The effect of culture on morphology, lipid and fatty acid composition, and polyunsaturated fatty acid metabolism of rainbow trout (Oncorhynchus mykiss) skin cells

Rainbow trout (Oncorhynchus mykiss) skin cell cultures were obtained by trypsinization of the tissue and grown in Leibovitz L-15 medium. Lipid class compositions, and fatty acid profiles of total lipids and individual phospholipid classes were determined at different times of culture. The metabolism of polyunsaturated fatty acids (PUFA) was investigated by incubating primary cultures after 7 and 14 days with [1-¹⁴C]18:2n-6 and [1-¹⁴C-]18:3n-3. The change in morphology between epithelial-like primary cultures and fibroblastic-like secondary subcultures was accompanied by alterations in the lipid composition. Polar lipids became predominant by 14 days in culture. The relative proportions of phosphatidylcholine (PC), the most abundant phospholipid, phosphatidylinositol and cholesterol increased significantly, while sphingomyelin decreased. Saturated fatty acids, 18:1n-9, n-6 and n-9PUFA were more abundant in total lipid in cultures at 14 days and 4 months than in cells initially isolated which contained higher percentages of longer chain monoenes and n-3PUFA. The changes in fatty acid composition with time in culture were observed in all the major phospholipid classes. Rainbow trout skin cells in culture desaturated and elongated both 18:2n-6 and 18:3n-3, with 20:4n-6 and 20:5n-3 being the most abundant products, respectively. PC presented the highest incorporation of radioactivity, especially following incubation with 18:3n-3. Lipid metabolism in general increased with the age of primary cultures, with both the amount of C18 PUFA incorporated and metabolized by desaturation/elongation significantly increased in 14 day cultures compared to 7 day cultures. Product/precursor ratios calculated for both n-6 and n-3 fatty acids showed that, while 6 desaturase activity was increased significantly with cell age, 5 desaturase activity was more affected by the fatty acid series, with 18:3n-3 being more readily transformed to 20:5n-3 than 18:2n-6 to 20:4n-6. Further desaturation of 20:5n-3 to hexaenes was low. Overall, the data suggested that the trout skin cell cultures were more similar to mammalian skin fibroblasts than mammalian epidermal/keratinocyte cultures.     

Inclusion of macroalgae meal (Macrocystis pyrifera) as feed ingredient for rainbow trout (Oncorhynchus mykiss): effect on flesh fatty acid composition

The use of macroalgae as an additional component in animal feeding has been studied. However, information on how it could influence muscle composition of fish body is scarce. This study evaluates four diets with different macroalgae inclusion levels (0%, 1.5%, 3% and 6%) to test the effect on body fatty acid composition of rainbow trout. Tanks with a volume of 600 L were stocked with 60.6 ± 7.9 g fish at a density of 45 individuals tank^?1 and fed for 124 days. At the end of the experiment there were not significant differences (P<0.05) in muscle proximate composition among fish fed the different treatments. However, it was determined that inclusion of 3% and 6% of macroalgae meal resulted in a significant increase (P<0.05) of polyunsaturated fatty acids (PUFAs) in muscle. In summary, macroalgae meal in rainbow trout diets do not enhance the quantity of protein and lipid contents at muscle level but an addition of 3–6% might contribute to increase the level of PUFAs, specially EPA, DHA and LIN. Thus, use of macroalgae meal might help to increase lipid quality content in the final product due the beneficial effects of PUFAs for human health.     

Effects of temperature and dietary n-3 and n-6 fatty acids on endocytic processes in isolated rainbow trout (Oncorhynchus mykiss,Walbaum) hepatocytes

Effects of different incubation temperatures (2, 8, 14 and 20°C) and hepatocyte membrane fatty acid composition on the rate of internalization and lysosomal degradation of the ligand, mannosylated albumin, that is taken up by receptor-mediated endocytosis, were investigated in rainbow trout (Oncorhynchus mykiss, Walbaum). The fish were kept at a water temperature ranging from 9 to 14°C and fed pelleted diets coated with either capelin oil (control), EPA/DHA-concentrate (rich in n-3 polyunsaturated fatty acids) or soybean oil (rich in n-6 unsaturated fatty acids) for at least 3 months prior to sampling. The endocytic uptake mediated by the mannose receptor was very efficient at all temperatures studied. Lysosomal degradation, on the other hand, came to a halt below 8°C. The activation energies for uptake and degradation were 54.6 and 164.2 kJ/mol respectively. No negative effects of increased amounts of either n-3 or n-6 fatty acids were observed on the endocytic parameters studied. On the contrary, multivariate analysis indicated a positive relationship between high levels of n-6 fatty acids and low unsaturation index in the phosphatidylcholine (PC) fraction of the hepatocytes and the internalization rate of 2°C, meaning that the rate of receptor-mediated endocytosis may be affected by membrane fatty acid composition.     

