Story Transcript
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 LAS PALMAS DE GRAN CANARIA 2014
Grupo de Investigación 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
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
B
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 (CBMULPGC). 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 (610 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
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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.
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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).
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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).
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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; RobertsonAnderson, 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
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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 ongrowing units) for the culture of native European and introduced Pacific ezo abalones,
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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 ecosector 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
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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 (seaweedabalone) 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.
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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 GorrostietaHurtado, 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 (BautistaTeruel 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
2 3 10
H. asinina
28
H. roei
29
19
16
4
H. midae
22
H. corrugata
30
27
18 20
12
5
H. discus hannai
13
H. rufescesn
14
H. sorenseni
1
H. laevigata
23 26
11
H. rubra
31
24
17
6
H. fulgens
7
H. diversicolor
25
8
H. iris
32
21
15
9
H. tuberculata
22
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)
Ulva spp
Green macroalgae
Undaria pinnatifida
Phyllospora comosa
Macrocistys pyrifera
Laminaria spp.
Eisenia spp
Egregia menziesii
Ecklonia maxima
Brown macroalgae
Palmaria spp.
Jeannerettia lobata
Gracilariopsis spp.
Gracilaria spp.
Gelidium spp.
Asparagopsis armata
Red macroalgae
Macraolgae
Abalone species
Table 2. Suitable wild and cultured macroalgae as food for different abalone species (Numbers indicate references which are listed below)
Introduction
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; ServiereZaragoza 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
5
5
10
6
6
5
7
4.5
3
6
5
3
1.3
5
6.7
48
33
39
40
48
45
43
4.3
1
9
6
16
10
6
Crude lipid Carbohydrate
44
42
6
Crude Fibre 3
Ash
11
8 5
Moisture GE (Kcal g-1)
4.5
P:e ratio*
47
3
4.7
3 85
4
2.9
4
4.5
4.5 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; GómezMontes 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-FishEfficiency-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
60
32
Soy bean meal
Casein
63
43
57
20-50
5
24-44
9-62
6
10
10
40
7
10
20
17
22-32
7-20
20
7-17
8
15-30
19-37
12-48
10
30
34
30
10
36-39
12
36
12
34
23
5
5
10
20
11
26
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)
Total protein
Whole egg
Egg albumin
Soya oil cake
37
47
Corn meal
Kelp meal
52
Vegetable meal
15
2
10
30
9
24-40
32
44
4
Torula yeast
27-47
41-71
3
24-48
0-50
8-41
2
Diet
Sunflower meal
Cotton seed meal
Spirulina spp.
Silage
Shrimp meal
47
1
Fish meal
Protein source and content
Nutrient
Table 4. Nutritional composition (% dry matter) of artificial diets tested for abalone: Protein sources and inclusion levels
Introduction
27
9-17
9-17
5-48
4-48
1-7
37
5
10
34
16
62
43
17
35
39-50
35
10
55
18
**
19
12
15
29
20
10
30
21
26-44
18
44
35
44
15
10
30
22
34-39
2.6
16-19
29-35
23
27
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)
Total protein
Seaweed 26
46
5
15
3
15
12
20
10
18-35
14
Kelp meal
20
35
10
13
12
7.5
35
7.5
12
Diet
Corn meal
Vegetable meal
Lupin meal
Soy bean protein isolate
Spirulina spp.
Shrimp meal
15
20
Soy bean meal
Casein
10
Fish meal
Protein source and content
Nutrient
Table 4. Continued.
Introduction
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)
docosapentaenoic acid (22:5n3) (Dunstan et al., 1999; Nelson et al., 2002).
29
and
6-8
0 and 3
4
6
1.5
1.5
5
4-5
0-3
6
3-4
2
7
1.5-5
8
0.5
0.5
9
3-5
10
5
1
1
11
1-19
0-16
12
5.3
13
0-10
1-5
1-5
14
30
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)
3
6-7
5 and 8
0-5
3
Total lipid
0.6-12
0-12
2
0.5
1.2-5
1 and 5
1
Diet
Corn oil
Squid oil
Soy bean oil
Cod liver oil
Sunflower oil / fish oil (1:1)
Corn oil / fish oil (1:1)
Soy bean oil / Pollock liver oil (3:2 + 1% Vit E)
Fish oil
Lipid source and content
Nutrient
Table 5. Nutritional composition (% dry matter) of artificial diets tested for abalone: Lipid sources and inclusion levels
Introduction
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; ServiereZaragoza et al., 2001; Naidoo et al., 2006).
