Improvements in the production technology of red porgy (Pagrus pagrus) larvae and fry: importance of rearing conditions and diet nutritional value on their quality

         Index I Index................................................................................................................................................I Acknowledgements.......................................................................................................................V List of figures...............................................................................................................................XI List of tables.............................................................................................................................XIV Abbreviations.............................................................................................................................XV Summary.................................................................................................................................XVII 1.-INTRODUCTION.......................................................................................................................1 1.1.-Aquaculture in the world...........................................................................................1 1.2.-Aquaculture in Spain.................................................................................................3 1.2.1.-General aspects..............................................................................................3 1.2.2.-Fry production...............................................................................................4 1.2.3.-Aquaculture in the Canary Islands................................................................5 1.3.-Importance of larval rearing.....................................................................................6 1.3.1.-Larval rearing techniques..............................................................................7 1.3.1.1.-Intensive systems............................................................................8 1.3.1.2.-Extensive systems..........................................................................10 1.3.1.3.-Semi-Intensive (Mesocosms) systems............................................12 1.3.1.3.1.-Naturalbloom...............................................................14 1.3.1.3.2.-Green water method......................................................14 1.3.1.3.3.-Pseudo green water........................................................15 1.3.1.3.4.-Clear water.....................................................................15 1.3.1.3.6.-Neo-greenwater..............................................................15 1.4.-Limiting factors in larval rearing............................................................................16 1.4.1.-Culture conditions related parameters ........................................................16 1.4.1.1.-Larval density................................................................................16 1.4.1.2.-Shape and tank volume.................................................................16 1.4.1.3.-Water renewal...............................................................................17 1.4.1.4.-Use of phytoplankton in the rearing tank................................ ....17 1.4.1.5.-Use of antibiotics..........................................................................18 1.4.2.-Physico-chemical parameters......................................................................18 1.4.2.1.-Temperature.................................................................................19 1.4.2.2.-Salinity..........................................................................................19 1.4.2.3.-Nitrogen excretion products.........................................................20 Index II 1.4.2.4.-Light..............................................................................................20 1.4.3.-Larval feeding and nutrition........................................................................21 1.4.3.1.-Prey density..................................................................................21 1.4.3.2.-Feeding sequence and co-feeding.................................................21 1.4.3.3.-Prey nutritional quality................................................................23 1.4.3.4.-Importance of lipids in the larval nutrition..................................24 1.5.-Problems in marine fish larval rearing..................................................................25 1.5.1.-Larval survival.............................................................................................25 1.5.2.-Incidence of morpho-anatomical disorders.................................................26 1.5.2.1.-Pigmentation anomalies...............................................................27 1.5.2.2.-Skeleton anomalies and factors related to their appearance.......27 1.5.2.2.1.-Environmental factors....................................................31 1.5.2.2.2.-Nutritional factors..........................................................31 1.5.2.2.3.-Genetic factors...............................................................33 2.-OBJECTIVES...........................................................................................................................35 3.-MATERIALS AND METHODS............................................................................................ 37 3.1.-Study localization......................................................................................................37 3.2.-Facilities description ................................................................................................37 3.2.1.-General overview.........................................................................................37 3.2.2.-Larval rearing tanks.....................................................................................40 3.2.2.1.-Tanks of 40.000 litresSemi-intensive system..............................40 3.2.2.2.-Tanks of 2.000 litres-Intensive system..........................................41 3.2.2.3.-Tanks of 500 litres-Intensive system.............................................42 3.2.2.4.-Tanks of 10.000 litres-Weaning and nursery................................42 3.3.-Studied species..........................................................................................................44 3.3.1.-Habitat.........................................................................................................44 3.3.2.-Geographical distribution............................................................................45 3.3.3.-Aquaculture perspectives.............................................................................45 3.4.-Experimental conditions..........................................................................................46 3.4.1.-Live food cultures.......................................................................................46 3.4.1.1.-Phytoplankton culture..................................................................46 3.4.1.2.-Rotifers culture.............................................................................50 3.4.1.2.1.-Stock maintenance and rotifers pre-culture...................50 Index III 3.4.1.2.2.-Mass culture..................................................................50 3.4.1.2.3.-Rotifers enrichment.......................................................52 3.4.1.3.-Brine shrimp production..............................................................55 3.4.1.3.1.-Hidratation....................................................................55 3.4.1.3.2.-Decapsulation................................................................56 3.4.1.3.3.-Storage..........................................................................57 3.4.1.3.4.-Hatching........................................................................60 3.4.1.3.5.-Enrichment....................................................................60 3.4.2.-Larval rearing..............................................................................................61 3.4.2.1.-Eggs............................................................................................ 61 3.4.2.2.-Semi-intensive larval rearing.......................................................63 3.4.2.3.-Intensive larval rearing.............................................................. 64 3.4.2.4.-Nursery……............................................................................... .67 3.5.-Measurements......................................................................................................... 69 3.5.1.-Growth in length........................................................................................ 69 3.5.2.-Growth in weight....................................................................................... 69 3.5.3.-Survival...................................................................................................... 70 3.5.4.-Histological studies.................................................................................... 71 3.5.5.-Osteological study...................................................................................... 72 3.5.6.-Characterisation of the different types of deformities............................... 74 3.5.7.-Meristic determinations.............................................................................. 75 3.6.-Biochemical analyses............................................................................................... 76 3.6.1.-Dry matter content.......................................................................................76 3.6.2.-Ash content................................................................................................. 76 3.6.3.-Protein content............................................................................................ 77 3.6.4.-Total lipids content..................................................................................... 77 3.6.5.-Fatty acids composition ..............................................................................77 3.7.-Statistical analysis .................................................................................................. 78 3.8.-Nomenclature of the referenced species.................................................................78 4.-STUDY I.............................................................................................................................. ....79 5.-STUDY II..................................................................................................................................99 6.-STUDY III..............................................................................................................................129 7.-STUDY IV..............................................................................................................................167 Index IV 8.-CONCLUSIONS...................................................................................................................207 9.-SPANISH SUMMARY........................................................................................................ 211 9.1.-Resumen……................................................................................................... 211 9.2.-Introducción............................................................................................................214 9.3.-Objetivos..................................................................................................................257 9.4.-Material y métodos.................................................................................................259 9.5.-Conclusiones............................................................................................................305 10.-REFERENCES.....................................................................................................................309 Acknowledgement V A Raquel, Javier y Carlos. Acknowledgement VI Agradecimientos El trabajo presentado no es fruto únicamente de un esfuerzo individual, sino del cúmulo de esfuerzos de un grupo de personas, que han contribuido de una u otra manera al desarrollo del mismo. Sirva este apartado para expresar mi más profundo agradecimiento a todas ellas. En primer lugar, a la Doctora Izquierdo como directora de este trabajo y principal impulsora del mismo. Por haberme dado la oportunidad de integrarme en este grupo de investigación del que es cabeza visible y referente mundial en el ámbito de la acuicultura marina. Igualmente, por haberme abierto innumerables puertas a los mejores grupos de trabajo y especialistas de este sector con los que poder discutir y compartir las experiencias realizadas. Por ser siempre mi referente como profesional y científico, por animarme a superarme y mejorar como investigador, sin olvidarme nunca de mojarme los pies. Por su apoyo incondicional y sus innumerables consejos sin los cuales este trabajo nunca se habría realizado. Gracias y un fuerte abrazo. A la Doctora Hernández-Cruz, por ser la primera en acercarme a la zootecnia de las larvas de peces, sus innumerables consejos, su cercanía y sus cuidados, casi como una madre, cuando tuve la oportunidad de convivir con ella en nuestra estancia en Grecia, han sido fundamentales para la realización de este trabajo. Gracias por todo y mucho más. Al Doctor Hipólito-Fernández-Palacios (Pipo), por ser uno de los últimos roqueros de la acuicultura, siempre un referente dentro de este grupo. Mi más sincera admiración por esa cabeza privilegiada y mi gratitud. A lo largo de estos años he podido disfrutar contigo, desde las mejores marchas, pasando por las fantásticas campañas de pesca, hasta compartir el despacho de al lado, desde donde, aunque sea a gritos tras el cristal, pedirte consejo sobre acuicultura y lo que sea. Espero poder seguir compartiendo y aprendiendo de tu experiencia por mucho tiempo. Muchas gracias y un casto abrazo. Al Doctor Juan Antonio Socorro, del que al nombrarlo es inevitable acordarme de mis comienzos en este mundo aún siendo un estudiante, nunca olvidaré los pateos desde el Cruce de Melenara después de clase para hacer unos bloques de parafina, que en aquella época, poco sentido tenían para mí. Tu enorme calidad humana y tu sonrisa siempre contagiosa, era de las pocas cosas que evitaba quedarme dormido en aquellas jornadas de trabajo, mientras Acknowledgement VII concentrado en el microscopio, me explicabas los entresijos del interior de una larva. A día de hoy gran amigo y otro gran maestro. Gracias Juanito. A D. Antonio Valencia, a quien desde su gran conocimiento práctico y experiencia en el cultivo de larvas, nunca agradecí lo suficiente su inmensa generosidad por enseñarme y compartir todos los secretos de este arte, gracias maestro, te llevo en el corazón. Al Doctor Daniel Montero, por soportarme como compañero de despacho y en tantas habitaciones de congresos con sus inseparables tapones. Mi más sincero agradecimiento, por sus consejos a la hora de enfocar la investigación, su experiencia y profesionalidad así como las horas que ha dedicado a revisar los diferentes trabajos incluidos en este documento. Sin duda tu ayuda ha sido inestimable. Muchas gracias, un fuerte abrazo Dani. A Dña. María del Pino Viera (Mapi) por su ayuda y su gran apoyo. Recordaré siempre el primer café griego que tomamos y lo fácil que fue la convivencia que compartimos en nuestra estancia en el IMBC, sin apenas conocernos y de la que salió como la hermana mayor que nunca tuve. Una compañera infatigable para sacar adelante todo lo que se le proponga. Un fuerte abrazo. A D. Miguel Medina, como pieza fundamental para que este gran engranaje del ICCM funcione (proveedores, facturas, agua, etc.) y más importante si cabe, por su apoyo personal y la amistad que se ha ido forjando durante todos estos años. Muchas gracias y un fuerte abrazo. No quisiera dejar pasar la oportunidad de expresar mi más sincero agradecimiento a todo el equipo de técnicos que a lo largo de estos años han compartido conmigo la experiencia de trabajar en la nave Mesocosmos (Moneiba, Rubén, Damian, Desiré, Emma) su profesionalidad, sus ganas de aprender e ilusión por sacar adelante las tareas, así como su capacidad para adaptarse a las circunstancias cambiantes dentro de un proyecto que debía compaginar investigación y producción. Gracias a todos. Por supuesto a todos los demás compañeros de la planta de cultivos (Ada; Daylos, Omar, Manolo y los que han ido pasando y ya no están) por su ayuda, generosidad y ganas de trabajar, el compañerismo para compartir buenos ratos y hacer más llevadero el trabajo diario. Gracias. Acknowledgement VIII A las técnicos de laboratorio de análisis (Carmen Quintana y Regina Morales) por su simpatía y buen hacer en todas las tareas de laboratorio y a las que siempre se pudo recurrir cuando fue necesaria su colaboración. Muchas gracias. A todos los demás miembros del GIA (Lucía, Lidia, José Vergara, María José, Rafa, Kiki), quienes siempre están ahí y a los que se puede recurrir para ayudar en lo que sea, de una u otra manera han contribuido al desarrollo de esta tesis. Muchas gracias. Al equipo de genética y en su representación el doctor Juan Manuel Afonso (Juanma), por su inestimable soporte para los estudios estadísticos, sus consejos y siempre cercana y generosa ayuda. Gracias y espero poder seguir trabajando juntos en nuevos proyectos. Igualmente agradecer al equipo de patología, Fernando Real, Daniel Padilla, Félix, Jimena, y los demás integrantes por su colaboración en el estudio de las patologías que nos han ido aconteciendo durante todos estos años. Muchas gracias y confió en seguir colaborando con ustedes. A las nuevas generaciones de doctores y doctorandos, en las que me incluyo, en primer lugar a los que integran el equipo de cultivo larvario como Tibi, Eyad, Francisco, Eneko y demás compañeros que han ido pasando por esta sección, gracias por vuestro apoyo y esos días de fin de semana en la nave. Igualmente a Dominique, por su colaboración en la gestión de los reproductores y como compañera de mareos en las campañas de captura de los mismos, de las que al final siempre salíamos más curtidos. Por supuesto al equipo de cultivo de Abalone, Gercende y Amaya, por su buen ánimo y apoyo cuando ha hecho falta. A todos los demás compañeros como Juan Estefanell, Fran, Silvia, Alex, Rachid y los que me pueda olvidar porque seguramente de alguna manera han contribuido a mejorar si no este trabajo en particular a mi desarrollo como investigador. También expresar, mi más sincero agradecimiento a todos los miembros del grupo de trabajo del proyecto Interactt y especialmente a los doctores Pascal Divanach, Nikos Papandroulakis y Panagiotis Anastasiadis, quienes contribuyeron de una manera definitiva a mi formación en el cultivo larvario de peces marinos, su buen hacer, consejos y cercanía, han sido fundamentales en mi posterior desarrollo profesional. Abbreviations XV Abbreviations ANOVA: Analysis normal variance APROMAR: Asociación Empresarial de productores de cultivos marinos (Association of marine aquaculture producers; Spain) FEAP: Federation of European aquaculture producers ARA: Arachydonic acid (20: 4n-6) BHT: Butilated hydroxitoluene BMP4: Bone morphogenesis proteins 4 DAH: Days after hatching DHA: Docosahexaenoic acid (22: 6n-3) DPA: Docosapentaenoic acid (22: 5n-6) DW: Dry weight EFA: Essential fatty acid EPA: Eicosapentaenoic acid (20: 5n-3) FAMES: Fatty acid methyl esters FAO: Food and Agriculture Organization FFA: Free fatty acid GC: gas chromatography HPLC: High polar liquid chromatography HPTLC: High polar think layer chromatography ICCM: Instituto Canario de Ciencias Marinas n-3 HUFA: n-3 series highly unsaturated fatty acid (20 or more carbons) LA: Linoleic acid (18: 2n-6) LNA: Linolenic acid (18: 3n-3) MAPA: Ministerio de Agricultura, Pesca y Alimentación (Ministry of Agriculture, Fisheries and food, Spain). NL: Neutral lipid PPAR: Peroxysome proliferators active receptors RXR: Retinoic acid X receptors SHH: Hox and sonic hedgehog genes TFA: Total fatty acid Units: Along the whole document, the international system of units (SI) was used Summary XVII Summary The main objective of this study was “to improve the production technology of red porgy (Pagrus pagrus) larvae and fry”. Specific objectives were divided in two complementary and consecutive phases; the first part was mainly based on a biological approach where specific aspects of the first development stages of this species were described. Followed by a second part where this knowledge was applied to develop a standard larval rearing protocol for commercial production of this species. In this sense, it was determined that red porgy larvae are visual feeders and hatch with an incomplete and nonfunctional visual and digestive system. Along