Dissertação-Maria-João-Xavier (versão final)Maria João Magalhães de Almeida Xavier Mestrado em...

Effect of dietary phospholipids and docosahexaenoic acid in growth performance, lipid profile and oxidative stress in Meagre (Argyrosomus regius) juveniles Maria João Magalhães de Almeida Xavier Mestrado em Recursos Biológicos Aquáticos Biologia da FCUP 2014/2015 Supervisor Prof. Doutor Pedro Pousão, Investigador Auxiliar, EPPO Co-Supervisor Prof. Doutor Aires Oliva-Teles, Professor Catedrático, FCUP

Todas as correções determinadas pelo júri, e só essas, foram efetuadas.

O Presidente do Júri,

Porto, /_ /

FCUP Effect of dietary phospholipids and docosahexaenoic acid in growth performance, fatty acid

composition and oxidative stress Argyrosomus regius

Acknowledgments I would like express my gratitude to my supervisor, Dr. Pedro Ferreira Pousão for the opportunity to work in the Olhão Aquaculture Research Station. For guiding me throughout the work of this dissertation and for all the support given and knowledge shared over this last year. A very special thanks to my co-supervisor Prof. Dr. Aires Oliva-Teles for his excellent guidance throughout this work. His immense scientific knowledge was an incentive to go further on and essential for the accomplishment of this dissertation. Thanks to the Dra. Narcisa Bandarra (L-IPMA/INRB, IP) for accepting me at IPMA laboratory and to guide me. To Dr. Jorge Dias and SPAROS Ltd, for the supply of diets used in this study, and their critical view have also contributed to the establishment of this research. I am especially grateful to all the members of EPPO for receiving me and providing me a stimulating and fun environment in which I had the opportunity to learn and grow. In particular to Ana Mendes for having guided me during the practical work. I would like to extend my thanks to Sara Costa for the entire accessibility and affection that has always shown. Throughout the time spent and for what taught me the laboratory level. A heartfelt thanks to all the members of Nutrimo laboratory and the Marine Zoology Station, for their help and friendship. Especially to Carolina Castro that taught me so much and spent the time to accompany me. To pilot project meagre cultivation in several production systems - "AQUACOR". PROMAR 31-03-05FEP-003 All my dear friends that were always present when I needed the most. A sincere thank you to Sofia Abrunheiro, Filipe Silva, Luis Esteves and Diana Moura for the work they have put in the review of this document. Last and most especially I would like to express my gratitude to my family who have always supported and encouraged me as well as for the unconditional love.



Abstract

Meagre (Argyrosomus regius, Asso, 1801) is a new species within in the

Mediterranean aquaculture industry and introduced to diversify the number of

cultured species. Meagre flesh is characterized by a high protein and low lipid

content, having a recommended nutritional quality. Moreover, it exhibit a high content

of polyunsaturated fatty acids (PUFA), mainly represented by a high proportion of n-3

PUFA, whose importance for human health, has been continuously increasing in the

last decades. These fatty acids (FA) are essential nutrients with a variety of important

structural, functional and signaling roles. Furthermore, highly unsaturated fatty acid

(HUFA) the phospholipids (PL) requirement has been documented to be essential

nutrients for larval and juvenile marine fish in regulation of a wide range of cellular

functions.

The present study was therefore designed and carried out to investigate the effects

of increased levels of PL (1.0 to 4.0 %) and docosahexaenoic acid (DHA) (1.0 to

2.0%) on the diet (at a constant lipid, protein and starch level), in growth

performance, fatty acid and lipid class composition of different organs and oxidative

state of meagre juveniles was also evaluated.

For that purpose, a total of 1800 juveniles, with mean initial weight 13.8± 2.0g, were

randomly assigned into 12 tanks, to perform analysis in triplicate for the 4 treatments

(diet A, B, C and D), fed, ad libitum, three times a day for 81 days.

Results showed that dietary enrichment of PL and DHA didn’t exert any influence in

the survival rate of juveniles of meagre. In terms of growth performance, specific

growth rate (SGR) and final body wet weight (FBW) were increased with the

combination of 4.0% PL and 2.0% DHA in the diet and the feed conversion ratio

(FCR) showed best results with higher PL percentage in the diet.

The use of 4.0% PL and 2.0% DHA diet promoted whole-body lipid retention as well

as higher energy content.

FA composition in the analyzed organs were correlated to the used diet. Therefore,

increasing DHA concentration in the diet resulted in a higher accumulation of DHA in

body tissues. DHA tissue distribution showed a higher accumulation in the brain than

in muscle or liver.

Finally, regarding the liver oxidative status, the liver lipid peroxidation levels observed

were similar in all diet treatments applied on this study. However, in diets with higher

PL percentage the activity of superoxide dismutase (SOD) and glucose-6-phosphate

dehydrogenase (G6PDH) was significantly increased.



In conclusion, this work showed that juveniles of meagre achieve an increasing

growth performance with an increase of 4.0% of PL in the diet. The increasing DHA

concentration in diet led to a higher retention of this essential FA in the muscle. No

major changes were also observed in lipid peroxidation levels related to an increase

of PL and DHA in the diet.

Keywords: Argyrosomus regius; Growth performance; Polyunsaturated Fatty Acids;

Docosahexaenoic acid; Phospholipids; Oxidative stress; Fatty acid composition.



Resumo Corvina (Argyrosomus regius, Asso, 1801) é uma nova espécie dentro da indústria

da aquicultura do Mediterrâneo, introduzida para diversificar o número de espécies

cultivadas. A qualidade nutricional da corvina é considerada muito boa,

caracterizada por um elevado teor de proteínas e baixo teor de lipídios. Além disso,

apresenta um elevado teor de ácidos gordos polinsaturados (PUFA), representado,

principalmente, por uma alta proporção de n-3 PUFA, cuja atenção tem aumentado

continuamente durante as últimas décadas para a saúde humana. Estes ácidos

gordos (FA) são nutrientes essenciais com uma variedade de papéis importantes a

nível estrutural, funcional e de sinalização. Tal como estes ácidos gordos

polinsaturados (HUFA) os requisitos de fosfolípidos (PL) têm sido documentados

como sendo nutrientes essenciais para larvas e juvenis de peixes marinhos ao nível

da regulação de uma vasta gama de funções celulares.

O presente estudo foi, por conseguinte, concebido e realizado para investigar os

efeitos do aumento dos níveis dietéticos de PL (1,0 a 4,0%) e ácido

docosahexanóico (DHA) (1,0 a 2,0%), com um nível de lípidos, proteínas e energia

constantes, no desempenho do crescimento. Também se avaliou o perfil dos ácidos

gordos e a classe de lípidos dos diferentes órgãos e estado oxidativo dos juvenis de

corvina.

Para esse objetivo, um total de 1800 juvenis, com peso médio inicial 13,8± 2,0g,

foram distribuídos ao acaso por 12 tanques, de forma a ter triplicados para os 4

diferentes tratamentos (dietas A, B, C e D) alimentados, ad libitum, três vezes ao dia

durante um período de 81 dias.

Os resultados mostraram que aumento de PL e DHA nas dietas não exerce

qualquer influência na taxa de sobrevivência dos juvenis de corvina. Quanto à

performance de crescimento, a taxa de crescimento específica e peso médio final

obtiveram um aumento com a combinação de 4,0% PL e 2,0% DHA na dieta e a

taxa de conversão alimentar mostrou melhores resultados com maior percentagem

de fosfolípidos na dieta.

A dieta com concentração de 4,0% de PL e 2,0% de DHA promoveu uma retenção

de lípidos no corpo inteiro, assim como um conteúdo energético mais elevado.



A composição de FA nos órgãos analisados refletiu a suplementação da dieta

aplicada. Assim, o aumento da concentração de DHA na dieta resultou numa maior

acumulação de DHA nos tecidos corporais analisados. A distribuição de DHA nos

tecidos mostrou uma acumulação mais elevada no cérebro quando comparado com

o tecido muscular e hepático.

Finalmente, em relação ao estado oxidativo do fígado, os níveis verificados de

peroxidação lipídica no fígado foram semelhantes em todos os tratamentos

dietéticos implementados no presente estudo. Contudo, nas dietas com maior

percentagem de PL a superóxido dismutase (SOD) e a glucose-6-phosphate

desidrogenase (G6PDH) mostrou uma maior atividade.

Em conclusão, os resultados deste trabalho mostram que os juvenis de corvina

obtiveram um aumento no desempenho de crescimento com um aumento de 4,0%

de PL na dieta. O aumento da concentração de DHA na dieta induziu uma maior

retenção deste FA no tecido muscular. Não foram observadas grandes alterações

nos níveis de peroxidação lipídica em relação ao aumento de PL e DHA na dieta.

Palavras-chave: Argyrosomus regius; Performance de crescimento; Ácidos gordos

polinsaturados; Ácido docosahexanóico; Fosfolípidos; Stress oxidativo; Composição

de ácidos gordos.



Index

1- INTRODUCTION .............................................................................................. 1

1.1- State of Aquaculture ............................................................................................................................ 1

1.2- Meagre ....................................................................................................................................................... 2

1.3- Meagre Aquaculture ............................................................................................................................. 3

1.4- Fish Nutrition .......................................................................................................................................... 5

1.5- Lipids .......................................................................................................................................................... 7

1.6- Fatty acids ................................................................................................................................................ 8

1.7- Phospholipids ...................................................................................................................................... 11

1.8- Oxidative Stress .................................................................................................................................. 12

1.9- Objectives .............................................................................................................................................. 14

2- MATERIAL AND METHODS ........................................................................ 15

2.1- Diets ......................................................................................................................................................... 15

2.2- Experimental conditions ................................................................................................................. 17

2.3- Sampling ................................................................................................................................................ 17

2.4- Analytical Methods ............................................................................................................................ 18 2.4.1- Zootechnical parameters .......................................................................................................................... 18 2.4.2- Proximate analysis ....................................................................................................................................... 18 2.4.3- Tissue lipids ..................................................................................................................................................... 19

2.4.3.1- Lipid classes .......................................................................................................................................... 19 2.4.3.2- Fatty-acids .............................................................................................................................................. 19

2.4.4- Oxidative stress ............................................................................................................................................. 20

2.5- Statistical analysis ............................................................................................................................. 22

3- RESULTS ....................................................................................................... 23

3.1- Diet composition ................................................................................................................................. 23

3.2- Growth trial ........................................................................................................................................... 24

3.3- Whole-body composition ................................................................................................................ 25

3.4- Lipid composition .............................................................................................................................. 26

3.5- Oxidative Stress .................................................................................................................................. 37

4- DISCUSSION ................................................................................................. 38



4.1- Conclusion ............................................................................................................................................ 44

5- REFERENCES ............................................................................................... 46

Figure Index FIGURE 1: FISHERY PRODUCTION IN LIVE WEIGHT EQUIVALENT FOR AQUACULTURE

(LEFT) AND CAPTURE (RIGHT). SOURCE: OECD AND FAO SECRETARIATS, 2014. ........................................................................................................................................................................................... 2

FIGURE 2: MEAGRE (ARGYROSOMUS REGIUS, ASSO, 1801) SOURCE: IRIDA’S RESEARCH FOR MEAGRE CULTURE ..................................................................................................... 3

FIGURE 3: GLOBAL PRODUCTION OF FISHMEAL AND FISH OIL. SOURCE: FAO, 2014 ... 7 FIGURE 4: THE METABOLIC PATHWAYS BY WHICH ESSENTIAL FATTY ACIDS ARE

CONVERTED TO LONGER CHAIN, MORE UNSATURATED DERIVATIVES. SOURCE: NARAYAN ET AL., 2015 .............................................................................................................. 9

FIGURE 5: PATHWAYS OF CONVERSION OF ARAQUIDONIC ACID 20:4 (N-6) AND EICOSAPENTAENOIC ACID 20:5 (N-3) TO EICOSANOIDS. SOUCE: LALL, 2000 ...... 10

FIGURE 6: VARIATION OF DIFFERENT FATTY ACIDS EICOSAPENTAENOIC ACID (EPA), DOCOSAHEXAENOIC ACID (DHA), ARACHIDONIC ACID (ARA), TOTAL SATURATED FATTY ACID (ΣSFA), TOTAL MONOUNSATURATED FATTY ACID (ΣMUFA) AND TOTAL POLYUNSATURETED FATTY ACID (ΣPUFA) IN THE LIVER, BRAIN AND MUSCLE (IN MG/G) OF ARGYROSOMUS REGIUS FED THE EXPERIMENTAL DIETS. RESULTS FROM TWO WAY ANOVA ARE REFLECTED; ASTERISKS INDICATE SIGNIFICANT DIFFERENCES AS *P<0.05; N.S. INDICATES NON SIGNIFICANT DIFFERENCES. ........................................................................................................ 33

FIGURE 7: VARIATION OF DIFFERENT LIPID CLASS, TRIACYLGLYCEROLS (TAG); FREE FATTY ACIDS (FFA); CHOLESTEROL (CH); DIACYLGLYCEROL (DAG); MONOACYLGLYCEROL (MAG); PHOSPHOLIPIDS (PL), IN THE LIVER, BRAIN AND MUSCLE (IN %) OF ARGYROSOMUS REGIUS FED THE EXPERIMENTAL DIETS. RESULTS FROM TWO WAY ANOVA ARE REFLECTED ASTERISKS INDICATE SIGNIFICANT DIFFERENCES AS *P<0.05; N.S. INDICATES NON SIGNIFICANT DIFFERENCES. .................................................................................................................................................... 36

Table Index TABLE 1: FORMULATION OF EXPERIMENTAL DIETS. .......................................................................... 16 TABLE 2: FATTY ACID COMPOSITION OF THE DIFFERENT EXPERIMENTAL DIETS

(RESULTS IN % AND MG/G FATTY ACID) .......................................................................................... 23 TABLE 3: LIPID CLASS OF DIFFERENT EXPERIMENTAL DIETS (RESULTS IN %) ............. 24 TABLE 4: GROWTH PERFORMANCE, FEED UTILIZATION, CONDITION AND HEPATIC

INDICES OF ARGYROSOMUS REGIUS FED THE EXPERIMENTAL DIET. .................... 25 TABLE 5: WHOLE-BODY COMPOSITION (DRY MATTER) OF ARGYROSOMUS REGIUS

FED THE EXPERIMENTAL DIET. .............................................................................................................. 26 TABLE 6: LIVER FATTY ACID COMPOSITION (MG/G FATTY ACIDS) OF ARGYROSOMUS

REGIUS FED THE EXPERIMENTAL DIETS. ....................................................................................... 27 TABLE 7: BRAIN FATTY ACID COMPOSITION (MG/G FATTY ACIDS) OF ARGYROSOMUS

REGIUS FED THE EXPERIMENTAL DIETS. ....................................................................................... 28 TABLE 8: MUSCLE FATTY ACID COMPOSITION (MG/G FATTY ACIDS) OF