32
5
5
18
47
*
5-11
4-18
28-82
42-44
13-23
21-49
9
4
31-48
0,5
5
13-29
10
39-47
7
2-3
12
11
5-6
14-23
12
4-18
11
46-50
9-43
9
6-16
5.85
47
9
5-20
20
8
5
33-47
6
20
26-88
7
4
01-1.3
40-50
6
4
7
0.5-31
16-27
6
Diet
6
1,2
43
13
15
2
6
19
14
33
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)
Ash
Cellulose
Crude fibre content
Total carbohydrate
Chemical binder
Gelatin
Agar
Sodium alginate
Seaweed
3-26
*
Starch (maize / rice)
Rice bran
19
*
Soybean meal / flour 5
15
10
12
5
*
20
4
Corn ( meal / gluten)
3
4
2
*
32-43
1
Wheat (flour / gluten)
Dextrin
Energy / binder source and content
Nutrient
Table 6. Nutritional composition (% dry matter) of artificial diets tested for abalone: Energy / binder sources and content
Introduction
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
Mineral Mixture
Composition
Composition (Total 100 g)
(mg) 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
0.009
MnSO4.4H2O
16.2
200
CuSO4.5H2O
3.1
Vitamin A
5000 U.I.
CoCl2.6H2O
0.1
Vitamin D
100 U.I.
KIO3
0.3
Cellulose
45
B12 Ascorbic acid
34
0.5
0.5
Choline chloride
0.02
0.1
0.3
4.4
4
0.4
9
0.1
0.5
5
2
3
10
5.5
4
1.5
11
0.08
0.2
0.35
0.01
0.1
4.2
2.9
1.3
12
0.08
0.23
0.4
0.11
4.6
3.3
1.3
13
35
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)
15
Bentonite
3
8
0.5
3
5
2
3
7
Diet
Mono calcium phosphate
Dicalcium phosphate
0.09
4
0.11
5.1
3.4
1.7
6
BTH
0.05
7
4
3
5
0.23
2
0.8
6
6
2
4
2
1
3
Sodium benzoate
Vitamin E
Vitamin C
Stay C
Alpha-Tocopherol
6
4
5.5
4
Mineral mix
2
2
Total
1.5
1
Vitamin mix
Vitamins and minerals
Nutrient
Table 8. Nutritional composition (% dry matter) of artificial diets tested for abalone. Ingredient with secondary nutritional contribution
Introduction
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
Diet vs macroalge
Uki et al., 1985a
2124
Artificial diets vs macroalgae
Fresh Macroalgae
Viana et al., 1993
Fleming, 1995
1020
14
T°
Fresh macroalgae
Mercer et al., 1993
Subject
Author
150 350
90
365
30-40
Days
H. rubra
H. fulgens
H. discus hannai
H. tuberculata
H. discus hannai
Species
310 260 101 50
P.comosa E.radiata
FI
U. australis
1.2
FCR
M.angustifolia
WS
540
37
P/E
1260
17
21
28
27
18
13
17
17
21
28
27
E
J. lobata
2
9 1 0 1
4
4
6
3
4
4
4
6
3
18
13
17
C
-28
0.1
1.2
2
-1
4.0
13
11
13
12
14
15
15
14
10
12
6
12
4
0.7
0.1
FCE
4.3
3.5
PER
Consumption and feed utilization efficacy
L.botryoides
44
Casein
13 35
C. crispus Fishmeal
M. pyrifera
12
6 10
L. digitata L. saccharina
Mixed diet
13
A. esculenta
13
13
C. crispus
U. lactuca
12
Mixed diet
17
3
13
U. lactuca
P. palmata
4
17
4
P. palmata
3
6 10
4
13
L. digitata
1
17
A. esculenta L. saccharina
5
L
28
P
