Effect of feeding gilthead seabream ("Sparus aurata") with vegetable lipid sources on two potential inmunomodulator products : prostanoids and leptins

INTERNATIONAL MASTER IN AQUACULTURE Las Palmas de Gran Canaria, Spain 2004 Effect of feeding gilthead seabream (Sparus aurata) with vegetable lipid sources on two potential inmunomodulator products: prostanoids and leptins Rachid Ganga Effect of feeding gilthead seabream (Sparus aurata) with vegetable lipid sources on two potential inmunomodulator products: prostanoids and leptins Rachid Ganga Trabajo realizado en el Instituto Canaria de Ciencias Marinas, España, bajo la dirección de la profesora, Marisol Izquierdo. y Presentado como requisito parcial la obtención del Título de Máster Universitario en Acuicultura, otorgado por la Universidad de las Palmas de Gran Canaria (ULPGC ), el Instituto Canaria de Ciencias Marinas (ICCM), y el Centro Internacional de altos Estudios Agronómicos Mediterráneos de Zaragoza (CIHEAM). Directora Autor Fdo.: Doña. Fdo.: Don Las Palmas de Gran Canaria, Marzo 2004 Acknowledgements I wish to express my warm thanks to the Centre Intemational des Hautes Etudes Agronomiques (CIHEAM) for giving me the fellowship allowing me the opportunity to improve my formation in aquaculture. I would like to express my profound gratitude to my supervisor Professor Marisol Izquierdo who has been my true support from the beginning to the end of this work. I will always appreciate and recognize the quality of your advices, scientific inspiration and great ideas. Biggest thanks to Professor Gordon Bell for the supervision of this work and for all the facilities he provided to make easy my work and my stay in Stirling, Thank you very mucho Special thanks to Fiona McGhee and James Dick, your help and your kindness were the key of the success of the analysis. Thanks to Dr. James Henderson for his help and his advices. I am also very grateful to the colleagues and staff at nutrition department in the Institute of Aquaculture, University of Stirling. Thanks to you Joanne, Yannis, Vasilis for the good days we shared in Stirling. Thanks to you Dr. Daniel Montero, you were always present and ready to help me when I asked for, thank you very mucho I wish to thank also Dr. Lidia Robaina, Dr. Maria Jose Caballero and Juan Socorro for their useful help and advices. I wish to thank also all those people who had liberally shared their sympathy, time, advices and friendship with me: Eyad and Manolo (we formed a good family), Ashraf, Mohcine, J ezabel, Marcos, David, Amaia, Eduardo, Tatiana, Valeria, Gercende, Mapi, Tibi, Ada, Moneíba, Carmen .... etc. Thanks to all the students of the third intemational master in aquaculture. Many thanks to the ICCM for all the facilities provided to carry out this study. T o my parents and brothers, words are not sufficient to express you my gratitude for all the sacrifices you have done for me. Aeknowledgements Contents List of figures List of tables List of abbreviations Abstraet Contents Introduction ................................................................................. 1 Introduction .. ................................................................................ 1 1Lipid sources ............................................................................. 1 2Overview ofthe fish immune system ................................................. .3 2-1 Nonspecific immune system ................................................... 3 2-2 Specific immune response .................................................... 10 3Lipids nutrition and fish health implications ........................................ .1 O 3-1 Dietary fatty acids, immune system and disease resistance ............ .11 3-2 Dietary fatty acids and eicosanoids production ........................... 13 4Leptin and immune system ............................................................. 15 5Objectives ... ............................................................................. 17 Materials & Methods ..................................................................... 18 1Animals and diets .. . .................................................................... 18 2Sampling procedure .......................................................................................... 21 3Blood leucocytes isolation ............................................................. 21 4Lipids extraction and analysis ......................................................... 22 5Extraction, separation and enzyme immunoassay of PGE isomers ........ ...... 23 5-1 Purification of eicosanoids .................................................. 23 5-2 Separation ofPGE 3 by HPLC ............................................... 23 5-3 Prostaglandins E2 and E3 immunoassay ...................................... 23 6Leptin immunoassay ................................................................... 26 6-1 PrincipIe of the assay ......................................................... 26 6-2 Reagents ............................................................................................. 26 6-3 Reagents preparation ........................................................................ 27 Con ten ts 6-3 Assay procedure .............................................................................. ... 27 7 - Statistical analysis ...................................................................... 28 Results ........................................................................................ 29 1Growth ..................... .................................. ... ......................... 29 2Fatty acids composition of plasma ................................................... 31 3Plasma prostaglandins ................................................................. 42 4Plasma leptin ................. ........... ... ................... . ..... ... ... .. . ........... 44 Discussion ............................................................................................................ 46 Conclusions ......................................................................................................... 57 References ........................................................................................................... 58 List offigures List of Fig ur es Page Figure 1: Sites of action for phospholipases in glycerol backbone of a phospholipid. The phospholipase A2 releas es the fatty acids occupying the sn-2 position ............. . .... .. ........ .. ...... . ....... . .... . .... ..... .. .. .. ... ... ... .. . ... ...... ....... .. 6 Figure 2: Arachidonic acid metabolism in mammalian cells. The major players in ARA metabolism following PLA2 mediated acylhydrolosis .. .. ....... ..... ...... ...... ... ....... .. 6 Figure 3: Pathways of conversion of 20:4(n-6) and 20:5(n-3) to eicosanoids ................... . ..... . ............ . ... .. ...... ............. ...... .... .... ............ . ....... ........ 7 Figure 4: Surnmary of eff ects of Leptin on Hypothalamus and immune system ....... .. 16 Fig. 5 : Feed intake in se abream along the last part of feeding period (5mm pellet) ... ............. .... .... ... ..... ....... . ... .. ... ... .. .. ........ . ......... . ............. 29 Figure 6: Specifi c gr o wth rates for seabream along the last part of the feeding period .............. .... ..... .... .......... .. ....... .. ....... . .... . ............ ... ........... 30 Figure 7: Effect of ex pe rimental diets on fish body weight. Values are means ± SD from all fish in each of the three tank replicates ......... . ....................... .. ............ 30 Figure 8: Levels of EPA and DHA in plasma polar lipids from sea bream fed experimental diets ..... .. ..... . .... .... ......... .. .... .... .. .... ..... .. ....... .... . ..... . ..... . .......... .... ....... 37 Figure 9: Levels of DHGLA and ARA in plasma total lipids from sea bream fed experimental diets .... ...... . .... .... . .... . ................... . ............. . ........ . ............. 38 Figure 10: Levels of EPA and DHA in plasma polar lipids from sea bream fed experimental diets .... .. .. ... ... ......... ... ........... .... ........ ... ........... .... ... ... . ... .... 38 Figure 11: Levels of DHGLA and ARA in plasma polar lipids from sea bream fed the 4 experimental diets .. ..... ... . ... ....... ............... ... ....... ............ ...... ... .... ... .. ..... 39 Figure 12: Levels of eicosanoids precursors: ARA, DHGLA and EPA in plasma polar lipids from sea bream fe d experimental diets .... .... . .... .. .... ... .... ....... ........ ... ...... 39 Figure 13: Levels of ARAlE P A and ARAIDHGLA in plasma polar lipids from sea bream fed experimental diets ................................ .. ........ ... .... ........... ... . .... .40 Figure 14: PGE2 concentration in plasma from sea bream fed the experimental diets ..... .. .. ... .... ..... . ...... .. ....... . ... ..... ... ... ..... ....... ............ . ......... .... ........ 42 Figure 15: PGE3 plasma concentration from sea bream fed the experimental diets .................. ..... ... ... .... .... .. . ... . .... ....... ............ ... ... . ... ..... .. ....... .... ... 43 List offigures Figure 16: The relation between PGE3 (pg/ml) and EPA (% in polar lipids) concentration in plasma from sea bream fed the experimental diets ................ ....... .. ...... . ... . .... . ....... ..... ... .......... ........... . ......... . ......... . 43 Figure 17: The relation between PGE 3 (pg/ml) and EPA (% in) concentration in PI from leukocytes of blood sea bream fed the experimental diets .................. .. ....................... ... ... ..... ... ... ......... ............................. 44 Figure 18: The levels of leptin concentration in plasma from sea bream fed the fOUT experimental diets .... . .......... .......... . ... ... .. ...... . ............. ....... ................ . ... 44 Figure 19: Relation between plasma leptin and conversion index in sea bream fed the fOUT experimental diets .... .... ... .... ... ........................ . ..... .......... .... .... .......... 45 Figure 20: Relation between liver weight and plasma leptin in sea bream fed the experimental diets ... ... ...... ..... .. ... . .... .... ................ .. ...... ........................... 45 List oftables List of t ab les Page - Tabl e 1: Tbe types and % of oils in tbe experiemental diets ................... 19 - Tab le 2: Mai n fatty acids of tbe different experimental diets 11 (g/lOO g fatty acid) (5mm) (Metbod Acid) ... ..................................... ................... 