MsC_Thesis_Miguel_Correia

Universidade de Lisboa
Faculdade de Ciências
Departamento de Biologia Animal
Alguns aspectos da alimentação choco (Sepia officinalis
Linnaeus, 1758) em cativeiro, nas fases iniciais do ciclo de
vida.
Miguel José Teodoro Correia
Mestrado em Biologia e Gestão dos Recursos Marinhos
2006

Universidade de Lisboa
Faculdade de Ciências
Departamento de Biologia Animal
Alguns aspectos da alimentação do choco (Sepia officinalis
Linnaeus, 1758) em cativeiro, nas fases iniciais do ciclo de
vida.
Miguel José Teodoro Correia
Mestrado em Biologia e Gestão dos Recursos Marinhos
Dissertação orientada pelo Prof. Doutor Henrique Cabral
e Prof. Doutor J. Pedro Andrade (Ualg)
2006

Índice
______________________________________________________________________
Índice
1 Resumo Geral --------------------------------------------------------------------------- pág.1
2 General Introduction ------------------------------------------------------------------- pág. 6
Introdução geral
3 1st
Manuscript - Effects of live prey availability on growth and survival in early
stages of cuttlefish Sepia officinalis (Linnaeus, 1758) life cycle. --------------- pág.9
3.1 Abstract ----------------------------------------------------------------------------- pág.9
Resumo
3.1.1 Keywords ------------------------------------------------------------------------ pág.10
Palavras chave
3.2 Introduction ----------------------------------------------------------------------- pág.10
Introdução
3.3 Material and methods ------------------------------------------------------------ pág.11
Material e método
3.4 Results ----------------------------------------------------------------------------- pág.14
Resultados
3.5 Discussion ------------------------------------------------------------------------- pág.17
Discussão
4 2nd
Manuscript – Effects of prey starvation on growth and survival of juvenile
cuttlefish Sepia officinalis (Linnaeus, 1758). -------------------------------------- pág.21
4.1 Abstract ---------------------------------------------------------------------------- pág.21
Resumo
4.1.1 Keywords ------------------------------------------------------------------------ pág.21
Palavras chave
4.2 Introduction ----------------------------------------------------------------------- pág.21
Introdução
4.3 Material and methods ------------------------------------------------------------ pág.22
Material e métodos
4.4 Results ----------------------------------------------------------------------------- pág.26
Resultados
4.5 Discussion ------------------------------------------------------------------------ pág.31
Discussão
5 Final considerations ------------------------------------------------------------------ pág.34
Considerações finais
6 References ----------------------------------------------------------------------------- pág.35
Referências bibliográficas
7 Agradecimentos ---------------------------------------------------------------------- pág. 40
___________________________________________________________________
Mestrado em Biologia e Gestão dos Recursos Marinhos – F.C.U.L.

Resumo Geral
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Resumo Geral
De todas as espécies de cefalópodes, o choco (Sepia officinalis Linnaeus, 1758) é
considerada a espécie mais estudada (Boletzky, 1983), e uma das mais fáceis de cultivar
e reproduzir em cativeiro (Pascual, 1978; Boletzky, 1983; Boletzky e Hanlon, 1983;
Forsythe et al., 1994; Lee et al., 1998; Domingues et al., 2001b, 2002, 2003a).
Esta espécie constitui um importante recurso piscícola e é altamente explorada em
vários países (Roper et al., 1984). O choco é vendido fresco ou congelado e é
grandemente consumido no Japão, República da Coreia, Itália, Espanha e Portugal. As
capturas totais registadas para esta espécie foram de 17017, 16535 e 15660 toneladas
para os anos 2002, 2003 e 2004, respectivamente (FAO, 2000). Em Portugal, os valores
registados para os mesmos anos foram de 1478, 1368 e 1809 toneladas respectivamente
(DGPA, 2004). Entre 2001 e 2003, o valor de mercado do choco variou entre 3.56 e
3.82 €.Kg-1
(DGPA, 2003). No entanto, o “choquinho” pode atingir valores de mercado
até aos 15 €.Kg-1
.
S. officinalis é a espécie de cefalópode mais facilmente cultivada em laboratório
(Forsythe et al., 1994; Domingues, 1999; Domingues et al., 2001a, 2001b, 2002,
2003a), tendo sido cultivada com sucesso durante muitos anos (Forsythe et al., 1994;
Domingues, 1999; Sykes et al., 2003). Actualmente, o cultivo desta espécie tem-se
desenvolvido devido o seu grande potencial para cultivo em larga escala (Domingues et
al., 2001a, 2001b, 2003a; Sykes, et al., 2003; Correia et al., 2005). O choco possui
várias características que o tornam altamente adequado para o cultivo em larga escala,
tais como elevada adaptabilidade à vida em cativeiro, ovos grandes, elevada taxa de
sobrevivência após eclosão, comportamento sedentário, elevada tolerância a grandes
densidades com pouco canibalismo, tolerante ao manuseamento, aceitação de presas
mortas e fácil reprodução em cativeiro (Domingues et al., 2002; Forsythe et al., 2002;
Sykes et al., 2006).
Nos últimos anos, vários estudos foram realizados no sentido de determinar a melhor
dieta disponível, de modo a obter taxas óptimas de crescimento e sobrevivência (Castro
et al., 1993; Domingues et al., 2001b, 2003a, 2003b, 2004, 2005). Várias dietas foram
testadas em recém-eclodidos, sendo que Paramysis nouvelli e Palaemonetes varians
obtiveram os melhores resultados (Domingues et al., 2004). Surimi e outras dietas
artificiais foram igualmente testadas mas com poucos ou nenhuns resultados (Castro,
___________________________________________________________________ 1

Resumo Geral
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1991; Castro et al., 1993; Castro e Lee, 1994; Domingues et al., 2005), sendo que
nenhuma dieta artificial foi ainda testada em recém-eclodidos. As duas primeiras
semanas após a eclosão são consideradas críticas para o sucesso do cultivo desta espécie
(Domingues et al., 2004) e até hoje, somente foi registado o uso de alimento vivo para
este período. Assim, devem ser canalizados esforços no sentido de determinar a melhor
dieta viva possível, bem como o protocolo alimentar, para esta fase de vida do choco.
Estudos anteriores efectuados sobre o uso de dietas vivas como alimento do choco são
na maioria qualitativos e não quantitativos, sendo que pouca informação existe sobre
relação entre alimento e crescimento. Esta informação é extremamente importante no
sentido de optimizar o cultivo desta espécie (Koueta e Boucaud-Camou, 1999), e de
acordo com estes autores a quantidade de alimento fornecido influencia a taxa de
alimentação, especialmente para chocos com 10 a 20 dias. Este resultado evidencia a
importância de uma correcta alimentação para este período de vida do choco.
O presente estudo é composto por duas experiências. Na primeira experiência
efectuada, testou-se o efeito da disponibilidade de dietas vivas no crescimento e
sobrevivência da espécie Sepia officinalis. Foi utilizado um total de 360 recém-
eclodidos, distribuídos aleatoriamente por 12 tanques de 10l de capacidade perfazendo
assim um total de 30 chocos/tanque. Testaram-se duas dietas diferentes, Paramysis
nouvelli (Dieta I) e Palaemonetes varians (Dieta II), fornecidas a 2 diferentes
quantidades (ad libitum e o dobro da quantidade fornecida aos tanques em ad libitum,
para o mesmo dia, relativos à biomassa de cada tanque). Efectuaram-se amostragens
semanais de modo a obter o peso individual de cada replicado, bem como o peso do
alimento não ingerido. A partir dos dados obtidos, calculou-se o peso médio; a taxa de
alimentação (TA=alimento consumido.d-1
/peso médio*100/número de indivíduos); a
taxa de conversão alimentar (TCA=((PM1-PM0)*100/(peso total de alimento consumido
entre pesagens/número de indivíduos) em que PM0 e PM1 representam o peso corporal
médio inicial e final respectivamente); a taxa de crescimento instantânea média (TCI)
(% PHC.d-1
)= (LnPM1-LnPM0)/t*100 em que PHC representa o Peso Húmido Corporal,
Ln o logaritmo neperiano e t o número de dias do período de tempo. Por outro lado
efectuou-se a comparação do crescimento através das curvas de crescimento obtidas do
tipo y=a*ebx
. Após a análise estatística dos resultados, foram encontradas diferenças
significativas no crescimento e peso médio, entre tratamentos alimentados com a Dieta
___________________________________________________________________ 2