the third and fourth days posthatch, major structural changes took place in the visual and digestive systems, with the definition and pigmentation of the primary photoreceptors (cones) and mouth opening. At this moment, the first digestive activity was detected in the midgut, which enables the larva to begin its exogenous feeding. The second most important change in relation to visual system development was detected around 20dah, when second photoreceptors nuclei appeared (rods), these structures prepared larvae with a greater sensitivity and visual acuity, allowing them to prey under restricted light conditions. These changes, agreed with the detection of the first gastric cells and the progressive migration of the larvae, from shallow to depth water, suggesting changes in feeding habits and culture conditions at this stage. On the other hand, the influence of the culture system over larval development was determined. These differences were showed in a lower growth, from the first week of life, when intensive systems were applied in comparison to semi-intensive ones. This fact is also translated to larval osteological development, since this is better correlated to larval sizes than age. Thus, larvae in more advanced ossification stages were observed in the semi-intensive systems in comparison to the intensive ones, for the same larval age. Furthermore, different patterns of growth throughout the culture period were identified and separated in three main stages, which differs depending on the rearing system. Accordingly, the first stage (5-15 dah) with high growth rates and very sensible to mortalities; a second period (15-30dah) related to completion of metamorphosis with lower growth rates and better control of mortalities and finally, a third post-metamorphic one (30-50dph) considered as a recovery phase in the intensive system with increased growth rates and high survival. Overall, red porgy grows much faster than other sparids such as Sparus aurata doubling the size of the juveniles of the later at 95 dah. Regarding to the meristic variations, intensification of the rearing system does not display any effect on the ribs number, but a higher incidence of individuals with an extra vertebra were Summary XVIII observed in the intensive systems. In addition, present results showed that regardless of the culture conditions, red porgy larvae and fry display a high incidence of morpho-anatomical deformities that might condition their commercial productivity under these culture conditions. In particular, culture intensiveness had a clear influence on the appearance of a lower number of normal shape larvae and this effect was reflected with an increase of larvae with cranial deformities and kyphosis, whereas this factor does not have a significant effect on the apparition of column deformities such as vertebral fusions and lordosis. These anomalies showed the highest impact in this species. Nevertheless, culture system affected vertebral fusions location, thus in the intensive systems reared larvae, most of the fused vertebrae were located in the caudal zone, whereas in the semi-intensive system reared larvae this anomaly were mainly located in the pre-hemal zone. To verify the potential effect of nutrition over this type of deformities, a comparative study were performed, feeding red porgy larvae with rotifers enriched with different enrichment emulsions under intensive culture conditions. The results of this study allowed concluding that an essential fatty acid supplementation in rotifers emulsions was decisive to reduce the incidence of skeletal deformities in this species. Thus, the increase of the DHA content in rotifers, in spite of not modifying the growth of the larvae, clearly affects the biochemical composition of the larva, improving the survival and reducing almost 50% the incidence of skeletal deformities. On the other hand, the presence of elevated content of other fatty acids like DPA (22:5n-6) might have negative consequences on the larval survival in this species. Additionally, in these studies, more abundant fatty acids in red porgy along larval development according to the rearing system were identified: 22:6 n-3, 16:0, 18:1 n-9, 18:0, 18:2 n-6, 20:5 n-3, 16:1 n-7,18: 1n-7, 18:3 n-3 and 20:4 n-6. Hence, the levels of DHA in the red porgy larvae between 12-20dah (15-20%) are higher to the supplemented through the diet when commercial enriching emulsions were used (9-10%), suggesting a selective accumulation of this EFA. The improvements in survival observed in the semi-intensive system could be related to a greater accumulation of DHA in these larvae, consequence of a longer duration of the yolk reserves in the larvae reared under this system, which in turn showed a better resistance to activity test. Indeed, this species under intensive culture conditions seems to be very sensitive to handling and stress. The results of this study allowed the improvement in the rearing protocols for red porgy, increasing the final total length and survival at 50dah from the initial trial to the last ones from 18.9 mm to 25.13 mm and from 4.9 to 12.5% in the intensive system and from 23.52 to 26.4 mm and 4.4 to 28.7% in the semi-intensive system. Finally, some general culture zoo-technical advices were established for red porgy larval rearing. Summary XIX In relation to the feeding sequence, present results suggest a prey density over 5 rot.ml-1 in semiintensive system and the extension in the length of rotifer feeding from 20 to 30 dah for both rearing systems facilitating the adaptation to feeding changes. In addition, since this species was found to be very voracious in preying Artemia, which was even defecated alive, the application of an early co-feeding and weaning, from 12-15dah, together with a reduction in the Artemia amount, markedly decreased mortalities and improved growth rates. Also, the use of continuous of photoperiod is suggested during the first 20 days, but reduced to 12:12 photoperiod and natural light in later stages, helps to reduce larval stress when the change from green to clear water are conducted. With all these information, it is possible to conclude that the best larval rearing protocol to sustain a regular and predictable red porgy fingerlings demand with a good biological performance for commercial application is the semi-intensive system technology, but even in this type of systems fry quality is seriously affected by the high incidence of skeletal deformities. However, this problem might be limited with specific formulation of the rotifers enrichment emulsions. Improvements in the production technology of red porgy (Pagrus pagrus) larvae and fry: Importance of rearing conditions and diet nutritional value on their quality INTRODUCTION Introduction 1 1. INTRODUCTION 1.1.–Aquaculture in the world Aquaculture has a long history of more than 4,000 years despite its significant contribution to the human feeding is only very recent. Aquaculture has experienced a great development in the last 30 years. Thus, world-wide production of fish, crustaceans and molluscs has increased from 3.9% of total fishing production in 1970 to 43% in 2006, being expected to reach 50% of total fishing production by the year 2025 (FAO, 2004). From 1970, aquaculture production has an average annual grown around 8.7% in comparison to 1.2% of fisheries production or 2.8% in terrestrial animal production. According to FAO previsions, aquaculture seems to be a feasible complement to fisheries in order to satisfy the high demand of marine products. In 2004 worldwide fish consumption rate was 16.6 kg/person/year, Spain being one of the most important fish consumer’s with 36.6 kg/person/year (MAP, 2007). Nevertheless, Spanish fish consumption depends on a fishing fleet that for many years has not been able to obtain the necessary captures to supply the national market. The overexploitation of the traditional fishing areas and the restrictions imposed by the changes in the policy and agreements of international fishing areas caused a chronic restriction in marine products supply, which in turns promoted high levels of fish imports. Hence, the increasing demand of food from marine origin ensures a successful future to aquaculture sector making this activity very attractive for investors, as well as an alternative to labor activity for the new generations. Nowadays, labor force working in aquaculture is estimated in more than 12 million people around the world (APROMAR, 2008). In industrialized countries, aquaculture as a complementary sector to fisheries comprises an intensive aquaculture with a high degree of specialization to produce fish, mollusks, crustaceans or plants under controlled conditions. More than 250 fish species and plants were reared around the world in 2006 with almost 50% of them being cultivated species (APROMAR, 2008). European aquaculture is mainly based on production of a reduced number of fish and mollusk species with a high commercial value. In marine fish (excluded salmonids) the last eight year production has experimented an important increase from 125,000 tons in year 2000, to more than 250,000 tons in 2008, this was mainly due to the intensive rearing of 3 species, gilthead seabream (Sparus aurata), European seabass (Dicentrarchus labrax), and turbot (Psetta maxima) (with a small contribution of Atlantic cod (Gadus morhua), meagre (Argyrosomus regius) and Atlantic bluefin tuna (Thunnus thynnus) fattening, included). Smaller productions of new species such as Atlantic cod (Gadus morhua), Senegalese sole (Solea senegalensis) and Atlantic halibut (Hippoglossus hippoglossus), are being included more recently (Table I), (FEAP, 2008). Introduction 2 Table I: Evolution of European aquaculture marine fish production in metric tons (t), (salmonids not included; source FEAP, 2008). Common name European seabass * Gilthead seabream* Turbot Northern bluefin tuna Atlantic cod * Atlantic halibut * Meagre White seabream Senegalese sole Total Year Dicentrarchus labrax Sparus aurata Psetta maxima Thunnus thynnus Gadus morhua Hippoglossus hippoglossus Argyrosomus regius Diplodus sargus Solea senegalensis 2000 57,811 57,272 4,872 6,082 27 135 - 400 - 126,599 2001 56,162 74,403 4,640 9,992 111 389 - 400 - 146,097 2002 61,093 79,367 5,320 8,816 255 350 - 400 60 155,661 2003 62,060 87,940 5,107 6,715 2,940 845 - 400 52 166,059 2004 68,679 88,522 6,076 11,792 3,200 884 211 400 75 179,839 2005 79,706 93,372 6,085 9,180 6,708 1,319 893 400 60 197,723 2006 97,336 119,099 7,101 8,730 8,570 1,839 1,425 400 80 244,580 2007 93,425 104,697 7,444 5,380 10,640 1,399 1,138 - 60 224,183 2008 102,765 128,943 9,067 5,680 11,680 1,260 2,235 - 90 261,720 *The production data includes some countries outside the European Union (Norway, Croatia, Iceland and Turkey). Table II: Evolution of European aquaculture marine fry production, in Millions (source FEAP, 2008 and authors data). Common name Scientist name Year 2000 2001 2002 2003 2004 2005 2006 2007 2008 Fry production (Millions) European seabass* Dicentrarchus labrax 214.2 207.9 204.0 220.5 349.8 374.6 389.7 419.7 457.7 Gilthead seabream* Sparus aurata 262.3 293.4 317.0 307.7 318.8 447.3 549.9 507.6 497.7 Turbot* Psetta maxima 4.0 4.0 5.2 5.2 - 6.8 8.5 6.1 13.0 Atlantic cod** Gadus morhua 0.04 0.17 0.40 4.59 5.00 10.48 12.83 - - Meagre* Argyrosomus regius - - - 1.3 1.1 3.4 2.4 2.2 3.0 White seabream Diplodus sargus 0.7 0.7 0.7 0.7 0.7 0.7 0.7 - - Senegalese sole Solea senegalensis - - - 0.6 0.6 0.6 1.1 0.6 0.6 Atlantic Halibut* Hippoglossus hippoglossus 1.0 - - 0.10 0.10 0.10 0.10 - - Red porgy Pagrus pagrus 1.1 - - 0.015 0.03 0.10 2.5 2.0 2.0 Sharpsnout seabream Puntazzo puntazzo 6.3 - - - - - 4.5 4.0 4.0 *The production data includes some countries outside the European Union (Norway, Croatia, Iceland and Turkey). **Rosenlund pers. com. Introduction 3 With the exception of the Atlantic blue fin tuna (Thunnus thynnus) fattening, whose production is based on wild fish capture and fattening in off-shore cages, the production increase in the rest of species has being associated to a better knowledge of reproductive biology and the development of the larval rearing techniques which allowed an important increased in fry availability (Table II). 1.2.-Aquaculture in Spain 1.2.1.-General aspects Spanish aquaculture is one of the most important of all the European Union with a production of 250-300 thousand tons/year, which accounts 3% of the worldwide production and 25% of the European production. To the traditional modalities of mussel (Mytilus galloprovincialis) culture in Galician coast and the extensive aquaculture in Andalucía, a new sector, highly industrialized and in constant expansion has joined from the early 90´s. Figure 1. Example of extensive traditional aquaculture in Spain: a) group of rafts for mussels (Mytilus galloprovincialis) production; b) collection of common cockles (Cerastoderma edule) in Ría de Muros y Noya (La Coruña, Spain). As example, the case of turbot (Psetta maxima) production in the north coast of Spain, with 7,512 tons production in 2008 that means 82.8% of the European production, and the Mediterranean and Canary Islands aquaculture mainly based on gilthead seabream (Sparus aurata) and European seabass (Dicentrarchus labrax) production with 24,790 and 11,760 tons in 2008 respectively (FEAP, 2008). In addition, the introduction of new species to marine aquaculture diversification has to be considered. In the year 2008, 72% of the world production Introduction 4 of meagre (Argyrosomus regius) (1,620 tons) was produced in Spain, followed by Italy and France. In addition, Spain is the single producer of blackspot seabream (Pagellus bogaraveo) with 134 tons also leads Senegalese sole production (Solea senegalensis) in Europe with 90 tons in 2008. Regarding Atlantic blue fin tuna fattening, Spain was the European leader with a 43.5% of the production in 2006 followed by Cyprus and Croatia. Figure 2. Examples of intensive modern aquaculture in Spain: a) off-shore cages for gilthead seabream (Sparus aurata) on growing in Telde, (Las Palmas, Spain); b) off-shore cages for turbot (Psetta maxima) on growing in the Ría de Vigo (Pontevedra, Spain). 1.2.2.-Fry production Actually, 16 commercial marine fish hatcheries produced 113.5 million of fry in 2008 in Spain (APROMAR, 2007), being this country the first European producer of turbot fry with 90.3 % of the production. Additionally, Spain produces 14.8 % of gilthead seabream fry and 5.6 % of sea bass fry (FEAP, 2008). To these data, should be added, the small amounts of fry from other marine species, produced in private companies or research centres: as an example should be considered the Senegalese sole production (600,000 fry in 2008), pollack (Pollachius pollachius) and red porgy (Pagrus pagrus) (100,000 fry each, in 2006) or black spot seabream production (16,000 fry in 2006). These experimental productions have promoted marine species diversification projects by commercial producers. Moreover, the introduction of new species in the Spanish farms was encouraged by experiences made at pilot scale for several species, i.e. sharp snout seabream (Puntazzo puntazzo) in Murcia (Hernández et al., 2003), common dentex (Dentex dentex) in Catalonia, Valencian Community and Balearic (Abellán, 2000), red banded Introduction 5 seabream (Pagrus auriga) in Andalusia and Canary Islands (Cárdenas et al., 2003), or white trevally (Pseudocaranx dentex) also in the Canary Islands (Roo et al., 2007b). At experimental level with a much lower degree of knowledge an even higher number of species are being tested such as amberjacks (Seriola dumerilli, Seriola rivoliana or Seriola fasciata), Atlantic bluefin tuna (Thunnus thynnus), pink dentex (Dentex gibbosus), striped red mullet (Mullus surmuletus), dusky grouper (Epinephelus marginatus) or European hake (Merluccius merluccius). 1.2.3.-Aquaculture in the Canary Islands The Canary Islands have very good perspectives of aquaculture expansion due to the exceptional climatic conditions and high quality of their coastal waters. Thus, the production values of the last years showed a continued growth of this activity that was increased from 456 tons in 1998 to more than 8,000 tons in 2006 (APROMAR, 2007). The most important reared species are gilthead seabream (Sparus aurata) with 65% of the production and sea bass with 35%, Canary Islands are the second Spanish producer of gilthead seabream with 26% of national production. Besides Canary Islands is the first national producer of European seabass with a production of 31.7% (APROMAR, 2008). Nevertheless, this activity is based in a reduce number of species, which is slowly changing with the introduction of small amounts of fry from other new species such as meagre, Senegalese sole and red porgy with different degree of success. Figure 3. a) Pre-growing of red porgy (Pagrus pagrus) on land; b) on-growing of red porgy in offshore cages. Like counterpoint, fits to mention that marine aquaculture in the Canary Islands continues depending on fry importation, in 2005, around 25 million fry were imported and only a small contribution were locally produced by the ICCM experimental hatchery. These facilities produce Introduction 12 and zooplankton concentration as well as migrations (Pitta et al., 1998). As a result, the success of these systems is usually higher than in the intensive systems. Frequently, species difficult to be reared under intensive conditions such as in the striped seabream (Lithoghnathus mormyrus) or sharp snout seabream (Puntazzo puntazzo) with small eggs size or reduced eggs availability are successfully produce in this system. In this type of system, fry quality is excellent and do not show problems such as abnormal swim bladder inflation, skeletal deformities, pigmentation anomalies or deviations in natural behaviour (Divanach et al., 1996). In the case of the European seabass, a common anomaly when larvae are reared under intensive systems is the apparition of abnormal sex proportion (higher male proportion), which is corrected in the extensive systems. Depending on the water volume, fry harvest can reach several hundreds of thousands or millions of fry. The challenge for its industrial success is the use of megavolumes (megacosmos) with several thousand millions of cubic meters (Gamble et al., 1985),or intermediate volumes (Mesocosm systems) being some commercial experiences conducted in Norway for cod production ((Drenner et al., 1990; Van der Meeren and Naas., 1997) (Figure 9). 1.3.1.3.-Semi-extensive (Mesocosms) systems These systems are commonly applied in semi-outer or inner conditions and are considered as intermediate systems between intensive and extensive (Divanach and Kentouri, 2000; Papandroulakis et al., 2004a). Its definition is relatively recent in the form for its present application and was defined by Divanach et al, (1998) after the study of the intrinsic qualities and failures of the original models (Grice and Reeves, 1982; Bever et al., 1985; Lalli, 1990). Mesocosms is a technology quite developed that takes the advantages of both extensive and intensive techniques without its corresponding disadvantages. The larval culture takes place at relatively low densities (2 - 10/litre), in relatively great volumes (30 - 100 m3) and with certain depth (1.5 – 2.5 m) tanks, located in well organized facilities which assures the quality, ergonomics and security in the culture achieving a high production of fry per tank (50,000 to 300,000 depending on the species) with a high productivity (> 2 million of fry/man/year) (Divanach et al., 2002). Introduction 13 Figure 10. Semi-extensive systems: a) Mesocosm facilities in the Canarian institute of Marine Sciences in Telde (Canary Islands; Spain); b) detail of larval rearing tank (40,000 l ) in the Institute of Marine Biology of Crete, (Heraklion, Greece). The environmental conditions in the tanks are a combination of natural and artificial, avoiding any strong seasonal climatic variation or geographic limitation and allowing the optimization of the operational costs (Divanach and Kentouri, 2000; Papandroulakis et al., 2004a). Long photoperiods (> 18 light hours) and warm temperatures (15-21ºC) are used for most of the species. The exogenous and endogenous combined feeding assures fulfill of larval nutritional requirements avoiding risks of nutritional deficiencies. This partial autonomy of the system is very important to obtain a good quality fry and helps to reduce operational cost during nights and weekends (Divanach et al., 2002). The results obtained with this technique for already culture marine species, as European seabass (Dicentrarchus labrax), gilthead seabream (Sparus aurata) or white seabream (Diplodus sargus) are frequently better than the one obtained with intensive or extensive techniques (Papandroulakis et al., 2004a). The fry survival after the weaning phase generally ranged between 40-90% from eggs stage. The percentage of total deformities is around 5 to 10%, swim bladder inflation goes up to 95% of the fry and larval growth is between 15 to 20 mg at the first month of life with low size dispersion and with animals that exhibit very homogenous behavior, with a low cannibalism incidence. Weaning is completed at 45-50 days of life (Divanach et al., 2002). This technology has been used successfully for the production of fry from more than 25 species of marine fish and 5 hybrids (Divanach et al., 2002). Most of them have been produced in mesocosms with extensive approach using pseudo-green water technique, as it is the case of the creek anchovy (Anchovy mitchilii), Atlantic cod (Gadus morhua), Atlantic herring (Clupea harengus), European plaice (Pleuronectes platessa), Atlantic halibut Introduction 14 (Hippoglossus hippoglossus), common sole (Solea solea), turbot (Psetta maxima). But others like the European seabass (Dicentrarchus labrax), gilthead seabream (Sparus aurata), sharpsnout seabream (Puntazzo puntazzo), white seabream (Diplodus sargus), common twobanded seabream (Diplodus vulgaris), annular seabream (Diplodus annularis), striped seabream (Lithognathus mormyrus), red porgy (Pagrus pagrus), common dentex (Dentex dentex), greater amberjack (Seriola dumerili), thick lipped grey mullet (Chelon labrosus), shi drum (Umbrina cirrosa L.), were reared under mesocosm technology with intensive approach (Divanach and Kentouri, 2000; Papandroulakis et al., 2004a, 2005; Koumoundouros et al., 2005; Zaiss et al., 2006; Ben Khemis et al., 2006). Within Mesocosms systems, two variants and four-sub variants according to the origin and quality of the preys were defined: In the extensive approach, the food chain is mainly endogenous and only complemented with exogenous feeding when symptoms of exhaustion and lack of food occur. In the intensive approach feeding is basically exogenous, although there is a certain capacity for endogenous food production due to the low larval density (low rates of feeding) and the presence of live phytoplankton in the tank. The four sub variants are characterized by the methods for nutritional chain improvement: 1.3.1.3.1.