Fish Physiology and Biochemistry 22: 97–107, 2000. © 2000 Kluwer Academic Publishers. Printed in the Netherlands.
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Recent advances in lipid nutrition in fish larvae M.S. Izquierdo, J. Socorro, L. Arantzamendi and C.M. Hern´andez-Cruz Grupo de Investigaci´on en Acuicultura, ICCM & ULPGC, Ciencias B´asicas, Tafira Baja, 35017 Las Palmas de Gran Canaria, Canary Islands, Spain (Fax:34-928-132908; E-mail:
[email protected]) Accepted: August 5, 1999
Key words: AA, absorption, DHA,digestion, EPA, fish larvae, lipid, nutrition
Abstract Due to the importance of dietary lipid utilization for larval rearing success, increasing attention has been paid during the last years to different aspects of larval lipid nutrition such as digestion, absorption, transport and metabolism, which are frequently studied by different research groups. The present study reviews the published information on these aspects, including some recent results obtained in our laboratory, that contribute to a better understanding of larval lipid nutrition. Neutral lipase activity was found in the digesta of larval gilthead seabream as early as first feeding, followed by a significant increase which reached up 8 times the initial levels at day 15 and was clearly influenced by the fatty acid composition of dietary lipids. Accordingly, the capacity for lipid absorption by the intestinal epithelium has been also observed at the onset of exogenous feeding, although the specific location in the different digestive tract segments differ with species. Whereas the capacity to absorb lipid increases with development in live prey-fed larvae, this improvemment is delayed in larvae fed formulated diet. Increasing dietary phosphatidyl cholines levels enhanced lipid absorption regardless of whether it is of soybean or marine origin, but the latter improved hepatic lipid utilization. Enzymatic, histological and biochemical evidences suggest that marine fish larvae are able to effectively digest and absorb n-3 HUFA-rich triacylglycerols, but feeding with phosphoacylglycerols, particularly if they are rich in n-3 HUFA, would enhance phosphoacylglycerols digestion and specially lipid transport alowing a better n-3 HUFA incorporation into larval membrane lipids and promoting fish growth. Although the essentiality of n-3 HUFA for larval marine fish has been studied extensively, only recently has the importance of dietary arachidonic acid in the larvae of few species been recognised. Evidences for competitive interactions among these essential fatty acids suggest that besides a minimum dietary requirement for each essential fatty acid, their relative ratios must also be considered. Abbreviations: AA – arachidonic acid; BAL – non-specific bile salt activated lipase; CE – cholesterol esters; CHO – cholesterol; DHA – docosahexaenoic acid; EPA – eicosapentaenoic acid; HUFA – highly unsaturated fatty acids; MPL – mammalian pancreatic lipase; PL – phosphoacylglycerides; PC – phosphatidylcholine; PE – phosphatidylethanolamine; PI – phosphatidyl inositol; PUFA – polyunsaturated fatty acids; TG – triacylglycerides; VLDL – very low density lipoprotein. Introduction The marked development of finfish culture, which at present accounts for more than half of the world’s total aquaculture production, is causing an increasing demand for good quality fish fry. Except for salmonid and few other non-salmonid species, fish fry production largely depends on rearing delicate fish larvae
which hatch at an early stage of development. The success of larval rearing is greatly influenced by first feeding regimes and the nutritional quality of starter diets, with dietary lipids being recognized as one of the most important nutritional factors that affect larval growth and survival (Watanabe et al. 1983). However, dietary lipid utilization by the larvae may be directly
98 or indirectly affected by several morphological and physiological changes that occurr during larval development. Among such changes, those that take place in the digestive system will clearly affect dietary nutrient utilization and the type of feed which should be offered to the larvae. For instance, although at the end of the larval lecithotrophic phase the enterocytes of seabass (Dicentrarchus labrax) larvae are functional, they are still poorly developed (Deplano et al. 1991), their size, number and expansion of organelles being increased in the following days. Throughout larval development the number of intestinal folds is also increased, the stomach is formed and its function