Effects of Partial Replacement of Fish Meal by Carob Seed Germ Meal on Growth Performance and Immune Parameters of Meagre (Argyrosomus Regius, Asso, 1801)

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2.º CICLO FCUP 2014

regius immune parameters of meagre ( seed germ mealgrowth on performance and

Effects Effects of partial replacement of fish meal by carob Effects of partial

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meal by carob seed germ meal on growth Argyrosomus performance and

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of meagre Carolina Isadora F. S.V. Barroso (Argyrosomus regius, Asso, 1801)

Carolina Isadora F. S. V. Barroso Dissertação de Mestrado apresentada à Faculdade de Ciências da Universidade do Porto em

Nutrição e Imunologia 2014

Effects of partial replacement of fish meal by carob seed germ meal on growth performance and immune parameters of meagre (Argyrosomus regius, Asso, 1801)

Carolina Isadora F. S. V. Barroso Mestrado em Recursos Biológicos Aquáticos Departamento de Biologia 2014

Orientador Doutor Benjamín Costas, Investigador Post-Doc, CIIMAR

Coorientador Doutora Paula Enes, Investigadora Post-Doc, CIIMAR Todas as correções determinadas pelo júri, e só essas, foram efetuadas.

O Presidente do Júri,

Porto, ______/______/______FCUP I CGM as a potential protein source in feeds for meagre

“Somos a memória que temos e a responsabilidade que assumimos. Sem memória, não existimos. Sem responsabilidade, talvez não mereçamos existir.” José Saramago, Cadernos de Lanzarote.

FCUP II CGM as a potential protein source in feeds for meagre

Acknowledgments

The first person I would like to express my enormous gratitude is to my supervisor Dr. Benjamín Costas, for the opportunity he gave me to carry out this work, for sharing scientific knowledge, for the availability and dedication since my first day at the Immunology Lab.

I want to make a very special thanks to Dra. Paula Enes, my co-supervisor, for her commitment and availability towards me and my work, even in the toughest moments.

A very special thanks to Marina Machado, Rita Azeredo and to Prof. Dr. António Afonso. For being always there, for all the support and motivation that gave me in the Immunology Lab.

To Prof. Dr. Aires Oliva-Teles, leader of NUTRIMU group and director of Master’s Degree in Biological Aquatic Resources, for providing all the necessary support that enabled me to perform this study.

To Dra. Patrícia Díaz-Rosales, Dra. Ana Couto and Dra. Helena Peres for promptly offering me their help during my samplings and analysis.

A special thanks to Erika Kanashiro for all of her assistance, both in Foz as in the Immunology Lab.

To Rita Pedrosa and Inês Guerreiro for all the help and for the good moments we spent in Foz.

To all NUTRIMU group.

To Mr. Pedro Correia for the technical and useful assistance so promptly given me during my work.

To CIIMAR, my “second home” since I started my work in the Immunology lab. A special thanks to Pedro Reis, for the good mood, for believing in me and in my work.

To Dra. Elisabete Matos of Soja de Portugal, for providing the ingredients of the experimental diets and, in association with Projectos IJUP-Empresas (PP-IJUP2012- SOJA DE PORTUGAL- 20), partially funded this work, making possible to achieve the proposed goals.

To Dr. Pedro Pousão-Ferreira of IPMA for the fish supply.

To my mom Fátima Salgado, my brother Luciano Barroso, my sister Ana Luísa Barroso, my brother in law Álvaro Vieira, my cousin Isabel Melo and my grandma Maria Joaquina Ferreira.

To my friends Vera Araújo, João Vale, Marta Teixeira, Ana Carolina Amorim, Sofia Ramos, Tiago Pereira, Joana Mendes, Agostinho Salgado, Mariana Lopes, Sara Sousa, Filipe Silva, João Brito e Rafael Abreu.

FCUP III CGM as a potential protein source in feeds for meagre

To my family, friends and coworkers: Thank you for the laughs, for your support and for always believing in me and for being there for me. I wouldn't be here without you pushing me every step of the way!

FCUP IV CGM as a potential protein source in feeds for meagre

Abstract Considering the Mediterranean market saturation with species like European seabass (Dicentrarchus labrax) and gilthead seabream (Sparus aurata) as well as the high cost and low availability of fish meal (FM), efforts have been made to introduce new candidate species and more economic and sustainable diets in the Mediterranean aquaculture. In this way, meagre (Argyrosomus regius) presents a high potential to be produced at large scale due to its high growth rate and easily adaptation to captivity, in addition to its high nutritional value. Information regarding meagre dietary requirements is scarce. However, some studies suggested that meagre has a high protein requirement (about 50-54%) and a low lipid level (about 12-17%). Several plant protein sources are currently introduced in aquafeeds to reduce dependence on FM, being soybean meal (SBM) among the most used due to its high protein content and relatively balanced amino acids profile. Few studies have been made to introduce carob seed germ meal (CGM) in aquafeeds, a by-product of carob seed processing industry. CGM provides about 45 to 50% of protein, similar to SBM, presents a lower price and it is produced locally in Portugal. Information regarding the effects of CGM on fish performance and immune system are scarce and no studies are available regarding the utilization of this plant protein source on dietary formulations for meagre. Therefore, meagre were fed four experimental diets with graded levels of CGM, including 0% (diet control), 7.5% (diet CGM7.5), 15% (diet CGM15), and 22.5% (diet CGM22.5) to evaluate effects of this particular ingredient on growth performance, feed utilization efficiency, whole-body composition, haematology and cell-mediated and humoral immune parameters of meagre. The trial lasted 8 weeks and the immune parameters were evaluated at weeks 1, 2 and 8. All the experimental diets were well accepted by the animals and no mortality was recorded during the course of the study. The values of feed efficiency, protein efficiency ratio and N retention decreased in fish fed diets with 15 and 22.5% of CGM incorporation compared to fish fed the control diet. However, growth performance was unaffected by dietary CGM inclusion. Haematocrit and mean corpuscular volume decreased from week 1 to week 2, thereafter increasing at week 8. Values of red blood cells counts augmented during feeding trial while total white blood cells showed the opposite pattern, with lower values at the end of the trial compared to week 1. Differential cell counting showed that lymphocytes were the most abundant leucocytes in meagre plasma, followed by thrombocytes, monocytes and neutrophils. The relative proportion of peripheral thrombocytes increased significantly from week 1 to week 2, decreasing at week 8, while lymphocytes followed a different pattern, decreasing from week 1 to week 2, and

FCUP V CGM as a potential protein source in feeds for meagre increasing at the end of the trial. Lymphocytes were also affected by dietary composition, with a significant decrease in fish fed diet with 7.5% of CGM incorporation compared to fish fed other dietary treatments. No significant effects were verified in monocytes and neutrophils proportion. While alternative complement pathway activity in plasma increased significantly in fish fed diets with CGM, plasma total immunoglobulins (Ig), nitric oxide, peroxidase and intestinal bactericidal activities decreased significantly over time. Anti-protease activity augmented from week 1 to week 2 and intestinal total Ig decreased from week 1 to week 2, increasing at the end of the trial. No significant effects were verified in plasma bactericidal activity. Results suggest that GCM incorporation did not induce an inflammatory response. The observed variations among sampling times could be related to the fast growth and development of meagre. Based on these results, it is possible to partially replace FM by CGM up to 7.5%, without compromising growth performance and health status of fish. However, higher incorporations may bring adverse effects on meagre growth and feed utilization efficiency. Due to the limited effects of CGM on meagre immune parameters, results suggest that this vegetable ingredient seems to be a promising alternative protein source to FM. Nevertheless, time-course studies with higher levels of incorporation as well as higher FM replacement are necessary to evaluate the effects of this plant feedstuff in a long-term growth trial. A challenge with pathogenic bacteria may bring further insights to evaluate the resistance of fish fed this alternative protein source during infection by opportunistic pathogens.

Keywords: Ceratonia siliqua; alternative protein sources; Sciaenidae; feed utilization; species diversification; immune responses

Resumo Dada a saturação de mercado de espécies como robalo (Dicentrarchus labrax) e dourada (Sparus aurata) no Mediterrâneo, e o elevado custo e baixa disponibilidade da farinha de peixe, têm sido feitos esforços para introduzir novas espécies e dietas mais económicas e sustentáveis na aquacultura Mediterrânica. Desta forma, a corvina possui um elevado potencial para ser produzida em grande escala, devido à sua elevada taxa de crescimento e fácil adaptação ao cativeiro, para além do seu elevado valor nutricional. É pouca a informação relacionada com as exigências nutricionais da corvina. No entanto, existem já alguns estudos que sugerem que a corvina necessita de um elevado requerimento proteico na dieta (50-54%), e um baixo nível em lípidos (12-17%).

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São várias as fontes vegetais utilizadas actualmente em dietas para reduzir a dependência em farinha de peixe, sendo a farinha de soja (SBM) uma das mais utilizadas, devido ao seu elevado teor proteico e um perfil em aminoácidos relativamente equilibrado. Poucos estudos têm sido feitos para introduzir a farinha de gérmen de semente de alfarroba (CGM) nos alimentos para peixes, um subproduto resultante da indústria do processamento da semente de alfarroba. A CGM fornece cerca de 45 a 50% de proteína, semelhante à SBM, sendo mais barata que esta e é produzida localmente em Portugal. É pouca a informação relacionada com os efeitos da CGM na performance e no sistema imunitário dos peixes e, para além disso, não há estudos sobre os efeitos da incorporação desta proteína alternativa em dietas para corvina. Desta forma, as corvinas foram alimentadas com quatro dietas experimentais, com diferentes níveis de CGM, incluindo 0% (dieta controlo), 7,5% (CGM7.5), 15% (CGM15) e 22,5% (CGM22.5) para avaliar os efeitos deste ingrediente no crescimento, eficiência de utilização do alimento e a composição corporal, bem como a hematologia e os parâmetros imunes celulares e humorais das corvinas. O ensaio durou 8 semanas e os parâmetros imunes foram avaliados na semana 1, 2 e 8. Todas as dietas experimentais foram bem aceites pelos peixes e não se verificou mortalidade durante o ensaio. Os valores de eficiência alimentar, taxa de eficiência proteica e retenção de azoto foram significativamente inferiores nos animais alimentados com as dietas incorporadas com 15% e 22,5% de CGM, comparando com o grupo controlo. No entanto, a performance de crescimento não foi afectada pela incorporação de CGM nas dietas. O hematócrito e o volume corpuscular médio diminuíram da semana 1 para a semana 2, aumentando na semana 8. Os valores das contagens totais de eritrócitos aumentaram durante o ensaio, enquanto os valores de leucócitos seguiram uma tendência oposta, com valores mais baixos às 8 semanas, comparando com a semana 1. A contagem diferencial de leucócitos mostrou que os linfócitos são os leucócitos mais abundantes, seguido dos trombócitos, monócitos e neutrófilos. A proporção relativa dos trombócitos aumentou da semana 1 para a semana 2, diminuindo na semana 8, enquanto os linfócitos seguiram uma tendência oposta, diminuindo da semana 1 para a semana 2, aumentando no final do ensaio. Os valores dos linfócitos foram também afectados pela composição da dieta, com uma diminuição significativa nos peixes alimentados com a dieta de 7,5% de incorporação de CGM, comparando com as restantes dietas experimentais. Não foram encontradas diferenças significativas na proporção relativa dos monócitos e neutrófilos.