-Natural-bloom This sub-variant is based on the generation of a natural trophic chain in the gross filtered seawater that is pumped. The water is fertilized with nutrients to promote phytoplankton growth in the case of very oligotrophic water phytoplankton inoculation with species from Nannochloropsis or Tetraselmis genus is also found. Water renewal is very low 1-2% day and after 10-15 days, an optimal zooplankton density is obtained. The fish larvae fed themselves on this zooplankton around 20-25 days, a critical time in their development. Afterwards they are ready to continue feeding on exogenous Artemia and to begin its weaning. 1.3.1.3.2.-Green water method It includes the generation of an endogenous trophic chain based in phytoplankton (Nannochloropsis sp.) and rotifers (Brachionus sp.) which are allowed to grow in filtered and Introduction 15 sterilized seawater. Water is fertilized with nutrients and inoculated with phytoplankton and rotifers which developed until they reach a 500,000 phytoplankton cells.ml-1 and >2 rot.ml-1. The water is not renewed until day 4 to 8 when larvae are introduced. When the food chain is correctly developed this last approximately 25 days before Artemia and weaning diets are necessary. 1.3.1.3.3.-Pseudo green water This technique is based on an exogenous food chain, that contains phytoplankton and rotifers, produced in parallel facilities and that are daily introduced according to the larval demand (Divanach et al., 2002). Two variations are found in this technique: 1. Pure: the tank is filled with sterilized seawater and the eggs or larvae are introduced. Later when the larvae initiate their heterotrophic life, phytoplankton and rotifers are added. 2. In the second variant, the first trophic chain is initiated with the introduction of phytoplankton and rotifers before eggs or larvae addition and, water is always renovated, increasing as larvae develops. Phytoplankton and rotifers, enriched to improve their nutritional value, are added 1-2 times a day. When the larva is able to feed on Artemia, addition of phytoplankton is discontinued and rearing continues in clear water. This technique it is used for species with a well known biological cycle as gilthead seabream (Sparus aurata). 1.3.1.3.4Clear water Here, phytoplankton is not added to the rearing water and, hence, high water renovation rates of more than 10%tank volume/hour is used to avoid proliferation of endogenous microorganisms. It is used with species which are able to accept Artemia from first feeding, like European seabass or associated to automatic feeding to provide food with an elevated nutritious quality (Divanach et al., 2002). 1.3.1.3.6Neo-green water These technique uses criopreserved, lyophilized or paste phytoplankton, its main limitation being the reduction of the beneficial value of using alive microalgae (Divanach et al., 2002). Introduction 16 1.4.–Limiting factors in larval rearing The selection of an appropriated rearing technology depends on the species specific characteristics and its susceptibility to different parameters affecting the larval rearing success. Some of these parameters, are related to culture conditions such as larval density, tank volume, water renewal, use of phytoplankton and drugs; whereas others refer to the physical-chemical conditions such as temperature, salinity, illumination (photoperiod, intensity and light quality) or waste products; and finally, to larval feeding and nutrition such as amount of food or live preys, prey and feeding sequence or the nutritional value of the food supplied. 1.4.1.-Culture conditions related parameters 1.4.1.1.-Larval density This factor markedly affects larval growth and survival and determines the type of rearing system selected as as it has been shown in many species like creek anchovy (Anchovy mitchilli), lined sole (Achirus lineatus) (Houde, 1977), cobia (Rachycentron canadum) (Hitzfelder et al., 2006; Holt et al., 2007) or meagre (Argyrosomus regius) (Estévez et al. 2007; Roo et al., 2007), common seabream (Pagrus pagrus) (Hernández-Cruz et al., 1999; Roo et al., 2005b), gilthead seabream (Sparus aurata) (Parra and Yufera, 1999; Roo et al., 2005a), or close species as common dentex, Dentex dentex (Giménez and Estévez, 2008a). Larval density and its effect on survival seems to be strongly associated to intracohort cannibalism (Baras and Jobling, 2002). Besides, the increase in larval density also reduces growth rates due to the increase in food competition and the stress caused by an elevated individuals concentration as it has been found in European seabass (Dicentrarchus labrax) or Canadian sole (Paralichthys dentatus) (Hatziathanasiou et al., 2002; Saillant et al., 2003; King et al., 2000). In other species as Atlantic cod (Gadus morhua) the influence of larval density on growth and survival is not so clear because is masked by other associated factors as food availability and water quality deterioration (Baskerville-Bridges and Kling., 2000). 1.4.1.2.-Shape and tank volume Tank volume strikingly affects larval rearing performance. For instance, early studies on gilthead seabream showed a positive correlation between the increase in tank volume and larval survival (Tandler and Sherman,1981). At present, commercially produced sparids are mainly reared in cylinder-conical shape tanks, usually made of fiberglass with a relatively big volume (1-15m3, Introduction 17 but frequently greater than 10m3). Moreover, the tank depth/surface can also affect larval growth and survival during the first stages of development, as occurs with gilthead seabream (Sparus aurata) seven bands grouper (Epinephelus septemfasciatus) or the weak “stinger” (Inimicus japonicus) (Salhi, 1997; Ruttanapornvareesakul et al., 2007). 1.4.1.3.-Water renewal The water renewal has been suggested to affect the incidence of column deformities (Kihara et al., 2002). Increase in water renewal enhances removal of live prey and microalgae affecting the quality of the available food and the water physical-chemical characteristics. On the contrary, poor water renewal boosts the accumulation of waste products (ammonium, nitrites, etc) and the amount of remaining live prey with a poor nutritional value (Reitan et al., 1993), negatively affecting larval growth and survival. However, during the first 10 days of life, water renewal reduces gilthead seabream (Sparus aurata) larvae growth and a closed system with no water exchange is recommended (Tandler and Helps, 1985; Tandler and Sherman, 1981; Hernández-Cruz et al,, 1990). After this period a water renewal higher than 25% .day-1 is recommended. Addition of phytoplankton together with water renewal also enhances gilthead seabream survival (Hernández-Cruz et al., 1994). In the last years, an increasing tendency to the use of sophisticated closed systems has being adopted among aquaculturists. In these systems water renewal come from the same systems passed through mechanical and biological filters, ensuring a good water quality and preys with a high nutritional value (Olivar et al., 2000; Faulk and Holt, 2005). 1.4.1.4.-Use of phytoplankton in the rearing tank The beneficial effect of phytoplankton addition in larval rearing tanks, called “green water technique” is supported by hundred of authors (Scott and Baynes, 1979; Hernández-Cruz et al., 1990; Koven et al., 1990; Salvesen et al., 2000). The positive effect of phytoplankton in the tank can be related to its contribution to water oxygenation; reduction of waste substances (ammonium and nitrites) produced by the larvae; control of bacterial flora; nutritional value of preys maintenance; light diffusion and prey color contrast. Algal species used in larval rearing have shown an important antibacterial activity that limits the growth of opportunistic bacteria. In the culture of the phytoplankton species used in the present study, Nannochloropsis sp. the main bacterial flora is composed by Alpha and a cluster of Cytophaga-Flavobacterium type bacteria, which acts as a biocontrol for other bacterial communities present in the culture tanks (Nakase Introduction 18 and Eguchi, 2007). Bacterial load increase in the larval rearing water results from repeated addition of phytoplankton contributing to the development of larval initial intestinal flora (Skjermo and Vadstein,1993). In addition, the presence of phytoplankton in the rearing water helps to maintain the nutritional value of the live prey. Enriched rotifers, loss 20% of the enrichment in nutritional quality when they are supply to larval tanks using green water technique, whereas in clear water systems the lose is several times higher (Reitan et al., 1993; Planas and Cunha, 1999). Thus, larvae reared in green water maintain the yolk sac reserve 2-3 days longer than in clear water and show a 3 times better growth rate and survival (Papandroulakis et al., 2001; Van der Meeren et al., 2007). Finally, phytoplankton also acts as a light filter, reducing light reflection in the tank walls. This effect, together with the use dark walls tanks (Hinshaw, 1985), contributes to enhance larval feeding activity of the larva (Naas et al., 1992, 1996; Ramos and Kobayashi, 1985; Muller-Feuga et al., 2003). At present, there is a tendency to use concentrated, pastes, lyophilized and frozen biomass of different phytoplankton strains. There are different comparative studies about the use of this type of products. For instance, Cañavate and Fernández-Diaz (2001) did not found differences in growth and survival when gilthead seabream (Sparus aurata) were reared with a microalgae frozen biomass in comparison with alive microalgae. Nevertheless, these authors pointed out water quality deterioration, increasing the ammonium level with the use of inert biomass. On the other hand, the use of this type of products may limit the beneficial effects of the live algae, such as the bacteriostatic effect or the bacterial flora colonization in the tanks and larvae intestine. 1.4.1.5.-Use of antibiotics The excess of bacterial load in the culture tank due to live prey supply and insufficient water renewal has lead to the use of antibiotics. A great controversy can be found in the ature about the use of these substances in the larval culture. For instance, whereas Hernández-Cruz et al. (1994) showed that the use of oxolinic acid has adverse results in larval survival, enriched rotifers treated with antibiotic showing a lower content of n-3 HUFA. Other studies associated the improvement in Australian trumpeter (Latris lineata) larval survival and growth to the use of the oxitetracicline (Bataglene et al., 2006). 1.4.2.-Physico-chemical parameters Several physico-chemical parameters potentially affect larval culture, such as temperature, salinity, illumination, waste products, gases or pH, among others. Introduction 19 1.4.2.1.-Temperature Within the physico-chemical parameters that affect larval rearing success, temperature is one of the most important for marine fish. Its effect can has been studied along both embryo and larval development. Incubation temperature during embryo development The development of fish embryos are strongly influenced by the incubation temperature. Too high temperature, accelerate the embryonic development, whereas low temperatures delayed it (Blaxter, 1988). For gilthead seabream, the embryo is able to adapt to a wide range of incubation temperatures that vary between 14 to 28 ºC, being the optimum around 19º C (Polo et al.,1991). The optimal incubation temperature is associated to that corresponding to the latitudes of the spawning grounds. In gilthead seabream, temperatures below 16 ºC and over 22º C reduced survival and increased bone deformities incidence. Similar results have been described for Japanese red seabream (Pagrus major) (Mihelakakis and Yoshimatsu, 1998). In the European seabass (Dicentrarchus labrax), a slight increase in incubation temperature had a positive effect in muscular growth during later stages (López-Albors et al., 2003). Larval rearing temperature Tandler and Sherman (1981), determined that gilthead seabream larval survival is not affected by temperature when they range from 17 to 23 ºC, despite highest survival was at 17ºC and larval growth increased with rearing temperature. For the same species, Polo et al, (1991) determined that 16 to 22 º C was a suitable range for the same species, the optimum temperature being at 19ºC. A positive correlation was found between temperatures outside this range and larval mortality and skeleton abnormalities incidence. In addition, Tandler et al, (1989), showed that elevated temperatures increased size dispersion, reducing larval survival and enhancing cannibalistic behavior from early stages of development. 1.4.2.2.-Salinity Despite this parameter rarely changes along larval rearing, certain type of facilities occasionally undergo salinity drops because of rain run-offs. Salinity affects eggs hatching and the incidence of skeletal deformities. For instance, in Southern black bream (Acanthopagrus butcheri), larval survival is reduced and column deformities increase when salinity drops below 15‰ (Haddy and Pankhurst, 2000). Moreover, skeleton deformities affected 100% of the population when the Introduction 20 salinity dropped to 5 ‰ from 35 ‰. Similar results have been shown gilthead seabream, when salinity drops from 37 to 32 ‰ during the first days of life. Besides, a significant increase in abnormal development of opercula can be obtained (author’s unpublished data). On the contrary, in European seabass (Dicentrarchus labrax) a salinity drop to 15 ‰ results in an increase of larval survival and improvement in the percentage of larvae with functional swim bladder, similar results were obtained in the case of flathead mullet (Mugil cephalus) (Harel et al., 1998). Finally, the combined effect of both temperature and salinity had a significant importance to obtain viable larvae. Thus, extreme values of 28ºC and 12 ‰ salinity increase the percentage of larvae with skeleton deformities in red seabream (Pagrus Major) (Mihelakakis and Yoshimatsu 1998). 1.4.2.3.-Nitrogen excretion products Ammonium from protein catabolism is the most important excretion product in fish, being toxic at high levels. The ammonium produced by unconsumed preys and organic detritus. Lethal concentrations for 12 day old gilthead seabream are around 20ppm, being no ionized ammonium (N-NH3) the most toxic form with 0.24 ppm a lethal concentration for 50% of the population (Lc 50) (Parra and Yúfera, 1999). In the case of nitrite (NO2-), a 4,500 ppm concentration causes a 100% mortality of the population and 1,997 ppm a 50%. Nevertheless, these levels were directly related to factors like pH, salinity and temperature as well as the reared species. Thus, lethal concentration of N-NH3 for 7 day old (days after hatching, dah) Senegalese sole (Solea senegalensis) larvae was about 80 ppm and 1.32 ppm 24h for Lc 50. Besides, the ammonium levels (0.05 – 0.015 ppm of NH3-H) have been found to be related to damages to cartilaginous structure in red seabream larvae (Mihelakakis and Yoshimatsu 1998). 1.4.2.4.-Light Most of marine fish larvae are visual feeders (Blaxter and Staines, 1970). Sparids larvae, such as gilthead seabream are visual feeders that need light to effectively attack its preys (Tandler and Mason 1983). Thus, during the first 12 days of life there is a positive correlation between the increase in photoperiod from 12 in 24 hours and larval survival in this species (Tandler and Helps, 1985). Similar results were reported by Barahona-Fernándes (1979) in European seabass larvae. Equally, an increase in light intensity from 205 to 1370 lux has been associated to a better growth and larval survival in gilthead seabream, being optimal light intensity between 1370- Introduction 21 5140 lux when a combination of artificial and natural light, under continuous photoperiod and green water systems, is used (Tandler and Mason, 1984). From 20dah there are no significant differences on growth and survival caused by the change of photoperiod from 12 to 24 h light. While, from 50-60 dah a reduction of photoperiod length improves growth rates in gilthead seabream (Tandler and Helps, 1985). The effect of tank wall coloration and light reflection are closely related to light conditions. Black tank walls when provided with an appropriate illumination seem to resemble better natural light conditions (Naas et al., 1996; Hinshaw, 1985). Illumination has been also related to larval skeletal development. As an example, the larvae of silver pearl fish (Carapus homei) need to pass through a complete dark period to complete metamorphosis. In nature, this species undergo a reduction in the number of vertebrae and a vertebral compression to adopt the definitive fry form (Parmentier et al., 2004). 1.4.3.-Larval feeding and nutrition First feeding is a critical stage in the larval rearing of marine fish. Marine fish larvae hatch with limited endogenous reserves and hence, the ability to catch and digest preys as soon as possible is critical for larval survival. But its small mouth size and the poor development of the digestive system restricts the type of food making first feeding a critical stage for the success of larval rearing. 1.4.3.1.-Prey density Optimum prey densities in intensive cultures to obtain good larval survival have been determined to be between 10-15 rot.ml-1 for gilthead seabream (Sparus aurata) reared at 100 larvae.l-1 (Tandler and Sherman, 1981) or 5-10 rot.ml-1 for common dentex (Dentex dentex) reared at 10 to 40 larvae.l-1 (Giménez and Estévez, 2008). When larvae are fed copepods densities are higher than 1nauplii.ml-1 in the culture of the creek anchovy (Anchovy mitchilli) and lined sole (Achirus lineatus) (Houde, 1977; O´Connell and Raymond, 1970) or about 4 nauplii.ml-1 in Californian anchovy (Engraulix mordax). 1.4.3.2.