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Dried Distillers Grains with

Solubles (DDGS): a potential

protein source in feeds for

aquaculture.

Rui Pedro Moreira de Magalhães

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Dried Distillers Grains with Solubles (DDGS):

a potencial protein source in feeds for

aquaculture.

Rui Pedro Moreira de Magalhães

Mestrado em Recursos Biológicos Aquáticos

Departamento de Biologia

2013

Orientador

Prof. Doutor Aires Oliva-Teles, Professor Catedrático, FCUP

Coorientador

Doutora Helena Peres, Investigadora Auxiliar, CIIMAR

FCUP 1 DDGS: a potential protein source in feeds for aquaculture

Todas as correções determinadas

pelo júri, e só essas, foram efetuadas.

O Presidente do Júri,

Porto, ______/______/_________


“With earth’s burgeoning human population to feed we must turn to the sea with understanding and new technology. We need to farm it as we farm the land.”

Jacques Cousteau

Acknowledgements

I would like to express my enormous gratitude to Prof. Dr. Aires Oliva-Teles, for the

great opportunity that gave me by accepting me as his MSc student and enable me to

perform this study. Sharing his immense knowledge and his excellent scientific

guidance were essential to the accomplishment of this dissertation.

I want to make a very special thanks to Drª. Helena Peres for her excellent guidance,

by sharing scientific knowledge and total availability to help in all steps along these two

years being indispensable for the realization of this thesis.

I want to leave my big thank you to my friend Filipe Coutinho for the ready assistance

in several taks, motivation and sharing of experiences that he has always provided.

Special thanks to Drª Amália for the precious teachings in the determination of

digestive enzymes activity.

I am also grateful to my other co-workers, Carolina Castro and Drª Paula Enes, who

promptly helped me whenever needed during this time.

Thanks to Mr. Pedro Correia for the technical and useful assistance.

Thanks to Dr. Pedro Pousão of the IPIMAR/CRIPSul for the fish supply.

A big thank you to my girlfriend Paula Castro for her unconditionally support and

motivate me in every moments.

A special thanks to my parents, José and Virginia Magalhães, for supporting me to

continue my education because without your support and love would not be possible to

accomplish this dissertation.


Abstract In the last decades, fish meal has been the main protein source in aquafeeds for

marine fish species. However, the increased demand, price, limited availability, market

supply fluctuations lead nutrition research to search for alternative protein sources to

fish meal. Within these alternative feedstuffs, distillers dried grains with solubles

(DDGS) have been emerging as an interesting protein source to include in the diets for

sparing fishmeal. However, few studies have been conducted in the area.

The exact levels of DDGS that can be included into the feeds for different cultivated

species aren’t known yet, neither the effects of its inclusion at physiological,

inflammatory or immunity levels. As a first approach for the evaluation of the potential

of DDGS in marine fish diets, in this study two digestibility trials were conducted to

evaluate apparent digestibility coefficient (ADC) of nutrients in DDGS from different

sources (DDGS1 - Biofuels de Castilla y Leon, Spain and DDGS2 - Pannonia Gold,

Hungry, respectively) in sea bass (Dicentrarchus labrax) and meagre (Argyrosomus

regius). Thereby, the objective of the present study was to evaluate the nutrient

availability of two different sources of corn DDGS and their effect on digestive enzyme

activities of two carnivorous species.

The ADC of protein in the tested ingredients was very high (92-98%) as well as the

ADC of lipids (82-89%). The ADC of dry matter and energy were moderate (57-66%

and 58-68%, respectively). The DDGS high fiber content can explain these moderate

energy digestibility values. The ADC of protein and phosphorus of the DDGS1 diet were

higher than the ADC of the reference diet, both in sea bass (92.85% and 55.63%,

respectivelly) and in meagre (90.96% and 60.36%, respectivelly). Different ADC values

of dry matter, protein, energy and lipids were obtained between the two tested

ingredients and the DDGS1 recorded the higher ADC values for the measured

parameters.

In both species, the highest activity of lipase was observed in animals fed the diet

containing DDGS2. In sea bass, amylase activity was higher in fish fed the diet

including DDGS2 than DDGS1 but did not differ significantly to fish fed the reference

diet. In meagre, animals fed the DDGS2 diet also showed a higher amylase activity

than those fed the DDGS1 diet, but the values were not significantly different.In the two

species, proteases activity didn´t differ significantly between diets.

There was no effect of diet on the proteolytic activity tested at various pH values.

These activity record peaks in the pH values tested (8,9 and 10) along the three

analyzed intestine sections.

In sea bass, the highest digestive enzymes activity was recorded in the medium and


distal intestine while in meagre maximum activity was registered in the medium

intestine.

Comparing the two species, proteolytic activity was higher in meagre while the highest

activity of amylase was recorded in sea bass.Overall, lipase activity was higher in sea

bass than in meagre, except in meagre juveniles fed diet2.

Based on the results of this work, it is concluded that distillers dried grains with

solubles (DDGS) seem to be a raw ingredient with high potential to be included in diets

for marine fish.

Keywords: Distillers dried grains with solubles (DDGS); European Sea Bass

(Dicentrarchus labrax); Meagre (Argyrosomus regius); Digestibility; Digestive enzymes

Resumo A farinha de peixe tem sido a principal fonte de proteína utilizada no fabrico de rações

para a alimentação de espécies marinhas durante a última década. Contudo, o

aumento da sua procura e preço, a sua reduzida disponibilidade, as flutuações do seu

fornecimento aos mercados e a imprevisibilidade destes, assim como as restrições do

uso de proteínas provenientes de fontes animais na formulação de rações, direccionou

uma grande parte da investigação efectuada em nutrição de animais aquáticos para a

procura de outras fontes proteicas abundantes, disponíveis e rentáveis. Deste modo,

os grãos secos de destilaria com solúveis (DDGS) perfilam-se como uma alternativa

proteica real e interessante à tão problemática farinha de peixe.

No entanto, poucos estudos foram efectuados na área, não se sabendo ainda quais

os níveis exactos que podem ser integrados em rações destinadas às diferentes

espécies cultivadas, nem quais poderão ser os efeitos que esta integração pode

provocar a nível imunitário. Assim, dois ensaios de digestibilidade foram efectuados

para avaliar o coeficiente de digestibilidade aparente dos nutrientes de DDGS1 e

DDGS2 provenientes de fontes diferentes (Biocarburantes de Castilla y Leon, Spain e

Pannonia Gold, Hungry, respectivamente) e o seu efeitos nas enzimas digrestivas em

robalo (Dicentrarchus labrax) e corvina (Argyrosomus regius, Asso, 1801). Os

resultados deste estudo mostraram que estas duas espécies carnívoras digerem muito

bem os ingredientes vegetais.

A digestibilidade da proteína nos ingredientes testados foi bastante elevada (92- 98%)

assim como a digestibilidade dos lípidos (82-89%). Os valores de digestibilidade da

matéria seca e da energia foram moderados (57-66% e 58-68%, respectivamente). O

elevado conteúdo em fibra dos DDGS pode explicar estes valores não tão elevados da

digestibilidade da energia. Os coeficientes de digestibilidade aparente (CDA) da


proteína (92,85%) e do fosforo (55,63%) em dietas contendo os DDGS1 foram

superiores aos CDA verificados na dieta de referência no robalo (91,88% e 31,65%,

respectivamente). Na corvina, os resultados obtidos foram semelhantes, com o CDA

da proteína (90,96 %) e do fosforo (60,36%) das dietas contendo DDGS1 a serem

superiores aos verificados em animais alimentados com as dietas controlo (88,99% e

23,63 %, respectivamente). Foram obtidos valores diferentes de CDA de matéria-seca,

proteína,energia e lípidos nos ingreditentes testados, sendo os valores mais elevados

observados no ingrediente DDGS1.

Nas duas espécies, a actividade mais elevada da lípase foi verificada em animais

alimentados com a dieta contendo DDGS2.. No robalo, a actividade da amílase foi mais

elevada em peixes alimentados com esta dieta não diferindo significativamente dos

peixes alimentados com a dieta de referência. No entanto, na corvina, os animais

alimentados com a dieta com incorporação de DDGS2 apresentaram uma maior

actividade da amílase, mas esta não foi significativamente diferente da actividade

registada nos animais alimentados com DDGS1. Nas duas espécies não foi verificado,

entre dietas, diferenças significativas na actividade proteolítica.

No robalo a actividade mais elevada das enzimas digestivas foi registada no intestino

médio e distal enquanto na corvina esta foi registada maioritariamente, no intestino

médio.

Comparando as duas espécies, a actividade proteolítica foi superior na corvina

enquanto a actividade mais elevada da amílase foi registada no robalo. Globalmente, a

actividade da lípase foi superior nos juvenis de robalo mas esta actividade foi superior

em juvenis de corvina alimentados com DDGS2.

Por fim, não se verificou qualquer influência da dieta na actividade proteolítica testada

a vários valores de pH. Sendo que esta registou picos de actividades nos valores de

pH testados (8,9 e 10) ao longo das três secções do intestino analisadas.

Concluo, com base nos resultados obtidos neste trabalho, que os os grãos secos de

destilaria com solúveis (DDGS) se perfilam como uma óptima matéria-prima para ser

incluída nas rações de aquacultura e que juntamente com outras matérias-primas de

origem vegetal, podem ser uma boa fonte proteica para a substituição parcial ou

completa da farinha de peixe nas rações utilizadas neste sector de produção animal.

Palavras-chave: Grãos secos de destilaria com solúveis (DDGS); Robalo

(Dicentrarchus labrax); Corvina (Argyrosomus regius); Digestibilidade; Enzimas

digestivas.


Contents Abstract ........................................................................................................................ 1

Resumo ........................................................................................................................ 2

Abbreviations ................................................................................................................ 6

Figures List ................................................................................................................... 6

Tables List .................................................................................................................... 6

Introduction ................................................................................................................... 7

Aquaculture development .......................................................................................... 7

Aquaculture in Portugal ............................................................................................. 8

Meagre (Argyrosomus regius, Asso, 1801) ............................................................. 10

Habitat and Biology .............................................................................................. 11

Production ........................................................................................................... 12

European Sea Bass (Dicentrarchus labrax, Linnaeus, 1758) ................................... 13

Habitat and Biology .............................................................................................. 13

Production ........................................................................................................... 13

Ingredients used in aquaculture feeds ..................................................................... 15

The importance of new ingredients in aquaculture feeds ......................................... 16

Dried Distillers Grains with Solubles (DDGS) .......................................................... 17

Physical characteristics ........................................................................................ 18

Chemical Composition ......................................................................................... 18

DDGS utilization in aquaculture ........................................................................... 19

Digestibility .............................................................................................................. 21

Digestive enzymes .................................................................................................. 23

Amylase ............................................................................................................... 23

Lipase .................................................................................................................. 25

Proteases ............................................................................................................ 26

Aims ........................................................................................................................... 28

Materials and methods ................................................................................................ 28

Ingredient composition ............................................................................................ 28

Experimental diets ................................................................................................... 29

Experimental animals .............................................................................................. 31

Experiment design................................................................................................... 31

Chemical analyzes performed in ingredients, diets and feces ................................. 33


Sample Preparation ............................................................................................. 33

Crude protein ....................................................................................................... 33

Crude lipids .......................................................................................................... 33

Dry matter ............................................................................................................ 34

Ash content .......................................................................................................... 34

Gross energy ....................................................................................................... 34

Starch content ..................................................................................................... 34

Chromic oxide ...................................................................................................... 35

Crude fiber ........................................................................................................... 35

Phosphorus ......................................................................................................... 36

Digestive Enzymes activities ................................................................................... 36

Statistics analyses ................................................................................................... 39

Results ....................................................................................................................... 40

Diets and ingredient digestibility .............................................................................. 40

Digestible basis ingredient composition ............................................................... 40

Experiment 1 – Sea bass ..................................................................................... 40

Experiment 2 - Meagre ........................................................................................ 41

Ingredient digestibility – comparison between species ......................................... 41

Digestive enzymes .................................................................................................. 42

Discussion .................................................................................................................. 48

Conclusions ................................................................................................................ 54

References ................................................................................................................. 55


Abbreviations ADC(s) - Apparent Digestibility

Coefficient(s)

ANF - Antinutritional Factor(s)

BW - Body Weight

DDGS - Distillers dried grains with

solubles

DE- Digestible Energy

DM - Dry Matter

DP - Digestible Protein

EFA - Essential Fatty Acid(s)

FA - Fat Acids

FM - Fish Meal

G - grams

HUFA - Highly Unsaturated Fatty Acids

Kg - kilograms

KJ - Kilojoules

L - Litre

Mg - miligrams

MJ - Milojoules

NSP - Non Starch Polysaccharides

P - Phosphorus

PUFA - Polyunsaturated Fatty Acids

Figures List Fig 1: Meagre (Argyrosomus regius, Asso)…………………………………………..10

Fig 2: European SeaBass (Dicentrarchus labrax, Linnaeus, 1758)………………..13

Fig 3: Meagre juveniles in experimental tanks………………………..……………...31

Fig 4: Thermo-regulated recirculation water system .………………………………31

Fig 5: Sampling collection of biological material…………….……………………….32

Fig 6: Collecting the sea bass intestine for determination of enzymatic activity….36

Fig 7: Enzymatic activity determination in microplates…………..………………….39

Fig 8: Variation in the activity of proteases in different sections of the intestine (anterior

intestine (IA), medium intestine (IM) and distal intestine (ID)) and different pH (8,9 and

10)………………………………………………………………………………………...........47

Tables List Table 1: Proximate composition (% dry matter) of the experimental ingredients….29

Table 2: Composition of the experimental diets………………………………..….…30

Table 3: Digestible basis ingredient composition…………………………………….40

Table 4: Apparent digestibility coefficients (ADC %) of nutrients and energy of the

experimental diets and test ingredients in sea bass…………………………………41

Table 5: Apparent digestibility coefficients (ADC%) of nutrients and energy of

experimental diets and test ingredients in meagre………………………………….42

Table 6: Specific activities of protease, lipase and amylase (mU mg protein-1) in

different intestine sections (anterior (IA), medium (IM), distal (ID) and total) of sea bass

fed the experimental diets………………………………………………………………43

Table 7: Specific activities of protease, lipase and amylase (mU mg protein-1) in

different intestine sections (anterior (IA), medium (IM), distal (ID) and total) of meagre

fed the experimental diets………………………………………………………………44

Table 8: Two-way ANOVA analysis of effect of intestine section and diet on the specific

activities of protease, lipase and amylased (mU mg protein-1) in seabass and

meagre…………………………………………………………………………………………45


Introduction

Aquaculture development

Aquaculture activity was originated in several Asian countries and introduced into

Europe in the middle ages by the common carp culture (Cyprinus carpio). Aquaculture

involves all human activities that use natural and / or artificial water bodies to produce

aquatic species consumed by man (Martín et al. 2005).

Global aquaculture production has been steadily growing in the new millennium, but at

a slower rate than in the 80’s and 90’s (Gjedrem et al. 2012). Since 1980, annual global

production has increased at an average rate of around 8% and this growth remains

higher than any other major animal food production sector (Campbell and Pauly 2012).

This global increase in production together with slowly declining of marine fisheries

catches (Watson and Pauly 2001) led to declare that aquaculture represents around

half of the seafood consumed by developed country’s markets (Loder et al. 2003). The

growth rate in farmed food fish production in these years outpaced the population

growth by 1.5 percent, resulting in a 7-fold increase in the average annual per capita

consumption of farmed fish, from 1.1 kg in 1980 to 8.7 kg in 2010 (FAO 2012). Indeed,

aquaculture is expected to resolve worldwide food issues (Cunningham et al. 2005). In

2010, total aquaculture production reached the historic mark of 79 million tons ($125

billion U.S dollars, approximately, 95 billion euros) or 60 million tons ($119 billion US

dollars, approximately 90 billion euros), excluding algae, aquatic plants and non-food

products (FAO 2012).

About 600 aquatic species are cultivated worldwide in a diverse variety of culture

systems, rearing densities and technological sophistication, in fresh, brackish or salt

water (FAO 2012). There is a global trend, driven mainly by demand from western

countries, on increasing the intensive production of omnivorous and carnivorous

species farmed in marine costal environments (Campbell and Pauly 2012). The

farming of these species, like salmon, sea bass and prawns is reliant on inputs of fish

meals and oils, water, land and energy (Trujillo et al. 2007). So, aquaculture has been

associated with negative impacts on marine and coastal ecosystem health (Liu et al.

2010).

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

of the world (Gjedrem et al 2012). In developed countries, growth rate of aquaculture

production decreased from 2.1 percent in 1990 to 1.5 in 2000, while in developing

countries it was observed a strong growth. Some countries in Asia, Pacific, sub-

Saharan Africa and South America have made great technological progress, in recent


years, and are dominating the production in these regions of the globe. In 2010, there

were 181 countries with record production (FAO 2012).

This global aquaculture distribution and production is vulnerable to several factors such

as natural, environmental, technological and socio-economic constraints. Countries

that are subject to natural disasters such as tropical storms, floods and earthquakes

often suffer considerable losses. Water pollution is also another threat of total

production loss in intensively urbanized areas. For instance, in 2010, China suffered an

output fall of 1.7 million tons with a value of U.S. $ 3.3 billion (2.5 billion euros) caused

by diseases (295 000 tonnes), natural disasters (1.2 million tonnes) and pollution (123

000 tonnes) (FAO 2012). In recent years, disease outbreaks are also causing

substantial or total losses of, for example, shrimp production in several countries

(Walker and Winton 2010).

Adequate feed supply is considered the major constraint to aquaculture development.

One third of all farmed fish food production, representing circa 20 million tons, is

achieved without providing artificial food (FAO 2012). However, the percentage of non-

fed aquaculture is declining gradually. This production represented 45% of world

aquaculture production, in 2005 (Tacon and Metian 2008), and currently represents

only 33.3%, mainly due to changes in Asia cultivation procedures (FAO 2012). This

rapid growth of fed species production is also due to the development, improvement

and availability of formulated feed for aquaculture finfishes and crustaceans.

Aquaculture in Portugal

Fisheries have been an important activity in Portugal since Neolithic period and

suffered a steady development along the several civilizations that inhabited the Iberian

Peninsula (Birmingham 2010). Fish culture may have been introduced in the Iberian

Peninsula by the Romans, who developed simple rearing techniques associated with

sea salt (Birmingham 2010). Salines may have been used for growing larvae and

juveniles, and oyster production (FAO 2012).

Aquaculture activity in Portugal has mainly been made on a familiar basis, never

reaching the economic importance of the fisheries industry. However, the production of

clams in Ria Formosa (Algarve) and oysters in Sado River estuary represents an

important incoming source for local communities. Oysters have been exported to

France since the 1950s, but in late 1970s its production declined due to an outbreak of

gills disease caused by poor water quality and iridovirus (Henriques 1998).


In Portugal, the first large scale industrial facility was built in late 1960s, in Paredes de

Coura to produce rainbow trout (Oncorhynchus mykiss). After 1986, economic

incentives from Portuguese government and European Community, enabled a great

aquaculture development complementing fisheries industry (INE 1998). At this time,

large number of salines was transformed into aquaculture facilities in Aveiro, Figueira

da Foz, Vale do Tejo, Setúbal and Algarve (Dinis 2010). Initially, fish production in

these salines was focused on two species: sea bass and sea bream.

Between 1986 and 1996, Portuguese fisheries production declined over 65,9%, due to

an increased interest in aquaculture (FAO 2013). Initially, producers possessed no

knowledge about aquaculture and many didn´t survive. In the late 80s, producers have

more business knowledge and adopted a professional approach. Consequently, the

production increased 27% between 1990 and 1997 (INE 1998).

In 2000, with massive quantities of fish coming from Greece, fish prices suffered a

significant decline. National producers faced major problems because of the low

competitiveness of production costs. Thus, Portuguese production has not grown

between 2000 and 2008 (FAO 2013).

In 2010, marine and brackish species production represents about 88% of total

production and bivalve molluscs (clams and oysters), gilthead sea bream (Sparus

aurata) and sea bass (Dicentrarchus labrax) are the most produced species (INE

2010).

Aquaculture represents only 3% of fish national production, a much lower number than

the European average (20%) (INE 2010). Indeed, total fisheries catch in Portugal

ensures consumption levels per capita of 23 kg / year, which is identical value to the

European average, but insufficient to meet the demand of 57 kg of fish / year per

capita. Several constrains to the development of Portuguese aquaculture production

has been contributing to this panorama. For instance, incorrect location of fish farms

which limits production expansion; licensing of aquaculture facilities involving a long

bureaucratic process; high land costs and competition with other users of the coastal

areas, such as tourism, urban development or environment protection are the main

constraints to aquaculture production.

In 2011, Portuguese aquaculture production was 9165 tons (FAO 2013). The

operational program for fisheries 2007-2013 predicted that in 2013 the sector would

reach 15000 tons, representing 8% of national fish production. These values were not

achieved but for this increase it would be necessary to promote intensive production


models, especially in offshore cages. As a consequence, in March 2008 was created a

zone designated Pilot Area of aquaculture production in Armona (APPA), located on

the southern coast of the country. This project consists in the installation of cages for

fish production and shellfish in long-lines. Thus, it is expected to achieve the goals that

were established initially for Portuguese aquaculture, though not until 2013.

Meagre (Argyrosomus regius, Asso, 1801)

Meagre belongs to Sciaenidae family,

which includes 270 species in 70 genera

(Ono et al. 1982; Chao 1986). Meagre has

a large head with an elongated and almost

fusiform body (Whitehead et al.,

1984/1986). Mouth is located in a terminal

position, it has no barbells and buccal

cavity is golden-yellow. Eyes are small and

the lateral line is black and evident,

extending to the caudal fin (FAO 2013).

The second dorsal fin is much longer than

the first one and the anal fin has a first short and spiny ray and a second one very thin

(FAO 2013). The caudal fin is truncated to s-shape and large. Scales are mostly

ctenoids except in the anterior area, nose and below the eyes, which present some

cycloid scales (Whitehead et al. 1984/1986). Various branched appendages and a pair

of muscles are present in the gas bladder and are associated with the lateral

musculature of the body (Tower 1908; Takemura et al. 1978). Those appendages can

vibrate producing a characteristic sound (a typical "grunt") that can be heard up to 30

meters (Tavolga 1971). Meagre has very large otoliths, its body color in vivo is silvery-

gray, with bronze dorsally traits and a reddish-brown fin base. The coloration becomes

brown after death.