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Enquanto a via alternativa do complemento aumentou significativamente nos peixes almimentados com dietas incorporadas com CGM, as imunoglobulinas (Ig) totais no plasma, o óxido nítrico, actividade da peroxidase no plasma e a actividade bactericida no intestino diminuíram significativamente ao longo do tempo. As anti-proteases no plasma aumentaram da semana 1 para a semana 2 e as Ig totais no intestino diminuiram da semana 1 para a semana 2, aumentado no final do ensaio. Não se verificaram diferenças significativas na actividade bactericida no plasma. Estes resultados demonstram que não ocorreu nenhuma resposta inflamatória. As variações encontradas durante os pontos de amostragem poderão estar relacionadas com o elevado crescimento e estado de desenvolvimento das corvinas. Baseado nestes resultados, é possível substituir parcialmente a farinha de peixe por farinha de gérmen de semente de alfarroba até 7,5% sem comprometer a performance de crescimento e o estado de saúde dos animais. No entanto, incorporações mais altas poderão trazer efeitos adversos no crescimento e eficiência de utilização do alimento das corvinas. Devido aos efeitos escassos que provocou no sistema imunitário das corvinas, estes resultados sugerem que este ingrediente vegetal parece ser uma potencial fonte proteica alternativa à FM. De qualquer forma, serão necessários estudos a longo prazo, com incorporações mais altas de CGM, bem como substituições mais elevadas de FM, para avaliar os efeitos desta matéria-prima vegetal num maior período de tempo. Infecções com uma bactéria patogénica poderão trazer uma nova perspectiva no que toca à resistência dos animais alimentados com esta fonte proteica alternativa durante uma infecção por um patogénio oportunista.

Palavras-chave: Ceratonia siliqua; fontes proteicas alternativas; Sciaenidae; utilização do alimento; diversificação de espéices; resposta imune

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Table of contents 1 Introduction……………………………………………………………………………. 1 1.1 World aquaculture……………………………………………………………... 1 1.2 European and Portuguese aquaculture…..………………………………… 2 1.3 Meagre (Argyrosomus regius, Asso, 1801)………………………………… 4 1.3.1 Biology of the species………………………………………………… 4 1.3.2 Production……………………………………………………………… 4 1.4 Carob seed germ meal……………………………………………………….. 6 1.5 Fish immune system …………………………………..……………………... 9 1.6 Effects of plant feedstuffs on the fish immune system…………………….. 11 2 Aims……………………………………………………………………………………. 13 3 Materials and Methods……………………………………………………………….. 13 3.1 Experimental diets…………………………………………………………….. 13 3.2 Fish and experimental conditions……………………………………………. 15 3.3 Sampling ...... 15 3.4 Proximate analysis…………………………………………………………….. 16 3.4.1 Crude protein….……………………………………………………….. 16 3.4.2 Crude lipids..…………………………………………………………… 17 3.4.3 Dry matter……………………………………………………………… 18 3.4.4 Ash content…………………………………………………………….. 18 3.5 Hematological study…………………………………………………………... 18 3.5.1 Haematocrit…………………………………………………………….. 18 3.5.2 Total erythrocyte and leucocyte counts……………………………… 19 3.5.3 Differential cell counting………………………………………………. 19 3.6 Humoral parameters………………………………………………………….. 20 3.6.1 Alternative complement pathway activity...... 20 3.6.2 Peroxidase activity...... 20 3.6.3 Plasma and intestinal total immunoglobulins….………...... 20 3.6.4 Plasma nitric oxide…..………………………………………………… 21 3.6.5 Plasma anti-protease activity…………………………………………. 21 3.6.6 Plasma and intestinal bactericidal activity…………………………… 22 3.7 Statistical analysis……………………………………………………………. 23 4 Results…………………………………………………………………………………. 23 4.1 Growth performance, feed utilization efficiency and whole-fish 23 composition……………………………………………………………………………… 4.2 Immune indicators…………………………………………………………….. 27 4.3 Humoral parameters…………………………………………………………. 31

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5 Discussion…………………………………………………………………………….. 34 6 Conclusions……………………………………………………………………………. 37 7 References…………………………………………………………………………….. 38

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Abbreviations

ACP – Alternative complement pathway CGM – Carob seed germ meal DGI – Daily growth index DM – Dry matter FE – Feed efficiency FM – Fish meal HSI – Hepatosomatic index Ht – Haematocrit IAA – Indispensable amino acid Ig – Immunoglobulins MCV – Mean corpuscular volume NO – Nitric oxide PER – Protein efficiency ratio RBC – Red blood cells SBM – Soy bean meal VI – Visceral index WBC – White blood cells

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Figures List

Fig 1. Total world capture fisheries and aquaculture production…………………... 1 Fig 2: Meagre (Argyrosomus regius, Asso,1801)...... 4 Fig 3: Commercial size of meagre………………………………………………….…. 5 Fig 4: Carob seed…………………………………………………………………….…. 8 Fig 5: Juvenile meagre in experimental tank………………………………………… 15 Fig 6: Experimental system……………………………………………………………. 15 Fig 7: Blood sampling collection………………………………………………………. 16 Fig 8: Meagre digestive tube, at the end of the trial…...…………………………… 16 Fig. 9: Kjeltec digestor and distillation and titration units…...……………………… 17 Fig. 10: Lipids extraction unit………………………...………………………………… 18 Fig. 11: Muffle furnace………………………………………………………………..… 18 Fig. 12a: Capillary tubes, sealing plasticine and graphic reader…………………... 19 Fig. 12b: Microhaematocrit centrifuge………………………………………………… 19 Fig. 13: Meagre gonads at the end of the growth trial……………………………... 23 Fig 14a: Meagre size at the beginning of feeding trial……………………………… 26 Fig 14b: Meagre size at the end of the trial………..…………………………………. 26 Fig 15a: Meagre lymphocytes……….………………………………………………… 29 Fig 15b: Meagre thrombocytes….…………………………………………………….. 29 Fig 15c: Meagre monocyte………………….………………………………………… 29 Fig 15d: Meagre neutrophil...... ……………….………………………………….. 29 Fig 16: Plasma alternative complement pathway activity……………………...…… 31 Fig 17a: Plasma peroxidase activity……………………………………….…..……… 32 Fig 17b: Plasma nitric oxide levels…………...……………………………………….. 32 Fig 17c: Plasma total immunoglobulins....…………………………..……………….. 32 Fig 17d: Plasma anti-protease activity……………...………………………....……... 32 Fig 18: Plasma bactericidal activity……………………………………..…………….. 33 Fig 19: Intestinal total immunoglobulins..…………………………………………….. 33 Fig 20: Intestinal bactericidal activity……………………………….…………………. 34

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Tables List

Table 1: Fish indispensable amino acid (IAA) and fish meal (FM), soybean meal (SBM) and carob seed germ meal (CGM) composition…………………………...... 7 Table 2: Composition of the experimental diets…………………………………….. 14 Table 3: Growth performance and feed utilization efficiency of meagre………….. 25 Table 4: Whole-fish composition and hepatosomatic and visceral indexes…….… 26 Table 5: Haematocrit (Ht), mean corpuscular volume (MCV), total red blood cells (RBC) and white blood cells (WBC) counts of juvenile meagre………...…………. 28 Table 6: Differential cell counting: relative proportion of thrombocytes, lymphocytes, monocytes and neutrophils of meagre….……………………………. 30

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1. Introduction 1.1 World aquaculture Aquaculture is the involvement of human activity in the production of aquatic species, using techniques designed for this purpose, with the intention of increasing the production beyond the natural capacities of the species (White et al., 2004). This activity has arisen in China, when populations became sedentary and used rice cultures for the production of common carp (Cyprinus carpio) (Rabanal, 1988). The Romans introduced this activity in Greece and Italy and later, aquaculture has been established in central Europe in palaces and monasteries provided with water areas, used for the culture of certain fish species, especially common carp and trout (genus Salmo) (Rabanal, 1988; Tidwell, 2012). In the last decades, the human population has been consuming more fish. World’s consumption of fish per capita increased from an average of 9.9 kg in the 1960s to 19.2 kg in 2012, varying according to region and type of fish (Tidwell, 2012; FAO, 2014). The overall fish availability is increasing to most consumers, but the pattern of world’s consumption per capita is uneven. For instance, in some African countries this pattern remained stable or is decreasing, while in some Eastern Asian countries consumption per capita is growing substantially. Developed countries have higher consumption rates, given the high imports, consequence of the declining domestic fishery production. On the other hand, the consumption on developing countries tends to be based on locally and seasonally available fish (FAO, 2014). Total of fisheries and aquaculture production reached, in 2012, 158 million tonnes, being 136 million for human consumption (Fig. 1). World fish aquaculture production continues to grow, expanded from 49.9 million tonnes produced to 66.6 million tonnes, between 2007 and 2012, representing an estimated value of US$137.7 (≈ 101.2 EUR) billion and is, nowadays, the animal food-producing sector that grew the most over the last decades (Walker and Winton, 2010; FAO, 2014).

Fig. 1: Total world capture fisheries and aquaculture production. Source: FAO, 2014.

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China is by far the major fish producer. In 2012, this country produced more than 41 million tonnes that means that Asia is the region that produced more fish (58 million tonnes). Followed by America with more than 3 million tonnes of fish produced; Europe, with more than 2.8 million tonnes; Africa, with more than 1.4 million tonnes and, finally, Oceania, with only 184 191 tonnes produced in 2012 (FAO, 2014). Nowadays about 600 aquatic species are produced in this sector, from fish to shellfish and crustaceans, from freshwater to seawater, using different ways of production, such as raceways, artificial lakes or earthen ponds (Cunningham, 2005; Walker and Winton, 2010). Aquaculture is now part of the economy in several countries, promoting their development and the creation of jobs. However, the great development of this activity brings environmental issues (Walker and Winton, 2010). Moreover, the great increase of fish production implies the manufacture of large quantities of aquafeeds, which needs great availability of ingredients, especially protein sources (Naylor et al., 2000). Fishmeal (FM) and fish oil are the main protein and lipid sources used in carnivorous fish diets. The fisheries products used to produce FMs were recorded in 1976 around 18.2 million tonnes. In 1994, this value reached 30.2 million tonnes and decreased to 14.8 million tonnes in 2010. Thereafter, this value increased up to 19 million tonnes in 2011 and then declined to 16.3 million in 2012. Consequently, the production of FM and fish oil followed the same trend, making it essential to turn to the use of alternative protein and lipid sources to solve this problem (Gatlin et al., 2007; FAO, 2012, 2014).

