Tesis Doctoral

Development of a Sustainable Grow-Out Technology

for Abalone Haliotis tuberculata coccinea (Reeve) as
a New Species for Aquaculture Diversification in the
Canary Islands

María del Pino Viera Toledo

Grupo de Investigación
LAS PALMAS DE GRAN CANARIA 2014
en Acuicultura
 Index

INDEX

Index I
Acknowledgements IV
List of figures VIII
List of tables X
Abbreviations XII
Abstract XIV

1. INTRODUCTION 1
1.1. Abalone in the world 1
1.2. Abalone in Europe 6
1.3. Abalone in the Canary Islands 8
1.4. Factors affecting abalone growth 9
1.4.1. Culture conditions related parameters 10
1.4.1.1. Initial size 10
1.4.1.2. Stocking density 10
1.4.1.3. Water flow 11
1.4.2. Physico-chemical parameters 12
1.4.2.1. Temperature 12
1.4.2.2. Light 13
1.4.2.3. Water quality 14
1.4.3. Grow-out culture systems 15
1.4.3.1. Land-based systems 15
1.4.3.2. Sea-based system 17
1.5. Abalone feeding and nutrition 19
1.5.1. General aspects 19
1.5.2. Abalone feeding practices 20
1.5.3. Abalone nutritional requirements 23

1.5.3.1. General composition of abalone manufacturated diets 23

1.5.3.2. Protein sources 23
1.5.3.3. Lipid sources 8
1.5.3.4. Energy / carbohydrate and binders sources 31

I
 Index

1.5.3.5. Vitamins and minerals 34

1.5.4. Abalone growth under culture conditions 36
1.6. Integrated multi-trophic aquaculture (IMTA) 42
1.6.1. General aspects 42
1.6.2. Seaweed-based integrated mariculture 43
1.6.3. Fish-seaweed- abalone integrated culture system 44

2. OBJECTIVES 46
3. MATERIALS AND METHODS 48
3.1. Location and general facilities 48
3.2. Abalone production 50
3.2.1. Brood-stock conditioning and selection 50
3.2.2. Spawning induction 50
3.2.3. Fertilization 51
3.2.4. Larval culture 52
3.2.5. Larval setlement 52
3.2.6. Post-larval and juvenile culture 53
3.3. Algal culture 54 B
3.3.1. Macroalgae species 54
3.3.2. Culture system (IMTA) 62
3.4. Artificial diets 64
3.4.1. Diet formulation 64
3.4.2. Diet preparation 64
3.4.3. Diet water stability 65
3.5. Experimental design 66
3.5.1. Land-based experimental set-up 68
3.5.2. Sea-based grow-out system 69
3.6. Biological parameters evaluation 72
3.6.1. Shell growth rate 72
3.6.2. Specific growth rate 72
3.6.3. Weight gain 72
3.6.4. Feed conversion ratio 73
3.6.5. Protein efficiency ratio 73

II
 Index

3.6.6. Feed intake 73

3.6.7. Condition index 74
3.6.8. Survival 75
3.7. Biochemical analysis 75
3.7.1. Dry matter content 75
3.7.2. Ash content 76
3.7.3. Protein content 76
3.7.4. Total lipid content 77
3.7.5. Fatty acids content 77
3.8. Statistical analysis 78

4. STUDY I 79

5. STUDY II 90

6. STUDY III 109

7. STUDY IV 133

8. CONCLUSIONS 150

9. SPANISH SUMMARY 153

9.1. Introducción 153

9.2. Objetivos 200
9.3. Material y Métodos 202
9.4. Conclusiones 227

10. REFERENCES 231

III
A mis padres, Víctor e Inma,
siempre a mi lado.
 Acknowledgement

AGRADECIMIENTOS

Esta tesis llega después de veinte años de trabajo en la acuicultura, en los que mi afán ha
sido aprender, avanzar, contribuir, compartir, ganarme la vida y divertirme. Parte de todo ello es
lo que se presenta ahora en esta tesis, resultado no sólo de un esfuerzo personal, sino también de
la contribución de otras muchas personas, que de una u otra manera me han acompañado en el
camino y a las que me gustaría agradecer.

En primer lugar, a la Dra. Marisol Izquierdo, la directora y principal impulsora de esta

tesis, que sin su insistencia, nunca me habría sentado a escribir. Por permitirme formar parte del
Grupo de Investigación en Acuicultura (GIA), y brindarme tantas oportunidades. Porque es un
honor y un respaldo tremendo contar con una investigadora de su talla con la que discutir tu
trabajo. Por ofrecer, junto a Ricardo, continuamente su casa para que su Grupo sea Grupo. Por
esos viajes plagados de conversaciones interesantes. Por meterse en continuos berenjenales con
tal de que sus “pollitos” se abran camino, y luego sentirse orgullosa de ellos…Muchas gracias
Marisol.

Al Dr. Hipólito Fernández-Palacios, mi co-direc y también alma mater del GIA. Porque
desde que le conocí contando huevos en aquella nave vieja (¡qué iba a estar haciendo si no!),
nunca ha dejado de estar ahí cuando hace falta, porque no hay asunto de acuicultura que le
preguntes que él no sepa, porque ir con él de congreso es comida rica y diversión asegurada,
porque por muy parquito que sea, es imposible no quererle. Muchas gracias D. Pipo y ¡Viva
España!.

Al Instituto Canario de Ciencias Marinas del Gobierno de Canarias (ICCM), donde he

desarrollado la mayor parte de mi vida profesional. En especial a dos de sus directores durante
los últimos años, el Dr. Octavio Llinás, por el que siempre me he sentido apoyada, y el Dr.
Eladio Santaella, artífice de mi “captación” para el cultivo de moluscos.

Al Dr. José Vergara, que allá por el 93 me dirigió la Tesis de Máster sobre un tema
novedoso por aquel entonces, piscicultura en jaulas flotantes, que tantas oportunidades
profesionales me ha dado.

A la Dra. Carmen Mª Hernández Cruz, que siempre ha estado a mi lado. Una mujer
trabajadora, eficiente, práctica y mejor persona. No olvido nuestra estancia en Creta; ni tantos
viajes en los que siempre ha sido un placer estar con ella.

Al Dr. Daniel Montero, compañero de carrera y amigo, él me animó a acercarme al

GIA, marcando el rumbo de mi vida para siempre. Por sus valiosos comentarios sobre mis
trabajos; por su valía científica; su sensibilidad y buen gusto; por la ilusión con que espero que

IV
 Acknowledgement

me enseñe sus nuevas fotos a la vuelta de cada viaje…porque sólo un canario de corazón como
él, puede mostranos lo mejor de nuestras Islas.

Al siempre sonriente Dr. Juan Socorro, con el que empecé en el GIA aprendiendo
histología por las tardes, aunque se riera de mí porque a veces no encotraba las larvas...

Al Dr. Ricardo Haroun, por empezar la línea de trabajo de cultivos integrados - abalón,
sentando las bases para la realización de esta tesis y por su amabilidad, siempre.

Al Dr. Juan Luis Gómez-Pinchetti y su equipo del Centro de Biotecnología Marina, por
su inestimable colaboración en los primeros trabajos de esta tesis, y demás proyectos y tesinas
en los que le hemos ido embarcando, por cedernos la Gracilaria que tan buenos resultados nos
ha dado, y por estar siempre dispuesto a echar una mano en lo que esté de su parte.

A la Dra. Lidia Robaina, por su valiosa ayuda en la formulación de las dietas, por su
compañerismo y buenos consejos a lo largo de tantos años.

Al Dr. Javier Roo, porque juntos hicimos posible que donde había un solar polvoriento
terminara habiendo peces. ¡Cuánto aprendimos en Creta trabajando como locos!, yo a base de
yogur griego, tú de mis tartas de manzana y ambos de nuestros giros pitas. Porque durante
cuatro años de duro trabajo compartido, no hubo más que armonía, compañerismo y buen
entendimiento. Nunca olvidaré la ilusión con la que nos regodeábamos en aquel primer lote de
peces producidos en Canarias, ¡y era el nuestro!, alevines superstar les llamaron en el periódico,
y para nosotros, ¡vaya si lo eran!. Estoy muy orgullosa (que no sorprendida) de lo que has
logrado en el Mesocosmos y ¡lo que queda!, porque alguien de tu brillantez y tesón, no tiene
límites.

A D. Antonio Valencia, que tanto me enseñó de cultivo larvario de peces y al que llevo
en el corazón.

Al resto de los senior del GIA, Dra. Lucía Molina, Dra. María José Caballero, Dr. Juan
Manuel Afonso, Dr. Rafael Ginés, Dra. María Jesús Zamorano. Es un auténtico privilegio
contar con un equipo tan preparado y siempre generoso en compartir conocimiento, y lo que
haga falta.

Mi más sincero agradecimiento a las otras “chicas haliotis”, Amaia, Bea y Desi. Su
trabajo, entusiasmo y entrega, han contribuído de forma muy especial a que los distintos
proyectos y por tanto la línea de investigación del abalón, hayan salido adelante.

A las técnicos de laboratorio de análisis, Regina, Yurena y en especial a Carmen

Quintana, por su buen hacer, amabilidad y disposición en la ¡búsqueda del DHA de las algas!.

V
 Acknowledgement

A todo el equipo de técnicos de la nave de cultivos con los que he compartido almuerzo
y búsqueda de cintillos todos estos años (Ada, Manolo, Rubén, Desiré, Damián....), y en
especial a mi querida Moneiba, con la tanto me reí trabajando en el Mesocosmos.

Son muchos los doctorandos, hoy doctores, con los que he tenido el placer de compartir,
viajar y aprender durante estos años en el ICCM, María, Martín, Leire, Agustín, Pedro, Gloria,
Domi, Valeria, Tibi, Silvia, Mónica, Alex, Rachid, Eyad, Fran, Juan Estefanell…, gracias a
ellos ir a Taliarte ha sido siempre mucho más que trabajo. Quiero agradecer especialmente a mi
peruana del alma, Tatiana Kalinowski, que siempre está cuando la necesito.

Al personal del ICCM, investigadores de otros departamentos (Solea, Alicia, Pepe

Ignacio, Chano..); personal de administración (Paula, Mabel..); mantenimiento (Sergio, Juan
Falcón, Pedro…); seguridad (Martín, Mario, Juan Carlos, Cristina..); limpieza (el trio más
simpático de España: Teri, María y Eulogia), y en especial Miguel Medina, del que siempre he
recibido un sí por respuesta (¡acompañado de un piropo cariñoso!). Cada uno desde su parcela,
ha contribuído a que el trabajo siempre saliera adelante.

Agradezco especialmente a D. Rafael Guirao y personal de Canexmar, S.L., su generosa

disposición y colaboración para realizar en sus instalaciones el experimento en el mar. Y a Tony
Legg de Jersey Sea Farm, por el suministro de las jaulas para abalón.

No quiero dejar de mencionar a la Dra. Supis Thongrod y personal del Prachuab

KhiriKhan Coastal Fisheries Research and Development Center de Tailandia, que tan
amablemente me recibieron, contribuyendo de una manera definitiva a mi formación en el
cultivo de abalón. Así mismo, a los investigadores del Instituto de Biología Marina de Creta
(IMBC), en especial los doctores Pascal Divanach, Nikos Papandroulakis y Aspasía Sterioti, por
su excelente formación en el cultivo larvario de peces marinos durante mi estancia en su centro
y a lo largo de todo el Proyecto Interactt.

A mis amigos y amigas, los de siempre, y los que la vida nunca deja de ponerte en el
camino: las niñas del cole, los de la facultad, la Expo, las Lindas de francés, Cáritas, las
cocinitas…Ellos hacen que la vida sea mucho más completa y divertida.

A mi familia majorera, con Tana a la cabeza, todo un ejemplo de juventud, esfuerzo y

curiosidad. Por acogerme entre ellos y formar ya parte de mi vida, como su maravillosa isla.

A mis tíos y primos del alma, tantos, y tan distintos, por su cariño y apoyo, porque están
ahí desde el principio y no soy capaz de imaginar una infancia y una vida sin ellos.

A mis queridos hermanos, Víctor y Panchi, con ellos comparto lo mejor que hay en mí y
también el amor por el mar, ¡Arguineguín nos marcó!. Y a mis sobris lindos, Víctor y Eduardo,
que siempre me reciben con el mejor de los abrazos.

VI
 Acknowledgement

Gercende, sin duda alguna, tú has sido la persona más importante en esta maravillosa
aventura del desarrollo del cultivo de abalón, en la que juntas, hemos trabajado mucho y nos
hemos divertido aún más. Cuando pienso en esas madrugadas de desoves con luna llena, los
muestreos y zafarranchos; la emoción con la que miramos las placas llenas de semillitas; esas
horas de discusión y trabajo para solicitar nuevos proyectos (y la alegría cuando los
conseguímos); las jornadas de reuniones con tantos socios distintos; la preparación de tantos
informes y congresos; esos viajes, bailes, charlas y tantas y tantas risas….no puedo dejar de
pensar que en ocasiones la vida te da buenas oportunidades, y creo sinceramente que a nosotras
nos la dio al reunirnos. Gracias por todo y mucho más. ¡Dame una R., dame una U, dame
una…..RUBIA!!!.

La acuicultura me ha dado mucho en la vida, tanto que ¡¡hasta me regaló un marido!!.

Quién podia imaginárselo, en aquella granja en medio de la nada, con mis botas y medio millón
de peces a medio criar, oliendo a pienso… ¡con tan poco glamour!. Pero apareció Héctor, y se
quedó, y con él la vida pasó a ser mejor y más intensa, el mundo más interesante, con más
lugares que descubrir, deportes que hacer, y… ¡cosas que encontrar!. Porque aunque no sepa
bailar, sé que es mi mejor pareja de baile. Te quiero.

Esta tesis está dedicada a mis padres. A mi madre, que tanto nos amó, por su lucha
inquebrantable por permanecer a nuestro lado, por transmitirme ese inmenso amor a la familia,
al placer de reunir a los amigos a la mesa y demostrarles el cariño a través de algo rico y
¡dintinto!, por ser una madre-madre, que me sigue acompañando en el camino. A mi padre, un
ejemplo de superación, de estudio, de amor por su profesión. Él me inculcó el “ímpetu juvenil”
necesario para llevar el trabajo adelante, porque “la vida es milicia”, y “el no, ya lo tenemos del
lado de acá”. Porque no puedo pensar en sus preciosas manos de cirujano sin emocionarme,
porque cuando desde que naces, te aman, te protegen y te miman de la manera que él lo hizo
con nosotros, nada te puede ir mal en la vida.

VII
 List of figures

LIST OF FIGURES

Figure 1. Newly settled, juvenile and adult abalone (H. tuberculata coccinea).
Figure 2. Abalone culinary products, pearl shell and handicraft.
Figure 3. Amas abalone divers (Japan).
Figure 4. Evolution of global abalone production from legal fisheries and aquaculture
during last decade (source FAO, 2012).
Figure 5. Distribution of H.tuberculata tuberculata and H. tuberculata coccinea
(Geiger, 2000).
Figure 6. PVC tiles used as shelters in donkey´s ear (A) and Canarian abalone (B)
culture.
Figure 7. Several on-shore systems for the culture of H. rufescens (A; Chile), H. discus
hannai (B; Korea), H. asinina (C; Thailand) and H. tuberculata (D; Ireland).
Figure 8. (A) Long-line culture of red abalone H. rufescens in Chile. (B) Represents a
six-tiered basket traditionally used for H. discus hannai farming in south China.
Figure 9. Sea-floor culture of European ormer in Jersey Islands (UK).
Figure 10. Cage culture of abalone H. tuberculata in Brittany (France).
Figure 11. Juvenile abalone (H. tuberculata coccinea) grazing on green algae.
Figure 12. Harvested Macrocystis pyrifera and Palmaria palmata to feed red abalone
(A; Chile) and European ormer (B; Brittany).
Figure 13. Fish-seaweed-abalone integrated culture system.
Figure 14. Top view of the Canary Islands and location of the GIA marine culture
facilities.
Figure 15. Brood-stock conditioning (A); larval rearing facilities (B); abalone nursery B
(C); diatoms production zone (D); grow out zone (E); feeding trials area (F) and
outdoor macroalgae culture systems (G, H).
Figure 16. Female (A) and male (B) H. tuberculata coccinea in stage 2-3 of the gonad
index.
Figure 17. Gametes expulsion from females (A) and males (B). B
Figure 18. Eggs are rinse to remove excess sperm.
Figure 19. Hatching out trochophore larvae equipped with cilia (A), third tubule
appearance on cephalic tentacles (B) (Courtois de ViÇose et al., 2007).
Figure 20. Vertical settlement plates.

A
VIII
 List of figures

Figure 21. Diatoms species fed to the abalone post- larvae: Proschkinia sp. (A),
Navicula incerta (B), Amphora sp. (C) and Nitzschia sp. (D) (Courtois de ViÇose et al.,
2012b).
Figure 22. Fishponds and semi-circular tanks for the cultivation of macroalgae (CBM-
ULPGC).
Figure 23. Diagram of the IMTA (ICCM-ULPGC).
Figure 24. Biofilter produced macroalgae and drying equipment.
Figure 25. Details of diets preparation: ingredients, processing and drying procedure.
Figure 26. Final product: vegetable-based experimental diets.
Figura 27a. Selection of experimental animals: Abalone sampling.
Figure 27b. Abalone distribution among experimental triplicates.
Figure 28. Feeding experimental abalones with macroalgae: Studies I and II (A), Study
III (B) and Study IV (C); or with artificial diets: Study IV (D).
Figure 29. Experimental set-up for the culture of juvenile abalones (Studies I and II).
Figure 30. Rearing system employed for the culture of 30 - 45 mm abalones (Study III).
Figure 31. CANEXMAR cages and location.
Figure 32. Details of the experimental abalone cages and shelters.
Figure 33. Scheme of the sea-based experimental set-up. A: aerial view of the fish farm
installation. B: detail of the ORTACS set-up.
Figure 34. ORTACS installation.
Figure 35. Underwater experimental devices next to fish cages.
Figure 36. Drying the diets leftover.
Figure 37. Condition index evaluation: abalone dissection and weighing.
Figure 38. Offshore grow-out system: (a) experimental abalone cages, (b) shelter, (c)
underwater experimental installation next to fish cages.
Figure 39. Linear growth in shell length (mm) and weight (g) of abalone H. tuberculata
coccinea initially measuring 30 (Trial I) and 40 mm (Trial II), fed with enriched mixed
diet of G. cornea and U. rigida at high and low stocking densities for 27 wks.

IX
 List of tables

LIST OF TABLES

Table 1. Taxonomic classification of abalone species

Table 2. Suitable wild and cultured macroalgae as food for different abalone species
Table 3. Proximate composition (% dry matter) and caloric content of artificial diets
tested for abalone
Table 4. Nutritional composition (% dry matter) of artificial diets tested for abalone:
Protein sources and inclusion levels
Table 5. Nutritional composition (% dry matter) of artificial diets tested for abalone:
Lipid sources and inclusion levels
Table 6. Nutritional composition (% dry matter) of artificial diets tested for abalone:
Energy / binder sources and content
Table 7. Vitamin and mineral mixture tested by Uki et al. (1985a)
Table 8. Nutritional composition (% dry matter) of artificial diets tested for abalone.
Ingredient with secondary nutritional contribution
Table 9. Summary of various nutritional studies regarding abalone growth performance
during the last three decades of abalone culture development
Tables 10-16. Summary of experimental macroalgae characteristics
Table 17. Proximate composition and caloric content of the three red macroalgae (g/100
g DW) (mean ± S.D.) fed to abalone along the experimental trial
Table 18. Growth, feed utilization and survival of juvenile Canarian abalone (H.
tuberculata coccinea) at the beginning of the experiment and after being fed the
selected macroalgae for 60 days under laboratory conditions
Table 19. Proximate composition and caloric content of the eight macroalgae treatments
(g/100 g DW) (Mean ± S.D.) fed to abalone along the experimental trial
Table 20. Growth performance, feed utilization and survival of juvenile abalone (H.
tuberculata coccinea) fed the selected 8 macroalgae diets for 12-weeks
Table 21. Fatty acid composition (% total Table 22. Proximate composition of foot
tissues of Haliotis tuberculata coccinea reared on the experimental diets (g/100 g DW)
(Mean ± S.D.) fatty acids) of the eight macroalgae treatments
Table 22. Proximate composition of foot tissues of Haliotis tuberculata coccinea reared
on the experimental diets (g/100 g DW) (Mean ± S.D.)

X
 List of tables

Table 23. Proximate and aminoacid composition of seaweed meals used in experimental
feeds for abalone H. tuberculata coccinea (%DW)
Table 24. Ingredients of the three experimental diets for abalone H. tuberculata
coccinea (DW basis)
Table 25. Proximate analysis of the fresh algae or experimental diets containing
different algal species (%DW)
Table 26. Fatty acid composition (% total fatty acids) of the fresh algae or experimental
diets containing different algal species*
Table 27. Survival and growth performance of abalone H. tuberculata coccinea fed for
6 months algae (fresh algae) or experimental diets containing different algal species *
(Mean ± S.D.)
Table 28. Consumption, feed efficiency and condition index of abalone H. tuberculata
coccinea fed for 6 months algae (fresh algae) or experimental diets containing different
algal species* (Mean ± S.D.)
Table 29. Proximate composition of viscera and muscle of Haliotis tuberculata
coccinea fed for 6 months algae (fresh algae) or experimental diets containing different
algal species*(g/100 g DW) (Mean ± S.D.) (Values in the same column with different
letters are significantly different. P< 0.05)
Table 30. Fatty acid composition (% total fatty acids) of the abalone tissues of Haliotis
tuberculata coccinea fed for 6 months fresh algae or experimental diets containing
different algal species*
Table 31. Proximate composition and caloric content of the macroalgal diets (g/100 g
DW) (Mean ± S.D.) fed to abalone along the experimental trials
Table 32. Survival and growth performance of abalone H. tuberculata coccinea, initially
measuring 30 mm (Trial I) and 40 mm (Trial II), fed with enriched G. cornea and U.
rigida at high and low stocking densities for 27 weeks
Table 33. Two-Way ANOVA analysis of variance for growth (size and weight) for the
27 wks grow-out culture period under the experimental densities
Table 34. Consumption and feed efficiency of abalone H. tuberculata coccinea. initially
measuring 30 mm (Trial I) and 40 mm (Trial II), fed with G. cornea and U. rigida at
different stocking densities (high and low) for 27 weeks

XI
 Abbreviationss

ABBREVIATIONS

ANOVA: Analysis normal variance

APROMAR: Asociación empresarial de productores de cultivos marinos de España
ARA: Arachydonic acid (20:4n-6)
BHT: Butilated hydroxitoluene
CANEXMAR: Canarias de Explotaciones Marinas
DGSL: Daily Growth Rate in shell lenght
DGW: Daily Growth Rate in weight
DHA: Docosahexaenoic acid (22:6n-3)
DPA: Docosapentaenoic acid (22:5n-3)
DW: Dry weight
EPA: Eicosapentaenoic acid (20:5n-3)
FA: Fatty acid
FAO: Food and Agriculture Organization
FCE: Feed Conversion Efficiency
FCR: Feed Conversion Ratio
FI: Feed intake
G: Gracilaria cornea
GN: Enriched Gracilaria cornea
GSI: Gonadosomatic index
H: Hypnea spinella
HN: Enriched Hypnea spinella
IUSA: Instituto Universitario de Sanidad Animal y Seguridad Alimentaria
L: Laminaria digitata
LA: Linoleic acid (18:2n-6)
Lys: Lysine
M: Mixed diet
Met: Methionine
MN: Enriched Mixed diet
MUFA: Monounsaturated fatty acids
P/E: Protein: energy ratio
P: Palmaria palmata

XII
 Abbreviationss

PER: Protein Efficiency Ratio

PUFA: Polyunsaturated fatty acids
S: Shell
SB: Soft body
SD: Standard deviation
SFA: Saturated fatty acids
SGR: Specific Growth Rate
SL: Shell length
TAN: Total ammonia nitrogen
TWBW: Total wet body weight
U: Ulva rigida
ULPGC: Universidad de Las Palmas de Gran Canaria
UN: Enriched Ulva rigida
Units: Along the whole manuscript, the international system of units (SI) was used
UV: Ultraviolet
WG: Weight gain
WS: Water Stability
WW: Wet weight

XIII
 Abstract

ABSTRACT

The overall aim of this thesis was “to develop grow-out technology for the local
abalone species, Haliotis tuberculata coccinea“, considered a new candidate for
Canarian aquaculture diversification. More specifically, algal and artificial diets
suitability, growth and survival, as well as various factors affecting the on-growing
success, were addressed through the performance of four different studies. Besides, this
general objective was undertaken through an environmental approach so as to maximize
the sustainability of the abalone production methods to be developed.

On one hand, it was determined that red macroalgae Hypnea musciformis,

Hypnea spinella and Gracilaria cornea are successfully produced in biofiltering
systems, and that their nutritional composition is similar to the one of other macroalgae
used as feed for abalone while matching abalone´s protein and lipid requirements,
promoting growth and survival. These red macroalgae are therefore considered suitable
feeds for juvenile Haliotis tuberculata coccínea. Nevertheless, H. spinella was found to
be the best growth promoting diet for this abalone juveniles due to the highest feed
intake and protein efficiency ratio observed. On the contrary, the harder texture of G.
cornea had a negative effect on feed consumption hence leading to the lowest growth
performance of juvenile Canarian abalone.

On the other hand, it was found that the rearing system employed to produce
macroalgae markedly affected their proximate composition, specifically protein content,
which was increased by 100-163% in algae reared in fishpond wastewater effluents
compared with those in fresh seawater. Furthermore, biofilter produced macroalgae
were discovered to greatly enhance abalone growth, revealing a strong potential to be
considered for future abalone culture. In addition, animals fed the mixed diets
performed significantly better than those fed a single algal diet, indicating that abalone
obtain a complete range of required nutrients by eating a mixed algal regime, and that
essential nutrients may become limiting when animal are fed single-species diets. Thus,
the dietary value of the macroalgal regimes tested can be divided into three categories:
best obtained with the mixed algal feeding regime, intermediate by using single Ulva
rigida or Hypnea spinella feeding regimes and the lowest by Gracilaria cornea.
Overall, the fatty acid profiles of the algae studied were characteristic of green and red

XIV
 Abstract

algae, palmitic acid being the most abundant SFA, the Chlorophyta Ulva rigida
showing predominat levels of C16 and C18 PUFAs and minimal levels of C20 fatty acids.
DHA was very low in all algae tested, hence this fatty acid do not appear to be essential
in H. t. coccinea, as all macroalgae tested supported optimal growth of this abalone
species.

Considering harvested macroalgae nutritional quality variability, and that most

of commercial formulated feeds are using fish meal as the main protein source, limiting
their utilization in ecologically sustainable aquaculture, a study was performed to
evaluate several vegetal-based formulated feeds for the culture of adult abalone Haliotis
tuberculata coccinea, with special emphasis on the determination of the suitability, as
potential feed ingredients, of the four species of macroalgae most commonly involved
in European abalone production. Feeding the enriched fresh algae produced a far better
growth for Canarian abalone than all the compound diets, further indicating the high
dietary value of the macroalgae reared in the IMTA system. The inclusion of Palmaria
palmata was found to improve growth, condition index and dietary protein utilization,
while the use of Laminaria digitata markedly reduced the efficiency of dietary protein.
The elevated contents, relative to their feeds, of ARA in the abalone fed the
experimental diets and EPA in abalone fed the fresh algae, denoted the presence of the
respective elongases Δ4 and Δ5 desaturases. However, the low content of DHA further
suggested that this fatty acid is not essential in abalone tissues. Overall, feeding H.
tuberculata coccinea with fishmeal-free formulated diets resulted in high survival and
good dietary protein utilization. However, further studies are required to improve the
growth obtained with this type of diets, especially concerning the use of different
seaweed combinations and inclusion levels, as well as the diet processing methods to
improve diets water stability.

Finally, the effect of stocking density on growth and survival of two different
size groups of Canarian abalone, as well as the potential of sea-based abalone farming
during the final grow-out culture phase were assessed. The results of this study revealed
that lower stocking density was the best suited to sustain abalone growth in both distinct
initial size groups of abalones, that exhibited an increase in feeding behaviour and feed
utilisation efficacy probably linked to a lower competition for space or food. Thus,
present results suggest stocking density of 100 abalone m-2 for 30-45 mm abalone, and

XV
 Abstract

of 30 abalone m-2 for 45 mm abalone onwards. Besides, the offshore mariculture system
as well as the biofilter produced macroalgae, were found to be suitable to sustain high
growth and survival of H. tuberculata coccinea that overall could reach
cocktail/commercial size of 45-60 mm in only 18-22 months.

These results provide crucial technical knowledge necessary to the development

of culture technology adapted not only to the local abalone species, but to the
archipelago conditions. With all these information, it is posible to conclude that H.
tuberculata coccinea can be efficiently produced in an integrated-culture system
suggesting that on-farm seaweed-abalone production could be a part of future
development of abalone industry in the Canary Islands.

XVI
 Introduction

Development of a Sustainable Grow-out Technology for Abalone

Haliotis tuberculata coccinea (Reeve) as a New Species for Aquaculture
Diversification in the Canary Islands

INTRODUCTION
 Introduction

1. INTRODUCTION

1.1. WORLD ABALONE: DESCRIPTION, TAXONOMIC POSITION, PRODUCTION

AND AQUACULTURE

Abalones are marine one-shelled gastropods that belong to the family Haliotidae
of the phylum Mollusca (Barkai and Griffiths, 1986) (Table 1; Fig.1). There are 56
currently described species, all belonging to the genus Haliotis, of world-wide
distribution in tropical to temperate waters (Geiger, 2000). They are usually found
between the intertidal and the littoral zone (Hone and Fleming, 1998), reaching their
maximum population density between 3–10 m where seaweeds, their natural food, grow
abundantly. All are benthic occurring on hard substrata of granite and limestone (Joll,
1996); however, newly settled abalone prefer to live on encrusting coralline algae (Hone
et al., 1997; Roberts, 2001), whereas juveniles (≥ 10 mm in size) and adults, graze
chiefly on attached or drifting algal material (Shepherd, 1973; Hanh et al., 1989;
Shepherd and Steinberg, 1992). Abalones are gonochoristic and broadcast spawners,
with males and females synchronously liberating their gametes in the water column for
reproduction (Stephenson, 1924; Crofts, 1929). Mature males and females can easily be
recognized by the differences in gonad colour (Bardach et al., 1972). Abalone species
that occur in temperate regions are bigger (20-30 cm) than those found in the tropics (6-
10 cm) (Hanh et al., 1989; Jarayabhand and Paphavasit, 1996).

Table 1. Taxonomic classification of abalone species

Phyllum: Mollusca Linnaeus, 1758

Class: Gastropoda Cuvier, 1797
Subclass: Prosobranchia H.M. Edwards, 1848
Order: Archaeogastropoda Thiele, 1929
Superfamily: Pleurotomarioidea Swainson, 1840
Family: Haliotidae Rafinesque, 1815
Genus: Haliotis Linnaeus, 1758

1
 Introduction

Figure 1. Newly settled, juvenile and adult abalone (H. tuberculata coccinea).

Abalone have traditionally been a highly prized delicacies worldwide, its flesh
being used for food whereas its shell, which has an iridescent interior, often is used for
implements, trade material and decoration (Howorth, 1988; Fig. 2).

Figure 2. Abalone culinary products, pearl shell and handicraft.

Despite the fact that the major world abalone consumers have traditionally been
Japan and China (early Japanese references to abalone divers date back to 30 A.D.; Fig.
3), abalone fisheries have also been the basis of social and economic development for
further coastal human settlements in several countries like United States of America,
Mexico, New Zealand, France, Australia or South Africa (Leighton, 1989; Guzmán del
Proó, 1992; Schiel, 1992; Mercer et al., 1993; Freeman, 2001; Troell et al., 2006), with
local communities depending heavily upon this resource.

2
 Introduction

Figure 3. Amas abalone divers (Japan).

The study of abalone biology and ecology started long ago (Stephenson, 1924;
Bonnot, 1930; Crofts, 1932), and has been further and intensively investigated in the
last 35 years as a result of the development of commercial fisheries and exponential
development of abalone aquaculture in the last two decades. Indeed, overexploitation,
illegal harvesting, disease, habitat degradation and inadequate enforcement policies,
have gradually decreased legal landings from abalone fisheries from almost 20.000 tons
(t) in the 1970s to less than 9.000t in 2008 (Cook and Gordon, 2010). As a consequence,
fisheries alone could gradually no longer meet the market demand for abalone and the
share of aquaculture production rapidly increased to cover the needs. In fact, although
abalone farming had begun in the 1950´s and early 1960´s in Japan and China (Elbert
and Houk, 1984; Leighton, 2000), the rapid development of abalone culture took place
in the 1990s, and is actually widespread in many countries worldwide. The major reason
behind this rapid increase is the lucrative market value for their large adductor muscle
or foot (Elliott, 2000), this increase being possible by the development of production
practices, especially for juvenile stages (Daume et al., 2004; Roberts et al., 2004).

Specifically, the global culture of abalone species has grown markedly in the
last decade, with production increased by more than 21 times (Fig. 4). In 2010, world
abalone production was 65.525t, valued about 451 million €, in contrast, only 8.656t
came from the fisheries sector which means that, approximately, 88% of the total
abalone legally consumed came from aquaculture (FAO, 2012).

3
 Introduction

Figure 4. Evolution of global abalone production from legal fisheries and aquaculture during
last decade (source FAO, 2012).

Among all abalone species, approximately 15 are globally exploited for

commercial purposes, either from fishery or through aquaculture (Bester et al., 2004;
Sales and Janssens, 2004; Hernández et al., 2009), being also the highest-priced
shellfish in the world, and additionally, the most important gastropods in aquaculture
(Naylor et al., 2000): Pacific ezo abalone (Haliotis discus hannai Ino, 1953), kuro
(Haliotis discus Reeve, 1846), tokobushi (Haliotis diversicolor Reeve, 1846) and
siebold´s abalone (Haliotis gigantea Gmelin, 1791) mainly found in Japan, China and
Korea; donkey´s ear abalone (Haliotis asinina Linnaeus, 1758) in Thailand and
Philippines; blacklip abalone (Haliotis rubra Leach, 1814), greenlip abalone (Haliotis
laevigata Donovan, 1808) and Roe´s abalone (Haliotis roei Gray, 1826) in Australia;
blackfoot paua (Haliotis iris Gmelin, 1791) and yellow foot paua (Haliotis australis
Gmelin, 1791) in New Zealand; perlemoen abalone (Haliotis midae Linnaeus, 1758) in
South Africa; green abalone (Haliotis fulgens Philippi, 1845), red abalone (Haliotis
rufescens, Swainson, 1822) and pink abalone (Haliotis corrugata Wood, 1828) in
Mexico and United States of America; black abalone (Haliotis cracherodii Leach,
1814) in United States of America; pinto abalone (Haliotis kamtschatkana Jonas, 1845)
from Alaska and Canada to United States of America (California); and European ormer
(Haliotis tuberculata Linneaus, 1758) in Great Britain and France (West Atlantic).

4
 Introduction

Regarding production countries, China is the largest world producer with a total
production of 56.511t in 2010 (FAO, 2012). The main cultured species is the preferred
and high valuable Pacific ezo abalone (H. discus hannai) (Wu et al., 2009), which
covers more than 95% of the total production. Small and lower value abalone H.
diversicolor, which was the most important culture species 10 years ago, is now only
culture in the subtropical and tropical China (Ke et al., 2012).

In Korea, production has grown to be the second one in the world, with over
5.000 abalone farmers engaged in from juvenile to grown up (Park and Kim, 2013).
Despite commercial abalone farming started just in the early 2000s, the development of
new aquaculture methods using sea cages, has dramatically increased the production
from 29t in 2001 to 6.228t in 2010 (FAO, 2012).

South Africa is the largest abalone (H. midae) producer outside Asia, with
production coming from both farmed and wild abalone. Current annual production is
close to 3.000t with aquaculture contributing 1.200t, legal fishery production 150t and
illegal fishery production estimated to be of the order of 1.500t (Britz, 2012).

Despite abalone are not Chilean native species, commercial abalone farming (H.
rufescens and H. discus hannai) has emerged during the 1990s mainly for exportation to
Asiatic countries, reaching a production of almost 800t (99% of H. rufescens) in 2010
(FAO, 2012).

Over 50% of the world´s wild caught abalone harvest comes from Australia,
where there is also a growing farming sector which produced 456t (H. rubra, H.
laevigata and hybrids) in 2010 (FAO, 2012).

Also, there are small industries in United States of America (250t), Taiwan
(171t) and New Zealand (80t, H. iris), that contribute to the world abalone production
(FAO, 2012).

Most of the market demand for abalone is in Asia, mainly in Japan (live, fresh
and frozen abalone), and Mainland China (canned product), whereas there are also well
established markets in Mexico, USA and Europe (Oakes and Ponte, 1996; Robertson-
Anderson, 2003). In addition, abalone shells are also sold for decoration purposes
(Gordon and Cook, 2001). As a result, most commercial cultivation of abalone species
is in Asia and abalone aquaculture and fishery harvests from other parts of the globe are
to a large extent intended for these markets. However, the combination of the highest

5
 Introduction

world supply during the past few years together with a huge increase in the availability
of illegal product as well as the world financial crisis, have led to an overall reduction of
abalone prices (Qi et al., 2010). Therefore, abalone producers are commanded to
improve profitability, through the use of more efficiency cultures systems, international
quality certification and both product and customers diversifications (Cook and Gordon,
2010).

1.2. ABALONE IN EUROPE

Only one species of abalone is present in Europe, the commonly known as the
Ormer, Haliotis tuberculata Linneaus, 1758 (Mgaya, 1995), with three sub-species
being initially described based on morphological characteristics: H. tuberculata
tuberculata Linneaus, 1758, in the Eastern Atlantic coast and the Mediterranean Sea and
both H. tuberculata coccinea Reeve, 1846, and the recently described H. tuberculata
fernandesi Owen and Afonso, 2012, mainly found in the Macaronesian Region (Clavier,
1992; Geiger, 2000; Geiger and Owen, 2012; Fig. 5).

Figure 5. Distribution of H.tuberculata tuberculata and H. tuberculata coccinea (Geiger,

2000).

The ormer, which reaches a maximum shell length of 14 cm (Roussel et al.,

2011), is an important commercial shellfish in the British Channel Islands (Bossy and
Culley, 1976) and on the Normandy and Brittany coast in France (Clavier, 1992), where

6
 Introduction

it occurs in sizeable densities and has been a traditional fishery products commanding
very high prices (Mgaya and Mercer, 1994).

Decreasing natural stocks and increasing value for the flesh, have led to an
interest in mariculture of this species since the late 1970s, with culture techniques
mostly developed in the Channel Islands (Bossy, 1989, 1990; Hayashi, 1982; Hjul,
1991), France (Koike, 1978; Koike et al., 1979; Flassch and Aveline, 1984) and Ireland
(Mercer, 1981; LaTouche and Moylan 1984; La Touche et al., 1993; Mercer et al.,
1993; Mgaya and Mercer 1994, 1995; Mai et al., 1995a,b, 1996), where both European
ormer and Pacific abalone were introduced during the late 1970ʼs and mid 1980ʼs
respectively (Leighton, 2008).

However, despite abalone being recognized as a prime candidate for European

aquaculture (Mgaya and Mercer, 1994), its availability is still severely restricted due to
lack of supplies from both wild resources and aquaculture production (Dallimore,
2010). Attempts are being made to increase aquaculture production, but progress is slow
and has been hampered by the lack of research and confusion in legislation (abalone is
placed in the same category as bivalves). Besides, feed type and sourcing as well as
sustainable culture technology, have been also identified as key areas which still remain
to be researched to assist the sector achieving sustainable growth and improving
economic competitiveness (SUDEVAB, 2007).

Abalone output in Europe is substantially focused on high quality, low volume

niche markets, such as organic or eco-certified products. Ireland, the Channel Islands
(Huchette and Clavier, 2004) and France are currently the only established producing
countries, with most farms growing abalone at sea, feeding them handpicked fresh
seaweed harvested from local shores. France, is the largest European abalone producer
with a total production of 10t in 2010 (FAO, 2012), most of them coming from one
main operator, which is the first abalone farm worldwide to gain organic certification
(100% sustainable harvested macroalgae; no pharmaceutical, chemical or artificial
fertilizers are used), selling live 6-7 cm sea-bred abalone at 69 € per kilogram (Legg et
al., 2012).

Regarding Spain, an onshore recirculation facility (both hatchery and on-

growing units) for the culture of native European and introduced Pacific ezo abalones,

7
 Introduction

has recently been started in Galicia, with an expecting production of 115 t in 2017
(GMA, 2014).

1.3. ABALONE (Haliotis tuberculata coccinea): CANDIDATE FOR CANARIAN

AQUACULTURE DIVERSIFICATION

The exceptional climate conditions and high quality of the Canary Island
coastal waters makes this Spanish region to have very good perspectives for aquaculture
expansion. Nevertheless, local commercial aquaculture is limited to a reduced number
of marine fish species such as gilthead seabream (Sparus aurata), European sea bass
(Dicentrachus labrax) and small amount of Senegalese sole (Solea senegalensis)
(APROMAR, 2012). Hence, species diversification being a challenge for further local
aquaculture development.

In the Canary Islands, the abalone H. tuberculata coccinea, is distributed from

the intertidal zone down to 15 m depth in semi-exposed and exposed areas. It grows to a
maximum size of about 8 cm in shell length and feeds on a diverse assemblage of
macroalgae (Espino and Herrera, 2002).

This abalone species has been locally exploited during decades, leading to an
overexploitation of its stocks, which are actually almost depleted (Pérez and Moreno,
1991; Espino and Herrera, 2002). Consequently, there is public interest in developing
the culture techniques of this species to both, supply the local market and to contribute
to the recovery of wild populations. Moreover, the potential of this species for
aquaculture production, relies also on the external growing demand for small “cocktail
size abalone“ (4-7 cm shell length) (Jarayabhand and Paphavasit, 1996; Najmudeen and
Victor, 2004), as well as on the high degree of development achieved in some other
species of the family Haliotidae, in particular of the currently produced European ormer
Haliotis tuberculata tuberculata L., with close biological characteristics.

Preliminary studies on H. t. coccinea, focusing on reproduction (Peña, 1985,

1986; Bilbao et al., 2004, 2010) and early life stages (Courtois de Viçose et al., 2007,
2009, 2010, 2012a, b), showed both the possibility of successfully reproducing of
Canarian abalone and the adequate spat production techniques adapted to this species,
hence suggesting it as a good candidate for aquaculture diversification.

8
 Introduction

However, little published data exist on the optimum conditions for juveniles
and adults production in the Canary Islands (Toledo et al., 2000). Thus, in order to
develop a grow-out technology adapted to this species, it is necessary to investigate in
the areas of nutrition and feeding, culture system and rearing conditions suitable for
abalone culture. Besides, since this culture frequently requires large quantities of wild
harvested macroalgae, not locally available, and alternative to this feed income should
be evaluated. Furthermore, work should also underway to develop production methods
which ensure the sustainability of this industry fitting into the strongly growing EU eco-
sector for shellfish products as well as the Abalone Aquaculture Dialogue standards
(WWF, 2010).

1.4. FACTORS AFFECTING ABALONE GROWTH

Abalone growth is generally low and variable (Day and Fleming, 1992; Britz
1996a; Sales and Britz, 2001), with typical growth rates of approximately 2-3 cm/year
and therefore, 2-5 years are required to produce a market-size abalone (Hahn, 1989;
Troell et al., 2006; Qi et al., 2010). Consequently, a proper grow-out techniques and the
resulting growth and survival of cultured abalone, are critical factors to maximize
economic success and production.

The selection of an appropriate culture technology depends on the abalone

species specific characteristics and its susceptibility to different parameters affecting the
on-growing success. Some of these parameters, are related to culture conditions such as
initial size, stocking density or water flow; to physical-chemical conditions such as
temperature, illumination (light and shading) or water quality; whereas others refer to
juvenile and adults feeding and nutrition such as nutritional requirement, feed quality
and quantity or feed type and sourcing; and finally, the economic viability of
commercial abalone farming is largely influenced by the culture system employed
during such a long lasting grow-out culture phase.

Besides, taking into account that sustainable, eco-friendly production methods

are to be a part of future expansion of the abalone industry (WWF, 2010), it is important
to adjust rearing techniques so as to maximise the sustainability of the on-growing
activity.

9
 Introduction

1.4.1. Culture conditions related parameters

1.4.1.1. Initial size

Immediately after settlement, abalone post-larvae feed on biofilm and mucus

trails (Shepherd, 1973; Saito, 1981) and once the radula is developed, at around 0.8 mm
shell length (SL) (Kawamura et al., 2001), juveniles start feeding on diatoms, turf,
crustose coralline algae and epiphytic bacteria (Dunstan et al., 1996,1998). Juveniles
maintain this diet until they are large enough to undergo the final diet transition from
diatoms to macroalgae (Jarayabhand and Paphavasit, 1996; Kawamura et al., 2001). The
size of individuals at this final transition varies among species, ranging from 5 to 10
mm for H. discus hannai (Kawamura et al., 2001), 7 to 8 mm for H. rufescens (Hahn,
1989), 10 to 20 mm for H. asinina and H. ovina (Jarayabhand and Paphavasit, 1996) or
10 mm for H. tuberculata (Mgaya and Mercer, 1994). In the case of Canarian abalone,
their size ranges from 6 to 10 mm (Courtois de ViÇose, personal comunication).

Stocking size has been regarded as a major factor affecting abalone grow-out
(Flemming and Hone, 1996; Wu et al., 2009), showing significant differences in growth
and survival among juvenile groups of different initial body sizes (Mgaya and Mercer,
1995; Nie et al., 1996; Leaf et al., 2007). These variations may be a result of genetic
differences related to body size (Sun et al., 1993; Wu et al., 2009), protein requirements
(Shipton and Britz, 2001), dietary protein and energy utilization (Britz and Hecht, 1997;
Shipton and Britz 2001; Green et al., 2011) or grazing efficiency and capability of the
radula (Jonhston et al., 2005).

1.4.1.2. Stocking density

Land-based abalone farming is highly capital intensive, and so the efficient use
of infrastructure is critical to profitability. Consequently, abalone farmers wish to use
high stocking densities in an attempt to maximize production. However, this factor
markedly affects abalone growth and survival, being determinant for growing abalone to
market size.
The negative effect of increasing stocking density on abalone growth seems to
be strongly associated with these grazing gastropods density-dependent competition for

10
 Introduction

space and/or food, as it has been shown for most of commercial abalone species
worldwide: H. tuberculata (Koike et al., 1979; Cochard, 1980; Mgaya and Mercer,
1995); H. cracherodii (Douros, 1987); H. discus hannai (Jee et al., 1988; Wu et al.,
2009; Wu and Zhang, 2013); H. rubra (Huchette et al., 2003a, b); H. asinina (Capinpin
et al., 1999; Fermin and Buen, 2002; Jarayabhand et al., 2010); Haliotis diversicolor
supertexta (Liu and Chen, 1999); H. rufescens (McCormick et al., 1992; Aviles and
Shepeherd, 1996, Valdés-Urriolagoitia, 2000); H. midae (Tarr, 1995); H. corrugata
(Badillo et al., 2007); H. kamtschatkana (Lloyd and Bates, 2008) and for H. iris (Heath
and Moss, 2009), in different culture systems.

Additionally, in optimizing production system, a number of factors which are

directly related to the stocking density must be also considered and of these water flow
(Vivanco-Aranda et al., 2011) and quality (Huchette et al., 2003b), culture system
(Badillo et al., 2007), size grading (Mgaya and Mercer, 1995; Heath and Moss, 2009;
Wu et al., 2009) and food quality and quantity (Lloyd and Bates, 2008; Tahil and Juinio,
2009), have in particular been emphasized.

The effects of stocking density on growth performance of H. tuberculata

coccinea have not yet been studied. However, initial densities of 43-175 abalones m2
tested in cage cultures of another warm water species such as H. asinina (Capinpin et
al., 1999) and of 83-386 abalones m2 for juveniles of a close related species such as H.
tuberculata (Koike et al., 1979; Mgaya and Mercer, 1995), showed a marked effect on
final growth of abalone. Indeed, a 3 times increase in initial stocking density may cause
a 52% reduction in growth for H. tuberculata (Mgaya and Mercer, 1995).

1.4.1.3. Water flow

Abalone culture is characterized by high water exchange rates (200% to 2.400%

per day) to keep optimum water quality parameters within levels recommended for
grow-out conditions (Badillo et al., 2007; Naylor et al., 2011). Moreover, increased
water flow is capable of impacting feed conversion ratio (FCR) positively by
stimulating feeding activity (Higham et al., 1998), hence enhancing abalone growth
(Shepherd, 1973; Mgaya and Mercer, 1995; Wassnig et al., 2010). Additionally, an
increase in water flow could be an effective means of counteracting the harmful effects

11
 Introduction

of high stocking density, helping farms to maximize financial returns (Wassnig et al.,
2010).

Besides, Tissot (1992) reported that suitable water velocities need to be provided
to induce sufficient mantle cavity circulation to allow optimal performance of abalone
species. However, in shallow raceway tanks (such as the so-called ‘slab’ tank), which
are common in commercial abalone farms (Hutchinson and Vandepeer, 2004; Wassnig
et al., 2010), high water flowing could also have a negative effect by causing increased
nutrient leaching from food pellets (Fleming et al., 1997; Marchetti et al., 1999) and by
washing feed downstream (Fleming et al., 1997). Thus, water velocity should also be
considered not to exceed a critical limit.

In a flow through systems, the cost associated with the maintenance of the
mentioned high water exchange rate (mainly pumping costs) accounts for between 15%
to 30% of the operational budgets (Neori et al., 2000; Badillo et al., 2007). Thus, in
order to not only reduce the production cost but the effluents nutrients loads to the
environments, several recirculating, serial-use raceways and also co-culture (seaweed-
abalone) recirculation systems, have been developed and even stated as suitable
commercial grow-out systems for several abalone species such as H. tuberculata
(Mgaya and Mercer, 1995; Schuenhoff et al., 2003), H. discus hannai (Nie et al., 1996;
Park et al., 2008; Demetropoulos and Langdon, 2004), H. rufescens and Haliotis
sorenseni, (Demetropoulos and Langdon, 2004), H. corrugata (Badillo et al., 2007), H.
asinina (Jarayabhand et al., 2010), H. midae (Naylor et al., 2011) or H. iris (Tait,
2012).

1.4.2. Physico-chemical parameters

1.4.2.1. Temperature

Temperature is the primary environmental controlling factor determining the

metabolic rate of poikilotherms (Fry, 1971) and therefore of fundamental importance to
the management of abalone on-growing, as it directly determines rates of gonad
development (Uki and Kikuchi, 1984; Hahn, 1989), feed consumption (Peck, 1989;
Britz et al., 1997; García-Esquivel et al., 2007), ammonia excretion (Barkai and
Griffiths, 1987; Lyon, 1995), growth rate and nutritional indices (Leighton, 1974; Uki

12
 Introduction

et al., 1981; Hahn, 1989; Peck, 1989; Britz et al., 1997; Gilroy and Edwards, 1998;
Hoshikawa et al., 1998; Lopez et al., 1998; Steinarsson and Imsland, 2003; Alcántara
and Noro, 2006; García-Esquivel et al., 2007); survival (Hahn, 1989), stress
susceptibility (Lee et al., 2001; Haldane, 2002; Malham et al., 2003), and the
development of temperature-based farm-management protocols (Vandepeer, 2006), and
is thus integral to the development of economically viable technology for abalone
culture.

Each abalone species has a preferred temperature range, but generally temperate
species can be found in water temperatures ranging from 8-18ºC, with subtropical
species found in ranges of 14-26ºC and tropical species located in temperatures around
16-30ºC. Gilroy and Edwards (1998) stated that in general, abalone have a conservative
thermal response and little tendency to adapt to chronically altered thermal
environments, hence rearing temperature should be as close to the preferred one as
possible.

Seawater temperature in the Canary Island ranges between 18 to 24ºC (Cuevas.,

2006), hence closer to the thermal range of subtropical and tropical species than the one
of its closely related European ormer, Haliotis tuberculata, reported as a temperate
species (Peck, 1989).

1.4.2.2. Light

Despite abalone habitat and behaviour may vary widely between species
(Shepherd, 1975), they are generally more active at night, light clearly influencing their
distribution and activities (Cochard, 1980; Huchette et al., 2003b; Morikawa and
Norman, 2003), hence their feeding behaviour (Tahil and Juinio-Menes, 1999).
Consequently, shading and refuges are useful management tools and either needed for
optimal growth (Maguire et al., 1996; Fig. 6). Moreover, providing shading and refuges
during higher stocking densities can lead to improved growth rates by both, increasing
the “preferred surface area” thus reducing abalone stacking, and limiting the impact of
photoperiod on the regulation of the feeding activity (Hindrum et al., 1999; Huchette et
al., 2003b). However, shading and refuges may also present several disadvantages,
including increase of labour cost for feeding and maintenance or reduction of good
water circulation (Maguire et al., 1996).

13
 Introduction

Figure 6. PVC tiles used as shelters in donkey´s ear (A) and Canarian abalone (B) culture.

1.4.2.3. Water quality

Abalone growth and health are reported to be inhibited by decreases in water

quality (Basuyaux and Mathieu 1999; Naylor et al., 2011). In an abalone grow-out
system, a reduction in water quality can result from the decomposition of faeces and
uneaten food (Yearsley et al., 2009), high levels of nitrogenous wastes excreted by the
animals (Barkai and Grifths, 1987) and from reduction of dissolved oxygen (Badillo et
al., 2007).

To ensure growth rates are maximized, it is generally assumed that DO content

of water should be kept at saturated levels (100%), and once oxygen levels are
maintained, level of nitrogenous wastes derived from excretion being probably the most
important parameter (Colt and Armstrong, 1981). Within the nitrogenous wastes,
ammonia is the major toxicant derived from protein catabolism (Kinne, 1976; Russo
and Thurston, 1991) and from bacterial activity (Reddy-Lopata et al., 2006), being a
stressor and even lethal factor in abalone aquaculture (Hargreaves and Kucuk, 2000;
Huchette et al., 2003a). Thus, a number of workers have investigated its influence on
the survival and growth of several abalone species including H. laevigata (Harris et al.,
1998, Hindrum et al., 2001), H. tuberculata (Basuyaux and Mathieu, 1999), H. rubra,
Huchette et al., 2003a), H. diversicolor supertexta (Cheng et al., 2004) or H. midae
(Reddy-Lopata et al., 2006; Naylor et al., 2011), reporting that, despite abalone can

14
 Introduction

adapt to sub-lethal levels of ammonia, a substantial reduction in growth is attained,

hence being essential to keep ammonia levels low. In abalone culture, ammonia levels
are regulated by the water exchange rate, which should be balanced carefully to meet
economical and physiological constraints (Ford and Langdon, 2000).

1.4.3. Grow-out culture systems

An ideal abalone culture system should promotes even distribution of animals,
ready access to feed, minimal contact of abalone and feed with faecal wastes, good
water flow and exchange, a means of accommodating and feeding abalone in
commercially viable densities and minimal human disturbance (McShane, 1988; Aviles
and Shepherd, 1996; Fleming and Hone, 1996). Thus, great varieties of both onshore
and offshore culture systems have been developed, system design being still a "work in
progress" resulting in farmers using a range of systems and achieving variable growth
rates.

1.4.3.1. Land-based systems

Onshore on-growing systems can include large deep concrete tanks, specialized
plastic tanks or outdoor ponds (McShane, 1988; Freeman, 2001; Alcántara and Noro,
2006; Fig. 7). However, since tank design influence both feed and behavior (Fleming
and Hone, 1996), these different systems vary considerably in effectiveness.
Over the past few years, a major objective of production techniques research has
focused on developing a tank system suitable for manufactured diets, which has become
in very shallow high flow rate tanks, which ensures that wastes can be easily flushed
from the system (Wassnig et al., 2010). Production in such a system is estimated at 1
t/tank/year (Morrison and Smith, 2000).

As a result of the high cost of water pumping, aeration, filtration and

temperature control, land-based production systems are low energy efficient, with major
drawbacks being the high start-up and operational costs (Wu et al., 2009; Wu and
Zhang, 2013). Another constraint for land-based operations is the shortage of suitable
sites, mainly due to the competition for space with other human activities.

15
 Introduction

Figure 7. Several on-shore systems for the culture of H. rufescens (A; Chile), H. discus hannai
(B; Korea), H. asinina (C; Thailand) and H. tuberculata (D; Ireland).

16
 Introduction

1.4.3.2. Sea-based system

In offshore farms, wave motion and tidal currents drive water exchange within
the offshore structures thus, growth might be affected by prevailing weather conditions
(Nagler et al., 2003). The advantages of sea-based abalone farming include potential
improvement of rearing conditions, minimal start-up and operational costs and easy
access for feeding, managing and harvesting (Jarayabhand and Paphavasist, 1996;
Leighton, 1989; Capinpin et al., 1999; Wu et al., 2009). Whereas drawbacks involved
high labour cost in keeping meshed areas clean, lack of control over environmental
conditions, lack of suitable conditions for spat grow-out and security issues (Hindrum et
al., 1996; Preece and Mladenov, 1999; Wu and Zhang, 2010, 2013).

In sea-based production systems, structures that afford shelter for abalone are
suspended below the water surface from long-line systems, floating docks or placed on
the sea-floor (Aviles and Shepherd, 1996; Capinpin et al., 1999; Fermin and Buen,
2002; Wu et al., 2009; Fig. 8-10). A variety of these offshore structures: barrels, PVC
tubes, net cages or plastic multitier baskets, have been tested for different abalone
species like H. rufescens (Benson et al., 1986); H. fulgens (Gonzáles-Avilés and
Shepherd, 1996); H. asinina, (Capinipin et al., 1999, Minh et al., 2010); H. iris (Preece
and Mladenov, 1999), H. diversicolor (Alcántara and Noro, 2006; Wu et al., 2009), H.
tuberculata (Bossy 1989, 1990; Hensey 1991, 1993; La Touche et al., 1993; Legg et
al., 2012) or hybrid abalone (H. rubra x H. laevigata, Mulvaney et al., 2012), showing
that abalone growth rate varies greatly as a result of the containment structure used for
grow-out.

Figure 8. (A) Long-line culture of red abalone H. rufescens in Chile. (B) Represents a six-tiered
basket traditionally used for H. discus hannai farming in south China.

17
 Introduction

However, the rapid expansion of rearing facilities in inner coastal bays has led to
the overcrowding of floating rafts, which reduces water flow, thereby affecting the
growth and survival of abalone (Fleming et al., 1997; Searcy-Bernal and Gorrostieta-
Hurtado, 2007; Wassnig et al., 2010). The development of floating cages submerged in
offshore waters has made cage culture possible for finfish (e.g., salmonids) in Europe
and Canada (Korsoen et al., 2009) and large yellow croaker Pseudosciaena crocea in
China (Lu et al., 2008). This culture technique combines high yield, easy husbandry and
low labour and energy costs (Mortensen et al., 2007). Thus, some novel submerge cages
have recently been tested in China, showing an overall better growth performance of
abalone H. discus hannai than the one attained in traditional suspended multi-tier
baskets (Wu and Zhang, 2013).

Figure 9. Sea-floor culture of European ormer in Jersey Islands (UK).

In general, sea-based culture technology has become the most popular grow-out
mode worldwide, because of its lower cost compared to the traditional land-based
farming, better quality products and higher sustainability in the long term (Ke et al.,
2012; Park and Kim, 2013; Legg et al., 2012).

18
 Introduction

Figure 10. Cage culture of abalone H. tuberculata in Brittany (France).

1.5. ABALONE FEEDING AND NUTRITION

The proper nutrition and the resulting growth of cultured abalone are critical
factors in the successful culture of this animal. Thus, as a first step in this thesis, an
extensive review of existing knowledge and ongoing research in Haliotids production
worldwide, mainly focused on their nutritional requirements and feeding practices, was
carried out.

1.5.1. General aspects

Abalone is an herbivorous gastropod that eats mainly seaweeds (Elliott, 2000;

Sales and Britz, 2001; Nelson et al., 2002; Tanaka et al., 2003) (Fig. 11). They start to
feed immediately after larval settlement (Tutschulte and Connell, 1988). As they grow,
they begin feeding on macroalgae and in the wild may change from one species to
another as they mature (Table 2).

19
 Introduction

Figure 11. Juvenile abalone (H. tuberculata coccinea) grazing on green algae.

The macroalgal preference of different abalone species varies worldwide

depending on their habitat and availability (Dunstan et al., 1996; Nelson et al., 2002).
Although a variety of macroalgae are naturally consumed (Britz, 1991), kelp, by far,
forms the primary food source for these herbivores (Rosen et al., 2000; Qi et al., 2010).

The natural feeding patterns of abalone change during different stages of their
life cycle (de Waal et al., 2003). This is attributed not only to the increased mouth size
(Fleming et al., 1996), but also to morphological changes of the radula as the abalone
grows (Steneck and Watling, 1982; Kawamura et al., 2001; Daume and Ryan, 2004;
Simental et al., 2004; Johnston et al., 2005). Changes in diet could also be due to
transformations in the gut micro-organisms, such as bacteria and enzymes within the
digestive system of abalone as they grow and thus enabling them to digest macroalgae
(Erasmus et al., 1997; Tanaka et al., 2003).

1.5.2. Abalone feeding practices

Abalone can consume seaweed at a rate close to 35% of its body weight per day
(Tahil and Juinio-Menez, 1999), hence sustaining of growth requires a large amount of
fresh macroalgae (Fig. 12). However, the limited availability of suitable seaweed, and
the low protein content of the harvestable ones, are major impediments for intensive
cultivation of this mollusk worldwide (Hahn, 1989). Moreover, macroalgae nutritional
quality and abundance varies greatly according to geographic location and time of
sampling (Dawczynski et al., 2007; Courtois de Viçose et al., 2012a), greatly affecting

20
 Introduction

growth rates and making it difficult to optimize feeding for abalone growers (Bautista-
Teruel and Millamena, 1999).

Owing to the relatively low and variable growth of abalone fed on macroalgae
(Britz, 1996a; Cook, 1991; Bautista-Teruel and Millamena, 1999; Tan and Mai 2001;
Moriyama and Kawauchi, 2004), and the logistical and supply problems associated with
the use of fresh seaweed, intensive abalone culture has become increasingly reliant upon
artificial feeds (Britz 1996a, b; Sales and Britz, 2001), which are formulated so as to
fulfill the nutritional requirements of each abalone species.

Feed options for abalone are also influenced by the choice of culture technique
with seaweed use generally better suited to offshore systems, whilst artificial feeds may
be more appropriate in land based operations (Kinkerdale et al., 2010; Qi et al., 2010).
In consequence, both seaweed and artificial dietary sources have their place and should
be considered on a site specific basis (Dlaza, 2006). Besides, feeding strategies and
recommended practices should be based upon not only nutritional factors but economic
success.

In commercial abalone farming, animals are generally fed 2-3 times per week or
every 2 days (Maguire et al., 1996).

Figure 12. Harvested Macrocystis pyrifera and Palmaria palmata to feed red abalone (A;
Chile) and European ormer (B; Brittany).

In the case of the Canary Islands, seaweed resources are less abundant than in
some other nutrient rich coastal areas and hence not harvested. Therefore, to replace
wild seaweed as a main diet component, the development of local abalone culture
industry, should be reliant on the use of cultured macroalgae and/or formulated feeds.