ARGYROSOMUS REGIUS FED THE EXPERIMENTAL DIETS. .............................................. 29 TABLE 9: LIVER FATTY ACID COMPOSITION (MG/G FATTY ACIDS) OF ARGYROSOMUS

REGIUS FED THE EXPERIMENTAL DIETS. ....................................................................................... 30 TABLE 10: BRAIN FATTY ACID COMPOSITION (% FATTY ACIDS) OF ARGYROSOMUS

REGIUS FED THE EXPERIMENTAL DIETS. ....................................................................................... 31



TABLE 11: MUSCLE FATTY ACID COMPOSITION (% OF TOTAL FATTY ACIDS) OF

ARGYROSOMUS REGIUS FED THE EXPERIMENTAL DIETS. .............................................. 32 TABLE 12: LIPID CLASS COMPOSITION (% TOTAL OF LIPID CLASS) AND FAT

DEPOSITION (%) OF LIVER, BRAIN AND MUSCLE OF ARGYROSOMUS REGIUS FED THE EXPERIMENTAL DIETS. ........................................................................................................... 35

TABLE 13: ACTIVITIES OF GLUTATHIONE PEROXIDASE (GPX; MU MG−1 PROTEIN), SUPEROXIDE DISMUTASE (SOD), GLUCOSE-6-PHOSPHATE DEHYDROGENASE (G6PDH), GLUTATHIONE REDUCTASE (GR), AND CATALASE (CAT) (U MG−1 PROTEIN), AND TBARS CONTENT (NMOL MDA G TISSUE−1) OF ARGYROSOMUS REGIUS FED THE EXPERIMENTAL DIETS ........................................................................................ 37



Abbreviations index ARA - Arachidonic Acid

ALA - α-Linolenic Acid

BW - Body Weight

BHT - Butylated Hydroxytoluene

CAT - Catalase

CH- Cholesterol

K - Condition Factor

DAG - Diacylglycerol

DHA - Docosahexaenoic Acid

DM - Dry Matter

EPA - Eicosapentaenoic Acid

EFA - Essential Fatty Acid

EPPO - Estação Piloto de Piscicultura

de Olhão

FA - Fatty Acids

FAME - Fatty Acid Methyl Esters

FCR - Feed Conversion Ratio

FBW - Final Body Wet Weight

FID - Flame Ionization Detector

FAO - Food and Agriculture

Organization

FFA - Free Fatty Acids

GC - Gas Chromatographer

G6PDH - Glucose-6-phosphate

Dehydrogenase

GPX - Glutathione Peroxidase

GR - Glutathione Reductase

HIS - Hepatosomatic Index

HUFA - Highly Unsaturated Fatty Acid

H2O2 - Hydrogen Peroxide

HO· - Hydroxyl Radicals

IPMA - Instituto Português do Mar e

da Atmosfera

LA - Linoleic Acid

LPO - Lipid Peroxidation

MDA - Malondialdehyde

MAG - Monoacylglycerol

MUFA - Monounsaturated Fatty Acids

MDA - Nanomoles Malondialdehyde

GSSG - Oxidized Glutathione

PL- Phospholipids

PUFA - Polyunsaturated Fatty Acids

ROS - Reactive Oxygen Species

GSH - Reduced Glutathione

SFA - Saturated Fatty Acids 1O2 -‐ Singlet Oxygen SGR - Specific Growth Rate

O2− - Superoxide Anions

SOD - Superoxide Dismutase

TLC - Thin Layer Cromatography

TBA - Thiobarbituric Acid

TBARS - Thiobarbituric Acid Reactive

Substances

TAG - Triacylglycerol

TCA - Trichloroacetic Acid

VFI - Voluntary Feed Intake



1

1- Introduction

1.1- State of Aquaculture Despite the increasing of world population, beyond 7 billion persons in 2015, the

world per capita apparent fish consumption increased from an average of 9.9 kg in

the 1960s to 19.2 kg in 2012. This was possible because global fish production has

grown steadily over the last five decades, with food fish supply increasing at an

average annual rate of 3.2 percent (FAO, 2014).

Fish and fishery products are among the most traded food commodities worldwide,

with trade volumes and values reaching new highs in 2011, and are expected to

carry on rising. However, this increase in fish demand causes huge pressure in wild

stocks leaving an increased percentage of overexploited fish stocks and decreased

proportion of non-fully exploited species. Of the world fish stocks assessed in 2011,

61.3 percent were fully-exploited and only 9.9 percent were under-exploited. While

capture fisheries production remains stable, aquaculture represents the only viable

solution to prevent the depletion of aquatic resources (FAO, 2014).

The contribution of farmed food fish has a record of 42.2 percent of the total 158

million tons of fish produced by capture fisheries (including for non-food uses) and

aquaculture in 2012 (FAO, 2014).

In terms of production, global aquaculture provided 90.4 million tons (live weight

equivalent) in 2012 (US$144.4 billion), including 66.6 million tons of food fish

(US$137.7 billion). According to the latest information, FAO estimated that world food

fish aquaculture production rose by 5.8 percent, to 70.5 million tons in 2013. The

global trend of aquaculture development is gaining importance in total fish supply,

and continues to grow strongly, yet at a slower rate (figure 1).

The development and distribution of aquaculture products is not uniform in all regions

of the world (Gjedrem et al., 2012). Asia accounted for 89 percent of world’s

aquaculture production by volume in 2010, with the share of freshwater aquaculture

gradually increasing to 65.6 percent in 2010, from around 60 percent in the 1990s.

Africa has increased its contribution to global production from 1.2 percent to 2.2

percent in the past ten years, mainly as a result of a rapid development in freshwater

fish farming in sub-Saharan Africa. Europe and Oceania had the lowest average

annual growth rates in the period 2000–2012, at 2.9 and 3.5 percent, respectively. In

North America aquaculture has ceased expanding in the recent past years, but in

South America it has shown strong and continuous growth, particularly in Brazil and



2

Peru. Some developed countries like Japan, The United States of America and

several European countries have reduced their aquaculture output in recent years,

mainly owing to competition from countries with lower production costs (FAO, 2014).

Figure 1: Fishery production in live weight equivalent for aquaculture (left) and capture (right). Source: OECD and FAO Secretariats, 2014.

1.2- Meagre Meagre, Argyrosomus regius (Asso, 1801), is a teleost fish of the family Sciaenidae,

which includes 70 genera and 270 species (Nelson, 1994) distributed in subtropical

waters such as Mediterranean and Black Sea and along the Atlantic coasts of

Europe and Africa (Poli et al., 2003). Meagre is nectobentonic, and inhabits inshore

and shelf waters close to the bottom, as well as surface and midwaters from 15 to

about 200 m in sandy bottoms and grassland of Posidonia (Whitehead et al., 1986).

This species exhibits a big head and an elongated, fusiform and slightly compressed

body. It has a dorsal silver-grey color with bronze trails and an evident lateral line

(figure 2). In the wild this species can grow up to 2 m and reach 50 kg, but it is more

frequent to capture individuals between 50 cm and 1 meter in length (Cárdenas,

2010).

A. regius has a relatively big terminal mouth, with small teeth disposed in several

series. It is a carnivorous species, very voracious and has a diet based on

polychaete worms, crustaceans, echinoderms, mollusks, and small fish (Jimenez et

al., 2005).

It is a gregarious species, moving in small groups, and displaying anadromous

behavior, migrating to spawn from deep waters to coastal areas like estuaries and

salt marshes between the months of April and June (Monfort, 2010). Areas that

remain important for spawning are the estuary of the river Gironde, Bay of Biscay,



3

France (Quéméner et al., 2002), river Tejo, Portugal and river Guadiana, Southern

Spain and Portugal (Gonzalez-Quiros et al., 2011). This species is capable of

reaching these areas because it is eurithermal and eurihaline, resisting sudden

changes in temperature from 2 to 38 °C and of salinity from 5 to 39 ‰.

The majority of Sciaenid species are iteroparous, reproducing more than once in a

lifetime and gonochoric, only have one distinct sex per organism.

Average world catches of A. regius were of 4408 tons per year from 2005 to 2007

(FAO, 2007). However, this value may have been higher as FAO data for some

countries were unavailable, or wrongly estimated.

Meagre is considered a highly susceptible species because it forms large spawning

aggregations and produces conspicuous sounds when migrating to shallow waters,

with substantial fishing effort during the reproductive season. Furthermore, the

spawning habitats of the species (river mouths and lagoons) often suffer serious

environmental deterioration (Quéro and Vayne, 1987; Sadovy and Cheung, 2003).

This reproductive behavior, coupled with the general worldwide tendency to overfish

top predators (Christensen et al., 2003), including adult and juvenile meagre

(Quéméner, 2002), and the lack of basic biological information about the species,

have raised concerns about the status of meagre stocks.

In Mediterranean waters, meagre populations have suffered an alarming decline in

numbers (Quéro and Vayne, 1987) and have disappeared from the Balearic Islands

(Western Mediterranean), where the species is considered to be in critical danger

(Mayol et al., 2000).

Figure 2: Meagre (Argyrosomus regius, Asso, 1801) Source: IRIDA’s research for meagre culture

1.3- Meagre Aquaculture The history of meagre aquaculture is quite recent, it is farmed in Europe since the

late nineties. The first commercial production was recorded in France in 1997. Since



4

then, production has expanded slowly in nearby regions, especially on the

Tyrrhenian side of the Italian coast and in Corsica.

Total aquaculture production has raised from a few tons in 2000 to 2200 tons in

Egypt, 1348 tons in Spain, 418 tons in France, 102 tons in Italy, and 44 tons in

Portugal, in 2009 (FAO, 2011).

Mediterranean mariculture has been dominated by the production of gilthead

seabream (Sparus aurata) and European seabass (Dicentrarchus labrax). Meagre

has potential characteristics suitable for aquaculture diversification, because it

adapts easily to captivity, exhibits high growth rates (>1 kg year per year), which is

much more than the growth potential of currently cultured species, it has an excellent

feed conversion rate (0.9–1.2 depending on the feed) (Quéro and Vayne, 1997;

Quèmèner, 2002) and tolerates wide ranges of temperature and salinity. It has a high

nutritional value owing to the fact it is a lean fish, with low lipid and high protein

quality flesh and long shelf-life (Quèmèner, 2002; Poli et al., 2003). Seabass and

seabream have two to five times more fat than meagre, however the profile of fatty

acids are similar to them, characterized by a high proportion of polyunsaturated fatty

acids (PUFAs), especially the n-3 series (among 20.7 and 26.7% in 100 grams of live

weight) (Rodriguez-Rua et al., 2009). In addition, meagre matures at a large size >4

kg (Schuchardt et al., 2007), which is larger than the harvest size, thus avoiding

problems of reduced growth associated with maturation. Although meagre fails to

undergo oocyte maturation spontaneously in confinement, recently developed

hormonal induction methods have proven effective in controlling spawning (Mylonas

et al., 2011; Duncan et al., 2012) and producing eggs of sufficient quality and

quantity for commercial hatchery production. Larvae and juveniles have been reared

with similar facilities and methodologies used for other marine fish species (Pousão-

Ferreira et al., 2013; Soares et al., 2015). This large size fish offer excellent

opportunities for processing such as fillets, loins, slices, etc. all items increasingly

demanded by the European market (Saavedra et al., 2015).

However, this species is generally unknown to fish consumers, apart from some local

areas, where it is landed from capture fisheries, western France, southern Portugal,

southern Spain and Egypt. So producers have to work out the marketing for the

successful introduction of this species on the market. During the last few years

market showed signs of saturation for this species, prices have been low and the

sector exhibited severe signs of crisis (Cardia and Lovatelli, 2007). Though culture of

new species has been suggested as a basic mean to overcome the problem of

Mediterranean aquaculture (Cardia and Lovatelli, 2007), and although meagre was



5

one of the most suitable candidate species, this apparently did not yet fulfil

consumers requirements.

The production of meagre is intensive, conducted both in land-based tanks and

cages. Production facilities are few and mainly distributed in southern France

(Camargue, Cannes, and Corsica) and Italy (La Spezia and Orbetello). In land-based

farms production is mainly achieved in circular or rectangular tanks with a water

depth of 1 meter and a volume of 500 m³, usually covered with PVC. Nowadays

meagre is mainly farmed in the sea, using circular or square surface cages of 500-

1000 m³. More recently, submerged cages have also successfully been used; these

2 000 m³ cages are submerged at 10-20 m depth, and an average stocking density

of 10-15/m³ is used (Monfort, 2010).

Commercial feed is supplied by all major aquafeed producers. Meagre feed is similar

to that used for other Mediterranean marine species. An extruded feed with 45-48

percent protein and up to 20-24 percent lipid is used. In land-based farms fish are fed

2 to 3 meals per day and in sea cages a single daily meal is often provided (Monfort,

2010). Little information exists about the dietary nutrient requirements that can be

used for the formulation of specialized diets for this species (Chatzifotis et al., 2012).

Chatzifotis et al. (2010), evaluated the effect of dietary lipid levels on growth and feed

utilization of meagre. Overall, results indicated that the best growth performance was

observed in fish fed with a diet including 17% lipids, whereas the increase of dietary

lipid level from 17% to 21% had a negative effect on growth.

Moedo et al. (2011) indicated that 60% of crude protein was the most suitable protein

level for meagre juveniles. Allied to these results the analysis of the proximal

composition of the whole body revealed that fish fed this diet had lower lipid content

and the highest percentage of protein content.

The mean market price of meagre coming from captured fisheries is between EUR 7-

12/kg and has increased since 1999. Now, the cost of production for meagre is

similar or lower to the cost of production of European seabass and gilthead

seabream, estimated to be approximately 3.9 kg (Monfort, 2010). Thus, from an

economical perspective, meagre still seems to be a promising aquaculture species.

1.4- Fish Nutrition For several years, the main concern in Nutrition studies was to accomplish the

minimal nutrients necessary to achieve good growth in different species of fish (NRC,

2011). However, nowadays, the role of nutrition is much more embracing than

designing fish performance trials, select appropriate diet formulations and feeding



6

levels. Nutrition plays an important role in aquatic environments by increasing

nutrient retention as well as in reproduction, fish health and welfare, or the capacity

of fish to respond to stressors and pathogens (Lall et al., 2000). Nutrition combines

efforts of various scientific areas, such as physiology, genetics, pathology, molecular

biology, which can contribute importantly for developing new knowledge in

aquaculture (Hardy, 1999).

The intensification of the production systems and technology was necessary for

aquaculture development. This was essentially a result of the formulation of new

diets that covered all nutritional requirements of the studied species (Allan et al.,

2006). As any other animal producing industry, aquaculture is focused on maximizing

growth rate and minimizing production costs: a rapid growth rate minimizes the time

to achieve the marketable size of the animals while decreasing production costs.

