justification of the nutrition work planned in diversify
Four types of feeds are used in aquaculture, differing in nutritional and physical characteristics, following important changes in nutritional requirements during development: enrichment products for live prey, and dry feeds for weaning, grow out and broodstock, all differing in their formulation and production technology. DIVERSIFY will (a) develop adequate first feeding regimes, (b) identify the optimum dietary nutrient levels required for weaning and (c) build the knowledge on nutritional requirements to develop sustainable and cost-effective grow out diets for the candidate species. DIVERSIFY will also study the requirements or feeding regimes to optimize reproductive success in some of the species.
Feed constitutes the largest running cost for European aquaculture, reaching up to 70% in cage-based facilities. Moreover, suboptimal commercial feeds and feeding protocols result in direct economic losses through feed waste, poor growth and water quality deterioration (Kaushik, 1998). Also, feed composition affects fish health and welfare (Kiron et al., 2010; Montero and Izquierdo, 2010) and affects markedly fillet quality (Izquierdo et al., 2003) and consumer acceptance (Rosenlund et al., 2010). Species-specific formulations for aquaculture fish can improve markedly reproductive performance and progeny quality (Izquierdo et al., 2001; Fernández-Palacios et al., 2011), larval development and survival (Hamre et al., in press) and fry quality (Izquierdo et al., 2010, 2012). Considering the specific bottlenecks defined for the proposed species and the nutritional knowledge available, particular tasks will be addressed in this proposal to provide specific solutions for the species considered.
In meagre, DIVERSIFY Partners have developed successful first feeding protocols (Roo et al., 2009a, 2010a; Fernández-Palacios et al., 2009; Hernández Cruz et al., 2007; Scabini et al., 2008) and used different commercial enrichment products for larval rearing (Fernández-Palacios et al., 2007; Roo et al., 2007; Scabini et al., 2008; Abreu et al., 2009, Vallés & Estévez, 2011). However, feeding meagre during weaning and grow out with diets developed for other marine fishes (i.e., gilthead sea bream and European sea bass) restricts the fast growing potential of this species (Robaina et al., in press), affects negatively the fish appearance and fillet quality (Gines et al., in press; Poli et al., 2003) and may be the cause of Systemic Granulomatosis (Ghittino et al., 2004). Lack of specific diets for meagre is due mainly to the very limited information regarding its nutritional requirements (Chatzifotis et al., 2010, 2012). Early studies denote that this species has high protein requirements, while dietary lipids are restricted around 17% (Chatzifotis et al., 2010, 2012). However, digestible energy (DE) and digestible protein (DP) requirements are not yet determined. Requirements for essential fatty acids (EFA), such as docosahexaenoic (DHA), eicosapentaenoic (EPA) and arachidonic (ARA) acids, which play important roles in fish growth (Izquierdo & Koven, 2011), health (Montero & Izquierdo, 2010) and fillet quality (Rosenlund et al., 2011), have not been studied in juvenile meagre. Finally, there is an almost complete lack of information on the requirements for essential amino acids (EAA, particularly in combination with plant protein sources), vitamins and minerals (Robaina et al., in press). DIVERSIFY Partners, building up on already existing knowledge, have selected the most relevant nutritional aspects (DE, DP, EFA, EAA) for dose-response studies, whereas vitamin and mineral requirements will be studied in multifactorial or whole micronutrient package approaches. The lack of meagre-specific weaning and grow out diets will be addressed in DIVERSIFY, in order to maximize growth potential, enhance fry quality and promote health of this species. The WP on Nutrition – meagre will determine requirements of specific nutrients generally important for growth, welfare and health to address two of the main specific bottlenecks identified for meagre: variable growth rates during the pre-growing phase and variable growth during on-growing phase. Variable growth rates constrain production planning and may be related to different causes including unsuitable feed formulations. In meagre, despite its great growth potential, still both growth and feed utilization rates may be poor (Chatzifotis et al., 2010; Estévez et al., 2011) and feeds must be improved to consistently obtain high growth rates. Indeed, diets specially formulated to satisfy nutritional requirements of related species such as the mulloway (Argyrosomus japonicus, Woolley et al., 2010) and brown meagre (Sciaena umbra, Chatzifotis et al., 2006) produce better growth and feed utilization rates in these related species than those obtained until present in meagre. Despite specific diets for meagre would produce more consistent growth rates (Martínez-Llorens et al., 2011), there is not enough information on the specific nutritional requirements of this species (Chatzifotis et al., 2010, 2012).
Building up on already existing knowledge available for other Sciaenids such as mulloway, brown meagre and, particularly, the abundant information on the requirements of the red drum (Sciaenops ocellatus), DIVERSIFY selected the most relevant nutritional aspects to promote meagre growth and welfare. According to previous studies in several Sciaenids (Daniels and Robinson, 1986; Gatlin, 1995; Pirozzi, et al., 2010a; Kaushik, unpublished data), meagre seems to have higher protein and lower lipid requirements than seabream and seabass, whose commercial diets are used by European farmers to produce up to 2,387 t of meagre. Optimum crude protein and crude lipid contents in diets for meagre have been estimated up to now to be around 50% (Chatzifotis et al., 2012) and 17% (Panagiotidoum et al., 2007; Chatzifotis et al., 2010; Chatzifotis et al., 2012), respectively. However, protein requirements could be even higher and lipid requirements lower, if this species, as it occurs in mulloway, has a limited capacity to spare dietary protein (Pirozzi et al., 2010b). Thus, in a closely related species, utilization efficiencies for DP and DE are independent of fish size, ration level or temperature (Pirozzi and Booth, 2009; Pirozzi et al., 2010), whereas the red drum shows lower protein efficiency ratios (McGoogan and Gatltlin III, 1999; Peng et al., 2008).
Despite essential fatty acids such as docosahexaenoic (DHA), eicosapentaenoic (EPA) and arachidonic (ARA) acids have been found to be essential for growth and welfare of several Sciaenids (Gatlin III, D.M., 2009), the requirements for these essential nutrients have not been yet studied in meagre. Another essential nutrient for fish growth is vitamin E (Hamre and Lie, 1995; Kocabas and Gatlin, 1999; Montero et al., 1998; Tocher et al., 2003; Lin and Shiau, 2005; Abdel-Hameid et al., 2012) and despite its optimum dietary levels have been determined in other Sciaenids (Peng et al., 2008; Peng, and Gattlin, 2009), its requirements are yet unknown in meagre. Finally, antioxidant nutrients such as vit C and selenium have been also found to be essential for growth of other species of the same family (Sealy and Gatlin, 2002), despite their importance for meagre has not been yet study. Among aminoacids, lysine is considered as an indispensable amino acid for proper growth being the first limiting EAA in protein sources commonly used in fish feeds based on plant feedstuffs (Wilson, 2003), but its requirement has not been yet determined in meagre. Lysine plays an essential role as precursor of carnitine, which carries long chain fatty acids into the mitochondria for β-oxidations of lipids to produce energy (Walton et al., 1984).