Casein, fishmeal E. biciclys
Treatment
Proximate composition and energy content
40-70
13
19
29
T0
25
0.8
0.9
3
W0
-1.7
0.1
0.3
0.7
-0.6
5.4
SGR
16
45
47
61
90
53
95
106
93-117
DGSL (μm d-1)
35
37
WG (%)
Growth and survival Survival (%)
Table 9. Summary of various nutritional studies regarding abalone growth performance during the last three decades of abalone culture development
Introduction
Protein sources; macroalgae
Protein; Energy ratio; Size
Fish meal replacemnt
Protein / Energy levels
Britz and Hetch, 1997
Guzmán and Viana, 1998
BautistaTeruel et al., 1999
Lipid levels
Subject
Britz, 1996b
Mai et al., 1995a
Author
2731
2215
18
19
13
T°
90
179
142
72
124
100
Days
H. asinina
H. fulgens
H. midae small H. midae large
H. midae
H. discus hannai
Artificial diets
H. tuberculata
29 32
Soya oil cake
Fish meal Viscera soybean meal Abfeed Fish meal Shrimp meal Soybean meal Garcilariopsis bailinae 8 6 6 6 6 0.5
36 38 22 28 31 17
6 6 10 6 6
35
48 47 40 33
40
2.2
3.2 3.1 3
90
76
1.4 1.2 1.4 1.2 1
34 44 44 44 39
Starch, fish, cellulose
3.4
10
7
0.9 2.3 1.8 1.5
1.6
2.8
1
Dried E. maxima
5
20
0.8
1
0.8
0.7
FCR
29
86
75 80 80 80 80 80
75 80 80 80 80 80
WS
P. corallorhiza
38
P/E
Torula yeast
3.6 3.6 4 3.6
4 4.2 4.3 4.4 4.6 4.6 3.7 4 4.2 4.3 4.4 4.6 4.6 3. 7
E
19
41
C
1.0
1.8
9.3 9.4 34 35 1.1
2.8
1.3
0.7
0.8
0.6
0.8
0.5
FI
FCE
0.1
2.2 2.3 2.5
3.6 2.3 2.2 2.5
3
2.2
3.3
3.9
3.4
6.5
4.7
PER
Consumption and feed utilization efficacy
Spirulina spp. 5
5
5
5
31
Casein Fishmeal
0.6 3 5 7 9 11.6 4 0.6 3 5 7 9 11.6
25 25 25 25 25 25 18 25 25 25 25 25 25 4
L
P
18
P. palmata
Atificial diets
P. palmata
Treatment
Species
Proximate composition and energy content
16
7
36
10
21
T0
0.7
7.3
0.2
1.8
0.4
0.6
W0
0.06
0.8 0.5 0.7 0.8
1.1
2.2 2.1 0.3 0.5 1
0.6
0.4
0.6
0.8
0.6
0.8
0.6
1
0.6 0.8 0.7 0.7 0.7 0.6 0.9 0.6 0.9 0.9 0.9 0.8 0.7
SGR
135
49 222 244 248
71
103 108 43 58 65
54
29
42
65
41
58
45
DGSL (μm d-1)
134
252 307 347
WG (%)
Growth and survival
85
85 95 85
98
93 95 95 95 97 95 97 87 92 98 90 88 92
Survival (%)
Introduction
Terresterial protein sources
Animal and plant protein sources
Protein / Energy level
Reyes and Fermín, 2003
BautistaTeruel et al., 2003
GómezMontes et al., 2003
Lipid levels
Macroalgae
DurazoBeltrán et al., 2004
Legrand, 2005
carbohydrate
Lipid /
Macroalgae and artificial diet
ServiereZaragoza et al., 2001
Thongrod et al., 2003
Subject
Author
717
20
2730
21
2831
2830
20
T°
183
60
196
60
90
120
106
Days
H. t. small
H. tuberculata large
H. fulgens
H. asinina
H. fulgens
H. asinina
H. asinina
H. fulgens
Species
Kelp; soybean; fish silage & protein P. palmata P. palmata + U. lactuca P. palmata P. p+ U. l.