20 - Table 3: Liver weight. hepatosomatic index (HSI) and eviscerated weight of gilthead sea br eam growth along the second part of Dietary Trial 11 .................... . .. .......... . ........................................... . .......... . ... . .......... 31 - Table 4 : Tota l li pids composition on fatty acids (% of total fatty acids) ........... 34 -Table 5 : plasma polar lipids composition on fatty acids (% of total fatty acids).35 - Table 6 : Plas ma ne utral lipids composition on fatty acids (% of total fatty acids) ............................................................................................................................... 36 - Tabl e 7: Tbe ration between fatty acid % in plasma lipid classes and dietary content on tbe same fatty acid (% Fatty acid in plasma / % fatty acid in diet ............. ..... .. ... .. ... ..................................................................... 37 - Table 8 : Fatty acids composition of leucocytes Phosphatidil Inositlol (PI) of sea bream fed experimental diets ................................................... . .............. 41 - Table 9: Concentration of total prostaglandins and PGE J in plasma from sea bream fed experimental diets .. .. ... ... . ....................................................... 42 Ab : antibody Ag: antigen ANOVA: analyse ofvariance ARA: arachidonic acid List of abbreviations BHT: Butylated hydroxytoluene CMI :cell-rnediated irnrnunity DHA: docosahexaenoic acid DHGLA: di-hornogarnalinolenic acid EFA: essential fatty acids EIA: enzyrne irnrnunoassay EPA: eicosapentaenoic acid FABP: fatty acid binding protein FO: fish oil GC: gas chrornatography HBSS: Rank's balanced salt solution HPLC: high-perforrnance li quid chrornatography HSI: hepatosornatic index HUFA: high unsaturated fatty acid LA: linoleic acid LNA: linolenic acid L Ts: leukotrienes LXs : lipoxins NL: neutrallipids PCI : prostacyclines PGE: prostaglandin E PI: phosphatidyl inositol PL: polar lipids PLA 2: Phospholipase A2 PUF A: polyunsaturated fatty acid Introduction bind with the pathogen which facilitate its uptake and removal by phagocyte cells. Other non-specific humoral factors in fish have been studied recently (Yan o, 1996; Balfry and Higgs, 2001), inc1uding various lytic substances (lysozyme hemolysins, chitinase, proteinases), agglutinins/precipitins (C-reactive protein, lectins, serum amyloid P-component, u-precipitin, natural precipitins, natural antibodie s, natural hemagglutinins), enzyme inhibitors (serine proteinase inhibitors, cyseine-proteinase inhibitors, metalloproteinase inhibitors, umacroglobulin), and pathogen growth inhibitors (interferon, transferrin, caeruloplasmin, metallothionein). The pro ces s of phagocytosis lS primordial in the immune system. Phagocytic cells are responsible for the c1earance of foreign substances and senescent blood cells from the body. Monocytes, macrophages, neutrophils, eisonophils, and thrombocytes all appear to be phagocytic in fish. Neutrophils are the most active phagocytes, because they are highly mobile (Secombes and Fletcher, 1992). Phagocytosis is initiated with a passive process of attachment to the pathogen (which can be facilitated by opsonization by such substances as complement and antibody), followed by ingestion of the foreign substance into the phagocyte (inc1ude creation of phagosome within the cytoplasm of the phagocyte), and the killing of the pathogen which can involve both oxygendependent and oxygen-independent processes (Secombes, 1996). Phagocytes activity is tig ht1 y regulated by cytokines and eicosanoids ( Secombes, 1996). Cytokines are a variety of soluble factors released from cells that facilitate interactions among immune cells and therby modulate the immune response (Balfry and Higgs, 2001). They have an involvement in inflammatory reactions (Secombes 1996 ). Several cytokines have been described in fish (Manning and Nakanishi, 1996) inc1uding: Interleukin-2 (IL-2), IL-3, IL-4, and IL-6. Interferon, tissue necrosis factor (TNF), transforming growth factor 131, migration inhibition factor, and the platelet aggregation factor are also produced in fish (Secombes, 1996). Other cytokines that are important mediators of the inflammatory reponse are eicosanoids (Secombes, 1996). Cytokine play a key role in the immune response. Antigen stimulated macrophages to synthesize 4 Introduction interleuki nl (I L-l ), process the antigen, and present it to T helper cells which in tum activate T cells to produce interleukin-2 and other lymphokines. These, in tum, activate B cells to produce antibodies. The production of growth promoting factors and both T and B cell proliferation is important in this sequence of events and PGE2 is involved in various phases of these reactions (Kinsella et al. , 1990). Eicosanoids are oxygenated derivatives of polysunsaturated fatty acids formed by the metabolism of membrane phospholipids by the action of phospholipases (Rowley et al. , 1995). In broad terms they are produced in response to stressful situations, both at a cellular and whole body level (Sargent et al., 1999). The principal substrate is arachidonic acid (ARA; 20:4n-6) with a 20-carbon backbone that have a potent proinflammatory effects (Secombes, 1996), but also eicosapentaenoic acid (EPA; 20:5n-3), docosahexaenoic acid (DHA; 22:6n -3 ) and di-homogamalinolenic (DHGLA; 20:3n-6) are important substrates in fi sh due to their high presence in membrane phospholipids of these organisms (Henderson & Sargent, 1985). Once leucocytes are stimulated by a pathogen, the phospholipase enzymes located in their membrane become activated (Figures. 1 ,2), and metabolize available phospholipids to generate nonesterified PUF A (ARA, EPA, DHA and DHGLA), which are converted by the actions of cy c100 xygenases and lipooxygenases enzymes to produce an array of eicosanoids (Fig .3 ). The types and the amounts of the foregoing PUF As produced, especia ll y ARA and EPA, determine the types and amounts of eicosanoids re le ased (Balfry and Higgs, 2001). It is reported that eicosanoids can be generated in a variety of different tissues in fish, inc1uding brain, gill, liver, spleen, and heart (Knight et al., 1995). 5 Introduction ' Phospholipase A2 ~OliyeAJ H C .. 1 -O-C-Rl " 2, R2-C-O-CH'.ú O' , , H20v-°TP-O-R3 ~ ~ \: Phospholipase D Phospholipase e Fig. l: Sites of action for phospholipases in glycerol backbone of a phospholipid. The phospholipase A2 releases the fatty acids occupying the sn-2 position (Capper and Marchall,2001) Pro&18110ids TlIomb\x1(UIK ProS1D.C) ' clin S. Lipoll)',nIlK (l.O S-HPETE J ~ LT~ S.lIETÉ t ~ LBT. LTC~ + LTD~ l2-liBTE Lys&-PtlOsilholípid ! A~c'rl Tr.!I.ru .. ~ Plaldd Actl~'aIing , Factor 1 P AF Rcc~plor Fig. 2: Arachidonic acid metabolism in mammalian cells. The major players in ARA metabolism following PLA2 mediated acylhydrolosis (Capper and Marchall, 2001) Eicosanoids have relatively a short half-life in vivo after their release (Rowley et al., 1995) and they are not stored in cell membrane but released soon after they are produced following cell stimulation and mobilization of phospholipase (fig.2). They include prostaglandins (PGs), prostacyclines (peI) and thromboxanes (TXs) derived from cyclooxygenase activity and leukotrienes( L Ts) and lipoxins (LXs) derived from lipoxygenase activity (Secombes, 1996). 6 Introduction Non esterified ARA, through the action of cyclooxygenase enzymes (Fig.3), yields 2-series prostanoids (Prostaglandins and thromboxanes) and, through the action of lipoxygenase enzymes produce 4-series leukotrienes and lipoxines (Fig.3). Altematively, the metabolic derivatives produced from nonesterified EPA are 3-series prostanoids and 5-series leukotreines and lipoxines. Besides, the l-series prostanoids are derived from DHGLA. These compounds are known to play essential roles in the regulation of many physiological and immunological processes in the body (Balfry and Higgs, 2001). 20:4(n-6) 20:5(0-3) CYCLO-OXYGE/;rASE . ~-' 12or 15202 O2 LIPOXYGENASE 2cPGH i Prostanoicls Hyd.ropcroxycicosatctr"acnoic acida (HPETEs) Hyd.ropcraxycicosapcntaenoic mcicls (HPEPEs) PGDl PGE2 PGF2 PGb TXAl PGDJ PGEl PGFl PGh TXAl y H2Ü HydroX}'cicoaa1ctracnoic acids (HETEa) HydroX}'cicos.apcntacnoic lICicls (HEPEs) LcukotriCDC A4 (L T~ Lcukotricnc AS (L TAs) Glutathione ~~ / tbO Lipoxin A " L' oxin B PcpI1do lcukotncncs !P L Te.¡ L TD4 L TE4 Lm LTDs L~ LcukotricneB4(LTB4) ~ Lcukotricne B5 (LTBs) Fig. 3: Pathways of conversion of20:4(n-6) and 20:5(n-3) to eicosanoids (Lall et al., 2000) The amounts of synthesized eicosanoids depends on: the avalability of precursors, the activity of phospholipase A2 (modulated by glucocorticoids and dexamethasone), the activity of cyclooxygenases (inhibited by non-steroidal antiinflammatory drugs) and lipoxygenases (Kinsella et al., 1990). Eicosanoids are important intercellular signaling agents which affect cells behaviour and cell to cell interactions. They modulate secretory, smooth muscle (contraction and 7 Introduction relaxat io n), and cascade-type reaction which are essential to normal health (Kinsella et a l. , 1990). Sorne immune modulatory effects of eicosanoids operate via cytokine expression in the animals (Rowley, 1995). The nature of dietary lipids and the concentration of essential fatty acids have a direct effect on the eicosanoid metabolism and consequently on immune function (Lall et al., 2000). The ef fect and the activity of eicosanoids depends on their relative concentratio n, POE2 at low concentration «109 M) activates certain cells of the immune system whereas high concentrations (> 1 0-8 M) can suppress the immune system by providing a ph ysiological feedback action (Kinsella et al., 1990). It has also been suggested that there are species differences in eicosanoid production (Rowley et a l. , 1995), thus, attention should be given when comparing eicosanoids results from different species. A deficiency of these compounds results in progressive impairment of function, while excessive or imbalanced production may result in a number of patho-physiological state (Kinsella et al., 1990). The chemotact ic activity of L T and LX is primordial in attracting more leucocytes to pathogens in the site of inflammation, this in itself may compromise bacterial c1earance rates. Besides, PO act also in blood vessels dilatation to allow the increased migration of leucocytes to inflammation site (Kinsella et a l. , 1990 ). Leukotrienes also facilitate stimulation of lymphocyte proliferation and cytokine release, L TB4 known to increase an activation marker (CD23) on resting B cells, to increase proliferation of