improved, and these changes in enterocytes and the digestive system imply an improvement in the digestion and absorption efficiency of the juvenile. Another interesting fact is the uptake of intact protein by pinocytosis that is observed in the the rectal epithelium and claimed to be an important nutrient uptake system in fish larvae. This could allow the direct absorption of enzymes or hormones present in live prey-fed to larvae and points out the posibility of including such proteins in formulated larval diets. Changes also occur in the endocrine system which regulates larval development and affects lipid metabolism. According to Tanaka et al. (1995), the development of the endocrine system seems to occur in three major phases: the embryonic phase, during which the organs are not functional and maternal hormones decline, the transition phase, during which the endocrine organs begin to form but hormone levels remain very low, and the transformation phase during which organ activity is accelerated and hormone levels show great fluctuations. Hormones such as thyroxine, cortisol, growth hormone and prolactin have all been shown to interfere with fish lipid metabolism. For instance, T4 treatment may stimulate fat absorptive functions in the larval digestive tract (Tanaka et al. 1995). The levels of these hormones in several tissues (pituitary, thyroid and interrenal glands and pancreatic islets) change during development, being very low or undetected at hatching but increasing as the larvae grows up to metamorphosis and showing different peak patterns among them (Hwang et al. 1992; Tanaka et al. 1995). As a consequence of these changes or others occurring in gas exchange, swimming and metabolism and the formation of various organs and systems, variations may be expected in the efficiency of digestion, absorption, transport or metabolic utilization of dietary lipids during larval development. In recent years there has been an increasing interest in all these
aspects of lipid nutrition in fish larvae, due to the importance of dietary lipid utilization for optimal larval growth and survival. Because these different aspects of lipid nutrition are frequently studied by different research groups, an overall view of the published information together with some recent results obtained in our laboratory may provide a better understanding of larval lipid nutrition.
Lipid digestion Because they have a less complex digestive system, larvae might be expected to have a lower digestion efficiency than adult fish. Nevertheless, digestive enzymes are present from the onset of exogenous feeding in most fish species. Although most of the recent studies of digestion in fish larvae focus on the enzymes that are involved in dietary protein and carbohydrate digestion, lipase/esterase activity was early reported in larvae of marine fish species such as seabass (Alliot et al. 1977) and turbot (Scophthalmus maximus) (Cousin et al. 1987). Several types of lipases have been recognized in the digestive tract of juvenile and adult fish. Among them, non-specific bile salt activated lipase (BAL) activity has been suggested to play an important role in digestion of neutral lipids in some fish species such as anchovy (Engraulis engrausicolus), striped bass (Morone saxatilis), pink salmon (Onchorhynchus gorbuscha), leopard shark (Triakis semifasciata), rainbow trout (Onchorhynchus mykiss), cod (Gadus morhua) and red seabream (Pagrus major). BAL has been purified from the pyloric caeca of cod (Gjellesvik et al. 1992) and its tertiary structure in Atlantic salmon (Salmo salar) has been also studied (Gjellesvik et al. 1994). Purified red seabream BAL shows an optimal pH in a range from 7.7 to 8.3 and its activity is increased by the addition of sodium cholate and sodium taurocholate, but not by sodium deoxycholate (Iijima et al. 1998). This type of enzyme catalyzes the hydrolysis of carboxyl ester bonds, not only of acyl-glycerols, but also of minor dietary fats including cholesterol esters (CE) and vitamin esters (Wang and Hartsuck 1993). There is also evidence of the presence of a mammalian type MPL in several species; this has been isolated in rainbow trout (Léger et al. 1977). This enzyme is activated by colipase in the presence of bile salts and it is specific for TG. A pancreatic lipase activity with an optimum pH of 7.3 at 37 ◦ C has
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Figure 1. Evolution of bile salt activated lipase activity (nanomoles 4-MU min−1 larvae−1 ) in the digestive tract of gilthead seabream during larval development.