Effect of diets containing laurel seed oil on growth and fatty acid composition of rainbow trout,Oncorhynchus mykiss

The aim of this study was to determine the effects of replacing fish oil (FO) with laurel seed oil (LSO), as an alternative plant lipid source in diets on the growth and fatty acid composition of rainbow trout (Oncorhynchus mykiss; 111.47 ± 0.2 g mean individual weight). At the end of the feeding trial, survival was 100% in all treatments. No significant differences were seen in growth between the dietary groups (P > 0.05). The protein, lipid and ash contents were not significantly different among the groups (P > 0.05); however, there was a significant difference in protein and ash content between the treatment groups and the initial, and between the 50LSO group and the initial group, respectively (P < 0.05). The viscerosomatic index (VSI) and hepatosomatic index (HSI) values were not affected by increasing LSO percentages in the diets. The n‐6 polyunsaturated fatty acid (PUFA) concentration increased with increasing LSO levels in the diets. In contrast, the n‐3 PUFA levels decreased with increasing LSO levels in the diets. The liver and muscle were used for the analysis of fatty acids. The highest level of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) concentrations was recorded in fillet of fish fed the FO diet and the lowest in those fed the 50LSO diet. However, EPA and DHA ratios in the liver of fish fed the 75LSO diet were higher than those in fillet of fish fed the FO and 50LSO diets. No significant differences were seen in fatty acid composition between the dietary groups (P > 0.05). Based on the results of growth performance and fatty acid composition of the experimental fish in this study, it can be concluded that the 75% concentration of laurel seed oil performed best among the diets tested in the experiment.     

The effects of phase‐feeding rainbow trout (Oncorhynchus mykiss) with canola oil and Alaskan pollock fish oil on fillet fatty acid composition and sensory attributes

Rainbow trout (186 g) were fed three test diets where the lipid source (150 g kg^?1) was either menhaden oil (MO), pollock oil (PO) or canola oil (CO) for eight weeks to an average weight of 370 g. The CO group was then divided into two groups, one continuing on the CO diet and the other switched to the PO diet (CO–PO). After nine additional weeks of feeding, the average fish weight approximately doubled (719–749 g). No significant differences were found in average final weight or fillet yield among dietary treatment groups. Fatty acid profiles of fillets from trout fed MO, PO or CO‐supplemented diets reflected the fatty acid profiles of the added oils, whereas the fatty acid profile of fillet from trout in the CO–PO group exhibited values similar to those of fish fed PO. The ratio of ω3 : ω6 FA was nearly 2.5 times higher in fillets from the CO–PO group compared to the CO group. Sensory analysis showed that panelists preferred CO‐fed fillets over those fed MO, PO, or CO–PO. Phase‐feeding CO and PO reduced fish oil use and resulted in fillets with double the content of Eicosapentaenoic acid (EPA) and Docosahexaenoic acid (DHA) over CO‐fed fish, similar to levels in MO‐fed fish.     

Identification and pathogenicity to rainbow trout, Oncorhynchus mykiss (Walbaum), of some aeromonads

Twelve strains of fish pathogenic aeromonads were identified by 16S rRNA sequencing as Aeromonas bestiarum , A. hydrophila , A. hydrophila subsp. dhakensis , A. salmonicida subsp. salmonicida , A. sobria biovar sobria and A. veronii biovar sobria. Following intramuscular injection, A. hydrophila subsp. dhakensis caused dark liquefying, raised furuncle-like lesions in rainbow trout within 48 h. Extracellular products of all cultures contained gelatinase and lecithinase, and most revealed lipase. Congo red absorption and siderophore production was recorded, but not so the suicide phenomenon or slime production. Sodium dodecyl sulphate polyacrylamide gel electrophoresis profile of the outer membrane proteins (OMP) revealed 10–25 bands, of which major bands were seen in the region of 32.5–47.5 and 62–83 kDa. Marked heterogenicity of the OMP and whole cell protein (WCP) profiles within and among the species was observed. Polypeptides of 83–173 kDa were detected in the WCP profile of the cultures, but they were not expressed in OMP fractions.     

