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 ______________________________________________________________________

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 ______________________________________________________________________

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 ______________________________________________________________________

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 ______________________________________________________________________

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 ______________________________________________________________________

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



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 ______________________________________________________________________

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



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



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

Mea

n W

ei

B 0.039(±0.002)XY=0.182(±0.007)*e

Y=0.164(±0,006)*e

0,038(±0,002)X A

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.



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



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

Mea

n W

eigh

t (g)

B Y=0.175(±0.007)*e

0.050(±0,002)X

Y=0.183(±0.006)*e

0.043(±0.002)X A

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 si

gnificantly 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



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



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



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 Manuscript – Material and methods ______________________________________________________________________

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.



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



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 37.0Soybean meal, 52%CP 8.0Corn gluten meal 0Wheat Gluten 8.0CaHPO4 1.0Lysine 0Methionine 0Vitamin premix 2.0Mineral premix 2.0Fish oil 1.0Soy lecithin 3.0Cholesterol 0.5Binder 2.0Wheat flour 35.5Total (g) 100 Analysed composition (g/100 g dry matter) Dry Matter (%) 91.5Crude Protein (%) 42.0Lipid (%) 9.09Ash (%) 9.15Phosphorus (%) 1.02Gross 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 Manuscript – Results ______________________________________________________________________

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

Wei

ght (

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.



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

(% b

w d

-1)

SPDPFP

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



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)

Feed

ing

rate

(% b

w d

-1)

SPDPFP

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



(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)

Food

Con

vers

ion

(%)

SPDPFP

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



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 Manuscript – Discussion ______________________________________________________________________

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



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



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


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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.

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