21
 Introduction

Table 2. Suitable wild and cultured macroalgae as food for different abalone species (Numbers indicate references which are listed below)

Abalone species
Macraolgae H.
H. H. H. H. H. H. H. H. H. H. H. H.
discus
asinina roei midae corrugata rufescesn sorenseni laevigata rubra fulgens diversicolor iris tuberculata
hannai
Red macroalgae
Asparagopsis armata 1
Gelidium spp. 2
Gracilaria spp. 3 4 5 6 7 8 9
Gracilariopsis spp. 10
Jeannerettia lobata 11
Palmaria spp. 12 13 14 15
Brown macroalgae
Ecklonia maxima 16
Egregia menziesii 17
Eisenia spp 18
Laminaria spp. 19 20 21
Macrocistys pyrifera 22 23 24 25
Phyllospora comosa 26
Undaria pinnatifida 27
Green macroalgae
Ulva spp 28 29 30 31 32
1. Shepherd and Cannon,1988.; 2. Troell et al., 2006 ; 3. Jackson et al.,2001 (G. edulis); 4. Sales and Britz, 2001 (G. gracilis); 5. Pang et al., 2006 (G. textorii); 6. Mcbride et al., 2001
(G. conferta); 7. Liao et al., 2003 (G. tenuistipitata); 8. Allen et al., 2006; 9. Neori et al., 1998; Mcbride et al., 2001(G. conferta); 10. Capinpin and Corre, 1996 (G. heteroclada);
Reyes and Fermin, 2003 (G. bailinae);11. Fleming, 1995; 12. Mercer et al., 1993 (P. palmata); Demetropoulos and Langdon, 2004 (P. mollis); 13& 14. Demetropoulos and Langdon,
2004 (P. mollis); 15. Mercer et al., 1993 (P. palmata); 16. Naidoo et al., 2006; 17. Nelson et al., 2002; 18. Uki et al., 1985a; 19. Troell et al., 2006 (L. pallida); 20. Qi et al., 2010 (L.
japonica); 21. Mercer et al., 1993 (L. digitata); 22. Badillo et al., 2007; 23 and 24. Serviere-Zaragoza et al., 2001; 25. Allen et al., 2006; 26. Vandepeer and van Barneveld 2003; 27.
Sakata et al., 1984; 28. Boarder and Shpigel, 2001 (U. rigida); 29. Naidoo et al., 2006; 30. Shuenhoff et al., 2003; 31 and 32. Mcbride et al., 2001 (U. lactuca)

22
 Introduction

1.5.3. Abalone nutritional requirements

The nutritional value of abalone food rations depends on many factors including
nutrient composition and bioavailability (Middlen and Redding, 1998; Serviere-
Zaragoza et al., 2001; Nelson et al., 2002; Bautista-Teruel et al., 2003); digestibility
(Sales and Britz 2001, 2002; Gomez-Montes et al., 2003); processing techniques (Booth
et al,. 2002; Sales and Britz, 2002); diet particle size (Southgate and Partridge, 1998);
feed pellet size and presentation (Fleming et al., 1996; Kinkerdale et al., 2010); the
presence of attractants (Fleming et al., 1996; Sales and Janssens, 2004); or texture and
palatability (Kautsky et al., 2001). Therefore, feed quality is based upon several linked
factors. Hence, no one factor should be considered alone.

1.5.3.1. General composition of abalone manufacturated diets

Artificial abalone diets to date are remarkably similar in their proximate

composition (Viera et al., 2009a). The caloric content (gross energy) is generally around
4 Kcal g-1 (Table 3). The moisture content of diets averages 10% (Table 3).The protein
content varies considerably from about 20-50 % (Table 4), averaging around 30%. Lipid
content tested ranges from 1.2-19%, averaging around 4% (Table 5). Carbohydrate
makes up the bulk of the diets, ranging from 21- 82%, averaging 45% (Table 6). Crude
fibre content is generally low (1-6%; Table 6) as the capacity of abalone to digest fibre
is limited (Fleming et al., 1996).

1.5.3.2. Protein sources, optimal inclusion levels and supplementation synthetic

amino acids

Among the different nutrients, abalone requires adequate levels of high quality
protein for soft tissue growth (Uki et al., 1985a; Mai et al., 1995a, b; Britz and Hecht,
1997; Bautista-Teruel and Millamena, 1999; Gómez-Montes et al., 2003; Reyes and
Fermín, 2003; Viana et al., 2007). This essential but expensive dietary component must
be optimally utilized by the abalone for growth rather than for energy. The main factors
affecting dietary protein utilisation are its digestibility and the balance and availability
of its amino acids, energy supplied being also considered important (Fleming et al.,
1996).

23
 Introduction

Table 3. Proximate composition (% dry matter) and caloric content of artificial diets
tested for abalone

References

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Crude protein 30 31 35 38 32 40 35 35 28 35 27 36 38 35 44

Crude lipid 5 5 10 6 6 5 7 4.5 3 6 5 3 1.3 5 6.7

Carbohydrate 48 33 39 40 44 42 48 45 43

Crude Fibre 6 4.3 1

Ash 3 11 8 9 6 16

Moisture 5 10 6

GE (Kcal g-1) 4.5 3 4.7 3 4 2.9 4 4.5 4.5

P:e ratio* 47 85 96

1. Uki et al., 1985b (Japan: H. discus hannai); 2. Mai et al., 1995b (Ireland: H. tuberculata and H. discus
hannai); 3. Viana et al., 1993 (México: H. fulgens); 4. Guzmán and Viana, 1998 (Mexico: H. fulgens); 5.
Bautista-Teruel and Millamena, 1999 (Philippines: H.asinina); 6. Jackson et al., 2001 (Australia: H.
asinina); 7. Serviere-Zaragoza et al., 2001 (Mexico: H. fulgens); 8. Shipton and Britz, 2001(South Africa:
H. midae); 9. Bautista-Teruel et al., 2003 (Philippines: H. asinina); 10. Gómez-Montes et al., 2003
(Mexico: H. fulgens); 11. Reyes and Fermín, 2003 (Philippines: H. asinina); 12. Sales et al., 2003 (South
Africa: H. midae); 13. Thongrod et al., 2003 (Thailand: H. asinina); 14. Naidoo et al., 2006 (South Africa:
H. midae); 15. Hernández et al., 2009 (Chile: H. rufescens). * Protein: energy ratio

The most common protein sources employed in abalone feeds includes fishmeal,
defatted soybean meal (Guzmán and Viana, 1998; Sales and Britz, 2002; Gómez-
Montes et al., 2003; Thongrod et al., 2003; Naidoo et al., 2006; García-Esquivel et al.,
2007), casein (Uki et al., 1985b; Viana et al., 1993; Mai et al., 1995b; Sales et al., 2003;
Vandepeer and van Barneveld, 2003) and Spirulina spp. (Uki et al., 1985b; Britz et al.,
1996a, Bautista-Teruel et al., 2003; Thongrod et al., 2003; Naidoo et al., 2006;Troell et
al., 2006). Few novel protein sources have also been tested at low inclusion proportions
(Table 4). To get advantage of the high nutritional value of algae, algal meals have been
occasionally included in abalone feeds (García-Esquivel et al., 2007; Viana et al., 2007).
Fish or abalone viscera silage have been also proposed as economic protein sources

24
 Introduction

(Viana et al., 1996; Guzmán and Viana, 1998). Besides, different protein sources may
be balanced by addition of synthetic amino acids such as methionine, threonine and
arginine in order to fulfil the essential amino acid requirements of these species (Mai et
al., 1995b; Guzmán and Viana, 1998; Serviere-Zaragoza et al., 2001; García-Esquivel et
al., 2007).

Among all the sources tested as a single protein source, fishmeal is the only one
that supports good growth performance (Fleming et al., 1996). However, concern over
the sustainability of fishmeal use in aquaculture has led the World Wildlife Fund to
place restrictions of its use in the new standards for sustainable abalone farming (WWF,
2010). For abalone fed diets containing fishmeal the standards require that it should not
take more than one kilogram of fish to produce a kilogram of abalone i.e. Forage-Fish-
Efficiency-Ratio (FFER) of 1 or lower. Moreover, abalone output in Europe is
substantially focused on organic or eco-certified products, implying that no fishmeal,
pharmaceuticals or fertilizers are used. Hence, developing a non-including fishmeal
artificial feed for abalone, would have marketing benefits not only for consumers, who
are increasingly environmentally sensitive, but also for producers, who could validate
the environmental and social sustainability of their farming operations (WWF, 2010).

25
 Introduction

Table 4. Nutritional composition (% dry matter) of artificial diets tested for abalone: Protein sources and inclusion levels

Nutrient Diet

1 2 3 4 5 6 7 8 9 10 11
Protein source and content
Fish meal 47 41-71 44 9-62 40 7-17 30 12-48 20
Soy bean meal 60 10 17 20 10 19-37 10
Casein 32 8-41 32
Shrimp meal 7-20
Silage 20
Spirulina spp. 43 15-30
Cotton seed meal 2 5
Sunflower meal 24-48 5
Torula yeast 57 24-40
Vegetable meal 52
Corn meal 47 10 10 15
Kelp meal 12 10 12
Soya oil cake 63
Egg albumin 37
Whole egg 34

Total protein 30 0-50 27-47 30 20-50 24-44 36-39 22-32 36 34 23

1. Uki et al., 1985b (Japan: H. discus hannai) ; 2. Mai et al., 1995b (Ireland : H. tuberculata and H. discus hannai); 3. Britz et al, 1996a (South Africa: H. midae); 4.
Britz et al, 1996b (South Africa: H. midae); 5. Fleming et al,, 1996 (review); 6. Britz and Hecht, 1997(South Africa: H. midae); 7. Guzmán and Viana, 1998 (México:
H. fulgens); 8. Bautista-Teruel and Millamena, 1999 (Philippines: H. asinina); 9. Serviere-Zaragoza et al., 2001 (México: H. fulgens); 10. Shipton and Britz,
2001(South Africa: H. midae); 11. Sales and Britz, 2002 (South Africa: H. midae)

26
 Introduction

Table 4. Continued.

Diet
Nutrient
12 13 14 15 16 17 18 19 20 21 22 23

Protein source and content

Fish meal 10 7.5 10 18-35 3 1-7 43 55 29 30 30 29-35

Soy bean meal 20 35 35 15 34 62 15 10

Casein 4-48 35 16-19

Shrimp meal 15 7.5 10 5

Spirulina spp. 20 20 10 10
Soy bean protein isolate 9-17 5
Lupin meal 10

Vegetable meal 46 39-50

Corn meal 12
Kelp meal 9-17 12 12 15 2.6

Seaweed **

Total protein 27 26-44 26 5-48 37 35 18 44 35 44 34-39

12. Bautista-Teruel et al., 2003 (Philippines: H. asinina); 13. Gómez-Montes et al., 2003 (Mexico: H. fulgens); 14. Reyes and Fermín, 2003 (Philippines: H. asinina);
15. Sales et al., 2003 (South Africa: H. midae); 16. Thongrod et al., 2003 (Thailand: H. asinina); 17. Vandepeer et al., 2003 (Australia: H. rubra and H. laevigata); 18.
Naidoo et al., 2006 (South Africa: H. midae); 19. Troell et al, 2006 (South Africa: H. midae); 20. García-Esquivel et al, 2007 (Mexico: H. fulgens); 21. Viana et al,
2007 (Mexico: H. fulgens); 22. Hernández et al., 2009 (Chile: H. rufescens); 23. Green et al., 2011 (South Africa: H. midae)

27
 Introduction

1.5.3.3. Lipid sources, optimal inclusion levels and essential fatty acids

Lipids are an important dietary constituent not only because of their high energy
value and source of essential fatty acids, that are necessary for cellular metabolism and
maintenance of the membrane structure (Corraze, 2001), but also because they contain
fat-soluble vitamins (Fleming et al., 1996). Moreover, lipids (especially long-chain
PUFA) are an important nutrient determining the flavour and odour of seafoods.
Therefore, several investigations have been conducted to evaluate the response of
abalone to various levels of dietary lipid (Uki et al., 1985a; 1986; Mai et al., 1995a;
Bautista-Teruel et al., 2011); whether abalone lipid requirement are met using fish oil in
artificial diets (Dunstan et al, 1996); the effect of protein-energy ratio on growth rate,
nutritional indices and body composition (Britz and Hecht, 1997; Bautista-Teruel and
Millamena, 1999; Gómez-Montes et al., 2003; Green et al., 2011); the role of lipid in
growth and gonadal maturation (Nelson et al., 2002), the tissues fatty acids composition
(Dunstan et al., 1996; Grubert et al., 2004; Li et al., 2002; Durazo and Viana, 2013;
Hernández et al., 2013) or the effect of lipid to carbohydrate ratio and energy content in
abalone diets (Thongrod et al., 2003).
Abalone species show a low lipid requirement, typical of herbivores molluscs
and fish (Mai et al., 1995a). This low lipid requirement has been associated by some
authors (Durazo-Beltrán et al., 2004) with a low use of dietary lipids as energy source
by abalone based upon its low metabolic rate. Furthermore, high levels of dietary lipid
(> 7%) seem to affect negatively abalone growth and reduce the uptake of other
nutrients in abalone diets as it has been shown for several species including H. laevigata
(Van Barneveld et al., 1998); H. tuberculata and H. discus hannai (Mai et al.,1995a); H.
midae (Britz and Hecht, 1997; Green et al., 2011), H. fulgens (Durazo-Beltran et al.,
2003, 2004); H. asinina (Thongrod et al., 2003) or H. corrugata (Montano-Vargas et
al., 2005).

Despite a wide range of total lipid content have been tested in abalone
formulated diets worldwide (2-19% DW; Table 5), in most cases the total lipid
comprised 3-5% of the diet (Uki et al., 1985a).

Lipid is supplied in artificial diets either as a fish/marine oil (Guzmán and

Viana, 1998; Sales and Britz, 2002; Thongrod et al., 2003; Green et al., 2011), a
vegetable oil (Shipton and Britz, 2001) or a combination of these (Mai et al., 1995a;

28
 Introduction

Britz, 1996a,b; Bautista-Teruel and Millamena, 1999; Shipton and Britz, 2001;
Bautista-Teruel et al., 2003; Gómez-Montes et al., 2003; Reyes and Fermín, 2003)
(Table 5). The lipid bound in fishmeal contributes to the total lipid content and in some
diets is the sole supply. To ensure that oils used do not become rancid, vitamin E, a
natural antioxidant, is commonly added to the lipids (Uki et al., 1985a, b).

A number of studies have found that the composition of fatty acids in the tissues
of macroalgal feeders, such as abalone, is very different to that of carnivorous and
plankton-feeders. Such differences between species have been attributed to the different
lipid composition of their respective diets (Dunstan et al., 1996; Grubert et al., 2004).

Uki et al. (1985a) estimated the requirement for n-3 PUFA to be about 1% of
diet containing 5% lipid (reviewed by Uki and Watanabe, 1992) which represents 20%
of the lipid.

In regards to abalone flesh, the lipid content is low and made up of the fatty
acids: palmitic acid (16:0), stearic acid (18:0), oleic acid (18:1n9), vaccenic acid
(18:1n7), arachidonic acid (20:4n6), eicosapentaenoic acid (20:5n3) and
docosapentaenoic acid (22:5n3) (Dunstan et al., 1999; Nelson et al., 2002).

29
 Introduction

Table 5. Nutritional composition (% dry matter) of artificial diets tested for abalone: Lipid sources and inclusion levels

Diet
Nutrient
1 2 3 4 5 6 7 8 9 10 11 12 13 14
Lipid source and content

Fish oil 2 0-16

Soy bean oil / Pollock liver oil 1 and
(3:2 + 1% Vit E) 5
Corn oil / fish oil (1:1) 0-12
Sunflower oil / fish oil (1:1) 0-5 0-3
0 and
Cod liver oil 1.5 3-5 1 1-5
3
Soy bean oil 1.5 0.5 1 1-5
Squid oil 0.5
Corn oil 0.5

Total lipid 1.2-5 0.6-12 5 and 8 6-8 6 4-5 3-4 1.5-5 3 6-7 5 1-19 5.3 0-10

1. Uki et al., 1985a, b (Japan : H. discus hannai); 2. Mai et al., 1995a (Ireland : H. tuberculata and H. discus hannai); 3. Britz, 1996a, b (South Africa: H. midae); 4. Guzmán
and Viana, 1998 (México: H.fulgens); 5. Bautista-Teruel and Millamena, 1999 (Philippines: H.asinina); 6. Shipton and Britz, 2001(South Africa:H. midae); 7. Sales and Britz,
2002 (South Africa:H. midae); 8. Viana et al, 2002 (Review); 9. Bautista-Teruel et al., 2003 (Philippines: H.asinina); 10. Gómez-Montes et al., 2003 (México: H.fulgens); 11.
Reyes and Fermín., 2003 (Philippines: H.asinina); 12. Thongrod et al., 2003 (Thailand: H. asinina); 13. Naidoo et al., 2006 (South Africa: H. midae); 14. Bautista-Teruel et
al., 2011 (Philippines: H. asinina)

30
 Introduction

1.5.3.4. Energy / carbohydrate and binders sources and content

Abalone consume a natural diet of 40-50% carbohydrate. Thus, high levels of

carbohydrate enhance growth (Thongrod et al., 2003) of abalone which have various
enzymes capable of hydrolysing complex carbohydrates (Fleming et al., 1996) and a
good capacity to synthesize non essential lipids from them. Consequently, energy is
supplied primarily as a carbohydrate, averaging 45% of the diet (Table 6).

Cheap cereal sources, such as wheat and corn flour, soybean meal, maize or rice
starch are frequently used as an energy source. Starches play a major role as both an
energy source and binder in many commercial and tested feeds (Table 6).

Despite the ability of abalone to utilise a wide variety of energy sources, being a
mollusc, the metabolic rate of abalone is low and therefore energy need are low. Caloric
content (gross energy) of the diets is generally around 4 Kcal g-1 (Table 3).

Aquatic animal feeds differ from conventional livestock feeds in that they
require a matrix in which the dietary nutrients are held. An additional requirement of the
feed by a slow feeder, such as abalone, is that water-soluble nutrient remain in the feed
and the food particles remain bound together so that pellets stay intact for at least two
days in water. Undoubtedly, artificial diets and macroalgae are very different in their
physical properties, such as texture and water stability. These properties affect feeding,
digestion, absorption, and subsequently growth of abalone. Thus, water stability is a
significant factor, being responsible for the different performances of aquatic animals
between feeding artificial diet and fresh one (Mai et al., 1995a). Therefore, achieving
this is crucial to the development of a successful feed for abalone (Fleming et al, 1996;
Hernández et al., 2009). The average stability of commercial abalone feeds is about 2-3
days (Fleming et al., 1996). Diets can lose approximately 30-40% of the dry matter
content after being immersed for 48 hours (Maguire, 1996; Bautista- Teruel et al.,
2003).

The most common forms of binder include starches, gluten, alginates or

seaweeds (Table 6). Gels are also used quite frequently in experimental diets, however
they are not seen to be economically viable for commercial feeds (Fleming et al., 1996).

Abalone have a limited ability to digest fiber, despite the presence of cellulases
in the gut. Some artificial diets contain fiber for binding purposes with the level as high

31
 Introduction

as 6% of the dry weight (Fleming et al., 1996; Guzmán and Viana, 1998; Serviere-
Zaragoza et al., 2001; Naidoo et al., 2006).

32
 Introduction

Table 6. Nutritional composition (% dry matter) of artificial diets tested for abalone: Energy / binder sources and content

Nutrient Diet
1 2 3 4 5 6 7 8 9 10 11 12 13 14

Energy / binder source and content

Dextrin 32-43

Wheat (flour / gluten) * 4 20 20 12

Corn ( meal / gluten) * 10 12 16-27

Soybean meal / flour * 15

Starch (maize / rice) * 5 19 0.5-31 26-88 9-43 13-29 14-23 19

Rice bran 3-26 5-20 2-3

Seaweed * 7 9 5 7

Sodium alginate 18 4

Agar 20

Gelatin 5 6 6 5-6 6

Chemical binder 0,5

Total carbohydrate 47 40-50 33-47 47 46-50 28-82 42-44 21-49 31-48 39-47 43

Crude fibre content 01-1.3 5.85 4 1,2

Cellulose 5 4 5 2

Ash 6-16 11 5-11 4-18 13-23 9 4-18 6 15

1. Mai et al., 1995a (Ireland: H. tuberculata and H. discus hannai); 2. Fleming et al., 1996 (review) ; 3. Guzmán and Viana, 1998 (México: H.fulgens); 4. Bautista-Teruel
and Millamena, 1999 (Philippines: H.asinina); 5. Serviere-Zaragoza et al., 2001(México: H.fulgens); 6. Shipton and Britz, 2001(South Africa: H. midae); 7. Sales et al., 2003
(South Africa: H. midae); 8. Bautista-Teruel et al., 2003 (Philippines: H.asinina); 9. Gómez-Montes et al., 2003 (México: H.fulgens); 10. Thongrod et al., 2003 (Thailand: H.
asinina); 11. Reyes and Fermín., 2003 (Philippines: H. asinina); 12. Durazo-Beltrán et al., 2004 (México: H. fulgens); 13. Naidoo et al., 2006 (South Africa: H. midae); 14.
García-Esquivel et al., 2007 (México: H.fulgens)

33
 Introduction

1.5.3.5. Vitamins and minerals

In the absence of information on the requirement by abalone for vitamins and

minerals, Uki et al. (1985a) test diets were based on the requirements for carp and
rainbow trout except choline chloride and vitamin E, added to lipid mixture to maintain
integrity, which were added independently (Table 7). The level of inclusion for the
mineral mix was 4% and for vitamins was 1.5% of the diet.

Most diet formulated since Uki´s experiments have copied these values (Table
8).

Table 7. Vitamin and mineral mixture tested by Uki et al. (1985a)

Vitamin Mixture  Composition Mineral Mixture  Composition

(mg) (Total 100 g)

Thiamine 6 NaCl 1.0

Riboflavin 5 MgSO4-7 H2O 15.0

Pyridoxine HCl 2 NaH2PO4-2H2O 25.0

Niacin 40 KH2PO4 32.0

Ca pantothenate 10 Ca(H2PO4)2.H2O 20.0

Inositol 200 Fe-citrate 2.5

Biotin 0.6 Trace element mixture* 1.0

Folic acid 1.5 Ca-lactate 3.5

PABA 20 Trace element mixture

Menadionine 4 ZnSO4.7H2O 35.3

B12 0.009 MnSO4.4H2O 16.2

Ascorbic acid 200 CuSO4.5H2O 3.1

Vitamin A 5000 U.I. CoCl2.6H2O 0.1

Vitamin D 100 U.I. KIO3 0.3

Cellulose 45

34
 Introduction

Table 8. Nutritional composition (% dry matter) of artificial diets tested for abalone. Ingredient with secondary nutritional contribution

Nutrient Diet
1 2 3 4 5 6 7 8 9 10 11 12 13

Vitamins and minerals

Vitamin mix  1.5 2 2 3 1.7 3 0.4 3 1.5 1.3 1.3

Mineral mix  4 4 6 4 3.4 2 4 2 4 2.9 3.3

Total 5.5 6 1 6 7 5.1 5 4.4 5 5.5 4.2 4.6

Choline chloride 0.5 0.5 0.8 0.11 0.3 0.1 0.11

Alpha-Tocopherol 0.01

Stay C 2 0.35 0.4

Vitamin C 0.1 0.5

Vitamin E 0.1

Sodium benzoate 2 0.23 0.2 0.23

BTH 4 0.05 0.09 0.02 0.08 0.08

Dicalcium phosphate 3 3

Mono calcium phosphate 0.5

Bentonite 15

1. Uki et al., 1985a,b (Japan: H. discus hannai); 2. Mai et al., 1995a (Ireland: H. tuberculata and H. discus hannai); 3. Britz et al., 1997 (South Africa: H. midae); 4. Guzmán and Viana, 1998
(México: H. fulgens); 5. Bautista-Teruel et al., 1999 (Philippines: H. asinina); 6. Serviere-Zaragoza et al., 2001(México: H. fulgens); 7. Reyes and Fermín, 2003 (Philippines: H. asinina); 8.
Sales et al., 2003 (South Africa: H. midae); 9. Thongrod et al., 2003 (Thailand: H. asinina); 10. Vandepeer et al., 2003 (Australia: H. rubra and H. laevigata); 11. Naidoo et al., 2006 (South
Africa: H. midae); 12. García-Esquivel et al., 2007(México: H. fulgens); 13. Viana et al., 2007 (México: H. fulgens)

35
 Introduction

1.5.4. Abalone growth under culture conditions

Review of abalone nutritional studies done worldwide demonstrate that there is

huge variation in growth performance of abalone, particularly in the first year up to 30
mm.
In general, currents research implies that abalone feeds are still under
development with further potential to improve the growth rates, health and quality of
abalone in cultivation systems. Moreover, this review has shown that optimal growth of
abalone is based upon several linked factors, in particular protein source and content,
carbohydrate and lipid levels in feed; water temperature and photoperiod; water quality;
rearing density, age, abalone species specific characteristics or culture system (Table 9).
Hence, to select an appropriate abalone grow-out technology several factors should be
considered.

36
 Introduction

Table 9. Summary of various nutritional studies regarding abalone growth performance during the last three decades of abalone culture development

Proximate composition and Consumption and feed

Growth and survival
energy content utilization efficacy
Author Subject T° Days Species Treatment DGSL WG Survival
P L C E P/E WS FCR FI FCE PER T0 W0 SGR
(μm d-1) (%) (%)
Uki et Casein, 1.2
Diet vs H. discus 28 5 0.1 3.5 93-117 37
al., 30-40 fishmeal 29 3
macroalge hannai
1985a E. biciclys 17 1 0.7 4.3 106 35
A. esculenta 13 4 17 4
L. digitata 6 3 13 12
H. L. saccharina 10 4 18 6
tuberculata P. palmata 17 3 27 12 0.9
U. lactuca 13 6 28 10
Mixed diet 12 4 21 14 95
Mercer Fresh C. crispus 13 4 17 15 53
14 365 19
et al., macroalgae A. esculenta 13 4 17 15
1993
L. digitata 6 3 13 14
L. saccharina 10 4 18 12
H. discus
P. palmata 17 3 27 13 0.8 90
hannai
U. lactuca 13 6 28 11 61
Mixed diet 12 4 21 13
C. crispus 13 4 17
Fishmeal 35 9 47
Artificial
Viana et 21- 1
diets vs 90 H. fulgens Casein 44 13 45
al., 1993 24 0
macroalgae
M. pyrifera 2 1 16
J. lobata 1260 4.0 5.4
L.botryoides 540 -1 -0.6
Fleming, Fresh 10- 150 U. australis 310 2 0.7
H. rubra 40-70 25
1995 Macroalgae 20 350 M.angustifolia 260 1.2 0.3
P.comosa 101 0.1 0.1
E.radiata 50 -28 -1.7

37
 Introduction

Proximate composition and energy Consumption and feed

Growth and survival
content utilization efficacy
Author Subject T° Days Species Treatment DGSL WG
P L C E P/E WS FCR FI FCE PER T0 W0 SGR Survival (%)
(μm d-1) (%)
25 0.6 4 75 0.6 93
25 3 4.2 80 0.8 95
25 5 4.3 80 0.7 95
H. Artificial diets
25 7 4.4 80 0.6 0.7 95
tuberculata
25 9 4.6 80 0.7 97
25 11.6 4.6 80 0.6 95
Mai et P. palmata 18 4 3.7 0.9 97
Lipid levels
al., 25 0.6 4 75 0.6 87
13 100
1995a 25 3 4.2 80 0.9 92
25 5 4.3 80 0.9 98
Atificial diets
H. discus 25 7 4.4 80 0.9 90
0.4
hannai 25 9 4.6 80 0.8 88
25 11.6 4.6 80 0.7 92
3.
P. palmata 18 4 1 98
7
Casein 31 5 0.7 0.5 4.7 0.6 45
Fishmeal 29 5 0.8 0.8 6.5 0.8 58
Soya oil cake 32 5 1 0.6 3.4 0.6 41
Protein
Britz, Spirulina spp. 19 5 0.8 0.8 3.9 0.8 65
sources; 19 124 H. midae 21 1.8
1996b macroalgae Torula yeast 29 5 1 0.7 3.3 0.6 42
P. corallorhiza 20 2.8 1.3 2.2 0.4 29
Dried E.
10 3.4 2.8 3 0.6 54
maxima
H. midae 34 6 3.6 1.4 9.3 3.6 2.2 103
Britz and Protein; 72 10 0.2
small Starch, fish, 44 6 3.6 1.2 9.4 2.3 2.1 108
Hetch, Energy 18
H. midae cellulose 44 10 4 1.4 34 2.2 0.3 43
1997 ratio; Size 142 36 7.3
large 44 6 3.6 1.2 35 2.5 0.5 58
Guzmán Fish meal 39 6 41 86 1 1.1 1 65
and Fish meal 22- Viscera
179 H. fulgens 36 8 40 76 1.6 1.8 7 1.1 71
Viana, replacemnt 15 soybean meal
1998 Abfeed 38 6 48 90 0.9 1.0 0.8 49
Fish meal 22 6 47 3.2 2.3 2.2 0.5 222 252 85
Bautista- Protein / 27- Shrimp meal 28 6 40 3.1 1.8 2.3 0.7 244 307 95
Teruel et Energy 90 H. asinina Soybean meal 31 6 33 3 1.5 2.5 16 0.7 0.8 248 347 85
31
al., 1999 levels Garcilariopsis
17 0.5 35 2.2 7 0.1 0.06 135 134 85
bailinae

38
 Introduction

Proximate composition and Energy Consumption and feed

Growth and survival
Author Subject T° Days Species Treatment content utilization efficacy
DGSL WG Surv.
P L C E P/E WS FCR FI FCE PER T0 W0 SGR
(μm d-1) (%) (%)
Eisenia
7.6 52 0.27 19 93
arborea
Macrocystis
Serviere- 12 63 0.7 46 93
Macroalgae pyrifera
Zaragoza Gelidium
and artificial 20 106 H. fulgens 17 0.4
et al., 18 33 0.32 23 89
diet robustum
2001
Phyyospadix
16 30 0.45 25 95
torreyi
Artificial diet 36 59 0.65 42 97
Carica papaya 25 5 41 3 6.0 40 14.7 1.8 71 59 80
Reyes Leucaena
Terresterial 25 5 47 3 13 37 14.7 0.9 47 28 100
and leucocephala
protein 28- Moringa
Fermín, 120 H. asinina 25 5 42 3 4 38 11.4 1.9 86 90 95
sources 30
2003 oliefera
Azolla pinnata 25 5 39 3 5 39 14 1.9 76 75 90
G. bailoinae 13 1 38 38 40 15.3 2.1 61 84 90
Fish meal 28 3 43 3 1 3.9 0.8 400 95
Bautista- Animal and Shrimp meal 27 3 42 3 1 4.2 0.9 454 95
28-
Teruel et plant protein 90 H. asinina 11 0.7
31 Soybean meal 28 3 44 3 1 4.1 0.7 326 85
al., 2003 sources
Sprirulina 27 3 42 3 1 4.6 0.9 421 85
26 6 1.6 4 62 1.6 76.4 3 1.3 61
Gómez- Fish, soybean, 31 6 1.4 4 74 1.4 93 3 1.5 7
Protein / 21 60
Montes et H. fulgens kelp, modified 35 6 1.4 4 85 1.4 110 3.1 12 0.2 1.9 92
Energy level
al., 2003 corn,starch 40 7 1.5 4 100 1.5 138 3.4 2.4 123
44 7 1.6 4 108 1.6 120 2.7 2.5 123
Soybean, 38 1 48 4.2 1.3 779 81
Thongrod Lipid / starch, 38 6 43 4.5 1.4 615 93
27- 11-
et al., 196 H. asinina Spirulina spp., 37 10 39 4.7 1.9 0.9 353 90
carbohydrate 30 12
2003 fish oil,
36 15 36 4.9 3.2 223 75
Gracilaria
37 19 31 5.2 7.4 97 76
Durazo- Kelp; soybean; 0.1 99.4 0.9 29 29
Beltrán et Lipid levels 20 60 H. fulgens fish silage & 39 4 36 6.4
al., 2004 protein 3 95.9 0.7 40 30
H. P. palmata 21 42 4.3 3.5 0.7 230
tuberculata P. palmata + 23 2
Legrand, 7- large 24 41 5.6 3.6 0.6
Macroalgae 183 U. lactuca
2005 17
P. palmata 21 42 2 3.7 1.3
H. t. small 10 0.1
P. p+ U. l. 24 41 3.9 5.8 1.2

39
 Introduction

Proximate composition and energy Consumption and feed

Growth and survival
content utilization efficacy
Author Subject T° Days Species Treatment DGSL WG Surv.
P L C E P/E WS FCR FI FCE PER T0 W0 SGR
(μm d-1) (%) (%)
Enriched U.
rigida + E. G. 66
gracilis+kelp
E. maxima +
60
Epiphiyte
Seaweed- Rotation 58
based diets;
Ecklonia
Macroalgae; 55
Naidoo maxima
Enriched
et al., macroalgae; 270 H. midae E. maxima + 35 7.5 53
2006 commercial Abfeed
feed Abfeed 35 5 45 49
Dried kelp
34
blade
Dried kelp
32
stipe
Dried Kelp
29
pellets
Fish, soybean,
kelp
20ºC / 00:24 0.8 3 109
García-
Esquivel Temperature 20- 20ºC / 12:12 0.9 2.6 82
180 H. fulgens 44 4 31 3.7
et al., Photoperiod 25
20ºC / 24:00 0.9 2.8 72
2007
25ºC/ 00:24 4 1.7 69
25ºC/ 12:12 1.4 1.8 55
25ºC/ 24:00 1.6 1.5 38
Formulated
44 6.7 34 4.5 96 33 1.8 50 471 85
diet
Formulated M. pyrifera 27 2.8 40 3.5 78 94 2.5 110 1004 90
Hernán-
diet; fresh
dez et 15 90 H. rufescens 6.6 0.06
macroalgae; P. columbina 12 0.6 45 2.6 44 93 1.8 70 548 93
al., 2009 mixed diet
P. columbina
+ formulated 2.3 87 728 80
diet

40
 Introduction

Proximate composition and energy Consumption and feed

Growth and survival
content utilization efficacy
Author Subject T° Days Species Treatment DGSL WG Surv.
P L C E P/E WS FCR FI FCE PER T0 W0 SGR
(μm d-1) (%) (%)
L. japonica 6.5 2.3 22 100
Seaweeds
suitability. G.
4.2 2.8 21 100
lemaneiformis
Qi et al., 10- H. discus S. pallidum 4.4 2.5 18 100
Long-line 120 76 62
2010 23 hannai
seaweed- L. j. + G. l. 6 2.3 26 100
abalone
culture L. j. + S. p. 5.8 1.8 20 100

34 2.8 4.5 1 0.4 3.2 0.7 43 100

36 5.3 4.6 100
H. midae
Lipid levels, 38 8.7 4.8 25 2.6 100
small
constant P/E 39 12.5 5 100
Fishmeal,
Green et ratio. 36 16.1 5.1 1.8 0.5 1.3 0.5 67 100
18 84 casein, kelp,
al., 2011 34 2.8 4.5 2 0.2 1.9 0.2 17 99
starch, fish oil
Recirculated 36 5.3 4.6 97
system H. midae
38 8.7 4.8 65 50 97
large
39 12.5 5 96
36 16.1 5.1 10 0.3 0.7 0.1 37 100
Cage Novel 61 45 78 >87.5
culture. Submerged 55 37 77 >87.5
cages >87.5
Wu and 48 32 63
Stocking 11- H. discus
Zhang, 180
density 21 hannai 61 45 64 >87.5
2013 Traditional
Multi Tier 55 37 60 >87.5
Size >87.5
baskets 48 32 53

FI= Feed intake expressed by % BW day-1 (Jackson, 2001) or mg abalone day-1 (Fleming, 1995); SGR = Specific growth rate; FCR=Food conversion ratio
expressed; FCE= Feed conversion efficiency (%); PER = Protein efficiency ratio; DGSL= daily growth rate in shell length, WG=Weight gain; E= gross energy
(Kcalg-1); P/E= protein: energy ratio; WS= Water stability (Guzmán and Viana, 1998; Hernández et al., 2009 (12 h inmersion); Mai, 1995a (48 h inmersion);

41
 Introduction

1.6. INTEGRATED MULTI-TROPHIC AQUACULTURE (IMTA)

1.6.1. General aspects

Rising global demand for seafood and declining catches have resulted in the
volume of mariculture doubling each decade, a growth expected to persist in the
decades to come (FAO, 2012). Such industry, whether semi-intensive or intensive,
thus release organic materials mainly as unconsumed feed, but also as undigested feed
residues and inorganic nutrient excretions (Msuya et al., 2006). Ammonium and
suspended solids are known to be the most significant polluting components of
aquaculture effluents (Tovar et al., 2000). If released directly to the sea, the organic
and nutrient loads cause eutrophication of the marine environment, which affects the
naturally growing marine organisms. In addition, a mariculture operation itself can be
affected by upstream impact of environmental degradation (Neori et al., 2004).

Awareness by scientists, industry, the public and politicians is that such

technologies with uncontrolled impact are no longer considered sustainable (Costa-
Pierce, 1996; Sorgeloos, 1999; Naylor et al., 2000; Chopin et al., 2001). Thus, the
treatment of aquaculture waters and the mitigation of the potential environmental
impacts of aquaculture is needed, and one way of achieving this goal is through the
Integrated Multi-Trophic Aquaculture (IMTA), where the excretion of one organism
in such system often supply food for another (Gordin et al., 1981; Edwards et al.,
1988). In addition, IMTA can be considered as a profitable environmental
management strategy (EMS).

The concept of IMTA is not only confined to open-water, marine systems

using finfish for the fed component and seaweeds and invertebrates for the extractive
component, but can also be applied to land-based, closed-containment and even
freshwater systems. What is important is that the appropriate co-cultured organisms
are chosen at multiple trophic levels based on their complementary functions, as well
as on their economic value (Chopin et al., 2012). Thus, today´s IMTA approaches,
integrate the culture of fish or shrimp with vegetables, microalgae, shellfish and/or
seaweeds, and can take place in coastal waters or in ponds and can be highly
intensified (Neori et al., 2004; Zhou et al., 2006; Cunha et al., 2012; Liping et al.,
2012).

42
 Introduction

1.6.2. Seaweed-based integrated mariculture

A primary role of biofiltration in shrimp/fish aquaculture is the treatment by

uptake and conversion of toxic metabolites and pollutants. Bacterial biofilters oxidize
ammonia to the much less toxic but equal polluting nitrate (Touchtte and Burkholder,
2000), while microalgae photosynthetically convert the dissolved inorganic nutrients
into particulate “nutrient packs” (Troell and Norberg, 1998), that are still suspended in
the water. In contrast, macroalgae not only can efficiently remove all forms of
inorganic nitrogenous waste products (Ryther et al., 1975; MacDonald, 1987;
Vandermeulen and Gordin, 1990; Cohen and Neori, 1991; Buschmann et al., 1994;
Demetropoulos and Langdon, 2004; Zhou et al., 2006), but also function as an oxygen
producer that provides oxygen for the animals (Schuenhoff et al., 2003; Neori et al.,
2003). The nutrients act as a fertilizer for the algae and a yield of useful biomass is
increased (Cohen and Neori, 1991; Muir, 1996; Neori et al., 1996, 2000; Shpigel and
Neori, 1996). Moreover, algae, and in particular seaweeds, are most suitable for
biofiltration because they probably have the highest productivity of all plants and can
be economically cultured (Gao and McKinley, 1994). Besides, seaweeds growth on
mariculture effluents has been also shown to be superior to that on fertilizer-enriched
clean seawater (Harlin et al., 1978; Vandermeulen and Gordin, 1990; Neori et al.,
1991; Viera et al., 2006, 2009b).

The choice of seaweed species for inclusion in an IMTA must first depend
upon meeting a number of basic criteria: high growth rate and tissue nitrogen
concentration; easy of cultivation and control of life cycle; resistance to epiphytes and
disease-causing organisms; and a match between the ecophysiological characteristics
and the growth environment. In addition, given the ecological damage that may result
from the introduction of nonnative organism, the seaweed should be a local species.
Beyond these basic criteria, the seaweed intended application (yield or
bioremediation) should also be taken into account, the optimal system including
seaweed with both applications (Neori et al., 2004). Besides, the market value of the
harvested biomass should be also considered (Buschmann et al., 1996).

Thus, the seaweed genera most common in mariculture biofiltration are Ulva:
U. lactuca (Cohen and Neori, 1991; Neori et al., 1991, 2000, 2003; Shpigel et al.,
1993; Schuenhoff et al., 2003; Vandermeulen and Gordin, 1990, Naidoo et al., 2006;

43
 Introduction

Robertson-Andersson et al., 2011; Ben-Ari et al., 2012), U. reticulata (Msuya et al.,

2006), U. rigida (Jiménez del Río et al., 1994, 1996; García, 1999; Toledo et al.,
2000; Viera et al., 2006, 2009b; Izquierdo et al., 2013) and Gracilaria: G.
lemaneiformis (Fei et al., 2000, 2002; Fei, 2004; Zhou et al., 2006; Yongjian et al.,
2008; Mao et al., 2009), G. chilensis (Buschmann et al., 1994, 1995, 1996, 2001;
Chow et al., 2001; Marquardt et al., 2010), G. changii (Phang et al., 1996), G.
parvispora (Nelson et al., 2001; Nagler et al., 2003), G. tenuistipitata (Haglund and
Pedersen, 1993), G. gracilis (Anderson et al., 1999; Njobeni, 2005; Hansen et al.,
2006; Naidoo et al., 2006), G. textorii (Pang et al., 2006), G.lichenoides (Xu et al.,
2008) or G. cornea (Viera et al., 2006, 2009b; Izquierdo et al., 2013). However,
despite Ulva industrial culture technology and nutrient uptake capacity are among the
highest known (Marínez-Aragón et al., 2002), there is a low commercial value for its
harvested biomass, in contrast, Gracilaria species further produce commercially
valuable bio-products such as agar-agar (Neori et al., 2004), though maximal growth
rates of these fleshy morphotypes are typically less than the flat sheets (Marinho-
Soriano et al., 2002; Nagler et al., 2003).

Other marketable genera such as Porphyra (Chopin et al., 1999; Carmona et

al., 2001; Fei, 2001, Yarish et al., 2001), Palmaria (Demetropoulos and Langdon,
2004; Matos et al., 2006), Hypnea (Langton et al., 1977; Viera et al., 2009b) or kelps
(Laminaria and Macrocystis) have also been successfully integrated into IMTA
systems (Chopin and Bastarache, 2002; Buschmann et al., 2008).

1.6.3. Fish - seaweed - abalone integrated culture system

The by-production of high quality seaweeds in the biofilters calls for the co-
culture of other high-valued marine macroalgivores, such as abalone. Already in the
1970s, Tenore (1976) published his pioneer study on the seaweed-abalone integrated
culture (Fig. 13). This was followed by the integrated abalone - Ulva and Gracilaria
systems developed in Israel (Shpigel et al., 1993, 1996a, b, 1999 ; Shpigel and Neori,
1996; Neori et al., 1998, 2000), of abalone and green algae in Japan (Sakai and Hirata,
2000), and of Palmaria – abalone in the USA (Evans and Langdon, 2000). All those
studies reported that abalone efficiently grow-out when fed with biofilter produced
macroalgae, probably related to its high protein content due to its production under

44
 Introduction

the high nitrogen culture conditions of the IMTA system (Neori et al., 1998; Shpigel
et al., 1999; Boarder and Shigel, 2001; Naidoo et al., 2006; Robertson-Andersson et
al., 2011).

Figure 13. Fish-seaweed-abalone integrated culture system.

45
Development of a Sustainable Grow-out Technology for Abalone
Haliotis tuberculata coccinea (Reeve) as a New Species for
Aquaculture Diversification in the Canary Islands

OBJECTIVES
 Objectives

2. OBJECTIVES

The overall aim of this thesis was “to develop grow-out technology for the
local abalone species, Haliotis tuberculata coccinea“, considered a new candidate
for Canarian aquaculture diversification. More specifically, algal and artificial diets
suitability, growth and survival, as well as various factors affecting the on-growing
success, were addressed in this thesis. Besides, this general objective was undertaken
through an environmental approach so as to maximize the sustainability of the
abalone production methods to be developed.

For that purpose, four different studies were undertaken:

Study I. Suitability of three red macroalgae as a feed for the abalone

Haliotis tuberculata coccinea Reeve.

This study aims to evaluate the suitability of three red macroalgae species
from genus reported to induce growth in other abalone species, as a potential feed for
the grow-out culture of juvenile Canarian abalone. Since the biomass of locally
harvested macroalgae is insufficient to sustain the commercial production of abalone,
experimental macroalgae were reared in an Integrated Multi-Trophic Culture System
(IMTA). This objective was addressed to identify suitable algal diets to promote high
growth and survival for this abalone species, and to investigate the dietary value of
the biofilter produced seaweeds. A two month feeding trial was conducted to achieve
this objective.

Study II. Comparative performances of juvenile abalone (Haliotis

tuberculata coccinea Reeve) fed enriched vs non-enriched macroalgae: Effect on
growth and body composition.

This study aims to evaluate the effect of several non-enriched versus farm-
grown green and red monoespecific and mixed macroalgae diets on the growth of
juvenile Canarian abalone. This objective was addressed in order to, not only find a
suitable feed to grow the animals, but to further evaluate the advantages of co-
culturing macroalgae alongside H. tuberculata coccinea in integrated aquaculture

46
 Objectives

systems, that will allow the sustainability of the future abalone production while
improving abalone growth performance. A three month feeding trial was conducted to
achieve this objective.

Study III. First development of various vegetable-based diets and their

suitability for abalone Haliotis tuberculata coccinea Reeve.

This study focused on the development and evaluation of several vegetal-

based formulated feeds for the culture of abalone Haliotis tuberculata coccinea, with
special emphasis on the determination of the suitability, as potential feed ingredients,
of the four species of macroalgae most commonly involved in European abalone
production. This objective was addressed in order to obtain fishmeal-free formulated
diets, adapted to abalone nutritional requirements that could provide the industry with
a readily available and more stable nutritional feed, while validating the
environmental and social sustainability of the farming operations. A six month
experiment was performed to achieve this objective.

Study IV. Grow out culture of abalone Haliotis tuberculata coccinea

Reeve, fed land-based IMTA produced macroalgae, in a combined fish/abalone
offshore mariculture system: effect of stocking density.

This study aims to describe the effect of stocking density, one of the key
important factors for growing abalone to market size, on growth and survival of two
different size groups of Canarian abalone. This objective was addressed in order to
identify certain culture conditions that improve abalone growth performance and
evaluate the potential of sea-based abalone farming during the final grow-out culture
phase. A six month offshore feeding experiment was performed to achieve this
objective.

47
 Material and Methods

Development of a Sustainable Grow-out Technology for Abalone

Haliotis tuberculata coccinea (Reeve) as a New Species for
Aquaculture Diversification in the Canary Islands

MATERIALS AND METHODS

 Material and Methods

3. MATERIALS AND METHODS

3.1. LOCATION AND GENERAL FACILITIES

The studies were carried out at the culture facilities of the Aquaculture
Research Group (GIA- ULPGC) in the Canarian Institute of Marine Sciences (ICCM),
located in Melenara, Telde, province of Las Palmas (Canary Islands, Spain), with a
geographic situation of 27º59’31’’N and 15º22’31’’W (Fig. 14).

Figure 14. Top view of the Canary Islands and location of the GIA marine culture facilities.

Abalone culture facilities consist in: a broodstock conditioning (Fig. 15-A),

spawning induction and larval rearing area (Fig. 15-B); area for post-larvae and
diatoms production (Fig. 15-C, D); grow out (Fig. 15-E) and feeding trial area (Fig.
15-F); and biofiltering units recycling fish tanks effluents for macroalgae production
(Fig. 15-G, H).

Both, ICCM abalone facilities and studies have been financed and developed
in the frame of several Canarian (PI 2007/034), Spanish (JACUMAR Oreja de mar,
2005/07; JACUMAR Multitrófico, TR 2003/08) and European projects
(SUDEVAB: FP 7-SME-2007-1/BSG-SME).

48
 Material and Methods

Figure 15. Brood-stock conditioning (A); larval rearing facilities (B); abalone nursery (C); B
diatoms production zone (D); grow out zone (E); feeding trials area (F) and outdoor
macroalgae culture systems (G, H).

49
 Material and Methods

3.2. ABALONE PRODUCTION B

3.2.1. Brood-stock conditioning and selection

Captive Haliotis tuberculata coccinea brood-stock were kept under natural

photoperiod conditions and ambient seawater temperature, in shaded 60-l tanks, in the
flow through broodstock conditioning system (Fig. 15-A). Inside each of the 24
broodstock holding tanks, 10-15 animals were kept under PVC tiles that provide them
shelter, and were separated from debris, on the tank’s bottom, through a perforated
divider. Males and females, differentiated by the colour of their gonads (creamy white
for the males and dark grey to violet in the females) (Fig. 16), were maintained in
separate holding tanks. Brood-stock was fed twice a week with a mix diet of Ulva
rigida and Gracilaria cornea produced in the biofiltering units. Abalone selected to
be induced to spawn were the ones showing mature gonads (GSI) in stage 3 or
between stage 2 and 3 (Ebert and Houk, 1984).

Figure 16. Female (A) and male (B) H. tuberculata coccinea in stage 2-3 of the gonad index.

3.2.2. Spawning induction

Mature males and females, with a male to female ratio of 1:2, were induced to
spawn, separately by sex, into spawning containers filled with 1-µm cartridge filtered
and UV sterilized seawater. Gametes from different sex were obtained separately in
order to control the ratio of gametes employed during fertilization (Fig. 17). During

50
 Material and Methods

spawning induction the containers were left in the dark. Two spawning induction
methods were employed to carry out these studies, the hydrogen peroxide one (Morse
et al., 1977) and the ultraviolet spawning induction method (Kikuchi and Uki, 1974).

Figure 17. Gametes expulsion from females (A) and males (B). B

3.2.3. Fertilization

Once the gametes were obtained, the released oocytes, which are negatively
buoyant, were siphoned fromA the spawning containers and passed through a 300 µm
mesh screen to retain faeces or other debris. The oocytes were collected in 10-l
containers and fertilized with a final sperm concentration of 105/ml during 30
minutes. After that, eggs were rinsed with fresh seawater to remove excess sperm and
fertilization rates were determined by recording the proportion of eggs showing
dividing cells 1 h after fertilization under the dissecting microscope (Mod. SL
260004, Optech, Duisburg, Germany) (Fig. 18). Fertilized eggs, were transferred to
the larval rearing tanks (Fig 15-B).

Figure 18. Eggs are rinse to remove excess sperm. B

51
 Material and Methods

3.2.4. Larval culture

Larval stages begins at the trocophore stage and is completed with the
formation of the fourth tubule on the cephalic tentacles, although larvae are
considered ready for settlement when the third tubule appears and larvae start to
explore the surface (Fig. 19).

Larvae were reared under natural photoperiod and ambient temperature, in a

flow through system in 100-l larval tanks at a density of 10-20 larvae/ml. Water
supplied was filtered through 1 µm cartridge and sterilized bay UV irradiation. The
oligotrophic larvae remained unfed until they are transferred for settlement to the
nursery tanks (Fig. 15-C). Settlement competency was observed between 62-72 hours
post-fertilization depending on the larval rearing temperature recorded throughout the
different abalone batches required for the feeding trials.

Figure 19. Hatching out trochophore larvae equipped with cilia (A), third tubule appearance
on cephalic tentacles (B) (Courtois de ViÇose et al., 2007).

3.2.5. Larval settlement

Settlement induction of H. tuberculata coccinea was performed using vertical

settlement plates located within baskets inside the 2500-l settlement tanks (Fig. 20).

52
 Material and Methods

Figure 20. Vertical settlement plates.

To induce the larval settlement, various substrates were used to colonize the
settlement plates: crustose coralline algae, benthic diatoms biofilms and spores of the
green algae Ulvella lens.

3.2.6. Post-larval and juvenile culture

Abalone post-larvae were fed four species of diatoms, Navicula incerta,

Proschkinia sp., Nitzschia sp. and Amphora sp. (Fig. 21). Diatoms were cultured in 40
l horizontally laid algal bags, at initial inoculums of 105 cells/ ml and grown for 5 days
in f/2 medium supplemented with silicate (1 mgl-1) (Guillard, 1975) at ambient
temperature and under continuous light of 62±8µmol photons m-2 s-1.

Figure 21. Diatoms species fed to the abalone post- larvae: Proschkinia sp. (A), Navicula
incerta (B), Amphora sp. (C) and Nitzschia sp. (D) (Courtois de ViÇose et al., 2012b).

53
 Material and Methods

Animals maintained this diets for 4-5 months, when juvenile abalone were
gradually switched to the green macroalgae Ulva rigida (Study I); Ulva rigida,
Hypnea spinella and Gracilaria cornea (Study II); or Ulva rigida and Gracilaria
cornea (Study III and IV), prior to the beginning of the feeding trials.

3.3. ALGAL CULTURE B

3.3.1. Macroalge species

Four fresh macroalgae species were used throughout the different abalone
feeding trials, the Rodophyta species: Gracilaria cornea J. Agardh, Hypnea spinella
(C. Agardh) Kützing and Hypnea musciformis (Wulfen) J.V. Lamoroux; and the
Chlorophyta Ulva rigida J. Agardh. Besides, the Rodophyta Palmaria palmata (L.)
Weber and Mohr, the Phaeophyta Laminaria digitata (Hudson) Lamoroux and the
Chlorophyta Ulva lactuca Linnaeus, were tested as ingredients in artificial diets
(Study III).

Macroalgae species were chosen according to their suitability to be included in

an IMTA as biofilters (Neori et al., 2004) as well as to be used as food for abalone
(Boarder and Shigel, 2001; Naidoo et al., 2006; Watson and Dring, 2011).

The most important characteristics of this species are shown in the following
Tables 10-16.

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 Material and Methods

Tables 10-16. Summary of experimental macroalgae characteristics

Hypnea spinella (C. Agardh) Kützing, 1847

Taxonomic Kingdom Plantae

classification Phylum Rhodophyta
Class Florideophyceae
Order Gigartinales
Family Cystocloniaceae
Genus Hypnea
Morphological Plant lax, from 3-30 cm long (most less than 15 cm) in tangled, bushy
characteristics clumps. Axes extend through entire length, but main branches absent,
dichotomous branching throughout. Axils rounded, with side
branches growing almost horizontally for 1-5 mm before bending up
or curling downward. Commonly yellowish but deep red when
shaded.

Habitat Lower intertidal and sub-littoral rocks.

Reproduction Asexual reproduction predominates over sexual reproduction.

Geographical Widely distributed in temperate (especially warm) seas.

distribution

Uses and compounds Edible species

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 Material and Methods

Hypnea musciformis (Wulfen) J.V. Lamoroux,

1853

Taxnomic Kingdom Plantae

classification Phylum Rhodophyta
Class Florideophyceae
Order Gigartinales
Family Cystocloniaceae
Genus Hypnea
Morphological Clumps or masses of loosely intertwined, cylindrical branches, 10 - 20
characteristics cm tall, 0.5 - 1.0 cm diameter, that become progressively more slender
towards tips. Firm, cartilaginous, highly branched. Branching is
variable and irregular, often tendril-like and twisted around axes of
other algae. The ends of many axes and branches are flattened with
broad hooks. Holdfasts are small, inconspicuous, or lacking. Usually
red, but can be yellowish brown in high light environments or nutrient
poor waters.

Habitat Common on calm intertidal and shallow subtidal reef flats, tidepools
and on rocky intertidal benches. Frequently epiphytic on reef algae
such as Sargassum echinocarpum and Acanthophora spicifera. In
bloom stage, may be found free-floating.

Reproduction Asexual reproduction predominates over sexual reproduction. There

are more vegetative than reproductive thalli under environmentally
stressful conditions for growth. Able to propagate vegetatively in all
size classes, with the greatest success observed in the smallest
fragments.

Geographical Worldwide: Mediterranean, Philippines, Indian Ocean, Caribbean to

distribution Uruguay.

Uses and compounds Biological, medical and pharmaceutical activity (antihelminthic).

Source of kappa carrageenan.

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 Material and Methods

Gracilaria cornea J. Agardh, 1852

Taxonomic Kingdom Plantae

classification Phylum Rhodophyta
Class Florideophyceae
Order Gracilariales
Family Gracilariaceae
Genus Gracilaria

Morphological Thallus erect, solitary or caespitose, cylindrical throughout,

characteristics arising from a small discoid holdfast. Purplish brown to dark
brown, sometimes greenish or yellowish; branches irregularly
alternated.

Habitat Can be found in protected, quiescent bays, as well as in high

energy coastline habitats. Grows free or attached to rocks or
other substrata.

Reproduction Sporophytes and gametophytes occur alternately in its life cycle.

Geographical Mainly warm-water specie. South America: Mexico, Brazil,

distribution Venezuela; Africa: Tanzania.

Uses and compounds Considerable economic importance as a source of food for both
humans and shellfish (abalone), playing a major role in the
production of agar and other hydrocolloids.

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 Material and Methods

Palmaria palmata, (L.) Weber & Mohr, 1805

Taxonomic Kingdom Plantae

classification Phylum Rhodophyta
Class Florideophyceae
Order Palmariales
Family Palmariaceae
Genus Palmaria
Morphological Dulse grows attached by its discoid holdfast to rocks. It has a
characteristics short stipe, the fronds are variable and vary in colour from deep-rose to
reddish-purple and are rather leathery in texture. The flat foliose blade
gradually expands and divides into broad segments ranging in size to
50 cm long and 3 cm–8 cm in width which can bear flat wedge-shaped
proliferations from the edge.

Habitat Easily found from mid-tide of the intertidal zone to depths of 20 m or

more in sheltered and exposed shores.

Reproduction Tetraspores occur in scattered reproductive tissue (sori) on the mature

blade, which is diploid. Spermatial sori occur scattered over most of the
frond of the haploid male plant. The female gametophyte is very small
stunted or encrusted, the carpogonia apparently occurring as single cells
in the young plants. The male plants are blade-like and produce
spermatia which fertilize the carpogonia of the female crust. After
fertilization the diploid plant overgrows the female plant and develops
into the tetrasporangial diploid phase attached to the female
gametophyte. The adult foliose tetrasporophyte produces tetraspores
meiotically. It is therefore usually the diploid tetrasporic phase or the
male plant which is to be found on the shore.

Geographical In Atlantic Europe from Portugal to the Balitc coasts, coasts of Iceland
distribution and the Faroe Islands. Shores of Arctic Russia, Arctic Canada, Atlantic
Canada, Alaska, Japan and Korea .

Uses and Food: additive, ground whole tissue; cosmetics; wellness and folk medicine;
compounds Biological, medical and pharmacological activity (antihelminthic). Animal
production (feed). Mineral supplement.

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 Material and Methods

Laminaria digitata (Hudson) Lamoroux, 1813

Taxonomic Kingdom Plantae

classification Phylum Heterokontophyta
Class Phaeophyceae
Order Laminariales
Family Laminariaceae
Genus Laminaria
Morphological Tough, leathery, dark brown seaweed that grows to two or three
characteristics metres. The holdfast which anchors it to the rock is conical and has
a number of spreading root-like protrusions rhizoids. The stipe or
stalk is flexible and oval in cross section. The blade is large and
shaped like the palm of a hand with a number of more or less
regular finger-like segments.

Habitat Is found mostly on exposed sites on shores in the lower littoral

where it may form extensive meadows. It has a high growth rate
(5.5% per day), and a carrying capacity of about 40 kg ww/m2. It
may reach lengths of about 4 m.

Reproduction Can be fertile throughout the year but highest in spring – April,
May onwards into summer, and lowest in Winter. Reproductive
tissue (sorus) appears as raised and darkened patches found on the
blades. Mature sorus, ready to release zoospores, is well raised from
the blade and several shades darker brown.

Geographical North west Atlantic from Greenland south to Cape Cod and in the
distribution north east Atlantic from northern Russia and Iceland south
to France. It is common round the coasts of the British Isles except
for much of the east coast of England.

Uses and Fertiliser; extraction of alginic acid; cosmetic; food industry and
compounds human and animal nutrition.

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 Material and Methods

Ulva rigida J. Agardh, 1823

Taxonomic Kingdom Plantae

classification Phylum Chlorophyta
Class Ulvophyceae
Order Ulvales
Family Ulvaceae
Genus Ulva
Species rigida

Morphological Bright green to dark green algae, gold at margins when

characteristics reproductive (may be colorless when stressed). Thin thallus,
sheet-like, variable in shape, up to 10-cm long. Blades ruffled
or flat, with small microscopic teeth on the margins. The
blades are two cells thick; the two layers easily separate into
single cell layers. Holdfasts consisting of small, tough rhizoids.

Habitat Commonly found on intertidal rocks, in tidepools, and on reef

flats. Often abundant in areas of fresh water runoff high in
nutrients such as near the mouths of streams and run-off pipes.
Also found in areas where nutrients are high, wave forces low
and herbivory reduced; it is tolerant to stressful conditions, and
its presence often indicates freshwater input or pollution.

Reproduction General for group: asexual reproduction may be by fission

(splitting), fragmentation or by zoospores (motile spores).
Sexual reproduction may be isogamous (gametes both motile
and same size); anisogamous (both motile and different sizes-
female bigger) or oogamous (female non-motile and egglike;
male motile)

Geographical Worldwide distribution

distribution
Uses and compounds Human and animal nutrition

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 Material and Methods

Ulva lactuca Linneaus, 1753

Taxonomic Kingdom Plantae

classification Phylum Chlorophyta
Class Ulvophyceae
Order Ulvales
Family Ulvaceae
Genus Ulva
Species lactuca

Morphological Thin flat alga growing from a discoid holdfast. The margin is
characteristics somewhat ruffled and often torn. It may reach 18 centimetres
(7.1 in) or more in length, though generally much less, and up
to 30 centimetres (12 in) across. The membrane is two cells
thick, soft and translucent, and grows attached, without astipe,
to rocks or other algae by a small disc-shaped holdfast. Green
to dark green in color, this species in the Chlorophyta is formed
of two layers of cells irregularly arranged, as seen in cross
section. The chloroplast is cup-shaped with 1 to 3 pyrenoids.

Habitat It is particularly prolific in areas where nutrients are abundant

Reproduction The sporangial and gametangial thalli are morphologically

alike. The diploid adult plant produces haploid zoospores by
meiosis, these settle and grow to form haploid male and female
plants similar to the diploid plants. When these haploid plants
release gametes they unite to produce the zygote which
germinates, and grows to produce the diploid plant.

Geographical Worldwide distribution

distribution
Uses and compounds Crop and biofilter of fishpond effluents

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 Material and Methods

3.3.2. Culture system: Integrated Multi-Trophic culture System (IMTA)

Except with the non enriched treatments (Study II), all experimental fresh
macroalgae were grown in a flow-through fish-seaweed integrated culture system.

In Studies I and II, algae were cultured at the Centro de Biotecnología Marina
(CBM-ULPGC, Gran Canaria, Spain) IMTA. Effluents were channelled from the
fishponds (Fig. 22-A) to a 11 m3 sedimentation pond for the removal of suspended
particles and then, pumped at a flow rate of 10 m3 h-1 to the seaweed tanks located in
a greenhouse, where maximum irradiance was approximately of 1600 µmol photons
m-2 s-1. Semi-circular fiberglass tanks with a surface of 1.8 m2 and a volume of 0.75
m3 were provided aeration through a bottom-central linear pipeline and were
employed for the cultivation of macroalgae (Fig. 22- B). In Study I, algal stocking
density for G. cornea, H. spinella and H. musciformis was 6 g l-1, whereas in Study
II, algal stocking density were 1, 3 and 4 g l-1 for U. rigida, H. spinella and G. cornea,
respectively. Water exchange rate in the seaweed culture tanks was 4 vol day-1 and
TAN (total ammonia nitrogen) inflow into the biofilter ranged between 10 and 400
µM.

Figure 22. Fishponds and semi-circular tanks for the cultivation of macroalgae (CBM-
ULPGC).

