Development of a method to assess binding of astaxanthin to Atlantic salmon Salmo salar L. muscle proteins

Several methods were examined to characterize the binding between astaxanthin and salmon muscle protein(s) in order to provide tools for evaluation of the role of muscle proteins on astaxanthin retention in Atlantic salmon Salmo salar L. flesh. The methods included gel filtration chromatography, displacement of a hydrophobic probe and ultrafiltration. With gel filtration chromatography, aggregation of astaxanthin under the experimental conditions was a major problem for the separation of bound astaxanthin from free astaxanthin because the apparent molecular weight of aggregated astaxanthin or astaxanthin micelles was in the range of protein–astaxanthin complexes. Displacement of the fluorescent probe 8‐anilino‐1‐naphthalenesulphonate (ANS) was not effective as astaxanthin quenched the fluorophore so that displacement could not be observed. An ultrafiltration method was developed using 200‐mM sodium cholate for dispersion of astaxanthin aggregates. This allowed unbound astaxanthin to be separated from bound astaxanthin using a 30‐kDa filter. After salmon muscle proteins were solubilized in different fractions by sequential extraction using low ionic strength solutions, the astaxanthin binding of different fractions was assessed using the ultrafiltration method. The significant difference (P<0.05) observed in the astaxanthin binding of the various fractions suggests an application of this assay to detect differences in affinity of proteins for astaxanthin. The results also suggest that proteins other than actomyosin or actin can bind astaxanthin in Atlantic salmon flesh. This method can be used for the identification of astaxanthin‐binding proteins in salmon flesh and other tissues.     

Calanus oil as a natural source for flesh pigmentation in Atlantic salmon (Salmo salar L.)

The present study was to understand how efficiently the astaxanthin in Calanus oil is utilized for flesh colouration in Atlantic salmon ( Salmo salar ). Postsmolts of the fish (309 g) were held at 7.9 °C and they were fed diets containing 20 or 60 mg astaxanthin per kilogram feed derived from a synthetic source or from Calanus oil for 181 days. Besides growth and feed intake assessments, at day 81 and 181, fish flesh were subjected to colour analysis and astaxanthin determination. Growth and feed performance did not vary between the groups. There were significant differences in the amount of astaxanthin in muscle between almost all groups both at day 81 ( P  < 0.05) and at day 181 ( P  < 0.001). However, a notable similarity between fish receiving 20 mg astaxanthin from the synthetic source and those receiving 60 mg astaxanthin from Calanus oil ( P  > 0.05) at day 181 indicated that comparable amounts were deposited only with the greater level of the natural source. Tristimuli colorimeter a* values support the analytical results at day 181. Although Calanus oil did serve as a natural dietary pigment source for farmed salmon, its inclusion level should provide more than 60 mg astaxanthin per kilogram feed to achieve colouration preferred by the market.     

Changes in intestinal fluid transport in Atlantic salmon (Salmo salar L) during parr-smolt transformation

We examined changes in fluid transport by the intestine of Atlantic salmon (Salmo salar L) undergoing parrsmolt transformation during springtime. In vitro measurements of fluid transport rate (J_v) across non-everted middle and posterior intestinal sac preparations were made in late April and early June 1990 and from February through June 1991 for juvenile smolting fish. Intestinal J_v was also compared between parr- and smolt-stage salmon in both years. To evaluate the osmoregulatory role of the intestine, J_v was measured for smolts adapted to seawater and their cohorts remaining in fresh water. The middle intestine of smolting fish underwent a significant decrease in fluid transport during the springtime, while posterior intestinal J_v significantly increased. Parr-stage fish decreased J_v in the middle intestine during springtime similar to smolts. However, the posterior intestinal J_v of smolts showed a significant increase over the parr around the peak smolt period in both years. Seawater-adapted smolts generally exhibited posterior intestinal J_v approximately double that of freshwater cohorts. A decrease over time shown for the middle intestine, together with the increased J_v in the posterior intestine preceding and after seawater entry, suggests the development of a functional regionalization during parr-smolt transformation, with the posterior intestine taking on increased importance in osmoregulation in seawater.     