intermediate B cells and increase Ig production from differentiated B cells, both enhancing antibody production (Ba lf ry and Higgs, 2001). Lipoxins (LX) are relatively less produced in mammals, whereas in fish they are major eicosanoids products (Pettit, Rowley and Secombes., 1989 ). Macrophages appear to be the major source of eicosanoids (Pettit et al., 1989; Blazer, 1992 ), although granulocytes, monocytes, and thrombocytes also contribute significantly. It is uncertain whether lymphocytes are capable of synthesizing eicosanoids, but their activity can be profoundly affected by eicosanoids generated by other leucocytes (Rowley et al., 1995). In the presence 8 Introduction of lymphocytes, macrophages produce increased levels of PGE2 in vitro (Kinsella et al., 1990). It is also reported that PGE2 affects the expression of certain antigen receptors on the surfaces of macrophages. Nevertheless, high level of PGE2 is a suppressor of macrophage antigen presenting function, and of T cell expression via suppression of IL-2 production which is essential for T and B cell proliferation (Kinsella et al. , 1990). Thus, PGE2 should be considered as a regulators but not a universal suppressor of T cell functions (Kinsella et al., 1990 ). Prostaglandins are also known to mediate fluid and electrolyte fluxes in fish gill and kidn ey and are important in adaptation to changes in salinity (Mustafa and Srivastva, 1989). In other hand, previous studies showed that eicosanoids (particularly those of the E series) are potent stimulators of corticosteroid secretion (Wales and Toole, 1987). Injection of arachidonic acid and prostaglandin E2 increased cortisol in plasma of hagfish (Wales, 1988) and are used to induce spawning in other fish species (Sargent et al. , 1989, 1995). While, PGE\ are known to inhibit aggregation of human platelets, relaxes uterine muscular arterioles, increases cAMP levels in several tissues, and is superior to PGE2 in suppressing the proliferation of synovial cells in vitro (Hummell, 19 93). The inflammato ry response in fish has been well described by Secombes (1996). According to Suzuki and Lida (1992), three major events happen during inflammatory responses: first there is an increased blood supply to the infected area, followed by an increased capillary permeability, and lastly there is a migration of leucocytes out of the capillaries and into the surrounding tissue. Once in the tissu e, they migrate toward the site of infection, attracted by a variety of hostand pathogen-derived molecules as described aboye. Neutrophils are the first to reach the site of inflammation, monocytes and macrophages joining later to remove and destroy the pathogen by phagocytosis (Balfry and Higgs, 2001). Eicosanoids and other cytokines play a primordial role in the development and control of in fl ammato ry reactions (Secombes, 1996). First, the response is initiated by performed mediators such as vasoactive amines. Later, newly synthesized ei cosanoids serve to attract and activate leukocytes. Once arrive to 9 Introduction the site of inflammation, leukocytes release mediators that regulate the response (Secombes et al., 1996). PGE2 is apparentIy produced by macrophages In response to lectin stimulation of lymphocytes and may indicate that macrophages are under feedback control by the lymphocytes (Secombes et al., 1996). In light of these reports, it appears that the balanced production of eicosanoids, many of which act antagonistically, modulates short-term local responses to injury and are required for normal health, tissue perturbations, and/or infection. Thus, knowledge of the factors regulating the eicosanoids synthesis is very important to improve fish immune system and health. 2-2 Specific immune response The specific immune response lS caracterized by the production of antibody (Ab) binding and neutralizing a specific foreign antigen (Ag), inhibiting its activity and virulence. Fish specific immune response seems to respond in a the similar manner to mammals, depending on the structure of the antibody (Ab) and the effector mechanisms involved. Ab/ Ag binding serves to enhance the phagocytosis and c1earance of pathogens from the body through the processes of opsonization, agglutination, and precipitation. It is also, characterised by production of memory cells which lead to fast and great response when there is subsequent exposure to the same antigen (Balfry and Higgs, 2001). The specific immune responses that are independent of Ab are collectively termed cell-mediated immunity (CMI). They inc1ude, T lymphocytes as they become activated and differentiated into different functional cell types. In fish, there are lymphocytes analogues to mammalian T cells and B cells (Balfry and Higgs, 2001). 3 -Lipids nutrition and fish health implications The influence of dietary components on fish health in cultured fishes has been recognized for many years. Nutritional status is considered one of the important factors that determine the ability of fish to resisto Fish diseases 10 Introduction commonly occur when fish are stressed due to a variety of factors inc1uding poor nutrition (Lall, 2000). 3-1 Dietary fatty acids, immune system and disease resistance Fish require three long chain polyunsaturated fatty acids (PUF A) for their normal growth, hea1th, development and reproduction: docosahexanoenoic acid (DHA), eicosapentaenoic acid (EPA) and arachidonic acid (ARA) (Sargent et al., 1995, 1997). In marine fish , EPA, DHA and , recently ARA are regarded as essential fatty acids (EFA) (Watanabe, 1993; Castell et al., 1994; Bell et al. , 1995a,b; Izquierdo, 1996; Bessonart et al., 1999) due to the inability of marine fish to effectively synthesise them from their precursors linolenic acid (l8:3n-3, LNA) and li noleic acid (l 8:2n-6, LA) by Ll 5 &Ll 6destaurases and elongase (Castel et al., 1994; Sargent et al., 1995, Izquierdo et al., 1996) in sufficient amounts to cover their requirements for best growth and hea1th. The enzymes performing the desaturation and elongation reactions are induced by the presence of precursors (LA and LNA), and inhibited by the presence of the end products (EPA, DHA and ARA). Hence, desaturase activity in salmon previously fed vegetable oil during the parr stage were reduced to very low levels upon feeding a diet containing fish o il simultaneous with transferring the smolts to seawater (Tocher et al., 2000). Increased hepatic fatty acid desaturase activities were also induced in rainbow trout fe d a diet containing olive oil compared to a diet containing fish oil (Buzzi et al., 1997 ). According to Balfry and Higgs (2001), there are three mechanisms by which dietary fatty acids may affect the fish immune system and disease resistance. The first is through their influence on cell membrane lipid composition, af fecting cell permeability and function, which has profound effects on disease resistance because many immune reponses are based on leucocytes cell membrane interactions (e. g. , phagocytosis, antigen-antibody binding, activation steps involving cytokine production). Sorne other studies report that changes in physical properties of membranes with aging on dietary lipids may reduce the sen si tivity for normal responses. The second mechanism is by altering 11 Introduction signal transductions partioning between and within cells (Kinsella et al., 1990). Finally, a third mechanism is through the production of immunologically active eicosan oi ds from non esterified ARA, EPA, DHA and DHGLA, these mediators are im portant in modulating the immune response as described aboye. The relationship between dietary levels of n-3 PUF As and immunosystem could differ among marine and freshwater environments. The focus of recent studies has been on dietary fat modulation of macrophage function because of the primordial role of this cell in immune responses and tumoricidal activity (Jakson, 1997). Inadequate le vels of dietary n-3 PUF As are also known to reduce antibody production and in vitro killing of bacteria by macrophages in rainbow trout (Kiron et al., 1995) and depleted altemative complement pathway activity in gilthead seabream (Montero et al., 1998). Unbalances in fatty acids contents in the ce ll membrane could influence membrane physical properties which affect phagocytic activity (Montero et al., 2003). The work of Blazer and colleagues (1991) showed that increasing dietary levels of n-3 fatty acids lead to increased activity of head kidney macrophages. Furthermore, Thompson et al. (1996) observed that Atlantic salmon fed diets with high n-3 to n-6 PUF A ratios had more chemo-attractive supematants and increased B lymphocyte response and survival following experimental challenges with Aeromonas salmonicida and Vibrio anguillarum indicating the immunostimulatory effects of dietary n-3 in these species. However, Bell et al. (1995a) have found that juvenile turbot fed diet deficient in n-6 PUF A had increased mortality and developed an extensive pathology in gill epithelium compared to those fed fish oil. In vitro, both enhancement and supression of T and B cell responses occur depending upon the concentrations and type of fat presented to the cells in culture (J ohnston and Marshall, 1984). Changes in dietary fats can alter composition of lymphocytes (Kinsella et al., 1990). ARA has been found to be preferentially retained in vanous specles together with DHA during starvation, suggesting a metabolic priority for its conservation (Izquierdo, 1996); its importance in improving growth, survival, reproduction, stress resistance and regulation of immune function has been 12 Introduction studied (Izquierdo, 1996; Bessonart et al., 1999; Koven et al., 2001, Bell et al., 2002; Fountoulakis et al., 2003). Furthermore, ARA incorporation into leukocytes was even more marked (Farndale et al., 1999), denoting the importance of this fatty acid in this all either as a precursor of eicosanoids or intracellular messenge r. It is now we ll established that fatty acids released from membrane phospholipids play important roles in cellular signaling. This has been demonstrated from multiple cell types and especially in cells participating in an active immune response. Recent studies in various cell types suggest that fatty acids, especia ll y unsaturated fatty acids, can modulate the activities of phospholipases in salmon (Bell et al., 1996) and ion channels and other biochemical mechanisms are involved in the coupling of stimuli to mammal cell responses (Massferrer, Ríos and Schwartzman, 1990). 