Soybean, starch, Spirulina spp., fish oil, Gracilaria
Fish, soybean, kelp, modified corn,starch
Eisenia arborea Macrocystis pyrifera Gelidium robustum Phyyospadix torreyi Artificial diet Carica papaya Leucaena leucocephala Moringa oliefera Azolla pinnata G. bailoinae Fish meal Shrimp meal Soybean meal Sprirulina
Treatment
62 74 85 100 108
6.0
1.6 1.4 1.4 1.5 1.6
FCE
42 41 42 41
24 21 24
39 38 43 42 44 42 1.6 1.4 1.4 1.5 1.6 48 43 39 36 31
42
21
3
5 1 3 3 3 3 6 6 6 7 7 1 6 10 15 19 0.1
25 13 28 27 28 27 26 31 35 40 44 38 38 37 36 37 39
5
25
47
4
3 3 3 3 4 4 4 4 4 4.2 4.5 4.7 4.9 5.2
3
3
3
99.4
39
95.9
2 3.9
5.6
4.3
1.3 1.4 1.9 3.2 7.4
5 38 1 1 1 1
4
13
3.7 5.8
3.6
3.5
0.7
0.9
76.4 93 110 138 120
59
36 25 5
30
16
25
33
18
63
3
FI
12
WS 52
41
P/E
7.6
5
E
3.9 4.2 4.1 4.6 3 3 3.1 3.4 2.7
PER
FCR
C
P
L
Consumption and feed utilization efficacy
Proximate composition and Energy content
10
23
36
1112
12
11
39 40
38
37
40
17
T0
0.1
2
6.4
0.9
0.2
0.7
14 15.3
11.4
14.7
14.7
0.4
W0
1.3 1.2
0.6
0.7
1.9 2.1 0.8 0.9 0.7 0.9 1.3 1.5 1.9 2.4 2.5
1.9
0.9
0.65 1.8
0.45
0.32
0.7
0.27
SGR
230
40
29
61 7 92 123 123
76 61
86
47
42 71
25
23
46
19
DGSL (μm d-1)
30
779 615 353 223 97 29
75 84 400 454 326 421
90
28
59
WG (%)
Growth and survival
81 93 90 75 76
90 90 95 95 85 85
95
100
97 80
95
89
93
93
Surv. (%)
Introduction
2025
15
Formulated diet; fresh macroalgae; mixed diet
Hernández et al., 2009
T°
Temperature Photoperiod
Seaweedbased diets; Macroalgae; Enriched macroalgae; commercial feed
Subject
GarcíaEsquivel et al., 2007
Naidoo et al., 2006
Author
90
180
270
Days
H. rufescens
H. fulgens
H. midae
Species
4
96
33
FI
FCE
3
PER
35
T0
7.5
W0
1.8
SGR
DGSL (μm d-1)
M. pyrifera
P. columbina + formulated diet
P. columbina
12
27
44
6.7
34
2.8 0.6
40 45
2.6
3.5
4.5
40
44
78
93
94
25ºC/ 24:00 Formulated diet
1.4 1.6
25ºC/ 12:12
0.9 0.9 4
44
20ºC / 24:00 25ºC/ 00:24
20ºC / 12:12
20ºC / 00:24
Fish, soybean, kelp
Dried kelp stipe Dried Kelp pellets
Dried kelp blade
Abfeed
E. maxima + Abfeed
2.6
1.5
1.8
2.8 1.7
6.6
31
0.06
3.7
2.5
2.3
1.8
87
70
110
50
38
55
72 69
82
109
29
32
34
49
53
55
0.8
FCR
58
45
WS
Rotation
5
P/E
Ecklonia maxima
35
E
60
C
E. maxima + Epiphiyte
L
728
548
1004
471
WG (%)
Growth and survival
66
P
Consumption and feed utilization efficacy
Enriched U. rigida + E. G. gracilis+kelp
Treatment
Proximate composition and energy content
80
93
90
85
Surv. (%)
Introduction
Size
Stocking density
Cage culture.
Recirculated system
Lipid levels, constant P/E ratio.
Long-line seaweedabalone culture
Seaweeds suitability.