1.2 European and Portuguese aquaculture In Europe, aquaculture is the animal food-producing sector that increased the most in the last decades as it happens elsewhere in the world. European aquaculture production of brackish and marine species increased from 1.6 million tonnes in 1990 to 2.8 million tonnes in 2012, being produced mainly some finfish and mollusks species, such as Atlantic salmon (Salmo salar), gilthead seabream (Sparus aurata), European seabass (Dicentrarchus labrax) and Pacific cupped oyster (Crassostrea gigas) (STECF, 2013). Norway has the biggest production of fish with more than 1 million tonnes, corresponding to 40% of the European fish production followed by Spain, the largest fish producer of the European Union, with more than 250 000 tonnes. In third place comes France, with about 220 000 tonnes of fish (STECF, 2013; FAO, 2014). Mediterranean aquaculture had a great development when certain technical difficulties, such as in reproduction and larvae culture, were overcome in the late 1980’s. In 1998,

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European seabass and gilthead seabream production had already exceeded 100 000 tonnes, reaching more than 170 000 tonnes in 2012. Despite of the studies and efforts that are being made to introduce new species in aquaculture, nowadays the Mediterranean production is based essentially in European seabass, gilthead seabream and turbot (Scophthalmus maximus) production (Barazi-Yeroulanos, 2010; FAO-FIGIS, 2014).

Portugal is a country with a strong fishing tradition and with great fish consumption (FAO, 2010). In 2012, aquaculture production in Portugal reached 10 318 tonnes, with an estimated value of more than 51 million EUR. The intensive farming of turbot is the most representative, with 5 301 tonnes produced (FAO-FIGIS, 2014). Portugal has a modest importance in the total aquaculture production in a European level and needs to import to meet the requirements of Portuguese consumers. For instance, from 1996 to 2004, gilthead seabream imports increased from 70 tonnes to 2 200 tonnes (Barazi-Yeroulanos, 2010). In 2010, Portugal imports reached more than 1.3 million EUR, being Spain one of the largest fish and mollusk provider (INE, 2010). Portugal is also involved in projects related with integrated multitrophic aquaculture, producing European seabass and gilthead seabream together with oyster (Crassostrea gigas) and clams, given their ability in filtering the organic material present in water. Moreover, the production of sole (Solea senegalensis) in recirculation systems is currently increasing in Portugal, with new strategies for optimize rearing conditions, fish welfare and the quality of products, being Aquacria Piscicolas S.A. and Safiestela - Sustainable Aqua Farming Investments, Lda leading companies (Sturrock et al., 2008; European Commission, 2014). In addition, the sole production market is being expanded to Asia; in 2013, the only sole hatchery operating in Portugal and producing eggs year-round sold juveniles (7 g weight) to Asia, and increased sales are expected in 2014 (Morais et al., 2014). However, it is still necessary to overcome some barriers. Licensing of production facilities and inappropriate location of fish farms are some constraints to the development of this industry (Sturrock et al., 2008).

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1.3 Meagre (Argyrosomus regius, Asso, 1801) 1.3.1 Biology of the species

Meagre (Fig. 2) is a carnivorous fish belonging to the Sciaenidae family, which includes 70 genus and 270 species (Quéméner et al., 2002). This fish inhabits in Mediterranean and Black Sea, being also found throughout the Atlantic cost (González-Quirós et al., 2011). It Fig. 2: Meagre (Argyrosomus regius, Asso, 1801). has an elongated body with the mouth located Source: Fish Base. http://www.fishbase.org in a terminal position, with lower jaw teeth in irregular rows, being one. enlarged, and without barbells (Cárdenas, 2010; FAO, 2013). The scales are mostly ctenoid, and the swimbladder is associated with 36-42 pairs of appendages and with a pair of sonic muscles that form characteristic sounds called “grunts” (Lagardère and Mariani, 2006; Cárdenas, 2010). The body is silver colored and has particular characteristics, such as the evident lateral line, extending to the caudal fin, the buccal cavity golden-yellow colored and its large otoliths. It is a fish of large dimensions, reaching up to about 2 m in length and 50 kg of weight. Meagre grows quite well in the summer and it optimal temperatures are between 17 and 21°C. Feeding activity of this species is much reduced when temperatures drop between 13 and 15⁰C (Poli et al., 2003; FAO, 2013). Meagre lives in depths of 15 to 300 m and has an anadromous migration. They approach the coast line in April, penetrate the estuaries to spawn and, from the end of May until the end of July, they leave estuaries to feed along the coast, being temperature the most important factor to determine meagre’s reproduction. During winter, they return to deeper waters (FAO, 1990, 2013). Meagre larvae consume its yolk sac in 96 hours when the mouth is already formed, being a species of fast larvae development. The juvenile feed small fish and shrimp (Mysidacea) and the adults feed pelagic fish, belonging to Clupeidae and Mugilidae groups (Quéro and Vayne, 1987; Cárdenas, 2010). Adult meagre, as typical carnivorous, has a short digestive tube, and the esophagus is small sized and large. The muscular stomach is bag-shaped and has 9 pyloric caeca which, together with intestine, have secretory and absorptive functions (Cárdenas, 2010).

1.3.2 Production

The production of meagre in aquaculture is quite recent, being France the first commercial producer of this species in 1997 (FAO, 2013). The world-wide of meagre

FCUP 5 CGM as a potential protein source in feeds for meagre production was very slow until 2009 and 2010, when the production increased from 4 184 tonnes to 14 596 tonnes, respectively. However, a decrease of the tonnes produced from 14 383 to 10 221 tonnes was verified in 2011 and 2012, respectively (FAO-FIGIS, 2014). Meagre production expanded slowly in Europe being the main producers France, Italy, Spain and Portugal. In 2002, meagre production in the European continent was 231 tonnes and the increase was slow until 2008, with increases of 753 tonnes in 2007 to 1 802 tonnes in 2008. However, 1 865 tonnes of meagre were produced in 2012, and a decrease in the production of this species was verified compared to 2011 (2 251 tonnes) (FAO-FIGIS, 2014). This species presents high potential to be produced in aquaculture, due to its high fertility, high growth rates and good conversion ratios, compared to European seabass and gilthead seabream (Estévez et al., 2011). Furthermore, meagre has a relatively high commercial value (6 to 10 EUR/kg). This species grows between 800 and 1 200 g in less than 2 years (Fig. 3), is excellent for filleting and is well accepted among consumers (Poli et al., 2003; Jiménez et al., 2005; Monfort, 2010; Chatzifotis et al., 2012; FAO, 2013). Meagre adapts easily to captivity in terms of resistance to a wide range of salinities (5-39‰) and temperatures (2-38 ºC) (Suquet et al., 2009).

Fig. 3: Commercial size of meagre. Adapted from Monfort (2010).

The on growing techniques for the production of meagre are similar to those used to produce European seabass and gilthead seabream. Spawning occurs spontaneously, by hormonal stimulation or through alteration of the conditions of production, such as the photoperiod and temperature, allowing an optimization of meagre production. Larvae are fed with rotifers and thereafter with artemia. Juveniles and adults are then fed with inert diets (Suquet et al., 2009; FAO, 2013). Records of diseases in meagre production are scarce. However, some authors reported parasitic infections, caused mainly by species belonging to the Monogenea group, while others reported diseases

FCUP 6 CGM as a potential protein source in feeds for meagre outbreaks caused by the bacteria Photobacterium damselae subsp. damselae (Merella et al., 2009; Ternengo et al., 2010; Labella et al., 2011). Although being resistant to captivity and handling, this fish easily loses its scales and its caudal fin is frequently damaged. Its eyes are also quite sensitive, suffering injuries that can even cause blindness (FAO, 2013). Meagre has lower values of muscle and mesenteric fat when compared, for instance, to European seabass, allowing a longer storage period under refrigerated conditions, therefore presenting an advantage for meagre production in aquaculture (Poli et al., 2003; Jiménez et al., 2005). Information related to the nutritional requirements of meagre is scarce and thus, diets currently used for meagre production are similar to those used for European seabass or gilthead seabream (Estévez et al., 2011). Given its high potential to diversify Mediterranean aquaculture, few studies have been made to determinate the requirements of this species and the ideal dietary formulation (Chatzifotis et al., 2010, 2012; Estévez et al., 2011). Chatzifotis et al. (2012) tested different inclusions of protein and lipids to determinate the optimal protein requirement and lipid level for meagre. Thus, higher growth and feed utilization efficiency were achieved with diets with 50-54% protein and 12-17% lipids.

1.4 Carob seed germ meal Plant feedstuffs provide a good alternative as dietary protein sources given their high availability and satisfactory price. However, the main limitation of using plant feedstuffs in aquafeeds is the presence of antinutritional factors as well as amino acids imbalances (Tacon, 1997; Gatlin et al., 2007). Soybean meal (SBM) is the main alternative protein source in fish diets. Soybean products are consistent in their chemical composition and economic as a protein source, especially in trout and salmon feeds (Hardy, 1996; Gatlin, et al., 2007). In contrast, the nutritional potential of certain leguminous by-products such as carob seed germ meal (CGM) has been rarely studied (Alexis et al., 1985, 1986; Filioglou and Alexis, 1989; Alexis, 1990; Martínez-Llorens et al., 2012). Compared with SBM, CGM presents similar protein content (45-50%) and also a balanced amino acid profile with the exception of methionine + cysteine that is limitative to fish requirements (Table 1). Additionally, CGM is less expensive than SBM within the European Union (approximately 300 versus 500 USD$/tonne in 2011, respectively) and is locally produced in Portugal (FAOSTAT, 2014).

FCUP 7 CGM as a potential protein source in feeds for meagre

Table 1: Fish indispensable amino acid (IAA) requirements and fishmeal (FM), soybean meal (SBM) and carob seed germ meal (CGM) composition (% dry mater, DM) (Adapted from Feedipedia: http://www.feedipedia.org/).

CP LYS THR M+C TRP ILE VAL LEU P+T HIS ARG CL CF NSP

Fish IAA Requirements Tannins 6.2 3.4 3.1 0.9 3.1 3.8 4.7 5.4 2.3 4.5 (Average values*) g/Kg DM

FM 65.5 7.4 4.2 3.6 1.1 4.3 5.1 7.3 7.0 3.0 5.8 11 - - -

SBM 47.1 6.1 3.8 2.8 1.2 4.7 4.7 7.4 8.3 2.7 7.5 1.8 4.9 22 3.6

CGM 49.4 5.4 3.3 2.4 1.0 3.5 3.9 6.0 5.9 2.6 8.6 5.6 4.4 18 4.5

CP: Crude Protein; LYS: Lysine; THR: Threonine; M+C: Methionine + Cysteine; TRP: Tryptophan; VAL: Valine; LEU: Leucine; P+T: Proline + Threonine; HIS: Histidine; ARG: Arginine; CL: Crude Lipids; CF: Crude Fiber; NSP: Non-starch polysaccharides. * Average values of fish IAA requirements based on IAA recommendations for Atlantic salmon (Salmo salar), rainbow trout (Oncorhynchus mykiss), common carp (Cyprinus carpio), hybrid tilapia (Oreochromis niloticus x Oreochromis aureus) and channel catfish (Ictalurus punctatus) (N.R.C., 2011).