Regarding Studies III and IV, U. rigida and G. cornea were grown in the
Grupo de Investigación en Acuicultura (GIA, Canary Islands, Spain) aquaculture
research facility, in a flow-through integrated system collecting wastewater from fish
and abalone ponds in a macroalgal biofilter (Fig. 23). Effluents were channelled from

62
 Material and Methods

the land-based facility tanks to a 11 m3 sedimentation pond for the removal of

suspended particles and then, pumped at a flow rate of 10 m3 h-1 to the seaweed tanks
located outdoor, where maximum irradiance was close to 1600 µmol photons m-2 s-1.
Circular plastic tanks with a volume of 1.5 m3 and aeration supplied by a bottom-
circular pipeline were used for the cultivation of macroalgae. Algal stocking densities
were 1 and 4 g l-1 for U. rigida and G. cornea, respectively. Water exchange rate in
the seaweed tanks was 12 vol day-1 and total ammonia nitrogen inflow into the
biofilter ranged between 10 and 30 µmoles.

Figure 23. Diagram of the IMTA (ICCM-ULPGC).

Each algal type was grown in triplicates with fortnightly seaweed harvests.
Prior to feed the experimental animals, freshly collected algae were blotted dry (AEG
SV 4528, Germany; Fig. 24) and accurately weighed (KERN EW 1500-2M, Balingen,
Germany).

Figure 24. Biofilter produced macroalgae and drying equipment.

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 Material and Methods

3.4. ARTIFICIAL DIETS

In order to test the nutritional value of different algae as ingredients for

sustainable abalone formulated feeds, three vegetal- diets were designed and tested for
six month in a land-based system (Study III). Processing of seaweed meals, diet
formulation and preparation are detailed in the chapters corresponding to this
experiment, only a general description of the methodology and materials used are
included in this section.

3.4.1. Diet formulation

Prior to diets preparation, seaweeds meals (U. lactuca (U), G. cornea (G), L.
digitata (L) and P. palmata (P)), and the rest of vegetable ingredients were analyzed
for proximate composition in GIA laboratories. Macroalgae ingredients were also
analyzed for aminoacid profile in LDG (Laboratorio de Diagnóstico General,
Barcelona, España).
Based on the results, three diets (UG, UGL and UGP) were formulated to
contain 35% protein, 4% lipid content and around of 4kcal g-1total energy, these
levels being reported as optimal for abalone growth. Vitamin and mineral mixture
were used as recommended by Uki et al. (1885a). All the experimental diets were
supplemented with synthetic L- methionine and lysine essential amino acids in order
to match the amino acid profile of abalone muscle which was used as a guide to
formulate the amino acid composition of the practical diets. Sodium alginate was used
as binder.

3.4.2. Diet preparation

Experimental diets were prepared by mixing pre-weighed finely ground

ingredients including vitamins and minerals to produce a homogeneous mixture. The
diets were processed through a pasta machine (Parmigiana, RV3, Italia) into 2 mm
thick strips from which 0.5 x 0.5 cm pieces were cut. The pellets obtained were dried
at 38ºC for 24 h, packed and stored at 4ºC until use (Fig. 25 and 26). Samples for
biochemical composition were taken and stored at -80ºC.

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 Material and Methods

Figure 25. Details of diets preparation: ingredients, processing and drying procedure.

3.4.3. Diet water stability

Final products were tested for their stability in seawater according to the
method of Hastings et al. (1971). Diet stability was determined at 17-h period (16:00-
9:00h). Percent water stability was computed as:

(𝐵𝐵 − 𝐴𝐴) (%𝑑𝑑𝑑𝑑𝑑𝑑 𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚)

% 𝑊𝑊𝑊𝑊𝑊𝑊𝑊𝑊𝑊𝑊 𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠 = 𝑥𝑥 100
𝐴𝐴 (%𝑑𝑑𝑑𝑑𝑑𝑑 𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚)

where B is the final weight of feed and A is the initial weight of feed

65
 Material and Methods

Figure 26. Final product: vegetable-based experimental diets.

3.5. EXPERIMENTAL DESIGN

Detailed experimental designs and samplings followed protocols are described in

the chapters corresponding to each experiment. Only, a general overview of the
methodology and materials used in the four feeding trials are included in this section.

To select the experimental animals, individuals were blot dried, weighed to the
nearest 0.1 mg (total wet body weight: TWBW) (KERN EW 1500-2M, Belingen,
Germany) and measured with manual caliper with 0.1 mm accuracy (total shell
length: SL) (Fig. 27a). Abalones were then distributed among replicates so that there
were no significant differences in SL or TWBW, and assigned to the specific
experimental units or cages (Fig. 27b).

Figura 27a. Selection of experimental animals: Abalone sampling.

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 Material and Methods

Figure 27b. Abalone distribution among experimental triplicates.

Each fresh algal diet was weekly supplied to the experimental abalones
(Studies I-IV) (Fig. 28-A, B, C), whereas the artificial ones (Study III), were offered
once daily from Monday to Saturday (Fig. 28-D). All of them were tested in
triplicates and supplied in excess to guarantee ad libitum feeding throughout the
experiments.

Figure 28. Feeding experimental abalones with macroalgae: Studies I and II (A), Study III
(B) and Study IV (C); or with artificial diets: Study IV (D).

67
 Material and Methods

In each trial, to calculate the feed intake, experimental units that contained
food but no abalone were used as controls for changes in algal and/or compound feeds
weight. In the land based trials, abalones were subjected to a natural photoperiod of
approximately 12 h L / 12 h D.
To assess abalone growth, both in length and weight, SL and TFBW of 100%
of the population in each experimental unit were monthly recorded in all the
experiments.
Cages were cleaned on a monthly basis to remove fouling organisms.

3.5.1. Land-based experimental set-up

In the first set of feeding studies with 11-12 mm juvenile abalones, the
experimental units consisted of a 1 l lidded (plastic net of 2 mm mesh) PVC plastic
container (20 x 14 cm), located in a 100 l cylindrical tank filled with flowing 50 µm
cartridge filtered seawater provided with constant aeration(Fig. 29) . Water flowed in
an approximately 2.4 l/min.

Figure 29. Experimental set-up for the culture of juvenile abalones (Studies I and II).

In Study III, the experimental unit consisted of a plastic bucket (15 x 16cm),
hung in a 100-l rectangular tank (100 x 40 x 25cm) filled with 50 µm filtered seawater
provide with constant aeration (Fig. 30-A, B). Water flowed in an approximately 2.8
l/min. Two PVC shelters were provided in each container.

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 Material and Methods

Figure 30. Rearing system employed for the culture of 30 - 45 mm abalones (Study III).

3.5.2. Sea-based grow-out system

The sea-based grow-out system was set up in a commercial open-sea cages

fish farm (CANEXMAR, S.L., Telde, Gran Canaria Island, Spain) (27º 57´ 31.7´´N,
15º 22´ 22.5´´W) (Fig. 31).

Figure 31. CANEXMAR cages and location.

Specially designed abalone sea cages (ORTACS, Jersey Sea Farms, St.
Martins, Ireland), were composed of a 33 l lidded black PVC meshed container, with
total underside surface area of 0.4 m2 and weighing 1.5 kg. Six black plastic discs
(12.0 cm Ø) were placed inside as shelters (Fig. 32). The total surface area for
attachment was 0.5 m2. The abalone cages were suspended from fish cages (25 m Ø)
mooring ropes and placed, approximately, 10 m below the water surface (Fig. 33-35).

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 Material and Methods

Figure 32. Details of the experimental abalone cages and shelters.

Figure 33. Scheme of the sea-based experimental set-up. A: aerial view of the fish farm
installation. B: detail of the ORTACS set-up.

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 Material and Methods

Figure. 34. ORTACS installation.

Figure 35. Underwater experimental devices next to fish cages.

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 Material and Methods

3.6. BIOLOGICAL PARAMETERS EVALUATION

3.6.1. Shell growth rate

Shell growth rate per day (μm d-1) was calculated for all treatments at the end
of the trials using the following equation:

(𝑺𝑺𝑺𝑺𝑺𝑺 − 𝑺𝑺𝑺𝑺𝑺𝑺)
𝑺𝑺𝑺𝑺𝑺𝑺𝑺𝑺𝑺𝑺 𝒈𝒈𝒈𝒈𝒈𝒈𝒈𝒈𝒈𝒈𝒈𝒈 𝒓𝒓𝒓𝒓𝒓𝒓𝒓𝒓 = 𝒙𝒙 𝟏𝟏𝟏𝟏𝟏𝟏𝟏𝟏
𝒕𝒕

where SL1 is the initial mean length of animals; SL2 is the animal final mean length at
time t (days of culture).

3.6.2. Specific growth rate (SGR)

This parameter indicates the increase in weight gain in relation to the number
of days of feeding period and it is expressed in percentage values:
(𝑳𝑳𝑳𝑳𝑾𝑾𝟐𝟐 −𝑳𝑳𝑳𝑳𝑾𝑾𝟏𝟏 )
SGR (% 𝒅𝒅−𝟏𝟏 ) = 𝒙𝒙 𝟏𝟏𝟏𝟏𝟏𝟏
𝒕𝒕

where W2 is the weight at time t (days of culture) and W1 is the initial weight.

3.6.3. Weight gain (WG)

In order to observe the potential differences in efficacy among the different

treatments, relative growth was evaluated. Weight gain was calculated as the relation
between the increases in biomass (g) compared to the initial weight (g). This
parameter could be expressed in absolute values as well as in percentage and it is
corrected in relation to the individual abalone weight through the following equation:

(𝒘𝒘𝟐𝟐 − 𝒘𝒘𝟏𝟏 )
𝑾𝑾𝑾𝑾 (%) = 𝒙𝒙 𝟏𝟏𝟏𝟏𝟏𝟏
𝒘𝒘𝟏𝟏

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 Material and Methods

3.6.4. Feed conversion ratio (FCR)

This parameter was evaluated in order to determine the efficiency of the

different feeding regimes to promote the abalone growth in terms of ingested food. It
is defined as the relation between the ingested food (g) and the generated biomass (g).

𝒕𝒕𝒕𝒕𝒕𝒕𝒕𝒕𝒕𝒕 𝒇𝒇𝒇𝒇𝒇𝒇𝒇𝒇 𝒊𝒊𝒊𝒊𝒊𝒊𝒊𝒊𝒊𝒊𝒊𝒊 (𝒈𝒈)

𝑭𝑭𝑭𝑭𝑭𝑭 =
𝒕𝒕𝒕𝒕𝒕𝒕𝒕𝒕𝒕𝒕 𝒘𝒘𝒘𝒘𝒘𝒘𝒘𝒘𝒘𝒘𝒘𝒘 𝒈𝒈𝒈𝒈𝒈𝒈𝒈𝒈 (𝒈𝒈)

3.6.5. Protein efficiency ratio (PER)

This parameter is based on the weight gain of the abalones in relation to their
protein intake during the trial period. It is calculated as follows:

𝒊𝒊𝒊𝒊𝒊𝒊𝒊𝒊𝒊𝒊𝒊𝒊𝒊𝒊𝒊𝒊 𝒊𝒊𝒊𝒊 𝒃𝒃𝒃𝒃𝒃𝒃𝒃𝒃 𝒘𝒘𝒘𝒘𝒘𝒘 𝒘𝒘𝒘𝒘𝒘𝒘𝒘𝒘𝒘𝒘𝒘𝒘(𝒈𝒈)

𝑷𝑷𝑷𝑷𝑷𝑷 =
𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑 𝒊𝒊𝒊𝒊𝒊𝒊𝒊𝒊𝒊𝒊𝒊𝒊 (𝒈𝒈)

3.6.6. Feed intake (FI)

In Studies I, II and IV, where just macroalgae diets were tested, to determine
feed intake, freshly collected algae to be fed to the abalone were blotted dry and
accurately weighed as well as the remaining algae at the end of the week. The weight
of unconsumed food was deducted from the total weekly ration. Besides, weight of
uneaten algae was corrected by calculating the natural weight variations of the algae
in the control units (without abalones) during the same feeding period.

In Study III, artificial feeds were offered once daily (ad libitum) in the
evening from Monday to Saturday. Any remaining diet was collected every day at
9:00 h except Sunday, by manually siphoning uneaten feed from tanks. Consumption
was estimated on a dry weight basis by relating the dry weight of the uneaten food to
the known dry weight of the feed provided (Fig. 36). Consumption data were
corrected for dry matter weight loss attributable to leaching, by allowing the diets to
leach over a 17-h period (16:00-9:00h) using a “control” rearing unit without abalone,
and drying the remaining diet until constant weight.

73
 Material and Methods

In all the experiments, average daily intake by individual abalone was

calculated by dividing the food eaten each week by the feeding days and the number
of abalones in each experimental unit.

Figure 36. Drying the diets leftover.

3.6.7. Condition index

At the end of experiments II and III, six and ten abalones respectively were
collected from each experimental unit, and the soft tissue (SB) was shucked from the
shell (S). Shell and meat were then weighed separately in order to calculate the
condition index as an indicator of the abalone nutritional status (Fig 37).

𝑺𝑺𝑺𝑺
𝑪𝑪𝑪𝑪 =
𝑺𝑺

Figure 37. Condition index evaluation: abalone dissection and weighing.

74
 Material and Methods

3.6.8. Survival

Dead abalones were daily recorded and, at the end of the trials, survival was
estimated taking into account daily mortality and final alive animals.

3.7. BIOCHEMICAL ANALYSIS

During the course of the different experiences, triplicate samples of each

feeding regimen (both fresh algae and artificial diets), artificial diets ingredients and
abalone (visceral mass and foot muscle) were collected to be analyzed for proximate
composition. Collection methods of the samples differed according to their nature. In
the case of fresh macroalgae, samples were cleaned, washed with fresh and distilled
water to remove salts and epizoos, frozen in -80ºC freezer and freeze- dried. The dried
samples were finely ground using an electric fine mill (sieve size <0.1 mm).
Once collected, all samples were frozen (-80 ºC) in hermetic bags under
nitrogen atmosphere until analysis. All samples were homogenized with mortar and
pestle before being weighed for further analysis. Determination of dry matter, ash,
protein and lipid content as well as fatty acids were performed. The biochemical
analyses were made in the laboratory of Instituto Universitario de Sanidad Animal y
Seguridad Alimentaria (IUSA; ULPGC).

3.7.1. Dry matter content

It was determined according to the Official methodology of the American
Chemical Analysis Association (AOAC, 2005). Dry matter content was determined
after drying the fresh known sample quantity (Pi) in an oven at 110ºC until constant
weight was obtained (Pf). Before being weighted, the samples were submitted to
desiccation for 30 min until reaching ambient temperature. The dry matter content
was calculated as follows:

(𝑷𝑷𝒊𝒊 − 𝑷𝑷𝒇𝒇)
% 𝑫𝑫𝑫𝑫 = 𝒙𝒙 𝟏𝟏𝟏𝟏𝟏𝟏
𝑷𝑷𝒊𝒊

75
 Material and Methods

3.7.2. Ash content

The ash content was determined gravimetrically after the incineration of a
well-known amount of sample (1-2 g) (Pi) in a Muffla oven, at 600ºC for 24 h. The
remaining amount of ashes was recorded and weighed until reaching constant weight
(Pf) according to the established recommendation (2005). Final ash content of the
sample was calculated according to the following formula:

𝑷𝑷𝑷𝑷 𝒙𝒙 𝟏𝟏𝟏𝟏𝟏𝟏
% 𝑨𝑨𝑨𝑨𝑨𝑨 =
𝑷𝑷𝑷𝑷

3.7.3. Protein content

The protein content, calculated from total content of the samples, was
determined by the Kjeldahl method. According to AOAC (2005), the technique
consists in the sulphuric acid (H2SO4) sample digestion at 420ºC in presence of a
copper catalyst for one hour. The end result is an ammonium sulphate solution
((NH4)2SO4). Excess base (NaOH) is added to the digestion product to convert NH4 to
NH3 which is recovered by distilling the reaction product. In the distillation unit
(Mod. Foss Tecator, 1002, Höganäs, Sweden) direct titration is performed, using boric
acid (H3BO3) as the receiving solution, to quantify the amount of ammonia in the
receiving solution. Titration is performed with HCl at 0.1.M. Analysis of a blank was
run in parallel to the samples analysis. In order to calculate the protein content, the
following expression was applied:
(𝑽𝑽𝒔𝒔 −𝑽𝑽𝒃𝒃 ) 𝒙𝒙 𝑵𝑵 𝒙𝒙 𝑷𝑷𝒎𝒎
% Protein = 𝒙𝒙 𝑭𝑭
𝑾𝑾

Where

Vs = ml acid titranl (HCl) for the blank

Vb = ml HCl for the sample
N = Normality of the acid titrant
Pm = Nitrogen molecular weight (14.007)
F = Empirical factor to covert the percent nitrogen in a sample to percent
protein, which has a value of 6.25
W = Weight of sample in milligrams

76
 Material and Methods

3.7.4. Total lipid content

Total lipids were extracted according to Folch et al. (1957). The methodology
commenced taking a sample amount between 50-200 mg and homogenise it in an
Ultra Turrax (IKA-Werke, T25 BASIC, Staufen, Germany) at 11,000 rpm for 5min in
a solution of chloroform-methanol (2:1) plus 0.01% of the antioxidant BHT. The
resulting solution was filtered at reduced pressure through glass wool and adding KCl
at 0.88%, to increase the water phase polarity. After decantation and centrifugation at
low speed (2000 rpm) for 5min, the watery and organic phases were separated. Once
watery phase was eliminated, N2 current was used to evaporate until completed
dryness. Finally total lipid content was gravimetrically determined.

3.7.5. Fatty acids content

Fatty acids in the lipid extracts were trans–esterified to methyl esters (FAMEs)
with 1% sulphuric acid: methanol complex (Christie, 1982) and preserved in N2
atmosphere. The mixture was left in shaking incubation for 16 hours at 50ºC then
cooled. Distilled water together with hexane diethyl ether 1:1 and BHT at 0.01% were
then added. The purified FAMEs samples were evaporated until complete dryness
with N2 and hence weighted. Finally the FAMEs were extracted into hexane and
stored at - 80ºC. Fatty acids were analyzed in a Thermo Finnigan- GC Focus gas
chromatograph (Mod. Shimadzu GC-14A; Analytical instrument division, Kyoto,
Japan) equipped with a flame ionization detector (260ºC) and a Supelcowax-10-fused
silica capillary column (Length: 28 m; 0.32 mm x 0.25 internal diameter; Supelco,
Bellefonte, USA), using helium as the carrier gas under the following gas pressures:
He 1 kg cm-2, H2 0.5 kg cm-2, N2 1 kg cm-2, air 0.5 kg cm-2. The conditions were the
following: injector temperature 180ºC for the first 10 min, increasing afterwards to
-1
215ºC at a rate of 2.5ºC min and maintained at 215ºC for 15 min. Fatty acids were
identified by reference to a well-characterized fish oil (EPA 28, Nippai, Ltd Tokyo,
Japan).

77
 Material and Methods

3.8. STATISTICAL ANALYSIS

All statistical analyses were applied using the Statgraphics Plus 5.1
(MANUGISTIES, Rockville, Maryland, USA) software. The data of each experiment,
proximate composition, as well as growth performance, survival or condition indexes
were statistically compared by means of T-Student (Sokal and Rolf, 1995) when two
treatments were established or with analysis of variance (ANOVAs) if the number of
treatments were greater. As general criteria 5% confidence level was applied. If
statistically significant differences with the ANOVA were detected, the differences
among means were detected with Tuckey HSD. Assumption of normality and
homogeneity of variance were assessed with standardized Skewness and Kurtosis and
Barlett´s test. When data did not follow a normal distribution, a non-parametric one-
way ANOVA on ranks of Kruskal-Wallis was applied (Zar, 1984).

78
Development of a Sustainable Grow-out Technology for Abalone Haliotis
tuberculata coccinea (Reeve) as a New Species for Aquaculture
Diversification in the Canary Islands

STUDY I: Suitability of Three Red Macroalgae as a Feed for

the Abalone Haliotis tuberculata coccinea Reeve.

Aquaculture 248 (2005) 75-82

 Study I Aquaculture 248 (2005) 75-82

Suitability of three red macroalgae as a feed for the abalone Haliotis

tuberculata coccinea Reeve

M.P. Vieraa, *, J.L. Gómez-Pinchettib, G. Courtois de Vicosea, A. Bilbaoa, S.

Suárezb, R.J. Harouna, M.S. Izquierdoa

a
Grupo de Investigación en Acuicultura (GIA), ULPGC and ICCM, P.O. Boz 56. 35200,
Telde, Las Palmas, Gran Canaria, Canary Islands, Spain
b
Grupo de Algología Aplicada. Centro de Biotecnología Marina, Universidad de Las Palmas
de Gran Canaria. Muelle de Taliarte s/n, 35214 Telde, Las Palmas, Canary Islands, Spain

Abstract

A 60-days feeding trial was conducted to assess the suitability of three red algae,
Hypnea spinella, Hypnea musciformis and Gracilaria cornea, as potential feed for the
abalone, Haliotis tuberculata coccinea R. Seaweeds were reared in a biofiltration unit with
fishpond waste water effluents. The three algal species were found to contain high protein
contents which would be related to their production under the high nitrogen conditions of the
biofilter system. Protein and carbohydrates contents were highest in H. musciformis and
lowest in G. cornea. Survival rates of juvenile abalone were very good, regardless of the
algae fed. Feed intake of H. spinella was highest, followed by H. musciformis. Growth rate of
abalone were in range obtained under commercial conditions, final shell length and weight
being significantly highest in animals fed H. spinella and lowest in those fed G. cornea.
Feeding G. cornea lead to the lowest growth performance due to the lowest fee intake,
whereas feed conversion ratios were significantly highest for H. musciformis and protein
efficiency ratios higher for both H. spinella and G. cornea. This study suggested the good
potential of any of three red seaweeds tested- successfully produced by the biofilter system,
their nutritional composition being similar to other macroalgae used as feed for abalone and
matching the abalone protein and lipid requirements- hence promoting growth and survival.
Nevertheless, the biofilter produced macroalgae H. spinella showed the highest dietary value
for juvenile of H. tuberculata coccinea.

Keywords: Haliotis tuberculata coccinea; Macroalgae; Seaweed biofilter; Polyculture; Feeding and
nutrition-molluscs

*
Corresponding autor. Tel + 34 928 132900/04; fax +34 928132908
E-mail address: mapi@iccm.rcanaria.es (M.P. Viera)

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 Study I Aquaculture 248 (2005) 75-82

4.1. INTRODUCTION

Commercial aquaculture in the Canary Island is limited to the production of

marine fish species such as gilthead seabream and European sea bass, with species
diversification being a challenge for further local aquaculture development. The
potential as a new specie for aquaculture of the Canarian abalone Haliotis tuberculata
coccinea R., whose natural population is becoming depleted due to overfishing, relies
on the high demand for abalone both in local and external markets, as well as on the
high degree of development achieved in some other species of the family Haliotidae
(Haliotis dicus hannai, Haliotis asinina, Haliotis rusfescens, etc), in particular of the
European ormer Haliotis tuberculata tuberculata L., with close biological
characteristics. Besides, there is a public interest in developing the culture techniques
of this species to contribute to the recovery of wild populations. Studies on H.
tuberculata coccinea are scarce, and have been focused only on spawning (Peña,
1985, 1986; Viera et al., 2003), ecology (Pérez and Moreno, 1991; Espino and
Herrera, 2002) and culture techniques (Toledo et al., 2000).
Food preferences differ among abalone species around the world, depending
on habitat and food availability (Barkai and Griffiths, 1986; Dunstan et al., 1996). In
the wild abalone consumes different macroalgae species, obtaining their required
nutrients from a combination of algal species. Although H. tuberculata coccinea feeds
on a diverse assemblage of macroalgae (Espino and Herrera, 2002), its nutritional
needs and the relative importance of these algae are unknown. Algal diets have been
extensively used for the production of abalone (Clarke, 1988). This herbivorous
gastropod can consume seaweed at a rate close to 35% of its body weight per day.
Hence sustaining of growth requires a large amount of fresh macroalgae (Tahil and
Juinio-Menez, 1999). Since wild macroalgae are not commercially valuable in the
Canary Islands, and hence not harvested, availability of this natural resource is scarce
and insufficient to sustain the commercial production of abalone, which would require
the replacement of wild seaweed as a main diet component.

In an aquaculture integrated system, nitrogenous enriched waste water of

intensively cultured organisms such as fish, shrimp and abalone may be transformed
into a valuable algal biomass, seaweeds production being and added income as feed
for shellfish (Evans and Langdon, 2000; Schuenhoff et al., 2003; Neori et al., 2004).
Besides, several studies have shown that culture of Ulva spp. in nutrient-rich waters

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 Study I Aquaculture 248 (2005) 75-82

increases its protein content from 11% to over 32% in dry weight (Shpigel et al.,
1999; Boarder and Schpigel, 2001). Thus, this biofilter produced Ulva have been
shown to provide good growth rate for H. tuberculata (Neori et al., 1998; Schpigel et
al., 1999), H. discus hannai (Corazani and Illanes, 1998) and H. roei (Boarder and
Schpigel, 2001). Previous experiences in our laboratory have also shown the potential
of using enriched Ulva rigida as a suitable feed for H. tuberculata coccinea (Viera
and co-workers, unpublished data). But in the wild, Canarian abalone seems to have a
higher preference for red algae (Espino and Herrera, 2002) as it has also been shown
for several abalone species such as H. iris and H. australis (Poore, 1972; Shepherd,
1973; Wells and Keesing, 1989; Shepherd and Steinberg, 1992; Fleming, 1995), H.
tuberculata or H. discus hannai (Mai et al., 1995a). Hence, in order to diversify the
offer of macroalgae to cultured abalone in the Canary Islands, this study focused the
evaluation of the suitability of three local red macroalgae cultivated in a biofiltration
unit as a potential feed for the Canarian abalone H. tuberculta coccinea.

4.2. MATERIAL AND METHODS

4.2.1. Algal culture

Gracilaria cornea J. Agardh, Hypnea spinella (C. Agardh) Kützing and

Hypnea musciformis (Wulfen) J. V. Lamoroux were grown in a flow- through
integrated system consisting of two intensive fishponds stocked with gilthead sea
bream and a macroalgal biofilter. Effluents were channelled from the fishponds to a
11 m3 sedimentation pond for the removal of suspended particles and, then, pumped
at a flow rate of 10 m3 h-1 to the seaweed tanks located in a greenhouse, were
maximum irradiance was close to 1600 µmol photons m-2 s-1. Semi-circular fiberglass
tanks with a surface of 1.8 m2, volume of 0.75 m3 and aeration supplied by a botton-
central linear pipeline were used for the cultivation of macroalgae. Algal stocking
densities were adjusted to the optimal values obtained from previous experiments (6 g
l-1). Water exchange rate in the seaweed tanks was 4 vol day -1
and TAN (total
ammonia nitrogen) inflow into the biofilter ranged between 10 and 400 µM.

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 Study I Aquaculture 248 (2005) 75-82

4.2.2. Abalone and rearing conditions

Abalones (H. tuberculta coccinea) were produced within the experimental

hatchery production unit of Instituto Canario de Ciencias Marinas, (Canary Islands,
Spain). A total of 360 juvenile abalones (40/replicate) with an average shell length
and weight of 11.18 ± 1.27 mm and 0.17 ±0.06 g. respectively, were selected for the
trial.
Animals were initially fed a diet of Navicula sp. and Nitschia sp. for 4-5
months. Feeding of all abalone was then switched to the green macroalgae Ulva
rigida cultured at the laboratory biofiltration system for a period of one month prior to
the beginning of the experiment.
Individuals were blotted dried, weighed to the nearest 0.1 mg 8total wet body
weight: TWBW), and measured with manual caliper with 0.1 mm accuracy (total shell
length: SL) and assigned to an experimental unit. Abalones were distributed among
replicates so that there were not significant differences in SL or TWBW. Each algal
type was fed for 8 weeks to the experimental abalone and tested in triplicates (9
experimental units plus 3 control units). The experimental unit consisting of a 1 l
lidded (plastic net of 2 mm mesh) PVC plastic container (20 x 14 cm), located in a
100- l cylindrical tank filled with 50 µm filtered seawater, provided with constant
aeration. Water flowed in at approximately 2.4 l/min. Water temperature ranged
between 23.9 and 25.4 ºC. Abalones were subjected to a natural photoperiod of
approximately 12 h l / 12 h D.

4.2.3. Growth and algal consumption

SL and TWBW of each animal were measured every four weeks.

Three experimental units that contained algae but no abalone were used as
controls for changes in algal weight. Feed was changed once a week during the
growth trial and supplied in excess to guarantee ad libitum feeding along the whole
experiment. To determine feed intake, freshly collected algae to be fed to the abalone
were blotted dry and accurately weighed as well as the remaining algae at the end of
the week. The weight of unconsumed food was deducted from the total weekly ration.

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 Study I Aquaculture 248 (2005) 75-82

Besides, weight of uneaten algae was corrected by calculating the natural change in
weight of the algae in the control units during the same feeding period.
The average daily intakes by individual abalones during the entire feeding trial
were calculated by dividing the algal biomass eaten each week by the feeding days
and the number of abalones in each experimental unit.
The following nutritional indices were calculated for all treatments ant the end
of the trial:

Shell growth rate = (L2 – L1) / t x 1000

Specific growth rate, SGR = (Ln W2 – LnW1) / t x 100
Weight gain (%) = ((W2 – W1)/ W1)) x 100
Food conversion ratio = total feed intake (g wet)/ total weight gain (g wet)
Protein efficiency ratio = increase in body wet weight (g) / protein intake (g)

Where L1 is the initial mean length of animals, L2 is the final mean length of animals,
Ln W2 is the natural logarithm of weight at time t (days of culture), and Ln W1 is the
natural logarithm of initial weight.

4.2.4. Nutritional analysis

Homogenized samples of each type of algae provided to the abalones were

analyzed in triplicate for nutrient composition. The samples collected were cleaned,
washed with freshwater to remove salt and epizoos, frozen in -80ºC freezer, freeze-
dried and finely ground using an electric fine mill (sieve size < 0.1 mm). The dry
matter was determined by incinerating samples at 600ºC for 24 h. Protein content was
analyzed according to AOAC (1995) standard methods. Total lipids were extracted
with chloroform-methanol (2:1) mixture as described by Folch et al. (1957).

4.2.5. Statistical analysis

Statistical analysis was conducted using one-way ANOVA and Tukey test for
multiple comparison of means with 5% significance level was applied (P < 0.05).
When data did not have normal distribution, a non- parametric one-way ANOVA on
ranks of Kruskal- Wallis was tested (Zar, 1984).

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 Study I Aquaculture 248 (2005) 75-82

4.3. RESULTS

4.3.1. Algal nutritional composition

Nutritional composition and caloric content of the three red seaweeds are
shown in Table 17. Protein energy ratios and gross energy were very similar among
the different algae. However, there were significant differences (P < 0.05) among the
proximate composition of the algae fed. Protein content was significantly highest in
H. musciformis, whereas G. cornea showed the lowest. Similarly, H. musciformis
showed the highest carbohydrate content and G. cornea the lowest. The three algal
species were low in lipid content, the highest being also found in H musciformis and
the lowest in H. spinella. The ash content varied inversely to protein and carbohydrate
contents and was highest in G.cornea and lowest in H. musciformis.

Table 17. Proximate composition and caloric content of the three red macroalgae
(g/100 g DW) (mean ± S.D.) fed to abalone along the experimental trial

Algal species

H. musciformis H. spinella G. cornea

Moisture 8.63±0.27a 8.11±0.17b 7.29±0.12c

Crude protein 27.13±0.2a 25.9±0.1b 21.54±0.08c

Crude lipid 2.45±0.08 a 1.99±0.01c 2.36±0.11b

Carbohydrate *** 32.79±0.33a 30.97±0.27b 27.7±0.1c

Ash 37.63±0.12c 41.14±0.28b 48.39±0.17a

GE ** (kcal g-1) 3.1 2.9 2.6

Protein: energy ratio* 87.4 89.3 82.7

Values in the same row with different letters are significantly different (P ˂0.05) n =3.
* Metabolizable energy was calculated based on the physiological values at 5.6 kcal g_1
protein, 9.5 kcal g_1 lipid and 4.1 kcal g_1 carbohydrates (Cho et al., 1982).
** Gross energy.
*** Calculated by difference (AOAC, 1995).

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Study I Aquaculture 248 (2005) 75-82

4.3.2. Growth and consumption

Survival, growth performance and feed utilization of juvenile H. tuberculata

coccinea fed the macroalgae H. musciformis, H. spinella and G. cornea are shown in
Table 18. Survival rates were very good and no significant differences were found
among the different feeding regimes. Daily feed intake on different algal rations
recorded for 8 weeks showed that all three macroalgae were very well accepted by the
abalone, but a significantly higher feed intake of H. spinella, followed by H.
musciformis, was registered. Hence, final shell length and weight were significantly
highest in animal fed H. spinella and lowest in those fed G. cornea. Accordingly,
shell growth rate, specific growth rate and weight gain followed the same pattern, all
these parameters being significantly highest in animals fed H. spinella and lowest in
those fed G. cornea.
Study of the feed efficacy of feed utilization showed that feed conversion
ratios were significantly higher for H. musciformis, whereas protein efficiency ratios
were higher for both H. spinella and G. cornea.

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 Study I Aquaculture 248 (2005) 75-82

Table 18. Growth, feed utilization and survival of juvenile Canarian abalone (H.
tuberculata coccinea) at the beginning of the experiment and after being fed the
selected macroalgae for 60 days under laboratory conditions

Algae species fed to the abalone

H. musciformis H. spinella G. cornea

Initial length (mm) 11.18±1.27 11. 2±1.29 11.17±1.27

Final length (mm) 15.92±1.52 b 18.54±1.73c 14.75±1.75a

Shell growth rate (µm day_1) 81.79±3.23b 126. 61±5.92c 61.71±1.73a

SGR (% day_1) 1.89±0.03b 2.66±0.11c 1.47±0.05a

Initial weight (g) 0.17±0.06 0.17±0.06 0.16±0.06

Final weight (g) 0.51±0.15b 0.78±0.21c 0.38±0.12a

Weight gain (%) 178.41±8.63b 348. 65±30.35c 123.64±4.43a

Feed intake (mg abalone-1 day -1) 165.54±4.96b 215.63±0.61c 78.98±1.79a

FCR 30.49±1.64b 20.94±1.49a 22.09±1.15a

PER 1.1±0.06a 1.68±0.1 b 1.75±0.09 b

Survival (%) 93.33±2.89 95.83±1.44 95.75±5.1

Values in the same row with different letters are significantly different (P ˂0.05) n =40x3.

4.4. DISCUSSION

The nutritional value of food rations depends on many factors including

nutrient composition, bioavailability, palability and digestibility (Serviere-Zaragoza et
al., 2001).This study examined the nutritional value of three algal diets for juvenile
abalone, H. tuberculata coccinea, by measuring biochemical composition and energy
content of the algae and relating this to growth performance and feed intake of the
animals.
The proximate composition of the algae was in the range of values reported
for other species of seaweeds used as feed for abalone (Jackson et al., 2001; Mcbride
et al, 2001). Protein content (22-27%DW) of the three algae was within the range for

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 Study I Aquaculture 248 (2005) 75-82

red seaweeds (10-47%) (Fleurence, 1999; Wong and Cheung, 2000) and was higher
than the level found in the red seaweed, Palmaria palmata (18.4%), which has been
found to be a good algal diet for H. tuberculata (Mercer et al., 1993; Mai et al.,
1995a). Furthermore, protein content of H. spinella and H. musciformis was higher
than that reported for other species of Hypnea (4.2-19% DW) (Portugal et al., 1983;
Wong and Cheun, 2000). These high levels of protein content observed in the algae of
the present study would be related to its production under the high nitrogen culture
conditions of the biofilter system, as it has been also observed in Ulva spp. cultured as
macroalgal biofilters (Tenore, 1976; Neori, 1996; Shpigel et al., 1996a, b, 1999).
Hence, protein content of the three macroalgae used in the present experiment would
match the protein requirements described for several species of Haliotis such as H.
discus (20%) (Ogino and Kato, 1964), H. discus hannai (20-30%), (Uki et al., 1986),
H. kamtschatkana (30%) (Taylor, 1992) or for H. tuberculata and H. discus hannai
(25-35%) (Mai et al., 1995b), although it would be lower than that required for H.
midae (47%) (Britz, 1996a).
All the algae were low in lipid (2-2.4% DW) but within the range of other
species of red seaweeds (1-3 % DW) reported previously (Mabeau and Fleurence,
1993; Wong and Cheun, 2000). Nevertheless, abalone species show low lipid
requirement, typical of herbivore molluscs and fish (Mai et al., 1995a). This low lipid
requirement has been associated by some authors (Durazo-Beltrán et al., 2004) with
the low used of dietary lipids as energy source by abalone based upon its low
metabolic rate. Indeed, high levels of dietary lipid seem to negatively affect abalone
growth (Thongrod et al., 2003). However, high levels of carbohydrate enhance growth
(Thongrod et al., 2003) of abalone which has various enzymes capable of hydrolysing
complex carbohydrates (Fleming et al., 1996) and a good capacity to synthesize non-
essential lipids from carbohydrates. In the present study, carbohydrate values (28-33%
DW) were high and comparable to those obtained for other algal species (Kaehler and
Kennish, 1996; Foster and Hodgson, 1998).
Ash content of the Hypnea species was higher than that of other species of the
same genus H. charoide (23-35% DW), H. pannosa (15% DW) and H. japonica (19%
DW) (Portugal et al., 1983; Wong and Cheun, 2000). High ash content in algae results
from calcium carbonate, which limit the other nutrient´s presence and reduce nutrient
digestibility (Horn, 1989; Hay et al., 1994).

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 Study I Aquaculture 248 (2005) 75-82

It is generally accepted that balanced levels of protein (> 15%), lipid (3-5%)
and carbohydrate (20-30%), with no toxic substances in natural algae, are essential for
optimal growth performance of abalone (Mercer et al, 1993). From this point of view,
the three algae tested appear to be able to match the abalone requirements.
Growth rates of abalone fed dietary treatments in this study were within the
range of 62-127 µm day-1, close to those obtained by other authors in similar species
(50-100 µm day-1; Viana et al., 1996, 2000; Guzmán and Viana, 1998; Gómez-
Montes et al., 2003) under similar experimental conditions and to that obtained under
commercial condition (80 µm day-1; Gómez–Montes et al., 2003). SGR values were
also high (1.5-2.7%) in comparison with those reported by Mai et al. (1996), who
studies the effect of five species of macroalgae (P. palmata, Alaria esculenta, Ulva
lactuca, Laminaria digitata and Laminaria saccharina) and reported SGR values of
1.03–1.31% for H. tuberculata and 0.7–1.25% for H. discus hannai.
Gracilaria species have been reported to promote high growth and survival in
other species of abalone such as H. asinina (Upatham et al., 1998; Bautista- Teruel
and Millamena, 1999; Capinpin et al., 1999; Reyes and Fermin, 2003). In the present
study feeding G. cornea lead to the lowest growth performance due to the lowest feed
intake registered, since feed utilization in terms of FCR and PER was as good as those
of the highest growing abalone fed H. spinella. Indeed, consumption rate in abalone
may be influenced by factors other than the nutritional quality of food, such as its
toughness or presence of antinutritional chemicals (Fleming, 1995). Hence, the harder
texture and ash content of G. cornea may have affected its consumption by H.
tuberculata since, in the wild, abalone prefer soft texture macroalgae (Shepherd and
Steinberg, 1992). This preference seem to be related to the little capacity of the
rhipidoglossan radula to penetrate the algal surface (Steneck and Watling, 1982) as its
teeth have limited ability to exert force against the substrate.

The high FCR of the three treatments agrees well with those reported for H.
discus hannai and H. tuberculata using seaweeds as food (Shpigel et al., 1999) and H.
asinina fed G. fisheri (Kunavongdate et al., 1995). The higher food conversion rate
(FCR) attained for abalone fed H. musciformis in the present study does not seem to
be explained by the general composition of the algae, or by the protein: energy ratio
but to a significantly lower protein efficiency ratio (PER) than the rest of the
treatments, suggesting that differences in response could be attributed to differential

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amino acid composition. Optimum growth is achieved through proper balance of

dietary nutrients and fulfillment of requirements of essential nutrients and energy
(Smith, 1989; Gómez-Montes et al., 2003).

In conclusion, this study suggested the good potential of using any of the three
red seaweeds tested -successfully produced by the biofilter system, their nutritional
composition being similar to other macroalgae used as feed for abalone and being
able to match the protein and lipid requirements of abalone- hence promoting good
growth and survival. Nevertheless, based on growth performance, food conversión
ratio, protein efficiency ratio and protein: energy ratio, the macroalgae H. spinella
produced by the biofilter showed the highest dietary value for juvenile of H.
tuberculata coccinea.

4.5. ACKNOWLEDGEMENTS

The authors would like to thank to Drs. S.Thongrod, L. Robaina and D.

Montero for their valuable comments on the manuscript. This study has been financed
by the Spanish Government in the frame of the National Plan for Development of
Marine Cultures (JACUMAR, TR 2003/08).

89
Development of a Sustainable Grow-out Technology for Abalone
Haliotis tuberculata coccinea (Reeve) as a New Species for
Aquaculture Diversification in the Canary Islands

STUDY II: Comparative Performances of Juveniles Abalone

(Haliotis tuberculata coccinea reeve) Fed Enriched vs Non-

Enriched Macroalgae: Effect on Growth and Body

Composition.

Aquaculture 319 (2011) 423-429

Study II Aquaculture 319 (2011) 423-429

Comparative performances of juveniles abalone (Haliotis tuberculata

coccinea reeve) fed enriched vs non-enriched macroalgae: Effect on
growth and body composition

Viera, M. P.a*, Courtois de Vicose, G.a, Gómez-Pinchetti, J.L.b, Bilbao, A a,

Fernández-Palacios, H.a, Izquierdo, M.S.a

a
Grupo de Investigación en Acuicultura, Instituto Canario de Ciencias Marinas &
Universidad de las Palmas de Gran Canaria P. O. Box 56, 35200 Telde, Canary Islands,
Spain
b
Grupo de Algología Aplicada. Centro de Biotecnología Marina, Universidad de Las Palmas
de Gran Canaria. Muelle de Taliarte s/n, 35214 Telde, Las Palmas. Canary Islands. Spain

Abstract

Abalone Haliotis tuberculata coccinea Reeve (1846), is a target species for

diversification of European aquaculture production. Taking into account that sustainable, eco-
friendly production methods are to be a part of future expansion of the abalone industry,
growth performance of juvenile abalone reared in an integrated culture system was evaluated
and compared with that of abalone fed non-enriched macroalgae. Four macroalgae treatments,
three monospecific: Ulva rigida (UN), Hypnea spinella (HN) and Gracilaria cornea (GN)
and a composite one (MN), were produced out of fishpond wastewater effluents, while other
four control treatments consisted of the same species reared in fresh seawater (U; H; G; M).
Seaweeds reared in fishpond wastewater effluents increased their protein content from 11-
17% to 29-34%. Lipids consisted mainly of saturated fatty acids (SFA) (43-60%), palmitic
acid being the most abundant fatty acid (40-47%). Highest EPA percentage was found in red
algae H. spinella (6.9%), being ten times higher than that of U. rigida (0.7%). All the algae
tested contained very low levels of arachinodic acid (0.1-1.6%) and docosahexaenoic acid
(0.5-3%). Protein levels in foot muscle (74-76%) did not differ significantly (P<0.05) among
treatments. Survival was generally high, ranging from 85 to 100%. Weight gain (17-561%)
and SGR (0.2-2.3%) were positively related to protein content; whereas, protein efficiency
ratio (PER) (0.5-3.7) was negative correlated. PE ratios increased by 82-159% (DW) as a
function of the enrichment among the different diets. Food conversion ratio (FCR) (7-188)
improved according to the increase in PER. Overall, biofilter-produced macroalgae showed a
significantly higher dietary value compared to the control treatments. Similarly, animals fed
the mixed diets performed significantly better than those fed a single algal diet. Feeding G.
cornea led to the lowest growth performance probably due to the lowest feed intake. The
results clearly indicate that H. tuberculata coccinea growout can efficiently take place in an
integrated-culture system suggesting that on-farm seaweed-abalone production could be part
of future development of the abalone industry in the Canary Islands.

Keywords: Haliotis tuberculata coccinea; macroalgae; seaweed biofilter; polyculture; feeding and
nutrition-molluscs.
*Corresponding author: María del Pino Viera-Toledo
Phone: (34) 928 132900/04 ; Fax: (34) 928 132908
Mail: mapi@iccm.rcanaria.es
Address: P.O. Box 56. Telde, 35200, Gran Canaria, Canary Islands, Spain

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5.1. INTRODUCTION

Abalone species (Haliotis spp.) are found worldwide and are becoming
important for aquaculture diversification due to their high market price and the over
exploitation of wild stocks. In Europe, abalone industry is currently focussed on the
production of the ormer Haliotis tuberculata Linnaeus (1758). Ireland, the Channel
Islands (Huchette and Clavier 2004) and France are currently the only established
producing countries. A subspecies of ormer, the abalone Haliotis tuberculata
coccinea Reeve (1846), is also considered a good candidate for European aquaculture,
as it is highly appreciated for its delicate taste, reaches a large enough size to be
commercialized and its culture techniques have been successfully developed (Toledo
et al., 2000; Viera et al., 2003, 2005, 2007, 2009a, b; Bilbao et al., 2004, 2010a, b;
Courtois de Viçose et al., 2007, 2009, 2010). A limiting factor for further expansion
of abalone aquaculture is the restricted availability of an economically and
environmentally sustainable feed, as this culture frequently requires large quantities of
wild harvested macroalgae. Such feed would be particularly important in areas where
wild algae are not commercially available (Viera et al, 2005).

Among different nutrients, protein constitutes the most costly component and
is a major determinant of the nutritional value in diets of the abalone (Uki et al.,
1985b; Uki and Watanabe, 1986; Mai et al., 1995b; Britz, 1996a, b; Britz and Hecht,
1997; Shipton and Britz, 2001; Bautista-Teruel et al., 2003; Gómez-Montes et al.,
2003; Reyes and Fermín., 2003; Sales et al., 2003). In an aquaculture integrated
system, nitrogen enriched waste water of intensively cultured organisms, may be
transformed into a valuable algal biomass, seaweeds production being an added
income as feed for shellfish (Evans and Langdon, 2000; Schuenhoff et al., 2003;
Neori et al., 2004). Besides, several studies have shown that the culture of macroalgae
in nutrient-rich waters increases their protein content (Shpigel et al., 1999; Boarder
and Shpigel, 2001; Robertson-Andersson et al, 2006; Viera et al, 2005; Naidoo et al,
2006). Thus, biofilter produced seaweed have been shown to support fast growth rates
for Haliotis tuberculata (Neori et al., 1998; Shpigel et al., 1999), Haliotis discus
hannai (Corazani and Illanes, 1998; Shpigel et al., 1999), H. roei (Boarder and
Shpigel, 2001) and H. tuberculata coccinea (Viera et al., 2005).

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Among the different macroalgae produced, Ulva spp. and Gracilaria spp. are
good candidates as feed for abalone since their mass production technologies are well
developed and their nutrient uptake capacities are among the highest known
(Martínez-Aragon et al., 2002). The valuable rhodophyte Hypnea sp. has also been
successfully cultured in mariculture biofilters (Harlin et al, 1978; Neori et al, 2004;
Viera et al, 2005).

In the wild, abalone consumes different macroalgae species, obtaining their

required nutrients from a combination of algal species. Although H. tuberculata
coccinea feeds on a diverse assemblage of macroalgae (Espino and Herrera, 2002), its
nutritional needs and the relative importance of these algae are unknown.

It is well recognised that consumer knowledge and attitudes to an aquaculture

product have a significant role to play in commercial success. As abalone in Europe
currently has low levels of production, it gives the sector an excellent opportunity to
set standards that will meet consumer expectations for sustainable, eco-friendly
production methods which fit into the strongly growing EU eco-sector for shellfish
products. Abalone producers may be enticed to adopt effluent treatment procedures
more readily if shown that enriched macroalgae can be suitable as a feed for local
abalone species promoting higher growth performance than the one achieved with
seaweeds harvested or reared in fresh seawater.

In this study, the comparative performance of juvenile abalone Haliotis

tuberculata coccinea fed on various enriched vs non enriched macroalgae was
examined. We determined: (1) Algal nutritional and fatty acid composition; (2)
Survival; (3) Growth (shell growth rate; specific growth rate and weight gain); (4)
Consumption (daily feed intake); (5) Feed efficacy of feed utilization (food
conversion ratio (FCR) and protein efficiency ratio (PER); (6) Biochemical
composition of the animals and (7) Soft-body to shell ratio (SB/S) of the abalone after
being fed these diets for 12 weeks. Performance promoted by the various diets was
related to a range of nutritional parameters including crude protein (CP), total lipid
(TL), gross energy (GE), protein-energy ratio (PE) and fatty acids (FA) of the diets
fed.

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5.2. MATERIAL AND METHODS

5.2.1. Algal culture

Ulva rigida J.Agardh, Hypnea spinella (C. Agardh) Kützing and Gracilaria
cornea J. Agardh were grown at the Centro de Biotecnología Marina (CBM-ULPGC),
Gran Canaria, Spain. Eight feeding regimes were evaluated: three monospecific ones
with macroalgae produced in fresh seawater Ulva rigida (U), Hypnea spinella (H) and
Gracilaria cornea (G), and a mixture of equal parts from the three algae (M); and the
same four feeding regimes using algae produced out of fishpond waste water effluents
(UN; HN; GN and MN). Effluents were channelled from the fishponds to a 11 m3
sedimentation pond for the removal of suspended particles and then, pumped at a flow
rate of 10 m3 h-1 to the seaweed tanks located in a greenhouse, where maximum
irradiance was approximately of 1600 µmol photons m-2 s-1. Semi-circular fiberglass
tanks with a surface of 1.8 m2 and a volume of 0.75 m3 were provided aeration
through a bottom-central linear pipeline and were employed for the cultivation of
macroalgae. Algal stocking densities were adjusted to the optimal values obtained
from previous experiments (1, 3 and 4 g l-1 for U. rigida, H. spinella and G. cornea,
respectively). Water exchange rate in the seaweed culture tanks was 4 vol day-1 and
TAN (total ammonia nitrogen) inflow into the biofilter ranged between 10 and 400
µM.

5.2.2. Abalone and feeding trial conditions

Abalone (Haliotis tubercalata coccinea) were produced within the

experimental hatchery production unit of the Instituto Canario de Ciencias Marinas
(Canary Islands, Spain).

Animals were initially fed a mixed diet of Navicula sp. and Nitzschia sp. for 4
to 5 months. Feeding of all abalone was then gradually switched to the green
macroalgae Ulva rigida cultured in the laboratory biofiltration system for a period of
1 month prior to the beginning of the experiment. A total of 600 juvenile abalones
(25/tank) with an average shell length and weight of 12.5 ± 1.6 mm and 0.27 ± 0.18 g,
respectively, were selected for the trial.

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Individuals were blot dried, weighed to the nearest 0.1 mg (total wet body
weight: TWBW), measured with manual calliper with 0.1 mm accuracy (total shell
length: SL) and assigned to an experimental unit. Abalones were homogeneously
distributed among tanks to avoid significant differences in SL or TWBW. Each
experimental algal regime (8 feeding diets tested in triplicates) was fed for 12 weeks
to the abalones and tested in triplicates in a flow-through system. Eight control units
containing the same feeding regimes without abalone, were used as controls to
estimate percentage of modification in algal weight (computed as: ((A-B) / A) x 100;
where B is the final weight of algae and A is the initial weight of algae in the control
unit determined at 1 week interval).

The experimental unit consisted in a 1 l lidded (plastic net of 2 mm mesh)

PVC plastic container (20 x 14 cm), located in a 100 l cylindrical tank filled with 50
µm filtered seawater provided with constant aeration. Seawater temperature ranged
between 22-24.5 ºC and flow was set at 2.4 l / min. Abalone were subjected to a
natural photoperiod of approximately 12 h L / 12 h D. Algae were supplied in excess
to guarantee ad libitum feeding and replaced once a week during the growth trial.

5.2.3. Growth and algal consumption

To determine feed intake, freshly collected algae were blotted dry and
accurately weighed as well as the remaining algae at the end of the week. The weight
of unconsumed food was deducted from the total weekly ration. Besides, weight of
uneaten algae was corrected by calculating the natural weight variations of the algae
in the control units during the same feeding period. Average daily intake by individual
abalone was calculated by dividing the algal biomass eaten each week by the feeding
days and the number of abalones in each experimental unit.

SL and TWBW of each animal were determined every four weeks. Besides,
the following indices were calculated for all treatments at the end of the trial:

Shell growth rate = (L2-L1) / days of culture x 1000

Specific growth rate, SGR= (LnW2-LnW1) / t x 100
Weight gain (%) = ((W2-W1) / W1) x 100
Food conversion ratio = total feed intake (g wet) / total weight gain (g wet)

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Protein efficiency ratio = (increase in body wet weight (g)) / (protein intake
(g))
where L1 is the initial mean length of animals; L2 is the final mean length of
animals; W2 is the weight at time t (days of culture), and W1 is the initial weight.

At the end of the experiment, ten abalones were collected from each
experimental unit, and the soft tissue was shucked from the shell. Shell and meat
were then weighed separately in order to calculate the condition index (wet weight
of soft flesh/wet weight of shell, SB/S in W/W) as an indicator of the abalone
nutritional status.

5.2.4. Proximate and fatty acid analysis

Homogenized samples of visceral mass and foot muscle of the selected

abalone and algae from each feeding regime were analyzed in triplicate for nutrient
composition. Fatty acids of the algae were also analyzed. The algae, abalone visceral
mass and foot muscle were cleaned, washed with freshwater and frozen at -80 ºC. Dry
matter was determined by drying samples at 110ºC until constant weight was attained.
Ash content was determined by incinerating samples at 600ºC for 24 h. Protein
content was analyzed according to AOAC (2005) standard methods. Total lipids were
extracted by a chloroform-methanol (2:1) mixture as described by Folch et al. (1957).
Fatty acids in the lipid extracts were transesterified to methyl esters (FAMEs) with
1% sulphuric acid: methanol complex (Christie, 1982). FAMEs samples were
extracted into hexane and stored at -80ºC. Fatty acids were analyzed in a Thermo
Finnigan- GC Focus gas chromatograph equipped with a flame ionization detector
(260ºC) and a capillary column (Supelcowax 28m x 0.32mm x 0.25 i.d.), using
helium as the carrier gas under the conditions described by Izquierdo et al. (1989).

5.2.5. Statistical analysis

All data were statistically treated by one-way ANOVA and Tukey’s test was
applied for multiple comparison of means at a 5% significance level (P< 0.05). When
data did not follow a normal distribution, a non-parametric one-way ANOVA on
ranks of Kruskal-Wallis was applied (Zar, 1984).

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5.3. RESULTS

5.3.1. Algal nutritional composition

Nutritional composition and caloric content of the eight seaweed treatments

are shown in Table 1. Gross energy values in diets ranged from 3.5-4.1(Kcal g-1).
Protein: energy ratios increased by 82-159% (dry weight) with the enrichment among
the different diets. Protein content was significantly higher (P<0.05) in the seaweeds
reared using fishpond waste water effluents, increasing their protein content from
11.3-16.6% to 29.3-33.8%. Lipid content ranged from 1.4 to 7.2%, being also
generally higher in enriched seaweeds, the highest being found in GN and the lowest
in H. spinella (H). The carbohydrate contents varied inversely to protein contents,
showing significantly higher values in non enriched treatments. No significant
differences were observed in the ash content for all diets.

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Table 19. Proximate composition and caloric content of the eight macroalgae treatments (g/100 g DW) (Mean ± S.D.) fed to abalone along
the experimental trial

Diets

Enriched Enriched Enriched Enriched

U. rigida H. spinella G. cornea Mixed diet
U. rigida H. spinella G. cornea Mixed diet

Moisture 82.09±0.8b 84.2±1ab 83.9±1.8ab 83.3±1.4ab 82.04±0.3b 83±2.9ab 84.94±0.6a 83.25±1.9ab

Crude protein 16.6±3.8 b 13.2±1.7b 11.27±1.1b 13.7±3b 33.76±0.5a 33.09±6a 29.35±2a 32.1±3.9a

Crude lipid 3.7±1abc 1.4±0.4c 5.4±3.5abc 3.4±2.5bc 4.4±0.8abc 6.6±2.6ab 7.21±2.7a 6.06±2.1a

Carbohydrate1 56.4±8.5ab 65.4±9.5a 58±8.5a 60.7±7.6a 40.5±6.1bc 39.2±11.1bc 31.8±11.3c 41.01±3.6c

Ash 25.2±6 21.4±7.9 24.1±3.6 23.6±5.9 21.5±5.2 21.3±2.7 32.3±7 25.24±7.3

GE2 (Kcal g-1) 3.5 3.5 3.6 3.6 3.9 4.1 3.6 4.04

Protein:energy ratio3 47.5 37.7 31.4 38.05 86.6 80.7 81.5 79.4

1
Calculated by difference (AOAC, 2005)
2
Gross energy
3
Metabolizable energy was calculated based on the physiological values at 5.6 Kcal g-1 protein, 9.5 Kcal g-1 lipid and 4.1 Kcal g-1 carbohydrates (Cho et al., 1982).
Values in the same row with different letters are significantly different (P<0.05) n=3

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5.3.2. Survival, growth, consumption and condition index

Growth performance, feed utilization and survival of juvenile H. tuberculata

coccinea fed the eight experimental diets are shown in Table 2. Survival was
generally high, ranging from 85 to 100% for those fed with G and H, respectively. In
general, abalone fed enriched algae performed better than those fed on non enriched
macroalgae, displaying higher shell growth rate, specific growth rate and weight gain.
Similarly, animals fed the mixed diets, both enriched and non enriched, performed
significantly better than those that were fed with a single algal diet. Daily feed intake
on different algal rations recorded for 12 weeks showed that, except with Gracilaria
treatments (G and GN), all diets were very well accepted by the abalone.
Nevertheless, a significantly (P<0.05) higher feed intake of the mixed diet, followed
by H. spinella and U. rigida was registered. Hence, at the end of the trial, animal fed
on the enriched mixed macroalgae diet (MN) presented a significantly (P<0.05)
higher growth performance, length (151±3.9 µm day-1), weight gain (561.3±20.3 %)
and specific growth rate (2.3%) than those on any other diets. For abalone fed non
enriched macroalgae, final shell length and weight were significantly highest in
animals fed the mixed diet (22.3±3.5mm and 1.4±0.6g), followed by U. rigida and H.
spinella, and were the lowest in those fed G. cornea (14.3±1.7mm and 0.3±0.1g).
Regarding feed utilization efficacy, except for Hypnea diets (H and HN), food
conversion ratio (FCR) values were inversely related to protein level, being
significantly lowest in animals fed UN and MN (7-10.1) and highest in those fed non-
enriched G. cornea (188.1±8.9). However, protein efficiency ratio (PER) values were
generally higher in abalone fed non-enriched macroalgae, declining from 3.7-1 to 2.4-
0.5, respectively. Soft-body to shell ratio were significantly influenced by the diets.
The lowest SB/S ratios (2.4) resulted from feeding both G. cornea diets, which
produced the poorest growth performance, whereas UN and both mixed diets,
produced the best growth performance, yielded the highest SB/S ratios (3.3-3.1).

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Table 20. Growth performance, feed utilization and survival of juvenile abalone (H. tuberculata coccinea) fed the selected 8 macroalgae diets for 12-weeks

Macroalgae treatments fed to the abalone

U. rigida H. spinella G. cornea Mixed diet Enriched Enriched Enriched Enriched

U. rigida H. spinella G. cornea Mixed diet
Initial length (mm) 12.6±1.6 12.5±1.6 12.6±1.6 12.5±1.6 12.5±1.5 12.6±1.6 12.5±1.6 12.5±1.5

Final length (mm) 19±3c 19.3±2.1c 14.3±1.7d 22.3±3.5b 21.6±2.3b 20.1±1.9c 14.4±1.8d 25.1±2.6a

Shell growth rate (μm d-1) 77.7±3.2c 81.9±7.8c 20.8±0.8d 118.4±7.9b 110.1±0.8b 90.5±8.4c 23.0±1.2d 150.9±3.9a

SGR (% day-1) 1.5±0.02c 1.4±0.2 c 0.2±0.0d 2±0.1b 1.9±0.0 b 1.4±0. 1c 0.3±0.04d 2.3±0.04a

Initial weight (g) 0.2±0.1 0.3±0.1 0.3±0.1 0.3±0.9 0.3±0.1 0.3±0.1 0.3±0.1 0.3±0.9

Final weight (g) 0.9±0.3c 0.8±0.4c 0.3±0.1d 1.4±0.6b 1.2±0.4b 0.9±0.3c 0.3±0.1d 1.8±0.6a

Weight gain (%) 239.9±6.5c 255.9±7 c 17±3.3 d 410±37.5 b 371.6±12.6 b 229.9±3 c 33±4.3d 561.3±20.3a

Feed intake (mg abalone-1day-1) 63.5±2.5d 147.4±10.0b 31.8±2.5e 168.7±18.3ab 81.0±0.8c 173.7±17.1ab 35.4±0.1e 189.9±5.0a

FCR 9.0±0.5e 21.0±2.8c 188.1±8.9a 12.9±0.6cd 6.8±0.3e 22.4±0.4c 57.1±6.0b 10.1±0.2e

PER 3.7±0.2a 2.3±0.3b 1±0.0 d 3.4±0.2 a 2.4±0.1b 0.8±0.01d 0.5±0.2e 1.8±0.04 c

CI (%) 2.9±0.4b 2.9±0.4b 2.4±0.5c 3.1±0.4ab 3.3±0.3a 2.8±0.4b 2.4±0.4c 3.3 ±0.3a

Survival (%) 93.3ab 100a 85.3b 97.3a 93.7ab 96ab 92ab 98.7a

Values in the same row with different letters are significantly different (P<0.05) n =25x3.

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5.3.3. Fatty acid composition of macroalgae

Fatty acid profiles of the eight macroalgae diets are summarised in Table 3.
The lipids of all algae tested consisted mainly of saturated fatty acids (SFA) (43-
60%), with palmitic acid (16:0) as the most abundant fatty acid (FA) (40-47%) of
total FAME. A higher amount (5-7.2%) of myristic acid (14:0) was detected in both
Rhodophyta species as compared to that in the green algae (1%). The green algae U.
rigida showed higher levels of C16 (58%) and C18 (32%) fatty acids and lower level of
C20 fatty acids (1.8-1.1%) than that of red algae. 18:1n-7 was the predominant mono-
unsaturated fatty acid of this Chlorophyta. All red algal treatments contained
considerable levels of mono-unsaturated fatty acid predominantly 18:1n-9. Linoleic
acid (18:2n-6) was present at similar levels in all selected algae, whereas linolenic
acid (18:3n-3) was higher in U. rigida. The levels of ∑ n-6 PUFA (7-9%) were
generally lower than those of ∑ n-3 PUFA (12-18%). Eicosapentanoic acid (20:5n-3)
(EPA) highest percentage was found in red algae H. spinella (6.3-6.9%), being ten
times higher than the one of U. rigida (0.7%). All macroalgae presented very low
level of arachinodic acid (0.1-1.6%) (ARA) and docosahexaenoic acid (DHA) (22:6-
n-3), with the highest percentage been found in enriched G. cornea with 3% of total
FAME.