Adult meagre has a relatively short digestive tract, typical of carnivorous fish,

representing about 70% of its body length. The esophagus is short and broad, with

muscular walls. The stomach has secretory function; it is muscular and with small bag

shape, in which the anterior portion fits both the esophagus and the intestines, forming

a posterior stomach that allows the storage of large size preys. The intestine is short

and has a wall of varying thickness. In the anterior portion of the intestine, near the

pyloric area of the stomach, exists 9 pyloric caeca (Gil et al. 2009), which together with

Fig 1: Meagre (Argyrosomus regius, Asso,

1801) Source: Claude Wacquant.


the intestine have secretory and absorptive functions. Meagre can reach 2 meters in

length and a weight of 100 kg (Froese and Pauly 2011).

Habitat and Biology

Meagre can be found in the Mediterranean Sea, Eastern Atlantic Ocean between

Senegal and the English Channel, in southern Norway, Denmark, Iceland and the

Black Sea (FAO 2013; González-Quirós et al. 2011). They migrate through the Suez

Canal to the Red Sea and can make the reverse Lessepsian migration (Chao 1990).

They live at moderate depths (15-300 m) over sand and rocks (Froese and Pauly 2011)

and are euryhaline and benthopelagic.

Meagre is a carnivorous fish and in the wild it feeds on Mysidacea, Decapoda and

Teleostei (Cabral and Ohmert 2001). The species reaches sexual maturity at 2-3 years

of age, depending on where they live, and make reproductive migrations towards the

coast (Maybank 2008) between April and July in the southern Mediterranean

(Whitehead et al., 1984/1986). Generally, they congregate inshore (Froese and Pauly,

2011), penetrate the estuaries (FAO 2013) and salt-marshes, and form large

agglomerates in muddy waters, aided by long grunts emitted by males (González-

Quirós et al., 2011).

This species spawning is seasonal, with a peak between April and June, and egg

fertilization is external (Chao, 1990). Matting elapses during an in-pair courtship in the

presence of typical deep sounds produced by the male by compression of the

abdominal muscles against the gas bladder (Lagardère and Mariani, 2006; González-

Quirós et al. 2011). From mid-June until the end of July they proceed from the

estuaries to coastal waters, where they feed in shallow waters until early autumn.

During winter they return to deep waters (FAO 2013). The major spawning places for

meagre in the North Atlantic and Mediterranean Sea are: Nile delta (Egypt), the Levrier

Bay (Mauritania) and the Gironde estuary (France) (Quéro 1989a and 1989b). A

female can reach 19 years of age and the male 16. A female of 1.2 meters can

produce 800,000 eggs per year.

Meagre is a gonochoric species that remains sexually undifferentiated up to nine

months of age. Histologically, sexual differentiation begins to be notorious from six

months of age (Schiavone et al. 2008) and usually occurs in females first than in

males. In captivity there is no record of natural spawns and viable egg production is

achieved only by artificial induction of reproduction with hormones administration

(Duncan et al. 2008). Then, spawning occurs spontaneously, at a temperature between

17° and 22°C, without requiring stripping or artificially fertilizing eggs, and eggs are not


released simultaneously but in multiple small batches (Mañanós et al. 2008). During

larval growth survival rates are encouraging (Pousão-Ferreira et al. 2010).

Production

Aquaculture of meagre has begun in the late 90s due to a consensus between Italian

and French producers. The first commercial production was performed in France, in

1997, with fingerlings produced in Sète. These fingerlings were then grown near

Orbetello lagoon, in Tuscany, on the west coast of Italy. This species production has

spread to other Mediterranean countries and increased rapidly. Global production was

4000 tons in 2008, more than 10000 tons in 2010 (Monfort 2010) and 13742 tons in

2011 (FAO 2013).

The intensive production of meagre is made in land-based tanks and in sea cages. The

juveniles supply comes from hatcheries located across the Mediterranean area.

Countries like Spain, France, Greece, Italy and Egypt stand out as main juvenile’s

producers (Suquet et al. 2009). Juveniles are stocked in small ponds or in cages with a

weight between 3 and 20 g and are maintained there for three months until reaching

100 g.

Rearing techniques are very similar to those for European sea bass and gilthead sea

bream (Chatzifotis et al. 2012). Nowadays, meagre on-growing is done mainly in sea

cages and the animals are fed extruded diets with 45-50% protein and 17-20% lipid

(Monfort 2010; Chatzifotis et al. 2012). Due to its high growth rate and its excellent feed

conversion ratio (FCR) (Calderón et al. 1997; Pastor et al. 2002) meagre is considered

one of the most promising species for aquaculture, being an attractive alternative to

diversify production. Meagre can increase 1 kg per year depending on growing

conditions, with FCR values from 0.9 to 1.6, depending on the feed used (Chatzifotis

et al. 2012).

Meagre is highly tolerant to environmental conditions changes and has good resistance

against environmental stress factors (Monfort 2010). In fact, they can tolerate wide

temperature (2-38 ºC) and salinity (5-39‰) variations and consequently can adapt to

different latitudes and farming conditions (Cittolin et al. 2008; Suquet et al. 2009;

Chatzifotis et al. 2010). However, more studies are needed to get further knowledge on

the characteristics and quality of the meat in relation to different diets (Piccolo et al.

2008), nutritional requirements and protocols for producing larvae (Roo et al. 2010;

Chatzifotis et al. 2010).


Fig 2: European SeaBass (Dicentrarchus labrax, Linnaeus,

1758). Source: Center for Genomic Regulation

European Sea Bass (Dicentrarchus labrax, Linnaeus, 1758)

European sea bass belongs to

Actinopterigii class, Teleostea

superorder, Perciformes order and

Moronidae family. It has an

elongated body with 8 to 10 dorsal

spines, 12 to 13 dorsal soft rays, 3

anal spines and 10 to 12 anal soft

rays. The posterior edge of the

operculum is finely serrated, and

the lower part possesses strong

denticles directed forward. It has 2

flat opercular spines and the mouth is moderately protractile. Vomerine teeth are

present anteriorly in a crescent band (Fishbase 2013). The juveniles have black spots

in the upper body.

Habitat and Biology

European sea bass is a euryhaline marine teleost species (Varsamos et al. 2001). It

has a geographic distribution that extends from Eastern Atlantic to Morocco, Canary

Islands and Senegal, to Black and Mediterranean Sea (FAO 2013). Sea bass inhabits

coastal zones, estuaries with various types of bottoms, ponds and even rivers. In the

summer months it enters the mouths of rivers; however, when the water temperature

drops it migrate to offshore and remains in deep waters during winter (FishBase 2013).

Seabass is a gonochoric species and spawning takes place between December and

March in the Mediterranean, to June in the Atlantic Ocean (Haffray et al. 2006). The

eggs, larvae and juveniles in the first 3 months are widely distributed and adults

migrate hundreds of kilometers along the coast (Haffray et al. 2006).

They are predators feeding on shrimp, mollusks and fish (Smith 1990). Some species

of nekton including zoobenthos (amphipods), benthic copepods, insects, fish eggs,

larvae, crabs, mysids, cladoceran, planktonic crustaceans, are also part of their diet

(FishBase 2013).

Production

The first mass-production techniques for this species were developed in France and

Italy in the late 1960s and 1970s. These techniques were then dispersed and further

developed by all Mediterranean countries (FAO 2013). European seabass is,


nowadays, one of the most important commercial species cultivated in the

Mediterranean Sea and was one of the first non-salmonid marine species to be

cultivated and commercialized in Europe. The main producers are: Greece, Italy,

Turkey and Spain (FAO 2013). The global aquaculture production of this species

reached 144 365 tonnes in 2011 (FAO 2013).

There are numerous studies on this species nutritional requirements. The optimal

dietary protein level for juvenile was established to be around 50% and is not affected

by water temperature (Hidalgo and Alliot 1988; Peres and Oliva-Teles 1999a). In

fingerlings, optimal growth was achieved with 45% (Perez et al. 1997) and no growth

differences were found with diets including either 43 or 52 % (Dias et al. 1998).

The optimum level of dietary lipids for juveniles was estimated to be 12.5% (Alliot et al.

1974). Growth performance was not affected by a dietary lipid range between 12 and

24%; however, incorporation of 30% of lipids in diets depressed growth rate (Peres and

Oliva-Teles 1999b). On the contrary, Lanari et al. (1999) observed higher growth

performance with 19% of lipids in diets than with 11 % or 15%. Similar results were

observed by Dias et al. (1998).

Starch may be used for partial replacement of protein and fat as energy source in feed

formulations, and it also contribute to improve the mechanical properties of pellets

(Lanari et al. 1999). Fish have limited capability to digest and metabolize carbohydrates

(Wilson 1994). In sea bass, crude starch digestibility decreases with the increase of

dietary carbohydrate level (Oliva-Teles 2000). Gelatinized starch is adequately

digested by seabass (Enes et al. 2011). No differences were found in the growth of

animals fed diets with 15 or 25 % crude or gelatinized starch (Gouveia et al. 1995) and

with 16-28% of gelatinized starch, but there was suppression of growth with 33% starch

(Perez et al. 1997).

Studies about requirement of minerals and vitamins are very scarce. A requirement of

vitamin C was demonstrated but not quantified (Alexis 1997; Henrique et al. 1998). It is

recommended to incorporate a minimum of 5 mg of ascorbic acid per kg of diet for

maximum growth and maintenance of normal skin and optimal collagen concentration

(Fournier et al. 2000).

The optimum protein to energy ratio of sea bass diets were estimated to be 19 mg/kJ in

diets with at least 21 MJ DE/kg (Dias et al. 1998). Lupastsch et al (2001) estimated the

requirements for maintenance to be 43.6 kJ DE BW (kg) -0.79 day-1 and 0.66 g DP BW

(kg) -0.69 day-1 .


Ingredients used in aquaculture feeds

In feeds manufacture, the ingredients are by-products from human food or are directly

produced for using in animal feeds. The ingredients are sources of proteins (amino

acids), fats, carbohydrates, vitamins and minerals (NRC 1993). They are also source of

energy although energy is not independent from nutrients.

The most important source of protein is fish meal which provides, depending on its

quality, a protein level ranging from 56% to 76%, with an appropriate essential amino

acids profile. It is also a good source of energy, essential fatty acids (EFA) and

minerals and is highly palatable and digestible for most fish (NRC 1993). However, fish

meal availability is limited due to fluctuations of production. Due to its high demand

(SOFIA 2006), it is also an expensive ingredient that usually contributes to the high

final price of fish feeds (Josupeit 2008). However, for carnivorous fish species only

selected ingredients with high protein content may be used as alternative protein

sources for fish, due to their high protein requirements (Hardy 2008). Among these

ingredients there are by-products of animal production, vegetable feedstuffs and single-

cell organisms. Comparatively to fish meal, alternative protein sources have some

nutritional disadvantages such as inadequate amino acid (AA) profile, low digestibility,

low palatability, and the presence of several anti-nutritional factors (Gatlin et al. 2007;

Lim et al. 2008b). The presence of contaminants like mycotoxins may also limit the use

of vegetable feedstuffs in aquafeeds (Hendricks 2002).

Partial replacement of fish meal by alternative protein sources has been achieved at

different levels in various species. However, dietary formulation with total or almost

total fish meal replacement usually leads to a depression of growth performance in

carnivorous species (Kaushik et al. 2004).

Soybean meal presents high protein content and a good amino acid profile and it is a

major alternative to fish meal in aquafeeds (Mohsen 1989). However, the increasing

prices in recent years tend to decrease its use (Josupeit 2008). Protein concentrate

obtained from oilseeds, such as rapeseed, cottonseed and sunflower, and from

cereals, like wheat gluten and maize gluten are also potential alternatives to fish meal

(Aslaksen et al. 2007). Their use is however limited due to incorrect balances of amino

acids profile (Pereira and Oliva-Teles 2003).

Incorporation of lipids and carbohydrates in diets is necessary to spare protein for

plastic purposes instead of being used for energetic purposes. Fats and oils are the

major sources of energy and also of EFA, with marine fish oils containing 10-25% of

HUFA (NRC 1993). Marine oils are derived from marine animals and are classified as


processed fish oils (menhaden oil), fish liver oils and marine mammal oils (Hertrampf

and Piedad-Pascal 2000). Fish oil is the main lipid source in aquafeeds but due to the

expected rates of aquaculture growth and fish oil decreased availability (SOFIA 2006)

its actual levels of incorporation will not be economically sustainable (Turchini et al.

2009). Its dietary incorporation is also limited to the possible presence of contaminants

such as dioxins or dioxin-like polychlorinated biphenyls (Bell et al. 2005). Indeed, in

farmed fish, fish oil is considered the main source of persistent organic pollutants

(Turchini et al. 2009). Plant oils such as soybean, canola (Glencross et al. 2003),

linseed (Bell et al. 2004), sunflower (Abowei and Ekubo 2011) and rapeseed or palm oil

(Karalazos 2007) have been used to replace fish oil, although their fatty acid

composition differ significantly because they contain low levels of n-3 HUFA (Oliva-

Teles 2012). As muscle FA profile tend to reflect the dietary FA profile, the dietary

incorporation of vegetal oils may affect the fatty acid composition of farmed fish (Bell et

al. 2001, 2003). This effect can be overcome by using diets rich in fish oil during the

finishing period as this, at least partially, allow to recover the “fishy” composition of fats

(Bell et al. 2004).

Carbohydrates are relatively inexpensive sources of energy that may spare protein

(more expensive) from being used as an energy source (Abowei and Ekubo 2011).

Fish use diets with no carbohydrates as efficiently as those including carbohydrates,

because fish do not have specific dietary carbohydrate requirements (Enes et al.

2009). Cereal grains have 62-72% starch and are important binders in steam-pelleted

and extruded feeds (NRC 1993). Many by-products of grain industry like wheat, oat,

corn, rice or rye are available as ingredients for animal feeds (Hardy and Barrows

2002). Plant feedstuffs such as peas, beans and chickpeas also contain large amounts

of starch that are used for fish as energy source (Booth et al. 2001). Raw starch is

considered a poor energy source (Peres and Oliva-Teles 2002). However,

technological treatment, involving heat and pressure, may increase its digestibility

(Peres and Oliva-Teles 2002).

Other ingredients used in low amounts in feeds are mineral and vitamin premixes, feed

binders, carotenoid supplements, drugs, antibiotics, probiotics, enzyme supplements,

preservatives, fiber, flavorings and water (Abowei and Ekubo 2011), but will not be

further detailed here.

The importance of new ingredients in aquaculture feeds

The rising prices due to increased demand and supply fluctuations of fishmeal, fish oil,

soybean meal, corn and wheat meal in global markets emphasizes the need to reduce


their incorporation in feeds. Production of fishmeal and fish oil has stabilized in the last

decades and natural phenomena like El Nino in 1997-1998 showed that availability of

these ingredients is unpredictable and therefore industry cannot rely solely on them.

Soybean production is limited in Europe due to climate and geographical constraints

and also due to the restriction of using genetically modified products adopted by the EU

countries in 2003. So, it is necessary to explore other plants cultivated in Europe and

around the Mediterranean zone and thereby decrease dependence on soybeans and

other ingredients produced outside the European continent.

Dried Distillers Grains with Solubles (DDGS)

Currently we are witnessing a concerted effort by nutritionists and feed formulators to

reduce aquafeeds costs by replacing fish meal and other expensive protein sources for

other lower cost vegetable sources.

Distiller's dried grains with solubles (DDGS) are the dry residue that remains after

fermentation of grain (corn, wheat, sorghum and barley) mash by selected enzymes

and yeasts to produce ethanol and carbon dioxide. When corn is used to produce

ethanol, approximately two thirds of the grain weight, corresponding to starch, is

fermented by yeast. The by-product will be used to produce distiller's dried grains with

solubles (DDGS). The yeast from the fermentation process remains in the finished co-

product.

Global production of ethanol to be used as vehicle fuel has rapidly increased in recent

years, in an attempt to reduce the use of petrol, since ethanol can be blended with

gasoline. According to the United States Department of Agriculture approximately 700

billion liters of starch-based ethanol (mostly from corn) will be used in the United States

by 2015 (Linwood and Baker 2011). As a result, the availability of these by-products

has increased considerably, and according to the Agricultural Marketing Resource

Center, in 2012/2013 its production in the United States amounted to 38.89 million

tons, more than doubling the 2007 production (14.5 million tons). Thus, as availability

increases, prices of distiller by-products become more competitive and this has

enhanced its use in animal feeds. Per unit of protein, DDGS are much less expensive

than conventional protein sources. However, their use in fish feeds is still limited,

although some studies showed that this ingredient can be a promising ingredient to be

used in fish feeds, namely for omnivore species.


Physical characteristics

Corn DDGS is a granular product with a color ranging from light yellow to dark brown.

Several factors can influence this color, including the amount of distiller's solubles

added to distiller's grains before drying, the drying temperature and duration. The color

of the raw material has little effect on the final color of DDGS (Rosentrater 2006; Liu

2009).

The main obstacle to the use of DDGS in diets for animals is the wide variation in the

nutritional content of DDGS produced in different manufactories due to differences in

ethanol processing methods (Liu 2009). The color is a strong indicator of the nutritional

value of DDGS, particularly of corn DDGS. An incorrect processing method (i.e. higher

drying temperatures) results in darker DDGS which have lower nutritional value

conditioning their use in animal feeds (Fastinger et al. 2006)

Chemical Composition

Corn DDGS have a protein content between 28 and 33%; lack of anti-nutritional

factors that are commonly found in most plant protein sources and have a reasonable

amount of fat (10.9 to 12.6%) (Lim et al. 2011). DDGS also have very low starch levels

as most of it is converted to ethanol during the fermentation process. Corn DDGS have

a higher crude fiber content than wheat DDGS and the neutral detergent fiber may

represent 29-39 % of the weight (Lim et al. 2011). The ash content is higher in wheat

than in corn DDGS.

Expressed as a percentage of crude protein, and relatively to soybean meal, corn and

wheat, DDGS are deficient in several essential amino acids (EAA), including lysine,

threonine, tryptophan, arginine, isoleucine, phenylalanine and methionine (Lim and

Yildirim-Aksoy 2008). The concentration of vitamins and minerals differs between

sources and batches of DDGS. Corn DDGS are rich in vitamin A, niacin and choline,

and contain several minerals, including phosphorus that is high bioavailable and

present in high levels (Lim et al. 2011).

When contaminated corn with mycotoxins is used for bioethanol production the

mycotoxins are accumulated in DDGS. The mycotoxins concentration in DDGS may be

3 to 3.5 times higher than values found in corn. Aflatoxins (B1, B2, G1 and G2),

deoxynivalenol (DON), fumonisins (B1, B2, B3) and T-2 may also be found in DDGS.

However, mycotoxins do not seem to be a major problem when corn is produced under

normal climacteric conditions (Zhang and Caupert 2012).


DDGS utilization in aquaculture

For omnivorous species, such as channel catfish (Ictalurus punctatus), DDGS may be

used to replace fish meal and soybean meal, up to 40% of diet, without lysine

supplementation (Webster et al. 1991, 1992, 1993; Lim et al. 2009) and with no

negative repercussions on growth performance. Higher dietary replacement levels

may be achieved with the adequate restoration of dietary essential amino acid profile,

by using amino acid supplements or combination among different protein sources

(Webster et al. 1991; Cheng and Hardy 2004b). Concordantly, for catfish, the dietary

lysine supplementation allowed an increased used of DDGS in the diet up to 70%

(Webster et al. 1991; Robinson and Li 2008). Also for catfish, Webster et al. (1992a)

states that a combination of DDGS with soybean meal (35% DDGS and 49% soybean

meal) can be used to totally replace fish meal in the diet, with or without lysine

supplementation and methionine.

In rocky mountain white tilapia (O. niloticus x O. aureus) a diet containing 30 % DDGS,

26 % meat and bone meal, and 16 % soybean meal provided good growth (Coyle et al.

2004). In Nile tilapia (Oreochromis niloticus), DDGS can be incorporated in diets at a

level of 20 % as a substitute of soybean meal and corn meal without affecting growth

performance and body composition (Schaeffer et al. 2009). For this species, this

feedstuffs can be also replaced in diets for 40 to 60 % of DDGS plus lysine

supplementation (Lim et al. 2007; Shelby et al. 2008). In the same species, Li et al.

(2011) found the same weight gain, feed efficiency ratio (FER), protein efficiency (PER)

in fish fed diets with up to 30% wheat DDGS or up to 40% with lysine supplementation.

In rainbow trout diets, Cheng and Hardy (2004b) states that DDGS can be used at

22.5% inclusion level or at 75 % with lysine and methionine supplementation. In the

same species a replacement of 25 % of fish meal can be achieved with a mixture of

corn DDGS and corn gluten meal (Stone et al 2005).

In yellow perch (Perca flavescens) a mixture of DDGS and soybean meal can be

incorporated up to 49.5 % without negative effects on growth (Schaeffer et al. 2011).

The same author (Webster et al. 1999) reported that sunshine sea bass (Morone

chrysops x M. saxatilis) can be fed with a diet of 29% soybean meal, 29% meat and

bone meal and 10% DDGS without negative effects on final weight, percent weight

gain, survival, specific growth rate and feed conversion ratio. High fiber content,

phytate and pigments, along with variation in chemical composition and physical

properties of DDGS may limit its use for some species (Belyea et al. 2004).


DDGS contains a substantial percentage of yeast cells. Ingledew (1999) estimated that

3.9% of the total biomass of this ingredient is yeast, representing 5.3% of total protein

present in DDGS. Li et al. (2010) showed improved weight gain and feed efficiency in

animals fed diets with 30% DDGS and attributed it to yeasts. Studies with sea bass

(Oliva-Teles and Goncalves 2001) and sunshine sea bass (Gause and Trushenski

2011) also showed growth improvement with diet yeast inclusion.

Few data has been published on amino acid availability of DDGS for aquatic species

(Webster et al. 2008). Cheng and Hardy (2000) were the first authors to assess the

bioavailability of amino acids and protein digestibility of DDGS. Results indicate for

rainbow trout an apparent digestibility of more than 80% for both amino acids and

protein, with the exception of cysteine (75.9%). In sunshine sea bass, bioavailability of

amino acids in DDGS was greater than 50% except for cysteine, histidine and valine;

protein digestibility was moderate (65%) compared to that reported for rainbow trout

(90%, Cheng and Hardy 2004; Metts et al. 2011).

Few studies have been performed to assess DDGS effects in fish immunity, although it

is recognized that diet modifications can positively or negatively affect fish immune

status and disease resistance (Lim et al. 2007). Yeasts are rich in protein, B vitamins,

β-glucans and nucleotides. These compounds, either in its purified form, as byproducts

of yeast, or present in living forms appear to stimulate the immune response in humans

and animals, including fish (Chen and Ainsworth 1992; Robertson et al. 1994; Li et al.

2004). Several studies point to an enhancement of non-specific immune response

when animals are fed with diets including β-glucans (Gatlin 2002 ; Oliva-Teles 2012).