The progeny of sex-inverted female Atlantic salmon (Salmo salar L.)

A group of Atlantic salmon juveniles received dietary 17α-methyl-testosterone (3 mg/kg of food) for 60 days after first-feeding. Many of these fish became sexually mature as males at 1.5 years of age, while they remained in freshwater. Supposed sex-inverted females among these were identified by their failure to strip manually. Milt from the excised gonads of five of these fish was used separately to fertilise mixed ova from normal adult females. The progeny of each male, when examined 8 weeks after first-feeding, all had typically female gonads (n = 50).     

Content of synthetic astaxanthin in escaped farmed Atlantic salmon, Salmo salar L., ascending Norwegian rivers

Abstract  Isomeric ratios of astaxanthin in eggs and alevins of Atlantic salmon, Salmo salar L., have proven useful in identifying female spawners of farmed origin, but the method underestimates the proportion of fish of farmed origin. The rate of underestimation was studied by analysing astaxanthin content in tissue of 55 farmed Atlantic salmon ascending two Norwegian rivers in the autumn of 1991. The astaxanthin content fell into two distinct classes. Fifty-one per cent of the adult escaped salmon had isomeric ratios similar to salmon fed synthetic astaxanthin, whereas all the remaining fish had ratios similar to wild fish. Discriminant analysis classified 96.4% of the fish with known astaxanthin content into the correct astaxanthin class on basis of tail-fin erosion, length, weight and gill-cover damage. This discriminant function was used to estimate the astaxanthin classification of 1017 farmed salmon caught in nine rivers during 1989–1991. The classification success varied among years from 52 to 64%. Corresponding numbers for females and males were 45–48% and 54–70%, respectively. Thus, estimates of spawning rates of farmed female salmon via astaxanthin content in eggs or alevins from redds should be adjusted accordingly. The observed isomeric ratios of astaxanthin in the escaped farmed salmon and the relationship with morphology indicates that a significant proportion of the escapees ascending rivers have spent more than 1 year in the wild after escape.     

Plasma protein changes in Atlantic salmon, Salmo salar L., with infectious salmon anaemia

Moderate to severe anaemia and hypoproteinaemia were reported in a Canadian outbreak of 'haemorrhagic kidney syndrome' in Atlantic salmon, later shown to be caused by a variant of infectious salmon anaemia virus (ISAV). The progressive anaemia associated with ISA has been previously reported, but hypoproteinaemia in salmon infected with European isolates of ISA virus has not been well documented. The present study showed a very significant positive correlation between decreasing haematocrit values and total plasma protein concentrations in Atlantic salmon infected with two Canadian and two Norwegian ISA viral isolates. However, variations in the concentration of individual plasma proteins, typical of acute phase responses in higher vertebrates, were not observed.     

Seasonal changes in selected muscle quality parameters in Atlantic salmon (Salmo salar L.) reared under natural and continuous light

In order to investigate how seasonal variation in growth affects selected fillet quality parameters, immature Atlantic salmon (Salmo salar L.) were reared under simulated natural photoperiod (SNP) for 12 months or continuous light (LL) from January to June followed by SNP until December. Photoperiod treatments advanced the growth rate pattern of the LL group compared with the SNP group and influenced macronutrient metabolism, evaluated both as trends in protein and lipid retention and in fillet lipid and protein levels. Good growth was associated with low fillet lipid and protein level, in addition to reduced levels of fillet tocopherol and astaxanthin, indicating increased oxidative stress. Elevated levels of thiobarbituric reactive substances (TBARs) further supported this. Slaughtering during periods of high growth may therefore reduce postmortem quality, both because of increased susceptibility to fillet lipid peroxidation and reduced astaxanthin levels, which were lowered in vivo and might consequently be depleted further after slaughter. Specialized use of antioxidant‐rich feed prior to slaughter is suggested if slaughtering is expected to occur during periods of high growth rate.     