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Table 21. Fatty acid composition (% total fatty acids) of the eight macroalgae treatments

Enriched Enriched Enriched Enriched

FA U. rigida H. spinella G. cornea Mixed diet
U. rigida H. spinella G. cornea Mixed diet

14:0 1.1 6 5.2 4.1 1 7.2 5 4.4

16:0 46.8 41.3 50.3 46.1 40.5 44.4 47.5 44.1

18:0 1.9 5.4 4.2 3.8 1.9 3.3 4.4 3.2

∑SFA 49.8 52.7 59.7 54.1 43.3 54.9 56.9 51.7

14:(1n-5) 1.64 2.42 0.47 1.51 1.57 3.24 0.44 1.75

16:(1n-7) 6.1 2.5 2.3 3.6 9.6 2.9 3.5 5.3

16:(1n-5) 0.2 0.3 1.8 0.8 3 0.3 0.6 1.3

18:(1n-9) 5 10.6 8.8 8.1 3.5 9.7 10.5 7.9

18:(1n-7) 9 2.5 3 4.8 8.3 3.1 3.5 5

22:(1n-11) 1.9 0.3 0.1 0.8 2 0.3 0.1 0.8

∑MUFA 23.9 18.6 16.5 19.7 27.9 19.5 18.6 22

16:(4n-3) 2.7 0.8 1.4 1.6 3.8 0.5 1.6 1.9

18:(2n-6) 7 7.7 5.3 6.7 6.1 5.5 6.1 5.9

18:(3n-3) 5.7 1.9 0.6 2.7 7 1.5 0.8 3.1

18:(4n-3) 3.3 1 0.7 1.6 4.2 0.9 0.6 1.9

20:(4n-6) 0.1 0.4 1.6 0.7 0.1 0.5 0.7 0.4

20:(4n-3) 0.4 2.9 7.1 3.4 0.3 3.8 4.5 2.9

20:(5n-3) 0.6 6.3 0.8 2.6 0.7 6.9 1.9 3.2

22:(5n-3) 1.6 0.5 0.3 0.8 1.5 0.4 0.5 0.8

22:(6n-3) 0.5 1.1 0.9 0.9 0.5 0.5 3 1.3

∑PUFA 21.8 22.5 18.7 21.0 24.2 20.4 19.6 21.4

Other 4.5 6.2 5.1 5.2 4.6 5.2 4.9 4.9

∑n-3-FA 14.7 14.5 11.8 13.6 18.0 14.5 12.9 15.1

∑n-6-FA 7.1 8.1 6.9 7.4 6.2 6.0 6.7 6.3

SFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids;
Other includes all components <1%: 14(1n-7), 15:00, 16:0ISO, 16:(2n-6), 16:(2n-4), 17:00, 16:(3n-
3), 16:(3n-1),16:(4n-1), 18:(1n-5), 18:(2n-4), 18:(3n-6), 18:(3n-4), 20:00, 20:(1n-9+n-7), 20:(1n-5),
20:(3n-6), 22:(1n-9), 22:(4n-6), 22:(5n-6)

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5.3.4. Effects of algal diets on the general biochemical composition of the

animals

Nutritional analysis of the abalone at the end of the experimental period

revealed that diet did significantly affect the soft body tissues of the animals (Table
4). Protein levels in foot muscle (74-76% DW) did not differ significantly (P<0.05)
among treatments. Generally the biofilter cultured algae produced better growth and
resulted in significantly lower moisture levels in viscera than others. Lipid levels in
viscera varied considerably within the feeding regimes, being higher in abalone fed
non enriched macroalgae. Abalone fed both enriched and non enriched macroalgae,
showed higher lipid levels, stored in the viscera (11-20%), rather than in the foot
muscle (5-8%), the highest being found in those fed with M and U diets. Ash content,
both in viscera and muscle, in animals fed non enriched algae, were generally higher
than that of abalone fed with macroalgae produced out of fishpond waste water
effluents.

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Table 22. Proximate composition of foot tissues of Haliotis tuberculata coccinea reared on the experimental diets (g/100 g DW) (Mean ± S.D.)

Diets Moisture Crude protein Crude lipid Carbohydrate1 Ash

Viscera Muscle Viscera Muscle Viscera Muscle Viscera Muscle Viscera Muscle

U. rigida 73.0±0.3b 75.7±0.1a 63.4±0.0b 74.0±0.1 19.4±1.8a 7.1±0.2ab 9.4±0.9d 9.9±0.4ab 10.1±0.7a 9.1±0.2c

H. spinella 73.7±0.9ab 75.3±0.3a 55.4±1.3d 74.5±1.5 16.8±1b 4.8±1.3b 18.8±2.1b 10.1±0.5ab 9.8±0.1ab 10±.1ab

G. cornea 75.3±2.4a 75.5±0.0a 63.1±0.1b 74.1±0.2 15.3±1.4bc 5.8±1.3ab 11.6±1.1c 10.6±1.7 ab 9.1±0.2abc 9.7±0.2bc

Mixed diet 73.8±0.6ab 74.5±0.4b 63.3±1.4bc 74.1±0.2 20.5±0.5a 6.7±1.2ab 7.7±1de 8.2±1.3c 8.4±0.2c 11.0±0.3a

Enriched
70.2±0.2c 73.9±0.3b 61.3±0.3bc 74.4±0.5 16.2±0.3b 5±0.2b 14.1±0.3c 12.6±0.7a 8.5±0.4c 8.1±0.3d
U. rigida
Enriched
73.2±0.1ab 75.4±0.3a 73.7±0.9a 76.3±1.2 12.8±0.4cd 7.3±0.3ab 4.9±0.1e 9.2±0.7 ab 8.9±0.5bc 6.9±0.4de
H. spinella
Enriched
71.4±0.1bc 74±0.3b 55±0.2d 76±1.6 11.5±0.6d 7.9±1.1a 24.4±0.1a 7.4±1.6c 9.5±0.1abc 9.3±0.6bc
G. cornea
Enriched
70.7±0.4c 75.6±0.1a 61.5±0.6c 75.9±2.6 12.2±1.1d 5.6±0.3ab 19.5±1.6b 10.6±3.1ab 7.1±0.3d 7.9±0.1de
mixed diet
1
Calculated by difference (AOAC, 2005)
Values in the same column with different letters are significantly different (Tukey test, P<0.05 n=3)

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5.4. DISCUSSION

Gross energy is the total amount of energy supplied by food and is an

important quantitative measurement of calorific value which may be an useful
indicator of the seaweed nutritional value (Lamarae and Wing, 2001; Hernández-
Carmona and Carrillo-Domínguez, 2009). Despite the ability of abalone to utilise a
wide variety of energy sources, being a mollusc, the metabolic rate of abalone is low
and consequently energy requirements are low. The caloric content (gross energy) of
the algae tested (3.5-4.1Kcal g-1) was in the range of values reported for other diets
used as feed for abalone generally reported around 4 Kcal g-1 (Shipton and Britz,
2001; Reyes and Fermín, 2003; García–Esquivel and Felbeck, 2006). Protein content
(11-17% DW) of the non enriched macroalgae was within the range of values
reported for other species of red and green seaweeds used as feed for abalone (13-
18% DW), (Mercer et al., 1993; Bautista-Teruel and Millamena, 1999; Wong and
Cheun, 2000; Jackson et al., 2001; Reyes and Fermín, 2003). The protein content of
seaweed species varies greatly and demonstrates a dependence on factors such as
season and growing conditions. The high protein content of macroalgae produced
using fishpond waste water effluents (29-34%) compared with those reared in fresh
seawater, would be related to its production under the high nitrogen culture conditions
of the biofilter system, as it has also been observed in previous research with Ulva
spp., Hypnea spp. and Gracilaria spp., used as macroalgal biofilters (Shpigel et al.,
1996a, b, 1999; Boarder and Shpigel, 2001; Robertson-Andersson, 2003; Viera et al,
2005; Njobeni, 2006). The crude lipid contents were low in the algae studied, ranging
between 1.4 to 7.2% DW, and being generally higher in enriched seaweeds, which is
comparable to the range reported for other macroalgae (0.6–6.15% DW) (Wong and
Cheun, 2000; Dawczynski et al., 2007; Hernández-Carmona and Carrillo-Domínguez,
2009). Nevertheless, abalone species show a low lipid requirement, typical of
herbivores molluscs and fish (Mai et al., 1995a). This low lipid requirement has been
associated by some authors with a reduced use of dietary lipids as energy source by
abalone based upon its low metabolic rate (Durazo-Beltrán et al., 2004). Studies on
the digestive enzymes of some species of Haliotis reveal that abalone have low
activities of lipases, chymotrypsim or aminopeptidase (García–Esquivel and Felbeck,
2006). Indeed, high levels of dietary lipid seem to affect negatively abalone growth

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(Thongrod et al., 2003). However, high levels of carbohydrate enhance growth of

abalone presenting high amylases activities and other carbohydrate digestive
enzymes, such as cellulase, agarase and alginate lyase, (Britzs, 1994; Mai et al, 1996;
Thongrod et al., 2003 García–Esquivel and Felbeck, 2006), as well as a good capacity
to synthesize non essential lipids from carbohydrates. Carbohydrates are the largest
component in many algae. In the present study, carbohydrate contents were high and
inversely related to protein contents with comparable values to those obtained for
other algal species (Kaehler and Kennish, 1996; Foster and Hodgson, 1998;
Hernández-Carmona and Carrillo-Domínguez, 2009). Ash was the second highest
fraction in all the diets, after carbohydrates, with similar values to those reported
previously for other species of the same genus (Wong and Cheun, 2000; Hernández-
Carmona and Carrillo-Domínguez, 2009).
Abalone fed enriched macroalgae diets showed better performance in terms of
growth rate per day, weight gain, increase in shell length and FCR values compared to
those fed non enriched diets. This could be related to the high protein content of
enriched macroalgae diets, suggesting that nitrogen may be a limiting factor for
growth in Haliotis spp. (Fleming, 1995; Boarder and Shpigel, 2001). Previous studies
support this view by stating that maximum growth can only be achieved when
sufficient protein, in the correct proportions of amino acid, is supplied in the feed.
Shpigel et al. (1996a, 1999), Boarder and Shpigel (2001), Viera et al. (2005) and
Naidoo et al. (2006) respectively, stated that satisfying growth of. H. tuberculata, H.
roei, H. t. coccinea and H. midae fed enriched macroalgae was attributable to a
consistent supply of high protein diet.
Besides, abalone fed mixed algal regimes, both enriched and non enriched,
performed significantly better than those that were fed with a single algal diet, in
agreement with studies showing that “mixed” diets produce better growth rates than
single-species diets (Naidoo et al, 2006). This suggest that abalone obtain a complete
range of required nutrients by eating a mixed algal regime and that essential nutrients
may become limiting in trials where animal are fed single-species diets. The growth
rate of abalone increased significantly with an increasing PE ratio up to a level of
32% protein / 6% fat. Similar observations have been reported for H. midae fed with
several dietary protein and energy levels (Britz and Hecht, 1997). With the exception
of animals fed both G. cornea treatments, growth rates values in this study (78-151

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μm day-1) were generally higher than both, those obtain by other authors with other
species (50-100 μm day-1; Viana et al., 1996, 2000; Guzmán and Viana, 1998;
Jackson et al., 2001; Gómez-Montes et al., 2003) under similar experimental
conditions, and those obtain under commercial conditions (80 μm day-1; Gómez-
Montes et al., 2003). SGR values in the present study (1.4 to 2.3%) were higher than
those reported by Mercer et al. (1993) and Mai et al. (1995b) in abalone fed several
species of macroalgae who reported SGR values of 0.8 % for H. tuberculata and 0.7-
1% for H. discus hannai, and similar to the results reported by Capinpin et al. 1996
(2.5%) for H. asinina fed Gracilariopsis heteroclada. In an aquaculture context, the
rate of weight gain is vitally important, particularly in the case of haliotids, as they are
relatively slow growing. Therefore, from an aquaculture perspective, an optimal
dietary protein level should be defined in terms of growth rate as well as dietary
ingredient cost. In the present study, except with G. cornea treatments, all feeding
regimes produced a similar or better weight gain (230-561%) than Gracilariopsis
bailinae (134%, Bautista-Teruel and Millamena, 1999) or compound diets (454%,
Bautista-Teruel et al., 2003) in H. asinina under similar conditions. Regarding G.
cornea, the lowest growth rate obtained with abalone fed this macroalgae, might be
due to the lowest feed intake registered. Indeed, consumption rate in abalone may be
influenced by various factors that may decrease algal palatability, such as, the
structure, growth form, and thallus toughness (Steneck and Watling, 1982). Hence,
the harder texture of G. cornea may have affected its consumption by H. t.coccinea,
since in the wild, abalone prefer soft textured macroalgae (Shepherd and Steinberg,
1992). This preference seem to be related to the little capacity of the rhipidoglossan
radula to penetrate the algal surface (Steneck and Watling, 1982) as its teeth have
limited ability to exert force against the substrate. However when the animals were
fed G. cornea as a proportion of a mixed diet, they exhibited excellent growth rates,
suggesting that this red seaweed provided essential nutrients not found in the other
algae fed.
Except in animals fed G. cornea, food conversion rate (FCR) values were
within the range of those reported for H. asinina, H. discus hannai, H. tuberculata or
H. t. coccinea fed seaweeds diets (Kunavongdate et al., 1995, Shpigel et al., 1999;
Viera et al., 2005). The lower FCR attained for abalone fed enriched seaweeds in the
present study seems to be explained, not only by the general composition of the algae,

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but also by a significantly higher protein:energy ratio than the rest of dietary
treatments. PER values observed on the non enriched treatments agreed well with
those reported for H. discus hannai (Uki et al, 1985a) and H. midae (Britz, 1996b) fed
seaweeds or H. midae and H. fulgens fed compound feeds (Britz and Hecht, 1997;
*Gómez-Montes et a., 2003). Protein efficiency ratios were generally lower in
abalone fed enriched macroalgae. Similar observations have been reported by Uki et
al., (1986), Britz (1996a) and Bautista-Teruel and Millamena (1999), who found that
the growth rate of H. discus hannai, H. midae and H. asinina respectively, increased
with an increase in protein content whereas the PER was negatively correlated with
protein level. Despite the poorer efficiency of protein conversion by abalone fed the
enriched macroalgae, higher weight gain and lower FCR were obtained with an
increasing protein level in the algal diet. This increase in protein level, linked to the
culture conditions of the integrated culture system, is of high economic significance
for the production.
Soft-body to shell ratios of the experimental animals (2.4-3.3) were similar
and even higher than those recorded by Mai et al. (1995a) for H. tuberculata (1.8-2.1)
and H. discus hannai (2-2.4) fed with P. palmata and various levels of dietary lipids
or by Sales et al. (2003) for H midae (2.9-3.2) fed different dietary crude protein
level. Furthermore, the high survival of abalone noted for all treatments may well
indicate a general balance of nutrients in the diets, although the low feed intake of
both G. cornea diets may not have been enough to sustain comparable growth of
abalone with those fed the rest of the experimental diets.
The algae studied presented typical fatty acid patterns of green and red algae
in agreement with previous macroalgal studies (Mai et al., 1996, Li et al., 2002).
Palmitic acid was the most abundant SFA, at similar contents of those reported for
other species of seaweeds (Jackson et al., 2001; Li et al., 2002; Nelson et al., 2002).
The fatty acid composition of the Chlorophyta Ulva rigida with predominat levels of
C16 and C18 PUFAs and minimal levels of C20 fatty acids followed a pattern similar to
the ones reported for other species of Ulvales such as U. lactuca (Mai et al., 1996)
and U. pertusa (Li et al., 2002). Accordingly, the higher level of 18:3-n-3 and 18:1-n-
7 relative to green algae, has been regarded as a characteristic of this phylum with a
particular taxonomic value in Chlorophyta species (Johns et al., 1979). All
macroalgae presented very low levels of 22:6-n3, with similar results found in other

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studies (Dawczynski et al., 2007). Hence DHA do not appear to be an essential FA in

H. t. coccinea as they were detected in a very low level in all macroalgae tested
despite they supported optimal growth of abalone. The growth promotion of EFA for
abalone is generally dependent upon a collective effect of certain combination of
different PUFA rather than on a single fatty acid.
Analyses of the abalone fed on the various diets showed that the biochemical
composition of various organs were markedly affected by the diets. Lipid content in
abalone tissues were generally higher than in their respective macroalgal diets
(Nelson et al., 2002). Foot muscle contained significantly lower lipid levels than
viscera indicating that selective storage of lipids occurs in the hepatopancreas/gonad
assemblage. Similar observations have been reported by Webber (1970), Mercer et al.
(1993) and Nelson et al. (2002), for H. cracheroidii, H. tuberculata and H. discus
hannai, and H. fulgens respectively.
In conclusion, the dietary value of the macroalgal regimes tested can be
divided into three categories based on the growth performances observed: Best
obtained with the mixed algal feeding regime, intermediate by using single Ulva
rigida or Hypnea spinella feeding regimes and the lowest by Gracilaria cornea.
Nevertheless, based on growth performance and nutritional indices, this study clearly
demonstrates that the macroalgae produced in a biofiltering system are enriched in
dietary protein and lipids and that their nutritional composition is matching the
protein, lipid and carbohydrate requirements of abalone resulting in satisfying growth
and survival of H. tuberculata coccinea. Results clearly indicate that H. tuberculata
coccinea can be efficiently grow-out in an integrated-culture system suggesting that
on-farm seaweed-abalone production could be a part of future development of
abalone industry in the Canary Islands.

5.4. ACKNOWLEDGEMENTS

The authors would like to thank to Dr. D. Montero for his valuable comments
on the manuscript. This study has been financed by the Spanish Government in the
frame of the National Plan for Development of Marine Cultures (JACUMAR, Oreja
de mar) and by the Canarian Government (PI 2007/034).

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Development of a Sustainable Grow-out Technology for Abalone Haliotis
tuberculata coccinea (Reeve) as a New Species for Aquaculture
Diversification in the Canary Islands

STUDY III: First Development of Various Vegetable-Based Diets

and their Suitability for Abalone Haliotis tuberculata coccinea

Aquaculture, submitted
Study III Aquaculture (2014), submitted

First development of various vegetable-based diets and their suitability for

abalone Haliotis tuberculata coccínea Reeve

M.P. Viera*, G. Courtois de Viçose, L. Robaina, M.S. Izquierdo

Grupo de Investigación en Acuicultura (GIA), Universidad de Las Palmas de Gran Canaria

(ULPGC). Las Palmas, Canary Islands, Spain

Abstract

To date, European abalone aquaculture relies mostly on locally harvested fresh

seaweeds which nutritional quality and abundance varies greatly, hence affecting abalone
growth. Abalone artificial diets generally include fishmeal, limiting their utilization in
ecologically sustainable aquaculture and affecting abalone quality and acceptance by the
consumers. A six month feeding trial was conducted to assess the nutritional value of four
different algae: Ulva lactuca (Chlorophyta), Gracilaria cornea (Rhodophyta), Laminaria
digitata (Phaeophyta) and Palmaria palmata (Rhodophyta), as ingredients to all-vegetal-
based formulated feeds for abalone Haliotis tuberculata coccinea (33.1 ± 0.8 mm and 4.7 ±
0.6 g). A mixed fresh algae diet of G. cornea and U. rigida, reared in an IMTA, served as
control. Survival rates were very high (95-98%), regardless of the diet fed. Enriched fresh
algae produced a far better growth for H. tuberculata coccinea (169% WG) than all the
artificial diets (49-84% WG). Comparison among abalone fed the different formulated diets
showed that the inclusion of P. palmata improved growth, condition index and dietary protein
utilization. On the contrary, the use of L. digitata markedly reduced the efficiency of dietary
protein since the protein-related nutritional index (PER), the percentage of protein deposited
in the foot muscle as well as the condition index recorded for animals fed this diet were the
lowest, despite a higher feed intake. Large differences were found in the FA profile of fresh
algae as compared with the three formulated diets, n-3/n-6 ratio being was much lower in the
latter ones and, consequently, in the foot tissues of abalone fed the control diet relative to
those fed the formulated ones. The elevated contents of 20:4n-6 in the abalone fed the
experimental diets and 20:5n-3 in abalone fed the fresh algae, as well as their respective
metabolites, suggest that abalone have the ability to desaturate and elongate LA to ARA and
ALA to EPA. Further studies are required to improve the growth obtained with this type of
diets, especially concerning the use of different seaweed combinations and inclusion levels, as
well as the diet processing methods to improve diets water stability.
Keywords: abalone; growth; artificial diets; seaweeds; IMTA

Corresponding author: M. P. Viera

Phone: (34) 928 132034
E-mail: mapi.viera@giaqua.org
Address: Muelle de Taliarte s/n, 35214, Telde, Gran Canaria, Canary Islands, Spain

109
Study III Aquaculture (2014), submitted

6.1. INTRODUCTION

The European native abalone species (Haliotis tuberculata spp.) is a highly

appreciated European shellfish product in both traditional markets (Mgaya and
Mercer, 1994) and novel premium quality abalone ones (Dallimore, 2010). However,
its availability is severely restricted due to lack of supplies from both wild resources
and aquaculture production. Due to abundance of macro-algae on Western Europe,
most European farms grow abalone feeding them locally harvested fresh seaweeds
(Fitzgerald, 2008). However, macroalgae nutritional quality and abundance varies
greatly according to geographic location and time of sampling (Dawczynski et al.,
2007), what greatly influences abalone growth rates, affecting the economic success
of the on-growing activity (Bautista-Teruel and Millamena, 1999).

Among the different nutrients, abalone requires adequate levels of high quality
protein for soft tissue growth (Uki et al., 1985a; Mai et al., 1995a, b; Britz and Hecht,
1997; Bautista-Teruel and Millamena., 1999; Gómez-Montes et al., 2003; Reyes and
Fermín, 2003; Viana et al., 2007). The most common protein sources employed in
abalone feeds include fishmeal, defatted soybean meal (Guzmán and Viana, 1998;
Sales and Britz, 2002; Gómez-Montes et al., 2003; Thongrod et al., 2003; Naidoo et
al., 2006; García-Esquivel et al., 2007), casein (Uki et al., 1985b; Viana et al., 1993;
Mai et al., 1995b; Sales et al., 2003; Vandepeer and van Barneveld, 2003) and
Spirulina spp. (Uki et al., 1985b; Britz et al., 1996a; Bautista-Teruel et al.,
2003;Thongrod et al., 2003; Naidoo et al., 2006; Troell et al., 2006). Few novel
protein sources have also been tested at low inclusion proportions (Vandepeer et al.,
1999, Vandepeer and van Barneveld, 2003; Shipton and Britz, 2001; Sales and Britz,
2002; Reyes and Fermín., 2003). To get advantage of the high nutritional value of
algae, algal meals have been occasionally included in abalone feeds (García-Esquivel
et al., 2007; Viana et al., 2007), reducing the cost and use of fresh algae. Fishery by-
products such as fish or abalone viscera silage have been also proposed as economic
protein sources (Lopez and Viana, 1995; Viana et al., 1996; Rivero and Viana, 1996;
Guzmán and Viana, 1998). Besides, different protein sources may be balanced by
addition of synthetic amino acids such as methionine, threonine and arginine in order
to fulfil the essential amino acid requirements of these species (Mai et al., 1995b;
Guzmán and Viana, 1998; Serviere-Zaragoza et al., 2001; García-Esquivel et al.,

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Study III Aquaculture (2014), submitted

2007). Among all the sources tested as a single protein source, fishmeal is the only
one that supports good growth performance (Fleming et al., 1996).

Studies with cultured abalone have demonstrated that diet can have a
significant effect on quality-related factors such as chemical composition, taste,
texture and colour (Dunstan et al., 1996; Chiou and Lai, 2002; Allen et al., 2006). In
particular, the lipid composition of abalone muscle is markedly affected by the diet
(Uki et al., 1986; Dunstan et al., 1996). Moreover, lipids (especially long-chain
polyunsaturated fatty acids, PUFA) are essential to determine the flavour and odour of
seafoods (Lindsay, 1988). Thus, the use of artificial feeds containing fishmeal could
give cultured abalone a much “fishier” flavour than diets containing vegetable sources
(Dunstan et al., 1996). Indeed, a way of improving market acceptability and product
quality includes feeding abalone on macroalgae immediately prior to sale (Dunstan et
al., 1996; Kinkerdale et al., 2010). Moreover, abalone output in Europe is
substantially focused on high quality and low volume niche markets, such as organic
or eco-certified products, implying that, among other requirements, no fishmeal,
pharmaceuticals or fertilizers are used. Hence, developing a vegetal based artificial
feed for European abalone, will have marketing benefits not only for consumers, who
are increasingly environmentally sensitive, but also for producers, providing them a
readily available and more stable nutritional feed, whereas validating the
environmental and social sustainability of their farming operations (SUDEVAB,
2007; WWF, 2010).
In the wild, abalone consumes a variety of seaweeds, obtaining its required
nutrients from a combination of algal species (Sales and Britz, 2001; Dlaza, 2008).
These seaweeds are selected mainly according to their abundance and availability in
the surrounding area (Nelson et al., 2002). In the case of the European abalone, red
algae such as Palmaria palmata and the green ones Ulva spp. are preferred.
Nevertheless, other coarser and abundant seaweeds like kelps Laminaria spp,. (Koike
et al., 1979; Mercer et al., 1993) are commonly used as a bulk feed in abalone
farms(Fitzgerald, 2008; Walsh and Watson, 2011). Earlier studies have shown that
fresh P. palmata, Ulva spp. or the rhodophyte Gracilaria spp. have a high dietary
value for H. tuberculata spp., (Culley and Peck, 1981; Mercer et al., 1993; Viera et
al., 2005, 2011), showing also that mixed diets produced far better growth rates than
single-species diets, which is generally accepted for most abalone species (Simpson

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Study III Aquaculture (2014), submitted

and Cook, 1998; Dlaza et al., 2008; Naidoo et al., 2006; Kinkerdale et al., 2010;
Robertson-Andersson et al., 2011 ).

Therefore, the objective of the present study was to test the nutritional value of
the most commonly used fresh abalone feed in Europe: Laminaria digitata
(Phaeophyta), Palmaria palmata (Rhodophyta), Ulva lactuca (Chlorophyta) and
Gracilaria cornea (Rhodophyta) as alternative ingredients to obtain more sustainable
feeds for abalone (Haliotis tuberculata coccinea) production in Europe.

6.2. MATERIAL AND METHODS

6.2.1. Processing of seaweed meals, diets formulation and preparation

Fresh G. cornea (G), U. lactuca (U), L. digitata (L) and P. palmata (P) (kindly
supplied respectively by the Centro de Biotecnología Marina (CBM-ULPGC), Gran
Canaria, Spain; South West Abalone Growers Association (SWAGA), Cornwall, UK;
Martin Ryan Institute (MRI), Galway, Ireland and France Haliotis (FRHAL),
Brittany, France), were cleaned, washed with freshwater to remove salts and epizoos
and oven dried at 35ºC. The dried samples were finely ground using an electric fine
mill (sieve size <0.1 mm) and frozen at -80 ºC until analysis. Algal meals were
analyzed for nutrient composition and aminoacid profile (Table 23).

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Study III Aquaculture (2014), submitted

Table 23. Proximate and aminoacid composition of seaweed meals used in

experimental feeds for abalone H. tuberculata coccinea (%DW)

G. cornea U.lactuca L. digitata P. palmata

Crude protein 20.3 17.0 8.4 15.0

Crude lipids 2.6 3.5 2.5 2.8
Carbohydrates* 36.3 57.4 65.7 67.5
Ash 40.7 22.1 23.6 14.7
Moisture 10.1 4.2 13.2 11.2

Amino acids
Alanine 8.5 7.2 10.7 7.2

Arginine 6.4 13.4 4.4 6.8

Aspartic 13.2 10.8 12.2 12.4

Cystine 0.0 0.0 15.7 0.8

Glutamic 12.8 13.8 10.1 12.4

Glycine 6.9 6.2 4.3 6.3

Histidine 2.6 1.4 2.9 2.1

Isoleucine 4.1 4.6 3.5 4.3

Leucine 6.7 7.4 5.7 6.8

Lysine 5.6 3.7 4.4 5.7

Methionine 1.9 2.4 2.0 2.4

Phenylalanine 5.1 4.8 3.5 4.6

Proline 4.9 4.4 4.0 5.0

Serine 5.7 5.3 3.8 5.2

Threonine 5.2 4.6 4.1 4.6

Tryptophan 1.2 0.9 1.5 2.9

Tyrosine 3.2 3.1 2.7 4.2

Valine 6.2 6.0 4.5 6.2

*
Calculated by difference (AOAC. 2005)

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Study III Aquaculture (2014), submitted

Based on these meals, three diets were formulated to contain 35% protein,
17% of which was contributed by the different selected seaweeds meals: U and G; U,
G and L and U, G and P (Tables 23 and 24). The remaining protein and energy
contents were supplied by soybean meal, corn gluten meal, spirulina and starch, such
that each experimental diet had a similar lipid content (4%) and total energy (4kcal g-
1
). These inclusion levels have been reported by Britz and Hetch (1997), Viana et al.
(2007) and Viera et al. (2011) as being optimal for abalone growth. Vitamin and
mineral mixtures were used as recommended by Uki et al. (1885a). All the
experimental diets were supplemented with synthetic L- methionine and lysine in
order to match the amino acid profile of abalone muscle which was used as a guide to
formulate the amino acid composition of the practical diets. Sodium alginate, which
has been suggested to increase protein efficiency when feeding with seaweed (Kemp
and Britz, 2012) was used as binder (Table 24).

Experimental diets were prepared by mixing pre-weighed finely ground

ingredients including vitamins and minerals to produce a homogeneous mixture. The
diets were then processed through a pasta machine (Parmigiana, RV3, Italia) into 2
mm thick strips from which 0.5 x 0.5 pieces were cut dried at 38ºC for 24 h and stored
at 4ºC until use.

A mixed fresh algae diet of G. cornea and U. rigida served as control.

Seaweeds were reared within the Grupo de Investigación en Acuicultura (GIA,
Canary Islands, Spain) aquaculture research facility, in a flow-through integrated
system collecting wastewater from fish and abalone ponds in a macroalgal biofilter
(Viera et al., 2014).

Experimental and control diets were sampled for proximate composition. The
algal samples were processed as indicated above and artificial diets were held under
the same conditions as algal samples until analysis.

All the diets were tested for their stability in seawater under the same
conditions as the feeding experiment in tanks without abalone, by measuring the loss
of dry matter of pellets after 17 h of immersion (16:00-9:00) and of natural diet for a
3-days period.

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Study III Aquaculture (2014), submitted

Table 24. Ingredients of the three experimental diets for abalone H. tuberculata coccinea
(DW basis)

Experimental diets1
Ingredients
UG UGL UGP

G. cornea 16 16 16
U. lactuca 27 15 15
L. digitata - 12 -
P. palmata - - 12
2
Spirulina 2 2 2
3
Soybean meal 22 22 22
Corn gluten4 18 18 18
5
Starch 4 4 4
6
Vitamin mix 2.5 2.5 2.5
Mineral mix6 1.5 1.5 1.5
7
Lys 1.35 1.35 1.35
7
Met 0.65 0.65 0.65
Na alginate 5 5 5
1
UG: U. lactuca + G. cornea; UGL: U. lactuca + G. cornea + L. digitata; UGP: U. lactuca +
G. cornea + P. palmata.
2
50.8% protein, 9.2% lipid.
3
48.2% protein, 3.6% lipid
4
78.3% protein, 6.3% lipid
5
8.4% protein, 4.7% lipid.α cellulosa. Sigmabrand
6
Uki et al.. 1985a
7
Sigma (UK)

6.2.2. Abalone and experimental procedure

Three hundred and sixty (30/replicate) 1-year-old H. tuberculata coccinea

individuals with an average shell length and weight of 33.1 ± 0.8 mm and 4.7 ± 0.6 g
respectively, were selected from the experimental hatchery production unit of GIA.
Animals were blot dried, weighed to the nearest 0.1 mg (total fresh body weight:
TFBW), measured with manual caliper with 0.1 mm accuracy (total shell length: SL)
and distributed among triplicates in a flow-through system. The experimental unit

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Study III Aquaculture (2014), submitted

consisting of a plastic bucket (15x16cm), hung in a 100-l rectangular tank

(100x40x25cm) filled with seawater provided with constant aeration. Water flowed at
2.8 l/min and two PVC shelters were provided in each container. Water temperature
ranged between 22.6-24.5ºC and abalones were subjected to a natural photoperiod of
approximately 12 h L / 12 h D. Animals were conditioned on the test diets for two
weeks before data collection begun. The feeding trial was run for 176 days and dead
abalones were daily recorded.

Artificial feeds were offered once daily (ad libitum) in the evening from
Monday to Saturday. Any remaining diet was collected every day at 9:00 h. except
Sunday, by manually siphoning uneaten feed from tanks. Consumption was estimated
on a dry weight basis by relating the dry weight of the uneaten food to the known dry
weight of the feed provided. Consumption data were corrected for dry matter weight
loss attributable to leaching, by allowing the diets to leach over a 17-hperiod (16:00-
9:00h) using a “control” rearing unit without abalone, and drying the remaining diet
until constant weight. To guarantee ad libitum feeding in the control diet, fresh algae
were supplied well in excess twice a week. To determine control feed intake, freshly
collected algae to be fed to the abalone were blotted dry and accurately weighed as
well as the remaining algae every third day. The weight of unconsumed food was
deducted from the total weekly ration. Besides, weight of uneaten algae was corrected
by calculating the natural change in weight of the algae in the control units during the
same feeding period. The average daily intakes by individual abalone during the
entire feeding trial were calculated by dividing the algal biomass eaten each week by
the feeding days and the number of abalones in each experimental unit.

SL and TFBW of each animal were monthly recorded. Abalone growth rates
(growth rate day-1), as well as the following indices, were calculated for all treatments
at the end of the trial:

Shell growth rate (μm d-1) = (SL2-SL1) / days of culture x 1000

Specific growth rate, SGR (%d-1) = (LnW2-LnW1) / days of culture x 100
Weight gain, WG (%) = ((W2-W1) / W1) x 100
Feed conversion ratio, FCR = total feed intake (g dry) / total weight gain (g
wet)

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Study III Aquaculture (2014), submitted

Protein efficiency ratio, PER = (increase in body wet weight (g)) / (protein
intake (g))

where L1 is the initial mean length of animals; L2 is the final mean length of animals;
W2 is the weight at time t (days of culture), and W1 is the initial weight.

Besides, six abalones were collected from each experimental unit, and the soft
tissue was shucked from the shell. Shell and meat were then weighed separately in
order to calculate the condition index (wet weight of soft flesh/wet weight of shell.
SB/S in W/W) as an indicator of the abalone nutritional status.Abalone tissues were
also sampled and frozen at -80 ºC until analysis.

6.2.3. Proximate, amino and fatty acid analysis

Homogenized samples of the seaweed meals, formulated diets, fresh algae and
abalone (visceral mass and foot muscle) were analyzed in triplicate for nutrient
composition. Amino acid was done on each experimental seaweed meal using an
HPLC amino acid analyzer. Protein content was determined by Kjedahl method
according to AOAC (2005) standard analyses. Total lipids were extracted with
chloroform–methanol (2:1) as described by Folch et al. (1957). Fatty acids in the lipid
extracts were transesterified to methyl esters (FAMEs) with 1% sulphuric acid:
methanol complex (Christie, 1982). FAMEs samples were extracted into hexane and
stored at -80ºC. Fatty acids were analyzed in a Thermo Finnigan- GC Focus gas
chromatograph equipped with a flame ionization detector (260ºC). FAMEs were
separated with capillary column (Supercowax 28m x 0.32mm x 0.25 i.d.) using
helium as the carrier gas under the conditions described by Izquierdo et al. (1989).
Fatty acids of the experimental and control diets and abalone were also analyzed. The
dry matter was determined by drying at 110ºC until constant weight (AOAC, 2005).
Ash content was determined by incinerating samples at 600ºC for 24 h (AOAC,
2005).

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Study III Aquaculture (2014), submitted

6.2.4. Statistical analysis

All data were tested for normality and homogeneity of variance. Means and
standard deviations (SD) were calculated for each parameter measured. At the end of
the trial, proximate composition of abalones, survival, growth performance and
nutritional indices were calculated and statistically treated by one-way ANOVA and
Tukey’s test was applied for multiple comparisson of means at a 5% significance
level (P< 0.05). All statistical analyses were applied using the Statgraphics Plus 5.1
(MANUGISTIES, Rockville, Maryland, USA) software.

6.3. RESULTS

6.3.1. Nutritional composition of diets and water stability

Proximate composition and caloric content of the formulated diets correspond

well to the intended compositions and levels based upon the dietary formulations
(Table 24). All artificial diets were isoproteic, containing approximately 35% crude
protein, and isocaloric with similar gross energy values and protein: energy (PE)
ratios. However, nutritional composition and caloric content varied between artificial
feeds and fresh algae, protein values and PE ratios being higher in formulated feeds as
compared to those of the control treatment, whereas the fresh algae showed higher
carbohydrate than that of the experimental ones. No differences were observed in
gross energy, lipid and ash contents among fresh algae and formulated diets (Table
25).

After 17h of immersion in seawater, pellets stability ranged between 58-73%,

diet UGL being 7-15% more water stable than other two artificial diets (Table 25).

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Study III Aquaculture (2014), submitted

Table 25. Proximate analysis of the fresh algae or experimental diets containing
different algal species (%DW)

Experimental diets2
Fresh algae1
UG UGL UGP

Crude protein 21.03±5.8 34.9 35.1 35.4

Crude lipid 5.3±1.5 4 4.1 3.9

Carbohydrates3 46±3.5 36.4 37.7 40.3

Ash 27.6±7.1 24.7 23.1 20.4

Moisture 83.3±3 20.6 16.2 19.3

GE (Kcal g-1)4 3.6±0.4 3.8 3.9 4

Protein : energy ratio5 58.3±10.6 91.8 90.0 88.5

% Water stability6 91.9 65.7 72.7 57.8

1
Fresh algae used: G. cornea and U. rigida reared in GIA integrated fish-seaweed culture system (n=4)
2
UG: U. lactuca + G. cornea; UGL: U. lactuca + G. cornea + L. digitata; UGP: U. lactuca + G.
cornea + P. palmata.
3
Calculated by difference (AOAC, 2005)
4
Calculated gross energy (Cho et al., 1982).
5
Calculated metabolisable energy (Cho et al., 1982)
6
Water stability of fresh algae was calculated for a 3-days period and of pellets for 17 h of immersion
(16:00-9:00)

6.3.2. Fatty acid composition of diets

Large differences were found in the FA profile of fresh algae as compared

with the three formulated diets (Table 26). Linoleic acid (18:2n-6) was 15 times more
abundant in formulated diets than in fresh algae. On the contrary, formulated diets
were lower in 16:0 and 16:1n-7 than in the fresh G. cornea and U. rigida.
Subsequently, the n-3/n-6 ratio was much lower in formulated diets. Differences in
FA profiles between formulated diets were less pronounced than those between fresh
algae and practical diets. Both control and formulated diets presented very low levels

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Study III Aquaculture (2014), submitted

of eicosapentanoic acid (EPA) 20:5n-3 and docosahexaenoic acid (DHA) 22:6n-3

(Table 26).

Table 26. Fatty acid composition (% total fatty acids) of the fresh algae or
experimental diets containing different algal species*

Experimental diets*
FA Fresh algae
UG UGL UGP

14:0 2.6 0.9 1.0 1.3

16:0 40.5 22.8 21.5 23.4
17:0 0.9 0.2 0.1 0.1
18:0 0.9 2.8 2.3 2.7
∑SFA** 44.9 26.6 25.0 27.5
14:1n-5 2.2 0.3 0.2 0.2
16:1n-7 9.0 1.3 1.1 1.0
16:1n-5 0.6 0.3 0.2 0.3
18:1n-9 3.4 12.5 12.7 12.6
18:1n-7 6.6 2.9 1.8 2.0
20:1n-9 0.2 0.5 0.5 0.5
∑MUFA*** 22.0 17.8 16.6 16.5
16:2n-4 0.8 0.2 0.1 0.2
16:3n-3 1.5 - - -
16:3n-1 - 0.6 0.3 0.4
16:4n-3 0.6 0.1 - 0.1
18:2n-6 2.7 44.9 46.3 45.6
18:2n-4 0.8 1.5 1.5 1.6
18:3n-3 3.7 4.0 4.0 3.9
18:4n-3 3.0 0.7 1.0 0.5
20:4n-6 1.2 0.1 0.2 0.1
20:3n-3 13.5 0.8 1.8 0.7
20:5n-3 2.4 0.5 1.1 0.7
22:5n-3 1.0 0.2 0.1 0.1
22:6n-3 0.3 0.6 0.3 0.5
∑PUFA**** 31.5 54.2 56.7 54.3
Others***** 1.6 1.4 1.7 1.7
∑n-3 25.9 6.9 8.3 6.4
∑n-6 3.9 45.0 46.5 45.8
n-3/n-6 6.7 0.2 0.2 0.1
* UG: U. lactuca + G. cornea; UGL: U. lactuca+ G. cornea + L. digitata; UGP: U. lactuca+
G. cornea + P. palmata. ** SFA, saturated fatty acids. *** MUFA, monounsaturated fatty
acids. **** PUFA, polyunsaturated fatty acids. ***** Other includes all components
<0.5%:15:0,16:(2n-6), 18:(1n-5), 20:0, 20:(2n-6), 20:(4n-3), 22:(1n-9). -, not detectable.

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Study III Aquaculture (2014), submitted

6.3.3. Abalone survival and growth

Survival rates were very high (95-98%), regardless of the diet fed. In general,
fresh algae produced a far better growth for H. tuberculata coccinea (169% WG) than
all the other experimental diets (49-84% WG) (Table 27). Comparison among abalone
fed the different formulated diets showed that growth was improved by the inclusion
of P. palmata, whereas the lowest growth was obtained by the inclusion of L. digitata.
Hence, at the end of the trial, animals fed diet UGP displayed the highest shell growth
rate, specific growth rate and weight gain, whereas those fed diet UGL showed the
lowest values (Table 27).

6.3.4. Feed intake, feed utilization and condition index

Regarding consumption, there was a significantly (P<0.05) higher feed intake

of the fresh algae, followed by diets UGL, UG and UGP (Table 26). In relation to
nutritional indices performances, except with UG and UGP diets, there were
significant differences among all diets for food conversion ratio (FCR), the highest
and lowest values recorded for abalone fed diet UGL and the fresh one, respectively.
Similarly, the trends in protein efficiency ratio (PER) were similar to those of FCR,
being better in animals fed fresh algae and worse in those fed diet including L.
digitata meal (Table 28). Regarding condition index, the lowest SB/S ratios (2.6)
resulted from feeding diet UGL, which produced the poorest growth performance,
whereas there were no significant differences among the rest of the treatments (3.0-
3.1) (Table 28).

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Table 27. Survival and growth performance of abalone H. tuberculata coccinea fed for 6 months algae (fresh algae) or experimental diets
containing different algal species * (Mean ± S.D.)

Survival Initial size Final size Shell growth rate Initial Final weight Weight gain
Treatment SGR (%d-1)
(%) (mm) (mm) (μm d-1) weight(g) (g) (%)

Fresh algae 97.8±3.8 33.1±0.1 44.2±0.9a 62.8±4.9a 4.7±0.2 12.6±0.8a 0.56±0.0a 168.8±15.4a
UG 98.3±2.4 33.1±0.1 37.9±0.1bA 27.3±0.8bB 4.7±0.2 7.8±0.1bB 0.27±0.0bcAB 61.6±6.2bAB
UGL 98.3±2.4 33.1±0.0 36.9±0.4bB 21.1±1.5bC 4.7±0.2 6.9±0.1bC 0.22±0.0cB 48.4±0.8bB
UGP 95±2.4 33.1±0.1 38.7±0.1bA 31.9±0.7bA 4.7±0.0 8.5±0.1bA 0.34±0.1bA 83.8±7.2bA

UG: U. lactuca + G. cornea; UGL: U. lactuca + G. cornea + L. digitata; UGP: U. lactuca + G. cornea + P. palmata
* Low case letters indicate significant differences among feed treatments including fresh algae, whereas upper case letters indicate differences only
among formulated feeds, P< 0.05.

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Study III Aquaculture (2014), submitted

Table 28. Consumption, feed efficiency and condition index of abalone H.

tuberculata coccinea fed for 6 months algae (fresh algae) or experimental diets
containing different algal species* (Mean ± S.D.)

Feed intake
Treatment FCR PER SB/S
(mg ind-1d-1)

Fresh algae 86.4±4.1a 2±0.2c 2.4±0.2a 3.1±0.3a

UG 68.2±0.9bB 4.1±0.4bB 0.7±0.1bAB 3.1±0.1aA

UGL 73.9±0.0bA 6±0.1aA 0.5±0.0bB 2.6±0.2bB

UGP 65.2±2.5bB 3.2±0.4bB 0.9±0.1bA 3.0±0.1aA

UG: U. lactuca + G. cornea; UGL: U. lactuca + G. cornea + L. digitata; UGP: U. lactuca

+ G. cornea + P. palmata

* Low case letters indicate significant differences among feed treatments including fresh
algae, whereas upper case letters indicate differences only among formulated feeds, P<
0.05.

6.3.5. Effect of diets on the general and fatty acid composition of animals

Nutritional analysis revealed that, except for moisture content, foot muscle
composition of H. tuberculata coccinea was significantly (P<0.05) affected by the
dietary treatments, whereas viscera composition did not differ significantly within the
feeding regimes (Table 29).The percentage of protein deposited was significantly
(P<0.05) the highest in abalone fed diet containing P. palmata meal (UGP), followed
by those fed fresh algae, and the lowest in those fed diet UGL. Overall, foot muscle
contained much lower lipid levels (5-7%) than viscera (19-20%), whereas the latter
showed much lower protein content. Abalone fed control diet showed the
significantly (P<0.05) lowest lipid levels stored in the foot muscle, the highest being
found in those fed diet UGL. Carbohydrate content in foot muscle was significantly
highest in animals fed diet UGP than that of abalone fed the rest of the feeding
regimes. Ash content was generally lower in animals fed fresh algae than that of
abalone fed the formulated diets (Table 29).

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Fatty acid profiles of the H. tuberculata coccinea foot tissues are summarised
in Table 30. The proportion of total saturated (41-49%), total monounsaturated (25-
29%) and total polyunsaturated (22-29%) fatty acids were remarkably similar among
all abalone tissues samples (both viscera and muscle). Palmitic acid (16:0) was the
major fatty acid in all tissues (29-34%). Other prominent FA included 18:0.18:1n-9
and18:1n-7 (Table 30). Abalone fed the practical diets showed accumulations of
linoleic acid (LA. 8-9%) in the foot muscle and elevated levels of its chain-elongation
product 20:2n-6 (3%) compared with the tissues of abalone fed fresh macroalgae (3%
LA and 0.2% 20:2n-6 respectively) (Table 30). In agreement with the dietary levels,
foot tissues of abalone fed fresh algae presented remarkably higher levels of n-3 fatty
acids, including 20:3n-3, 20:4n-3, 22:5n-3, and specially, eicosapentanoic acid (EPA)
20:5n-3 (3.2-5%) compared with those showed by abalone fed artificial diets (0.8-
1.7%). Abalone fed all diets showed elevated levels of ARA relative to their feeds.
For all treatments, the proportion of ARA was higher in the foot muscle than in the
viscera, resulting in relatively higher ARA: EPA ratio in the foot, being also higher in
abalones fed artificial diets relative to those fed the control one. Docosahexaenoic
acid (DHA) (22:6-n-3) was a minor FA (<1%) in all samples.

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Study III Aquaculture (2014), submitted

Table 29. Proximate composition of viscera and muscle of Haliotis tuberculata coccinea fed for 6 months algae (fresh algae) or experimental
diets containing different algal species*(g/100 g DW) (Mean ± S.D.) (Values in the same column with different letters are significantly different.
P< 0.05)

Diets Moisture Crudeprotein Crudelipid Carbohydrate Ash

Viscera Muscle Viscera Muscle Viscera Muscle Viscera Muscle Viscera Muscle

Fresh algae 70.0±0.9 71.9±0.8 57±1.4 74.6±0.2b 19.2±1.1 4.6±0.1c 15.8±1.2 15.1±0.7a 8±0.9 5.7±0.5b

UG 70.2±0.1 73.1±0.4 56.8±2.4 73.7±0.5bc 19.8±0.0 6.2±0.0ab 15.3±2 13±0.2a 8.2±0.4 7±0.2a

UGL 72.9±0.6 72.8±0.1 57.1±2.2 72.3±0.6c 20.2±1.5 6.8±0.3a 12.9±2.8 14.5±0.8a 9.9±1.1 6.4±0.1ab

UGP 71.1±2.2 72.4±0.9 58.3±2 80.7±0.1a 20.4±2.4 5.7±0.2b 12.3±2.8 6.8±0.3b 9±1.7 6.7±0.1ab

UG: U. lactuca + G. cornea; UGL: U. lactuca + G. cornea + L. digitata; UGP: U. lactuca + G. cornea + P. palmata.

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Study III Aquaculture (2014), submitted

Table 30. Fatty acid composition (% total fatty acids) of the abalone tissues of
Haliotis tuberculata coccinea fed for 6 months fresh algae or experimental diets
containing different algal species*

Experimental diets*

FA Fresh algae UG UGL UGP

Viscera Muscle Viscera Muscle Viscera Muscle Viscera Muscle

14:0 5.3 2.0 4.6 2.6 4.1 3.1 4.7 3.2
15:0 0.9 1.7 0.5 0.9 - 0.9 - 1.1
16:0 31.0 34.4 32.3 30.9 30.1 32.1 29.5 33.7
18:0 3.6 10.0 4.0 10.7 4.1 11.0 4.6 10.6
20:0 4.0 - 3.2 - 3.2 - 3.0 -
∑SFA** 44.8 48.1 44.6 45.1 41.5 47.1 41.8 48.6
16:1n-7 2.8 1.5 2.1 1.7 1.7 2.0 1.8 1.6
18:1n-9 10.1 7.5 15.5 11.6 15.1 12.5 14.1 11.6
18:1n-7 11.8 10.3 8.6 5.8 7.4 5.6 7.2 6.5
20:1n-9+n-7 1.2 4.2 1.9 3.2 2.5 3.1 2.5 3.6
20:1n-5 0.5 0.5 0.3 1.8 0.2 1.9 0.3 1.6
22:1n-11 1.2 0.3 0.2 0.4 0.4 0.5 1.2 0.6
22:1n-9 0.1 0.4 0.1 0.5 0.2 0.5 0.3 0.6
∑MUFA*** 27.6 24.8 28.8 25.0 27.6 26.2 27.5 26.1
18:2n-6 2.7 3.3 13.0 9.2 16.4 8.5 15.7 8.6
18:3n-3 4.6 2.9 1.0 0.8 1.1 0.7 1.0 0.6
18:4n-3 2.2 1.4 - 0.1 0.1 0.1 0.1 0.1
20:2n-9 0.3 0.1 0.6 - 0.8 - 07 -
20:2n-6 0.6 0.2 5.0 3.3 4.1 2.8 4.1 2.7
20:3n-6 0.3 0.1 - 0.5 0.6 0.3 0.6 0.4
20:4n-6 5.2 8.2 2.8 8.1 3.5 6.7 3.2 6.2
20:3n-3 0.6 0.1 0.2 0.2 0.1 0.1 0.1 0.1
20:4n-3 1.3 0.3 - 0.1 - 0.1 0.1 -
20:5n-3 5.1 3.2 1.0 1.7 1.2 1.6 1.6 0.8
22:4n-6 0.2 0.3 1.3 1.4 1.0 1.2 0.9 1.1
22:5n-3 3.1 4.6 0.6 2.3 0.4 1.7 1.1 1.2
22:6n-3 0.5 0.2 - 0.6 - 0.5 0.2 0.2
∑PUFA**** 26.8 24.9 25.9 28.4 29.2 24.0 29.4 21.6
Other***** 0.8 2.3 0.7 1.5 1.7 2.7 1.4 3.7
∑n-3 17.4 12.6 2.8 5.8 2.8 4.9 4.2 3.0
∑n-6 9.1 12.1 22.6 22.6 25.6 19.1 24.5 18.6
n-3/n-6 1.92 1.04 0.12 0.26 0.11 0.25 0.17 0.16
ARA/EPA 1.0 2.6 2.8 4.8 3.1 4.1 2.0 7.9
* UG: U. lactuca + G. cornea; UGL: U. lactuca + G. cornea + L. digitata; UGP: U. lactuca + G.
cornea + P. palmata. ** SFA, saturated fatty acids. *** MUFA, monounsaturated fatty acids. ****
PUFA, polyunsaturated fatty acids.***** Other includes all components <0.5%: 14(1n-7), 14(1n-5),
16:0ISO, 16:(1n-5), 16:(2n-6), 17:00, 16:(3n-4), 16:(3n-3), 18:(1n-5), 18:(3n-6), 18:(3n-4), 20:(1n-5). –
not detectable.

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6.4. DISCUSSION

Compound feeds have been reported to outperform fresh algae diets in abalone
culture (Viana et al., 1993; Britz, 1996b; Corazani and Illanes, 1998; Bautista-Teruel
and Millamena. 1999; Coote et al., 2000). In those studies, the low protein content
and less balanced amino acid profile of the seaweed could not be sufficient to support
abalone rapid growth, suggesting a high requirement of good quality protein in this
species. Moreover, it has been suggested that this could be the reason of such a long
time to get abalone to marketable size (2-5 years) using macroalgae (Hahn, 1989;
Robertson-Andersson, 2003; Johnston et al., 2005). However, in the present study,
feeding the fresh algae resulted in maximum growth of abalone, indicating the high
dietary value of the macroalgae reared in the IMTA. Indeed, the type of macroalgae
consumed can significantly affect abalone growth offering different proportions of
their nutrients requirement. The good growth performance attained for these large
abalones fed enriched seaweeds in the present study (169±15% WG) seems to be
explained, not only by the high protein content of the macroalgae produced under the
high nitrogen culture conditions of the biofilter system (Boarder and Shpigel, 2001;
Viera et al., 2005, 2011; Naidoo et al., 2006; Roberstson-Andersson et al., 2006,
2011), but also by the rest of their nutrients composition and energy content, which
matched abalone nutritional requirements (Fleming et al., 1996; Jackson et al., 2001;
McBride et al., 2001; Sales and Janssens, 2004). Besides, the combination of both red
and green algae species, each one with the typical biochemical composition of its
phylum (Li et al., 2002; Dawczynski et al., 2007; Kinkerdale et al., 2010), may have a
complementary effect to fulfil the micronutrient requirements of abalone, contributing
to the good growth rates obtained. In fact, abalone fed mixed algal regimes, have been
reported to perform significantly better than those fed with a single algal diet (Mercer
et al., 1993; Dlaza, 2008; Naidoo et al., 2006; Roberstson-Andersson et al., 2011;
Viera et al., 2011). Analysis of the proximate composition of the compound diets
showed no differences in protein, lipid, gross energy values, protein:energy (PE)
ratios and only a small difference in carbohydrates content, all of them being also
within the limits recommended for several abalone species (Fleming et al., 1996;
Jackson et al., 2001; McBride et al., 2001).

In comparing the artificial treatments, abalone growth was influenced by the

type of seaweed meal incorporated into its diet. Animals fed diet containing P.

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Study III Aquaculture (2014), submitted

palmata as specific seaweed meal, performed better than the others and, particularly,
those fed diets containing L. digitata. These results agree well with those obtained by
Mercer et al. (1993) and Mai et al. (1995a, 1996) who reported the best performance
on close European ormer, H. tuberculata fed P. palmata and the lowest on those fed
L. digitata and L. sacharina. SGR values in this study (0.2-0.3%) were higher than in
abalone H. roei fed various compound diets as reported by Boarder and Shpigel
(2001), who found SGR values of 0.1 %, under similar experimental conditions.
However, growth rates (21-32μm day-1) were generally lower (21-56μm day-1) than
those obtain by Britz and Hetch (1997) in similar size H. midae fed with fish meal-
based diets containing different proportions of protein and energy. Previous studies
have shown that abalone fed formulated diets based on animal protein sources or a
combination of plant and animal protein ones, yield better growth rates than those fed
diets with protein sources of plant origin only (Britz, 1996b; Viana et al., 1996;
Boarder and Shpigel, 2001; Bautista-Teruel et al., 2003). In agreement, Dlaza et al.
(2008) reported the poorest performance (27μm day-1) of post-weaning abalone H.
midae when fed an all-seaweed-based formulated feed compared to that recorded for
those fed several fishmeal-based protein diets (46-61 μm day-1), claiming that
seaweed proteins were less readily absorbed than animal based protein.

Laminaria digitata including diet (UGL) was found to be water stable

retaining at 73% of dry matter after the 17 h test for stability. However, diets UG and
UGP leached a considerable proportion of their dry matter (34-42%), indicating low
diet water stability, when compared with other compound diets (17-25-%, Jackson et
al., 2001; 35-37%; Bautista- Teruel et al., 2003). This could be related to the higher
moisture content of the former (19-20%) related to those previously tested (9-12%,
Bautista-Teruel and Millamena, 1999; Jackson et al., 2001). Despite diet UGL
showed the highest water stability, it also provided the lowest growth performance,
whereas the one including P. palmata with the lowest water stability, produced the
highest growth. Moreover, feed intake was also highest in abalone fed UGL, whereas
protein deposition and the protein-related nutritional index (PER) were lowest. Thus,
dietary inclusion of Laminaria digitata, despite it did not negatively affected feed
water stability, feed intake or dietary protein or essential aminoacid contents,
significantly reduced dietary protein efficiency, suggesting a reduction in protein
digestibility that could be partly responsible for the low growth obtained. Indeed,

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brown algae are reported to be digested more slowly than red algae for several
abalone species (H. midae, Day and Cook, 1995; H. rubra, Foale and Day, 1992).
Nevertheless, this growth and PER reduction could be also related to an specific
leaching of dietary proteins even though dry matter leaching was low, as suggested by
other authors (Viana et al., 1996; Edward and Cook, 1999; Jackson et al., 2001).

Animals fed fresh macroalgae ate significantly more than animals fed practical
diets. This result could be due to the significantly lower PE ratio of the fresh algae
related the artificial feeds, since feed intake is the main compensatory mechanism of
herbivorous animals to satisfy energy requirement and being protein and
carbohydrate, rather than lipid, the principal energy sources in abalone (Durazo-
Beltrán et al., 2004; Viana et al., 2007). Additionally, previous investigations have
demonstrated that dietary protein source may affect feed consumption in Haliotids
(Uki and Watanabe, 1986; Viana et al., 1994), hence different attractiveness and
palatability between fresh algae and artificial diets could have also affected
consumption. The food conversion ratio (FCR) attained for the control treatment,
agrees well with those obtained previously for this (H. tuberculata coccinea, Viera et
al., 2005, 2011) and other abalone species fed macroalgae (H. asinina, Kunavongdate
et al., 1995; H. discus hannai and H. tuberculata, Shpigel et al., 1999). However,
FCR (3-6) obtained for abalone fed formulated diets were higher than reported values
(0.7- 1.8; Britz, 1996b; Shipton and Britz, 2001; Bautista-Teruel et al., 2003; García-
Esquivel et al., 2007), probably due to the loss of unconsumed feed that could not be
recovered during the study. Thus, some unconsumed or rasped pellets could have
broken up and drained out of the tank. Similar observations and even higher FCR
results (3-13) have been reported for H. asinina fed formulated diets (Reyes and
Fermin, 2003; Thongrod et al., 2003).

In the present study, PER was significantly better in abalone fed fresh algae,
hence, suggesting a higher protein utilization efficiency. However, the CI (SB/S) of
animals reared on fresh macroalgae was similar to those of animals fed diets UG and
UGP, indicating that all of them produced healthy animals of a high product quality.
Moreover, protein deposition being higher in animals fed UGP and similar in those
fed UG, related to that obtained with fresh algae, further indicate the good protein
quality of those diets, as tissue deposition cannot occur unless the requirements for
essential amino acids are met (Durazo-Beltrán et al., 2003). CI values (2.6-3.1) were

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Study III Aquaculture (2014), submitted

similar and even higher than those recorded for Viera et al. (2011) for this specie (2.4-
3.3) or by Mai et al. (1995a) for H. tuberculata (1.8-2.1) and H. discus hannai (2-2.4)
fed with P. palmata and various levels of dietary lipids or by Sales et al. (2003) for H
midae (2.9-3.2) fed different dietary crude protein level.

Total lipid in the foot muscle were relatively low in agreement to their natural
diets and to previous reports on both wild and cultured abalone (Nelson et al., 2002;
Durazo-Beltrán, et al., 2004; Grubert et al., 2004). Abalone seem very efficient in
assimilating lipids from their diets and, thus, diets with only 3-5% lipid have been
recommended to promote high growth rate of abalone (Fleming et al., 1996; Bautista-
Teruel and Millamena, 1999; Shipton and Britz, 2001; Johnston et al., 2005; Green et
al., 2011).Viscera contained much higher lipid levels than muscle in agreement with
previous studies (Webber, 1970; Mercer et al.,1993; Nelson et al., 2002; Viera et al.,
2011), and denoting the lipid storage function of HG (Uki et al., 1986b; Dunstan et
al., 1996). In Haliotids, lipids are essential nutrients for growth and gonad maturation,
but not a primary source of energy (Nelson et al., 2002). Moreover, muscle in the
abalone foot is a major energy consumer due to daily movements and strong shell
adhesion properties, free aminoacids playing an important role in rapid energy
production for shell adhesion, while the energy for slow locomotion mainly comes
from carbohydrates (Mercer et al., 1993).

Overall, FA results for H. tuberculata coccinea in the present study were

similar to those for the foot muscle of H. laevigata and H. rubra (Dunstan et al.,
1996; Grubert et al., 2004), H. asinina (Jackson et al., 2001; Thongrod et al., 2003)
and H. fulgens (Nelson et al., 2002; Durazo-Beltrán et al., 2004) fed macroalgae and
/or artificial diets, where the main FA were 16:0, 18:0, 18:1n-7, 18:1n-9, 18:2n-
6,18:3n-3,20:4n-6, 20:5n-3 and 22:5n-3, and may reflect biochemical and dietary
similarities of wild-caught representatives from Haliotidae. However, abalone fed
fresh algae showed higher levels of n-3 fatty acids, specially, EPA 20:5n-3 in
comparison to abalone fed formulated diets. The main PUFA in the formulated diets
were n-6, while those in fresh algae were the more highly unsaturated n-3 PUFAs.
Similarly, the ratio of n-3/n-6 was higher in the foot tissues of abalone fed fresh algae
relative to those fed formulated diets, hence reflecting not only the composition of the
diets (Uki et al., 1986b; Dunstan et al.,1996; Nelson et al., 2002) but also their highest
nutritional value, since growth enhancement of H. tuberculata L. appeared to depend

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Study III Aquaculture (2014), submitted

largely on n-3 PUFA, mainly 20:5n-3 and 18:3n-3 playing an important role in
accelerating the growth of this species (Mai et al., 1996).

In relation to the dietary levels, the elevated contents of 20:4n-6 in the abalone
fed the experimental diets and 20:5n-3 in abalone fed the fresh algae, as well as their
respective metabolites 20:2n-6, 20:3n-6, 20:4n-3, suggest that abalone have the ability
to desaturate and elongate LA to ARA and ALA to EPA. This conversion of C18
PUFA to the C20 and C22 PUFAs they require, is present in many marine
invertebrates including other Haliotis species, these herbivorous marine animals
converting much more efficiently than carnivorous ones (Uki et al., 1986). Both
control and formulated diets as well as all abalone tissues presented very low levels of
DHA 22:6-n3 (Mai et al., 1996; Dawczynski et al., 2007; Viera et al., 2011; Courtois
de Viçose et al., 2012a), indicating that the composition of abalone is quite different
to that of other marine animals, which have this fatty acid as one of the main tissue
essential PUFA. Furthermore, it has been suggested that abalone are unusual
compared with other marine animals in the importance of DPA rather than DHA
(Viana et al., 1993; Dunstan et al., 1996).

In summary, this study has shown that feeding H. tuberculata coccinea with
seaweeds based diets without fishmeal inclusion resulted in high survival and good
dietary protein utilization. The inclusion of P. palmata in these types of diets is
recommended to improve growth, condition index and dietary protein utilization. On
the contrary, the use of L. digitata should be avoid, at least in the level included in the
present study, since markedly reduced growth performance and increased conversion
index, reducing the efficiency of dietary protein. Further studies are required to
improve the growth obtained with this type of diets, especially concerning the use of
different seaweed combinations and inclusion levels, as well as the diet processing
methods to improve stability.

