Effects of salinity on the growth and lipid composition of Atlantic salmon (Salmo salar) and turbot (Scophthalmus maximus) cells in culture

The direct effects of osmotic pressure (salinity) on growth performance and lipid composition were investigated in fish cells in culture. Cell lines from a relatively stenohaline marine species, turbot (Scophthalmus maximus) (TF) and an anadromous species, Atlantic salmon (AS) were cultured in media supplemented with NaCl to produce osmotic pressures varying from 300 to 500 mOsm kg⁻¹. The growth rates of the two cell lines were affected in a similar manner by the salinity of the media with the rank order for both peak cell numbers and growth rates up to the day of peak cell number being 300 > 350 > 400 > 450 > 500 mOsm kg⁻¹. Cell death occurred in both cell lines in older cultures at all salinities with the greatest loss of viable cells in media of 300 and 350 kg⁻¹. However, there were quantitative and qualitative differences between the cell lines in their lipid metabolism in response to the salinity of the media. The lipid content expressed per cell showed a positive correlation between lipid per cell and salinity in TF cells, but this was less apparent in AS cells. The percentage of total polar lipid classes increased with increasing salinity in TF cells due mainly to graded increases in the percentages of choline phospholipids. In contrast, there were no significant differences in the proportions of polar and neutral lipid classes with salinity in AS cells. The only significant effect of salinity in AS cells was a decreased proportion of dimethylacetals in total lipid at the highest salinity. The same significant effect of salinity on dimethylacetal content of total lipid was observed in TF cells. However, in addition there was a graded decrease in the percentage of 18:2n-9 in TF cell total lipid with increasing salinity. This was accompanied by increased percentages of total n-3 and n-6 PUFA with higher proportions of both groups of PUFA at 450 and 500 compared with 300 mOsm kg⁻¹. The results show that environmental salinity, in the absence of hormonal or other physiological stimuli, has direct effects on the growth and lipid metabolism of fish cells and that these effects differ in cells from different fish species.     

The incorporation and metabolism of polyunsaturated fatty acids in phospholipids of cultured cells from chum salmon (Oncorhynchus keta)

The incorporation and metabolism of (n-3) and (n-6) polyunsaturated fatty acids were studied in a cell line derived from chum salmon heart (CHH-1). Supplementing media with 25 M fatty acid considerably altered the cellular fatty acid composition but did not affect the lipid class composition or cause the appearance of cytoplasmic lipid droplets. CHH-1 cells exhibited considerable -6-desaturase activity but showed no preference between (n-3) and (n-6)PUFA substrates. CHH-1 cells also possess -5-desaturase activity which showed preference towards (n-3)PUFA, but -4-desaturase activity was totally absent. Elongation of 20-carbon PUFA was especially active in CHH-1 cells with 22-carbon PUFA being specifically incorporated into PE and PS lipid classes. The fatty acid composition of PI indicated specific incorporation of 20-carbon PUFA into this lipid class. Supplementation with 22:6(n-3) generated fatty acid compositions more closely resembling those of intact salmonid hearts. Substantial chain shortening of 22:6(n-3) to 20:5(n-3) occurred.Abbreviations BHT butylated hydroxytoluene - BSA bovine serum albumin - CL cardiolipin - FCS fetal calf serum - PA phosphatidic acid - PC phosphatidylcholine - PE phosphatidylethanolamine - PI phosphatidylinositol - PS phosphatidylserine - PUFA polyunsaturated fatty acid - SM sphingomyelin     

Polyunsaturated fatty acid metabolism in a cell culture model of essential fatty acid deficiency in a freshwater fish, carp (Cyprinus carpio)