3-2 Di etary fatty acids and eicosanoids production To date, there is accumulating evidence that alterations In the phospholipid composition led to changes in eicosanoid precursors, as was reflected by variations in amounts of derived eicosanoids (Bell, Sargent and Raynard, 19 92). Therefore, by altering the dietary lipid content it is possible to alter the type and level of eicosanoids generated, which can have profound effects on fish health (Balfry and Higgs, 2001). Considering the fact that fish tissues are naturally abundant in n-3 PUF A (Henderson and Tocher, 1987), this offer a useful model system for studying the production and interaction of eicosanoids de ri ved from both ARA and EPA (Bell et al. , 1992). Numerous studies have demonstrated the effect of dietary fats on the fatty acid composition of ti ss ues and cells and the subsequent effects on eicosanoid production. For exampl e, Bell et al. (1993) have found that Atlantic salmon postsmolts fed a diet which contain high linolenic acid content leads to decreased levels of ARA-derived PGE2 and TXB2, which resulted in incresed antiinflammatory activity and marked reduction in the severity of cardiac lesions. Further, it was demonstrated that lipid composition, can be modified by changes 13 Materials & Methods subsequentIy n-3/n -6 ratio lower than in diet 2. Diet 4 was lowest in EPA, DHA and ARA and highest in 18 :2 n-6 and monoenoic (oleic acid). Conversion index (C I), specific growth rate (SGR) and hepatosomatic index (HSI) were calculated according to the following formulae: Fish oH CI= Feed Intake/ Weigbt Gain S GR= [(Ln Final Weigbt - Ln Initial Weigbt)/ t)]*100 With t= experimental period (days) HIS = Liver weigbt / total weigbt Table 1 : The types and % of oHs in the experiemental diets FO 60L 60R 100 L 1 00 40 40 - Rapeseed oH - 10 24 17 Linseed oH - 35 24 58 Pa lm oH - 15 12 25 19 Materials & Methods Table 2: Ma in fatty acids of the different experimental diets 11 (g/lOO g fatty acid) (5mm) (Method Acid) Diet 1 Diet2 Diet3 Diet4 % Lipids (d.w) 20.24 21.36 22.79 25.14 14:0 5.92 3. 05 2. 93 0. 79 15:0 0.50 0.28 0.24 0. 08 16:0 19 .30 14 .99 15 . 95 15.92 16:10-7 7.21 3. 93 3.23 1.23 16:20-6 - - - - 17:0 0.26 0. 15 0.13 0. 02 16:40-3 0.1 7 0. 04 0.03 0.02 18:0 3.3 7 3.21 3.36 3.40 18: 1 0-9 11. 71 24.61 29.89 32 . 98 18:20-6 5. 84 11.87 12 .50 13 . 67 18:30-6 0. 07 0.10 0. 08 - 18:40-6 0 .1 7 0. 03 0. 03 - 18:30-3 1.62 14 .36 12 . 25 23 . 02 18:40-3 2.1 8 1.28 0.87 0. 17 20:0 0.21 0.24 0.32 0. 23 20:10-9 2.38 2.05 1.98 2 .23 20:10-7 - - - - 20:20-6 0.1 5 0.10 0. 08 0. 03 20:40-6 0.66 0.34 0.28 0. 06 20:40-3 0.54 0.27 0.21 0.05 20:30-3 0.08 0. 05 0. 04 0.02 20:50-3 11. 90 6.10 4. 86 1.06 22:0 0. 08 0. 10 0.24 0.13 22:10-11 2.98 2.35 2.20 2 .41 22:10-7 -0.02 - - 22:40-6 0.1 8 -0. 03 0. 01 22:50-6 0.24 0. 12 0.11 - 22:40-3 --0.03 0. 02 22:50-3 1.17 0.56 0.47 0. 08 22:60-3 14.1 4 7.36 3.21 2. 10 Saturated 30. 01 22.22 23.33 20 . 63 Moooeooics 27. 70 33 . 12 37.44 38.85 L 0-3 32.23 30.27 25 . 22 26. 57 L 0-6 7.3 7 12.59 13.13 13 . 78 L 0-9 14 .56 26.81 31.91 35.22 L 0-3 RUFA 27.84 14.34 11.82 3. 33 0-3/0-6 4.3 7 2.40 1.92 1.93 20 Materials & Methods 2Sampling proce dure At the end of the feeding trial, day 281 , fish were individually sampled from each tank. B lo od was collected from caudal veins in heparinised seringues from 6 fish per each tank (1 8 fish per diet) and transferred to an eppendorf tube coated lithium heparin as an anticoagulant. The blood was centrifuged immediately at 3000 rpm for 10 min to sediment the cells. One milliliter of plasma was removed, 50 ¡..t I/ mI of 2M formic acid was added and the acidified samples were frozen in liquid nitro gen (-80 OC) before the eicosanoids analysis. 250 ¡..tI of plasma were removed and stored at -80 oc for leptin analysis and the remainder plasma was pooled per each tank and stored at -80 oC for fatty acid analysis. 3Blood leucocytes isolation This experiment was done at the end of the second trial II only for three diet since fish fed 100 L were dead. A modification of the method developed by Izquierdo & Lall, 2002 for halibut leukocytes isolation was adapted to seabream. Seven mI of blood were collected in heparisined syringues from the caudal vein of 9 fish per diet and transferred to c1ean glass tube kept in ice. The blood was centrifuged at 500 x g for 10 min at 4 oC. Cells were diluted in 10 mI of HBSS, Ca-Mg free and centrifuged at 500 x g for 10 min at 4 oC. Cells were separated in to 2 samples, each sample w as diluted in 6 mI of HBSS, Ca-Mg free; and layered carefully onto 6 mI of 46% Percoll and centrifuge at 450 x g for 40 min at 4 o C. The cells were collected from the white layer (intermediate) and diluted in 10 mI of HBSS, Ca-Mg free and centrifuged at 500 x g for 10 min at 4 oC . Cells were diluted in 6 mI HBS S, Ca-Mg free and layered carefully onto 6 mI of 46 % Percoll and centrifuged at 450 x g for 40 min at 4 oC. Cells were collected and diluted in 10 of HBSS, Ca-Mg fre e; and centrifuged at 500 x g for 10 min at 4 oC. Cells were diluted in 6 mI of HBSS, Ca-Mg free and layered carefully onto 6 mI 46 % and centrifuged at 450 x g for 40 min at 4 oC. The leucocytes (white intermediate layer) ce ll s were collected and washed with 10 mI of HBSS, Ca-Mg free. The leucocytes obtained from 3 fish were pooled and were suspended in 4 21 Materials & Methods mI of HB SS , 2 mI of chloroform were added and the sample is stored at - 80 oC prior to li pids extraction. 4Lipids ex traction and analysis Extraction of total lipid from plasma samples, diets and leucocytes was performed by the method of Folch et al. (1957) using a mixture of chloroform: methanol (2:1)(v:v) containing 0.01% BHT, as an antioxidant, and KCI (0.88 %); 300 111 of pooled plasma were used. Vigorous vortex mixer followed by centrifugation to assist separation of chloroform and aqueous layers extracted the lipids from plasma, diet and leukocytes samples. The lower layers is filtered through Whatman filter paper and dried under a flow of nitro gen, total lipids were weighted. Neutr al and polar fractions were separated by adsorption chromatography on silica cartridges Sep-pak, (Waters, Milford, MA) as described by Juaneda and Rocquelin (1985). Individual phospholipid c1asses from leucocytes total lipids were isolated according to Bell et al. (1997) by loading 3-4 mg of totallipid onto a 2 cm origin of a 20X 20 cm * TLC plate and eluting with methyl acetate/isopropanol/chloroform/methanol/0.25% (w/v> aqueous KC 1 (25 :25 :25: 10:9 by voL). Individual phospholipids were identified by spraying the plate with 0.1 % 2',7' -dichlorofluorescein in 97% methanol containing 0.05% BHT and the lipid c1asses visualised under UV light. Lipid c1asses were scraped from the silica and acid-catalysed transmethylation performed ovemight at 50°C as described by Christie (1982). Fatty acid methyl esters were produced from aliquots of total lipids extracted from diet and plasma samples by acid-catalyzed transmethylation performed ovemight at 50 °C as described by Christie. (1982). Fatty acids methyl esters were separated and quantified by Gas-chromatography (Shimadzu C-R5A, 30m*0,32mm Silice column with Supleco-l0) according to conditions described by (Izquierdo et al., 1990). Individual methyl esters were identified by comparison with known standards and published data. 22 Materials & Methods 5Extraction, separation and enzyme immunoassay of PGE isomers 5-1 Purification of eicosanoids The frozen plas ma for eicosanoids analysis was thawed and eentrifuged at 1000 x g for 5min to precipitate the debris. The supernatants were extraeted using oetadeeyl s il yl ( CI8 ) "Sep-Pak" mini-eolumns (Millipor(UK), Watford) as deseribed in deta il by Bell et al. (1994). 200 ~l of supernatants was applied to the eolumn, whieh had been pre-washed with 5 mI methanol and 10 mI distilled water. The eolumn was washed sueeessively with 10 mI distilled water, 5 mI of 15% (v/v) ethanol and 5 mI hexane/ehloroform (65:35, v/v) before elution of prostanoids with 10 mI of ethyl aeetate. This extraet was dried under nitrogen and resuspended in 100 ¡.tI methanol and stored in a small glass vial in the deepfreezer (- 4 OC) before analyzing by immunoassay. 5-2 Sepa ration of PGE 3 by HPLC PGE3 was separated by reverse-phase HPLC using a Spherisorb 5 ¡.tm C18 (ODS2) eolumn. The ehromatographie system was equipped with Waters Model M-45 pu mps and Waters 680 monitored at 196nm using a Pye-Unieam LC-UV detector to determine elution of prostaglandin standards. An isoeratie solvent system wa s used eontaining 17 mM phosphorie aeid/aeetonitrile (70:30, v/v) at a flow of 0,75ml/min. The remaining 50 ¡.tI of purified eieosanoids from plasma extraet was in je eted to the eolumn and 2.25 mI fraetions were eolleeted using an LKB 2112 "Redirae". Fraetions eorresponding to PGE3 were applied to a C18 "Sep-Pak" whieh had been-prewashed as deseribed above, and the prostaglandin eluted in 5 mI ethyl aeetate. Samples were dried under nitro gen and redissolved in 100 ¡.tI of immunoassay buffer. Mesurement of PGE3 was performed using enzyme immunoassay (EIA) kits for PGE2 aeeording to the same protoeol deseribed above ( SPI-bio, Gif sur Yvette, Franee). The speeifieity of the kit antibodies used in this immunoassay with PGE2 is 100% but it'is only 43% with PGE3, whieh is eounted when ealeulating results. 5-3 Pr ostaglandins E2 and E3 immunoassay For prostaglandins E 2: 50 ¡.tI of the methanol extraet were taken, dried under nitro gen and re-dissolved in 500 ¡.tI of EIA buffer and stored in the the 23 Materials & Methods fridge ( 4°C). This assay is based on the competition between PGE2 and a PGE2acetylcholinesterase (AchE) conjugated PGE2 (tracer) for a limited amount of PGE2 monoclonal antibody. Because the concentration of PGE2 tracer is constant while the concentration of PGE2 varies depending on the sample, the amount of the PGE2 tracer that is able to bind to the PGE2 monoclonal antibody is inversely-proportional to the concentration of PGE2 in the well. This antibody PGE2 complex binds to goat polyclonal anti-mouse IgG that has been previously attached to the well. The plate is washed to remove any unbound reagents and then Ellman's Reagent (which contain the substrate to AchE) is added to the well. The product of this enzymatic reaction has distinct yellow color and absorbs strongly at 412 nm. The intensity of this color, determined spectrometrically, is proportional to the amount ofPGE 2 tracer bound to the well, which is inversely proportional to the amount of free PGE2 present in well during the incubation. All assay-specific reagents were prepared before starting the assay. EIA Buffer: Diltuting the contents of the vial of EIA Buffer Concentrate with 90 mI of UltraPure water. Wash Buffer: Diluting the content of Wash Buffer Concentrate1:400 with UltraPure water and adding Tween (0.5mllliter of Wash Buffer). Prostaglandin E2 standard: The concentrate of The PGE2 standard (10 ng) is reconstituted with 1ml of EIA Buffer. Eight clean tubes (#1-8) were prepared; 360 /-11 EIA Buffer to tube # 1 and 200 /-11 EIA Buffer to tubes # 2-8. 