FCUP 8 CGM as a potential protein source in feeds for meagre

Carob tree (Ceratonia siliqua) (Fig. 4) grows in Mediterranean countries. Carob world production in 2012 was estimated at approximately 163 000 tonnes and was mainly produced in countries such as Spain, Italy and Portugal (FAOSTAT, 2014). This seed is formed by a dark husk, an endosperm, with high galactomannans Fig. 4: Carob seed hanging in carob tree. Source: content, and a germinal portion or germ. WWF Oasis of Monte Arcosu. The endosperm is processed by chemical or thermodynamic methods, producing a gum that is used in food and non-food industries. This process results in the carob seed germ which, after milling and drying, is used in animal feeds or dietetic foods (Drouliscos and Malefaki, 1980; Dakia et al., 2007). The method of processing the carob seed may cause some effects in chemical composition of this by-product. It has been demonstrated that with an acid pre-treatment, the lipid and protein content decreased, when compared to boiling water pre-treatment. The acid used in the treatment can reach the germen and partially hydrolyze it, causing the degradation of lipids and proteins (Dakia et al., 2007). As already mentioned, the main limitation on the use of plant feedstuffs in aquafeeds is the presence of several antinutrients. Antinutritional factors are defined as endogenous constituents of feedstuffs that are capable to reduce feed intake, growth, nutrient utilization, disturb the normal function of internal organs and affect the resistance to diseases (Krogdahl et al., 2010). Among the most important are protease inhibitors, non-starch polysaccharides, phytates and tannins (Francis et al., 2001). In what concerns to CGM, the high content of tannins (4.5 g kg-1 dry matter) seems to be the limiting factor to the high levels of inclusion in fish diets (Nachtomi and Alumot, 1963). This antinutrient form a complex group of water soluble polyphenolic compounds, with similar chemical and physical properties. Tannins are formed by two distinct groups, the hydrolysable tannins and the condensed tannins, both with toxic and antinutritional properties, when are ingested by animals (Acamovic and Brooker, 2005). Tannins can interfere with growth, by reducing the palatability of feed, as well as interfering with digestion, showing anti-trypsin and anti-amylase activities. These antinutrients are also capable to bind to digestive enzymes, reducing its hydrolytic action, or directly to proteins (Alexis et al., 1986; Filioglou and Alexis, 1989; Alexis, 1990). Hydrolysable tannins, contrary to condensed ones, are easily degraded, forming small compounds

FCUP 9 CGM as a potential protein source in feeds for meagre that can reach the bloodstream and at the end of a certain period of time, can cause toxicity (Francis et al., 2001).

1.5 Fish immune system The fish immune system is classically divided into non-specific or innate and specific or acquired responses. However, it is currently known that both systems are actually two combined responses: the innate precedes the specific, activates and determines the nature of this response, cooperating in order to maintain the homeostasis of the animal (Magnadóttir, 2006). Fish live in an environment that can comprise high concentrations of inflammatory agents. In normal conditions, fish are capable to defend themselves of these invaders, given their complex innate immune system, formed by physical, cellular and humoral barriers, constituting a relatively fast response and independent upon recognition of the invader. After an inflammatory stimulus, the innate immune machinery takes action, through production of several microbial substances, acute phase proteins, complement activation, release of cytokines, inflammation and phagocytosis. When the first physical barriers to foreign agents are compromised, an influx of phagocytic cells is induced, with potent bactericidal activity (Ellis, 1999, 2001). Contrary to mammals, fish lack bone narrow and lymphoid nodes. Instead, the major lymphoid organs of fish are head-kidney, thymus, spleen and mucosa-associated lymphoid tissues, which include tegument, gills and intestine (Press and Evensen, 1999; Rombout et al., 2011). The mucosa-associated lymphoid system possesses all the necessary cells to respond to an inflammatory agent, such as lymphocytes, neutrophils and macrophages. Particularly in the digestive tract, it is also possible to find varied sub-populations of immunoglobulins (Ig) (Rombout et al., 2011). There are several types of leucocytes involved both in innate and acquired defense, which include monocytes/macrophages, granulocytes (i.e. basophils, eosinophils and neutrophils), lymphocytes and thrombocytes (Ellis, 1977). Thrombocytes are involved in blood clotting and, contrary to mammal platelets, which are fragments of cells, thrombocytes are complete cells which appears as round until fusiform (Esteban et al., 2000; Tavares-Dias and Oliveira, 2009; Robert and Ellis, 2012). Beyond the clotting function, it is believed that thrombocytes have phagocytic capacity. However, this function is not totally accepted by scientific community since available information is incomplete and somewhat contradictory (Mesenguer et al., 2002). Still, Tavares-Dias et al. (2007) showed that thrombocytes in fact have some degree of phagocytic function

FCUP 10 CGM as a potential protein source in feeds for meagre after finding glycogen granules for the power supply during phagocytosis, as well as cellular debris and vesicles containing degraded material. Neutrophils and macrophages are the professional phagocytes and present different characteristics. The most important role of phagocytic cells in host defense is to prevent or limit the initial spread of the infectious organism and its growth (Neumann et al., 2001). Neutrophils are present in blood, but are rare in tissues and body cavities; on the contrary, macrophages are present in all body cavities (Ellis, 1977; Afonso et al., 1998b). Macrophages are typically mononuclear and generally peroxidase negative and esterase positive. They secrete varied reactive oxygen and nitrogen species with capacity to kill several pathogens. These cells also have varied receptors for antibodies and components from the complement system (Secombes and Fletcher, 1992; Secombes, 1996). Neutrophils are usually polymorphonuclear. Their granules generally stain for varied dyes and enzymes (including peroxidase) and can also be used for its identification. Neutrophils are phagocytes highly mobile producing, as macrophages, reactive species, in addition to substances such as peroxidase and lysozyme, with antimicrobial and anti-viral properties (Ellis, 1977; Secombes, 1996; Saurabh and Sahoo, 2008). It is a cell type with a short lifespan, resulting in a great increase of neutrophils in the first hours after infection, and persisting up to 5 days. Macrophages and neutrophils can be identified at light microscopy through esterase and peroxidase labeling, respectively (Ellis, 1977; Afonso et al., 1998a). The complement system is a key component of fish innate immune system, being composed of around 35 proteins and involved in processes such as microbial killing, phagocytosis and antibody production (Holland and Lambris, 2002). Complement system participates in the inflammatory response by attracting phagocytic cells to the inflammatory site and through pathogen opsonisation, increasing the uptake of phagocytes (Ellis, 2001; Holland and Lambris, 2002). It is divided in the classic, lytic and alternative pathways, being the latter of great importance in fish (Yano, 1996; Ellis, 2001; Holland and Lambris, 2002). The alternative complement pathway (ACP) is considered one of the most important innate immune mechanisms in fish, being its activity modulated with the inclusion of certain nutrients in diets (Holland and Lambris, 2002; Boshra and Sunyer, 2006). The ACP is directly activated by microorganisms, through lipopolysaccharides that constitutes cellular wall of Gram-negative bacteria and is characterized by the absence of antigen/antibody complexes and regarded as an innate immune mechanism (Montero et al., 1998; Holland and Lambris, 2002).

FCUP 11 CGM as a potential protein source in feeds for meagre

Nitric oxide (NO) is a cell proliferation inhibitor and mediator of innate anti-microbial activity, interfering with the resistance to various pathogens (Saeij et al., 2000). It is produced through stimulation of inducible nitric oxide synthase (iNOS) by pro- inflammatory cytokines and bacterial lipopolysaccharides in both fish and mammals macrophages (Evans et al., 1996; Tafalla and Novoa, 2000). It was already reported that dietary treatment influences iNOS and, consequently, the production of NO (Kiron, 2012). Lymphocytes are cells highly differentiated and are the most important cells of specific response, being also called immunocompetent cells. This cellular type interferes in three different steps of specific response: in cellular immunity, through activation and mediation of certain cell types, especially macrophages; in humoral immunity, through the production of Ig; and immunological memory, through adaptive changes in lymphocytes, in order to recognize an antigen that previously invaded the host (Ellis, 1977; Secombes and Ellis, 2012). This cellular type is found in bloodstream and in lymphoid organs, being also possible to distinguish T and B lymphocytes (Ellis, 1977, 1999; Secombes and Ellis, 2012). The acquired system is an important key against repeated infections, by creating memory cells (lymphocytes) and specific receptors, such as T-cell receptors (TCR) and antibodies or Ig (Holland and Lambris, 2002). After antigen’s stimulation, there is a period of time until antibodies arise, during that time the specialized cells in their production take action. Thereafter, Igs are capable of neutralizing these antigens or act as an opsonin, leading to some degree of enhancement of phagocytic activity (Secombes and Ellis, 2012). Teleost immune molecules are more diverse than in mammals, however, the opposite is verified for Ig. Thus, only three Ig classes have been identified: IgM, IgD and IgT. While IgM are highly found in plasma, but in smaller amount in mucus, lgT has a great importance in mucosal immune response. IgD function in teleost fish is not fully understood and, although is part of the adaptive system, this Ig can be involved at the crossroads between the innate and the adaptive systems (Hansen et al., 2005; Solem and Stencik, 2006; Zhang et al., 2010; Ramirez- Gomez et al., 2012; Sunyer, 2013).

1.6 Effects of plant feedstuffs on the fish immune system Many studies regarding the incorporation of plant feedstuffs in fish diets are focused on growth performance of different fish species (Francis et al., 2001; Gatlin et al., 2007; Nasopoulou and Zabetakis, 2012). Fish nutritional status can be considered as a major factor that influences and modulates immune mechanisms, acting as

FCUP 12 CGM as a potential protein source in feeds for meagre immunostimulators or immunosupressors (Sakai, 1999; Trichet, 2010; Harikrishnan et al., 2011; Kiron, 2012). The endogenous sources of nutrients provide the essential requirements for immune system’s effectiveness. Immunonutrition aims to supply additional resources to the animal that would support one or more of the immune processes, to finally obtain an improved degree of protection. The incorporation of plant protein sources in carnivorous fish diets, especially from a source that is not present in the food chain of the animal, can lead to imbalances and, as a consequence, immune function is affected (Kiron, 2012). It is currently known that some plant feedstuffs can damage the gastrointestinal tract, with an associated diffuse lymphoid system, causing an inflammatory response (Baeverfjord and Krogdahl, 1996; Bakke-McKellep et al., 2000; Urán et al. 2008, 2009; Rombout et al., 2011). These damages can be characterized, for instance, by a shortening of heights of the mucosal folding, a loss of the normal vacuolization of the absorptive cells in the intestinal epithelium and the increase of leucocyte population in the lamina propria, including lymphocytes, polymorphonuclear leucocytes and macrophages, and also diffuse Ig (Baeverfjord and Krogdahl, 1996; Bakke-McKellep et al., 2000). Several studies have already addressed the effects of FM replacement by plant protein sources in humoral immune parameters and immune regulatory genes, especially in well-established aquaculture species, such as salmonids, gilthead seabream or common carp (Burrells et al., 1999; Bransden et al., 2001; Estévez et al., 2011; Pakravan et al., 2012; Hernández et al., 2013; Fuentes-Appelgren et al., 2014). Sitjà-Bobadilla et al. (2005) tested a mixture of plant protein sources on gilthead seabream humoral immune parameters and reported an increase in complement activity with 50% of replacement and decreased values of this humoral parameter with 75% and 100% of FM replacement, while the values of peroxidase activity progressively increased with the degree of plant protein sources, peaked highest values with a total FM replacement. In contrast, Bransden et al. (2001) found no effects on immune parameters in Atlantic salmon fed with diets incorporated with 40% of lupin (Lupinus angustifolius) meal. More recently, Sahlmann et al. (2013) tested the early responses of Atlantic salmon fed 20% SBM diet and verified an early immune response, with an up-regulation of several immune related genes in the first 3 to 5 days following first feeding. Few studies have been made to evaluate the effects of CGM on growth performance, haematological and inflammatory response of fish fed with this by-product. It has already been reported an effect of different CGM inclusion levels, of about 37% to 53%, in haematocrit and haemoglobin values of rainbow trout (Oncorhynchus mykiss) and described a negative correlation between these parameters and CGM content after a

FCUP 13 CGM as a potential protein source in feeds for meagre long-term feeding trial (Alexis et al., 1986; Alexis, 1990). More recently, Martínez- Llorenz et al. (2012) tested increased inclusion levels of CGM in diets for gilthead seabream and the worst growth performance and the significant gut histological alterations were verified with a 53% of CGM inclusion, after 12 weeks of feeding. Since there are no studies regarding the use of CGM as a partial FM substitute in diets for meagre, it is interesting to evaluate the inclusion of this by-product at different levels, and its consequences on growth and health status of this species.