Proliferation of an essential fatty acid deficient cell line from carp (EPC-EFAD; epithelioma papillosum carp-essential fatty acid deficient) is stimulated by supplementing the cells with C₂₀, but not C₁₈ polyunsaturated fatty acids (PUFA). It is hypothesized that the differential ability of the PUFA to stimulate proliferation of the EPC-EFAD cells may be related to the extent of the cells' ability to desaturate and elongate C₁₈ PUFA. In the present study, the metabolism of ¹⁴C-labeled C₁₈ and C₂₀ PUFA was investigated in EPC-EFAD cells in comparison with normal EPC cells. The incorporation of all the PUFA was significantly greater in EPC-EFAD cells but the rank order, 20:5n-3 > 18:3n-3 = 18:2n-6 >20:4n-6 was the same in both cell lines. The proportion of radioactivity from all labeled PUFA recovered in phosphatidylethanolamine and total polar lipids was significantly lower in EPC-EFAD cells compared to EPC cells, whereas the proportion of radioactivity recovered in all the other phospholipid classes and total neutral lipid was greater in EPC-EFAD cells. Both cell lines desaturated[1-¹⁴C]18:3n-3 and [1-¹⁴C]20:5n-3 to a greater extent than the corresponding (n-6) substrates but the desaturation of all the ¹⁴ C-labeled PUFA was significantly greater in EPC-EFAD cells compared to EPC cells. The results showed that, although essential fatty acid deficiency had several significant effects on PUFA metabolism in EPC cells, the fatty acid desaturation/elongation pathway was not impaired in EPC-EFAD cells and so they can desaturate 18:3n-3 to 20:5n-3 and 22:6n-3, and 18:2n-6 to 20:4n-6. However, 20:4n-3 and 20:3n-6, and not 20:4n-6 and 20:5n-3, were the predominant C₂₀ PUFA produced by the elongation and desaturation of [1-¹⁴C]18:3n-3 and [1-¹⁴C]18:2n-6, respectively. Therefore, the previously reported inability of 18:3n-3 and 18:2n-6, compared to 20:5n-3 and 20:4n-6, to stimulate proliferation of the cells is apparently not due to a general deficiency in the fatty acid desaturation pathway in EPC-EFAD cells but may be related to potential differences in eicosanoid profiles in cells supplemented with C₁₈ PUFA compared to C₂₀ PUFA.     

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.     

The Effect of Culture System on Proximate Composition and Amino and Fatty Acid Profiles of Peled Coregonus peled Fillets

Proximate composition and amino and fatty acid profiles of peled fillets of both sexes reared in a pond-based system (PS) and intensively cultured in a recirculating aquaculture system (RS) were compared. Significantly higher values of dry matter, fat, and caloric value were found in RS fish, irrespective of sex. Fish from the PS group showed a higher percentage of saturated fatty acids and polyunsaturated fatty acids (PUFA) than those of the RS group. Percentages of n-3 PUFA were lower in the RS group compared to the PS group, while percentages of n-6 PUFA were higher in RS, irrespective of sex. The ratio of n-3:n-6 PUFA was significantly higher for the RS group. Fillets of the PS group showed significantly higher EPA and DHA percentages compared to RS fish. Significant differences were found in amino acid profile, with the PS group showing higher content, especially of serine, glutamic acid, methionine, leucine, and lysine.     

Fatty Acid Porfiles of Farm-Raised Catfish Fillet:

Fatty acid profiles of the total lipids and the phospholipid classes of farm-raised channel catfish (Ictalurus punctatus) fillets were analyzed. Monoenes represented 49.22 mol% of the total lipid fatty acids while polyunstaturated fatty acids (PUFA) accounted for 19.37 mol%. The total n-3 PUFA content was low (4.22 mol%). The total saturated fatty acids as 31.44 mol %. Catfish contained phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), lysophosphatidylcholine (LPC), and sphingomyelin (SPH). Analysis for the individual phospholipid class fatty acid profiles indicated that PI had a high concentration of 18:0 (33.20 mol%) and 20:4 n-6 (13.08 mol%). Phosphatidylethanolamine had the highest concentrations of combined eicosapentaenoic acid, and docoshahexaenoic acid, of 17.37 mol%. Higher ratios of n-3/n-6 PUFA were found in the phospholipid classes than the total lipids. LPC and SPH did not contain measurable PUFA of the n-3 series. The level of 18:1 n-9 in PC, PE, and LPC was approximately 31 mol% whereas in PI, PS, and SPH, the level was approximately 16 mol%. The understanding of the basic distribution of fatty acids in the individual phospholipid classes of channel catfish may be an essential first step in explaining the protective role of 18:1 n-9 and n-3 PUFA against thrombotic and cardiovascular disorder in humans.     