However, a prolonged feeding with high levels of β-glucan seems to increase Atlantic

salmon and gilthead sea bream susceptibility to bacterial infections (Robertsen et al.

1990; Couso et al. 2003).

Nucleotides correspond to 12-20% of total N in yeasts (Oliva-Teles 2012). Nucleotides

are associated to increase innate defense mechanisms and disease resistance in fish

(Li et al. 2004). A supplementation with a mix of nucleotides seems to increase the

number of villi in the intestines of mice (Uauy et al. 1990) and Atlantic salmon (Salmo

salar) (Burrells et al. 2001). Thus, the surface of the intestine is increased and the

nutrients seem to be absorbed more efficiently.

However, DDGS inclusion in Nile tilapia diets do not seems to affect hematological

parameters (white blood cell count, red blood cells, hemoglobin and hematocrit) or

immune responses, such as serum proteins, lysozyme activity and antibody production

against Streptococcus iniae (Lim et al. 2007). However, diets with 10 to 40% DDGS


significantly increased immunoglobulin and hematocrit percentage compared to

animals fed with control diet (0% DDGS) (Lim et al. 2009).

Lim et al. (2009) noticed that juvenile catfish fed diets incorporating between 20 and

40% DDGS (1.14 g to 2.28 g glucans/kg diet) showed a significant increase in plasma

immunoglobulins but no changes in serum proteins, lysozyme activity, alternative

complement, production of superoxide anion and rate of macrophages chemotaxis. An

increase in plasma immunoglobulins may be responsible for a decrease or delay of

mortality in fish fed diets containing DDGS. Shoemaker et al. (2007) refers that fish

immunoglobulins are capable of specifically binding to epitopes of the bacterial surface.

Further, the same author asserts that these are potential activators of the complement

system and are effective opsonins and agglutinins that facilitate the removal of

pathogens. Consequently, more research is necessary to evaluate the potential of

incorporating DDGS in diets and its influence on animal’s immunity.

Some studies indicate that diets with DDGS may have high levels of pigments. Levels

of lutein and zeaxanthin over 7-10 mg per feed kg can be deposited as visible pigments

in catfish fillets (Lee 1987; Lim et al. 2009). Li et al. (2011) state that DDGS levels

above 30% may result in a pronounced deposition of yellow pigments that may reduce

the commercialization value of fish. However, DDGS obtained as byproducts of ethanol

production can be incorporated at higher percentages without causing excessive

pigmentation since the pigments are effectively removed by ethanol extraction (Li et al.

2011).

Digestibility

The bioavailability of nutrients and energy in feeds for fish may be defined in terms of

digestibility. Digestibility indicates the fraction of nutrient or energy in the ingested diet

that is not excreted in feces. Apparent digestibility does not take into account nutrient

losses of endogenous origin which are part of feces. “True” or “correct” digestibility

excludes the endogenous losses from the feces. The apparent digestibility has a more

practical importance than true digestibility because the endogenous losses are minor if

the animal is not fed (Lovell 1998).

The first task to assess the potential of any new ingredient for inclusion in aquaculture

diets is the determination of its apparent digestibility (Cho et al. 1982; Bureau et al.

2002). The apparent digestibility coefficients (ADC) of ingredients are necessary to

formulate commercial and experimental diets on a digestible basis rather than on a

gross basis.


Several methods of direct or indirect quantification of digestibility have been used with

fish. The direct method involves measuring directly all of food ingested and all the

feces excreted. The indirect method eliminates the need to quantitatively collect all the

feces the fish produce (Vandenberg and Noüe 2001). It involves the quantification of

the ratios of nutrient to some indigestible indicator in the feed and feces. This digestion

indicator must be indigestible, unaltered chemically, nontoxic to the fish, easily

quantified and capable to pass through the gut uniformly with other ingesta (Ward et al.

2005). As the dietary ingredients are absorbed in the gut, the ratio of nutrient to the

indicator will be lower in feces than in fed. Some internal indicators used are ash, crude

fiber or plant chromagens, while external indicators are additives such as chromium

oxide or yttrium oxide (Carter et al. 2003).

The collection of fecal material in fish is a difficult process and can substantially

influence apparent digestibility coefficients for nutrients in feed ingredients (Amirkolaie

et al. 2005). The methods of fecal collection must ensure that the results are accurate,

repeatable and harmless to the fish (Austreng 1978; Cho et al. 1982; Allan et al. 1999).

Methods for collecting feces include suctioning of fecal material, dissection (Windell et

al. 1978), manual stripping (Glencross et al. 2005), settling columns (Cho et al. 1982)

and netting (fecal matter is sieved continuously by a net present at the water outlet)

(Choubert et al. 1979).

The removal of chyme before being completely digested and the contamination of

feces with physiologic fluids and intestinal epithelium, that would have been

reabsorbed by the fish before natural defecation, are the main disadvantages of direct

feces collection from the intestine. This affects the reliability of these methods and in

general leads to underestimation of digestibility (Amirkolaie et al. 2005)

ADC in which feces came in contact with water before collection may present

overestimated values than those obtained by stripping or dissection. This is due to

disintegration or separation of feces, or leaching of nutrients and/or marker from the

fecal matter (Cho and Kaushik 1990). However, these methods are widely accepted

because they facilitate repeated measures and lower handling stress. Specific devices

to collect fecal material were created by Ogino et al. (1973), Cho et al.(1975) and

Choubert et al. (1979). Ogino et al. (1973) collected feces by passing the effluent water

from the tanks through a filtration column. The Guelph system uses sedimentation

collumns without significant nutrient leaching (Cho and Slinger, 1979). Choubert et al.

(1979) developed a mechanically rotating screen to filter fecal material from the water

(INRA system).


Smith (1971) developed a metabolic chamber to collect fecal material voided naturally

into the water. In this method the fish is restrained and need to be force-fed. This

technique imposes a high stress level on the fish and the digestibility values were

questionable (Cho et al. 1982).

A major problem to evaluating the digestibility of ingredients in fish is their refusal to eat

most of these ingredients separately or, in some cases, the reduced voluntary feed

intake and nutrient utilization due to nutrient unbalances, amino acids deficiencies,

presence of anti-nutritional factors and reduced palatability (Tacon 1995; Booth et al.

2001). To avoid these problems, the most appropriate approach is the substitution

method, where the test diet comprises a portion of a reference diet (usually 70%) and a

portion of the test ingredient (usually, 30%) (Glencross et al. 2007).

Digestive enzymes

Digestive enzymes are crucial for digestive processes, allowing protein, carbohydrate

and fat degradation into smaller and simple molecules. These molecules can then be

absorbed and transported into tissues, by the circulatory system, and used for growth,

tissues repair and reproduction (Furné et al. 2005).

There are several factors that affect the activity of digestive enzymes. These include

diet composition (Debnath et al. 2007; Santigosa et al. 2008; Chatzifotis et al. 2008;

Cedric 2009), age (Kuz´mina 1996b) and environment conditions (Zhi et al. 2009). The

structure of digestive tract and digestive enzymes distribution are closely related (i.e.

the gastrointestinal tract of herbivorous species is longer than the carnivorous ones)

(De Almeida et al. 2006). Quantifying the activity of digestive enzymes is a useful way

to provide information on the nutritional value of diets and possible interaction between

anti-nutritional factors and digestive enzymes of fish when fed formulated diets (Refstie

et al. 2006, Corrêa et al. 2007).

Amylase

All fish species seem to possess the enzymatic apparatus necessary to hydrolyze and

absorb simple and complex carbohydrates. Digestion and absorption takes place by

the same routes in herbivores, omnivores and carnivores species. The α-amylase (EC

3.2.1.1) is a key enzyme for carbohydrate digestion. It acts on complex

polysaccharides, like starch and glycogen, hydrolyzing them up into maltotriose and

maltose, a combination of branched oligosaccharides and some glucose

(Papoutsoglou and Lyndon 2003).


In mammals, amylase is produced by the salivary and pancreatic cells, while in fish it

has been identified in pancreatic juice, in the stomach and in the intestines but the

main producers seems to be the pancreas and the liver (Klahan et al. 2009). Amylase

activity has been found in most tested tissues, including the heart (Hokari et al. 2003).

Together with other pancreatic enzymes, α-amylase activity is detected in the lumen of

the intestine, in the chyme and connected to mucosal membranes (Hoehne-Reitan et

al. 2001; Krogdahl 2004). The highest values of enzyme activity are usually reported in

the pyloric caeca (Correa et al. 2007, Gai et al. 2012). Pérez-Jiménez et al. (2009)

found increased activity in pyloric caeca of juvenile dentex (Dentex dentex) fed

increasing levels of carbohydrates and low levels of lipids in the diet. This is plausible

since starch digestion and glucose absortion occur mainly in the anterior part of the

intestine (Lundstedt et al 2004).

The pancreatic tissue is considered to be the source of amylase detected in the

medium and distal intestine; however, the origin of amylase present in the proximal

portions of the gastrointestinal tract is not properly documented (Krogdahl 2004).

Amylase present in the proximal regions may however be of pancreatic origin because

the species in question mostly lack distinct stomach pouches. Thus, it was not possible

to identify the origin of amylase and distinguish the contribution of dietary amylase or

the reflux of lower intestine. Moreover, some enzymes may be supplied by the diet or

produced by intestinal bacteria, and these exogenous contributions can be substantial

(Caruso et al. 2009).

Surprisingly, little is known about amylases and other carbohydrases and even less

about its regulation. Herbivorous and omnivorous species appear to digest starchy

components of vegetable feedstuffs more efficiently than carnivorous species (De

Almeida et al. 2006; Al-Tameemi et al. 2010). In herbivorous (Boops boops) and

omnivorous species (Cyprinus carpio, Carassius auratus, Tinca tinca and Pagellus

erythrinus), α-amylase activity is higher than in carnivores species (Oncorhyncus

mykiss, Sparus aurata, Anguilla anguilla) (Fernandez et al. 2001; Hidalgo et al. 1999;

De Almeida et al. 2006). Thus, feeding habits have great impact on amylase activity

(Horn et al. 2006). As demonstrated in European sea bass, rainbow trout and

paddlefish (Polyodon spathula), amylase activity in fish is directly related to the level of

carbohydrates in the diet and feeding intensity (Corrêa et al. 2007; Caruso et al. 2009;

Ji et al. 2012). However, it appears that some ingredients such as rice protein

concentrate included in large quantities in the feed may exert an inhibitory effect on

amylase activity, probably due to the presence of anti-nutritional factors (Gai et al.

2012).


The characteristics of amylase differ among species in relation to optimum pH for

maximum activity and temperature stability. The optimum pH for maximum activity of

intestinal amylase has been shown to be 6.0-7.5 in different species (Li et al. 2006).

Papoutsoglou and Lyndon (2005) observed in vitro that higher levels of carbohydrates

and lower temperatures decrease amylase activity. Gilthead sea bream seems to have

the ability to regulate the amylolitic activity to compensate differences in temperatures

(Couto et al. 2012). Fish amylases also show differences in the dependence on ions

and ion concentrations (Munilla-Moran and Saborido-Rey 1996a).

Lipase

The general digestive process of lipids involves their extracellular hydroliysis in the

stomach, intestine and cecal lumen by a variety of lipases and colipases (Higgs and

Dong 2000).

Lipase (E.C.3.1.1.3) catalyses the breakdown of triacylglycerol into diacylglycerol and

monoacylglycerol (Savona et al. 2011). Lipase activity in fish has been found in

pancreas extracts, pyloric ceca and upper intestine but can extend to the distal part of

the intestine, decreasing progressively its activity (Klahan et al. 2009). This lipase

activity may have pancreatic or gastric origin, resulting in adsorption of enzyme in the

intestinal mucosa or can be secreted by intestinal cells (Wong 1995).However, the

pyloric ceca and anterior intestines seem to be the primordial sites of lipid hydrolysis

(Halver and Hardy 2002). Nevertheless, lipase activity can increase distally in intestine

of some fish species perhaps as a lipid-scavenging mechanism (German and Bittong,

2009). Despite of all fat-digestive enzymes are known to act in alkaline media (7-9)

(Tramati et al. 2005; Klahan et al. 2009), in some species like the Siberian sturgeon

there is hydrolysis by lipase in the stomach (Halver and Hardy 2002). It is improbable

that lipolytic activity found in the stomach to be of pancreatic origin, suggesting that this

organ is also a source of lipases; also, it can never be excluded any possible lipolytic

activity from bacteria present in the digestive tract of fish (Caruso 2009). In Diplodus

puntazzo, lipase activity was detected in all regions of the gut, indicating a uniform

distribution in the entire gut system (Tramati et al. 2005). Lipase may play a relatively

minor role in lipid digestion in some fish species (Savona et al. 2011).

Several authors suggested that lipase presence is greater in carnivorous than in

omnivores or herbivores fish because carnivorous species consume fat-rich food

(Tengjaroenkul et al., 2000; Furné et al., 2005). This fact might suggest that the type of

diet could influence the production of lipases in adult fish (Ji et al. 2012; Kuz'mina et al.

2008). The largest class of lipids present in fish diets is triacylglycerol class. The lipase


activity is directly related to triglycerides (TG) and phospholipids (PL) levels present in

the diet as shown in sea bass larvae fed diets which contain different levels of these

lipid fractions (Cahu et al. 2003; Zambonino Infante and Cahu 2007 ; Savona et al.

2011).

Proteases

The digestive proteases are polyfunctional enzymes that catalyze the hydrolytic

degradation of proteins (Garcia-Carreno and Hernandez-Cortes 2000). Proteases also

activate the released zymogens of a large number of digestive enzymes into their

active form (Bureau et al. 2002). In the EC system for the enzymatic nomenclature, all

proteases (peptide hydrolases) belong to the subclass 3.4, which is divided into 3.4.11-

19 (exoproteases) and 3.4.21-24 (endoproteases) (Nissen, 1993). Endoproteases

disrupt in the middle of polypeptide chain, while exoproteases hydrolyze the free ends

of peptide chains (Bureau et al. 2002). The exopeptidases, particularly

aminopeptidases, are mostly intracellular or membrane bound and cuts amino acids

from the amino end of a peptide chain one at a time (Bureau et al. 2002). The high

catalytic efficiency at low temperatures and low thermal stability are some of the

differences in properties of proteases from marine and terrestrial animals (Klomklao et

al. 2005).

In fish digestive organs have been found proteases such as pepsin, gastricsin, trypsin,

chymotrypsin, collagenase, elastase, carboxypeptidase and carboxyl esterase (Dimes

1994; Simpson 2000). In the fish digestive system, the two major groups of proteases

are pepsin and trypsin. Pepsin has been identified as the major acidic protease in fish

stomach and is the first proteolytic enzyme to break large peptide chains

(Tengjaroenkul et al. 2000).Trypsin and chymotrypsin are the major alkaline proteases

in the intestine (Caruso 2009; Natalia et al. 2004).

The distribution of proteases varies between species and organs. Several studies show

high activity in the acidic pH region, in the stomach, and in the alkaline pH region, in

the intestine (Alarcón 1998). Proteolitic activities at low pH (1-3) have been reported in

species with a clear stomach region and high pepsin secretion (Chong et al. 2002).

Pepsins from several important species like tilapia (Yamada et al. 1993), dentex

(Jimenez et al. 2009) or European seabass have been documented (Eshel et al. 1993).

Pyloric caeca may be related to the need of retaining feed for the acid secretion

neutralization. However, this gastrointestinal region is also associated to alkaline

proteases storage until the acid bolus is neutralized by other pancreatic secretions

(Alarcón et al. 1998). In fact, high proteases activities at alkaline pH (8-10) have also


been reported in several fish species like rainbow trout (Kristjansson and Nielsen 1992)

and seabass (Eshel et al.1993)

The expression of proteases in higher trophic level species increases with age

(Kuz'mina et al. 2008) and in lower trophic level species this enzymatic activities are

brought down (Ugolev and Kuz'mina 1993a). Usually, carnivorous fish species have a

short intestine, with higher protease activity than herbivores (Lazzari et al. 2010) and

the level of proteolytic activity seems to be related with the fish species growth rate

(Hidalgo et aI. 1999). Some authors suggested that vegetable protein sources included

in the diet can decrease proteolytic activity (Venou et al. 2003; Santigosa et al. 2008).

The pancreas secretes enzymes with alkaline protease activity as inactive proenzymes

mixed with digestive juice with a basic pH. A peptide located in the active site need to

be released for the activation of this enzyme. In fish, this family of alkaline enzymes

includes the serine proteases (trypsin, chymotrypsin, elastase and collagenase). In

pancreatic tissue most of the trypsin and chymotrypsin are present as trypsinogen and

chymotrypsinogen. Trypsinogen is activated by enterokinase with a release of a

peptide located at the active site. The active trypsin then activates other digestive

enzymes like chymotrypsin (Bureau et al. 2002). These enzymes become active in

existing pyloric caeca and proximal intestine and hydrolyze the protein (Santigosa et al.

2008).

Trypsin, specifically hydrolyses the carboxyl side of arginine and lysine peptide bond

(Klomklao et al. 2006a).This digestive enzyme, contributes to 40-50 % of the overall

protein digestion activity in carnivorous fishes (Eshel et al. 1993). The optimal pH range

for this enzyme is between 7.0 and 9.0 (Jimenez et al., 2009). At low pH values the

enzyme denatures, alters its conformation and, therefore, cannot bind correctly to the

substrate (Klomklao et al. 2006a).

Chymotrypsin has much broader specificity than trypsin. This digestive enzyme

hydrolyses peptide bonds close to hydrophobic amino acids such tyrosine,

phenylalanine and tryptophan (Neurath 1989). Although the activation pH is similar to

trypsin, chymotrypsin seems to be present at pH values between 7.0 and 8.0 in several

fish species (Jimenez et al. 2009).

It is known that trypsin and chymotrypsin secretion occur in response to food ingestion

and they complete the protein hydrolysis when the food reaches the intestine (Savona

et al. 2011). Trypsin activities were found generally higher in carnivorous species while

chymotrypsin activities were higher in omnivorous and herbivorous species (Jonas et


al. 1983; Chong et al. 2002). Nevertheless, both types of proteases play a cooperative

role in protein digestion at intestinal tract (Chong et al., 2002).

Aims Currently, the aquafeed industry seeks to reduce the inclusion of fishmeal in diets. This

approach is justified by the high price of this raw material, the fluctuations of its

availability in the market and environmental concerns. Thus, feedstuffs such as Dried

Distillers Grains with Solubles (DDGS) emerged as potential substitutes.

This study has the following main objectives:

- Evaluate the apparent digestibility coefficient of dry matter, protein, lipids and

energy of DDGS in European sea bass and meagre.

- Assess the effect of DGGS incorporation in the diets in digestive enzymes

activity: amylase, lipase and total proteases.

- Determine if the DDGS inclusion in the diet might have some influence on

proteolytic activity at different pH (8,9 and 10).

Materials and methods

Ingredient composition

The proximate composition of ingredients tested in this trial is present in table 1.

Proximate composition and energy content of both sources of DDGS were very similar.

Dry matter content was similar in all raw materials, but protein content was

considerably higher in fishmeal that in DDGS. Lipid and gross energy content was

higher in DDGS than in fishmeal. Ash content was markedly higher in fishmeal and

concurrently phosphorus content was also higher. Crude fiber and starch in both DDGS

was quite similar, but slightly higher in DDGS2. On the concrary, acid and neutral

detergent fibers contents were higher in DDGS1.


Table 1: Proximate composition (% dry matter) of the experimental ingredients

1 Steam Dried LT, Pesquera Diamante, Peru

2DDGS1: corn dried distillers grains with soluble from Biocarburantes de Castilla y Leon, Spain

3DDGS2: corn dried distillers grains with soluble from Pannonia Ethanol, Hungry

Experimental diets

A reference diet was formulated containing 45% protein, 16% lipids, and 1% chromium

oxide, included as an inert digestibility marker. Two experimental diets were then

formulated containing a mixture of 70% of the reference diet and 30% of the test

ingredient, i.e. corn DDGS obtained from two different producers. DDGS were supplied

by Pannonia Gold, Hungary (www.pannoniagold.com), and by Biocarburantes Castilla

y Leon, Spain (www.abengoabioenergy.com). All dietary ingredients were finely ground

and well mixed. Mixtures were then dry pelleted without steam using a laboratory pellet

mill (California Pellet Mill, Crawfordsville, IN, USA) through 3 mm die. After dried in an

oven for 24h at 35ºC, pellets were sieved and stored in a freezer until use. The

ingredients and proximate composition of the experimental diets are presented in table

2.

Due to inherent feed formulation, diet composition of reference and test diets was

considerably different. The protein content was higher in the reference diet than that in

the experimental diets (DDGS1 and DDGS2). Reference and DDGS2 diets had similar

lipid content, which was higher than in DDGS1 diet. Gross energy content was similar

among diets. Ash content was higher in reference diet and by consequence also its

phosphorus content. Crude fiber content was almost equal in DDGS1 and DDGS2 diets.

Chromium oxide and starch were higher in reference diet than in the experimental

diets.

Dry

matter Protein Lipids

Gross Energy (kJ/g)

Ash Phosphorus Crude fiber

Acid detergent

fiber

Neutral detergent

fiber Starch

Fish meal

1

89.0 70.7 9.2 17.9 19.2 2.5 - - - -

DDGS12 89.2 30.4 11.8 20.0 4.7 0.5 7.2 14.6 42.4 0.5

DDGS23 88.7 29.4 12.8 19.6 4.9 0.78 7.8 13.8 39.3 2.9


Table 2: Composition of the experimental diets.

1 Steam Dried LT, Pesquera Diamante, Peru.

2Cerestar, France.

3 Vitamins (mg kg

−1 diet): retinol, 18,000 (IU kg

−1 diet); calciferol, 2000 (IU kg

−1 diet); alpha tocopherol, 35; menadion

sodium bis., 10; thiamin, 15; riboflavin, 25; Ca pantothenate, 50; nicotinic acid, 200; pyridoxine, 5; folic acid, 10;

cyanocobalamin, 0.02; biotin, 1.5; ascorbyl monophosphate, 50; inositol, 400. 4

Minerals (mg kg−1

diet): cobalt sulphate, 1.91; copper sulphate, 19.6; iron sulphate, 200; sodium fluoride, 2.21;

potassium iodide, 0.78; magnesium oxide, 830; manganese oxide, 26; sodium selenite, 0.66; zinc oxide, 37.5; dibasic

calcium phsophate, 5.9 (g kg−1

diet); potassium chloride, 1.15 (g kg−1

diet); sodium chloride, 0.4 (g kg−1

diet). 5 Binder (Aquacube. Agil, England).


