Potential use of meat and bone meal in diets for gilthead seabream (Sparus aurata) juveniles

Potential use of meat and bone meal in diets for gilthead seabream (Sparus aurata) juveniles Sara Patrícia Pinto Moutinho Dissertação de Mestrado apresentada à Faculdade de Ciências da Universidade do Porto, Universitat Politècnica de València Recursos Biológicos Aquáticos 2015 Potential use of meat and bone meal in diets for gilthead seabream (Sparus aurata) juveniles Sara Patrícia Pinto Moutinho MSc FCUP UPV 2015 2.º CICLO Potential use of meat and bone meal in diets for gilthead seabream (Sparus aurata) juveniles Sara Patrícia Pinto Moutinho Mestrado em Recursos Biológicos Aquáticos Departamento de Biologia 2015 Orientador Doutora Helena Peres, Investigadora Auxiliar, CIIMAR Coorientador Professor Miguel Jover Cerdá, Professor Catedrático, UPV Coorientador Doutora Silvia Martínez-Llorens, Investigadora, UPV Todas as correções determinadas pelo júri, e só essas, foram efetuadas. O Presidente do Júri, Porto, ______/______/_________ Acknowledgements First of all, I would like to express my sincere gratitude to Prof. Dr. Aires OlivaTeles, not only for accepting me as a Master student and giving me the opportunity to begin my journey in this field but also for all his support, advice and scientific knowledge shared during this year. Secondly, I would like to thank Prof. Miguel Jover Cerdá, of the Polytechnic University of Valencia, for accepting and allowing me to develop my thesis in collaboration with the GAB research group. A very special thank you to Prof. Silvia Martínez-Llorens whose care, guidance and knowledge ensured the success of this work. I must also thank Prof. Ana Tomás Vidal, Raquel, Ana, Guillem and fellow colleagues at UPV for helping me whenever needed. To “La Résistance”, a very kind thank you for the friendship that made my stay in Valencia a memorable experience. A very special thank you to Cláudia Serra, for the dedication and patience and whose teachings were essential to make this work possible. To all my colleagues at NUTRIMU, thank you. I would especially like to express my gratitude towards Dra. Helena Peres, as I am sure that without her tremendous support, encouragement, knowledge, dedication, and kindness, none of this would have been possible. I would especially like to thank her for the recognition in my work, motivating me to do better and pursue my goals. Finally, I would like to show my sincere appreciation to my dear friends, family and my parents, Luciano e Maria, for all the love and support and for always being there for me whenever I needed the most and also to Gaspar, for being there in the final stages of my work. To everyone who, in some way, contributed to the success of this thesis, thank you. FCUP Potential use of meat and bone meal in diets for gilthead seabream (Sparus aurata) juveniles 1 Abstract Fishmeal (FM) has been the preferred protein source for aquafeeds, in particular for carnivorous species. However, current FM inclusion levels threaten the expansion of the intensive production of those species. In this context, research has been focusing on evaluating more cost-effective and sustainable alternative ingredients to FM. Meat and bone meal (MBM) is a desirable product for carnivorous fish diets as it generally possesses a high protein content, relatively balanced amino acid profile, high digestibility and palatability and lacks anti-nutritional factors. Also, it is produced worldwide with a steady availability. Recent unban of the use of these ingredients inside the European Union emphasizes the necessity to evaluate it as new potential ingredient for FM replacement. Gilthead seabream (Sparus aurata) is an important economic species in Mediterranean aquaculture but overproduction, associated with increasing price of feeds, led to a decrease in profitability of the intensive production of this species. Therefore, present study aimed to evaluate FM replacement with MBM on growth, digestibility, feed efficiency utilization and gut microbiota of gilthead seabream juveniles. Three experimental diets were formulated (45% CP; 20% CL): a control diet (FM100), with FM as the main protein source, and MBM50 and MBM75 where FM was replaced at 50% and 75%, respectively. Triplicate groups of juvenile gilthead seabream (25 ± 0.72 g) were fed for 83 days with the experimental diets. A 50% substitution did not significantly affect growth (DGI of 2.48 and 2.51 for FM100 and MBM50 diets, respectively), feed utilization efficiency (FCR of 1.51 and 1.53; PER of 1.51 and 1.50, for FM100 and MBM50 diets, respectively). However, a 75% substitution led to a significant decrease on growth rate (DGI of 2.25) and feed utilization (FCR of 1.72; PER of 1.29), although feed intake (g kg ABW-1 day-1) was significantly higher (26.1 compared to 24.2 for diet MBM50). Whole-body composition was mostly unaffected by the experimental diets with the exception of lipid and energy content, which were significantly lower in fish fed the diet MBM75. Protein and essential amino acid retention were unaffected by the experimental diets while lipid and energy retention were significantly reduced with the increase of FM substitution. Crude protein digestibility was high (>89%) and unaffected by the experimental diets while energy digestibility was significantly higher for diet MBM50 (95.2 %), compared to the control (82 %). ADCs of essential amino acids were high (>92%) for all experimental diets and statistically similar or higher for diet MBM50, compared to the control diet, but lower for MBM75 when compared to MBM50. FCUP Potential use of meat and bone meal in diets for gilthead seabream (Sparus aurata) juveniles 2 MBM significantly modulated gastrointestinal microbiota with a decrease in operational taxonomic units (OTUs) and species richness but an increase in replicate similarity with increasing MBM inclusion rate. MBM appeared to promote the development of Vibrio, Bacillus and Mycobacterium genera while colonization by Staphylococcus and Corynebacterium genera appeared to decrease. Overall, results indicate that half of FM could be replaced by MBM, in diets for gilthead seabream juveniles, without compromising growth performance and feed utilization with good results in nutrient and EAA digestibility and retention. Further studies are required to study fatty acid profile, digestibility and retention, and the effect of dietary MBM inclusion on general intestine health fish, fish wellbeing and immune status as well as on flesh quality traits of gilthead seabream. Keywords: Gilthead seabream; alternative protein sources; meat and bone meal; amino acids; digestibility; microbiota Resumo A farinha de peixe (FP) é a principal fonte proteica em dietas para aquacultura, em particular para espécies carnívoras. No entanto, os atuais níveis de incorporação da FP ameaçam a expansão da produção aquícola destas espécies. Sendo assim, a investigação tem centrado esforços no estudo de novos ingredientes, alternativos à FP, economicamente mais viáveis e sustentáveis. A farinha de carne e osso (MBM) é um ingrediente com elevado potencial para incorporação em dietas para peixes carnívoros, dado o seu elevado teor em proteína, perfil de aminoácidos relativamente equilibrado, elevada digestibilidade, boa palatabilidade e ausência de fatores anti-nutricionais. Para além disso, a MBM é produzida mundialmente e com disponibilidade contínua. A recente reautorização do uso destes ingredientes na alimentação de peixes na União Europeia salienta a necessidade da avaliação do seu potencial como alternativa à FP, em espécies produzidas na Europa. A dourada (Sparus aurata) é uma espécie de grande importância económica na aquacultura Mediterrânica mas o excesso de produção, associada ao aumento do preço das dietas, levou a uma diminuição na rentabilidade da produção intensiva desta espécie. Neste contexto, o presente estudo teve como objetivo avaliar o efeito da substituição da FP por MBM no crescimento, digestibilidade, eficiência de utilização do alimento e microbiota gastrointestinal de juvenis de dourada. Foram formuladas 3 dietas experimentais, com 45% de proteína bruta e 20% de lípidos totais, fazendo variar a taxa de incorporação da MBM: dieta controlo (FM100), com FP como FCUP Potential use of meat and bone meal in diets for gilthead seabream (Sparus aurata) juveniles 3 a principal fonte proteica, e MBM50 e MBM75 onde FP foi substituída em 50 e 75%, respetivamente. Cada uma das dietas foi fornecida, em triplicado, a grupos de juvenis de dourada (peso médio inicial 25 ± 0.72 g), durante 83 dias. A substituição de 50% da FM por MBM não afetou significativamente o crescimento (DGI de 2.48 e 2.51 para as dietas FM100 e MBM50, respetivamente) ou a eficiência de utilização do alimento (FCR de 1.51 e 1.53; PER de 1.51 e 1.50 para as dietas FM100 e MBM5, respetivamente). No entanto, uma substituição de 75% da FP acarretou uma diminuição significativa da taxa de crescimento (DGI de 2.25) e da utilização do alimento (FCR de 1.72; PER de 1.29), apesar da ingestão voluntária de alimento (g kg ABW-1 day-1) ter sido significativamente maior (26.1 comparado a 24.2 para a dieta MBM50). A composição corporal não foi, de uma forma geral, afetada pelas dietas experimentais, com a exceção do teor em lípidos e energia, que foram significativamente mais baixos nos peixes alimentados com a dieta MBM75. A eficiência de retenção proteica e aminoacídica