Effects of essential fatty acid-deficient diets on growth, mortality, tissue histopathology and fatty acid compositions in juvenile turbot (Scophthalmus maximus)

Three diets in which the lipid component was supplied either as fish oil (FO), linseed oil (LO) or olive oil (OO) were fed to duplicate groups of juvenile turbot (Scophthalmus maximus) of initial weight 1.2 g for a period of up to 12 weeks. The latter two diets resulted in a significant reduction in specific growth rate and an increased mortality compared to the FO (control) fed fish. A liver histopathology was evident in around half of the fish fed the LO and OO diets but was absent in fish fed FO. The lesion showed indications of cellular alterations consisting of foci of densely basophilic cells but without evidence of inflammatory activity. The total lipid fatty acid composition of the carcass from fish fed LO had increased percentages of 18:2n-6 and 18:3n-3, but decreased percentages of all other polyunsaturated fatty acids (PUFA) including the physiologically important 20:4n-6, 20:5n-3 and 22:6n-3, compared to fish fed FO. Almost 2/3 of the total fatty acids in the carcass of OO-fed fish were monounsaturated while the percentages of total saturated fatty acids and all other PUFA, except 18:2n-6, were significantly reduced compared to fish fed FO. Broadly similar effects on total lipid fatty acid composition were observed in liver. In the liver glycerophospholipid classes of fish fed LO, percentages of 18:2n-6, 18:3n-3 and 20:3n-3 were significantly increased whereas all C20 and C22 PUFA, with the exception of 20:5n-3 in PI, were significantly reduced compared to fish fed FO. The liver glycerophospholipids of fish fed OO all showed significantly increased total monounsaturates, 18:2n-6, 20:2n-6, 18:2n-9 and 20:2n-9 as well as reduced percentages of 20:4n-6 and 22:6n-3, compared to fish fed FO. The brain glycerophospholipids showed broadly similar changes in response to dietary treatment although the magnitude of fatty acid alterations was less than those observed in liver. The greater mortalities in the OO-fed fish compared to the LO-fed fish suggests that incorporation of 18:3n-3 into tissue phospholipids can offset losses of long-chain PUFA more effectively than incorporation of 18:1n-9. However, levels of dietary long-chain PUFA must be optimised to allow normal growth and development. We conclude that the very low flux through the fatty acid desaturase/elongase pathways in turbot is not up-regulated by diets deficient in 20:5n-3 and 22:6n-3.     

Fatty acid profiles of two freshwater fish larvae (gudgeon and perch) reared with Brachionus calyciflorus Pallas (rotifer) and/or dry diet

The efficiency of the rotifer Brachionus calyciflorus Pallas as a nutritional source for rearing larvae was studied in a coldwater cyprinid, the gudgeon Gobio gobio (L.), and in a percid, the perch Perca fluviatilis L., through their composition in fatty acids. Rotifer intake affected the fatty acid profiles of the larvae significantly, with an especially remarkable presence of the linoleic family. In gudgeon fed with rotifers, the polyunsaturated fatty acids (PUFA) reached 10.98% of the dry weight of the sample. This rate was highly influenced by the presence of the acids C18:2n-6 and C22:6n-3 which represented 66% of the total PUFA. In perch fed exclusively with rotifers, the PUFA represented 7.27% of the dry weight. In both cases, the ratio n-3/n–6 decreased by 75% and 73% after 10 days of feeding with B. calyciflorus. This variation was probably due to the exogenous supply in acids of the linoleic family through the rotifers and to the fact that these two species of fish seem to favour the mobilization of the n-3 PUFA such as C22:6n-3 for growth and survival. Moreover, with the utilization of rotifers. the reactions of elongation and desaturation from the C18:2n-6 and C18:1n-9 seemed to be much more important in the larvae. Lastly, the transition from a diet based on rotifers to one made up exclusively of frozen Artemia nauplii led to a significant reduction of fatty acids in fish. It reached 60.2% and 26.5% of the total fatty acids in the gudgeon and perch, respectively, and was observed especially at the level of the PUFA. On the other hand, a slight increase of the ratio n-3/n-6 was pointed out in the perch fed a mixed diet (co-feeding with rotifers and dry food), a phenomenon probably due to the reduction of C18:2n-6 in the larvae.     