been described in Dolphin fish (Coryphaena hippuras) (Divakaran and Ostrowsky 1990). Phospholipase A2 (PLA2) activity has also been found in several fish species (Izquierdo and Henderson 1998). PLA2 catalyses the hydrolysis of the fatty acid ester bond at the sn-2 position of phosphoacylglycerides (PL) and produces free fatty acids and lysophospholipids. It is secreted in the intestinal tract as an inactive proenzyme (zymogen) being activated by tryptic proteolysis. It catalyzes both dietary and biliary PL and it is Ca2+ dependent. PLA2 has been partially purified from red seabream hepatopancreas (Iijima et al. 1990) and pyloric caeca (Iijima et al. 1997) showing several isoforms. In pyloric caecae PLA2 activity is optimal in a pH range of 8 to 9, the enzyme being active only in the presence of bile salt and stimulated by the addition of sodium deoxycholate (Iijima et al. 1997). Despite such major advances in our understanding of lipid digestion in juvenile or adult fish, knowledge of lipolytic enzymes in young larvae is scarce, particularly of luminal digestion of dietary lipids. Very low lipase activity has been detected in a freshwater fish, the walleye pollock (Theragra chalcogramma), at the start of exogenous feeding (Oozeki and Bailey 1995). Esterase activity showing an optimum pH about 6.0 has also been found in larval turbot by Munilla et al. (1993). Lipase activity has been reported to increase in larval turbot throughout early development (Cousin et al. 1987) and both neutral lipase and phospholipase activities increase with weight in very young fish, as has been shown also in turbot (Izquierdo and Henderson 1998). Results of our recent studies of the luminal digestion of dietary lipids, applying the method devel-
oped by Izquierdo and Henderson (1998), failed to demonstrate any phospholipase A2 activity in gilthead seabream (Sparus aurata) larvae at first feeding, which could be linked to a limited sensitivity of the method to PLA2. On the contrary, the method was proved to be very effective in detecting neutral lipase activity in the digesta of larvae as early as first feeding (day 4). During the first days of larval development a significant increase was found in the lipolytic activity (BAL) which reached up to 5 and 8 times the initial levels in days 9 and 15, respectively (Figure 1). Although some authors state that a general lack of digestive capacity is not true for first feeding larvae (Segner et al. 1993) others suggest that the larvae rely mainly on the digestive enzymes of their prey (Munilla et al. 1993), and consider this as one of the beneficial effects of feeding larvae with live prey versus artificial diets. Several authors have included digestive enzymes in microdiets with variable degrees of success in terms of improving larval growth and survival. Thus, the addition of porcine pancreatic enzymes, including MPL, to formulated diets improved the lipid deposition in 20-34 day-old seabass (Dicentrarchus labrax) larvae, especially when larvae were also fed Artemia, although it did not affect larval growth (Kolkowski et al. 1997). Koven et al. (1993) observed that bovine MPL supplementation to microdiets for 21–45 day-old gilthead seabream larvae increased lipid absorption only in the older fish (32–45 days). These authors suggested that the level of dietary lecithin was insufficient or that the intestinal length was too short in early larval stages. It is worth noting that in a 45 day-old gilthead seabream, the digestive system is completely developed and the presence of functional gastric glands will determine changes in the luminal conditions in comparison with a 21 day-old larvae. Indeed, highest MPL activity requires, under the lumen conditions of the fish gut, an optimum pH lower than that found in the 21 day-old larvae. Although MPL has been detected in some species, this type of enzyme does not seem to be the main responsible for the digestion of neutral lipid in marine fish. Fats containing PUFAs, predominant in the marine environment and essential for marine fish, are more resistant to hydrolysis by MPL. In contrast, specificity for PuFAs has been demonstrated in cod lipase (BAL) (Gjellesvik et al. 1992). Thus, although porcine pancreatic lipase preferentially hydrolyzes 18:1n-9 followed by 18:2n-6 and 18:3n-3 esters, fish BAL preferentially hydrolyzes 20:4n-6 and 20:5n-3, followed by 18:2n-6 and then 18:1n-9 and
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Figure 2. Effect of feeding gilthead seabream with rotifers rich in docosahexaenoic, eicosapentaenoic or oleic acids on bile salt activated lipase activity (nanomoles 4-MU min−1 larvae−1 ) in the larval digestive tract.
