Effects of partial - COnnecting REpositories · 2017. 12. 21. · Effects of partial replacement of fish meal by carob seed germ meal on growth performance and immune parameters of

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 Dissertação de Mestrado apresentada à

Faculdade de Ciências da Universidade do Porto em

Nutrição e Imunologia

2014

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2014

2.º

CICLO

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 CGM as a potential protein source in feeds for meagre

I

“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.


II

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.


III

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!


IV

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


V

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%).


VI

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.


VII

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


VIII

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 composition………………………………………………………………………………

23

4.2 Immune indicators…………………………………………………………….. 27

4.3 Humoral parameters…………………………………………………………. 31


IX

5 Discussion…………………………………………………………………………….. 34

6 Conclusions……………………………………………………………………………. 37

7 References…………………………………………………………………………….. 38


X

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


XI

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


XII

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


1

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.


2

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,


3

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).


4

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

has an elongated body with the mouth located

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

Fig. 2: Meagre (Argyrosomus regius, Asso, 1801). Source: Fish Base. http://www.fishbase.org

.

http://www.fishbase.org/


5

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).

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

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


6

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).


7

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: 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).

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

Fish IAA Requirements

(Average values*) 6.2 3.4 3.1 0.9 3.1 3.8 4.7 5.4 2.3 4.5

Tannins

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

http://www.feedipedia.org/


8

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

content, and a germinal portion or germ.

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

Fig. 4: Carob seed hanging in carob tree. Source: WWF Oasis of Monte Arcosu.


9

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


10

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 anti-

microbial 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).


11

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


12

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


13

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


14

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).


15

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.

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).

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


16

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).

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

Fig. 7: Blood sampling collection.

Fig. 8: Meagre digestive tube, at 8 weeks of feeding trial.


17

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.

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.

Fig. 9: Kjeltec digestor, distillation and titration units


18

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.

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

Fig. 10: Lipids extraction unit.

Fig. 11: Muffle furnace.


19

room temperature. Afterwards, the percentage of red blood cells in the volume of blood

was determined using a graphic reader (Fig. 12a).

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

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

b a


20

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


21

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

microplates. Prior to analysis, a sodium bicarbonate (NaHCO3, 5 mg mL-1, pH 8.3,

Sigma-Aldrich, Steinheim, Germany) buffer was prepared. Briefly, 10 µL of plasma

were incubated with the same volume of a trypsin solution (5 mg mL-1 in NaHCO3) for

10 min at 22 ºC in polystyrene microtubes. To the incubation mixture, 100 µL of

phosphate buffer (NaH2PO4, 13.9 mg mL-1, pH 7.0) and 125 µL of azocasein (20 mg

mL-1 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


22

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


23

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

the trial. At the end of 8 weeks of feeding

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

Fig 13. Meagre gonads at the end of the feeding trial.


24

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.


25

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

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 weight

1/3 - initial body weight

1/3) / 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.

Diets One-Way Anova (p)


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

Daily growth index (%)1 3.64±0.16 3.66±0.08 3.37±0.32 3.37±0.07 0.159

Feed intake (g kg ABM†-1

day-1

) 17.0±0.5 16.3±0.4 16.8±0.9 17.1±0.3 0.372

Feed efficiency2 1.25±0.01

a 1.32±0.01

b 1.25±0.01

a 1.22±0.02

a 0.000

Protein efficiency ratio3 2.39±0.02

b 2.52±0.02

c 2.36±0.02

ab 2.33±0.03

a 0.000

N intake (g kg ABM†-1

day-1

) 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±0.6

b 38.6±1.1

a 38.7±0.02

a 0.000


26

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

Anova (p) Control CGM7.5 CGM15 CGM22.5

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±0.2

a 4.2±0.1

ab 4.2±0.2

ab 4.4±0.0

b 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.

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

a b


27

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.


28

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.

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).

Parameters Diets


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

1Two-way Anova

Variation Source Time Diet Interaction Time Diets

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 - - - -


29

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.

b a

d c

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.


30

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.

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, while different lower case letters denotes significant differences between diets (Tukey test P < 0.05).

Diets


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

Variation Source Time Diet Interaction Time Diets

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 - - - - - - -


31

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).


32

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


33

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).


34

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


35

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


36

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


37

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


38

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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