6.5. ACKNOWLEDGEMENTS

The authors especially thank Dr. J.L. Gómez-Pinchetti from Centro de

Biotecnología Marina, A. Fitzgerald, from South West Abalone Growers Association,
Dr. A. Soler from Martin Ryan Institute and Dr. S. Huchette from France Haliotis,
who kindly supplied the macroalgae experimental meals. This study has been

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financed by the FP 7-SME-2007-1/BSG-SME. Project Sustainable Development of

European SMEs engaged in Abalone Aquaculture – (SUDEVAB).

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Development of a Sustainable Grow-out Technology for Abalone
Haliotistuberculata coccinea (Reeve) as a New Species for Aquaculture
Diversification in the Canary Islands

STUDY IV: Grow Out Culture of Abalone Haliotis tuberculata

coccinea reeve, fed land-based IMTA produced Macroalgae, in a
combined Fish/Abalone Offshore mariculture system: effect of
stocking density

Aquaculture Research DOI: 10.1111/are. 12467

Study IV Aquaculture Research (2014) DOI: 10.1111/are. 12467

Grow out culture of abalone Haliotis tuberculata coccinea Reeve, fed land-
based IMTA produced macroalgae, in a combined fish/abalone offshore
mariculture system: effect of stocking density

María del Pino Viera, Gercende Courtois de Viçose, Hipólito Fernández-Palacios

& Marisol Izquierdo

Grupo de Investigación en Acuicultura (GIA), Universidad de Las Palmas de Gran

Canaria (ULPGC). Las Palmas, Canary Islands, Spain

Abstract

Haliotis tuberculata coccinea has been identified as a target species for European
aquaculture development, in order to fulfil the rising demand for abalone. The effects of
different stocking densities on the growth performance, feed utilization and survival of two
different initial size groups (30 and 40 mm) of abalones, during the final grow-out to
cocktail/market size (45-60 mm), were determined over a 6 month period. Trials were
performed in abalone cages installed in a commercial open-sea cages fish farm. Animals were
fed the red algae Gracilaria cornea and the green one Ulva rigida, both obtained from a land-
based integrated multi-trophic aquaculture system. Survival rates were very high (94-98%)
regardless the density employed. Sustained high linear growth was recorded both in shell and
weight. However, a 17-19% reduction in weight gain was obtained by doubling the initial
stocking density, suggesting a higher competition for space or food. Nevertheless, the high
growth performance (70-94 μm d-1; 250-372% weight gain) and survival attained, even at
high densities, denoted the suitability of the offshore mariculture system as well as the
biofilter produced macroalgae for grow out culture of H. tuberculata coccinea that overall
could reach cocktail/commercial size in only 18-22 months.

Keywords: abalone; growth; density; seaweeds; integrated aquaculture; sea-based culture

Corresponding author: M P Viera

Phone: (34) 928 132034
E-mail: mapiviera@gmail.com
Address: Muelle de Taliarte s/n, 35214, Telde, Gran Canaria, Canary Islands, Spain

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Study IV Aquaculture Research (2014) DOI: 10.1111/are. 12467

7.1. INTRODUCTION

The ormer, Haliotis. tuberculata is the only European abalone species

commercially exploited (Roussel et al., 2011). Ireland, the Channel Islands (Huchette
and Clavier, 2004) and France are currently the only established producing countries,
with several farms growing abalone at sea feeding them locally harvested fresh
seaweeds (Fitzgerald, 2008). The native abalone species in the Canary Islands, H.
tuberculata coccinea Reeve (1846) (Geiger, 2000), has also been identified as a target
species for aquaculture development, contributing to fulfil abalone’s increasing
demand on European markets (Dallimore, 2010) and to diversify the Canarian
aquaculture industry, currently limited to the production of marine fish species (Viera
et al., 2005). Moreover, since this abalone species grows to a maximum size of about
7-8 cm in shell length (Espino and Herrera, 2002), its potential for aquaculture
production, further relies on the international growing demand for small “cocktail
size” abalone (4-7 cm shell length) (Jarayabhand and Paphavasit, 1996; Najmudeen
and Victor, 2004).

Previous studies on H. tuberculata coccinea have successfully contributed to

the development of production techniques for this species (Viera et al., 2005; Bilbao
et al., 2010b; Courtois de Viçose et al., 2010, 2012a, b; Viera et al., 2011). However,
a commercial grow-out system is still to be developed.

As abalone growth is generally low, the economic viability of commercial

abalone farming depends largely on the rearing system employed during the long
lasting grow-out culture period (Demetropoulos and Langdon, 2004; Badillo et al.,
2007). Commercial cage culture is considered the faster sector in aquaculture (Tacon
and Halwart, 2007). The main advantage of cage culture is that both the initial capital
investment and operational cost are considerable lower than in land-based facilities
(Beveridge, 2004; Wu et al., 2009). Hence, many fish, crustacean and even benthic
cephalopod species have been cultured in cages at commercial or experimental scale
(Chua and Tech, 2002; Estefanell et al., 2012). Indeed, cage culture is practised in
many abalone producing countries (Capinpin et al., 1999; Wu and Zhang, 2013).

Another key factor for the successful culture of abalone species is the
availability of suitable feed (Shipton and Britz, 2001; Reyes and Fermín, 2003; Viana
et al., 2007). Abalone production requires large quantities of macroalgae, not always

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Study IV Aquaculture Research (2014) DOI: 10.1111/are. 12467

available in all production locations. Among different nutrients, protein constitutes

the most costly component and is a major determinant of the nutritional value in
abalone diets (Coote et al., 2000; Sales et al., 2003). Algal production in a land-based
integrated multi-trophic aquaculture (IMTA) system, mainly representatives from
Ulva and Gracilaria genus, can provide a highly nutritious feed for abalone whilst
reducing nutrient discharge levels (Neori et al., 2004).

Stocking density has also been found to be a determinant factor to optimize

abalone growth up to market size. Hence, its effect on growth have been reported for
most of commercial abalone species worldwide: H. tuberculata (Koike et al., 1979;
Cochard, 1980; Mgaya and Mercer, 1995); H. cracherodii (Douros, 1987); H. discus
hannai (Jee et al., 1988; Park et al., 2008; Wu et al., 2009; Wu and Zhang, 2013); H.
rubra (Huchette et al., 2003a,b); H. asinina (Capinpin et al., 1999; Fermin and Buen,
2002; Jarayabhand et al., 2010); H. diversicolor supertexta (Liu and Chen, 1999); H.
rufescens (McCormick et al., 1992; Aviles and Shepherd, 1996, Valdés-Urriolagoitia,
2000); H. midae (Tarr, 1995); H. corrugata (Badillo et al., 2007) and for H.
kamtschatkana (Lloyd and Bates, 2008).

In a quest to develop a commercial abalone grow-out system, it is of interest to

consider the incorporation of abalone production within the conventional fish sea
cages facilities. It would allow a significant improvement in the economic return for
the producers, while also avoiding the risk of single species production. Furthermore,
the algal production in a land-based IMTA system, would allow a development of a
not only more cost-effective production system, but an environmentally friendly
aquaculture.

Thus, the present research aimed to determine, during the final abalone grow-
out to market/cocktail size (30-60 mm), the effects of initial stocking densities on
growth, feed consumption and survival of two distinct initial size groups of H.
tuberculata coccinea, fed biofilter produced macroalgae, in an offshore mariculture
system.

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Study IV Aquaculture Research (2014) DOI: 10.1111/are. 12467

7.2. MATERIAL AND METHODS

7.2.1. Algae

Ulva rigida J.Agardh and Gracilaria cornea J. Agardh were grown in the
Grupo de Investigación en Acuicultura (GIA, Canary Islands, Spain) aquaculture
research facility, in a flow-through integrated system collecting wastewater from fish
and abalone ponds in a macroalgal biofilter. Effluents were channelled from the land-
based facility tanks to a 11 m3 sedimentation pond for the removal of suspended
particles and then, pumped at a flow rate of 10 m3 h-1 to the seaweed tanks located
outdoor, where maximum irradiance was close to 1600 µmol photons m-2 s-1. Circular
plastic tanks with a volume of 1.5 m3 and aeration supplied by a bottom-circular
pipeline were used for the cultivation of macroalgae. Algal stocking densities were 1
and 4 g L-1 for U. rigida and G. cornea, respectively. Water exchange rate in the
seaweed tanks was 12 vol day-1 and total ammonia nitrogen inflow into the biofilter
ranged between 10 and 30 µmoles.

To assess the feed quality, homogenized samples of each algae were analyzed
in triplicates for proximate composition. The algae were cleaned, washed with
freshwater and frozen at -80 ºC. Dry mass was determined by drying samples at
110ºC until constant weight was attained. Ash content was determined by incinerating
samples at 600ºC for 24 h. Protein content was analyzed by the Kjeldahl method in
agreement with AOAC (2005) and total lipids were extracted by a chloroform-
methanol (2:1) mixture as described by Folch et al., (1957).

7.2.2. Abalone

Experimental abalones (Haliotis tuberculata coccinea) were produced in the

experimental hatchery production unit of GIA in two different artificial spawning
events (March and June 2009).Until reaching initial stocking sizes (30 and 40 mm),
abalones were fed several diatoms species for 4 to 5 months, followed by the green
algae U. rigida and the red one G. cornea for 7-10 months.

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Study IV Aquaculture Research (2014) DOI: 10.1111/are. 12467

7.2.3. Offshore grow-out system

The sea-based grow-out system was set up in a commercial open-sea cages

fish farm (CANEXMAR, S.L., Telde, Gran Canaria Island, Spain) (27º 57´31.7´´N,
15º22´22.5´´W). This area is regarded as highly exposed, submitted to wave height of
0.2-3.05 m and maximum current of 41-50cms-1, mainly NE, during the experimental
period (Mar 3rd / Sep 9th, 2010) (network of state ports, Spanish Government,
http://www.puertos.es). Seawater ranged between 19 and 22.9 ºC. Photoperiod was
approximately13h L / 11 h D.

Specially designed abalone sea cages (ORTACS, Jersey Sea Farms, St.
Martins, Ireland), were composed of a 33 L lidded black PVC meshed container with
total underside surface area of 0.4 m2 and weighing 1.5 kg. (Fig.38a). Six black
plastic discs (12.0 cm Ø), stacked together forming a shelf-like device connected by
tube-shaped rods, were placed inside as shelters (Fig. 38b). The total surface area for
attachment was 0.5 m2. The abalone cages were suspended from fish cages (25 m Ø)
mooring ropes and placed, approximately, 10 m below the water surface (Fig.38c).

Figure 38. Offshore grow-out system: (a) experimental abalone cages, (b) shelter, (c)
underwater experimental installation next to fish cages.

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Study IV Aquaculture Research (2014) DOI: 10.1111/are. 12467

7.2.4. Culture trials

Two different grow-out trials were simultaneously performed for 27 weeks at

the experimental sea-based system. In Trial I, the growth of 450 1-year-old abalones,
with an average initial shell length of 29.7 ± 1 mm and weight of 3.3 ± 0.5 g, was
tested at two different densities of 200 and 100 individuals m-2 (100 and 50 abalones
per cage). In Trial II, the growth of 135 15-months-old abalones, with an initial
average shell length of 39.5 ± 1.1 mm and weight of 7.8 ± 1.1 g, was tested at two
different densities of 60 and 30 individuals m-2 (30 and 15 abalones per cage). All
treatments, further mentioned as I200, I100, II60 and II30 respectively, were tested in
triplicates. Besides, 3 cages containing the same feeding regime but without abalones
was used as control to determine algal mass variations independently of abalone
grazing.

The experimental abalones were blot dried, weighed to the nearest 0.1 mg
(total fresh body weight: TFBW), measured with a manual calliper with 0.1 mm
accuracy (total shell length: SL) and assigned to the experimental cages. Abalones
were homogeneously distributed among cages to avoid significant differences in SL
or TFBW.

A pre-determined amount of algae were weighed and provided to guarantee ad

libitum feeding, being replaced once a week during the growth trials. Cages were
monthly cleaned to remove fouling organisms. At the time of feeding, mortality was
determined in each cage.

To determine feed intake, the weight of unconsumed food was deducted from
the total weekly ration. Besides, weight of uneaten algae was corrected by calculating
the natural weight variations of the algae in the control cages without abalone during
the same feeding period.

To assess the effects of density on growth performance, SL and TFBW of

100% of the population in each cage were recorded on weeks 5, 9, 17, 22 and 27
respectively. Daily growth rate (both for length and weight) as well as the following
indices were calculated for all treatments at the end of the trial:

DGSL (μm d-1) = GSL / n

DGW (mg d-1) = GW / n
Specific growth rate, SGR (%d-1) = (LnW2-LnW1) / n x 100

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Weight gain, WG (%) = ((W2-W1) / W1) x 100

Feed conversion ratio, FCR = total feed intake (g wet) / total weight gain (g
wet)
Protein efficiency ratio, PER = (increase in body wet weight (g)) / (protein
intake (g))
Feeding rate (% body weight day-1) = FCR*SGR

where GSL is increase in shell length (μm); GW is increase in weight (mg); n is

days of rearing; W2 is the weight at time n, and W1 is the initial weight.

7.2.5. Statistical analysis

All data were tested for normality and homogeneity of variance. Means and
standard deviations (SD) were calculated for each parameter measured. At the end of
the trial, proximate composition of the diets, survival, growth performance and
nutritional indices of each trial were calculated and compared statically by means of
T-Student test (Sokal and Rolf, 1995). To compare the regression models (weight and
length as a function of time) for each trial, the test of homogeneity of regression
slopes was performed between both treatments. Additionally, a two-factors ANOVA
has been applied to growth parameters (size and weight) in order to evaluate the effect
of the fixed factors “time” and “density”. Significant differences were considered for
P< 0.05. All statistical analyses were applied using the Statgraphics Plus 5.1
(MANUGISTIES, Rockville, Maryland, USA) software.

7.3. RESULTS

7.3.1. Nutritional composition of biofilter seaweeds

Nutritional composition and caloric content varied considerably between both

seaweeds (Table 31), gross energy values, protein, lipid and carbohydrate levels being
higher (P<0.05) in U. rigida as compared to those of the red algae, whereas the G.
cornea showed higher ash content than that of the green one. No significant
differences were observed in protein: energy ratios for both seaweeds.

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Table 31. Proximate composition and caloric content of the macroalgal diets (g/100 g
DW) (Mean ± S.D.) fed to abalone along the experimental trials

Macroalgae

Ulva rigida Gracilaria cornea

Moisture 79.6±0.2b 84.9±0.1a

Crude protein 25±0.2a 18.8±1.7b

Crude lipid 6.3±0.2a 4.3±0.9b

Carbohydrate* 45.2±1.6a 41.9±0.7b

Ash 23.5±1.6a 35.1±0.9b

GE**(Kcal g-1) 3.9±0.1a 3.2±0.0b

Protein : energy ratio*** 65±0.9 59.2±5

*
Calculated by difference (AOAC, 2005).
**
Calculated gross energy (Cho et al., 1982).
***
Metabolizable energy was calculated based on the physiological values at 5.6 Kcal g-1
protein, 9.5 Kcal g-1 lipid and 4.1 Kcal g-1 carbohydrates (Cho et al., 1982).

Values in the same row with different letters are significantly different (P<0.05)

n=3

7.3.2. Abalone survival and growth

In general, survival rates were very good (94-98%) and no significant

differences were found between different densities within each trial or different initial
sizes between trials (Table 32). Overall, there was a sustained linear growth in shell
(r2=0.96-0.98) and weight (r2=0.99) at both abalone sizes throughout the experimental
period (Fig.39). The regression slopes analysis (t-student, dif=4) showed a significant

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effect of density on growth both in shell length and weight in trial I (t4=-3. 34798, p=0
.0286; t4= -21.5088, p=0.000028) and II (t4=-4.2733, p=0.0129; t4=-4.801,
p=0.008642) respectively.

Two-way ANOVA analysis showed a highly significant effect of time and

density on abalone shell length and weight with a significant interaction between both
factors (Table 33). Overall, Student’s t-test showed that growth performance, in terms
of shell length, body weight, SGR, weight gain and daily growth rates (both shell and
weight), were significantly affected by stocking densities being higher in abalone
reared at low density in both trials (Table 32).

Thus, at the end of the Trial I, animal cultured at low density (I100) presented a
significantly (P<0.05) higher growth performance in shell growth rate (16%), weight
growth rate (21%), specific growth rate (10%) and weight gain (19%) than those at
high density.

Accordingly, in Trial II, where abalones had a higher initial size than in Trial
I, at the end of the experimental period abalones reared at a lower density (II30)
showed significantly (P<0.05) higher daily growth rate, both in shell length (14%)
and weight (16%), specific growth rate (11%) and weight gain (17%) than those at
high density (Table 32).

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Figure 39. Linear growth in shell length (mm) and weight (g) of abalone H. tuberculata coccinea initially measuring 30 (Trial I) and 40 mm (Trial II), fed
with enriched mixed diet of G. cornea and U. rigida at high and low stocking densities for 27 wks.

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Table 32. Survival and growth performance of abalone H. tuberculata coccinea, initially measuring 30 mm (Trial I) and 40 mm (Trial II), fed
with enriched G. cornea and U. rigida at high and low stocking densities for 27 weeks

Treatment Survival Initial size Final size DGSL Initial Final DGW SGR Weight gain
(%) (mm) (mm) (μm d-1) weight (g) weight (g) (mg d-1) (% d-1) (%)

Trial I

I200 94.3±1.2 29.7 ± 1 43±0.5b 70.1±2.6b 3.3 ± 0.5 13.0±0.2b 51.7±1.3b 0.74±0.02b 302.6±12b

I100 96.7±1.2 29.7 ± 1 45.4±0.6a 82.7±3.2a 3.3 ± 0.5 15.8±0.1a 65.8±0.7a 0.82±0.0a 371.9±4.3a

Trial II

II60 97.8±1.9 39.5 ± 1.1 54.6±0.7b 79.2±3.4b 7.8 ± 1.1 27.3±0.8b 103.4±3.4b 0.66±0.01b 250.5±6.2b

II30 97.8±3.8 39.5 ± 1.1 57.3±0.8a 94.1±4.3a 7.8 ± 1.1 30.8±1.3a 122.5±7.3a 0.74±0.03a 302.3±22.4a

GSL= daily growth rate in shell length, GW= daily growth rate in weight, SGR= Specific growth rate
Within each trial, values in the same column with different letters are significantly different (P<0.05); I200: n =100x3; I100: n =50x3; II60: n =30x3;
II30: n =15x3.

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Table 33. Two-Way ANOVA analysis of variance for growth (size and weight) for the
27 wks grow-out culture period under the experimental densities

Effect SS d.f. MS F p

Trial I

Lenght Intercept 50415.22 1 50415.22 235096.9 0.000000

Time 855. 16 5 171.03 796.6 0.000000

Density 14.19 1 14.19 66.2 0.000000

Time * Density 4.75 5 0.95 4.4 0.005317

Error 5.15 24 0.21

Weight Intercept 2587. 418 1 2587. 418 46341.81 0.000000

Time 488.136 5 97.627 1748.55 0.000000

Density 9.201 1 9.201 164.8 0.000000

Time * Density 7.206 5 1.441 25.81 0.000000

Error 1.34 24 0.056

Trial II

Lenght Intercept 81628.3 1 81628.3 166244.4 0.000000

Time 1109.87 5 221.97 452.1 0.000000

Density 5.37 1 5.37 10.9 0. 002967

Time * Density 7.74 5 1.55 3.2 0.025107

Error 11.78 24 0.49

Weight Intercept 11165.44 1 11165.44 19528.16 0.000000

Time 1854. 82 5 370.96 648.81 0.000000

Density 19.33 1 19.33 33.81 0.000005

Time * Density 16.6 5 3.32 5.81 0.001189

Error 13.72 24 0.57

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7.3.3. Feed intake and feed utilization

In Trial I, daily feed intake on algal rations, recorded for 27 weeks, was
significantly (P<0.05) higher in abalones cultured at lower density (I100) (Table 34). In
relation to feed utilization efficacy, food conversion ratio (FCR) values were
significantly affected by stocking density being lower in animals cultured at low
density. Similarly, protein efficiency ratio (PER) values were significantly (P<0.05)
improved in abalone cultured at low density (Table 34).

Regarding Trial II, II30 treatment abalones showed a significantly (P<0.05)

higher feed intake than those stocked at double density (Table 34). However, there were
no differences in FCR nor in PER between high (60 abalones m-2) and low (30 abalones
m-2) stocking densities.

Table 34. Consumption and feed efficiency of abalone H. tuberculata coccinea.

initially measuring 30 mm (Trial I) and 40 mm (Trial II), fed with G. cornea and U.
rigida at different stocking densities (high and low) for 27 weeks

Treatment Feed intake Average feeding rate FCR PER

(mg ab. d-1) (% BW d-1)

Trial I

I200 1114.9±6.7b 16.2±0.3 22±0.8a 1.1±0.04b

I100 1255.1±11.2a 15.9±0.4 19.4±0.6b 1.3±0.04a

Trial II

II60 2245.1±110.8b 14.5±0.5b 21.9±1.0 1.1±0.05

II30 2840±183.5a 17.2±0.9a 23.4±0.4 1.1±0.02

FCR=Feed conversion ratio. PER=Protein efficiency ratio

Within each trial. values in the same column with different letters are significantly different
(P<0.05); I200: n =100x3; I100: n =50x3; II60: n =30x3; II30: n =15x3.

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7.4. DISCUSSION

Optimum abalone growth is achieved through proper balance of dietary nutrients

and fulfillment of requirements of essential nutrients and energy (Gómez-Montes et al.,
2003). Hence, the sustained growth attained by experimental abalones throughout both
long-term trials (189 days), may well indicate an optimum nutritional quality of the
algae produced in the land-based IMTA system. Indeed, as shown by the proximate
composition analysis, both U. rigida and G. cornea were high in protein (19-25%)
content, which would be related to their production under the rich nitrogen culture
conditions of the integrated mariculture system (Boarder and Shpigel, 2001; Robertson-
Andersson, 2003). Furthermore, not only protein but also very high carbohydrates (42-
45%) and low lipid levels (4-6%), as well as energy content (3-4 Kcal g-1) of the algae
tested, matched abalone nutritional requirements (Mercer et al., 1993; Fleming et al.,
1996). Besides, the combination of both green and red algae, each one with typical
characteristic of its phylum (Table 1; Li et al., 2002; Dawczynski et al., 2007) may also
have complemented the micronutrient requirements of abalone, contributing to the good
growth rates obtained. Indeed, abalone fed mixed algal regimes, have been reported to
perform significantly better than those that are fed with a single algal diet (Naidoo et al.,
2006; Viera et al., 2011). Growth rates values in this study (70-94 μm d-1) were higher
than those obtained by Reyes and Fermín (2003) and Naidoo et al. (2006) (55-66 µm
day-1) in similar size H. asinina and H. midae fed with macroalgae. Moreover, they
were within the range considered acceptable for commercial culture (80 µm day-1;
Gómez – Montes et al., 2003).

Average feeding rates (16% BW d-1; 30-57 mm shell length; 30-200 abalones m-
2
) were good and comparable to those of the tropical fast growing H. asinina fed
Gracilaria bailinae under similar experimental conditions (16% BW d-1; 30-55 mm
shell length, 43-175 ind m-2) (Capinpin et al., 1999). Moreover, feed conversion ratios
(19-23) were also good and similar to those previously obtained for other abalone
species fed macroalgae (H. asinina, Kunavongdate et al., 1995; H. discus hannai and H.
tuberculata, Shpigel et al., 1999).

Increasing initial stocking density negatively affected H. tuberculata coccinea

growth, regardless the initial size tested. This negative effect of increased stocking
density, has also been found in other abalone species in net cages (Jee et al., 1988;
Mgaya and Mercer, 1995; Capinpin et al. 1999) and different culture systems (Marsden

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and Williams, 1996; Valdés-Urriolagoitia, 2000; Huchette et al., 2003a, b; Badillo et al.,
2007; Lloyd and Bates, 2008; Jarayabhand et al., 2010), where a wide range of stocking
sizes (7-65mm) and densities (43- 4100 abalones m-2) have been tested. In particular,
initial densities of 43-175 abalones m-2 tested in cage cultures of another ”cocktail size”
species such as H. asinina (Capinpin et al., 1999) and of 83-386 abalones m-2 for
juveniles of a close related species such as H. tuberculata (Koike et al., 1979; Mgaya
and Mercer, 1995), showed a marked effect on final growth of abalone. In the present
study, doubling the stocking density from 30 to 60 abalones m-2 caused a 14% reduction
in growth (mm month-1), whereas doubling from 100 to 200 further reduced growth in a
16%. In agreement, growth reduction in other studies ranged from 14% to 52% (mm
month-1) due to 2- to 60-fold increases in density (Huchette et al., 2003a). Particularly, a
3 times increase in initial stocking density may cause a 52% reduction in growth for H.
tuberculata (Mgaya and Mercer, 1995).

Results obtained in the present study when initial stocking densities were
increased suggest that these grazing gastropods show a density-dependent competition
for space or food (Marshall and Keogh, 1994; Capinpin et al., 1999; Huchette et al.,
2003a, b). In both trials, abalones stocked at a lower density showed a significantly
higher food consumption than those stocked at double density, suggesting that at high
density, abalone in the cage would tend to stack, hence restricting movement during
feeding and affecting their feed intake even though the food supply is in sufficient
quantity (Douros, 1987; Lloyd and Bates, 2008). A similar reduction in feeding
behaviour has been found in other abalone species (Mgaya and Mercer, 1995; Tarr,
1995; Marsden and Williams, 1996), particularly when they are fed algae (Tahil and
Juinio-Menes, 1999). Additionally, the highest stocking density (200 abalones m-2) in
the present study, not only decreased feed intake in the smaller size abalones, but also
increased FCR and reduced PER, denoting a reduction in feed utilisation efficacy.

Despite the fact that stocking density may have a negative effect on abalone
survival (Nie et al., 1996), in the present study, and in agreement with previous
research, survival was not influenced by density (Mgaya and Mercer, 1995; Capinpin et
al., 1999; Jarayabhand et al., 2010). The high survival rates (94-98%) registered for all
treatments, regardless the density conditions, may well indicate not only the mentioned
general balance of nutrients in the enriched mixed diet but also the generally good
culture conditions that could have contributed to the well-being of the abalone. Indeed,

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besides feeding regime and stocking density, several factors are important in providing
the optimal conditions for growth and survival in abalone such as water flow, water
quality, light, shading and culture system (Capinpin et al., 1999; Huchette et al., 2003a;
Demetropoulos and Langdon, 2004; Badillo et al., 2007; Wassnig et al., 2010). Abalone
growth and health are reported to be inhibited by decreases in water quality (Basuyaux
and Mathieu, 1999; Naylor et al., 2011) which can result from the decomposition of
faeces and uneaten food (Yearsley et al., 2009), high levels of nitrogenous wastes
excreted by the animals (Barkai and Grifths, 1987; Reddy-Lapata et al., 2006),
reduction of dissolved oxygen (Badillo et al., 2007; Naylor et al., 2012) or from low
water pH exposure (Naylor et al., 2012). Hence, the high water exchange rate linked to
the oceanic conditions of the open water experimental set-up, were consequently
appropriate for H. tuberculata coccinea culture, maintaining water quality even as
stocking densities increased (Wassnig et al., 2010). Abalone are generally more active
at night, light and shading clearly influence their distribution and activities (Cochard,
1980; Huchette et al., 2003b; Morikawa and Norman, 2003), hence their feeding
behaviour (Tahil and Juinio-Menes, 1999). Since the cages were located at 10 m depth,
the influence of light on the regulation of abalone´s feeding activity inside the
experimental cages, could have been limited. Additionally, the refuges provided could
have led to an improvement of growth rates by increasing the “preferred surface area”
(Hindrum et al., 1999). Regarding culture system, growth performance of H.
tuberculata coccinea cultured in the tested offshore cages, was significantly better than
the one obtained under similar experimental conditions in a land-based system (60.2 μm
d-1; WG: 151%; Viera et al., submitted).

In summary, data demonstrated the suitability of the enriched mixed macroalgae

diet together with the offshore mariculture system, both essential factors contributing to
successful grow-out of H. tuberculata coccinea that could reach the
cocktail/commercial size of 45-60 mm in only 18-22 months. However, doubling the
initial densities in both experimental trials reduced growth in a 17-19% (WG) at the end
of the experimental period.

The stocking densities tested and providing the best growth rates are within the
range of those currently applied in commercial sea-based system for H. tuberculata (30-
125 abalones m-2 for 30- ≥ 65mm; Huchette, personal communication). Therefore, as the
choice of stocking density is essentially a trade–off between maximum growth, optimal

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biomass gain and economic considerations, cost-benefit analysis would be required to

determine the optimum stocking density when setting up abalone on-growing
operations.

7.5. ACKNOWLEDGEMENTS

The authors especially thank Rafael Guirao, and the rest of the staff from
CANEXMAR, S.L., who have kindly cooperated in this study hosted within their
facilities. They also acknowledge Tony Legg, from Jersey Sea Farms, for kindly
supplying the ORTACS. They would also like to thank Desirée Chacón and Beatriz
Sosa for their support through maintenance tasks in harsh conditions and to Dr.
Fernando Tuya for his assistance with the statistical analysis. This study has been
financed by the Spanish Government in the frame of the National Plan for Development
of Marine Cultures (Jacumar Multitrófico).

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Development of a Sustainable Grow-out Technology for Abalone Haliotis
tuberculata coccinea (Reeve) as a New Species for Aquaculture
Diversification in the Canary Islands

CONCLUSIONS
 Conclusions

8. CONCLUSIONS

Study I: Suitability of three red macroalgae as a feed for the abalone Haliotis
tuberculata coccinea Reeve.

1. Biofilter produced Hypnea musciformis, Hypnea spinella and Gracilaria

cornea showed adequate nutritional composition, to be potentially used as feed
for juvenile Haliotis tuberculata coccinea. High protein contents were found
for the three algal species investigated, which would be related to their
production under the high nitrogen conditions of the IMTA system.

2. H. spinella was found to be the best growth promoting diet for H. tuberculata
coccinea due to the highest feed intake and protein efficiency ratio observed.
The harder texture of G. cornea had a negative effect on feed consumption
hence leading to the lowest growth performance of Canarian abalone.

3. Overall growth rate of abalone were within the range of those obtained under
commercial conditions this fact, linked to the high survival attained, suggesting
the suitability of the three red seaweeds tested, successfully produced in the
IMTA system, for the grow-out culture of H. tuberculata coccinea.

Study II: Comparative performances of juveniles (Haliotis tuberculata coccinea

Reeve) fed enriched vs non-enriched macroalgae: effect on growth and body
composition.

4. The rearing system employed markedly affected seaweeds proximate

composition, specifically protein content, which was 100-163% increased in
those reared in fishpond wastewater effluents compared with those in fresh
seawater.

5. The fatty acid profiles of the algae studied were characteristic of green and red
algae, palmitic acid being the most abundant SFA, the Chlorophyta Ulva rigida
showing predominat levels of C16 and C18 PUFAs and minimal levels of C20

150
 Conclusions

fatty acids. DHA was very low in all algae tested, hence this fatty acid do not
appear to be essential in H. t. coccinea, as all macroalgae tested supported
optimal growth of this abalone species.

6. Abalone fed enriched macroalgae diets showed far better performance

compared to those fed non enriched diets, suggesting that nitrogen may be a
limiting factor for growth in H. tuberculata coccinea.

7. Animals fed the mixed diets, both enriched and non enriched, performed
significantly better than those that were fed with a single algal diet, indicating
that abalone obtain a complete range of required nutrients by eating a mixed
algal regime and that essential nutrients may become limiting when animal are
fed single-species diets.

8. Abalone biochemical composition was markedly affected by the diets. Lipid

content in abalone tissues were generally higher than in their respective
macroalgal diets. Foot muscle contained significantly lower lipid levels than
viscera indicating that selective storage of lipids occurs in the
hepatopancreas/gonad assemblage.

9. The dietary value of the macroalgal regimes tested can be divided into three
categories: Best obtained with the mixed algal feeding regime, intermediate by
using single Ulva rigida or Hypnea spinella feeding regimes and the lowest by
Gracilaria cornea.

10. Growth results clearly indicate that H. tuberculata coccinea can be efficiently
grow-out in an integrated-culture system suggesting that on-farm seaweed-
abalone production could be a part of future development of abalone industry
in the Canary Islands.

Study III: First development of various vegetable-based diets and their suitability
for abalone Haliotis tuberculata coccinea.

151
 Conclusions

11. Feeding the fresh algae produced a far better growth for H. tuberculata
coccinea than all the compound diets, indicating the high dietary value of the
macroalgae reared in the IMTA system.

12. The inclusion of P. palmata was found to improve growth, condition index and
dietary protein utilization, while the use of L. digitata markedly reduced the
efficiency of dietary protein.

13. The elevated contents, relative to their feeds, of ARA in the abalone fed the
experimental diets and EPA in abalone fed the fresh algae, denoted the
presence of the respective elongases Δ4 and Δ5 desaturases. However, the low
content of DHA further suggested that this fatty acid is not essential in abalone
tissues.

14. Overall, feeding H. tuberculata coccinea with vegetable-based artificial diets

resulted in high survival and good dietary protein utilization.

Study IV: Grow out culture of abalone Haliotis tuberculata coccinea Reeve, fed
land-based IMTA produced macroalgae, in a combined fish/abalone offshore
mariculture system: Effect of stocking density.

15. Doubling stocking density markedly reduced growth performance in both

distinct initial size groups of abalones, showing a reduction in feeding
behaviour and feed utilisation efficacy mainly linked to a higher
competition for space or food.

16. Recommended stocking density for 30-45 mm abalone is 100 abalone m-2,
while for 45 mm abalone until commercial size is 30 abalone m-2.

17. The offshore mariculture system as well as the biofilter produced

macroalgae, were found to be suitable to sustain high growth and survival of
H. tuberculata coccinea that overall could reach cocktail/commercial size of
45-60 mm in only 18-22 months.

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Desarrollo Sostenible de la Tecnología de Engorde de la Oreja de Mar
Haliotis tuberculata coccinea (Reeve) como Nueva Especie para la
Acuicultura en Canarias

RESUMEN EN ESPAÑOL
 Spanish summary

9.1. INTRODUCCIÓN

1.1. LA OREJA DE MAR: DESCRIPCIÓN, TAXONOMÍA, PRODUCCIÓN Y

ACUICULTURA

La oreja de mar o abalón es un molusco gasterópodo univalvo que pertenece a la

familia Haliotidae (Barkai y Griffiths, 1986) (Tabla 1; Fig.1). En la actualidad, hay
descritas 56 especies, todas pertenecientes al género Haliotis (Geiger, 2000).
Normalmente se localizan entre la zona intermareal y litoral (Hone y Fleming, 1998),
alcanzando las densidades máximas entre los 3–10 m donde las algas, su alimento
natural, crece abundantemente. Todos son bentónicos, estando los ejemplares recién
asentados normalmente asociados a sustratos de algas coralinas (Hone et al., 1997;
Roberts, 2001), mientras que los juveniles (≥ 10 mm de talla) y adultos, ramonean
principalmente sobre algas fijadas o a la deriva por las corrientes (Shepherd, 1973;
Hanh et al., 1989; Shepherd y Steinberg, 1992). Son animales gonocóricos, con machos
y hembras que liberan sincrónicamente sus gametos a la masa de agua para la
reproducción (Stephenson, 1924; Crofts, 1929). El sexo de los ejemplares maduros se
puede diferenciar fácilmente por la diferencia en el color de las gónadas (Bardach et al.,
1972). Las especies de abalón de las regiones templadas alcanzan un mayor tamaño (20-
30 cm) que las especies tropicales (6-10 cm) (Hanh et al., 1989; Jarayabhand y
Paphavasit, 1996).

Tabla 1. Clasificación taxonómica de las especies de abalón

Filum: Mollusca Linnaeus, 1758

Clase: Gastrópoda Cuvier, 1797
Subclase: Prosobranquia H.M. Edwards, 1848
Orden: Archaeogastropoda Thiele, 1929
Superfamilia: Pleurotomarioidea Swainson, 1840
Familia: Haliotidae Rafinesque, 1815
Género: Haliotis Linnaeus, 1758

153
 Spanish summary

Figura 1. Ejemplares de abalón (H. tuberculata coccinea), recién asentados, juvenil y

adulto.

A nivel mundial la oreja de mar se ha considerado tradicionalmente un producto

de alta calidad y valor, usándose su carne como alimento mientras que su concha, que
tiene un interior nacarado, se usa normalmente con fines decorativos, en artesanía e
incluso en joyería (Howorth, 1988; Fig. 2).

Figura 2. Productos derivados del abalón: culinarios, perlas y artesanía.

A pesar de que los mayores consumidores mundiales de oreja de mar han sido
tradicionalmente Japón y China (las primeras referencias de la pesca de abalón datan del
30 A.D.; Fig. 3), las pesquerías de este molusco también han sido una fuente tradicional
de desarrollo económico y social de otras poblaciones en diferentes países como
Estados Unidos de América, México, Nueva Zelanda, Francia, Australia o Sudáfrica
(Leighton, 1989; Guzmán del Proó, 1992; Schiel, 1992; Mercer et al., 1993; Freeman,
2001; Troell et al., 2006), con ciertas comunidades locales muy vinculadas a la
explotación de este recurso.

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 Spanish summary

Figura 3. Amas, buceadoras - pescadoras de abalón (Japón).

A pesar de que el estudio sobre la biología y ecología de la oreja de mar

comenzó hace mucho tiempo (Stephenson, 1924; Bonnot, 1930; Crofts, 1932), su
investigación se ha ido intensificando en los últimos 35 años, en primer lugar como
consecuencia del desarrollo de las pesquerías y en las dos últimas décadas, por el
desarrollo exponencial de la acuicultura de este molusco. De hecho, debido a la
sobreexplotación de los recursos, planificación incorrecta, destrucción del hábitat,
enfermedades o políticas inadecuadas, las pesquerías legales de abalón sufrieron un
descenso desde casi 20.000 toneladas (t) en los 70 a menos de 9.000t en 2008 (Cook y
Gordon, 2010). Es más, a pesar de que los cultivos de abalón habían empezado a finales
de los años 50 y principio de los 60 en Japón y China (Elbert y Houk, 1984; Leighton,
2000), en los años 90 tuvo lugar un rápido desarrollo en muchos países del mundo, con
el fin de satisfacer la creciente demanda que no estaba siendo abastecida por el sector
pesquero. Este aumento fue posible gracias al desarrollo de ciertas prácticas de
producción, especialmente para la etapas juveniles, que permitieron la rápida expansión
de la industria (Daume et al., 2004; Roberts et al., 2004).

En concreto, el cultivo mundial de abalón ha crecido de forma espectacular en la

última década, multiplicándose por más de 21 veces (Fig. 4). En 2010, la producción
mundial de abalón fue de 65.525t, con un valor de aproximadamente 451 millones €, de
ellas, tan sólo 8.656t procedían del sector pesquero, lo que significa que
aproximadamente un 88% del consumo de abalón de procedencia legal fue abastecido
por el sector acuicola (FAO, 2012).

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Figura 4. Evolución de la producción global de abalón procedente de pesquerías

legales y acuicultura durante la última década (source FAO, 2012).

De entre todas las especies de abalón, aproximadamente 15 son explotadas

comercialmente, tanto por el sector pesquero como acuícola (Bester et al., 2004; Sales y
Janssens, 2004; Hernández et al., 2009): Abalón ezo pacifico (Haliotis discus hannai
Ino, 1953), kuro (Haliotis discus Reeve, 1846), tokobushi (Haliotis diversicolor Reeve,
1846) y abalón gigante (Haliotis gigantea Gmelin, 1791) principalmente en Japón,
China y Corea; abalón tropical (Haliotis asinina Linnaeus, 1758) en Tailandia y
Filipinas; abalón negro (Haliotis rubra Leach, 1814), abalón verde (Haliotis laevigata
Donovan, 1808) y abalón de Roe (Haliotis roei Gray, 1826) en Australia; paua de pie
negro (Haliotis iris Gmelin, 1791) y paua de pie amarillo (Haliotis australis Gmelin,
1791) en Nueva Zelanda; perlemoen (Haliotis midae Linnaeus, 1758) en Sudáfrica;
abalón verde (Haliotis fulgens Philippi, 1845), abalón rojo (Haliotis rufescens,
Swainson, 1822) y abalón rosa (Haliotis corrugata Wood, 1828) en México y Estados
Unidos de América; abalón negro (Haliotis cracherodii Leach, 1814) Estados Unidos
de América; abalón pinto (Haliotis kamtschatkana Jonas, 1845) desde Alaska y Canada
hasta Estados Unidos de América (California) y el abalón europeo (Haliotis tuberculata
Linneaus, 1758) en Gran Bretaña y Francia (Atlántico occidental).

En cuanto a los países productores, China es el mayor productor mundial con

una producción de 56.511t en 2010 (FAO, 2012). La principal especie de cultivo es el
valioso abalón del Pacífico (H. discus hannai) (Wu et al., 2009), que abarca más del

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95% de la producción. Sin embargo, la especie de menor tamaño y menos apreciada

H. diversicolor cuyo cultivo era el más importante hasta hace diez años de, se limita en
la actualidad a la China subtropical y tropical (Ke et al., 2012).

En Corea, el ascenso de la producción lo ha situado como el segundo productor

mundial, con más de 5.000 granjeros dedicados al cultivo de abalón (Park y Kim, 2013).
A pesar de que el cultivo de abalón en Corea comenzó tan sólo a principio de los 2000,
el desarrollo de nuevas metodologías de cultivo, principalmente la implantación de
jaulas en el mar, ha dado lugar a un incremento espectacular de la producción que pasó
de 29t en 2001 a 6.228t en 2010 (FAO, 2012).

Sudáfrica es el mayor productor de abalón (H. midae) fuera de Asia, con una
producción procedente tanto del sector pesquero como de la acuicultura. La producción
anual ronda las 3.000t, de las que 1.200t son aportadas por el sector acuicola, 150t por
las pesquerías legales, mientras que se estima que unas 1.500t son capturadas de forma
ilegal (Britz, 2012).

A pesar de que la oreja de mar no es nativa de Chile, durante los años 90 se

desarrolló el cultivo comercial de este molusco (H. rufescens y H. discus hannai)
principalmente para su exportación a mercados asiáticos, alcanzando una producción de
casi 800t (99% de H. rufescens) en 2010 (FAO, 2012).

Más del 50% de la pesca mundial de abalón procede de Australia, donde

también existe un sector acuícola creciente que produjo 456t (H. rubra, H. laevigata e
híbridos) en 2010 (FAO, 2012).

Además, hay pequeñas industrias en los Estados Unidos de América (250t),

Taiwan (171t) y Nueva Zelanda (80t, H. iris), que contribuyen a la producción mundial
de oreja de mar (FAO, 2012).

En cuanto a los países consumidores, la mayor demanda de abalón se localiza en

Asia, principalmente en Japón (vivo, fresco y congelado), y la China peninsular
(enlatados), además también existen mercados estables en México, EUA y Europa
(Oakes y Ponte, 1996; Robertson-Anderson, 2003). Como consecuencia, la mayoría de
los cultivos de abalón en el mundo se localizan en Asia, y es a este continente donde se
destina la mayoría de la producción mundial de este molusco. Sin embargo, la alta
producción mundial durante los últimos años unido a la disponibilidad de abalón
procedente de capturas ilegales, así como la crisis financiera mundial, ha llevado a una

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reducción generalizada del precio de abalón (Qi et al., 2010). Por lo tanto, los
productores de abalón están encaminados a buscar vías para aumentar su productividad
a través del uso de sistemas de cultivos más eficientes, certificaciones internacionales de
calidad o diversificación de productos y mercados (Cook y Gordon, 2010).

1.2. LA OREJA DE MAR EN EUROPA

En Europa solo está presente una especie de abalón, la conocida popularmente

como ormer, Haliotis tuberculata Linneaus, 1758 (Mgaya, 1995), con tres sub-especies
descritas a partir de sus características morfológicas: H. tuberculata tuberculata
Linneaus, 1758, en el costa del Atlántico oriental y el Mar Mediterráneo, y H.
tuberculata coccinea Reeve, 1846 junto a la recientemente descrita H. tuberculata
fernandesi Owen y Afonso, 2012, que se distribuyen fundamentalmente en la region
macaronésica (Clavier, 1992; Geiger, 2000; Geiger y Owen, 2012; Fig. 5).

Figura 5. Distribución de H.tuberculata tuberculata y H. tuberculata coccinea

(Geiger, 2000).

El ormer, que alcanza un tamaño máximo de 14 cm (Roussel et al., 2011), es una

especie de molusco de bastante importancia comercial en las Islas del Canal (Bossy y
Culley, 1976) y las costas de Normandía y Bretaña en Francia (Clavier, 1992), donde se
encuentran poblaciones importantes y donde ha sido un recurso tradicional de alto valor
(Mgaya y Mercer, 1994).

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Debido a la sobreexplotación de los stocks naturales y al aumento del valor del

producto, a partir del final de la década de los 70 hubo un aumento del interés sobre el
cultivo de esta especie, lo que motivó el desarrollo de la tecnología de producción
principalmente en las Islas del Canal, (Bossy, 1989, 1990; Hayashi, 1982; Hjul, 1991),
Francia (Koike, 1978; Koike et al., 1979; Flassch y Aveline, 1984) e Irlanda (Mercer,
1981; LaTouche y Moylan 1984; La Touche et al., 1993; Mercer et al., 1993; Mgaya y
Mercer 1994, 1995; Mai et al., 1995a,b, 1996), donde tanto el abalón europeo como el
japonés fueron introducidos al final de los 70 y mitad de los 80 (Leighton, 2008).

Sin embargo, a pesar de haber sido reconocida como especie candidata para la
acuicultura europea hace ya dos décadas (Mgaya y Mercer, 1994), su disponibilidad en
Europa es aún insuficiente debido a la escasez de suministro tanto del sector pesquero
como del acuícola (Dallimore, 2010). Si bien se han realizado ciertos intentos para
aumentar la producción acuícola, el avance ha sido lento, dificultado entre otro motivos,
por la escasez en la investigación o por problemas derivados de confusion en la
legislación del recurso (la oreja de mar está situada en la misma categoría que los
bivalvos). Además, aspectos nutricionales como el tipo de alimento y sus fuentes, así
como el desarrollo de tecnologías sostenibles de producción, han sido también
identificados como áreas claves en las que avanzar para alcanzar mejores crecimientos y
métodos de producción que redunden en la competitividad del sector productivo
(SUDEVAB, 2007).

La producción de abalón en Europa está actualmente enfocada hacia un producto

de alta calidad, pequeño volumen y que ocupa su nicho de mercado, tales como los
productos con certificación orgánica o ecológica. Irlanda, las Islas del Canal (Huchette y
Clavier, 2004) y Francia son actualmente los únicos países productores, con la mayoría
de las granjas engordando el abalón en el mar, y basando su alimentación en macroalgas
cosechadas manualmente en el entorno próximo. Francia, es el principal productor
europeo con una producción de 10t en 2010 (FAO, 2012), la mayoría de la cual procede
de la misma empresa, que es además la primera granja de abalón en el mundo en tener
certificación ecológica (100% de las macroalgas cosechadas de forma sostenible;
ausencia de medicamentos, químicos o fertilizantes). Dicha granja vende ejemplares
vivos de 6-7 cm engordados en el mar a 69 € por kg (Legg et al., 2012).

En cuanto a España, en Galicia recientemente ha empezado a funcionar una

instalación en tierra, en circuito cerrado (criadero y engorde) para el cultivo de la

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especie europea H. tuberculata tuberculata y la japonesa (introducida) H. discus

hannai, esperando alcanzar una producción de 115 t en 2017 (GMA, 2014).

1.3. OREJA DE MAR (Haliotis tuberculata coccinea): CANDIDATA PARA LA

DIVERSIFICACIÓN DE LA ACUICULTURA EN CANARIAS

Las excepcionales condiciones climáticas y alta calidad de las aguas canarias

hacen que esta region española cuente con unas muy buenas perspectivas para la
expansión de la acuicultura. A pesar de ello, el sector acuícola en el archipiélago se
limita exclusivamente a la producción de especies de peces marinos tales como dorada
(Sparus aurata), lubina (Dicentrachus labrax) y una pequeña cantidad de lenguado
senegalés (Solea senegalensis) (APROMAR, 2012). Por tanto, la diversificación de las
especies cultivadas es uno de los retos más importantes del sector productivo de
Canarias.

En las Islas Canarias, la oreja de mar H. tuberculata coccinea, conocida

popularmente como almeja canaria, se distribuye desde la zona intermareal hasta unos
15 m de profundidad en areas expuestas y semi expuestas. Alcanza un tamaño máximo
de 8 cm de longitud de concha y se alimenta de mezclas de macroalgas (Espino y
Herrera, 2002).

La almeja canaria ha sido explotada a nivel local durante las ultimas décadas, lo
que ha llevado a la sobreexplotación del recurso hasta su casi esquilmación (Pérez y
Moreno, 1991; Espino y Herrera, 2002). Como consecuencia, existe un público interés
en el desarrollo de la tecnología de cultivo de esta especie, tanto de cara al suministro de
la población local como para contribuir a la recuperación de las poblaciones naturales.
Es más, el potencial de esta especie para su producción en acuicultura, radica también
en la creciente demanda externa por ejemplares pequeños del denominado “tamaño
cocktail” (4-7 cm de longitud de concha) (Jarayabhand y Paphavasit, 1996; Najmudeen
y Victor, 2004), así como en el alto grado de desarrollo alcanzado en el cultivo de otras
species de la familia Haliotidae, en particular del ya mencionado abalón europeo
Haliotis tuberculata tuberculata L., de características biológicas muy parecidas.

Estudios previos realizados en H. t. coccinea sobre reproducción (Peña, 1985,

1986; Bilbao et al., 2004, 2010) y etapas tempranas de desarrollo (Courtois de Viçose et

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al., 2007, 2009, 2010, 2012a, b), han permitido tanto la reproducción de esta especie en
cautividad, como las técnicas de producción de semillas adaptadas a esta especie,
señalándola por tanto, como una buena candidata para la diversificación del sector
acuícola canario.

Sin embargo, apenas existen trabajos sobre las condiciones óptimas para el
cultivo de juveniles y adultos en las Islas Canarias (Toledo et al., 2000). Por tanto, para
desarrollar la tecnología de engorde adaptada a esta especie, es necesario investigar en
las areas de nutrición y alimentación, así como en las condiciones y sistemas de cultivo
idóneos para su implantación en las islas. Además, considerando que el cultivo de este
molusco frecuentemente require altas cantidades de macroalgas, no disponibles
localmente, para el desarrollo de la producción es también necesario evaluar otras
alternativas como fuentes de alimentación. Es más, los trabajos también deben estar
encaminados hacia el desarrollo de métodos de producción que aseguren la
sostenibilidad de la industria, ajustados a la cada vez más restrictiva legislación europea
para la producción de mariscos y también a la normativa para la producción
internacional de abalón (WWF, 2010).

1.4. FACTORES CONDICIONANTES EN EL ENGORDE DE LA OREJA DE MAR

De forma general, la oreja de mar presenta un crecimiento lento y variable (Day

y Fleming, 1992; Britz 1996a; Sales y Britz, 2001), con tasas de aproximadamente 2-3
cm/año, requeriéndose por tanto de 2-5 años para alcanzar las talla comercial (Hahn,
1989; Troell et al., 2006; Qi et al., 2010). En consecuencia, una tecnología de engorde
adecuada y el resultante crecimiento y supervivencia de los animales en cultivo, son
factores críticos para maximizar el éxito de la producción.

La selección de la tecnología de cultivo dependerá de las características propias

de la especie y de su susceptibilidad hacia los diferentes factores que condicionan el
éxito del proceso de engorde. Algunos de estos factores están relacionados con las
condiciones generales de cultivo; con los parámetros físicos-químicos; mientras que
otros están relacionados con la alimentación de los juveniles y adultos. Finalmente, la
viabilidad económica de la producción de abalón se verá especialmente condicionada
por el sistema de cultivo empleado durante la larga fase de engorde.

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1.4.1. Parámetros generales

1.4.1.1. Talla inicial

Justo después del asentamiento, las postlarvas de abalón se alimentan de
películas bacterianas y huellas de mucus segregados por los propios animales
(Shepherd, 1973; Saito, 1981), y una vez que la rádula está desarrollada, lo que tiene
lugar alrededor de los 0.8 mm de longitud de concha (SL) (Kawamura et al., 2001), los
juveniles empiezan a ramonear sobre diatomeas bentónicas, algas coralinas incrustantes
y películas bacterianas (Dunstan et al., 1996, 1998). Los juveniles mantienen dichas
dietas hasta alcanzar el tamaño suficiente que les permita realizar la transición final de
las diatomeas a las macroalgas (Jarayabhand y Paphavasit, 1996; Kawamura et al.,
2001). El tamaño en el que los animales realizan esta fase final varía entre las distintas
especies, estando situado entre los 5 y 10 mm para el H. discus hannai (Kawamura et
al., 2001), 7 y 8 mm para H. rufescens (Hahn, 1989), 10 y 20 mm para H. asinina y H.
ovina (Jarayabhand y Paphavasit, 1996) o 10 mm para H. tuberculata (Mgaya y Mercer,
1994). En el caso del abalón presente en Canarias, la talla se encuentra entre 6 y 10 mm
de longitud (Courtois de ViÇose, comunicación personal).

La talla de los ejemplares en cultivo ha sido señalada como uno de los factores
más influyentes en el crecimiento del abalón (Flemming y Hone, 1996; Wu et al.,
2009). Estas variaciones pueden deberse a diferencias genéticas relacionadas con el
tamaño de los ejemplares (Sun et al., 1993; Wu et al., 2009), con los requerimientos de
proteínas (Shipton y Britz, 2001), con la utilización de las proteínas y la energía (Britz y
Hecht, 1997; Shipton y Britz 2001; Green et al., 2011) o bien con diferencias en la
capacidad de la rádula para ramonear el alimento (Jonhston et al., 2005).

1.4.1.2. Densidad de cultivo

La densidad de cultivo, además de ser un factor que condiciona el tipo de

sistema, ha sido señalado como unos de los factores de mayor influencia sobre el
crecimiento y supervivencia de la oreja de mar, siendo por tanto un factor determinante
para alcanzar la talla commercial. El efecto negativo provocado por el incremento de la
densidad, parece estar estrechamente relacionado con la alta competencia tanto por el
espacio como por el alimento que estos animales presentan, y al estrés inherente a la

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gran acumulación de individuos, tal y como se ha demostrado para la mayoría de las

especies comerciales de abalón: H. tuberculata (Koike et al., 1979; Cochard, 1980;
Mgaya y Mercer, 1995); H. cracherodii (Douros, 1987); H. discus hannai (Jee et al.,
1988; Wu et al., 2009; Wu y Zhang, 2013); H. rubra (Huchette et al., 2003a, b); H.
asinina (Capinpin et al., 1999; Fermin y Buen, 2002; Jarayabhand et al., 2010); Haliotis
diversicolor supertexta (Liu y Chen, 1999); H. rufescens (McCormick et al., 1992;
Aviles y Shepeherd, 1996, Valdés-Urriolagoitia, 2000); H. midae (Tarr, 1995); H.
corrugata (Badillo et al., 2007); H. kamtschatkana (Lloyd y Bates, 2008) y for H. iris
(Heath y Moss, 2009).

Si bien la influencia de la densidad de cultivo sobre el crecimiento del abalón en

Canarias aun no ha sido estudiado, densidades de cultivos experimentales de 43-175
abalones m2 en jaulas de otra especie de talla pequeña como H. asinina (Capinpin et al.,
1999), y de 83-386 abalones m2 para juveniles del abalón europeo (Koike et al., 1979;
Mgaya y Mercer, 1995), mostraron una marcada influencia en la talla final de los
ejemplares. De hecho, el triplicar la densidad de cultivo puede causar una reducción del
52% en el crecimiento de H. tuberculata (Mgaya y Mercer, 1995).

1.4.1.3. Renovación de agua

El cultivo de la oreja de mar se caracteriza por necesitar una alta renovación del
medio (200% a 2.400% por día) para mantener la calidad del agua dentro de los
parámetros recomendados (Badillo et al., 2007; Naylor et al., 2011). Lo que es más, una
adecuada renovación influye positivamente sobre el factor de conversion del alimento
(FCR) mediante la estimulación de la actividad alimenticia (Higham et al., 1998),
aumentando por tanto el crecimiento del abalón (Shepherd, 1973; Mgaya y Mercer,
1995; Wassnig et al., 2010). Adicionalmente, el aumentar la renovación del agua puede
ser un medio eficiente de mitigar los efectos nocivos de las altas densidades de cultivo
contribuyendo a los beneficios de la actividad (Wassnig et al., 2010).

Además, Tissot (1992) estableció que para un crecimiento óptimo del abalón es
necesario una adecuada velocidad del agua que permita una circulación suficiente a
través del manto de los animales. Sin embargo, en el caso de los tanques poco
profundos (como los denominados tanques ‘slab’), muy extendidos en granjas
comerciales (Hutchinson y Vandepeer, 2004; Wassnig et al., 2010), una alta renovación

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puede tener efectos negativos al favorecer la lixiviación de nutrientes del pienso

(Fleming et al., 1997; Marchetti et al., 1999) y propiciar también la pérdida de los
mismos como efecto de la corriente (Fleming et al., 1997). Por tanto, la velocidad del
agua es un factor a tener en cuenta para no supercar ciertos límites.

En sistemas de cultivos abiertos, el coste asociado al mantenimiento de una alta

tasa de renovación (principalmente bombeo) corresponde entre el 15% y 30% del total
de los gastos operacionales (Neori et al., 2000; Badillo et al., 2007). Así, y con el fin de
no solo reducir costes sino también la emisión de nutrientes al medio, se han
desarrollado diversos sistemas de recirculación, tanques en serie, o incluso sistemas de
cultivos multitróficos (algas-abalón) en recirculación, como sistemas de engorde para
diferentes especies tales como H. tuberculata (Mgaya y Mercer, 1995; Schuenhoff et
al., 2003), H. discus hannai (Nie et al., 1996; Park et al., 2008; Demetropoulos y
Langdon, 2004), H. rufescens y Haliotis sorenseni, (Demetropoulos y Langdon, 2004),
H. corrugata (Badillo et al., 2007), H. asinina (Jarayabhand et al., 2010), H. midae
(Naylor et al., 2011) o H. iris (Tait, 2012).

1.4.2. Parámetros físico-químicos

1.4.2.1. Temperatura

La temperatura es uno de los parámetros ambientales más influyentes en la tasa

metabólica de los organísmos poiquilotermos (Fry, 1971) y por tanto, de vital
importancia en el manejo del engorde del abalón, ya que afecta directamente a la tasa de
desarrollo gonadal (Uki y Kikuchi, 1984; Hahn, 1989), al consumo de alimento (Peck,
1989; Britz et al., 1997; García-Esquivel et al., 2007), a la excreción de amonio (Barkai
y Griffiths, 1987; Lyon, 1995), al crecimiento e indices nutricionales (Leighton, 1974;
Uki et al., 1981; Hahn, 1989; Peck, 1989; Britz et al., 1997; Gilroy y Edwards, 1998;
Hoshikawa et al., 1998; Lopez et al., 1998; Steinarsson y Imsland, 2003; Alcántara y
Noro, 2006; García-Esquivel et al., 2007); supervivencia (Hahn, 1989), susceptibilidad
al estrés (Lee et al., 2001; Haldane, 2002; Malham et al., 2003) o a la definición de los
protocolos de cultivo a implementar (Vandepeer, 2006).

Aunque cada especie de abalón tiene un rango de temperatura óptimo, de forma

general las especies templadas se encuentran en rangos de temperatura de entre 8-18ºC,

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las subtropicales entre 14-26ºC y las tropicales entre los 16-30ºC. Gilroy y Edwards
(1998) establecieron que en general, la oreja de mar tiene una respuesta térmica
conservativa y poca capacidad de adaptación a los cambios termales, por lo tanto, la
temperatura del cultivo debería ser lo más cercana posible a la óptima de la especie.

La temperatura del agua de mar en Canarias se encuentra entre 18 y 24ºC

(Cuevas., 2006), por tanto, más parecida a la natural de las especies tropicales y
subtropicales que a la cercana Haliotis tuberculata, considerada una especie templada
(Peck, 1989).

1.4.2.2. Luz

A pesar de que el habitat y el comportamiento varía según las especies de abalón

(Shepherd, 1975), en general su actividad es mayor durante la noche, existiendo una
gran influencia de la luz sobre su distribución y actividades (Cochard, 1980; Huchette et
al., 2003b; Morikawa y Norman, 2003), y por tanto sobre sus pautas alimenticias (Tahil
y Juinio-Menes, 1999). En consecuencia, el aporte de refugios o sombreos en los
cultivos son herramientas útiles en incluso necesarias para un óptimo crecimiento
(Maguire et al., 1996; Fig. 6). Es más, la provision de sombras y refugios en
condiciones de alta densidad, puede ayudar a favorecer el crecimiento de los animales
mediante, por un lado, el aumento de “las zonas preferidas” reduciendo por tanto el
apilamiento de los animales, y por otro, limitando la influencia del fotoperíodo en la
regulación de la actividad alimenticia (Hindrum et al., 1999; Huchette et al., 2003b). Por
el contrario, el sombreo y los refugios pueden también presentar ciertas desventajas,
como son el incremento de la mano de obra en las labores de alimentación y
mantenimiento o la reducción de la circulación (Maguire et al., 1996).

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Figura 6. Tejas de PVC empleadas como refugios en los cultivos de abalón en

Tailandia (A; H. asinina) y en Canarias (B; H. tuberculata coccinea).

1.4.2.3. Calidad del agua

Es ampliamente conocida la influencia negativa del descenso de la calidad de

agua sobre el crecimiento y la salud de la oreja de mar (Basuyaux y Mathieu 1999;
Naylor et al., 2011). En el engorde de abalón, este deterioro puede deberse a la
presencia de heces y restos de comida (Yearsley et al., 2009), a altos niveles de
compuestos nitrogenados resultantes de la excreción de los animales (Barkai y Grifths,
1987) o a una disminución de la concetración de oxígeno (Badillo et al., 2007).

De forma general, para lograr tasas de crecimiento máximas se asume que el

oxígeno disuelto del agua debe de mantenerse a niveles de saturación (100%), y una vez
que estos niveles están logrados, el parámetro de cultivo más importante pasa a ser la
presencia en el agua de compuestos nitrogenados derivados de la excreción (Colt y
Armstrong, 1981). Entre estos compuestos, es especialmente tóxico el amonio resultante
del propio catabolismo de los animales (Kinne, 1976; Russo y Thurston, 1991) y de la
actividad bacteriana (Reddy-Lopata et al., 2006), siendo un factor estresante e incluso
letal en el cultivo de este molusco (Hargreaves y Kucuk, 2000; Huchette et al., 2003a).
Debido a ello, existen numerosos trabajos sobre la influencia de este compuesto sobre la
supervivencia y crecimiento de diversas especies de abalón como son H. laevigata
(Harris et al., 1998, Hindrum et al., 2001), H. tuberculata (Basuyaux y Mathieu, 1999),
H. rubra, Huchette et al., 2003a), H. diversicolor supertexta (Cheng et al., 2004) o H.

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midae (Reddy-Lopata et al., 2006; Naylor et al., 2011), en los que de forma general se
concluye que, a pesar de que los abalones se pueden adaptar a niveles sub-letales de
amonio en el agua, sí tiene lugar una reducción sustancial del crecimiento, siendo por
tanto esencial el mantener niveles bajos de amonio. En los cultivos de oreja de mar, los
niveles de amonio se regulan a través de la renovación del agua, debiéndose además
lograr un equilibrio entre las demandas tanto fisiológicas como económicas (Ford y
Langdon, 2000).