-Feeding sequence and co-feeding Adequate prey sequence is a critical factor for the success of larval rearing techniques in new species. Mouth size and types of sustainable and mass produced available preys are limiting aspects. Therefore, eurihaline rotifers from the genus Brachionus with several morphotypes and Introduction 28 Matsuoka, 2003), common dentex (Dentex dentex) (Koumoundouros et al., 2001a), common pandora (Pagellus erythinus) (Boglione et al., 2003; Sfakianakis et al., 2004), sharpsnout seabream (Diplodus puntazzo) (Boglione et al., 2003; Favaloro and Mazzola, 2003) and white seabream (Diploduss sargus) (Koumoundouros et al., 2001b; Sfakianakis et al., 2003). In these species, the most important skeleton anomalies with important economic implications are those affecting the opercula complex, neurocranium and vertebral column. Deformities associated to neurocranium affect to the opercula complex and jaws and they are frequently found in commercial hatcheries. The anomalies of the opercula complex can be present in one or both sides of the fish and consist in a folding or an incomplete formation of the opercula complex (Koumoundourous et al., 1997b) (Figure13). Commonly this anomaly is associated with branchial arcs malformations (Sadler et al., 2001) and appear from very early stages of development being able to affect up to 98.3% of the population (Beraldo et al., 2003). Figure 13. Red porgy (Pagrus pagrus) fry and meagre (Argyrosomus regious) (a,c) with opercula complex anomaly; b) detail of the opercula folding. Jaw deformities include lower or upper jaw torsions and different degrees of prolongation (Cobcroft et al., 2001), being sometimes associated to lethal effects (Barahona-Fernandes, 1982) (Figure 14). Introduction 29 Figure 14. Mandible deformities: a,b) red porgy; c) normal shape gilthead seabream, In general, ontogeny development of the jaw structures and fins appears at very early stages but is not completed to older stages. Fin deformities are characterized by torsion, partial or total lack and can reach up to 65% of the population in gilthead seabream (Sparus aurata) larvae (Koumoundouros et al., 1997b) although they have a minor relevance at commercial scale (Figure 15). Figure 15. Anomalies in the pectoral fins radios: a) gilthead seabream; b) red porgy. Vertebral column abnormalities are among the most important skeleton aberrations found in aquaculture, not only by their severe effect on fish morphology but also by its clear influence on growth productive characters (Gjerde et al., 2005; Kause et al., 2005). The main column Introduction 30 deformities are scoliosis, lordosis, kyphosis and vertebral fusions, sometimes several of them combined in the same fish (Afonso et al., 2000) (Figure 15). Figure 16. Column anomalies in red porgy (Pagrus pagrus). Column abnormalities have been well documented in freshwater (Akiyama et al., 1985; Madsen and Dalsgaard, 1999; Witten et al., 2005, 2006) and marine fish species (Paperna 1978; Takashima 1978; Barahona-Fernandes, 1982; Taniguchi et al., 1984; Daoulas et al., 1991; Andrades et al., 1996; Koumoundouros et al., 1997b, 2001a b, 2002; Faustino and Power 1998, 2001; Afonso et al., 2000; Boglione et al., 2001; Faustino 2002 ; Gavaia et al., 2002; Kihara et al., 2002; Moteki 2002; Favaloro and Mazzola 2003; Boglione et al., 2003; Matsuoka, 2003; Sfakianakis et al., 2004). Appearance of skeletal deformities in fish has been related to environmental, nutritional and genetical factors. Environmental factors associated to deformities include abiotic (light intensity, dissolved oxygen, temperature, pH, salinity, tank hydrodynamic, rearing system), biotic (bacteria, virus, fungi and parasites) and xenobiotic (algaecides, fungicides, herbicides, insecticides, industrial effluents and heavy metals). Among nutritional factors, the importance of essential fatty acids, phospholipids, amino acids, proteins and vitamins has being mainly emphasized. In relation to genetic factors both unigenic and polygenic have been described. Introduction 31 1.5.2.2.1-Environmental factors Light, temperature or salinity has been described as main environmental factors inducing skeleton deformities incidences in marine fish (Battaglene and Talbot, 1990; Polo et al., 1991; Mihelakakis and Yoshimatsu, 1998; Cobcroft et al., 2001; Sfakianakis et al., 2004). Other causes of damage to the larval skeleton integrity include mechanical-shock during embryonic or early larval development. Anomalous development of the swing bladder (Chatain, 1989,1990; Andrades, 1993;) and tank hydrodynamics variations (Chatain, 1994; Divanach et al., 1997; Koumoundouros et al., 1997a, b; Kihara et al., 2002) have been reported as causes of lordosis episodes. In addition, culture system, and particularly the intensification of the rearing techniques, has been described as a modulator of skeletal deformities occurrence. Thus, extensive and semiintensive systems have been associated to lower incidence of skeletal deformities than in intensive systems (Divanach and Kentouri, 1983; Divanach et al., 1996; Boglione et al., 2001; Koumoundouros et al., 2001a; Sfakianakis et al., 2004; Roo et al., 2005b; Giménez and Estévez, 2008b). Information about the effect of biotic environmental factors on skeletal deformities is scarce, being parasites one of the most studied factors. Parasites from mixosporea family have been described as skeletal deformities promoters in rainbow trout (Oncorhyncus mykiss), Korean amberjack (Seriola quinqueradiata) and perca (Perca fluviatilis) (Lom et al., 1991). Among xenobiotic factors related to skeletal deformities incidence, pesticides (Chun et al., 1981; Thi Hong Lien et al., 1997), herbicides (Koyama 1996), hydrocarbons (Grady et al., 1992), organic and organoclorade compounds (Lindesjöö et al., 1994) and metals (Slominska and Jezierska, 2000) have been found clearly correlated. Excess of antibiotics to treat several types of diseases has been also related to the induction of skeletal deformities in fish (Toften and Jobling, 1996). 1.5.2.2 2.-Nutritional factors In addition to environmental factors, nutritional factors such as dietary hormones, vitamins and fatty acids contents might directly act on bone and cartilage metabolism. Other indirect metabolic factors related to these processes are prostaglandins, cytoquines and growth factors (Watkins and Seifert, 2000a). In fish, nutritional factors such as excess or deficiencies of essential fatty acids and vitamins in broodstock diets and/or during the first stages of growth can Introduction 32 markedly alter the osteological development of embryo and larvae (Kanazawa et al., 1983; Akiyama et al., 1986; Knox et al., 1988; Chatain and Ounais-Guschemann, 1990; Afonso et al., 2000; Cahu et al., 2003; Saele et al., 2003; Hamre et al., 2005). Although, in fish it has not been shown a clear relation between dietary and cartilage and bone fatty acid composition, studies in other vertebrates as chickens and rats have shown that variations in the relation of n-3/n-6 series in the diet, alters bone and cartilages fatty acids composition (Xu et al., 1994; Watkins et al., 1991, 2000b; Liu et al., 2004). Nevertheless, skeletal deformities appearance in marine fish larvae have been suggested to be induced by alterations in the dietary essential fatty acids profile (Gapasin and Durai, 2001). However, the mechanisms by which fatty acids control the osteological development are clearly understood. Some authors have suggested that certain fatty acids regulate bone development by acting as gene modulators through specific nuclear active receptors, such as peroxysome proliferators active receptors (PPAR) that are join to DNA molecule as heterodymers with retinoic acid X receptors (RXR). These receptors act like transcription ligand-activated factors (Mangelsdorf et al., 1994) regulating genes involved in skeletal development during the larval ontogenesis. In this way, indirectly, essential fatty acids regulate bone development related genes expression and affect skeletogenesis. Still studies must be conducted to clearly elucidate the role of essential fatty acids in bone development and malformations occurrence in order to design effective diets, which are able to prevent these alterations (Cahu et al., 2003). More recently it has been suggested that an excess of certain essential fatty acids in the diets accelerates the osteoblast differentiation through a hyper regulation of α-retinoic X receptors and bone morphogenesis proteins (BMP4), inducing the occurrence of an excess of vertebrae in European seabass (Dicentrarchus labrax) (Villeneuve et al., 2005, 2006). In addition, essential amino acids, such as tryptophan, deficiencies have been long ago associated to deformities induction (Walton et al., 1984; Akiyama et al., 1985, 1986; Wilson 1989). Moreover, the inclusion of peptides (protein hydrolasates) in the diet along the larval development noticeably reduce skeletal malformations in European seabass larvae, (Cahu et al., 1999). Besides, as it is well known in other mammals, unbalanced vitamin C contents induce bone malformations in fish (Halver, 1989; Dedi et al., 1995; Takeuchi et al., 1995, 1998). Finally, there is also an important terathogenic ability in retinoic acid along human and animal embryogenesis, measured through the expression of Hox and Sonic Hedgehog (SHH) genes, and in fish a dietary excess of retinoic acid or some of its molecular derivatives Japanese flounder Introduction 33 larvae (Paralichthys olivaceus) increases the occurrence of vertebrae compression (Takeuchi et al., 1998). 1.5.2.3.3.-Genetic factors Andrades et al. (1996) related that most of the lordotic larvae observed (Sparus aurata), probably are lordotic larvae survivors, suggesting that primary causes of the lordosis appearance could be, among others, from genetic origin that can affect eggs during the embryonic development. Afonso et al. (2000), described the relation of a severe column deformity with the simultaneous appearance of lordosis, scoliosis and kyphosis to familiar association. Besides Astorga et al (2003a, b) demonstrated the effect of consanguinity in the appearance of the skeletal deformities of gilthead seabream, throughout crossing normal individuals with different levels of consanguinity (F=0.125; F=0.25), to the ages of 4, 14 and 35 days post-appearance and to the 194 days of age. Improvements in the production technology of red porgy (Pagrus pagrus) larvae and fry: Importance of rearing conditions and diet nutritional value on their quality OBJECTIVES Objectives 35 2.-OBJECTIVES The main objective of this study was “to improve the production technology of red porgy (Pagrus pagrus) larvae and fry”. Specific objectives are grouped in two complementary and consecutive phases: the first part was mainly based in a biological approach where specific aspects of the first developmental stages of this species such as visual and digestive system, osteology and apparition of skeletal deformities according with the rearing system and nutritional value of the live preys were described; whereas the second part, taking advantage of this knowledge aimed to improve the larval rearing protocol for this species. These objectives are addressed in four different studies included in the thesis: Study I. Development of red porgy larvae Pagrus pagrus (Linnaeus, 1758) visual system in relation with changes in the digestive tract and feeding habits. This study aims to describe in red porgy larvae, the development of the visual system and its correlation with that of the digestive system. This objective was addressed in order to obtain basic information in relation to some general culture conditions as photoperiod and light intensity changes, duration of green water use or feeding sequence. An experiment was done to achieve this objective. Study II. Osteological development and occurrence of skeletal deformities in red porgy Pagrus pagrus (Linnaeus, 1758) larvae cultured under different rearing techniques. This study aims to describe the effect of the intensification of the larval rearing techniques in relation to the osteological development and the incidence of skeletal malformations in red porgy. This objective was addressed in order to obtain basic information about the osteological development pattern and the incidence of the skeletal anomalies in this species that will allow determining the fry quality and suitability of the rearing techniques. Two experiments were done to achieve this objective. Objectives 36 Study III. Effect of DHA content in rotifers on the occurrence of skeletal deformities in red porgy Pagrus pagrus (Linnaeus, 1758). This study aims to describe the effect of certain nutritional factors on growth, survival and the incidence of skeletal malformations in the first stages of development of red porgy. This objective was addressed in order to identify the nutritional factors affecting larval performance and to improve the enrichment products for live preys fed to this species. Two experiments were done to achieve this objective. Study IV. Advances in rearing techniques of red porgy Pagrus pagrus, (Linnaeus, 1758): Comparison between intensive and semi-intensive larval rearing systems. This study aims to improve the larval rearing protocol for this species. To achieve this objective three experiments were conducted focusing the effect of light regimes, prey density and cofeeding protocol and comparing larval rearing systems. Improvements in the production technology of red porgy (Pagrus pagrus) larvae and fry: Importance of rearing conditions and diet nutritional value on their quality MATERIALS AND METHODS Materials and methods 43 Figure 26. Longitudinal section of the weaning and nursery rearing tanks. Figure 27. Longitudinal section, top view and different details of the water inlet and out lets of the weaning and nursery rearing tanks. Materials and methods 44 3.3.-Studied species Red porgy (Pagrus pagrus), is a teleost, belonging to Sparidae family, its detailed taxonomic classification is presented bellow. Phyllum: Chordata Superclass: Gnathostomata Class: Osteichthyes Orden: Perciformes Suborden: Percoidei Family: Sparidae Genus: Pagrus Species: Pagrus pagrus Figure 28. Adult fish, eggs, red porgy larvae and fry; on-land comercial facilities for red porgy on growing test. 3.3.1 Habitat Red porgy is an euritermal species that adapts to an wide range of temperatures and a great variety of habitats. It is generally found in rocky bottoms in a depth around 50 m, although small size individuals are frequently found in shallow waters (10-30 m). In the wild, red porgy diet is mainly based on mollusc and crustaceans, but in captivity it easily adapts to pellets diets. This is a protogynous species which develops female gonads during the first years of life and whose Materials and methods 45 spawning season commences in December or January in the western Atlantic (Ciechomski and Weiss, 1973) and can be prolonged until spring in the Canary Islands (Pajuelo and Lorenzo, 1996). In this geographical location and under captivity, spawning season starts at the beginning of March and is extended until the end of May without photo-thermal manipulation (Cejas et al., 1997). 3.3.2 Geographical distribution This species has been reported in different regions of the Mediterranean and Adriatic Sea, in the Atlantic, from British Islands to the south of Angola, including the Canary Islands, Azores and Madeira. It is also possible to locate it in the west Atlantic from New York to the south of Argentina (Manooch and Hassler, 1978) (Figure 29). Figure 29. Geographical distribution of Pagrus pagrus around the world (Fish Base, 2008). 3.3.3 Aquaculture perspectives Pagrus pagrus is one of the marine fish species proposed as potential candidate for the marine aquaculture diversification of the Atlantic coast of Spain and Mediterranean sea (Kentouri et al., 1995; Hernandez-Cruz et al., 1999). It has a high market price and a good acceptance what are indispensable conditions for a new species introduction. Commercial on-growing of common seabream, is still very limited, although some production statistic data were reported in Greece in 1999 with 100 tons (FEAP, 2006). Additionally, during the development of this study different on-growing experiences were carried out both offshore and inland in collaboration with local Materials and methods 46 commercial farms. Fry transport conditions have been described by Pavlidis et al., (2003), and recently data of fry production were reported in Greece with 2 million fry in years 2006, 2007 and 2008 (FEAP, 2008). while no data of on growing production is yet available. On pilot scale, the productions obtained in the Canary Islands have been increased in the last years, with 12.000 fry in 2002 to reach values close to 100.000 regularly produced in 2005 and 2006. Different studies of basic biology have described in detail the ontogeny of different organs (Socorro et al., 2001, Roo et a.,, 1999, Darias et al., 2005, 2007) also the detailed description of the osteological development of common seabream has been published by Socorro (2006). Studies on larval rearing of this species show different degree of success according to the culture conditions applied (Kentouri et al., 1995; Hernández-Cruz et al., 1999; Mihelakakis et al., 2001; Papandroulakis et al., 2004). 3.4.-Experimental conditions 3.4.1.-Live food cultures “Live food”, a term used to define the live organisms that are used as feed for marine fish larvae, were produced under the specific conditions within the different units. 3.4.1.1.-Phytoplankton culture For all the experimental trials, the Eustigmatophycea microalgae, Nannochloropsis sp. was used. The most important characteristics of this species were described by Maruyama et al., (1989) are shown in table III and Figure 30. Table III: Biological characteristics of Nannochlropsis sp. (Maruyama et al.,1989). Dimensions: 2-4 μm maximun diameter. Cellular shape: circular or oval shaped. Chloroplast shape: cup or oval shaped. Propagation: Binary fission Endoplasmic reticulum in the chloroplast: present. Tylacoids disposition: 3-tylacoids. Pigments: chlorophyll a, carothene, violaxanthyn, vauqueriaxanthyn. Materials and methods 47 Figure 30: Cell organization of Nannocloropsis sp. and cells vision in Neubauer chamber. The culture system employed for massive phytoplankton production was a “Bach type”. This was conducted in the live food production area, under volumes increasing from 50 to 230 and 460 l, using transparent polyethylene bags (Figure 31). Figure 31. Phytoplankton culture bags of 50, 230 and 460 l volume. Water was mechanically filtered through a polyester reinforced fibre glass filter (Mod.00689; Astral pool, Barcelona, Spain) filled with a sand bed of diverse sizes. Water was also sterilized by UV radiation at 254nm wavelength with industrial equipment (Mod. M-3PE-300; Wedeco AG, Herford, Germany). Natural salinity (37 ‰) was reduced by addition of fresh water to reach a final salinity of 25 ‰, which was verified by means of a portable refractometer (Mod. SZJ-S, Madrid, Spain). To avoid cells sedimentation, air was supplied throughout ceramics diffusers Materials and methods 48 settled at 15 cm of the bag bottom. Culture was performed under continuous photoperiod, combining natural with artificial illumination by means of fluorescent lights (Mod. TLD 58W/54-765, Philips, France), maintaining a minimum light intensity during the night of 9,500lux measured with a digital Luxometer (Mod. HT170N; Italy). Regardless the culture volume, the culture protocol followed a similar pattern: inoculation of microalgae (initial concentration of 1.5 x 106 cells.ml-1) in the bag or column previously filled with seawater (25 ‰) and fertilized with a commercial product (Nutri-Phyt; Easy Algae S.L, Puerto de Santa María, Spain) the first day of culture. The growing period lasted 8 to 10 days on average, when culture maximum density was reached (35 to 80 x 106cells.ml-1 ) depending on the culture volume, being generally lower in the larger volumes. At this stage, total volume was harvested and a new cycle started. Figure 32. Growth curve and correlation between cell concentration and culture turbidity. Culture evolution was daily checked by a photometric evaluation testing turbidity with a portable photometer (Mod.PF-11; Macherey-Nagel; Durew, Germany) and individual counts with haemocytometer (Mod. Neubauer, Germany) also, samples of different bags were randomly collected, to determine the cells concentration and to detect the presence of biological contamination agents in the culture media (Figure 32). Occasionally, contamination with the ciliate Euplotes sp. (Figure 33), was observed, being air supply the most common source of contamination. To prevent ciliates contamination, the addition of formaldehyde (38%) (Panreac, Spain) at a dose of 0.05 ml.l-1 of culture, gave good results killing 100% of the polluting agents without compromising phytoplankton cell viability. Concentration(Cells.ml-1) 0 20x10640x10660x10680x106100x106 Turbidity (FAU) 0 200 400 600 800 1000 1200 1400 Concentration vs Turbidity Regresión Days Siembra123456789 Concentration ( Cells.ml-1) 0 20x106 40x106 60x106 80x106 100x106 Nannochloropsis sp. Inoculum Materials and methods 49 Figure 33. Polluting organism (Euplotes sp) found in Nannochloropsis sp. culture. The phytoplankton produced, was used for different purposes within the hatchery process. Thus, 50l bags were commonly used to inoculate and up-scale phytoplankton to 230 l bags. Microalgae coming from 230 l bags were used to inoculate 460 l bags as well as for rotifers strain maintenance. Finally, the phytoplankton produced in 460 l bags was used for green water techniques in larvae culture and occasionally for rotifers feeding. 