Transport stress in Atlantic salmon (Salmo salar L.): anaesthesia and recovery

The effects of metomidate anaesthesia on levels of plasma cortisol, glucose, haematocrit and chloride in Atlantic salmon (1⁺) (Salmo salar L.), after a 2‐h transport and during a 48‐h recovery period were investigated. The use of metomidate anaesthesia during transport led to a reduced release of cortisol and significantly lower levels of plasma cortisol after a 48‐h recovery period. Plasma glucose did not return to basal level after a 48‐h recovery period, indicating that even longer recovery may be needed for the fish to return to a pre‐stress state. The results show that metomidate anaesthesia combined with a recovery period lessens the stress burden imposed by hauling and transport.     

Maintenance ration, protein synthesis capacity, plasma insulin and growth of Atlantic salmon (Salmo salar L.) with genetically different trypsin isozymes

Growth was found to be associated with the changes of trypsin activity in the pyloric caecal tissues and the level of plasma insulin in Atlantic salmon (Salmo salar L.). A decrease in trypsin activity accompanied by an increase in plasma insulin was detected one month before an enhanced growth was observed. There were significant relationships between weight specific consumption rate, plasma insulin levels and fish growth. The correlation of weight specific consumption rate was higher with growth rate (R²=0.7, p<0.0001) than with plasma insulin concentration (R²=0.4, p<0.0001).When the comparison was made between Atlantic salmon carrying and lacking the trypsin variant TRP-2*92, the fish with the variant had lower maintenance ration (p<0.05), higher capacity for protein synthesis in the white muscle (p<0.02), and a greater ability to utilize the feed at a restricted ration than the fish without the variant. In Atlantic salmon lacking the variant, both plasma insulin concentrations and growth rates were significantly lower (p<0.05) in the fish fed 0.5% bw day^–1 than those fed 1% bw day^–1. Whilst the growth rates of TRP-2*92 salmon fed the different rations became similar one month after similar levels of plasma insulin were observed between them. The TRP-2*92 salmon may be defined as a high protein growth efficiency fish with low protein turnover rate.Genetic variation in trypsin isozyme pattern affects feed utilization, plasma insulin levels and growth in Atlantic salmon.     

Motoricity of Atlantic salmon embryos (Salmo salar L.) at different temperatures

Atlantic salmon eggs were fertilized and incubated in two temperature series at 3 and 8°C. Four motor criteria (heart rate, embryo motion, motion of the pectorals and motion of the mouth-gill apparatus) and oxygen consumption at different stages of embryogenesis were studied. At certain intervals the temperature was changed rapidly (within 30 min) to 3, 6, 8, 10, 12, 14 or 16°C and the detectable changes in the above motor criteria were recorded. Heart rate and embryo motion were the first recognizable motor components. The heart responded particularly sensitively to temperature shock with a change in heart rate, whereas temperature changes were not clearly reflected in embryo motion because space within the egg membrane was restricted owing to embryonal growth. Motion of the pectorals and mouth-gill apparatus did not start until shortly before hatching and achieved maximal values at 8–10°C. Pulse rates for all of the motor criteria except embryo motion were distinctly higher for eggs incubated at 8°C than for those incubated at 3°C.     

Protease activities, plasma free amino acids and insulin at different ages of Atlantic salmon (Salmo salar L.) with genetically different trypsin isozymes

We show significant relationships between digestion rate of dietary protein, absorption/transport rate of amino acids, and plasma insulin concentration. Digestion rate of dietary protein was indicated by protease activity ratio of trypsin to chymotrypsin in the pyloric caeca. Plasma total free amino acids and/or the ratio of essential to non-essential free amino acids indicated absorption/transport rate of amino acids. These relationships are associated with and likely to be primarily affected by genetic variation in the expression of different isozymes of trypsin, a key enzyme for feed utilization and growth, affecting digestion, absorption/transport rate of amino acids and plasma insulin levels in Atlantic salmon (Salmo salar L.).     