Resumo Geral
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I. Do mesmo modo, para a mesma dieta, os tratamentos apresentaram diferenças
significativas na taxa de alimentação (TA) (na semana 1 e 2) e na taxa de crescimento
instantânea (TCI) (na semana 1).
Quanto aos tratamentos alimentados com a Dieta II, foram encontradas diferenças
significativas na taxa de conversão alimentar (TCA), TCI e TA (a partir da semana 2),
no entanto, nenhumas diferenças significativas foram encontradas no crescimento. Os
resultados obtidos nesta experiência indicam que, no âmbito da produção do choco, nas
fases iniciais do ciclo de vida, os tanques de cultivo devem ter presentes uma
determinada quantidade de dietas vivas de modo a promover o aumento das taxas de
alimentação e assim optimizando o crescimento dos indivíduos cultivados. A
quantidade de alimento a fornecer por tanque deverá ser ajustada tendo em conta o tipo
de presa. Torna-se assim essencial a investigação nesta área de modo a determinar a
quantidade óptima de alimento a ser fornecido por volume de tanque de cultivo.
Relativamente à segunda experiência, pretendeu-se estudar os efeitos da qualidade de
dietas vivas no crescimento e sobrevivência de juvenis de S. officinalis. A dieta utilizada
foi a camarinha P. varians. Foi utilizado para esta experiência um total de 90 chocos,
com um mês de idade, distribuídos aleatoriamente por 9 tanques de 10l de capacidade e
divididos em três grupos de 3 tanques. O primeiro grupo foi alimentado com camarinha
capturada no próprio dia (DP), o segundo foi fornecido camarinha armazenada durante
5 dias em tanques de 200 litros (SP), sem alimento, e finalmente o terceiro grupo em
que foi fornecido camarinha alimentada com uma dieta artificial (FP). A dieta artificial
usada para alimentar FP foi elaborada com base em dietas para camarão obtidas por
outros autores (Oliva-Teles, 1985; Sudaryono et al., 1995; Mu et al, 1998; Floreto et al.,
2000; Glencross et al., 2002; Gong et al., 2000; Kureshy e Davis, 2000; Wouters et al.,
2001). Efectuaram-se amostragens semanais de modo a obter o peso individual de cada
replicado, bem como o peso do alimento não ingerido. A partir dos dados obtidos,
calculou-se o peso médio; a taxa de alimentação (TA=alimento consumido.d-1
/peso
médio*100/número de indivíduos); a taxa de conversão alimentar (TCA=((PM1-
PM0)*100/(peso total de alimento consumido entre pesagens/número de indivíduos) em
que PM0 e PM1 representam o peso corporal médio inicial e final respectivamente); a
taxa de crescimento instantânea média (TCI) (% pc d-1
)= (LnPM1-LnPM0)/t*100 em
que pc representa o Peso húmido Corporal, Ln o logaritmo neperiano e t o número de
___________________________________________________________________ 3

Resumo Geral
______________________________________________________________________
dias do período de tempo. Por outro lado efectuou-se a comparação do crescimento
através das curvas de crescimento obtidas do tipo y=a*ebx
. Por fim procedeu-se à
análise estatística de modo a determinar possíveis diferenças entre tratamentos.
A média de TCI para SP, DP e FP foi de 2.8 ± 1.0% do peso corporal por dia (pc d-1
),
3.3 ± 1.1% pc d-1
e 4.9 ± 0.5% pc d-1
, respectivamente. Não foram encontradas
diferenças significativas entre SP vs DP (P>0.05). Por outro lado, foram encontradas
diferenças significativas entre DP vs FP e SP vs FP (P<0.05).
Os valores médios de TA foram de 9.3 ± 2.4% do pc d-1
, 9.0 ± 1.6% pc d-1
e 15.5 ±
0.9% pc d-1
, para SP, DP e FP, respectivamente. Não foram encontradas diferenças
significativas entre SP vs DP (P>0.05). Por outro lado, foram encontradas diferenças
significativas entre DP vs FP e SP vs FP (P<0.05).
A média de TCA foi de 40.2 ± 13.3%, 46.4 ± 16.2% e 38.7 ± 5.6% para SP, DP e FP,
respectivamente. Não foram encontradas nenhumas diferenças significativas entre todos
os grupos (P>0.05).
A mortalidade mais elevada foi registada em FP (7 indivíduos, sendo que 6
pertenciam a um só replicado). SP registou 2 mortos e DP 1 único morto.
Os resultados obtidos indicam que o armazenamento de presas (P. varians), sem
alimentação durante 5 dias, não é adequado no âmbito da alimentação de juvenis de
choco. Por outro lado, visto que os chocos alimentados com presas alimentadas a ração
obtiveram os melhores resultados, esforços têm de ser realizados no sentido de elaborar
uma dieta artificial que promova um maior crescimento nos chocos, sem custos
económicos acrescidos. Assim, o armazenamento de presas deve ser tido em conta de
modo a promover a redução de custos e de recursos humanos aquando da produção de
choco. Assim, devem ser efectuados estudos no sentido de determinar uma dieta
artificial adequada e economicamente viável de modo a promover o crescimento óptimo
dos indivíduos produzidos.
Assim, como conclusão, este trabalho evidenciou a importância de uma correcta
alimentação nas primeiras semanas de vida do choco, sendo que vários estudos devem
ser realizados no sentido de determinar a quantidade óptima de alimento a ser fornecido,
de modo a assegurar um crescimento óptimo de todos os indivíduos em cultivo. Por
outro lado, o armazenamento de presas vivas deve ser considerado, desde que seja
acompanhado com uma correcta alimentação das mesmas. Assim, futuros estudos
devem ter como objectivo a determinação de uma dieta artificial que seja
___________________________________________________________________ 4

Resumo Geral
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economicamente viável no sentido de tornar o armazenamento de presas uma solução a
considerar, beneficiando da redução de mão-de-obra e de custos que esta implica.
___________________________________________________________________ 5

General Introduction
______________________________________________________________________
Cephalopods play a very important role in marine food webs, as active predators of
shrimp, crabs and fish; and also as prey of marine mammals, aquatic birds and fish
(Roper et al., 1984). Cephalopods have a high market value (Rocha et al., 1999), and
many species are caught for human consumption all over the world (González et al.,
1996), thus being a very important food source for humans (Boletzky & Hanlon, 1983).
Its nervous system has been recently studied due to its homology to mammals
physiologic systems (Lee, 1994), and used as models for biologic research and medicine
in the neuroscience field, biochemical nutrition and immunology (Oestmann et
al.,1997). This has decisively contributed to the development of cephalopod culture in
the early 1980´s (Boletzky & Hanlon, 1983; Hanlon et al., 1991; Lee, et al., 1998).
When compared with fishes, their direct competitors in the food web, these organisms
have considerably higher growth rates. This fact is due to the lack of internal and
external skeleton, a very efficient energetic use of proteins (up to 90%), and a high
feeding rate (up to 50% of body weight per day in some species, especially in early
stages of life cycle) (Lee, 1994).
Cephalopods have high growth rates, ranging from 3 to 15% of body weight per day
during their life cycle (Lee, 1994), but can be as high as 20% in the first weeks
(Domingues et al., 2001a); high conversion rates (Domingues et al., 2003b, 2004) and
short life cycles (Forsythe & Van Heukelem, 1987; Domingues et al., 2001a, 2002).
Thus, the importance of cephalopod culture has being consolidating in the past few
years (Lee et al., 1998), and the potential for commercial aquaculture of some species
has been recognized.
Amongst all cephalopod species, cuttlefish (Sepia officinalis Linnaeus, 1758) (figure
1) is considered to be the most studied (Boletzky, 1983), and one of the easiest to breed
and maintain in captivity in worldwide laboratories (Pascual, 1978; Boletzky, 1983;
Boletzky & Hanlon, 1983; Forsythe et al., 1994; Lee et al., 1998; Domingues et al.,
2001b, 2002, 2003a).
This species constitutes a very commercially important fishing resource, and is
heavily exploited in several countries such as Italy, France, England, Western Africa
and Senegal (Roper et al., 1984). However, this resource’s fishery has increased
significantly in Morocco in the past few years (Roper et al., 1984). Cuttlefish is sold
___________________________________________________________________ 6

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fresh or frozen and is highly consumed Japan, Korea Republic, Italy, Spain and
Portugal. Total captures for this species were 17017, 16535 and 15660 tons for 2002,
2003 and 2004, respectively (FAO, 2000). In Portugal, cuttlefish captures for the same
years were of 1478, 1368 and 1809 tons respectively (DGPA, 2004). Between 2001 and
2003, the market price of cuttlefish ranged from 3.56 to 3.82 €.Kg-1
(DGPA, 2003).
Nevertheless, the “choquinho” can achieve a market value as high as 15 €.Kg-1
.
___________________________________________________________________ 7
2cm
Figura 1 – Sepia officinalis
S. officinalis is one of the cephalopod species most easily reared in captivity (Forsythe
et al., 1994; Domingues, 1999; Domingues et al., 2001a, 2001b, 2002, 2003a). This
species has been successfully cultured for many years (Forsythe et al., 1994;
Domingues, 1999; Sykes et al., 2003). Nowadays, the rearing of this species has been
developed due to its great potential for large scale culture (Domingues et al., 2001a,
2001b, 2003a; Sykes, et al., 2003; Correia et al., 2005). The cuttlefish presents several
characteristics that make it highly suitable for large scale culture, such as high
adaptability to captivity, large eggs, high hatchling survival, sedentary behaviour,
tolerance to high culture densities with little or even no cannibalism, handling, shipping,
acceptance of dead prey and easy reproduction in captivity (Domingues et al., 2002;
Forsythe et al., 2002; Sykes et al., 2006).
For the past few years, several experiments have been done to determine the best
available diet, in order to obtain optimal growth and survival (Castro et al., 1993;
Domingues et al., 2001b, 2003a, 2003b, 2004, 2005). Several diets were tested on
cuttlefish hatchlings, with Paramysis nouvelli and Palaemonetes varians promoting the