22:6n-3 (Iijima et al. 1998). Our studies of the digestion of dietary lipids in gilthead seabream larvae have shown an increased neutral lipase activity when larvae where fed rotifers containing TG rich in EPA or DHA instead of monoenoic fatty acids (Figure 2). These results agree well with those of Iijima et al. (1998) and suggest that BAL is present in the digestive system of gilthead seabream larvae since early exogenous feeding and its activity is regulated by dietary lipids. In juveniles, lipase production is increased in mahseer (Tor khudree) when dietary sardine oil is increased (Muzaffar-Bazaz and Keshavanath 1993) and in carp (Catla catla) (Mukhopadhyay and Rout 1996) intestinal lipase activity is also enhanced by dietary n-3 PUFA (18–22 carbon atoms). Only recently, the distribution patterns of lipase activity along the various digestive tract segments of juveniles and adult fish have been studied and seem to differ with species (Koven et al. 1994a,b; Chakrabarti et al. 1995; Izquierdo and Henderson 1998). Neutral lipase activity is widely distributed along the digestive tract (Chakrabarti et al. 1995; Izquierdo et al. 1997; Izquierdo and Henderson 1998), with a lower proportion of the gut’s total activity in the stomach (Borlongan 1990; Koven et al. 1994b). In contrast with the higher lipolytic activity found in the anterior than posterior gut in milkfish (Chanos chanos) (Borlongan 1990), cod, gilthead seabream and red porgy (Pagrus pagrus) (Izquierdo et al. 1997), in species such as turbot (Koven et al. 1994b; Izquierdo and Henderson 1998) the major proportion of neutral lipase total activity occurs in the posterior region (Figure 3). Izquierdo and Henderson (1998) found that phospholipase activity was also higher in the posterior than
Figure 3. Neutral lipase distribution along different segments of digestive tract in juveniles of several marine fish species (Izquierdo et al. 1997; Izquierdo and Henderson 1998).
in the anterior intestine of adult turbot. These authors claim that the overall short digestive tract of turbot, with a more extensive folding of the mucosa and significant absorption further in the gut than in fish with a long gut, would be responsible for the higher neutral lipase and phospholipase activities found in the posterior intestine of this species. Interestingly this pattern of distribution of lipolytic activities found in adult turbot, almost disappears in juveniles (Izquierdo and Henderson 1998), suggesting a lower functional differentiation of the different digestive tracts in young fish.
Lipid absorption and transport Lipid absorption in fish resembles that of mammals. After intraluminar hydrolysis, dietary fat is uptake on the epithelial cells of the intestine by diffusion of a micellar form of monoglycerides and fatty acids. Reacilation is known to occur by two pathways: 1) the monoglyceride pathway in the smooth endoplasmic reticulum which synthesizes triglycerides and 2) the L-α-glycerophosphate pathway in both the rough and the smooth endoplasmic reticulum which synthesizes both TGs and PLs (Sire et al. 1981). Absorbed lipids are finally discharged into the submucosa as VLDL or chylomicron-like particles, the relative importance of both reacilation pathways and lipoproteins synthesis in fish has been discussed by several authors (Sire et al. 1981; Léger et al. 1988) and seems to be affected by dietary lipids (Sire and Vernier 1981; Caballero et al. unpublished data).