Health problems in meagre may be related to the use of inadequate feeds, as it occurs in other aquaculture produced species (Cooke and Sneddon, 2007, Montero and Izquierdo, 2010). Farmers are also aware of the harmful consequences of nutritionaly-unbalanced diets on fish welfare (Conte, 2004). In turn, poor welfare conditions could not only markedly reduce meagre growth as it has been seen in a closely related species (Pirozzi et al., 2009), but also negatively affect immune system and disease resistance. Imbalances in nutrition negatively affect fish welfare and health in many fish species (Lall, 2000). The importance of nutrition on welfare and health involve almost all physiological functions related to health, including a direct effect on modulation of the immune system, stress response, mechanisms of defense against infection or tissue integrity (Waagbø, 2006). Among different nutrients, deficiency in essential fatty acids or antioxidants including vitamin E and vitamin C, negatively affect fish welfare, increasing plasma cortisol levels and affecting fish behaviour and stress responses (Montero et al., 1998; Montero et al., 1999). Besides unbalanced levels of these nutrients cause inmune deficiencies, pathological features in several tissues and reduce disease resistance in several fish species (Montero and Izquierdo, 2010; Betancor et al., 2012a, 2012b) including some Sciaenids (Sealey and Gatlin, 2002). Amino acids and their metabolites have been characterized as important regulators of main physiological pathways that are required for fish maintenance, growth performance, feed utilization, protection from oxidative stress and resistance to environmental stressors and pathogenic organisms (Wilson and Halver, 1986; Li et al., 2009). Among them, lysine has a role in calcium absorption and formation of collagen, a substance important for bones and connective tissues including skin and cartilage (Civitelli et al., 1992), being essential for the health status of these tissues. Despite the importance of those nutrients on fish health, the nutritional requirements of meagre have not been yet defined particularly in relation to welfare and health issues.
Being a carnivorous species, meagre should have a high requirement for digestible protein (DP), which may be affected by fish size and temperature. Previous work has been carried out in a closely related species, the mulloway in Australia by Pirozzi et al (2010), but data obtained from mulloway cannot be directly transferred to meagre since both species are cultured under greatly different temperature conditions. In this sense, mulloway may be considered as a warm water species with an optimal temperature range comprised between 25 to 26.4ºC (Bernatzeder & Britz, 2007), whereas meagre is a temperate species inhabiting the Mediterranean and Black Sea, and along the eastern Atlantic coast, where the best temperature for rearing meagre lies between 17 and 21ºC. As nutrient utilization efficiencies have been shown to be influenced by many different factors such as species effects, fish size and temperature (Bendiksen et al. 2003; Moreira et al. 2008), it is clear that optimal DP:DE data obtained for mulloway cannot be transferred to the meagre industry without previous experimental validation. Thus, a species-specific research on the nutrition of this species (DP:DE) is needed in order to optimize actual feeding practices and reduce one of the main bottlenecks identified by the industry during the on-growing phase of this species, which is the highly variable growth rates that may be linked to unsuitable feed formulation (of which DP:DE is the key aspect determining growth), among other potential reasons that will also be investigated (mainly potential genetic effects).
Contrary to meagre, the greater amberjack exhibits high mortalities during larval development, thus juvenile availability is a major bottleneck to its industrial production, in addition to reproduction control. Previous studies conducted by the Partners on ontogenic development (Abreu et al., 2009) and larval rearing techniques (Roo et al., 2010b, Grossi et al., 2009) have shown that larval greater amberjack perform poorly when fed the available commercial enrichment products. However, the elevation of certain nutrients in experimental enrichment products (Fernández-Palacios, unpublished data) increased up to 5 fold larval survival (Yamamoto et al., 2008). A recent review on larval nutrition (Hamre et al., in press), as well as preliminary studies on greater amberjack (Yamamoto et al., 2009) point to the importance of EFA, vitamins E and C, carotenoids and taurine (Tau) as essential nutrients during fish development. DIVERSIFY Partners will study these nutrients through dose-response and multifactorial approaches, in order to develop specific live food enrichments and improved weaning diets. Regarding grow out, information on greater amberjack nutritional requirements is scarce (Aly et al., 1999 (lipid sources); Talbot et al., 2000 (Lipid levels); Takakuwa et al., 2006 (DP/DE); Vidal et al., 2008 (DP/DE); Uyan et al., 2009 (phospholipid levels)). However, the close congener yellowtail (S. quinqueradiata) has been studied extensively (i.e., Furutani et al., 2012 (alternative ingredients); Matsunari et al., 2005 (Tau); Ren et al., 2008 (Vit C)) and high requirements in protein, DHA, Tau and antioxidants are foreseen for greater amberjack. Therefore, DIVERSIFY partners will build up on already existing knowledge available for this and other Seriola species (Watanabe and Kiron, 1994; Sakai et al., 1998; Takeuchi, 2001; Liu, 2001; Uyan et al., 2008; Kolkovski et al., 2009, 2010; Miki et al., 2011), to specifically address lack of knowledge on nutrients that are particularly important for larval development, juvenile performance and reproduction.