Mercury comparisons between farmed and wild Atlantic salmon (Salmo salar L.) and Atlantic cod (Gadus morhua L.)

Wild and farmed Atlantic salmon ( Salmo salar L.) and Atlantic cod ( Gadus morhua L.) were collected to assess changes in mercury with size in wild vs. farmed fish. Mercury concentrations were compared with Health Canada and United States Environmental Protection Agency consumption guidelines. Lipid dilution of mercury was examined by comparing lipid-extracted (LE) and non-lipid-extracted (NLE) flesh samples in both farmed and wild fish. Mercury concentrations in the flesh and liver of farmed salmon were significantly lower than concentrations in wild salmon of similar fork length ( P <0.001), possibly due to growth dilution in rapidly growing farmed fish. Mercury concentrations were higher in LE tissue compared with NLE ( P <0.05), suggesting lipid dilution of mercury in farmed fish with a high lipid content. Farmed cod, which do not grow more rapidly than wild cod, did not have significantly different flesh and liver concentrations compared with wild cod of similar fork length ( P >0.05). Between species of farmed fish, cod had significantly higher mercury concentrations than salmon ( P <0.05), but neither farmed nor wild salmon mercury concentrations exceeded federal consumption guidelines. These results suggest that rapid growth rates and a high lipid content may play important roles in regulating concentrations of contaminants such as mercury.     

Soybean meal-induced enteritis in Atlantic salmon (Salmo salar L.) at different temperatures

This study evaluates the effect of temperature on the development of intestinal disorders when Atlantic salmon are fed soybean meal (SBM). In this study 20% of the dietary fishmeal was replaced by solvent-extracted Hipro SBM. Atlantic salmon reared at two different water temperatures (8 °C and 12 °C), were fed a control diet and an experimental diet for 20 days. Samples were taken at days 7 and 20. The extent of the morphological changes was assessed using a semi-quantitative scoring system developed for this purpose. The study demonstrates that enteritis is affected by temperature. The intestinal disorders were more severe in fish reared at 12 °C compared with those reared at 8 °C. It can be concluded from this study that temperature changes the speed but not the type of SBM-induced enteritis expressed as a delay on the response when Atlantic salmon are kept at lower temperatures.     

Evaluation of feed protein quality by measuring plasma free amino acids in Atlantic salmon (Salmo salar L.) after dorsal aorta cannulation

Two successive experiments were conducted in order to assess plasma free amino acid (FAA) profiles as a method for evaluating protein quality of fish feeds for Atlantic salmon, Salmo salar (L.). In experiment 1, the importance of meal size and inter‐fish variation was assessed by using dorsal aorta cannulated fish and diets that contained different sources of fishmeal (menhaden versus herring) which in the case of herring, had been dried at either 70 or 100 °C. In experiment 2, an attempt was made to mimic a production situation by comparing the FAA profiles in salmon fed diets containing two commercially available fishmeals that had been produced in accordance with industrial standards (Norse‐LT94® and NorSeaMink®; Norsildmel AL, Fyllingsdalen, Norway). FAA profiles in plasma 6 h after feeding were compared with feed true protein digestibility as determined in mink. Cannulated fish, held in individual tanks, were hand‐fed twice daily to pellet rejection (satiety) and daily records of the actual rations consumed were maintained. A total of 24 different amino acids and other amino‐containing compounds were detected using high‐pressure liquid chromatography. Morning and evening meal size showed significant correlations. Meal size had a significant effect on blood levels of the majority of essential free amino acids (EAA) as well as the total sum of FAA (TFAA). In experiment 1, a marked inter‐individual effect was found, possibly because of incipient sexual maturation. FAA profiles were therefore corrected for meal size by linear regression while repeated sampling via the permanently implanted cannula allowed paired comparisons of the different test diets, minimizing inter‐individual variation. Significant differences in plasma FAA profile, EAA and TFAA were detected between fish fed all diets in both experiments. The preceding parameters for fish ingesting each feed were directly related to their respective mink protein digestibility in experiment 1, but not in experiment 2. Our results show that dietary protein quality can be differentiated by the aforementioned protocol, and by using fish with a low metabolic rate feed qualities could be ranked correctly as in experiment 1.     