However, despite all technologic advances aquaculture’s future is threatened as far

the production of fishmeal and fish oil remains stagnant. Being highly dependent on

nutrient inputs, aquaculture main challenges for the next years must focus on the

development of new alternative ingredients to feed cultivated species (Lane et al.,

2014).

Important ingredients like fishmeal and fish oil are hugely produced worldwide,

approximately 6 million and 1 million tonnes respectively are produced annually

(figure 3) (Tacon, 2003). Even so, these ingredients cannot sustain the continued

increase in demanding from aquaculture products. Understanding the nutritional

requirements of aquaculture target species and exploring new ingredients and their

capacity to fulfil the needs of the species is imperative (Allan et al., 2006). Fish

growth and overall production are influenced by many factors, with feeding the fish

occupying the first place in the running costs of production. The most expensive

nutrient is protein and therefore, it is necessary that diets be formulated with

optimized quantities of protein that meet but not exceed fish protein needs, for

reducing feed costs (Rahimnejad et al., 2015). In aquaculture, the objective is to

provide species with the quantities of protein necessary to satisfy the requirements of

the fish in essential amino acids for a maximum performance and growth (Chi et al.,

2010). Excess of dietary protein can lead to higher excretion of ammonia by the fish,

as well as making the diets more expensive than needed. As a consequence of

increase levels of ammonia, the water quality gets poorer (Rahimnejad et al., 2015).

Diet efficiency is one of the central issues in fish nutrition and this factor is very

influence by the ratio protein/energy, as well as, the specie or life stage of the fish. If



7

the protein is in excess or deficient in a diet, it will either be used as an energy

source or have a negative influence in growth (NRC, 2011)

It has been demonstrated that protein utilization efficiency can be improved by using

lipids and carbohydrates for sparing protein for energy purposes (Moedo et al., 2011;

Cho and Kaushik, 1990; Kaushik and Medale, 1994). In this regard, lipids are a more

efficient source of energy than carbohydrates because lipids are readily metabolized

by fish, especially by carnivorous species (NRC, 2011).

Figure 3: Global production of fishmeal and fish oil. Source: FAO, 2014 1.5- Lipids Lipids are generally described as hydrophobic compounds with high solubility in

organic solvents, and usually containing fatty acids covalently bond to glycerol

molecules or amino groups, forming glycerides in the first case and sphingolipids in

the latter (Tocher, 2003). Lipids include, as major groups, triacylglycerols (TAG), wax

esters, sterols and phospholipids (PL). As for most vertebrates, lipids also constitute

a great source of energy for fish and participate in the growth, reproduction and other

important metabolic functions. They are important constituents of biological

membranes, precursors of many hormones and signaling molecules like eicosanoids

and others. The term 'eicosanoids' is used to denote a group of oxygenated, twenty

carbon fatty acids, and the pathways leading to it are known collectively as the

'arachidonate cascade'. Almost all tissues produce eicosanoids, and they have a

wide range of physiological actions in blood clotting, immune and inflammatory

responses, cardiovascular tone, renal and neural functions, and reproduction

(Schmitz and Ecker, 2008).

Lipids are also a source of essential fatty acids (EFA), like n-3 and n-6

polyunsaturated fatty acids that cannot be synthesized de novo in animal cells and



8

must therefore be obtained from the diet (Turchini et al., 2009). In fish diets, n-3

PUFA are mainly provided by fish oils, which contain highly levels of these EFA. Of

all animals currently farmed, fish are the richest source of n-3 PUFA, which has

fundamental implications for human nutrition (Huang et al., 2007).

About 15-30% of fish diets are lipids, and the increase of dietary lipids contributes to

reducing diet costs, by diminishing protein content (Craig and Helfrich, 2009), and

maximizing protein retention and fish performance (Cho and Kaushik, 1990). An

increase of lipids in the diet can however lead to higher body fat deposition

(Chatzifotis et al., 2010; Luo et al., 2005), and induce metabolic alterations, including

fatty liver (Dos Santos et al., 1993), abnormal oxidative status (Rueda-Jasso et al.,

2004), impairment of nutritional value and transformation yield, or affect organoleptic

and physical properties of the fillets (Austreng and Krogdahl, 1987; Gjedrem, 1997;

Hillestad et al. 1998) which may reduce its commercial value (Martino et al., 2002).

So it is fundamental knowing the optimal levels of dietary lipids that promotes

maximum efficiency of fish growth, development and flesh quality (Wang et al.,

2005).

1.6- Fatty acids Fatty acids (FA) are hydrocarbon chains of varying length with one end of the chain

terminated by a methyl group and the other end by a reactive carboxyl group. The

hydrocarbon chain can be saturated or unsaturated. Unsaturated fatty acid contain

double bonds between pairs of adjacent carbon atoms and can be classified

depending on the number of double bonds as monounsaturated (MUFAs), which

contain just one double bond, and polyunsaturated that contain more than one

double bond. FA are the major constituent of triacylglycerides and phospholipids but

can be found either as free molecules, although at small amounts.

Generally, there are only two essential fatty acids. These are linoleic and α-linolenic

acids that can be elongated and desaturated by the animals to the functional FA.

However, marine fish can only met their FA requirements with highly unsaturated

fatty acids (HUFAs) of the n-3 series: eicosapentaenoic (EPA; 20:5(n-3)) and

docosahexaenoic (DHA; 22:6(n-3)), as they lack or have a very low activity of 5-

desaturase, thus preventing the elongation and desaturation of the precursor α-

linolenic acid (figure 4) (Owen et al., 1975; Watanabe, 1982; Sargent et al., 1989).

The spatial conformation of DHA is different from that of EPA as a result of its carbon

backbone length and degree of unsaturation, so EPA has 20 carbon atoms and 5



9

double bonds (20:5) and DHA has a longer chain, 22 carbon atoms and 6 double

bonds (22:6) (Gorjão et al., 2009).

Figure 4: The metabolic pathways by which essential fatty acids are converted to longer chain, more unsaturated derivatives. Source: Narayan et al., 2015

These two HUFAs play diverse roles in cells, controlling and regulating growth

performance, survival, stress resistance, cell membrane fluidity, immune function,

nervous system development, vision and pigmentation (Bell et al., 1986)

Recent studies have also shown that n-3 long chain PUFA decrease fat

accumulation, increase fatty acid β-oxidation capacity and modulate the expression

of lipid metabolism-related genes, which are essential for lipid synthesis and play key

roles in the catabolism and storage of fatty acids (Kjær et al., 2008; Todorčević et al.,

2009; Ji et al., 2011).

On the other hand, HUFAs are more susceptible to peroxidation and trigger much

more deleterious effects than other dietary fatty acids do. So, high levels (about 5%

dry weight basis) of dietary HUFAs were reported to cause poor growth, low feed

conversion (Ibeas et al., 2000), impaired mitochondrial function and β-oxidation

activity in grass carp, rainbow trout (Oncorhynchus mykiss) and severe oxidative

stress in Atlantic salmon (Du et al., 2006, 2008; Kjær et al., 2008; Østbye et al.,

2009; Todorčević et al., 2009).



10

EPA, DHA and ARA, are precursors for the synthesis of bioactive lipid mediators,

eicosanoids, that include prostaglandins, leukotrienes, lipoxins and thromboxanes.

The principal substrate is arachidonic acid, but both eicosapentaenoic acid and

docosahexaenoic acid are also important substrates (Henderson and Sargent, 1985).

There are three major pathways within the eicosanoid cascade, including the

cyclooxygenase, lipoxygenase, and epoxygenase pathways (figure 5). ARA and EPA

compete for the cyclooxygenases and lipoxygenases that produce, respectively, 2-

series prostanoids and 4-series leukotrienes from ARA, and 3-series prostanoids and

5-series leukotrienes from EPA. Eicosanoids produced from ARA are generally more

biologically active than those produced from EPA and the respective eicosanoids

compete for the same cell membrane receptors. Wherefore ARA produces more

potent inflammatory and pro-aggregatory eicosanoids while EPA has an antagonist

response (Tocher, 2010).

Figure 5: Pathways of conversion of araquidonic acid 20:4 (n-6) and eicosapentaenoic acid 20:5 (n-3) to eicosanoids. Souce: Lall, 2000

Therefore, adequate EPA:ARA ratios are important because one of these EFAs can

regulate the efficacy of the other (Izquierdo and Koven, 2011; Koven et al., 2001).

The EFA requirements of fish vary both qualitatively and quantitatively, and the

optimal level of for marine fish is known to be around 0.5-1% of dry matter for EPA



11

and DHA (NRC, 2011).

Thus, several studies have been made to evaluate the ideal proportion of EFA in the

diet. Some studies with larvae of red and gilthead seabream and juvenile striped

jack, demonstrated that including more DHA promoted growth, feed efficiency and

vitality more effectively than EPA (Watanabe et al., 1989; Izquierdo et al., 1989;

Takeuchi et al., 1990; Rodriguez, 1994). Besides, elevated levels of dietary EPA

relative to DHA were shown to have a negative impact on larval neural function,

growth and survival (Copeman et al. 2002).

Given that DHA is naturally found at very high levels in the neural tissue, it is thought

to play a critical role in neural membrane structure and function, including visual

acuity for optimum hunting success (Copeman et al., 2002) and escape swimming

with improved higher burst speed (Benítez-Santana et al., 2014).

In contrast, fresh water fish like rainbow trout, Oncorhynchus mykiss, that has an

enzymatic system of desaturases and elongases very active are able to

biosynthesize EPA and DHA so diets containing similar levels of EPA or DHA

demonstrated that growth was independent of dietary type of n-3 HUFA; however a

synergistic effect on growth and feed efficiency was observed when these acids were

combined (Takeuchi and Watanabe, 1977). So, it is important to continue studies in

this area to better understand the complex interactions between EFA.

1.7- Phospholipids Berg et al. (2002) refer that the three principal lipids present in the membranes are

phospholipids, glycolipids and cholesterol, being phospholipids the most abundant.

Similarly to other animals, the biomembranes of fish tissues contain

phosphatidylcholine as the major phospholipid followed by

phosphatidylethanolamine, and minor components like phosphatidylserine,

phosphatidylinositol, cardiolipin, and sphingomyelin.

Phospholipids are composed by fatty acids, a group were fatty acids are attached,

usually glycerol, a phosphate, and an alcohol connected to the phosphate. The fatty

acids create a hydrophobic barrier, while the other components of the phospholipids

(the hydrophilic group) are able to keep an interaction with the environment (Berg et

al 2002).

Phospholipids have PUFA preferentially located on the sn-2 position of their glycerol

backbone. Saturated and monounsaturated fatty acids are preferentially located on

the sn-1 position of phospholipids and the sn-1 and sn-3 positions of triacylglycerols.

Fish phospholipids characteristically contain a ratio of n-3/n-6 PUFA of 10-15:1



12

(Ackman, 1980).

Tocher et al. (2008) referred the important role of PL in the structure and function of

biomembranes, in digestion and intestinal absorption of different components of the

diet such as long chain fatty acids, proteins, vitamins and minerals. Also, PL

contribute to optimize fish growth and survival, and decrease some deformities in

different ages and types of fish.

The optimization of the quantity of PL in a diet may enhance lipid deposition,

increasing the energy available for growth and ovaries development (Kanazawa et

al., 1985; Teshima et al., 1986; Alava et al., 1993; Cahu et al., 1994; Kontara et al.,

1997; Gong et al., 2000; Gonzalez- Felix et al., 2002). Furthermore, PL may exert

beneficial effects by providing essential nutrients, e.g. essential fatty acids,

phosphorous, choline and inositol (Tocher, 1995; Lall, 2002; Tocher et al., 2008). In

previous studies, King et al. (1992) showed that phospholipids also have an

antioxidant property, as well as feed-attractant properties (Harada, 1987).

It has been suggested that fish at larval stages, are not capable of synthesizing PL at

a sufficient rate to meet the requirements for cell formation and its components

during the initially period of larval growth (Izquierdo and Koven, 2011; Kanazawa,

1993; Geurden et al., 1997a). Due to this suggestion, several fish larvae receive an

abundance of phospholipids in their natural diets, whether from yolk sac lipids or

from natural prey (Tocher et al., 2008).

Recommended requirements for PL ranged from 8% to 12% dry diet in larval fish

(Cahu et al., 2009). Although the PL requirement generally decreases with age or

developmental stage, decrease to around 2 to 4% for juvenile (Tocher et al, 2008).

Requirement for dietary phospholipids has not been established for adult fish. There

is some confusion over whether excessive levels of dietary phospholipids can be

detrimental to fish and crustaceans. Increasing the dietary PL level beyond the

required level did not affect survival or growth in various studies (Conklin et al., 1980;

Kanazawa, 1993; Geurden et al., 1995b).

1.8- Oxidative Stress Generation of reactive oxygen species (ROS) is an unavoidable consequence in

aerobic organisms. ROS are naturally produced during oxidative metabolism and

include superoxide anions (O2−), hydrogen peroxide (H2O2), hydroxyl radicals (HO·),

and singlet oxygen (1O2) (Livingstone, 2001).



13

In normal situations, a balance exists between the production of ROS and antioxidant

processes, but if the balance is not perfect increased levels of oxidative damage can

occur in organisms and increase lipid peroxidation (LPO), protein oxidation, and DNA

damage that can deleteriously affect cell viability by causing membrane damage and

enzyme inactivation (Livingstone, 2001). To maintain homeostasis and prevent

oxidative stress, living organisms have evolved antioxidant defense mechanisms that

include both enzymatic and non-enzymatic components. The main enzymes with

antioxidant activity are: superoxide dismutase (SOD), which catabolizes the

dismutation of superoxide radicals to molecular oxygen and hydrogen peroxide;

catalase (CAT), which degrades hydrogen peroxide into molecular oxygen and

water; and glutathione peroxidase (GPX), which reduces both hydrogen peroxide

and organic peroxides to water and corresponding alcohols, respectively, by a

glutathione-dependent reaction (Storey, 1996; Morales et al., 2004; Pérez-Jiménez

et al., 2009). Other enzymes, such glutathione reductase (GR) and the rate-limiting

enzyme of the pentose phosphate pathway, glucose-6-phosphate dehydrogenase

(G6PDH), are considered to play a crucial role on the modulation of the activity of the

two H2O2 scavenging pathways of cells (Mourente et al., 2002; Morales et al., 2004).

GR, at the expense of NADPH, regenerates the reduced glutathione (GSH),

substrate for GPX from oxidized glutathione (GSSG); and G6PDH generates the

NADPH that is crucial for the normal functioning of the antioxidant enzymes (CAT,

GPX and GR) (Storey, 1996; Morales et al., 2004).

Several circumstances can interact with the antioxidant defense response in fish.

Factors intrinsic to the fish itself, such as age, phylogenetic position, and feeding

behavior, as well as environmental factors such as the type of diet supplied, daily or

seasonal changes in temperature, dissolved oxygen, toxins present in the water,

pathologies, or parasites, can either fortify or reduce antioxidant defenses (Felton,

1995; Martínez-Álvarez et al., 2005).