não foi afetada pelas dietas experimentais enquanto a lipídica e energética foram significativamente reduzidas com o aumento da substituição da FP. O coeficiente de digestibilidade aparente da matéria seca, proteína, lípidos energia e aminoácidos foi avaliada através de um ensaio de digestibilidade. A incorporação de MBM não alterou significativamente a digestibilidade da proteína, que foi elevada (> 89%), mas aumentou a digestibilidade da energia, sendo esta significativamente maior para a dieta MBM50 (95.2%), comparativamente ao controlo (82%). Os coeficientes de digestibilidade aparente dos aminoácidos essenciais foram elevados (> 92%), para todas as dietas experimentais e estatisticamente semelhantes ou superiores para a dieta MBM50, comparativamente ao controlo, mas mais baixas para a dieta MBM75 quando comparadas à MBM50. A incorporação de MBM nas dietas modulou significativamente o microbiota gastrointestinal, verificando-se um decréscimo em unidades taxonómicas operacionais (OTUs) e riqueza de espécies. A inclusão de MBM parece promover o desenvolvimento das bactérias dos géneros Vibrio, Bacillus e Mycobacterium, enquanto a colonização pelos géneros Staphylococcus e Corynebacterium diminui. De um modo geral, estes resultados indicam que metade da FP pode ser substituída por MBM, em dietas para juvenis de dourada, sem comprometer a desempenho de crescimento, eficiência de utilização do alimento, digestibilidade e retenção dos nutrientes e energia. Contudo, futuros estudos são necessários para avaliar o efeito da inclusão de MBM em dietas na saúde intestinal, bem-estar e estado imune de douradas, bem como avaliar a sua repercussão na qualidade da carne da dourada. Palavras-chave: Dourada; fontes proteicas alternativas; farinha de carne e osso; aminoácidos; digestibilidade; microbiota FCUP Potential use of meat and bone meal in diets for gilthead seabream (Sparus aurata) juveniles 4 Contents Abstract ........................................................................................................................ 1 Resumo ........................................................................................................................ 2 Tables list ..................................................................................................................... 6 Figure list ...................................................................................................................... 6 Abbreviations ................................................................................................................ 7 Introduction ................................................................................................................... 8 The State of Aquaculture .......................................................................................... 8 The State of European and Mediterranean Aquaculture ........................................... 9 Gilthead seabream (Sparus aurata L.).................................................................... 10 Biology ............................................................................................................ 10 Aquaculture production .................................................................................... 11 Feed formulation in aquaculture ............................................................................. 12 Ingredients for aquafeeds: current situation ............................................................ 14 Plant protein sources in gilthead seabream ............................................................ 16 Animal by-products protein sources ....................................................................... 17 The use of animal by-products in aquaculture ........................................................ 19 Objectives of this study .......................................................................................... 20 Materials and methods ................................................................................................ 24 Diet composition ..................................................................................................... 24 Growth trial ............................................................................................................. 25 Fish sampling ......................................................................................................... 26 Sampling for GIT microbiota analyses .................................................................... 26 Digestibility trial ...................................................................................................... 26 Chemical analyses ................................................................................................. 27 Amino acid determination ....................................................................................... 27 PCR-DGGE (Polymerase Chain Reaction - Denaturing Gradient Gel Electrophoresis) ................................................................................................................................ 28 FCUP Potential use of meat and bone meal in diets for gilthead seabream (Sparus aurata) juveniles 5 DNA extraction from GIT samples ................................................................... 28 Polymorphism analyses of 16S rRNA genes by denaturing gradient gel electrophoresis (DGGE) .................................................................................. 28 Data and statistical analyses .................................................................................. 29 Ethics statements ................................................................................................... 30 Results ....................................................................................................................... 30 Growth trial: performance and feed utilization efficiency ......................................... 30 Digestibility trial: ADC (%) of diets and amino acids ............................................... 31 Whole body composition and biometric parameters ............................................... 32 Nutrient and amino acid budget .............................................................................. 32 Modulation of gilthead seabream gut microbiota .................................................... 36 Discussion .................................................................................................................. 41 Conclusion .................................................................................................................. 48 References ................................................................................................................. 50 FCUP Potential use of meat and bone meal in diets for gilthead seabream (Sparus aurata) juveniles 12 (Basurco et al. 2011) or the Egyptian “hosha”, which are natural traps that take advantage of the juveniles’ trophic migration from the sea to coastal lagoons. For a very long time, marine rearing of this species depended on the collection of wild juveniles and it was only up until the 1980’s that intensive fish rearing systems were developed, mostly due to successful artificially breeding techniques derived by a shortage of fry and juveniles, establishing the beginning of mass production of gilthead seabream (Moretti et al. 1999). Since then, this species has become one of the main products of European aquaculture. Currently, grow out of gilthead seabream is performed on floating cages at open sea while most of the reproduction and growth phase is in intensive land systems (Merinero et al. 2005). In 2011, the EU produced around 74 000 tons of gilthead seabream, providing a revenue of 370 million euros. However, this represented a decrease of 19% in volume and 6% in value when compared to 2010 (EUMOFA 2014). Greece was the largest contributor, responsible for 67% of all volume produced. In 2013, Portugal reported a production of 1 201 tons of gilthead seabream (INE 2015). Improvements of rearing techniques in the last several years, such as feeding systems automation, harvesting procedures and health management, have resulted in an overproduction of gilthead seabream that is having a toll on prices in the main European markets (Flos et al. 2002). From 1996 to 2005, production in Mediterranean countries rose from 30 000 tones to 90 000 tones (Martínez-Llorens et al. 2008) and this, associated with the decrease in sale price, has forced farmers to control the production costs in order to improve profitability (Merinero et al. 2005). Feed formulation in aquaculture About 40% of total aquaculture production is dependent on the supply of exogenous feeding (Deutsch et al. 2007). This is known as intensive aquaculture production and allows producers more control over the quality of the final product and more control over the culture conditions. From the period 1995-2008, the industrial aquafeed production increased more than threefold, as a consequence of the increase in intensive aquaculture, growing from 7.6 MT to 29.2 MT, at an average rate of 11% per year, and it is expected to reach 71 MT by 2020 (FAO 2011). Currently, the challenge in fish nutrition research is the formulation of sustainable diets, less dependent on marine ingredients that support maintenance, growth, reproduction, health and well-being of the animal, at a reduced cost, while providing food with a good