2. Aims Considering the position of aquaculture in the world food sector, it is widely recognized that this sector should become a more sustainable industry. Species diversification is essential for a further growth in aquaculture production, as well as the replacement of FM by alternative protein sources in aquafeeds. Thus, this study aims to improve aquaculture sustainability and profitability. The main scientific question to be addressed is how to produce low FM diets for meagre that support rapid, efficient and cost- effective growth, without compromising fish health. Use of an emerging feedstuff - CGM, was the nutritional strategy assessed with this aim. Meagre was the species of choice due to its carnivorous feeding habits and its potential for Mediterranean aquaculture diversification. Major goals to be achieved include the evaluation of three different dietary CGM inclusion levels (7.5%, 15% and 22.5%) on meagre growth performance, feed utilization efficiency, body composition, haematology and cell- mediated and humoral immune parameters.

3. Materials and methods 3.1 Experimental diets Four experimental diets were formulated to be isonitrogenous (53% crude protein) and isolipidic (18% crude lipids). FM and CGM were used as the main protein sources and fish oil as the main lipid source. The experimental diets included 0% (diet control), 7.5% (diet CGM7.5), 15% (diet CGM15) and 22.5% (diet CGM22.5) of CGM. No adjustment to dietary essential amino acid profile was needed (Table 2). Prior to incorporation in the experimental diets, CGM was autoclaved at 110 ºC and 0.4 bar, during 10 min, and then dried in an oven (60 ºC) for 24 h in order to inactivate some antinutrients. The amount of tannins was analyzed prior and after autoclaving (Silliker Portugal S.A., Canelas, Portugal). All dietary ingredients were finely ground, well mixed and dry pelleted in a laboratory pellet mill (California Pellet Mill, CPM

FCUP 14 CGM as a potential protein source in feeds for meagre

Crawfordsville, IN, USA) through a 3 mm die. The pellets were then dried in an oven at 40 ºC for 24 h and stored at -20 ºC until used.

Table 2: Ingredient composition and proximate analysis of the experimental diets.

Diets Control CGM7.5 CGM15 CGM22.5 Ingredients (% dry weight basis) Fish meala 66.4 61.8 57.1 52.4 Wheat mealb 18.6 15.7 12.8 9.9 Fish oil 11.5 11.6 11.7 11.8 Carob seed germ mealc - 7.5 15.0 22.5 Vitamin premixd 1.0 1.0 1.0 1.0 Mineral premixe 1.0 1.0 1.0 1.0 Choline chloride 0.5 0.5 0.5 0.5 Binderf 1.0 1.0 1.0 1.0 Proximate analysis (% dry weight basis) Dry matter 92.4 94.7 92.2 93.5 Crude protein 52.5 52.5 53.2 52.5 Crude fat 17.8 17.1 17.9 18.0 Ash 14.5 13.7 13.9 13.3 Starch 10.9 8.3 8.2 6.2 Gross Energy (kJ g−1 DM)g 22.0 22.1 22.3 22.3 DM, dry matter; CP, crude protein; GL, gross lipid. aPesquera Diamante, Steam Dried LT. Austral Group, S.A. Peru (CP: 71.4% DM; GL: 9.3% DM). bSorgal, S.A. Ovar, Portugal (CP: 13.9% DM; GL: 1.8% DM). cSorgal, S.A. Ovar, Portugal (CP : 50.0% DM ; GL : 5.2% DM). dVitamins (mg kg-1 diet): retinol, 18 000 (IU kg-1 diet); cholecalciferol, 2 000 (IU kg-1 diet); alpha tocopherol, 35; menadion sodium bisulphate, 10; thiamin, 15; riboflavin, 25; Ca pantothenate, 50; nicotinic acid, 200; pyridoxine, 5; folic acid, 10; cyanocobalamin, 0.02; biotin, 1.5; ascorbyl monophosphate, 50; inositol, 400. eMinerals (mg kg-1 diet): cobalt sulphate, 1.91; copper sulphate, 19.6; iron sulphate, 200; sodium fluoride, 2.21; potassium iodide, 0.78; magnesium oxide, 830; manganese oxide, 26; sodium selenite, 0.66; zinc oxide, 37.5; potassium chloride, 1.15 (g kg-1 diet); sodium chloride, 0.44 (g kg-1 diet). fAquacube. Agil, England (guar gum, polymethyl carbamide, manioc starch blend, hydrate calcium phosphate).

FCUP 15 CGM as a potential protein source in feeds for meagre gGross energy calculated based on theoretical values (CP: 5.65 Kcal g-1 = 23.6 KJ g-1; GL: 9.45 Kcal g-1 = 39.5 KJ g-1; Carbohydrates: 4.15 Kcal g-1 = 17.2 KJ g-1)

3.2 Fish and experimental conditions This experiment was directed by trained scientists (following FELASA category C recommendations) and conducted according to the European Union Directive 2010/63/EU on the protection of animals for scientific purposes. Meagre juveniles were obtained from IPMA (Olhão, Portugal) and transported to the Marine Zoology Station’s facilities (Porto University, Porto, Portugal). After transportation to the experimental facilities fish were submitted to a quarantine period of 30 days. During this period fish were fed with a commercial diet (56% protein and 22% lipids). Thereafter, 12 groups of 18 fish with an initial mean body weight of 35 ± 0.01 g were established and each diet randomly assigned to triplicate tanks (Fig. 5). Fish were fed by hand, 6 days a week until visual satiation during 8 weeks. Utmost care was taken to assure that all feed supplied was consumed. The experimental system consisted of a thermoregulated recirculating seawater system (Fig. 6), equipped with a battery of fiberglass tanks (100 L capacity) supplied with continuous flow of filtered seawater (6.0 L min-1). During feeding trial, temperature averaged 23.6 ± 0.9 ⁰C; salinity 37.5 ± 0.8 g L-1 and dissolved oxygen kept near saturation (7.0 mg L-1). Fish were subject to a 12 h light / 12 h dark photoperiod regime provide by artificial illumination.

Fig. 5: Juvenile meagre in experimental tank. Fig. 6: Experimental system.

3.3 Sampling Fish in each tank were bulk-weighed at the beginning and at the end of the trial, after 1 day of feed deprivation. For that purpose, fish were slightly anaesthetised with 0.3 ml L- 1 ethylene glycol monophenyl ether (1000 ppm; Sigma-Aldrich, Steinheim, Germany).

FCUP 16 CGM as a potential protein source in feeds for meagre

Six fish from the initial stock population and 3 fish from each tank were sampled at the end of the trial and pooled for whole-body composition analysis. Whole-fish, viscera and liver weights of these fish were recorded for determination of hepatosomatic (HSI) and visceral indices (VI). At 1 and 2 weeks and at the end of feeding trial, 3 fish of each tank were also collected, after 1 day of feed deprivation, to remove blood and intestine samples for the immunological study (Fig. 7). Blood samples were withdrawn from caudal vessel with heparinized syringes, placed in heparinized eppendorf tubes and used to determine hematocrit, mean corpuscular volume, total leucocyte and erythrocyte counts and to prepare the blood smears. The remaining blood was used to collect plasma, by centrifugation at 10000 x g for 10 min. The plasma was then stored at −80 °C until further analysis, described below. The whole digestive tube was cut, without pyloric caeca, and kept at −80 °C until further analysis (Fig. 8).

Fig. 7: Blood sampling collection. Fig. 8: Meagre digestive tube, at 8 weeks of feeding trial.

3.4 Proximate analysis 3.4.1 Crude protein Prior the analysis whole-fish were dried in an oven at 80 ⁰C, until constant weight, and then ground to obtain a homogeneous sample. The protein content (Nitrogen (N) x 6.25) of ingredients, diets and whole-fish was determined using the Kjeldahl method, after acid digestion of samples, using a Kjeltec digestor and distillation units (Tecator Systems, Höganäs, Sweden, models 1015 e 1026, respectively) (Fig. 9). This method assumes that one protein contains 16% of N (100/16=6.25). Approximately 0.5 g of each sample was weighed, in duplicate, into distillation tubes. Then, were added 2 Kjeldahl tablets containing selenium (Se), as a catalyc, plus 15 mL of sulfuric acid. Samples were digested 1 h in the digestor unit at 450 ⁰C. At the end of digestion, the organic N was converted in ammonium sulfate. After digestion and cooling, samples

FCUP 17 CGM as a potential protein source in feeds for meagre were then distilled in the distillation unit. Water and sodium hydroxide (NaOH, 40%) were added to each digestion tube and distillation was performed using saturated boric acid for sequestering ammonium. The final step consisted in quantifying N by titration with hydrochloric acid (HCl, 0.2N), in the presence of a methyl orange pH indicator.

Fig. 9: Kjeltec digestor, distillation and titration units

3.4.2 Crude lipids The lipid content of ingredients, diets and whole fish was determined by the Soxtec method, using a continuous extraction with petroleum ether in a Soxtec system (Tecator Systems, Höganäs, Sweden; extraction unit model 1043 and service unit model 1046) (Fig. 10). Approximately 0.5 g of each sample was added, in duplicate, in a cartridge and placed in the extraction unit. Then, was added in the extraction cups, previously identified and weighted, 50 mL of petroleum ether and positioned in the extraction unit. Samples were then boiled for 1 h in petroleum ether, rinsed for 2 h and the extracted lipids were collected in the extraction cups. After extraction, the solvent was evaporated and the extracted material was dried in an oven at 105 ⁰C and weighed. The lipid content was determined through the difference in weight of extraction cups before and after the extraction process.

FCUP 18 CGM as a potential protein source in feeds for meagre

Fig. 10: Lipids extraction unit.

3.4.3 Dry matter To determine moisture of ingredients, diets and whole-fish, was added in crucibles previously identified and weighted, 0.5 g of each sample, in duplicate. Then, samples were dried in an oven, at 105 ºC until constant weight. The moisture content was determined through weight loss of samples plus crucibles.

3.4.4 Ash content After moisture determination, the same samples were incinerated in a muffle furnace (Fig. 11), at 450 ºC, during 24 h. The ash content is the inorganic matter present in samples after this process.

Fig. 11: Muffle furnace.

