Gabriel Mourente

Increased activities of hepatic antioxidant defence enzymes in juvenile gilthead sea bream (Sparus aurata L.) fed dietary oxidised oil: attenuation by dietary vitamin E

Abstract Previously, we had shown that altering the highly unsaturated fatty acid (HUFA)/vitamin E ratios in gilthead sea bream livers significantly affected their peroxidation status, with fish fed a diet rich in HUFA and low in vitamin E showing significantly higher values of lipid peroxidation products, without, however, significant effects on liver antioxidant defence enzyme activities. The aim of the present trial was to further characterise the biochemical indicators of peroxidative stress in juvenile gilthead sea bream. A high pro-oxidative stress was induced by feeding diets containing around 7% of the dry weight as n −3 HUFA. The potential peroxidative stress was increased by oxidising the oil, increasing the peroxide value of the oil some 10-fold. These oils were fed without or with supplemental vitamin E (α-tocopheryl acetate at 200 mg kg −1 dry diet) giving four diets in total. Fish were sampled after 30 and 60 days of feeding the experimental diets. None of the diets had any serious deleterious effects on growth and mortality of the fish during the trial. Similarly, there were few significant effects due to dietary oxidised oil or supplementary vitamin E on liver lipid and fatty acid profiles and, in particular, the proportions of HUFA were not decreased by dietary oxidised oil. The vitamin E content of the liver reflected the vitamin E content of the diets but was also affected by dietary oxidised oil being reduced by oxidised oil in fish fed diets without supplemental vitamin E but, unexpectedly, increased by oxidised oil in fish fed diets supplemented with vitamin E. Liver thiobarbituric acid reactive substances (TBARS) levels were significantly lower in fish fed diets supplemented with vitamin E whereas dietary oxidised oil had no major effect on lipid peroxidation products. Catalase (CAT) and superoxide dismutase (SOD) activities were both increased in fish fed dietary oxidised oil and reduced by supplementary vitamin E after 30 days feeding. In contrast, glutathione peroxidase (GPX) was less affected by the diets, and the activities of glutathione-S-transferase (GST) and glutathione reductase (GR) were only reduced by dietary vitamin E after 60 days of feeding. However, all the enzyme activities were significantly affected by the duration of feeding, but the number of interactions between the three factors (time, oil and vitamin E) showed that the relationships were complicated. In conclusion, the present study showed that feeding diets containing oxidised oil significantly affected the activities of liver antioxidant defence enzymes and that dietary vitamin E partially abrogated these effects. Growth and survival of the fish were relatively unaffected suggesting that the responses in gilthead sea bream offered effective protection. However, the duration of feeding the diets of high pro-oxidative stress was observed to have a hitherto unknown effect, possibly the result of an adaptive process, but which requires further investigation.

Effects of dietary docosahexaenoic acid (DHA; 22:6n-3) on lipid and fatty acid compositions and growth in gilthead sea bream (Sparus aurata L.) larvae during first feeding

Abstract The effects of dietary DHA levels on lipid class compositions, total lipid and phosphoglyceride fatty acid compositions and growth were investigated in gilthead sea bream, Sparus aurata , larvae during the first 2 weeks after hatching. Different dietary levels of DHA were supplied to the larvae in the rotifer, Brachionus plicatilis , previously enriched with (a) Nannochloropsis gaditana , (b) a mixture of N. gaditana, Isochrysis galbana and Rhodomonas sp. or, (c) Protein Selco (Artemia Systems). The results showed that growth performance of gilthead sea bream larvae at first feeding was influenced by both total n -3HUFA and DHA contents of the diet, the best growth rate being achieved with a high n -3HUFA content and a high DHA:EPA ratio. Lipid contents, nutritional status, and total lipid and phophoglyceride DHA levels of the larvae were directly correlated with dietary DHA levels. The fatty acid compositional data suggested that dietary EPA could not contribute significantly to tissue DHA levels in larval gilthead sea bream, presumably due to low Δ 4 desaturase activity.