The desaturation and elongation of 14C-labelled polyunsaturated fatty acids by pike (Esox lucius L.) in vivo

To examine the ability of pike (Esox lucius L.) to modify exogenous PUFA by desaturation and elongation, ¹⁴C-labelled 18:2(n-6), 18:3(n-3), 20:4(n-6) and 20:5(n-3) were injected intraperitoneally and the distribution of radioactivity in tissue lipid classes and liver PUFA measured. In all tissues examined, radioactivity from all ¹⁴C-PUFA was recovered in many classes of acyl lipids and the level of recovery generally reflected the relative abundance of the lipid classes. Triacylglycerols, CGP and EGP usually contained high levels of all incorporated ¹⁴C-PUFA. PI contained higher levels of radioactivity from ¹⁴C-20:4(n-6) than from other injected substrates. In liver lipid, the 6 desaturation products of ¹⁴C-18:2(n-6) and ¹⁴C-18:3(n-3) contained no measurable radioactivity although the elongation products of the 6 desaturation products were labelled, as were the direct elongation products of these injected substrates. No radioactivity from ¹⁴C-18:2(n-6) or ¹⁴C-18:3(n-3) was detected in C₂₀ or C₂₂ products of 5 and 4 desaturation. Almost all radioactivity from injected ¹⁴C-20:4(n-6) was recovered in this PUFA. Of the total radioactivity from ¹⁴C-20:5(n-3) incorporated into liver lipid, 7% was present as 24:5 and 16.4% was recovered in hexaenoic fatty acids. In liver, 24:5(n-3) and 24:6(n-3) each accounted for 1% of the mass of total fatty acids and were located almost exclusively in triacylglycerols. The presence of radioactivity in these C₂₄ PUFA suggests that in pike the synthesis of 22:6(n-3) from 20:5(n-3) may proceed without 4 desaturase via the pathway which involves chain shortening of 24:6(n-3). It is concluded that under the circumstances employed in this study pike, do not exhibit 5 desaturase activity and are unable to synthesize 20:4(n-6) and 20:5(n-3) from 18:2(n-6) and 18:3(n-3), respectively. This suggests that pike may require 20:4(n-6) and 20:5(n-3) preformed in the diet.Abbreviations CGP choline glycerophospholipids - CL cardiolipin - EGP ethanolamine glycerophospholipids - PG phosphatidylglycerol - PI phosphatidylinositol - PS phosphatidylserine - PUFA polyunsaturated fatty acids - SM sphingomyelin - TLC thin-layer chromatography     

Incorporation and metabolism of (n-3) and (n-6) polyunsaturated fatty acids in phospholipid classes in cultured turbot (Scophthalmus maximus) cells

The incorporation and metabolism of various (n-3) and (n-6) polyunsaturated fatty acids (PUFA) supplemented to the culture medium was investigated in a turbot cell line (TF). The distribution, and the occurrence and extent of further metabolism of incorporated PUFA via desaturation/elongation mechanisms in specific phospholipid classes was determined from the different fatty acid compositions. The cells contained Δ6 and Δ4 desaturase activities but were generally deficient in C18–20 elongase activity. Δ5 Desaturase activity was generally masked by this deficiency but was present. The compositional data indicated that there was a high degree of specificity between individual phospholipid classes and particular fatty acids probably driven by the specificities of the acylating enzymes. The highest percentages of the supplemented acids were generally observed in the phosphatidic acid/cardiolipin fraction (PA/CL), suggesting a role for PA in the incorporation of the supplemented acids into the phospholipid pool. PI had a characteristic composition consistent with a putative role as a pool of precursor fatty acid for eicosanoid synthesis. Mechanisms were evident for generating and/or maintaining this composition.     

The conversion of linoleic acid and linolenic acid to longer chain polyunsaturated fatty acids by Tilapia (Oreochromis) nilotica in vivo

Tilapia (Oreochromis) nilotica were fed either a commercial diet containing 2.2% (n-3) and 0.5% (n-6) polyunsaturated fatty acids (PUFA), or a diet containing 1.0% methyl linoleate as the only PUFA. The fatty acid composition of tissue lipids generally reflected that of the diet. Fish from both dietary groups were injected intraperitoneally with ¹⁴C-labelled linoleic acid, 18:2 (n-6), or linolenic acid, 18:3 (n-3), and the distribution of radioactivity in tissue lipids examined. The conversion of both 18:2 (n-6) and 18:3 (n-3) to longer chain PUFA was lower in fish fed the commercial diet than in those fed the diet containing only 18:2 (n-6). Half of the radioactivity from both substrates recovered in liver polar lipids was present in C20 and C22 PUFA with fish maintained on the experimental diet. It is concluded that T. nilotica is capable of elongating and desaturating both 18:2 (n-6) and 18:3 (n-3), but that this conversion is suppressed by dietary longer chain PUFA. NERC Unit of Aquatic Biochemistry     