3.5 Hematological study 3.5.1 Hematocrit Hematocrit (Ht) was measured using the microhematocrit method, described by Goldenfarb et al. (1971). Briefly, homogenized blood was added into heparinized capillary tubes (Fig12a) and then centrifuged at 10000 x g during 10 min (Fig. 12b) and

FCUP 19 CGM as a potential protein source in feeds for meagre room temperature. Afterwards, the percentage of red blood cells in the volume of blood was determined using a graphic reader (Fig. 12a).

a b

Fig. 12: Microhaematocrit material. a: Capillary tubes, sealing plasticine and graphic reader. b: Microhaematocrit centrifuge.

3.5.2 Total white blood cells and red blood cells counts Total white blood cells (WBC) and red blood cells (RBC) counts were performed using a Neubauer chamber. Homogenized blood previously collected was added into a heparinized Hank’s Balanced Salt Solution (HBSS) to final dilutions of 1:20 and 1:200 for WBC and RBC counts, respectively. The mean corpuscular volume (MCV) was calculated according the following equation:

MCV (µm3) = (Ht/RBC)*10, where Ht is haematocrit (%) and RBC is red blood cells (106 / µL)

3.5.3 Differential cell counting The blood smear preparations were fixed with formol-ethanol (10% of 37% formaldehyde in absolute ethanol) and stained with Wright’s stain (Haemacolor; Merck, Lisboa, Portugal). Detection of peroxidase activity to label neutrophils was carried out by a procedure described by Afonso et al. (1998a) and the peripheral leucocytes were differentially counted in thrombocytes, lymphocytes, monocytes and neutrophils and the relative and total abundance was calculated. At least 200 cells per blood smear were counted.

3.6 Humoral parameters

FCUP 20 CGM as a potential protein source in feeds for meagre

3.6.1 Alternative complement pathway activity Alternative complement pathway activity was estimated according to Sunyer and Tort (1995). The following buffers were used: GVB (Isotonic veronal buffered saline), pH 7.3, containing 0.1% gelatin; EDTA–GVB, as the previous one but containing 20 mM EDTA (ethylenediaminetetraacetic acid); Mg–EGTA–GVB, which is GVB with 10 mM Mg2+ and 10 mM EGTA (Ethylene glycol-bis(2-aminoethylether)-N,N,N’N’-tretraacetic acid) and rabbit red blood cells (RaRBC; Probiologica Lda, Lisbon, Portugal). RaRBC was washed 4 times in GVB by centrifugation, at 3000 x g, during 5 min, and resuspended in GVB to a concentration of 2.8 × 108 cells mL-1. Then, 10 μL of RaRBC suspension was added to 40 μL of serially diluted plasma in Mg–EGTA–GVB buffer in conical 96-well plates. Samples were incubated at room temperature for 100 min with occasional shaking. The reaction was stopped by adding 150 μL of cold EDTA–GVB. Microplates were centrifuged at 1000 x g during 2.5 min and then 100 μL of supernatant was transferred to flat 96-well plates. The extent of hemolysis was estimated by measuring the optical density (OD) of the supernatant at 414 nm. The ACH50 units were deﬁned as the concentration of plasma giving 50% hemolysis of RaRBC. All analyses were conducted in triplicates.

3.6.2 Peroxidase activity The peroxidase activity was measured using the procedure described by Quade and Roth (1997). Briefly, 15 μL of plasma were diluted with 135 μL of HBSS without Ca+2 and Mg+2 in flat-bottomed 96-well plates. Then, 50 μL of 20 mM 3,3',5,5'- tetramethylbenzidine hydrochloride (TMB; Sigma-Aldrich, Steinheim, Germany) and 50

μL of 5 mM H2O2 were added. The color-change reaction was stopped after 2 min by adding 50 μL of 2 M sulphuric acid and the OD was read at 450 nm in a Synergy HT microplate reader, Biotek (Potton, Bedfordshire, England). The wells without plasma were used as blanks. The peroxidase activity (units mL-1 plasma) was determined defining one unit of peroxidase as that which produces an absorbance change of 1 OD.

3.6.3 Plasma and intestinal total Immunoglobulins Prior to total Ig determination, digestive tube was homogenized in sodium phosphate buffer (0.05M; pH 6.2) and centrifuged at 3000 x g for 10 min and 4 °C. Then, the supernatant was collected and placed in 2 mL eppendorf tubes and stored at -80 °C until the analysis. Plasma and intestine total Ig were determined using the method described by Siwicki and Anderson (1993). This technique was based on the measurement of total protein content of samples using the bicinchoninic acid (BCA) Protein Assay Kit (Pierce #23225, Rockford, USA) for microplates prior and after

FCUP 21 CGM as a potential protein source in feeds for meagre precipitating Ig, using a 12% polyethylene glycol (PEG; Sigma-Aldrich, Steinheim, Germany) solution. The difference in protein content is considered the total Ig content. Bovine serum albumin (BSA) was used as standard during protein quantification. Briefly, plasma or gut supernatant were added to 50 µL PEG and incubated for 2 hours at room temperature. Afterwards, samples were centrifuged at 2000 x g for 10 min and room temperature. Ten µL of either plasma or gut supernatant were then diluted in distilled water (1:50 v/v) and the total protein content was assessed according to kit’s recommendations.

3.6.4 Nitric Oxide Total nitrite plus nitrate in plasma was analysed in duplicates using a nitrite/nitrate colorimetric method kit (Roche Diagnostics GmbH, Mannheim, Germany), adapted for 96-well microplates. Briefly, 100 µL of diluted plasma samples were added to a microplate. Then, 50 μL of reduced nicotinamide adenine dinucleotide phosphate (NADPH) and 4 μL of the enzyme nitrate reductase (NR) were added to each well in order to reduce nitrate to nitrite. The plates were then incubated for 30 min at 25°C. Afterwards, 50 μL of sulfanilamide and 50 μL of N-(1-naphthyl)-ethylene-diamine dihydrochloride were added and incubated for 10 min at 25 °C in dark. The absorbance was then read at 540 nm after the 2 incubation periods. Water instead of plasma was used as blank. Total nitrite was determined by comparison with a sodium nitrite standard curve. Since nitrite and nitrate are endogenously produced as oxidative metabolites of the messenger molecule NO, these compounds are considered as indicative of NO production (Saeij et al., 2003)

3.6.5 Anti-protease activity The method described by Ellis (1990) was modified and adapted for 96-well -1 microplates. Prior to analysis, a sodium bicarbonate (NaHCO3, 5 mg mL , pH 8.3, Sigma-Aldrich, Steinheim, Germany) buffer was prepared. Briefly, 10 µL of plasma -1 were incubated with the same volume of a trypsin solution (5 mg mL in NaHCO3) for 10 min at 22 ºC in polystyrene microtubes. To the incubation mixture, 100 µL of -1 phosphate buffer (NaH2PO4, 13.9 mg mL , pH 7.0) and 125 µL of azocasein (20 mg -1 mL in NaHCO3) were added and incubated for 1h at 22 ºC. Finally, 250 µL of trichloroacetic acid were added to each microtube and incubated for 30 min at 22 ºC. The mixture was centrifuged at 10 000 x g for 5 min at room temperature. Afterwards, 100 µL of the supernatant was transferred to a 96 well-plate that previously contained 100 µL of NaOH (40 mg mL-1) per well. The OD was read at 450 nm. Phosphate buffer in place of plasma and trypsin served as blank whereas the reference sample was

FCUP 22 CGM as a potential protein source in feeds for meagre phosphate buffer in place of plasma. The percentage of trypsin activity inhibition was calculated as follows:

% non-inhibited trypsin = Sample Absorbance x 100 / Absorbance of the reference sample % inhibited trypsin= 100 - % non-inhibited trypsin

3.6.6 Plasma and intestinal bactericidal activities Photobacterium damselae subsp. piscicida (Phdp) strain PP3 was kindly provided by Dr. Ana do Vale (Institute for Molecular and Cell Biology, University of Porto, Portugal) and isolated from yellowtail (Seriola quinqueradiata; Japan) by Dr Andrew C. Barnes (Marine Laboratory, Aberdeen, UK) and was used in the bactericidal activity assay. Bacteria were cultured for 48 h at 25 °C on tryptic soy agar (TSA; Difco Laboratories, Detroit, USA) and then inoculated into tryptic soy broth (TSB; Difco Laboratories, Detroit, USA), both supplemented with NaCl to a final concentration of 1% (w/v) (TSA-1 and TSB-1, respectively). Bacteria in TSB-1 medium were then cultured during 24 h at the same temperature, with continuous shaking (100 rpm). Exponentially growing bacteria were collected by centrifugation at 3500 × g for 30 min, resuspended in sterile HBSS and adjusted to 1 × 106 cfu mL-1. Plating serial dilutions of the suspensions onto TSA-1 plates and counting the number of cfu following incubation at 25 °C confirmed bacterial concentration of the inoculum. Plasma and intestinal bactericidal activities were determined following the method of Stevens et al. (1991) with modifications. Briefly, 20 µL of plasma or gut supernatant were added to duplicate wells of a U-shaped 96-well plate. HBSS was added to some wells instead of plasma or gut supernatant and served as positive control. To each well, 20 µL of Phdp (1 × 106 cfu mL-1) were added and the plate was incubated for 2.5 h at 25 ºC. To each well, 25 µL of 3-(4,5 dimethyl-2-yl)-2,5-diphenyl tetrazolium bromide (1 mg mL-1; Sigma-Aldrich, Steinheim, Germany) were added and incubated for 10 min at 25 ºC to allow the formation of formazan. Plates were then centrifuged at 2000 × g for 10 min and the precipitate was dissolved in 200 µL of dimethyl sulfoxide (Sigma- Aldrich, Steinheim, Germany). The absorbance of the dissolved formazan was measured at 560 nm. Bactericidal activity is expressed as percentage, calculated from the difference between bacteria surviving compared to the number of bacteria from positive controls (100%).

% viable bacteria = Sample Absorbance x 100 / Absorbance of the reference sample % no viable bacteria (bactericidal activity) = 100 - % viable bacteria

FCUP 23 CGM as a potential protein source in feeds for meagre

3.7 Statistical analysis All results are expressed as means ± standard deviation (SD). Growth parameters were analyzed by One-Way Analysis of Variance (One-Way ANOVA) whereas immune parameters were analyzed by Two-Way ANOVA, with dietary treatment and time as main factors. Data were tested for normality by the Shapiro-Wilk test and homogeneity of variances by the Levene’s test. When normality was not verified values were transformed prior to ANOVA. In two-way ANOVA, a one-way ANOVA was performed for each factor if interaction was significant. Significant differences among groups were determined by the Tukey’s multiple range test. Differences were considered statistically significant when P ≤ 0.05. All data were analyzed with the Statistical Package for Social Science (IBM SPSS© Statistics, IBM Corp., Armonk, NY).

4. Results

All the experimental diets were well accepted by juvenile meagre. Fish easily adapted to the experimental system and even after manipulation due to sampling procedures, meagre resumed ingestion of diets, though it was generally lower than in the other days of the feeding trial. No mortality occurred during Fig 13. Meagre gonads at the end of the feeding the trial. At the end of 8 weeks of feeding trial. trial, it was possible to verify a great development of fish gonads (Fig. 13). The amount of tannins in CGM was not substantially reduced after autoclaving (1200 mg kg-1 and 1150 mg kg-1, prior and after autoclaving, respectively). However, it is possible that an oxidation of molecules and its consequent inactivation may have occurred.