nutritional value for humans. FCUP Potential use of meat and bone meal in diets for gilthead seabream (Sparus aurata) juveniles 13 Compared to terrestrial animals, aquaculture feeds possess a wide range of nutritional composition where ideal values in nutrients vary among the different species and life stages, and according to other factors such as production and environmental constrains, markets or manufacture’s preferences and economic climate (Bureau 2006). Marine fish, due to developing in an aquatic environment where carbohydrates sources are scarce, have a digestive and metabolic system better adapted to use protein and lipids as energy source (Lovell 1998), which is required for life-sustaining processes, such as maintenance, movement, and tissue synthesis. Currently, commercial feeds for marine carnivorous fish contain between 40-50% protein and 12-26% lipids (Cerdá 2012). Fish also seem to use protein more efficiently than terrestrial animals, due to a more effective nitrogen excretion through the gills that requires much less energy than excretion as urea or uric acid (Lovell 1998). Fish, like other animals, do not have a true protein requirement but have a requirement for a well-balanced mixture of essential (EAA) and non-essential amino acids (NEAA). Amino acids are the structural components of proteins and are used, among other functions, to synthesize new protein and new muscle and a balanced amino acid profile is essential for fish growth and wellbeing (NRC 2011). Dietary incorporation of lipids and carbohydrates is important to promote dietary protein sparing, so that protein is solemnly used for muscle growth instead of energetic purposes, as protein is considered to be most expensive component in a feed (Forster and Dominy 2006). It is important to note that the metabolic capacity to use either of these non-protein energetic components differs among fish species and feeding habits and environments, being necessary knowledge on the species’ specific ability of its utilization. Also, feeding is considered to be one of the main factors that can significantly modulate the gut microbial community in farmed fish (Estruch et al. 2015). The gastrointestinal tract (GIT) is a complex system not only involved in digestion and nutrient absorption but also in the animal’s immune response and disease resistance (Cerezuela et al. 2012), which are largely influenced by the GIT microbiota (Silva et al. 2011). Bacteria are the most commonly found microorganisms in the GIT of fish (Rawls et al. 2004; Nayak 2010) with colonization beginning in the larval stages and consequent establishment, composition, and diversity being modulated through the rest of the fish’s life cycle by many endogenous and exogenous factors including habitat, environmental/culture conditions, age and diet (Nayak 2010; Tapia-Paniagua et al. 2010). Therefore, it is important to study how dietary manipulations can modulate the diversity and composition of the GIT microbiota and its relationship with the host. This FCUP Potential use of meat and bone meal in diets for gilthead seabream (Sparus aurata) juveniles 14 information might be used as a strategy to improve nutrition but also as a way to prevent diseases (Navarrete et al. 2009) since a disruption in this delicate balance may lead to alterations in immune regulatory response, increasing disease susceptibility or reducing functionality (Perez et al. 2010; Dimitroglou et al. 2011; Tapia-Paniagua et al. 2011). Up to this point, little is known about the diversity and functional role of gilthead seabream GIT microbial communities (Kormas et al. 2014). Most of the studies performed so far on several fish species are based on culture dependent methods which are laborious and time consuming (Tapia-Paniagua et al. 2010), and do not allow a true evaluation of the microbiota diversity and composition, as many bacteria species are unculturable (Navarrete et al. 2009; Feng et al. 2010). The devolvement of molecular culture-independent approaches, such as PCR-DGGE (Polymerase Chain Reaction - Denaturing Gradient Gel Electrophoresis), has allowed a more complete and rapid assessment of the composition, diversity and functional relationships of the microbiota in fish GIT (Clements et al. 2014). PCR-DGGE is based on the amplification of equally sized PCR products of a hypervariable region of a highly conserved bacterial gene (V3region of the 16S rRNA gene), which are then separated by electrophoresis on a denaturing gradient gel, based on their differential denaturation profile, i.e. their differential polymorphisms (Ercolini 2004). These DNA fragments can then be further analyzed, through sequencing of the excised gel bands and subsequent identification (Hovda et al. 2007). Ingredients for aquafeeds: current situation In order to formulate adequate feeds for farmed species, the industry has relied heavily on ingredients of marine origin such as fishmeal (FM) and fish oils. According to FAO (2012), fishmeal is “the crude flour obtained after milling and drying fish or fish parts, and it is produced from whole fish, remains or other fish by-products resulting from processing” and it is the preferred ingredient as it can support rapid fish growth and feed conversion, constituting the major dietary protein source in commercial aquafeeds for marine species (Gao et al. 2013). It has a high protein content, high nutrient digestibility and palatability, balanced amino acid profile, lack of anti-nutritional factors and it is generally widely available (Forster and Dominy 2006; Gatlin et al. 2007). It is also a highly tradable product, and its production can be an important source of revenue for some countries (FAO 2014). Although in 2012, 35% of the worlds’ FM was produced from fish waste, a significant proportion derives from capture fisheries of small pelagic fish, such as FCUP Potential use of meat and bone meal in diets for gilthead seabream (Sparus aurata) juveniles 15 anchoveta, sardines, herring and mackerel (FAO 2014; Sun et al. 2014). As the main producing countries, responsible for 2/3 of the trade, Peru and Chile, are affected by natural phenomena, such as El Niño, the catches of these species are bound to fluctuate, affecting supply and prices of FM (Hardy 2000). In fact, over the last decade, the prices of this commodity have significantly risen associated with the increasing demand and scarcity, both for terrestrial and aquatic production, going from a little over 500 US$/ton in 2003 to almost 2000 US$/ton in 2013 (FAO 2014). Due to this, there has been general concern about future supply of FM and its sustainable use in feeds as the aquaculture sector remains the largest consumer, currently using 60% of total production (Karapanagiotidis 2014). At current inclusion rates, annual FM production is not enough to support the predicted growth of intensive rearing systems. Also there is a perceived inefficiency in catching fish, processing it into FM and then fed it back to fish (Yu 2004). Taking everything into account, it is, therefore, necessary to improve feeding strategies as an industry that is dependent on FM is vulnerable to collapse through the loss of profit margins (Read and Fernandes 2003). In Mediterranean intensive aquaculture, feeding and diet formulation can account for as much as 45% of the overall production costs (Williams et al. 2003). FM supplies the largest portion of dietary protein for carnivorous fish in aquaculture (Kokou et al. 2012) and for some farmed species, inclusion levels are still around 50% (Glencross et al. 2007). In fact, intensive production of European sea bass and gilthead seabream still requires continuous supply of high quality marine products (Karapanagiotidis 2014) and, particularly for gilthead seabream production, feeding costs can go as high as 49% of total production costs (Merinero et al. 2005). One way to increase profitability and reduce the environmental impact of FM is through the optimization of the nutrient levels in diets (Williams et al. 2003), for example, by achieving an optimal protein/energy ratio (García-Gallego et al. 1998), or by strategically replace FM with other less expensive protein sources (Martínez-Llorens et al. 2008). Research is ongoing to find the best sustainable replacements for FM, without compromising growth, quality and welfare of farmed fish while still ensure the best economic returns. Throughout the years, there has been a great number of potential ingredients evaluated to be included in feeds for farmed species. According to Gatlin et al. (2007), a suitable replacer for FM must be widely available at a competitive price, easy to handle, ship, store and incorporate in fish diets. In terms of nutritive value, it must be low in fiber and carbohydrates, high in protein, with a balanced amino acid profile, as well as be highly digestible and palatable. Many of these ingredients are more complex than FM FCUP Potential use of meat and bone meal in diets for gilthead seabream (Sparus aurata) juveniles 16 and require thorough evaluation in order to determine their nutritional value, appropriate incorporation levels and nutritional limitations, and its practicality to include in commercial feed formulation (Glencross et al. 2007). Due to the high protein requirements of carnivorous marine species, such as gilthead seabream, the number of potential alternatives is restricted to ingredients with high protein content and