101 At the onset of exogenous feeding the larvae of several species appear to be able to absorb lipid by the intestinal epithelium. Specific locations in the different digestive tract segments seems to differ with species. Thus, whereas in coregonid larvae lipid absorption has been located mainly in the anterior part (Segner et al. 1989), in gilthead seabream it could be observed along the whole prevalvular intestine (authors’ unpublished data). Similar to gilthead seabream, in seabass larvae lipid absorption is found in the prevalvular intestine, but it is particularlly marked in the distal portion (Deplano et al. 1991). This distal location remains throughout larval development and it has been related by some authors, to the incomplete differentiation of the digestive tract, especially to the absence of a functional stomach (Deplano et al. 1991). Despite this precocious ability of seabass larvae to absorb lipids, at the end of lecithotrophic phase enterocytes are functional, but poorly developed, showing a scarcity of endoplasmic reticulum and Golgi apparatus as described by Deplano et al. (1991). Thus, only a small proportion of the absorbed lipids is incorporated into lipoprotein particles, suggesting a reduced lipid transport capacity. Along subsequent days of zooplankton feeding, a larger number of fat vacuoles is observed. The lipid transport efficiency of larval seabass seems to improve from day 9 onwards, when a clear intensification of lipoprotein synthesis is apparent together with an increase in glycogen deposition in the liver (Deplano et al. 1991). From day 18, Deplano et al. (1989) suggested an enhanced capacities for lipoprotein synthesis and transport than in the adult sea bass, based on the extreme development of the rough endoplasmatic reticulum and the Golgi system. However, if seabass larvae are fed artificial feed, lipid transport seems to be reduced due to the poor development of the endoplasmic reticulum and Golgi system in the enterocytes (Deplano et al. 1991). Lipid transport from the enterocytes to the hepatocytes of larvae fed inert diets has been suggested to be increased by dietary PL supplementation in prawn (Pennaeus japonicus) (Teshima et al. 1986a,c), carp (Cyprinus carpio) (Fontagné 1996) and some marine fish (Kanazawa 1993a). Effect of dietary PLs There is a general beneficial effect of PL addition to microdiets on the larval growth and survival of several fish species such as ayu (Plecoglossus altivelis) (Kanazawa et al. 1985), carp (Radünz-Neto et al.
1994), Japanese flounder (Paralichthys olivaceous), knife jaw (Oplegnatus fasciatus) (Kanazawa et al. 1983) and red seabream (Kanazawa 1993a). PCs and PIs have been claimed by several authors to be the main components in PL that are responsible for such effects, although the particular action of each lipid class seems to differ. Thus, PC seems more effective in promoting growth than PI in larval Japanese flounder (Kanazawa 1993a) and carp whereas PI seems to be more effective than PC in enhancing survival and preventing skeletal deformities in larval carp (Guerden et al., 1995a,b). The beneficial effect of both PC and PI in diets for carp larvae does not seem to be related to a correction of inositol or choline deficiency or to a potential emulsifying effect (Guerden et al. 1995a,b). The addition of soybean lecithin to larval diets enhances ingestion rates in prawn (Teshima et al. 1986b,c) and gilthead seabream. Koven et al. (1993) have shown that the dietary increase of PL enhances microdiet ingestion by gilthead seabream larvae. Total lipid digestibility is increased by the addition of soybean PC to diets for carp (Fontagné et al. 1998); this could be related to an enhancement of the PLA2 activity in view of the preferential affinity of the enzyme for PC (Iijima et al. 1998). Dietary PL also seems to have a marked effect on lipid transport. Feeding gilthead seabream larvae with diets without lecithin supplementation produces accumulation of lipidic vacuoles in the basal zone of the enterocyte and esteatosis in the hepatic tissue. Both responses are markedly reduced by the addition of 2% soybean lecithin, denoting an enhancement in the lipid transport activity in gut and liver (authors’ unpublished data). PL deficiency has been associated with an accumulation of fat droplets in the enterocytes of the anterior intestine of larval carp, suggesting that PLs have a specific role for VLDL synthesis as in mammals (Fontagné 1996). The accumulation of fat droplets is reduced by feeding PC, whereas PI is unable to prevent such alterations (Fontagné 1996). The enhancement of lipoprotein synthesis by dietary PC may be related to its predominance in fish lipoproteins as compared with other PLs (95% of total PL in lipoprotein) and a possible stimulation of apoprotein secretion as occurs in mammals (Field and Mathur 1995). Biochemical evidence of lipid transport enhancement by dietary PL has been found by Teshima et al. (1986b,c). These authors showed an increased lipid transport through the hemolymph, denoted by increased PC and cholesterol (CHO) content and also by