3.4.1.2.-Rotifers culture The rotifer Brachionus plicatilis was used in larval rearing experiences; adult individuals had an average total length of 240µm, being classified as L morphotype. Figure 34. Rotifers (Brachionus plicatilis) used in larval cultures. Materials and methods 50 Culture process comprised several phases (stock maintenance, pre-culture and massive culture) to ensure the quickly re-start of the culture after population failure or accident (Dhert, 1996). 3.4.1.2.1.–Stock maintenance and rotifers pre-culture Rotifer strain was maintained in 2 to 5l containers filled with Nannochloropsis sp. at a density of 20-25 x 106 cells.ml-1 with an initial concentration of 2-5 rot.ml-1. Culture was supplied with smooth ventilation in a temperate room under continue photoperiod. Once the culture reached 75-100 rot.ml-1, after 5-7 days, total volume was harvested and this was used to inoculate a greater volume, normally, 20 l bottles repeating afterwards the described protocol. Figure 35: Rotifers pre-culture bottles. 3.4.1.2.2.-Mass culture Rotifers production was carried out on cylinder conical fibre glass tanks with a total capacity of 1700 l (Figure 36) filled with a mixture of fresh and seawater to attain a salinity of 25 ppt. General culture conditions are presented in Table IV. Table IV: Culture conditions for rotifers mass production at ICCM experimental hatchery. Water: UV sterilized Temperature: 20-25ºC Salinity: 25 ppt Illumination: Natural light and photoperiod. Aeration: Strong, a single air diffuser settled at 80% of tank height. Oxygen: Pure O2 controlled injection when values drop under 3.5ppm. Materials and methods 51 Production cycles lasted 8 days (Table V). Initial rotifers density was 265 indv.ml-1; from day 4th of culture, harvest of 400 l volume was performed in alternative days, harvested volume being replaced by a mix of seawater and fresh water to reach a 25 ‰ salinity; the 8th day, the total volume was harvested and a new production cycle started. Routinely, after rotifers harvesting, a 1 min freshwater bath was applied to kill potential contaminants such as ciliates. Average total density and percentage of ovigerous females were daily calculated after individual counts (n=3) of 0.5 ml collected with a Micropipette (Mod. Eppendorf Research 100-1000 µl; Hamburg, Germany) from a sample randomly collected from the culture tank. A visual observation of the rotifers mobility was conducted to classify them into three categories: high, medium or low. The content of potential contaminants such as ciliates or copepods was classified in high, medium or low and water quality was checked reflecting the presence of debris and the viscosity increase in a visual estimation (clean, medium or dirty). Similarly, oxygen and temperature were registered twice a day at 9:00 and 15:00 hours by means of a portable probe (Mod. Handy Polaris, OxyGuard; Birkerød, Denmark). Rotifers feeding consisted in lyophilized baker yeast (Saccharomyces cerevisiae) supplied at a dose of 0.4g/106rotifers. The first day of culture yeast was supplemented with lyophilized microalgae 0.1g/106rotifers. Food was added manually at 09:00 and 15:00 hours, and automatically at 21:00 and 03:00 hours. Figure 36: Rotifers culture tanks and food automatic distribution system. Materials and methods 52 Table V. Average rotifers production sheet, according to described methodology (n=9). Rotifers production sheet Day Density (rot/ml) Volume (m3) Total (mill) Harvest (rot/ml) Egg bearing females (%) Food ration (g/mill) Food (g/tank) Food/fed (g/ration) O2 (ppm) Tª (ºC) 1 262.1 1.7 446 19.3% 0.5 225 37.5 7.0 22.9 2 251.5 1.7 428 22.1% 0.4 165 27.4 6.3 22.3 3 281.9 1.7 479 23.3% 0.4 197 32.9 4.3 22.1 4 307.6 1.7 523 72.4 17.9% 0.4 224 37.4 4.3 22.0 5 292.6 1.7 497 19.8% 0.4 187 31.1 5.1 22.0 6 293,0 1,7 498 68.9 21.6% 0.4 198 32.9 4.1 22.2 7 269.9 1.7 459 19.2% 0.4 166 27.6 3.9 22.4 8 265.1 1.7 451 451 17.8% - - 4.9 22.3 Rotifers harvesting was performed with a socket bag of 63 µm net size or with a rotifers concentrator made with the same net size. Figure 37. Rotifers harvesting and detail of harvesting bag. 3.4.1.2.3.-Rotifers enrichment Rotifer Brachionus plicatilis reared under the, previously described protocols are deficient in highly-unsaturated fatty acid (HUFA) and other nutrients, needing to be enriched previously to be fed to the larvae. Enrichment was performed in cylinder-conical fibre glass tanks 500 l Material and methods 59 Salt storage solution Artemia (kg) Salt (kg) Seawater (l) Final volume(l) Final concentration (g/l) 0.25 0.29 1.00 1.79 139.4 0.50 0.59 2.00 3.59 139.4 1.0 1.17 4.00 7.17 139.4 1.5 1.76 6.00 10.76 139.4 2.0 2.34 8.00 14.34 139.4 2.5 2.93 10.0 17.93 139.4 3.0 3.52 12.0 21.52 139.4 4.0 4.69 16.0 28.69 139.4 5.0 5.86 20.0 35.86 139.4 6.0 7.03 24.0 43.03 139.4 7.0 8.20 28.0 50.20 139.4 7.5 8.79 30.0 53.79 139.4 Notes Sea water: 4 l of sea water ( 37% ) per kg of dry cists. Salt: 330 g per l minus quantity of salt added thought sea water. Final Volume = Seawater + salt + cists (2 x dry weight) Concentration: Decapsulated cists/Final Volume Storage: in the dark and at low temperature (4ºC). Made by: Modification date: J.Roo 04/10/2008 Materials and methods 60 3.4.1.3.4.-Hatching Artemia was hatched according to the larval feeding needs. The protocol included tank disinfection and filtered and sterilized seawater filling. Cylinder conical tanks of 1700 l total volume were used and strong air supply and continue photoperiod was applied. Tanks were equipped with a heater to maintain water temperature at 28-29ºC. A density of 1.8 g of cists.l-1, were used, obtaining average hatchability rates of 95% after 24h and Artemia nauplii of 450650μm depending on the Artemia strain which were used in the transition feeding from rotifers to Artemia metanauplii (Figures 41). Figure 41. a) Artemia introduction in the hatching tanks; b) hatching tanks; c) newly hatched Artemia nauplii. 3.4.1.3.5.-Enrichment To improve nutritional quality of Artemia, an enrichment procedure was necessary. For this purpose, cylinder-conical fibre glass tanks 1700 l total volume were employed, strong aeration from the tank bottom and 24 h photoperiod were applied. Besides, an internal heater was used to maintain a culture temperature of 25-26ºC. Just hatched Artemia nauplii were introduce in the enrichment tank at a concentration of 250,000-300,000 nauplii.l-1. In all experimental trials the Easy DHA Selco (Inve, Dendermonde, Belgium) enrichment product was used. The enrichment last for 18-24 h with a concentration 0.6 gr.l-1, added in a single dose at the beginning of the enrichment process (h=0). Enriched nauplii were harvested in a sock net with 125µm mesh size, washed with seawater to eliminate residual lipid particles and concentrated in a 20l beaker to be counted and check the Materials and methods 61 enrichment success (Figure 42). Enriched nauplii were added to the larval tank both manually or by means of automatic distributors. Figure 42. a) Enrichment tank; b) enriched Artemia cold storage; c) success enrichment checking. 3.4.2.-Larval rearing 3.4.2.1 Eggs Marine fish eggs are in a biological stage that makes them easy to manipulate and transport. Nevertheless, inadequate manipulation can promote high embryos mortality or later negative effects such as low hatching rates, high larval mortality during the first days of culture or the appearance of different morpho-anatomical anomalies. Consequently, handling of fish eggs must be careful to avoid mortalities and to obtain high quality larvae. In the different trials of larval culture, eggs natural spawned from broodstock belonging to ICCM and the Instituto Español de Oceanografía, Centro Oceanográfico de Canarias (IEO-COC) were used. In both facilities, broodstocks were fed twice per week with commercial diets, complemented once a week with fresh fish and mollusc like cuttlefish, squid and mussels. Materials and methods 62 Figure 43. Adult red porgy (Pagrus pagrus) from ICCM broodstock. Fertilized eggs display positive buoyancy and they were gathered in a mesh collector of 500 µm, which receives tank water overflow (Figure 44a). Once harvested, eggs were placed in a decantation cone where different fractions were separated, the floating fraction mainly composed by viable eggs and the non floating one mainly composed by nonviable eggs (Figure 44b). When the origin of eggs was IEO-COC, they were transferred by airplane or boat, inside reinforced plastic containers of 20 l capacity inside individual expanded polystyrene boxes, which protect them and maintain stable temperature conditions during transport (Figure 44c). In each container, an average of 250,000 eggs at a concentration of about 25,000 eggs.l-1 were introduced. About 50% of the volume was filled with seawater an 50% with saturated oxygen gas (Figure 44d). Figure 44. a) Eggs collector, b) Decantation cone; c,d) Eggs transport preparation. Materials and methods 63 After arrival to the facilities, eggs were acclimatized to the local seawater physical-chemical parameters and a new separation to eliminate dead or damaged eggs during the transport was performed. Viable eggs were volumetrically counted and introduced in the rearing tanks (Figure 45). At the same time, six small cylindrical containers provided with water exchange were settled to calculate hatching rates and larval survival by day 3 after hatching before mouth opening (Figure 46). Figure 45. a) Eggs count; b) viable eggs; c) eggs introduction in the rearing tanks. Figure 46. a) Containers for hatching rates and larval survival determinations; b) dead eggs and unviable newly hatched larvae; c) good quality red porgy larvae. 3.4.2.2.-Semi-intensive larval rearing Semi-intensive system (SMIS) larval rearing was performed in the 40,000 l tanks previously described. In all rearing trials, fertilized eggs were stocked at a density of 5-6 eggs.l-1. Seawater Materials and methods 64 was previously filtered by a sand filter and pass through a UV sterilizer. Water renewal was increased from 10% of the total volume daily to more than 25% per h-1 at 30 dah. Water salinity was kept almost constant (37±0.5 ‰) in all the trials while temperature varied as specified in each trial. Larval culture was conducted under continuous photoperiod and changed to natural photoperiod in later stages (conditions being specify in each trial). Semi-intensive tanks were equipped with 4 fluorescent lights (Mod TLD 36W/54, Philips, France) that in combination with natural light coming from a translucent roof maintained an average light intensity between 1,000-3,500 lux measured in the water surface. Green water technique was used adding living phytoplankton (Nannochloropsis sp.) to maintain a concentration of 250 ± 100 x 103 cells.ml-1 in the rearing tanks. Feeding protocol included the use of rotifers (Brachionus plicatilis) at 4-5 rot ml-1 enriched with the different commercial or experimental emulsions from day 3 after hatching (dah) until larvae reached 8.0 mm total length around 25dah. Specific details of feeding periods are given in each trial. Rotifers were added to the larval tank twice a day (09:00; 15:00 hours). At 13dah, Artemia nauplii Instar I (AF type, INVE Aquaculture, Dendermonde, Belgium). were added once a day (11:00), in all the tanks at (0.25 A0 ml-1). From 15 to 17 dah Artemia Instar II enriched with Easy DHA Selco ® (INVE, Belgium), were added three times a day (09:00;15:00; 20:00 hours) at (0.50 A1 l-1) being the last day fed automatically distributed (Table VII). Prey concentration in the tanks was determined samplings the tank water collected twice a day (08:00; 14:00 hours) previously to the new fed addition. From 5dah till 25dah a surface skimmer were used to eliminate the lipid film derived from feeding enriched preys. Generally, larvae were fed commercial diets from 20 dah (Genma Micro;Skretting, France), manually supplied for the first 3 days and by means of automatic feeders (T-Drum feeders, Arvotec, Noruega) afterwards (Table VII). After 50dah the whole population was transferred to 10,000 l tanks, and kept under the same rearing conditions (1-10 indv l-1) in a flow through water system until 95dah when all the fish were individually counted and skeletal characterization was performed. 3.4.2.3.-Intensive larval rearing Larval rearing experiments with intensive technology were performed in 2,000 or 500 l tanks previously described. This technology is widely used in commercial hatcheries, with different variants according to general culture conditions such as tank size and form, water turnover, position and number of air diffusers, feeding sequence, prey enrichment products and/or weaning Materials and methods 65 diets. Generally this technique is based on the use of a high larval density that commonly varies between 50 and 150 larvae l-1.For all the rearing trials fertilized eggs were stocked at a density of 100-125 eggs.l-1. Seawater was previously filtered by a sand filter and pass through a UV sterilizer. Water renewal was decreased from 25% per h-1 at hatching to 10% of the total volume daily in the first larval stages, being progressively increased to more than 25% per h-1 at 30 dah. Water salinity was kept almost constant (37±0.5‰) for all the experiences, while temperature and oxygen varies along the trials and was daily measured. Larval culture was conducted under continuous photoperiod and changed to natural photoperiod in later stages (these conditions can vary in the different trials as it is specified in each of them). Intensive system rearing tanks were equipped with a single central light (Mod TLD 36W/54, Philips, France) that in combination with natural light coming from a translucent roof maintained an average light intensity between 1500-3500 lux measured in the water surface. With this technology, green water technique was used as well, adding living phytoplankton (Nannochloropsis sp.) to maintain a concentration of 250 ± 100 x 103 cells ml-1 in the rearing tanks. Feeding protocol includes the use of rotifers (Brachionus plicatilis) at 5-10 rot ml-1 enriched with the different commercial or experimental emulsions from day 3 after hatching (dah) until larvae reached 8.0 mm total length and rotifers were fed twice a day (09:00; 15:00 hours). At 13dah, Artemia Nauplii Instar I (AF Type, INVE Aquaculture, Dendermonde, Bélgica) was added once a day (11:00) at (0.25 A0 ml-1) .From 1517 dah Artemia Instar II enriched with Easy DHA Selco ® (INVE, Belgium), were added three times a day (09:00;15:00 and 20:00 hours) at (0.50-1.5 A1l-1) being the last feed of the day automatically distributed (Table VII). Prey concentration in the tanks was measured by sampling the tank water twice a day (08:00; 14:00 hours) before new fed addition. From 5 dah untill 25 dah a surface skimmer was used to eliminate the lipid film introduce with enriched live prey. Generally, larvae were fed commercial diets from 20 dah (Genma Micro, Skretting, Francia), manually supplied for the first 3 days and by means of automatic feeders (T-Drum feeders, Arvotec, Noruega), afterwards (Table VII). After 50 dah the whole population was transferred to 10,000 l tanks, and kept under the same rearing conditions (1-10 ind l-1) in a flow through water system until 95dah when all the fish were individually counted and skeletal characterization was performed. Materials and methods 66 Table VII. General culture conditions and feeding sequence during red porgy larval rearing Age (dah) Degree day Length (mm) Light Filters Water flow Tank cleaning Water Feeding sequence Eggs intro Natural Photoperiod Superior filter (mesh 315µm) 25%/day Clear water Endogenous feeding Haching 2 50 3.00 Photoperiod 24 h Ligth (15003000)(Natural+artifitial) 10%-25%/day Green water (250-350.000.cells.ml-1) Rotifers( 5-10) Indv.ml-1) 5 80-90 3.1-3.5 Surface cleaning 10 125-135 4.0-5.1 Nauplii (0,025-0,25) Manual dry Fed Co-feeding Protocol B 25%-50%-75%- 100%/day 15 175-190 5.5-6.4 Both 180-200 20 225-250 6.5-8.0 Down filter (mesh 500 µm) Metanauplii (0,25 -1 indv.ml-1) Manual dry Fed Co-feeding protocol A Photoperiod 12:12 (1500-3000 Lux) (Natural+artifitial light) 260-275 5%-15%-25%/hour Manual+ automatic dry fed (8-12% biomass day) 25 280-290 7.5-11.5 30 350 8.5-12.5 Bottom siphoning Clear water 35 375-400 9.5-14.5 Down filter (mesh, 1000µm) 40 450 15.0-18.5 Automatic dry fed (8-12% biomass day) 45 500 17-22.5 50 530-550 19.0-30.0 Materials and methods 67 3.4.2.4.-Nursery The weaning and nursery phase for all the trials described in the present study were carried out in identical conditions, regardless of the culture technology used during the larval phase but maintaining fish stocks separated according to the culture conditions employed during the larval phase. Tanks of 10,000 capacity previously described were used, (Figure 47). During this stage, unfiltered seawater was used, with a water turnover of 50 % tank volume h-1. Salinity was almost constant during all the experiences (37 ‰) and dissolved oxygen varied around 5.5 to 7.5 ppm being controlled and supply when necessary by means of an automatic control system (Mod. Multi-channel, OxyGuard; Birkerød, Denmark) (Figure 47). The temperature was daily measured with a fluctuation between 20-22ºC in the different experiments. The illumination was a combination of natural and artificial light with a photoperiod of 12 to 18 h. Feeding in this phase was exclusively based on commercial diets (Genma; Skretting; France) combining manual and automatic feeding (Mod. T-Drum feeders, Arvotec; Norway), (Figure 47). Figure 47. a) Dissolved oxygen control unit; b) automatic feeder. During this phase when the tank population weight dispersion was higher than 30%, fry grading was performed using commercial bar graders (Mod. Rods, Catvis; Holland) according to the fry size (Figure 48). Materials and methods 68 Figure 48. a) Fry grading procedure; b) grids detail; c) red porgy fry. Tank bottom cleaning was daily performed by siphoning uneaten food and dead fish; that were individually counted for survival adjustment. Materials and methods 75 Figure 52. Deformity visual evaluations, X-ray plate’s preparation and mammography obtained to conduct detailed studies. 3.5.7.-Meristic determinations In a specific trial, meristic determinations of total number of vertebrae (including urostyle) and number of pleural ribs were carried out in 95 dah juveniles (n=300) from each treatment. According with Matsuoka (2003), all vertebrae with 2 neural spines and/or 2 hemal spines were considered to be formed by fusion and counted as two vertebrae. Figure 53. Vertebrae and ribs identification. Materials and methods 76 3.6.-Biochemical analyses During the course of the different experiences, samples of products used for the production and enrichment of the live prey were taken (rotifers and Artemia), dry feed samples and live preys before and after their enrichment were also taken. In addition, larval samples of different ages from each rearing tank and treatments were collected. Once collected, samples of live prey and fish larvae were placed on a 63µm mesh, washed with fresh and distilled water and residual water blotted with dry paper. Samples were frozen (-80ºC) in hermetic bags under nitrogen atmosphere, for its later analysis. The biochemical analyses were made in the laboratory of Instituto Universitario de Sanidad Animal y Seguridad Alimentaria (IUSA; ULPGC). Determinations of dry matter, ash, protein and lipid content as well as fatty acids were performed. All the analyses were performed at least in triplicate. 3.6.1.-Dry matter content It was determined following the Official methodology of the American Chemical Analysts Association of (AOAC, 1995). Dry matter content was determined after drying the fresh known sample quantity (Pi) in an oven at 105 ºC until constant weight was obtained (Pf). Before being weighted, the samples were introduce in a desecator for 30min to ambient temperature adaptation and finally they were weighted to obtain final data. The dry matter content was obtained by the following expression. 3.6.2.-Ash content. The ash content was determined after incineration of a well-known amount of sample (Pm) in a Muffla oven, at 450ºC during 24 hours, remaining ashes amount was recorded (Pc) and weight until constant weight according to the AOAC (1995). Final ash content was obtained applying the following expression: %DM = ((Pi - Pf) × 100)/Pi %Ash = (100 x Pm)/Pc Materials and methods 77 3.6.3.