Page 1
MS
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Dried Distillers Grains with
Solubles (DDGS): a potential
protein source in feeds for
aquaculture.
Rui Pedro Moreira de Magalhães
Dri
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2013
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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
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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, ______/______/_________
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FCUP 2 DDGS: a potential protein source in feeds for aquaculture
“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
Page 5
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.
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FCUP 1 DDGS: a potential protein source in feeds for aquaculture
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
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FCUP 2 DDGS: a potential protein source in feeds for aquaculture
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
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FCUP 3 DDGS: a potential protein source in feeds for aquaculture
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.
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FCUP 4 DDGS: a potential protein source in feeds for aquaculture
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
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FCUP 5 DDGS: a potential protein source in feeds for aquaculture
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
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FCUP 6 DDGS: a potential protein source in feeds for aquaculture
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
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FCUP 7 DDGS: a potential protein source in feeds for aquaculture
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
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FCUP 8 DDGS: a potential protein source in feeds for aquaculture
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).
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FCUP 9 DDGS: a potential protein source in feeds for aquaculture
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
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FCUP 10 DDGS: a potential protein source in feeds for aquaculture
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.
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FCUP 11 DDGS: a potential protein source in feeds for aquaculture
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
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FCUP 12 DDGS: a potential protein source in feeds for aquaculture
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).
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FCUP 13 DDGS: a potential protein source in feeds for aquaculture
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,
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FCUP 14 DDGS: a potential protein source in feeds for aquaculture
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 .
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FCUP 15 DDGS: a potential protein source in feeds for aquaculture
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
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FCUP 16 DDGS: a potential protein source in feeds for aquaculture
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
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FCUP 17 DDGS: a potential protein source in feeds for aquaculture
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.
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FCUP 18 DDGS: a potential protein source in feeds for aquaculture
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).
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FCUP 19 DDGS: a potential protein source in feeds for aquaculture
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).
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FCUP 20 DDGS: a potential protein source in feeds for aquaculture
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
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FCUP 21 DDGS: a potential protein source in feeds for aquaculture
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.
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FCUP 22 DDGS: a potential protein source in feeds for aquaculture
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).
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FCUP 23 DDGS: a potential protein source in feeds for aquaculture
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).
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FCUP 24 DDGS: a potential protein source in feeds for aquaculture
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).
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FCUP 25 DDGS: a potential protein source in feeds for aquaculture
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
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FCUP 26 DDGS: a potential protein source in feeds for aquaculture
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
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FCUP 27 DDGS: a potential protein source in feeds for aquaculture
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
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FCUP 28 DDGS: a potential protein source in feeds for aquaculture
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.
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FCUP 29 DDGS: a potential protein source in feeds for aquaculture
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
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FCUP 30 DDGS: a potential protein source in feeds for aquaculture
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).
6DDGS1: corn dried distillers grains with soluble from Biocarburantes de Castilla y Leon, Spain
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
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FCUP 31 DDGS: a potential protein source in feeds for aquaculture
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.
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FCUP 32 DDGS: a potential protein source in feeds for aquaculture
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.
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FCUP 33 DDGS: a potential protein source in feeds for aquaculture
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
Page 39
FCUP 34 DDGS: a potential protein source in feeds for aquaculture
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.
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FCUP 35 DDGS: a potential protein source in feeds for aquaculture
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
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FCUP 36 DDGS: a potential protein source in feeds for aquaculture
( ) (( )
)
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
Page 42
FCUP 37 DDGS: a potential protein source in feeds for aquaculture
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
Page 43
FCUP 38 DDGS: a potential protein source in feeds for aquaculture
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
Page 44
FCUP 39 DDGS: a potential protein source in feeds for aquaculture
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).
Page 45
FCUP 40 DDGS: a potential protein source in feeds for aquaculture
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
1DDGS1: corn dried distillers grains with soluble from Biocarburantes de Castilla y Leon, Spain
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
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FCUP 41 DDGS: a potential protein source in feeds for aquaculture
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.