40 /-11 of the bulk standard (10 ng/ml) is transferred to the tube # 1 and mix thoroughly. Serially, the standard was diluted by removing 200 /-11 from # 1 and placing in tube # 2, mixing. Next, removing 200 /-11 from tube 2 to tube # 3, mixing. The process was repeated for tubes # 3-8. These diluted standards should not be stored for more than 24 hours. Prostaglandin E2 AchE: The concentrate is diluted with 6 mI EIA Buffer, The tracer dye is added at a final dilution of 1: 100. 24 Materials & Methods Prostaglandin E2 Monoclonal Antibody: The concentrate is diluted with 6 mI EIA, the dye to the reconstituted antiserum is added at a final dilution of 1 :100. Once the immunoassay kits was opened, each plat contained eight Blanks (Blk), one non-specific bindings wells (NSB), one maximum binding wells (BO), and eight point standard curve (S 1-S8). A test with different samples dilutions was run to choose the best (80 ~l) . Pipetting the reagents: different tips are used to pipet the buffer, standard, sample, tracer, and antibody. 1EIA Buffer: 1 00 ~l EIA buffer were added to non-specific Binding (NSB) wells and 50 ~l to maximum binding (BO) wells. 2Prostaglandin E2 Standard: 50 ~l from tube # 8 to the lowest standar well (S8). 50 ~l from tube # 7 were added to the next standard well (S7). The same procedure was used until all standards were aliquoted. 3Samples: 80 ~l of sample were added per well. 4Prostaglandin E2 AchE Tracer: 50 ~l were added to each well except the Blank (Blk) wells. 5Prostaglandin E2 Monoclonal Antibody: 50 ~l were added to each well except the Non Specific Binding (NSB), and the Blank (Blk) wells. 6The plate was covered with pastic film and incubated for 18 hours at 4°C wich increase the sensitivity ofthe assay. 7Developing the plate: Before developing the plate, Ellman's Reagent is reconstituted with 20 mI of Ultrapure water. This should be prepared and used the same day it is prepared, and protected from light when not in use. The wells were emptied and rinsed five times with Wash Buffer. 200 ~l of Ellman's Reagent were added to each well and 5 ~l of tracer to the Total Activity wells. The plate is covered with plastic film and developed by using an orbital shaker and dark during 75 mino 8Reading the plate: Is accomplished at a wavelength between 405. 9Calculating the results : The results were calculated manualy as follows: Average the absorbace readings from Blk wells. 25 Substract average Blk from all the other wells. Substract NSB from all th other wells. Materials & Methods Calculate %BIBO (% Sample standard BoundlMaximum Bound) for the remaining wells. The standard curve is traced ( %BIBO with standard concentration on PGE2 en pg/ml). The concentration of each sample in PGE2 is calculated. 6Leptin irnrnunoassay 6-1 -Principie of the assay This assay uses a cuantitative sandwich enzyme immunoassay technique. A monoc1onic antibody for leptine is applied to the plates. A monoc1onal antibody (Human leptin antibody) specific for leptin had been pre-coated onto a microplate. Standards and samples are pipetted ino the wells and any leptin present is bound by the immobilized antibody. After washing away any unbound substances, an enzyme-linked monoc1onal antibody specific for leptin is added to the wells. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of leptin bound in the initial step. The color development is stopped and the intensity of the color is measured. 6-2 Reagents Leptine microplates: 96well. Mircoplate de polystyrene ( 12 columns of 8 well) with monoclínic antibody leptins. Leptin conjugate (Part 890574): 21 mI de antibody monoc1onic against Leptin of rate with preservatif. Leptin estandard (Part 890575): 10 ng de recombinant leptin human in buefferd proteins with preservatif. Análisis diluente RDl-19 ( Part 895467): 11ml de buffered protein base with preservatif. Calibrator diluente RD5P (5x) Concentrate (Part 895151): 21ml de solución concentrate of buffered protein base with preservatif. 26 '-- Materials & Methods Concentrate wash buffer (Part 895003): 21 mI of 25-fold concentrate of solution buffered surfactant with preservatif. Dye reactivif A (part 895000): 12,5 mI of estabilized hyrdogen preoxidase. Dye reactif B (Part 895002): 12,5 mI de estabilized chromogen ( tetramethy I benzidine ). Stop solution: 6 mI of 2 N Sulfiric acid. 6-3 Reagents preparation All reagents are brought to the room temperature before the assay. Wash Buffer: The concentrate vial is mixed gently and diluted into 1 :25 with deionized water. 500 mI ofwash buffer were prepared. Substrate solution: Color reagent A and B were mixed together in egual volumes and put in dark within 15 minutes before using. Calibrator Diluent RD5P (IX): the concentrat is diluted to 1:5 . 100 mI were prepared. Leptin standard:It is prepared 15 minutes before use. The concentrat is mixed gently and diluted with 1 mI of deionized water. This reconstitution produces a stock solution of 10.000 pg/ml. 8 tubes standard are prepared as expained aboye ( Prostaglandin standard). 6-4 Assay procedure The frozen plasma were thawed and centrifuged at 1000 x g for 5 min to precipitate the debris. 100 111 of assay diluent RD 1-19 were added to each well. 100 111 of Standard are added to each stadanrd well. -100 111 of control were added to each well followed by 200 111 of Plasma sample. Multi-channel pipette was used. The plate is covered and incubated for 2 hours at room temperature. All the wells were aspirated and washed with 400 111 of Wash buffer. This process is repeated 4 times. At the last wash, the wells were bolted against clean paper towels to remove any remaining wash buffer 27 Materials & Methods 200 ¡..tI of Leptin Conjugate were added to each well, the pIate was covered with new adhesive strip and incubated for 1 hour at room tem perature. The wells were aspirated and washed as in step 5. 200 ¡..tI of Substrate Solution were added to each well and the plate was incubated for 30 minutes at room temperature and protected from light. 50 ¡..tI of Stop SoIution were added to each well. The plate is developped within 30 minutes in shaker and the optical density of each well was determined using a micropIate reader set to 450 nm. Calculation of results: The standard curve is drawn by ploting optical density for the standards versus the concentration of the standards. The data is linearized using a logaritmic transformation. The equation of standard curve is determined and the final Leptin concentration in plasma is determined by simple application of their absorbance in the standard curve equation. 7Statistical analysis Significance of difference (P<0.05) between dietary treatments was determined by one-way analysis of variance (ANOV A) followed by Duncan multiple comparison test . Analyses were performed using a SPSS software (SPSS for windows 11.0). 28 Results Table 4: Plasma totallipids composition of fatty acids (% of total fatty acids) Fattyacids FO 60 L 60R 100 L 12 :0 0. 05 0.10 + 0.04 0.86 + 0.69 1.31 + 1.20 14 :0 1.60+0.14 1.29 + 0.26 1.03 + 0.07 0.91 + 0.44 15:0 0.26 + 0.04 0.16+0 .05 0.13 + 0.01 0.13 + 0.07 16 :0 21.47 + 1.24 20.50 + 4.90 19.06 + 1.92 20.35 + 1.98 16:ln-7 2. 85+0 . 15 2.21 + 0. 01 1.19+0.15 0. 83 + 0.09 16: ln5 0.11+ 0.00 0.08 + 0.00 0.06 + 0.02 0.04 + 0. 01 16:2n-3 0.26 + 0.02 0. 15+0.01 0. 11 + 0. 01 0.04 + 0.04 16:2n-4 1.25+0.11 0.81 + 0.01 0.62 + 0.04 0.30 + 0. 11 17 :0 0.32 + 0.04 0.21 + 0.03 0. 16+0 . 01 0.07 + 0.05 17: I 0.30 + 0.03 0.05 + 0.03 0.160 . 01 0.12+0 .06 16:4n-3 0.14+0.00 0.06 + 0.04 0.04 + 0.01 0. 03 + 0.00 16:4n-1 0.10+0.04 0.04 0.05 0.04 + 0.04 18 :0 4.83 + 0.07 5.86 + 2.20 5. 91 + 0.45 6. 90+1.21 18: I n-9 + 18 : I n-7 11.47+0 .07 19. 53 + 2.40 20.77 + 1.08 20.90 + 0. 72 18: In-5 0.13 + 0.00 0.67 + 0.74 0.12+0.01 0.10 + 0. 03 18:2n-9 0.14 + 0.01 0. 14+0.01 0.15 + 0.02 0.14+0.01 18:2n-6 3.10+O.l6 e 7.89 + 1.37b 9.56 + 1.48b 13.36 + 1.44a 18:3n-9 0.12+0.00 0.06 + 0.03 0.04 + 0.01 0.06 + 0.01 18:3n-6 0.11 + 0.00 0.09 + 0.03 0.06 + 0.00 0.12+0 . 01 18 :4n-6 0.09 + 0.00 0.08 + 0.04 0.12+0 . 03 0. 03 + 0. 01 18:3n-3 0.62 + 0. 05 e 5.90 + 0.71 ab 4.27 + 0.44b 7.27 + 2.28a 18:4n-3 0.41 + 0.08 0.24 + 0.09 0. 19 + 0.00 0.41 + 0.04 18:4n-1 0.06 + 0. 01 0.03 + 0.01 0. 03 + 0.01 0.02 + 0.02 20:0 0.08 + 0.00 0. 10 + 0.04 0. 15 + 0.05 0.16 + 0.07 20: 1 n-9 0.80 + 0.10 1.03 + 0.22 1.00+0 . 10 0. 92+0 . 19 20:ln-7 0.09 0.07 + 0.00 0.08 + 0.02 0.05 + 0.02 20: I n-5 - - -0.07 + 0.06 20:2n-9 0.07 + 0.00 0.04 + 0.01 0.13 + 0.02 0.07 + 0. 05 20:2n-6 0.16+0.03 b 0 .2 9 + 0.08 ab 0.22 + 0.14 ab 0.44+0 . 16 a 20:3n-9 0.03 + 0. 01 0.02 + 0.00 0.36 + 0.09 0.10 + 0.02 20:3n-6 0.10+0 . 03 b 0.15 + 0.02b 0.23 + 0.04 ab 0.40 + 0.17" 20:4n-6 1.33 + O. ll a 0.84 + 0.15b 0. 81 + 0.02b 0.70 + 0. 03 b 20:3n-3 0.10 + 0. 03 b 0.39 + 0.08a 0.32 + 0. 05 a 0. 12 + O.OOb 20:4n-3 0.69 + 0. 03 0.46 + 0.14 0.45 + 0.07 0.56 + 0.16 20:5n-3 10 .39 + 0.73a 6.20+2 . 15 b 6.07 + 0.31 b 3.56 + 0.62c 22:0 0.07 + 0.01 0. 10 + 0.05 0.09 + 0.06 0.09 + 0.02 22:ln-11 0.52 + 0.09 0.54 + 0.07 0.43 + 0.01 0.41 + 0.05 22: I n-9 0.21 +0 . 03 0.26 + 0.03 0.27 + 0.02 0.25 + 0.02 22:ln-7 0.09 + 0.06 0.07 + 0.05 0.04 + 0.02 0.02 + 0. 01 22:4n-6 1.99+0 . 16 0.04 + 0.01 0.05 + 0.03 0.05 + 0.03 22:5n-6 0.410.01 0. 28 + 0.05 0. 31 + 0.02 0. 18+0.02 22:4n-3 0.05 + 0.01 0.04 + 0.00 0.06 + 0. 03 0.04 + 0.00 22:5n-3 2. 53 + 0.10 1.72 + 0.57 1.90 + 0.07 1.20+0.10 22:6n-3 30.51 + O.l1a 21.26 + 8.51 b 22.46 + 1.36 ab 17.15 + 2.03b Total saturates 28.57 + 1.40 28.31 + 7.57 27.49 + 1.30 29.97 + 4.79 Total monoenes 15.36 + 0.34b 24 .5 1 + 1.96a 24.21 + 1.20a 23.81 + O.72 a n-3 45.44 + 0. 71 a 36.42 + 10 .71 ab 35.93 + 1.34 ab 30.36 + 3.78b n-6 7.29 + 0.07d 9.66 + 1.30c 11.39 + 1.68b 15.46 + 1.30a n-9 11.90 + 0.07d 19 . 83 + 2.36c 22.81 + 1.1 Oa 21.40 + 0.80b n-3 HUFA 40.90 + 0.62 30.07 + 11.30 31.30 + 1.60 22.59 + 1.68 EPAIDHA 0.34 + 0.03a 0.30 + 0.02a 0 .2 7 ab 0.21 + 0.06b ARA/EPA O. 13 b 0.14+0.03 b 0.13 +O. Olb 0.20 + 0.04a DHGLA/ARA 0.08 ± 0.02b 0.19 ± 0.07b 0.28 ± 0.12b 0.57 ± 0.06a 34 Results Table 5 : Plasma polar lipids composition on fatty acids (% oftotal fatty acids): Fattyacids FO 60 L 60R 100 L 12:0 1.59 + 0.36 1.61 + 0.72 1.44+0.51 1.38 + 0.85 14:0 2.15 + 0.16 1.32 + 0.74 l.01 + 0.19 0.90 + 0.34 15 :0 0.39 + 0.09 0.23+0.10 0.30 + 0.08 0.15+0.04 16 :0 22.91 + 0.93 18.76 + 4.33 23.30 + 1.29 21.97 + 1.78 16:1n-7 2.99 + 0. 