1.4.3. Sistemas de engorde

El sistema de cultivo ideal para el engorde de oreja de mar debe ser aquel que
favorezca una buena destribución de los animales, fácil acceso al alimento, mínimo
contacto entre los animales en cultivo y sus heces, un buen flujo de agua y renovación,
un medio de acomodación y alimentación de los animales en densidades de cultivo
comerciales y un mínimo de manejo (McShane, 1988; Aviles y Shepherd, 1996;
Fleming y Hone, 1996). Así, son numerosos los sistemas de cultivo desarrollados hasta
el momento tanto en tierra como en el mar, actividad que aún continua, resultando en
una gran disparidad de sistemas en las distintas granjas dando lugar por tanto a
crecimientos muy dispares.

1.4.3.1. Sistemas en tierra

Los sistemas de engorde en tierra abarcan desde tanques de cemento largos y
profundos, a tanques plásticos específicos para el abalón o estanques exteriores
(McShane, 1988; Freeman, 2001; Alcántara y Noro, 2006; Fig. 7). Sin embargo, dada la
influencia que el tipo de tanque ejerce tanto sobre la alimentación como sobre el
comportamiento del abalón (Fleming y Hone, 1996), la efectividad lograda en los
distintos sistemas varía considerablemente.
En los últimos años, las investigaciones relacionadas con las técnicas de
producción han estado principalmente encaminadas al desarrollo de tanques aptos para
el uso de dietas compuestas, lo que ha dado lugar a tanques muy poco profundos con
elevadas tasas de renovación que favorecen la eliminación de los deshechos del sistema
(Wassnig et al., 2010). La producción en esos tipos de tanques se estima en 1
t/tanque/año (Morrison y Smith, 2000).

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A consecuencia de los elevados gastos derivados del bombeo, aireación,

filtración o control de temperatura, los sistemas de engorde en tierra son muy poco
eficientes desde el punto de vista energético, siendo los gastos de instalación y
operacionales las principales desventajas de este tipo de sistemas (Wu et al., 2009; Wu y
Zhang, 2013). Otro de los problemas asociados a estos emplazamientos, es la escasez
de terrenos apropiados en zonas costeras, en la mayoría de las ocasiones además en
competencia de usos con tras actividades humanas.

Figura 7. Diversos sistemas de granjas en tierra para el cultivo de H. rufescens (A;

Chile), H. discus hannai (B; Corea), H. asinina (C; Tailandia) y H. tuberculata
(D; Irlanda).

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1.4.3.2. Instalaciones en el mar

En las granjas en mar abierto, la corriente y el oleaje son los responsables del
intercambio de agua dentro las estructuras y por tanto, son las condiciones reinantes las
que afectan al crecimiento de los animales en cultivo (Nagler et al., 2003). Las ventajas
de este tipo de sistema para el cultivo de abalón incluyen una mejora potencial de las
condiciones generales del cultivo, menores costes de inversion y de funcionamiento, y
fácil acceso para la alimentación, mantenimiento y cosecha (Jarayabhand y Paphavasist,
1996; Leighton, 1989; Capinpin et al., 1999; Wu et al., 2009). Mientras que las
desventajas serían un mayor requerimiento de mano de obra para las labores de limpieza
de las unidades de cultivo, la falta de control sobre los parámetros medioambientales, la
escasez de emplazamientos adecuados para el engorde de las semillas o la mayor
inseguridad de las instalaciones (Hindrum et al., 1996; Preece y Mladenov, 1999; Wu y
Zhang, 2010, 2013).

Existe también una gran variedad de sistemas en las instalaciones en el mar,

pudiendo las unidades de cultivo estar suspendidas de long-lines, de plataformas
flotantes o situadas directamente sobre el lecho marino (Aviles y Shepherd, 1996;
Capinpin et al., 1999; Fermin y Buen, 2002; Wu et al., 2009; Fig. 8-10). Así mismo,
para diferentes especies de abalón tales como H. rufescens (Benson et al., 1986); H.
fulgens (Gonzáles-Avilés y Shepherd, 1996); H. asinina, (Capinipin et al., 1999, Minh
et al., 2010); H. iris (Preece y Mladenov, 1999), H. diversicolor (Alcántara y Noro,
2006; Wu et al., 2009), H. tuberculata (Bossy 1989, 1990; Hensey 1991, 1993; La
Touche et al., 1993; Legg et al., 2012) o híbridos de abalón (H. rubra x H. laevigata,
Mulvaney et al., 2012), se han probado una gran diversidad de unidades de cultivo:
barriles, tubos de PVC, jaulas de red o cestas plásticas apiladas, mostrando de forma
general una gran influecia de los sistemas de engorde sobre las tasas de crecimiento de
los animales. Sin embargo, la rápida expansion de instalaciones de cultivo en bahías
costeras cerradas, ha dado lugar a una sobrecarga de las estructuras flotantes con la
consiguiente reducción de la circulación del agua, afectando finalmente al crecimiento y
la supervivencia de los animales (Fleming et al., 1997; Searcy-Bernal y Gorrostieta-
Hurtado, 2007; Wassnig et al., 2010). Por otro lado, el desarrollo de la tecnología de
jaulas flotantes en mar abierto ha permitido una gran expansion del cultivo de peces
como salmónidos en Europa y Canadá (Korsoen et al., 2009) o corvina Pseudosciaena
crocea en China (Lu et al., 2008). Esta técnica de cultivo combina una alta producción,

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fácil manejo y bajo coste energético (Mortensen et al., 2007). Así, recientemente se han
probado en China jaulas sumergibles, mostrando uno crecimiento general de H. discus
hannai superior a los obtenidos con los sistemas tradicionales de cestas suspendidas
apiladas (Wu yd Zhang, 2013).

Figura 8. (A) Jaulas suspendidas de long-lines para el cultivo de abalón rojo H.

rufescens en Chile. (B) Sistema tradicional de cestas apiladas para el cultivo de H.
discus hannai en China.

A modo de resúmen, se podría decir que los sistemas de cultivo en el mar para
el engorde del abalón se han convertido en los más extendidos a nivel mundial, debido a
un menor coste respecto a los tradicionales cultivos en tierra, mayor calidad de los
productos y mayor sostenibilidad a largo plazo (Ke et al., 2012; Park y Kim, 2013; Legg
et al., 2012).

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Figura 9. Diversos sistemas de cultivo fondeados en el lecho marino para el abalón

europeo en la Isla de Jersey (Reino Unido).

Figura 10. Cultivo del abalón europeo H. tuberculata en jaulas suspendidas en Bretaña
(Francia).

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1.5. NUTRICIÓN Y ALIMENTACIÓN EN LA OREJA DE MAR

Dada la extrema importancia de una adecuada gestión de la alimentación para el

óptimo desarrollo de la tecnología de engorde de la oreja de mar, en la presente tesis se
ha llevado a cabo una extensa revisión acerca del conocimiento e investigaciones en
curso, relativos la producción mundial de Haliotis spp., con especial atención hacia sus
requerimientos nutricionales y estrategias de alimentación.

1.5.1. Aspectos generales

La oreja de mar es un gasterópodo herbívoro que se alimenta de microalgas en

sus etapas iniciales y macroalgas en su etapa adulta (Elliott, 2000; Sales y Britz, 2001;
Nelson et al., 2002; Tanaka et al., 2003) (Fig. 11), comenzando a alimentarse
inmediatamente después del asentamiento (Tutschulte y Connell, 1988). Cuando crece,
se alimenta de macroalgas pasando de una especie a otra conforme va madurando
(Tabla 2).

Figura 11. Semillas de abalón (H. tuberculata coccinea) ramoneando sobre alga
verde.

Dependiendo del habitat y la disponibilidad en el medio, las prefenrencias por

los distintos tipos de algas varían para cada especie de abalón. (Dunstan et al., 1996;
Nelson et al., 2002). Aunque de forma natural consumen una gran variedad de algas
(Britz, 1991), las laminarias son, de lejos, la principal fuente de alimento de estos
herbívoros (Rosen et al., 2000; Qi et al., 2010).

El patron de alimentación de la oreja de mar va cambiando a lo largo de las

distintas etapas de su ciclo vital (de Waal et al., 2003). Esto es debido no sólo al

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aumento del tamaño de la boca (Fleming et al., 1996), sino también a los cambios
morfológicos de la rádula conforme el animal crece (Steneck y Watling, 1982;
Kawamura et al., 2001; Daume y Ryan, 2004; Simental et al., 2004; Johnston et al.,
2005). Así mimo, los cambios en la dieta también son debidos a cambios en la flora
intestinal y en la actividad enzimática en el digestivo del animal según va creciendo, lo
que les permite digerir las macroalgas (Erasmus et al., 1997; Tanaka et al., 2003).

1.5.2. Alimentación de la oreja de mar

La oreja de mar puede consumir algas a una tasa cercana al 35% de su peso al
día (Tahil y Juinio-Menez, 1999), por lo que la biomasa algal requerida para sostener el
engorde del abalón es muy elevada (Fig.12). Sin embargo, la limitada disponibilidad de
algas adecuadas en el medio natural, además de su generalmente bajo contendido en
proteínas, suponen un impedimento importante para el cultivo intensivo de este molusco
a nivel mundial (Hahn, 1989). Es más, la abundancia y calidad nutricional de las algas
varía mucho en función del lugar y época de cosecha (Dawczynski et al., 2007; Courtois
de Viçose et al., 2012a), afectando por tanto a las tasas de crecimiento dificultando a los
granjeros una óptima gestión de la producción (Bautista-Teruel y Millamena, 1999).

Debido al crecimiento bajo y variable de los animales cuando se alimentan con

macroalgas (Britz, 1996a; Cook, 1991; Bautista-Teruel y Millamena, 1999; Tan y Mai
2001; Moriyama y Kawauchi, 2004), unido a los problemas de logística y suministro
asociados al uso de algas frescas, el cultivo intensivo del abalón se sustenta cada vez
más en el uso de dietas artificiales (Britz 1996a, b; Sales y Britz, 2001), que son
formuladas para satisfacer los requerimientos nutricionales de cada una de las especies
en cultivo.

La selección del tipo de alimento también va a depender del sistema de cultivo,

las macroalgas generalmente empleadas para las instalaciones en el mar, mientras que
los piensos pueden ser más adecuados para sistemas en tierra (Kinkerdale et al., 2010;
Qi et al., 2010). A modo de resumen se podría decir que ambas fuentes de alimentación
tienen su lugar, y deben de ser elegidas en función de las características específicas de
cada granja (emplazamiento, condiciones de cultivos, disponibilidad de algas del medio
etc.) (Dlaza, 2006). Además de estos factores nutricionales, para la selección del sistema

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alimentación de la granja, han de tenerse en cuenta así mismo criterios económicos que
redunden en el éxito de la empresa.

De forma general, en las granjas de abalón los animales se alimentan 2-3 veces a
la semana o cada 2 días (Maguire et al., 1996).

Figura 12. Macrocystis pyrifera y Palmaria palmata cosechadas del medio natural para
alimentar al abalón rojo (A; Chile) y al europeo (B; Bretaña).

En el caso de las Islas Canarias, la biomasa algal del medio es mucho menos
abundante que en otras zonas costeras de aguas ricas en nutrientes, lo que hace inviable
su cosecha. Por lo tanto, para el desarrollo comercial de esta industria es necesario
reemplazar las algas extraídas del medio como fuente principal de la dieta, debiendo
basarse por tanto en el cultivo de algas y/o el uso de piensos compuestos.

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Tabla 2. Algas del medio y cultivadas, idóneas para el cultivo de distintas especies de abalón (Los números indican los trabajos abajo referenciados)

Especies de abalón
Macraolga H.
H. H. H. H. H. H. H. H. H. H. H. H.
discus
asinina roei midae corrugata rufescesn sorenseni laevigata rubra fulgens diversicolor iris tuberculata
hannai
Alga roja
Asparagopsis armata 1
Gelidium spp. 2
Gracilaria spp. 3 4 5 6 7 8 9
Gracilariopsis spp. 10
Jeannerettia lobata 11
Palmaria spp. 12 13 14 15
Alga parda
Ecklonia maxima 16
Egregia menziesii 17
Eisenia spp 18
Laminaria spp. 19 20 21
Macrocistys pyrifera 22 23 24 25
Phyllospora comosa 26
Undaria pinnatifida 27
Alga verde
Ulva spp 28 29 30 31 32
1. Shepherd y Cannon, 1988.; 2. Troell et al., 2006 ; 3. Jackson et al.,2001 (G. edulis); 4. Sales y Britz, 2001 (G. gracilis); 5. Pang et al., 2006 (G. textorii); 6. Mcbride et al., 2001 (G.
conferta); 7. Liao et al., 2003 (G. tenuistipitata); 8. Allen et al., 2006; 9. Neori et al., 1998; Mcbride et al., 2001(G. conferta); 10. Capinpin y Corre, 1996 (G. heteroclada); Reyes y
Fermin, 2003 (G. bailinae); 11. Fleming, 1995; 12. Mercer et al., 1993 (P. palmata); Demetropoulos and Langdon, 2004 (P. mollis); 13 y 14. Demetropoulos and Langdon, 2004 (P.
mollis); 15. Mercer et al., 1993 (P. palmata); 16. Naidoo et al., 2006; 17. Nelson et al., 2002; 18. Uki et al., 1985a; 19. Troell et al., 2006 (L. pallida); 20. Qi et al., 2010 (L. japonica);
21. Mercer et al., 1993 (L. digitata); 22. Badillo et al., 2007; 23 y 24. Serviere-Zaragoza et al., 2001; 25. Allen et al., 2006; 26. Vandepeer y Van Barneveld 2003; 27. Sakata et al.,
1984; 28. Boarder y Shpigel, 2001 (U. rigida); 29. Naidoo et al., 2006; 30. Shuenhoff et al., 2003; 31 y 32. Mcbride et al., 2001 (U. lactuca)

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1.5.3. Requerimientos nutricionales de la oreja de mar

El valor nutricional de las dietas de abalón depende de muchos factores como

son su composición, disponibilidad (Middlen y Redding, 1998; Serviere-Zaragoza et al.,
2001; Nelson et al., 2002; Bautista-Teruel et al., 2003); digestibilidad (Sales y Britz
2001, 2002; Gomez-Montes et al., 2003); técnicas de procesado (Booth et al,. 2002;
Sales y Britz, 2002); tamaño de las partículas (Southgate y Partridge, 1998);
presentación y tamaño del grano (Fleming et al., 1996; Kinkerdale et al., 2010); la
presencia de atractantes (Fleming et al., 1996; Sales y Janssens, 2004) o su textura y
palatibilidad (Kautsky et al., 2001). Por lo tanto, para determinar la calidad de una dieta
son numerosos los factores que en conjunto, han de considerarse.

1.5.3.1. Composición general de las dietas procesadas para la oreja de mar

En la actualidad, la composición proximal de las dietas es muy parecida a la

propia del abalón (Viera et al., 2009a). El contenido calórico (energía bruta) se sitúa
generalmente alrededor de 4 Kcal g-1 (Tabla 3). La humedad promedio es del 10%
(Tabla 3). El contenido en proteínas de las dietas varía considerablemente entre 20-50 %
(Tabla 4), con un promedio del 30%. Mientras que el el rango de los lípidos testados se
sitúa entre 1.2-19%, con un 4% de promedio (Tabla 5). Los carbohidratos constituyen el
componente mayoritario de las dietas, oscilando entre 21- 82%, con un valor medio
aproximado del 45% (Tabla 6). El percentage de fibra es generalmente bajo (1-6%;
Tabla 6) debido a la baja capacidad del abalón para digerirla (Fleming et al., 1996).

1.5.3.2. Fuentes de proteínas, niveles óptimos de inclusión y suplementación de

aminoácidos

Entre los dintintos nutrientes, la oreja de mar require para un óptimo crecimiento
niveles adecuados de protenias de alta calidad (Uki et al., 1985a; Mai et al., 1995a, b;
Britz y Hecht, 1997; Bautista-Teruel y Millamena, 1999; Gómez-Montes et al., 2003;
Reyes y Fermín, 2003; Viana et al., 2007). En consecuencia, para el uso eficiente de
este componente dietético esencial pero caro, el abalón debe emplearlo para crecer y no
como fuente energética. Los factores más importantes que afectan a la utilización de la
proteína son su digestibilidad, el balance y disponibilidad de los aminoácidos, así como
la relación proteína-energía (Fleming et al., 1996).

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Tabla 3. Composición proximal (% peso seco) y contenido calórico de diversas dietas

artificiales testadas para el abalón

Referencias

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Proteínas 30 31 35 38 32 40 35 35 28 35 27 36 38 35 44

Lípidos 5 5 10 6 6 5 7 4.5 3 6 5 3 1.3 5 6.7

Carbohidratos 48 33 39 40 44 42 48 45 43

Fibra 6 4.3 1

Cenizas 3 11 8 9 6 16

Humedad 5 10 6

EB (Kcal g-1) 4.5 3 4.7 3 4 2.9 4 4.5 4.5

P: e ratio* 47 85 96

1. Uki et al., 1985b (Japón: H. discus hannai); 2. Mai et al., 1995b (Irlanda: H. tuberculata y H. discus
hannai); 3. Viana et al., 1993 (México: H. fulgens); 4. Guzmán y Viana, 1998 (México: H. fulgens); 5.
Bautista-Teruel y Millamena, 1999 (Filipinas: H.asinina); 6. Jackson et al., 2001 (Australia: H. asinina); 7.
Serviere-Zaragoza et al., 2001 (México: H. fulgens); 8. Shipton y Britz, 2001(Sudáfrica: H. midae); 9.
Bautista-Teruel et al., 2003 (Filipinas: H. asinina); 10. Gómez-Montes et al., 2003 (México: H. fulgens);
11. Reyes y Fermín, 2003 (Filipinas: H. asinina); 12. Sales et al., 2003 (Sudáfrica: H. midae); 13. Thongrod
et al., 2003 (Tailandia: H. asinina); 14. Naidoo et al., 2006 (Sudáfrica: H. midae); 15. Hernández et al.,
2009 (Chile: H. rufescens). * Ratio proteína: energía.

La fuentes de proteínas más empleadas en las dietas de abalón incluyen la harina

de pescado, harina de soja desengrasada (Guzmán y Viana, 1998; Sales y Britz, 2002;
Gómez-Montes et al., 2003; Thongrod et al., 2003; Naidoo et al., 2006; García-Esquivel
et al., 2007), caseína (Uki et al., 1985b; Viana et al., 1993; Mai et al., 1995b; Sales et
al., 2003; Vandepeer y van Barneveld, 2003) y Spirulina spp. (Uki et al., 1985b; Britz
et al., 1996a, Bautista-Teruel et al., 2003; Thongrod et al., 2003; Naidoo et al., 2006;
Troell et al., 2006). También se han probado otras fuentes novedosas en pequeñas
proporciones (Tabla 4). En ocasiones, para aprovechar el alto valor nutricional de las
algas, éstas han sido incluídas también en las dietas (García-Esquivel et al., 2007; Viana
et al., 2007). Otros compuestos que también se han propuesto como fuentes económicas

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de proteínas han sido ensilados de vísceras de peces o del propio abalón (Viana et al.,
1996; Guzmán y Viana, 1998). Además, en ocasiones, con el fin de aportar los
aminoácidos esenciales para estas species, las dietas deben ser suplementadas con
aporte de aminoácidos sintéticos tales como metionina, treonina o arginina (Mai et al.,
1995b; Guzmán y Viana, 1998; Serviere-Zaragoza et al., 2001; García-Esquivel et al.,
2007).

De entre todas las fuentes testadas, la harina de pescado es la única que da lugar a
un buen crecimiento cuando se incluye como única fuente proteica (Fleming et al.,
1996). Sin embargo, aspectos concernientes al uso sostenible de la harina de pescado en
la acuicultura, han llevado al Fondo Mundial para la Naturaleza a establecer
restricciones en cuanto a su uso dentro de la normativa para la producción internacional
de abalón (WWF, 2010). Así, para abalones alimentados con dietas que contengan
harina de pescado, dicha normativa establece que no debe emplearse más de un kilo de
pescado para producir un kilo de este molusco. Cabe añadir que hasta el momento, la
producción europea de oreja de mar está principalmente enfocadada hacia productos
orgánicos o con certificación ecológica, lo que implica que en su proceso de cultivo, no
se emplean harinas de pescado, medicamentos o fertilizantes. En este sentido, el
desarrollo de dietas compuestas para abalón que no contengan harina de pescado,
tendría ventajas comerciales no sólo para los consumidores europeos, cuya
preocupación por el medio ambiente es cada vez mayor, sino también para los
productores que podrían validar la sostenibilidad ambiental y social de su proceso
productivo (WWF, 2010).

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Tabla 4. Composición nutricional (% peso seco) de las dietas compuestas ensayadas para el abalón: Fuentes proteicas y niveles de inclusión

Nutriente Dieta

1 2 3 4 5 6 7 8 9 10 11
Fuente proteica y contenido
Harina de pescado 47 41-71 44 9-62 40 7-17 30 12-48 20
Harina de soja 60 10 17 20 10 19-37 10
Caseína 32 8-41 32
Harina de langostino 7-20
Ensilaje 20
Spirulina spp. 43 15-30
Harinas de semillas de algodón 2 5
Harina de girasol 24-48 5
Levadura de tórula 57 24-40
Harinas vegetales 52
Harina de maiz 47 10 10 15
Harina de laminarias 12 10 12
Extracto d e aceite de soja 63
Albúmina de huevo 37
Huevos enteros 34

Proteína total 30 0-50 27-47 30 20-50 24-44 36-39 22-32 36 34 23

1. Uki et al., 1985b (Japón: H. discus hannai) ; 2. Mai et al., 1995b (Irlanda : H. tuberculata y H. discus hannai); 3. Britz et al, 1996a (Sudáfrica: H. midae); 4. Britz et
al, 1996b (Sudáfrica: H. midae); 5. Fleming et al,, 1996 (revisión); 6. Britz y Hecht, 1997 (Sudáfrica: H. midae); 7. Guzmán y Viana, 1998 (México: H. fulgens); 8.
Bautista-Teruel y Millamena, 1999 (Fipipinas: H. asinina); 9. Serviere-Zaragoza et al., 2001 (México: H. fulgens); 10. Shipton y Britz, 2001(Sudáfrica: H. midae); 11.
Sales y Britz, 2002 (Sudáfrica: H. midae).

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Tabla 4. (Continuación)

Dieta
Nutriente
12 13 14 15 16 17 18 19 20 21 22 23

Fuente proteica y contenido

Harina de pescado 10 7.5 10 18-35 3 1-7 43 55 29 30 30 29-35

Harina de soja 20 35 35 15 34 62 15 10
Caseína 4-48 35 16-19

Harina de langostino 15 7.5 10 5

Spirulina spp. 20 20 10 10
Proteína de soja aislada 9-17 5
Harina de lupino 10

Harinas vegetales 46 39-50

Harina de maíz 12
Harina de laminarias 9-17 12 12 15 2.6

Algas -

Total protein 27 26-44 26 5-48 37 35 18 44 35 44 34-39

12. Bautista-Teruel et al., 2003 (Filipinas: H. asinina); 13. Gómez-Montes et al., 2003 (México: H. fulgens); 14. Reyes y Fermín, 2003 (Filipinas: H. asinina); 15. Sales et
al., 2003 (Sudáfrica: H. midae); 16. Thongrod et al., 2003 (Tailandia: H. asinina); 17. Vandepeer et al., 2003 (Australia: H. rubra y H. laevigata); 18. Naidoo et al., 2006
(Sudáfrica: H. midae); 19. Troell et al, 2006 (Sudáfrica: H. midae); 20. García-Esquivel et al, 2007 (México: H. fulgens); 21. Viana et al, 2007 (México: H. fulgens); 22.
Hernández et al., 2009 (Chile: H. rufescens); 23. Green et al., 2011 (Sudáfrica: H. midae).

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1.5.3.3. Fuentes lipídicas, niveles de inclusion óptimos y ácidos grasos

esenciales.

Los lídidos son un constituyente esencial de la dieta no sólo por su alto valor
energético y como fuente de ácidos grasos, imprescindibles para el metabolismo celular
y mantenimiento de la estructura de las membranas (Corraze, 2001), sino también como
fuente de vitaminas liposolubles (Fleming et al., 1996). Además, los lípidos
(especialmente los ácidos grasos ploiinsaturados de cadena larga) son determinantes en
el sabor y olor de los alimentos marinos. En consecuencia, se han llevado a cabo
numerosas investigaciones referentes a este nutriente para evaluar, entre otro aspectos,
la respuesta del abalón a distintos niveles de lípidos dietéticos (Uki et al., 1985a; 1986;
Mai et al., 1995a; Bautista-Teruel et al., 2011); la obtención de los lípidos necesarios
con la inclusion de aceite de pescado en las dietas (Dunstan et al, 1996); el efecto del
ratio proteína-energía sobre el crecimiento, indices nutricionales y composición corporal
(Britz y Hecht, 1997; Bautista-Teruel y Millamena, 1999; Gómez-Montes et al., 2003;
Green et al., 2011); el papel de los lípidos en el crecimiento y la maduración gonadal
(Nelson et al., 2002), la composición en ácidos grasos de los tejidos (Dunstan et al.,
1996; Grubert et al., 2004; Li et al., 2002; Durazo y Viana, 2013; Hernández et al.,
2013) o la influencia en el crecimiento del ratio lípidos: carbohidratos y la energía bruta
de las dietas de abalón (Thongrod et al., 2003).
En general, al igual que en otro moluscos y peces herbívoros, el requerimiento
lipídico de la oreja de mar es muy bajo (Mai et al., 1995a), lo que según diversos
autores está relacionado con el bajo uso de los lípidos como fuente energética derivado
de su baja tasa metabólica (Durazo-Beltrán et al., 2004). Y lo que es más, niveles altos
de lípidos en la dieta (>7%) parecen afectar de forma negativa al crecimiento del abalón
reduciendo la captación de otros nutrientes tal y como se ha visto para diversas especies
como H. laevigata (Van Barneveld et al., 1998); H. tuberculata y H. discus hannai
(Mai et al.,1995a); H. midae (Britz y Hecht, 1997; Green et al., 2011), H. fulgens
(Durazo-Beltran et al., 2003, 2004); H. asinina (Thongrod et al., 2003) o H. corrugata
(Montano-Vargas et al., 2005).

A pesar de que el rango de los niveles lipídicos testados en las dietas de abalón
ha sido muy amplio (2-19% PS; Tabla 5), en la mayoría de los casos los lípidos
constituyen unicamente un 3-5% de la dieta (Uki et al., 1985a).

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En cuanto a las fuentes, los lípidos en la dietas artificiales son aportados como
aceite de pescado/marina (Guzmán y Viana, 1998; Sales y Britz, 2002; Thongrod et al.,
2003; Green et al., 2011), aceite vegetal (Shipton y Britz, 2001) o una combinación de
ambas (Mai et al., 1995a; Britz, 1996a,b; Bautista-Teruel y Millamena, 1999; Shipton y
Britz, 2001; Bautista-Teruel et al., 2003; Gómez-Montes et al., 2003; Reyes y Fermín,
2003) (Tabla 5). En ocasiones, la misma grasa contenida en la harina de pescado es el
único aporte en la dieta. Para prevenir el enranciamiento del aceite, normalmente se le
añade vitamina E, (Uki et al., 1985a, b).

Son numerosos los trabajos que han encontrado que la composición en ácidos
grasos de los tejidos de los animales que se alimentan de algas, como el abalón, es muy
distintas de la de los carnívoros o aquellos que se alimentan de plancton, lo que se ha
relacionado como un reflejo de la diferente composición de sus respectivos regímenes
dietéticos (Dunstan et al., 1996; Grubert et al., 2004).

Uki y colaboradores (1985a) estimaron que el nivel de n-3 PUFA de una dieta
que contiene un 5% de lípidos debe ser aldedor del 1% (revisado por Uki y Watanabe,
1992) lo que representa un 20% del total de los lípidos.

En lo que respecta a la carne del abalón, el nivel de lípidos también es bajo,

siendo los siguientes los ácidos grasos mayoritarios: ácido palmítico (16:0), ácido
esteárico (18:0), ácido oléico (18:1n9), ácido vaccénico (18:1n7), ácido araquidónico
(20:4n6), ácido eicosapentaenoico (20:5n3) y ácido docosapentaenoico (22:5n3)
(Dunstan et al., 1999; Nelson et al., 2002).

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Tabla 5. Composición nutricional (% peso seco) de dietas artificiales testadas para el abalón: Fuentes lipídicas y niveles de inclusión

Dieta
Nutriente
1 2 3 4 5 6 7 8 9 10 11 12 13 14
Fuente lipídica y contenido

Aceite de pescado 2 0-16

Aceite de soja / Aceite de hígado
de abadejo (3:2 + 1% Vit E)
1y5

Aceite de maíz / aceite de 0-12

pescado (1:1)

Aceite de girasol/ aceite de 0-5 0-3

pescado (1:1)

Aceite de hígado de bacalao

0y3 1.5 3-5 1 1-5

Aceite de soja
1.5 0.5 1 1-5

Aceite de calamar
0.5

Aceite de maíz 0.5

Lípidos totales 1.2-5 0.6-12 5y8 6-8 6 4-5 3-4 1.5-5 3 6-7 5 1-19 5.3 0-10

1. Uki et al., 1985a, b (Japón : H. discus hannai); 2. Mai et al., 1995a (Irlanda: H. tuberculata y H. discus hannai); 3. Britz, 1996a, b (Sudáfrica: H. midae); 4. Guzmán y
Viana, 1998 (México: H.fulgens); 5. Bautista-Teruel y Millamena, 1999 (Filipinas: H.asinina); 6. Shipton y Britz, 2001 (Sudáfrica: H. midae); 7. Sales y Britz, 2002
(Sudáfrica: H. midae); 8. Viana et al, 2002 (Revisión); 9. Bautista-Teruel et al., 2003 (Filipinas: H.asinina); 10. Gómez-Montes et al., 2003 (México: H.fulgens); 11. Reyes y
Fermín., 2003 (Filipinas: H.asinina); 12. Thongrod et al., 2003 (Tailandia: H. asinina); 13. Naidoo et al., 2006 (Sudáfrica: H. midae); 14. Bautista-Teruel et al., 2011
(Filipinas: H. asinina)

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1.5.3.4. Energía / carbohidratos: fuentes y contenidos

La dieta del abalón en el medio natural contiene entre un 40-50% de

carbohidratos. Por tanto, la inclusion de altos niveles de carbohidratos en la dieta
favorece el crecimiento de este molusco (Thongrod et al., 2003) que tiene diversas
enzimas capaces de hidrolizar carbohidratos complejos (Fleming et al., 1996), y una
buena capacidad para sintetizar lípidos no esenciales a partir de ellos. En consecuencia,
la energía en las dietas de abalón se suministra principalmente a través de los
carbohidratos, cuya proporción ronda el 45% de la dieta (Tabla 6).

Las fuentes más empleadas como aporte energético son los cereales tales como
harina de trigo o maíz, harina de soja y almidón de maíz o de arróz. En relación a los
almidones, en la mayoría de las dietas experimentales y también en las comerciales,
éstos juegan un importante doble papel no sólo como fuente de energía, sino también
como aglutinante (Tabla 6).

A pesar de la habilidad del abalón para utilizar un amplio rango de fuentes

energéticas, al tratarse de un molusco, su metabolismo es bajo y en consecuencia
también lo es su requerimiento energético. Generalmente, el contenido calórico (energía
bruta) de las dietas se sitúa alrededor de las 4 Kcal g-1 (Tabla 3).

El alimento de los animales acuáticos se diferencia del empleado en las

ganaderías convencionales, en que éstos necesitan una matriz que incluya los distintos
nutrientes. Otro requerimiento adicional de las dietas de animales lentos para comer,
como es el caso de la oreja de mar, es que tanto los nutrientes solubles en agua, como el
resto de partículas se han de mantener en el alimento, de modo que los granos de pienso
se conserven intactos en el agua durante al menos dos días. Indudablemente, las
propiedades físicas tales como textura o establidad en el agua, difieren mucho entre las
dietas artificiales y las macroalgas. Estas características afectan a la alimentación,
digestión, absorción y por tanto, al crecimiento de los animales. Así, la estabilidad de la
dieta en el agua es una cualidad muy importante, siendo responsable de los diferentes
rendimientos obtenidos entre los animales que se alimentan de algas frescas y aquellos
que lo hacen de piensos compuestos (Mai et al., 1995a). En este sentido, alcanzar la
estabilidad es crucial para el éxito en el desarrollo de un pienso (Fleming et al, 1996;
Hernández et al., 2009). La estabilidad promedio de las dietas comerciales es alrededor

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 Spanish summary

de 2-3 días (Fleming et al., 1996), pudiendo perder entre un 30-40% de su peso seco tras
48 horas de inmersión (Maguire, 1996; Bautista- Teruel et al., 2003).

Las fuentes más comunes de aglutinantes incluyen el almidón, gluten, alginatos

y algas (Tabla 6). Los geles también se han testasdo frecuentemente en dietas
experimentales, si bien su elevado precio no parece indicar su inclusion en dietas
comerciales (Fleming et al., 1996).

A pesar de la presencia de celulasas en el intestino, la capacidad del abalón para

digerir la fibra es limitada. Algunas dietas incluyen fibra a modo de aglutinante, con
unos contenidos máximos del 6% del peso seco (Fleming et al., 1996; Guzmán y Viana,
1998; Serviere-Zaragoza et al., 2001; Naidoo et al., 2006) (Tabla 6).

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Tabla 6. Composición nutricional (% peso seco) de las dietas artificales testadas para el abalón: Energía / aglutinantes fuentes y niveles

Nutriente Dieta
1 2 3 4 5 6 7 8 9 10 11 12 13 14

Energía/aglutinantes fuentes y
niveles
Dextrina 32-43

Trigo (harina /gluten) * 4 20 20 12

Maíz (harina / gluten) * 10 12 16-27

Harina de soja * 15

Almidón (arróz / maíz) * 5 19 0.5-31 26-88 9-43 13-29 14-23 19

Salvado de arróz 3-26 5-20 2-3

Algas * 7 9 5 7

Alginato de sodio 18 4

Agar 20

Gelatina 5 6 6 5-6 6

Aglutinantes químicos 0,5

Carbohidratos totales 47 40-50 33-47 47 46-50 28-82 42-44 21-49 31-48 39-47 43

Fibra 01-1.3 5.8 4 1,2

Celulosa 5 4 5 2

Cenizas 6-16 11 5-11 4-18 13-23 9 4-18 6 15

1. Mai et al., 1995a (Irlanda: H. tuberculata y H. discus hannai); 2. Fleming et al., 1996 (revisión) ; 3. Guzmán y Viana, 1998 (México: H.fulgens); 4. Bautista-Teruel y Millamena, 1999
(Filipinas: H.asinina); 5. Serviere-Zaragoza et al., 2001(México: H.fulgens); 6. Shipton y Britz, 2001(Sudáfrica: H. midae); 7. Sales et al., 2003 (Sudáfrica: H. midae); 8. Bautista-Teruel et al.,
2003 (Filipinas: H.asinina); 9. Gómez-Montes et al., 2003 (México: H.fulgens); 10. Thongrod et al., 2003 (Tailandia: H. asinina); 11. Reyes y Fermín., 2003 (Filipinas: H. asinina); 12. Durazo-
Beltrán et al., 2004 (México: H. fulgens); 13. Naidoo et al., 2006 (Sudáfrica: H. midae); 14. García-Esquivel et al., 2007 (México: H.fulgens)

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1.5.3.5. Vitaminas y minerales

En ausencia de información referente al requerimiento de vitaminas y minerales

del abalón, las dietas experimentadas por Uki y colaboradores (1985a), se basaron en
los requerimientos de la carpa y trucha arcoiris, con excepción del cloruro de colina y
vitamina E, añadidos para mantener la mezcla de lípidos que se añadió de forma
independiente (Tabla 7). El nivel de inclusion fue del 4% y 1.5% de la dieta para la
mezcla de minerales y vitaminas respectivamente.

A partir de entonces, la mayoría de las dietas formuladas para el abalón incluyen

dichos valores (Tabla 8).

Tabla 7. Mezclas de vitaminas y minerals ensayadas por Uki y col. (1985a)

Mezcla de vitaminas  Composiciónn Mezcla de minerales  Composición

(mg) (Total 100 g)

Tiamina 6 NaCl 1.0

Riboflavina 5 MgSO4-7 H2O 15.0

HCl Piridoxina 2 NaH2PO4-2H2O 25.0

Niacina 40 KH2PO4 32.0

Pantotenato cálcico 10 Ca(H2PO4)2.H2O 20.0

Inositol 200 Citrato de Fe 2.5

Biotina 0.6 Mezcla de trazas* 1.0

Ácido fólico 1.5 Lactato-Ca 3.5

PABA 20 Mezcla de elementos

trazas
Menadiona 4
ZnSO4.7H2O 35.3
B12 0.009
MnSO4.4H2O 16.2
Ácido ascórbico 200
CuSO4.5H2O 3.1
Vitamina A 5000 U.I.
CoCl2.6H2O 0.1
Vitamina D 100 U.I.
KIO3 0.3

Celulosa 45

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 Spanish summary

Tabla 8. Composición nutricional (% peso seco) de dietas artificiales testadas para el abalón. Ingredientes secundarios

Nutriente Dieta
1 2 3 4 5 6 7 8 9 10 11 12 13

Vitaminas y minerales
Mezcla de vitaminas  1.5 2 2 3 1.7 3 0.4 3 1.5 1.3 1.3

Mezcla de minerales  4 4 6 4 3.4 2 4 2 4 2.9 3.3

Total 5.5 6 1 6 7 5.1 5 4.4 5 5.5 4.2 4.6

Cloruro de colina 0.5 0.5 0.8 0.11 0.3 0.1 0.11

Alfa-Tocoferol 0.01

Estabilizante de vit C 2 0.35 0.4

Vitamina C 0.1 0.5

Vitamina E 0.1

Benzoato de Na 2 0.23 0.2 0.23

BTH 4 0.05 0.09 0.02 0.08 0.08

Fosfato dicálcico 3 3

Mono fosfato de Ca 0.5

Bentonita 15

1. Uki et al., 1985a,b (Japón: H. discus hannai); 2. Mai et al., 1995a (Irlanda: H. tuberculata y H. discus hannai); 3. Britz et al., 1997 (Sudáfrica: H. midae); 4. Guzmán y Viana, 1998
(México: H. fulgens); 5. Bautista-Teruel et al., 1999 (Filipinas: H. asinina); 6. Serviere-Zaragoza et al., 2001(México: H. fulgens); 7. Reyes y Fermín, 2003 (Filipinas: H. asinina); 8.Sales et
al., 2003 (Sudáfrica: H. midae); 9. Thongrod et al., 2003 (Tailandia: H. asinina); 10. Vandepeer et al., 2003 (Australia: H. rubra y H. laevigata); 11. Naidoo et al., 2006 (Sudáfrica: H.
midae); 12. García-Esquivel et al., 2007(México: H. fulgens); 13. Viana et al., 2007 (México: H. fulgens)

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 Spanish summary

1.5.4. Crecimiento de la oreja de mar en condiciones de cultivo

La revisión de los trabajos de nutrición realizados a nivel mundial, evidencian

una enorme variación en el crecimiento de los animales en cultivo, particularmente
durante el primer año de vida y hasta los 30 mm de talla.
En general, las investigaciones en curso demuestran que el alimento para abalón
está aún en pleno desarrollo, tratándose de alcanzar un mayor potencial para mejorar el
crecimiento, salud y calidad de los animales en cultivo. Es más, la revisión realizada
demuestra que un crecimiento de abalón óptimo está basado en la conjunción de
muchos factores, en particular el contenido en proteínas y la fuente proteica; niveles de
carbohidratos y lípidos; temperatura y fotoperíodo; calidad del agua; densidad de
cultivo; edad; características específicas de la especie o sistema de cultivo (Tabla 9). Por
tanto, para la selección de la tecnología de engorde adecuada, son muchos los factores
que han de entrar en consideración.

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 Spanish summary

Tabla 9. Resumen de varios estudios nutricionales en relación al crecimiento del abalón, durante las últimas 3 décadas de desarrollo del cultivo

Composición proximal y Consumo y eficacia de

Crecimiento y supervivencia
contenido energético utilización del alimento
Autor Asunto Tª Días Especie Tratamiento DGSL WG Superv.
P L C E P/E WS FCR FI FCE PER T0 P0 SGR
(μm d-1) (%) (%)
Uki et Caseína, harina de 1.2
Dieta vs H. discus 28 5 0.1 3.5 93-117 37
al., 30-40 pescado 29 3
macroalga hannai
1985a E. biciclys 17 1 0.7 4.3 106 35
A. esculenta 13 4 17 4
L. digitata 6 3 13 12
H. L. saccharina 10 4 18 6
tuberculata P. palmata 17 3 27 12 0.9
U. lactuca 13 6 28 10
Mixed diet 12 4 21 14 95
Mercer C. crispus 13 4 17 15 53
Algas frescas 14 365 19
et al., A. esculenta 13 4 17 15
1993
L. digitata 6 3 13 14
L. saccharina 10 4 18 12
H. discus
P. palmata 17 3 27 13 0.8 90
hannai
U. lactuca 13 6 28 11 61
Mixed diet 12 4 21 13
C. crispus 13 4 17
Harina de
35 9 47
Dietas pescado
Viana et 21- 1
artificiales vs 90 H. fulgens Caseína 44 13 45
al., 1993 24 0
macroalgas
M. pyrifera 2 1 16
J. lobata 1260 4.0 5.4
L.botryoides 540 -1 -0.6
Fleming, 10- 150 U. australis 310 2 0.7
Alga sfrescas H. rubra 40-70 25
1995 20 350 M.angustifolia 260 1.2 0.3
P.comosa 101 0.1 0.1
E.radiata 50 -28 -1.7

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 Spanish summary

Tabla 9. Continuación
Composición proximal y contenido Consumo y eficacia de
Crecimiento y supervivencia
energético utilización del alimento
Autor Asunto Tª Días Especie Tratamiento DGSL WG
P L C E P/E WS FCR FI FCE PER T0 P0 SGR Superv. (%)
(μm d-1) (%)
25 0.6 4 75 0.6 93
25 3 4.2 80 0.8 95
Dietas 25 5 4.3 80 0.7 95
H.
artificiales 25 7 4.4 80 0.6 0.7 95
tuberculata
25 9 4.6 80 0.7 97
25 11.6 4.6 80 0.6 95
Mai et Niveles de P. palmata 18 4 3.7 0.9 97
al., lípidos 25 0.6 4 75 0.6 87
13 100
1995a 25 3 4.2 80 0.9 92
Dietas 25 5 4.3 80 0.9 98
H. discus artificiales 25 7 4.4 80 0.9 90
0.4
hannai 25 9 4.6 80 0.8 88
25 11.6 4.6 80 0.7 92
3.
P. palmata 18 4 1 98
7
Caseína 31 5 0.7 0.5 4.7 0.6 45
Harina
29 5 0.8 0.8 6.5 0.8 58
pescado
Fuentes Harina soja 32 5 1 0.6 3.4 0.6 41
Britz,
proteicas; 19 124 H. midae Spirulina spp. 19 5 0.8 0.8 3.9 21 1.8 0.8 65
1996b macroalgas Levadura
29 5 1 0.7 3.3 0.6 42
tórula
P. corallorhiza 20 2.8 1.3 2.2 0.4 29
E. maxima 10 3.4 2.8 3 0.6 54
H. midae 34 6 3.6 1.4 9.3 3.6 2.2 103
Britz y Proteína; 72 Almidón, 10 0.2
pequeño 44 6 3.6 1.2 9.4 2.3 2.1 108
Hetch, Energía 18 pescado,
H. midae 44 10 4 1.4 34 2.2 0.3 43
1997 ratio; Talla 142 celulosa 36 7.3
grande 44 6 3.6 1.2 35 2.5 0.5 58
Harina pescado 39 6 41 86 1 1.1 1 65
Guzmán Sustitución
22- Víscera, harina
y Viana, harina de 179 H. fulgens 36 8 40 76 1.6 1.8 7 1.1 71
15 de soja
1998 pescado
Abfeed 38 6 48 90 0.9 1.0 0.8 49
Harina pescado 22 6 47 3.2 2.3 2.2 0.5 222 252 85
Harina de
Bautista- Proteína / 27- 28 6 40 3.1 1.8 2.3 0.7 244 307 95
90 langostino
Teruel et Niveles 31 H. asinina 16 0.7
Harina soja 31 6 33 3 1.5 2.5 0.8 248 347 85
al., 1999 energía
Garcilariopsis
17 0.5 35 2.2 7 0.1 0.06 135 134 85
bailinae

191
 Spanish summary

Autor Asunto Tª Días Especie Tratamiento Composición proximal y contenido Consumo y eficacia de Crecimiento y supervivencia
energético utilización del alimento
DGSL WG Superv..
P L C E P/E WS FCR FI FCE PER T0 P0 SGR
(μm d-1) (%) (%)
Eisenia
7.6 52 0.27 19 93
arborea
Macrocystis
Serviere- 12 63 0.7 46 93
Macroalgas pyrifera
Zaragoza
y dieta 20 106 H. fulgens Gelidium 17 0.4
et al., 18 33 0.32 23 89
artificial robustum
2001
Phyyospadix
16 30 0.45 25 95
torreyi
Dieta artificial 36 59 0.65 42 97
Carica papaya 25 5 41 3 6.0 40 14.7 1.8 71 59 80
Leucaena
Reyes y Fuentes de 25 5 47 3 13 37 14.7 0.9 47 28 100
leucocephala
Fermín, porteínas 28-
120 H. asinina Moringa
2003 terrestres 30 25 5 42 3 4 38 11.4 1.9 86 90 95
oliefera
Azolla pinnata 25 5 39 3 5 39 14 1.9 76 75 90
G. bailinae 13 1 38 38 40 15.3 2.1 61 84 90
Harina pescado 28 3 43 3 1 3.9 0.8 400 95
Fuentes de Harina de
Bautista- 27 3 42 3 1 4.2 0.9 454 95
proteínas 28- langostino
Teruel et 90 H. asinina 11 0.7
animales y 31
al., 2003 Harina soja 28 3 44 3 1 4.1 0.7 326 85
vegetales
Spirulina 27 3 42 3 1 4.6 0.9 421 85
26 6 1.6 4 62 1.6 76.4 3 1.3 61
Pescado, soja, 31 6 1.4 4 74 1.4 93 3 1.5 7
Gómez- Proteínas /
21 60 kelp, maíz
Montes et Niveles de H. fulgens 35 6 1.4 4 85 1.4 110 3.1 12 0.2 1.9 92
modificado,
al., 2003 energía 40 7 1.5 4 100 1.5 138 3.4 2.4 123
almidón
44 7 1.6 4 108 1.6 120 2.7 2.5 123
Soja, almidón 38 1 48 4.2 1.3 779 81
Thongrod Lípidos / Spirulina spp., 38 6 43 4.5 1.4 615 93
27- 11-
et al., 196 H. asinina aceite de 37 10 39 4.7 1.9 0.9 353 90
carbohidratos 30 12
2003 pescado,
36 15 36 4.9 3.2 223 75
Gracilaria
37 19 31 5.2 7.4 97 76
Durazo- Laminarias; 0.1 99.4 0.9 29 29
Niveles de
Beltrán et 20 60 H. fulgens soja; ensilado 39 4 36 6.4
lípidos 3 95.9 0.7 40 30
al., 2004 pescado
H. P. palmata 21 42 4.3 3.5 0.7 230
tuberculata P. palmata + 23 2
Legrand, 7- grande 24 41 5.6 3.6 0.6
Macroalgas 183 U. lactuca
2005 17 H. P. palmata 21 42 2 3.7 1.3
tuberculata 10 0.1
pequeña P. p+ U. l. 24 41 3.9 5.8 1.2

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Composición proximal y contenido Consumo y eficacia de

Crecimiento y supervivencia
energético utilización del alimento
Autor Asunto Tª Días Especie Tratamiento DGSL WG Superv.
P L C E P/E WS FCR FI FCE PER T0 P0 SGR
(μm d-1) (%) (%)
U. rigida
enriquecida +
66
E. G.
gracilis+kelp
E. maxima +
60
Epífitos
Dietas
basadas en Rotación 58
algas;
Ecklonia
Naidoo Macroalgas; 55
maxima
et al., Macroalgas 270 H. midae 35 7.5
enriquecida; E. maxima +
2006 53
dieta Abfeed
comercial Abfeed 35 5 45 49
Hojas kelp
34
secas
Estipes kelp
32
seco
Pienso de Kelp
29
seco
Pescado, soja,
kelp
20ºC / 00:24 0.8 3 109
García-
Esquivel Temperatura 20- 20ºC / 12:12 0.9 2.6 82
180 H. fulgens 44 4 31 3.7
et al., Fotoperiodo 25
20ºC / 24:00 0.9 2.8 72
2007
25ºC/ 00:24 4 1.7 69
25ºC/ 12:12 1.4 1.8 55
25ºC/ 24:00 1.6 1.5 38

Dieta artificial 44 6.7 34 4.5 96 33 1.8 50 471 85

Dieta M. pyrifera 27 2.8 40 3.5 78 94 2.5 110 1004 90
Hernán-
artificial;
dez et 15 90 H. rufescens 6.6 0.06
algas fresca; P. columbina 12 0.6 45 2.6 44 93 1.8 70 548 93
al., 2009 dieta mixta
P. columbina
+ dieta 2.3 87 728 80
artificial

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Composición proximal y contenido Consumo y eficacia de

Crecimiento y supervivencia
calórico utilización del alimento
Autor Asunto Tª Días Especie Tratamiento DGSL WG Superv.
P L C E P/E WS FCR FI FCE PER T0 P0 SGR
(μm d-1) (%) (%)
L. japonica 6.5 2.3 22 100
Idoneidad G.
4.2 2.8 21 100
algal lemaneiformis
Qi et al., 10- H. discus S. pallidum 4.4 2.5 18 100
120 76 62
2010 Cultivo algas 23 hannai
en long-line L. j. + G. l. 6 2.3 26 100
- abalón
L. j. + S. p. 5.8 1.8 20 100

34 2.8 4.5 1 0.4 3.2 0.7 43 100

36 5.3 4.6 100
Niveles de H. midae
38 8.7 4.8 25 2.6 100
lípidos, ratio pequeño Harina
39 12.5 5 100
P/E pescado,
Green et 36 16.1 5.1 1.8 0.5 1.3 0.5 67 100
constante 18 84 caseína, kelp,
al., 2011 almidón, aceite 34 2.8 4.5 2 0.2 1.9 0.2 17 99
Sistema de de pescado 36 5.3 4.6 97
H. midae
recirculación 38 8.7 4.8 65 50 97
grande
39 12.5 5 96
36 16.1 5.1 10 0.3 0.7 0.1 37 100
Cultivo en Jaulas 61 45 78 >87.5
jaulas sumergibles 55 37 77 >87.5
Wu y 48 32 63 >87.5
Densidad de 11- H. discus 61 45 64 >87.5
Zhang, 180
cultivo 21 hannai 55 37 60 >87.5
2013
Cestas apiladas >87.5
Talla 48 32 53

P= protenías; L= lípidos; C= carbohidratos; E= energía bruta (Kcalg-1); P/E= ratio proteína: energía; WS= Estabilidad en el agua (“Water Stability”) (Guzmán y Viana, 1998;
Hernández et al., 2009 (12 h inmersión); Mai, 1995a (48 h inmersion); FCR=Tasa de conversion del alimento (“Feed Conversion Ratio”); FI= Alimento ingerido (“Feed
intake”) expresado en % BW día-1 (Jackson, 2001) o por mg abalón día-1 (Fleming, 1995); FCE= Eficiencia de conversion del alimento (“Feed Conversion Efficiency”) (%);
PER = Tasa de eficiencia proteica (“Protein Efficiency Ratio”) ; T0 = talla inicial; P0 = peso inicial; SGR = Tasa específica de crecimiento (“Specific Growth Rate”); DGSL=
crecimiento diario en talla (“Daily Growth Rate, in shell lenght”; WG= Peso ganado (“Weight gain”); Superv.= Supervivencia.

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1.6. ACUICULTURA INTEGRADA DE CULTIVOS MULTI-TRÓFICOS (AIMT)

1.6.1. Aspectos generales

La creciente demanda de alimentos de origen marino junto a la disminución de

las capturas, han dado lugar a un incremento de los cultivos marinos que se duplican
cada diez años, crecimiento que se espera persista en las próximas décadas (FAO,
2012). La industria acuícola, tanto semi-intensiva como intensiva, libera al medio
materia orgánica, principalmente pienso sin consumir, pero también compuestos
inorgánicos derivados de la excreción de los organísmos en cultivo (Msuya et al., 2006).
De entre todos ellos, el amonio y los sólidos en suspension son los compuestos
contaminantes más importantes (Tovar et al., 2000), ya que si son liberados
directamente al mar, puede causar la eutrofización del medio circundante, afectando por
tanto al crecimiento natural de los organísmos. Es más, la propia instalación acuícola
puede verse afectada por el deterioro de la calidad del medio en donde desarrolla su
actividad (Neori et al., 2004).

En la actualidad, existe un amplio conceso por parte de la comunidad científica,

la propia industria, la opinion pública o los politicos, en cuanto a catalogar como
insostenibles aquellas tecnologías que no controlen sus emisiones al medio (Costa-
Pierce, 1996; Sorgeloos, 1999; Naylor et al., 2000; Chopin et al., 2001). En
consecuencia, es necesario el tratamiento de los efluentes y la mitigación del potencial
impacto medioambiental de la acuicultura, y una de las formas de lograrlo es a través de
la Acuicultura Integrada de Cultivos Multi-Tróficos (AIMT), donde la excreción
procedente de unos niveles tróficos, sirve de alimento para otros (Gordin et al., 1981;
Edwards et al., 1988). Además, la implantación de este tipo de acuicultura puede
considerarse como una Estrategia de Gestión Ambiental.

El concepto de AIMT no hace referencia únicamente a sistemas de cultivos

marinos abiertos, en los que los peces son el eslavón que genera el efluente y las algas y
organísmos invertebrados el componente biofiltrador, sino que también puede
extenderse a sistemas en tierra, en circuito cerrado e incluso a cultivos de agua dulce.
Lo que es importante es la correcta selección de organísmos de diferentes niveles
tróficos basada en la complementariedad de sus funciones, a la vez que en su valor
económico (Chopin et al., 2012). Así, en la actualidad los AIMT integran el cultivo de

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peces o langostinos con algas, microalgas, mariscos y/o algas, pudiendo desarrollarse
tanto en aguas costeras como en estanques, siendo válida incluso para cultivos
superintensivos (Neori et al., 2004; Zhou et al., 2006; Cunha et al., 2012; Liping et al.,
2012).

1.6.2. Acuicultura integrada basada en macroalgas

El papel principal de la biofiltración en los cultivos de langostinos o peces, es el

tratamiento mediante absorción de los metabolitos tóxicos y contaminantes. Las
bacterias biofiltrantes reducen el amonio a nitrato, mucho menos tóxico pero igualmente
contaminante (Touchtte y Burkholder, 2000), mientras que las microalgas, a través de la
fotosíntesis, convierten los nutrientes inorgánicos disueltos en materia particulada
(Troell y Norberg, 1998), que sigue permaneciendo en el agua. Por el contrario, las
macroalgas no sólo son capaces de eliminar de forma eficiente todas los compuestos
nitrogenados inorgánicos de los efluentes (Ryther et al., 1975; MacDonald, 1987;
Vandermeulen y Gordin, 1990; Cohen y Neori, 1991; Buschmann et al., 1994;
Demetropoulos y Langdon, 2004; Zhou et al., 2006), sino que también actúan como
productores de oxígeno que aportan a los animales (Schuenhoff et al., 2003; Neori et al.,
2003). Dichos nutrientes actúan como fertilizantes para las algas, incrementando así su
biomasa (Cohen y Neori, 1991; Muir, 1996; Neori et al., 1996, 2000; Shpigel y Neori,
1996). Es más, las algas, especialmente las marinas, son las más adecuadas para
sistemas de biofiltración ya que probablemente, son las plantas de mayor productividad
pudiendo además producirse de forma económica (Gao y McKinley, 1994). Además,
diversos autores han constatado que la calidad de las algas cultivadas en efluentes de
cultivos, es superior a la de aquellas cultivadas en agua marina limpia con adición de
fertilizantes (Harlin et al., 1978; Vandermeulen y Gordin, 1990; Neori et al., 1991;
Viera et al., 2006, 2009b).

En cuanto a la selección de la especie algal para su inclusion en un sistema

integrado, ésta debe de cumplir unos criterios básicos: alta tasa de crecimiento y
concentración de nitrógeno en los tejidos; facilidad de cultivo y ciclo de vida
controlado; resistencia a epífitos y a organísmos patógenos; y adaptación entre las
características ecofisiológicas del alga y el medio de crecimiento. Además, teniendo en
cuenta el daño ecológico que podría derivarse de la introducción de especies exóticas, el
alga a elegir debería ser así mimo una especie local. Más allá de estos criterios básicos,

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también dede considerarse la futura aplicación del alga a integrar en el cultivo

(producción de biomasa o biorremediación), siendo lo óptimo aquel alga que satisfaga
ambas aplicaciones (Neori et al., 2004). Por último, el precio de mercado de la biomasa
producida también es un factor a considerar (Buschmann et al., 1996).

Así, en acuicultura marina, los géneros algales más comunmente utilizado como
organismos biofiltrantes son el género Ulva: U. lactuca (Cohen y Neori, 1991; Neori et
al., 1991, 2000, 2003; Shpigel et al., 1993; Schuenhoff et al., 2003; Vandermeulen y
Gordin, 1990, Naidoo et al., 2006; Robertson-Andersson et al., 2011; Ben-Ari et al.,
2012), U. reticulata (Msuya et al., 2006), U. rigida (Jiménez del Río et al., 1994, 1996;
García, 1999; Toledo et al., 2000; Viera et al., 2006, 2009b; Izquierdo et al., 2013) y
Gracilaria: G. lemaneiformis (Fei et al., 2000, 2002; Fei, 2004; Zhou et al., 2006;
Yongjian et al., 2008; Mao et al., 2009), G. chilensis (Buschmann et al., 1994, 1995,
1996, 2001; Chow et al., 2001; Marquardt et al., 2010), G. changii (Phang et al., 1996),
G. parvispora (Nelson et al., 2001; Nagler et al., 2003), G. tenuistipitata (Haglund y
Pedersen, 1993), G. gracilis (Anderson et al., 1999; Njobeni, 2005; Hansen et al., 2006;
Naidoo et al., 2006), G. textorii (Pang et al., 2006), G.lichenoides (Xu et al., 2008) o G.
cornea (Viera et al., 2006, 2009b; Izquierdo et al., 2013). Sin embargo, a pesar de que la
tecnología de cultivo industrial y la capacidad de captación de nutrientes del género
Ulva son de los mayores conocidos (Marínez-Aragón et al., 2002), el valor comercial de
la biomasa resultante es bajo. Por el contrario, de las especies del género Gracilaria, a
pesar de que presentan crecimientos generalmente menores (Marinho-Soriano et al.,
2002; Nagler et al., 2003), sí se obtienen bio-productos de altísimo valor commercial,
como es el caso del agar-agar (Neori et al., 2004).

Otros géneros de valor comercial como Porphyra (Chopin et al., 1999; Carmona
et al., 2001; Fei, 2001, Yarish et al., 2001), Palmaria (Demetropoulos y Langdon, 2004;
Matos et al., 2006), Hypnea (Langton et al., 1977; Viera et al., 2009b) o laminarias
(Laminaria and Macrocystis), también han sido satisfactoriamente integrados en
sistemas multitróficos (Chopin y Bastarache, 2002; Buschmann et al., 2008).

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1.6.3. Sistemas de cultivos integrados peces-macroalgas-oreja de mar

La producción de algas de alta calidad en sistemas de biofiltración hace

oportuno el co-cultivo de otros organísmos herbivoros de alto valor comercial, como es
el caso de la oreja de mar. Ya en los años 70, Tenore (1976) publicó un primer trabajo
acerca del cultivo integrado de algas-abalón (Fig. 13). Este trabajo fue seguido por los
implementados en Israel que integraban abalón - Ulva y Gracilaria (Shpigel et al.,
1993, 1996a, b, 1999; Shpigel y Neori, 1996; Neori et al., 1998, 2000), en Japón con
abalón y algas verdes (Sakai yHirata, 2000), o Palmaria – abalón en los EUA (Evans y
Langdon, 2000). De forma general, dichos trabajos evidenciaron un buen crecimiento de
la oreja de mar cuando se alimenta con algas producidas en sistemas de biofiltro,
probablemente debido al alto contenido en proteínas de las algas cultivadas en las altas
concentraciones de nitrógeno del sistema integrado (Neori et al., 1998; Shpigel et al.,
1999; Boarder y Shigel, 2001; Naidoo et al., 2006; Robertson-Andersson et al., 2011).

Figura 13. Esquema de un sistema de cultivo integrado peces-algas-abalón.

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9.2. OBJETIVOS

El objetivo general de la presente tesis fue el “desarrollo de la tecnología de

engorde de la oreja de mar, Haliotis tuberculata coccinea“, como nueva especie para
el sector acuícola de Canarias. De forma específica se evaluó la idoneidad de distintas
dietas, tanto algales como artificiales, el crecimiento y la supervivencia de los
ejemplares, así como el efecto de distintos parámetros sobre el rendimiento del proceso
productivo. Además, la tecnología de cultivo a desarrollar se abordó desde un enfoque
tal que garantice la sostenibilidad mediambiental de los procesos productivos a
implementar.

A tal fin, se llevaron a cabo cuatro estudios:

Estudio I. Idoneidad de tres algas rojas como alimento para la oreja de

mar Haliotis tuberculata coccinea Reeve.

El objetivo de este estudio fue evaluar la viabilidad de tres algas rojas,

pertenecientes a géneros ampliamente utilizados para el cultivo de distintas especies de
abalón, como alimento potencial para el engorde de juveniles de la especie presente en
Canarias. Debido a que la biomasa macroalgal existente en el medio no es suficiente
para sustentar una producción comercial de este molusco, las macroalgas
experimentales se cultivaron en un Sistema Multi-Trófico de Acuicultura Integrada
(AMTI). Este objetivo se abordó a fin de identificar algas capaces de promover un alto
crecimiento y supervivencia para la especie local, así como para investigar el valor
nutricional de las algas producidas en sistemas de biofiltros. Para alcanzar dicho se llevó
a cabo un ensayo nutricional de dos meses de duración.

Estudio II. Crecimiento comparativo de juveniles de oreja de mar (Haliotis

tuberculata coccinea Reeve) alimentados con algas enriquecidas vs sin enriquecer:
Efecto sobre el crecimiento y la composición corporal.

El propósito del presente trabajo fue evaluar el efecto de diferentes dietas

algales: rojas y verdes, monoalgales y mixtas, sin enriquecer y cultivadas en efluentes
de cultivos marinos, sobre el crecimiento de juveniles de la oreja de mar de Canarias.
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Dicho objetivo se abordó no sólo con el fin de identificar dietas idóneas para el
crecimiento de los animales, sino también para evaluar las ventajas del cultivo multi-
trófico de algas y H. tuberculata coccinea en sistemas integrados, que permitan tanto la
sostenibilidad de la futura producción de abalón como una mejora en el crecimiento.

Estudio III. Desarrollo de las primeras dietas vegetales y su idoneidad para

el cultivo de Haliotis tuberculata coccinea Reeve.

El objetivo de este trabajo fue el desarrollo y evaluación de varias dietas

compuestas, exclusivamente vegetales, para el cultivo del abalón Haliotis tuberculata
coccinea, con particular interés en determinar la potencialidad de la inclusión de las
cuatros especies de macroalgas más comúnmente utilizadas en la producción de abalón
en Europa. El objetivo descrito se abordó con el fin de obtener dietas sin harinas de
pescado, adaptadas a los requerimientos nutricionales del abalón, que permitan al sector
productor disponer de fuentes nutricionales estables que además validen la
sostenibilidad medioambiental de sus procesos productivos. Para la implementación de
este estudio se llevó a cabo un experimento en tierra de seis meses.