digestibility (Yiğit et al. 2012). Plant protein sources in gilthead seabream Due to its wide availability, competitive price and relatively constant nutritional composition (Pereira and Oliva-Teles 2002), plant protein sources have been the subject of study for a great variety of farmed species. Because gilthead seabream is an economically important species in Mediterranean aquaculture (Kokou et al. 2012), there has been substantial effort to evaluate plant protein ingredients for FM replacement. Some of the studied plant ingredients include soybean meal (Robaina et al. 1995; Kissil et al. 2000; Kissil and Lupatsch 2004; Martínez-Llorens et al. 2007; Kokou et al. 2012), corn gluten meal (Robaina et al. 1997; Kissil and Lupatsch 2004; Yiğit et al. 2012), hazelnut meal (Emre et al. 2008), lupin seed meal (Robaina et al. 1995; Pereira and Oliva-Teles 2004), wheat gluten (Kissil and Lupatsch 2004), pea protein concentrate (Sánchez-Lozano et al. 2010), pea seed meal (Pereira and Oliva-Teles 2002), carob seed germ meal (Martínez-Llorens et al. 2012), rapeseed protein concentrate (Kissil et al. 2000), as well as mixtures of several plant ingredients (Venou et al. 2003; Kissil and Lupatsch 2004). Despite its potential, plant protein sources have only seldom been able to fully replace FM (Pereira and Oliva-Teles 2002), or even being used at high levels in feeds as fish performance has been inversely related to the inclusion levels of test ingredients (Pereira and Oliva-Teles 2003). This trend also follows for most carnivorous fish species (Gómez-Requeni et al. 2004; Karapanagiotidis 2014). Overall, when using plant protein sources individually, FM can be replaced up to 60% for Sparidae fish, while with mixtures, substitution levels can go up to 75% but with major negative effects on fish’s health (Oliva-Teles et al. 2011). The high content of carbohydrates present in plants protein sources limits its use as most fish species, in particular carnivorous, cannot use them effectively as energy source (Li et al. 2010). Enes et al. (2011) suggested that diets for gilthead seabream juveniles should not include more than 20% digestible carbohydrates, as higher dietary inclusions may depress growth and feed utilization. Furthermore, the presence on anti-nutritional factors FCUP Potential use of meat and bone meal in diets for gilthead seabream (Sparus aurata) juveniles 17 in many plant sources has negative physiological effects, by damaging the gastrointestinal tract and reducing nutrient digestibility, growth performance, and increasing disease susceptibility (Baeza-Ariño et al. 2014). Also, some plant protein sources appear to be limited in some EAA, such as lysine, methionine and tryptophan (NRC 1993; Martínez-Llorens et al. 2012) that can restrain growth and protein accretion (Kissil et al. 2000). However, this limitation can be overcome by using a mixture of complementary protein sources (Pereira and Oliva-Teles 2004) or by dietary supplementation with crystalized amino acids, making the feed more complex and, sometimes, more expensive. The aquaculture industry also competes in the international market for the use of these plant ingredients along with the animal husbandry sector, biofuel production and direct use for human consumption (Karapanagiotidis 2014). Additionally, sustainability issues arise where there is growing pressure to develop environmentally friendly aquafeeds as some of the most used plant sources are produced in tropical countries, causing destruction of the rainforest for soy production in South America or the native forest in south-east Asia for palm (Karapanagiotidis 2014). Animal by-products protein sources Animal by-products, also called processed animal proteins (PAP) or rendered animals ingredients, have received great attention worldwide as they appear to be more practical and cost-effective alternative to FM for aquaculture feeds (Booth et al. 2012). These products are usually the result of processed slaughterhouses leftovers and in the EU, around 17 MT of these by-products are produced annually, corresponding to 3 million metric tons of protein (Woodgate and Veen 2004). Depending on the raw materials used to manufacture these ingredients, they can have various designations such as meat meal (MM), meat and bone meal (MBM), poultry by-product meal (PBM), feather meal (FeM), blood meal (BM) and even milk-byproducts and gelatin (Hardy and Barrows 2002). MM or MBM are widely used animal by-products, derived from slaughtered farmed livestock (cattle, swine, sheep, and/or poultry) (FAO 2011), and typically possess high protein content (50-85%) with a relatively good amino acid profile, (Wilson 2002), moderate fat level (7-15%), high ash content (10 to 40%), high digestibility, low carbohydrate content, and lack anti-nutritional factors (Hu et al. 2013). Meat meals are also a good source of calcium and trace minerals (Rossi and Davis 2014) and can be used as a source of phosphorous (P), even at low inclusion levels (Suloma FCUP Potential use of meat and bone meal in diets for gilthead seabream (Sparus aurata) juveniles 18 et al. 2013). It is a product produced worldwide with a steady availability, allowing a more flexible feed formulation and advantageous in countries where FM is not locally available. Nutritional quality of animal by-products are greatly influenced by the quality and specific combination of the raw materials as well as by the processing methods used to manufacture these products (Forster and Dominy 2006; Rossi and Davis 2014) which can result in an inconsistent and unpredictable final product, being more variable than between fish meals. Meat meals are typically produced by a dry-rendering process where the raw material is cooked by dry heat at 135-140ºC in a steam jacked cooker until all the moisture is evaporated, followed by fat removal by draining off and screw press and grinding. Variation in the proportion of bone and soft tissues used contribute to large variations in meal quality and their classification as MM (<55% protein and <4.4 P) or MBM (<55% protein and <4.4% P) (Bureau et al. 2000). Compared to FM, protein digestibility of meat meals is generally lower, up to 20% for meals with high ash and fat content, and may be low in some EAA such as lysine, methionine, phenylalanine, isoleucine and/or histidine (Millamena and Golez 2001; Hu et al. 2008a; Wang et al. 2008), mainly caused by heat-damaged protein due to excessive heat during processing (Allan et al. 2000). In fact, an unbalance of EAA content may lead to poor results when high substitution levels are applied (Tidwell et al. 2005; Forster and Dominy 2006). Indigestible inorganic matter content, depending on the quantity of bone in the raw material, may also be a limiting factor when using MM and MBM as high content may impair digestibility, reducing nutrient and energy availability. Robaina et al. (1997) determined that ash levels in MBM exceeding 12.5% could lead to a decrease in protein digestibility in gilthead seabream. Animal by-products, particularly meat meals, usually possess high levels of saturated fatty acids (FA) and 18:2 n-6 polyunsaturated FA but are low in n-3 highly unsaturated FA (eicosapentaenoic, docosahexaenoic, and arachidonic acids) that are required by marine fish (Millamena 2002; Hu et al. 2013). Unbalances in saturated and unsaturated FA content can lead to body lipid accumulation and morphological alterations (Robaina et al. 1997) as well as contribute to reduced palatability of these diets for fish. Robaina et al. (1997) found that gilthead seabream fed increasing levels of MBM showed hepatocyte necrosis in the liver and a progressive decrease in lipid digestibility. FCUP Potential use of meat and bone meal in diets for gilthead seabream (Sparus aurata) juveniles 19 The use of animal by-products in aquaculture The use of animal by-products in aquafeeds is highly variable depending on the region. In the EU, its use was prohibited in 1990-2000, by the EU Commission Regulation (EC No. 999/2001) due to the arising of bovine spongiform encephalopathy (BSE) in ruminants in Western Europe in the 1980-1990’s. BSE is a disease caused by prion protein and is also linked to a human disease, Creutzfeldt-Jakob disease, believed to be transmitted from infected cows to humans (Friedland et al. 2009), including its PAP byproducts derived from infected ruminants. In 2013, however, this prohibition was lifted allowing the use of only PAPs derived from non-ruminant animals (Category 3, including poultry, feather meal, porcine and porcine blood meal, PAP) for feeding of aquaculture animals (EU Commission Regulation, EC No. 56/2013). This opened doors to a whole new range of ingredients that can be used in aquafeeds inside the EU. Up to this point limited work has been carried out towards the use of animal byproducts in gilthead seabream feeds. Table 1 summarizes all the literature found. Table 1: Studies performed to evaluate fishmeal replacement with different animal by-products ingredients in gilthead seabream. Animal byproduct1 IBW (g)2 Dietary protein content (%DM) Recommended substitution level (%DM) Reference MBMa 5 51.8 40 Davies et al. (1991) MBMb 5 49.2 36 Alexis (1997) PMM 1.1 44.9 100 MBMc 40 43.9 20 Robaina et al. (1997) PBM 1.55 44.0 75 Nengas et al. (1999) BMd 33/179 46.6 15 Martínez-Llorens et al. (2008) 1 MBM: Meat and bone meal; MBMa (46-59% CP; 9-12% CL; 25-33% Ash); MBMb (60% CP; 9% CL; 25% ash); MBMc (64% CP; 10.3% CL; 25.4% ash). PMM: Poultry meat meal; PBM: Poultry by-product meal; BMd: Blood meal (98.8% CP; 0.2% CL; 1.0% ash). 