______________________________________________________________________
highest growth rates (Domingues et al., 2004). Surimi and other artificial diets were
also tested but with very little success (Castro, 1991; Castro et al., 1993; Castro and
Lee, 1994; Domingues et al., 2005), and up to now no artificial diet was ever recorded
to be used on hatchlings. The first two weeks after hatching are considered to be
important to insure culture success (Domingues et al., 2004) and only the use of live
food, for cuttlefish this age, has been recorded. Therefore, efforts must be made to
assess best live diet and feeding protocol, especially for the early stages of cuttlefish life
cycle.
Previous investigations concerning the use of live diets as food source for cuttlefish
were mostly qualitative and little data is available concerning feed ration and
growth/ration relations. This information is very important in order to optimize
cuttlefish culture (Koueta and Boucaud-Camou, 1999), and according with these
authors the amount of food offered affects the food ingestion, especially in reared
cuttlefish from 10 to 20 days. This result highlights the importance of proper feeding
conditions for cuttlefish during the first two or three weeks of its life cycle.
The present thesis is composed by two papers. The first study aimed to determine the
influence of food availability on growth and survival of newly-born cuttlefish for two
different diets. This information is essential to promote optimal growth on critical
phases of cuttlefish life cycle, thus obtaining better fitted specimens and so contributing
to the production success of this species. The second paper addresses the influence of
prey starvation on growth and survival of juvenile cuttlefish. Up to now no artificial diet
was ever recorded to be used on hatchlings and, therefore, tests must be made to
determine the viability of live prey culture to sustain production, especially for the early
stages of cuttlefish. The results obtained in this study are important to determine the
viability of stocking large quantities of prey under starvation, when feeding cuttlefish,
thus reducing the production costs when rearing this species at a commercial level.
___________________________________________________________________ 8

1th
Manuscript - Abstract
______________________________________________________________________
3. Effects of live prey availability on growth and survival in early
stages of cuttlefish Sepia officinalis (Linnaeus, 1758) life cycle
3. 1. Abstract
The effects of live prey availability on growth and survival of Sepia officinalis
were studied. A total of 360 cuttlefish hatchlings were used, distributed in twelve 10
litre tanks (277 cuttlefish m-2
). Two experiments were performed, being each
experiment composed by two treatments. Cuttlefish in first experiment were fed with
live mysids Paramysis nouvelli (Diet I). In the first treatment of this experiment,
cuttlefish were fed enough live diet to enable satiation (Diet I A). Second treatment
(Diet I B) was fed the double quantity that was provided to first treatment, of the same
live diet (i.e. if 20% body weight day-1
was used for first treatment, 40% bw d-1
would
be used for second treatment). Cuttlefish in second experiment were fed with live
Atlantic ditch shrimp Paleomonetes varians (Diet II). In this experiment the number of
specimens, as well as the experimental design was the same as described above for the
first experiment.
Mean values of feeding rate (FR) for the first experiment were 3.4 ± 3.1% and
14.8 ± 3.3%, while for the second experiment, FR values were 9.6 ± 1.5% and 13.4 ±
0.9%, for first and second treatment, respectively. Average instantaneous growth rates
(IGR) were 7.0 ± 0.9% and 7.7± 1.8% for Diet I A and Diet I B, respectively; and 4.3 ±
1.5% and 4.8 ± 1.6%, for Diet II A and Diet II B, respectively.
Cuttlefish fed Diet I showed statistical differences (P<0.05) in FR on week 1 and
week 2, while IGR showed statistical differences (P<0.05) in week 1. Statistical
differences were found in IGR, food conversion (FC) and FR (P>0.05) in cuttlefish fed
Diet II, from week 2 onwards. Nevertheless no statistical differences were found in
growth (P>0.05). Final biomass was overall higher for cuttlefish in the second
treatment, for both diets tested (16.68 ± 0.39g and 13.10 ± 1.24 g for Diet I B and II B,
respectively).
Results indicate that prey availability influences growth and final biomass, no
matter the prey used. Therefore, a certain amount of prey should be always present in
culture tanks, in order to promote higher feeding rates and thus providing optimal
growth, especially in early stages of cuttlefish life cycle
___________________________________________________________________ 9

1th
Manuscript – Introduction
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3. 1. 1. Keywords: Cuttlefish; Cuttlefish culture; Hatchlings; Live diet; Growth;
Prey availability
3. 2. Introduction
Cephalopod’s potential for commercial aquaculture has been increasing in the
past few years (Boucaud-Camou, 1990; Hanlon et al., 1991; Lee et al., 1998) due to
several important characteristics, such as high growth rates between 3 and 10% body
weight per day (bw d-1
) (Lee, 1994), which can be higher than 20% bw d-1
for the early
stages of their life cycle (O’Dor and Wells, 1987; Domingues et al., 2001a); high food
conversion rates (Domingues et al., 2003a, 2003b, Correia et al., 2005); feeding rates
between 20 and 50% bw d-1
(Boucher-Rodoni et al., 1987) and short life cycle (Forsythe
and Van Heukelem, 1987; Domingues et al., 2001a, 2002).
Cuttlefish, Sepia officinalis has been cultured in laboratory for many years
(Boletzky and Hanlon, 1983; Forsythe et al., 1991, 1994; Lee et al., 1991; Domingues
et al., 2001b, 2002). This species possesses several characteristics that make it highly
suitable for large scale culture (Forsythe et al., 1994, Domingues et al., 2002; Sykes et
al., 2006).
In order to obtain high survival rates and optimal growth in early stages of
cuttlefish life cycle, adequate feeding must be provided. For the past few years, several
experiments have been done to determine the best available diet, in order to obtain
optimal growth and survival (Castro et al., 1993; Domingues et al., 2001b, 2003a,
2003b, 2004, 2005). Several live diets were tested on cuttlefish hatchlings, with
Paramysis nouvelli and Palaemonetes varians promoting the highest growth rates
(Domingues et al., 2004). Surimi and other artificial diets were also tested but with very
little success (Castro, 1991; Castro et al., 1993; Castro and Lee, 1994; Domingues et al.,
2005), and up to now no artificial diet was ever recorded to be used on hatchlings.
Therefore, efforts must be made to assess best live diet and feeding protocol.
___________________________________________________________________ 10
Previous investigations concerning the use of live diets as food source for
cuttlefish were mostly qualitative and little data is available concerning food ration and
growth/ration relations. This information is very important to optimize cuttlefish culture
(Koueta and Boucaud-Camou, 1999), and according with these authors the amount of

1th
Manuscript – Material and methods
______________________________________________________________________
food offered affects the food ingestion rate, especially in reared cuttlefish from
10 to 20 days. The results obtained by these authors highlight the importance of proper
feeding conditions for cuttlefish during the first two or three weeks of its life cycle.
This study aimed to determine the influence of food availability on growth and
survival of newly-born cuttlefish fed with two different live diets. This information is
essential to promote optimal growth in critical phases of cuttlefish life cycle, thus
obtaining better fitted specimens and so contributing to the success of cuttlefish culture.
3. 3. Material and methods
Two experiments were conducted at the Ramalhete Aquaculture Field Station of
the University of the Algarve, located in the Ria Formosa marine lagoon (South
Portugal), in a flow-through culture system that was composed of 12 rectangular tanks
(12 cm water depth and 10 litre of volume) (figure 1). Salinity varied between 36 ± 1 ‰
and water flow was of 10 L h-1
per tank. Temperature averaged 18.5 ± 0.5 ºC. Tanks
were illuminated from above with fluorescent light, with an intensity of 600 lux at the
water surface and a photoperiod controlled by a timer at 12L:12D. Water quality
parameters kept stable throughout the experiment. Ammonia values were always bellow
detectable levels, nitrate <0.3 mg l-1
and nitrite <12.5 mg l-1
. Hatchlings used in this
study were obtained from a natural breeding broodstock kept in the facilities where
these experiments took place.
Experiment 1 (Diet I – Paramysis nouvelli)
At the beginning of the experiment, mean initial weight of cuttlefish was 0.12 ±
0.01 g, for both Diet I A and Diet I B. No significant differences (P>0.05) were found
between weights of first and second treatment replicates.
In the first experiment, the effect of prey availability was tested using live
mysids (P. nouvelli) (Diet I) as food source. A total of 180 hatchlings were randomly
distributed in six tanks, obtaining a final density of 30 cuttlefish tank-1
. Two treatments
were used in this experiment. Triplicates were used for each treatment. Cuttlefish in
each tank was weighed on a weekly basis.
In the first treatment (Diet I A), hatchlings were fed once a day and enough live
diet to enable individual satiation between feeding intervals. Food provided, was
___________________________________________________________________ 11