Estudio IV. Cultivo de la oreja de mar Haliotis tuberculata coccinea Reeve,

alimentadas con algas procedentes de Sistemas Multi-Tróficos Integrados (AMTI),
en una instalación de peces/ abalón en mar abierto: Efecto de la densidad.

La finalidad de este estudio fue evaluar el efecto de la densidad de cultivo, uno

de los parámetros más influyentes en el engorde de oreja de mar, sobre el crecimiento y
la supervivencia de ejemplares de oreja de mar de dos tallas distintas. Se abordó dicho
objetivo con el fin de identificar ciertas condiciones de cultivo que mejoren el
rendimiento del proceso, al tiempo que evaluar la potencialidad del cultivo de abalón en
mar abierto durante la última fase del proceso de engorde. A tal fin se realizó un ensayo
de engorde en jaulas en mar abierto durante seis meses

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9.3. MATERIAL Y MÉTODOS

3.1. EMPLAZAMIENTO E INSTALACIONES GENERALES

Las experiencias que se describen en esta tesis, (con excepción de la

implementada en mar abierto; 3.5.2.), han sido realizadas en la nave de cultivos del
Grupo de Investigación en Acuicultura (GIA- ULPGC), en el Instituto Canario de
Ciencias Marinas (ICCM). Dicho instituto está emplazado en Melenara, municipio de
Telde, isla de Gran Canaria, provincia de Las Palmas (Islas Canarias, España),con una
situación geográfica de latitud 27º59’31’’N y longitud 15º22’31’’W (Fig. 14).

Figura 14. Fotografía desde satélite de las Islas Canarias y ubicación del ICCM (Foto del
Google Earth).

Las instalaciones de cultivo de oreja de mar comprenden las siguientes zonas:

acondicionamiento de reproductores (Fig. 15-A), inducción a la puesta y cría larvaria
(Fig. 15-B); cultivo de post-larvas y producción de diatomeas (Fig. 15-C, D); engorde
(Fig. 15-E) y ensayos de nutrición (Fig. 15-F); y unidades de biofiltración y
reutilización de los efluentes de la nave de cultivo para la producción de macroalgas
(Fig. 15-G, H).

Tanto las instalaciones destinadas al cultivo del abalón del ICCM, como los
estudios realizados, han sido financiados por proyectos de Canarias (PI 2007/034),
España (JACUMAR Oreja de mar, 2005/07; JACUMAR Multitrófico, TR 2003/08) y
Europa (SUDEVAB: FP 7-SME-2007-1/BSG-SME).

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Figura 15. Acondicionamiento de reproductores (A); cultivo larvario (B); post-larvas y pre- B
engorde (C); cultivo de diatomeas (D); zona de engorde (E); zona experimental (F) producción
de macroalgas en el exterior (G, H).

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3.2. PRODUCCIÓN DE OREJA DE MAR B

3.2.1. Acondicionamiento y selección de repodructores

Los reproductores de Haliotis tuberculata coccinea se mantuvieron en

cautividad en tanques de 60 l cubiertos y flujo abierto, bajo condiciones naturales de
fotoperíodo y temperatura (Fig. 15-A). Se estabularon entre 10-15 ejemplares
(dependiendo del tamaño) dentro de cada uno de los 24 tanques, y se colocaron tejas de
PVC a modo de refugios. Los reproductores se separaron de los desechos, a través de la
colocación de una pared perforada en la parte inferior del tanque. Machos y hembras,
diferenciados por el color de las gónadas (de color blanco cremoso para los machos y de
gris oscuro a violeta par las hembras) (Fig. 16), se mantuvieron por separado en los
tanques de acondicionamiento. De forma general, los animales se alimentaron dos veces
por semana con una dieta mixta de Ulva rigida y Gracilaria cornea, producidas en el
sistema de biofiltros. Los ejemplares seleccionados para ser inducidos para los distintos
desoves fueron siempre los que presentaban gónadas maduras en estadío 3 o entre 2 y 3
de maduración (Ebert y Houk, 1984).

Figura 16. Hembra (A) y macho (B) de H. tuberculata coccinea en estadío 2-3 de
maduración gonadal.

3.2.2. Inducción a la puesta

Los machos y hembras maduros, con un ratio macho/hembra de 1:2, fueron

inducidos a desovar por separado, en recipientes con agua de mar filtrada a 1µm
mediante filtros de cartucho y esterilizada por UV. Los gametos de distinto sexo se

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obtuvieron por separado con el fin de controlar el ratio de gametos empleado durante la
fecundación para así tener un mejor control sobre el proceso de fertilización (Fig. 17).
Durante la inducción al desove los recipientes se mantuvieron en oscuridad. Se
utilizaron dos métodos de inducción, el método del peróxido de hidrógeno (Morse et al.,
1977) y el del ultravioleta (Kikuchi y Uki, 1974).

Figura 17. Desove de hembras (A) y machos (B). B

3.2.3. Fertilización

Una vez obtenidos los gametos, los ovocitos expulsados (depositados en el

fondo), fueron sifonados de los recipientes de desove y tamizados a través de una malla
A
de 300 µm con el fin de retener las heces y restos de residuos. Los ovocitos se
recogieron en un cubo de 10 l y fueron fertilizados a una concentración de
espermatozoides final de 105/ml durante 30 minutos. A continuación, los huevos se
lavaron con agua de mar esterilizada para eliminar el exceso de esperma (Fig. 18), y se
determinaron las tasas de fertilización mediante el registro bajo una lupa (Mod. SL
260004, Optech, Duisburg, Alemania), de la proporción de los huevos que presentaban
células en división 1 h después de la fertilización. Los huevos fertilizados se
transfirieron a los tanques de cultivo larvario (Fig 15-B).

Figura 18. Enjuague de los huevos para eliminar el exceso de esperma. B

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3.2.4. Cultivo larvario

Las distintas fases larvarias de la oreja de mar comienzan con la larva trocófora
y acaban con la formación del cuarto túbulo en los tentáculos cefálicos, si bien las larvas
se consideran preparadas para el asentamiento cuando el tercer túbulo aparece, y las
larvas empiezan a explorar la superficie (Fig. 19).

Las larvas de H. tuberculata coccinea se cultivaron bajo fotoperíodo natural y en

flujo abierto, en tanques larvarios de 100 l a una densidad de 10-20 larvas/ml. El agua
suministrada a los tanques larvarios se filtró a través de filtros de cartucho de 1 µm y se
esterilizó por radiación UV. Las larvas se mantuvieron sin comer en los mismos tanques
hastas que fueron transferidas a los tanques de nursery, una vez listas para la fijación y
posterior metamorfosis (Fig. 15-C). La competencia para la fijación se observó entre 62
y 72 horas después de la fertilización, en función de la temperatura del cultivo larvario
de los distintos lotes utilizados en los diferentes experimentos.

Figura19. Larva trocófora con cilios (A), aparición del tercer túbulo en los tentáculos cefálicos
(B) (Courtois de ViÇose et al., 2007).

3.2.5. Fijación larvaria

La inducción a la fijación de las larvas de H. tuberculata coccinea, se realizó en

places verticales de fijación, agrupadas y situadas dentro de los tanques de asentamiento
de 2500 l (Fig. 20).

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Figura 20. Placas verticales para la fijación de las larvas.

Para la inducción al asentamiento, las placas se colonizaron con distintos tipos

de sustratos: algas coralinas incrustantes, diatomeas bentónicas y esporas del alga verde
Ulvella lens.

3.2.6. Cultivo de postlarvas y juveniles

Las postlarvas de abalón se alimentaron con cuatro cepas de diatomeas:

Navicula incerta, Proschkinia sp., Nitzschia sp. y Amphora sp. (Fig. 21). Dichas
microalgas se cultivaron en bolsas horizontales de 40 l, a una densidad de inóculo
inicial de 105 cel/ ml, en medio f/2 suplementado con silicatos (1 mgl-1) (Guillard,
1975), a temperatura ambiente y bajo luz continua de 62±µmol fotones m-2 s-1. Las
diatomeas se cosecharon tras 5 días de cultivo, correspondiente a la fase de crecimiento
exponencial.

Figura 21. Especies de diatomeas suministradas a las postlarvas: Proschkinia sp. (A),
Navicula incerta (B), Amphora sp. (C) y Nitzschia sp. (D) (Courtois de ViÇose et al., 2012b).
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Los animales mantuvieron estas dietas durante 4-5 meses, cuando de forma
gradual, ya juveniles, se les comenzó a alimentar con macroalgas: Ulva rigida (Estudio
I); Ulva rigida, Hypnea spinella y Gracilaria cornea (Estudio II); o Ulva rigida y
Gracilaria cornea (Estudios III y IV).

3.3. CULTIVO ALGAL B

3.3.1. Especies de macroalgas

A lo largo de los experimentos se cultivaron cuatro especies de macroalgas: las

rodofitas: Gracilaria cornea J. Agardh, Hypnea spinella (C. Agardh) Kützing e Hypnea
musciformis (Wulfen) J.V. Lamoroux; y la clorofita Ulva rigida J. Agardh. Además, el
alga roja Palmaria palmata (L.) Weber y Mohr, la parda Laminaria digitata (Hudson)
Lamoroux y la verde Ulva lactuca Linnaeus, se testaron como ingredientes en dietas
compuestas (Estudio III).

Las citadas macroalgas fueron seleccionadas debido a su idoneidad para ser

incluídas en AIMT como biofiltros (Neori et al., 2004), y también como alimento para
la oreja de mar (Boarder y Shigel, 2001; Naidoo et al., 2006; Watson y Dring, 2011).

3.3.2. Sistema de cultivo: Acuicultura Integrada de Cultivos Multitróficos

(AIMT)

Excepto aquellos tratamientos experimentales en el que las algas no fueron

enriquecidas (Estudio II), el resto de las algas frescas utilizadas en los distintos ensayos
de nutrición, fueron producidas en un sistema integrado peces-macroalgas.

En los Estudios I y II, las algas se cultivaron en el sistema multitrófico integrado

del Centro de Biotecnología Marina (CBM-ULPGC, Gran Canaria, España). En dicho
sistema, el efluente de los tanques de peces (Fig. 22-A) fue canalizado a un tanque de
sedimientación de 11 m3 con el fin de eliminar el material en suspension, y de ahí,
bombeado con un flujo de 10 m3 h-1 a los tanques de algas situados en un invernadero,
donde la radiación máxima fue de aproximadamente µmol fotones m-2 s-1. Para el
cultivo de las algas se emplearon tanques de fibra de vidrio semicirculares de 1.8 m2 de

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superficie y 0.75 m3 de volumen, y aireación a través de un tubo lineal situado en en

centro del fondo (Fig. 22- B). En el Estudio I, las algas G. cornea, H. spinella e H.
musciformis se inocularon a una densidad de 6 g l-1, mientras que en el II, la densidad de
inóculo fue de 1, 3 y 4 g l-1 para U. rigida, H. spinella y G. cornea, respectivamente. La
tasa de renovación del agua en los tanques algales fue de 4 vol día-1 y el TAN (N-
amonio total) que entró en los biofiltros osciló entre 10 y 400 µM.

Figura 22. Tanques de peces y tanques semicirculares para el cultivo de algas (CBM-ULPGC).

En cuanto a los Estudios III y IV, las algas U. rigida y G. cornea fueron
cultivadas en el sistema de cultivo multitrófico integrado en las instalaciones de cultivo
del Grupo de Investigación en Acuicultura (GIA, Islas Canarias, España). En dicho
sistema, los efluentes generales de la nave fueron conducidos a un tanque de
sedimentación de 11 m3 para eliminar las material en suspension, y de allí bombeados a
tanques exteriores donde la radiación máxima es de alrededor de 1600 µmol fotones m-2
s-1. El cultivo de las algas se realizó en tanques plásticos circulars de 1.5 m3 de
volumen, y la aireación se suministró mediante manguera porosa circular situada en el
fondo del tanque (Fig. 23). Las algas se inocularon a una densidad de 1 y 4 g l-1 para U.
rigida y G. cornea, respectivamente. La tasa de renovación del agua de los tanques
algales fue de 12 vol día-1, y TAN (N-amonio total) que entró a los tanques osciló entre
los 10 y 30 µmoles.

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Figura 23. Esquema del Sistema Multitrófico Integrado (ICCM-ULPGC).

Las algas se cultivaron por triplicados y se cosecharon quincenalmente. Antes de

alimentar a los animales, las algas se secaban en una centrífuga (AEG SV 4528,
Alemania; Fig. 24) y se pesaba la cantidad necesaria de cada tratamiento para cada
réplica (KERN EW 1500-2M, Balingen, Alemania).

Figura 24. Biomasa algal producida en los biofiltros y centrífuga para el secado.

3.4. DIETAS ARTIFICIALES

Con el fin de evaluar el valor nutricional de diversas macroalgas como

ingredientes en dietas de abalón, se diseñaron tres dietas exclusivamente con
ingredientes vegetales, y se suministraron durante seis meses en un experimento en
tanques en tierra (Estudio III). El procesado de las harinas de las algas, así como la
formulación y preparación de las dietas, se detalla en los capítulo correspondiente a

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dicho experimento, en esta sección, sólo se incluye una descripción general de la

metodología y materiales empleados.

3.4.1. Formulación de las dietas

Antes de la preparación de las dietas, en el laboratorio del GIA se analizó la
composición proximal tanto de las harinas de las algas experimentales (U. lactuca (U),
G. cornea (G), L. digitata (L) y P. palmata (P)), como de la del resto de los
ingredientes. Así mismo, se analizó el perfil de aminoácidos de las algas en el
Laboratorio de Diagnóstico General de Barcelona, España, (LDG).
A raíz de los resultados obtenidos, y teniendo en cuenta los valores descritos en
la literatura como óptimos para el crecimiento de la oreja de mar, se formularon tres
dietas (UG, UGL y UGP) de modo que contuviesen un 35% proteínas, 4% lípidos y una
energía bruta de alrededor de 4 kcal g-1. La mezcla de vitaminas y minerales añadida fue
la recomendada por Uki y col. (1985a). Con el fin de ajustar el perfil de aminoácidos de
las dietas a la del músculo del abalón, que se tomó como patrón, todas las dietas se
suplementaron con los aminoácidos sintéticos L-metionina y lisina. Como aglutinante se
usó el alginato de sodio.

3.4.2. Preparación de las dietas

Para preparar los tratamientos experimentales, una vez molidos todos los
ingredientes, se pesaron y mezclaron hasta homogeneizarlos. Las dietas se procesaron a
través de una máquina de pasta industrial (Parmigiana, RV3, Italia), de la que se
obtuvieron unas cintas de 2 mm de grosor, que se cortaron en cuadrados de
aproximadamente 0.5 x 0.5 cm. Una vez procesados, los piensos se secaron en una
estufa a 38ºC durante 24 h, se empaquetaron al vacío, y se almacenaron en una cámara a
4ºC hasta ser usados (Fig. 25 y 26). Se recogieron muestras para el análisis proximal y
se conservaron a -80ºC.

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Figura 25. Detalles del procesado de las dietas: ingredientes, preparación y secado.

3.4.3. Estabilidad de las dietas en el agua

La estabilidad de las dietas en el agua se evaluó mediante el método de Hastings

y col. (1971). Se determinó para un período de 17-h (16:00-9:00h). El porcentaje de
estabilidad de las dietas se calculó de la manera siguiente:

(𝐵𝐵 − 𝐴𝐴) (% 𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚 𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠)

% 𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸 𝑑𝑑𝑑𝑑 𝑒𝑒𝑒𝑒 𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎 = 𝑥𝑥 100
𝐴𝐴 (% 𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚 𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠)

donde B es el peso final del alimento y A es el peso inicial.

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Figura 26. Producto final: dietas experimentales con ingredientes exclusivamente vegetales.

3.5. DISEÑO EXPERIMENTAL

Los detalles tanto del diseño experimental como de los protocolos de muestreo
se describen de forma detallada en cada uno de los capítulos correspondientes a los
distintos experimentos. En esta sección, unicamente se describen, de forma general, los
materiales y métodos empleados en dichos estudios.

Para seleccionar los ejemplares experimentales, los animales se secaron, pesaron

con una balanza con un rango de 0.1 mg (Peso húmedo total, “total wet body weight”:
TWBW) (KERN EW 1500-2M, Belingen, Alemania) y se midieron con un calibrador
de 0.1 mm de precisión (Longitud total de la concha, “total shell length”: SL) (Fig. 27a).
Una vez seleccionados, se ditribuyeron entre las réplicas de modo que no hubiese
diferencias significativas en talla o peso, y se asignaron a cada unidad experimental
(Fig. 27b).

Figura 27a. Selección de los animales experimenales: muestreo de los ejemplares.

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Figura 27b. Réplicas de los animales seleccionados.

Las dietas algales se suministraban semanalmente a los animales experimentales

(Estudios I-IV) (Fig. 28-A, B, C), mientras que las artificiales (Estudio III), se
suministraban diariamente en una sola toma de lunes a sábado (Fig. 28-D). Todas se
testaron por triplicado y se suministraban en exceso para garantizar una alimentación a
saciedad.

Figura 28. Réplicas de las dietas algales: Estudios I y II (A), Estudio III (B) y Estudio IV (C);
o con dietas compuestas: Estudio IV (D).

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Para una estimación real del consumo de alimento, se usaron como control
unidades experimentales con alimento pero sin animales. En los experimentos en tierra,
los animales estuvieron bajo un fotoperíodo natural de aproximadamente 12 h L / 12 h
D.
Para evaluar el crecimiento, tanto en talla como en peso, se midió mensualmente
el SL y TFBW del 100% de la población de cada réplica.
En el experimento en el mar, las jaulas se limpiaron mensualmentede con el fin
de evitar la acumulación de fouling.

3. 5.1. Instalación experimental de los ensayos en tierra

La unidad experimental empleada para los juveniles de 11-12 mm de longitud en

los dos primeros experimentos, consistió en un tubo de PVC (20 x 14 cm) de 1 l de
capacidad, provisto con tapas de malla plástica en los extremos (luz de malla de 2 mm).
Dichas unidades se introdujeron en tanques cilíndricos de fibra de vidrio de 100 l,
llenos de agua de mar filtrada a través de un cartucho de 50 µm, y aireación constante.
El flujo de agua fue de aproximadamente 2.4 l/min. (Fig. 29).

Figura 29. Unidad experimental y tanque empleado para el cultivo de los juveniles (Estudios I
y II).

En el Estudio III, la unidades experimentales consistieron en unas cestas

plásticas (15x16cm), suspendidas dentro de una cajas rectangulares de 100-l de
capacidad (100x40x25cm), provistos de agua de mar filtrada a 50 µm, flujo abierto y
aireación constante (Fig. 30-A, B). El flujo de agua fue de aproximadamente 2.8 l/min.
(Fig. 29). En cada cesta se introdujeron dos tejas de PVC a modo de refugio.

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Figura 30. Sistema de cultivo empleado para los ejemplares de 30 - 45 mm (Estudio III).

3.5.2. Instalación para el ensayo de cultivo en el mar

El sistema experimental para el engorde del abalón en el mar se instaló en una

piscifactoría commercial de jaulas flotantes (CANEXMAR, S.L., Telde, Gran Canaria,
España) (27º 57´ 31.7´´N, 15º 22´ 22.5´´W) (Fig. 31).

Figura 31. Localización y jaulas de la piscifactoría CANEXMAR en Gran Canaria.

Las jaulas marinas, diseñadas expresamente para el cultivo de la oreja de mar

(ORTACS, Jersey Sea Farms, St. Martins, Irlanda), consistían en unos dispositivos
negros de PVC perforados, con una capacidad 33 l y 1.5 kg de peso. Cada jaula disponía
de una tapa, y una superficie interior total 0.4 m2. En el interior de cada ortac, se
colocaron seis discos negros (12.0 cm Ø) a modo de refugios (Fig. 32). La superficie
total disponible para la adeherencia de los animales fue de 0.5 m2 por jaula. Las jaulas

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se suspendieron desde las estachas de las jaulas de peces (25 m Ø), situándose
aproximadamente a 10 m de la superficie del agua (Fig. 33-35).

Figura 32. Detalle de las jaulas experimentales y de los refugios.

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Figura 33. Diagrama de la instalación en el mar para el engorde de abalón. A: Planta de las
jaulas de peces. B: Detalle de la disposición de los ortacs.

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Figura. 34. Equipamiento e instalación en el mar de las jaulas de abalón.

Figure 35. Imágenes de los ortacs sumergidos junto a las jaulas de peces (Fotos de Elodie
Turpin).

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3.6. EVALUACIÓN DE LOS PARÁMETROS BIOLÓGICOS

3.6.1. Crecimiento en talla

La tasa de crecimiento en talla al día (μm d-1), se calculó para cada muestreo y al
final de cada uno de los ensayos mediante la siguiente ecuación:

(𝑺𝑺𝑺𝑺𝑺𝑺 − 𝑺𝑺𝑺𝑺𝑺𝑺)
𝑻𝑻𝑻𝑻𝑻𝑻𝑻𝑻 𝒅𝒅𝒅𝒅 𝒄𝒄𝒄𝒄𝒄𝒄𝒄𝒄𝒄𝒄𝒄𝒄𝒄𝒄𝒄𝒄𝒄𝒄𝒄𝒄𝒄𝒄 = 𝒙𝒙 𝟏𝟏𝟏𝟏𝟏𝟏𝟏𝟏
𝒕𝒕

donde SL1 es la talla media inicial de los animales; SL2 es la talla media final al tiempo t
(días de cultivo).

3.6.2. Tasa de crecimiento específico (SGR)

También se calculó la Tasa de crecimiento especifíco en peso, que indica el

incremento en peso ganado en relación al número de días experimentales y se expresa
en porcentajes:
(𝑳𝑳𝑳𝑳𝑾𝑾𝟐𝟐 −𝑳𝑳𝑳𝑳𝑾𝑾𝟏𝟏 )
SGR (% 𝒅𝒅−𝟏𝟏 ) = 𝒙𝒙 𝟏𝟏𝟏𝟏𝟏𝟏
𝒕𝒕

W2 es el peso al tiempo t (días de cultivo) y W1 es el peso inicial.

3.6.3. Peso ganado (WG)

Se calcula a partir de la relación entre el incremento en peso y el peso inicial,

expresado en porcentaje mediante la siguiente ecuación:

(𝒘𝒘𝟐𝟐 − 𝒘𝒘𝟏𝟏 )
𝑾𝑾𝑾𝑾 (%) = 𝒙𝒙 𝟏𝟏𝟏𝟏𝟏𝟏
𝒘𝒘𝟏𝟏

3.6.4. Tasa de conversión del alimento (FCR)

Este parámetro se calculó con el fin de determinar la eficiencia de los diferentes

regímenes alimenticios para promover el crecimiento del abalón en relación al alimento

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ingerido. Se define como la relación entre el alimento consumido y la generación de

biomasa (g):

𝒂𝒂𝒂𝒂𝒂𝒂𝒂𝒂𝒂𝒂𝒂𝒂𝒂𝒂𝒂𝒂 𝒄𝒄𝒄𝒄𝒄𝒄𝒄𝒄𝒄𝒄𝒄𝒄𝒄𝒄𝒄𝒄𝒄𝒄 𝒕𝒕𝒕𝒕𝒕𝒕𝒕𝒕𝒕𝒕 (𝒈𝒈)

𝑭𝑭𝑭𝑭𝑭𝑭 =
𝒕𝒕𝒕𝒕𝒕𝒕𝒕𝒕𝒕𝒕 𝒅𝒅𝒅𝒅 𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑 𝒈𝒈𝒈𝒈𝒈𝒈𝒈𝒈𝒈𝒈𝒈𝒈 (𝒈𝒈)

3.6.5. Tasa de eficiencia proteica (PER)

Este parámetro relaciona el incremento en peso de los animales durante el

período experimental, con la proteína ingerida. Se calcula de la forma siguiente:

𝒊𝒊𝒊𝒊𝒊𝒊𝒊𝒊𝒊𝒊𝒊𝒊𝒊𝒊𝒊𝒊𝒊𝒊𝒊𝒊 𝒆𝒆𝒆𝒆 𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑 𝒉𝒉ú𝒎𝒎𝒎𝒎𝒎𝒎𝒎𝒎(𝒈𝒈)

𝑷𝑷𝑷𝑷𝑷𝑷 =
𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑í𝒏𝒏𝒏𝒏 𝒊𝒊𝒊𝒊𝒊𝒊𝒊𝒊𝒊𝒊𝒊𝒊𝒊𝒊𝒊𝒊 (𝒈𝒈)

3.6.6. Consumo de alimento (FI)

Para evaluar el consumo de alimento en los estudios I, II y IV, donde sólo se

alimentó con algas frescas, antes de cada toma semanal, el alga se cosechó, se secó y se
pesó para su reparto en cada réplica, pesándose así mismo el alga sobrante al final de la
semana. El peso del sobrante se corrigió mediante el cálculo de la variación natural del
alga en las réplicas de control (sin animales), durante el mismo período experimental.

En el Estudio III, las dietas artificiales se suministraron por la tarde, en una sola
toma de lunes a sábado (ad libitum). El pienso sobrante se sifonaba todas las mañanas a
las 9:00 h, con excepción de los domingos. La estimación del consumo se hizo en peso
seco mediante la relación del peso seco del pienso sobrante con el peso seco del pienso
suministrado (Fig. 36). El dato del consumo se corrigió restándole la pérdida debida a la
lixiviación, que se estimó a partir de dejar el pienso durante el mismo período de 17-h
(16:00-9:00h), en las unidades de control sin animales, y secando la dieta sobrante hasta
peso constante.
En todos los ensayos, el consumo de alimento por individuo y día, se calculó
dividiendo el alimento ingerido cada semana entre el número de días y entre los
ejemplares de cada réplica.

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Figura 36. Secado del pienso sobrante para la estimación del consumo.

3.6.7. Índice de condición

Al final de los experimentos II y III, se recolectaron respectivamente seis y

diez individuos de cada réplica, separando el animal (SB) de la concha (S). Para
calcular el índice de condición, como parámetro indicador del estatus nutricional de
los animales, la concha y la carne (vísceras y pie), se pesaron por separado, y el
índice se calculó mediante la siguiente relación (Fig 37):

𝑺𝑺𝑺𝑺
𝑪𝑪𝑪𝑪 =
𝑺𝑺

Figura 37. Evaluación del índice de condición: disección y pesado del abalón.

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3.6.8. Supervivencia

La mortalidad se estimó diariamente (Estudios I-III) y semanalmente (Estudio

IV). Al final del experimento, se calculó la supervivencia mediante la relación entre
los individuos vivos y la mortalidad de cada réplica.

3.7. ANÁLISIS BIOQUÍMICOS

Durante el transcurso de las diferentes experiencias, se tomaron por triplicado

muestras de los diferentes tratamientos experimentales (algas frescas y dietas
compuestas), así como ingredientes y animales experimentales (víceras y músculo) para
el análisis de su composición proximal. Los métodos de recolección de las muestras
difieren en función de su naturaleza. En el caso de las algas frescas, una vez recogidas,
se lavaban con agua dulce y luego destilada para eliminar la sal y posible epizoos, a
continuación se eliminaba la mayor cantidad de agua posible con papel secante y se
procedía a su congelación a -80ºC así como posterior liofilización y molienda (<0.1
mm).
Una vez recogidas, todas todas las muestras se congelaban a -80 ºC en bolsas
herméticas bajo atmósfera de nitrógeno. Antes de los análisis, las muestras se
homogeneizaron con mortero y luego se pesaban. Se hicieron determinaciones del
contenido en humedad, cenizas, proteínas, lípidos totales y ácidos grasos. Los análisis
bioquímicos se realizaron en el laboratoprio del Instituto Universitario de Sanidad
Animal y Seguridad Alimentaria (IUSA; ULPGC).

3.7.1. Determinación de la humedad

Se determinó siguiendo la métodología oficial de análisis internacional (AOAC,
2005). El procedimiento consiste en secar en una estufa a 110ºC una cantidad conocida
de muestra fresca (Pi) hasta obtener un peso constante (Pf). Posteriormente, la muestra
se introduce en el desecador 30 minutos y se realiza la pesada. El porcentaje de
humedad de la muestra se obtiene mediante la expresión:

(𝑷𝑷𝒊𝒊 − 𝑷𝑷𝒇𝒇)
% 𝑯𝑯 = 𝒙𝒙 𝟏𝟏𝟏𝟏𝟏𝟏
𝑷𝑷𝒊𝒊

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3.7.2. Determinación de las cenizas

El contenido em cenizas se determinó gravimétricamente después de la
incineración de una cantidad conocida de muestra (1-2 g) (Pi) en un horno mufla a una
temperatura de 600ºC durante 24 h. La cantidad de cenizas remanente (Pf) se pesó hasta
alcanzar un peso constante, de acuerdo a las recomendaciones establecidas por la
AOAC (2005). El contenido de cenizas se calculó según la siguiente fórmula:

𝑷𝑷𝑷𝑷 𝒙𝒙 𝟏𝟏𝟏𝟏𝟏𝟏
% 𝑨𝑨𝑨𝑨𝑨𝑨 =
𝑷𝑷𝑷𝑷

3.7.3. Determinación de las proteínas

El contenido proteico se calculó a partir del contenido de nitrógeno total de la

muestra, según el método de Kjeldahl. De acuerdo con la AOAC (2005), la técnica
consiste en la digestion de la muestra con ácido sulfúrico (H2SO4) a 420ºC en presencia
de un catalizador de cobre durante una hora, seguido de una destilación con NaOH al
40%, utilizando ácido bórico saturado (H3BO3) como sustancia receptora en una unidad
detiladora (Mod. Foss Tecator, 1002, Höganäs, Suecia). Finalmente se realiza una
valoración con HCl 0.1.M. El cálculo del porcentaje en proteínas se realiza de la forma
siguiente:
(𝑽𝑽𝒔𝒔 −𝑽𝑽𝒃𝒃 ) 𝒙𝒙 𝑵𝑵 𝒙𝒙 𝑷𝑷𝒎𝒎
% Proteina = 𝒙𝒙 𝑭𝑭
𝑾𝑾

Siendo:

Vs = Volumen de (HCl) usado en la valoración en ml

Vb = Media de la valoración de los patrones en ml
N = Normalidad del HCl
Pm = Peso molecular del nitrógeno (14.007)
F = Factor de conversión empírico para convertir el porcentaje de nitrógeno de la
muestra en porcentaje de proteínas, tiene un valor de 6.25
W = Peso de la muestra en mg

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3.7.4. Determinación de lípidos totales

La extracción de los lípidos se realizó según el método de Folch et al. (1957). El
método consistió en tomar una cantidad de muestra de entre 50-200 mg, que se
homogeneizó en un Ultra Turrax (IKA-Werke, T25 BASIC, Staufen, Alemania) a
11,000 rpm durante 5 min en una solución de 5 ml de cloroformo: metanol (2:1) con
0.01% de BHT. La solución resultante se filtró a presión reducida a través de fibra de
vidrio, añadiendo KCl al 0.88% para aumentar la polaridad de la fase acuosa. Por
decantación y tras un centrifugado a 2000 rpm durante 5 min, la fase orgánica y la
acuosa se separaron. Una vez retirada la fase acuosa, la fase resultante se evaporó a
sequedad con una corriente de N2. Finalmente, el contendido en lípidos totales se
determinó por gravimetría.

3.7.5. Determinación de ácidos grasos

Los lípidos totales extraídos, se trasesterificaron a esteres metílicos (FAMEs)
con un 1% de ácido sulfúrico: metanol (Christie, 1982) y se conservaron en atmósfera
de N2. La mezcla se dejó incubando a 50ºC durante 16 horas y se enfrió. Se le añadió
agua destilada ultrapura y hexano: dietileter 1:1 y BHT al 0.01%. Los FAMEs
purificados se evaporaron a sequedad con N2 y se pesaron. Por último, los FAMEs
fueron extraídos en hexano y se conservaron a - 80ºC hasta el momento de su
identificación mediante un cromatógrafo de gases (Mod. Shimadzu GC-14A; Analytical
instrument division, Kyoto, Japón), equipado con un detector de ionización en llama
(260ºC) y un integrador Shimazu (CR-5A). Los FAMESs fueron separados en columna
capilar de sílice fundida con supelco-10 como fase estacionaria (Longitud: 28 m; 0.32
mm x 0.25 diámetro interno; Supelco, Bellefonte, USA), actuando el helio como gas
portador bajo las presiones de los gases siguientes: He 1 kg cm-2, H2 0.5 kg cm-2, N2 1
kg cm-2, aire 0.5 kg cm-2. Las condicioenes fueron las siguientes: temperatura del
inyector 180ºC durante 10 min, aumentando a 215ºC a una tasa de incremento de 2.5ºC
min -1, para luego mantenerse a una temperatura final de 215ºC durante 15 min. La
identificación de los ácido grasos se llevó a cabo mediante la utilización de EPA 28
(EPA 28, Nippai, Ltd Tokyo, Japón), como aceite estándar.

224
 Spanish summary

3.8. ANÁLISIS ESTADÍSTICO

El análisis estadístico se realizó mediante el programa Statgraphics Plus 5.1

(MANUGISTIES, Rockville, Maryland, USA). Los datos de cada experimento,
composición proximal, crecimiento, supervivencia e indices nutricionales, se
compararon estadísticamente mediante test de la T-Student (Sokal y Rolf, 1995),
cuando había sólo dos tratamientos o con un análisis de varianza (ANOVA) si era
mayor el número de tratamientos ensayado. Como criterio general se tomó el 5% como
nivel de significación. Una vez detectadas diferencias significativas con la ANOVA, las
diferencias entre medias fueron puestas de manifiesto mediante el test de comparación
multiple de Tuckey. La anormalidad y homogeneidad de varianza fueron evaluados con
Skewness y Kurtosis estandarizados y la prueba de Barlett. Cuando los datos no tenían
distribución normal, se aplicó el test no-paramétrico de Kruskal-Wallis (Zar, 1984).

225
 Spanish summary

9.4. CONCLUSIONES

Estudio I. Idoneidad de tres algas rojas como alimento para la oreja de

mar Haliotis tuberculata coccinea Reeve.

6. La composición nutricional de las macroalgas producidas en sistemas de

biofiltros Hypnea musciformis, Hypnea spinella y Gracilaria cornea, evidenció
la potencialidad de dichas macroalgas para ser usadas como alimento de
juveniles de Haliotis tuberculata coccinea. Las tres algas analizadas mostraron
un alto contenido en proteínas, que estaría relacionado con las altas
condiciones de nitrógeno del sistema integrado (AMTI).

7. H. spinella fue la mejor dieta para el crecimiento de la oreja de mar, debido a

un mayor consumo y eficiencia protéica. La mayor dureza de G. cornea tuvo
un efecto negativo en la ingesta lo que dió lugar al peor crecimiento.

8. De forma general, las tasas de crecimiento de la oreja de mar estuvieron en el

rango de las obtenidas en condiciones comerciales este hecho, unido a la alta
supervivencia registrada, sugiere la idoneidad de las tres algas rojas testadas
para el engorde de H. tuberculata coccinea.

Estudio II. Crecimiento comparativo de juveniles de oreja de mar (Haliotis

tuberculata coccinea Reeve) alimentados con algas enriquecidas vs sin enriquecer:
Efecto sobre el crecimiento y la composición corporal.

9. El sistema de cultivo empleado influyó de forma significativa sobre la

composición proximal de las algas, particularmente en el contenido en
proteínas, que registró un incremento del 100-163% en aquellas cultivadas en
efluentes de cultivos marinos respecto a las cultivadas en agua de mar.

10. El perfil de ácidos grasos de las algas estudiadas fue el característico de las
algas verdes y rojas siendo el ácido palmítico el más abundante, en la
clorofita Ulva rigida, predominaron los ácidos grasos poliinsaturados de C16
y C18 siendo mínimos los C20. Los niveles de DHA fueron muy bajos en todas

226
 Spanish summary

las algas estudiadas, por tanto este ácido graso no parece ser esencial para H.
t. coccinea, ya que todas ellas dieron lugar a un óptimo crecimiento.

6. Las orejas de mar alimentadas con las algas cultivadas en efluentes de cultivos
marinos tuvieron un crecimiento mucho mayor que aquellas alimentadas con
las no enriquecidas, lo que sugiere que el nitrógeno puede ser un factor
limitante para el crecimiento de H. tuberculata coccinea.

7. Los animales alimentados con las dietas mixtas, tanto enriquecidas como sin
enriquecer, tuvieron un crecimiento mucho mayor que los alimentados con
dietas monoalgales, indicando que el abalón satisface sus requerimentos
nutricionales a partir de la ingesta de varias dietas, pudiendo verse limitados
cuando se suministra una unica dieta.

8. Las dietas ingeridas afectaron de forma significativa a la composición

bioquímica de los animales. El contenido en lípidos de los tejidos fue
generalmente mayor que los de las dietas. El nivel de lípidos en el músculo fue
significativamente menor que el de las vísceras, indicando que éstos son
almacenados en el conjunto (apéndice conical) hepatopáncreas/gónada.

9. El valor dietético de los regímenes alimenticios testados pueden dividirse en

tres categorías: El mejor es el de las dietas mixtas, intermedio el de las dietas
monoalgales de U. rigida o H. spinella y el menor el de G. cornea.

10. Los resultados indican de forma clara que la oreja de mar H. tuberculata
coccinea puede crecer de forma eficiente en un sistema de cultivo integrado,
sugiriendo que este tipo sistemas multitróficos algas-abalón, podría formar
parte del desarrollo de la producción industrial de este molusco en las Islas
Canarias.

Estudio III. Primer desarrollo de dietas vegetales y su idoneidad para el

cultivo de Haliotis tuberculata coccinea Reeve.

227
 Spanish summary

11. La alimentación con algas frescas dio lugar a un crecimiento mucho mayor que
el obtenido con cualquiera de los piensos compuestos, indicando el alto valor
nutricional de las algas producidas en el sistema integrado.

12. La inclusion de P. palmata mejoró el crecimiento, índice de condición y

utilización de la proteína de la dieta, mientras que la inclusion de L. digitata
redujo ésta última de forma significativa.

13. Los elevados contenidos, en relación a las dietas, de ARA en los animales
alimentados con las dietas experimentales y de EPA en los alimentados con
algas frescas, denotaron la presencia de las respectivas elongasas Δ4 y Δ5
desaturasas. Sin embargo el bajo contenido de DHA sugirió de nuevo la no
esencialidad de este ácido graso.

14. De forma general, la alimentación de H. tuberculata coccinea con dietas

exclusivamente vegetales dio lugar a una alta supervivencia y una buena
utilización de las proteínas de la dieta.

Estudio IV. Cultivo de la oreja de mar Haliotis tuberculata coccinea Reeve,

alimentadas con algas procedentes de Sistemas Multi-Tróficos Integrados (AMTI),
en una instalación de peces/ abalón en mar abierto: Efecto de la densidad.

15. Al doblar la densidad de cultivo se redujo significativamente el crecimiento

en los dos grupos de tallas de ejemplares de abalón, sugiriendo una mayor
competencia por el espacio y alimento que se refleja tanto en una reducción
de la ingesta como en la eficiencia de utilización del alimento.

16. Para el rango de talla de 30-45 mm, se recomiendan densidades de cultivos

de alrededor de 100 abalones m-2, mientras que para los mayors de 45 mm y
hasta talla commercial, la densidad optima estaría en torno a los 30 abalones
m-2.

17. Tanto el sistema de cultivo en mar abierto como la alimentación con

macroalgas cultivadas en sistemas integrados mostraron su idoneidad para

228
 Spanish summary

obtener altos crecimientos y supervivencias de H. tuberculata coccinea que,

de forma general podría alcanzar la talla commercial de 45-60 mm en tan
sólo 18-22 meses.

229
Development of a Sustainable Grow-out Technology for Abalone
Haliotis tuberculata coccinea (Reeve) as a New Species for Aquaculture
Diversification in the Canary Islands

REFERENCES
 References

10. REFERENCES

Alcántara, L., Noro, T., 2006. Growth of the abalone Haliotis diversicolor (Reeve) fed
with macroalgae in floating net cage and plastic tank. Journal of Shellfish
Research 37, 708-717.
Allen, V.J., Marsden, I.D., Ragg, N.L.C., Gieseg, S., 2006. The effect of tactile
stimulants on feeding, growth, behaviour, and meat quality of cultured blackfoot
abalone, Haliotis iris. Aquaculture 257, 294-308.
Anderson, R.J., Smit, A.J., Levitt, G.J., 1999. Upwelling and fish factory waste as
nitrogen sources for suspended cultivation of Gracilaria gracilis in Saldanha
Bay, South Africa. Hydrobiologia 399, 455- 462.
AOAC, 1995. Official Methods of Analysis of the Association of Analytical Chemistry,
15th ed. Washington, DC, 1018 pp.
AOAC, 2005. Official Methods of Analysis of the Association of Analytical Chemistry.
Washington, DC 1018 pp.
APROMAR, 2012. La Acuicultura Marina en España 2012. www.apromar.es.
Aviles, J. G. G., Shepherd, S.A., 1996. Growth and survival of the blue abalone Haliotis
fulgens in barrel at Cedros Island, Baja California, with a review of abalone
barrel culture. Aquaculture 140, 169-176.
Badillo, L., Segovia, M., Searcy-Bernal, R., 2007. Effect of two stocking densities on
the growth and mortality of the pink abalone Haliotis corrugata in recirculating
and flow-trough systems. Journal of Shellfish Research 26 (3), 801-807.
Bardach, J.E., Ryther, J.H., McLarney, W.O., 1972. Aquaculture - The farming and
husbandry of freshwater and marine organisms. John Wiley and Sons Inc., New
York. 868 pp.
Barkai, R., Griffiths, L.,1986. Diet of the South African abalone Haliotis midae. South
African Journal of Marine Science 4, 37-44.
Barkai R., Griffiths L., 1987. Consumption, absorption efficiency, respiration and
excretion in the South African abalone Haliotis midae. South African Journal of
Marine Science 5, 523-529.
Basuyaux, O., Mathieu, M., 1999. Inorganic nitrogen and its effect on growth of the
abalone Haliotis tuberculata Linnaeus and the sea urchin Paracentrotus lividus
Lamarck. Aquaculture 174, 95-107.

231
 References

Bautista – Teruel, M.N., Millamena, O.M., 1999. Diet development and evaluation for
juvenile abalone, Haliotis asinina: protein/energy levels. Aquaculture 178, 117-
126
Bautista-Teruel, M.N., Fermin, A.C., Koshio, S.S., 2003. Diet development and
evaluation for juvenile abalone, Haliotis asinina: animal and plant protein
sources. Aquaculture 219, 645-653.
Bautista-Teruel, M.N., Koshio, S.S., Ishikawa, M., 2011. Diet development and
evaluation for juvenile abalone, Haliotis asinina Linne: Lipid and essential fatty
acid levels. Aquaculture 312, 172-179.
Ben-Ari, T., Ben-Ezra, D., Odintzov, V., Shauli, L., Shpigel, M., 2012. Lowering
aeration regimes in Ulva cultivation: an energy approach for treating mariculture
effluents. In: Proceedings of European Aquaculture Society (EAS) and the
World Aquaculture Society (WAS) conference (AQUA 2012), Prague, Czech
Republic, 1-5 Sep, 2012, pp. 64.
Benson, J.A., 1986. Polyculture of abalone and algae in the sea. US Department of
Commerce, National Technical Information Service Report (unpublished), 21
pp.
Bester, A. E., Slabbert, R., D’amato, M. E., 2004. Isolation and characterization of
microsatellite markers in South African abalone (Haliotis midae). Molecular
Ecology Notes 4, 618-619.
Beveridge, M., 2004. Cage Aquaculture, 3rd ed. Blackwell Publishing Ltd, Oxford, UK,
368 pp.
Bilbao, A, Viera, M.P., Courtois de Vicose, G., Socorro, J., Fernández-Palacios, H.
2004. Characterization of females broodstocks Haliotis tuberculata coccinea R.
European Aquaculture SocietySpecial publication, 34, 168.
Bilbao, A., Courtois de Viçose, G., Viera, M.P., Sosa, B., Fernández-Palacios, H.,
Hernández, C. 2010a. Efficiency of clove oil as anesthetic for abalone (Haliotis
tuberculata coccinea, Reeve). Journal of Shellfish Research, 29 (3), 679-682.
Bilbao, A., Tuset, V, Viera, M.P., Courtois de Viçose, G., Fernández-Palacios, H.,
Haroun, R., Izquierdo, M., 2010b. Reproduction, fecundity and growth of
abalone (Haliotis tuberculata coccinea, Reeve 1846) in the Canary Islands.
Journal of Shellfish Research, 29 (4), 959-967.

232
 References

Boarder, S.J., Shpigel, M., 2001. Comparative performances of juvenile Haliotis roei
fed on enriched Ulva rigida and various artificial diets. Journal of Shellfish
Research 20(2), 653-657.
Bonnot, P., 1930. Abalones in California. California Fish and Game 16, 15-23.
Booth, M. A., Allan, G. L., Evans, A. J., Gleeson, V. P., 2002. Effects of steam
pelleting or extrusion on digestibility and performance of silver perch Bidyanus
bidyanus. Aquaculture Research 33, 1163-1173.
Bossy, S. F. 1989. Provisional results of ormer (Haliotis tuberculata) ongrowing trials
in cages off Jersey, Channel Islands. European Aquaculture Society Special
Publication 10, 41-2.
Bossy, S. F. 1990. Ongrowing of the ormer Haliotis tuberculata in Jersey, Channel
Islands. European Aquaculture Society Special Publication 12, 172-3.
Bossy, S.F., Culley, M.B., 1976. Abalone fishing in Britain´s Channel Islands. Fish.
New International 15(9), 55-62.
Britz, P. J., 1991. Global status of abalone aquaculture. In: Cook P A (ed) Perlemoen
farming in South Africa. Mariculture Association of Southern Africa, pp 20-26.
Britz, P.J., 1994. The development of an artificial feed for abalone farming. South
African Journal of Science 90, 6-7.
Britz, P.J., 1996a. Effect of dietary protein level on growth performance of South
African abalone, Haliotis midae, fed fishmeal-based semi-purified diets.
Aquaculture 140, 55-61.
Britz, P.J., 1996b. The suitability of selected protein sources for inclusion in formulated
diets for the South African abalone, Haliotis midae. Aquaculture 140, 63-73.
Britz, P.J., 2012. The status of the South African abalone sector. 8th International
Abalone Symposium, May 2012, Hobart, Tasmania. pp 100.
Britz, P.J., Hecht, T., 1997. Effect of dietary protein and energy levels on growth and
body composition of South African abalone, Haliotis midae. Aquaculture 156,
195-210
Britz, P. J., Hecht, T., Mangold, T. S. 1997. Effect of temperature on growth, feed
consumption and nutritional indices of Haliotis midae fed a formulated diet.
Aquaculture 140, 75-85.
Buschmann, A.H., Mora, O.A., Gómez, P., Böttger, M., Buitano, S., Retamales, C.,
Vergara, P.A., Gutierrez, A., 1994. Gracilaria chilensis outdoor tank cultivation

233
 References

in Chile: use of land-based salmon culture effluents. Aquacultural Engineering

13, 283– 300.
Buschmann, A.H., Westermeier, R., Retamales, C.A., 1995. Cultivation of Gracilaria in
the seabottom in southern Chile: a review. Journal of Applied Phycololy 7, 291–
301.
Buschmann, A.H., Troell, M., Kautsky, N., Kautsky, L., 1996. Integrated tank
cultivation of salmonids and Gracilaria chilensis (Gracilariales Rhodophyta).
Hidrobiologia 326/327, 75-82.
Buschmann, A.H., Troell, M., Kaustby, N., 2001. Integrated algal farming: a review.
Can. Biology Marine 42, 93-90.
Buschmann, A.H., Varela, M., Hernández-González, M., Huovinen, P., 2008.
Opportunities and challenges for the development of an integrated seaweed-
based aquaculture activity in Chile: determining the physiological capabilities of
Macrocystis and Gracilaria as biofilters. Journal of Applied Phycology 20 (5),
571-577.
Capinpin, E.C, Corre, K.G., 1996. Growth rate of the Philippine abalone Haliotis
asinina fed an artificial diet and macroalgae. Aquaculture, 144,81-89
Capinpin, E.C.J., Toledo, J.D., Encena, V.C.I., Doi, M., 1999. Density dependent
growth of the tropical abalone Haliotis asinina in cage culture. Aquaculture 171,
227-235.
Carmona, R., Kraemer, G.P., Zertuche, J.A., Chanes, L., Chopin, T., Neefus, C.,
Yarish., C., 2001. Exploring Porphyra species for use as nitrogen scrubbers in
integrated aquaculture. Journal of Phycology 37, 9-10 (suppl.).
Cheng, W., Hsiao, I.S., Chen, J.C., 2004. Effect of ammonia on the immune response of
Taiwan abalone Haliotis diversicolor supertexta and its susceptibility to Vibrio
parahaemolyticus. Fish Shellfish Immunology 17, 193–202.
Chiou, T.K., Lai, M.M., 2002. Comparison of taste components in cooked meats of
small abalone fed different diets. Fisheries Science 68, 388-394.
Cho, C.Y., Slinger, S.J., Bayley, H.S., 1982. Bioenergetics of salmonid fishes: energy
intake, expenditure and productivity. Comparative Biochemistry and Physiology
73B, 25-41.
Chopin, T., Bastarache, S., 2002. Finfish, shellfish and seaweed mariculture in Canada.
Bulletin of the Aquaculture Association of Canada 102, 119-124.

234
 References

Chopin, T., Yarish, C., Wilkes, R., Belyea, E., Lu, S., Mathieson, A., 1999. Developing
Porphyra/salmon integrated aquaculture for bioremediation and diversification
of the aquaculture industry. Journal of Applied Phycology 11, 463– 472.
Chopin, T., Bushmann, A.H., Halling, C., Troell, M., Kautsky, N., Neori, A., Kraemer,
G.P., Zertuche-González, J.A., Yarish, C., Neefus, C., 2001. Integrating
seaweeds into marine aquaculture sytems: a key towards sustainability. Journal
of Phycology 37, 975-986.
Chopin, T., Bakhsh, H.K., Elliot, J., Ang, K.P., Taylor, C., Dickie, M., Ketchum, G.,
Powell, F., Lyons, T., 2012. Integrated Multi-Trophic Aquaculture (IMTA) can
also be developed for the first phase of salmon aquaculture taking place in
landbased, closed-containment, freshwater hatcheries. In: Proceedings of
European Aquaculture Society (EAS) and the World Aquaculture Society
(WAS) conference (AQUA 2012), Prague, Czech Republic, 1-5 Sep, 2012,
pp.213.
Chow, F.Y., Macchiavello, J., Cruz, S.S., Fonck, E., Olivares, J., 2001. Utilization of
Gracilaria chilensis (Rhodophyta: Gracilariaceae) as a biofilter in the depuration
of effluents from tank cultures of fish, oysters, and sea urchins. Journal of the
World Aquaculture Society 32 (2), 215– 220.
Christie, W. W., 1982. Lipids Analysis. Christie, W. W. (Ed.). Pergamon Press, Oxford,
UK.
Chua, T.E., Tech, E., 2002. Introduction and history of cage culture. In: by, P.T.K.,
Woo, D.W., Bruno, L.H.S., Lim (Eds.), Diseases and Disorders of Finfish in
Cage Culture. CABI Publishing, Wallingford, 1-39.
Clarke, S.M., 1988. Abalone mariculture: the significance of algae. Austasia
Aquaculture 3, 9 –11.
Clavier, J., 1992. The ormer (Haliotis tuberculata) fishery of France and the Channel
Islands. In: S.A. Shepherd, M.J. Tegner and S.A. Guzmán del Próó (Editors),
Abalone of the World: Biology, Fisheries and Culture. Proceeding of the First
International Symposium on Abalone, 21-25 November 1989, La Paz México.
Fishing News Books, Osney Mead, Oxford, Uk, pp. 454-457.
Cochard J. C., 1980. Recherche sur les facteurs déterminants la sexualité et la
reproduction chez Haliotis tuberculata L. Thèse 3ème cycle. Brest. Université
de Bretagne Occidentale. pp 163.

235
 References

Cohen, I., Neori, A., 1991. Ulva lactuca biofilters for marine fishponds effluents.
Botánica Marina 34, 475–482.
Colt, J.E., Armostrong, D.A., 1981. Nitrogen toxicity to crustaceans, fish and mollusks.
Bioengineering Symposium for Fish Culture. Publication, vol. 1, 34-47.
Cook, P. A., 1991. The potential for abalone culture in South Africa. In: Cook P A (ed)
Perlemoen farming in South Africa. Mariculture Association of Southern Africa,
pp 27-32.
Cook, P.A., Gordon, H.R., 2010. World abalone supply, markets, and pricing. Journal
of Shellfish Research 29 (3), 569-571.
Coote, T.A., Hone, P.W., van Barneveld R.J., Maguire, G.B., 2000. Optimal protein
level in semi-purified diet for juvenile Haliotis laevigata. Aquaculture Nutrition
6, 213-220.
Corazani, D., Illanes, J.E., 1998. Growth of juvenile abalone, Haliotis discus hannai Ino
1953 and Haliotis rufescens Swainson 1822, fed with different diets. Journal of
Shellfish Research 17 (3), 663– 666.
Corraze, G., 2001. Lipid nutrition. In: Guillaume J, Kaushik S, Bergot P, Metailler R
(eds.), Nutrition and Feeding of Fish and Crustaceans. Springer, Chichester, UK,
pp. 111–130.
Costa-Pierce, B.A., 1996. Environmental impact of nutrients from aquaculture: towards
the evolution of sustainable aquaculture systems. In: Baird, J.D., Beveridge,
M.C.M., Kelly, L.A., Muir, J.F. (Eds.), Aquaculture and Water Resource
Management, Blackwell, UK, pp. 81– 109.
Courtois de Vicose, G., M. P. Viera, A. Bilbao, M. S. Izquierdo, 2007. Embriology and
complete larval development of Haliotis tuberculata coccinea Reeve: an indexed
micro-photografic sequence. Journal of Shellfish Research 26, 1-8.
Courtois de Vicose, G., Porta, A., M. P. Viera, A. Bilbao, Fernández-Palacios, H, M. S.
Izquierdo, 2009. Potential value of Navicula incerta, Proschkinia sp., Nitzschia
sp. and Amphora sp. as feed for Haliotis tuberculata coccinea post-larvae: effect
of inoculums density on algal growth rates. Aquaculture Society Special
Publication 38, 56-59.
Courtois de Vicose, G., Viera, M. P., Bilbao, A., Izquierdo, M.S., 2010. Larval
settlement of Haliotis tuberculata coccinea in response to different inductives
cues and the effect of larval density on settlement, early growth and survival.
Journal of Shellfish Research 29 (3), 587-591.

236
 References

Courtois de Vicose, G., Viera, M.P., Huchette, S., Izquierdo, M.S., 2012a. Improving
nursery performances of Haliotis tuberculata coccinea: Nutritional value of four
species of benthic diatoms and green macroalgae germlings. Aquaculture, 334-
337, 124-131.
Courtois de Vicose, G., Porta, A., Viera, M.P., Fernández-Palacios, H., Izquierdo, M.S.,
2012b. Effects of density on growth rates of four benthic diatoms and variations
in biochemical composition associated to growth phase. Journal of Applied
Phycology 24, 1427-1437.
Crofts, D. R., 1929. Haliotis. Liverpool Marine Biology Committee. Memorial 39, 1-
174.
Crofts, D. R., 1932. Haliotis. In: LMBC (Ed.), Memoirs on Typical British Marine
Plants and Animals. University of Liverpool, Liverpool, pp.174.
Cuevas, E., 2006. Evolución futura del clima canario. El cambio climático en Canarias.
Ciclo de Conferencias organizado por la Academia Canaria de Ciencias. Museo
de la Ciencia y el Cosmos, La Laguna 20 de octubre. http://acanacie.
Webs.ull.es/cuevas.pdf
Culley, M.B., Peck, L.S., 1981. The feeding preferences of the ormer, Haliotis
tuberculata (L.), in: Rheinheimer, G. et al. (Ed.) (1981). Lower Organisms and
their Role in the Food Web: Proceedings of the 15th European Marine Biology
Symposium, Kiel, Damp 2000, Federal Republic of Germany (September 29-
October 3, 1980). Kieler Meeresforschungen. Sonderheft, 5: pp. 570-572.
Cunha, M.E., Quental-Ferreira, H., Leão, A.C., Matias, A., Matias, D., Joaquim, S.,
2012. Integrated Multitrophic Aquaculture (IMTA) in earthen ponds. In:
Proceedings of European Aquaculture Society (EAS) and the World Aquaculture
Society (WAS) conference (AQUA 2012), Prague, Czech Republic, 1-5 Sep,
2012, pp.238.
Dallimore, J., 2010. Developing Marketing Data for Abalone in Europe. SUDEVAB-
Symposium, Brest -October 2010.
Daume, S., Huchette, S., Ryan, S., Day, R.W., 2004. Nursery culture of Haliotis rubra:
the effect of cultured algae and larval density on settlement and juvenile
production. Aquaculture 236, 221–239.
Daume, S., Ryan, S., 2004. Nursery culture of the abalone Haliotis laevigata: larval
settlement and juvenile production using cultured algae or formulated feed.
Journal of Shellfish Research 23,1019–1026.

237
 References

Dawczynski, C., Shubert, R., Jahreis, G., 2007. Amino acids, fatty acids, and dietary
fibre in edible seaweeds products. Food Chemistry 103, 891-899.
Day, R. W., Fleming, A. E., 1992. The determinants and measurement of abalone
growth En: abalone of the world: biology, fisheries and culture. In: S. A.
Shepherd, M. J. Tegner & S. A. Guzmán del Próo, editors. First international
symposium on abalone, La Paz, Mexico, 1989. Oxford: Blackwell Scientific
Publications Ltd. 141–165 pp.
Day, R.W., Cook, P., 1995. Bias towards brown algae in determining diet and food
preferences: the South African abalone Haliotis midae: Marine and Freshwater
Research 46 (3), 623-627.
de Waal, S.W.P., Branch, G.M., Navarro, R., 2003. Interpreting evidence of dispersal
by Haliotis midae juveniles seeded in the wild. Aquaculture 221, 299 – 310
Demetropoulos, C., Langdon, C., 2004. Pacific dulse (Palmaria mollis) as a food and
biofilter in recirculated, land-based abalone culture systems. Aquaculture
Engineering 32, 57-75.
Dlaza, T.S., 2006. Growth of juvenile abalone under aquaculture conditions. Ph Thesis.
Department of Biodiversity & Conservation Biology. University of the Western
Cape, South Africa, 182 pp.
Dlaza, T. S., Maneveldt, G.W., Viljoen, C., 2008. The growth of post-weaning abalone
(Haliotis midae Linnaeus) fed commercially available formutaled feeds
supplemented with fresh wild seaweed. African Journal of Marine Science 30
(1), 199-203.
Douros, W.J., 1987. Stacking behaviour of an intertidal abalone: an adaptive response
or a consequence of space limitation?. Journal of Experimental Marine Biology
and Ecology 108, 1-14.
Dunstan, G.A., Baillie, H.J, Barret, S.M., Volkman, J.K., 1996. Effect of diet on the
lipid composition of wild and cultured abalone. Aquaculture 140, 115-127.
Dunstan, G.A., Brown, M.R., Augerinos, M., Johns, D.R., Knuckey, R., 1998.
ʻFormulated feeds for juvenile abalone: based on natural feeds (diatoms and
crustose coralline algae).ʼ In: Hone, P. (Ed). Proceedings of the 5th Annual
Abalone Aquaculture Workshop, July, 1997, Hobart, Tasmania, Fisheries
Research and Development Corporation, pp 16-21.

238
 References

Durazo-Beltrán, E., Viana, M.T, 2013. Fatty acid profile of cultured green abalone
(Haliotis fulgens) exposed to lipid restriction and long-term starvation. Ciencias
Marinas, 39 (4), 363–370. http://dx.doi.org/10.7773/cm.v39i4.2283.
Durazo-Beltrán, E., D´Abramo, L.R., Toro-Váquez, J.F., Vásquez-Peláez, C., Viana,
M.T., 2003. Effect of triacylglycerols in formulated diets on growth and fatty
acid composition in tissue of green abalone (Haliotis fulgens). Aquaculture 224,
257–270.
Durazo-Beltrán E., Viana, M.T., D´Abramo L.R., Toro-Váquez J.F., 2004. Effect of
starvation and dietary lipid on the lipid and fatty acid composition of muscle
tissue of juvenile green abalone (Haliotis fulgens). Aquaculture 238, 329-341.
Edwards, P., Pullin, R.S.V., Gartner, J.A., 1988. Research and education for the
development of integrated crop –livestock– fish farming systems in the tropics.
ICLARM Studies and Reviews, vol. 16. International Center for Living Aquatic
resources Management, Manila, p. 53.
Edwards, S., Cook, K., 1999. Rapid leaching of minerals from abalone dietary binders
is not determined by ionic mobility and competition between cations. Dry matter
loss is not a good indicator. Proceeding of the 6th Annual Abalone Aquaculture
Workshop. Sydney, pp. 235.
Ebert, E.E., Houk, J.L., 1984. Elements and innovations in the cultivation of red
abalone Haliotis rufescens. Aquaculture, 39, 375-392.
Elliott, N.G., 2000. Genetic improvement programmes in abalone: what is the future?.
Aquaculture Research, 13: 51-59.
Erasmus, J.H., Cook, P.A., Coyne, V.E., 1997. The role of bacteria in the digestion of
seaweed by the abalone Haliotis midae. Aquaculture, 155, 377–386.
Espino, F., Herrera, R. 2002. Seguimiento de poblaciones de especies amenazadas 2002
(Haliotis tuberculata coccinea, Nordsieck, 1975) Gran Canaria. Informe final
presentado por Gesplan y la Consejería de Política Territorial y Medio Ambiente
(Viceconsejería de Medio ambiente de Gran Canaria y Dirección General de
Política Ambiental). Informe no publicado. 52 pp.
Estefanell, J., Roo, J., Guirao, R., Izquierdo, M.S., Socorro, J., 2012. Benthic cages
versus floating cages in Octopus vulgaris: biological performance and
biochemical composition feeding on Boops boops discarded from fish farms.
Aquaculture Engineering 49, 46-52.

239
 References

Evans, F., Langdon, C.J. 2000. Co-culture of dulse Palmaria mollis and red abalone
Haliotis rufescens under limited flow conditions. Aquaculture 185, 137-158.
FAO, 2012. The State of World Fisheries and Aquaculture 2012. FAO Fisheries and
Aquaculture Department. Food and Agricultural Organization of the United
Nations. Rome. pp 230.
Fei, X.G., 2001. Seaweed cultivation in large scale- a possible solution to the problem
of eutrophication by removing nutrients. Proceedings of the second Asian
Pacific Phycological Forum. Hydrobiologia, 167-175 (special volume).
Fei, X.G., 2004. Solving the coastal eutrophication problem by large scale seaweed
cultivation. Hydrobiologia 512, 145– 151.
Fei, X.G., Bao, Y., Lu, S., 2000. Seaweed cultivation—traditional way and its
reformation. Oceanologia et Limnologia Sinica 31, 575–580.
Fei, X.G., Tseng, C.K., Pang, S.J., Lian, S.X., Huang, R.K., Chen, W.Z., 2002.
Transplant of Gracilaria lemaneiformis by raft culture on the sea along fish
cages in southern China. Proc. World Aquaculture Society, April 23–27, 2002.
World Aquaculture Soc, Baton Rouge, LA, USA, p. 219 (636).
Fermin, A., Buen, S.M., 2002. Grow-out culture of tropical abalone, Haliotis asinina
(Linnaeus) in suspended mesh cages with different shelter surface areas.
Aquaculture International 9, 499-508.
Fitzgerald, A., 2008. Abalone Feed Requirements. South West abalone Growers
Association (SWAGA). Report for Seafish, pp. 34.
Flassch, J.P., Aveline, C., 1984. Production de jeunes ormeaux à la station
expérimentale d´Argenton. Centre Natiotal pour l¨Explotation des Oceans,
Rapports Scientifiques et Techniques, 50-68.
Fleming, A.E., 1995. Growth, intake, feed conversion efficiency and chemosensry
preference of the Australian abalone, Haliotis rubra.Aquaculture 132, 297-311.
Fleming, A. E., Hone, P. W., 1996. Abalone aquaculture. Aquaculture 140, 1-4.
Fleming, A.E., Van Barneveld, R.J., Hone, P.W., 1996. The development of artificial
diets for abalone: a review and future directions. Aquaculture 140, 5-53.
Fleming, A.E., Hone, P., Higham J., 1997. The effect of water velocity on consumption
and growth of greenlip abalone in tanks. In: Proceedings of the 4th Annual
Abalone Aquaculture Workshop, Port Fairy, Victoria, Australia, July 1997 (ed.
by P.W. Hone & A. Fleming). Fisheries Research and Development
Corporation, Canberra, Australia. pp. 23–30.