2 IBW (g): Initial body weight Also, for other aquaculture fish species worldwide, research has demonstrated that these ingredients have potential to be included in aquafeeds. Table 2 summarizes the studies that have been performed using animal by-products ingredients as fishmeal replacement. FCUP Potential use of meat and bone meal in diets for gilthead seabream (Sparus aurata) juveniles 20 Objectives of this study The aim of this study is to evaluate the effects on growth performance, wholebody composition, digestibility, nutrient and amino acid retention, and gut microbiota modulation in gilthead seabream fed diets formulated to replace 50 and 75% of fishmeal (FM) by meat and bone meal (MBM). FCUP Potential use of meat and bone meal in diets for gilthead seabream (Sparus aurata) juveniles 21 Table 2: Studies performed with several farmed species using animal by-product ingredients for fishmeal replacement. Species Habitat/ Trophic level1 IBW (g)2 Animal by-product (% DM)3 Diet protein content (%DM) Recommended FM substitution levels (% DM) Reference African catfish (Clarias gariepinus) FW 3.8 ± 0.4 93.1 PBM 25.3 100 Goda et al. (2007) 93.9 MBM 25.8 75 Australian short-finned eel (Anguilla australis australis) FW 4.3 ± 0.5 2.23 MM 43.6 23 Engin and Carter (2005) Australian silver perch (Bidyanus bidyanus) FW 3.0 ± 0.34 12.2 MM 34 (DP4 basis) 52 Stone et al. (2000) Australian snapper (Pagrus auratus) Marine 3.6 ± 0.2 14.0 MM 53.6 35 Booth et al. (2012) PBM 80.8 36 18% PBM + 15% BM + 5% MM 52.7 84 Black sea bream (Acanthopagrus schlegelii) Marine 3.2 ± 0.45 7.90 Enzyme treated PBM 41.8 16 Gao et al. (2013) Bluegill (Lepomis macrochirus) FW 3.2 ± 0.2 22.0 38% MBM 44.0 100 Masagounder et al. (2014) Climbing perch (Anabas testudineus) FW 3.0 ± 0.4 0.53 18% MBM 40.2 67 Kader et al. (2011a) Cuneate drum (Nibea miichthioides) Marine 4.0 ± 0.7 28.0 17% PBM + 9% MBM + 3% BM 42.8 80 Guo et al. (2007) 17% PBM + 6% MBM + 3% BM + 3% FeM 43.0 80 Florida pompano (Trachinotus carolinus) Marine 3.5 ± 0.6 2.99 10% MBM 37.1 67 Rossi and Davis (2014) 5.87 14% MBM + 0.6% Tau 39 100 Gibel carp (Carassius auratus gibelio) FW 2.5 ± 0.0 15.3 8% PBM + 2% BM + 4% MBM 37.9 67 Hu et al. (2008a) 13.5 12% PBM + 6% MBM + Lys and Met 37.9 67 Hu et al. (2008b) 4.88 PBM 37.9 67 Yang et al. (2006) FCUP Potential use of meat and bone meal in diets for gilthead seabream (Sparus aurata) juveniles 28 cysteic acid after oxidation with performic acid. Amino acids were separated by HPLC with a C-18 reverse-phase column Waters Acc. Tag (150 mm x 3.9 mm). PCR-DGGE (Polymerase Chain Reaction - Denaturing Gradient Gel Electrophoresis) DNA extraction from GIT samples DNA extraction from GIT samples, from a pool of 2 fish per tank to reduce variation, was performed according to Pitcher et al. (1989) with some modifications. Briefly, approximately 300 mg of each sample were resuspended in 1 mL of TE buffer (10 mM Tris, 1 mM EDTA, pH 8) vigorously mixed and pelleted by centrifugation at 13000 g for 5 min. After 2 washes with 1 mL TE, cell pellet was resuspended in 200 µL of TE containing 50 mg/mL of lysozyme and incubated for 30 min at 37 ºC. A second 30 min incubation at 37ºC was performed with the addition of 10mg/mL RNAse, followed by a 30 min incubation at 55ºC with 20 mg/mL Proteinase K and 10% SDS. After 10 min on ice in the presence of 500 µL of GES (Pitcher et al. 1989) and 250 µL of ammonium acetate (7.5 M), a phenol-chloroform extraction was performed by adding 500 µL phenolchloroform-isoamyl alcohol (25:24:1). The aqueous phase was re-extracted with 500 µl of chloroform-isoamyl alcohol (24:1) and the DNA of the subsequent aqueous phase was precipitated with 0.6 volumes of isopropanol. After 10 min centrifugation at 13000 g, the DNA pellet was washed with ice-cold 70 % ethanol and dried at room temperature. DNA was resuspended in 50 µL ultrapure water. Polymorphism analyses of 16S rRNA genes by denaturing gradient gel electrophoresis (DGGE) Bacterial 16S rRNA gene fragments were amplified by a touchdown PCR on a T100TM Thermal Cycler (Bio-Rad), using primers 16S-358F (which has a GC clamp at the 5’ end) and 16S-517R (Muyzer et al. 1993), yielding a 233bp DNA fragment. PCR mixtures (50 µL) contained 24.75 µL of water (Sigma), 10 µL of GoTaq Buffer 5X (PROMEGA), 5 µL of each dNTPs (2 mM, PROMEGA), 2.5 µL of each primer (10 µM Forward and Reverse), 0.25 µL of GoTaq polymerase (PROMEGA), and 5 µL of DNA template were subjected to a touchdown PCR. A 94ºC incubation for 5 min was followed by 10 cycles of 64ºC, 1 min, 65ºC, 1 min and 72ºC, 3 min. The annealing temperature was decreased at every cycle 1ºC, until reaching 55ºC. Thus, final 20 cycles of 94ºC for 1 min, 55ºC for 1 min and 72ºC for 3 min. Final extension was at 72ºC, 10 min. PCR FCUP Potential use of meat and bone meal in diets for gilthead seabream (Sparus aurata) juveniles 29 products were resolved by electrophoresis on 1 % (w/v) agarose gels containing Gel Red (Biotium) to check for product size. 300 ng of each PCR product were loaded on an 8% polyacrylamide gel composed by a denaturing gradient of 40 to 80% 7M urea/40%formamide. Electrophoresis occurred on a DCode™ universal mutation detection system (Bio-Rad), during 16h at 60°C, 65V in 1×TAE buffer. Gels were stained for 1 hour with SYBR-Gold Nucleic Acid Gel Stain, and imaged on a Gel Doc EZ System (Bio-Rad) with the Image Lab software v4.0.1 (Bio-Rad). Selected bands were excised from the gel and eluted in 20 µl ultrapure water prior to DNA re-amplification using the same oligonucleotide primers as above, but without the GC clamp. Amplicons were sequenced to identify microbiota OTUs (Operational Taxonomic Units). Phylogenetic analysis, to identify the closest known species, was done by comparison with sequences in the GenBank non-redundant nucleotide database using BLAST (http://www.ncbi.nlm.nih.gov). Only sequences higher than 100 bp reads and 80–100% query coverage were considered a valid identification. Data and statistical analyses Before analysis, all data obtained was checked for normality (Kolmogorov– Smirnov test) and homogeneity of variances (Levene’s test). If necessary, variables were normalized by log transformation or arcsin square root transformation, for data expressed as decimal fractions or percentage, respectively. DGGE banding patterns were transformed into presence/absence matrices and band intensities measured using Quantity One 1-D Analysis Software v4.6.9 (Bio-Rad Laboratories, Lda. Amadora, Portugal). Relative similarities between dietary treatments and replicates were calculated using Primer software v7.0.5 (PRIMER-E Ltd, Ivybridge, UK). Similarity percentages (SIMPER) were used to represent the relative similarities between treatments. Species richness was assessed using Margalef’s measure of richness, and species diversity was assessed by the Shannon–Weaver index. Clustering of DGGE patterns was achieved by construction of dendrograms using the Unweighted Pair Groups Method with Arithmetic Averages (UPGMA). Statistical analysis of data was done by one-way analysis of variance (ANOVA). Newman–Keuls test was used to assess significant differences among diets at 0.05 significant levels (Stat graphics, Statistical Graphics System, Version plus 5.1, Herndon, VA, USA). DGGE parameters were subjected to a two-way ANOVA, with section and diet as fixed factors. FCUP Potential use of meat and bone meal in diets for gilthead seabream (Sparus aurata) juveniles 30 Ethics statements The experimental protocol was reviewed and approved by the Committee of Ethics and Animal Welfare of the Universitat Politècnica de València (UPV), following the Spanish Royal Decree 53/2013 on the protection of animals used for scientific purposes (BOE 2013). Results Growth trial: performance and feed utilization efficiency At the end of the growth trial, survival rate was high (>95%) and unaffected by the experimental diets. Growth performance and feed utilization of fish fed the experimental diets are presented in Table 5. Diet MBM50 showed similar results to the control diet with no significant differences in terms of final body weight, weight gain, daily growth index, feed conversion ratio and protein efficiency ratio. However, increasing the inclusion rate of MBM to 75%, growth performance and feed utilization were significantly reduced. Feed intake was significantly higher for diet MBM75 when compared to the control diet but not statistically different from MBM50. Table 5: Growth performance and feed utilization efficiency of gilthead seabream fed the experimental diets1. FM100 MBM50 MBM75 SEM Initial Body Weight (g) 24.3 23.8 24.3 0.3 Final Body Weight (g) 121.4a 121.7a 108.2b 2.4 Weight Gain (%)2 399.4a 412.4a 346.1b 12.5 Daily growth index3 2.48a 2.51a 2.25b 0.05 Feed intake (g kg ABW -1day-1)4 24.2b 24.6ab 26.1a 0.4 Feed conversion ratio5 1.51b 1.53b 1.72a 0.04 Protein efficiency ratio6 1.51a 1.50a 1.29b 0.04 Survival Rate (%) 100 94.8 99.3 1.5 1 Means in the same row with different superscript letters are significantly different (p<0.05). SEM: pooled standard error of the mean. Average body weight (ABW): initial body weight (IBW) + final body weight (FBW)/2. 