A main bottleneck for the mass production of high quality greater amberjack juveniles is the low survival and growth obtained during larval development and metamorphosis (Yamamoto et al., 2009). However, larval greater amberjack perform very poorly when fed the available commercial enrichment products for live preys. The partners are familiar with the abundant information produced in Japan and Australia regarding larval rearing of greater amberjack (Yamamoto et al., 2009), but very few studies have determined the specific nutritional requirements of this species testing a sufficient number of diets varying only the target nutrient. Thus, despite Yamamoto et al., (2009) pointed out the importance of essential fatty acids, antioxidants such as vitamins E and C, carotenoids and taurine as essential nutrients for greater amberjack larval development, the specific requirements for these nutrients have not been yet determined. For instance, Matsunari and coworkers (2012), studied the effect of four enrichment products for rotifers, such as Chlorella or a commercial emulsion that besides having different docosahexaenoic acid (DHA) content also differ in many other nutrients and other compounds that also may affect larval performance such as fat-soluble vitamins, pigments, antioxidants, minerals, etc. Moreover, despite they tested a range of DHA (0.0-1.9 mg g), DHA requirements for other Seriola species (i.e., yellowtail, S. quinqueradiata) are known to be the highest among all studied species (Takeuchi, 2001), well above the levels studied by Matsunari and co-workers (2012). Indeed, essential fatty acid requirements in rotifers may be as high as to 3.9% in the fast growing larvae of yellowtail (Kolkovsky et al., 2010). The relevance of DHA in diets for marine fish larvae has been well documented (Watanabe et al., 1989; Izquierdo, 1996; Sargent et al., 1999) and its positive effect on survival has been related to its important role in stress control (Watanabe et al., 1993; Izquierdo, 2005, Ganga et al., 2006), immune system development (Montero et al., 2003) and improvement of health and bacterial resistance in fish larvae (Brandsden et al., 2003). Moreover, DHA has been found to increase eye diameter and density of photoreceptors in gilthead seabream larvae (Izquierdo et al., 2000) and, in agreement, visual capacity was found to be reduced in yellowtail fed DHA-deficient diets (Masuda et al., 1999). Although both arachidonic and eicosapentaenoic acids are also considered essential and play important roles in fish metabolism, not only their absolute amounts but also their relative proportions are determinant of larval growth and survival as it has been seen in other species (Izquierdo and Koven, 2010). For instance in gilthead seabream, dietary ARA is more efficiently incorporated into larval tissues than EPA (Atalah et al., 2011) and therefore EPA/ARA ratios become lower in larval tissues than in diets. Similarly, increased dietary EPA has been found to reduce dietary DHA incorporation into larval tissues, higher EPA/DHA ratios decreasing larval growth (Rodríguez et al., 1997, 1998). Up to date, the optimum EPA/ARA and EPA/DHA ratios have not been yet determined in enrichment products for greater amberjack, despite their importance for larval performance. The high polyunsaturated fatty acid requirements forseen in greater amberjack increase the risk of lipid peroxidation in this species. For instance, in yellowtail lipid peroxidation as a consequence of an imbalance between polyunsaturated fatty acids and antioxidants damages the biomembranes, producing several pathological conditions (Sakai et al. 1998) and causing irreversible changes in the developing tissues of marine fish larvae. Therefore, the enrichments must contain high levels of antioxidants, such as vit E (Betancor et al., 2011; Robaina et al., in press) and carotenoids. Despite taurine requirements have not been determined in greater amberjack larvae, the requirement of this aminoacid for very young juveniles of yellowtail (Matsunari et al., 2005) suggest its importance in early larval stages of Seriola spp as proposed by Yamamoto et al. (2009).
Regarding on-growing, despite no specific diets are produced in Europe for this species, commercial diets for other Seriola spp are available in the Pacific region, although they are far from satisfying the requirements of this species as the legal demand of Australian farmers has pointed out. There are some published studies on greater amberjack nutriton during on-growing periods regarding dietary lipid sources (Aly et al., 1999) optimum lipid levels (Talbot et al., 2000), digestible protein/ energy ratios (Takakuwa et al., 2006; Vidal et al., 2008 (DP/DE) or phospholipid levels (Uyan et al., 2008) but a closely related species (yellowtail) has been extensively studied. For instance, in yellowtail the effect of alternative ingredients (Furutani et al., 2012), taurine (Matsunari et al., 2005), vit C (Kanazawa et al., 1992; Ren et al., 2008), vit E (Shimeno et al., 1991; Sakai et al., 1998) or P (Sarker et al., 2009). As the greater amberjack is a highly carnivorous species, high protein requirements are expected. Poor sustainability of fishmeal is encouraging the use of alternative plant proteins. When high plant protein diets are fed the first limiting essential aminoacid is frequently lysine (Wilson, 2003), but its requirement has not been yet determined in greater amberjack.
Reproduction success in terms of gonad development, fecundity, fertilization or hatching rates is markedly affected by broodstock diets in many fish species including Seriola spp. (Watanabe and Kiron, 1994; Fernadez-Palacios et al., 2011). Previous studies in this (Roo et al., in press) and other Seriola spp suggested that high protein, DHA and carotenoids are required for the reproductive success, but precise levels have not been investigated yet (Rodríguez-Barreto et al., 2012; Roo et al., in press). For instance, dietary protein and essential fatty acids markedly affect gamete quality in yellowtail (Verakunpiriya et al., 1997a, Watanabe et al., 2000). Taurine has been also identified as an essential component in broodstock diets for yellowtail necessary to improve fecundity, percentage of viable eggs and fertilization rates (Matsunari et al., 2006). Finally, carotenoids also play an important role in yellowtail reproduction (Verakunpiriya et al., 1997a, 1997b; Vassallo-Agius et al., 2001a). For instance, dietary astaxanthin increased fecundity but did not improve the egg quality in the yellowtail (Verakunpiriya et al., 1997b). Despite the importance of these nutrients for reliable reproduction of other Seriola species, their optimum levels and ratios among them in diets for greater amberjack broodstock have not been yet studied.
Regarding pikeperch, studies on percid larvae suggest that supplementation of diets by phospholipids or specific vitamins may decrease scoliosis and lordosis rates and increase larval resistance to osmotic stress (Kestemont et al., 1996; Hamza et al., 2008; Henrotte et al., 2010), but the optimal levels for major essential nutrients are still unknown for pikeperch and thus very important to increase quality of the produced larvae. Besides pike perch eggs have a high DHA content, which could be related to its strict carnivorous behavior or reflect the evolution of this species from marine water fish. Recent studies have suggested (Lund & Steenfeldt, 2011; Lund et al., 2012), that lack of LC-PUFAs especially DHA during live feed first feeding (i.e. within 25 days post hatch) both may have immediate and long term negative consequences on stress sensitivity and mortality in pikeperch larvae and in juveniles.