7DDGS2: corn dried distillers grains with soluble from Pannonia Gold, Hungry.

Diets

Reference DDGS1 DDGS2

Ingredients (% dry weight)

Fish meal1 63.2 44.2 44.2

Pre-gelatinized corn starch2 22.1 15.4 15.4

Cod liver oil 10.2 7.2 7.2

Vitamin premix3 1.0 0.7 0.7

Choline chloride (50%), 0.5 0.4 0.4

Mineral premix4 1.0 0.7 0.7

Binder5 1.0 0.7 0.7

Chromic oxide 1.0 0.7 0.7

DDGS1 6 − 30 −

DDGS2 7 − − 30

Proximate composition (% dry matter)

Dry matter 95.5 93.8 94.6

Protein 45.6 41.2 41.3

Lipids 15.9 13.6 15.4

Chromium oxide (Cr2O3) 0.82 0.53 0.59

Energy (kJ/g) 20.0 20.7 20.8

Ash 14.8 11.5 11.6

Phosphorus 1.77 1.58 1.49

Crude fiber - 2.0 2.1

Starch 13.6 12.7 9.0


Fig 3: Meagre juveniles in experimental tanks.

Fig 4: Thermo-regulated recirculation water system

Experimental animals

Two digestibility trials were performed with

European sea bass (Dicentrarchus labrax) and

meagre (Argyrosomus regius) at the Marine

Zoology Station, Porto University. Both fish

species were provided from IPMA, Algarve.

After arriving at the experimental unit, fish were

kept in the quarantine system for 3 weeks.

Then, fish were transferred to the experimental

system and acclimatized to the rearing

conditions for 15 days. During this period fish were fed a commercial diet two times a

day.

Experiment design

Experimental system consisted in a thermo-

regulated recirculation water system, equipped

with twelve 60 L fiberglass tanks, designed

according to Cho et al. (1982). A feces settling

column was connected to the outlet of each tank. A

continuously water-flow was established, at a rate

of about 4.5 L/min. During the trial, water

temperature averaged 22 ºC, salinity averaged 38

‰ and dissolved oxygen was kept above 90% of

saturation.

Nine groups of six European sea bass, with an average weight of 206 g, were

established. Diets were randomly assigned to triplicate tanks and fish were fed to

apparent satiation, twice a day (9.30 a.m and 16.30 p.m). The first 7 days of the

experimental period were used for fish adaption to the diets and then feces were

collected once a day for 24 days. Before the morning meal, feces accumulated in each

settling column were collected, centrifuged (3000 g), pooled for each tank and stored at

- 20 ° C until analysis. Thirty minutes after the afternoon meal, tanks, water pipes and

settling columns were thoroughly cleaned to remove excess feed and feces.

After the experimental period with sea bass juveniles, groups of nine meagre juveniles,

with an average body weight of 78.8 g, were randomly distributed to the same tanks

and the same experimental protocol used for sea bass was utilized.


Apparent digestibility coefficients (ADC) of protein, lipid, dry matter, energy and

phosphorus was determined by the following formula (Maynard and Loosli 1979):

( (

( )))

The apparent digestibility coefficients of the test ingredients were calculated according

to Bureau et al. (1999) as follows:

( ) (

)

Where Dcontrol = % nutrient (or kJ/g) of control diet mash (dry matter basis) and Dingr= %

nutrient (or kJ/g) of test ingredient (dry matter basis).

Fig 5: Sampling collection of biological material.

At the end of each digestibility trial, and to ensure a full intestine at the sampling time

fish were fed in a continuous manner during the sampling collection day. Then, two fish

per tank were randomly sampled, euthanized with a sharp blow to the head and

immediately eviscerated. Digestive tract was excised, and adherent adipose and

connective tissue were carefully removed. The intestine was divided in three different

portions: anterior, middle and distal. The distal part was distinguished from de mid

intestine by the increase in intestinal diameter, darker mucosa and annular rings. The

anterior and medium portions were obtained by division of the remaining intestine into

two parts. The anterior intestine represents the portion, with the pyloric caeca, directly

after the stomach. The different portions of intestine were immediately frozen in liquid

nitrogen and stored at -80°C until measurement of enzyme activity.


Chemical analyzes performed in ingredients, diets and feces

Sample Preparation

Prior to analysis, ingredients, diets and feces were dried, in an oven at 105 ºC until

constant weight and, then, finely ground to obtain a homogeneous sample.

Crude protein

The protein content (N x 6.25) of ingredients, diets and feces were determined by

Kjeldahl method following acid digestion, using a Kjeltec digestor and distillation units

(Tecator Systems, Höganäs, Sweden; model 1015 and 1026, respectively). Crude

protein was calculated by multiplying the total nitrogen content by the factor 6.25

(16gN/100 protein). Crude protein was determined by estimating the total nitrogen

content of the material, assuming that all nitrogen is proteinaceous in origin.

Approximately 400 mg of sample was added to the tubes of digestion. Two tablets

containing 1 g of sodium sulphate (Na2SO4) plus 0.05 g of selenium (Se) were added to

each sample as catalyst. The samples were digested for one hour at 450 °C with 15 ml

concentrated sulfuric acid (H2SO4) which converts organic nitrogen to ammonium

sulfate. After cooling, 20 ml of distilled water was added to each tube and the samples

were distilled in the Kjeltec distillation unit. The solution was neutralized with 50 ml

sodium hydroxide (40%) and ammonium salt converted to ammonia. Ammonia reacts

with the boric acid and the amount of ammonia was determined by titration with

hydrochloric acid (HCL) (0.2 N), in the presence of a methyl orange pH indicator.

Crude lipids

Soxtec method

The lipid content of ingredients and diets was determined by the Soxtec method,

involving a continuous extraction with petroleum ether in a Soxtec system (Tecator

Systems, Höganäs, Sweden; extraction unit model 1043 and service unit model 1046).

Approximately 500 mg of sample was placed in a thimble and positioned in the

extraction unit. Samples were boiled for 30 min in petroleum ether, rinsed for 2 hours

and the extracted lipids were completely collected in the extraction cups. After

extraction the solvent was evaporated and the extracted material weighed after drying.

Lipid content was estimated through the difference in weight of the cups before and

after extraction.

Folch method

Lipid content in feces was determined according to the gravimetric method described

by Folch et al. (1957). 200 mg of feces were weighed to a tube and 5ml of chloroform /

methanol (2:1, v / v) added; mixed in a vortex for 3 minutes and then centrifuged for 5


minutes at 2000 rpm. The upper phase was discarded in a pre-weighted centrifuge

tubes, 1 ml of water was added and homogenized in a vortex. The tubes were then

centrifugated at 3000 r.p.m. during 10 minutes. The top layer was removed and about 1

ml of Folch reagent (chloroform / methanol / NaCl 0.9%, 3:48:47) was added. The top

layer was discarded again. Then, 0.2ml of methanol was added to the remaining

solution and it was placed in an oven at 50 °C overnight. In the morning, the samples

were cooled in a desiccator and then weighed. The lipid content was calculated as the

difference in weight between the tubes before and after extraction.

Dry matter

Approximately 500 mg of sample was placed in pre-weighed crucibles. The moisture

content was determined by total weight loss of the sample, expressed as a percentage

of the original weight, after drying at 105°C until constant weight.

Ash content

After determining the moisture content, samples were placed in a muffle furnace and

the ash content was determined as the inorganic residue obtained after incineration at

450°C for 16 hours.

Gross energy

The gross energy content in a sample is defined by -Δ Uc, which is the combustion

energy at constant volume (kJ / g) (Rossini, 1956). Energy content of the samples was

determined using an adiabatic bomb calorimeter (PARR Instruments, Moline, IL, USA;

PARR model 1261). Approximately 200-500 mg of sample, depending of the predicted

caloric value, was weighed, pelletized and combusted under a pressurized (2.53x106

Pa) oxygen atmosphere in the bomb. After combustion, the temperature in the 2 L

water jacket surrounding the stainless steel bomb rised and it was measured and used

to calculate the energy content in the sample. The apparatus was calibrated with

benzoic acid, the conversion factor of 1 cal = 4.1814 Joule was applied.

Starch content

The starch content was measured by an enzymatic procedure, according to Thivend et

al. (1972). Quantitative enzyme hydrolyzes of starch by amyloglucosidase was

performed in dimethyl sulfoxide (DMSO) buffer solvent and the glucose released was

measured using a Spinreact commercial kit (ref: 1001200). In this kit hexoquinase (HK)

catalyzes the phosphorylation of glucose to glucose-6-phosphate (G6P) by ATP. The

glucose-6-phosphate is then reduced to 6-phosphogluconate in the presence of

glucose-6-phosphate dehydrogenase (G6P-DH) with the subsequent reduction of

NAD+ to NADH.


Glucose + ATP Glucose-6-phosphate + ADP

G6P + NAD+ 6-Phosphogluconate + NADH + H+

Chromic oxide

Chromium oxide content in diets and feces was quantified by acid digestion, according

to Furukawa and Tsukahara (1966). 50 mg of sample were weighed and placed in 100

ml volumetric flasks. Then, 5 ml of HNO3 was added and flasks content was digested in

a heating mantle at 220 ° C until the acid volume was reduced to half (approximately

30 min). Then, another 5 ml of HNO3 was added to ensure a complete digestion of the

organic matter. The flasks were allowed to cool and subsequently,3 ml of perchloric

acid (HClO4) were added to the cold flasks. If flasks are still hot and/or an incomplete

digestion of organic matter has occurred, an explosive reaction may happen. The

flasks are placed again on the heating mantle until the green solution becomes lemon

yellow. Then, flasks were cooled and its content was poured into 25 ml volumetric

flasks. The spectrophotometer was adjusted with the blank and then reading of the

samples at 350 nm was performed. With the standard line obtained by oxidation with

the acid technique, chromium oxide content of the sample was calculated using the

following standard line:

Where y represents optical density at 350 nm and x represents the content of

chromium oxide of the sample in mg/100 ml.

Crude fiber

Crude fiber is the organic residue that is obtained after treating the samples with diluted

sulfuric acid, diluted sodium hydroxide and a lipid diluent. For that purpose, 0.5 g of

sample (C) was weighed and placed in a digestion flask. Thereafter 50 ml of boiling

sulfuric acid was added and the flask placed in a heating mantle for 30 minutes. Then,

the flask content was transferred to centrifuge tubes and centrifuged for 15 minutes.

The precipitate was again placed in the digestion flasks and 50 ml of boiling sodium

hydroxide solution was added and the flask placed in a heating mantle for another 30

minutes. Thereafter, the flask content was filtered with a filter funnel and the flask walls

washed twice with boiling water. The filtered sample was washed with 30 ml of boiling

petroleum ether. The filter was placed in the oven for 24 hours and in the next day was

cooled in the desiccator (A), weighed and placed in muffle furnace for 16 hours. In the

next morning, was cooled in the desiccator and weighed again (B). The crude fiber

content was determined with the following equation:

HK G6P - DH


( ) (( )

)

Phosphorus

Total phosphorus content in ingredients, diets and feces were determined based on a

colorimetric method (Silva and Queiroz 2002). Briefly, phosphorus content was

dissolved in aqueous solution and in contact with ammonium molybdate produced

ammonium phosphomolybdate. This compound was reduced in the presence of

ascorbic acid, forming colloidal oxides proportionally to the concentration of

phosphorus in the solution. Excess ammonium molybdate does not react with ascorbic

acid. The quantity of phosphorus was determined by measuring the intensity of blue

color, which is produced by the formation of colloidal oxides. The color intensity

developed by phosphomolybdate depends on the acidity and temperature of the

solution, being stable in acidic solution.

Digestive Enzymes activities

For the enzymatic activity measurement, each intestine was homogenized in ice, with

an Ultra Turrax, and centrifuged at 3,300 g, for 10 min at 4°C. The supernatant was

collected and stored at -80°C, until analyses. All enzyme activities were determined

using a PowerWavex microplate scanning spectrophotometer (Bio-Tek Instruments,

USA).

Fig 6: Collecting the sea bass intestine for determination of enzymatic activity.

Total protein activity

Total protease activity was measured by the casein-hydrolysis method according to

Hidalgo et al. (1999) at three different pH values, within the physiological range of

digestive tract (Hidalgo et al. 1999; Furné et al. 2005). For each pH, the following

buffers were used: 0.1M tris HCL buffer, for pH 8.0 and 9 and 0.1 M glycine-NaOH for


pH 10. The reaction mixture containing casein (1% w/v; 0.125 ml), buffer (0.125 ml)

and homogenate supernatant (0.05 ml) was incubated for 1 hour at 37°C and stopped

by adding 0.6 ml trichloroacetic acid (8% w/v) solution. After being kept for 1 h at 2°C,

samples were centrifuged at 1800 g for 10 min and the supernatant absorbance was

read at 280 nm against blanks. A control blank for each sample was prepared adding

the supernatant from the homogenates after incubation. Tyrosine solution was used to

establish a calibration curve. One unit of enzyme activity was defined as the amount of

enzyme needed to catalyze the formation of 1.0 µmol of tyrosine per min.

Amylase activity

Amylase (E.C.3.2.1.1) activity was measured using a Spinreact kit (ref. 41201). The

method comprises in the hydrolyzis of 2-chloro-4-nitrophenyl-α-D-maltotrioside by α-

amylase; this reaction releases 2-chloro-4-nitrophenol(CNP) and forms 2-chloro-4-

nitrophenyl-α-D-maltoside (CNPG2), maltotriose (G3) and Glucose(G). The rate of 2-

chloro-4-nitrophenol formation, measured photometrically, is proportional to the

catalytic concentration of α-amylase present in the sample.

10 CNPG3 9 CNP + 1 CNPG2 + G3 + G

The reaction mix consisted of 200 µl of amylase reagent (2-chloro-4-nitrophenyl-α-D-

maltotrioside, CNPG3) and 10 µL of sample homogenate. This mixture was incubated

at 37 ºC during 30 seconds and absorbance (ΔDO/min) was read at 1 minute intervals

during 3 minutes at 405 nm and 37°C.

Lipase activity

Lipase (EC 3.1.1.3) activity was measured using a Spinreact Kit (Ref. 1001274). This is

a new procedure at the Nutrimu laboratory, so it was needed to validate and adjust this

method to fish intestine samples. Validation was accomplished by ensuring linearity of

lipase activity in the same sample with different dilutions and comparing it to the

quantification of the calibrator activity using the molar extinction coefficient of the

reaction product (Methylresorufin) and the theorical activity expected in the kit

calibrator.

In this method, the pancreatic lipase, along with the colipase, desoxycholate and

calcium ions, hydrolyses the substrate 1-2-O-dilauryl-rac-glycero-3-glutaric acid-(6'-

methylresorufin)-ester.

Amilase


1-2-O-dilauryl-rac-glycero-3-glutaric-(6' -methylresorufin)-ester

1-2-O-dilauryl-rac-glycerol + Glutaric-6'-methylresorufin-ester (no stable)

Glutaric acid + Methylresorufin

The rate of methylresorufin formation was quantified photometrically and it is

proportional to the concentration of catalytic lipase present in the sample homogenate.

The reaction mix consists in 200µl of R1(TRIS pH 8.3, colipase, desoxycholate and

taurodesoxycholate), 40 µl of R2 (tartrate pH 4,0, lipase substrate and calcium chloride

(CaCl2)) and 20 µl of sample. This mixture was incubated for 30 seconds and the

sample absorbance (ΔDO/min) was then read at 10 seconds intervals, during 11

minutes, at 580 nm and 37°C.

Specific enzymatic activity

Enzyme activity of total proteases, amylase and lipase was expressed as specific

activity (units per milligram of soluble protein; one unit (U) of activity was defined as

µmol of product generated per minute). Soluble protein concentration was determined

using Bradford's method (1976), with bovine serum albumin solution as standard.

Amylase and lipase activities were determined using the formula:

( )

Where (Δ DO/ Δ t ) is the decrease or increase of optical density / minute, Vt is the total

reaction volume, f is the correction factor for the dilution of the extract, Ex is the molar

extinction coefficient, 10-3 is the conversion factor of liter to milliliter, 10-9 is the

conversion factor from mol to nmol, Ve is the volume of added extract in ml, d is the

length of the light beam through the microplate (0.79 for lipase activity and 0.675 for

amylase activity) and P is the mg of protein per ml.

OH-

Lipase


Fig 7: Enzymatic activity determination in microplates.

Statistics analyses

All statistical analyses were done using the SPSS 21.0 software package for Windows.

Data are presented as means ± standard error of the mean (S.E.M). Before analysis,

ADC values and enzymatic activity were subjected to arcsin square root transformation

and ln or √x transformation, respectively, to meet ANOVA requirements

(homoscedasticity and normality).

Differences in ADC data was accomplished by one-way analysis of variance (ANOVA)

for each species. Differences in ADC between species, diets and ingredients were

performed by two-way ANOVA. For each species, differences between diets and

intestine section, in digestive enzymes, were analyzed using a two-way ANOVA. If

interaction was significant, one-way ANOVA was performed for diets and intestine

sections. To test differences on enzyme activities between species and dietary

treatment two-way ANOVA was performed. For each species and each intestine

section, differences on total protease activity between pH and dietary treatment diet

were analyzed by a two-way ANOVA. When p-values were significant (P<0.05), means

were compared with Tukey´s HSD test (Tukey, 1949).


Results

Diets and ingredient digestibility

Digestible basis ingredient composition

On a digestible basis the experimental ingredients have a much lower dry matter and

gross energy composition but the protein and lipids content remain almost the same

(table 3). The table data were obtained from the nutrient digestibility values media of

the two tested species.

Table 3: Digestible basis ingredient composition


2DDGS2: corn dried distillers grains with soluble from Pannonia Ethanol, Hungry

Experiment 1 – Sea bass

The ADC of diets and ingredients for sea bass are presented in table 4. In this species,

ADC of dry matter, energy and lipids of the reference diet was significantly higher than

in the experimental diets (Table 4). ADC of protein and phosphorus was significantly

higher in DDGS1 diet (92.85% and 55.63%, respectively) than in the reference diet

(91.88% and 31.65%, respectively). Regarding ingredient digestibility, DDGS1 showed

higher ADC for all analysed parameters than DDGS2.

Dry

matter Protein Lipids

Gross Energy (kJ/g)

DDGS11 57.5 27.6 10.4 13.5

DDGS22 50.5 27.0 10.8 11.9


Table 4: Apparent digestibility coefficients (ADC %) of nutrients and energy of the

experimental diets and test ingredients in sea bass1.

Sea Bass

Ref. diet

DDGS1

diet DDGS2

diet SEM

ADC diets

Dry matter 83.66b 77.76a 75.92a 1.24

Protein 91.88a 92.85b 91.93a 0.31

Energy 93.93b 86.11a 84.94a 1.33

Lipids 98.93b 96.55a 95.93a 0.43

Phosphorus 31.65a

55.63b

44.34ab

4.32

ADC ingredient

Dry matter 63.27b 56.66a 2.28

Protein 96.26b 92.09a 1.17

Energy 67.88b 63.58a 1.44

Lipids 89.01b 87.23a 1.04 1One-way ANOVA: Means in the same row with different superscript letters are significantly different (P < 0.05).

2DDGS1,2: corn dried distillers grains with soluble from Biocarburantes de Castilla y Leon, Spain and Pannonia Gold,

Hungry, respectively

Experiment 2 - Meagre

The ADC of diets and ingredients for meagre is presented in table 5. ADC of dry

matter, energy and lipids of the reference diet was significantly higher than that of the

experimental diets. ADC of protein was higher in DDGS1 diet (90.96%) than in the

reference diet (88.99%). ADC of phosphorus was higher in the experimental diets

(60.36% and 52,04%) than in the reference diet (23.6%). Regarding ingredient

digestibility, DDGS1 showed higher ADC for all parameters analysed than DDGS2.

Ingredient digestibility – comparison between species

The ADC of dry matter, protein and energy of the reference diet were significantly

higher in seabass than in meagre. Regarding ADC of DDGS, no significant differences

were observed between species.


Table 5: Apparent

digestibility coefficients (ADC%) of nutrients and energy of

experimental diets and test ingredients in meagre

1.

1One-way ANOVA: Means in the same row with different superscript letters are significantly different (P <0.05).


Hungry, respectively.

Digestive enzymes

The proteolytic, amylolytic and lipolytic activities (mU mg protein-1) in the anterior,

medium, distal and total intestine of seabass and meagre juveniles fed the

experimental diets are present in tables 6 and 7, respectively. These tables express

proteolytic activity measured at pH 8. Total intestine activity was obtained by the sum

of the activity in anterior, medium and distal intestine.

Irrespectively of dietary treatment and species, digestive enzymes activity was higher

in medium intestine than in the other intestine regions. In sea bass, total protease

activity was not different among groups, while total lipase activity was higher in fish

feed with DDGS2 diet than the other diets. Total amylase activity was higher in fish fed

DDGS2 diet than DDGS1 diet but did not differ from that of fish fed the reference diet.

In meagre juveniles there were no differences between diets in total proteolytic activity.

Total lipase activity was higher in fish fed DDGS2 diet than the other diets, while total

amylase activity was higher in fish fed DDGS2 diet than the reference diet.

Meagre

Ref. diet

DDGS1

diet DDGS2

diet SEM

ADC diets

Dry matter 79.91b 75.77a 73.41a 1.03

Protein 88.99a 90.96b 89.66ab 0.35

Energy 91.96b 84.60a 81.92a 1.53

Lipids 98.35b 95.83ab 94.59a 0.62

Phosphorus

23.63a

60.36b

52.04b

5.75

ADC ingredient

Dry matter 65.63b 57.25a 2.17

Protein 97.87b 91.85a 1.6

Energy 67.40b 58.04a 2.60

Lipids 87.86b 82.01a 2.59


Table 6: Specific activities of protease, lipase and amylase (mU mg protein-1

) in different intestine sections (anterior (IA), medium (IM), distal (ID) and total) of sea bass fed the experimental diets

1.

Sea bass

Ref. diet DDGS1 diet DDGS2 diet SEM

Proteases

IA 55.18 72.16 54.72 5.76

IM 152.6 58.98 139.75 13.34

ID 51.13 37.79 61.31 5.67

Total2 258.92 168.94 250.71 15.58

Lipase

IA A1.04 A1.39 A1.57 0.2 IM BC8.15ab AB3.43a BC18.27b 2.19 ID AB2.12 AB2.44 AB7.75 0.18 Total2 C11.31a B7.26a C27.59b 0.37

Amylase

IA A29.61 AB39.45 A38.85 4.82

IM AB63.67ab A27.77a B109.42b 12.27

ID A12.42 A10.45 A20.31 3.16

Total2 B100.81ab B77.68a B168.58b 15.37 15.37

Two-way ANOVA

section Diet Interaction Section

IA IM ID Total

Proteases *** N.S N.S A B A C Lipase *** *** ** - - - - Amylase *** ** * - - - -

1Two-way ANOVA: N.S: non-significant (P > 0.05); ** P < 0.01; *** P < 0.001). If interaction was significant, a One-Way

ANOVA was performed for each factor (diet and section). Means in same row with different superscript letters represent significant differences between diets and in the same column means with different capital letters represent significant differences between intestine sections (P < 0.05) 2Total intestinal tract: sum of the activity in anterior, medium and distal intestine sections.