1th
______________________________________________________________________
adjusted each day by observation of the remaining prey in the tanks. Food percentages
(% body weight day-1
) of prey provided for each tank, were recorded daily. During the
total duration of the experiment, prey was always present in the tanks. Food quantity
provided for each tank was based on respective cuttlefish biomass, in each weighing
interval. After each weighing period and knowing the total biomass present in each
tank, food rations were re-calculated. Before each weighing period, all remaining prey
in each tank was removed and weighed, to determine exactly the weight of prey
consumed in a week period. Food percentage (% bw d-1
) given for first treatment, at the
beginning of the experiment, was of 20% bw d-1
which is considered to be an adequate
ration, according to Domingues et al. (2001b, 2002, 2003a; 2003b, 2004).
In the second treatment (Diet I B), hatchlings were fed the double of food
percentage given to the first treatment (i.e. if 10% bw d-1
was given to first treatment,
20% bw d-1
would be given to second treatment).
Fig. 1 – Rearing system representing the schematics of the experiment tanks; (1) inflow pipes; (2) outflow
pipes; (3) rearing tanks; (4) settling tank; (5) outflow during semi-open system; (6) filtering tank; (7) bio-
filter; (8) protein skimmer, (9) reservoir tank; (10) leveller; (11) inflow during semi-open system after
passing through a ultra-violet light filter; (12) water pump; (13) inflow to the other rearing tanks.
Experiment 2 (Diet II – Palaemonetes varians)
At the beginning of the experiment, mean initial weight of cuttlefish was 0.19 ±
0.01 g and 0.19 ± 0.01 g, for Diet II A and Diet II B, respectively. No significant
___________________________________________________________________ 12

1th
______________________________________________________________________
differences (P>0.05) were found between weights of first and second treatment’s
replicates.
In the second experiment, the effect of prey availability was tested using live
Atlantic ditch shrimp (P. varians) (Diet II) as food source. In this experiment the
number of specimens, as well as the experimental design was the same as described
above for the first experiment.
Prey was captured daily in ponds surrounding the culture facility, using bottom
hand held trawling nets. Both experiments lasted 3 weeks.
Data analysis
Growth between first and second treatment in each experiment was compared
through the analysis of the growth curves, using a multiple regression analysis (Zar,
1999). Since cuttlefish growth is exponential during its early the life cycle (Domingues
et al., 2002; Sykes et al., 2003; Correia et al., 2005), growth data was converted to
natural logarithm and linear regression models were used for comparison between
treatments of the same diet. Mean weight was used to calculate the Mean Instantaneous
Growth Rate (% bw d-1
) (IGR) = ((lnW2-lnW1)/t*100), where W2 and W1 are the final
and initial weights of the cuttlefish, respectively, ln the natural logarithm and t the
number of days of the time period. Comparisons (one-way ANOVA) (Zar, 1999) were
done using all individual weights from each replicate in each treatments of the same diet
tested. If during that period no differences were found in the three replicates of each
density, all weights of the three replicates were pooled and t-test (Zar, 1999) was used
to compare weights of all individuals in the two treatments. Feeding Rate (% bw d-1
)
(FR) was calculated in all weighing periods using the expression (FI/Average
W(t))*100, where FI is the food ingested and average W(t) is the average wet weight of
the cuttlefish during the time period (t). Food Conversion (FC) was determined using
the expression (W2-W1)/FI, where W2-W1 is the weight gained by the cuttlefish during
the time period. Biomass (g) (B) present in each tank was calculated at each weighing
period. For every weighing period, and for each diet, feeding rates of the three replicates
of one treatment were compared to those from the other, using a t-test (Zar, 1999).
___________________________________________________________________ 13

1th
Manuscript – Results
______________________________________________________________________
Mean cumulative mortality (mean percentage of increasing values of mortality)
was calculated for every weighing period. The t-test (Zar, 1999) was used to determine
differences between diet densities.
In all test procedures, data was tested for normality and homogeneity, and
whenever one of these requisites was not present, alternative non-parametric tests (Zar,
1999) were used.
3. 4. Results
Experiment 1
The average weight of cuttlefish fed with Diet I A and Diet I B, at the end of the
experiment was 0.47 ± 0.07 g and 0.56 ± 0.08 g, respectively (figure 2). Significant
differences in growth were found only for cuttlefish fed with Diet I (P<0.05), from
week 1 onwards (table 1).
The growth curve for cuttlefish fed with Diet I is described by the expression
W=0.164(±0.006)*e0.038(±0.002)D
(R2
=0.57) and W=0.182(±0.007)*e0.039(±0.002)D
(R2
=0.55), for first and second treatment, respectively; where W is average wet weight
(g) and D represents time (days). Statistical differences were found between growth
curves (F=15.897; P<0.05).
0,0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
1 8 15 22
Days
MeanWei
B 0.039(±0.002)X
Y=0.182(±0.007)*e
Y=0.164(±0,006)*e
0,038(±0,002)XA
ht(g)g
___________________________________________________________________ 14
Fig. 2. Mean growth in weight (g) of cuttlefish fed with Diet I A and Diet I B. Dots represent
average weight of cuttlefish in that replicate. The exponential curves were adjusted to the
average weights.

1th
______________________________________________________________________
Average growth rates were 7.0 ± 0.9% and 7.7± 1.8% for Diet I A and Diet I B,
respectively. Highest IGR values for cuttlefish fed with Diet I were obtained at week 1
(8.0 ± 0.5% and 10.0 ± 0.2% bw d-1
, for first and second treatment, respectively). For
both feeding quantities, growth rate was not significantly different for Diet I (P>0.05),
except for week 1 (table 1).
Mean values of feeding rate for this experiment were 13.4 ± 3.1% and 14.8 ±
3.3% for first and second treatment, respectively. Highest values of FR were obtained
for Diet I A and Diet I B at week 1 (17.0 ± 0.7% and 18.7 ± 0.2%, respectively). No
statistical differences were found for cuttlefish fed with Diet I (P>0.05), except for week
2 (table 1).
Average food conversion values were 40.6 ± 12.6% and 38.8 ± 7.2%, for Diet I
A and Diet I B, respectively. Highest values of FC were obtained at week 2 for
cuttlefish fed with Diet I for first and second treatment (56.4 ± 4.7% and 47.0 ± 2.0%,
respectively). Food conversion showed no statistical differences between first and
second treatment, for cuttlefish fed with Diet I (P>0.05), except for week 2. Statistical
differences were found for Diet II, from week 2 onwards (table 1).
Final biomass obtained for first and second treatment was 14.01 ± 0.39g and
16.68 ± 0.39g, respectively.
Mortality was only registered for cuttlefish in the first treatment (a total of 3
deaths).
Experiment 2
The average weight obtained for cuttlefish fed with Diet II A and Diet II B was
0.46 ± 0.10 g and 0.51 ± 0.12 g, respectively (figure 3)
The growth curve for cuttlefish fed with Diet II is described by the expression
W=0.183(±0.006)*e0.043(±0.002)D
(R2
=0.72) and W=0.175(±0.007)*e0.050(±0.002)D
(R2
=0.75), for first and second treatment, respectively. No statistical differences were
found between growth curves (F=1.839; P>0.05), for first and second treatment,
respectively.
Mean values of growth rates for this experiment were 4.3 ± 1.5% and 4.8 ±
1.6%, for Diet II A and Diet II B, respectively. Highest IGR values for cuttlefish fed
with Diet II were obtained at week 2 (6.0 ± 0.2% and 7.0 ± 0.2% bw d-1
, for first and
___________________________________________________________________ 15

1th
______________________________________________________________________
second treatment, respectively). Statistical differences between treatments (P<0.05)
were found from week 2 onwards (table 1).
0,0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
1 8 15 22
Days
MeanWeight(g) B Y=0.175(±0.007)*e
0.050(±0,002)X
Y=0.183(±0.006)*e
0.043(±0.002)XA
Fig. 3. Mean growth in weight (g) of cuttlefish fed with Diet II A and Diet II B. Dots
represent average weight of cuttlefish in that replicate. The exponential curves were adjusted
to the average weights.
Average feeding rates obtained were 9.6 ± 1.5% and 13.4 ± 0.9%, for first and
second treatment, respectively. Individuals fed with Diet II A and Diet II B presented
higher feeding rates at week 1 of the experiment (11.3 ± 1.1% and 14.0 ± 0.9% bw d-1
,
respectively). Significant differences were found (P<0.05) between treatments
throughout the experiment (table 1).
Mean food conversion rates were 38.5 ± 10.7% and 30.2 ± 10.8%, for Diet II A
and Diet II B, respectively. Higher values of FC were obtained at week 2 for cuttlefish
fed with Diet II for first and second treatment (51.0 ± 2.2% and 44.5 ± 1.2% bw d-1
,
respectively). Statistical differences were found (P<0.05) for Diet II, from week 2
onwards (table 1).
Final biomass obtained for first and second treatment was 11.15 ± 2.20g and
13.10 ± 1.24g, respectively.
The highest mortality was obtained in cuttlefish fed with Diet II (total deaths of
9 and 3 for the first and second treatment, respectively).
___________________________________________________________________ 16