-Protein content The protein content, calculated from the total nitrogen content of the samples, was determined by Kjeldahl method. According to AOAC (1995), the technique consists in the sulfuric acid sample digestion at 420ºC in presence of a copper catalyst for one hour. This is, followed by a distillation with NaOH using saturated boric acid 40% as receptor substance in the distilling unit (Mod. Foss Tecator, 1002, Höganäs, Sweden). Finally a valuation with HCl 0.1 M is made. In order to calculate the protein content, the following expression was applied: Where V = HCl volume for valuation in ml P = Measured of the valoration of the patterns in ml N = Normality of the HCl Pm= Nitrogen molecular weight (14.007) F = Conversion empirical factor that has a value of 6.25 Ms = Sample weight in mg 3.6.4.-Total lipids content Total lipids were extracted according to Folch et al. (1957). The methodology commenced taking a sample amount between 50-200mg and homogenise it in an Ultra Turrax (IKA-Werke, T25 BASIC, Germany, Staufen) at 11,000 rpm during 5min in a solution of 5ml of Chloroform: Methanol (2: 1) with 0,01% of BHT. The resulting solution was filtered at reduced pressure through glass wool and adding KCl at 0.88%, to increase the water phase polarity. After decantation and centrifugation at 2000rpm during 5min the watery and organic phases were separated. Once watery phase was eliminated, N2 current was used to evaporate until completed dryness. Finally total lipid content was gravimetrically determined. 3.6.5.-Fatty acids content The extracted total lipids were trans-esterified according to the method of Christie (1989). In this procedure a solution of Toluene with BHT and another with Methanol and Sulphuric acid at 1% was added to the sample. The mixture was strongly shaken to improve lipids dissolution. Afterwards, the container was filled with N2 and sealed. This mixture was left in shaking incubation for 16 hours at 50ºC. After this time, the sample was let cool and pure distilled water and Hexane: Dyetil 1:1 Ether with BHT at 0.01% were added. The purified FAMES were %Protein =(V-P) x N x Pm x F/M Materials and methods 78 evaporated to dryness with N2 and hence weighed. Finally the FAMES concentration was dilute with hexane and stored in vials in a -80ºC fridge. Fatty acid methyl esters were analyzed using a gas chromatograph (Mod. Shimadzu GC-14A; Analytical instrument division, Kyoto, Japon), with a flame ionization detector and a Supelcowax-10 fused-silica capillary column 30m length x 0.32 mm I.D. (Supelco, Inc., Bellefonte, EE.UU). Helium was used as carrier gas with the following gas pressures: He 1kg.cm-2, H2 0.5 kg.cm-2, N2 1 kg.cm-2, air 0.5 kg.cm-2. Conditions were: injector temperature 250 ºC, column temperature 180 ºC during 10 min, increasing afterwards to 215ºC at a rate of 2.5ºC min-1 and maintained at 215ºC for 15 min. Fatty acids were identified by reference to a wellcharacterized fish oil (EPA 28). 3.7.-Statistical analysis Results are expressed as mean ± standard deviation. The statistical analyses were performed with program SPSS Version 14.0 (SPSS Chicago, Illinois, 1999). The data of each experiment were compared statistically by means of T-Student test (Sokal and Rolf, 1995) when two treatments were established or with variance analysis (ANOVA) if the number of treatments were greater. As general criteria 5% confidence level was applied. If statistically significant differences with the ANOVA were detected, the differences among means were detected the Tukey multiple means comparison test. When the variances were heterogeneous and/or the data were not normally distributed, these were tried to make them homocedastic and the data normally distributed transforming them to logarithms or with arc sine function. If heterogeneity or the normal distribution were not obtained, the non-parametric test of Kolmogorov-Smirnov when two treatments comparison or if a greater nº of treatments the Games-Howell test was used. Finally, to study the fry quality log-linear analysis, with Pearson χi (Sokal and Rolf., 1995) were applied. 3.8.-Nomenclature of the referenced species The common names in English of the species referenced in this work were taken from FAO “Fishbase” data base. Improvements in the production technology of red porgy (Pagrus pagrus) larvae and fry: Importance of rearing conditions and diet nutritional value on their quality Study I.-Development of red porgy larvae Pagrus pagrus (Linnaeus, 1758) visual system in relation with changes in the digestive tract and feeding habits Aquaculture 179 (1999) 499-512 Study I Aquaculture 179 (1999) 499-512 79 Corresponding author. Tel: +34 928 13 29 00/04; fax: +34 928 13 20 08. E-mail address: [email protected] Development of red porgy Pagrus pagrus (Linnaeus, 1758) visual system in relation with changes in the digestive tract and larval feeding habits Francisco J. Roo a, Juan A. Socorro a, María S. Izquierdo b, María J. Caballero b, Carmen M. Hernández-Cruz b, Antonio Fernández c, Hipólito Fernández-Palacios a. a Instituto Canario de Ciencias Marinas, Gobierno de Canarias, PO Box 56, E-35200 Telde, Las Palmas, Canary Islands, Spain b Dpto. de Biología, Universidad de Las Palmas de Gran Canaria, PO Box 550, E-35017 Tafira Baja, Las Palmas, Canary Islands, Spain c Dpto. de Morfología, Facultad de Veterinaria, Universidad de Las Palmas de Gran Canaria, Francisco Inglott Artiles,12 35016-Las Palmas, Canary Islands, Spain Abstract Red porgy Pagrus pagrus larvae, like other sparids such as red sea bream are visual feeders. The normal development of the visual system is essential for successful prey capture and predator avoidance, leading to increased larval growth and survival. The aim of this work is to characterise the development of visual organs in relation to changes in the digestive system and feeding habits. Twenty five larvae from hatching to day 29 were daily collected from the rearing tank, fixed in formalin, embedded in paraffin, 5 µm sectioned and stained with haematoxylin and eosin (H&E) and Periodic Acid Shift Reactive-Haematoxiline (PAS-Hx). Light microscopy was used to study changes in ocular morphology with respect to digestive system development. At hatching, eye and digestive system of Pagrus pagrus larvae have no function. However, at day 3 posthatch, when the mouth opens, the larvae must be ready for prey capture and digestion. Despite this, few day 3 larvae had food in the digestive tract. At day 4 photoreceptors were well developed in the eye, pigmentation pattern was complete and thus the visual system was completely ready for prey capture. This development coincided with detection of digestive activity in the midgut and most of larvae starting to take food. The results of this study suggest that the adequate development of the visual system is important to establish the start of exogenous feeding. Besides, the appearance of rod cells increase larval photosensitivity and suggest that changes in lighting regimes could be necessary throughout the larval phase. Keywords: Pagrus pagrus, Red porgy, New species, Visual system, Digestive tract, Histology Abbreviations: A: amacrine cells; B: bipolar cells; C: cornea; CR: cartilaginous ring; DHA: docosahexaenoic acid; EFA: essential fatty acids; EPA: eicosapentaenoic acid; G: ganglion cell layer; HG: hindgut; H: Horizontal cells; INL: inner nuclear layer; IPL: inner plexiform layer; ILCV: ileocecal valve; LEC: lens epithelial cells; L: lens; LRM: lens retractor muscle; MG: Midgut; ON: optic nerve; ONL: outer nuclear layer; OPL: outer plexiform layer; P: pigments; PN: photoreceptor nuclei; PRES: photoreceptors external segments; PRIS: photoreceptors inner segments; RN: rod nuclei. Study I Aquaculture 179 (1999) 499-512 80 1. Introduction Red porgy Pagrus pagrus (Linnaeus, 1758) (Osteichthyes, Sparidae) is a species found on Eastern Atlantic coasts: Gibraltar Straits to 15°N (occasionally to 20°N) including Madeira and the Canary Islands; in the Mediterranean and northward to the British Isles, (Bauchot, 1987; Bauchot and Hureau, 1990). It is found over hard (rock and rubble) or sandy bottoms (the young frequently found on seagrass beds) down to about 250 m depth, often above 150 m (Franquet and Brito, 1995). Red porgy is a characteristic pelagic spawner producing pelagic eggs (1.00 + 0.02mm diameter). Spawning occurs between January and April, mainly when the water temperature reaches 18 to 22 ºC (Hernández-Cruz et al., 1990). This species is being currently investigated as a potential aquaculture candidate (Kentouri et al., 1995; Stephanou et al., 1995; Hernández-Cruz et al., 1997; Socorro et al., 1997). At hatching the larvae has a non functional visual system, together with a poorly structured mouth and limited locomotion control similar to sea bream Sparus aurata (Blaxter, 1986). Larvae hatched with relatively small yolk reserves and, as a result, they must start feeding soon after hatching. This requires the development of organs and systems involved in the capture and digestion of the food. Digestive and visual systems must be developed at the onset of feeding (Govoni et al., 1986) Fish larvae are usually visual feeders, indicating that vision plays an important role in larval orientation at this stage (Blaxter, 1986). In sparids such as Pagrus major (Kawamura,1984) and Pagrus auratus (Pankhurst,1996) the most important changes in the eye structure occur in the lecitotrophic larvae as a preparation for prey capture. These changes should be coincident with digestive system development to start feeding as in Sparus aurata (Sarasquete et al., 1995; Hernández-Cruz et al., 1990). Other aspects of visual performance in relation to feeding have been investigated, especially the reception distance for food, which increases with body length (Blaxter, 1986). This study aimed to shows the parallelism between the development of the digestive and visual system and the importance of both for the beginning of prey capture and predator avoidance. It has implications for hatchery management because an appropriate choice of prey size and lighting regimes will contribute to the success of larval rearing. Study I Aquaculture 179 (1999) 499-512 81 2. Materials and methods The experiment was carried out from March 27th to April 26th (1998) at the Canary Institute of Marine Sciences. Eggs obtained from a wild stock of Pagrus pagrus by spontaneous spawning were distributed into 500 l fibreglass tank (100 eggs.l-1) filled with seawater. Larval development occurred between 20.5 to 21.8 ºC. Natural photoperiod of approximately 12h light was used. Larvae were feed with rotifers Brachionus plicatilis (L strain) from day 4 after hatching, at a concentration of 10 rot.ml-1 in the larval culture tank. Rotifers were mass-cultured using bakers yeast and Nannochloropsis sp. (Eustigmatophycea). After day 20, Artemia nauplii were offered to larvae. Larval rearing procedure followed protocols described by Hernández-Cruz et al, (1990). Newly hatched larvae of 2.77mm total length were reared up to twenty-nine days and sampled daily. For that purpose, 20 larvae total length (TL), standard length (SL), head height (HH) and eye diameter (ED) were measured using a profile projector (Nikon V-12A). 2.1. Light microscopy Another 25 red porgy larvae were fixed in 10% buffered formaldehyde for histological studies. All fish were sacrificed during the photo phase so that the eyes were adapted to light at the time of fixation. Larvae were then dehydrated in an ethanol series, embedded in paraffin, serially sectioned at 4-5μm, stained with haematoxylin and eosin (H&E) and Periodic Acid Shift Reactive-Haematoxylin (PAS-Hx) (García del Moral, 1993). 3. Results At hatching, the eye lens (L) of P. pagrus showed undifferentiated cells having a spherical distribution. Retinal cells (R ) are arranged radially and optic nerve were observed. At this stage, visual pigments were not present and some precursors of the photoreceptors nuclei cells (PN) were beginning to develop in the external part of the retina (Plate I). In the digestive tract, larvae had a simple undifferentiated straight gut linked to a non-structured mouth and anus. The lens structure of one day old larva had two differentiated layers: an external layer of epithelial cells (LEC) surrounded by a non nucleated lens fibers layer formed by highly modified epithelial cells. On day 1 the retina also showed well defined layers, outer nuclear layer (ONL) composed by columnar nuclear bodies of photoreceptors (presumptive cones) and an inner nuclear layer (INL). A ganglion cell layer (G) formed by spherical nuclei of ganglion cells. Inner (IPL) and outer plexiform layers (OPL) showed the first signs of formation. At this stage several nuclei of pigment cells (P) were seen up the outer nuclear layer and a cartilaginous ring (CR) was Study I Aquaculture 179 (1999) 499-512 82 present (Plate II). At this time in the foregut a short brush border was observed, with a digestive lumen being found in some larvae from the foregut to the hindgut. In two-day-old larvae, the nuclei photoreceptor in the outer nuclear layer increased in length. Inner and outer plexiform layers were well differentiated and a single layer of horizontal cells were present, down to the external plexiform layer. There was an increase in pigment contents. Lens retractor muscle was sketched and iris structure was better defined than days before. In the lens structure, there was an increase in the content of lens fibers and the lens epithelial cells were slender. The digestive tract, initially straight, presented at this stage a small curvature in the last third of its length. The hindgut, which was not pressured by the yolk sac, presented a clear digestive lumen. The brush border in the hindgut was less evident than in the foregut. The third day after hatching the lens was well defined by a single epithelial cell layer with PAS affinity, which covered the lens fibres. Photoreceptor cells were completely differentiated. Internal segment (PRIS) and external segment (PRES) contained visual pigments at this stage. Pigmented epithelial (PE) was well defined. Lens retractor muscle (LRM) was present and reached the lens, and the iris (I) was well defined (Plate III). At day 3 post-hatch, the digestive system showed an increase in length at the anterior zone by a multiplication of the cell layers. In other parts of the digestive gut there was only a simple epithelium layer. At this time, the ileocecal valve (ILCV) was formed, which separates midgut (MG) and hindgut (HG) (Plate IV). The brush border was seen from the most anterior part of foregut to the end of the intestinal tract. From day 3 post-hatch, the larval digestive tube could be divided into several parts: an oesophagus composed of a simple plane epithelium layer; a foregut formed by a simple cubical epithelium layer; a midgut where the epithelium layer was columnar, and a hindgut from the ileocecal valve composed of a columnar epithelium also. By the fourth day, a clear differentiation of the retina layers was found. Pigmentation was completed and the eyes appeared like an adult fish eye (Plate V). The eye diameter of red porgy continued increasing in accordance with body size increment. In this way, during retinal growth, cones and other cell types were added concentrically at the retinal germinal margin. Horizontal and amacrine cells were clearly observed within the inner nuclear layer. There was an increase in length in the outer and inner segment of the photoreceptors. Study I Aquaculture 179 (1999) 499-512 83 On day 4, the digestive tract was developed and had several invaginations and an abundant brush border with columnar epithelium. The midgut of four old day larvae had digestive activity showing PAS affinity in the brush border. The ileocecal valve was better developed than on the third day. The larvae had started to ingest food and the anus was opened. Food in the digestive tract was seen in most of the larvae at this time. During this time there was an increase in eye diameter which was significantly related to larval total length r2 = 0.987 and head height r2= 0.989 (Figures 13). At day 10 after hatch, the sclera (S) started to differentiate but there were not significant changes in the retina. Over the following days, an increase in length of the eye occurred but one important change in the visual system occurred at 20 day post-hatch. At this time spherical nuclei were seen in the basal zone of ONL, this type of cells were rod precursor nuclei (RN) which in following days increased in number (Plate VI). At the same time there were changes in the digestive tract with gastric glands appearing. On day 25 post-hatch there was a clear increase in rods nuclei cells and a single layer of them were seen under the cones nuclear layer (Plate VII). Cornea, sclera and iris were totally developed now. From day 25 post-hatch there was an increase in length but no more changes in eye structure were observed. 4. Discussion Red porgy eye structure was similar to those described by Kawamura, (1984).for P.major, lens with undifferentiated cells disposed spherically, retinal cells on radial disposition and nerve optic cup were observed at this stage. The eye was not pigmented and some precursors of the photoreceptors nuclei cells were sketched in the external part of the retina. One-day-old larvae had all the structural elements necessary for visual function, but most of them were not complete. This was an indication that the eye was about to be functional, Kawamura, (1984) found that the visual system of P. major is functional at 36 h after hatch when visual cells and pigments are present and nerve optic fibbers connect with the optic tectum. In red porgy, the visual system could not be functional at 28 h, principally because the pigmentation pattern, responsible for photon absorption was very sparse at this stage. The digestive system had started to differentiate. The third day after hatching was one of the most important days for the larva, which need to be ready for prey capture and digestion. Although histologically the larvae appears to be prepared and had their mouth opened, only a few of them had food in the digestive tract at this time. This fact could be due to a learning delay between the larvae. Study I Aquaculture 179 (1999) 499-512 90 Plate III. Light micrograph of a longitudinal section through the eye of P. pagrus larvae at 3rd day after hatching (3.77mm TL) (H&E). Pigmentation pattern was complete (PE), and the iris (I) was present, cornea (C) and lens epithelial cells (LEC) was well differentiated, lens retractor muscle (LRM) was patent. Study I Aquaculture 179 (1999) 499-512 91 Plate IV. Light micrograph of a longitudinal section through the digestive system of P. pagrus larvae at 3rd day after hatching. Midgut(MG),ileocecal valve (ILCV), hindgut (HG), (3.77mm TL)(H&E). Study I Aquaculture 179 (1999) 499-512 92 Plate V. Diagram of P. pagrus retina layers. Pigment epithelium layer (PE),outer nuclear layer (ONL),outer plexiform layer (OPL),inner nuclear layer (INL),horizontal cells (H),amacrine cells (A),bipolar cells (B), inner plexiform layer (IPL), ganglion cell layer (G), optic nerve (ON). ONL OPL INL IPL ON Study I Aquaculture 179 (1999) 499-512 93 Plate VI. Light micrograph of a longitudinal section through the retina of P. pagrus larvae at day 20 after hatching (6.29mm TL). In the retina at day 20 an spherical nuclei were seen added to the basal zone of ONL, this type cells were rod precursor nuclei (RN) and in following days increased in number, sclera was present too (S) (H&E). Study I Aquaculture 179 (1999) 499-512 94 Plate VII. Light micrograph of a longitudinal section through the retina of P. pagrus larvae at day 25 after hatching (6.29mm). Rod precursor nuclei layer (RN) (H&E). Study I Aquaculture 179 (1999) 499-512 95 List of figures. Figure 1. Daily evolution of different morphological parameters, total length, standard length, head height and eye diameter in Pagrus pagrus larvae. 14 +Totallon¡¡th 12 •SIlIndard length '* He><! heI¡¡ht --- [] El" diam....- .... _- --- • 10 ---- --.-.- - E- ........ -•• _ ... __ . E8 -_. o• - ~ ..... _-- -:•• --- •• c, ---- j• 611• e11 •"'""""" .. " ... •• ~ ----- • -•• 4oo¡¡1•1 • ••** 2 ** ** ... .Jr*~*******~: ****** ee eeeeeee e ee O DODlJDO O5 10 15 20 25 30 Days Study I Aquaculture 179 (1999) 499-512 96 Figure 2. Relationship between the total length (TL) and the eye diameter (DE) for P. pagrus larvae. ,. , .• oo. 01"". "no" I '.' ~ •• "17 N.'" '.' I, ,. •,. • O, O, O ~ , , • • W" " T""'lenglh ("""1 Study I Aquaculture 179 (1999) 499-512 97 Figure 3. Relationship between the head height (HH) and the eye diameter (DE) for P. pagrus larvae. [O_.OO1O.<U>OII .... ~·o_ "_,. , .• , .. í '.' I, •• ... ,o .