Table 7: Specific activities of protease, lipase and amylase (mU mg protein-1

) in different intestine sections (anterior (IA), medium (IM), distal (ID) and total) of meagre fed the experimental diets

1

1Two-way ANOVA: N.S: non-significant (P > 0.05); ** P < 0.01; *** P < 0.001). If interaction was significant, a One-Way

ANOVA was performed for each factor (diet and section). Means in same row with different superscript letters represent significant differences between diets and in the same column means with different capital letters represent significant differences between intestine sections (P < 0.05). 2Total intestinal tract: sum of the activity in anterior, medium and distal intestine sections.



Meagre

Ref.

diet

DDGS1

diet

DDGS2

diet SEM

Proteases

IA A147.89a AB185.50ab A223.33b 12.58 IM B348.34 B398.72 A345.65 33.6 ID A170.03 A133.74 A166.18 23.64

Total2 C666.25 C717.96 B798.65 36.07

Lipase

IA 1.27 1.60 1.76 0.19 IM 3.06 6.03 9.23 0.74 ID 2.25 1.52 3.76 0.35

Total2 6.58 9.16 14.75 0.98

Amylase

IA AB17.32 A15.30 A22.17 2.24 IM B21.72a B37.07b B42.64b 2.93 ID A10.25 A7.35 A14.25 1.31

Total2 C49.29a C59.72ab C79.06b 4.34

Two-way ANOVA Section

Diet

interaction Section Diet

IA IM ID Total Ref DDGS

1 DDGS

2

Proteases *** ** *** - - - - - - - Lipase *** ** N.S A A A B a a b

Amylase *** ** * - - - - - - -


A comparative analysis of total proteases, amylase and lipase activity(mU mg protein-1)

between sea bass and meagre fed the experimental diets is present in the table 7. This

table expresses proteolytic activity measured at pH 8.

Independently of diet, proteolytic activity was higher in meagre than in sea bass while

amylase activity was higher in sea bass. Lipase activity was also higher in sea bass

than in meagre, though differences between species were not very important.

Overall, and independently of species, lipase and amylase activity was higher in fish

fed diet DDGS2 diet than the other diets, and no differences among diets was observed

for protease activity.

Table 8: Two-way ANOVA analysis of effect of intestine section and diet on the specific

activities of protease, lipase and amylased (mU mg protein-1

) in seabass and meagre1.

Sea Bass Meagre Sea bass / Meagre Reference Diet

Proteases 258.92 666.25 0,39 Lipase 11.31 6.58 1,72 Amylase 100.81 49.29 2,05

DDGS1 Diet Proteases 168.94 717.96 0,24 Lipase 7.26 9.16 0,79 Amylase 77.68 59.72 1,30

DDGS2 Diet Proteases 250.71 798.65 0,31 Lipase 27.59 14.75 1,87 Amylase 168.58 79.06 2,13

Two-Way ANOVA

Species Diet Interaction

Reference diet

DDGS1 diet

DDGS2

diet

Proteases *** N.S N.S - - -

Lipase * *** N.S a a b

Amylase *** ** N.S a a b 1Two-way ANOVA:N.S:non-significant (P>0.05);** P<0.01; *** P < 0.001.

3 Total intestinal tract comprises the sum of anterior intestine, medium intestine and distal intestine activities.




In both species a similar profile activity of proteases relating to pH values was

observed in the anterior intestine (Fig. 8). In the anterior intestine of sea bass higher

protease activity occurred at lower pH values (8 and 9). However, in the medium

intestine the highest proteolytic activity was found at pH values of 8 and 10. In the

anterior intestine of meagre the highest proteolytic activity was observed at pH 8 and 9,

while in the medium intestine that was found at pH 9 and 10. In both species, protease

activity in the distal intestine was not affected by pH.

Diet influenced protease activity in the two species, except in the distal intestine of sea

bass and the medium intestine of meagre. In the anterior intestine of sea bass the

highest proteolytic activity was observed in DDGS1 diet, while in the medium intestine

fish fed this diet showed the lowest protease activity. On the distal intestine there are

no longer significant differences in proteolytic activity between diets. In the anterior

intestine of meagre proteolytic activity was higher in DDGS2 diet but not significantly

different from proteolytic activity of DDGS1 diet while in the distal intestine there was a

decrease in activity in the DDGS1 diet. There were no interactions between pH and diet

in the protease activity in any intestine section.


Fig 8.Variation in the activity of proteases in different sections of the intestine (anterior intestine (IA), medium

intestine (IM) and distal intestine (ID)) and different pH (8,9 and 10).

20

45

70

95

120

8 9 10

mU

mg

Pro

tein

-1

pH

Sea Bass (IA)

diet2

ref.diet

diet3

50

110

170

230

290

8 9 10

mU

mg

Pro

tein

-1

pH

Meagre (IA)

diet2

ref.diet

diet3

0

60

120

180

240

8 9 10

mU

mg

pro

tein

-1

pH

Sea bass (IM)

diet2

ref.diet

diet3300

410

520

630

740

8 9 10

mU

mg

Pro

tein

-1

pH

Meagre (IM)

diet2

ref diet

diet3

0

20

40

60

80

100

8 9 10

mU

mg

pro

tein

-1

pH

Sea bass ID

diet2

ref.diet

diet3

100

140

180

220

260

8 9 10

mU

mg

Pro

tein

-1

pH

Meagre (ID)

diet2

ref.diet

diet3


Discussion Fishmeal is the most commonly protein source used in aquafeeds due to its high

protein content, essential amino acid profile, n-3 HUFA contents, high digestibility and

palatability and no anti-nutritional factors. The aquaculture industry urgently needs to

find alternative ingredients to reduce or eliminate the use of fish meal in aquafeeds.

New feedstuffs are required due to fish meal production fluctuations, constant demand

(SOFIA 2006), restrictions on the use of animal origin protein in feed formulation and

elevated costs that most contribut to the fish feeds final price (Josupeit 2008).

Evaluation of diets and ingredients digestibility has a huge relevance for the

development of a new aquaculture feed and to determine the potential of new

ingredients to incorporate those diets (Glencross et al. 2007). Currently, the modern

fish diets are formulated on digestible nutrient and energy basis, and it is impossible to

do this if accurate and precise digestibility data is not available (Glencross 2008).

The nutritional value and nutrient content of corn DDGS may be greatly affected by

source, quality of the grains, fermentation efficiency, temperature and time of expose to

drying process and quantity of distiller´s solubles added (Lim et al. 2011). Thus, it is

imperative to evaluate the nutritional value of this feedstuff, particularly its digestibility

value, rather than simply applying standard values for the raw materials before

considering their incorporation in fish diets. Indeed, although in this study the two

sources of corn DDGS used had similar proximal composition, it is known that chemical

composition of DDGS is more variable that in the original cereal, depending on the

DDGS processing technology (Schaeffer et al. 2011).

To evaluate DDGS digestibility, it was followed a method, previously suggested by Cho

et al. (1982), in which 30% of the test ingredient (DDGS) is mixed with 70% of a

reference diet. This procedure assumes that diet digestibility is additive, i.e. it is equal

to the sum of the digestibility of individual diet ingredients, and don´t exist any

interaction between ingredients or nutrients. Thus, it is presupposed that the ADCs of

the ingredients are constant, regardless of its inclusion level and that they are not

affected by other ingredients or the inclusion levels of the other ingredients. In practice,

there are interactions between ingredients and effects of these interactions on diet

digestibility and ingredients availability may be difficult to predict (Gregory et al. 2012).

In the present study, to reduce potential interference of these factors and others, such

as temperature, feed transit, fecal digestion volume and evacuation time, the reference

diet was formulated with very low content of indigestible constituents (Refstie et al.


2006; Adamidou et al. 2009). Indeed, a reference diet with high content of indigestible

fraction may compromise the applicability of this method for the evaluation of

ingredients digestibility. The adequacy of the formulated reference diet used is attested

by its high nutrient and energy ADC in both species. In this study, it was used a low-

temperature fishmeal, which preserves protein structure and amino acid profile (Davies

et al. 2011), leading to a high protein and energy digestibility of the reference diet.

Comparatively to the reference diet, DDGS diets had lower ADCs, except for protein

which was higher in DDGS1 diet. This lower ADC may be related to the carbohydrate

fraction of DDGS, particularly non starch polysaccharides (NSPs) and fiber. Many fish

species, especially carnivorous species, have a moderate capacity to digest and

metabolize carbohydrates and cannot digest fiber or NSP (Enes et al. 2011). Non-

digestible carbohydrates, if present in high quantity, may also impair digestibility of

other nutrients by reducing gut-retention time of feed and time available for nutrients

absorption (Stone 2003; Enes et al. 2011). The mechanism responsable for this effect

is primarily physical. Nutrient absorption is maximized when food has sufficient gut-

retention time to complete digestion and properly nutrients absorption (Gregory et al.

2012). However, nutrients can be absorbed only if they come into contact with

enterocytes.

In most carnivorous fishes, the intestinal tract is short, less than the length of the body,

which limit the retention time of the food. This is the case of the two target species of

this study, seabass and meagre, that have carnivorous feeding habits and short

intestine length. The high DDGS fiber content, containing more that 15% of acid and

40% of neutral detergent fiber, probably explain the decrease of dry matter and energy

digestibility in both species. Also, the high amount of NSPs present in corn DDGS

(averaging 19% soluble, and more than 17% insoluble NSP; Widyaratne and Zijlstra

2007), may also contribute to the low digestibility of dry matter and energy in DDGS

diets. Indeed, NSPs are not digested by fish and have been associated to low diet

digestibility (Francis et al. 2001). This is related with a mechanism that involves the

binding of the nutrient with bile salts, changes in viscosity and the digesta rate of

passage and / or digestive enzymes obstruction. Dietary NSP may also affect lipid

digestibility as it interfere with the micelle formation in the gastrointestinal tract (Enes et

al. 2012). The impact of NSP in digestibility depends of its functional properties on the

intestine microbiota, as NSP may be partially digested by the intestinal bacteria (Ringo

et al. 2010). A limited incorporation of low molecular weight NSP may act as prebiotic

with beneficial proprieties in fish throught the stimulation of growth and/or activity of

intestine bacteria (revised by Ringo et al. 2010). In white sea bream, for instance, it


was observed that dietary incorporation of guar gum up to 12% did not compromise

growth, feed utilization or intestine health (Enes et al. 2012). Also in red drum, it was

observed that an adequate amount on NSP in the diet acted as prebiotic enhancing

nutrient and energy digestibility (Burr et al. 2008)

The inclusion of vegetable oils in carnivorous fish diets can affect the digestive and

absortive processes (Santigosa et al. 2010). Thus, modifications in the enterocyte

membranes composition have been described in fish fed with vegetable oils (Sitjà –

Bobadilla et al. 2005). These modifications can compromise the intestinal function,

reducing lipid digestibility (Geurden et al. 2009). These facts can explain the lower

lipids digestibility found in the tested diets since part of fish oil lipids were replaced by

vegetable oils from DDGS.

Comparatively to the reference diet, dietary incorporation of DDGS1 increased protein

digestibility. This may be due to yeast fermentation process, as circa 4% of DDGS

biomass or 5.5% of DDGS protein has yeast origin (Ingledew et al. 1999; Zhou and

Davies 2010).

Approximately 60 to 70% of phosphorus (P) in cereals is bound to phytate, which is

poorly available for fish (Oliva-Teles et al. 1998). The high digestibility of P in DDGS

indicates that the availability of P in this ingredient is improved due to the phytate-P

hydrolysis. During the fermentation process of DDGS, phyatate-P may be partially

degraded, increasing P availability (Widyaratne and Zijlstra 2007). The higher P

digestibility in DDGS diets can also be related to the total dietary P content, as it is

known that ADC of phosphorus is influenced by dietary inclusion level (Buyukates et al.

2000). This also suggests that fishmeal replacement by DDGS may improve P

excretion management, preventing eutrophication of water courses. Li et al (2008a,b)

documented that several marine yeast strains isolated from the gut of sea cucumber

(Holothuria scabra) and marine fish (Hexagrammos otakii and Synecogobius hasts)

had the ability to produce large amount of extra-cellular phytase. They claimed that

such marine yeasts might play an important role in phytate degradation within marine

animals gut. By consequence, the yeasts present in DDGS may have helped to

degrade the phosphorus contained in the form of phytate and thereby increase its

digestibility.

DDGS digestibility studies in fish are relatively scarce, as this is a new ingredient for

aquafeeds, even though DDGS has long been used in terrestrial animal feeds. The

present study was the first to evaluate the digestibility of DDGS in sea bass and

meagre. DDGS digestibility was similar for both species, averaging 94.5% for protein,


86.5% for lipids and 64% for energy. These values are close to those reported for

rainbow trout (ADC of 90 and 82% for protein and lipids, respectively; Cheng and

Hardy 2004) and for Florida Pompano (63-66% energy digestibility; William et al.

2008). However, other authors reported much lower digestibility values for DDGS in

hybrid sea bass (Morone saxatilis x M chrysops) (ADC of 65 and 69 % for protein and

lipids, respectively; Thompson et al. 2008; Metts et al. 2011). Differences in DDGS

digestibility may depend of species (Thodesen et al. 2001), DDGS processing methods

or composition of the diets.

Comparatively to raw corn meal, DDGS seems to have higher digestibility. For

instance, Venou et al. (2003) in gilthead sea bream, found ADC values of protein, lipid

and energy of diets incorporating raw corn were much lower than those obtained in this

study, but ADC values were similar with extruded corn. In rainbow trout fed 10, 20 and

30% of corn meal, protein digestibility was 87.6, 90.1 and 90.2%, respectively (Ufodike

and Matty 1989). In seabass, ADC of DDGS seems to be similar to that of soybean

meal (ADC of dry matter, protein and energy averaging 65.5, 89.8 and 69.3%

respectively; Gomes da Silva and Oliva-Teles 1998). In meagre juveniles, the ADC of

protein of corn meal, corn gluten meal and soybean meal (99.6%, 89% and 92.9%,

respectively) were similar to that obtained with DDGS in this study. However,

comparatively to DDGS, the ADC of energy and dry matter were higher in corn gluten

meal (77.8% and 78.9%, respectively) and soybean meal (73.6% and 65.1%,

respectively) but lower in corn meal (40.6 % and 49.8 %, respectively) (Olim 2012).

Even though seabass and meagre are species of a high trophic level, their digestive

capacity to utilize DDGS seems very promising.

Enzymatic investigations are crucial to clarify the effect of fishmeal replacement by

plant protein feedstuffs in fish diets, allowing evaluating if the metabolic functionality of

intestine was modified by DDGS incorporation (Palmegiano et al. 2006; Corrêa et al.

2007) as any changes in digestive enzyme level may influence digestion and

absorption of the food (Lemieux et al. 1999). Several factors may affect digestive

enzyme production in fish, such as feeding habits, food preferences, diet formulations

and anti-nutritional factors (ANFs) (Pavasovic et al. 2007).

A bibliographic comparison of digestive enzymatic activity is not easy due to different

protocols used (Hidalgo et al. 1999) and different distribution patterns of digestive

enzymes among species (Corrêa et al., 2007). In fish, nutrient absorption is known to

take place in anterior intestine and, to a lower extent, in the posterior intestine (Gai et

al. 2012). In this study, results showed generally higher levels of proteases, lipase and


amylase activity in medium and distal intestine. This uncommon elevated digestive

enzyme activity in the mid and distal intestine observed both in seabass and meagre

may be due a possible drag of the secreted mucous to this parts of the digestive tract,

as previously observed in two other carnivorous species, the rainbow trout (Gai et al.

2012) and common dentex (Pérez-Jiménez et al. 2009).

In seabass, total proteases activity was not significantly affected by the dietary

treatment, but in meagre protease activity in the anterior intestine was significantly

increased by dietary DDGS incorporation. In other species like Scylla serrate

(Pavasovic et al. 2004), Gadus morhua (Refstie et al. 2006) and Litopenaeus vannamei

(Rivas-Vega et al. 2006) proteases activity was not affected by dietary soy bean meal

at different inclusion levels. Proteases activity may be also influenced by nutrient

quality and quantity (Le Moullac et al. 1996). For instance, in seabream and rainbow

trout, fishmeal replacement by plant protein mixtures decreased trypsin and

chymotrypsin activity (Santigosa et al. 2008). However, fish may develop

compensatory mechanism and adapt its digestive physiology in response to changes in

dietary profile (Daprá et al. 2009; Lin and Luo 2011), which are in accordance to the

increase of proteases activity observed in meagre.

Amylase activity is directly related to dietary starch levels, as demonstrated in

European seabass and rainbow trout (Cahu and Zambonino Infante 1994; Corrêa et al.

2007). In the present study dietary starch was higher in the reference diet than in the

DDGS diets. Nevertheless, in meagre, higher amylase activity was observed in DDGS

diets. One possible explanation for this result is that the yeasts present in DDGS could

have worked as probiotic enhancing amylase activity and improving the starch

digestibility.

Indeed it has been suggested that an increase in the dietary lipids amount and

composition may lead to an increase in lipolytic activity (Barrington et al. 1962; Ghosh

1976; Borlongan 1990; Bazaz and Keshavanath 1993). Lipase activity is closely linked

to dietary triglycerides (TG) and phospholipids content, as previously reported for

seabass lavae (Zambonino Infante and Cahu 2007). Thus differences in the fatty acid

composition of the diets may contribute to explain differences in lipase activity in DDGS

diets (Kenari et al. 2011). Corn oils are mainly composed by complex mixtures of TG

(generally 95-97 %), before refining. After the refining process, TG increase up to 99%

(Gunstone 2011). In DDGS, a refining product, lipid and TG are concentrated as the

starch faction is removed during the fermentation processes, concentrating all other

nutrients of the ingredient. Oil is typically present at a concentration of approximately


10% in DDGS, and contains high levels of polyunsaturated fatty acids (PUFA),

particularly linoleic acid and unsaturated fatty acids (UFA) like oleic acid (Wang et al.

2007). This high content in free unsaturated fatty acids may be due to the high

temperatures of processing, combined with moisture content, acids and bases used for

pH adjustment and the high temperatures used during evaporation of thin stillage and

grains drying (Jill 2012). Unsaturated fatty acids have lower melting points than

saturated fatty acids, resulting in higher digestibility coefficients of unsaturated

vegetables oils (Olsen and Ringo 1997). The specificity of pancreatic lipase activity is

related to the acyl chain length and the degree of unsaturation, which has been shown

to have a higher preference for PUFA as substrate resulting in high lipids digestibility in

diets (Iijima et al. 1998).

In this work lipase activity seemed to be higher in mid and distal intestinal in booth

species. Lipolytic activity in fish is generally greater in the proximal part of intestine and

pyloric caeca, deceasing progressively to the end of the gut, but it can be extended into

the lower parts of intestine (Tocher 2003). However, in others carnivorous species such

as turbot (Koven et al. 1994a) and plaice (Olsen and Ringo 1997), lipolitic activity was

also found to be higher in the distal part of intestine. This may be a physiologic

adaptation to a short digestive tract and few pyloric caeca as in turbot.

Results of this study strengthen the hypothesis that fishes can modulate digestive

enzyme activities in response to changes in dietary composition (Fountoulaki et al.

2005). Several authors argue that the presence of lipases is higher in carnivoures fish

than in omnivores and herbivores fish (Tengjaroenkul et al. 2000; Furné et al. 2005). In

nature, carnivorous fish ingest large amounts of lipids and thereby lipase is needed in

higher amounts for digesting it (Chakrabarti et al. 1995). Concordantly, present results

also indicate that the two species have similar capacity to digest lipids. Even though,

optimal dietary lipid levels seem to differ between the two species, with values of up to

22% in seabass (Peres and Oliva-Teles 1999; Montero et al. 2005) and up to 17% in

meagre (Chatzifotis et al. 2012).

In the wild seas bass and meagre have similar eating habits, being described as

carnivorous and predatory species. However, the two species have different trophic

level (4.3 for meagre and 3.8 for sea bass according to Fishbase). Most authors

consider that proteolytic activity is not strictly dependent on nutritional habits being

amylase much more dependent on that (Hidalgo et al.1999). In this study, substantial

higher proteolytic activity was however observed in meagre than in seabass. This

higher protease activity in meagre can be related to growth rate, as meagre is a fast


growth fish species, reaching 1.2 kg in less than 2 years (Chatzifotis et al. 2012). Direct

relationship between digestive proteolytic activity and the growth rate is often observed

(Hidalgo et al.1999).

Generally, the amount of specific enzymes is directly related to the capacity of

digesting a nutrient. Sea bass had a higher amylase activity than meagre and this is in

accordance to its ability to digest starch. Indeed, seabass digests efficiently processed

starch, with a digestibility average of 90% (Enes et al. 2011).

Many studies in teleost fish reported that the pH 8-10 is the optimal range for alkaline

proteases in the pyloric caeca and intestine (Chong et al. 2002; Tramati et al. 2005).

Present results showed that DDGS dietary incorporation did not influence the optimum

pH for protease activity in the intestine. In both species, higher proteolitic activity was

attained in anterior intestine at pH 8-9 and in medial intestine at pH 8-10. Similar

results were reported for carp (Jonas et al. 1983), rainbow trout and Atlantic salmon

(Torrissen 1984), halibut and turbot (Glass et al. 1989), European sea bass and striped

sea bass (Eshel et al. 1993), goldfish (Hidalgo et al. 1999), and dentex (Jimenez et al.

2009). These results suggest at least two major groups of alkaline proteases. The high

activity of intestinal proteases at pH 7.0-9.0 was related to trypsin activity in

carnivorous species like Solea solea (Clark et al. 1985) and S. formosus (Natalia et al.

2004). In contrast, chymotrypsin despite having a similar pH of activation appears to be

more active at pH 7.0-8.0. An increase in trypsin activity may occur in animals fed with

vegetable feedstuffs due to long term compensation mechanisms, as described in trout

(Krogdahl et al. 1994). Changes in trypsin and chymotrypsin activities imply differential

availability of oligopeptides and amino acids, which can cause an amino acid

imbalance under these feeding circumstances. The high proteolytic activity at pH 10

may be associated with collegenase or elastase activities which are related to the

higher pH of 9.5 in Dover sole (Clark et al. 1985).