Comparative study of antioxidant defence mechanisms in marine fish fed variable levels of oxidised oil and vitamin E

The aim of the study was to compare theantioxidant systems in juvenile marine fish ofcommercial importance to European aquaculture,namely turbot (Scophthalmus maximus),halibut (Hippoglossus hippoglossus) andgilthead sea bream (Sparus aurata). Thepresent dietary trial was specifically designedto investigate the antioxidant effects ofvitamin E under moderate oxidising conditions,including high dietary levels of highlyunsaturated fatty acids and the feeding ofoxidised oils. The objective was to induce astressful pro-oxidant status to enablecharacterisation of the biochemical responsesto peroxidative stress without causingunnecessary suffering to the experimentalanimals or high mortalities during the trials. Both sea bream and turbot showed excellentgrowth, whereas growth was poorer in halibut.Dietary oxidised oil significantly reducedgrowth in turbot and especially in halibut, butnot in sea bream. Vitamin E improved growth insea bream fed oxidised oil but not in turbot orhalibut. However, vitamin E supplementationappeared to improve survival in all threespecies. In sea bream and turbot, liverantioxidant defence enzyme activities weregenerally increased by feeding peroxidised oiland reduced by vitamin E. Conversely, inhalibut, the liver antioxidant defence enzymeactivities were not increased by feedingperoxidised oil and only superoxide dismutasewas reduced by feeding vitamin E. Consistentwith these data, feeding oxidised oil increasedlipid peroxidation products in halibut, butgenerally not in sea bream or turbot.Furthermore, lipid peroxidation products weregenerally reduced by dietary vitamin E in bothsea bream and turbot, but not in halibut. Therefore, halibut liver antioxidant defenceenzymes did not respond to dietary oxidised oilor vitamin E as occurred in turbot and,especially sea bream. This resulted inincreased levels of lipid peroxides in halibutcompared to turbot and sea bream in fish givendietary oxidised oil. In addition, supplementalvitamin E did not reduce lipid peroxides inhalibut as it did in turbot and sea bream. Theincreased peroxidation stress in halibut mayaccount for their poorer growth and survival incomparison to turbot and especially sea bream.Halibut were reared at a lower temperature,although relatively high for halibut, thaneither turbot or sea bream but they were alsoslightly younger/smaller fish and possibly,therefore, more developmentally immature, andeither or all of these factors may be importantin the lack of response of the liver enzymes inhalibut.

Biochemical composition and fatty acid content of fertilized eggs, yolk sac stage larvae and first-feeding larvae of the Senegal sole (Solea senegalensis Kaup)

Abstract Changes in biochemical composition and fatty acid content were investigated during the early development of the Senegal sole ( Solea senegalensis Kaup). The pattern of lipid utilization in this rapidly developing marine flatfish species favored neutral lipids, particularly triacylglycerol and sterol ester fractions. Fertilized eggs and yolk sac larvae were richer in neutral lipids, which decreased during development. In contrast, a significant increase occurred to proportions of phospholipids, mainly due to significant increases in minor classes such as phosphatidylserine, phosphatidylinositol and phosphatidic acid/cardiolipin, whereas major phospholipid classes such as phosphatidylcholine and phosphatidylethanolamine remained constant during development. Saturated and monounsaturated fatty acids such as 16:0, 16:1 n −7, 18:1 n −9 and 18:1 n −7 were utilized to a greater extent than polyunsaturated fatty acids as energy substrates. A requirement for long-chain polyunsaturated fatty acids such as eicosapentaenoic acid (20:5 n −3) and docosahexaenoic acid (22:6 n −3) is likely since no evidence of bioconversion from their precursors was found. A requirement for arachidonic acid (20:4 n −6) is also suggested as it is specifically retained throughout development.

Relationships between antioxidants, antioxidant enzyme activities and lipid peroxidation products during early development in Dentex dentex eggs and larvae

Abstract The overall aim is the characterization of the antioxidant systems in marine fish during early development in order to enhance growth and quality of early life stages by avoiding oxidation problems that may cause pathologies and disease. The present study determined the activities of the antioxidant system enzymes and levels of vitamin E, lipid peroxidation products and fatty acids during early development without exogenous feeding in the marine fish, Dentex dentex. Samples of fertilized eggs just prior to hatch, newly hatched larvae and unfed larvae up to day 9 after hatch were analysed on a daily basis. The larvae did not grow during the experiment and by day 9 the surviving larvae weighed only a third of the newly hatched larvae. Lipid was a major energy source during this period of starvation as evidenced by greatly decreased amounts of total lipid due to a preferential utilisation of neutral lipids. All groups of fatty acids (saturated, monounsaturated and polyunsaturated) were consumed throughout the starvation period. Vitamin E levels were relatively constant until significantly declining by days 8 and 9. Similarly, levels of lipid-soluble fluorescent products (LSFP) were relatively constant until day 6 and then significantly decreased from days 7 to 9. In contrast, the level of malondialdehyde (MDA) was very high in eggs but was 3-fold lower in newly hatched larvae and then decreased over the whole time-course and was also significantly lower on days 8 and 9. The activities of enzymes of the antioxidation system were expressed in both eggs and early larvae and varied during the developmental period with levels of catalase and glutathione peroxidase (GPX) being initially low in eggs whereas glutathione-S-transferase (GST) and superoxide dismutase (SOD) were higher in eggs than in larvae. Overall, the activities of catalase and GPX increased, whereas GST and total SOD activities decreased, during the early stages of development during a period where there was no exogenous dietary input into the larvae. Glutathione reductase activity was very low and showed an increasing trend initially until days 2–3 before declining and varying over the remaining period. In conclusion, we determined the prooxidant (polyunsaturated fatty acid) status, antioxidant status (vitamin E), peroxidation status (MDA and LSFP) and the activities of the antioxidant enzyme systems in a single integrated study in unfed marine fish larvae during early development.