Subject
1121
18
1023
T°
180
84
120
Days
H. discus hannai
H. midae large
H. midae small
H. discus hannai
Species
Traditional Multi Tier baskets
Novel Submerged cages
Fishmeal, casein, kelp, starch, fish oil
34 36 38 39 36 34 36 38 39 36
2.8 5.3 8.7 12.5 16.1 2.8 5.3 8.7 12.5 16.1
4.5 4.6 4.8 5 5.1 4.5 4.6 4.8 5 5.1
E
P/E
WS
10
1.8 2
1
FCR
0.3
0.5 0.2
0.4
2.8
1.8
2.5 2.3
2.3
6.5
FCE
4.2
FI
5.8
C
L. j. + S. p.
L
4.4 6
P
L. japonica G. lemaneiformis S. pallidum L. j. + G. l.
Treatment
0.7
1.3 1.9
3.2
PER
Consumption and feed utilization efficacy
48
61 55
48
61 55
65
25
76
T0
32
45 37
32
45 37
50
2.6
62
W0
0.1
0.5 0.2
0.7
SGR
53
64 60
63
37 78 77
67 17
43
20
18 26
21
DGSL (μm d-1) 22
WG (%)
Growth and survival
>87.5 >87.5 >87.5
100 100 100 100 100 99 97 97 96 100 >87.5 >87.5 >87.5
100
100 100
100
Surv. (%) 100
41
(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);
expressed; FCE= Feed conversion efficiency (%); PER = Protein efficiency ratio; DGSL= daily growth rate in shell length, WG=Weight gain; E= gross energy
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
Wu and Zhang, 2013
Green et al., 2011
Qi et al., 2010
Author
Proximate composition and energy content
Introduction
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 (CostaPierce, 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 (MarinhoSoriano 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 coculture 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 farmgrown 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 coculturing 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 vegetalbased 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); diatoms production zone (D); grow out zone (E); feeding trials area (F) and outdoor macroalgae culture systems (G, H).
49
B
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). B
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. 51
B
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.
54
Material and Methods
Tables 10-16. Summary of experimental macroalgae characteristics
Hypnea spinella (C. Agardh) Kützing, 1847
Taxonomic classification
Kingdom Plantae Phylum Rhodophyta Class Florideophyceae Order Gigartinales Family Cystocloniaceae Genus Hypnea
Morphological characteristics
Plant lax, from 3-30 cm long (most less than 15 cm) in tangled, bushy 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 distribution
Widely distributed in temperate (especially warm) seas.
Uses and compounds
Edible species
55
Material and Methods
Hypnea musciformis (Wulfen) J.V. Lamoroux, 1853
Taxnomic classification
Kingdom Plantae Phylum Rhodophyta Class Florideophyceae Order Gigartinales Family Cystocloniaceae Genus Hypnea
Morphological characteristics
Clumps or masses of loosely intertwined, cylindrical branches, 10 - 20 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 distribution
Worldwide: Mediterranean, Philippines, Indian Ocean, Caribbean to
Uses and compounds
Biological, medical and pharmaceutical activity (antihelminthic).
Uruguay.
Source of kappa carrageenan.
56
Material and Methods
Gracilaria cornea J. Agardh, 1852
Taxonomic classification
Kingdom Plantae Phylum Rhodophyta Class Florideophyceae Order Gracilariales Family Gracilariaceae Genus Gracilaria
Morphological characteristics
Thallus erect, solitary or caespitose, cylindrical throughout, 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 distribution
Mainly warm-water specie. South America: Mexico, Brazil,
Uses and compounds
Considerable economic importance as a source of food for both
Venezuela; Africa: Tanzania.
humans and shellfish (abalone), playing a major role in the production of agar and other hydrocolloids.
57
Material and Methods
Palmaria palmata, (L.) Weber & Mohr, 1805
Taxonomic classification
Kingdom Plantae Phylum Rhodophyta Class Florideophyceae Order Palmariales Family Palmariaceae Genus Palmaria
Morphological characteristics
Dulse grows attached by its discoid holdfast to rocks. It has a 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 distribution
In Atlantic Europe from Portugal to the Balitc coasts, coasts of Iceland and the Faroe Islands. Shores of Arctic Russia, Arctic Canada, Atlantic Canada, Alaska, Japan and Korea .
Uses and compounds
Food: additive, ground whole tissue; cosmetics; wellness and folk medicine; Biological, medical and pharmacological activity (antihelminthic). Animal production (feed). Mineral supplement.