Interestingly, despite pikeperch is generally considered a freshwater fish, this species inhabits brackish waters (Baltic Sea ≤ 10 ppt.) and estuaries, sharing several characteristics with marine fish. Thus, pikeperch egg/larvae tissue LC PUFA composition and requirements resemble those of marine fish (Lund et al., 2011). Also in common with marine fish, pikeperch has the ability to hypo-osmoregulate keeping their body fluid osmolality below that of the environment (Scott et al., 2008). Laboratory studies revealed a great tolerance to saline waters, tolerating direct transfer to 16 ppt salinity and simulated tidal cycles of 33 ppt even though in both cases it induced increased cortisol levels (Brown et al., 2001). Thus during exposure to low salinity between 6- and 12 ppt pike perch are able to manipulate their nitrogen metabolism (Sadok et al., 2004). Despite previous studies have demonstrated a strong effect of salinity on fatty acid requirements and metabolism in other species such as Atlantic salmon (Tocher et al. 1995), the salmoniform fish Galaxias maculatus (Dantagnan et al., 2007) or the Mexican silverside, Chirostoma estor (Fonseca- Madrigal et al., 2012), the ability of pikeperch to regulate its fatty acid metabolism and LC- PUFA synthesis in combination with salinity has not been investigated. Nevertheless, this species has the ability to elongate and desaturate precursor fatty acids for n-3 PUFA synthesis (Schulz et al., 2005). Commercial production of pikeperch is practiced in freshwater, but low salinity initiate physiological changes that could affect growth rate and development, and therefore it is interesting to better understand the interactive effect of salinity and nutrition on stress resistance in pikeperch. Therefore, DIVERSIFY will study the effect of selected dietary nutrients on pike perch larval development and performance, and particularly of EFA on long-term stress sensitivity.
Despite the fact that Atlantic halibut commercial rearing has started many years ago, early weaning still constitutes a main bottleneck. Feeding on-grown Artemia may improve halibut weaning, contributing to complete larval metamorphosis and pigmentation (Olsen et al., 1999) and leading to stronger juveniles. There is great interest in Atlantic halibut larval rearing using RAS technologies, where a different microbiota might have a positive effect on intestinal health (Nayak, 2010) and contribute with essential nutrients such as DHA, EPA or certain vitamins (Ray et al., 2012). However there is a lack of specific studies to determine their importance in Atlantic halibut productive systems. Among minerals, the importance of iodine for larval rearing has been emphasized (Morris et al., 2011; Ribeiro et al., 2011). The slow growth in late larval stages could be overcome by early weaning. Most often, weaning of Atlantic halibut occurs only at 70 days post first-feeding (dpff), but attempts have been made to introduce formulated diets from 20 and 50 dpff, with varying results. The first problem arising is that the larvae refuse to eat formulated feed (Harboe, Hamre and Erstad, unpublished results). It has frequently been observed, however, that they ingest inert particles such as Artemia cysts and pollen from pinewood, the main similarity being that both particles have neutral buoyancy and a bright color. Previous experiments have also shown better feed ingestion with floating compared to sinking feed particles. Furthermore, the structure of the visual system of halibut larvae indicates that they hunt prey in the horizontal plane (Helvik pers. com.), favoring feed intake when particles stay in the same position in the water column for some time. Additionally the type of feed could also affect digestive capacity determined as proteases, carbohydrases and lipases activities (Caruso et al., 2009) or even ATPase activity, which in gut is essential to ensure the ion gradient necessary for nutrient uptake.
Another strategy to alleviate the slow growth in the later larval stages is to use on-grown Artemia. Ongrown Artemia are larger, contain more protein and phospholipids and have a different micronutrient status from Artemia nauplii (Hamre and Harboe, NIFES, preliminary results). They also have a lower shell to nutrient content. This may explain why Atlantic halibut larvae fed on-grown Artemia develop into juveniles with better pigmentation and eye migration than Atlantic halibut fed Artemia nauplii (Olsen et al., 1999; Hamre and Harboe, NIFES, preliminary results). The industry is considering implementing this knowledge in the production line, but will need further documentation.
Atlantic halibut larvae kept in a RAS system will encounter matured water, which will affect their gut flora (Nayak, 2010) in a way that probably has a positive effect on intestinal health. Gnotobiotic and conventional studies indicate the involvement of gut microbiota in nutrition and epithelial development (Nayak, 2010). Gastrointestinal bacteria may also produce essential nutrients such as vitamins and polyunsaturated fatty acids, and enzymes that can aid digestion (Ray et al., 2012). These considerations favor the hypothesis that the general nutrient absorption and retention in the fish is affected by RAS. Iodine retention must have an extra focus, since NO3- at levels found commonly in recirculation systems block iodide uptake by the sodium iodide symporter and may cause goiter in the fish (Morris et al., 2011; Ribeiro et al., 2011).
The third important bottelneck in halibut production is slow growth after weaning. One possible reason for this is a suboptimal diet. We have shown that juvenile Ballan wrasse increase the growth rate by up to 40% when lipids are added as phospholipids (PL) in stead of triacylglycerols (TAG, Sæle et al., ubpublished), while requirements for PL in A. halibut juveniles are not known. DIVERSIFY will develop a new production strategy for on-growing Artemia and subsequently test them to improve weaning performance of Atlantic halibut juveniles. In addition, new information will be gathered on the effects of RAS vs Flow Through Systems (FTS) in Atlantic halibut larval development.
Studies on wreckfish nutritional requirements and optimum diets are missing, since control of reproduction and reliable supply of eggs has not been achieved yet (Fauvel et al., 2008; Papandroulakis et al., 2004). Nevertheless, some information is available from studies on feeding habits of wild populations, biochemical composition of eggs, larvae and juveniles, or results obtained in other relative species (Anderson et al., 2012). Therefore, studies on nutrition of this species will focus mainly on broodstock feeds for enhancing fecundity and spawn quality, and the development of adequate live prey enrichments for wreckfish larvae, as first steps for the development of proper nutrition and culture of this serranid species.
Preliminary studies suggest that rotifer enrichment with EPA and DHA improve grey mullet larvae performance (Eda et al., 1990; Tamaru et al., 1992), although the optimum levels and ratios have not been determined yet. Requirements of EFA in fish seem to be dependent on salinity conditions (Dantagnan et al., 2010). Interestingly, older grey mullet juveniles seek out less saline coastal environments and show best weight gain in low salinity or freshwater lakes and ponds. Another nutrient that may be closely related is Tau, which may improve bile salt-assisted lipid transport and metabolic regulation (Hansen & Mortensen, 2012). Importantly, the larvae of many marine species lack a key enzyme in Tau synthesis (Yokoyama et al., 2001), whereas this amino acid is present only in trace levels in rotifers (Van der Meeren et al., 2008). Moreover, fishmeal replacement by plant protein in grow-out diets reduces markedly Tau levels and its supplementation improves greatly juvenile growth performance (Lunger et al., 2007; Chatzifotis et al., 2008). Having this in mind, DIVERSIFY will focus on the requirements of grey mullet for EFA and Tau, to improve enrichment products, weaning, grow out and broodstock diets.