Effects of dichlorvos and formalin on fatty acid metabolism of rainbow trout (Oncorhynchus mykiss) skin cells in primary culture

The effects of sub-lethal doses of dichlorvos and formalin, antimicrobial/parasitic agents used in aquaculture, on lipid composition and metabolism of rainbow trout skin cells in primary culture were investigated. [1-¹⁴C]Stearic (18:0), [1-¹⁴C]lin 18:2n-6) and [1-¹⁴C]linolenic (18:3n-3) acids were used as tracers to determine effects on fatty acid incorporation and metabolism. Formalin increased cell numbers and reduced the lipid content of the cells and the incorporation of radioactive fatty acids. The effects of dichlorvos were qualitatively similar but quantitatively less. Formalin induced relatively small but significant changes in lipid class composition including a decreased proportion of phosphatidycholine with increased proportions of sphatidylethanolamine and phosphatidylserine. Dichlorvos had no significant effect on lipid class compositions. The trout primary skin cells expressed substantial 9, 6 and 5 fatty acyl desaturase activities. Although, as expected, the cells were m active towards [1-¹⁴C]18:3n-3, the cells were unusually active towards [1-¹⁴C]18:2n-6. Both dichlorvos and, especially, formalin appeared to significantly inhibit 9 and 6 desaturation. Changes in the distribution of radioactivity between individual spholipid classes was also influenced by formalin and dichlorvos, and this may be related to changes in desaturase activity. This study has shown that topically active agents used in aquaculture, formalin and dichlorvos, had a range of effects on the rainbow trout skin cell cultures that may affect cell proliferation and lipid and fatty acid metabolism. Both agents significantly inhibited desaturation of fatty acids, particularly of 18:2n-6 to 20:4n-6 and, as 20:4n-6 is a major eicosanoid precursor ish and considering the importance of eicosanoids in the biochemistry of skin, it is suggested that these agents may have direct effects on fish skin that could have important consequences for fish health in general.     

A test of a feed budget model for rainbow trout, Oncorhynchus mykiss (Walbaum)

A feed budget model, which enables farmers to adapt a general model to local conditions, was tested on rainbow trout, Oncorhynchus mykiss (Walbaum). The model has two major components; component 1 is used to calculate the energy required for one unit of biomass gain (g or kg) and component 2 estimates the daily growth increment (g or kg). Both components can be adjusted in relation to prevailing environmental conditions and, together, they express the daily energetic requirement of the specific species or strain. Fish fed using the model as a guide had a high rate of growth and good feed conversion. The model accurately predicted growth over the experiment, and is presented as a means of calculating the daily feed budget in fish farming. Growth of fish fed using self-feeders is presented for comparison with the results obtained using the model.     

Arginine effects on biochemical composition of sperm in rainbow trout,Oncorhynchus mykiss

The present study investigated the effect of arginine on seminal plasma composition in rainbow trout. Male rainbow trout broodstocks (2500 ± 200 g) were fed five practical diets (each consisting of three triplicates) supplemented with Arginine at 0.50%, 1.50% and 2.00%. The control group were fed without arginine. Broodstock feeding lasted for 90 days, and then fish semen was sampled. Results indicated no significant differences in LDH, ALP, Fe²⁺ and phosphorous content among the different treatments. The lowest levels of AST and ALT and the highest levels of Ca²⁺ and Mg²⁺ ions were observed in the treatment fed with 1.50% arginine, which showed significant differences from other treatments (P < 0.05). Moreover, the amount of Cl^?, Na⁺ and K⁺ ions was significantly increased in the seminal plasma in fish fed diets containing arginine in comparison with the control. As the amount of arginine was increased, the levels of uric acid became significantly greater in contrast to urea and glucose levels. The highest amounts of cholesterol, fructose and total protein were observed in treatments fed on 2.00%, 0.50% and 1.00% arginine, respectively, showing significant differences from other treatments (P < 0.05). The highest pH value was assayed in the 1.50% arginine treatment. Results indicated that arginine had a potential efficacy on semen quality in rainbow trout broodstocks.     