Essential fatty acid deficiency in freshwater fish: the effects of linoleic, α-linolenic, γ-linolenic and stearidonic acids on the metabolism of [1-14C]18:3n-3 in a carp cell culture model

The desaturation of [1-¹⁴C]18:3n-3 to 20:5n-3 and 22:6n-3 is enhanced in an essential fatty acid deficient cell line (EPC-EFAD) in comparison with the parent cell line (EPC) from carp. In the present study, the effects of competing, unlabeled C₁₈ polyunsaturated fatty acids (PUFA), linoleic (18:2n-6), -linolenic (18:3n-3), -linolenic (18:3n-6) and stearidonic (18:4n-3) acids, on the metabolism of [1-¹⁴C]18:3n-3 were investigated in EPC-EFAD cells in comparison with EPC cells. The incorporation of [1-¹⁴C]18:3n-3 in both cell lines was significantly reduced by competing C₁₈ PUFA, with the rank order being 18:4n-3>18:3n-3 = 18:2n-6>18:3n-6. In the absence of competing PUFA, radioactivity from [1-¹⁴C]18:3n-3 in EPC cells was predominantly recovered in phosphatidylethanolamine followed by phosphatidylcholine. This pattern was unaffected by competing n-6PUFA, but n-3PUFA reversed this pattern as did essential fatty acid deficiency in the presence of all competing PUFA. The altered lipid class distribution was most pronounced in cells supplemented with 18:4n-3. Competing C₁₈ PUFA significantly decreased the proportions of radioactivity recovered in 22:6n-3, pentaene and tetraene products, with the proportions of radioactivity recovered in 18:3n-3 and 20:3n-3 increased, in both cell lines. However, the inhibitory effect of competing C₁₈ PUFA on the desaturation of [1-¹⁴C]18:3n-3 was significantly greater in EPC-EFAD cells. The magnitude of the inhibitory effects of C₁₈ PUFA on [1-¹⁴C]18:3n-3 desaturation was dependent upon the specific fatty acid with the rank order being 18:4n-3>18:3n-3>18:2n-6, with 18:3n-6 having little inhibitory effect on the metabolism of [1-¹⁴C]18:3n-3 in EPC cells. The differential effects of the C₁₈ PUFA on [1-¹⁴C]18:3n-3 metabolism were consistent with mass competition in combination with increased desaturation activity in EPC-EFAD cells and the known substrate fatty acid specificities of desaturase enzymes. However, the mechanism underpinning the greater efficacy with which the unlabeled C₁₈PUFA competed with [1-¹⁴C]18:3n-3 in the desaturation pathway in EPC-EFAD cells was unclear.     

Essential fatty acids in Atlantic salmon: time course of changes in fatty acid composition of liver, blood and carcass induced by a diet deficient in n-3 and n-6 fatty acids

Atlantic salmon fry (4 g) were fed a semi-synthetic diet devoid of n-3 and n-6 polyunsaturated fatty acids (PUFA) for 4 months. External signs of nutritional imbalance during the period were slow growth rate and increased mortality. Some symptoms of essential fatty acid deficiency appeared in the fatty acid composition of the blood and liver during the fourth month. At that time, the percentages of n-3 and n-6 PUFA of triacylglycerols (TAG) were nearly exhausted, and the percentages of 22:6 n-3 and of 20:5 n-3 in phospholipids (PL) showed a marked decrease. This decrease in the PUFA level of the PL was paralleled by the appearance of 20:3 n-9, whereas in the TAG an increase in the percentage of 18:1 n-9 was observed. After 4 months the monounsaturated fatty acid 18:1 n-9 constituted nearly 50% of the fatty acids in the TAG fraction of the liver. The time course of the changes in fatty acid composition of liver and blood lipids was quite similar, whereas the carcass lipid composition appeared to respond slowly to a diet devoid of essential fatty acids.     