Feed constitutes the largest running cost for European aquaculture, reaching up to 70% in cage-based facilities. Moreover, suboptimal commercial feeds and feeding protocols result in direct economic losses through feed waste, poor growth and water quality deterioration (Kaushik, 1998). Also, feed composition affects fish health and welfare (Kiron et al., 2010; Montero and Izquierdo, 2010) and affects markedly fillet quality (Izquierdo et al., 2003) and consumer acceptance (Rosenlund et al., 2010). Species-specific formulations for aquaculture fish can improve markedly reproductive performance and progeny quality (Izquierdo et al., 2001; Fernández-Palacios et al., 2011), larval development and survival (Hamre et al., in press) and fry quality (Izquierdo et al., 2010, 2012). Considering the specific bottlenecks defined for the proposed species and the nutritional knowledge available, particular tasks will be addressed in this proposal to provide specific solutions for the species considered.
In meagre, DIVERSIFY Partners have developed successful first feeding protocols (Roo et al., 2009a, 2010a; Fernández-Palacios et al., 2009; Hernández Cruz et al., 2007; Scabini et al., 2008) and used different commercial enrichment products for larval rearing (Fernández-Palacios et al., 2007; Roo et al., 2007; Scabini et al., 2008; Abreu et al., 2009, Vallés & Estévez, 2011). However, feeding meagre during weaning and grow out with diets developed for other marine fishes (i.e., gilthead sea bream and European sea bass) restricts the fast growing potential of this species (Robaina et al., in press), affects negatively the fish appearance and fillet quality (Gines et al., in press; Poli et al., 2003) and may be the cause of Systemic Granulomatosis (Ghittino et al., 2004). Lack of specific diets for meagre is due mainly to the very limited information regarding its nutritional requirements (Chatzifotis et al., 2010, 2012). Early studies denote that this species has high protein requirements, while dietary lipids are restricted around 17% (Chatzifotis et al., 2010, 2012). However, digestible energy (DE) and digestible protein (DP) requirements are not yet determined. Requirements for essential fatty acids (EFA), such as docosahexaenoic (DHA), eicosapentaenoic (EPA) and arachidonic (ARA) acids, which play important roles in fish growth (Izquierdo & Koven, 2011), health (Montero & Izquierdo, 2010) and fillet quality (Rosenlund et al., 2011), have not been studied in juvenile meagre. Finally, there is an almost complete lack of information on the requirements for essential amino acids (EAA, particularly in combination with plant protein sources), vitamins and minerals (Robaina et al., in press). DIVERSIFY Partners, building up on already existing knowledge, have selected the most relevant nutritional aspects (DE, DP, EFA, EAA) for dose-response studies, whereas vitamin and mineral requirements will be studied in multifactorial or whole micronutrient package approaches. The lack of meagre-specific weaning and grow out diets will be addressed in DIVERSIFY, in order to maximize growth potential, enhance fry quality and promote health of this species. The WP on Nutrition – meagre will determine requirements of specific nutrients generally important for growth, welfare and health to address two of the main specific bottlenecks identified for meagre: variable growth rates during the pre-growing phase and variable growth during on-growing phase. Variable growth rates constrain production planning and may be related to different causes including unsuitable feed formulations. In meagre, despite its great growth potential, still both growth and feed utilization rates may be poor (Chatzifotis et al., 2010; Estévez et al., 2011) and feeds must be improved to consistently obtain high growth rates. Indeed, diets specially formulated to satisfy nutritional requirements of related species such as the mulloway (Argyrosomus japonicus, Woolley et al., 2010) and brown meagre (Sciaena umbra, Chatzifotis et al., 2006) produce better growth and feed utilization rates in these related species than those obtained until present in meagre. Despite specific diets for meagre would produce more consistent growth rates (Martínez-Llorens et al., 2011), there is not enough information on the specific nutritional requirements of this species (Chatzifotis et al., 2010, 2012).
Building up on already existing knowledge available for other Sciaenids such as mulloway, brown meagre and, particularly, the abundant information on the requirements of the red drum (Sciaenops ocellatus), DIVERSIFY selected the most relevant nutritional aspects to promote meagre growth and welfare. According to previous studies in several Sciaenids (Daniels and Robinson, 1986; Gatlin, 1995; Pirozzi, et al., 2010a; Kaushik, unpublished data), meagre seems to have higher protein and lower lipid requirements than seabream and seabass, whose commercial diets are used by European farmers to produce up to 2,387 t of meagre. Optimum crude protein and crude lipid contents in diets for meagre have been estimated up to now to be around 50% (Chatzifotis et al., 2012) and 17% (Panagiotidoum et al., 2007; Chatzifotis et al., 2010; Chatzifotis et al., 2012), respectively. However, protein requirements could be even higher and lipid requirements lower, if this species, as it occurs in mulloway, has a limited capacity to spare dietary protein (Pirozzi et al., 2010b). Thus, in a closely related species, utilization efficiencies for DP and DE are independent of fish size, ration level or temperature (Pirozzi and Booth, 2009; Pirozzi et al., 2010), whereas the red drum shows lower protein efficiency ratios (McGoogan and Gatltlin III, 1999; Peng et al., 2008).
Despite essential fatty acids such as docosahexaenoic (DHA), eicosapentaenoic (EPA) and arachidonic (ARA) acids have been found to be essential for growth and welfare of several Sciaenids (Gatlin III, D.M., 2009), the requirements for these essential nutrients have not been yet studied in meagre. Another essential nutrient for fish growth is vitamin E (Hamre and Lie, 1995; Kocabas and Gatlin, 1999; Montero et al., 1998; Tocher et al., 2003; Lin and Shiau, 2005; Abdel-Hameid et al., 2012) and despite its optimum dietary levels have been determined in other Sciaenids (Peng et al., 2008; Peng, and Gattlin, 2009), its requirements are yet unknown in meagre. Finally, antioxidant nutrients such as vit C and selenium have been also found to be essential for growth of other species of the same family (Sealy and Gatlin, 2002), despite their importance for meagre has not been yet study. Among aminoacids, lysine is considered as an indispensable amino acid for proper growth being the first limiting EAA in protein sources commonly used in fish feeds based on plant feedstuffs (Wilson, 2003), but its requirement has not been yet determined in meagre. Lysine plays an essential role as precursor of carnitine, which carries long chain fatty acids into the mitochondria for β-oxidations of lipids to produce energy (Walton et al., 1984).