Page 47
FCUP 42 DDGS: a potential protein source in feeds for aquaculture
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).
2DDGS1,2: corn dried distillers grains with soluble from Biocarburantes de Castilla y Leon, Spain and Pannonia Gold,
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
Page 48
FCUP 43 DDGS: a potential protein source in feeds for aquaculture
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.
3DDGS1,2: corn dried distillers grains with soluble from Biocarburantes de Castilla y Leon, Spain and Pannonia Gold,
Hungry, respectively.
Page 49
FCUP 44 DDGS: a potential protein source in feeds for aquaculture
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.
3DDGS1,2: corn dried distillers grains with soluble from Biocarburantes de Castilla y Leon, Spain and Pannonia Gold,
Hungry, respectively.
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 *** ** * - - - - - - -
Page 50
FCUP 45 DDGS: a potential protein source in feeds for aquaculture
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.
3DDGS1,2: corn dried distillers grains with soluble from Biocarburantes de Castilla y Leon, Spain and Pannonia Gold,
Hungry, respectively.
Page 51
FCUP 46 DDGS: a potential protein source in feeds for aquaculture
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.
Page 52
FCUP 47 DDGS: a potential protein source in feeds for aquaculture
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
Page 53
FCUP 48 DDGS: a potential protein source in feeds for aquaculture
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.
Page 54
FCUP 49 DDGS: a potential protein source in feeds for aquaculture
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
Page 55
FCUP 50 DDGS: a potential protein source in feeds for aquaculture
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,
Page 56
FCUP 51 DDGS: a potential protein source in feeds for aquaculture
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
Page 57
FCUP 52 DDGS: a potential protein source in feeds for aquaculture
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
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FCUP 53 DDGS: a potential protein source in feeds for aquaculture
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
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FCUP 54 DDGS: a potential protein source in feeds for aquaculture
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.
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FCUP 55 DDGS: a potential protein source in feeds for aquaculture
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.
Page 61
FCUP 56 DDGS: a potential protein source in feeds for aquaculture
• 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.
Page 62
FCUP 57 DDGS: a potential protein source in feeds for aquaculture
•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.
Page 63
FCUP 58 DDGS: a potential protein source in feeds for aquaculture
• 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.
Page 64
FCUP 59 DDGS: a potential protein source in feeds for aquaculture
• 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.
Page 65
FCUP 60 DDGS: a potential protein source in feeds for aquaculture
• 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.
Page 66
FCUP 61 DDGS: a potential protein source in feeds for aquaculture
• 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.
Page 67
FCUP 62 DDGS: a potential protein source in feeds for aquaculture
• 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.
Page 68
FCUP 63 DDGS: a potential protein source in feeds for aquaculture
• 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.
Page 69
FCUP 64 DDGS: a potential protein source in feeds for aquaculture
• 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.
Page 70
FCUP 65 DDGS: a potential protein source in feeds for aquaculture
• 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.
Page 71
FCUP 66 DDGS: a potential protein source in feeds for aquaculture
• 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.
Page 72
FCUP 67 DDGS: a potential protein source in feeds for aquaculture
• 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
Page 73
FCUP 68 DDGS: a potential protein source in feeds for aquaculture
• 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
Page 74
FCUP 69 DDGS: a potential protein source in feeds for aquaculture
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
Page 75
FCUP 70 DDGS: a potential protein source in feeds for aquaculture
• 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
Page 76
FCUP 71 DDGS: a potential protein source in feeds for aquaculture
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.
Page 77
FCUP 72 DDGS: a potential protein source in feeds for aquaculture
• 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.
Page 78
FCUP 73 DDGS: a potential protein source in feeds for aquaculture
• 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.
Page 79
FCUP 74 DDGS: a potential protein source in feeds for aquaculture
• 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.
Page 80
FCUP 75 DDGS: a potential protein source in feeds for aquaculture
• 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.
Page 81
FCUP 76 DDGS: a potential protein source in feeds for aquaculture
• 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.