Habitat related variations in fatty acids of catadromous Galaxias maculatus

The fatty acid profile of the galaxiid fish Galaxias maculatus (Salmoniformes) collected from an estuarine habitat (a river mouth), a freshwater creek and two land-locked freshwater lakes in Victoria, Australia, were investigated with a view to evaluating habitat influences on the fatty acid profile. Fish from the freshwater creek were unpigmented, fresh-run juveniles (referred to as whitebait), the others adult fish. The fatty acids that predominated in adult G. maculatus muscle were 16:0 and 22:6(n-3), and 16:0 and 18:1(n-9), respectively. In whitebait 22:6(n-3), 20:5(n-3) and 16:0 were predominant. Hopkins estuary fish had the highest amount of 18:2(n-6) but the lowest 18:3(n-3). In all galaxiid populations, polyunsaturated fatty acids (PUFA) were the main group of fatty acids in total lipid in muscle, and accounted for more than 40 %, followed by saturates and monoenes. In landlocked populations, the amount of monoenes was lower than in other galaxiid populations. G. maculatus whitebait had significantly lower amount of saturates and the highest amount of n-3 fatty acids. The ratio of n-3 to n-6 PUFA ranged from 4.6 (Hopkins estuary) to 7.6 (whitebait), and the former differed significantly from all the other populations. The main fatty acids predominating in the liver of galaxiids from landlocked (Lake Bullen Merri) and estuarine populations were 16:0, 22:6(n-3) and 18:1 (n-9), and generally reflected the corresponding pattern in muscle. A principal component analysis of the fatty acid composition of muscle samples confirmed and highlighted the distinct fatty acid profile of the populations investigated, when three groupings could be discerned. The study suggests that in addition to the diet, other habitat related factors may influence the fatty acid profile in catadromous G. maculatus.     

Chemical changes in fed and starved larval trout cod, Maccullochella macquarensis during early development

The changes in proximate composition, amino acid (total and free) and fatty acid content of artificially propagated trout cod, Maccullochella macquariensis larvae from five mothers hatched, weaned and reared separately, each in two groups, one fed with Artemia naupli and the other starved, for 15 days (after yolk resorption), are presented. There was no significant change in the proximate composition of fed larvae with devlopment, but in starved larvae the protein (linearly) and lipid (curvi-linearly) content decreased significantly as starvation progressed. The essential amino acids (EAA) and non- essential amino acids (NEAA) found in highest amounts in trout cod larvae were lysine, leucine, threonine and arginine, and alanine, serine and glutamic acid, respectively. In fed larvae the total amino acid (TAA), TEAA and TNEAA content did not vary significantly as development progressed. In starved larvae the TAA, EAA and NEAA content, as well as all the individual amino acids decreased significantly (P<0.05) from the levels in day of hatch and/or yolk-sac resorbed larvae. The greatest decrease occurred in the TEAA content (7.38±0.76 at day of hatch to 1.96±0.09 15 day starved in moles larva^–1; approximately a 74% decrease), whereas the decrease in TNEAA was about 38%. Unlike in the case of TAA distinct changes in the free amino acid (FAA) pool were discernible, from day of hatch and onwards, in both fed and starved trout cod larvae. In both groups of larvae the most noticeable being the decrease of % FEAA in TFAA, but not the % FAA in TAA. Four fatty acids together, accounted for more than 50% of the total in each of the major fatty acid categories in all larvae sampled; 16: 0, 18:1n-9, 22: 6n-3 and 20: 4n-6, amongst saturates, monoenes, n-3 PUFA and n-6 PUFA, respectively. Twelve fatty acids either decreased (14: 0, 16: 1n-7, 20: 1n-9, 20: 4n-6, 20: 5n-3, 22: 5n-3 and 22: 6n-3) or increased (18: 2n-6, 18: 3n-3, 18: 3n-6, 18: 4n-3 and 20: 3n-3) in quantity, after 15 days of feeding, from the base level in day of hatch and/ or yolk- sac resorbed larvae. The greatest increase occurred in 18: 3n-3 from 6.4±0.1 to 106.2±13.1 g mg lipid^–1 larva^–1, and the greatest decrease occurred in 22: 6n-3 (181.2±12.4 to 81.4±6.2 g mg lipid^–1 larva^–1). In starved larvae, at the end of 15 days, all the fatty acids, except 18: 0, 20: 3n-3 and 20: 4n-6, decreased significantly (P<0.05) from the levels in day of hatch and/or yolk- sac resorbed larvae.     