Health problems in meagre may be related to the use of inadequate feeds, as it occurs in other aquaculture produced species (Cooke and Sneddon, 2007, Montero and Izquierdo, 2010). Farmers are also aware of the harmful consequences of nutritionaly-unbalanced diets on fish welfare (Conte, 2004). In turn, poor welfare conditions could not only markedly reduce meagre growth as it has been seen in a closely related species (Pirozzi et al., 2009), but also negatively affect immune system and disease resistance. Imbalances in nutrition negatively affect fish welfare and health in many fish species (Lall, 2000). The importance of nutrition on welfare and health involve almost all physiological functions related to health, including a direct effect on modulation of the immune system, stress response, mechanisms of defense against infection or tissue integrity (Waagbø, 2006). Among different nutrients, deficiency in essential fatty acids or antioxidants including vitamin E and vitamin C, negatively affect fish welfare, increasing plasma cortisol levels and affecting fish behaviour and stress responses (Montero et al., 1998; Montero et al., 1999). Besides unbalanced levels of these nutrients cause inmune deficiencies, pathological features in several tissues and reduce disease resistance in several fish species (Montero and Izquierdo, 2010; Betancor et al., 2012a, 2012b) including some Sciaenids (Sealey and Gatlin, 2002). Amino acids and their metabolites have been characterized as important regulators of main physiological pathways that are required for fish maintenance, growth performance, feed utilization, protection from oxidative stress and resistance to environmental stressors and pathogenic organisms (Wilson and Halver, 1986; Li et al., 2009). Among them, lysine has a role in calcium absorption and formation of collagen, a substance important for bones and connective tissues including skin and cartilage (Civitelli et al., 1992), being essential for the health status of these tissues. Despite the importance of those nutrients on fish health, the nutritional requirements of meagre have not been yet defined particularly in relation to welfare and health issues.
Being a carnivorous species, meagre should have a high requirement for digestible protein (DP), which may be affected by fish size and temperature. Previous work has been carried out in a closely related species, the mulloway in Australia by Pirozzi et al (2010), but data obtained from mulloway cannot be directly transferred to meagre since both species are cultured under greatly different temperature conditions. In this sense, mulloway may be considered as a warm water species with an optimal temperature range comprised between 25 to 26.4ºC (Bernatzeder & Britz, 2007), whereas meagre is a temperate species inhabiting the Mediterranean and Black Sea, and along the eastern Atlantic coast, where the best temperature for rearing meagre lies between 17 and 21ºC. As nutrient utilization efficiencies have been shown to be influenced by many different factors such as species effects, fish size and temperature (Bendiksen et al. 2003; Moreira et al. 2008), it is clear that optimal DP:DE data obtained for mulloway cannot be transferred to the meagre industry without previous experimental validation. Thus, a species-specific research on the nutrition of this species (DP:DE) is needed in order to optimize actual feeding practices and reduce one of the main bottlenecks identified by the industry during the on-growing phase of this species, which is the highly variable growth rates that may be linked to unsuitable feed formulation (of which DP:DE is the key aspect determining growth), among other potential reasons that will also be investigated (mainly potential genetic effects).
Contrary to meagre, the greater amberjack exhibits high mortalities during larval development, thus juvenile availability is a major bottleneck to its industrial production, in addition to reproduction control. Previous studies conducted by the Partners on ontogenic development (Abreu et al., 2009) and larval rearing techniques (Roo et al., 2010b, Grossi et al., 2009) have shown that larval greater amberjack perform poorly when fed the available commercial enrichment products. However, the elevation of certain nutrients in experimental enrichment products (Fernández-Palacios, unpublished data) increased up to 5 fold larval survival (Yamamoto et al., 2008). A recent review on larval nutrition (Hamre et al., in press), as well as preliminary studies on greater amberjack (Yamamoto et al., 2009) point to the importance of EFA, vitamins E and C, carotenoids and taurine (Tau) as essential nutrients during fish development. DIVERSIFY Partners will study these nutrients through dose-response and multifactorial approaches, in order to develop specific live food enrichments and improved weaning diets. Regarding grow out, information on greater amberjack nutritional requirements is scarce (Aly et al., 1999 (lipid sources); Talbot et al., 2000 (Lipid levels); Takakuwa et al., 2006 (DP/DE); Vidal et al., 2008 (DP/DE); Uyan et al., 2009 (phospholipid levels)). However, the close congener yellowtail (S. quinqueradiata) has been studied extensively (i.e., Furutani et al., 2012 (alternative ingredients); Matsunari et al., 2005 (Tau); Ren et al., 2008 (Vit C)) and high requirements in protein, DHA, Tau and antioxidants are foreseen for greater amberjack. Therefore, DIVERSIFY partners will build up on already existing knowledge available for this and other Seriola species (Watanabe and Kiron, 1994; Sakai et al., 1998; Takeuchi, 2001; Liu, 2001; Uyan et al., 2008; Kolkovski et al., 2009, 2010; Miki et al., 2011), to specifically address lack of knowledge on nutrients that are particularly important for larval development, juvenile performance and reproduction.