1th
Manuscript – Discussion
______________________________________________________________________
___________________________________________________________________ 17
Prey Sampling
Week 1 Week 2 Week 3
Mean Weight (g)
Diet I A 0.12 ± 0.02 0.21 ± 0.04* 0.34 ± 0.06* 0.47 ± 0.07*
Diet I B 0.12 ± 0.01 0.24 ± 0.05* 0.38 ± 0.08* 0.56 ± 0.08*
Diet II A 0.19 ± 0.02 0.25 ± 0.04 0.38 ± 0.07 0.46 ± 0.10
Diet II B 0.19 ± 0.02 0.24 ± 0.04 0.40 ± 0.08 0.51 ± 0.12
IGR (%bw.d-1
)
Diet I A 8.0 ± 0.5* 6.8 ± 0.5 6.1 ± 0.2
Diet I B 10.0 ± 0.2* 6.6 ± 0.4 6.5 ± 0.4
Diet II A 4.0 ± 0.9 6.0 ± 0.2* 2.9 ± 0.3*
Diet II B 3.8 ± 0.0 7.0 ± 0.2* 3.7 ± 0.3*
FC (%)
Diet I A 37.0 ± 1.6 56.4 ± 4.7* 30.4 ± 1.8
Diet I B 38.5 ± 0.9 47.0 ± 2.0* 30.8 ± 1.9
Diet II A 31.9 ± 9.5 51.0 ± 2.2* 32.5 ± 3.3*
Diet II B 22.6 ± 2.8 44.5 ± 1.2* 23.5 ± 0.1*
FR (%bw.d-1
)
Diet I A 16.6 ± 0.1* 9.9 ± 0.4* 13.3 ± 0.4
Diet I B 18.7 ± 0.2* 11.2 ± 0.3* 14.6 ± 1.2
Diet II A 11.3 ± 1.1* 9.6 ± 0.2* 8.0 ± 0.0*
Diet II B 14.0 ± 0.9* 12.4 ± 0.0* 13.7 ± 0.1*
Table 1 - Mean weight, growth rate (IGR), feeding rate (FR) and food conversions (FC) of
cuttlefish hatchlings, fed mysids (Diet I) and Atlantic ditch shrimp (Diet II), first (A) and second
treatment (B), during 3 weeks.
* Values were significantly different within that period (P<0.05).
3. 5. Discussion
It is generally accepted that reared species fed with live diets, should have
always prey available in culture tanks, to enable satiation thus promoting optimal
growth. Yet, it is sometimes difficult to maintain proper food quantities in the tanks, in
order to sustain satiation. Several biotic and abiotic factors may produce variations in
feeding rate and prey consumption per day, and thus punctual lack of prey in tanks may
occur. Since cuttlefish is a visual predator and attack is promoted by prey movement
(Cole and Adamo, 2005), it is essential that a minimum prey amount, between feeding
periods, is left in culture tanks. The critical phase for cuttlefish survival is considered to
occur three days after hatching, period between hatchling’s yolk consumption and first

1th
______________________________________________________________________
prey captured (Vecchione, 1987; Koueta and Boucaud-Camou, 1999). Therefore, proper
conditions must be provided to ensure the success of first live prey consumption.
Our results indicate that cuttlefish grew bigger when fed with mysids than fed
with Atlantic ditch shrimp, which is consistent with results obtained by Domigues et al.
(2004). These authors obtained better results in growth rate and feeding rates for
cuttlefish fed with mysids P. nouvelli when compared to other live diets, for the first
weeks of cuttlefish life cycle. This experiment ended at day 22 (week 3) due to the
relation between prey size (P. nouvelli) and cuttlefish size. It can therefore be
considered that mysids should not be used on cuttlefish older than 3 weeks, since it
would require the capture of a higher number of preys, and therefore more energy
would be expended, with probable costs in the food conversion rate. Thus, from that
point onwards, bigger prey should be used. Domingues et al. (2004) suggested the use
of Atlantic ditch shrimp when feeding cuttlefish aged 2 weeks. We decided to include a
second prey species in this study (P. varians) to evaluate if the cuttlefish behaviour
would be affected not only by the prey availability but also by the prey type. Our results
clearly indicate that no matter the prey used (in this case P. nouvelli and P. varians),
cuttlefish will grow bigger if prey is available in higher quantities. This might be
explained by the fact that, higher prey availability reduces the competition between
individuals, increases hunting success, and less energy is spent chasing prey.
In both experiments, feeding rates ranged between 8.0 and 18.7% bw d-1
which
is in agreement with those reported by Pacual (1978), DeRusha et al. (1989), Koueta
and Boucaud-Camou (1999, 2001), Forsythe et al. (2002) and Domingues et al. (2003a,
2003b, 2004). Feeding rates showed statistical differences between cuttlefish fed Diet II
A and Diet II B, throughout the experiment. Koueta and Boucaud-Camou (1999)
suggested that a high prey density in culture tanks might trigger capture, raising the
probability of visual stimulation. These authors tested three different daily fixed
percentages of food (21%, 30% and 35%), and obtained significant differences in
feeding rate between higher and lower food percentages.
Cuttlefish fed with Diet I showed statistical differences in feeding rate, between
first and second treatment, until week 2, while no statistical differences were found in
growth rate, except for week 1. This result indicates that cuttlefish fed with Diet I
undergo a more energy consuming hunt, since they need a higher amount of prey due to
the relatively small size of mysids, to cope with its energetic needs. Thus, mysids do not
___________________________________________________________________ 18

1th
______________________________________________________________________
seem to be an adequate prey for cuttlefish aged 2 weeks or older. This agrees with
results reported by Domingues et al. (2004).
Highest values of feeding rate and instantaneous growth rate were found at week
1 of the experiment for second treatment of both diets tested. This result agrees with
Dickel et al. (1997) that reported the decrease with age of the trigger effect promoted by
prey presence in tanks, due to the maturation of the short-term memory processes.
Average food conversions ranged from 23 to 56 % and fall within the values
reported by Pacual (1978), DeRusha et al. (1989), Koueta and Boucaud-Camou (1999,
2001), Forsythe et al. (2002) and Domingues et al. (2003a, 2003b, 2004). Food
conversion was overall higher for cuttlefish in first treatment of both diets tested.
Statistical differences were found in food conversion between cuttlefish fed Diet II A
and Diet II B only, from week 2 onwards which might indicate that although cuttlefish
fed Diet I B and Diet II B increased their feeding rate and therefore their growth rate,
extra energy was used for prey capture, thus decreasing mean food conversion rate.
Nevertheless, this fact did not influenced growth of cuttlefish fed Diet I B and Diet II B,
when comparing with first treatment of both diets tested. This suggests that, although a
decrease in food conversion was observed, the higher feeding rate shown by cuttlefish
in second treatment of both diets, promoted higher growth rates and mean weights.
Instantaneous growth rate ranged between 2.9 and 10.0% bw d-1
, which was
similar to the results obtained by Koueta and Boucaud-Camou (1999) and Grigoriou
and Richardson (2004) for the same temperature range. Cuttlefish fed with Diet I
showed statistical differences in mean weight from week 1 onwards. This result could
be explained by the higher feeding rate shown by cuttlefish in second treatment, for that
period of time. This difference in feeding rate could be responsible for the difference in
mean weights for week 1, and seems to have been enough to contribute to the
statistically differences in mean weight shown by cuttlefish in second treatment,
throughout the experiment. In contrast, cuttlefish fed with Diet II did not show any
significant difference in mean weight and between growth curves through statistical
analysis, which might be explained by the high standard deviation obtained in both
treatments. This fact may be due to the feeding hierarchy and the presence of slow and
fast growers as reported by Mathers (1986).
Results obtained in this study indicate that a certain minimum quantity of prey
should always be present in the culture tanks, in order to provide optimal growth,
___________________________________________________________________ 19

1th
______________________________________________________________________
especially in early stages of cuttlefish life cycle. Studies should be performed to assess
proper food quantities in order to obtain optimal feeding rates and therefore higher
growth rates. Feeding quantities should be adjusted depending on the prey used, in order
to optimize cuttlefish growth.
___________________________________________________________________ 20