102 PC and CHO deposition in muscle of larval prawn fed with increased PL levels. Enhanced lipid transport, in turn, could be responsible for the improved lipid retention found in various species. Thus, increased dietary PL improves lipid retention, particularly PC and CHO, in larval prawn (Teshima et al. 1986a), increases labelled oleic acid incorporation in gilthead seabream (Koven et al. 1993) and enhances PL and TL retention as well as DHA incorporation into polar lipids of seabream larvae (Salhi et al. 1995). The fatty acid composition of PL also seems to determine the beneficial effect of these lipid classes. While PC and PI containing PUFAs improved growth and survival of larval ayu, growth was not improved by chicken-egg PC or dipalmitoil PC (Kanazawa et al. 1985). In a similar way, in larval Japanese flounder, soybean lecithin has been found to be more effective in promoting growth than chicken egg lecithin (Kanazawa 1993a). In larval gilthead seabream, the accumulation of lipidic vacuoles in the basal zone of the enterocyte caused by feeding diets without lecithin supplementation disappeared when 0.1% PC was added to the diet regardless of its (squid or soybean) origin. However, squid PC was more efficient in reducing hepatic steatosis than soybean PC (Figure 10), suggesting a combined effect of dietary PC and n-3 HUFA to further enhance hepatic lipid utilization. Indeed both types of molecules have been found to promote lipoprotein synthesis. Efficiency of dietary PL versus TG Dietary n-3 HUFA from PL or from TG seems to be more efficient in preventing essential fatty acid deficiency than free fatty acids (FFA). Thus, reduced larval growth obtained with rotifers enriched with fatty acid methyl esters instead of triglycerides has been related with the n-3 HUFA incorporation mainly into the FFA fraction in the rotifers rather than in the TG and PL fractions (Izquierdo 1988; Izquierdo et al. 1989). These authors suggested that besides the dietary fatty acid content, the molecular form in which they are present in the diet is also important for good growth and survival of larval marine fish. Indeed, the incorporation of dietary FFA into larval lipids seems to be lower than that of TG or PL. Studies with labelled dietary lipids have shown that the incorporation of dietary oleic acid in a free fatty acid form into gilthead seabream larval lipids is lower than the incorporation of oleic acid from triolein, when diets contained ma-
rine TG (Salhi et al. unpublished data). Besides, the incorporation of dietary PL into larval lipids is higher than that of dietary free fatty acids (Salhi et al. unpublished data) in agreement with previous studies (Izquierdo 1988), regardless of the type of dietary lipid used. The incorporation of different free fatty acids also seems to differ, that of EPA being higher than that of oleic acid. After feeding larval gilthead seabream with marine PL, there was a higher incorporation into larval PL of labelled dietary EPA than labelled oleic acid (Salhi et al. unpublished data). This seems to reflect the higher afinity of enzymes involved in PL synthesis and transport for EPA. Some authors have also suggested a more rapid digestion and a more effective incorporation of n-3 HUFA PL in comparison with n-3 HUFA TG (Ackman and Ratnayake 1989). Feeding larval gilthead seabream with microdiets that contain marine PL instead of marine TG significantly improves growth despite the slightly lower dietary n-3 HUFA levels of the former (1.5 versus 1.8%, respectively) (Salhi et al. 1999). Larvae that were fed marine TG showed an accumulation of lipid vacuoles in the basal zone of the enterocyte and hepatic steatosis, denoting the good absorption of dietary TG but also a reduced lipid transport to peripheral tissues, whereas feeding with marine PL markedly reduced lipid accumulation in both type of tissues. A higher lipid content due to accumulation of TG and CEs was found in larvae fed marine TG, whereas in larvae fed marine PL relative proportions of PC and PE were higher and richer in n-3 HUFA (Salhi et al. 1999). Enzymatic, histological and biochemical evidence suggests that marine fish larvae are able to effectively digest and absorb n-3 HUFA rich TG, but feeding with PL, particularly if they are rich in n-3 HUFA, will enhance PL digestion and specially lipid transport allowing a better n-3 HUFA incorporation into larval membrane lipids and promoting fish growth. Metabolic utilization of dietary lipids Dietary essential fatty acids Essentiality of n-3 HUFA for marine fish larvae has been reviewed by several authors (Watanabe and Kiron 1994; Izquierdo 1996; Sargent et al. 1999). Marine fish eggs are characterized by the high contents in DHA and EPA among other fatty acids, which are retained throughout embryonic development, particularly DHA, suggesting the importance of these
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Figure 4. Effect of dietary arachidonic acid levels on survival of larval gilthead seabream (Bessonart et al. 1999).