88 1.27 + 0.64 1.43 + 0.66 1.05 + 0.69 16:1n-5 0.15 + 0.04 0.13 + 0.00 0.04 + 0.02 0.08 + 0.02 16:2n-3 0.22 + 0.00 0.17+0 . 01 0.05 + 0.03 0.10+0.01 16:2n-4 0.57 + 0.01 0.60 + 0.29 0.38 + 0.02 0.32 + 0.05 17 :0 0.35 + 0.01 0.20 + 0.03 0.05 + 0.03 0.04 + 0.04 17 : 1 0.29 + 0.08 0.08 + 0.02 0.21 + 0.04 0.14 + 0.02 16:4n-3 0.12+0.00 0.09 + 0.03 0.05 + 0.03 0.04 + 0.04 16:4n-l 0.27 + 0. 01 0.26 + 0.08 0.32 + 0.02 0.26 + 0.06 18:0 6 .3 4 + 0. 13 6.12 + 2.42 7.16+1.34 7.24 + 0.49 18:1n-9 + 18:1n-7 10.47+0 . 96 16.15+4.81 14 . 28 + 0.30 15.73 + 1.12 18:1n-5 0. 14+0.01 0.16+0.06 0.06 +0. 03 0.08 + 0.07 18:2n-9 0.12+0.01 0.11+0.11 0.05 + 0.02 0.12 + 0.03 18:2n-6 6.04 + 2.64b 8.47 + 2. 78 ab 8.07 + 1.89 ab 11.75 + 1.63a 18:3n-9 0.07 + 0.01 0.06 + 0.02 0. 01 + 0.01 0. 03 + 0. 03 18:3n-6 0.09 + 0.00 0.07 + 0.02 0. 05 + 0.02 0. 10+0 . 03 18:4n-6 0.10 + 0.02 0. 08 + 0. 05 0.03 + 0.01 0.03 + 0.02 18:3n-3 0.59 + O. 23 c 3.05 + 0. 95 ab 2.19 + 0.06bc 4.98 + 1.54a 18:4n-3 0.10 + 0.00 0.11 + 0.08 0.04 + 0.05 0.06 + 0.06 18 :4n-l 0 .14+0.0 0 0.04 + 0.06 0.07 + 0.11 0. 12+0 .02 20 :0 0.21 + 0.09 0 .2 0 + 0.13 0.19 + 0.14 0.12+0 . 01 20:1 n-9 0.53 + 0.04 0.70 + 0.13 0.67 + 0.01 0. 81 + 0. 25 20:1n-7 0.08 + 0.00 0. 08 + 0.01 0.06 + 0.01 0.08 + 0. 03 20: ln-5 -0. 12+0.01 - - 20:2n-9 0.02 + 0.00 0.06 + 0.05 0.04 + 0.06 0. 10 + 0. 03 20:2n-6 0.16+0.01 0.32 + 0.01 0.36 + 0.06 0.46 + 0.09 20:3n-9 0.01 + 0.01 0.01 +0.01 -0.02 + 0.01 20:3n-6 0.12 + 0. 02 b 0.26 + O.07"b 0.31+0.12 a 0.31 + 0.06a 20:4n-6 1.28 + 0.15a 1.02 + 0.15 ab 1.05 + 0.32 ab 0. 65 + 0.12b 20:3n-3 0.09 + O.OOb 0.34 + 0.02a 0.38+0.16 a 0.32 + 0.2a 20:4n-3 0.26 + 0.04 0.39 + 0.06 0.38+0 . 13 0.39 ± 0. 08 20:5n-3 9.02 + 0.90a 7.53 + 1.16 ab 6.87 + 0.82b 4.52 + 1.04b 22:0 0.17 + 0.02 0. 13 + 0. 02 0.17 + 0.08 0. 11 + 0.00 22:1n-ll 0.32 + 0.04 0.29 + 0.14 0.31 + 0. 01 0.39 + 0.20 22:1n-9 0.13 + 0.00 0.15 + 0.06 0.06 + 0. 08 0. 17 + 0.04 22:1n-7 0.15+0 . 05 0.09 + 0.07 0. 03 + 0.04 0.04 + 0.04 22:4n-6 0.20 + 0. 01 0.12 + 0. 06 0.05 + 0.07 0.07 + 0.02 22:5n-6 0.47 + 0. 02 0. 64 + 0.28 0. 51 + 0.30 0.29 + 0.04 22:4n-3 0.11 + 0.02 0.09 + 0.03 0.02 + 0. 03 0.04 + 0.03 22:5n-3 1.89 + 0.22 2.04 + 0.16 1.96 + 0.34 1.47+0 .07 22:6n-3 28.63 + 3.56 26.28 + 3.20 26.03 + 4.86 22.95 + 1.77 Total saturates 34.11 + 0.06 28.56 + 5.07 33.62 + 3.37 31.92 + 2.06 Total monoenes 15 .24 + 2.09 19.22 + 5.93 17.12 + 1.19 18.58 + 0.76 n-3 4 1.01 + 4.47 40.09 + 3.47 37.97 + 6.08 34.87 + l.21 n-6 8.44 + 2.41 10 . 98 + 2.23 10.42 + 1.26 13.67 + 1.75 n-9 11.43 + 1.01 17 . 24+5.08 15.12 + 0.52 16.97 + 1.03 n-3 HUFA 39.99 + 4.71 36. 68 + 4. 53 35 .64 + 6.28 29.69 + 2.65 n-3 /n-6 4.86 + 1.37" 3.65 + 1.08ab 3.64 + 1.03 ab 2.58 + 0.40b EPAlDHA 0.31+0.01 0.29 + 0.01 0.26 + 0.02 0.20 + 0.03 ARAlEPA 0.14 + 0.00 0. 14 + 0.00 0.15+0.03 0.14 + 0.01 DHGLA/ARA 0.09 + 0.004c 0.26 + 0.09b 0.28 + 0. 02 b 0.49+0 . 15 a AH Values are means ± SD from three fish. Values in the same row with different superscript letters are significantly different. 35 Results Table 6 : Plasma neutrallipids composition on fatty acids (% of total fatty acids) Fattv acids FO 60 L 60R 100 L 12:0 4.70 + 2.09 3.14 + 1.09 3.34 + 0.69 2.97 + 0.90 14:0 5.52 + 3.28 2.42 + 0.55 2.31 + 0.46 2.43 + 0.40 15:0 0.58 + 0.10 0.41 + 0.25 0.32 + 0.09 0.22 + 0.05 16 :0 22.36 + 0 .53 16.96 + 0.36 18.15+1.11 18.77+1.07 16:1n-7 4. 13+1.11 2.55 + 0.40 1.99+0.15 2.09 + 1.13 16:1n-5 0.21 ± 0.05 0.18±0.07 0.14 ± 0.03 0. 12±0.03 16:2n-3 0. 17 ± 0. 02 0.20 ± 0.04 0.16±0.03 0.16±0.05 16 :2n-4 0.87 ± 0.73 1.03 ± 0.05 0.63 ± 0.42 0.74 ± 0.24 17 :0 0. 35 + 0. 01 0. 23 + 0.07 0. 18 + 0.02 0.16+0.05 17:1 0. 68 + 0.05 0.46 + 0.17 0. 33 + 0.09 0.26 + 0.10 16 :4n-3 0.26 + 0.04 0.13 + 0.00 0.13 + 0.02 0.70 + 0.08 16:4n-l 0. 81 ± 0. 11 0. 53 ± 0.09 0.70 ± 0.13 0. 73 ± 0.00 18 :0 4. 10 ± 0.97 5.70 ± 0.23 4.89 ± 0.55 5.07 ± 0.64 18:1n-9 + 18 :1n-7 16.46 + 2.34 22. 78 + 0.72 27.31 + 0.60 24. 65 + 3. 37 18 :1 n-5 0.29+0.10 0.26+0.12 0.19+0.05 0.17+0.04 18:2n-9 0.13 + 0.04 0.17 + 0.02 0.15 + 0.03 0.16+0.01 18 :2n-6 6.34 + 2. 35 b 13.37 + 3.18a 11.09 + 3.26 ab 10 . 61 + 0.52 ab 18:3n-9 0. 14+0.02 0. 12 + 0.07 0.08 ± 0.03 0.06 ± 0.00 18 :3n-6 0. 14±0 .06 0.07 ± 0.00 0.16 ± 0.03 0.23 ± 0.5 18 :4n-6 0.24 + 0.07 0.17 + 0.07 0.10+0.03 0.14+0 . 03 18 :3n-3 1.06 + 0.09c 7.09 + 0.44ab 6.39 + 0.33b 9. 11 + 3.71a 18:4n-3 0.40 + 0. 03 0.50 + 0.05 0.36 + 0.13 0.46 + 0.09 18:4n-l 0.06 + 0.03 0.28 + 0.06 0.38 + 0.05 0.38 + 0.02 20:0 0. 18 + 0. 08 0.40 ± 0.11 0.28±0 . 10 0.28±0.16 20: 1 n-9 1.51±0.18 1.46 ± 0.04 1.54±0.13 1.33 ± 0.06 20:ln-7 0. 19 + 0.00 0.12+0.00 0.12 + 0.01 0.04 + 0.03 20:1n-5 0.02 + 0. 01 0.02 + 0.03 0. 13 + 0.09 0.09 + 0.04 20:2n-9 0.09 +0. 01 0.18 + 0.05 0.18 + 0. 01 0.37 + 0.04 20:2n-6 0.18 + 0.02 0. 32 + 0.16 0.27 + 0.02 0.28 + 0.03 20:3n-9 0. 02 + 0.02 0. 14 + 0.06 0.03 ± 0.00 0.08 ± 0.00 20:3n-6 0.09 + 0.03 0.10+0.03 0.17 + 0. 11 0.16+0.05 20:4n-6 0. 52 + 0. 14 0.30 + 0.01 0.34 + 0.10 0.49 + 0.23 20:3n-3 0.38 + 0. 02 0.38 + 0. 11 0.35 + 0.07 0.36+0.10 20:4n-3 0.42 + 0.22 0.33 + 0. 05 0.30 + 0.08 0.25 + 0.03 20:5n-3 6.19+1.73 a 3.27 + 0.17b 2.85 + 0.59b 2.68 ± 1.64b 22:0 0.30 + 0.00 0.19 ± 0.00 0.21 ± 0.04 0.16±0.07 2 2:ln-l1 1.12 + 0.04 0.89 + 0.13 0.83 + 0.04 0.70 + 0. 05 22:1n-9 0.53 + 0.11 0.50 + 0.10 0.56 + 0.08 0.53 + 0.02 22:1n-7 0.15 + 0.07 0.14+0.10 0.08 + 0.03 0.05 + 0.00 22:4n-6 0.31 + 0.06 0.14+0.01 0.09 + 0.07 0.16 + 0.03 22:5n-6 0.37 + 0. 08 0.21 +0.01 0.22 + 0.01 0.22 ± 0.01 22:4n-3 0. 21 + 0.06 0.14+0.00 0.12 ± 0.07 0. 23 ± 0.03 22:5n-3 2.37 + 0. 65 1.36 + 0.05 1.32 + 0.20 1.02 + 0.44 22:6n-3 14.85 + 4.02a 10.67 + 1.00b 10.54 + 2.07b 10 . 15+2.09 b Total saturates 38.08 + 6.91 29.45 + 0.70 29.68 + 1.68 29. 84 + 1.42 Total monoenes 25.28 + 3.47 29.36 + 0.69 33.22 + 0.62 29.81 ± 1.96 n-3 26.32 + 6. 13 24.05 + 1.40 22.52 ± 3.01 24.92 ± 1. 54 n-6 8.19+1.90 b 14 . 68 ± 3. 05 a 12.43±3.14 ab 12.93 ± 1.30 ab n-9 18 . 88+2 . 71 25.35 + 1.02 29.84 + 0.77 26.99 + 2.99 n-3 HU FA 24.42 + 6. 54 16.13 + 1.04 15.49 + 2. 96 14.58 + 4.26 n-3 / n-6 3.39 + 1.53a 1. 68 + 0.44b 1.81 + 0.73b 1. 93 + 0. 08 b EPA/DHA 0.4 2 + 0. 00 0. 11 + 0. 04 0. 27 + 0. 02 0.2:'i + 0. 11 ARA / EPA O OR O OQ 01? 01R 36 Results Table 7 : The ration between fatty acid % in plasma lipid classes and dietary content on the same fatty acid (% Fatty acid in plasma / % fatty acid in diet) Fatty aeid in plasma lipid elasses FO Total 0.98 Oleie Aeid (OA) Polar 0.89 Neutral 1.41 Total 1.11 Palmitie aeid Polar 1.19 Neutral 1.16 Total 2.02 Araehidonie aeid (ARA) Polar 1.94 Neutral 0.79 Total 0.87 EPA Polar 0.76 Neutral 0.52 Total 2.16 DHA Polar 2.02 Neutral 1.05 35 30 '" 25 ! 20 ~ ~ ] 15 ~ ... .. ~ 10 5 O EPA Fatty acid 60 L 0.79 0.66 0.93 1.37 1.25 1.13 2.47 3.00 0.88 1.02 1.23 0.54 2.89 3.57 1.45 a b DHA 60 R 0.70 0.48 0.91 1.19 1.46 1.14 2.89 3.75 1.21 1.26 1.41 0.59 7.00 8.11 3.28 100 L 0.63 0.48 0.75 1.28 1.38 1.18 11.67 10.83 8.17 3.36 4.26 2.53 8.17 10.93 OFO .60 L 060 R 0100 L 4.83 Fig. 8: Levels of EPA and DHA in plasma total lipids from sea bream fed experimental diets. Values are ± SD for 3 pools of fish per treatment. Values for each fatty acid having a different column letter are significantly different (P< 0. 05 ). 37 "- '- '- '- Results 1,6 a 1,4 1,2 '" b "O OFO '¡¡ 1 " ~ .60 L :: 0,8 !! 060 R o a ~ 0,6 0100 L 't' 0,4 ab 0,2 O DGHLA ARA Fatty acid Fig. 9: Levels of DHGLA and ARA in plasma total lipids from sea bream fed experimental diets. Values are ± SD for 3 pools of fish per treatment. Values for each fatty acid having a different column letter are significantly different (P< 0.05). 35 lfI 30 ~ u 25 ni OFO ~ 20 l1li60 L ~ ñi 15 060 R ... a o 0100 L ... .... 10 o ~ o 5 O EPA DHA Fattyacid Fig. 10: Levels of EPA and DHA in plasma polar lipids from sea bream fed experimental diets. Values are ± SD for 3 polls offish per treatment. Values for each fatty acid having a different column letter are significantly different (P< 0.05). 38 DHGLA ARA Fatty acid OFO b . 60 L 060 R 0100 L Results Fig. 11: Levels of DHGLA and ARA in plasma polar lipids from sea bream fed the 4 experimental diets.Values are means ± SD for 3 pools of fish per treatment. Values for each fatty acid having a different column letter are significantly different ( P<O.05). 1, 65 1,5 1,35 1 ,2 :g 1,05 ~ 0,9 :: 0,75 o ~ 0,6 10,5 9 7,5 ~ 'v 6 ~ ~ 4,5 'o ~ 0,45 3 0,3 _ _____ , t 1,5 0,15 ........- 0 +--- - +---------1--- --+- -- -+ 0 Diets -+- ARA -+- DHGLA -'- EPA Fig. 12: Levels of eicosanoids precursors: ARA, DHGLA and EPA in plasma polar lipids from sea bream fed experimental diets. Values are means ± SD for 3 pools of fish per treatment. Values for each fatty acid having a different column letter are significantly different ( P<O.05). 39 ..: Q. ¡.¡ ~ ..: 0,2 a 0,15 0,1 0,05 b 12 10 4 '--:-.--C-: A R::-CAlE = P ::-CA ---' -+- AAlDHG LA 2 ° +----+----+----+----+ ° «0 ~~ ~ Diets Results Fig. 13: Levels of ARAIEPA and ARAIDHGLA in plasma polar lipids from sea bream fed experimental diets. Values are means ± SD for 3 pools of fish per treatment. Values for each fatty acid having a different column letter are significantIy different ( P<0.05). The fatty aeid eomposition of PI from leueoeytes is shown in table 8. Oleie acid was the predominant fatty acid in this lipid class and was more higher (up to 3.5 fold) in fish fed 60 L followed by fish fed 60 R (up to 3 fold) than FOfed fish. PI from leueoeytes eontained more saturates, monoenes and n-6 eompared with total and polar plasma lipids. By eontrast, this phospholipid class eontained lower levels of n-3 and n-3 HUF A giving a very low n-3/n-6 ratio. Fish fed fish oil ineorporated signifieantly more 12:0, 14:0, 15:0, 17:0 and 20:0. Fish fed fish oil had more DHA, EPA and ARA eompared to those fed vegetable oils, as well as 18:3n-6 and 20:3n-9. Oleie acid was markedly higher in PI from leueoeytes of fish fed vegetable oils eompared to FO-fed fish. AAIEPA was fairly eonstant among the treatments and higher than in polar and total plasma lipids. EPA/DHA was also higher than in plasma lipid. EPA/DHA eontents were lower in eomparison with plasma polar lipid ones, and was eonstant among dietary treatments. 40 Results Table 8: Fatty acids composition of leucocytes Phosphatidil Inositlol (PI) of sea bream fed experimental diets Fatty acid FO 60L 60 R 12:0 7. 61 ± 0. 94 2.69± 0.65 2.26 ± 1.31 14:0 8.18 ± 1.05 2.65 ± 0.56 2.68 ± 0.21 14: 1 1.61 ± 0. 11 1.80 ± 0.22 1.69 ±0.16 15:0 1.14 ±0.17 0.28 ± 0.10 0.46 ± 0. 08 16:0iso 5.53± 1.35 1. 52 ± 0.33 1.70±0.18 16:0 10.28± 2.72° 18.80±0.10 " 17.77 ± 1.33" 16: l n7 1.66 ± 0.20 3.55 ± 0.08 2. 