58
Material and Methods
Laminaria digitata (Hudson) Lamoroux, 1813
Taxonomic classification
Kingdom Plantae Phylum Heterokontophyta Class Phaeophyceae Order Laminariales Family Laminariaceae Genus Laminaria
Morphological characteristics
Tough, leathery, dark brown seaweed that grows to two or three 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 distribution
North west Atlantic from Greenland south to Cape Cod and in the 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 compounds
Fertiliser; extraction of alginic acid; cosmetic; food industry and human and animal nutrition.
59
Material and Methods
Ulva rigida J. Agardh, 1823
Taxonomic classification
Kingdom Plantae Phylum Chlorophyta Class Ulvophyceae Order Ulvales Family Ulvaceae Genus Ulva Species rigida
Morphological characteristics
Bright green to dark green algae, gold at margins when 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 sizesfemale bigger) or oogamous (female non-motile and egglike; male motile)
Geographical distribution
Worldwide distribution
Uses and compounds
Human and animal nutrition
60
Material and Methods
Ulva lactuca Linneaus, 1753
Taxonomic classification
Kingdom Plantae Phylum Chlorophyta Class Ulvophyceae Order Ulvales Family Ulvaceae Genus Ulva Species lactuca
Morphological characteristics
Thin flat alga growing from a discoid holdfast. The margin is 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 distribution
Worldwide distribution
Uses and compounds
Crop and biofilter of fishpond effluents
61
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 (CBMULPGC).
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 bottomcircular 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. 63
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. 64
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 (16:009:00h). Percent water stability was computed as: % 𝑊𝑊𝑊𝑊𝑊𝑊𝑊𝑊𝑊𝑊 𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠 =
(𝐵𝐵 − 𝐴𝐴) (%𝑑𝑑𝑑𝑑𝑑𝑑 𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚) 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.
66
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.
68
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). 69
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. 70
Material and Methods
Figure. 34. ORTACS installation.
Figure 35. Underwater experimental devices next to fish cages.
71
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:
𝑺𝑺𝑺𝑺𝑺𝑺𝑺𝑺𝑺𝑺 𝒈𝒈𝒈𝒈𝒈𝒈𝒈𝒈𝒈𝒈𝒈𝒈 𝒓𝒓𝒓𝒓𝒓𝒓𝒓𝒓 =
(𝑺𝑺𝑺𝑺𝑺𝑺 − 𝑺𝑺𝑺𝑺𝑺𝑺) 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: SGR (% 𝒅𝒅−𝟏𝟏 ) =
(𝑳𝑳𝑳𝑳𝑾𝑾𝟐𝟐 −𝑳𝑳𝑳𝑳𝑾𝑾𝟏𝟏 ) 𝒕𝒕
𝒙𝒙 𝟏𝟏𝟏𝟏𝟏𝟏
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:
𝑾𝑾𝑾𝑾 (%) =
(𝒘𝒘𝟐𝟐 − 𝒘𝒘𝟏𝟏 ) 𝒙𝒙 𝟏𝟏𝟏𝟏𝟏𝟏 𝒘𝒘𝟏𝟏
72
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).
𝑭𝑭𝑭𝑭𝑭𝑭 =
𝒕𝒕𝒕𝒕𝒕𝒕𝒕𝒕𝒕𝒕 𝒇𝒇𝒇𝒇𝒇𝒇𝒇𝒇 𝒊𝒊𝒊𝒊𝒊𝒊𝒊𝒊𝒊𝒊𝒊𝒊 (𝒈𝒈) 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:
𝑷𝑷𝑷𝑷𝑷𝑷 =
𝒊𝒊𝒊𝒊𝒊𝒊𝒊𝒊𝒊𝒊𝒊𝒊𝒊𝒊𝒊𝒊 𝒊𝒊𝒊𝒊 𝒃𝒃𝒃𝒃𝒃𝒃𝒃𝒃 𝒘𝒘𝒘𝒘𝒘𝒘 𝒘𝒘𝒘𝒘𝒘𝒘𝒘𝒘𝒘𝒘𝒘𝒘(𝒈𝒈) 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). 𝑪𝑪𝑪𝑪 =
𝑺𝑺𝑺𝑺 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 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; GómezMontes 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 88
Study I
Aquaculture 248 (2005) 75-82
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 NonEnriched 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, ecofriendly 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 1117% 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