Conclusions

The ADC of the diets, except for phosphorus, was higher in sea bass than in meagre.

However, both sea bass and meagre digested well DDGS. ADC of protein (92 % and

98%) and of lipid (82 and 89 %) of the tested ingredients was high. ADC of dry matter

and energy were moderate (57 and 66%; 58 and 58%, respectively). The high fibre

content of DDGS may explain this moderate dry matter and energy digestibilities.


Due to differences in DDGS processing, it was possible to observe differences in dry

matter, protein and energy ADC between the two types of DDGS tested, reinforcing the

importance of a good characterization of raw materials before using them in formulated

diets. Diets with DDGS have lower ADC of dry matter, energy and lipids than the

reference diet. However the protein ADC in DDGS1 and phosphorus in both tested

diets was higher than in the reference diet.

The enzymatic activity data showed that DDGS affected the digestive enzymatic ability

of both species, especially lipase and amylase activity.

In summary, DDGS seems to have high potential to be included in diets for sea bass

and meagre.

References • Abowei, J.F.N., Ekubo, A.T. (2011). A Review of Conventional and Unconventional

Feeds in Fish Nutrition. British Journal of Pharmacology and Toxicology, 2(4), 179-191.

• Adamidou, S., Nengas, I., Alexis, M., Foundoulaki, E., Nikolopoulou, D., Campbell, P.,

Karacostas, I., Rigos, G., Bell, G. J., Jauncey, K. (2009). Apparent nutrient digestibility

and gastrointestinal evacuation time in European seabass (Dicentrarchus labrax) fed

diets containing different levels of legumes. Aquaculture , 289, 106-112.

• Agricultural Marketing Resource Center (2013). Estimated U.S. Dried Distillers

Grains with Solubles (DDGS) Production & Use.

http://www.extension.iastate.edu/agdm/crops/outlook/dgsbalancesheet.pdf

• Alarcón, F.J., Diaz, M., Moyano, F.J., Abellá, E. (1998). Characterization and

functional properties of digestive proteases in two sparids; gilthead seabream (Sparus

aurata) and common dentex (Dentex dentex). Fish Physiology and Biochemistry, 19,

257–267.

• Alexis, M. (1997). Fish meal and fish oil replacers in Mediterranean marine fish diets.

Feeding Tomorrow’s Fish. Cahiers Options Méditerranéennes. 22, 183-204.

• Allan, G.L. Rowland,.S.J., Parkinson, S., Stone, D.A.J & Jantrarotai, W. (1999).

Nutrient digestibility for juvenile silver perch Bidyanus bidyanus : development of

methods. Aquaculture, 170, 131-145.

• Alliot, E., Febvre, A., Marquet, R., Metailler, R., Pastoureaud, A. (1974). Automated

programmefor dry granules distribution. Effect of daily ratios on seabass(Dicentrarchus

labrax). In Laubier L., Reyss D., Cent. Oceanol. Bretagne, France (Eds.). Paris:

CNEXO.


• Al-Tameemi, R., Aldubaikul, A., Salman N.A., (2010). Comparative study of α-

amylase activity in three Cyprinid species of different feeding habits from Southern

Iraq. Turkish Journal of Fisheries and Aquatic Sciences, 10, 411-414.

• Amirkolaie, A.K., El-Shafai, S.A., Eding, E.H., Schrama, J.W. & Verreth, J.A. (2005)

.Comparison of faecal collection method with high and low-quality diets regarding

digestibility and faecal characteristics measurements in Nile tilapia. Aquacult. Res., 36,

578– 585.

• Aslaksen, M.A., Kraugerud, O.F., Penn, M., Svihus, B., Denstadli, V., Jorgensen,

H.Y.,Hillestad, M., Krogdahl, A., Storebakken, T. (2007). Screening of nutrient

digestibilities and intestinal pathologies in Atlantic salmon,Salmo salar , fed diets with

legumes, oilseeds, or cereals. Aquaculture, 272, 541-555.

• Austreng, E. (1978) Digestibility determination in fish using chromic oxide marking

and analysis of contents from different segments of the digestibility tract. Aquaculture

13, 265-272.

• Barrington, E.J.W. (1962). Digestive enzymes. In: Lowenstein.O (Editor), Advances in

Comparative Physio-logy and Biochemistry, Vol. 1, Page 1. Academic Press, New

York.

• Bazaz, M.M. & Keshavanath, P. (1993). Effect of feeding different levels of sardine oil

on growth, muscle composition and digestive enzyme-activities of mahseer,

TorKhudree. Aquaculture, 115, 111.

• Bell, J.G., Henderson, R.J., Tocher, D.R., Sargent, J.R. (2004). Replacement of

dietary fish oil with increasing levels of linseed oil: Modification of flesh fatty acid

compositions in Atlantic salmon (Salmo salar ) using a fish oil finishing diet. Lipids. 39,

223-232.

• Bell, J.G., McEvoy, J., Tocher, D.R., McGhee, F., Campbell, P.J., Sargent, J.R.

(2001).Replacement of fish oil with rapeseed oil in diets of Atlantic salmon (Salmo

salar) affects tissue lipid compositions and hepatocyte fatty acid metabolism. J. Nutr.

131, 1535–1543.

• Bell, J.G., McGhee, F., Campbell, P.J., Sargent, J.R. (2003). Rapeseed oil as an

alternative to marine fish oil in diets of post smolt Atlantic salmon (Salmo salar):

changes in flesh fatty acid composition and effectiveness of subsequent fish oil ‘wash

out’. Aquaculture. 218, 515–528.

• Bell, J.G., McGhee, F., Dick, J.R., Tocher, D.R. (2005). Dioxin and dioxin-like

polychlorinated biphenyls (PCBs) in Scottish farmed salmon (Salmo salar ): effects of

replacement of dietary marine fish oil with vegetable oils. Aquaculture 243, 305-314.

• Belyea RL, Rausch KD, Tumbleson ME. (2004). Composition of corn and distillers

dried grains with solubles from dry grind ethanol processing. Bio resource

Technology.66, 207–212.


•Birmingham. (2010). Portuguese Aquaculture: current status and future perspectives.

Aquaculture europe.

• Booth, M.A., Allan, G.L., Frances, J., Parkinson, S. (2001). Replacement of fish meal

in diets for Australian silver perch, Bidyanus bidyanus: IV. Effects of dehulling and

protein concentration on digestibility of grain legumes. Aquaculture. 196, 67-85.

• Borlongan, I.G. (1990). Studies on the digestive lipases of milkfish, Chanos

chanos.Aquaculture, 89, 315.

• Bureau D.P., Kaushik S.J. & Cho C.Y. (2002). Bioenergetics. In: Fish Nutrition, 3rd

Edition (ed. by J.E. Halver & R.W. Hardy), pp. 1–59. Academic Press, San Diego, CA.

• Bureau, D.P., Harris, A.M., Cho, C.Y. (1999). Apparent digestibility of rendered animal

protein ingredients for rainbow trout (Oncorhynchus mykiss). Aquaculture. 180, 345–

358.

• Burr, G., Hume, M., Neill, W.H., Gatlin, D.M. (2008). Effects of prebiotics on nutrient

digestibility of a soybean-meal-based diet by red drum Sciaenops ocellatus (Linnaeus).

Aquac Res., 39, 1680-1686.

• Burrells C.,Williams P.D., Southgate P.J. & Wadsworth S.L. (2001). Dietary

nucleotides: a novel supplement in fish feeds. 2. Effects on vaccination, salt water

transfer, growth rates and physiology of Atlantic salmon (Salmo salar L).

Aquaculture.199, 171-184.

• Buyukates, Y., Rawles, S.D., Gatlin III, D.M. (2000). Phosphorus fractions of various

feedstuffs and apparent phosphorus availability to channel catfish. N. Am. J. Aquacult.,

62, 184–188.

• Cabral, H., Ohmert, B. (2001). Diet of juvenile meagre, Argyrosomus regius, within

the Tagus Estuary. Cahiers-de-Ecologie Marine, 24, 289–293.

• Cahu, C. L. and J. L. Zambonino Infante. (1994). Early weaning of sea bass

(Dicentrarchus labrax) larvae with a compound diet: effect on digestive enzymes.

Comparative Biochemistry and Physiology, 109A, 213–222.

• Cahu, C. L., J. L. Zambonino Infante and V. Barbosa. (2003). Effect of dietary

phospholipid level and phospholipid: neutral lipid value on the development of sea bass

(Dicentrarchus labrax) larvae fed a compound diet. British Journal of Nutrition, 90,21–

28.

• Calderón, J. A., J. C. Esteban, M. A. Carrascosa, P. L. Ruiz y F. Varela. (1997).

Estabulación y crecimiento de un lote de reproductores de corvina (Argyrosomus

regius (A.)). En: Actas VI Congreso Nacional de Acuicultura (9-11 de julio, 1997.

Cartagena, Murcia, España). J. de Costa, E. Abellán, B. García, A. Ortega y S. Zamora

(eds.), pp. 365-370. Ministerio de Agricultura, Pesca y Alimentación. Madrid.

• Campbell, B., Pauly, D. (2012). Mariculture: A global analysis of production trends

since 1950. Marine Policy. 39. 94–100.


• Carter, C.G., Lewis, T.E. & Nichols, P.D. (2003). Comparison of cholestane and

yttrium oxide as digestibility markers for lipid components in Atlantic salmon (Salmo

salar L.) diets. Aquaculture, 225, 341–351.

• Caruso G., Denaro M.G., Genovese L. (2009). Digestive Enzymes in Some Teleost

Species of Interest for Mediterranean Aquaculture. The Open Fish Science Journal, 2,

74-86.

• Cedric, JS. (2009). Digestive enzyme response to natural and formulated diets in

cultured juvenile spiny lobster, Jasus edwardsii. Aquaculture, 294,271–281.

• Center for Genomic Regulation. (2011). Molecular Mechanism Links Temperature

With Sex Determination in Some Fish Species. Obtained in 21 April 2013 in

http://pasteur.crg.es/portal/page/portal/Internet/06_NOTICIAS/HIDE-

PRESSRELEASES/The%20molecular%20mechanism%20that%20links%20temperatur

e%20with%20sex%20determination%20in%20some%20species%20has%20been%20

found

• Chakrabarti, I., Gani, M.A., Chaki, K., Sur, R., Misra, K., (1995). Digestive enzymes in

11 freshwater teleost fish species in relation to food habit and niche segregation.

Comp. Biochem. Physiol, 112 A, 167–177.

• Chao NL. (1986). A synopsis on zoogeography of the Sciaenidae. In Uyeno T, R Arai,

T Taniuchi, K Matsuura, eds. Indo-Pacific fish biology: Proceedings of the Second

International Conference on Indo-Pacific Fishes. Tokyo: Ichthyological Society of

Japan, pp. 570-589.

• Chao, L. T., 1990. FishBase. Argyrosomus regius. Froese, R., Pauly, D. (Eds.),World

Wide Web electronic publication. Retrieved November 30, 2011, from FishBase:

www.fishbase.org

• Chatzifotis S, Polemitou I, Divanach P, Antonopoulou E (2008) Effect of dietary

taurine supplementation on growth performance and bile salt activated lipase activity of

common dentex, Dentex dentex, fed a fish meal/soy protein concentrate-based diet.

Aquaculture, 275, 201–208.

• Chatzifotis, S., Panagiotidou, M., Papaioannou, N., Pavlidis, M., Nengas, I., Mylonas,

C. C. ( 2010). Effect of dietary lipid levels on growth, feed utilization, body composition

and serum metabolites of meagre (Argyrosomus regius) juveniles. Aquaculture, 307,

65-70.

• Chatzifotis,S., Panagiotidou, M., Divanach, P. (2012). Effect of protein and lipid

dietary levels on the growth of juvenile meagre (Argyrosomus regius). Aquacult. Int.,

20, 91–98.

• Chen, D. and A. Ainsworth. 1992. Glucan administration potentiates immune defense

mechanisms of channel catfish, Ictalurus punctatus, Rafinesque. Journal of Fish

Diseases, 15,295–304.

http://www.fishbase.org/


• Cheng, Z.J. & Hardy, R.W. (2000). Nutritional value of diets containing distillers dried

grain with solubles for rainbow trout, Oncorhynchus mykiss. J. World Aquac. Soc., 15,

101–113.

• Cheng, Z.J. & Hardy, R.W. (2004b). Nutritional value of diets containing distillers dried

grains with solubles for rainbow trout, Oncorhynchus mykiss. J. Appl. Aquac., 15, 101–

113.

• Cho, C. Y., and Slinger, S. J. (1979). In “Finfish Nutrition and Fishfeed Technology”

(J. E. Hlaver, and K. Tiews, eds.), Vol. II, pp. 239–247. Heenemann, Berlin.

• Cho, C. Y., Bayley, H. S., and Slinger, S. J. (1975). “Proc. 28th Annu. Meet. Can.

Conf. Fish. Res.” Vancouver, BC.

• Cho, C.Y. & Kaushik, S.J. (1990) Nutritional energetics in fish: energy and protein

utilization in rainbow trout (Salmo gairdneri). World Rev. Nut. Diet. Karger, Basel., 6,

132–172.

• Cho, C.Y., Slinger, S.J & Bayley, H.S. (1982). Bioenergetics of salmonid fishes,

energy intake, expenditure and productivity. Comparitive Biochemestry and Physiology,

73B, 23-42

• Chong, A.S.C., Hashim, R., Chow-Yang, L. and Ali, A.B. (2002). Partial

characterization and activities of proteases from the digestive tract of discus fish

(Symphysodon aequifasciata). Aquaculture, 203, 321-333.

• Choubert, G., De La Noue, J. & Luquet, P. (1979) Continuous quantitative automatic

collector for fish faeces. Prog. Fish-Cult., 41, 64–67.

• Cittolin, G., Borgoni, N., Angelini, M., Lorenzini, C. (2008). L'allevamento di

Argyrosomus regius. Acquacoltura in Toscana. Studi e analisi di settore , 49-61.

• Clark, J., Macdonald, N.L. and Stark, J.R. (1985). Metabolism in marine flatfish. III.

Measurement of elastase activity in the digestive tract of dover sole (Solea solea L).

Comparative Biochemistry and Physiology B: Biochemistry and Molecular Biology, 91,

677-684.

• Claude, W. (2009). Argyrosomus regius. Obtained in 20 April 2013 in

http://doris.ffessm.fr/photo_gde_taille_fiche2.asp?varpositionf=&varSQL=SELECT%20

*%20FROM%20fiche_liste%20where%20fiche_numero%20=%201230&varposition=1

&varSQLphoto=SELECT%20*%20FROM%20vue_photos%20where%20photo_fiche%

20=%201230%20ORDER%20BY%20photo_ordre&fiche_numero=1230&origine=

• Corrêa, C. F., L. H. Aguiar, L. M. Lundstedt and G. Moraes. (2007). Responses of

digestive enzymes of tambaqui (Colossoma macropomum) to dietary corn starch

changes and metabolic inferences. Comparative Biochemistry and Physiology, 147A,8

57–862.

• Couso, N., R. Castro, B. Magarinos, A. Obach, and J. Lamas. (2003). Effects of oral

administration of glucans on the resistance of gilthead seabream to pasteurellosis.

Aquaculture, 219,99–109.


• Couto A., Enes P., Peres H., Oliva-Teles A. (2012). Temperature and dietary starch

level affected protein but not starch digestibility in gilthead sea bream juveniles. Fish

Physiol Biochem, 38, 595–601.

• Coyle, S.D., Mengel, G.J., Tidwell, J.H. & Webster, C.D. (2004). Evaluation of growth,

feed utilization, and economics of hybrid tilapia, Oreochromis niloticus x Oreochromis

aureus, fed diets containing different protein sources in combination with distillers dried

grains with solubles. Aquac. Res., 35, 1–6.

• Cunningham, L. (2005). Assessing the contribution of aquaculture to food security: a

survey of methodologies. FAO Fish Cir, 1010-1034.

• Daprá, F., Gai, F., Costanzo, M.T., Maricchiolo, G., Micale, V., Sicuro, B., Caruso, G.,

Genovese, L., Palmegiano, G.B. (2009). Rice protein-concentrate meal as a potential

dietary ingredient in practical diets for blackspot seabream Pagellus bogaraveo: a

histological and enzymatic investigation. Journal of Fish Biology., 74, 773-789

• Davies, S. J., Abdel-Warith, A. A., Gouveia, A. (2011). Digestibility Characteristics of

Selected Feed Ingredients for Developing Bespoke Diets for Nile Tilapia Culture in

Europe and North America. Journal of the World Aquaculture Society, 42, 388-398.

• De Almeida L.C., Lundstedt L.M., Moraes G. (2006) Digestive enzyme responses of

tambaqui (Colossoma macropomum) fed on different levels of protein and lipid.

Aquaculture Nutrition, 12, 443-450.

• Debnath D, Pal AK, Sahu NP, Yengkokpam S, Baruah K, Choudhury D,

Venkateshwarlu G. (2007). Digestive enzymes and metabolic profile of Labeo rohita

fingerlings fed diets with different crude protein levels. Comp Biochem Physiol, 146 B,

107–114.

• Dias, J., Alvarez, M.J., Diez, A., Arzel, J., Corraze, G., Bautista, J.M., Kaushik, S.J.

(1998).Regulation of hepatic lipogenesis by dietary protein/energy in juvenile European

seabass (Dicentrarchus labrax). Aquaculture, 161, 169-186.

• Dimes, L. E., Garcia Carreno, F. L., Haard, N. F. (1994). Estimation of protein

digestibility--3. Studies on the digestive enzymes from the pyloric ceca of rainbow trout

and salmon. Comp. Biochem. Physiol., A, 109A, 349-360.

• Dinis. (2010). Focus on Portugal: Current status and future perspectives. Aquaculture

europe, Vol 35.

• Direcção Geral das Pescas e Aquicultura (2007). Programa Operacional Pescas

2007-2013. Ministério da Agricultura , do DesenvolvimentoRural e das pescas.

• Duncan, N., Estevez, A., Padros, F., Aguilera, C., Montero, F. E., Norambuena,F.,

Carazo, I., Carbo, R., Mylonas, C. C. (2008). Acclimation to captivity and GnRHa-

induced spawning of meagre (Argyrosomus regius). Cybium , 32(2), 332-333.

• Enes P., Panserat S., Kaushik S. & Oliva-Teles A. (2009). Nutritional regulation of

hepatic glucose metabolism in fish. Fish Physiology and Biochemistry, 35, 519–539.


• Enes, P., Panserat, S., Kaushik, S., Oliva-Teles, A. (2011). Dietary Carbohydrate

Utilization by European Sea Bass (Dicentrarchus labrax L.) and Gilthead Sea Bream

(Sparus aurata L.) Juveniles. Reviews in Fisheries Science, 19, 201-215.

• Enes, P., Perez-Jimenez, A., Peres, H., Couto, A., Pousao-Ferreira, P., Oliva-Teles,

A. (2012). Oxidative status and gut morphology of white sea bream, Diplodus sargus

fed soluble non-starch polysaccharide supplemented diets. Aquaculture, 358, 79-84.

• Eshel, A., Lindner, P., Smirnoff, P., Newton, S., Harpaz, S. (1993), Comparative study

of proteolytic enzymes in the digestive tracts of the European sea bass and hybrid

striped bass reared in freshwater. Comp. Bioch. Physiol, 106A, 627–634.

• FAO. (2012). The State of World Fisheries and Aquaculture: 2012. Rome

• FAO. © 2005-2013. Cultured Aquatic Species Information Programme. Dicentrarchus

labrax. Cultured Aquatic Species Information Programme. Text by Bagni, M. In: FAO

Fisheries and Aquaculture Department [online]. Rome. Updated 18 February 2005.

[Cited 1 April 2013].

http://www.fao.org/fishery/culturedspecies/Dicentrarchus_labrax/en

• FAO. © 2013. Fishery and Aquaculture Country Profiles. Portugal (2005). Fishery and

Aquaculture Country Profile fact sheets. In: FAO Fisheries and Aquaculture

Department [online]. Rome. Updated 1 November 2005. [Cited 17 May 2013].

http://www.fao.org/fishery/facp/174/en

• FAO. © 2005-2013. Cultured Aquatic Species Information Programme. Argyrosomus

regius. Cultured Aquatic Species Information Programme. Text by Stipa, P.; Angelini,

M. In: FAO Fisheries and Aquaculture Department [online]. Rome. Updated 10

February 2005. [Cited 1 April 2013].

http://www.fao.org/fishery/culturedspecies/Argyrosomus_regius/en

• Fastinger ND, Latshaw JD, Mahan DC. (2006). Amino acid availability and true

metabolizable energy content of corn distillers dried grains with solubles in adult

Cecectomized Roosters. Poultry Science, 85, 1212–1216.

• Fernandez, I., Moyano, F.J., Diaz, M. & Martinez, T. (2001). Characterization of

alpha-amylase activity in five species of Mediterranean sparid fishes (Sparidae,

Teleostei). J. Exp. Mar. Biol. Ecol., 262, 1–12.

• Fishbase, 2013. http://www.fishbase.gr/summary/Argyrosomus-regius.html

• Fishbase, 2013. http://www.fishbase.org/summary/63

• Folch, J., Lees, M., Sloane-Stanley, G.H. (1957). A simple method for the isolation

and purification of total lipids from animal tissues. Journal of Biological Chemistry, 226,

497–509.

• Fountoulaki, E., Alexis, M. N., Nengas, I. & Venou, B. (2005). Effect of diet

composition on nutrient digestibility and digestive enzyme levels of gilthead sea bream

(Sparus aurata L.). Aquaculture Research, 36, 1243–1251.

http://www.fishbase.org/summary/63


• Fournier, V., Gouillou-Coustans, M.F., Kaushik, S.J. (2000). Hepatic ascorbic acid

saturation is the most stringent response criterion for determining the vitamin C

requirement of juvenile European sea bass (Dicentrarchus labrax). J. Nutr., 130, 617-

620.

• Francis, G., H. P. S. Makkar, and K. Becker. (2001). Antinutritional factors present in

plant-derived alternate fish feed ingredients and their effects in fish. Aquaculture, 199,

197–227.

•Froese, R., Pauly, D. (Eds.), 2011. FishBase. Argyrosomus regius. World Wide Web

electronic publication. Retrieved November 30, 2011, from FishBase:

www.fishbase.org

• Furné M, Hidalgo MC, López A, García-Gallego M, MoralesAE, Domezain A,

Domezainé J, Sanz A. (2005). Digestive enzyme activities in Adriatic sturgeon

Acipenser naccarii and rainbow trout Oncorhynchus mykiss. A comparative Study.