PUFA are particularly susceptible to lipid peroxidation (Halliwell and Chirico, 1993),

due to the high number of double bonds. Fish tissues and commercial diets for

cultured fish often contain high levels of PUFA (Stephan, Guillaume and Lamour

1995), and increasing dietary n-3 PUFA can elevate the concentrations of these FA

in fish tissues. Lipid peroxidation can be a major contributor to the loss of cell

function through cellular membrane disruption, by the decrease of membrane fluidity

and activation of calcium-dependent proteases and lipases that lead to an increase

of membrane permeability (Hermes-Lima et al. 1995).



14

To determine the oxidative stress levels in fish it is usually measured the antioxidant

enzymes activities and malondialdehyde (MDA) or levels of end-product of lipid

oxidation, such as thiobarbituric reactive species (TBARS) (Tsangaris et al., 2011).

These biomarkers are however non-specific and non-reliable indicators of oxidative

stress (Knight et al., 1988).

Most of the studies on oxidative stress have been conduced mostly in humans and

rodents. Some reports suggested that 1% EPA + DHA in the diet could lead to

oxidative damage in rats, showing higher thiobarbituric acid–reactive substance

levels and reduced superoxide dismutase activity and glutathione levels (Park et al.,

2009). On the other hand, Popović (2013) showed opposite results; after treatment of

aged male rats with fish oil obtained an increase of SOD, CAT activities and

decreased lipid peroxidation

In fish species like juvenile of grass carp, Ctenopharyngodon idellus, SOD activity

increased significantly with increasing dietary HUFA content, which is consistent with

the level of MDA (Ji et al., 2011) and in Atlantic salmon (Salmo salar) SOD activity

also increased with more dietary EPA and DHA. Dietary PL supplement also

significantly increased SOD activity and reduced the activities of catalase and

glutathione peroxidase in the whole body of Misgurnus anguillicaudatus larvae (Gao

et al., 2014).

1.9- Objectives

As mentioned above, meagre is a species with high potential for aquaculture, but it is

still necessary to improve knowledge on meagre nutrition to enhance fish

performance.

Thus, this study aimed to determine the growth performance and body composition

of meagre fed four diets with different proportions of phospholipids and DHA.

Moreover, the effect of the dietary treatments on fatty acid and lipid class

composition of the muscle, liver and brain was also evaluated, as well as dietary

effects on hepatic oxidative damage and antioxidant mechanisms.

The overall objective of this study was to address two key issues: if the increase of

phospholipids promotes growth performance and if increased levels of DHA in the

diet promotes increased deposition of DHA in muscle.



15

2- Material and methods

2.1- Diets

Four experimental diets (A, B, C and D) were formulated to contain two different

proportions of DHA (1.0 and 2.0%) and phospholipids (1.0 and 4.0%). The

percentage of protein, lipids and starch were constant in all diets.

The variation in the content of phospholipids was achieved by altering the

concentration of lecithin in the diet, and the variation of the DHA was made by

changing the concentration of tuna and fish oil. The experimental diets used in this

work were formulated by SPAROS Lda. (Olhão, Portugal).

All ingredients and theoretical composition of the different diets are presented in

table 1. Additionally, analyzed concentration of lipids and lipid composition are shown

in table 2.



16

Table 1: Formulation and proximal chemical composition of the experimental diets.

Ingredients A B C D % % % % Fishmeal 70 LT 15,000 15,000 15,000 15,000 Fish protein concentrate 5,000 5,000 5,000 5,000 Squid meal 7,000 7,000 7,000 7,000 Krill meal 6,000 6,000 6,000 6,000 Feathermeal hydrolisate 4,000 4,000 4,000 4,000 Porcine blood meal 2,000 2,000 2,000 2,000 Poultry meal 65 9,500 9,500 9,500 9,500 Soy protein concentrate 10,000 10,000 10,000 10,000 Wheat Gluten 10,500 10,500 10,500 10,500 Corn gluten 10,000 10,000 10,000 10,000 Gelatinized pea starch 6,000 6,000 6,000 6,000 Fish oil 6,800 0,000 4,700 1,000 Tuna oil 1,800 10,600 0,000 2,500 Algatrium DHA70 0,000 0,000 0,400 1,600 Krill oil 0,000 0,000 2,500 2,500 Soybean oil 2,000 0,000 0,000 0,000 Soy lecithin 0,000 0,000 3,000 3,000 Vitamin & Mineral Premix 1,000 1,000 1,000 1,000 Binder 1,400 1,400 1,400 1,400 Antioxidants & Preservative 0,500 0,500 0,500 0,500 MCP 1,500 1,500 1,500 1,500 Total 100,000 100,000 100,000 100,000 Theoretical (as fed basis) A B C D Crude protein 56,4 56,4 56,4 56,4 Crude fat 16,5 16,5 16,4 16,4 Fiber 0,6 0,6 0,6 0,6 Starch 3,6 3,6 3,6 3,6 Ash 6,0 6,0 6,0 6,0 Gross Energy 19,1 19,1 19,0 19,0 Lysine 2,9 2,9 2,9 2,9 Methionine + Cystine 1,8 1,8 1,8 1,8 Total Phosphorus 1,0 1,0 1,0 1,0 Calcium 1,1 1,1 1,1 1,1 Eicosapentaenoic Acid (EPA) 1,0 1,0 1,1 1,1 Docosahexaenoic Acid (DHA) 1,0 2,0 1,0 2,1 Total Phospholipids (PL) 1,0 1,0 4,0 4,0



17

2.2- Experimental conditions

This experiment was conducted for 8 weeks, beginning on September 15th until

November 13th, in the facilities of Instituto Português do Mar e da Atmosfera (IPMA),

Estação Piloto de Piscicultura de Olhão (EPPO), Portugal with juveniles of meagre

Argyrosomus regius bred in captivity at IPMA.

A total of 1800 juveniles from the same cohort were randomly assigned to 12

fiberglass tanks (150 juveniles per tank) with 1500 liters of capacity. An open

circulation system was established with water previously filtered and pumped from

Ria Formosa.

Diets were tested in triplicate tanks with fish sorted manually to have an initial weight

of 13.8± 2.0g. Previously to the start of the trial fish were acclimatized for 2 weeks in

order to adapt to the experimental conditions.

During the experiment photoperiod was maintained at 14 hours light: 10h dark,

dissolved oxygen between 3 to 6 mg/l, salinity ranged from 34 to 36 psu and water

temperature between 19 to 24ºC. A heater assured that water temperature did not

decrease from 19ºC to assure that fish ingestion were adequate.

Tanks maintenance was done daily, and included the monitoring of water

temperature and dissolved oxygen. Regularly, the flow rate of each tank was also

verified to maintain a water renovation rate of 60% per hour.

The meagre were fed by hand, 3 times a day, to apparent satiety at 9h, 13h and 17h,

seven days a week.

2.3- Sampling

During the trial fish were sampled at 3 times: at the start of the experiment, at the fifth

week, and the end of the experiment.

In the first sampling, 31 fish from the stock population were weighed, measured and

any deformations registered. Six of these fish were frozen for proximal analysis of the

carcass. Samples of muscle, brain and liver from another six fish, were collected for

fatty acid and lipid class determination. Liver weight was also measured to calculate

the hepatosomatic index.

In the second sampling, 31 fish from each tank were measured and weighed and any

deformations registered. The remaining fish of each tank were weighed in groups of

10 fish.



18

At the end of the trial, all fish from each tank were weighed, measured and any

deformations registered. Six fish from each tank were immediately frozen for whole

body composition analysis. Two pools of four other fish per thank were sampled for

liver, brain and muscle tissues for lipid class and fatty acid analysis. A portion of liver

was also used for oxidative stress analysis. All livers were weighted for determination

of the hepatosomatic index.

Before sampling fish were starved (for 4h) and lightly anaesthetised (ethylene glycol

monophenyl ether, 0.3 ml l-1). Immediately after collection, all tissue samples were

placed in liquid nitrogen and then frozen at -80ºC until analysed.

2.4- Analytical Methods

2.4.1- Zootechnical parameters

All data referring to the growth performance, nutrient retention and physiological

status of meagre were evaluated based on the following formulas:

• Specific growth rate (SGR): 100 × (ln FBW- ln IBW))/ Nº of days

• Feed conversion rate (FCR): Total dry feed intake (g)/ Wet weight gain (g)

• Voluntary feed intake (VFI): (Total dry feed intake (g)/ ((Initial biomass (g) +

Final biomass (g))/2) × 100)/ Nº of days)

• Protein efficiency rate (PER): Wet weight gain (g)/ Protein intake (g)

• Nutrient Retention (% nutrient intake): Total dry nutrient gain (g)/ Total dry

nutrient intake (g)

• Energy Retention (% intake): Total energy gain (kJ)/ Total energy intake (kJ)

• Hepatosomatic index (HSI): (Liver wet weight (g)/ whole-body wet weight (g))

× 100

• Condition factor (K): (Body weight (g)/ Total body length (cm)) × 1000)

2.4.2- Proximate analysis

Frozen fish carcasses were transferred to the facilities of Faculdade de Ciências da

Universidade do Porto (FCUP) for analysis. All proximal analyses were performed in

duplicate.



19

Before analyses, fish were dried at 70 ºC and then ground and stored until posterior

analysis. Dry matter (DM) was determined by drying the samples at 105°C until

constant weight; ash by incineration in a muffle furnace at 450 °C for 16 h; protein

content (N×6.25) by the Kjeldahl method after acid digestion using Kjeltec digestion

and distillation units (Tecator Systems, Höganäs, Sweden; model 1015 and 1026,

respectively); lipid by petroleum ether extraction (Soxtec HT System) and gross

energy by direct combustion in an adiabatic bomb calorimeter (PARR Instruments,

Moline, IL, USA; PARR model 1261).

2.4.3- Tissue lipids

The samples were frozen in dry ice and carried out to the IPMA facilities at Lisboa for

analyses. Samples were freeze-dried and then grinded. Two pools of each sample

tissue were obtained and analysed in triplicate.

2.4.3.1- Lipid classes

Total lipids were extracted from 1 g of the hogenized tissue, using chloroform:

methanol (2:1 v:v), according to Folch et al. (1957). Solvent was evaporated under

nitrogen flushing to quantify the total lipids extracted from the samples. Lipids were

re-dissolved at a concentration of 10 mg/ml of chloroform for determining lipid class

composition.

The lipid classes were classified through thin-layer chromatography (TLC) by the

method of Olsen and Henderson (1989). Total lipid samples (10 µl) were applied in

20×20 cm TLC silica gel plates (VWR, Lutterworth, UK) and running in a solvent

mixture comprising n-hexane, diethyl ether and formic acid (50:50:2, by vol.). Excess

solvent was evaporated via air drying and vacuum desiccation. Lipid classes were

visualised by spraying with solution of phosphomolybdic acid (10%) in ethanol and

charring plates at 160ºC for 20min. The relative percentage of the lipid classes were

quantified by densitometry (GS-800 Calibrated Densitometer) with a software

program from Quantity One 4.5.2 (PDI).

2.4.3.2- Fatty-acids

The fatty acid profile was determined according to Lepage and Roy (1986) modified

by Cohen et al. (1988). This method consists in the transesterification, in acidic

medium, of fatty acid methyl esters (FAME). The fatty acids were transesterified



20

using a mixture of acetylchloride and methanol (1:19, v/v) and 10 mg/ml of

tricosanoic acid (23:0), as internal standard. FAME were analyse throw injection of 2

µl into a gas chromatograph (GC; Varian Star CP-3800) equipped with an auto

sampler and a flame ionization detector (FID) at 250ºC. The separation was

performed in a polyethylene glycol capillary column DB-wax (0.25 mm internal

diameter 30 m polar capillary precolumn × 0.25 µM layer thickness) and helium as

carrier gas. Helium was used as carrier gas at a flow rate of 1 mL min-1, to perform

the separation in a capillary column DB-Wax (30 m length × 0.32 mm internal

diameter; 0.25 µm film thickness; Hewlett Packard, Albertville, MN, USA)

programmed at 180 ºC for 5 min, raised to 220 ºC at 4 ºC min-1, and maintained at

220 ºC for 25 min, with the detector and the split injector (100:1) at 250 ºC. FAME

identification was carried out by comparing their retention time with those of Sigma

standards. The quantification of the different fatty acids as a function of its peak area,

and the peak area of internal standard (23: 0) of the heavy mass of the sample and

the total area of fatty acid in the sampleusing Varian software . Fatty acids present

in the sample were identified by comparing the retention time obtained for each one

and that of the standard pattern Sigma-Aldrich (Supelco Analytical). The accuracy of

this methodology was assessed by testing certified reference materials in the same

conditions as the samples, and the results obtained in this study showed to be in

agreement with the certified values.

2.4.4- Oxidative stress

In order to have representative liver samples to perform the enzymatic analysis, 2

pools of 4 livers each per tank were used. Each liver pool was homogenized (dilution

1:4) in ice-cold buffer (100 mM Tris-HCl, 0.1 mM EDTA and 0.1% triton X-100 (v/v),

pH 7.8). All procedures were performed on ice. Homogenates were centrifuged at

30.000g for 30 min at 4°C. After centrifugation, the resultant supernatants were

collected and aliquots were stored at −80°C until analysis.

All antioxidant enzyme assays were carried out at 25°C. The enzymatic reactions

were started by addition of the tissue extract, except for SOD were xanthine oxidase

was used. The specific assay conditions were as follows:

• SOD

Superoxide dismutase (SOD; EC 1.15.1.1) activity was measured

spectrophotochemically at 550 nm by the ferricytochrome c method using

xanthine/xanthine oxidase as the source of superoxide radicals (McCord and



21

Fridovich, 1969). The reaction mixture consisted of 50mM potassium

phosphate buffer (pH 7.8), 0.1 mM EDTA, 0.1 mM xanthine, 0.012 mM

cytochrome C, and 0.025 IU mL−1 xanthine oxidase. Activity was reported as

units per mg of protein. One unit of activity was defined as the amount of

enzyme necessary to produce a 50% inhibition of the ferricytochrome C

reduction rate.

• CAT

Catalase (CAT; EC 1.11.1.6) activity was determined according to the method

of Aebi (1984) by measuring the decrease of hydrogen peroxide

concentration at 240 nm. The reaction mixture contained 50 mM potassium

phosphate buffer (pH 7) and 10 mM H2O2 freshly added.

• GPX

Glutathione peroxidase (GPX; EC 1.11.1.9) activity was assayed as

described by Flohé and Günzler (1984). The glutathione disulfide (GSSG)

generated by GPX was reduced by glutationa redutase (GR), and NADPH

consumption rate was monitored spectrophomotetrically at 340 nm. The

reaction mixture consisted of 50 mM potassium phosphate buffer (pH 7.1), 1

mM EDTA, 3.9 mM GSH (reduced glutathione), 3.9 mM sodium azide, 1 IU

mL−1 glutathione reductase, 0.2 mM NADPH and 0.05 mM H2O2.