Effects of formalin treatment on epithelial structure and mucous cell densities in rainbow trout, Oncorhynchus mykiss (Walbaum), skin

Formalin bath treatments of rainbow trout are widely used for control of ectoparasitic infections in fish farms and the effects of this chemical on parasite infections have been studied. In addition, tolerance of the host to various concentrations of formalin are also known. However, effects of formaldehyde on the skin epithelium integrity and mucous cell density still need elucidation. The present study revealed that exposure of rainbow trout fry 2 months post-hatching to various concentrations of formalin affected the mucous cell dynamics of the fish. Limited exposure (50 ppm, 1 h) stimulated mucous cell proliferation as indicated by the slight increase in densities of Alcian blue positive cells in tail fin epidermis. In contrast, high concentrations (200-300 ppm, 1 h) or lengthy exposure (24 h) at lower concentrations caused a decrease in mucous cell density. Scanning electron microscopy revealed effects on the epithelial structure of fish fins. Blebbing of epithelial cell membranes was the first sign of injury. The dilated openings of mucous cells indicated increased release of mucus. Highly irregular organization of the cells followed, with regional differences occurring in different parts of fins. The implications of formalin bathing for susceptibility or resistance of fish to subsequent pathogen exposure are discussed.     

Lipid-stimulated somatostatin secretion in rainbow trout,Oncorhynchus mykiss

Previous work has shown that somatostatins (SS) affect teleost lipid metabolism indirectly by inhibition of insulin (INS) and directly by stimulation of hepatic lipolysis. In the present study, rainbow trout (Oncorhynchus mykiss) were used to characterize further the lipid-SS relationship by evaluating how lipid, contributes to SS secretion bothin vivo andin vitro. In vivo hyperlipidemia was induced for up to 3 h by short-term (2 min) infusion of a triacylglycerol (TG)-rich lipid emulsion (20% Intralipid^®). Plasma total lipid concentration increased 118 and 155% over control levels 1 h and 3 h, respectively, after infusion; much of this increase was due to elevated plasma fatty acids (FA), which increased 39 and 520%, respectively, over the same time-frame. The hyperlipidemic pattern was attended by a significant increase in the plasma concentration of SS. The specific effects of fatty acids were evaluated on isolated Brockmann bodies. Palmitic acid and oleic acid stimulated SS release 378 and 82%, respectively, over baseline levels. These results indicate that lipids, and in particular fatty acids, modulate SS secretion in rainbow trout.     

Use of probiotics to control furunculosis in rainbow trout, Oncorhynchus mykiss (Walbaum)

Aerobic heterotrophic bacteria were isolated from the intestinal contents of Atlantic salmon, Salmo salar , rainbow trout, Oncorhynchus mykiss , and turbot, Scophthalmus maximus , on tryptone soya agar and De Man Rogosa and Sharpe agar, of which 11 of 177 (6% of the total) of the isolates were antagonistic to Aeromonas salmonicida . Four of these cultures, which were identified tentatively as A. hydrophila , Vibrio fluvialis , Carnobacterium sp. and an unidentified Gram-positive coccus, were beneficial to fish when fed singly or as an equi-mixture. Feed supplemented with the putative probiotics indicated survival of the organisms in the gastrointestinal tract for 7 days. Feeding with the probiotics for 7 and 14 days led to better survival following challenge with A. salmonicida . There was no indication of serum or mucus antibodies to A. salmonicida , but there was an increased number of erythrocytes, macrophages, lymphocytes and leucocytes, and enhanced lysozyme activity in the fish.     

Relationships between growth and disease resistance in rainbow trout,Oncorhynchus mykiss (Walbaum)

Rainbow trout from 23 families were evaluated for growth and resistance to the bacterial coldwater disease (BCWD) caused by Flavobacterium psychrophilum and infectious haematopoietic necrosis (IHN) caused by IHN virus. Average family weights were between 161 and 263 g with an average of 225 g at 213 days post‐fertilization with specific growth rates ranging from 2.37 to 2.88. Per cent survival of fish challenged with F. psychrophilum was between 18% and 100%, while for those challenged with IHNV, the range was between 12% and 93%. Significant positive correlations were found for end body weight and resistance to IHN (P < 0.05) and for early body weight and resistance to BCWD (P < 0.1). However, no significant correlations were detected between resistance to both pathogens or disease resistance and overall genetic diversity or diversity within the major histocompatibility locus.     