• w o., o , o o., , 1,5 2 U Iioad height{nvn) ,"• Study II. Osteological development and occurrence of skeletal deformities in red porgy Pagrus pagrus (Linnaeus, 1758) larvae cultured under different rearing techniques Journal of fish Biology (2008), (submitted) Improvements in the production technology of red porgy (Pagrus pagrus) larvae and fry: Importance of rearing conditions and diet nutritional value on their quality .' .. Study II Journal of fish Biology (2008), (submitted) 99 Corresponding author. Tel: +34 928 13 29 00/04; fax: +34 928 13 20 08. E-mail address: [email protected] Osteological development and occurrence of skeletal deformities in red porgy Pagrus pagrus (Linnaeus, 1758) larvae cultured under different rearing techniques. Francisco J. Roo, Juan A. Socorro, Carmen M. Hernández-Cruz, Hipólito FernándezPalacios, María S. Izquierdo Grupo de Investigación en Acuicultura (ICCM & ULPGC), PO Box 56, E-35200 Telde, Las Palmas, Canary Islands, Spain Abstract Red porgy is a candidate species for aquaculture diversification, because of its relative fast growth and good adaptability to culture conditions. Standard techniques and feeding products for massive larval rearing have to be improved and their relation to fry quality and skeleton anomalies occurrence have not been studied yet. The objective of the present study was to describe the osteological development and the occurrence of skeletal deformities in Pagrus pagrus larvae in relation to the intensification of the rearing system. Eggs obtained from natural spawning, were cultured under two different rearing systems: intensive (100 eggs.l-1) in 2 m3 and semi-intensive (mesocosm) system, (5 eggs.l-1) in 40 m3 cylinderconical tanks. Fish samples were periodically collected along the development from hatching to juveniles (95days after hatching). Osteological development, meristic counts and the presence of skeleton abnormalities were evaluated. Thus, the general pattern of the osteological development for red porgy was similar between fish from both culture systems. However, the ontogeny of the skeleton structures differed between the rearing systems. In addition a significant interaction was found between meristic counts (total number of vertebrae) and the type of rearing system used, fish from the intensive system showing a higher number of fish with an extra vertebrae (10 abdominal+15 caudal). Furthermore, despite the external appearance of the juveniles being similar to wild standards, X-ray studies revealed a high number of fish with skeleton abnormalities regardless of the rearing system (Semi-intensive:38.8%; Intensive:46.5 %). in addition, no significant interaction was found between the highest incidence skeleton anomalies (lordosis and presence of fused vertebrae) and rearing system. However, cranial abnormalities and kyphosis incidence were significantly higher in intensive system cultured red porgy. Moreover, the position of fused vertebrae in this fish was located mainly in the caudal area instead of pre-hemal area for semi-intensive system reared red porgy. Present results, report the osteological description and described for first time the most important skeletal malformation associated with this specie. This information is a useful tool to adapt and improve larval rearing protocols for Pagrus pagrus. Keywords: Pagrus pagrus, Larvae, Development, Skeleton, Abnormalities, Rearing techniques, Mesocosms, Culture system Abbreviations: ARA: Arachydonic Acid, BMP4: Bone morphogenetic protein 4; DAH: days after hatching; DHA: Docosahexaenoic Acid; DPA: Docosapentaenoic acid; EFA: Essential fatty acids; EPA: Docosapentaenoic Acid; FA: Fatty acid; HUFA: Highly unsaturated fatty acids; RXR: Retinoid X receptor; SGR: specific growth rate; TFA: Total fatty Acids; Study II Journal of fish Biology (2008), (submitted) 106 than related fish from lower latitudes and warmer waters (Iguchi et al., 2006). On the contrary, Georgakopoulou et al. (2007) did not find any vertebral number variation when European sea bass larvae were reared under a range of 15-20ºC. Salinity also seems to affect vertebral number as it has been reported by Boumaiza et al. (1981) for A. fasciatus. Besides, genetically distinct populations as well as siblings may have divergent responses in the number of vertebrae to the same environmental factor (Ali& Lindsey, 1974; Beacham and Murray, 1986). However, in the present study there were no significant differences in water temperature, salinity or genetic background, between the two culture systems assayed and only the culture intensiveness differed. Nevertheless, despite both rearing systems used the same type of food, prey density (Semiintensive: 3-5 vs Intensive: 7.5-10 rot.ml-1) differs and the nutritional quality of the rotifers may become different, once they are in the rearing tank (authors unpublished data). Indeed, supernumerary vertebra in European sea bass larvae has been related to nutritional unbalances such as an excess of HUFA which accelerated the osteoblast differentiation process through the up-regulation of Retinoid X receptor α and BMP4, (Villeneuve et al., 2005, 2006). Skeletal abnormalities were higher in the intensive system and similar to those reported for other species such as Sparus aurata, (Boglione et al., 2001), Diplodus puntazzo and Pagellus erythinus (Boglione et al., 2003; Sfakianakis et al., 2004), Pagrus major (Hattori et al., 2003), and Solea senegalensis (Gavaia et al., 2002). The effect of the rearing system in relation to system intensification has been pointed out as a cause of skeletal abnormalities in other species, (Divanach et al., (1996), Koumoundourous et al., (1997a,b, 2001), Boglione et al., (2001), (Sfakianakis et al., 2004) and Roo et al., (2005). Nevertheless, deformities found in semiintensive reared fish were also considered very high in comparison with the values obtained in other species produced by this system. For instance, historical data from the last six years of studies with Sparus aurata in our facilities showed a percent of deformities 3 to 6 fold times higher in larvae reared in the intensive system than semi-intensive ones (Roo et al., 2005), whereas in red porgy deformities were only a 8 % higher in the intensive system suggesting the effect of different causative factors, in addition to the rearing system. The impact of cranial abnormalities and kyphosis increased in fish reared under the intensive system. These anomalies have been described in other species such as larval European sea bass, Dicentrachus labrax, (Barahona-Fernandes,1982), striped trumpeter, Latris lineata, (Cobcroft et al., 2001), bluefin tuna, Thunnus orientalis, (Shimizu and Takeuchi, 2002) or yellowtail kingfish, Seriola lalandi (Cobcroft et al., 2004). Particularly, the occurrence of cranial abnormalities, such as lack or folding of opercula, upperlower jaw reduction or twisted jaw are very common in different fish species (Fraser and de Nys, Study II Journal of fish Biology (2008), (submitted) 107 2005). Beraldo et al. (2003) reported the presence of the opercular fold as the first sign of opercular abnormality in 25-40days old gilthead sea bream. Reared red porgy presented a very low incidence of opercula deformities that could be related to the long opercula spines found in this species from early stages, which would restrict the folding of the operculum into the gill chamber. Other cranial deformities mainly related to upper-jaw reduction and cross bite jaw were increased in red porgy reared in the intensive system and could be related to nutritional (highly unsaturated fatty acids, dietary phospholipids and vitamins) and mechanical (nose-walling effect) factors (Kanazawa et al., 1983; Cobcroft et al., 2001; Roo et al., 2008). Kyphosis in reared larvae has been cited in different culture species, like gilthead seabream or European sea bass. Koumoundouros et al., (2002) reported the presence of kyphosis in European sea bass larvae closely associated to branchiostegal ray anomalies. Both skeletal elements are membrane bones, which develop at the same ontogeny phase and probably are sensitive to the same causative factors, being nutrition proposed as an important factor affecting this type of anomaly (Koumoundouros et al., 2002). Kyphosis has been associated with swim bladder overinflation and overfilling of the digestive tract, which induce a dorsal curvature of the notochord in the region above the swim bladder because of excessive pressure on this region (Grotmol et al., 2005). In the present study, red porgy larvae showing abnormally large swim bladder were rarely found and most of them, floating in the water surface, were removed by floating skimmers or dead in 1-2 days. Hence, swim bladder over-inflation is unlike to be a main cause of the kyphosis incidence found in red porgy. However, red porgy larvae are very voracious and frequently show an abdomen distended by the digestive tract overfilling with large quantities of Artemia, which press the cephalic vertebrae and might contribute to a greater kyphosis incidence (Figure 9g). In both rearing systems the most common skeleton anomalies were, vertebral column disorders, lordosis and fused vertebrae. Tank hydrodynamic has been considered the most important causative factor for haemal lordosis (Chatain, 1994; Andrades et al., 1996; Divanach et al., 1997). In agreement with this hypothesis, lordosis apparition in red porgy was located between the 8th and 12th vertebra, which is the vertebral region, which supports the highest muscle pressure during swimming (Kihara et al., 2002). No effect of the culture system was found in the incidence of lordosis, denoting a similar quality in tank hydrodynamic among systems. Additionally, most of the lordotic specimens showed the presence of another abnormalities such as fused vertebrae, reductions or excessive number of vertebrae, as previously described in Pagrus major (Matsuoka, 2003; Hattori et al., 2003). Fused vertebra is one of the most important types of deformity for juvenile quality, since it alters fish shape and length. The high incidence of fused vertebrae found in red porgy was in agreement Study II Journal of fish Biology (2008), (submitted) 108 with the results found for other species such as Sparus aurata, (Boglione et al., 2001), Pagrus major (Hattori et al., 2003) and Senegalese sole (Gavaia et al., 2002). Despite no effect of the rearing system on the percentage of fish bearing this deformity, a significant relationship was found with the location in the vertebral column. Thus, whereas in the intensive system vertebral fusions were located along the whole vertebral column, particularly in the caudal region, in the semi-intensive system fusions were mainly localized in the pre-hemal vertebrae. Besides, a higher number of vertebral fusions in the caudal region an extra number of vertebrae appeared in this region in the intensive system, suggesting a common factor negatively affecting vertebrae formation during the time of ossification of this vertebral region. Since ossification of vertebrae follows an antero-posterior pattern which lasts from day 20 to day 30 in the semi-intensive system, with ossification of hemal vertebrae around day 25, and until day 35 in the intensive system, these differences in ossification timing and vertebral fusion could be related with the changes in feeding protocol and larval feeding habits which occurred during this period. Feeding with dry diets started on day 20, with a marked increase in dietary intake from day 25 to 30, together with a progressive reduction in Artemia ingestion. Since semi-intensive larvae grew, developed and ossified their vertebrae faster, the earlier ability of these larvae to ingest dry feed could be related to the differences in the localization of vertebral anomalies among both systems. Indeed, our previous studies have found a direct relationship between vertebral fusion and nutritional factors in red porgy (Roo et al., 2008). Several nutritional factors have been found to be related with the apparition of fused vertebrae. For instance, inadequate amounts of certain types of vitamin A in Artemia have been found to be associated with higher incidence of vertebral fusions (Dedi et al., 1995; Takeuchi et al., 1998) and skeleton development (Estévez and Kanazawa, 1995; Takeuchi et al., 1995; Ronnestad et al., 1998; Suzuki et al., 2000; Moren et al., 2004) new reference Mazurais . Other nutritional factors involved in osteological development are the essential fatty acids (Cahu et al., 2003; Hamre et al., 2005; Lall and Lewis-McCrea, 2007). Not only the quantity but also the ratios between DHA and EPA to ARA are considered important for normal growth and development of fish larvae (Izquierdo, 1996; Rainuzzo et al., 1997; Sargent et al., 1999). Although it has been found that dietary lipids affect body fatty acid composition (Izquierdo et al., 1996) their effect on cartilage and bone fatty acid composition in fish has not been clearly determined. In chickens and rats dietary n-6/n-3 PUFA alters the fatty acid composition of bone and cartilage and modulates eicosanoid production (Watkins et al., 2000b; Xu et al., 1994), which in turn modulates the development and resorption of bones. Fish bone has similar cell types (Witten, (1997); Witten et al., 2000) and chemical composition to other vertebrates (Meunier 1989; Witten, 1997). In the present study, changes in type of feed Study II Journal of fish Biology (2008), (submitted) 109 (rotifers, Artemia and dry diet) implied changes in dietary fatty acid composition. Also, a significant drop in DHA:EPA ratios of prey FA composition occurred when Artemia was introduced and large differences in ARA concentration and DHA/ARA and ARA /EPA were found among rotifers, Artemia and dry diet (Table 1). In agreement with these results, low levels of DHA were associated with a significantly higher incidence of vertebral fusions in red porgy (Roo et al., 2008). Both n-3 HUFA and particularly DHA have been suggested to be important factors for correct osteological development (Gapasin and Duray 2001, Hamre et al., 2005). 5. Conclusions This osteological description and first report of the most important skeletal malformation associated with this specie is a useful tool to adapt and improve larval rearing protocols for Pagrus pagrus. The influence of the rearing system on the apparition of skeletal deformities and their location, suggest a relationship among feeding sequence, osteological development and deformities. Present results showed that despite the fact that a semi-intensive system can be successfully applied for red porgy fry to achieve semi-industrial production with good result in terms of growth, the high incidence of vertebral fusions observed, in this rearing system suggest different causative factors apart from system intensification. Many aspects of red porgy biology remain unknown and further studies are needed in order to reduce the incidence of skeleton anomalies to achieve cost-effective juvenile production of this species at a comercial scale. 6. Acknowledgements Funding was partially provided by the Spanish Ministries of Science and Education (AGL2003– 09131) and the Ministry of Agriculture, Fisheries and Food (Jacumar: Promoción del cultivo de nuevas especies de espáridos: Ensayos piloto y transferencia tecnológica). J.Roo, thanks the financial support provided by the Spanish Ministry of Science and Education and European Social Funds through the program “Incorporación de técnicos de apoyo de infraestructuras”. Thanks, also to D. Montero for his valuable comments and critical reading of the manuscript. Study II Journal of fish Biology (2008), (submitted) 110 7. References Afonso, J.M., Montero, D., Robaina, L., Astorga, N., Izquierdo, M.S., Ginés, R., 2000. Association of a lordosis-scoliosis-kyphosis deformity in gilthead sea bream (Sparus aurata L.) with family structure. Fish Physiol. Biochem. 22, 159-163. Akiyama, T., Murai, T., Nose, T., 1986. 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Aquaculture 199, 333–352. Moren, M., Opstad, I., Berntssen, M.H.G., Zambonino Infante, J.L., Hamre, K., 2004b. An optimum level of vitamin A supplements for Atlantic halibut (Hippoglossus hippoglossus L.) juveniles. Aquaculture 235, 587–599. Moteki, M., 2002. Morphological aspects of feeding and improvement in feeding ability in the early larval stages of red sea bream Pagrus major. Fisheries Science 68, 996–1003. Polo, A., Yufera, M., Pascual, E., 1991. Effects of temperature on eggs and larval development of Sparus aurata L. Aquaculture 92, 367-375. Papandroulakis, N., Kentouri, M., Divanach, P., 2004. Biological performance of red porgy (Pagrus pagrus) larvae under intensive rearing conditions with the use of an automated feeding system. Aquac. Int. 12, 191–203. Pavlidis, M., Angellotti, L., Papandroulakis, N., Divanach, P., 2003. Evaluation of transportation procedures on water quality and fry performance in red porgy (Pagrus pagrus) fry. Aquaculture 218, 187–202. Study II Journal of fish Biology (2008), (submitted) 115 Rainuzzo, J.R., Reitan, K.I., Olsen, Y., 1997. The significance of lipids at early stages of marine fish: a review. Aquaculture 155, 103–115. Richards, W.J. (Ed.)., 2006. Early stages of Atlantic fishes: an identification guide for the western central North Atlantic. CRC Press, Boca Raton, USA. ISBN 0-8493-1916-1. CRC Marine Biology Series 2, 2640 pp. Ronnestad, I., Helland, S., Lie,O., 1998. Feeding Artemia to larvae of Atlantic halibut (Hippoglossus hippoglossus L.) results in lower larval vitamin A content compared with feeding copepods. Aquaculture 165, 159-164. Roo, F.J., Socorro, J.A., Izquierdo, M.S., Caballero, M.J., Hernández-Cruz, C.M., Fernández, A., Fernández-Palacios, H., 1999. Development of red porgy Pagrus pagrus visual system in relation with changes in the digestive tract and larval feeding habits. Aquaculture 179, 499–512. Roo, F.J., Hernández-Cruz C.M., Fernández-Palacios, H., Izquierdo, M. S., 2005. Development of skeletal deformities in gilthead sea bream (Sparus aurata) reared under different larval culture and dietary conditions. In: Hendry, G. Van Stappen, M. Wille and P. Sorgeloos (Eds). Larvi’05–fish & Shellfish Larviculture Symposium.c.i. Oostende, Belgium. European Aquaculture Society, Special Publication 36, Roo, F.J., Hernández-Cruz C.M., Socorro, J.A., Fernández-Palacios, H., Montero, D. Izquierdo, M.S., 2008. Effect of DHA content in rotifers on the occurrence of skeletal deformities in red porgy Pagrus pagrus (Linnaeus, 1758). Doi:10.1016/j.aquaculture.2008.10.010. Rotllant, J., Tort, L., Montero, D., Pavlidis, M., Martinez, M., Wenderlaar Bonga, S.E., Balm, P.H.M., 2003. Background colour influence on the stress response in cultured red porgy Pagrus pagrus. Aquaculture 223, 129–139. Saele, O., Solbakken, J.S., Watanabe, K., Hamre, K., Pittman, K., 2003. The effect of diet on ossification and eye migration in Atlantic Halibut larvae (Hippoglossus hippoglossus L.). Aquaculture 220, 683-696. Sargent, J.R., McEvoy, L.A., Bell, J.G., 1999. Requirements, presentation and sources of polyunsaturated fatty acids in marine fish larval feeds. Aquaculture 155, 119-129. Sfakianakis, D.G., Koumoundouros, G., Anezaki, L., Divanach, P. and Kentouri, M., 2003. Development of saddleback-like syndrome in reared white seabream Diplodus sargus, L. Aquaculture 217, 673-676. Sfakianakis, D.G., Koumoundouros, G., Divanach, P., Kentouri, M. 2004. Osteological development of the vertebral column and of the fins in Pagellus erythrinus, (L. 1758): Study II Journal of fish Biology (2008), (submitted) 122 Number of pleural ribs VII VIII Frequency (%) 0 20 40 60 80 100 Semi-intensive system Intensive system a Number of vertebrae 23V 24V 25V Frequency (%) 0 20 40 60 80 100 Semi-intensive system Intensive system b ab a b a b Figure 5a,b. Percent frequency distribution of pleural ribs (a) and number of vertebrae (b) according to the rearing system; Data are given as mean, (n=150 fish per tank), from two consecutive production cycles comparing both rearing systems.VII and VIII denotes the number of pairs of pleural ribs and 23V, 24V, 25V denotes the number of vertebrae). (*) Different letters over each column, denotes significant differences (P<0.05). Study II Journal of fish Biology (2008), (submitted) 123 X-ray characterization Normal Fused Vert Lordosis Cranial Short Vert Kyphosis Others Frequency (%) 0 20 40 60 80 Semi-intensive system Intensive system aba b a b Figure 6. Incidence of skeleton abnormalities in the red porgy Pagrus pagrus seedlings reared in different systems Data are given as mean values of two replicates for each production cycles, (n=300 per culture system). (*) Different letters over each column, denotes significant differences (P<0.05). Study II Journal of fish Biology (2008), (submitted) 124 Fused vertebrae location Cephalic Pre-hemal Hemal Caudal Frequency (%) 0 20 40 60 80 100 Semi-intensive system Intensive system a b a b a Lordosis location Cephalic Pre-hemal Hemal Caudal Frequency (%) 0 20 40 60 80 100 Semi-intensive system Intensive system b Figure 7a,b. Location in the vertebral column of fused vertebrae (a) and lordosis (b) in red porgy Pagrus pagrus seedlings. Data are given as mean values of two replicates for each of the two production cycles presented data (n=300 per culture system). (*) Different letters over each column, denotes significant differences (P<0.05). Study II Journal of