• Lipid Peroxidation:

The concentration of thiobarbituric acid reacting substances (TBARS) was

determined as a marker of lipid peroxidation (LPO) following the methodology

described by Buege and Aust (1978). An aliquot of the supernatant from the

homogenate (100 µL) was mixed with 500 µL of a previously prepared

solution containing 15% (w/v) trichloroacetic acid (TCA), 0.375% (w/v)

thiobarbituric acid (TBA), 80% (v/v) hydrochloric acid 0.25 N and 0.01% (w/v)

butylated hydroxytoluene (BHT). The mixture was heated to 100 °C for 15

min. Then, after being cooled at room temperature and centrifuged at 1.500 g

for 10 min, supernatant was removed and absorbance measured at 535 nm.

Concentration was expressed as nanomoles malondialdehyde (MDA) per

gram of tissue, calculated from a calibration curve.

• G6PDH



22

Glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) activity was

assayed as previously described by Morales et al. (1990), using a reaction

mixture containing 50 mM imidazole–HCl buffer (pH 7.4), 5 mM MgCl2, 2 mM

NADP and 1 mM glucose-6-phosphate.

• GR

Glutathione reductase (GR; EC 1.6.4.2) activity was determined

spectrophotometrically at 340 nm by measuring the oxidation of NADPH as

described by Morales et al. (2004). The reaction mixture consisted of 0.1 M

sodium phosphate buffer (pH 7.5), 1 mM EDTA, 0.63 mM NADPH, and 0.16

mM GSSG.

Except for SOD, which units of expression were already described, all enzyme

activities were expressed as units (CAT) or milliunits (GK, HK, PK, FBPase, 21 GDH,

ASAT, ALAT, G6PDH, ME, FAS, GPX and GR) per milligram of hepatic soluble

protein (specific activity).

Protein concentration was determined according to Bradford (1976) using Sigma

protein assay kit and bovine serum albumin as standard. One unit of enzyme activity

was defined as the amount of enzyme required to transform 1 µmol of substrate per

minute under the above assay conditions.

2.5- Statistical analysis Data are expressed as mean ± standard error of the mean (S.E), and all data

analysis was done with the software used SPSS 19.0. Levene and Shapiro-Wilk tests

were performed before analysis to verify whether the data were homogeneous and

were normally distributed, respectively. When values were significant, data was

transformed using ln, √x or arcsin square root . Statistical evaluation of growth trial,

lipid composition and oxidative stress data was done by two-way analysis of variance

(ANOVA). The significance level of 0.05 was used for rejection of the null hypothesis.



23

3- Results

3.1- Diet composition Table 2 presents the fatty acids composition of different diets. All diets had relatively

similar amounts of total SFA, MUFA and PUFA, although diet A had a slightly higher

level of MUFA than diet D (32.2 and 26.8% respectively DW) and the amount of

PUFA was higher in D diet (37.2% DW) than in the other diets.

Lipid classes of the four diets are presented in table 3. TAG is the highest proportion

in all diet, despite its percentage being lower in diets C (63.2% DW) and D (59.8%

DW) than in the other diets. This is due to the diets supplementation with 4.0% PL as

well as the high among of DAG (table 3).

Table 2: Fatty acid composition of the different experimental diets (results in % and mg/g fatty acid)

Fat acid (%) Diet A Diet B Diet C Diet D

Fat acid (mg/g) Diet A Diet B Diet C Diet D

14:00 6.9±0.7 6.1±1.8 7.4±0.2 6.3±0.5

14:00 11.6±2.7 10.2±5.5 12.6±0.4 11.0±1.8

16:00 20.6±0.9 23.5±3.8

22.5±0.1 22.7±0.5

16:00

34.8±6.4 38.8±15.9 38.1±0.5 39.4±3.9

18:00 3.1±0.0 4.2±0.1 2.8±0.0 3.1±0.1

18:00 5.1±0.7 6.8±1.5 4.8±0.0 5.4±0.3

Σ SFA 32.5±1.8 36.7±6.4 34.2±0.

5 34.0±1.3

Σ SFA

54.8±10.5 60.7±24.8 57.9±1.2 59.0±6.5

16:1(n9+n7) 6.5±0.3 6.5±1.2 6.0±0.1 5.3±0.2

16:1(n9+n7) 10.9±2.1 10.8±4.6 10.2±0.2 9.2±1.0

18:1(n9+n7+n5) 17.7±0.2 19.2±0.3

17.4±0.1 17.8±0.2

18:1(n9+n7+n5

29.8±4.2 31.1±7.5 29.5±0.0 30.8±2.0

20:1(n9+n7) 4.0±0.3 1.8±0.4 3.5±0.0 1.9±0.0

20:1(n9+n7) 6.6±0.4 2.8±0.1 6.0±0.0 3.3±0.2

Σ Mufa 32.2±1.2 28.9±2.4

30.7±0.2 26.8±0.5

Σ Mufa

54.1±7.0 46.9±12.8 52.0±0.3 46.5±3.4

18:2n6 14.5±0.2 7.9±0.2

14.4±0.1 14.3±0.2

18:2n6

24.3±3.2 12.9±3.5 24.3±0.1 24.8±1.6

18:3n3 LNA 1.8±0.0 1.0±0.0 1.7±0.0 1.6±0.0

18:3n3 LNA 3.0±0.4 1.6±0.4 2.9±0.1 2.8±0.2

20:4n6 ARA 0.6±0.0 1.3±0.2 0.4±0.0 0.6±0.0

20:4n6 AA 1.0±0.1 2.1±0.2 0.7±0.1 1.1±0.1

20:5n3 EPA 5.3±0.3 5.1±0.8 5.8±0.1 5.6±0.2

20:5n3 EPA 8.9±0.7 8.1±0.8 9.9±0.1 9.6±0.4

22:6n3 DHA 5.8±0.7 12.4±4.2 5.6±0.0 10.4±0.4

22:6n3 DHA 9.6±0.3 19.2±1.7 9.5±0.1 18.0±0.8

Σ Pufa 33.4±1.4 32.6±6.5 33.1±0.

4 37.2±1.0

Σ Pufa

56.3±5.9 52.1±8.7 56.6±0.7 65.3±3.6

DHA/EPA 1.1±1.9 2.4±5.0 1.0±0.0 1.9±1.8

DHA/EPA 1.1±0.4 2.4±2.2 1.0±0.4 1.9±1.9

EPA/ARA 8.8±30.4 3.9±4.4

13.3±3.3

9.1±107.6

AA

8.8±5.5 3.9±3.3 13.3±2.5 9.1±5.1

DHA/LNA 3.2±34.0

12.5±149.7 3.3±0.0 6.5±20.7

DHA/LNA

3.2±0.6 12.0±4.6 3.3±0.6 6.5±3.2

Σ n3 PUFA 16.4±1.1 20.5±5.5

16.7±0.2 20.9±0.7

Σ n3 PUFA

27.8±2.1 32.6±3.5 28.8±0.4 37.1±1.8

Σ n6 PUFA 15.5±0.2 10.2±0.8

15.0±0.2 15.3±0.3

Σ n6 PUFA

26.0±3.3 16.4±4.1 25.4±0.2 26.6±1.7

Ratio n3/n6 1.1±5.4 2.0±7.1 1.1±1.0 1.4±2.8

Ratio n3/n6 1.1±0.6 2.0±0.9 1.1±2.7 1.4±1.0

Σ(Sfa+Mufa+Pufa)

98.4±0.0 98.5±0.2

98.3±0.2 98.5±0.0

Σ(Sfa+Mufa+Pufa)

165.3±23.3

159.6±40.3

166.6±1.3

170.8±13.2

SFA, Saturated Fatty Acids; MUFA, Monounsaturated Fatty Acids; LNA, α-Linolenic acid; ARA, Arachidonic Acid; EPA, Eicosapentaenoic acid; DHA, Docosahexaenoic acid; PUFA, Polyunsaturated Fatty Acids.



24

Table 3: Lipid class of different experimental diets (results in %)

Diet

Lipid Class (%) A B C D

TAG 73.9±1.2 70.6±1.5 63.2±1.5 59.8±3.0

FFA 16.0±0.1 16.8±0.8 18.2±1.0 23.4±1.5

CH 3.4±0.6 4.2±0.6 5.7±1.1 4.2±0.8

DAG 3.7±0.6 4.7±0.9 8.4±0.8 8.0±0.7

MAG 0.8±0.1 1.0±0.2 1.6±0.2 1.7±0.4

PL 2.2±0.0 2.6±0.3 2.9±0.2 2.9±0.4

Mean values and standard error of the mean (±SE) are presented for each parameter (n=3); TAG, Triacylglycerols; FFA, Free fatty acids; CH, Cholesterol; DAG, Diacylglycerol; MAG, Monoacylglycerol; PL, Phospholipids.

3.2- Growth trial Mortality during this experiment was low and it was not affected by diets (Table 4).

The main cause of death was associated to fish jumping out of the tanks.

Average body weight of the fish increased more than fourfold the original weight,

from an initial average body weight of 18.2±2.7g to an average body weight of

96.9±15.6g at the end of the experiment.

Final weight and specific growth rate were significantly affected (P<0.05) by dietary

treatments (Table 4). Final body weight was higher in fish fed the diets with more

phospholipids. In fish fed the diets with higher PL levels, growth further improved with

the increase of dietary DHA. Specific growth rate was higher in fish fed the PL

supplemented diets, but DHA supplementation did not affect SGR. Nevertheless, a

significant interaction between DHA and PL was noticed, due to the higher SGR in

fish fed the diet fortified with PL and the higher DHA level.

There were no differences between groups on voluntary feed intake, however, feed

conversion ratio was affect by dietary phospholipids, with lower FCR being observed

with the diets supplemented with higher amounts of phospholipids. Dietary levels of

DHA did not affect FCR.

The condition factor increased with the increase of dietary phospholipids and the

hepatossomatic index was slightly lower with diet including lower PL and higher DHA

content (Table 4).



25

Table 4: Growth performance, feed utilization, condition and hepatic indices of Argyrosomus regius fed the experimental diet.

Treatment

Analysis of variance

A B C D PL DHA

PL x DHA

Inicial body weight (g) 18.2±2.7 - - - Week 5 body weight (g) 60.2±9.8 60.8±10.4 67.7±12.0 69.4±13.9 * ns ns Final body weight (g) 84.4±0.4 83.2±1.7 105.8±1.1 114.3±1.7 * * *

VFI 2.9±0.2 2.9±0.2 2.4±0.0 2.7±0.6 n.s n.s n.s

SGR 2.7±0.0 2.7±0.0 3.1±0.0 3.2±0.1 * n.s *

FCR 1.3±0.2 1.3±0.1 1.0±0.0 1.1±0.2 * n.s n.s

Mortality (%) 2.0±1.3 2.7±3.1 1.8±1.0 2.0±2.0 n.s n.s n.s

Inicial HSI 2.1±0.3 - - - Final HSI 2.2±0.1 1.8±0.0 2.2±0.0 2.2±0.2 * * *

Inicial CF 11.3±1.0 - - - Final CF 11.5±0.1 11.3±0.5 11.8±0.1 11.8±0.3 * ns *

Mean values and standard error of the mean (±SE) are presented for each parameter (n=3). In the right moiety of the table, results from two way ANOVA are reflected; asterisks indicate significant differences as *P<0.05; n.s. indicates non significant differences.

3.3- Whole-body composition Regarding carcass composition, only protein showed no differences among

treatments at the end of the trial (Table 5).

The dry matter content of the carcasses increased considerably with an increase of

PL in the diet.

Increase of the PL content in the diets, promoted an increase of the whole-body

lipids, which was higher in fish fed the diet with higher level of DHA.

Carcass energy content was not affected by dietary PL or DHA, although a

significant interaction between them was detectable, showing that dietary 4.0% PL

and 2.0% DHA promote higher energy content.



26

Table 5: Whole-body composition (% dry matter) of Argyrosomus regius fed the experimental diet.

Treatment


Inicial A B C D PL DHA

PL x DHA

Dry matter (%) 14.0±0.5 23.2±2.7 22.6±4.2 26.2±2.8 31.4±5.5 * n.s n.s Ash (%) 14.6±0.9 11.7±1.6 14.2±2.7 12.5±2.2 10.9±1.5 n.s n.s * Protein (%) 69.3±2.5 72.1±2.5 74.2±5.4 72.3±6.0 72.8±6.8 n.s n.s n.s Lipids (%) 16,4±0,1 18.4±2.0 14.6±3.6 21.3±5,2 30.9±5.7 * n.s * Energy (KJ/g) 26.4±0.1 33.4±1.8 31.1±1.1 31.8±1.2 35.4±1.6 n.s n.s *


3.4- Lipid composition The fatty acid profile of meagre fed the four diets is shown in tables 6 and 9 for liver,

tables 7 and 10 for brain, and tables 8 and 11 for muscle.

The fatty acid profile was similar to that of the diets in all organs, with special focus to

the brain, that not being a lipid accumulation organ kept a very constant composition.

The brain was the organ that showed the highest levels of DHA (both in % and in

mg/g).

In general, fish fed the diets with higher DHA content (mg/g) had higher deposition of

this fatty acid in all organs. However, high supplementation of PL in the diet led to a

decrease of DHA in the liver.

The concentration of EPA in the tissues (mg/g) was always lower than that of the

diets. An increase of PL in the diet increased EPA retention in the muscle, while in

the liver the deposition of EPA was higher in fish fed diets containing 2.0% of DHA,

but only at the lower dietary PL level.



27

Table 6: Liver fatty acid composition (mg/g fatty acids) of Argyrosomus regius fed the experimental diets.