4.1 Growth performance, feed utilization efficiency and whole-fish composition Data concerning growth performance and feed utilization efficiency are described in Table 3. Meagre body weight increased from an initial value of 35.0 g (Fig. 14a) to 149.9 g, 150.8 g, 137.6 g and 137.3 g, for fish fed the control, CGM7.5, CGM15 and CGM22.5 diets, respectively (Fig. 14b). Although not statistically significant, final body weight, weight gain and daily growth index of fish fed the CGM15 and CGM22.5 diets were slightly lower compared to control and CGM7.5 diets. Feed intake and N intake were similar among experimental treatments. In contrast, feed efficiency (FE) and

FCUP 24 CGM as a potential protein source in feeds for meagre protein efficiency ratio (PER) were significantly affected by dietary composition. FE and PER increased in fish fed CGM7.5, compared to fish fed the control diet, while fish fed the CGM22.5 diet showed the lowest values. N retention (% N intake) was also significantly affected by dietary composition, being the lowest values obtained with the two highest CGM inclusion levels compared to the control and CGM7.5 diets.

FCUP 25 CGM as a potential protein source in feeds for meagre

Table 3: Growth performance and feed utilization efficiency of meagre fed the experimental diets, at 8 weeks of growth trial.

Diets One-Way Anova (p) Control CGM7.5 CGM15 CGM22.5

Initial body weight (g) 35.0±0.0 35.0±0.0 35.0±0.0 35.0±0.0 0.874 Final body weight (g) 149.9±7.6 150.8±3.8 137.6±14.4 137.3±3.1 0.153

Weight gain (% initial weight) 328.2±21.6 331.0±10.8 293.0±41.0 292.4±8.8 0.152

1 Daily growth index (%) 3.64±0.16 3.66±0.08 3.37±0.32 3.37±0.07 0.159 †-1 -1 Feed intake (g kg ABM day ) 17.0±0.5 16.3±0.4 16.8±0.9 17.1±0.3 0.372 Feed efficiency2 1.25±0.01a 1.32±0.01b 1.25±0.01a 1.22±0.02a 0.000

Protein efficiency ratio3 2.39±0.02b 2.52±0.02c 2.36±0.02ab 2.33±0.03a 0.000

†-1 -1 N intake (g kg ABM day ) 1.43±0.04 1.37±0.04 1.43±0.08 1.43±0.02 0.347 N retention (g kg ABM†-1 day-1) 0.59±0.02 0.58±0.02 0.55±0.04 0.55±0.01 0.114

N retention (% N intake) 41.6±0.5b 42.4±0.6b 38.6±1.1a 38.7±0.02a 0.000

Data are expressed as means ±SD (n=3). Different letters denotes significant differences within experimental diets (One-way Anova; Tukey test; p≤ 0.05). 1DGI: ((final body weight1/3 - initial body weight1/3) / time in days)  100. 2FE: (wet weight gain / dry feed intake). 3PER: (wet weight gain / crude protein intake). †Average body weight: (initial body weight + final body weight) / 2.

FCUP 26 CGM as a potential protein source in feeds for meagre

The whole-fish composition of meagre fed the experimental diets are present in Table 4. At the end of the growth trial, no significant differences were found in dry matter, crude protein, crude lipid, ash content and in hepatosomatic index among groups fed the experimental diets. Visceral index was affected by dietary composition, resulting in significantly higher values of fish fed CGM22.5 diet, compared to the control group, while fish fed diets CGM7.5 and CGM15 showed no changes, compared to fish fed the control diet.

Table 4: Whole-fish composition and hepatosomatic and visceral indexes of meagre fed the experimental diets, at 8 weeks of growth trial.

Initial Diets One-Way Control CGM7.5 CGM15 CGM22.5 Anova (p) Whole-fish Dry matter (%) 24.6 26.0±1.0 25.3±0.3 24.7±0.6 25.1±0.7 0.205 Protein (%) 15.8 16.9±0.1 16.5±0.1 16.2±0.4 16.3±0.1 0.991 Lipid (%) 5.9 7.8±1.3 7.5±0.4 7.7±0.5 7.8±0.5 0.954 Ash (%) 4.2 3.7±0.3 3.9±0.1 3.7±0.1 3.8±0.3 0.714 Indexes Hepatosomatic1 - 1.7±0.2 1.7±0.0 1.6±0.1 1.6±0.0 0.334 Visceral2 - 4.0±0.2a 4.2±0.1ab 4.2±0.2ab 4.4±0.0b 0.043 Data are mean values ±SD (n=3). Significant differences within the diets are indicated by different letters (One-way Anova; Tukey test, p≤ 0.05). 1HSI: (liver weight / body weight)  100. 2VI: (viscera weight / body weight)  100.

a b

Fig 14. a: Meagre size at the beginning of feeding trial. b: Meagre size at the end of 8 weeks of study.

FCUP 27 CGM as a potential protein source in feeds for meagre

4.2 Immune indicators The values of Ht, MCV and total RBC and WBC counts are described in Table 5. Ht significantly decreased from week 1 to week 2, peaking highest values at week 8. MCV showed significantly higher values at week 1, decreasing at week 2 and increased later, at week 8. No dietary effect was verified in these parameters. Absolute values of RBC counts increased significantly from week 1 to week 2, presenting no changes from week 2 to week 8. On the contrary, the values of WBC decreased significantly from week 1 to week 2 and similar numbers were verified at week 2 and 8. It was not verified an effect of diet composition in these haematological parameters.

FCUP 28 CGM as a potential protein source in feeds for meagre

Table 5: Haematocrit (Ht), mean corpuscular volume (MCV), total red blood cells (RBC) and white blood cells (WBC) counts of juvenile meagre fed the experimental diets, after 1 (W1), 2 (W2) and 8 (W8) weeks of the growth trial.

Diets Parameters Control CGM7.5 CGM15 CGM22.5 W1 23.7±1.9 24.9±2.8 25.2±2.8 23.9±1.6 Ht (%) W2 21.1±1.7 20.4±1.8 21.2±2.2 20.6±1.9 W8 26.6±1.9 25.8±2.0 24.9±2.0 25.6±1.7 W1 115.1±11.0 120.3±18.1 120.4±16.7 110.3±12.6 MCV (µm3) W2 84.3±13.7 82.5±13.5 82.9±15.7 77.0±9.2

W8 104.7±15.1 92.3±15.9 101.6±23.8 103.5±22.0

W1 2.1±0.2 2.1±0.2 2.1±0.3 2.1±0.2

RBC (x106 mm-3) W2 2.5±0.3 2.4±0.3 2.6±0.4 2.7±0.4

W8 2.7±0.3 2.9±0.5 2.7±0.6 2.7±0.4

W1 2.7±0.8 2.7±0.7 2.4±0.2 2.7±0.6

WBC (x104 mm-3) W2 2.2±0.4 2.3±0.4 2.2±0.3 2.2±0.3

W8 2.3±0.5 2.6±0.5 2.2±0.4 2.7±0.7

1 Two-way Anova Time Diets Variation Source Time Diet Interaction W1 W2 W8 Control CGM7.5 CGM15 CGM22.5 Ht (%) 0.000 0.835 0.360 B A C - - - - MCV (µm3) 0.000 0.623 0.603 C A B - - - - RBC (×106 mm-3) 0.000 0.750 0.766 A B B - - - - WBC (×104 mm-3) 0.031 0.152 0.729 B A AB - - - -

Mean values and standard deviation (±SD) are presented for each parameter (n=9). 1Two-way ANOVA; means with different capital letters represent significant differences between times (Tukey test P < 0.05).

FCUP 29 CGM as a potential protein source in feeds for meagre

Lymphocytes were usually observed as small and round cells (Fig. 15a) and were the most abundant leucocyte, followed by thrombocytes, which were observed mostly as spindle-shaped (Fig. 15b). Monocytes were large cells with a blue cytoplasm and kidney-shaped purpled nucleus (Fig.15c), while neutrophils presented positive peroxidase granules labeled with the Antonow technique (Fig. 15d). The relative percentage of peripheral thrombocytes and lymphocytes were significantly affected by time (Table 6). While thrombocytes showed significantly lower values at week 8 compared to weeks 1 and 2, the percentage of lymphocytes showed the opposite pattern with the highest values at week 8 compared to weeks 1 and 2. Circulating lymphocytes were also affected by dietary treatments, showing fish fed the CGM7.5 diet the lowest values compared to fish fed the control diet. Regarding peripheral monocyte and neutrophil numbers, no significant differences on time nor dietary treatment were found in their relative proportion.

a b

c d

Fig. 15: Peripheral leucocytes of meagre. Fig. 15a: Lymphocytes. Fig. 15b: Spindle-shaped thrombocytes. Fig. 15c: Monocyte. Fig. 15d: Neutrophil labeled with Antonow reagent. Blood smear stained with Wright’s stain.

FCUP 30 CGM as a potential protein source in feeds for meagre

Table 6: Differential cell counting: relative proportion (% of total) of thrombocytes, lymphocytes, monocytes and neutrophils of meagre fed the experimental diets, at 1 (W1), 2 (W2) and 8 (W8) weeks of growth trial.

Diets Control CGM7.5 CGM15 CGM22.5 W1 28.4±4.9 36.7±11.0 30.8±7.5 31.7±4.6 Thrombocytes (%) W2 38.2±11.7 35.3±7.2 35.3±7.1 31.6±6.4 W8 20.4±4.5 24.5±4.4 19.7±5.7 20.8±3.7 W1 63.4±10.8 49.6±9.0 60.2±7.7 57.5±9.4 Lymphocytes (%) W2 49.4±11.6 51.9±7.2 54.4±10.5 56.9±6.9 W8 69.1±5.3 62.1±2.0 71.3±3.7 67.1±5.6 W1 8.36±4.24 8.85±2.78 5.89±1.80 6.72±3.00 Monocytes (%) W2 6.97±3.78 7.66±3.89 9.16±1.64 8.10±4.44 W8 7.62±3.10 9.02±4.97 6.08±3.23 8.73±2.85 W1 10.5±4.5 10.7±2.7 12.2±2.7 11.6±5.3 Neutrophils (%) W2 11.8±7.2 12.5±3.7 10.3±3.7 12.2±4.0 W8 9.7±2.3 9.7±2.2 9.6±3.0 10.1±3.3

1Two-way Anova

Time Diets Variation Source Time Diet Interaction W1 W2 W8 Control CGM7.5 CGM15 CGM22.5 Thrombocytes (%) 0.000 0.143 0.430 B B A - - - - Lymphocytes (%) 0.000 0.004 0.089 A A B b a b ab Monocytes (%) 0.849 0.569 0.229 ------Neutrophils (%) 0.134 0.396 0.643 ------

FCUP 31 CGM as a potential protein source in feeds for meagre

4.3 Humoral parameters Fig. 16 shows changes in ACP activity due to both experimental diets (p= 0.000) and time (p=0.025). In the first week, fish fed the CGM7.5 and CGM15 diets presented higher values compared to the control diet. In Week 2, ACP activity increased in fish fed CGM22.5 compared to fish fed the other experimental diets, presenting also the highest values observed in the experiment. At the end of the trial, no effect of diet composition was found in this humoral parameter. ACP activity of meagre fed diets CGM7.5 and CGM22.5 was significantly altered during the trial. Fish fed diet with 7.5% of CGM incorporation had decreased ACP activity values from week 1 to week 2, and showed similar values in week 2 and week 8. Values of meagre fed diet CGM22.5 increased from week 1 to week 2, decreasing to values similar to the other diets, at week 8. The interaction between time and diet was statistically significant (p= 0.000).