A main bottleneck for the mass production of high quality greater amberjack juveniles is the low survival and growth obtained during larval development and metamorphosis (Yamamoto et al., 2009). However, larval greater amberjack perform very poorly when fed the available commercial enrichment products for live preys. The partners are familiar with the abundant information produced in Japan and Australia regarding larval rearing of greater amberjack (Yamamoto et al., 2009), but very few studies have determined the specific nutritional requirements of this species testing a sufficient number of diets varying only the target nutrient. Thus, despite Yamamoto et al., (2009) pointed out the importance of essential fatty acids, antioxidants such as vitamins E and C, carotenoids and taurine as essential nutrients for greater amberjack larval development, the specific requirements for these nutrients have not been yet determined. For instance, Matsunari and coworkers (2012), studied the effect of four enrichment products for rotifers, such as Chlorella or a commercial emulsion that besides having different docosahexaenoic acid (DHA) content also differ in many other nutrients and other compounds that also may affect larval performance such as fat-soluble vitamins, pigments, antioxidants, minerals, etc. Moreover, despite they tested a range of DHA (0.0-1.9 mg g), DHA requirements for other Seriola species (i.e., yellowtail, S. quinqueradiata) are known to be the highest among all studied species (Takeuchi, 2001), well above the levels studied by Matsunari and co-workers (2012). Indeed, essential fatty acid requirements in rotifers may be as high as to 3.9% in the fast growing larvae of yellowtail (Kolkovsky et al., 2010). The relevance of DHA in diets for marine fish larvae has been well documented (Watanabe et al., 1989; Izquierdo, 1996; Sargent et al., 1999) and its positive effect on survival has been related to its important role in stress control (Watanabe et al., 1993; Izquierdo, 2005, Ganga et al., 2006), immune system development (Montero et al., 2003) and improvement of health and bacterial resistance in fish larvae (Brandsden et al., 2003). Moreover, DHA has been found to increase eye diameter and density of photoreceptors in gilthead seabream larvae (Izquierdo et al., 2000) and, in agreement, visual capacity was found to be reduced in yellowtail fed DHA-deficient diets (Masuda et al., 1999). Although both arachidonic and eicosapentaenoic acids are also considered essential and play important roles in fish metabolism, not only their absolute amounts but also their relative proportions are determinant of larval growth and survival as it has been seen in other species (Izquierdo and Koven, 2010). For instance in gilthead seabream, dietary ARA is more efficiently incorporated into larval tissues than EPA (Atalah et al., 2011) and therefore EPA/ARA ratios become lower in larval tissues than in diets. Similarly, increased dietary EPA has been found to reduce dietary DHA incorporation into larval tissues, higher EPA/DHA ratios decreasing larval growth (Rodríguez et al., 1997, 1998). Up to date, the optimum EPA/ARA and EPA/DHA ratios have not been yet determined in enrichment products for greater amberjack, despite their importance for larval performance. The high polyunsaturated fatty acid requirements forseen in greater amberjack increase the risk of lipid peroxidation in this species. For instance, in yellowtail lipid peroxidation as a consequence of an imbalance between polyunsaturated fatty acids and antioxidants damages the biomembranes, producing several pathological conditions (Sakai et al. 1998) and causing irreversible changes in the developing tissues of marine fish larvae. Therefore, the enrichments must contain high levels of antioxidants, such as vit E (Betancor et al., 2011; Robaina et al., in press) and carotenoids. Despite taurine requirements have not been determined in greater amberjack larvae, the requirement of this aminoacid for very young juveniles of yellowtail (Matsunari et al., 2005) suggest its importance in early larval stages of Seriola spp as proposed by Yamamoto et al. (2009).
Regarding on-growing, despite no specific diets are produced in Europe for this species, commercial diets for other Seriola spp are available in the Pacific region, although they are far from satisfying the requirements of this species as the legal demand of Australian farmers has pointed out. There are some published studies on greater amberjack nutriton during on-growing periods regarding dietary lipid sources (Aly et al., 1999) optimum lipid levels (Talbot et al., 2000), digestible protein/ energy ratios (Takakuwa et al., 2006; Vidal et al., 2008 (DP/DE) or phospholipid levels (Uyan et al., 2008) but a closely related species (yellowtail) has been extensively studied. For instance, in yellowtail the effect of alternative ingredients (Furutani et al., 2012), taurine (Matsunari et al., 2005), vit C (Kanazawa et al., 1992; Ren et al., 2008), vit E (Shimeno et al., 1991; Sakai et al., 1998) or P (Sarker et al., 2009). As the greater amberjack is a highly carnivorous species, high protein requirements are expected. Poor sustainability of fishmeal is encouraging the use of alternative plant proteins. When high plant protein diets are fed the first limiting essential aminoacid is frequently lysine (Wilson, 2003), but its requirement has not been yet determined in greater amberjack.
Reproduction success in terms of gonad development, fecundity, fertilization or hatching rates is markedly affected by broodstock diets in many fish species including Seriola spp. (Watanabe and Kiron, 1994; Fernadez-Palacios et al., 2011). Previous studies in this (Roo et al., in press) and other Seriola spp suggested that high protein, DHA and carotenoids are required for the reproductive success, but precise levels have not been investigated yet (Rodríguez-Barreto et al., 2012; Roo et al., in press). For instance, dietary protein and essential fatty acids markedly affect gamete quality in yellowtail (Verakunpiriya et al., 1997a, Watanabe et al., 2000). Taurine has been also identified as an essential component in broodstock diets for yellowtail necessary to improve fecundity, percentage of viable eggs and fertilization rates (Matsunari et al., 2006). Finally, carotenoids also play an important role in yellowtail reproduction (Verakunpiriya et al., 1997a, 1997b; Vassallo-Agius et al., 2001a). For instance, dietary astaxanthin increased fecundity but did not improve the egg quality in the yellowtail (Verakunpiriya et al., 1997b). Despite the importance of these nutrients for reliable reproduction of other Seriola species, their optimum levels and ratios among them in diets for greater amberjack broodstock have not been yet studied.
Regarding pikeperch, studies on percid larvae suggest that supplementation of diets by phospholipids or specific vitamins may decrease scoliosis and lordosis rates and increase larval resistance to osmotic stress (Kestemont et al., 1996; Hamza et al., 2008; Henrotte et al., 2010), but the optimal levels for major essential nutrients are still unknown for pikeperch and thus very important to increase quality of the produced larvae. Besides pike perch eggs have a high DHA content, which could be related to its strict carnivorous behavior or reflect the evolution of this species from marine water fish. Recent studies have suggested (Lund & Steenfeldt, 2011; Lund et al., 2012), that lack of LC-PUFAs especially DHA during live feed first feeding (i.e. within 25 days post hatch) both may have immediate and long term negative consequences on stress sensitivity and mortality in pikeperch larvae and in juveniles.