2 Weight gain, % = [(Final weight – Initial weight) / Initial weight] x 100 3 DGI = [(Final weight1/3 – Initial weight1/3) / days] x 100 4FI = Total dry feed intake / Average body weight / days 5 FCR = Dry feed intake (g) / Weight gain (g) 6PER = weight gain / crude protein intake FCUP Potential use of meat and bone meal in diets for gilthead seabream (Sparus aurata) juveniles 31 Digestibility trial: ADC (%) of diets and amino acids The apparent digestibility coefficients (ADC) of diets and amino acids were evaluated (Table 6). ADC of dry matter, crude protein and energy were the highest for diet MBM50, averaging 89, 97 and 95%, respectively. ADC of crude protein was not statistically affected by the experimental diets while ADC of dry matter was significantly lower for both MBM75 (68%) and the control (75%) diets. ADC of energy was lower for fish fed diet MBM75 (87%) but not significantly different from the control diet (82%). The ADCs of EAA were all high (>93%) but significantly lower for fish fed diet MBM75 when compared to diet MBM50, which were significantly higher. Digestible amino acid content of the experimental diets is presented in Table 7. Table 6: Apparent digestibility coefficients (ADC, %) of the experimental diets1. FM100 MBM50 MBM75 SEM Dry matter 74.6b 86.9a 68.4b 3.3 Crude Protein 95.7 96.7 88.6 1.9 Energy 82.2b 95.2a 87.0ab 2.5 Amino acids Essential amino acids Arg 95.7ab 96.7a 94.1b 0.5 His 95.2b 97.8a 95.2b 0.6 Ile 95.9ab 97.2a 94.0b 0.6 Leu 95.9ab 97.0a 93.9b 0.6 Lys 97.3ab 97.9a 95.4b 0.5 Met 96.7a 96.6a 92.5b 0.9 Phe 94.4a 97.0a 93.3b 0.7 Thr 95.5ab 96.6a 93.6b 0.6 Val 95.6ab 96.8a 93.8b 0.6 Non-essential amino acids Ala 95.5 96.0 93.3 0.7 Asp 92.0b 96.4a 93.0b 0.8 Cys 91.1ab 92.6a 87.6b 0.6 Glu 96.5a 96.7a 93.9b 0.7 Gly 92.2 94.4 91.9 0.6 Pro 95.9a 95.0ab 91.8b 0.8 Ser 95.0ab 95.9a 92.9b 0.6 Tyr 97.1a 97.2a 94.1b 0.7 1 Means in the same row with different superscript letters are significantly different (p<0.05). SEM: pooled standard error of the mean ADC (%) = 100 -100 x [(marker in diet/marker in feces) x (AA in feces/AA in diet)] FCUP Potential use of meat and bone meal in diets for gilthead seabream (Sparus aurata) juveniles 32 Table 7: Digestible amino acids content (g 100 g-1 DM) of the experimental diets1. Diet Essential amino acids Non-essential amino acids Arg His Ile Leu Lys Met Phe Thr Val Ala Asp Cys Glu Gly Pro Ser Tyr FM100 2.84 1.15 1.80 3.12 2.97 1.20 1.78 1.75 2.26 2.38 3.42 0.27 6.01 2.40 1.70 1.58 1.24 MBM50 4.80 0.80 1.35 2.56 2.50 1.02 1.41 1.33 1.85 2.42 2.97 0.27 5.39 3.66 2.13 1.39 0.95 MBM75 3.41 0.84 1.30 2.60 2.37 0.95 1.37 1.50 1.96 2.94 3.27 0.30 6.07 4.99 2.65 1.63 0.93 1 AA in the diet (g 100g-1) x ADC (%) of AA / 100 Whole body composition and biometric parameters At the end of the growth trial, whole-body composition was unaffected by the dietary inclusion of MBM, with the exception of crude lipid and energy content, which were significantly lower for fish fed the diet MBM75 (Table 8). There were no significant differences in whole-body amino acid composition (g 100 g-1) and in the measured biometric parameters of gilthead seabream fed the different experimental diets. Nutrient and amino acid budget Nitrogen, lipid and energy balance of fish fed the experimental diets are presented in Table 9. Results show that the inclusion of MBM did not significantly affect nitrogen retention (% intake), while daily nitrogen intake was significantly higher for fish diet MBM75. Daily lipid and energy intake were significantly higher for diet MBM75 and lower for diet MBM50 but neither were significantly different from the control diet. Lipid retention (% intake) was significantly higher for fish fed diet MBM50 but lower for diet MBM75 than that of the control diet. Compared to the control diet, energy retention was significantly lower for fish fed the diet with the highest inclusion of MBM while with MBM50 it was not significantly different. Amino acid budget is presented in Table 10 and in Fig. 4, it is represented the efficiency of EAA retention (% intake) of fish fed the different experimental diets. Results show no significant changes in EAA retention, daily or per percentage of intake, for the different experimental diets. FCUP Potential use of meat and bone meal in diets for gilthead seabream (Sparus aurata) juveniles 33 Table 8: Whole-body composition and biometric parameters of gilthead seabream fed the experimental diets1. FM100 MBM50 MBM75 SEM Whole-body composition (% wet weight) Dry matter (%) 35.0 34.6 33.1 0.4 Crude protein 16.1 15.9 15.9 0.2 Crude lipid 16.1a 16.4a 14.5b 0.3 Ash 2.10 1.91 2.01 0.1 Energy (kJ g-1) 9.94a 9.68a 8.67b 0.23 Essential amino acid (g 100g-1 wet weight) Arg 1.24 1.14 1.05 0.08 His 0.34 0.32 0.28 0.11 Ile 0.60 0.58 0.56 0.16 Leu 1.08 1.04 0.99 0.22 Lys 1.13 1.05 0.97 0.18 Met 0.49 0.51 0.53 0.07 Phe 0.55 0.52 0.51 0.13 Thr 0.61 0.54 0.56 0.13 Val 0.74 0.72 0.70 0.13 Non-essential amino acids (g 100g-1 wet weight) Ala 0.92 0.84 0.87 0.21 Asp 1.32 1.35 1.27 0.19 Cys 0.10 0.11 0.12 0.02 Glu 2.11 2.04 1.97 0.27 Gly 1.11 0.91 1.00 0.81 Pro 0.64 0.52 0.61 0.32 Ser 0.57 0.55 0.53 0.09 Tyr 0.44 0.38 0.39 0.10 Biometric indices Condition factor (g cm-3)2 1.74 1.81 1.75 0.03 Visceral index (%)3 8.57 8.53 9.31 0.22 Hepatosomatic index (%)4 2.75 2.87 2.40 0.32 Visceral fat index (%)5 1.23 1.17 1.22 0.13 1 Means in the same row with different superscript letters are significantly different (p<0.05). SEM: pooled standard error of the mean. 2 CF = [Wet weight (g) / Length3 (cm)] x 100 3 VSI = [Visceral weight (g) / wet weight (g)] x 100 4 HSI = [Liver weight (g) / wet weight (g)] x 100 5 VFI= [Visceral fat (g) / wet weight (g)] x 100 FCUP Potential use of meat and bone meal in diets for gilthead seabream (Sparus aurata) juveniles 34 Table 9: Nitrogen, lipid and energy budget of gilthead seabream fed the experimental diets1. FM100 MBM50 MBM75 SEM Nitrogen Intake (g kg ABW-1 day-1) 1.71b 1.72b 1.89a 0.03 Retention (g kg ABW-1 day-1) 0.43 0.42 0.40 0.01 Retention (% intake) 25.0 24.5 21.3 0.8 Lipid Intake (g kg ABW-1 day-1) 5.17a 4.68b 5.37a 0.11 Retention (g kg ABW-1 day-1) 2.88a 2.94a 2.47b 0.08 Retention (% intake) 55.8b 62.9a 46.0c 2.6 Energy Intake (kJ kg ABW-1 day-1) 5.47a 5.03b 5.64a 0.10 Retention (kJ kg ABW-1 day-1) 1.72a 1.67a 1.40b 0.06 Retention (% intake) 31.5a 33.2a 24.9b 1.4 1 Means in the same row with different superscript letters are significantly different (p<0.05). SEM: pooled standard error of the mean. Nutrient intake (g kg ABW-1 day-1) = [Nutrient intake (g DM) / 1000)] / (ABW (g) x number of days) Nutrient retention (g kg ABW-1 day-1) = [((FBW x final whole-body nutrient content) – (IBW x initial whole-body nutrient content) / 1000] / [ABW x number of days] Nutrient retention (% intake) = Nutrient retention / Nutrient intake x 100 Average body weight (ABW): initial body weight (IBW) + final body weight (FBW) / 2. Fig. 4: Retention (%) of ingested essential amino acid in gilthead seabream fed the experimental diets. 0 10 20 30 40 Arg His Ile Leu LysMet Phe Thr Val Essential amino acid retention (% intake) FM100 MBM50 MBM75 FCUP Potential use of meat and bone meal in diets for gilthead seabream (Sparus aurata) juveniles 35 Table 10: Amino acid budget of gilthead seabream fed the experimental diets1. FM100 MBM50 MBM75 SEM Essential amino acids Arg Int (mg kg-1 day-1) 719.5b 801.2a 857.3a 22.6 Ret (mg kg-1 day-1) 205.2 187.9 158.1 13.2 Ret (%Int) 28.7 23.6 18.5 2.3 His Int (mg kg-1 day-1) 291.3a 209.7c 231.9b 12.6 Ret (mg kg-1 day-1) 58.2 53.6 43.1 5.7 Ret (%Int) 20.2 25.6 18.6 2.3 Ile Int (mg kg-1 day-1) 453.9a 355.8b 364.1b 16.5 Ret (mg kg-1 day-1) 96.6 94.4 84.6 5.4 Ret (%Int) 21.5 26.7 23.3 1.51 Leu Int (mg kg-1 day-1) 810.9a 673.1b 728.1b 22.2 Ret (mg kg-1 day-1) 176.3 169.7 150.4 10.5 Ret (%Int) 21.9 25.3 20.7 1.5 Lys Int (mg kg-1 day-1) 739.6a 653.2b 652.4b 17.0 Ret (mg kg-1 day-1) 186.6 170.4 145.2 15.0 Ret (%Int) 25.5 26.1 22.3 2.1 Met Int (mg kg-1 day-1) 301.4a 270.7b 269.7b 6.4 Ret (mg kg-1 day-1) 84.0 88.8 88.5 2.1 Ret (%Int) 27.9 33.0 32.9 1.2 Phe Int (mg kg-1 day-1) 447.4a 370.2b 386.7b 12.8 Ret (mg kg-1 day-1) 90.2 85.5 77.1 5.3 Ret (%Int) 20.3 23.1 20.0 1.4 Thr Int (mg kg-1 day-1) 444.3a 352.6b 420.0a 14.7 Ret (mg kg-1 day-1) 99.4 86.0 86.3 6.3 Ret (%Int) 22.5 24.9 20.6 1.7 Val Int (mg kg-1 day-1) 572.7a 487.8b 547.5a 14.4 Ret (mg kg-1 day-1) 121.6 116.9 106.7 6.6 Ret (%Int) 21.4 23.7 19.5 1.4 Non-essential amino acids Ala Int (mg kg-1 day-1) 603.9b 643.7b 827.6a 35.6 Ret (mg kg-1 day-1) 153.9 139.3 135.9 7.0 Ret (%Int) 25.7a 21.7ab 16.4b 1.8 Asp Int (mg kg-1 day-1) 900.4a 788.7b 924.1a 23.7 Ret (mg kg-1 day-1) 216.2 221.3 192.2 12.4 Ret (%Int) 24.2 28.1 20.9 1.8 Cys Int (mg kg-1 day-1) 72.6b 75.0b 91.1a 3.1 Ret (mg kg-1 day-1) 16.7 17.5 18.6 0.6 Ret (%Int) 23.1 23.4 20.4 1.0 Gly Int (mg kg-1 day-1) 629.6c 989.2b 1427.9a 116.3 Ret (mg kg-1 day-1) 192.0 152.2 162.5 14.9 Ret (%Int) 30.8a 15.4ab 11.4b 3.8 Glu Int (mg kg-1 day-1) 1507.2b 1423.3b 1697.8a 45.3 Ret (mg kg-1 day-1) 342.9 332.9 299.2 19.5 Ret (%Int) 22.9 23.4 17.7 1.7 Pro Int (mg kg-1 day-1) 429.6c 572.0b 757.0a 48.0 Ret (mg kg-1 day-1) 110.2 86.5 98.3 5.3 Ret (%Int) 25.8a 15.2b 13.0b 2.3 Ser Int (mg kg-1 day-1) 402.1b 371.0b 460.2a 14.1 Ret (mg kg-1 day-1) 92.4 88.4 79.7 6.9 Ret (%Int) 23.2 23.9 17.3 2.1 Tyr Int (mg kg-1 day-1) 309.3a 248.7b 258.9b 10.0 Ret (mg kg-1 day-1) 69.2 59.0 56.6 3.9 Ret (% Int) 22.5 23.5 21.9 1.4 1 Means in the same row with different superscript letters are significantly different (p<0.05). SEM: pooled standard error of the mean; Int: intake; Ret: retention. FCUP Potential use of meat and bone meal in diets for gilthead seabream (Sparus aurata) juveniles 36 Modulation of gilthead seabream gut microbiota The microbial community profiling of the stomach (STO) and intestinal samples (AI, MI, PI) recovered from gilthead seabream fed the experimental diets was studied by polymorphism analyses of the variable V3 region of the 16S rRNA gene using DGGE. Similar banding patterns between the 3 replicates for each diet were not always evident, with one replicate constantly failing to cluster with the other 2 in the Bray–Curtis dendrogram (Fig. 5). The figure further shows that the bacterial communities obtained from the AI of fish fed diet MBM75 seem to be more closely related (percentages of similarity around 70% between 2 out of 3 samples) than those recovered from fish fed the control and MBM50 diets, which seem to diverge more (percentages of similarity below 50% between 2 out of 3 samples for each diet). Nevertheless, variations on the average number of OTUs (Operational Taxonomic Units), microbial richness, microbial diversity and similarity indices between samples were detected with statistical significance between experimental diets and between gastrointestinal sections (Table 11). With exception on the average number of OTUs, there were significant differences (p<0.05) on the indices of microbial richness, microbial diversity and similarity between the different gastrointestinal samples analyzed. The AI samples presented the highest microbial richness, the PI samples the highest microbial diversity, while the STO samples revealed the lowest microbial diversity and richness. PI and STO samples where the ones with higher similarity between replicates, that is, were the most homogeneous samples (Table 11). Replacement of FM by MBM lead to a significant decrease (p<0.01) on the average number of OTUs and on the microbial richness, and to a significant increase on the SIMPER similarity (p<0.001) (Table 11). Sequence analysis from the DGGE bands (Fig. 5, Table 12) showed that the detectable dominant bacteria present in the stomach and intestines of gilthead seabream fed the experimental diets were most closely related to uncultured bacteria (bands 13, 15, and 16) or bacteria belonging to the Corynebacterium (bands 6, 8, 12, and 18), Staphylococcus (bands 1, 10, and 11), Vibrio (bands 9 and 14), Weissella (bands 4 and 5) or Bacillus (bands 2 and 3) genus. Mycobacterium (band 7) and uncultured Plantibacter (band 17) were also detected. FCUP Potential use of meat and bone meal in diets for gilthead seabream (Sparus aurata) juveniles 37 Fig. 5: PCR-DGGE fingerprints of the microbiota found in stomach and intestinal sections recovered from gilthead seabream fed the experimental diets. Black numbers on top of the figure represent the different samples analyzed (from a pool of two fish each) while red numbers inside the figure correspond to bands removed for sequencing, which results are presented in Table 12. FCUP Potential use of meat and bone meal in diets for gilthead seabream (Sparus aurata) juveniles 44 Unbalanced levels of EAA in diets have been reported as one of the causes for growth depression in several farmed fish when fed animal by-products (García-Gallego et al. 1998; Millamena 2002; Sun et al. 2014; Xavier et al. 2014), as protein deposition is closely related to weight gain. García-Gallego et al. (1998) reported that, for European ell, MM diets led to lower feed intake and utilization due to some EAA deficiency. However, in the present study, almost all EAA of the experimental diets exceeded the requirement levels for gilthead seabream, as determined by Peres and Oliva-Teles (2009), with the exception of methionine and phenylalanine + tyrosine, in accordance with previous studies using animal by-products (Nengas et al. 1999; Wang et al. 2008). Besides the rendering composition, the technological processes may also condition protein digestibility, as heat and other processes can damage protein (Booth et al. 2005; Rossi and Davis 2014; Xavier et al. 2014). Tidwell et al. (2005) reported that when FM was replaced by 50% with MBM, growth reduction of largemouth bass was attributed not to the dietary amino acid composition but to their availability. Lysine is considered to be one of the first limiting amino acids in alternative ingredients to FM in aquafeeds (Kaushik and Seiliez 2010; NRC 2011) and, in processed animal ingredients, lysine is considered to be the amino acid most sensitive to heat damage during the rendering process (Nengas et al. 1999). Indeed, lysine availability may greatly differ among different batches of MBMs, ranging from 73 to 91% (Parsons et al. 1997). In the present study, even though lysine availability of the MBM ingredient was not determined, the obtained lysine digestibility coefficient and lysine retention efficiency for diet MBM50 suggested that amino acid availability of the MBM ingredient was little affected by the rendering process. In the present study, EAA retention was not different for the three experimental diets. Lysine intake was significantly lower for the MBM diets but, for diet MBM50, the retention efficiency of lysine was even slightly higher (2.3%) that the control diet, suggesting a similar lysine efficiency utilization at a 50% replacement level. However, for diet MBM75, lysine retention was 12.5% lower than that of control diet. Despite the significant higher arginine intake for fish fed MBM diets, arginine retention in fish fed diet MBM75 decreased about 35.5%, though not statistically significant, compared to the non-MBM control diet. This lower arginine retention efficiency may be due to the high arginine content of the MBM based diets (>7 g 16 N-1). A reduction of arginine utilization efficiency with an increase in intake is indeed to be expected due to a reduction of the absorption rate or to an increased metabolic utilization for other purposes than muscle growth, or both (Peres and Oliva-Teles 2008). FCUP Potential use of meat and bone meal in diets for gilthead seabream (Sparus aurata) juveniles 45 At the end of this trial, whole body composition was unaffected by the dietary MBM inclusion, with the exception of crude lipid and energy which were significantly lower for fish fed diet MBM75. Nutrient deposition in the body is related to the efficiency of its retention and, in this trial, whole-body crude protein and nitrogen retention efficiency were not significantly affected by the experimental diets although daily nitrogen intake was significant higher for diet MBM75. However, whole-body lipid content and retention, as well as energy retention, decreased in fish fed the diet with the highest level of MBM, suggesting a lower lipid and energy utilization efficiency with increasing MBM, as diets had similar crude lipid content (approximately 20% DM) and intake was significantly higher. Also for gilthead seabream, Robaina et al. (1997) reported a decrease, though not statistically significant, in both lipid digestibility and whole-body lipid content in gilthead seabream with increasing dietary MBM. Similar results were also obtained by Ai et al. (2006) where diets with more than 45% MBM caused a decrease in whole-body lipid content in large yellow croaker. On the contrary, juvenile snapper had a slight increase, although significant, of whole-body lipid content as dietary MBM increased (Booth et al. 2012), while other studies shown no significant differences in whole-body composition of fish fed diets with different levels of animal by-products (Bureau et al. 2000; Bharadwaj et al. 2002; Goda et al. 2007; Jamil et al. 2007), suggesting that the lipid utilization efficiency is influenced by either the species, quality of the ingredient or both. Even though GIT microbiota modulation action due to the dietary incorporation of plant ingredients (Heikkinen et al. 2006; Refstie et al. 2006; Ringø et al. 2006b; Dimitroglou et al. 2010; Silva et al. 2011) and pre and probiotics (Dimitroglou et al. 2010; Cerezuela et al. 2012; Cerezuela et al. 2013; Kormas et al. 2014) has been previously reported, from all the literature found, this is the first study evaluating the effect of dietary animal by-products inclusion. In the present study, the inclusion of MBM in the diets for gilthead seabream modulated its GIT microbiota with significant changes in composition, and richness, while diversity was not significantly affected. A 50% FM replacement with MBM did not cause significant changes on the microbiota parameters analyzed, when compared to the non-MBM control diet, whereas an increase of the substitution level to 75% lead to a significant decrease in GIT microbial richness and OTUs, and to a significant increase in the similarity between replicates (i.e. homogeneity between individuals under the same treatment). Results from this study indicate that a 75% FM replacement with MBM may increase fish susceptibility to diseases as reduction of GIT microbial richness and diversity is often associated with higher susceptibility to diseases in both humans and animals (de Vos and de Vos 2012; Thomas et al. 2014). Also, FCUP Potential use of meat and bone meal in diets for gilthead seabream (Sparus aurata) juveniles 46 reduced diversity can compromise intestinal functionally as a diverse microbiota allows better adaptation to changing environmental conditions, such as those in aquaculture production (Cerezuela et al. 