fatty acids for the developing embryo (Rainuzzo et al. 1993; Lie 1993). AA, EPA and, especially DHA are also preferentially retained in the larval lipids at the expense of other fatty acids during periods of starvation (Tandler et al. 1989), allowing the preservation of valuable essential components of biological membranes during such critical period. A deficiency in n-3 HUFA delays fish growth, induces mortalities and reduces resistance to stress (Izquierdo 1996) as it has been found in juveniles (Montero et al. 1998). In addition, it has been associated with some anatomical alterations such as hypomelanosis on the ocular side of flatfish (Kanazawa 1993b), disgregation of gill epithelium and hydrops. Some of these signs appear to be associated to other nutritional disorders such as insufficient fat-soluble vitamins or PL contents in diet (Izquierdo 1996). Based upon these effects, the optimum dietary levels for n-3 HUFA have been determined for larvae of several species, and they differ over a range between 0.3 and 39 g kg−1 (formulated diet or live prey) dry weight (Izquierdo 1996). DHA is known to have a higher efficiency as an essential fatty acid than EPA (Watanabe et al. 1989; Watanabe 1993), the former being particularly accumulated in the olfactory nerve, retina and central nervous system (Sargent et al. 1993). Although the essentiality of n-3 HUFA for larval marine fish has been extensively studied, only recent studies have demonstrated the importance of dietary AA in the larvae of a few species. In turbot juveniles, the higher growth found in fish fed chicken egg yolk PL has been related to a higher AA content (Castell et al. 1994). But the higher incorporation of AA into the polar lipid fraction of larval gilthead seabream caused by an increase in dietary soybean lecithin in diets containing marine PL, did not further promote
Figure 5. Effect of dietary DHA levels on gilthead seabream growth and incorporation of DHA and EPA into larval lipids (Dietary EPA and AA: 0.7 and 0.06% d.w., respectively).
Figure 6. Effect of dietary EPA levels on gilthead seabream growth and incorporation of DHA larval lipids (Dietary DHA and AA: 0.7 and 0.06% d.w., respectively).
fish growth (Salhi et al. 1995). In larvae of this species, dietary AA seems to be important for survival, but is not as efficient in improving growth as DHA or EPA. Thus, the elevation of AA levels from 0.1 to 1.0% in microdiets for larval gilthead seabream, significantly improved larval survival (Figure 4), but only
Figure 7. Effect of dietary EPA levels on gilthead seabream growth and incorporation of DHA and EPA into larval lipids (Dietary DHA and AA: 1.1 and 0.7% d.w., respectively).
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Figure 8. Effect of dietary AA levels on gilthead seabream growth and incorporation of DHA and EPA into larval lipids (dietary DHA and EPA: 1.2 and 0.7% d.w., respectively)
slightly enhanced growth (Bessonart et al. 1999). Larval total and polar lipids, particularly PC, increased with dietary AA levels, as well as dietary AA incorporation into neutral lipids (NL) and PL. AA seems also to play an important role in flatfish pigmentation (Estévez et al. 1997). Effect of different proportions among essential fatty acids Besides a minimum dietary requirement for each essential fatty acid, the relative proportions among the different PUFAs in the larval tissues seems to be related to the best growth rates. If competitive interactions among these fatty acids exist, it implies that there is a need to control both the dietary proportions as well as absolute amounts. As it has been shown for adult red seabream (Iijima et al. 1998), competition among AA, EPA and DHA could even begin from the digestion processes, since BAL has shown to have a higher affinity for EPA or AA than for DHA esterified to dietary neutral lipids. Since the acylases and transacylases that esterify fatty acids into the different phosphoacylglycerides have preferences for some fatty acids, competition among them is obvious. For instance, the affinity of PC and, especially, PE synthetases for DHA, particularly in the 2n position, seems to be very strong. On the one hand, elevation of DHA in diets for gilthead seabream from 0.7 to 2.6% at a constant EPA level (0.7%), increased the DHA content in the 2n position of larval PL and PE, whereas it only inhibited EPA incorporation into the 2n position of PE and not in the other lipid classes, larval growth being significantly improved (Figure 5). On the other hand, the elevation of dietary EPA levels from 0.3 to 1.1%, at
Figure 9. Digestive tract of larval gilthead seabream fed diets with(a) and without (b) 2% soybean lecithin dietary supplementation (Hematoxiline & Eosine staining. Lipid vacuoles were confirmed by Black Sudan techniques).