2nd
Manuscript - Abstract
______________________________________________________________________
4. Effects of prey condition on growth and survival of juvenile
cuttlefish, Sepia officinalis (Linnaeus, 1758).
4. 1. Abstract
The effect of prey condition on growth and survival of juvenile cuttlefish (Sepia
officinalis) were studied. Tanks were divided in three groups of three tanks each.
Cuttlefish in the first group were fed live Atlantic ditch shrimp Palaeomonetes varians
freshly captured from the wild (DP), second group were fed five days stocked and
starved P. varians, (SP), while in the third group, cuttlefish were fed five days stocked
P. varians fed with ration (FP). Mean instantaneous growth rate (IGR) was 2.8 ± 1.0%
bw d-1
, 3.3 ± 1.1% bw d-1
and 4.9 ± 0.5% bw d-1
for SP, DP and FP, respectively. Final
biomass reported was 47.6 ± 10.2g, 58.4 ± 5.2g and 60.0 ± 31.7g when fed SP, DP and
FP, respectively. Through the growth curve analysis, statistical differences were found
between every group tested (P<0.05). No statistical differences were found in food
conversions between all groups tested (P>0.05). Nevertheless, statistical differences
(P<0.05) were found in mean instantaneous growth rate (IGR) between DP vs FP and
SP vs FP. Results indicate that prey starvation (up to 5 days) should not be considered
when feeding juvenile cuttlefish. Nevertheless, prey stocking should be taking in
consideration, if proper artificial diet is provided, in order to obtain optimal cuttlefish
growth. In this study, cuttlefish fed with fed P. varians obtained higher growth rates and
better fitted individuals.
4. 1. 1. Keywords: Cuttlefish; Culture; Hatchlings; Live diet; Growth; Prey
condition
4. 2. Introduction
Cephalopod aquaculture has increased in recent years (Lee et al., 1998), based
on the potential of some species for commercial culture (Boucaud-Camou, 1990;
Hanlon et al., 1991; Lee et al., 1998). Cephalopods have high growth rates, between 3
and 10% body weight per day (bw d-1
) (Lee, 1994), which can be higher than 20% bw d-
1
for the early stages of their life (O’Dor and Wells, 1987; Domingues et al., 2001a).
These are related to high food conversion rates such as reported by Domingues et al.
___________________________________________________________________ 21

2nd
Manuscript - Introduction
______________________________________________________________________
(2003a, 2003b) and Correia et al. (2005), high feeding rates between 20 and 50% bw d-1
(Boucher-Rodoni et al., 1987), and short life cycles (Forsythe and Van Heukelem,
1987; Domingues et al., 2001a, 2002).
The European cuttlefish Sepia officinalis has been maintained, reared and
cultured in the laboratory for many years (Richard, 1975; Pascual, 1978; Boletzky,
1979, 1983; Boletzky and Hanlon, 1983; Forsythe et al., 1991; Forsythe et al., 1994;
Lee et al., 1991; Domingues et al., 2001b, 2002). This species presents several
characteristics that make it highly suitable for large scale culture, such as high
adaptability to captivity, large eggs, high hatchling survival, sedentary behaviour,
tolerance to high culture densities with little or even no cannibalism, handling, shipping,
acceptance of dead prey and easy reproduction in captivity (Domingues et al., 2002;
Forsythe et al., 2002; Sykes et al., 2006).
To insure the culture success of this species, adequate feeding must be provided,
and for the past few years, several experiments have been done to determine the best
available diet, in order to obtain optimal growth and survival (Castro et al., 1993;
Domingues et al., 2001b, 2003a, 2003b, 2004). Several diets were tested on cuttlefish
hatchlings, with Paramysis nouvelli and Palaemonetes varians obtaining the highest
growth rates (Domingues et al., 2004). Surimi and other artificial diets were also tested
with very little success (Castro, 1991; Castro et al., 1993; Castro and Lee, 1994;
Domingues et al., 2005). Up to now no artificial diet was ever recorded to be used on
hatchlings. Therefore, tests must be made to determine the viability of live prey culture
to sustain cuttlefish production, especially for the early stages of cuttlefish life cycle.
The present study aimed to determine the influence of prey quality on growth
and survival of juvenile cuttlefish. This information is important to determine the
viability of stocking large quantities of prey, when feeding cuttlefish.
4. 3. Material and methods
Cuttlefish production and experimental conditions
Experiments were conducted at the Ramalhete Aquaculture Field Station of the
University of the Algarve (South Portugal), in a flow-through culture system and water
flow of 10 l h-1
per tank. Nine rectangular tanks (38 x 28.5cm) with 12cm water depth
___________________________________________________________________ 22

2nd
______________________________________________________________________
and 10 l volume (Figure 1) were used. Average temperature was 18.5 ± 0.5 ºC and
salinity varied between 36 ± 1 ‰. Tanks were illuminated from above with fluorescent
light, with an intensity of 600 lux at the water surface and a photoperiod controlled by a
timer at 12L:12D. Water quality parameters kept stable throughout the experiment.
Ammonia values were always bellow detectable levels, nitrate <0.3 mg l-1
and nitrite
<12.5 mg l-1
. Hatchlings used in this study were obtained from a natural breeding
broodstock kept in the facilities where these experiments took place. Cuttlefish used in
this study were one month old.
___________________________________________________________________ 23
Fig. 1 – Rearing system representing the schematics of the experiment tanks; (1) inflow pipes; (2) outflow
pipes; (3) rearing tanks; (4) settling tank; (5) outflow during semi-open system; (6) filtering tank; (7) bio-
filter; (8) protein skimmer, (9) reservoir tank; (10) leveller; (11) inflow during semi-open system after
passing through a ultra-violet light filter; (12) water pump; (13) inflow to the other rearing tanks.
Three treatments have been carried out. In the first treatment, cuttlefish were fed
live Atlantic ditch shrimp (Palaemonetes varians) captured in the same day (DP) in
ponds surrounding the culture facility, using bottom hand held trawling nets. In the
second treatment, live P. varians were previously captured and kept for five days in 200
litre tanks with no food provided (SP). Finally, in the third treatment, live P. varians
were captured and kept in 200 litre tank during a five day period, fed with an
experimental artificial diet for shrimp (FP). In all the treatments performed, animals
were fed ad libitum.

2nd
______________________________________________________________________
Prey quantity provided in each tank was based on respective cuttlefish biomass,
in each weighing period. During the interval of weighing periods, food provided to each
group was adjusted by observation of the remaining prey in the tanks. In each weighing
period, all remaining prey in each tank was weighed, to determine exactly the weight of
consumed prey. The cuttlefish in each replicates were weighed on a weekly basis and
mortality was registered. This experiment lasted for 4 weeks (29 days).
Artificial diet formulation
The artificial shrimp diet used in this study met the known nutritional
requirements for shrimp, since, the ingredients and nutritional characteristics of the
formulated diets conform the values mentioned in the available literature (e.g. Oliva-
Teles, 1985; Sudaryono et al., 1995; Mu et al, 1998; Floreto et al., 2000; Glencross et
al., 2002; Gong et al., 2000; Kureshy and Davis, 2000; Wouters et al., 2001). Diet
ingredients and chemical analysis are presented in (Table 1). The diet was steam
pelleted using a laboratory pellet mill (California Pellet Mill, San Francisco, CA).
Pellets were dried overnight under forced air at 35ºC, and stored at 4ºC until used.
The artificial diet and shrimp carcass were analyzed for dry matter and ash
contents according to the methods of AOAC (1995), crude protein (N×6.25) by
Kjeldahl method using a Kjeltech auto-analyzer (Model 1030, Tecator, Höganäs,
Sweden), and total lipid with chloroform:methanol extraction according to the method
of Bligh and Dyer (1959). Gross energy (GE) content of samples was measured using
an automated bomb calorimeter (Model 1272, Parr Instruments, Moline, IL). Digestible
energy was calculated using apparent digestible coefficients according to Cuzon and
Guillaume (in D’Abramo, 1997): 17.2 kJ for carbohydrates, 39.5 kJ for lipids and 21.3
kJ for proteins.
___________________________________________________________________ 24

2nd
______________________________________________________________________
Table 1 – Ingredients and chemical analysis of
diet used to feed Atlantic ditch shrimp
(Palaemonetes varians)
Composition of experimental
diets
Ingredient
Diet 1
g/100 g
Herring meal 37.0
Soybean meal, 52%CP 8.0
Corn gluten meal 0
Wheat Gluten 8.0
CaHPO4 1.0
Lysine 0
Methionine 0
Vitamin premix 2.0
Mineral premix 2.0
Fish oil 1.0
Soy lecithin 3.0
Cholesterol 0.5
Binder 2.0
Wheat flour 35.5
Total (g) 100
Analysed composition (g/100 g dry matter)
Dry Matter (%) 91.5
Crude Protein (%) 42.0
Lipid (%) 9.09
Ash (%) 9.15
Phosphorus (%) 1.02
Gross Energy (kJ g-1
) 18.45
Statistical analysis
Growth was compared using a multiple regression analysis of the growth curves
obtained in each experiment (Zar, 1999). Since cuttlefish growth is exponential in the
early stages of their life cycle (Domingues et al., 2002; Sykes et al., 2003; Correia et
al., 2005), growth data was converted to natural logarithm and linear regression models
were used for comparison between groups of the same diet. Also, growth curves where
obtained through the use of regression model of type (y=a*exp(bx)) in Sigmaplot® v.10
software. Mean weight was used to calculate the Mean Instantaneous Growth Rate (%
bw d-1
) (IGR) = ((lnW2-lnW1)/t*100), where W2 and W1 are the final and initial
weights of the cuttlefish, respectively, ln the natural logarithm and t the number of days
between samplings. One-way ANOVA’s (Zar, 1999) were done using all individual
___________________________________________________________________ 25