240
 References

Fleurence, J., 1999. Seaweed proteins: biochemical, nutritional aspects and potential
uses. Trends in Food Science and Technology 10, 25–28.
Foale, S., Day, R., 1992. Recognizability of algae ingested by abalone. Australian
Journal of Marine and Freshwater Research 43 (6), 1331-1338.
Folch, J. Lees, M., Sloane-Stanley, G.H., 1957. A simple method for the isolation and
purification of total lipids from animal tissue. Journal of Biological Chemistry
226, 479-509.
Ford, E., Langdon, C.J., 2000. Co-culture of dulse Palmaria mollis and red abalone
Haliotis rufescens under limited flow conditions. Aquaculture 185 (1-2), 137-
158.
Foster, G.G., Hodgson, A.N., 1998. Consumption and apparent dry matter digestibility
of six intertidal macroalgae by 11 (Mollusca: Vetgastropoda: Turbinidae).
Aquaculture 167, 211-227.
Freeman, K.A., 2001. Aquaculture and related biological attributes of abalone species in
Australia- a review. Fisheries Research Report. Department of Fisheries, Perth,
Western Australia. No. 128.
Fry, F.E.J., 1971. The effect of environmental factors on the physiology of fish. In.W.S.
Hoar and D.J. Randall (Editors), Fish Physiology, 9, Academic Press New York,
pp 1-98.
Gao, K., McKinley, K.R., 1994. Use of macroalgae for marine biomass production and
CO2 remediation- a review. Journal of Applied Phycology 6, 45-60.
García, R.M.I. 1999. Utilización de Ulva como biofiltro en la Acuicultura. Estudio
comparativo de la retención de compuestos nitrogenados. Tesis doctoral.
Universidad de las Palmas de Gran Canaria, España. pp 172.
García-Esquivel, Z., Felbeck, H., 2006. Activity of digestive enzymes along the gut of
juvenile red abalone, Haliotis rufescens, fed natural and balanced diets.
Aquaculture 261, 615-625.
García-Esquivel, Z., Montes-Magallón, S., González-Gómez, M.A., 2007. Effect of
temperature and photoperiod on the growth, feed consumption, and biochemical
content of juvenile green abalone, Haliotis fulgens, fed on a balanced diet.
Aquaculture 262, 129-141.
Geiger, D.L., 2000. Distribution and biogeography of Haliotidae (Gastropoda: veti
gastropoda), worldwide. Bolletinno Malacologico. Vol. 35, 57-120.

241
 References

Geiger, D.L., Owen, B., 2012. Abalone: Worldwide Haliotidae. Hackenheim:

Conchbooks. viii + 361 pp.
Gilroy, A., Edwards, S., J., 1998. Optimum temperature for growth of Australian
abalone: preferred temperature and critical thermal maximum for blacklip
abalone, Haliotis rubra (Leach) and greenlip abalone, Haliotis laevigata
(Leach). Aquaculture Research 29, 481 – 485.
GMA, 2014. http://gmaquaculture.com.
Gómez-Montes, L., García-Esquivel, Z., D´Abramo, L.R., Shimada, A., Vásquez-
Peláez, C., Viana, M.T., 2003. Effect of dietary protein: energy ratio on intake,
growth and metabolism of juvenile green abalone Haliotis fulgens. Aquaculture
220, 769-780.
González-Aviles, J.G., Shepherd, S. A., 1996. Growth and survival of the blue abalone
Haliotis fulgens in barrels at Cedros Island, Baja California, with a review of
abalone barrel culture. Aquaculture 140, 169-176.
Gordin, H., Motzkin, F., Huges-Games, W.L., Porter, C. 1981. Seawater mariculture
pond and integrated system. European Mariculture Society Special Publication
6, 1-13.
Gordon, H.R., Cook, P.A., 2001. World supply, market and pricing: Historical, current
and future. Journal of Shellfish Research 20 (2), 567–570.
Green, A.J., Jones, C.L.W., Britz, P.J., 2011. The protein and energy requirements of
farmed South African abalone Haliotis midae L. cultured at optimal and elevated
water temperatures. Aquaculture Research 42 (11), 1653-1663.
Grubert, M.A., Dunstan, G.A., Ritar, A.J., 2004. Lipid and fatty acid composition of
pre- and post spawning blacklip (Haliotis rubra) and greenlip (Haliotis
laevigata) abalone conditioned at two temperatures on a formulated diets.
Aquaculture 242, 297-311.
Guillard, R.R.L., 1975. Culture of phytoplankton for feeding marine invertebrates. In:
Smith W.L. and Chanley M.H. (Eds.). Culture of Marine Invertebrates Animals.
Plenum Press, New York, USA. pp.29-60.
Guzmán del Proó, S. A. 1992. A review of the biology of abalone and its fishery in
México. In: S. A. Shepherd, M. J. Tegner & S. A. Guzmán del Proó, editors.
Abalone of the world: biology, fisheries and culture.Oxford: Fishing News
Books, pp. 341–360.

242
 References

Guzmán, J.M., Viana, M.T., 1998. Growth of abalone Haliotis fulgens fed diets with
and without fishmeal compared to a commercial diet. Aquaculture 165, 321-331.
Haglund, K., Pedersen, M., 1993. Outdoor cultivation of the subtropical marine red alga
Gracilaria tenuistipitata in brackish water in Sweden. Growth, nutrient uptake,
co-cultivation with rainbow trout and epiphyte control. Journal of Applied
Phycology 5, 271– 284.
Hahn, K.O., 1989. Handbook of culture of abalone and other marine gastropods. CRC
Press, Boca Ratón, Florida. 348 pp.
Haldane, C., 2002. Measuring stress in greenlip abalone (Haliotis laevigata) using
biochemical indicators. Honors thesis, Flinders University of South Australia.
Hansen, J.P., Robertson-Andresson, D., Troell, M., 2006. Control of the herbivorous
gastropod Fissurella mutabilis (Sow.) in a land-based integrated abalone–
seaweed cultura. Aquaculture 255, 384–388.
Hargreaves, J.A., Kucuk, S., 2000. Effects of diel un-ionized ammonia fluctuation on
juvenile hybrid striped bass, channel catfish, and blue tilapia. Aquaculture 195
(1-2), 163-181.
Harlin, M.M., Thorne-Miller, B., Thursby, G.B., 1978. Ammonium uptake by
Gracilaria sp (Florideophyceae) and Ulva lactuca (Clorophyceae) in closed
system fish culture. In: Jensen, A., Stein, J.R. (Eds), Proc. IXth Int. Seaweed
Symposium Science Press., Princeton, pp. 285-293
Harris, J.O., Maguire, G.B., Edwards, S., Hindrum, S.M., 1998. Effect of ammonia on
the growth rate and oxygen consumption of juvenile greenlip abalone Haliotis
laevigata Donovan. Aquaculture 160, 259–272.
Hastings, W.H., Meyers, S.P., Butler, D.P., 1971. A commercial process for water-
stable fish feeds. Feedstuffs 43(47), 38.
Hay, M.E., Kappel, Q.E., Fenical, W., 1994. Synergism in plant defenses against
herbivores: interactions of chemistry, calcification and plant quality. Ecology,
75, 1714– 1726.
Hayashi, I., 1982. Small scale laboratory culture of the ormer, Haliotis tuberculata.
Journal of the Marine Biological Association of the United Kingdom 62, 835 44.
Heath, P., Moss, G., 2009. Farming the Abalone. Aquaculture, 290 (1-2), 80-86.
Hensey, J., 1991. Abalone. Aquaculture Ireland 49, 28.
Hensey, J., 1993. Abalones- the Irish ongrowing experience. In M.S. van Patten (ed.),
Irish-American technical exchange on the aquaculture of abalone, sea urchins,

243
 References

lobsters and kelp. Abstracts of an international workshop. Connecticut Sea Grant

College Program, publication no. CT-SG-93-05. pp. 5-6.
Hernández, J., Uriarte, I., Viana, M.T., Westermeier, R., Farías, A., 2009. Growth
performance of weaning red abalone (Haliotis rufescens) fed with Macrocystis
pyrifera plantlets and Porphyra columbina compared with a formulated diet.
Aquaculture Research 40, 1694–1702.
Hernández, J., Matus de la Parra, A., Lastra, M., Viana, M.T., 2013. Effect of lipid
composition of diets and environmental temperature on the performance and
fatty acid composition of juvenile European abalone (Haliotis tuberculata L.
1758). Aquaculture 412-413, 34-40.
Hernández-Carmona, G., Carrillo-Domínguez, S., 2009. Monthly variation in the
chemical composition of Eisenia erborea J.E. Areschoug. Journal of Applied
Phycololy 21, 607-616.
Higham, J., Hone, P., Clarke, S., Baudinette, R., Geddes, M., 1998. ʻThe effect of flow
on growth in juvenile Greenlip abalone, Haliotis laevigata (Donovan).ʼ In:
Hone, P. (Ed). Proceedings of the 5th Annual Abalone Aquaculture Workshop,
July, 1997, Hobart, Tasmania. Fisheries Research and Development
Corporation. Pp. 116-124.
Hindrum, S., Edwards, S., Burke, C., Johns, D., Maguire, G., 1999. Exploring the
dynamic relationship between water quality, stocking density and refuge
provision for greenlip abalone (Haliotis laevigata). In: Burke, C.M., Harris, J.O.,
Hindrum, Edwards, S.J. and Maguire, G.B., (Eds). Enviromental Requirements
of Abalone. Final Report Proj. ect 97/323. Fisheries Research and Development
Corporation, Canberra, Australia. Tasmanian Aquaculture and Fisheries
Institute, Hobart.
Hindrum, S., Burke, C., Edwards, Johns, D., 2001. Effects of combined exposure to
elevated ammonia and low dissolved oxygen levels in greenlip (Haliotis
laviegata Donovan) and blacklip (H. rubra) abalone. 1. Growth and mortality
data from simulated system failure. Journal of Shellfish Research 20, 679-684.
Hjul, P., 1991. Abalone farmer has hatchery in from garden. Fish Farming International
18 (11), 24-25
Hone, P.W., Fleming, A.E., 1998. ʻAbalone.ʼ In: K. Hyde, (Ed), 1998. The new rural
industries – A handbook for farmers and investors. Rural Industries and
Development Corporation, Canberra. Pp. 85-92.

244
 References

Hone, P.W., Madigan, S.M., Fleming, A.E., 1997. Abalone hatchery manual for
Australia. South Australian Research and Development Institute, Adelaide. 33
pp.
Horn, M.N., 1989. Biology of marine herbivorous fishes. Oceanography and Marine
Biology Annual Reviews 27, 167– 272.
Hoshikawa, H., Sakai, Y., Kijima, A., 1998. Growth characteristics of the hybrid
between pinto abalone, Haliotis kamtschatkana Jonas, and ezo abalone, H.
discus hannai Ino, under high and low temperature. Journal of Shellfish
Research 17, 673 – 677.
Howorth, P.C., 1988. The abalone book. Naturegraph Publishers. Happy Camp. CA. 80
pp.
Huchette, S. M. H., Clavier, J., 2004. Status of the ormer (Haliotis tuberculata L.)
industry in Europe. Journal of Shellfish Research 23, 951–955.
Huchette, S.M.H., Koh, C.S., Day, R.W., 2003a. Growth of juvenile blacklip abalone
(Haliotis rubra) in aquaculture tanks: effect of density and ammonia.
Aquaculture 219, 457-470.
Huchette, S.M.H., Koh, C.S., Day, R.W., 2003b. The effect of density on the behaviour
and growth of juvenile blacklip abalone (Haliotis rubra). Aquaculture
International 11, 411-428.
Hutchinson, W., Vandepeer, M., 2004. Water quality: Effects and management on
abalone farms. South Australian Research and Development Institute (SARDI)
Aquatic Sciences, Adelaide. Pp. 65.
Izquierdo, M. S., Watanabe, T., Takeuchi, T., Arakawa, Kitajima, C., 1989.
Requirements of larval red seabream Pagrus major for essential fatty acids.
Nippon Suisan Gakkaishi 55, 859-867.
Izquierdo, M. S., Viera, M.P., Courtois de ViÇose, G., Haroun, R., Fernández-Palacios,
H., 2013. Nutritional value of Ulva lactuca, Gracilaria cornea, Laminaia
digitata and Palmaria palmata for European abalone. 10th International
Phycological Congress. Orlando, USA.
Jackson, D., Williams, K.C., Degnan, B.M., 2001. Suitability of Australian formulated
diets for aquaculture of the tropical abalone Haliotis asinina Linnaeus. Journal
of Shellfish Research 20, 627-636.
Jarayabhand, P., Paphavasit, N., 1996. A review of the culture of tropical abalone with
special reference to Thailand. Aquaculture 140,159-168.

245
 References

Jarayabhand, P., Kruiroongroj, W., Chaisanit, Ch., 2010. Effect of stocking density on
growth performance of Thai abalone, Haliotis asinina, Linnaeus 1758, reared
under a semiclosed recirculating land-based system. Journal of Shellfish
Research 29 (3), 593-597.
Jee, Y.J., Yoo, S.K., Rho, S., Kim, S.H., 1988. The stocking density and growth of
young abalone Haliotis discus hannai Ino cultured in the hanging net cage.
Bulletin of Fisheries Research and Development Agency 42, 59-69.
Jiménez del Río, M., Ramazanov, Z., García-Reina, G., 1994. Optimization of yield and
biofiltering efficiencies of Ulva rigida cultivated with Sparus aurata
wastewaters. Scientia Marina 58(4), 329-335.
Jiménez del Río, M., Ramazanov, Z., García-Reina, G. 1996. (Ulvales, Chlorophyta)
tanks culture as biofilters for dissolved inorganics nitrogen from fishpond
effluents. Hydrobiologia 326/327, 62-66.
Johns, R.B., Reid Nichols, P.D., Perry, G.J., 1979. Fatty acid composition of ten marine
algae from Australian waters. Phytochemistry 18 (5), 799-802.
Johnston, T., Moltschaniswskyj, N., Wells, J., 2005. Development of the radula and
digestive system of juvenile blacklip abalone (Haliotis rubra): Potential factors
responsible for variable weaning success on artificial diets. Aquaculture 250,
341-355.
Joll, L., 1996. ʻAbalone in the wild- life history and habitat in WA.ʼ In: Forster, A. (Ed).
Proceedings of the Abalone Aquaculture Workshop, December, 1995, Albany,
Western Australia. Aquaculture Development Council and Fisheries Department
of Western Australia. pp 11-13.
Kaehler, S., Kennish, R., 1996. Summer and winter comparisons in the nutritional value
of marine macroalgae from Hong Kong. Botanica Marina 39, 11-17.
Kautsky, N., Folke, C., Rönnback, P., Troell, M., Beveridge, M., Primavera, J., 2001.
Aquaculture. Encyclopedia of Biodiversity 1, 185-198.
Kawamura, T., Takami, H., Roberts, R. D., Yamashita, Y., 2001. Radula development
in abalone, Haliotis discus hannai, from larva to adult in relation to feeding
transitions. Fisheries Science 67, 596-605.
Ke, C., You, W., Luo, X., 2012. Current status of abalone aquaculture industry in
China. Proceedings of the 8th International Abalone Symposium, May 2012,
Hobart, Tasmania. pp. 56.

246
 References

Kemp, J.O.G., Britz P.J., 2012. Abalone nutrition from an aquaculture perspective: a
review. Proceedings of the 8th International Abalone Symposium, May 2012,
Hobart, Tasmania. pp 62.
Kinne, O. (Ed.), 1976. Marine Ecology, vol. 3. Wiley, London.1293 pp.
Kikuchi, S. Uki, N., 1974. Technical study on artificial spawning of abalone (genus
Haliotis). 2-Effect of irradiated sea water with U.V. rays on inducing spawning.
Bulletin of Tohoku Regional Fisheries Research Laboratory 33, 79-96.
Kirkendale, L., Robertson-Andersson D.V., Winberg, P.C., 2010. Review on the use
and production of algae and manufacturated diets as feed for sea-based abalone
aquaculture in Victoria. Report by the University of Wollongon, Shoalhaven
Marine and Freshwater Centre, Nowra, for the Department of Primary
Industries, Fisheries Victoria, 198 p.
Koike, Y., 1978. Biological and ecological studies on the propagation of the ormer,
Haliotis tuberculata Linnaeus. I: Larval development and growth of juveniles.
La Mer (Bulletin de la Société Franco Japonaise d'Océanographie) 16 (3), 124-
36.
Koike, Y., Flassch, J., Mazurier, J., 1979. Biological and ecological studies on the
propagation of the ormer, Haliotis tuberculata Linnaeus. II: Influence of food
and density on the growth of juveniles. La Mer (Bulletin de la Société Franco-
Japonaise d'Océanog raphie) 17 (1), 43-52.
Korsoen, J., Dempster, T., Fjelldal, P.G., Oppedal, F., Kristiansen, T.S., 2009. Long-
term culture of Atlantic salmon (Salmo salar L.) in submerged cages during
winter affects behavior, growth and condition. Aquaculturre 296, 373-381.
Kunavongdate, P., Sakares, W., Muangsakorn, S., 1995. Experimental rearing on
abalone, Haliotis asinina Linneus with three species of seaweed. Technical
Paper, 39. Department of Fisheries, Ministry of Agriculture and Cooperatives,
Thailand.
Lamare, M.D., Wing, S.R., 2001. Caloric content of New Zealand marine macrophytes.
New Zealand Journal of Marine Freshwater Research 35, 341-355.
Langton, R.W., Haines, K.C., Lyon, R.E., 1977. Ammonia nitrogen produced by the
bivalve mollusc Tapes japonica and its recovery by the red seaweed Hypnea
musciformis in a tropical mariculture system. Helgolander Wissenschaftjiche
Meeresuntersuchungen 30, 217-229.

247
 References

LaTouche, B., Moylan, K., 1984. Abalone farming in Ireland. Aquaculture Ireland 16,
12-14.
LaTouche, B., Moylan, K. 1986. Abalone. Aquaculture Ireland 28, 12-13.
LaTouche, B., Moylan, K., Twomey, W., 1993. Abalone on-growing manual.
Aquaculture Explained, 14. Dublin. Irish Sea Fisheries Board (BIM)
Aquaculture Technical Section, Dun Laoghaire, Co. Dublin. 39 pp.
Leaf, R.T., Rogers-Bennett, L., Haaker, P.L., 2007. Spatial, temporal, and size-specific
variation in mortality estimates of red abalone, Haliotis rufescens, from mark-
recapture data in California. Fisheries Research 83, 341-350.
Lee, K.K., Liu, P.C., Chen, Y.C., Huang, C.Y., 2001. The implications of ambient
temperature with the outbreak of vibriosis in cultured small abalone Haliotis
diversicolor supertexta Lischke. Journal of Thermal Biology 26, 585-587.
Legg, T., Lesage, X., Huchette, S., 2012. Technical and economical analysis of sea-
based abalone farming systems in Europe. Proceedings of the 8th International
Abalone Symposium, Hobart, Tasmania. pp 51.
Leighton, D.L., 1974. The influence of temperature on larval and juvenile growth in
three species of southern California abalones. Fishery Bulletin of the United
States 72, 1137-1145.
Leighton, D.L., 1989. Abalone (Genus Haliotis) mariculture on the North American
Pacific Coast. Fisheries Bulletin 87(3), 689-702.
Leighton, D.L., 2000. The Biology and Culture of the California Abalones. Dorrance
Publishing Co., Pittsburgh, PA . 216 pp.
Leighton, P., 2008. Abalone hatchery manual. Aquaculture Technical Section,
Aquaculture Development Division, Bord Iascaigh Mhara 5, 1.
Li, X., Fan, F., Han, L., Lou, Q., 2002. Fatty acids of some algae from the Bohai Sea.
Phytoquemistry 59, 157-161.
Liao, Ch-M., Ling, M-P., Chen, J-Sh., 2003. Appraising zinc bioaccumulation in
abalone Haliotis diversicolor supertexta and alga Gracilaria tenuistipitata var.
liui by probabilistic analysis. Aquaculture 217, 285-299.
Lindsay, R.C., 1988. Flavor chemistry and seafood quality factors. Proc. Ocean´88,
Maine Technology, Baltimore, MD, 31 October to 2 November, pp. 61-65.
Liping, L., Hayse-Gregson, K., Diana, J.S., Weimin, W., Biyu, S., Zongwen, W., 2012.
Impacts of integrated cage culture system on Water Quality in Deep Water
Lakes. Proceedings of the European Aquaculture Society (EAS) and the World

248
 References

Aquaculture Society (WAS) conference (AQUA 2012), Prague, Czech Republic,

1-5 Sep, 2012. Pp. 654.
Liu, K.M., Chen W.K., 1999. Examining the effect of stocking density and depth on
growth of intensive cultured abalone, Haliotis diversicolor supertexta Lischke,
Journal of the Fisheries Society of Taiwan 26, 23-33.
Lloyd, M., Bates, A., 2008. Influence of density-dependent food consumption, foraging
and stacking behaviour on the growth rate of the Northern abalone, Haliotis
kamtschatkana. Aquaculture, 277, 24-29.
Lopez, L.M., Viana, M.T., 1995. Determination of the quality of food elaborated from
unheated and heated fish silage for the juveniles abalone Haliotis fulgens.
Ciencias Marinas 21 (3), 331–342.
Lopez, L.M., Tyler, P.A., Viana, M.T., 1998. The effect of temperature and artificial
diets on growth rates of juvenile Haliotis tuberculata (Linnaeus, 1758). Journal
of Shellfish Research 17, 657 – 662.
Lu, H., Xu, J., Vander Haegen, G., 2008. Supplementing Marine Capture Fisheries in
the East China Sea: Sea Ranching of Prawn Penaeus orientalis, Restocking of
Large Yellow Croaker Pseudosciaena crocea, and Cage Culture. Reviews in
Fisheries Science, Volume 16 (1-3), 366-376.
Lyon, R.G., 1995. Aspects of the physiology of the South African abalone, Haliotis
midae L., and implications for intensive abalone culture. M.Sc. Thesis, Rhodes
University, Grahamstown, South Africa, 85 pp.
Mabeau, S., Fleurence, J., 1993. Seaweed in food products: biochemical and nutritional
aspects. Trends in Food Science and Technology, 4, 103–107.
Maguire, G.B., Johns, D., Hindrum, S., Cropp, M., 1996. Effects of shading and refuges
on the growth of juvenile Greenlip abalone Haliotis laevigata. In: Hone, P. (Ed).
Proceedings of the 3rd Annual Abalone Aquaculture Workshop. Port Lincoln,
South Australian. Fisheries Research and Development Corporation. pp 40-45.
Mai, K., Mercer, J.P., Donlon, J., 1995a. Comparative studies on the nutrition of two
species of abalone, Haliotis tuberculata L. and Haliotis discus hannai Ino: III.
Response of abalone to various levels of dietary lipid. Aquaculture, 134, 65–80.
Mai, K., Mercer, J.P., Donlon J., 1995b. Comparative studies on the nutrition of two
species of abalone, Haliotis tuberculata L. and Haliotis discus hannai Ino. IV.
Optimum dietary protein level for growth. Aquaculture 136, 165-180.

249
 References

Mai, K., Mercer, J.P., Donlon J., 1996. Comparative studies on the nutrition of two
species of abalone, Haliotis tuberculata L. and Haliotis discus hannai Ino. V.
The role of polyunsaturated fatty acids of macroalgae in abalone nutrition.
Aquaculture 139, 77-89
Malham, S. K., Lacoste, A., Gelebart, F., Cueff, A., Poulet, S. A., 2003. Evidence for a
direct link between stress and immunity in the mollusk Haliotis tuberculata.
Journal of Experimental Zoology 295A, 136- 144.
Mao, Y.Z., Yang, H.S., Zhoy, Y., Ye, N.H., Fang, J.G., 2009. Potential of the seaweed
Gracilaria lemaneiformis for integrated multi-trophic aquaculture with scallop
Chlamys farreri in North China. Journal of Applied Phycology 21(69), 649-656.
Marchetti, M., Tossani, N., Marchetti, S., Bruce G., 1999. Leaching of crystalline and
coated vitamins in pelleted and extruded feeds. Aquaculture 171, 83-92.
Marinho-Soriano, E., Morales, C., Moreira, W.C., 2002. Cultivation of Gracilaria
(Rhodophyta) in shrimp pond effluents in Brazil. Aquaculture Research 33,
1081– 1086.
Marquardt, R., Schubert, H., Varela, D.A., Huovinen, P., Henríquez L., Buschmann,
A.H., 2010. Light acclimation strategies of three commercially important red
algal species. Aquaculture 299 (1-4), 140-148.
Marsden, I.D., Williams, P.M.J., 1996. Factors affecting the grazing rate of the New
Zeland abalone Haliotis iris Martyn. Journal of Shellfish Research 15, 401-406.
Marshall, P.A., Keogh, M.J. 1994. Asymetry in intraspecific competition in the limpet
Cellana tramoserica (Sowerby). Journal of Experimental Marine Biology and
Ecology 177, 121-138.
Martínez-Aragón, J.F., Hernández, I., Pérez Llorens, J.L., Vázquez, R., Vergara, J.J.,
2002. Biofiltering efficiency in removal of dissolved nutrients by three species
of estuarine macroalgae cultivated with seabass (Dicentrarchus labrax) waste
waters:1. Phosphate. Journal of Applied Phycology 14, 365-374.
Matos, J., Costa, S., Rogríguez A., Pereira, R., Sousa Pinto, I., 2001. Experimental
integrated aquaculture of fish and red seaweeds in Northern Portugal.
Aquaculture 252, 31–42.
Mcbride S.C., Rotem, E., Ben-Ezra, D., Shpigel, M., 2001. Seasonal energetics of
Haliotis fulgens (Philippi) and Haliotis tuberculata (L.). Journal of Shellfish
Research 20, 659-665.

250
 References

McCormick, T.B., Arguirre, A., Mill, T.S., Herbison, K.T., 1992. Growth and survival
of red (Haliotis rufescens), green (Haliotis fulgens) and pink (Haliotis
corrugata) abalone in land-based cultivation systems. Fisheries Research Paper,
Department of Fisheries (South Australia) 24, 49-60.
McDonald, M.E., 1987. Biological removal of nutrients from wastewater: an algal-fish
system model. In: Reddy, K.R., Smith, W.H. (Eds.), Aquatic Plants for Waste
Water Resource Recovery, Magnolia Publishing, Orlando, Florida, USA, pp.
959– 968.
McShane, P.E., 1988. ʻWorld developments in abalone culture.ʼ In: L. Evans and D.
OʼSullivan (Eds). In: Proceedings 1st Australian Shellfish Aquaculture
Conference, Perth, Western Australia. (Editors L.H. Evans and D. O'Sullivan),
pp. 212-223.
Mercer, J P., 1981. Low cost spat production units. In G. Gardner (ed.) Proceedings of
the Twelfth Annual Shellfish Conference, London. Shellfish Association of
Great Britain. Pp. 69-78.
Mercer, J.P., Mai, K.S., Donlon, J., 1993. Comparative studies on the nutrition of two
species of abalone, Haliotis tuberculata coccinea Linneaeus and Haliotis discus
hannai Ino. I. Effects of algal diets on growth and biochemical composition.
Invertebrate Reproduction and Development 23, 75-88.
Mgaya, Y. D., 1995. Synopsis of biological data on the European abalone (Ormer)
Haliotis tuberculata Linnaeus, 1758 (Gastropoda, Haliotidae) FAO-FISH-
SYNOP. 1995 no. 156, 28 pp.
Mgaya, Y.D., Mercer, J.P., 1994. A Review of the Biology, Ecology, Fisheries and
Mariculture of the European Abalone Haliotis tuberculata Linnaeus 1758
(Gastropoda: Haliotidae). Biology and Environment: Proceedings of the Royal
Irish Academy, Vol. 94B, No. 3, 285-304.
Mgaya, Y.D., Mercer, J.P, 1995. The effect of size grading and stocking densities on
growth performance of juvenile abalone, Haliotis tuberculata Linnaeus.
Aquaculture 136, 297-312.
Midlen, A., Redding, T.A., 1998. Environmental Management for Aquaculture,
Aquaculture Series 2. Kluwer Academic Publishers. Dordrecht, Boston, London.
Pp 215.

251
 References

Minh, N.D., Petpiroon S., Jarayabhand P., Meksumpun S., Tunkijjanukij S., 2010.
Growth and survival of abalone, Haliotis asinina Linnaeus 1758, reared in
suspended plastic cages. Kasetsart Journal: Natural Science 44, 621–630.
Montano-Vargas, J., Viana, M.T., D’Abramo, L.R., Armando, S., Vásquez-Peláez, C.,
2005. Growth and energy utilization of juvenile pink abalone Haliotis corrugata
fed diets containing different levels of protein and two starch: lipid ratios.
Journal of Shellfish Research 24, 1179-1187.
Morikawa, Y., Norman, C.P., 2003. Perception of light intensity by Haliotis discus
discus based on locomotor activity patterns. Fisheries Science 69, 478-486.
Moriyama, S., Kawauchi, H., 2004. Somatic growth acceleration of juvenile abalone,
Haliotis discus hannai, by immersion in and intramuscular injection of
recombinant salmon growth hormone. Aquaculture 229, 469-478.
Morrison, J., Smith, B., 2000. ʻTank design changes direction.ʼ In: Fleming, A. (Ed).
AB-brief, 4(1), 5-5.
Morse, D. E., Duncan, H., Hooker, N., Morse, A., 1977. Hydrogen peroxide induces
spawning in mollusks, with activation of prostaglandin endoperoxide synthetase,
Science, 196, 298-300.
Mortensen, A., Toften, H., Aas K., 2007. Cage culture of spotted wolffish (Anarhichas
minor Olafsen). Aquaculture 264, 475–478.
Msuya, F.E., Kyewalyanga, M.S., Salum, D., 2006. The performance of the seaweed
Ulva reticulata as a biofilter in a low-tech, low-cost, gravity generated water
flow regime in Zanzibar, Tanzania. Aquaculture 254, 284–292.
Muir, J.F., 1996. A systems approach to aquaculture and environmental management.
In: Baird, J.D., Beveridge, M.C.M., Kelly, L.A., Muir, J.F. (Eds.), Aquaculture
and Water Resource Management. Blackwell Science, UK, pp. 19– 47.
Mulvaney, W., Jahangard, S., Krsinich, A., Ingram, B., Winberg, P., 2012. Growth of
hybrid abalone (H. rubra Leach x H. laevigata Leach) in offshore cages.
Proceedings of AQUA 2012, Prague, Czech Republic.pp
Nagler, P.L., Glenn E.P., Nelson S.G., Napoleon, S., 2003. Effects of fertilization
treatment and stocking density on the growth and production of the economic
seaweed Gracilaria parvipora (Rhodophyta) in cage culture at Molokai, Hawaii.
Aquaculture 219, 379-391.

252
 References

Naidoo, K., Maneveldt, G., Ruck, K., Bolton, J., 2006. A comparison of various
seaweed-based diets and formulated feed growth rate of abalone in a land-based
aquaculture system. Journal of Applied Phycology 18, 437-443.
Najmudeen, T. M., Víctor, A.C.C., 2004. Reproductive biology of the tropical abalone
Haliotis varia from Gulf of Mannar. Journal of Marine Biological Association of
India, 46 (2), 154 – 161.
Naylor, R., Goldberg, R., Primavera, J., Kautsky, N., Beveridge, M., Clay, J., Folke, C.,
Lubchenco, J., Mooney, J., Troell, M., 2000. Effect of aquaculture on world fish
supplies. Nature 405, 1017–1024.
Naylor, M.A., Kaiser, H., Jones, C.L.W, 2011. Water quality in a serial-use raceway
and its effect on the growth of South African abalone, Haliotis midae Linnaeus,
1758. Aquaculture Research 42, 918-930.
Naylor, M.A., Kaiser, H., Jones, C.L.W., 2012. The effect of free ammonia nitrogen, pH
and supplementation with oxygen on the growth of South African abalone,
Haliotis midae L. in an abalone serial-use raceway with three passes.
Aquaculture Research 45, 213–224.
Nelson, M.M., Leighton, D.L., Phleger, C.F., Nichols P.D., 2002. Comparison of
growth and lipid composition in the green abalone, Haliotis fulgens, provided
specific macroalgal diets. Comparative Biochemistry and Physiology Part B 131,
695-712.
Nelson, S.G., Glenn, E.P., Conn, J., Moore, D., Walsh, T., Akutagawa, M., 2001.
Cultivation of Gracilaria parvispora (Rhodophyta) in shrimp-farm effluent
ditches and floating cages in Hawaii: a two-phase polyculture system.
Aquaculture 193, 239– 248.
Neori, A., 1996. The type of N-supply (ammonia or nitrate) determines the performance
of seaweed biofilters integrated with intensive fish culture. The Israeli Journal of
Aquaculture-Bamidgeh, 48, 19– 27.
Neori, A., Cohen, I., Gordin, H., 1991. Ulva lactuca biofilters for marine fish-pond
effluents: II. Growth rate, yield and C:N ratio. Botanica Marina 34, 483– 489.
Neori, A., Krom, M.D., Ellner, S.P., Boyd, C.E., Popper, D., Rabinovitch, R., Davison,
P.J., Dvir, O., Zuber, D., Ucko, M., Angel, D., Gordin, H., 1996. Seaweed
biofilters as regulators of water quality in integrated fishseaweed culture units.
Aquaculture 141, 183–199.

253
 References

Neori, A., Ragg, N.L.C., Shpigel, M., 1998. The integrated culture of seaweed, abalone,
fish and clams in modular intensive land-based systems: II. Performance and
nitrogen partitioning within an abalone (Haliotis tuberculata) and macroalgae
culture system. Aquaculture Engineering 17(4), 215-239.
Neori, A., Shpigel, M., Ben-Ezra, D., 2000. A sustainable integrated system for culture
of fish, seaweed and abalone. Aquaculture 186, 279-291.
Neori, A., Msuya, F.E., Shauli, L., Schuenhoff, A., Kopel, F., Shpigel, M., 2003. A
novel three-stage seaweed (Ulva lactuca) biofilter design for integrated
mariculture. Journal of Applied Phycology 15, 543– 553.
Neori, A., Chopin, T., Troell, M., Buschmann, A.H., Kraemer, G.P., Halling, C.,
Shpigel, M., Yarish, C., 2004. Integrated aquaculture: rationale, evolution and
state of the art emphasizing seaweed biofiltration in modern mariculture.
Aquaculture 231, 361-391.
Nie, Z.Q., Ji, M.F., Yan, J.P., 1996. Preliminary studies on increased survival and
accelerated growth of overwintering juvenile abalone, Halioti discus hannai Ino.
Aquaculture 140, 177-186
Njobeni A., 2006. The cultivation of Gracilaria gracilis (Rhodophyta) in an integrated
culture system for the production for abalone feed and the bioremediation of
aquaculture effluent. MCs Dissertation, Cape Town University, Cape Town. Pp.
222.
Oakes, F., Ponte, R., 1996. The abalone market: opportunities for cultured
abalone. Aquaculture 140, 187–195.
Ogino, C., Kato, N., 1964. Studies on the nutrition of abalone-II. Protein requirements
for growth of abalone, Haliotis discus. Bulletin of the Japanese Society for the
Science of Fiseries 30, 523– 526 (in Japanese with English abstract).
Pang, S.J., Xiao, T., Bao, Y., 2006. Dynamic changes of total bacteria and Vibrio in an
integrated seaweed–abalone culture system. Aquaculture 252, 289– 297.
Park, Ch. J., Kim., S.Y. 2013. Abalone aquaculture in Korea. Journal of Shellfish
Research 32(1), 17-19. Doi: http.//dx.doi.org/10.2983/035.032.0104.
Park, J., Kim, H., Kim, P., Jo, J., 2008. The growth of disk abalone, Haliotis discus
hannai at different culture densities in a pilot-scale recirculating aquaculture
system with a based culture tank. Aquaculture Engineering 38,161-170.
Peck, L.S., 1989. ʻFeeding, growth and temperature in the ormer, Haliotis tuberculata
(L.).ʼ Progress in Underwater Science, 14, 95-107.

254
 References

Peña, J., 1985. Primeras experiencias de inducción a la puesta en Haliotis coccinea

canariensis Nordsieck, 1975 (Gastropoda, Prosobranchia). Informes Técnicos
del Instituto de Investigaciones Pesqueras130, 3-12.
Peña, J., 1986. Preliminary study on the induction of artificial spawning in Haliotis
coccinea canariensis Nordsieck (1975). Aquaculture, 52, 35-41.
Pérez, J., Moreno, E., 1991. Invertebrados Marinos de Canarias. En: Cabildo Insular de
Gran Canaria, editor. Las Palmas de Gran Canaria. 335 pp.
Phang, S.M., Shaharuddin, S., Noraishah, H., Sasekumar, A., 1996. Studies on
Gracilaria changii (Gracilariales Rhodophyta) from Malaysian mangroves.
Hydrobiologia 326/327, 347– 352.
Poore, G.C.B., 1972. Ecology of New Zealand abalones, Haliotis species (Mollusca:
astropoda): I. Feeding. New Zealand Journal of Marine and Freshwater Research
6, 11 –22.
Portugal, T.R., Ladines, E.O., Ardena, S.S., Resurreccio´n, L., Medina, C.R., Matibag,
P.M., 1983. Nutritive value of some Philippine seaweeds: Part II. Proximate,
amino acids and vitamin composition. Philippine Journal of Nutrition, 166– 172.
Preece, M.A., Mladenov, P.V., 1999. Growth and mortality of the New Zealand abalone
Haliotis iris Martyn 1784 cultured in offshore structures and fed artificial diets.
Aquaculture Research 30, 865-877.
Qi, Z., Liu, H., Li, B., Mao, Y., Jiang, Zhang, J., Fang, J., 2010. Suitability of two
seaweeds, Gracilaria lemaneiformis and Sargassum pallidum, as feed for the
abalone Haliotis discus hannai Ino. Aquaculture 300, 189–193.
Reddy-Lopata, K., Auerswald, L., Cook, P., 2006. Ammonia toxicity and its effect on
the growth of the South African abalone Haliotis midae Linnaeus. Aquaculture
261, 678-687.
Reyes, O.S., Fermin, A.C., 2003. Terrestrial leaf meals or freshwater aquatic fern as
potential feed ingredients for farmed abalone Haliotis asinina (Linnaeus 1758).
Aquaculture Research 34, 593-599.
Rivero, L.E., Viana, M.T., 1996. Effect of pH, water stability and toughness of artificial
diets on the palatability for juvenile abalone Haliotis fulgens. Aquaculture 144,
353–362.
Roberts, R., 2001. A review of settlement cues for larval abalone (Haliotis spp.).
Journal of Shellfish Research, 20, 571-586.

255
 References

Roberts, R. D., Kaspar, H. F., Barker. R. J., 2004. Settlement of abalone (Haliotis iris)
larvae in response to five species of coralline algae. Journal of Shellfish
Research, 23, 975–987.
Robertson-Andersson, D.V., 2003. The cultivation of Ulva lactuca (Chlorophyta) in an
integrated culture system for the production for abalone feed and the
bioremediation of aquaculture effluent. MCs Dissertation, Cape Town
University, Cape Town.
Robertson-Andersson D.V., Leitao, D., Bolton, J.J., Anderson, R.J., Njobeni, A., Ruck,
K., 2006. Can kelp extract (Kelpac) be useful in seaweed mariculture?. Journal
of Applied Phycology 18, 315-321.
Robertson-Andersson, D.V., Maneveldt, G.W., K. Naidoo, 2011. Effects of wild and
farm-grown macroalgae on the growth of juvenile South African abalone
Haliotis midae Linnaeus. African Journal of Aquatic Science, 36 (3), 331-337.
Rosen, G., Langdon, Ch. J., Evans, F., 2000. The nutritional value of Palmaria mollis
cultured under different light intensities and water exchange rates for juvenile
red abalone Haliotis rufescens. Aquaculture 185, 121–136.
Roussel, S., Huchette, S., Clavier, J., Chauvaud, L., 2011. Growth of the European
abalone (Haliotis tuberculata L.) in situ: seasonality and aging using stable
oxygen isotopes. Journal of Sea Research 65 (2), 213-218.
Russo, R.C., Thurston, R.V., 1991. Toxicity of ammonia, nitrite, and nitrate to fishes.
In: Brune, D.E., Tomasso, J.R. (Eds.), Aquaculture and Water Quality. The
World Aquaculture Society, Baton Rouge, LA, USA, pp. 58-89.
Ryther, J.H., Goldman, J.C., Gifford, J.E., Huguenin, J.E., Wing, A.S., Clarner, J.P.,
Williams, L.D., Lapointe, B.E., 1975. Physical models of integrated waste
recycling—marine polyculture systems. Aquaculture 5, 163–177.
Saito, K., 1981. The appearance and growth of 0-year-old Ezo abalone. Japanese
Society for the Science of Fiseries 47, 1393-1400.
Sakai, K., Hirata, H., 2000. Integrated culture of Ulva and abalone. In: Notoya, X. (Ed.),
Utilization of Ulvacea for Mitigation Fish Farms Seizando, Tokio.
Sakata, K., Itoh, T., Ina, K., 1984. A new bioassay method for phagostimulants for a
young abalone, Haliotis discus Reeve. Agricultural and Biological Chemistry 48
(2), 425-429.

256
 References

Sales, J., Britz, P.J., 2001. Research on abalone (Haliotis midae L.) cultivation in South
Africa. Aquaculture Research 32 (11), 863–874.
Sales, J., Britz, P.J., 2002. Influence of ingredient particle size and inclusion level of
pre-gelatinised maize starch on apparent digestibility coefficients of diets in
South African abalone (Haliotis midae L.). Aquaculture 212, 299-309.
Sales, J., Janssens, J. J., 2004. Use of feed ingredients in artificial diets for abalone: a
brief update. Nutrition Abstracts and Reviews Series B74: 13N-21N.
Sales, J., Truter, P.J., Britz P.J., 2003. Optimum dietary crude protein level for growth
in South African abalone (Haliotis midae L.). Aquaculture Nutrition 9, 85-89.
Schiel, D.R.T., 1992. The paua (abalone) fishery of New Zealand. In: S.A. Shepherd,
M.J. Tegner and S.A. Guzmán del Proó, editors. Abalone of the world: Biology,
fisheries and culture. Oxford, UK: Blackwell Scientific. Pp. 427-437.
Schuenhoff, A., Shpigel, M., Lupatsch, I., Ashkenazi, A., Msuya, F.E., Neori, A., 2003.
A semi-recirculating, integrated system for the culture of fish and seaweed.
Aquaculture 221, 167-181.
Searcy-Bernal, R., Gorrostieta-Hurtado, E., 2007. Effect of darkness and water flow
rate on survival, grazing and growth rate of abalone Haliotis rufescens
postlarvae. Journal of Shellfish Research 26 (3), 789-794.
Serviere-Zaragoza, E., Mazariegos-Villareal, A., Ponce-Dıaz, G., Montes-Magallon, S.,
2001. Growth of juvenile abalone, ´Haliotis fulgens Philippi, fed different diets.
Journal of Shellfish Research 20, 689–693.
Shepherd, S.A., 1973. Studies on southern Australian abalone (Genus Haliotis): I.
Ecology of five sympatric species. Australian Journal of Marine and Freshwater
Research 24, 217– 257.
Shepherd, S.A., 1975. Distribution habit and feeding habits of abalone. Australian
Fisheries 34, 12–15.
Shepherd, S.A., Cannon, J., 1988. Studies on southern Australian abalone (Genus
Haliotis) X. Food and feeding of juveniles. Journal of the Malacalogical Society
of Australia 9, 21-26.
Shepherd, S.A, Steinberg, P.D., 1992. Food preference of three Australian abalone
species with the review of the algal food of abalone. In: Shepherd, S.A., Tegner,
M.J., Guzmán del Próo, S.A. (Eds.), Abalone of the world. Biology, Fisheries
and Culture. Blackwell Scientific Publications, Oxford, pp. 169-181.

257
 References

Shipton, T.A., Britz, P.J., 2001. The partial and total replacement of fishmeal with
selected plant sources in diets for the South African abalone Haliotis midae L.
Journal of Shellfish Research 20 (2), 637-645.
Shpigel, M., Neori, A., 1996. Abalone and seaweeds intensive cultivation in integrated
land-based mariculture system: I. Proposed design and cost analysis.
Aquaculture Engineering 15, 313-326.
Shpigel, M., Neori, A., Popper, D.M., Gordin, H., 1993. A proposed model for
‘‘environmentally clean’’ landbased culture of fish, bivalves and seaweeds.
Aquaculture 117, 115– 128.
Shpigel, M., Marshall, I., Lupatsch, J.P., Mercer, J.P., Neori, A., 1996a. Acclimatation
and propagation of the abalone Haliotis tuberculata in a land-based culture
system in Israel. Journal of World Aquaculture Society 27, 435-442.
Shpigel, M., Neori, A., Marshall, A., 1996b. The suitability of several introduced
species of abalone (Gastropoda: Haliotidae) for land-based culture with pond-
grown seaweed in Israel. Israeli Journal of Aquaculture- Bamidgeh 48, 192-200.
Shpigel, M., Ragg, N.C., Lupatsch, I., Neori, A., 1999. Protein content determines the
nutritional value of the seaweed Ulva lactuca for the abalone Haliotis
tuberculata and H. discus hannai. Journal of Shellfish Research 18, 223– 227.
Simental-Trinidad, J. A., Sánchez-Saavedra, M. D. P., Flores-Acevedo, N., 2004.
Growth and survival of juvenile red abalone (Haliotis rufescens) fed with
macroalgae enriched with a benthic diatom film. Journal of Shellfish Research
23(4), 995-999.
Simpson, B.J.A., Cook, P.A., 1998. Rotation diets: A method of improving growth of
cultured abalone using natural algal diets. Journal of Shellfish Research 17(3),
635-640.
Smith, R.R., 1989. Nutritional energetics. In: Halver, J.E. (Ed.), Fish Nutrition, (2nd
ed.)Academic Press, San Diego, CA, USA, pp. 1– 29.
Sokal, R.R., Rolf, S.J.,1995. Biometry. The Principles And Practice Of Statistics In
Biological Research. 3rd edition. 419 pp. New York, W.H. Freeman and
Company.
Sorgeloos, P., 1999. Challenges and opportunities for aquaculture research and
development in the next century. World Aquaculture Magazine 30, 11– 15.

258
 References

Southgate, P.C., Partridge, G.J., 1998. Developments of Artificial Diets for Marine
finfish larvae: Problems and Aspects. In: De Silva SS (ed) Tropical Mariculture,
Academic Press, California, 487 pp.
Steinarsson, A., Imsland, A.K., 2003. Size dependent variation in optimum growth
temperature of red abalone (Haliotis rufescens). Aquaculture 224, 353–362.
Steneck, R.S., Watling, L., 1982. Feeding cababilities and limitations of herbivorous
mollusc: a functional group aproach. Marine Biololy 68, 299-319.
Stephenson, T. A., 1924. Notes on Haliotis tuberculata. I. Journal of the Marine
Biological Association of the United Kingdom 13, 480-495.
SUDEVAB, 2007. Sustainable Development of European SMEs engaged in Abalone
Aquaculture – SUDEVAB. FP 7-SME-2007-1/BSG-SME.
Sun, Z., Li, N., Song, Z., Zhao, Y., Guan, X., 1993. The condition of triploid induction
in abalone Haliotis discus hannai and its indoor raised experiment. Journal of
Fisheries of China 17(3), 243-248.
Tacon, A.G.J., Halwart, M., 2007. Cage aquaculture: a global overview. In: Halwart M.,
Soto D. & Arthur J.R. (Eds.), Cage Aquaculture-Regional Reviews and Global
Overview, FAO Fisheries Technical Paper. No. 498. Rome. 240 pp.
Tahil, A.S., Juinio-Menes, M.A., 1999. Natural diet, feeding periodicity and functional
response for food density of the abalone, Haliotis asinina L. (Gastropoda).
Aquaculture Research 30, 95-107.
Tait, M., 2012. From the trenches- a story by development of New Zealand Abalone Ltd
in intensive recirculating aquaculture. Proceedings of the 8th International
Abalone Symposium, Hobart, Tasmania. pp 50.
Tan, B., Mai, K., 2001. Effects of dietary vitamin K on survival, growth, and tissue
concentrations of phylloquinone (PK) and menaquinone-4 (MK-4) for juvenile
abalone, Haliotis discus hannai Ino. Journal of Experimental Marine Biology
and Ecology 256, 229-239.
Tanaka, R., Sugimura, I., Sawabe, T., Yoshimizu, M., Ezura, Y., 2003. Gut microflora
of abalone Haliotis discus hannai in culture changes coincident with a change in
diet. Fish Science 69, 951–958.
Tarr, R.J.Q., 1995. Growth and movement of the South African abalone Haliotis midae:
a reassessment. Marine and Freshwater Research 46, 583-590.
Taylor, B., 1992. Abalone nutrition: optimum protein levels in artificial diets for
Haliotis kamtschatkana. Journal of Shellfish Research 11, 556.

259
 References

Tenore, K., 1976. Food chain dynamiolcs of abalone in a polyculture system.

Aquaculture 8, 23-27
Thongrod, S., Tamtin, M., Boonyaratpalin, M., 2003. Lipid to carbohydrate ratio in
donkey´s ear abalone (Haliotis asinina, Linne) diets. Aquaculture 225, 165-174.
Tissot, B. N., 1992. Water movement and the ecology and evolution of the Haliotidae.
In: Shepherd S A, Tegner M J and del Proo G (ed’s.), Abalone of the World –
Biology, fisheries and culture. Fishing News Books. Pp. 34 – 45.
Toledo, P., Haroun, R., Fernández-Palacios, H., Izquierdo, M., Peña, J., 2000. First
culture experiences of Haliotis coccinea canariensis in a biofilter system.
Journal of Shellfish Research 19, 493-541.
Touchette, B.W., Burkholder, J.M., 2000. Overview of the physiological ecology of
carbon metabolism in seagrass. Journal of Experimenatl Marine Biology and
Ecology 250, 169-205.
Tovar, A., Moreno, C., Manuel-vez, M.P., García-Vargas, M., 2000. Environmental
impact of intensive aquaculture in marine waters. Water Research 34, 334-342.
Troell, M., Norberg, J., 1998. Modelling output and retention of suspended solids in an
integrated salmon –mussel culture. Ecological Modelling 110, 65-77.
Troell, M., Robertson-Andersson, D., Anderson, R.J., Bolton, J.J., Maneveldt, G.,
Halling C., Probyn, T., 2006. Abalone farming in South Africa: An overview
with perspective on kelp resources, abalone feed, potential for on-farm seaweed
production and socio-economic importance. Aquaculture 257, 266-281.
Tuschulte, T.C., Connell, J.H., 1988. Feeding behavior and algal food of three species
of abalones (Haliotis) in southern California. Marine Ecology Progress Series,
49, 57-64.
Uki, N., Kikuchi, S., 1984. Regulation of maturation and spawning of an abalone,
Haliotis (Gastropoda) by external enviroment factors. Aquaculture 39, 247-261.
Uki, N., Watanabe, T., 1986. Effect of heat-treatment of dietary protein sources on their
protein quality for abalone. Bulletin of the Japanese Society of Scientific
Fisheries 52(7), 1199-1204.
Uki, N., Watanabe, T., 1992. Review of the nutritional requirements of abalone
(Haliotis spp.) and development of more efficient diets. En: Shepherd, S.A.,
Tegner, M.J. Guzmán-del Proo, S.A. (Eds.), Abalone of the World: Biology,
Fisheries and Culture. Fishing News Books, Oxford, pp. 504-517.

260
 References

Uki, N., Grant, J.F., Kikuchi, S., 1981. Juvenile growth of the abalone, Haliotis discus
hannai, fed certain benthic micro algae related to temperature. Bulletin of
Tohoku Regional Fisheries Research Laboratory 43, 59-64.
Uki, N., Kemuyama, A., Watanabe, T., 1985a. Development of semipurified test diets
for abalone. Bulletin of the Japanese Society of Scientific Fisheries 51(11),
1825-1833.
Uki, N., Kemuyama, A., Watanabe, T., 1985b. Nutritional evaluation of several protein
sources in diets for abalone Haliotis discus hannai. Bulletin of the Japanese
Society of Scientific Fisheries 51(11), 1835-1839.
Uki, N., Kemuyama, A. Watanabe, T., 1986. Optimum protein level in diets for
abalone. Bulletin of the Japanese Society of Scientific Fisheries 52, 1005-1012
(in Japanese, with English abstract).
Upatham, E.S., Sawatpeeran, S., Kruatrachue, M., Chitramvong, Y.P., Singhagraiwan,
T., Jarayabhand, P., 1998. Food utilization by Haliotis asinina L. Journal of
Shellfish Research 17, 771– 776.
Valdés-Urriolagoitia, A. A., 2000. Efecto de tres densidades de cultivo en la
sobrevivencia y crecimiento de juveniles de abulón rojo Haliotis rufescens en un
laboratorio comercial. Tesis de Licenciatura, UABC. Ensenada, México, 52pp
Van Barneveld, R.J., Fleming, A.E., Vandepeer, M.E., Kruk, J.A., Hone, P.W., 1998.
Influence of dietary oil type and oil inclusion level in manufactured feeds on the
digestibility of nutrients by juvenile greenlip abalone (Haliotis laevigata).
Journal of Shellfish Research 42, 1501-1508.
Vandepeer, M.E., 2006. Preventing summer mortality of abalone in aquaculture systems
by understanding interactions between nutrition and water temperature. South
Australian Research and Development Institute (SARDI), Aquatic Sciences
Publication No. RD02/0035-2, 54 pp.
Vandepeer, M.E., Van Barneveld, R.J., 2003. A comparison of the digestive capacity of
blacklip (Haliotis rubra) and greenlip (Haliotis laevigata) abalone. Journal of
Shellfish Research 22 (1), 171-175.
Vandepeer, M. E., Hone, P.W., Van Barneveld, R.J., Havenhand, J.N., 1999. The
utility of apparent digestibility coefficients for predicting comparative diet
growth performance in juvenile greenlip abalone Haliotis laevigata. Journal of
Shellfish Research 18, 235-241.

261
 References

Vandermeulen, H., Gordin, H., 1990. Ammonium uptake using Ulva (Chlorophyta) in
intensive fishpond systems: mass culture and treatment of effluent. Journal of
Applied Phycology 2, 363– 374.
Viana, M.T., 2002. Avances en la nutrición, fisiología digestiva y metabolismo del
abulón. In: Cruz-Suárez, L.E., Ricque-Marie, D., Tapia-Salazar, M., Gaxiola-
Cortés, M., Simoes, N. (Eds.). Avances en Nutrición Acuícola VI. Memorias del
VI Symposium Internacuional de Nutrición Acuícola. 3 al 6 de Septiembre del
2002. Cancún, Quintana Roo, México.
Viana, M.T., López, L.M., Salas, A., 1993. Diet development for juvenile abalone
Haliotis fulgens: Evaluation of two articicial diets and macroalgae. Aquaculture
117, 149-156.
Viana, M.T., Trujano, M., Solana-Sansores, R., 1994. Palatability and attraction
activities in juveniles of abalone H. fulgens: nine ingredients used in artificial
diets. Aquaculture 127, 19-28.
Viana, M.T., López, L.M., García-Esquivel, Z., Méndez, E., 1996. The use of silage
from fish and abalone viscera as an ingredient for abalone feed. Aquaculture
140, 87-98.
Viana, M.T., Jarayabhand, P., Menasveta, P., 2000. Evaluation of an artificial diet for
use in the culture of the tropical abalone Haliotis ovina. Journal of Aquaculture
in the Tropics 15, 71-79.
Viana, M.T., D´Abramo, L.R., González, M.A., García-Suárez, J.V., Shimada, A.,
Vázquez-Peláez, C., 2007. Energy and nutrient utilization of juvenile green
abalone (Haliotis fulgens) during starvation. Aquaculture 264, 323-329.
Viera, M.P., Courtois de Vicose, G., Fernández-Palacios, H., Roo, J., Valencia, A.,
2003. Inducción al desove de la almeja canaria Haliotis tuberculata coccinea
mediante el método del Peróxido de Hidrogeno. In: Actas del IX Congreso
Nacional de Acuicultura. Consejería de Agricultura y Pesca. Junta de
Andalucía, 289.
Viera, M.P., Gómez-Pinchetti J.L., Courtois de Viçose, G., Bilbao, A., Suárez, S.,
Haroun, R.J., Izquierdo, M.S., 2005. Suitability of three red macroalgae as a
feed for the abalone Haliotis tuberculata coccinea Reeve. Aquaculture 248, 75-
82.
Viera, M.P., Bilbao, A., Suárez Alvarez, S., Courtois de Viçose, G, Gómez-Pinchetti
J.L, Hernández-Cruz, C.M., Haroun, R.J., 2006. Yield, biofiltering efficiencies

262
 References

and nutritional composition of Gracilaria cornea J. Agardh and Ulva rigida C.

Agardh cultivated with marine fish waste waters: VI Internacional Abalone
Symposium, Chile.
Viera, M.P., Gómez-Pinchetti J.L., Courtois de Viçose, G., Bilbao, A., Izquierdo, M.S.,
2007. Crecimiento comparativo de juveniles de oreja de mar Haliotis
tuberculata coccinea Reeve alimentados con macroalgas cultivadas en agua de
mar y en efluentes marinos. Antonio Cerviño Eiroa, Alejandro Guerra Díaz y
Carmen Pérez Acosta (EDS). ISBN TOMO I: 978-84-611-9086-7. 1, 627-630.
Viera, M.P., Courtois de Viçose, G., Bilbao, A., Fernández-Palacios, H., Robaina, L.,
Izquierdo, M.S., 2009a. Requerimientos nutricionales y dietas artificiales en el
cultivo de la oreja de mar Haliotis spp.: Revisión. Libro de Actas del XII
Congreso Nacional de Acuicultura, 1:46-47. D. Baez, M. Villaroel y S.
Cardenas. Madrid (ISBN: 978-84-937611-0-3)
Viera, M.P., Fidalgo, P., Haroun, R.J, Courtois de Viçose, G., Bilbao, A., Gómez-
Pinchetti J.L., Izquierdo, M.S., 2009b. Effect of different rearing conditions on
the growth performance of the macroalgae Ulva rigida, Hypnea spinella and
Gracilaria cornea. Proceedings of the 7th International Abalone Symposium,
Pattaya-Thailand, pp. 107.
Viera, M. P., Courtois de Vicose, G., Gómez-Pinchetti, J.L., Bilbao, A., Fernández-
Palacios, H., Izquierdo, M.S., 2011. Comparative performances of juvenile
abalone (Haliotis tuberculata coccinea Reeve) fed enriched vs non-enriched
macroalgae: Effect on growth and body composition. Aquaculture 319, 423-429.
Viera, M. P., Courtois de Vicose, G., Fernández-Palacios, H., Izquierdo, M.S., 2014.
Grow out culture of abalone Haliotis tuberculata coccinea Reeve, fed land-
based IMTA produced macroalgae, in a combined fish/abalone offshore
mariculture system: effect of stocking density. Aquaculture Research, DOI:
10.1111/are.12467
Vivanco-Aranda, M., Gallardo-Escárate, C.J., del Río-Portilla, M.A., 2011. Low-
density culture of red abalone juveniles, Haliotis rufescens Swainson 1822,
recirculating aquaculture system and flow-through system. Aquaculture
Research 42, 161-168.
Walsh, M., Watson, L., 2011. A Market Analysis towards the Further Development of
Seaweed Aquaculture in Ireland. Irsh Sea Fisheries Board. Pp. 52.

263
 References

Wassnig, M., Roberts, R.D., Krsinich, A., Day, R.W., 2010. Effects of water flow rate
on growth rate, mortality and biomass return of abalone in slab tanks.
Aquaculture Research 41, 839-846.
Watson, L., Dring, M., 2011. Business Plan for the Establishment of a Seaweed
Hatchery & Grow-out Farm. Part II. Irish Sea Fisheries Board., unpublished
report. 37pp.
Webber, H.H., 1970. Changes in metabolite composition during the reproductive cycle
of the abalone Haliotis cracheroidii (Gastropoda: Prosobranchiata). Phisiology
2, 213-231.
Wells, F.E., Keesing, J.K., 1989. Reproduction and feeding in the abalone Haliotis roei
Gray Australian Journal of Marine and Freshwater Research 40, 187-197.
Wong, K.H., Cheung, P.C.K., 2000. Nutritional evaluation of some subtropical red and
green seaweeds Part I- proximate composition, amino acid profiles and some
physico-chemical properties. Food Chemistry 71, 475-482
Wu, F., Zhang G., 2010. Preliminary study on over summering of juvenile hybrid
Pacific abalone in east Fujian inner bay. Journal of Marine Science of China 33,
9–14 (in Chinese with English abstract).
Wu, F., Zhang, G., 2013. Suitability of cage culture for Pacific abalone Haliotis discus
hannai Ino production in China. Aquaculture Research 44, 485-494.
WWW (World Wildlife Fund, Inc.), 2010. Abalone Aquaculture Dialogue Standards.
Abalone Aquaculture Dialogue. 34pp.
Wu, F., Liu, X., Zhang, G., Wang, Ch., 2009. Effects of the initial size, stocking density
and sorting on the growth of juvenile Pacific abalone, Haliotis discus hannai
Ino. Aquaculture Research 40, 1103-1110.
Xu, Y.J., Fang, J.G., Wei, W., 2008. Application of Gracilaria chilenoides
(Rhodophyta) for alleviating excess nutrients in aquaculture. Journal of Applied
Phycoloogy 20 (2), 199-203.
Yarish, C., Neefus, C.D., Kraemer, G.P., Chopin, T., Nardi, G., Curtis, J., 2001.
Development of an integrated recirculating aquaculture system for nutrient
bioremediation in urban aquaculture. The Connecticut Sea Grant College grant
R/A-34, NOAA OAR’s National Marine Aquaculture Initiative project number
NA16RG1635.

264
 References

Yearsley, R.D., Jones, C.L.W., Britz, P., 2009. Effect of settled sludge on dissolved
ammonia concentration in tanks used to grow abalone (Haliotis midae L.) fed a
formulated diet. Aquaculture Research 40, 166-171.
Yongjian, X., Fang, J., Tang, Q., Lin, J., Le, G., Liao, L. 2008. Improvement of Water
Quality by the Macroalga, Gracilaria lemaniformis (Rhodopyhta), near
Aquaculture Effluent Outlets, Journal of the World Aquaculture Society 39 (4),
549-554.
Zar, J.H., 1984. Biostatistical Analysis, 2nd ed., Prenting Englewood Cliffs, New Jersey.
Zhou Y., Yang, H., Hu, H., Liu, Y., Mao, Y., Zhou, H., Xu, X., Zhang, F., 2006.
Bioremediation potential of the macroalga Gracilaria lemaneiformis
(Rhodophyta) integrated into fed fish culture in coastal waters of north China.
Aquaculture 252, 264– 276.

265