constant DHA and AA levels (0.7 and 0.06%, respectively), was not able to displace DHA from these lipid classes in larval gilthead seabream. On the contrary, it enhanced the incorporation of DHA in the 2n position of PC and PE, whereas EPA increased in PC but was even decreased in PE (Figure 6). As a consequence, larval growth was slightly improved. Further elevation of dietary EPA to 1.7% at constant DHA and AA levels of 1.1 and 0.7% (1-0.5 DHA/EPA), respectively, displaced DHA from the 2n position of the PC and increased the EPA in the 2n position of the PE, growth being reduced (Figure 7). This suggests the importance of a balanced proportion between 20 and 22 carbon fatty acids. Other evidence of the deleterous effect of EPA have been found by Takeuchi et al. (1992); these authors fed striped jack with increased dietary EPA (0.8 to 1.7%) (without DHA) and formed significantly reduced growth although survival was very similar between larvae fed any of the two levels (Takeuchi et al. 1992).
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Figure 10. Hepatic tissue of larval gilthead seabream fed diets containing 0.1% soybean (a) or 0.1% squid (b) PC (Hematoxiline & Eosine staining. Lipid vacuoles were confirmed by Black Sudan techniques).
In a similar way, the elevation of dietary AA from 0.1 to 1.5% (DHA:1.2%; EPA:0.7%) not only did not displace DHA and EPA from larval PL but it increased the incorporation of the three fatty acids into PC and PE in the 2n position (Figure 8). Interestingly, EPA levels were always higher than AA in these lipid classes, regardless the low dietary EPA levels, demonstrating the preferential incorporation of EPA to PC and PE. The increased incorporation of EPA into body lipids have been also found in larval Japanese flounder fed increased dietary AA levels (Estévez et al. 1997). The effect of further elevation of dietary AA and a possible competition with EPA for phosphatidylinositol synthesis deserves further studies in marine larvae. Moreover, competition for incorporation into the different lipid classes will probably show differences for each tissue since fatty acid composition of each lipid class markedly differs among cellular types (Lie et al. 1992). For instance, in juvenile turbot, the elevation of dietary AA up to 0.78% reduced the incorporation of EPA into the liver and brain PI (Bell et al. 1995).
Another well known level of competition between AA and EPA is the synthesis of eicosanoids. Both AA and EPA are adequate substrates for lipoxygenases to produce leukotrienes from 4- and 5-series, respectively, and for cyclo-oxygenases to produce 2and 3-series prostanoids, respectively. Lipoxygenases seem to have a higher affinity for EPA whereas AA is a preferred substrate for cycloxygenase, relative levels of both fatty acids in PI of different tissues regulating the proportional synthesis of each series (Hwang 1989). Products of each fatty acids have different physiological activities and in turn compete between them for cell receptors. Finally, the n-6 and n-3 fatty acid series also compete for desaturases and elongases for the synthesis of HUFA fatty acids. This type of competition has not received so much interest in marine fish due the low activity of those enzymes in such species, although it should be considered in particular formulated diets and feeding regimes. Evidence for such competitive interactions among essential fatty acids suggest that in orther to estimate the dietary requirements further research must be devoted considering not only their absolute amounts in formulated diets or live preys, but also the dietary proportions among them.
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