Liver:

Treatment


Fat acid (mg/g) Initial A B C D PL DHA PL x DHA

14:00 22.5±1.6 18.1±4.7 12.8±1.7 16.9±1.8 12.4±1.0 n.s * n.s

16:00 102.2±9.7 93.2±14.1 83.1±4.9 92.2±7.5 86.9±2.3 n.s * n.s

18:00 21.5±2.4 23.5±1.6 20.1±0.4 25.8±2.4 26.0±0.2 * n.s *

Σ SFA 156.6±14.7 143.0±21.9 126.4±9.1 141.9±12.4 138.8±15.5 n.s n.s n.s

16:1(n9+n7) 53.4±3.5 37.7±7.9 32.6±2.4 32.4±3.1 27.8±1.0 * * n.s

18:1(n9+n7+n5) 74.1±4.7 97.7±10.5 87.9±3.2 89.6±5.1 88.3±0.8 n.s * n.s

20:1(n9+n7) 4.6±0.2 18.1±1.1 7.3±1.7 13.8±0.2 10.1±2.9 n.s * *

Σ Mufa 137.9±8.9 155.9±19.8 129.9±7.5 138.0±8.7 128.3±4.7 * * n.s

18:2n6 25.0±1.4 60.8±10.0 35.4±1.7 51.6±1.9 44.4±10.4 n.s * *

18:3n3 LNA 6.8±0.3 6.4±1.0 3.8±0.2 5.4±0.2 4.8±0.2 n.s * *

20:4n6 ARA 3.8±0.1 2.3±0.2 5.3±0.3 1.8±0.4 2.2±0.4 * * *

20:5n3 EPA 36.7±1.0 15.5±1.4 17.5±1.1 16.7±0.3 16.6±0.1 n.s * *

22:6n3 DHA 38.0±1.7 26.8±2.3 57.7±6.1 26.9±0.8 38.6±2.4 * * *

Σ Pufa 138.4±6.5 135.4±18.1 145.7±11.5 124.9±4.7 130.1±16.4 * * n.s

DHA/EPA 1.0±1.7 1.7±1.6 3.3±5.4 1.6±3.0 2.3±28.0 * * *

EPA/ARA 9.7±13.3 6.7±8.8 3.3±3.6 9.3±0.6 7.7±0.2 n.s n.s n.s

DHA/LNA 5.6±5.4 4.2±2.3 15.2±32.5 5.0±4.0 8.1±10.4 * * *

Σ n3 PUFA 100.1±4.0 61.4±6.4 89.5±8.4 62.5±1.6 73.7±4.2 * * *

Σ n6 PUFA 32.7±1.7 66.4±10.4 46.2±2.6 d 55.5±2.6 49.1±11.2 * * *

Ratio n3/n6 3.1±2.4 0.9±0.6 1.9±3.3 1.1±0.6 1.5±0.4 * * n.s

Σ (Sfa+Mufa+Pufa) 432.9±19.3 434.4±59.7 402.0±28.1 404.8±25.8 397.1±36.7 n.s n.s n.s

Mean values and standard error of the mean (±SE) are presented for each parameter (n=3). In the right moiety of the table, results from two way ANOVA are reflected; asterisks indicate significant differences as *P<0.05; n.s. indicates non significant differences; SFA, Saturated Fatty Acids; MUFA, Monounsaturated Fatty Acids; LNA, α-Linolenic acid; ARA, Arachidonic Acid; EPA, Eicosapentaenoic acid; DHA, Docosahexaenoic acid; PUFA, Polyunsaturated Fatty Acids



28

Table 7: Brain fatty acid composition (mg/g fatty acids) of Argyrosomus regius fed the experimental diets.

Brain:

Treatment



14:00 0.9±0.4 2.4±0.5 1.7±0.6 2.1±0.4 2.0±0.2 n.s * n.s

16:00 37.2±9.8 55.4±2.3 52.2±2.3 51.6±5.4 53.6±1.6 n.s n.s n.s

18:00 26.6±2.5 30.8±0.4 30.8±0.8 30.1±1.8 31.3±0.8 n.s n.s n.s

Σ SFA 68.0±13.4 91.8±3.5 88.2±4.0 86.5±7.8 89.6±2.8 n.s n.s n.s

16:1(n9+n7) 4.7±1.4 10.6±0.7 9.4±0.8 9.1±1.2 9.2±0.4 n.s n.s n.s

18:1(n9+n7+n5) 39.6±6.2 65.2±3.2 63.9±3.5 61.6±6.0 63.6±2.7 n.s n.s n.s

20:1(n9+n7) 1.2±0.2 5.0±0.6 3.4±0.2 3.7±0.2 3.4±0.3 * * *

Σ Mufa 51.2±9.0 82.8±4.9 78.4±4.7 76.2±7.7 77.9±3.8 n.s n.s n.s

18:2n6 2.7±1.0 13.4±2.3 8.5±1.2 9.5±1.1 9.4±0.7 * * *

18:3n3 LNA 0.6±0.5 1.1±0.2 0.7±0.1 0.7±0.1 0.7±0.1 * * *

20:4n6 ARA 3.0±0.3 4.2±0.1 5.1±0.0 3.8±0.1 4.0±0.1 * * *

20:5n3 EPA 7.8±0.3 9.9±0.5 8.8±0.5 10.0±0.7 9.9±0.1 n.s n.s n.s

22:6n3 DHA 40.9±5.0 48.7±1.7 51.8±1.3 50.9±1.4 51.5±1.1 n.s n.s n.s

Σ Pufa 70.4±9.6 101.4±7.3 100.7±4.9 99.7±6.0 100.5±3.3 n.s n.s n.s

DHA/EPA 5.3±14.8 4.9±3.1 5.9±2.8 5.1±2.0 5.2±21.1 n.s * n.s

EPA/ARA 2.6±1.1 2.3±6.2 1.7±22.5 2.6±11.0 2.5±0.4 * * *

DHA/LNA 67.8±10.7 45.1±7.1 75.5±10.8 71.8±15.9 75.3±15.9 * * *

Σ n3 PUFA 63.1±7.7 79.6±4.0 81.9±3.2 83.2±4.2 83.9±1.8 * n.s n.s

Σ n6 PUFA 6.9±1.6 18.8±2.9 15.4±1.5 14.2±1.6 14.2±1.3 * * *

Ratio n3/n6 9.1±4.7 4.2±1.4 5.3±2.1 5.9±2.6 5.9±1.4 * * n.s

Σ (Sfa+Mufa+Pufa) 189.6±19.5 276.0±6.8 267.2±8.9 262.5±16.4 268.0±4.9 n.s n.s n.s




29

Table 8: Muscle fatty acid composition (mg/g fatty acids) of Argyrosomus regius fed the experimental diets.

Muscle:

Treatment



14:00 1.0±0.2 1.8±0.4 1.4±0.3 1.7±0.3 2.6±1.1 n.s n.s *

16:00 7.1±1.3 12.9±1.8 11.7±1.1 12.3±1.4 16.0±4.5 n.s n.s n.s

18:00 2.1±0.3 3.7±0.2 3.6±0.1 3.3±0.3 3.8±0.7 n.s n.s n.s

Σ SFA 10.9±1.8 19.4±2.7 17.8±1.7 18.2±2.0 23.4±6.6 n.s n.s n.s

16:1(n9+n7) 1.7±0.3 2.9±0.6 2.5±0.3 2.5±0.4 3.3±1.2 n.s n.s n.s

18:1(n9+n7+n5) 5.4±0.7 12.0±1.0 10.7±0.3 11.0±1.2 13.4±3.3 n.s n.s n.s

20:1(n9+n7) 0.5±0.0 2.9±1.1 1.0±0.1 1.9±0.1 1.8±0.3 n.s * *

Σ Mufa 8.1±1.1 18.2±2.7 14.5±0.7 15.6±1.7 18.9±4.9 n.s n.s *

18:2n6 1.7±0.2 7.3±0.7 4.0±0.1 7.1±0.7 8.6±2.1 * n.s *

18:3n3 LNA 0.4±0.0 0.7±0.1 0.4±0.1 0.7±0.1 0.8±0.2 * n.s *

20:4n6 ARA 0.6±0.1 0.8±0.1 1.1±0.0 0.6±0.0 0.7±0.1 * * *

20:5n3 EPA 4.5±0.3 3.5±0.3 2.9±0.0 3.7±0.3 4.1±0.8 * n.s n.s

22:6n3 DHA 7.4±0.3 8.5±0.7 10.5±0.5 8.1±0.3 10.5±1.4 n.s * n.s

Σ Pufa 17.0±1.2 23.6±2.4 21.8±1.0 23.2±1.8 28.5±5.5 * n.s *

DHA/EPA 1.7±1.0 2.5±2.7 3.6±10.9 2.2±1.0 2.5±1.7 * * *

EPA/ARA 7.3±6.9 4.4±2.4 2.8±1.1 6.5±11.9 6.3±9.3 * * *

DHA/LNA 16.9±7.6 12.1±6.1 27.4±7.2 11.9±3.5 12.5±6.0 n.s n.s n.s

Σ n3 PUFA 13.9±0.9 14.4±1.4 15.2±0.7 14.6±0.9 18.1±3.0 n.s * n.s

Σ n6 PUFA 2.8±0.3 8.4±0.9 5.6±0.2 7.8±0.8 9.5±2.3 * * *

Ratio n3/n6 5.0±2.9 1.7±1.4 2.7±3.7 1.9±1.1 1.9±1.3 * * * Σ (Sfa+Mufa+Pufa) 36.0±3.7 61.2±7.9 54.1±3.3 57.0±5.6 70.7±17.0 n.s n.s n.s




30

Table 9: Liver fatty acid composition (mg/g fatty acids) of Argyrosomus regius fed the experimental

diets.

Liver:

Treatment


Fat acid (%) Initial A B C D PL DHA PL x DHA

14:00 5,1±0,4 4,0±0,5 3,1±0,2 4,0±0,2 3,0±0,2 n.s * n.s

16:00 23.0±1,1 20,5±0,6 20,2±0,1 22±0,6 21,4±0,4 * n.s n.s

18:00 4,8±0,4 5,2±0,6 4,9±0,3 6,2±0,2 6,4±0,1 * n.s n.s

Σ SFA 35,2±2,1 31,5±2,0 30,5±0,6 33,8±1,2 32,4±0,8 * * n.s

16:1(n9+n7) 12,0±0,3 8,3±0,7 7,9±0,1 7,7±0,3 6,8±0,2 * * n.s

18:1(n9+n7+n5) 16,7±0,5 21,6±0,6 21,3±0,5 21,4±0,4 21,7±0,1 n.s n.s n.s

20:1(n9+n7) 1,0±0,1 4,0±0,3 2,0±0,1 3,3±0,2 2,7±0,3 n.s * *

Σ Mufa 31,0±0,9 34,5±1,6 31,7±0,7 32,9±0,9 31,8±0,6 * * *

18:2n6 5,6±0,1 13,4±0,7 8,5±0,1 12,3±0,3 12,4±0,0 * * *

18:3n3 LNA 1,5±0,0 1,4±0,0 0,9±0,0 1,3±0,0 1,2±0,0 * * *

20:4n6 ARA 0,9±0,0 0,5±0,0 1,3±0,0 0,4±0,1 0,6±0,0 * * *

20:5n3 EPA 8,3±0,6 3,5±0,2 4,2±0,0 4,0±0,2 4,1±0,0 * * *

22:6n3 DHA 8,6±0,8 6,0±0,6 14,0±0,6 6,5±0,3 9,5±0,6 * * *

Σ Pufa 31,2±1,9 30,0±1,8 35,3±0,8 29,9±1,1 33,4±1,0 * * *

DHA/EPA 1,0±1,3 1,7±3,2 3,3±17,3 1,6±2,1 2,3±15,2 * * *

EPA/ARA 9,7±13,5 6,7±4,9 3,3±4,3 9,3±2,2 6,9±2,9 n.s n.s n.s

DHA/LNA 5,6±242,2 4,3±12,5 15,3±79,4 5,0±9,0 7,9±22,8 * * *

Σ n3 PUFA 22,6±1,6 13,7±1,0 21,7±0,7 15,0±0,7 18,2±0,9 * * *

Σ n6 PUFA 7,4±0,1 14,7±0,8 11,2±0,1 13,2±0,5 13,5±0,1 * * *

Ratio n3/n6 3,1±11,2 0,9±1,3 1,9±5,0 1,1±1,4 1,3±12,4 * * n.s

Σ (Sfa+Mufa+Pufa) 97,4±0,2 96,0±5,4 97,4±2,1 96,6±3,3 97,6±2,4 * * n.s Mean values and standard error of the mean (±SE) are presented for each parameter (n=3). In the right moiety of the table, results from two way ANOVA are reflected; asterisks indicate significant differences as *P<0.05; n.s. indicates non significant differences; SFA, Saturated Fatty Acids; MUFA, Monounsaturated Fatty Acids; LNA, α-Linolenic acid; ARA, Arachidonic Acid; EPA, Eicosapentaenoic acid; DHA, Docosahexaenoic acid; PUFA, Polyunsaturated Fatty Acids



31

Table 10: Brain fatty acid composition (% fatty acids) of Argyrosomus regius fed the experimental diets.

Brain:

Treatment



14:00 0,5 0,8±0,2 0,6±0,2 0,7±0,1 0,7±0,1 n.s * n.s

16:00 19,0 19,3±0,7 18,7±0,3 18,6±1,4 19,2±0,3 n.s n.s n.s

18:00 13,7 10,7±0,4 11,1±0,6 10,9±0,4 11,2±0,1 n.s n.s n.s

Σ SFA 34,7 31,9±1,4 31,7±1,1 31,2±2,0 32,7±1,4 n.s n.s n.s

16:1(n9+n7) 2,4 3,7±0,2 3,4±0,2 3,3±0,3 3,3±0,1 * n.s n.s

18:1(n9+n7+n5) 20,3 22,7±0,6 22,9±0,7 22,7±0,9 22,7±0,6 n.s n.s n.s

20:1(n9+n7) 0,6 1,7±0,2 1,2±0,1 1,4±0,0 0,9±0,2 * * n.s

Σ Mufa 26,2 28,8±1,1 28,1±1,0 28,0±1,3 27,5±1,0 n.s n.s n.s

18:2n6 1,4 4,6±0,7 3,1±0,3 3,4±0,3 2,9±0,6 * * *

18:3n3 LNA 0,3 0,4±0,1 0,2±0,0 0,3±0,0 0,2±0,0 * * *

20:4n6 ARA 1,5 1,5±0,0 1,8±0,1 1,4±0,1 1,1±0,3 * n.s *

20:5n3 EPA 4,0 3,4±0,1 3,2±0,1 3,7±0,2 3,1±0,8 n.s * n.s

22:6n3 DHA 21,3 17,0±0,9 18,7±0,3 18,6±1,6 18,5±0,6 n.s n.s n.s

Σ Pufa 36,3 35,3±2,7 36,3±1,3 36,5±2,9 35,7±3,8 n.s n.s n.s

DHA/EPA 5,3 5,0±8,0 6,0±3,9 5,1±8,3 5,9±0,8 n.s n.s n.s

EPA/ARA 2,6 2,3±2,2 1,7±1,0 2,7±2,3 2,8±2,2 n.s * n.s

DHA/LNA 71,2 45,4±11,9 76,6±7,0 72,1±96,0 76,1±27,6 * * *

Σ n3 PUFA 32,7 27,7±1,7 29,6±0,7 30,6±2,3 30,2±2,3 * n.s n.s

Σ n6 PUFA 3,5 6,5±0,9 5,5±0,5 5,1±0,6 4,7±1,3 * * *

Ratio n3/n6 9,2 4,3±1,8 5,4±1,3 6,0±4,1 6,5±1,7 * * n.s

Σ (Sfa+Mufa+Pufa) 97,3 96,0±0,1 96,0±0,1 95,7±0,1 95,9±0,1 n.s n.s n.s




32

Table 11: Muscle fatty acid composition (% of total fatty acids) of Argyrosomus regius fed the experimental diets.