Aquaculture, 250,391–398

• Furukawa, A. and Tsukahara, 5. (1966). On the acidic digestion method for the

determination of chromic oxide as an index substance in the study of digestibility of fish

feeds. Bulletin of the Japanese Society of Scientific Fisheries, 32, 502-506.

• Gai F., Gasco L., Dapra F.,Palmegiano G. B.,Sicuro B. (2012). Enzymatic and

Histological Evaluations of Gut and Liver in Rainbow Trout, Oncorhynchus mykiss, Fed

with Rice Protein Concentrate-based Diets. Journal of the World Aquaculture Society,

43, 218-229.

• Garcia-Carreno, F.C. and Hernandez-Cortes, P. 2000. Use of protease inhibitors in

seafood products. In Seafood Enzymes: Utilization and Influence on Postharvest

Seafood Quality, N.F. Haard and B.K. Simpson editors. Marcel Dekker, New York., pp.

531-540.

• Gatlin D.M., Barrows F.T., Brown P., Dabrowski K., Gaylord T.G., Hardy R.W.,

Herman E., Hu G.S., Krogdahl A., Nelson R., Overturf K., Rust M., Sealey W.,

Skonberg D., Souza E.J., Stone D., Wilson R. & Wurtele E. (2007). Expanding the

utilization of sustainable plant products in aquafeeds: a review. Aquaculture Research,

38, 551–579.

• Gatlin, D. M. (2002). Nutrition and fish health. Fish Nutrition, 3, 671-702

• Gause, B., Trushenski, J. (2011). Replacement of Fish Meal with Ethanol Yeast in the

Diets of Sunshine Bass. American Fisheries Society. 73, 97–103.

• German, D.P, Bittong, R.A. (2009). Digestive enzyme activities and gastrointestinal

fermentation in wood-eating catfishes. J Comp Physiol B, 179, 1025-42.

• Geurden I., Jutfelt F., Olsen R. & Sundell K. (2009). Avegetable oil feeding history

a¡ects digestibility and intestinal fatty acid uptake in juvenile rainbow trout

Oncorhynchus mykiss. Comparative Biochemistry and Physiology Part A, 152, 552-

559.


• Ghosh, A. (1976). J. Inland. Fish. Soc. India, 8, 137.

• Gil MM, Grau A, Riera l. (2009). Atlas histológico del tracto digestivo de la corvina de

cría, Argyrosomus regius. pp. 150-151 en Beaz D, Villarroel M, Cárdenas S. eds XII

Congreso Nacional de Acuicultura: Com la acuicultura alimentamos tu salud. MARM,

SEA y FOESA. Madrid, España.

• Gjedrem, T., Robinson, N., Rye, M. (2012). The importance of selective breeding in

aquaculture to meet future demands for animal protein: A review. Aquaculture, 350,

117-129.

• Glass, H.J., MacDonald, N.L., Moran, R.M., Stark, J.R., 1989. Digestion of protein in

different marine species. Comp. Biochem. Physiol., 94B, 607–611.

• Glencross, B., Evans, D., Dods, K., McCafferty, P., Hawkins, W., Maas, R. & Sipsas,

S. (2005). Evaluation of the digestible value of lupin and soybean protein concentrates

and isolates when fed to rainbow trout, Oncorhynchus mykiss, using either stripping or

settlement faecal collection methods. Aquaculture, 245, 211–220.

• Glencross, B.D. (2008). A factorial growth and feed utilisation model for barramundi,

Lates calcarifer, based on Australian production conditions. Aquac. Nutr., 14, 360–373.

• Glencross, B.D., Booth, M., Allan, G.L. (2007a). A feed is only as good as its

ingredients—a review of ingredient evaluation for aquaculture feeds. Aquaculture

Nutrition, 13, 17–34.

• Glencross, B.D., Hawkins, W.E., Curnow, J.G. (2003). Restoration of the fatty acid

composition of red seabream (Pagrus auratus) using a fish oil finishing diet after grow-

out on plant oil based diets. Aquac. Nutr., 9, 409-418.

• Gomes da Silva, J., Oliva-Teles, A. (1998). Apparent digestibility coefficients of

feedstuffs in seabass (Dicentrarchus labrax) juveniles. Aquatic Living Resources, 11,

187-192.

• Gonzáles-Quirós, R., Del Árbol, J., Del Mar García-Pacheco, M., Silva-García, A. J.,

Naranjo, J. M., Morales-Nin, B. (2011). Life-history of the meagre Argyrosomus regius

in the Gulf of Cadiz (SW Iberian Peninsula). Fisheries Research , 109, 140-149.

• Gouveia, A., Oliva Teles, A., Gomes, E. and Peres, M.H. (1995) The effect of two

dietary levels of raw and gelatinized starch on growth and food utilization by the

European seabass. In: Actas del V Congresso Nacional de Acuicultura (eds. F.

Castelló i Orvay, and A. Calderer i Reig), Publ. Universitat de Barcelona, pp. 516–521.

• Gregory, P., Robert, C. (2012). Plant Products Affect Growth and Digestive Efficiency

of Cultured Florida Pompano (Trachinotus carolinus) Fed Compounded Diets. PLoS

ONE 7(4): e34981.doi:10.1371/journal.pone.0034981

• Gunstone, Frank D. (2011). The World’s Oils and Fats. In: Fish Oil Replacement and

Alternartive Lipid Sources. (ed. by Giovanni M. Turchini, Wing-Keong Ng and Douglas

R. Tocher). pp 61-95. CRC Press, Boca Ranton.


• Haffray, P., Tsigenopoulos, C.S., Bonhomme, F., Chatain, B., Magoulas, A., Rye,

M.,Triantafyllidis, A., Triantaphyllidis, C. (2006). European seabass – Dicentrarchus

labrax. http://genimpact.imr.no/__data/page/7650/european_sea_bass.pdf

• Halver, J.E. and R.W. Hardy. (2002). Fish Nutrition. Academic Press. United States of

America. 824 p.

• Hardy R.W. (2008). Farmed fish diet requirements for the next decade and

implications for global availability of nutrients. In: Alternative Protein Sources in

Aquaculture Diets (ed. by C. Lim, C.D. Webster&C.S. Lee), pp. 1–15. Haworth Press,

New York.

• Hardy, R.W., Barrows, F.T. (2002.) Diet formulation and manufacture. In: Halver, J.E.

and Hardy, R.W. 3rd ed. Fish Nutrition. Elsevier Science, Academic Press, pp. 2-54

• Hendricks J.D. (2002). Adventitious toxins. In: Fish Nutrition, 3rd edn (ed. by J.E.

Halver & R.W. Hardy), pp. 601–649. Academic Press, San Diego, CA.

• Henrique, M.M.F., Gomes, E.F., Gouillou-Coustans, M.F., Oliva-Teles, A., Davies,

S.J. (1998) .Influence of supplementation of practical diets with vitamin C on growth

and response to hypoxic stress of seabream,Sparus aurata. Aquaculture, 161, 415-

426.

• Henriques, M. A. (1998). Manual de aquacultura. Lisboa, Portugal.

• Hertrampf, J.W., Piedad-Pascal, F. (2000). Pulses. In: Handbook on Ingredients for

Aquaculture Feeds. Kluwer Academic Publishers, Dordrecht, The Netherlands, pp. 281

(Marineoils), pp. 338-349 (Pulses).

• Hidalgo, F., Alliot, E. (1988). Influence of water temperature on protein requirement

and proteinutilization in juvenile seabass, Dicentrarchus labrax. Aquaculture, 72, 115-

129.

• Hidalgo, M.C., Urea, E., Sanz, A. (1999). Comparative study of digestive enzymes in

fish with different nutritional habits. Proteolytic and amylase activities. Aquaculture,

170, 267-283.

• Higgs, D. M., and Dong, F. M. (2000). In “The Encyclopedia of Aquaculture” (R. R.

Stickney, ed.), p. 496. John Wiley & Sons, New York.

• Hoehne-Reitan, K., Kjorsvik, E. & Gjellesvik, D.R. (2001). Development of bile salt-

dependent lipase in larval turbot. J. Fish Biol., 58, 737–745.

• Hokari, S., Miura, K., Koyama, I., Kobayashi, M., Matsunaga, T., Iino, N., Komoda, T.,

(2003). Expression of alpha-amylase isozymes in rat tissues. Comp. Biochem. Physiol.,

B 135, 63–69.

• Horn, M.H., Gawlicka, A.K., German, D.P., Logothetis, E.A., Cavanagh, J.W. and

Boyle, K.S. (2006). Structure and function of the stomachless digestive system in three

related species of New World silverside fishes (Atherinopsidae) representing herbivory,

omnivory, and carnivory. Mar. Biol., 149, 1237-1245.


• Iijima N.,Tanaka S. & OtaY. (1998). Puri¢cation and characterization of bile salt-

activated lipase from the hepatopancreas hepatopancreas of red sea bream, Pagrus

major. Fish Physiology and Biochemistry, 18, 59-69.

• INE/DGPA (2010). Estatísticas da Pesca. Lisboa: Instituto Nacional de Estatística.

Lisboa, Portugal.

• INE/DGPA. (1998). Estatísticas da Pesca. Lisboa: Instituto Nacional de Estatística.

Lisboa, Portugal.

• Ingledew, W. M. (1999). Yeast— could you base business on this bug? Pages 27–47

in T. P. Lyons and K. A. Jacques, editors. Under the microscope— focal points for the

new millennium— biotechnology in the feed industry. Proceedings of Alltech’s 15th

annual symposium. Nottingham University Press, Nottingham, UK.

• Ji H.,Sun, H. T., Xiong, D. M. (2012) Studies on activity, distribution, and zymogram of

protease, alpha-amylase, and lipase in the paddlefish Polyodon spathula. Fish

Physiology and Biochemistry, 38, 603-613.

• Jill K . Winkler-Moser. (2012). Lipids in DDGS In: Distillers Grains. Production,

Properties, and Utilization. Pages : 179–191. CRC Press, New York.

• Jonas, E., Ragyanszki, M., Olah, J., Boross, L., (1983). Proteolytic digestive enzymes

of carnivorous (Silurus glanis L.), herbivorous (Hypophthalmichthys molitrix Val.) and

omnivorous (Cyprinus carpio L.) fishes. Aquaculture, 30, 145–154.

• Josupeit, H. (2008). Fishmeal market report for FAO Globefish. Fishmeal prices up,

but might decline soon.

http://www.thefishsite.com/articles/452/fismeal-market-report.

• Karalazos, V. (2007). Sustainable alternatives to fishmeal and fish oil in fish nutrition:

effects on growth, tissue fatty acid composition and lipid metabolism. Thesis submitted

in Institute of Aquaculture, University of Stirling.

• Kaushik S.J., Coves D., Dutto G. & Blanc D. (2004). Almost total replacement of fish

meal by plant protein sources in the diet of a marine teleost, the European seabass,

Dicentrarchus labrax. Aquaculture, 230, 391–404.

• Kenari, A. A., Sotoudeh, E., Rezaei, M. H. (2011). Dietary soybean

phosphatidylcholine affects growth performance and lipolytic enzyme activity in

Caspian brown trout (Salmo trutta Caspius) alevin. Aquaculture Research, 42, 655-

663.

• Klahan R., Areechon N., Yoonpundh R., Engkagul A. (2009). Characterization and

Activity of Digestive Enzymes in Different Sizes of Nile Tilapia (Oreochromis niloticus

L.). Kasetsart J. (Nat. Sci.), 43, 143-153.

• Klomklao, S., Benjakul, S., Visessanguan, W., Kishimura, H. and Simpson, B.K.

(2006a). Purification and characterization of trypsin from the spleen of tongol tuna

(Thunnus tonggol). Journal of Agricultural and Food Chemistry, 54, 5617-5622.


• Klomklao, S., Benjakul, S., Visessanguan, W., Simpson, B.K. and Kishimura, H.

(2005). Partitioning and recovery of proteinase from tuna spleen by aqueous two-phase

systems. Process Biochemistry, 40, 3061-3067.

• Koven, W. M., R.J. Henderson, and J.R. Sargent. (1994a). Lippid digestion in turbot

(Scophthalmus maximus). 1: Lipid class and fatty acid composition of digesta from

different segments of the digestive tract. Fish physiol. Biochem., 13, 69-79.

• Kristjansson, M. and Nielsen, H.H. (1992). Purification and characterization of two

chymotrypsin-like, proteases from the pyloric caeca of rainbow trout (Oncorhynchus

mykiss). Comp. Biochem. Physiol., 101B, 247–257.

• Krogdahl, A., Lea, T.B., Olli, J.J., (1994). Soybean proteinase inhibitors affect

intestinal trypsin activities and amino-acid digestibilities in rainbow trout (Oncorhynchus

mykiss). Comp. Biochem. Physiol., 107A, 215–219.

• Krogdahl,Ǻ ., Sundby, A. & Olli, J.J. (2004) Atlantic salmon (Salmo salar, L) and

rainbow trout (Oncorhynchus mykiss) digest and metabolize nutrients differently

depending on water salinity and dietary starch level. Aquaculture, 229, 335–360.

• Kuz' mina, V.V. (1996b). Influence of age on digestive enzyme activity in some

freshwater teleosts. Aquaculture, 148, 25-37.

• Kuz' mina, V.V. (2008). Classical and Modern Concepts in Fish Digestion, J.E.P.

Cyrino, D.P. Bureau, R.G. Kapoor (Eds.), In: Feeding and digestive functions in fish.

• Lagardére, J. P., Mariani, A. (2006). Spawning sounds in meagre Argyrosomus regius

recorded in the Gironde estuary, France. Journal of Fish Biology , 69, 1697-1708.

• Lanari, D., Poli, B.M., Ballestrazzi, R., Lupi, P., D'Agaro, E., Mecatti, M. (1999). The

effects of dietary fat and NFE levels on growing European seabass (Dicentrarchus

labrax L.).Growth rate, body and fillet composition, carcass traits and nutrient retention

efficiency. Aquaculture, 179, 351-364

• Lazzari R., Neto J. R., Pedrón F. A., Loro V. L., Pretto A., Gioda R. G. (2010). Protein

sources and digestive enzyme activities in jundiá (Rhamdia quelen). Sci. Agric.

(Piracicaba, Braz.), 67, 259-266.

• Le Moullac, G., Klein, B., Sellos, D., Van Wormhoudt, A. (1996). Adaptation of trypsin,

chymotrypsin and amylase to casein level and protein source in Panaeus vannamei

(Crustacea, Decapoda). J. Exp. Mar. Biol. Ecol., 208, 107–125.

• Lee P.H. (1987) Carotenoids in cultured channel catfish. PhD dissertation. Auburn

University, AL, USA.

• Lemieux, H., Blier, P., Dutil, J.D., 1999. Do digestive enzymes set a physiological limit

on growth rate and food conversion efficiency in the Atlantic cod (Gadus morhua)? Fish

Physiol. Biochem., 20, 293–303.

• Li M.H., Robinson E.H., Oberle D.F. & Lucas P.M. (2010). Effects of various corn

distillers by-products on growth and feed efficiency of channel catfish, Ictalurus

punctatus. Aquaculture Nutrition, 16,188-193.


• Li, J. S., Li, J. L., Wu, T. T. (2006). Ontogeny of protease, amylase and lipase in the

alimentary tract of hybrid Juvenile tilapia (Oreochromis niloticus x Oreochromis

aureus). Fish Physiology and Biochemistry. 32, 295-303.

• Li, M. H., Oberle, D. F., Lucas, P. M. (2011). Evaluation of corn distillers dried grains

with solubles and brewers yeast in diets for channel catfish Ictalurus punctatus

(Rafinesque). Aquaculture Research, 42, 1424-1430.

• Li, P., Gatlin, D.M. (2004). Dietary brewers yeast and the prebiotic Grobiotic (TM) AE

influence growth performance, immune responses and resistance of hybrid striped

bass (Morone chrysops × M. saxatilis) to Streptococcus iniae infection. Aquaculture,

231, 445–456.

• Li, X.Y., Chi, Z.M., Liu, Z.Q., Yan, K. & Li, H. (2008a). Phytase production by a marine

yeast Kodamea ohmeri BG3. Appl. Biochem. Biotechnol., 149, 183–193.

• Li, X.Y., Liu, Z.Q. & Chi, Z.M. (2008b). Production of phytase by a marine yeast

Kodamaea ohmeri BG3 in an oats medium: optimization by response surface

methodology. Bioresour. Technol., 99, 6386–6390.

• Lim C, Yildirim-Aksoy, M. (2008). Distillers dried grains with solubles as an alternative

protein source in fish feeds. Proceedings of the 8th International Symposium on Tilapia

in Aquaculture, 12–14 October 2008. Cairo, Egypt, pp. 67–82.

• Lim C., Webster C.D. & Lee C.S. (2008b). Alternative Protein Sources in Aquaculture

Diets. Haworth Press, New York, NY.

• Lim, C., J. C. Garcia, M. Yildirim-Aksoy, P. H. Klesius, C. A. Shoemaker, and J. J.

Evans. (2007). Growth response and resistance to Streptococcus iniae of Nile tilapia,

Oreochromis niloticus, fed diets containing distiller’s dried grains with solubles. Journal

of World Aquaculture Society, 38,231–237.

• Lim, C., M. Yildirim-Aksoy, and P. H. Klesius. (2009). Growth response and

resistance to Edwardsiella ictaluri of channel catfish, Ictalurus punctatus, fed diets

containing distiller’s dried grains with soluble. Journal of the World Aquaculture Society,

40, 182–193.

• Lim, Chhorn., Li, Erchao., Klesius, Phillip H. (2011). Distiller’s dried grains with

solubles as an alternative protein source in diets of tilapia. Reviews in Aquaculture, 3,

172-178.

• Lin, S. M., Luo, L. (2011). Effects of different levels of soybean meal inclusion in

replacement for fish meal on growth, digestive enzymes and transaminase activities in

practical diets for juvenile tilapia, Oreochromis niloticus x O. aureus. Animal Feed

Science and Technology, 168, 80-87.

• Linwood A. Hoffman, Allen Baker. (2011). Estimating the Substitution of Distillers’

Grains for Corn and Soybean Meal in the U.S. Feed Complex / FDS-11-I-01. A Report

from the Economic Research Service. Economic Research Service/USDA


• Liu, KS. (2009). Effect of particle size distribution, compositional and color properties

of ground corn on quality of distillers dried grains with solubles (DDGS). Bio resource

Technology, 100, 4433–4440.

• Liu, Y.,Sumaila, UR. (2010). Estimating pollution abatement costs of salmon

aquaculture: a joint production approach.Land Econ, 86(3), 569–84.

• Loder, N. (2003). The promise of a bluer evolution. Economist. Issuedate: August 7th,

2003. p.19–21.

• Lovell, T. (1998). Digestion and metabolism In:Nutition and feeding of fish, 2º edition.

pp,71-89. Kluwer Academic Publishers, 101 Philip Drive, Assinippi Park, Norwell,

Massachusetts.

• Lundstedt, L.M., Fernando Bibiano Melo, J., Moraes, G. (2004). Digestive enzymes

and metabolic profile of Pseudoplatystoma corruscans(Teleostei: Siluriformes) in

response to diet composition. Comp. Bioch. Physiol. Part B: Bioch. Molecular Biol.,

137, 331-339.

• Lupatsch, I., Kissil, G.WM., Sklan, D. & Pfeffer, E.(2001). Effects of varying dietary

protein and energy supply on growth, body composition and protein utilization in

gilthead seabream (Sparus aurata L.) Aquaculture Nutrition, 7, 71-80.

• Lupatsch, I., Kissil, G.Wm., Sklan, D. (2001). Optimization of feeding regimes for

European seabass (Dicentrarchus labrax): a factorial approach. Aquaculture, 202, 289-

302.

• Mañanós, E., Duncan, N., Mylonas, C. C., 2008. Reproduction and control of

ovulation, spermiation and spawning in cultured fish. In Cabrita, E., Robles, V.,

Herráez, P. (Eds.), Methods in Reproductive Aquaculture: Marine and Freshwater

Species. CRC Press, pp. 3-65.

• Martín, J. M.V., Tabraue R. J. H. e y Henríquez M. N. G. (2005). Evaluación de

Impacto Ambiental de Acuicultura en Jaulas en Canarias. Oceanográfica, 110pp.

• Maybank, M., 2008, August 27. ReproFish: Argyrosomus regius. Retrieved March 30,

2013, from ReproFish: http://www.reprofish.eu

• Maynard, L.A. & Loosli, J.K. (1979) Animal Nutrition, 6th edn. McGraw-Hill Book Co,

New York, NY. Tukey, J.W., 1949. Comparing individual means in the analysis of

variance. Biometrics, 5, 99–114.

• Metts, L.S., Rawles, S. D., Brady, Y. J., Thompson, K. R., Gannam, A. L., Twibell, R.

G., Webster, C. D. (2011). Amino acid availability from selected animal- and plant-

derived feedstuffs for market-size sunshine bass (Morone chrysops x Morone saxatilis).

Aquaculture Nutrition, 17,123-131

• Mohsen, A.A. (1989). Substituting animal protein sources into corn-soybean meal

catfish diets. M.S. thesis. Auburn University, Auburn, Alabama.

• Monfort, M. C. (2010). Present market situation and prospects of meagre

(Argyrosomus regius), as an emerging species in Mediterranean aquaculture. Studies


and reviews. General Fisheries Commission for the Mediterranean, No. 89. FAO,

Rome.

• Montero D., Robaina L., Caballero M.J., Gine´s R., Izquierdo M.S. (2005). Growth,

feed utilization and flesh quality of European sea bass (Dicentrarchus labrax) fed diets

containing vegetable oils: A time-course study on the effect of a re-feeding period with

a 100% fish oil diet. Aquaculture, 248,121– 134.

• Munilla-Moran, R. & Saborido-Rey, F. (1996a). Digestive enzymes in marine species.

I. Proteinase activities in gut from redfish (Sebastes mentella), seabream (Sparus

aurata) and turbot (Scophthalmus maximus). Comp. Biochem. Physiol. B, 113, 395–

402.

• N.R.C. (1993) Nutrient Requirements of Fish. National Academy Press, Washington,

DC, 114 pp.

• Natalia, Y., Hashim, R., Ali, A., Chong, A.(2004). Characterization of digestive

enzymes in a carnivorous ornamental fish, the Asian bony tongue Scleropages

formosus (Osteoglossidae). Aquaculture, 233, 305-320.

• Neurath, H. (1989). The diversity of proteolytic enzymes. In: Beynon, R.J., Bond, J.S.

(Eds), Proteolytic Enzymes. A Practical Approach. IRL Press, Oxford, pp. 1–15.