Interestingly, despite pikeperch is generally considered a freshwater fish, this species inhabits brackish waters (Baltic Sea ≤ 10 ppt.) and estuaries, sharing several characteristics with marine fish. Thus, pikeperch egg/larvae tissue LC PUFA composition and requirements resemble those of marine fish (Lund et al., 2011). Also in common with marine fish, pikeperch has the ability to hypo-osmoregulate keeping their body fluid osmolality below that of the environment (Scott et al., 2008). Laboratory studies revealed a great tolerance to saline waters, tolerating direct transfer to 16 ppt salinity and simulated tidal cycles of 33 ppt even though in both cases it induced increased cortisol levels (Brown et al., 2001). Thus during exposure to low salinity between 6- and 12 ppt pike perch are able to manipulate their nitrogen metabolism (Sadok et al., 2004). Despite previous studies have demonstrated a strong effect of salinity on fatty acid requirements and metabolism in other species such as Atlantic salmon (Tocher et al. 1995), the salmoniform fish Galaxias maculatus (Dantagnan et al., 2007) or the Mexican silverside, Chirostoma estor (Fonseca- Madrigal et al., 2012), the ability of pikeperch to regulate its fatty acid metabolism and LC- PUFA synthesis in combination with salinity has not been investigated. Nevertheless, this species has the ability to elongate and desaturate precursor fatty acids for n-3 PUFA synthesis (Schulz et al., 2005). Commercial production of pikeperch is practiced in freshwater, but low salinity initiate physiological changes that could affect growth rate and development, and therefore it is interesting to better understand the interactive effect of salinity and nutrition on stress resistance in pikeperch. Therefore, DIVERSIFY will study the effect of selected dietary nutrients on pike perch larval development and performance, and particularly of EFA on long-term stress sensitivity.
Despite the fact that Atlantic halibut commercial rearing has started many years ago, early weaning still constitutes a main bottleneck. Feeding on-grown Artemia may improve halibut weaning, contributing to complete larval metamorphosis and pigmentation (Olsen et al., 1999) and leading to stronger juveniles. There is great interest in Atlantic halibut larval rearing using RAS technologies, where a different microbiota might have a positive effect on intestinal health (Nayak, 2010) and contribute with essential nutrients such as DHA, EPA or certain vitamins (Ray et al., 2012). However there is a lack of specific studies to determine their importance in Atlantic halibut productive systems. Among minerals, the importance of iodine for larval rearing has been emphasized (Morris et al., 2011; Ribeiro et al., 2011). The slow growth in late larval stages could be overcome by early weaning. Most often, weaning of Atlantic halibut occurs only at 70 days post first-feeding (dpff), but attempts have been made to introduce formulated diets from 20 and 50 dpff, with varying results. The first problem arising is that the larvae refuse to eat formulated feed (Harboe, Hamre and Erstad, unpublished results). It has frequently been observed, however, that they ingest inert particles such as Artemia cysts and pollen from pinewood, the main similarity being that both particles have neutral buoyancy and a bright color. Previous experiments have also shown better feed ingestion with floating compared to sinking feed particles. Furthermore, the structure of the visual system of halibut larvae indicates that they hunt prey in the horizontal plane (Helvik pers. com.), favoring feed intake when particles stay in the same position in the water column for some time. Additionally the type of feed could also affect digestive capacity determined as proteases, carbohydrases and lipases activities (Caruso et al., 2009) or even ATPase activity, which in gut is essential to ensure the ion gradient necessary for nutrient uptake.
Another strategy to alleviate the slow growth in the later larval stages is to use on-grown Artemia. Ongrown Artemia are larger, contain more protein and phospholipids and have a different micronutrient status from Artemia nauplii (Hamre and Harboe, NIFES, preliminary results). They also have a lower shell to nutrient content. This may explain why Atlantic halibut larvae fed on-grown Artemia develop into juveniles with better pigmentation and eye migration than Atlantic halibut fed Artemia nauplii (Olsen et al., 1999; Hamre and Harboe, NIFES, preliminary results). The industry is considering implementing this knowledge in the production line, but will need further documentation.
Atlantic halibut larvae kept in a RAS system will encounter matured water, which will affect their gut flora (Nayak, 2010) in a way that probably has a positive effect on intestinal health. Gnotobiotic and conventional studies indicate the involvement of gut microbiota in nutrition and epithelial development (Nayak, 2010). Gastrointestinal bacteria may also produce essential nutrients such as vitamins and polyunsaturated fatty acids, and enzymes that can aid digestion (Ray et al., 2012). These considerations favor the hypothesis that the general nutrient absorption and retention in the fish is affected by RAS. Iodine retention must have an extra focus, since NO3- at levels found commonly in recirculation systems block iodide uptake by the sodium iodide symporter and may cause goiter in the fish (Morris et al., 2011; Ribeiro et al., 2011).
The third important bottelneck in halibut production is slow growth after weaning. One possible reason for this is a suboptimal diet. We have shown that juvenile Ballan wrasse increase the growth rate by up to 40% when lipids are added as phospholipids (PL) in stead of triacylglycerols (TAG, Sæle et al., ubpublished), while requirements for PL in A. halibut juveniles are not known. DIVERSIFY will develop a new production strategy for on-growing Artemia and subsequently test them to improve weaning performance of Atlantic halibut juveniles. In addition, new information will be gathered on the effects of RAS vs Flow Through Systems (FTS) in Atlantic halibut larval development.
Studies on wreckfish nutritional requirements and optimum diets are missing, since control of reproduction and reliable supply of eggs has not been achieved yet (Fauvel et al., 2008; Papandroulakis et al., 2004). Nevertheless, some information is available from studies on feeding habits of wild populations, biochemical composition of eggs, larvae and juveniles, or results obtained in other relative species (Anderson et al., 2012). Therefore, studies on nutrition of this species will focus mainly on broodstock feeds for enhancing fecundity and spawn quality, and the development of adequate live prey enrichments for wreckfish larvae, as first steps for the development of proper nutrition and culture of this serranid species.
Preliminary studies suggest that rotifer enrichment with EPA and DHA improve grey mullet larvae performance (Eda et al., 1990; Tamaru et al., 1992), although the optimum levels and ratios have not been determined yet. Requirements of EFA in fish seem to be dependent on salinity conditions (Dantagnan et al., 2010). Interestingly, older grey mullet juveniles seek out less saline coastal environments and show best weight gain in low salinity or freshwater lakes and ponds. Another nutrient that may be closely related is Tau, which may improve bile salt-assisted lipid transport and metabolic regulation (Hansen & Mortensen, 2012). Importantly, the larvae of many marine species lack a key enzyme in Tau synthesis (Yokoyama et al., 2001), whereas this amino acid is present only in trace levels in rotifers (Van der Meeren et al., 2008). Moreover, fishmeal replacement by plant protein in grow-out diets reduces markedly Tau levels and its supplementation improves greatly juvenile growth performance (Lunger et al., 2007; Chatzifotis et al., 2008). Having this in mind, DIVERSIFY will focus on the requirements of grey mullet for EFA and Tau, to improve enrichment products, weaning, grow out and broodstock diets.