Muscle:

Treatment



14:00 2,7 2,8±0,4 2,4±0,4 2,8±0,2 3,4±0,6 * n.s n.s

16:00 19,3 20,1±1,5 21,0±1,1 20,6±0,6 21,5±1,1 n.s n.s n.s

18:00 5,8 5,9±0,7 6,5±0,5 5,7±0,1 5,2±0,3 * n.s *

Σ SFA 29,4 30,4±3,0 31,9±2,2 30,4±0,9 31,5±2,1 n.s n.s n.s

16:1(n9+n7) 4,6 4,5±0,5 4,5±0,4 4,1±0,2 4,4±0,5 n.s n.s n.s

18:1(n9+n7+n5) 14,6 19,0±0,2 19,4±0,3 18,5±0,3 18,4±0,2 * n.s n.s

20:1(n9+n7) 1,2 3,6±0,6 1,9±0,2 3,1±0,1 2,4±0,1 n.s * *

Σ Mufa 22,0 27,4±1,4 26,1±0,9 26,2±0,7 25,7±0,9 * * n.s

18:2n6 4,6 11,4±0,7 7,2±0,1 11,9±0,2 11,8±0,2 * * *

18:3n3 LNA 1,2 1,1±0,1 0,7±0,1 1,1±0,1 1,1±0,0 * * *

20:4n6 ARA 1,7 1,3±0,2 1,9±0,1 1,0±0,1 0,9±0,1 * * *

20:5n3 EPA 12,1 5,5±0,1 5,3±0,2 6,4±0,1 5,7±0,3 * * n.s

22:6n3 DHA 20,1 13,9±0,3 19,0±0,6 14,2±1,1 14,9±1,5 * * *

Σ Pufa 46,3 37,9±1,9 39,4±1,4 39,6±1,8 39,6±2,4 n.s n.s n.s

DHA/EPA 1,7 2,5±5,0 3,6±3,1 2,2±14,7 2,6±6,1 * * *

EPA/ARA 7,3 4,4±0,4 2,8±1,8 6,5±1,3 6,3±2,7 * * *

DHA/LNA 17,0 12,9±2,9 28,2±4,9 12,4±19,0 13,0±37,4 n.s n.s n.s

Σ n3 PUFA 37,9 23,4±0,7 27,5±1,1 25,2±1,4 25,3±2,1 n.s * *

Σ n6 PUFA 7,5 13,2±1,0 10,1±0,2 13,2±0,3 13,0±0,3 * * *

Ratio n3/n6 5,0 1,8±0,8 2,7±4,7 1,9±4,1 1,9±7,1 * * * Σ (Sfa+Mufa+Pufa) 97,7 95,7±6,3 97,5±4,6 96,2±3,4 96,9±5,4 n.s * *

Mean values and standard error of the mean (±SE) are presented for each parameter (n=3). In the right moiety of the table, results from two way ANOVA are reflected; asterisks indicate significant differences as *P<0.05; n.s. indicates non significant differences; SFA, Saturated Fatty Acids; MUFA, Monounsaturated Fatty Acids; LNA, α-Linolenic acid; ARA, Arachidonic Acid; EPA, Eicosapentaenoic acid; DHA, Docosahexaenoic acid; PUFA, Polyunsaturated Fatty Acids.

The level of ARA (mg/g) was low in all tissues, and it was positively related to the

level of DHA and negatively related to the level of PL in the diets. Diet

supplementation with DHA and PL did not affect deposition of total SFA in the

different organs (mg/g). Total MUFA deposition in the liver (mg/g), decreased with

the ingestion of diets with higher DHA and increased with higher PL dietary content.

As can be seen in figure 6 an increase of dietary PL reduced the PUFA in the liver

and the opposite is true for DHA. However higher dietary PL supplementation

promoted increased PUFA in the muscle and this effect was more preponderant with

the presence of 2.0% DHA in the diet.



33

PL n.s * * DHA * * * PL*DHA * * *

PL n.s * * DHA n.s * * PL*DHA n.s n.s n.s

PL n.s n.s * PL n.s n.s n.s DHA n.s n.s * DHA n.s n.s n.s PL*DHA n.s n.s * PL*DHA n.s n.s n.s

PL * n.s * PL n.s n.s * DHA n.s * * DHA n.s n.s n.s PL*DHA n.s n.s * PL*DHA n.s * * Figure 6: Variation of different fatty acids Eicosapentaenoic acid (EPA), Docosahexaenoic acid (DHA), Arachidonic acid (ARA), Total saturated fatty acid (ΣSFA), Total monounsaturated fatty acid (ΣMUFA) and Total polyunsatureted fatty acid (ΣPUFA) in the liver, brain and muscle (in mg/g) of Argyrosomus regius fed the experimental diets. Results from two way ANOVA are reflected; asterisks indicate significant differences as *P<0.05; n.s. indicates non significant differences.

0,0 10,0 20,0 30,0 40,0 50,0 60,0 70,0

EPA DHA ARA

Liver (mg/g):

A

B

C

D 0,0 30,0 60,0 90,0 120,0 150,0 180,0 210,0

Σ SFA Σ MUFA Σ PUFA

Liver (mg/g):

A

B

C

D

0,0 10,0 20,0 30,0 40,0 50,0 60,0

EPA DHA ARA

Brain (mg/g):

A

B

C

D 0,0 20,0 40,0 60,0 80,0 100,0 120,0


Brain (mg/g):

A

B

C

D

0,0

10,0

20,0

30,0

40,0

50,0

EPA DHA ARA

Muscle (mg/g):

A

B

C

D 0,0

10,0

20,0

30,0

40,0


Muscle (mg/g):

A

B

C

D



34

The lipid class profiles of meagre fed the four different diets is shown in figure 7. PL

is more constant than TAG once these are the major components of cellular

membranes. PL only increased in muscle and liver with the increment of DHA and

decrease of PL in the diets, respectively.

On the other hand, the TAG levels in tissues are highly variable, depending on diet.

TAG is the dominant lipid class in liver and muscle, while in the brain the major

constituent is cholesterol. TAG in the liver and brain were higher in fish fed the diets

with 4.0% PL and decreased with the dietary increase of DHA. In the muscle, TAG

increased with the increase of PL in the diet, and the best TAG deposition was in

meagre feed diets with 4.0% PL and 1.0% DHA content.

FFA in liver and brain showed an opposite behavior; in the liver, higher DHA diet

boost the concentration of FFA in the tissues and a higher PL diet lower it, while in

the brain occurred the opposite. In the muscle, highest concentration of FFA was

observed in fish fed the diet containing less PL and DHA, although lowest

concentration of FFA was obtained in fish fed the diet with lower DHA and higher PL.

In the liver, CH was not affected by dietary composition while in the muscle CH level

decreased with the increase of dietary DHA and PL. In the brain, CH increased with

the increase of dietary DHA and decreased with the increase of dietary PL.

MAG in the muscle increased with as dietary DHA decreased, although it decreased

with more PL in the diet. In the brain, concentration of MAG was higher in fish fed the

diet with less DHA and PL and decreased when fish was fed diets with higher DHA

and less PL.

Fat percentage, shown in table 12, is higher in the liver than in the others organs,

wherein the muscle shows the lowest fat percentage. The amount of fat in the brain

seems not be affected by the diets, presenting constant concentration.

The liver and muscle of the meagre fed the diet with less DHA and PL presented a

severe decrease of fat deposition.



35

Table 12: Lipid class composition (% total of lipid class) and fat deposition (%) of liver, brain and muscle of Argyrosomus regius fed the experimental diets.

Treatment


Liver:

Inicial A B C D Pl DHA Pl x DHA

TAG (%) 76.4±2.7 87.5±1.9 85.6±1.0 89.9±0.6 86.5±2.1 * * n.s

FFA (%) 11.0±2.1 4.1±1.0 5.0±0.5 3.0±0.2 4.8±0.9 * * n.s

CH (%) 8.4±0.5 5.7±1.0 5.7±0.8 4.6±0.4 5.8±1.1 n.s n.s n.s

PL (%) 2.8±0.3 2.6±0.2 3.0±0.4 2.5±0.2 3.0±0.3 n.s * n.s

Fat (%) - 30.7 65.1 62.5 66.3 - - -

Muscle:

Inicial A B C D Pl DHA Pl x DHA

TAG 58.1±3.8 55.8±0.7 71.6±2.8 73.1±1.3 71.7±0.9 * * *

FFA 11.7±1.6 19.0±0.8 8.4±1.1 6.4±0.8 9.5±0.2 * * *

CH 22.5±1.8 16.3±1.1 13.1±1.0 13.9±0.7 12.9±0.6 * * *

MAG 1.7±0.5 3.4±0.3 1.7±0.1 2.0±0.4 1.7±0.2 * * *

PL 6.0±1.1 5.5±1.2 5.2±2.4 4.6±0.8 4.1±0.4 * n.s n.s

Fat (%) - 4.8 7.1 6.3 7.5 - - -

Brain: Inicial A B C D Pl DHA Pl x DHA

TAG - 21.3±2.2 16.6±1.7 31.0±2.1 24.0±1.2 * * n.s

FFA 297±1.2 8.5±1.3 6.0±0.7 9.9±0.8 6.3±0.8 * * n.s

CH 29.7±1.2 51.3±2.5 64.6±2.6 45.2±2.0 53.4±2.6 * * *

MAG 3.4±0.4 4.3±0.6 1.8±0.5 2.2±0.4 2.4±0.1 * * *

PL 10.3±1.3 14.6±1.7 11.0±0.7 11.8±1.6 13.9±2.6 n.s n.s *

Fat (%) - 49.3 46.8 47.7 48.1 - - -

Mean values and standard error of the mean (±SE) are presented for each parameter (n=3). In the right moiety of the table, results from two way ANOVA are reflected; asterisks indicate significant differences as *P<0.05; n.s. indicates non significant differences; TAG, triacylglycerols; FFA, free fatty acids; CH, cholesterol; MAG, Monoacylglycerol; PL, phospholipids.



36

PL * * n.s n.s DHA * * n.s * PL*DHA n.s n.s n.s n.s PL * * * * n.s DHA * * * * n.s PL*DHA n.s n.s * * * PL * * * * * DHA * * * * n.s PL*DHA * * * * n.s Figure 7: Variation of different lipid class, triacylglycerols (TAG); free fatty acids (FFA); cholesterol (CH); Monoacylglycerol (MAG); phospholipids (PL), in the liver, brain and muscle (in %) of Argyrosomus regius fed the experimental diets. Results from two way ANOVA are reflected; asterisks indicate significant differences as *P<0.05; n.s. indicates non significant differences.

0,0

20,0

40,0

60,0

80,0

100,0

TAG FFA CH PL

Liver (%):

A

B

C

D

0,0 20,0 40,0 60,0 80,0

TAG FFA CH MAG PL

Brain (%):

A

B

C

D

0,0

20,0

40,0

60,0

80,0

TAG FFA CH MAG PL

Muscle (%):

A

B

C

D



37

3.5- Oxidative Stress Dietary DHA did not affect oxidative stress enzymes activity. SOD and G6PDH were

the only antioxidant enzymes affected by PL (Table 13). Increase of dietary PL

increased the activity of these two enzymes. TBARS content was not affected by the

increase of PL or DHA in the diet.

Table 13: Activities of glutathione peroxidase (GPX; mU mg−1 protein), superoxide dismutase (SOD), glucose-6-phosphate dehydrogenase (G6PDH), glutathione reductase (GR), and catalase (CAT) (U mg−1 protein), and TBARS content (nmol MDA g tissue−1) of Argyrosomus regius fed the experimental diets

Treatment


Oxidative enzimes A B C D PL DHA

PL x DHA

SOD 314.5±155.3 293.0±138.1 547.4±266.2 549.5±211.6 * ns ns GPX 353.1±70.6 263.1±64.1 359.1±140.3 334.6±51.9 ns ns ns G6PDH 91.6±19.0 81.0±12.7 111.7±9.5 125.3±24.7 * ns ns GR 11.3±0.6 11.2±1.5 9.9±1.7 11.2±1.5 ns ns ns CAT 255.4±23.2 284.4±34.2 271.2±61.1 278.1±41.2 ns ns ns TBARS 5.5±0.8 6.4±1.9 6.2±1.6 5.6±0.6 ns ns ns




38

4- Discussion

Mortality was very low and it was not affected by dietary supplementation with DHA

and PL. Studies in different species and stages of development also showed that

DHA and PL do not promote mortality (Hamza et al., 2008; Wold et al., 2007; Wu et

al., 2011; Coutteau et al., 1996; Wu et al., 2007; Li et al., 2014; Geurden et al., 1997;

Villalta et al., 2005; Morton et al., 2014; Lund et al., 2007; Om et al., 2001). Although

improvements in survival rates have been registered in larvae fed diets

supplemented with PL and n-3 HUFA (Zhao et al., 2013; Kanazawa, 1997; Das et al.,

2007; Kontara et al., 1997; Gao et al., 2014; Rezek et al., 2010; Furuita et al., 1996)

in this study mortality was already very low with all diets.

Data of the present trial indicates that an increase of dietary phospholipids up to

4.0% promoted growth performance, increasing FBW and SGR, and decreased

FCR. Most available literature report a positive effect of dietary PL in improving

growth of larvae and juveniles of several species of fish and crustaceans, such as

dojo loach Misgunnus anguillicaudatus larvae (Gao et al., 2014), European sea bass

Dicentrarchus labrax and turbot Scophthalmus maximus juveniles (Geurden et al.,

1997a), common carp Cyprinus carpio (Geurden et al., 1997b), yellow croaker

Larmichthys crocea larvae (Zhao et al., 2013), juvenile swimming crab Portunus

trituberculatus (Li et al., 2014), Atlantic cod Gadus morhua larvae (Wold et al., 2007),

pikeperch Sander Lucioperca larvae (Hamza et al., 2008), tilapia Oreochronnis

niloticus (Atar et al., 2009), shrimp Litopenaeus vannamei juveniles (Niu et al., 2011)

and blunt snout bream Megalobrama ambblycephala fingerlings (Li et al., 2015).

Therefore supporting that dietary PLs may be required for good physiological

function and development of some fish species due the fact that larvae and juveniles

may have low ability to biosynthesize PL de novo (Kanazawa, 1993; Geurden et al.,

1995). It is also possible that PL are essential for an efficient lipoprotein synthesis,

promoting transport of dietary fatty acids and lipids from the gut to the rest of the

body (NCR, 2011). On the contrary, Gibbs et al. (2009) showed a negative

correlation between dietary PL level and weight gain. So, the role of dietary PL in

larval and early juvenile nutrition are not yet fully understood and more studies are

needed to clarify it.