Changes in the content of total lipid, lipid classes and their fatty acids of developing eggs and unfed larvae of the Senegal sole,Solea senegalensis Kaup

Total lipids, lipid classes and their associated fatty acids were quantified in developing eggs, yolk-sac larvae and starving larvae (from day 1 to day 5 after hatching) of the Senegal sole,Solea senegalensis Kaup. Larvae during early development and starvation consumed about 0.6% of its dry weight per day, mainly due to lipid catabolism. There was a net consumption of approximately 1.7% total lipid per day, and a net energy utilization of 1.3 kcal g−1 dry weight biomass day−1, mostly derived from lipid depletion. The overall decrease of total neutral lipids (mainly triacylglycerols and sterol esters) was 3.4 faster than that of total polar lipids (primarily phosphatidylcholine), with rates of 29.2 and 8.7 μg mg−1 dry weight biomass day−1, respectively. There was a concomitant increase in PE, PS and phosphatidic acid during the period under study. Total saturated and total monounsaturated fatty acids were catabolized (primarily 16∶0 and 16∶1 (n-7)) as energy substrates at rates of 7.4 and 10.9 μg mg−1 total lipid day−1, whereas total PUFAs were conserved. DHA was specifically retained in PE, whereas EPA and DHA were catabolized in PC and triacylglycerol. Total DMA and AA contents in total lipid increased during early development and starvation. The data denote a pattern of lipid metabolism during early development of Senegal sole similar to that of other marine larval fish, with eggs containing high amounts of total lipids (presence of oil globule/s), from temperate waters and with short developmental periods; the pattern contrasts with fish larvae from eggs of cold water fish species that contain low levels of total lipids (lack of oil globule/s) and have long developmental periods.

Effects of weaning onto a pelleted diet on docosahexaenoic acid (22: 6 n-3)levels in brain of developing turbot (Scophthalmus maximus L)

The brain lipid and fatty acid compositions of turbot, Scophthalmus maximus (L.), were determined in unweaned fish and in fish from the same batch that had been weaned 1 week earlier. Fish were maintained on the same dietary regime until the time of weaning. Immediately prior to weaning fish were fed enriched Artemia. At weaning, one group of fish was fed a dry pelleted food whereas the other group remained on enriched Artemia. The dry pelleted diet contained 2-fold more eicosapentaenoic acid (EPA, 20:5 n-3) and 13-fold more docosahexaenoic acid (DHA, 22:6 n-3) per mg dry weight than the Artemia. In weaned turbot, there were significant increases in brain dry weight (21.6% greater than in unweaned fish,P < 0.05) and in the percentage of total polar classes in total lipid, due to a significant increase in the percentage of phosphatidylcholine (PC). There were no significant differences in the other lipid classes, with the exception of phosphatidylinositol (PI) which was significantly lower (P < 0.05) in brain of weaned fish. The most striking effect of weaning on brain fatty acid composition was the rapid and specific incorporation of DHA into brain phosphoglycerides. The accumulation of DHA was highly significant in all phosphoglyceride classes, with the levels of DHA increased by factors of 52% in total lipid, 86% in PC, 62% in phosphatidylethanolamine (PE), 43% in phosphatidylserine (PS) and 31% in PI. The rapid incorporation of 22:6 n-3 in turbot brain lipids was discussed with respect to the roles of this fatty acid in neutral tissues during development. The implication for the aquaculture of this species is that brain DHA levels may be directly related to larval performance, with the low levels of DHA in the brains of unweaned fish an important factor in the high mortality of larvae experienced during the stage when live feeds are being offered.

Effects of dietary polyunsaturated fatty acid/vitamin E (PUFA/tocopherol ratio on antioxidant defence mechanisms of juvenile gilthead sea bream (Sparus aurata L., Osteichthyes, Sparidae)).