Infectious pancreatic necrosis in Atlantic salmon, Salmo salar L

A clinical and histopathological review was carried out of 21 outbreaks of acute infectious pancreatic necrosis (IPN) in Scottish Atlantic salmon, Salmo salar L., farms (13 marine and eight fresh water) during 1991-2004. A distinctive syndrome was evident in both post-smolts in sea water and fry in fresh water, where liver lesions, which had not previously been associated with IPN, became a consistent finding in addition to the more typical pancreatic and intestinal changes. Initial cases were described in post-smolts in Shetland, but by the end of the period of investigation this type of pathology had extended down the West coast of Scotland and into Ireland. Limited viral strain analysis suggested that similar strains were involved in both fresh water and sea water and that these differed from earlier isolates from rainbow trout, Oncorhynchus mykiss (Walbaum). In fresh water, recovered fish frequently developed a greatly distended intestine associated with accumulation of undigested food. In sea water, after the initial, often significant (50% or more), losses, there were many fish which failed to grow and became chronically emaciated and prone to sea louse infection. Although use of transfer diets containing immune enhancers and the selection of IPN resistant broodstock has reduced losses the disease remains a serious cause of economic loss.     

Optical properties of the pineal window of Atlantic salmon (Salmo salar L.)

The spectral composition and intensity of light penetrating different parts of fresh preparations of the upper part of the skull of Atlantic salmon (Salmo salar) (fork length 25–30 cm) was investigated. All measurements were made in an aqueous medium, by moving the tip of an optical fibre in a three dimensional lattice below preparations that were illuminated by a parallel light source from above. The intensity of the transmitted light showed a well-defined maximum just below the pineal groove. Light that penetrated the skull from a source vertically above was refracted to produce a focusing point in the approximate position of the pineal body. Light projected from angles of 45° relative to the vertical position was only slightly (25%) attenuated, thus indicating a wide acceptance angle. There was an almost uniform transmission of light (of 3%) between 500 and 700 nm. The transmittance of UV light (350 nm) was about 10% of that of green light. These differences are small when compared with the intensity range reported for the pineal light receptors. The transmission properties are discussed in relation to the known diurnal changes in the spectral composition of natural light.     

Growth and survival of Atlantic salmon, Salmo salar L. fed different dietary levels of astaxanthin. Juveniles

Atlantic salmon, Salmo salar L., juveniles, with a mean initial weight of 1.75 g, were fed casein-based purified diets which had been supplemented with different levels of astaxanthin for a 10-week period. The astaxanthin content of the diets ranged from 0 to 190 mg kg^?1 dry diet. The growth and survival of the juveniles were recorded throughout the experiment. The proximate composition, astaxanthin and vitamin A content were determined from whole-body samples at the start and termination of the experiment. The dietary treatment was found to affect growth significantly (P < 0.05). A reduction in the mean weight of the juveniles was observed in the groups fed the diets without astaxanthin supplementation. There was no difference in growth rate between the fish in the groups fed the diets containing 36 or 190 mg astaxanthin kg^?1 dry diet, whereas the fish in the group fed the diet containing 5.3 mg astaxanthin kg^?1 dry diet had a lower growth rate. There was a tendency to higher survival in the groups fed the diets containing astaxanthin when compared with the groups fed the non-supplemented diets. The moisture and ash contents were significantly lower and the lipid content was higher in the groups fed the astaxanthin-supplemented diets. The astaxanthin and the vitamin A concentrations in the fish were found to be dependent upon the dietary astaxanthin dose; the highest values were found in the fish fed the diet with the highest astaxanthin content. These results strongly indicate that astaxanthin functions as a provitamin A for juvenile Atlantic salmon. The body storage of vitamin A increased in the fish fed the diets containing astaxanthin. However, the increase was low in the fish fed the diet containing 5.3 mg astaxanthin kg^?1 dry diet.