2nd
______________________________________________________________________
weights from each replicate in each group of the same diet tested. If during that
period no differences were found in the three replicates of each diet, all weights of the
three replicates were gathered and student t-tests (Zar, 1999) were used to compare
weights of all individuals in the two groups. Feeding Rate (% bw d-1
) (FR) was
calculated for every weighing period using the expression (FI/Average W(t))*100,
where FI is the food ingested and average W(t) is the average wet weight of the
cuttlefish during the time period (t). Food Conversion (FC) was determined using the
expression (W2-W1)/FI, where W2-W1 is the weight gained by the cuttlefish between
sampling. Food conversion and feeding rates from each treatment were compared using
a student t-test (Zar, 1999).
Mean cumulative mortality (mean percentage of increasing values of mortality) was
calculated for each sampling period. Student t-tests (Zar, 1999) were used to determine
differences between diet densities.
Normality and homogeneity of each sample were analysed and, when either of
the requisites was not verified, Mann-Whitney non-parametric tests (Zar, 1999) were
performed. Significant differences were considered for P<0.05.
4. 4. Results
Figure 2 shows the average weight increase of cuttlefish fed starved prey (SP),
daily captured prey (DP) and 5 day fed prey (FP).
___________________________________________________________________ 26
0,0
2,0
4,0
6,0
8,0
10,0
1 8 15 22 29
Days
Weight(g)
SP
DP
FP
y=2.407±0.107(0.027±0.002)x
y=2.510±0.138(0.032±0.003)x
y=2.275±0.112(0.034±0.002)x
Fig. 2. Growth in weight (g) of cuttlefish fed starved prey (SP) and daily
captured prey (DP). Dots represent average weight of cuttlefish in that
replicate. The exponential curves were adjusted to the average weights.

2nd
______________________________________________________________________
The growth curve for cuttlefish fed SP is described by the expression
W=2.407(±0.107)*e0.026(±0.002)D
(R2
=0.925), cuttlefish fed DP is described by the
expression W=2.510(±0.138)*e0.032(±0.003)D
(R2
=0.920), and cuttlefish fed FP is
described by the expression W=2.275(±0.112)*e0.034(±0.002)D
(R2
=0.951), where W is
average wet weight (g) and D represents time (days). At the end of the experiment,
cuttlefish fed Atlantic ditch shrimp SP, DP and FP averaged 5.06 ± 0.51 g, 6.04 ± 0.19 g
and 8.84 ± 0.69 g, respectively.
Statistical differences were found in the growth curve between SP vs DP
(F=4.498; P=0.021); DP vs FP (F=61.832; P=0.000); and SP vs FP (F=167.089;
P=0.000).
Instantaneous Growth Rates (IGR)
Average IGR were of 2.8 ± 1.0% bw d-1
, 3.3 ± 1.1% bw d-1
and 4.9 ± 0.5% bw
d-1
for SP, DP and FP, respectively (Figure 3).
0%
1%
2%
3%
4%
5%
6%
7%
week 1 week 2 week 3 week 4
Time (weeks)
IGR(%bwd
-1
)
SP
DP
FP
Fig. 3 - Average feeding rate (% bw d-1
) of cuttlefish fed 5 day starved prey (SP), daily captured prey (DP) and
5 day fed prey (FD). Vertical lines represent standard deviations.
Highest value of growth rate was obtained for cuttlefish fed FP on week 2 of the
experiment (5.3 ± 0.4% bw d-1
). The lowest growth rate was found on week 3 for
___________________________________________________________________ 27

2nd
______________________________________________________________________
cuttlefish fed SP (1.7 ± 0.4% bw d-1
) (Table 2). Tukey HSD test revealed no significant
differences between SP vs DP (P>0.05), while significant differences where found
between DP vs FP; and SP vs FP (P<0.05) (Table 3).
Feeding Rates (FR)
Mean feeding rates obtained in this experiment were 9.3 ± 2.4% bw d-1
, 9.0 ±
1.6% bw d-1
and 15.5 ± 0.9% bw d-1
for SP, DP and FP respectively (Figure 4).
0%
4%
8%
12%
16%
20%
Time (weeks)
Feedingrate(%bwd-1
)
SP
DP
FP
Fig. 4 - Average feeding rate (% bw d-1
) of cuttlefish fed 5 day starved prey (SP), daily captured prey (DP) and
5 day fed prey (FD). Vertical lines represent standard deviations.
Highest FR values were obtained for cuttlefish fed FP on week 1 (16.6 ± 0.3%
bw d-1
), whereas lowest value were found on week 4 (6.2 ± 2.1% bw d-1
) for cuttlefish
fed SP (Table 2). No statistical differences were found between FR for cuttlefish fed SP
vs DP (P>0.05), while statistical differences were found between DP vs FP and SP vs
FP (P<0.05) (Table 3).
Food Conversion Rates (FC)
Food conversion rates averaged 40.2 ± 13.3%, 46.4 ± 16.2% and 38.7 ± 5.6% for
SP, DP and FP respectively (Figure 5). Highest values of FC were obtained on week 4
___________________________________________________________________ 28

2nd
______________________________________________________________________
(64.7 ± 5.5%) for cuttlefish fed DP (Table 2). Lower values were obtained on week 3
(27.6 ± 12.5%) for SP (Table 2). Statistical analysis of FC through Dunn’s multiple
comparison test shown no statistical differences between every group tested (P>0.05)
(Table 3).
0%
20%
40%
60%
80%
Time (weeks)
FoodConversion(%)
SP
DP
FP
Fig. 5 - Average food conversion (%) of cuttlefish fed 5 day starved prey (SP), daily captured prey (DP) and 5
day fed prey (FD). Vertical lines represent standard deviations.
Biomass (B)
At the end of the experiment, cuttlefish reached a total biomass of 47.6 ± 10.2g,
58.4 ± 5.2g and 60.02 ± 31.7g when fed SP, DP and FP, respectively.
Mortality
The highest mortality was found in cuttlefish fed FP (total of 7 dead specimens
with 6 on one of the replicates). Mortality of cuttlefish fed SP resumed to 2 dead
animals (in same replicate) whereas for DP, only 1 animal died during the entire
experiment.
___________________________________________________________________ 29

2nd
______________________________________________________________________
Table 2 - Growth rate (IGR), feeding rate (FR) and food conversion (FC) estimates
for cuttlefish aged one month, fed 5 days starved prey (SP) , daily captured prey (DP)
and 5 day fed prey (FP) during 36 days (4 weeks).
Prey Days
Week 0 Week 1 Week 2 Week 3 Week 4
Mean Weight (g)
SP 2.29 ± 0.05 3.07 ± 0.14 3.68 ± 0.27 4.13 ± 0.20 5.06 ± 0.51
DP 2.38 ± 0.15 3.30 ± 0.12 4.09 ± 0.13 4.68 ± 0.20 6.04 ± 0.19
FP 2.24 ± 0.04 3.01 ± 0.08 4.37 ± 0.26 6.11 ± 0.48 8.84 ± 0.69
IGR (%bw d-1
)
SP 4.2 ± 0.5 2.6 ± 0.4 1.7 ± 0.4 2.8 ± 0.8
DP 4.6 ± 0.6 3.1 ± 0.3 1.9 ± 0.7 3.6 ± 0.2
FP 4.2 ± 0.3 5.3 ± 0.4 4.8 ± 0.3 5.3 ± 0.7
FC (%)
SP 45.4 ± 5.6 31.4 ± 5.7 27.6 ± 12.5 56.5 ± 6.6
DP 54.6 ± 5.4 37.7 ± 2.0 28.8 ± 11.1 64.7 ± 5.5
FP 31.6 ± 2.3 41.6 ± 5.0 37.1 ± 3.1 44.4 ± 6.8
FR (%bw d-1
)
SP 11.4 ± 0.2 10.8 ± 0.5 8.8 ± 2.1 6.2 ± 2.1
DP 10.4 ± 0.4 10.1 ± 0.6 8.6 ± 0.6 6.9 ± 0.6
FP 16.6 ± 0.3 15.3 ± 0.8 15.6 ± 0.5 14.5 ± 2.9
Table 3 – Statistical analysis of growth rate (IGR), feeding rate
(FR) and food conversion (FC) for cuttlefish aged one month, fed
5 days starved prey (SP) , daily captured prey (DP) and 5 day fed
prey (FP) during 36 days (4 weeks).
Prey P value
SP vs DP DP vs FP SP vs FP
IGR (%bw d-1
) 0.4115 0.0009* 0.0001*
FC (%) 0.9980 0.4221 1.0000
FR (%bw d-1
) 0.9291 0.0001* 0.0001*
* Values were significantly different within that period (P<0.05).
___________________________________________________________________ 30