FCUP 32 CGM as a potential protein source in feeds for meagre

Plasma peroxidase activity (Fig. 17a), NO levels (Fig. 17b) and total Ig (Fig. 17c) decreased significantly from weeks 1 and 2 to week 8 (p= 0.001, p= 0.000 and p= 0.000, respectively), regardless of dietary treatment. No effect of diet composition (p= 0.958, p= 0.919 and p= 0.221, respectively) as well as interaction between diet and time was observed (p= 0.309, p= 0.060 and p= 0.319, respectively). Plasma anti-protease activity of fish fed the experimental diets varied significantly over time (p= 0.000), increasing from week 1 to week 2 and showing similar values at week 8, compared to week 2 (Fig. 17d). No diet effect and no interaction between diet and time was verified in this humoral parameter (p= 0.328 and p= 718, respectively). The effect of CGM incorporation in experimental diets was not verified (p= 0.078) in plasma bactericidal activity, as well as time and the interaction between time and diet (p= 716 and p= 414, respectively).

a b

FCUP 33 CGM as a potential protein source in feeds for meagre

Intestinal total Ig values were significantly affected by time (p= 0.003) (Fig. 19), decreasing from week 1 to week 2 increasing then, at week 8. No diet effect and the interaction between time and diet were not statistically significant. (p= 0.299 and p= 104, respectively).

Intestinal bactericidal activity (Fig. 20) decreased significantly from weeks 1 and 2 to week 8 (p= 0.004), regardless of dietary composition. Diet effect and the interaction effect between diet and time were not observed (p= 0.915 and p= 0.243, respectively).

FCUP 34 CGM as a potential protein source in feeds for meagre

5. Discussion Information regarding the adequate protein and lipid levels to include in meagre diets is scarce. Chatzifotis et al. (2012) tested different dietary protein (40, 45, 50 and 54%) and lipids (12, 15, 17 and 20%) levels on juvenile meagre growth performance. The best results were achieved with a protein inclusion levels from 50% up to 54% and a lipid level of 17%. In the present study, a dietary protein and lipid levels of 53% and 18%, respectively, allowed a satisfactory growth performance, which is in agreement with the previous study. The effects of dietary replacement of FM by CGM were only evaluated in two fish species. Alexis (1990) in rainbow-trout, tested different dietary CGM inclusion levels (22, 42 and 58%) and verified a decrease in weight gain, FE and protein retention with increasing CGM levels. In the present study, the lowest values for FE, PER and N retention were recorded with 15 and 22.5% CGM dietary inclusion levels. Moreover, it was also observed a tendency for a decrease of meagre growth with CGM15 and CGM22.5 diets. However, Martínez-Llorens et al. (2012) reported that CGM can be include at levels up to 34% in diets for gilthead seabream without compromising growth performance and nutritive parameters. Raw plant materials are known for having high concentrations of antinutrients, such as protease inhibitors, lectins, saponins and tannins (Francis et al., 2001). It has already been demonstrated that antinutrients present in vegetable protein sources may reduce fish growth by reducing the palatability of feed and interfering with the processes of digestion and absorption (Alexis, 1986; Tacon, 1997). CGM contains antinutritional factors, primarily tannins. Tannins can bind to feed proteins, making then inaccessible

FCUP 35 CGM as a potential protein source in feeds for meagre to digestive enzymes, thus reducing its digestibility. Moreover, tannins can also bind to digestive enzymes, altering its activity. In fact, a decrease of trypsin activity was observed in rainbow trout small intestine fed a diet with 20% of CGM (Filioglou and Alexis, 1989). As a consequence, a reduction of protein and dry matter digestibility was also recorded (Filioglou and Alexis, 1989). In the present study, the lower FE, PER and N retention obtained with the CGM15 and the CGM22.5 diets could be due to tannins present in CGM (~1.2 g Kg-1), suggesting that its inactivation through heat treatment was not efficient. High dietary CGM levels (58%) decreased whole-body protein and increased whole- body lipid contents in rainbow trout (Alexis, 1990). In contrast, in gilthead sea bream, dietary CGM had no effect on fish protein content, and a dietary inclusion level of 17 or 52% of CGM lead to lower whole-fish body lipids compared to the FM control group (Martínez-Llorens et al., 2012). Present data pointed to an absence of effect of dietary CGM on fish whole-body composition, although higher VI were recorded in fish fed the highest CGM inclusion level.

To the best of our knowledge, data regarding the effects of FM replacement by CGM on the fish immune system is limited. Furthermore, there are no studies concerning the utilization of this alternative protein source on meagre diets. It is currently recognized that a poor nutrition can cause an immunosupression, even in the absence of an inflammatory agent, as well as environmental and genetic aspects, transport, confinement and handling, leading to a disorder in fish physiology (Schreck, 1996; Montero et al., 1999; Ortuño et al., 2001; Tort, 2011). Heat stable antinutrients can imbalance fish immune functions, resulting in increased lymphocyte, neutrophil and macrophage numbers in lamina propria, as well as increased expression of anti- and pro-inflammatory genes and humoral and antibody responses (Baeverfjord and Krogdahl, 1996; Urán et al., 2008; Kiron, 2012). Diets containing SBM and soybean components usually induce an inflammatory response in the gastrointestinal tract, with mobilization of several leucocytes, including phagocytic cells, to the inflammatory focus (Baeverfjord and Krogdahl, 1996). Neutrophil and macrophage numbers of rainbow trout increased in fish fed diets with different levels of SBM incorporation, including 20% to 30% of incorporation, suggesting an inflammatory response to soybean proteins (Rumsey, et al., 1994; Baeverfjord and Krogdahl, 1996; Bakke-McKellep et al., 2000; Krogdahl et al., 2000; Urán et al., 2008). In the present study, a relative lymphopenia in fish fed CGM7.5 diet was observed at the end of feeding trial, suggesting a mobilization of this cell type. Since the relative proportion of neutrophils and monocytes were similar at the three sampling points with no effect of dietary

FCUP 36 CGM as a potential protein source in feeds for meagre treatments, the hypothesis of cell migration due to inflammation is not possible. These phagocytes play an important role during an inflammatory response, with their highest bactericidal and phagocytic activities, preventing the initial extend of the inflammatory agent (Secombes and Fletcher, 1992; Afonso et al., 1998a; Neumann et al., 2001). In fact, plasma peroxidase and NO levels are consistent with this hypothesis since they were not affected by dietary CGM. The lower proportion of lymphocytes in fish fed the CGM7.5 was already observed at the beginning of the growth trial, compared to fish fed the other experimental diets, and this difference may have resulted in significant changes at the end of the experiment. Previous studies show multiple effects on ACP activity with the use of different nutrients and different percentages of incorporation, resulting in increased or decreased values, in addition to the expression of genes that encode complement components, in several fish species (Burrells et al., 1999; Bransden et al., 2001; Lin and Shiau, 2003; Sitjà-Bobadilla et al., 2005; Costas et al., 2014; Fuentes-Appelgren et al., 2014). In the present study, increased values of ACP activity in fish fed diets containing CGM were observed, especially in fish fed CGM22.5, suggesting a stimulation of complement system and its consequent production. In gilthead seabream, Sitjà-Bobadilla et al. (2005) reported increased ACP values with 50% of FM replacement, while those values reduced with 75% and a 100% of FM replacement. Costas et al. (2014) found higher values of ACP activity with diets containing saponins and phytosterols, antinutrients found in soybean products, resembling an increase of leucocytes, its migration to the inflammatory site and suggesting a role of complement system in this response. Complement system is formed by a complex enzyme cascade of glycoproteins, mostly synthesized in their proenzyme form (Kiron, 2012). Fish complement components allows for a wider recognition of foreign surfaces when compared to mammals, being easily influenced by external factors including nutritional status. Once activated, complement fragments opsonize the inflammatory agents, increasing the uptake of phagocytes and recruitment of inflammatory cells (Holland and Lambris, 2002; Boshra et al., 2006; Kiron, 2012). In the present study, no correlation was reported between ACP activity and phagocytic cells. In fact, present data indicates that no inflammatory injury was caused by CGM incorporation in the experimental diets, since there was no diet effect on the main phagocytic cells and in anti-protease, peroxidase and plasma bactericidal activities. Thus, a stimulation of complement system occurred however, in the absence of a real inflammatory response, the uptake of phagocytic cells to the inflammatory focus was not verified. Erythrocytes have respiratory gas exchange as the major function (Thomas and Egée, 1998). There are several external factors that influence blood oxygen carrying-capacity

FCUP 37 CGM as a potential protein source in feeds for meagre and the values of haemoglobin and Ht which includes variations in temperature and salinity (Nikinmaa and Salama, 1998; Brauner and Val, 2005). Prior to the experiment, meagre were kept in the quarantine system under temperatures, salinities and densities different from those used in the experimental system. Indices such as Ht or MCV are used as indicators of anaemia or systemic responses to external stimuli (Sarma, 1990; Tavares-Dias e Moraes, 2007). Increased values of Ht are reported in a response not only to the factors mentioned above, but also to transport, handling and confinement, were already reported (Frisch and Anderson, 2000). This increase suggests an approach to increasing the blood’s oxygen carrying (Montero et al., 1999). Thus, the alterations found in the first two weeks of trial in these haematological parameters, over time and regardless the dietary treatment are possibly related to the different culture conditions prior and during growth trial. Most fish species are harvested before or when are fully developed, since growth performance is higher prior to sexual maturity. Thus, it is important to document age- related variations in haematological and immune parameters of juvenile fish, since this is usually the age when fish are breed in aquaculture (Hrubec et al., 2001). Fish age and size are important factors that may alter some immune indicators. In larval stage, fish size is important for maturation of the immune system. In fully developed fish, immune parameters can vary with increasing age, depending on the parameter studied and fish species (Tatner, 1996; Magnadóttir, 2010). In this study, significant variations in WBC values, thrombocytes and lymphocytes, as well as in the humoral parameters determined in the present study (plasma and intestinal total Ig, anti-proteases, NO levels, peroxidase and intestinal bactericidal activities) over time and regardless the dietary composition were reported. Present results suggest that these changes in immune parameters are related with meagre great growth performance and fast development.

6. Conclusions The results of the present study showed that CGM can be included at levels up to 7.5% in diets for short-term feeding of meagre without compromising growth performance and feed utilization efficiency. On the other hand, higher CGM inclusion levels (15 and 22.5%) negatively affect FE and PER and in long-term feeding will possible lead to a lower growth performance. Dietary inclusion of CGM resulted in reduced effects on immune parameters evaluated in the present study and, in addition to the high survival and satisfactory growth with experimental diets, probably have limited health implications. According to these results, CGM seems to be a good alternative to FM in aquafeeds. Nevertheless, further

FCUP 38 CGM as a potential protein source in feeds for meagre studies with higher percentages of CGM and FM replacement, as well as challenges with pathogenic bacteria, would be useful to evaluate the consequences on growth performance and if animals fed with this protein source are resistant to diseases.

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