2012). Besides the dietary effect on general GIT microbiota, significant differences in microbiota composition of different GIT sections (stomach, anterior, middle or posterior intestine) were observed, with an increase in the microbial diversity and richness towards the end of GIT. The lower microbial diversity and richness observed in the stomach might be explained by the harsh acidic stomach environment, which does not allow the establishment of bacteria unable to growth at low pH (Navarrete et al. 2009). Indeed, the pH variation of the different GIT compartments of juvenile fish can act as a selective mechanism, allowing colonization of some species and not others (Grisez et al. 1997). Besides the pH effect, the availability of digested nutrients, which is higher in the intestine than in the stomach, might also help to explain the higher microbial richness encountered in the last sections of the GIT, independent of the diet (Navarrete et al. 2009). PCR-DGGE, followed by DGGE bands sequencing, is a powerful tool to determine the predominant bacteria present in GIT samples (Tapia-Paniagua et al. 2010). In present study, the predominant bacteria found from the sequenced bands belonged to the phyla Firmicutes (38.9%), followed by Actinobacteria (27.8%), uncultured bacteria (22.2%) and Proteobacteria (11.1 %). This is in accordance with previous studies where Proteobacteria, Firmicutes, and Actinobacteria prevailed in the gut of wild, organically or conventionally reared gilthead seabream, determined by pyrosequencing (Kormas et al. 2014), as well as in other farmed species such as grass carp (Han et al. 2010), yellow grouper (Zhou et al. 2009; Feng et al. 2010) and olive flounder (Kim and Kim 2013). Bacteroidetes, a predominant group found in gilthead seabream by Kormas et al. (2014), was not detected in this study. Among the phyla described, the detectable predominant bacteria present in the stomach and intestine of gilthead seabream fed the experimental diets were most closely related to bacteria belonging to the Staphylococcus, Vibrio, Corynebacterium, Weissella, or Bacillus genera. A similar study reported Diaphorobacter as the dominant genus in wild and commercially reared gilthead seabream but this was not the case in the present study (Kormas et al. 2014). Vibrio spp. and Bacillus spp. are particularly common genera found in the GIT of fish (Perez et al. 2010; He et al. 2013). In this study, Vibrio is the only genus that appears both in the intestine and stomach. While in the intestine it seems to appear in just in one replicate (middle intestine of fish fed diet MBM75), in the stomach, Vibrio appears to be FCUP Potential use of meat and bone meal in diets for gilthead seabream (Sparus aurata) juveniles 47 absent in the control diet and its bands become more pronounced with increasing MBM, suggesting that the inclusion of this ingredient promoted its appearance. Other authors have reported Vibrio as the dominant genus in juveniles and adult marine fish gut (Grisez et al. 1997; Tapia-Paniagua et al. 2010), but this was not observed in present study. Additionally, Vibrio is a common genus in aquatic environments, and its predominance in the stomach could also be attributed to the ingestion of the surrounding water since it is recognized that bacteria from water can survive and multiply in the digestive tract (Navarrete et al. 2009). Despite some species of this genus being pathogenic for fish (Heikkinen et al. 2006; Feng et al. 2010), others, such as V. alginolyticus, are beneficial for seabream larvae, competing with opportunist pathogenic bacteria (Grisez et al. 1997). Similarly, MBM inclusion appears to potentiate the appearance of Bacillus subtilis as these bands become more pronounced (or only appear) in fish fed diet MBM75 in all intestinal sections. Other authors have also reported an increase in the presence of Bacillus spp. in the intestinal microbiota of rainbow trout fed diets with SBM (Heikkinen et al. 2006) and in Atlantic salmon fed diets with chitin (Askarian et al. 2012). Although Cerezuela et al. (2013) reported negative changes in the intestinal morphology of gilthead seabream when supplementing with a particular strain of B. subtilis, other strains are currently being used as probiotics in humans and animals (Cutting 2011), with different studies reporting the probiotic proprieties in fish, by enhancing fish immune response, growth performance and disease resistance (Nayak 2010; Sun et al. 2010; He et al. 2011, 2013; Liu et al. 2012). The inclusion of MBM also appears to promote the development of Mycobacterium spp. as it is clearly more pronounced, though replicates were not homogenous, in the posterior intestine of fish fed diet MBM75. Bacteria from this genus are known to cause fish mycobacteriosis, a chronic disease characterized by the presence of numerous variable sized granulomas in tissues (Righetti et al. 2014), that can lead to high mortality rates in a variety of fish species worldwide (Stine et al. 2005; Sonda-Santos and Lara-Flores 2012) and can be pathogenic for humans due to its zoonotic potential and resistance to water disinfectants (Yanong et al. 2010). From all the literature found, there has been no report of the presence of Mycobacterium spp. in the GIT other than in fish with mycobacteriosis related symptomatology (Stine et al. 2005; Yanong et al. 2010; Sonda-Santos and Lara-Flores 2012; Righetti et al. 2014; Zhang et al. 2015). However, in this study, despite the presence of Mycobacterium spp. in the intestine of gilthead seabream fed diet MBM75, fish did not appear to show symptoms of the disease. FCUP Potential use of meat and bone meal in diets for gilthead seabream (Sparus aurata) juveniles 48 Some studies have also reported presence of Staphylococcus spp. in the GIT of fish (Ringø et al. 2006a; Ringø et al. 2006b; Bakke-McKellep et al. 2007; Askarian et al. 2012; Cantas et al. 2012) as well as Corynebacterium spp. (Al-Harbi and Naim Uddin 2004; Wu et al. 2010). In present study, Staphylococcus spp. and Corynebacterium spp. were present in all intestinal sections, but more predominantly in fish fed the non-MBM control diet, indicating that inclusion of MBM may reduce fish colonization by species of these genus. This might be beneficial since these genus are often associated with pathogenic species for humans and animals (Thomas et al. 2014). In particular C. aquaticum is considered to be pathogenic for fish, such as striped bass and rainbow trout, and mice (Baya et al. 1992). Finally, the genus Weisella was present in all intestinal sections and more predominant in fish fed the non-MBM control diet, suggesting that gradual inclusion of MBM also led to its disappearance. While some strains of these genus are considered to be pathogenic for farmed rainbow trout (Figueiredo et al. 2012), other strains of Weissella are receiving attention as potential probiotics (Fusco et al. 2015) and Weissellin A, a protein produced by these bacteria, has shown to have antimicrobial properties, suitable for food and feed preservation (Papagianni and Papamichael 2012). Conclusion The future of aquaculture nutrition will rely on the search for alternative protein sources for FM replacement as current inclusion rates threaten the expansion of the industry. Results from present study indicate that MBM is a promising ingredient and that a 50% substitution did not compromise growth performance and feed utilization of gilthead seabream juveniles. However, a substitution up to 75% MBM led to a decrease in growth, lipid and energy retention and EAA digestibility. Although ADCs of EAA were high for all experimental diets (>92%), they were significantly reduced by the inclusion of MBM. The reduced performance of 75% MBM diet may be attributed to its high ash content and high levels of saturated fats that may have compromised nutrient digestibility. Species diversity was not affected by the MBM inclusion level. However, only the 50% substitution with MBM maintained the OTUs and species richness unaltered, indicating that higher levels might compromise the GIT microbiota stability. Also, MBM FCUP Potential use of meat and bone meal in diets for gilthead seabream (Sparus aurata) juveniles 49 appeared to promote the development of Bacillus genus, a group of organisms commonly associated with beneficial effects in animal health, namely as probiotics but also Vibrio and Mycobacterium genus, often associated with pathogenic bacteria. Overall, MBM has the potential to be included in diets for gilthead seabream juveniles but better characterization of this product is required in order to improve utilization and feeding strategies. The next step in this research could be the evaluation of the performance of a MBM intermediate inclusion level (between the 50 and 75% FM replacement level). Also the effect of dietary inclusion of MBM on general intestine health fish, fish wellbeing and immune status, as well as on flesh quality traits of gilthead seabream deserves further research. FCUP Potential use of meat and bone meal in diets for gilthead seabream (Sparus aurata) juveniles 50 References Ai, Q., Mai, K., Tan, B., Xu, W., Duan, Q., Ma, H., and Zhang, L. (2006). "Replacement of fish meal by meat and bone meal in diets for large yellow croaker, Pseudosciaena crocea." Aquaculture, 260(1-4), 255-263. Al-Harbi, A. H., and Naim Uddin, M. (2004). 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