• Nissen, J.A. (1993). Proteases. In Enzymes in Food Processing, T. Nagodawithana

and G. Reed, editors. Academic Press, Inc. New York, pp. 159-203.

• NRC. (1998). Pages 110–123 in Nutrient Requirements of Swine. 10th rev. ed. Natl.

Acad. Press, Washington, DC.

• Ogino, C., Kakino, J., and Chen, M. S. (1973). Bull. Jap. Soc. Sci. Fish., 39, 519–523.

• Olim, C.P.R. Apparent digestibility coefficient of feed ingredients for juvenile meagre

(Argyrosomus regius, Asso 1801) [Dissertation]. Porto: Faculdade de Ciências da

Universidade do Porto; 2012.

• Oliva-Teles A. & Goncalves P. (2001) Partial replacement of fishmeal by brewers

yeast (Saccharomyces cerevisiae) in diet for sea bass (Dicentrarchus labrax) juveniles.

Aquaculture, 202, 269-278.

• Oliva-Teles, A. (2000). Recent advances in European seabass and gilthead

seabream nutrition. Aquac. Internat., 8, 477-492.

• Oliva-Teles, A. (2012). Nutrition and health of aquaculture fish. Journal of Fish

Diseases, 35, 83–108.

• Oliva-Teles, A., Pereira, J.P., Gouveia, A., Gomes, E., 1998. Utilisation of diets

supplemented with microbial phytase by seabass (Dicentrarchus labrax) juveniles.

Aquat Living Resour., 11, 255-259.

• Olsen, R.E. and E. Ringoe (1997) Lipid digestibility in fish. A review Recent Res

Devel. In Lipids Res, 1, 199-265


• Ono, R. D. and Poss, S. G. (1982). Structure and innervation of the swim bladder

musculature in the weakfish, Cynoscion regalis (Teleostei: Sciaenidae). Can. J. Zool.,

60, pp. 1955 – 1967.

• Palmegiano, G. B., F. Daprà, G. Forneris, F. Gai, L. Gasco, K. Guo, P. G. Peiretti, B.

Sicuro and I. Zoccarato. (2006). Rice protein concentrate meal as a potential ingredient

in practical diets for rainbow trout (Oncorhynchus mykiss). Aquaculture, 258,357–367.

• Papoutsoglou ES., Lyndon AR. (2005). Effect of incubation temperature on

carbohydrate digestion in important teleosts for aquaculture. Aquacult Res, 36, 1252–

1264.

• Papoutsoglou, E. S. & Lyndon, A. R. (2003). Distribution of a-amylase along the

alimentary tract of two Mediterranean fish species, the parrotfish Sparisoma cretense

L. and the stargazer, Uranoscopus scaber L. Mediterranean Marine Science, 4, 115–

124.

• Pastor, E., Grau, A., Massuti, E., Sánchez de Lamadrid, A (2002). Preliminary results

of growth of meagre, Argyrosomus regius (Asso, 1801) in sea cages and indoor tanks.

In: Aquaculture Europe 2002: Seafarming- Today and tomorrow. EAS Special

Publication, 2002, 32, p. 422-423.

• Pavasovic, A., Anderson, A.J., Mather, P.B., Richardson, N.A. (2007). Influence of

dietary protein on digestive enzyme activity, growth and tail muscle composition in

redclaw crayfish, Cherax quadricarinatus (von Martens). Aquac. Res., 38, 644–652.

• Pavasovic, M., Richardson, N.A., Anderson, A.J., Mann, D., Mather, P.B. (2004).

Effect of pH, temperature and diet on digestive enzyme profiles in the mud crab, Scylla

serrata. Aquaculture, 242, 641–654.

• Pereira, T.G., Oliva-Teles, A. (2003). Evaluation of corn gluten meal as a protein

source in dietsfor gilthead seabream (Sparus aurataL.) juveniles. Aquac. Res., 34,

1111-1117.

• Peres, H., Oliva-Teles, A. (1999a). Effect of dietary lipid level on growth performance

and feedutilization by European seabass juveniles (Dicentrarchus labrax). Aquaculture,

179,325-334.

• Peres, H., Oliva-Teles, A. (1999b). Influence of temperature on protein utilization in

juvenile European seabass ( Dicentrarchus labrax). Aquaculture, 170, 337-348.

• Peres, H., Oliva-Teles, A. (2002). Utilization of raw and gelatinized starch by

European seabass( Dicentrarchus labrax) juveniles. Aquaculture, 205, 287-299.

• Perez, L., Gonzalez, H., Jover, M., Fernandez-Carmona, J. (1997). Growth of

European seabass fingerlings (Dicentrarchus labrax) fed extruded diets containing

varying levels of protein, lipid and carbohydrate. Aquaculture, 156, 183-193.

• Pérez-Jiménez, A., G. Cardenete, A. E. Morales, A. García-Alcázar, E. M. Abellán

and M. C. Hidalgo. (2009). Digestive enzymatic profile of Dentex dentex and response


to different dietary formulations. Comparative Biochemistry and Physiology Part A,

154,157–164.

• Piccolo, G., Bovera, F., De Riu, N., Marono, S., Salati, F., Cappuccinelli, R., Moniello,

G. (2008). Effect of two different protein/fat ratios of the diet on meagre (Argyrosomus

regius) traits. Italian Journal of Animal Science , 7, 363-371.

• Pousão-Ferreira P., Ribeiro L, Soares F, Nicolau L, Mendes A C, Castanho S, Barata

M, Dâmaso-Rodrigues L, Cabrita E, Dinis M T. (2010). Adaptation to captivity and

spawning induction of meagre (Argyrosomus regius) at ipimar aquaculture research

station. Aquaculture Europe 10, Porto, Portugal, 2010.

• Quéro, J. C. (1989b). Le maigre, Argyrosomus regius (Asso) (Pisces, Sciaenidae) en

Méditerranée occidentale. Bulletin de la Société zoologique de France , 14, 81-89.

• Quéro, J. C., 1989a. Sur la piste des maigres Argyrosomus regius (Pisces,

Sciaenidae) du Golfe de Gascogne et de Mauritanie. Océanis , 15, 161-170.

• Refstie, S., B. Glencross, T. Landsverk, M. Sørensen, Lilleeng E., W. Hawkins and Ǻ.

Krogdahl. (2006). Digestive function and intestinal integrity in Atlantic salmon (Salmo

salar) fed kernel meals and protein concentrates made from yellow or narrow-lea fed

lupins. Aquaculture, 261,1382–1395.

• Ringo, E., Olsen, R.E., Gifstad, T.O., Dalmo, R.A., Amlund, H., Hemre, G.I., Bakke,

A.M. (2010). Prebiotics in aquaculture: a review. Aquacult Nutr., 16, 117-136.

• Rivas-Vega, M.E., Goytortúa-Bores, E., Ezquerra-Brauer, J.M., Salazar-García, M.G.,

Cruz-Suárez, L.E., Nolasco, H., Civera-Cerecedo, R. (2006). Nutritional value of

cowpea (Vigna unguiculata L. Walp) meals as ingredients in diets for Pacific white

shrimp (Litopenaeus vannamei Boone). Food Chem. 97, 41–49.

• Robertsen, B., G. Roerstand, R. E. Engstad, and J. Raa. (1990). Enhancement of

non-specific disease resistance in Atlantic salmon, Salmo salar L., by a glucan from

Saccharomyces cerevisiae cell walls. Journal of Fish Diseases, 3,391–400.

• Robertson, B., R. E. Engstand, and J. B. Jorgensen. (1994). β-Glucan as

immunostimulants. Pages 83–99 in J. Stolen and T. C. Fletcher, editors. Modulators of

fish immune responses. SOS, Fair Haven, New Jersey, USA.

• Robinson, E. H., and M. H. Li. (2008). Replacement of soybean meal in channel

catfish, Ictalurus punctatus, diets with cottonseed meal and distiller’s dried grains with

solubles. Journal of the World Aquaculture Society, 39,521–527.

• Roo, J., Hernández-Cruz, C. M., Borrero, C., Schuchardt, D., Fernández-Palacios, H.

(2010). Effect of larval density and feeding sequence on meagre (Argyrosomus regius;

Asso, 1801) larval rearing. Aquaculture, 302, 82-88.

• Rosentrater, K.A. (2006). Some physical properties of distillers dried grains with

solubles (DDGS). Applied Engineering and Agriculture, 22, 589–595.

• Rossini, F. D., ed.,(1956). Experimental thermochemistry, Vol. 1, Interscience, New

York.


• Santigosa E., García-Meilán I., Valentín J. M., Navarro I., Pérez-Sánchez J., Gallardo

M.A. (2010). Plant oils’ inclusion in high fish meal-substituted diets: effect on digestion

and nutrient absorption in gilthead sea bream (Sparus aurata L.). Aquaculture

Research, 42, 962-974.

• Santigosa E., Sánchez J., Médale F., Kaushik S., Pérez-Sánchez J., Gallardo MA.

(2008). Modifications of digestive enzymes in trout (Oncorhynchus mykiss) and sea

bream (Sparus aurata) in response to dietary fish meal replacement by plant protein

sources. Aquaculture, 282,68–74.

• Savona B., Tramati C., Mazzola A. (2011). Digestive Enzymes in Larvae and

Juveniles of Farmed Sharpsnout Seabream (Diplodus puntazzo) (Cetti, 1777). The

Open Marine Biology Journal, 5, 47-57.

• Schaeffer, T. W., Brown, M. L., Rosentrater, K. A. (2011). Effects of Dietary Distillers

Dried Grains with Solubles and Soybean Meal on Extruded Pellet Characteristics and

Growth Responses of Juvenile Yellow Perch. North American Journal Of Aquaculture,

73, 270-278.

• Schaeffer, T.W., Brown, M.L., Rosentrater, K.A. (2009). Performance characteristics

of Nile tilapia (Oreochromis niloticus) fed diets containing graded levels of fuel-based

distiller’s dried grains with solubles. J. Aquacult. Feed Sci. Nutr. 1, 78–83.

• Schiavone, R., Zilli, L., Vilella, S. (2008). Sex differentiation and serum levels of sex

steroids in Meagre (Argyrosomus regius). Comparative Biochemistry and Physiology ,

S16-S17.

• Shelby, R.A., Lim, C., Yildrim-Aksoy, M., Klesius, P.H. (2008). Effect of distillers dried

grains with solubles-incorporated diets on growth, immune function and disease

resistance in Nile tilapia (Oreochromis niloticus L.). Aquac. Res. 39, 1351–1353.

• Shoemaker, C., P. H. Klesius, and C. Lim. 2007. Immunity in fish. Pages 53–65 in L.

E. Pezzato, M. M. Barros, and W. M. Furuya, editors. Proceeding, 2nd symposium on

nutrition and fish health; November 14–16, 2007, Botucatu, Brazil. Departamento de

Melhoramento e Nutrição Animal, Faculdade de Medicina Veterinaria e Zootecnia,

Universidade Estadual Paulista, Botucatu, São Paulo, Brazil.

• Silva, D.J.; Queiroz, A.C. Análise de alimentos. Métodos químicos e biológicos. 3 ed.

Viçosa: Editora UFV, 2002. 235p

• Simpson, B.K. (2000). Digestive proteinases from marine animals. In Seafood

Enzymes: Utilization and Influence on Postharvest Seafood Quality, N.F. Haard and

B.K. Simpson, editors. Marcel Dekker, New York., pp. 531-540.

• Sitjà -Bobadilla A., Peña-Lopez S., Go¤mez-Requeni P., Médale F., Kaushik S. &

Pérez-Sanchez J. (2005). Effect of fish meal replacement by plant protein sources on

non-specific defencemechanisms and oxidative stress in gilthead seabream (Sparus

aurata). Aquaculture, 249,387- 400.


• Smith, C.L. (1990). Moronidae. In: J.C. Quero, J.C. Hureau, C. Karrer, Post and L.

Saldanha(eds.) Check-list of the fishes of the eastern tropical Atlantic (CLOFETA).

JNCIT,Lisbon; SEI, Paris; and UNESCO, Paris. Vol. 2, pp. 692-694.

• Smith, R. R. (1971). Prog. Fish. Cult.,33, 132–134.

•SOFIA, 2006. The state of world fisheries and aquaculture. Food and Agriculture

Organizationof the United Nations. Rome, 2007.

• Stone DAJ., Hardy RW., Barrows FT., Cheng ZJ. (2005). Effects of extrusion on

nutritional value of diets containing corn gluten meal and corn distiller’s dried grain for

rainbow trout, Oncorhynchus mykiss. J Appl Aquacult, 17,1–20.

• Stone, D.A.J., Allan, G.L. & Anderson, A.J. (2003), Carbohydrate utilization by

juvenile silver perch, Bidyanus bidyanus (Mitchell). II. Digestibility and utilization of

starch and its breakdown products. Aquaculture Research, 34, 109-122.

• Suquet, M., Divanach, P., Hussenot, J., Coves, D., Fauvel, C. (2009). Pisciculture

marine de «nouvelles espèces» d’élevage pour l’Europe. Cah Agric, 18,148–156.

• Tacon, A. & M. Metian. 2008. Global overview on the use of fish meal and fish oil in

industrially compounded aquafeeds: Trends and future prospects. Aquaculture, 285,

146–158.

• Tacon, A.G.J. (1995). Fishmeal Replacers: Review of Antinutrients Within oilseeds

and Pulses – A Limiting Factor for the Green Revolution? In: Proceedings of Feed

Ingredients Asia 95, September 19-21, 1995, Singapore, 23-48. Turret Group Plc.

• Takemura A, T Takita, K. Mizue. (1978). Underwater calls on the Japanese marine

drum fishes (Sciaenidae). Bull. Jpn. Soc. Sci. Fish., 44, pp. 121-125.

• Tavolga WN. (1971). Sound production and detection. WS Hoar, DS Randall, eds. In

Fish physiology, Vol. 5. New York: Academic Press, pp. 135-205.

• Tengjaroenkul B., Smith BJ., Caceci T., Smith SA. (2000). Distribution of intestinal

enzyme activities along the intestinal tract of cultured Nile tilapia, Oreochromis niloticus

L. Aquaculture, 182,317–327.

• Thivend, P., Christiane, M. & Guilbot, A. (1972). Determination of starch with

glucoamylase. Methods Carbohydr. Chem., 6, 100-105.

• Thodesen, J., Storebakken, T., Shearer, K.D., Rye, M., Bjerkeng, B. & Gjerde, B.

(2001). Genetic variation in mineral absorption of large Atlantic salmon (Salmo salar)

reared in seawater. Aquaculture, 194, 263–271.

• Thompson, K.R., Rawles, S.D., Metts, L.S., Smith, R., Wimsatt, A., Gannam, A.L.,

Twibell, R.G., Johnson, R.B., Brady, Y.J. & Webster, C.D. (2008). Digestibility of dry

matter, protein, lipid, and organic matter of two fish meals, two poultry by-product

meals, soybean meal, and distillers dried grains with solubles in practical diets for

sunshine bass, Morone chrysops x M. saxatilis.J. World Aquac. Soc., 39, 352–363.


• Tocher, D.R. (2003). Metabolism and functions of lipids and fatty acids in teleost fish.

Rev. Fish. Sci., 11, 107-184.

• Torrissen, K.R. (1984). Characterization of proteases in the digestive tract of Atlantic

salmon (Salmo salar) in comparison with rainbow trout (Salmo gairdneri). Comp.

Biochem. Physiol. 77B, 669–674.

• Tower RW. (1908). The production of sound in the drum fishes, the sea-robin and the

toadfish. Ann. NY Acad. Sci., 18, pp. 149 - 180.

• Tramati, C., Savona, B., Mazzola, A. (2005). A study of the pattern of digestive

enzymes in Diplodus puntazzo (Cetti, 1777) (Osteichthyes, Sparidae): evidence for the

definition of nutritional protocols. Aquac. Int., 13, 89–95.

• Trujillo, P. (2007). A global analysis of the sustainability of marine aquaculture. Master

of science, resource management & environmental sciences dissertation. Vancouver,

BC: University of British Columbia 127p.

• Tukey, J.W., (1949). Comparing individual means in the analysis of variance.

Biometrics, 5, 99–114.

• Turchini G.M., Torstensen B.E. & Ng W.-K. (2009). Fish oil replacement in finfish

nutrition. Reviews in Aquaculture, 1, 10–57.

• Uauy R., Stringel G.,Thomas R. & Quan R. (1990). Effect of dietary nucleotides on

growth and maturation of the developing gut in the rat. Journal of Pediatric

Gastroenterology and Nutrition, 10, 497-503.

• Ufodike, E. B. C., Matty, A. J. (1989). Effect of potato and corn meal on protein and

carbohydrate digestibility by rainbow trout. Progressive Fish-Culturist, 51, 113-114.

• Ugolev AM, Kuz’mina VV (1993) Digestion processes and adaptation in fish.

Hydromrteoizdat, Sanct Petersburg, Russia, pp (In Russian)

• Vandenberg, G. W., De la Noue, J. (2001). Apparent digestibility comparison in

rainbow trout (Oncorhynchus mykiss) assessed using three methods of faeces

collection and three digestibility markers. Aquaculture Nutrition, 7, 237-245.

• Varsamos, S., Connes, R., Diaz, J., Barnabé, P., Charmantier, G. (2001). Ontogeny

of osmoregulation in the European sea bass Dicentrarchus labrax L.. Mar. Biol. 138,

909– 915.

• Venou, B., Alexis, M. N., Fountoulaki, E., Nengas, I., Apostolopoulou, M., Castritsi-

Cathariou, I.(2003). Effect of extrusion of wheat and corn on gilthead sea bream

(Sparus aurata) growth, nutrient utilization efficiency, rates of gastric evacuation and

digestive enzyme activities. Aquaculture, 225, 207-223.

• Walker J. P., Winton J. R. (2010). Emerging viral diseases of fish and shrimp. Vet Res.,6, 41-51. • Wang, L., C.L. Weller, V.L. Schlegel, T.P. Carr, and S.L. Cuppett. (2007). Comparison

of supercritical CO2 and hexane extraction of lipids from sorghum distillers grains. Eur.

J. Lipid Sci. Tech., 109(6),567-574.


• Ward, D. A., Carter, C. G., Townsend, A. T. (2005). The use of yttrium oxide and the

effect of faecal collection timing for determining the apparent digestibility of minerals

and trace elements in Atlantic salmon (Salmo salar, L.) feeds. Aquaculture Nutrition,

11, 49-59.

• Watson, R., Pauly, D. (2001).Systematic distortions in world fisheries catch trends.

Nature, 414(6863), 534–536.

• Webster, C.D., Thompson, K.R., Metts, L.S. & Muzinic, L.A. (2008). Use of distillers

grain with solubles and brewery by-products in fish and crustacean diets. In: Alternative

Protein Sources in Aquaculture Diets (Lim, C., Webster, C.D. & Lee, C.-S. eds), pp.

475–499. Hayworth Press, Taylor & Francis Group, New York, NY, USA.

• Webster, C.D., Tidwell, J.H. & Goodgame, L.S. (1993). Growth, body composition,

and organoleptic evaluation of channel catfish fed diets containing different

percentages of distillers grains with solubles. Prog. Fish Cult., 55, 95–100.

• Webster, C.D., Tidwell, J.H. & Yancey, D.H. (1991). Evaluation of distillers grains with

solubles as a protein source in diets for channel catfish. Aquaculture, 96, 179–190.

• Webster, C.D., Tidwell, J.H., Goodgame, L.S., Yancey, D.H. & Mackey, L. (1992b).

Use of soybean meal and distillers grains with solubles as partial or total replacement

of fish meal in diets for channel catfish, Ictalurus punctatus. Aquaculture, 106, 301–

309.

• Webster, C.D., Tiu, L.G., Morgan, A.M. & Gannam, A.L. (1999). Effect of partial and

total replacement of fish meal on growth and body composition of sunshine bass

Morone chrysops x M. saxatilis fed practical diets. J. World Aquac. Soc., 30, 443–453.

• Webster, C.D., Yancey, D.H. & Tidwell, J.H. (1992a) Effect of partially or totally

replacing fish meal with soybean meal on growth of blue catfish (Ictalurus furcatus).

Aquaculture, 103, 141–152.

• Whitehead, P., Bauchot, M.-L., Hureau, J.-C., Nielsen, J., Tortonese, E. (1984/1986).

Marine Species Identification Portal. Fishes of the NE Atlantic and the Mediterranean.

E. Bio Informatics (Ed.), UNESCO, Producer. Retrieved March 30, 2013, from Marine

Species Identification Portal: http://speciesidentification.org

• Widyaratne, G.P., Zijlstra, R.T. (2007). Nutritional value of wheat and corn distiller’s

dried grain with solubles: Digestibility and digestible contents of energy, amino acids

and phosphorus, nutrient excretion and growth performance of grower-finisher pigs.

Can. J. Anim. Sci., 87, 103–114.

• Williams, TN. (2008). An assessment of alternative feed ingredients in practical diets

for Florida pompano (Trachinotus carolinus) held in low salinity recirculating systems.

Master of Science thesis, University of Maine, Orono, Maine, USA.

• Wilson, R.P. (1994) Utilization of dietary carbohydrate by fish. Aquaculture, 124, 67–

80.

http://speciesidentification.org/


• Windell, J.T., Foltz, J.W. & Sarokon, J.A. (1978). Method of faecal collection and

nutrient leaching in digestibility studies. Progress in Fish Culture, 40, 51–55.

• Wong, D.W.S. (1995). Food Enzyme : Structure and Mechanism. Chapman and Hall.

New York. 390 p.

• Yamada, A., Takano, K. and Kamoi, I. (1993). Purification and properties of protease

from tilapia stomach. Nippon Suisan Gakkaishi, 59, 1903–1908.

• Zambonino Infante, J. L., Cahu, C. L. (2007). Dietary modulation of some digestive

enzymes and Metabolic processes in developing marine fish: Applications to diet

formulation. Aquaculture, 268, 98-105.

• Zhang, Y., Caupert. (2012). Survey of mycotoxins in US distiller´s dried grains with

solubles from 2009 to 2011. Journal of Agricultural and Food Chemistry, 60, 539-545.

• Zhi BJ., Liu W., Zhao CG., Duan YY. (2009). Effects of salinity on digestive enzyme

and alkaline phosphatase activity of young chum salmon (Oncorhynchus keta

Walbaum). J Shanghai Ocean Univ (in Chinese), 18, 289–294.

• Zhou, P., Zhou, P., Davis, D. A., Lim, C., Yildirim-Aksoy, M., Paz, P., Roy, L. A.

(2010). Pond Demonstration of Production Diets Using High Levels of Distiller's Dried

Grains with Solubles with or without Lysine Supplementation for Channel Catfish. North

American Journal of Aquaculture, 72, 361-367.

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