces probably affect this distribution.     

Fertility of gynogenetic Atlantic cod (Gadus morhua L.)

In order to investigate whether meiotic gynogenetic Atlantic cod is fertile and able to produce viable offspring, meiotic gynogenetic females were produced in spring 2010 by activating cod eggs using irradiated sperm. The extrusion of the second polar body was prevented by the application of hydrostatic pressure (56.6 MPa) 36 min after fertilization. In February 2012, their mean round weight was 972 g, and 2580 g in March 2013. In 2012, when the fish were 2 years old, about 52% were mature, 33% were immature, and 13% had undifferentiated gonads. One year later, 77% were mature, 11% were immature, and 11% had undifferentiated gonads. Several of the mature females had malformed gonads, with only one developed ovary lobe or with the two lobes fused. The mean gonadosomic index (GSI) of the 2‐year‐old mature females was 5.2%, with an estimated relative fecundity of 581 000 eggs kg ovary‐free wet weight^?1. Females were stripped for eggs when 2 and 3 years old (2012 and 2013), and fertilized with sperm from normal males. Offspring were obtained from 12 of 17 and 12 of 15 egg batches incubated in 2012 and 2013, respectively, proving that the gynogenetic females are fertile. Furthermore, larvae in all but one of the hatched groups from 2013 had commenced feeding 2 h after being startfed using rotifers 4 days after hatch, indicating viable offspring.     

Immune parameters of immunised cod (Gadus morhua L.)

The immune response of cod (Gadus morhua L.) is unusual in that specific antibody response is limited or absent. In the present study cod was immunised with haptenated and non-haptenated protein antigen at two different temperatures and the antibody response monitored over a period of 18 months. Other humoral parameters of immunological importance were also analysed, namely total immunoglobulin concentration, anti-protease and spontaneous haemolytic activity. No specific antibody response was detected but increased activity of non-specific anti-TNP antibodies was observed 10-12 weeks after immunisation, irrespective of the antigen used. This antibody activity was attributed to the adjuvant used (FCA) and did not cross react with other antigens tested. Other parameters were probably not influenced by the immunisation but seasonal fluctuations were indicated. The immunoglobulin level appeared to peak in August-September and the anti-protease activity and the haemolytic activity in October-January.     

Allometric growth in juvenile cod (Gadus morhua) and possible effects on cannibalism

Cod juveniles of about 20 mm had the lowest potential predator: prey length ratio based on mouth gape of the predator and the body height of the prey. This implies that the cannibalistic potential is maximized in this size range.     

Survival, activity and feeding ability of Baltic cod (Gadus morhua) yolk-sac larvae at different salinities

Due to unfavourable conditions (declines in salinity and water oxygen content) in the spawning areas, there has been a considerable decrease in the Baltic cod stock since the beginning of the 1980s, and consequently a decrease in catches. In order to examine the feasibility of introducing yolk-sac larvae in areas of low salinity to improve the stock, laboratory experiments were performed on the effects of salinity on the survival, level of activity and feeding ability of larvae. Yolk-sac larvae from spawning cod caught off northern Gotland, Sweden, were exposed to four different salinities: 10 and 15%○ (salinities of the main spawning areas); and 5 and 7%○ (salinities in the Bothnian Sea and the Baltic proper respectively).
The survival of yolk-sac larvae was high at all salinities, even though there was an indication of higher mortality at low salinities in less viable larval groups. No differences were found in swimming speed or feeding ability at the four salinities, but a significant difference in vertical distribution was recorded. There were significant differences in survival, vertical distribution and feeding ability among larval groups, which indicates that larval quality or viability is of greater importance for larval survival than salinity, in the range of 5–15%○.     