Muscle fatty acid composition and oxidative stress indices of Arctic charr, Salvelinus alpinus (L.), in relation to dietary polyunsaturated fatty acid levels and temperature

The influence of feeding high levels of polyunsaturated fatty acids (PUFA) on muscle fatty acid composition and indices of oxidative damage was examined in Arctic charr, Salvelinus alpinus (L.). All diets contained 100 g kg^?1 lipid of dry weight. Two diets contained marine fish oils giving a PUFA level of 250 g kg^?1 and 500 g kg^?1 of lipid. The remaining two diets contained vegetable oils high in either 18:2n-6 or 18:3n-3, giving a PUFA level of more than 500 g kg^?1 of dietary lipid. The charr were maintained at 8°C until their weight doubled, and were then transferred to 0.8°C for 30 days. Growth was similar in all groups. The fatty acid compositions of muscle were influenced by dietary PUFA but were less diverse than those of the diets. The overall pattern of fatty acid compositions indicated preferential desaturation and elongation of n-3 PUFA coupled with selective oxidation of 18:2n-6. Total n-3 PUFA content in TAG was always lowered compared with the diet, suggesting a specific mechanism for the removal of these fatty acids. Subjecting the fish to low temperature increased PUFA content in muscle of charr fed the 250 g kg^?1 marine n-3 PUFA diet, but had no effect on the other treatments. For fish at 8°C, no significant differences were found between groups in terms of haematocrit, plasma alanine aminotransferase (ALAT), and plasma and muscle thiobarbituric acid reactive substances (TBARS), although there was a tendency towards increased levels of TBARS in the group receiving 500 g kg^?1 marine n-3 PUFA of lipid. Subjecting the muscle to forced oxidative conditions resulted in increases in TBARS in all groups, particularly those fed 500 g kg^?1 marine n-3 PUFA. Lowering the environmental temperature corresponded with a further increase in the plasma ALAT and muscle TBARS in this group. It is concluded that feeding diets containing high levels of long-chain n-3 PUFA may be detrimental to the fish's health and flesh quality, particularly at low environmental temperatures.     

Metabolism of 18:4n-3 (stearidonic acid) and 20:4n-3 in salmonid cells in culture and inhibition of the production of prostaglandin F2α (PGF2α) from 20:4n-6 (arachidonic acid)

Arachidonic acid (AA; 20:4n-6) is the precursor of a range of highly biologically active derivatives, collectively termed eicosanoids, including prostaglandins, thromboxanes, leukotrienes and lipoxins, that act as autocrine hormones regulating many physiological processes including haemostasis, reproduction, immune and inflammatory responses. Eicosapentaenoic (EPA; 20:5n-3) and dihomo-γ-linolenic (20:3n-6) acids modulate eicosanoid metabolism by both inhibiting the conversion of AA to eicosanoids whilst simultaneously being converted to eicosanoids with different, often attenuated, properties compared to their AA homologues. Eicosatetraenoic acid (20:4n-3) is a naturally occurring C₂₀ polyunsaturated fatty acid (PUFA), present in fish oil at levels of around 1–2%, that has been suggested to be the active metabolite responsible for the anti-inflammatory effects of plant oils containing stearidonic acid (18:4n-3). However, the biochemical properties of 20:4n-3 in terms of cellular biology have rarely been investigated, partly due to difficulties in obtaining the fatty acid in high purity. In this paper, we describe methods for the medium scale laboratory preparation of high purity 20:4n-3, and investigate its metabolism in fish cell culture systems which normally contain significant amounts of n-3 PUFA. Thus the incorporation and metabolism of 18:4n-3 and 20:4n-3, and their distribution in phospholipid classes was studied in an established cell line from Atlantic salmon (Salmo salar) (AS), and the effects of 20:4n-3 on eicosanoid production studied in freshly isolated macrophages from rainbow trout (Oncorhynchus mykiss). Both 18:4n-3 and 20:4n-3 were preferentially esterified into phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine in contrast with the accumulation of AA in phosphatidylinositol. Incorporated 18:4n-3 was readily converted to 20:4n-3, and both fatty acids were further desaturated and elongated to EPA and 22:5n-3 but not 22:6n-3. Supplementation with 20:4n-3 decreased the conversion of AA into prostaglandins, as demonstrated by the decreased levels of PGF_2α produced in trout macrophages supplemented with 20:4n-3 and AA compared to cells supplemented with AA alone. In addition, 20:4n-3 was converted into eicosanoids in fish cells as indicated by the presence of Δ^17,18 12-HETE, Δ^17,18 PGE1 and Δ^17,18 PGF_1α in extracts from rainbow trout macrophages incubated with 20:4n-3.     