Our results showed that dietary supplementation with 2.0% DHA did not seem to

affect the performance of meagre. On the contrary, Mourente et al. (1993) observed

that the best growth rate of Sparus aurata larvae was achieved with an high n-3

HUFA content and an high DHA/EPA ratio and Nevejan et al. (2003) showed that



39

dietary supplementation of 20% and 40% lipid emulsion rich in EPA and DHA led to a

significantly better daily growth rate of scallop larvae, Argopecten purpuratus. Rezek

et al. (2010) also showed that growth (body length and body weight) was higher in

larvae of black sea bass, Centropristis striata, fed preys enriched with 10% DHA

against 0% DHA, and Om et al. (2001) showed that fortification with 3% EPA and

DHA increased the weight gain and feed conversion efficiency of juvenile black sea

bream Acanthopagrus schlegeli. Ji et al. (2011) observed in juvenile grass carp,

Ctenopharyngodon idellus, that weight gain, specific growth rate, feed efficiency and

protein efficiency improved by increasing dietary HUFAs content from 0% to 0.52%

and declined thereafter. These results support the hypothesis that HUFA

administration may improve larval development through the presence of better

structured mitochondria, a higher synthesis of energy compounds and coenzymes

with a central position in the metabolism. This higher energy status was confirmed by

better growth performance and a shorter larval phase of clownfish, Amphiprion

ocellaris, fed with a live prey enriched with DHA in relation to control group (non

enriched live prey) (Olivotto et al., 2011).

Similar to our results, Morton et al. (2014); Villalta et al. (2005); Furuita et al. (1996)

and Rodriguez et al. (1998) also observed that there was no significant relationship

between dietary DHA and growth (juvenile barramundi Lates calcarifer, in larvae

senegal sole Solea senegalensis, juvenile red sea bream Pagrus major and larvae of

Sparus aurata), and Lund et al., 2007 also showed that growth rate in common sole

larvae was not related to ARA or dietary EFA combinations.

In the present work we found that there was a significant interaction between dietary

DHA and PL, and the best final weight was achieved with the diet with higher levels

of DHA and PL. This may be due the fact that incorporation more PL in the diet

provided more PL to be used as component of the membranes, which is essential for

rapid growth of juveniles, and more DHA in the diet would improve the incorporation

of DHA in PL membranes. Both DHA and EFA are important precursor of

eicosanoids that can exert anti-inflammatory response thus promoting the heath of

the fish and enhancing the growth of the fish. Results of Félix et al. (2002) are

partially in disagreement with our results, indicating that no significant interaction

exist between PL and DHA on growth of Litopenaeus vannamei juveniles; however,

diets containing both PL and DHA showed significant higher instantaneous growth

rate. In Penaeus japonicus post-larvae growth increased with increasing levels of n-3

HUFA and the addition of soybean phosphatidylcholine to the diet further improved

growth, and no interaction between these two dietary supplements was observed



40

(Kontara, 1997). Although some reports showed a possible interaction between PL

and DHA, in larvae of red sea bream, Pagrus major, the presence of 1-2% DHA and

5% PL improved growth performance (Kanazawa, 1997). So our results also seem to

support the beneficial effects of supplementing diets with PL and DHA to achieve

maximum growth of meagre juveniles.

The HSI is usually used as indicator of the nutritional state of fish once the liver is a

storage organ of glycogen and fat in several species. An increase in HSI was

observed in juveniles fed with the diets with higher PL content, supporting that PL

might improve lipid digestion (NCR, 2011). This may explain the increase of fat

deposition registered in this organ (63.3%). Our results contrast with those made in

juveniles of swimming crab Portunnus trituberculatus that don’t shown any influence

in HSI (Li et al., 2014) and in blunt snout bream Megalobrama amblycephala

fingerlings that had lower HSI in fish fed PL supplemented diets (Li et al., 2015). The

authors suggest that dietary PL could have promoted liver fat metabolism.

Besides, our results showed that diets with more DHA lead to a decrease in HSI

although this decrease was not observed when more PL was added to the diet. Wu

et al. (2007) tested diets with PL and HUFAs and didn’t have significant differences

in HSI in Chinese mitten crab Eriocheir sinensis. Jingying et al. (2014) demonstrated

in juveniles starry flounder Platichthys stellatus that HSI was higher with a DHA/EPA

ratio of 0.64 and it was lower with a DHA/EPA ratio of 1.18, although differences

were not statistically significant.

Proximal composition of meagre in this trial was also analysed, and this factor is

known to be influence by endogenous and exogenous factors. The levels of protein

are primarily related to the size of the fish (endogenous factor), while the lipid content

is associated with exogenous factors such as diet (Shearer, 1994). So this data

showed that protein content of fish carcase was not significantly affected by the

different diets and the lipid levels of the whole-body was affected by the

supplementation of PL and DHA. The same was observed in the energy content of

the carcass, showing that supplementation of 4.0% of PL and 2.0% of DHA in the

diet leads a more energy. Whole-body dry-matter significantly increased only in high

PL diets. This increase is related with increase of whole-body lipids. Data also

showed that diet supplementation with 4.0% of PL increased the CF. This may be

due the fact that fish growth more with more dietary PL.



41

Researchers, in different species, have observed that inclusion of more PL in the diet

increased lipids content (Gao et al., 2014; Zhao et al., 2013; Niu et al., 2011; Atar et

al., 2009). In addition, Zhao et al. (2013) reported an increase of protein in carcass

with more PL. In contrast, Li et al. (2015) showed that while crude protein content

increased with incremental dietary PL levels and the crude lipid content decreased.

Regarding dietary DHA, it seems that an increase of this FA promoted the effect of

PL in the energy and lipid content of the carcass. Mourete et al., 1993 also showed

that dietary DHA promoted an increase of total lipids content. We were unable to find

bibliography about interaction between PL and DHA.

Results of this study indicate that fish fed with more PL in the diet were more

susceptible to oxidative stress, as there was an increase of antioxidant enzymes

activity like SOD and G6PDH. However TBARs content, who is considered a

valuable indicator of oxidative damage of cellular components, was similar in all

treatments. So, fish fed the diet with 4.0% PL needed more antioxidant defenses to

maintain the same state of lipid peroxidation.

Few data exists on the effect of dietary PL supplementation on oxidative stress in

fish. Results of Li et al. (2015) contrast with ours as they show an increase of liver

CAT, SOD and GPX activities with incremental dietary PL level up to 6%, and

TBARS values negatively correlated with the activities of these enzymes. The

authors concluded that more dietary PL might minimize oxidative stress damage.

Gao et al. (2014) showed as well that 6% of PL in the dietary might influence larvae

antioxidant enzymes activities, such as induced of SOD and decrease of CAT and

GPX activities.

Although the susceptibility of each PUFA to oxidation is linearly proportional to the

degree of unsaturation (NCR, 2011), in this work diet supplementation with DHA at 1-

2% didn’t influence hepatic oxidative stress of meagre juveniles. There is one report

that also didn’t observe an increase of oxidative damage with high fish oil intake

(Ando et al., 2000), but the majority of bibliography seems to be in disagreement with

our data. Mourent et al. (2002) showed that a high pro-oxidative stress was induced

by feeding diets containing around 7% of dry weight as n-3 HUFA. In juvenile grass

carp, Ctenopharyngodon idellus the activities of SOD and the amount of TBARS

increased with increasing dietary HUFAs content up to 0.83% (Ji et al., 2011). The

results of Todorčević et al. (2009) indicate an increase of SOD activity in the DHA

and EPA groups showing that high dietary supplementation of DHA and EPA

resulted in oxidative stress. Betancor et al. (2012) showed that a high dietary content



42

of DHA, up to 5% DM, induced higher incidence of muscular lesions and TBARS

content.

The data of the present study are consistent with the well-established fact that the

fatty acid composition of fish tissue reflects that of the diet on which they have been

reared (Hamza et al., 2008; Villalta et al., 2005; Li et al., 2015). However, it is also

evident that fish actively modify dietary fatty acids by selective metabolism to

maintain narrowly defined levels in the membranes, reflecting a homeostatic

relationship in which fish tries to maintain optimal membrane function (Olsen and

Hunderson, 1997). In this study, the brain was the organ that maintained a more

constant composition. The same was observed by Betancor et al. (2014). Also, the

brain was the organ where higher concentrations of DHA were detected, regardless

of the diet. This is in agreement to what had been observed by Mourente (2003).

In the present study, it was shown that higher DHA supplementation in the diet

promoted higher deposition of this FA in the different organs. Similar results were

obtainde in larvae of Senegal sole Solea senegalensis (Villalta et al., 2005); in

shrimp Litopenaeus vannamei juveniles (González-Félix et al., 2002); in juveniles of

barramundi Lates calcarifer. High DHA retention were also observed when fish were

fed diets low in DHA, suggesting high conservation of this FA or possible synthesis of

DHA from EPA (Morton et al., 2014). Om et al. (2001) showed that EPA and DHA in

muscle, liver and adipocytes in juvenile of Acanthopagrus schlegeli were markedly

increased by diet fortification with EPA and DHA, and Ji et al., 2011 demonstrated

that levels of EPA and DHA increased with increasing dietary HUFAs content.

In this work, percentage of DHA in the tissues was higher than in the diet and the

opposite was observed for EPA, results that are in accordance with those of Morton

et al. (2014) and Betancor et al. (2014), and indicating the important role of this FA in

cell membranes.

Diet supplementation with PL had significant interaction with DHA content in the liver,

which contrasts with results of González-Félix et al. (2002) which indicate that PL

doesn’t interact with EFA.

Our results showed that PL and DHA supplementation didn’t exert any significant

influence in deposition of SFA but decreased MUFA contents in the liver of meagre.

Zhao et al. (2013) obtained similar results in yellow croaker, where there were no

significant differences in SFA, mainly 16:0, 18:0 and 20:0, in both polar and neutral

lipid fractions of larvae, and MUFA in both polar and neutral lipid fractions decreased

as dietary PL increased. Furthermore, Villalta et al. (2005) showed that with PL and



43

DHA supplementation there was a MUFA decrease in the head and carcass. Li et al.

(2014) observed that muscle had higher HUFA levels, especially for EPA, and lower

MUFA levels. Jingjing et al. (2014) showed that saturated FA in liver were all

decrease with the increase of dietary DHA/EPA and the sum of monounsaturated FA

remained constant, excluding that 18:1 n-9, which was decreased by dietary

treatments. Results from Betancor et al. (2014) indicate that levels of SFA and MUFA

in liver PL were noticeably lower than those of the feed. Om et al., 2001 observed

that the saturated and monounsaturated fatty acid levels in the EPA/DHA-fortified

group were lower than those in the control group. Ji et al., 2011 observed that the

levels of 20:1 n-9, total saturated and total monounsaturated fatty acids in muscle

decreased with increasing dietary HUFAs content. Zhao et al. (2013) showed that

fish larvae fed live prey (control) had higher SFA composition than larvae fed

microdiets with PL supplementation. MUFA of fish larvae fed live prey were

comparable to those in treatments with 26.0 and 38.5 g kg−1 PL, but higher than in

treatments with 57.2 g kg−1 or higher PL. Olsen and Henderson, (1997) observed no

differences in saturated and monounsaturated fatty acids between dietary groups

containing different levels of PUFA.

In general, results of all these authors suggest that supplementation of PL and/ or

HUFAS promotes the accumulation of HUFA, by shifting the metabolism to a

preferential use of SFA and MUFA as energy sources for β-oxidation.

In this study, PL and DHA seemed to promote an increase of PUFAs in the muscle of

meagre. The increase of PL concentration in the diet promoted an increase of total n-

3 and n-6 PUFA while DHA only promoted an increase of total n-3 PUFA. This is in

agreement with a competitive interaction with the same metabolic pathways of

degradation, elongation and desaturation by both n-3 and n-6 FA. Dietary PL also

improved intestinal absorption of total PUFA. This results seen to be in agreement

with Ji et al.,(2011) that observed increased PUFAs, n-3 PUFAs, n-3 HUFAs and n-

3/n-6 fatty acids in the muscle in fish fed diets with high dietary HUFAs. Kontara et al.

(1997) also observed that inclusion of soybean phosphatidylcholine (SPC) in the diet

resulted in increased lipid and n-3 HUFA in shrimp tissues compared to that of

shrimp fed PL-free diets containing similar levels of total and n-3 HUFA; Geurden et

al. (1997b) concluded that the major effect of diet PL supplementation on fish FA

composition was that total fatty acids were richer in n-6 and n-3 HUFA when fish

were fed dietary PL. Hamza et al. (2008) observed that the major effect of dietary PL

supplementation on larval FA composition was an increase in the percentage of total

n-6 and decrease of n-3 FA (especially 20:5n-3 and 22:n-3). On the contrary, Zhao et



44

al. (2013) observed that n−3 PUFA decreased but n−6 PUFA increased with the

increase of dietary PL in microdiets.

As far as we know, there is not much bibliography on the effect of DHA and PL on

lipid class composition. In this study, was notorious the influence of DHA and PL on

different lipid class in muscle, brain and liver, although studies made by Vagner et al.

(2013) and Betancor et al. (2014) showed no difference among diets with respect to

lipid classes.

In our work, an increase of dietary PL decreased the PL content and promoted TAG

retention in the three tissues of meagre juveniles. This may be due to the fact that

dietary PL promoted the incorporation of TAG, acting like lipid emulsification,

enhancing the transport an absorption of lipids and PL, that are structural

components of membranes, on the contrary of TAG that are manly used for energy

reserve. Our results agree with those of Gao et al. (2014) that showed that the whole

body PL decreased with incremental dietary PL levels, and those of Li et al. (2014)

that observed an increase of TAG levels in the hemolymph with diet PL

supplementation.

Our results also showed a decrease of CH content in the muscle and brain of

meagre fed diets with more PL. This is not in accordance with NCR (2011) that

assign PL as having an important role as component of high density lipoproteins, that

transport CH from the gut to the tissues. Li et al (2014) showed that CH levels

increased with soybean lecithin supplementation although with egg yolk lecithin

supplementation the levels of CH displayed a tendency to decrease.

4.1- Conclusion

Meagre is a very promising species for aquaculture as it has a fast growth rate fish,

and this work confirms this, as in two months juveniles quadruplicated their initial

weight and their FCR was similar or better than that of other cultured Mediterranean

species, such as Dicentrachus labrax and Sparus aurata.

The increase of PL in the diets improved growth performance and feed utilization, as

well as whole body lipids.



45

One of the concerns that fish nutrition faces is the attempt of replacement of fishmeal

and fish oil feeds for alternative sources, and their impacts on DHA and EPA

contents in fish. The principal provider of n-3 HUFA in the diets is fish oil, and during

recent decades the importance of these fatty acids in human heath has been

continuously increasing. This study shows that inclusion of DHA in the diet improves

the retention of this FA in the muscle, thereby obtaining fish with higher nutritional

value for human consumption. These results may be very useful for production of

finishing diets.

No major changes were also observed in lipid peroxidation levels related to an

increase of PL and DHA in the diet.



46

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