Lipid peroxidation, specifically polyunsaturated fatty acid (PUFA) oxidation is highly deleterious, resulting in damage to cellular biomembranes, and may be a principal cause of several diseases in fish including jaundice and nutritional muscular dystrophy. Tissue lipid PUFA content and composition are critical factors in lipid peroxidation, as is the level of endogenous antioxidant molecules such as vitamin E. The primary objective of the present study was the characterization of antioxidant systems in a cultured juvenile marine fish, gilthead sea bream (Sparus aurata) with the underlying aim to understand how to avoid oxidation problems that may cause pathologies and disease and so to enhance growth and quality of early ongrowing stages. Juvenile sea bream were fed diets having either high or low levels of fish oil and supplemented or basal levels of vitamin E with PUFA/vitamin E ratios ranging from 117±12 in the diet with low PUFA supplemented with vitamin E to 745±48 in the diet with high PUFA with no additional vitamin E. None of the diets had serious deleterious effects on growth or survival of the fish, but the different dietary regimes were effective in significantly altering the PUFA/vitamin E ratios in the fish livers with values ranging from 5.7±0.4 in fish fed the diet with low PUFA supplemented with vitamin E to 91.1±13.2 in fish fed the diet with high PUFA with no additional vitamin E. This had effects on the peroxidation status of the fish as indicated by the significantly altered levels of in vivo lipid peroxidation products measured in liver, with fish fed the diet rich in PUFA and low in vitamin E showing significantly higher values of thiobarbituric acid reactive substances (TBARS) and isoprostanes. The isoprostane levels generally followed the same pattern as the TBARS levels supporting its value as an indicator of in vivo oxidative stress in fish, as it is in mammals. However, few significant effects on antioxidant enzyme activities were observed suggesting that more severe conditions may be required to affect these activities such as increasing the PUFA/vitamin E ratio or by increasing peroxidative stress through the feeding of oxidized oils.

In vivo metabolism of [1-14C]linolenic acid (18:3(n − 3)) and [1-14C]eicosapentaenoic acid (20:5(n − 3)) in a marine fish: Time-course of the desaturation/elongation pathway

The metabolism (via the desaturation/elongation pathways) of [1-14C]18:3(n-3) and [1-14C]20:5(n-3) in a marine fish, gilthead sea bream (Sparus aurata L.), were investigated over 8 days to determine the time-courses for the production of delta 6 and delta 5-desaturase products and 22:6(n-3). Fish were starved for 1 week prior to, and during, the period of the experiment. The recovery of radioactivity from [1-14C]20:5(n-3) in tissue lipids exceeded that of [1-14C]18:3(n-3) at all time points. The recoveries of both fatty acids decreased by 85-89% between days 2 and 8, indicating that substantial loss of radioactivity due to beta-oxidation occurred. Incorporation of 18:3(n-3) and 20:5(n-3) was predominantly into triacylglycerol but during the time-course of the experiment there were decreased percentages of radioactivity from both labelled fatty acids recovered in triacylglycerol with concomitant increased percentages recovered in phospholipids indicating preferential oxidation of fatty acids in triacylglycerol and/or redistribution of incorporated fatty acids. Recovery of radioactivity in 22:6(n-3) was 10-fold greater with [1-14C]20:5(n-3) than with [1-14C]18:3(n-3). However, there were few consistently significant trends in the levels of components of the desaturation/elongation pathways during the time-course of the experiment. In particular, the relative recovery of radioactivity in 22:6(n-3) did not increase during the experiment with either substrate. Substantial amounts of radioactivity were found in 24:5(n-3) and 24:6(n-3), particularly after injection with [1-14C]20:5(n-3), indicating that the conversion of 20:5(n-3) to 22:6(n-3) in sea bream may occur by a pathway utilizing delta 6-desaturase activity rather than by a delta 4-desaturation.

Incorporation and metabolism of 14C-labelled polyunsaturated fatty acids in juvenile gilthead sea bream Sparus aurata L. in vivo

The incorporation, and the capacity for desaturation and elongation in vivo, of intraperitoneally-injected, 14C-labelled n−3 and n−6 C18 and C20 PUFAs were investigated in juvenile gilthead sea bream, Sparus aurata. The results indicate that juvenile gilthead sea bream have only limited ability to convert CH PUFAs to C20 and C22 HUFAs in vivo. The data are consistent with the results from nutritional studies on larvae, postlarvae and fingerlings that have shown that gilthead sea bream require the provision of preformed eicosapentaenoic and docosahexaenoic acids in the diet. The impairment in the desaturase/elongase pathway was quantitatively and qualitatively similar to that found in turbot, Scophthalmus maximus, being at the level of the Δ5-desaturase. The low activity of Δ5-desaturase combined with the consistent finding that arachidonic acid is selectively retained in membrane phosphatidylinositol suggests that, in addition to eicosapentaenoic and docosahexaenoic acids, gilthead sea bream may also have a requirement for preformed arachidonic acid in the diet.