Effects of ascorbic acid enrichment by immersion of rainbow trout (Oncorhynchus mykiss, Walbaum 1792) eggs and embryos

This study was conducted to examine the effects of different forms and concentrations of ascorbic acid (vitamin C), and different enrichment times (24 and 48 h post ovulation) on egg, embryo and alevin ascorbate concentrations and survival of rainbow trout (enrichment was at the ova stage). In experiments 1 and 2, fertilized eggs were immersed in water containing ascorbate at 0 (control), 100, 1000 mg L^?1 l ‐ascorbic acid (AA) and 2000 mg L^?1 l ‐ascorbyl monophosphate (AP). In experiment 3, 0 (control), 500 and 1000 mg L^?1 AA neutralized (N) with NaOH, 1000 mg L^?1 AA non‐neutralized (NN), 1000 and 2000 mg L^?1 AP immersions were used. The mean total ascorbic acid (TAA) and dehydroascorbic acid (DHA) concentrations were measured before fertilization, at 3 and 24 h after fertilization, at the eyed stage, and in hatched alevins. We observed significant differences in TAA concentration at different immersion levels at 3 and 24 h after fertilization. Survival decreased significantly depending on the level of vitamin C, pH of the solutions and immersion time. We suggest that when broodstock rainbow trout do not have enough vitamin C in their ovaries, immersion of eggs in 1000 mg L^?1 of neutralized AA may be useful.     

The effects of Spirulina platensis meal on proximate composition,fatty acid profile and lipid peroxidation of rainbow trout (Oncorhynchus mykiss) muscle

This study evaluated the effects of diets containing 0, 25, 50, 75 and 100 g kg^?1 Spirulina platensis on proximate composition, fatty acid profile and lipid peroxidation of rainbow trout (Oncorhynchus mykiss). Supplementation of S. platensis did not change moisture and protein contents, but fish fed 50 and 100 g kg^?1 S. platensis had lower muscle lipid content than those fed control diet (P < 0.05). Fish fed 100 g kg^?1 of S. platensis contained lower percentages of saturated and monounsaturated fatty acid and a higher percentage of polyunsaturated fatty acid than those fed control diet (P < 0.05). The n‐3/n‐6 ratio of the fatty acid increased and muscle atherogenic and thrombogenic indices were significantly decreased as the dietary supplement of S. platensis increased. Furthermore, lipid peroxidation of the fillet significantly decreased with increasing dietary S. platensis at 4 °C and at ?20 °C (P < 0.05). The results of this study show that supplementation of S. platensis to the diet improves muscle quality of the rainbow trout.     

Immunization of rainbow trout, Oncorhynchus mykiss (Walbaum), with a low molecular mass fraction isolated from Flavobacterium psychrophilum

Flavobacterium psychrophilum, the causative agent of rainbow trout fry syndrome has become a widespread fish pathogen in freshwater aquaculture worldwide. In this study, a low molecular mass fraction (P25-33), with an approximate weight of 25-33 kDa, was identified among F. psychrophilum strains in an immunoblotting analysis with anti-F. psychrophilum sera. The immunogenic efficacy of the isolated and extracted P25-33 was investigated in two intraperitoneal immunization trials with rainbow trout, Oncorhynchus mykiss (Walbaum). The first trial included immunizations using P25-33 with Freund's complete adjuvant (FCA) and the second trial included immunizations using P25-33, formalin-inactivated whole and sonicated F. psychrophilum cell preparations without FCA. In both trials, antibody titres against F. psychrophilum were analysed with an enzyme-linked immunosorbent assay and the efficacy of the immunizations was determined by a challenge with F. psychrophilum. The P25-33 was shown to give rise to a protective immune response in rainbow trout after immunization with FCA, but not without FCA when a low concentration of P25-33 was used. Instead formalin-inactivated whole and sonicated cells of F. psychrophilum were able to protect the immunized fish more effectively when immunized without FCA. The results suggest that whole or sonicated F. psychrophilum cells could be better candidates for a cost-effective water-based injection vaccine than the immunogenic fraction.     

Digestibility, growth and excretion response of rainbow trout (Oncorhynchus mykiss Walbaum) to feeds of different ingredient particle sizes

The effects of feed ingredient particle size on apparent digestibility, growth and excretion responses of rainbow trout were investigated in pilot production systems. Particle size of the feed ingredients (ground using a hammer mill with 0.6 and 3.0 mm screen sizes) had no significant effect (P > 0.05, d.f. = 1/6) on the apparent digestibility of dry matter, phosphorus and crude protein in cold‐extruded diets. Feed ingredient particle size had an impact on the feed conversion ratio over an 85‐day feeding trial when key ingredients (78.65% of diet) were ground using a pulverizer and a hammer mill with 3.0 mm screen size and heat extruded. No significant differences (P > 0.05, d.f. = 1/12) were observed in the excretion ratios of TAN (total ammonia nitrogen), TKN (total Kjeldahl nitrogen), TP (total phosphorus), BOD₅ (5‐day biochemical oxygen demand) and TSS (total suspended solids) among three feed types, which included a wide ingredient particle size distribution.