Reversible inactivation of alkaline phosphatase from Atlantic cod (Gadus morhua) in urea

Alkaline phosphatase (AP) from Atlantic cod (Gadus morhua) is a zinc and magnesium containing homodimer that requires the oligomeric state for activity. Its kinetic properties are indicative of cold-adaptation. Here, the effect of urea on the structural stability was studied in order to correlate the activity with metal content, the microenvironment around tryptophan residues, and events at the subunit interface. At the lowest concentrations of urea, the first detected alteration in properties was an increase in the activity of the enzyme. This was followed by inactivation, and the release of half of the zinc content when the amount of urea reached levels of 2 M. Intrinsic tryptophan fluorescence and circular dichroism ellipticity changed in the range 2.5 to 8 M urea, signaling dimer dissociation, followed by one major monomer unfolding transition at 6-8 M urea as indicated by ANS fluorescence and KI fluorescence quenching. Gibbs free energy was estimated by the linear extrapolation method using a three-state model as 8.6 kcal/mol for dimer stability and 11.6 kcal/mol for monomer unfolding giving a total of 31.8 kcal/mol. Dimer association had a very small ionic contribution. Dimers were stable in relatively high concentration of urea, whereas the immediate vicinity around the active site was vulnerable to low concentrations of urea. Thus, inactivation did not coincide with dimer dissociation, suggesting that the active site is the most dynamic part of the molecule and closest related to cold-adaptation of its enzymatic activity.     

Directional hearing of cod (Gadus morhua) under approximate free field conditions

Summary The cod,Gadus morhua L., can hear the direction of a sound source of 75 Hz in the transversal plane. At the experimental site in the middle of a fjord, local depth 35 m, the sound sources were situated at radial distances of 4 to 5.3 m from the subject in its netting cage. Both in two-alternative and four alternative choice experiments, in which an acoustic discrimination of separate similar sound sources was required, reward conditioning yielded positive results. The bearing of the sound source was decisive for the discrimination and not the identity of the sound sources used (intensity or timbre deviations). If the reverberation characteristics of the fjord at the receiving point were strongly dependent on the spatial position of the source, the results of the choice experiments might represent a demonstration of pseudo-directional hearing. However, from acoustic measurements of the time averages of the sound parameters such an anisotropy for sources with different bearing was not apparent (Fig. 5). Furthermore, in a well-trained cod, in which the pars inferior of only one single labyrinth was put out of function by surgery, the discrimination of the bearing of the sound sources was abolished but not the detection of sound as such (see Table 5). Detection of the direction of the sound therefore seems to be a function of the labyrinths and not of the lateral line system (directionalhearing). Estimates are given of the minimal angle that can be distinguished (about 22° for a 50% detectability likelihood; Fig. 6), and of the minimal acoustic level necessary for acoustic orientation (about — 13 dB re 1 bar; see Fig. 9). Finally an extension to an existing detection model of directional hearing is proposed. It receives empirical support in the next paper (Schuijf and Buwalda, 1975).Professor S. Dijkgraaf's readiness to develop the necessary operations and perform them in Norway in 1971 and 1972 is gratefully acknowledged. I thank Mr. G. Aase, Mr. K. Olsen and Dr. G. Sætersdal, Fiskeridirektoratets Havforskningsinstitutt (Bergen, Norway), for offering facilities to continue Kjell Olsen's study on directional responses of cod to sound. The contributions and the enormous efforts of my former student companions Drs M. A. van Arkel and Drs. M. E. Siemelink were decisive for success in the field. Guests from Utrecht supported the project in different directions, especially in constructing. I am grateful to Drs. R. J. A. Buwalda, Prof. S. Dijkgraaf, Drs. E. Meelis (statistics), Dr. F. J. Verheijen and Ir. D. W. van Wulfften Palthe (acoustics), all of whom made valuable comments on various aspects of the paper, and to Drs. J. W. Baretta for his aid enabling a quick start in 1971. The Netherlands Organization for the Advancement of Pure Research (Z.W.O.) for this study financed two journies to Norway and one to Scotland.     

Ecological implications of Echinorhynchus gadi parasitism of Baltic cod (Gadus morhua)

Of 533 Baltic cod (body length 21–93 cm) 530 were infected with Echinorhynchus gadi at infection intensities from 1 to 490 per host. The heavy infection of large cod (normally not feeding on amphipods) may be explained by a transfer of parasites from prey fishes to the predating large cod.