17±1.15 16: In-5 1.24 ± 0.20 0.36 ± 0.10 1.28 ± 1.66 16: 2n-3 0.30 ± 0. 25 n.d. 0. 07±0 . 12 17:0 1.37 ± 1.02 0.13±0.ll 0.07 ±0.12 16:2 n -4 0.29 ± 0.22 0. 15 ± 0. 13 0.09 ± 0.16 16: 3n-4 0.39 ± 0. 55 0.22 ± 0.20 O.10 ±0.17 16: 4n-3 n.d. 0.08±0 . 13 n. d. 16:4nl 3. 73 ± 1.99 0.82 ± 0.17 0.86 ± 0.08 18:0 7.28 ± 0. 85 5. 0l± 0. 73 7. 03 ± 0.66 18 : 1 n-9 7. 84 ± 2.4r 31.13 ± 1.64" 25.93 ± 1.85° 18: In-7 0. 52 ± 0.74 1.96±0 . 14 1.76 ±0.18 18: In-5 0. 19± 0.26 0.16±O.15 0. 06±0.10 18: 2n-9 0.23 ± 0.33 0.08 ± 0.14 0. 31 ± 0.31 18:2n-7 0.60 0. 87 ± 0.33 0.64 ± 0.38 18: 2n-6 7.34 ± 2.84° 7.09 ± 1.16° 9.36 ± 2.59" 18: 3n-6 6. 81 ± 1.74" 2.5l± 1.19° 2.45 ± 0.94° 18: 4n-6 n.d. n.d. 0.06 ± 0. 11 18: 3n-l 0.48 ± 0.68 n.d. 0.08±O.13 18: 3n-3 0.74 ± 1.05" 5.44 ± 5.98" 5. 95 ± 4. 33" 18 : 4n-3 1.54± 0. 84 0.59 ± 0.27 0. 59 ± 0.20 18 : 4n-l 0.71± 1.01 0. 64 ± 0.40 0.68 ± 0.22 20:0 2. 50 ± 2.24 0.47 ± 0.09 0. 58 ± 0.06 20:1 n-9 + 20:1n7 1.53 ± O 1.57 ± 0.09 2.00 ± 0.55 20: 2n-9 0.29 ± 0.41 0.60 ± 0.60 n.d. 20: 2n-6 1.10 ± 1.55 0.30 ± 0.06 0.09± 0. 15 20: 3n -9 3.49 ± 1.63 " 1.05 ± 0.44° 1. 05 ± 0.34° 20: 4n-6 1.15 ±0.41" 0. 36 ± 0.16° 0. 7l± 0.08° 20: 3n-3 n.d. 0.08 ± 0.13 n.d. 20: 4n-3 n.d. 0. 14±0 . 12 0.06 ± 0. 11 20: 5n-3 4.90 ± 1.91" 2.42 ± 0.20° 2.90 ± 0.36b 22:0 n.d. n.d. 0.07 ± 0. 11 22: ln-Il+n -9 1.47 ± 0.42 0.78 ± 0.07 1.39 ± 0. 58 22: l n9 0. 93 ± 1. 33 n.d. 0. 09±0 . 16 22: ln-7 n.d. 0. 08 ± 0.14 n. d. 22: 3n-6 n. d. n.d. 0. 05 ± 0.09 22: 5n-3 0.28 ± 0.40 0.45±0.12 0.47 ± 0.02 22:0 n.d. n.d. 0. 05 ± 0.09 22: 6n-3 4.19 ± 1.77 3. 17 ± 0. 82 4.40 ± 0.83 Saturados 40.32 ± 8. 85 31.55 ± 1.86 32 . 73 ± 1.19 Monoenoicos 18.22 ± 3. 95 41.40 ± 1.95 36.46 ± 1.95 n-3 13.38 ± 2.08 12 .37 ± 5.95 14.45 ± 3. 53 n-6 17.58 ± 3. 85" 10.25 ± 1.88° 12 . 75 ± 3.77° n-9 15.28 ± 1.55b 34.43 ± 1.47" 29.45 ± 1.00" n-3HUFA 10.59 ± 1.17" 6.26 ± 1.200 7. 84 ± 1.160 AAlEPA 0.24 ± 0.01 0. 15 ± 0.07 0. 25 ± 0.05 EPAlDHA 1.19 ± 0. 85 0. 76±0.16 0.67± 0.09 n-3/ n6 0.77± 0.05° 1.32 ± 0. 91" 1.24 ± 0.55"° 41 Results 3Plasma prostaglandins The concentrations of PGE2 and isolated PGE3 in plasma are shown in Table 9. The concentration of PGE2 was no significantly different between the four treatments with the lowest value in fish fed FO (33.72 pg/ml) and the highest in fish fed 60 R (44.74 pglmI) (Figure 14). However, the concentration of PGE3 was significantly decreased with reducing EPA input in diet. Fish fed FO had significantly higher levels compared to fish fed 60 L and 60 R. Fish fed 100 L presented significantly the lowest plasma PGE3 level compared to the other treatments (Figure 15). Ta ble 9: Concentration of PGE2 and PGE3 in plasma from sea bream fed experimental diets Prostaglandins FO 60L 60R 100 L PGEz (pg/rnl) 33.72 ± 6. 52 41.87 ± 7.18 44.74 ± 16.47 35 . 15 ± 4. 58 PGE 3 (pg/rnl) 49 . 03 ± 0.49 a 41. 7 O ± 3.44 ab 40.77 ± 6 ab 33 . 89 ± 2. 55 b PGE3IPGEz 1.45 ± 0. 33 1.00 ± 0.22 0. 91 ± 0.46 0.96 ± 0.12 Values are means ± SD from three fish. Values in the same row with different superscript letters are significantIy different. The specificity ofthe antibody was 100 % for PGE2• 43 % for PGE3 and only 18.7 % for PGE¡. 70 60 ]' 50 .... c:. ;:;- 40 '-l ~ 30 " E ] 20 ... 10 O +- ~~ --,- ~~ L-~~ --L- ~~ --L- ~ Diel Fig. 14: PGE2 concentration in plasma fmm sea bream fed the experimental diets. Values are means ± SD from three fish per treatment. 42 '- Results 60 50 ~ ab ab ~ 40 ,e, b § 30 ... , ~ 20 ¡¡; 10 O ~O b~\.> b~~ \.>0 \.~~ Diel Fig. 15: PGE3 plasma concentration from sea bream fed the experimental diets. Columns assigned a different letter are significantly different (P<O.05). The study of correlation between POE3 and it's precursor (EPA) m plasma, is shown in Figure 16. A high correlation (0.98) was found between plasma POE3 and EPA concentration with a positive relation (Y=3.26X + 18.61). High correlation (0.96) was also found between plasma POE3 and EPA concentration in PI leucocytes (Y= 3.30 X + 32.59) (Fig. 17). 50 48 46 -44 ..§ ~ 42 .5 &l 40 " ... ~ 38 " ¡¡: 36 34 32 30 4 • 6 8 10 EPA in plasma (%. in polar lipids) • PGE3 -Lineal (PGE3) y = 3,26x + 18,61 R2 = 0,97 Fig.16: The relation between PGE3 (pg/ml) and EPA (% in polar lipids) concentration in plasma from sea bream fed the experimental diets 43 Discussion (the product of .1 5_ desaturase from 20:3n-6 and 20:4n-3 respectively) have decreased in the plasma of fish fed vegetable oils comparing with fish fed the cont ro l di et. This can be explained by the low activity of .1 5 -desaturase in marine fish (Sargent et al., 1995, Henderson and Tocher 1987; Tocher, 1993). At the same time, the lack of .1 5 -desaturase in this fish specie enabled DHGLA to accumulate in plasma membrane while the concentration of ARA remained low, which agree with the results found in turbot by Bell et al.(l995). DHA was the major PUF A in all lipid c1asses. The concentration of DHA in fish plasma w as hi gher than their dietary input. This fatty acid was especially esterified in PL with hi gh ratio comparing to NL. Hence, the ratio DHA in plasma PLI DHA in diet rise from 2.02 (in fish fed fish oil) to 10.93 (in fish fed 100 % vegetable oils); this can be explained by the preference of plasma cells phospho li pi ds to assimilate this fatty acid, through its high affinity with fatty acid binding protein (F ABP) (Sire and Vernier., 1981) and with the enzyme 1lisofosfatidilacilCoA transferase (Gurr and Harwood, 1991). Similar results were found in cod (Waagbo et al., 1995), European sea bass (Farndale et al., 1999) and sea bream (Caballero, 2002; Montero et al., 2003). Previous other studies have also demonstrated that DHA was preferentially retained under dietary essential fatty acids deficiency (Izquierdo et al., 1996, 2001; Montero et al. , 2001), showing the im portance of this fatty acid as a main structural component of fish membran es (Watanabe, 1993; Sargent et al., 1995). The plasma content in EPA followed broadly the dietary input, this fatty acid was especially incorporated in plasma PL and its concentration was significantly decreased in fish fed blends of vegetable oils, particularly with 100% substitution levels. This fatty acid was especially incorporated into PL c1ass with ratio of incorporation increased with increasing dietary LA and LNA, rising between 0.76 in fish fed control diet and the highest ratio (4.26) in fish fed 100% vegetable oils (with the lowest dietary EPA content). This may suggest a selective incorporation of this fatty acid in plasma membrane which agree with the results found sea bass leucocytes (Farndale et al., 1999) and sea bream 49 Discussion (Caballero, 2002). Together with DHA, EPA has been recorded to play important structural function in membranes (Sargent et al., 1995). ARA concentration in fish plasma was markedly decreased in plasma of fish fe d vegetable oils with the lowest concentration found in fish fed 100 % blend vegeta bl e oil. Deposition of ARA in both polar and neutral lipids was related to dietary fatty acid levels. However, the type of deposition in the two lipid classes was different as indicated by the ratio of tissue to dietary level. In NL, this ratio was small and did not change much from high to low dietary concentrations among the three first diets, but in fish fed 100 % vegetable oil this ratio markedly increased. By contrast, a stronger deposition was noted for low dietary concentrations in the case of PL, with ratio rising between 1.94 in fish fed control diet to 10.83 in fish fed 100% blend of vegetable oils. This finding agrees well with results found by Farndale et al., (1999), Montero et al., (2003) and Fountoulaki et a l. , (2003) indicating the importance of this fatty acid for the proper cell function. The relative deposition of ARA in PL indicates that this fatty acid is under a specific control (Fountoulaki et al., 2003). Waagbo et al. , (1995) have also found that in cod head kidney, the ratio ARA in macrophages/ARA in diet varied between 5 and 14. Other studies demonstrated that t hi s fatty acid w as retained in case of fatty acid deficiency (Izquierdo et al. , 1996) and was incorporated and retained in phosphatidyl inositol of membrane from sea bream ( Mourente & Tocher, 1993) and turbot (Linares and Henderson, 1991), it is also considered as the main PUF A in this phospholipid class (Bell and Sargent., 2002). Henc e, evidences suggest that PI accumulate selectively 20 PUF A suggesting that this phospholipid class may be a source of precursors for the synthesis of eicosanoids (Bell, 1995). PI of blood leucocytes was very rich in oleic acid in fish fed diet 60 L and 60 R, despite the different levels in the diet, suggesting the competition of this fatty acid with palmitic acid in its frequent 1 sn-position of this phospholipid class, explaining the low content in the latter fatty acid. Comparing the fatty acid composition of PI and total polar lipids one, it appears that PI is less rich in PUF A and the PUF A represented only around 30 50 '--o Discussion % of the tot al content of this fatty acid c1ass, this might be explained by the competition of other fatty acids to esterification in sn-2 of PI. It is well established that the deposition of fatty acids in tissue lipids is strongly affected by dietary fatty acids (Bell et al., 1994; Sargent et al., 1995). In this stud y, n-3 HUF A were much higher in plasma of fish fed FO and decreased with increasing dieta ry LA and LNA input, but it was proportionally much higher than in dieto Nevertheless, plasma n-6 content was significantly different among treatments according to dietary input, with the lower level in fish fed FO diet and the highest in plasma of fish fed 100% vegetable oils. DHA, EPA, ARA and DHGLA were more abundant in polar than in neutral lipids, which confirm the role of polar li pids especially PI in accumulating C 20 fatty acids (Bell et al., 1994). The existence of a competition between EPA and ARA during phospholipid esterification has been suggested to occur in fish (Bell and Dick, 1990). In this stud y, the fatty acids composition of PI from leucocytes, have shown a difference compared with plasma phospholipids contents. Even despite the diets 60 L and 60 R were very rich LA and LNA, those fatty were not very abundant in PI, whereas oleic acid was very high. By contrast with plasma phospholipids content, PI have not shown higher levels on DHGLA, but 20:3n-9 was strongly incorporated. N-3/n-6 was also significantly higher in fish fed vegetables oils than in those fed control dieto There was high content in saturate and monoenes in fish fed vegetables oils, which may be explained by their importance as a source of energy and their