2nd
______________________________________________________________________
4. 5. Discussion
Atlantic ditch shrimp P. varians has been indicated by several authors as the
best live food available to feed cuttlefish, since it has been considered to have a high
nutritional value, due to its biochemical composition (Domingues et al., 2003a, 2004).
This species showed higher growth and feeding rates when compared to other live prey
(Domigues et al., 2004) and is considered the best food source for cuttlefish ageing 2
weeks (Domingues et al., 2004; Forsythe et al., 2004). Nevertheless, Atlantic ditch
shrimp must be collected from the wild, since the economical and practical viability of
this species culture has only been evaluated at a research scale (Palma et al., submitted).
Due to the location of our research facilities, P. varians can be easily obtained from the
wild, in large quantities, in the nearby ponds. Nevertheless, seasonal prey availability
fluctuations may occur, thus limiting proper animal feeding. Also, obligatory daily
capture of live prey implies the necessity of hiring extra staff which contributes to
increase the production costs and contributes to over 50% in labour (Lee, 1994).
When considering the economical scope of cuttlefish aquaculture, food is one of
the most important factors that contribute to the optimal growth and survival of farmed
individuals. Since live P. varians promote the best results as main food source, efforts
have been recently made to assess culture viability of this species in a cuttlefish
aquaculture system. This work was assessed to cope with the lack of life food
production studies and poor results obtained with artificial or frozen diet, until present.
The objective is to determine the viability of stocking large quantities of live prey, thus
reducing the production costs of S. officinalis.
The density used in this experiment (93 cuttlefish m-2
) is considered to be an
adequate density for specimens ageing between 30 and 60 days. Forsythe et al. (2002)
suggested an optimal culture density of 400 cuttlefish m-2
, while Sykes et al. (2003)
reported that cuttlefish could sustain culture densities up to 500 cuttlefish m-2
.
Mean feeding rates obtained in this experiment varied from 9.0 ± 1.6% bw d-1
and 15.5 ± 0.9 % bw d-1
and are in agreement with the results obtained by other authors,
when using the same live diet (Pacual, 1978; DeRusha et al., 1989; Koueta and
Boucaud-Camou, 1999, 2001; Forsythe et al., 2002 and Domingues et al., 2003a,
2003b, 2004).
___________________________________________________________________ 31

2nd
______________________________________________________________________
Average overall food conversions varied between 38.7 ± 5.6% and 46.4 ±
16.2%, and fell within the values reported by Pacual (1978), DeRusha et al. (1989),
Koueta and Boucaud-Camou (1999, 2001), Forsythe et al. (2002) and Domingues et al.
(2003a, 2003b, 2004). Food conversion rates did not showed statistical differences
between the groups tested. Nevertheless, cuttlefish fed FP showed a more constant
behaviour in food conversion rates, when comparing with food conversion of the two
other tested groups. Variations observed in food conversion of cuttlefish fed SP and DP
seems to be due to natural variation of prey nutritional quality in the wild. This fact is
supported by the similarity of food conversion behaviour throughout the experiment.
Food conversion in FP showed a more constant behaviour throughout time. This can be
due to the direct effect of the artificial diet provided to the prey, which enabled the
cuttlefish to obtain the proper biochemical requisites and thus, promoting a constant
food conversions.
At the end of the experiment, overall mean biomass was higher for cuttlefish
fed FP (69.0 ± 31.7g). Nevertheless, cuttlefish fed FP presented a high standard
deviation due to the high mortality that occurred in one of the group’s replicates.
Although cuttlefish fed DP had higher values of final biomass when comparing to with
SP (58.4 ± 5.2g vs 47.6 ± 10.2g), no statistical differences in biomass (P>0.05) between
these two groups. This fact may be due to the high standard deviation showed by
cuttlefish fed DP. This deviation could be explained by the existence of fast and slow
growers suggested by Mathers (1986). Thus, the observed variation in the individual
final weight of the specimens fed SP can be a direct cause of individual differences in
the nutrient intake efficiency and therefore relative growth rate. Individual food
conversion rates should be investigated when feeding with a starved live diet to
determine the nature of this variability.
The IGR values obtained in this study (between 2.82 ± 1.04% bw d-1
for SP and
4.90 ± 0.51% bw d-1
for FP) were similar to those reported by other authors (Koueta and
Boucaud-Camou, 2001; Forsythe et al., 2002 and Grigoriou and Richardson, 2004) at
similar temperatures. When analysing FP´s IGR through the entire experience, it can be
observed a more constant behaviour, when comparing with the other two groups, fact
that can be explained by the stabilizing effect on prey biochemical profile due to the
artificial diet provided. This diet has improved shrimp nutritional value when
comparing with the freshly captured prey, therefore becoming an important factor to
___________________________________________________________________ 32

2nd
______________________________________________________________________
promote optimal cuttlefish growth. In contrast, when analysing the IGR of the two other
groups tested (SP and DP), it can be observed a direct relation due to the variations of
prey nutritional value in the wild. This variation can be explained by the food
availability in the wild, being more evident when in starvation.
Significant differences were found when comparing growth curves and mean
weight of cuttlefish fed SP and DP, which indicates that 5-days starved preys promote
lower growth on juvenile cuttlefish thus being unsuitable as food source to obtain
optimal cuttlefish growth. In contrast, statistical differences were found between DP and
FP which indicates that feeding captured prey from the wild, promotes better results in
growth. This result is an important finding to enable future studies to focus on proper
live prey feeding, since no encouraging results have been obtained from the use of
artificial diets. Efforts should be made to determine optimal live prey feeding diet,
taking into account both biochemical and economical factors.
Domingues et al. (2003b) report the use of frozen shrimp as food source for
cuttlefish, having no influence on growth when compared with live shrimp.
Nevertheless, frozen shrimp showed a reduction on polar lipid contents on frozen
shrimp and an increase on free fatty acids percentages, associated to oxidation processes
(Domingues et al., 2004). In addition, frozen shrimp was recorded to leave more
detritus at the bottom of the tanks which leads to higher maintenance costs, and could
be associated to skin diseases due to mal-nutrition (Loi and Tublitz, 1998; Domingues
et al., 2003b, 2004).
Results obtained in this experiment indicate that 5-days starved prey produce
poorer results compared with live captured prey, when culturing cuttlefish. In contrast,
live fed prey promoted better overall results, which highlights the importance of live
prey to be properly fed. The artificial diet produced and used in this study was made out
of plain ingredients and no rendered ingredients were used. Nonetheless it promoted, in
a short term period, an improvement in the shrimp nutritional values. Other artificial
diets should be tested in order to promote optimal prey nutritional quality and thus,
contributing to a higher cuttlefish growth. This procedure should be taken in to
consideration when culturing cuttlefish and economical viability of artificial diets use
on live prey, should be assessed.
___________________________________________________________________ 33

Final considerations
______________________________________________________________________
5. Final considerations
This work has highlighted the importance of a proper feeding protocol, for the first
two weeks of cuttlefish life cycle. The proper balance between live diet provided and
prey consumed, by cultured individuals, must be achieved. Future studies must be done
to determine optimal daily feeding quantities, in order to obtain optimal growth rates
and to insure higher survival rates. Thus, contributing to a higher profit, and enabling to
obtain larger animals in a shorter time period.
Also, since the viability of this new technology is highly dependable on the
economical factors, and until present no artificial diet has provided encouraging results,
live prey stocking should be considered. This procedure is advantageous due to its
considerable reduction of labour and general costs. Nevertheless this procedure should
only be considered if proper food is provided to the stocked live prey. Therefore, future
efforts should be done in order to determine an economically viable artificial diet, thus
making prey stocking an option to be considered, in a large scale aquaculture.
___________________________________________________________________ 34

References
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___________________________________________________________________ 39

Agradecimentos
______________________________________________________________________
7. Agradecimentos
O autor gostaria de agradecer ao Professor Doutor Henrique Cabral pela sua
colaboração na revisão de toda a tese. Por outro lado, o autor gostaria de agradecer
igualmente a colaboração do Professor Doutor José Pedro Andrade na revisão da tese e
por ter fornecido as condições necessárias, essenciais à correcta realização de todas as
experiências que compõem esta tese de dissertação. Finalmente o autor gostaria de
agradecer à participação activa do Doutor Jorge Palma na concepção das experiências
bem como nos ajustes fundamentais para a correcta realização das mesmas, bem como
na revisão da dissertação.
___________________________________________________________________ 40

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