Nutritional Quality and Safety of the Important Commercial Cuttlefish Sepia lycidas

Proximate composition, amino acid, fatty acid composition, and contaminants (As, Cd, Hg, and Pb) of the kisslip cuttlefish Sepia lycidas were determined and compared with the recommendations and maximum allowed levels. The ratio of essential amino acids (EAA) to nonessential amino acids (NEAA) was 0.82. All of the amino acid scores were > 100, except for valine (93), and the highest essential amino acid score was obtained for tryptophan (327). The fatty acids profile was dominated by polyunsaturated fatty acids (PUFA; 45.01% of total fatty acids), and docosahexaenoic acid (DHA; 24.54% of total fatty acids) was the dominant fatty acid. S. lycidas was rich in n-3 PUFA, and the ratio of n-3/n-6 PUFA was 2.48. Regarding contaminants, As, Cd, and Pb concentrations in S. lycidas were 2.2, 0.28, and 0.02 mg kg^?1, respectively. Moreover, Hg values were below the detection limit (0.01 mg kg^?1).     

Effect of a dietary phospholipid supplementation on growth and fatty acid composition of European sea bass (Dicentrarchus labrax L.) and turbot (Scophthalmus maximus L.) juveniles from weaning onwards

Two 40-day feeding trials using extruded diets were conducted to assess the effect of a dietary phospholipid (PL) supplementation on growth, survival and fatty acid composition of European sea bass (Dicentrarchus labrax) and turbot (Scophthalmus maximus) from weaning onwards. Two dietary treatments (FO and PL) were tested; both had an identical extruded basis (92.5% total diet weight) coated with a different lipid fraction (7.5% total diet weight). Diet PL contained 2% egg yolk PL (69% pure). In diet FO the PL was replaced by hydrogenated coconut oil. The isolipidic diets contained an equal amount of fish oil ethyl esters providing 1.6% (% diet dry weight) of n-3 highly unsaturated fatty acids (HUFA). A diet water stability test showed no effect of the PL supplementation on the leaching of the dietary fatty acids. In both fish species weight, but not survival, significantly increased as a result of PL supplementation. Weaning onto the experimental diets resulted in similar changes in the relative percent levels of fatty acids in both species. In general, the percentage of saturated fatty acids levelled off after a rapid increase, while monoenes increased after an initial decrease. Total n-3 polyunsaturated fatty acids (PUFA) decreased and total n-6 PUFA remained almost constant. The major effect of the dietary PL on fish fatty acid composition was a 50% increase in n-6 and n-3 HUFAs compared to the PL-free FO diet. The rise in n-6 HUFA may have reflected the higher moiety in the dietary PL. On the other hand this was not the case for the n-3 HUFA since they represented only low levels in the PL fraction (0.1%) compared to that provided by the ethyl esters (1.6%) suggesting a more efficient incorporation of the PL n-3 HUFA than of the ethyl ester n-3 HUFA. A second hypothesis is that the dietary PL may have favored the incorporation of the dietary ethyl ester n-3 HUFA.     

Dynamics of total lipids and fatty acids during embryogenesis and larval development of Eurasian perch (Perca fluviatilis)

Total lipid and fatty acid compositions were determined during embryogenesis and larval development in Eurasian perch (Perca fluviatilis). During embryonic development, perch did not catabolize lipids and fatty acids as an energy source. However, during larval development, there was an exponential relationship between the decrease in total lipids and the duration of starving (r ²=0.9957) and feeding (r ²=1). The duration of the starving period (10 days post hatching) was shorter than the feeding period (35 days post hatching). In both starved and fed larvae, there is an apparent preference in utilization of polyunsaturated fatty acids followed by monounsaturated fatty acids. Saturated fatty acids were utilized by neither fed perch larvae nor by starved perch larvae. In starved larvae, palmitoleic 16:1(n-7) and oleic 18:1(n-9) acids were the preferentially monounsaturated fatty acids catabolized and their contribution as energy source from total fatty acids catabolized over the first week was 37.6%. In fed larvae, these 2 nutrients were also the most monounsaturated fatty acids utilized as energy source and possibly also as precursors for others monounsaturated fatty acids biosynthesis. During the same period and among (n-6) polyunsaturated fatty acids, starved perch utilized less linoleic 18:2(n-6) and arachidonic 20:4(n-6) acids than fed larvae despite the fact that the starved perch were in more unfavorable nutritional conditions. In the case of (n-3) fatty acids, starved larvae utilized more linolenic acid 18:3(n-3) and less eicosapentaenoic 20:5(n-3) acid and docosahexaenoic 22:6(n-3) acid than fed perch. Starved larvae probably spared 20:5(n-3) and 22:6(n-3) for physiological functions.