62 SEPTEMBER 2013 • WORLD AQUACULTURE • WWW.WAS.ORG Along with proteins, lipids are the major organic constituents of fish, with carbohydrates quantitatively less prominent. Lipids are an important source of metabolic energy, components of biological membranes and precursors of essential metabolites (Sargent et al. 2002). Most lipids are comprised of fatty acids (FA) and fish are rich in long-chain polyunsaturated fatty acids (LCPUFA), which have chain lengths of 20 or more carbons and three or more ethylene (double) bonds. To achieve normal growth and development, including reproduction, fish require three LC-PUFA: docosahexaenoic acid (DHA; 22:6n-3), eicosapentaenoic acid (EPA; 20:5n-3) and arachidonic acid (ARA; 20:4n-6). The essentiality of these fatty acids is corroborated by selective retention throughout embryonic development (Rainuzzo et al. 1993) or at the expense of other fatty acids during periods of starvation (Tandler et al. 1989). Moreover, these fatty acids are considered essential in marine fish species because of the limited activity of Δ6- and Δ5-desaturases and elongases, enzymes capable of synthesizing ARA, EPA and DHA when their precursors are included in the diet, in contrast to freshwater species. The Importance of DHA The particular structure of DHA provides this fatty acid with many important functions in fish metabolism (Izquierdo 2005). It is incorporated into cell membranes – regulating membrane integrity and function – and is an important component of phosphoglycerides in larvae. DHA is also retained in starved or low-essential fatty acid fed fish, possibly due to a lower cell oxidation rate of DHA than other fatty acids (Koven et al. 1989). However, in terms of its essentiality, DHA seems to have a greater potential than EPA to promote growth and stress resistance in red sea bream, among other species (Watanabe and Kiron 1994) and, compared to other LC-PUFA, the requirement for DHA is more limiting for growth and survival (Izquierdo 1996). The importance of DHA for marine larvae starts prior to hatching. Eggs include an adequate DHA content (Laurel et al. 2011) to ensure proper embryonic and larval development, as controlled by broodstock feeding (Fernández-Palacios et al. 2011). Thus, DHA, vital for early survival and development of newly hatched larvae, is determined by the lipids derived directly from the dietary input to broodstock in the period preceding gonadogenesis (Sargent 1995). However, the DHA content of marine fish larvae rapidly decreases during the first ten days after hatching (Watanabe 1993). Therefore, DHA must be supplied to maintain adequate levels of this LC-PUFA in developing larvae (Table 1). The high DHA requirement is reflected in larval tissue composition. In particular, DHA is incorporated into developing optical and neural tissues (Mourente 2003) where, at this stage, it accounts for a higher proportion of neural tissue in the relatively small body mass of larvae. Oxidative Stress in European Seabass Larvae Mónica B. Betancor, Marisol Izquierdo and Mª José Caballero Effects of Free Radical Damage Excessive levels of DHA may lead to deleterious alterations in fish, especially when the increase in LC-PUFA is not accompanied by adequate quantities of antioxidants. The high degree of unsaturation of DHA makes it prone to attack by reactive oxygen species (ROS) that, in turn, may cause lipid oxidation. Thus, to avoid adverse effects and improve performance, supplementation of antioxidants such as vitamin E is necessary when high levels of n-3 LC-PUFA are incorporated into larval diets. For instance, growth and tolerance to salinity stress of beluga larvae fed with Artemia enriched with LC-PUFA was better when 20 percent α-tocopherol was included in the enrichment media (Jalali et al. 2008). Similarly, in vivo and in vitro oxidation of lipids in turbot larvae muscle is reduced when the diet is supplemented with α-tocopherol (Stéphan et al. 1995). In juvenile and adult fish, free radical damage can lead to diseases such as haemolysis (Kawatsu 1969), anaemia (Cowey et al. 1984), jaundice (Sakai et al. 1989), liver degeneration (Cowey et al. 1984) or skeletal alterations (Lewis-McCrea and Lall 2007). Among these skeletal alterations, one of the most frequently described in juvenile fish and adults is muscular dystrophy (Lovell et al. 1984, Gatlin et al. 1986, Frischknecht et al. 1994, Bowater and Burren 2007). Correspondingly the incidence of muscular dystrophy in European seabass (Dicentrarchus labrax) larvae increased with graded levels of dietary DHA (from 1 to 5 percent) (Betancor et al. 2011). This study reported for the first time the appearance of nutritional muscular dystrophy in marine fish larvae. A previous work (López-Albors et al. 1995) described an early myopathy affecting the myotomes of recently hatched European seabass larvae. However, the authors did not perform any biochemical analysis and were therefore unable to relate the appearance of lesions with a lack of any antioxidant nutrient or an excess of dietary lipid. ROS Detoxification: Antioxidant Defense System An antioxidant is any substance that, when present at low concentrations compared to those of an oxidizable substrate, is able to interact with free radicals to terminate the reaction (Halliwell and Gutteridge 1990). The antioxidant systems in living organisms may be divided into two types: one represented by enzymes (antioxidant enzymes; AOE), such as catalase (CAT), superoxide dismutase (SOD) or glutathione peroxidase (GPX); and the other by low molecular weight molecules, such as vitamins C and E, selenium or carotenoids that must be provided through the diet. As previously mentioned, increases in dietary DHA must be accompanied by increases in the antioxidant content. In this sense, for a given dietary DHA content, an increase in vitamin E from 1500 to 3000 mg/kg reduces the incidence of muscular lesions and increased growth and survival following application
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