estherification in the sn-l position of this phospholipid c1ass. ARAIEPA was constant among treatments but was higher than in phospholipids content, which confirm the specificity of PI in accumulating ARA compared to the other phospholipids c1a sses. EPAIDHA was also higher than in plasma polar lipids. ARA is known to turn over rapidly in response to external signals for prostaglandins synthesis (Sargent et al. , 1989). There was also slightly higher levels of saturates and higher levels of polyunsaurates in plasma PL compared to NL in accordance with previous studies with freshwater fish (Henderson and Tocher, 1987) a nd Mediterranean species (Ibeas et al. , 1996). There was also 51 Discussion increased levels of mono enes from control to 100% vegetable oils fed fish. Thus, the substitution of dietary FO by blend of vegetable oils in this experiment have demonstrated a pr ofound modifications in plasma fatty acid composition. Long-chain PUF A possess a wide range of cellular functions, one of their most important is to supply precursors for the production of eicosanoids, which are bioactive fatty acid metabolites that can modulate many immune functions. Feeding vegetable oil could cause significant reductions in nonspecific immune parameters including haematocrit, total white blood cell and red blood cell counts, and macropahage respiratory burst in salmon (Good et al., 2001), and influence phagocytic activity of head kidney macrophages from sea bream (Montero et al., 2003). Fish offer a particularly useful model system for studying the production and interaction of eicosanoids derived from ARA and EPA because their tissues are naturally abundant in n-3 PUF A (Henderson and Tocher, 1987 ). Feeding dietary lipids with varying amounts of n-3 and n-6 PUF A, derived from fi sh and vegetable oils, have been demonstrated to affect the fatty acid composition, and in particular the incorporation of C 20 eicosanoid precursors, in gill and kidney of salmon (Bell et al., 1996a). The dietary treatments employed in the present study have resulted in profound alterations of ARAIDHGLA rat io , but not in ARA/EPA one. ARA, EPA and DHGLA are all precursors for eicosanoid production (Bell et al., 1994), changes in the ratios of these HUF A have important consequences for the quantity and spectrum of eicosanoids produced by the turbot (Bell et al 1998). The lowest ARAIDHGLA ratio was noted in phospholipids from fish fed 100% vegetable oils (2.21) followed by those fed 60 L and 60 R with only 60% ofFO substitution (3.94 and 3.44 respectively) and the highest ARAIDHGLA ratio in phospholipids of fish fed FO (l0.56), thu s, there was a significant decrease (up to 5 fold) in this ratio due the increase in DHGLA concentration in fish fed vegetable oils compared to fish fed control diet. The basal concentrations of eicosanoids in plasma from sea bream fed dietary treatments were also affected by feeding vegetable oils. The methods used to measure PGE2 and PGE3 concentrations in this study were different, 52 Discussion which may make di fficult the comparison of their concentrations. PGE2 was determined by direct quantification with EIA after purification. However, PGE3 was firstly separated with HPLC, coUected, purified and, lastly, quantified with EIA. The slightly similar amount of ARA-derived eicosanoids (PGE2) , regardless the diet fed, could be a reflection of the constant eicosanoids precursor (ARAIEPA) in plasma phospholipids (around 0.14) among fish fed the different dietary treatment s. Comparing with previous studies, feeding increasing dietary ARA level lead to significant increase in PGE2 concentration in heart, brain and kidney in turbot (BeU et al. , 1995). In vitro culture of turbot (Scophtalmus maximus) astrocytes have demonstrated that ARA is the preferred substrate for prostaglandin production, even when cultures are supplemented with other precursors fatty acids, and despite the excess of DHGLA or EPA in ceUular lipid, demonstrating that PGE2 was the major produced prostaglandin (Bell et al., 1994). Thus, in this stud y, despite a significant reduction in plasma ARA in vegetable oils fe d fi sh, PGE2 concentration remained constant which might be explained by the par aUe l reduction in EPA concentration leading to a constant ARAIEP A (eicosanoids precursors). Furthermore, the production of PGE3 was significantly different am ong fish fed the four experimental diets and strongly correlated with plasma polar lipid (0.98) and PI from leucocytes concentrations on EPA. Fish fe d FO, with high content in EPA, showed the highest PGE3 concentration (49.03 pg/ml of plasma), whereas fed 100% vegetable oils showed the lowest (33.89 pg/ml of plasma). However, in contrast with previous studies "in vitro" conducted in juvenile turbot (Scophtalmus maximus) and salmon (Salmo salar L.) (B eU et a l. , 1994; Henderson et al., 1996), PGE2 w as not the major prostaglandin p ro duced in fish plasma, since in fish fed FO, PGE3 was higher than PGE2 with a ratio PGE3IPGE2 reaching 1.45. Among the different fish species studied to date, differences in the relative concentrations of these substances have been found. The production of these compounds have been also reported to be di fferent among tissues of the same species. Thus, PGE2 production was hi gher in kidney macrophages than in blood leukocytes in turbot 53 IX. Discussion (Tafalla et al., 1999). Further studies of prostanoid production in different tissues and species are required to clarify this effect. Feeding vegetable oils lead to a decrease in plasma EPA which in tum give caused a reduction in plasma PGE3 concentration. Phospholipid fatty acid composition is determinant of the physical properties of cell membranes which can influence the activities of membrane-associated proteins and enzymes (Spector and Y orek, 1985) and thereby influence the catalytic activity of the phospholipase (Bell et al. , 1996). The production of plasma PGE2 was not significantly di ff erent among the four treatments. Previous study, in rain bow trout, demonstrated that blood erythrocytes are not capable of eicosanoid synthesis (Pettit, Rowley and Barrow, 1989), thus plasma eicosanoids reflect principally leucocyt es products. Despite the plasma PGE2 levels were not significantly different, the lower production in fish fed only fish oil fed fish may be explained by the higher content of EPA in the PI of this fish, since this fatty acid is . a potent competitor of AA. Besides the slight increase of PGE2 between 60 L to 60 R agrees we ll with the slight increase in PI content on AA and the lower EPA leve ls in comparison with fish fed fish oil. In the present study, we found that substitution of fish oil with vegetable oils in diets for sea bream can affect profoun dl y plasma PUF A composition which in tum affect 2and 3series prostaglandins production. In our experiment, it is notable in total and in polar lipid fatty acid composition that the amount of DHGLA was increased up to 4-fold in fish given the highest dieta LA (diet with 100 % vegetable oils); this fatty acid (as precursor of PGE\) is known to inhibit the production of ARA-derived lipoxygenase metabolites (Miller et al., 1990) by increasing the synthesis of camp (Horrobin 1980 ). Supplementation with both DHGLA and EPA significantly reduced ARA-derived prostaglandins production in salmon (Bell et al., 1994) which indicate a competitive inhibition by those fatty acids at the cyclooxygenase active site (Bell et al., 1994). AIso, phospholipase activity, which is the key of release of eicosanoid precursors fatty acids, is affected by the dietary fatty acids. In salmon fed diet a reduced n-3/n-6 PUF A ratio, the ~ 54 Discussion phosph ol ipase A activity was increased in cardiac tissue compared to those fed fish oil (Bell et al., 1993). Feeding fish with borage oil (rich in 18:2n-6 and 18:3n-6 precursors of DHGLA) decreased significantly PGE2 concentration (Bell et al., 1995). The ability of DHGLA and EPA to attenuate the production of ARA derived eicosanoids is fundamental in the control of pathophysiological processes in numerous in fl ammatory conditions occurring in human (Horrobin, 1992). In this study we did not analysed plasma PGE¡ concentration, therefore we can not elucidate the effect of this slight increase in plasma DHGLA on PGE¡ production. It is notable in total and polar lipid fatty acid composition that the amount of DHGLA was increased in fish given the highest dietary LA (diet with 100 % LO), evidences exist suggesting that increase in concentration of DHGLA may increase PGE¡ production and consequently reducing PGE2 one. This fatty acid is known to inhibit also the production of ARA-derived lipoxygenase metabolites (Miller et a l. , 1990). Thus, it might be an increase in plasma PGE¡ in fish feed vegetable oils compared with those fed FO. PGE2 is a mediator of inflammatory activity (Kinsella et al 1990), and is fundamental in the control of pathophysiological processes prevalent in numerous inflammatory conditions occurring in human populations (Horrobin, 1992). But high production of these compounds by leucocytes may be responsible for the severity of lesion (Bell et al., 1993). L TB4 (derived from the same precursor) is a pa rticularly powerful chemoatractic agent for neutrophils (Strasser et al., 1985 ), active in stimulating cellular uptake of ca1cium in human (Hunt and Rowley, 1986) and is able to increase lymphocytes proliferation (Secombes et al., 1994). While L TBs (derived from EPA) has similar activity but it is around 30 times less potent (Lee et al., 1984). There is good evidence that eicosanoids promote inflammatory processes and they are vital for the activation, proliferation and differentiation of B and T lymphocytes during the immune response (Yamaoka et al., 1989). The reduction in production of prostaglandins, or alteration in the spectrum and efficacy of prostaglandins produced, may be sufficient to alter immune cell composition and function ( Kinsella et al., 1990), and cause increased inflammatory activity. 55 Discussion In summary, the present study have clearly demonstrated that inclusion of higher levels of vegeta bl e oils in diets for sea bream may profoundly affect the fatty acid composition of their plasma and leukocytes, especially HUF A and consequently the production of eicosanoids in these cells. Thus, in sea bream, which seems to have a low /15 than /1 6 desaturase activity, there is a potential to provide membrane phospholipids with a high variability in the ratio of eicosanoids precursors, DHGLA/ARAIEPA. 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