fish Biology (2008), (submitted) 125 Figure 8. Soft X-ray study in Pagrus pagrus fingerlings at 95 dah with a mean weight of 3 g: (a) fish with normal skeleton development and meristic characters identified: Vn: Vertebrae number; I-VIII: Pleural rib number; (b,c,d) fish exhibiting fused vertebrae (Fun-n) in different regions (n=involved vertebrae number); (e) fish exhibiting acute lordosis between vertebrae 13th to 17th (Lo13-17); (f) detail of cranial (Cr) deformity, fish with upper jaw reduction (Cr); (g) fish showing kyphosis (Ki) (arrow head); (h) example of fish with a vertebral shortening between 6th to 10th vertebrae (VShort (6-10)). Study II Journal of fish Biology (2008), (submitted) 126 Figure 9. Pagrus pagrus larvae at 20dah: (a) fish with normal skeleton development, (SB: Swim bladder); (b) fish showing initial kyphosis (arrowhead) potentially caused by gut overfilling with Artemia. Study II Journal of fish Biology (2008), (submitted) 127 List of tables Table 1. Proximal composition (lipid, protein and ash content; mg/g dry weight) and fatty acid composition (% total fatty acids) of the live preys used, rotifers, Artemia (InstarII) and microdiet fed to the larvae. Data are given as mean±SD. Live preys Rotifers Artemia Microdiet % Lipids (dw) 22.35 ± 2.30 a 25.45 ± 2.15 b 17.09±0.83 c % Protein (dw) 52.27 ± 4.34 a 55.38 ± 2.25 ab 57.66±1.09 b % Ash (dw) 1.20 ± 0.27a 0.65 ± 0.06 b 17.09±0.83 c ∑ saturated (1) 23.93 ± 0.63 24.14 ± 3.60 27.19±1.58 ∑ mono-unsaturated (2) 39.26 ± 1.36 a 32.89 ± 1.73 b 27.59±1.08 c ∑ n-3 (3) 21.01 ± 0.52 a 31.40 ± 3.14 b 20.68±2.89 a ∑ n-6(4) 10.95 ± 1.23 a 9.06 ± 0.48a 22.83±3.57 b ∑n-9(5) 22.78 ± 0.68 a 21.41 ± 0.59 ab 20.01±1.06 b ∑n-3HUFA(6) 18.55 ± 0.56 18.36 ± 2.85 15.78±2.92 16:00 14.60 ± 0.96 a 16.19 ± 2.66 ab 17.48±1.30 b 16:1 n-7 11.51 ± 1.20 a 3.47 ± 0.31 b 3.72±0.79 b 18:00 5.46 ± 0.56 6.47 ± 1.65 4.76±0.71 18:1 n-9 19.12 ± 0.55 a 19.06 ± 0.82 a 12.23±0.56 b 18:1 n-7 3.09 ± 0.17a 5.84 ± 0.25 b 2.79±0.85 a 18:2 n-6 7.77 ± 1.30a 5.47 ± 0.37a 21.64±3.95 b 18:3 n-3 1.35 ± 0.39a 11.31 ± 1.47 b 3.51±0.68 c 20:1 n-9 2.19 ± 0.10a 1.50 ± 0.09a 3.56±0.67 b ARA (20:4n-6) 1.83 ± 0.23a 1.62 ± 0.16a 0.55±0.15 b EPA (20:5n-3) 6.63 ± 0.34 7.56 ± 0.82 5.98±1.50 DHA (22:6n-3) 10.11 ± 0.32 9.11 ± 1.82 8.61±1.16 EPA/ARA 3.68 ± 0.59a 4.67 ± 0.05a 11.04±1.21 b DHA/EPA 1.53 ± 0.05a 1.20 ± 0.12 b 1.48±0.23 ab DHA/ARA 5.61 ± 0.85a 5.59 ± 0.60a 16.29±3.02 b oleic/DHA 1.89 ± 0.07a b 2.15 ± 0.37 b 1.44±0.24 a oleic/n-3HUFA 1.03 ± 0.04 1.05 ± 0.13 0.80±0.18 n-3/n-6 1.94 ± 0.21a 3.46 ± 0.26 b 0.94±0.27 c dw: dry weight. Values (mean ± S.D) followed by different superscript letters within a row for the same trial were significantly different ( P <0.05). (1) Includes 12:0, 14:0, 15:0, 16:0, 17:0, 18:0, 20:0, 22:0 and 24:0; (2) Includes 14:1n-5, 14:1n-7, 16:1n-9, 16:1n-7, 16:1n-5, 18:1n-9, 18:1n-7, 18:1n-5, 20:1n-9, 20:1n7, 20:1n-5, 22:1n-11, 22:1n-9 and 22:1n-7; (3) Includes 16:2n-3, 16:3n-3, 16:4n-3, 18:3n-3, 18:4n-3, 20:3n-3, 20:4n-3, 20:5n-3, 22:4n-3, 22:5n-3 and 22:6n-3; (4) Includes 16:2n-6, 18:2n-6, 18:3n-6,18:4n-6, 20:2n-6, 20:3n-6, 20:4n-6, 20:5n-6, 22:3n-6, 22:4n-6 and 22:5n-6; (5) Includes 16:1n-9, 18:1n-9, 18:2n-9, 18:3n-9, 20:1n9,20:2n-9, 20:3n-9 and 22:1n-9; (6) Includes 20:3n-3, 20:4n-3, 20:5n-3, 22:4n-3, 22:5n-3 and 22:6n-3. Study II Journal of fish Biology (2008), (submitted) 128 Table 2: Specific growth rate from 5 to 50 days after hatching. Specific growth rate in length (%) Age (days) 5-15 15-30 30-50 Average Semi-Intensive 7.10±0.14% 3.03±0.4% 4.60±0.35% 4.74±0.10% Intensive 5.77±0.31% 2.66±0.28% 4.37±0.12% 4.16±0.17% Study III.-Effect of DHA content in rotifers on the occurrence of skeletal deformities in red porgy Pagrus pagrus (Linnaeus, 1758) Aquaculture (2008), doi:10.1016/ j.aquaculture.2008.10.010 Improvements in the production technology of red porgy (Pagrus pagrus) larvae and fry: Importance of rearing conditions and diet nutritional value on their quality Study III Aquaculture (2008), doi:10.1016/j.aquaculture.2008.10.010 129 Corresponding author. Tel: +34 928 13 29 00/04; fax: +34 928 13 20 08. E-mail address: [email protected] Effect of DHA content in rotifers on the occurrence of skeletal deformities in red porgy Pagrus pagrus (Linnaeus, 1758) Francisco J. Roo, Carmen M. Hernández-Cruz, Juan A. Socorro, Hipólito FernándezPalacios, Daniel Montero, María S. Izquierdo Grupo de Investigación en Acuicultura (ICCM & ULPGC), PO Box 56, E-35200 Telde, Las Palmas, Canary Islands, Spain Abstract Despite being proposed as a potential candidate for diversification of marine aquaculture, limited larval survival and the occurrence of elevated levels of skeletal deformities restrict the commercial production of red porgy. The present study was conducted to determine the effect of rotifer enrichment, particularly on DHA, on growth, survival and occurrence of skeleton deformities in this species. The study included two trials, in one the viability of commercial emulsions for rotifer enrichment was evaluated whereas in another the effect of the elevation of DHA in rotifer emulsions on the larvae was studied. No significant differences were found in growth between larvae fed different rotifers in both trials suggesting that 1.9% dw DHA is enough to fulfil the DHA requirements of red porgy larvae for maximum growth. However, a marked positive effect of rotifer DHA content supplementation on larval survival was found. A low larval survival was found when rotifers had a high DPA content, in agreement with the results obtained for other Sparids. This fatty acid was accumulated in red porgy larval tissues in high levels (0.79 % dw DPA) when rotifers rich in DPA were the main source of food. Although the external appearance of the juveniles seemed to be normal, X-ray observations showed elevated levels of bone abnormalities associated, in both trials, to low DHA content in the live prey. Among different anomalies, the presence of fused vertebrae was the most frequent deformity for both rearing trials. Although DHA content in the rotifers used for feeding was the only difference a 50% reduction in the number of deformed fish for each type of deformity studied was obtained when the larvae fed higher DHA levels, denoting the important role of this FA in the prevention of deformities at the rotifer feeding stage. Further studies are needed to elucidate the importance of essential fatty acids on the development of bone deformities in fish, since the functions of HUFA are different, and their absolute levels and ratios among them can lead to very different effects in fish metabolism, including bone formation. Keywords: Red porgy, Pagrus pagrus, Larviculture, Enrichment, Essential fatty acids, Skeletal deformities. Abbreviations: FA: Fatty acid; TFA: Total fatty Acids; EFA: Essential fatty acids; HUFA: Highly unsaturated fatty acids; DHA: Docosahexaenoic Acid; EPA: Docosapentaenoic Acid; ARA: Araquidonic Acid, DPA: Docosapentaenoic acid; VA: Vitamin A; PGE2: Prostaglandin E2 ; PGE3: Prostaglandin E3; LT4: Leukotrienes T4; LTC4 : Leukotrienes C4 ; LTD4: leukotrienes D4 ; IGF: Insulin-like Growth Factor; BMP: Bone Morphogenetic Proteins; BMP4:Bone morphogenetic protein 4 ; BGP: Bone Gla protein; Oc: Osteocalcin; MGP; matrix Gla protein; RXR: Retinoid X receptor; RARγ:Retinoic Acid Receptor; IL-1: Interleukins 1; IL-6: Interleukins 6 and TNF: Tumour necrosis factors; PPAR: Peroxisome proliferators activated receptors; dah: days after hatching; SGR: specific growth rate. Study III Aquaculture (2008), doi:10.1016/j.aquaculture.2008.10.010 130 1.Introduction Red porgy has been proposed as a potential candidate for diversification of marine aquaculture (Kentouri et al., 1995; Hernández-Cruz et al., 1999) and some on-growing experiences have already been conducted in different Mediterranean and Atlantic areas (Author´s unpublished data). Previous studies focused on the ontogeny of different organs and tissues (Roo et al., 1999; Socorro et al., 2001; Darias et al., 2005) and improvements on larval rearing techniques (Kentouri et al., 1995; Hernández-Cruz et al., 1999; Mihelakakis et al., 2001; Papandroulakis et al., 2004). However, limited larval survival together with high occurrence of skeletal deformities (over 50% of the population) under intensive or semi-intensive systems, constitute major bottlenecks for the production of this species at commercial scale (Author´s unpublished data). There are several studies on the osteological development and skeleton anomalies of different sparids such as Sparus aurata (Koumondouros et al., 1997a,b; Boglione, 2001; Faustino, 2002), Pagrus major (Moteki, 2001; Kihara et al., 2002; Matsuoka, 2003), Dentex dentex (Koumoundouros et al., 1999, 2001a), Pagellus erythinus (Boglione et al., 2003; Sfakianakis et al., 2004), Diplodus puntazzo (Boglione et al., 2003; Favaloro and Mazzola, 2003) and Diploduss sargus (Koumoundouros et al., 2001b; Sfakianakis et al., 2003). Detailed information about osteological development has also been reported for red porgy by Socorro (2006), although the causes for the high incidence of deformities remain unclear. Different authors have studied the relationship between skeletal deformities and environmental factors (light, temperature, salinity), mechanical shock during embryo or larval development, tank currents and the type of rearing system (Battaglene and Talbot, 1990; Polo et al.,1991; Chatain, 1994; Divanach et al., 1997; Koumoundouros et al., 1997a,b; 2001a; Mihelakakis and Yoshimatsu, 1998; Cobcroft et al., 2001; Boglione et al., 2001; Kihara et al., 2002; Sfakianakis et al., 2004; Giménez and Estévez, 2005 and Roo et al., 2005). Besides, a genetic component has been also found to contribute to the apparition of some types of acute deformities (Afonso et al., 2000). Several biologically active compounds such as hormones, prostaglandins, cytokines, growth factors and certain nutrients can contribute as biological regulators to control the metabolism of specific bones and cartilages (Watkins and Seifert, 1997, 2000a,b). Thus, nutritional imbalances on early life stages, such as deficiencies in vitamins, aminoacids or essential fatty acids in feeding regimes of broodstock or larvae may also alter the osteological development of reared larvae (Kanazawa et al., 1983; Akiyama et al., 1986, Knox et al., 1988; Chatain and Ounais–Guschemann, 1990; Cahu et al., 2003, Saele et al., 2003; Hamre et al., 2005). Spanish summary 232 oligotróficas incluso se procede a introducir un inóculo de fitoplancton con especies de los géneros Nannochloropsis o Tetraselmis. La renovación en este sistemas es muy baja 1-2% día, y después del llenado del tanque, esta se mantiene en torno a 10-15 días para permitir un óptimo desarrollo del zooplancton. Las larvas se alimentaran de este zooplancton alrededor de 20-25 días, tiempo crítico en el desarrollo de la larva y tras el cual ya están preparadas para continuar alimentándose de Artemia y comenzar su destete. Sin embargo, el uso de este sistema puede encontrarse a veces con dificultades como bloom inesperado de otros organismos como medusas, anélidos, o incluso ciertos ciliados parásitos que pueden dar al traste con la producción. 2.3.1.3.2.-Método de agua verdeFilosofía extensiva Al igual, que el descrito anteriormente, consiste en la generación de una cadena trófica, sin embargo, esta será de origen endógeno (Divanach et al., 2002). En este caso, el tanque se llena con agua de mar previamente filtrada y esterilizada. Se fertiliza con nutrientes y se introduce un inóculo de fitoplancton y rotíferos permitiendo que se desarrollen ambos hasta una media de 500.000 cells/ml y >2 rot/ml. No se renueva el agua del tanque, manteniendo el cultivo de 4 a 8 días hasta que se introducen los huevos o larvas. Cuando la cadena alimenticia se desarrolla correctamente esta dura aproximadamente 25 días pasando a usarse Artemia y dietas de destete y circuito abierto de agua. Los inconvenientes de este sistema estriban en los desequilibrios que se pueden establecer a consecuencia de una siembra demasiado baja, los rotíferos alcanzan un bloom rápidamente consumiendo el fitoplancton y pudiéndose dar condiciones de bajada de pH y oxígeno durante la noche. Por otro lado un exceso en el número de larvas puede conllevar un agotamiento prematuro de las presas y el bloom de fitoplancton remanente puede dar lugar a una hiper-oxigenación superficial en el tanque y condiciones anóxicas en el fondo (baja penetración de luz) lo que puede originar enfermedades en las larvas, como la sobresaturación de gases o la mortalidad por hipoxia. En la segunda, la filosofía intensiva, la alimentación es básicamente exógena pero presenta una cierta capacidad de producción endógena debido a la baja densidad de las larvas (baja tasas de alimentación), presencia de fitoplancton en el tanque o a la introducción de inóculos de otros tipos de presas como pueden ser copépodos marinos. Spanish summary 233 2.3.1.3.4.- Agua pseudo verdeFilosofía intensiva En ella, el cultivo larvario se desarrolla en un medio con una composición más estable que los anteriores, que contiene fitoplancton y rotíferos de origen exógeno, producidos en instalaciones paralelas y que se aportan diariamente según la demanda de las larvas (Divanach et al., 2002). De esta técnica existen dos variantes: Puro: el tanque se llena con agua de mar esterilizada y se introducen los huevos o larvas recién eclosionadas. Posteriormente cuando las larvas inician su vida heterotrófica, se adicionan el fitoplancton y los rotíferos. En la segunda variante se inicia primero la cadena trófica añadiendo fitoplancton y rotíferos antes de sembrar los huevos o larvas. En este caso, el periodo de vida autotrófico de la larva puede verse perjudicado por la presencia de un medio muy poblado. En estos casos, el circuito de agua es siempre abierto y su renovación se incrementa a medida que la larva se desarrolla. La adición de fitoplancton y rotíferos se realiza 1-2 veces al día y se utilizan rotíferos previamente enriquecidos con preparados comerciales para mejorar su valor nutritivo. Cuando la larva alcanza el tamaño necesario para poder alimentarse de Artemia cesa el uso del fitoplancton y el cultivo se continúa en técnica de agua clara. Suele utilizarse con especies de las que se conoce bien su ciclo biológico como es el caso de la dorada (Sparus aurata), y presentan unos rendimientos muy elevados. 2.3.1.3.5.-Agua claraFilosofía intensiva. Es un método en el cual no se utiliza fitoplancton y esta siempre asociado a altas tasas de renovación de agua >10%/h lo que imposibilita el desarrollo de microorganismos endógenos. Se utiliza generalmente con especies, como la lubina, que aceptan rápidamente los nauplios de Artemia en su primera alimentación. O bien asociado a sistemas de alimentación automática capaces se suministrar alimento con una calidad nutritiva elevada (Divanach et al., 2002). 2.3.1.3.6.-Técnicas neo-verde. Son técnicas que todavía están siendo testadas en muchas instalaciones a nivel experimental, utilizando generalmente fitoplancton crioconservado o producido en sistemas de cultivos heterotróficos, como pastas y liofilizados de microalgas (Divanach et al., 2002).. La principal limitación de este tipo de técnica se basa en la perdida de los valores beneficiosos que se apuntan con el uso de microalgas vivas . Spanish summary 234 2.4.-Factores condicionantes en el cultivo larvario Se han realizado infinidad de estudios conducentes a determinar cuáles son los parámetros óptimos para el cultivo larvario de las especies marinas. Hay que destacar que existe una clara influencia de la especie a cultivar sobre la tecnología más apropiada para la misma. Entre los parámetros que se han considerado como más importantes están aquellos que se pueden englobar dentro de las condiciones generales de cultivo: densidad de siembra, volumen del tanque, renovación del medio, presencia de fitoplancton o uso de antibióticos. Los parámetros físicoquímicos que han sido más estudiados como temperatura, salinidad, iluminación (fotoperiodo, intensidad y calidad de luz) o los productos de excreción. Y finalmente, los que afectan a la nutrición larvaria: cantidad de presas aportadas, secuencia alimentaria y el considerado como uno de los factores más importantes como es el valor nutricional del alimento suministrado. 2.4.1.-Parámetros generales 2.4.1.1.-Densidad larvaria La influencia de la densidad de cultivo, además de ser un factor que condiciona el tipo de sistema, ha sido señalado como parámetro condicionante del crecimiento y la supervivencia durante la etapa larvaria de peces marinos, como se ha señalado en el caso de la anchoa de caleta (Anchoa mitchilli), lenguado americano (Achirus lineatus) (Houde,1977), cobia (Rachycentron canadum) (Hitzfelder et al., 2006, Holt et al., 2007) o corvina (Argyrosomus regius) (Estevez et al, 2007; Roo et al., 2007). En este mismo sentido, se han pronunciado otros autores que han trabajado con la misma especie objeto de este estudio como el bocinegro (Pagrus pagrus) (Hernández-Cruz et al., 1999; Roo et al., 2005b), dorada, (Sparus aurata) (Parra y Yúfera, 1999; Roo et al., 2005a), o especies cercanas como dentón, (Dentex dentex) (Giménez y Estevez, 2008a). Uno de los factores asociados al aumento de densidad, que puede condicionar la supervivencia es el canibalismo intracohorte como señalan Baras y Jobling, (2002). Sin embargo, en lubina (Dicentrarchus labrax) y lenguado canadiense (Paralichthys dentatus) se asocia el incremento de la densidad larvaria únicamente a la disminución del crecimiento como consecuencia de la gran competencia por el alimento y el estrés inherente a la gran acumulación de individuos (Hatziathanasiou et al., 2002; Saillant et al., 2003; King et al., 2000). Trabajos más recientes con una especie como bacalao (Gadus morhua) muestran que la densidad de cultivo no parece ser un factor que limite el crecimiento y la supervivencia, porque su influencia se ve enmascarada por otros factores asociados a la alta densidad de cultivo, como son la Spanish summary 235 disponibilidad del alimento y el riesgo en el deterioro de la calidad de agua a altas densidades (Baskerville-Bridges B. y Kling, J. L., 2000). 2.4.1.2.-Forma y volumen del tanque. Se ha encontrado que el volumen del tanque de cultivo tiene diferentes efectos en los sistemas de cría de larvas de espáridos como la dorada, así Tandler y Sherman (1981) encuentran una correlación positiva, entre el incremento del volumen del tanque de cultivo y la supervivencia. En la actualidad, la mayoría de criaderos que se dedican a la producción intensiva de espáridos utilizan un modelo de tanque cilindro cónico de fibra de vidrio y con volúmenes relativamente grandes 1-15m3 al menos en las primeras etapas larvarias, siendo la tendencia generalizada el uso de volúmenes mayores de 10m3. Del mismo modo, al igual que con el volumen, se ha encontrado que la relación profundidad/superficie del tanque puede afectar a la supervivencia larvaria en las primeras etapas de desarrollo, como se ha visto en el mero de siete bandas (Epinephelus septemfasciatus) y el escorpénido “devil stinger” (Inimicus japonicus) (Ruttanapornvareesakul et al., 2007). 2.4.1.3.-Renovacion de agua. Un exceso de renovación de agua, del tanque, afectará la aparición de deformidades de columna (Kihara et al., 2002) ó la rápida eliminación de presas o microalgas que estén presentes en el tanque, limitando la calidad del alimento suministrado y las condiciones físico-químicas del medio. Por el contrario, un defecto en la renovación, puede contribuir a la acumulación de metabolitos (amonio, nitritos, etc.) en el sistema de cultivo, o la acumulación de presas vivas con un valor nutritivo escaso (Reitan et al., 1993) que puede tener efectos negativos en las tasas alimenticias de la larva y consecuentemente en el crecimiento y supervivencia de las mismas. Autores como Tandler y Helps, (1985), Tandler y Sherman, (1981) o Hernández-Cruz et al, (1990), correlacionan el uso de sistema de circuito cerrado con un mejor crecimiento de la larva de dorada (Sparus aurata), durante los 10 primeros días de vida. Sin embargo, sugieren una renovación del 25%.día-1 a partir del día 20 de vida. Otros trabajos muestran que la combinación de circuito abierto y agua verde mejora significativamente la supervivencia en los cultivos de dorada (Hernández-Cruz et al., 1994). En los últimos años, se ha establecido una tendencia creciente al uso de sofisticados sistemas de circuito cerrado, donde se realiza una renovación del medio con agua del propio sistema pasada por filtros mecánicos y biológicos que aseguran el Spanish summary 236 mantenimiento de una buena calidad de agua y el valor nutritivo de las presas vivas (Olivar et al., 2000, Faulk y Holt, 2005,). 2.4.1.4.-Presencia de fitoplancton en el tanque de cultivo larvario Numerosos trabajos defienden el uso de fitoplancton o la denominada técnica de agua verde para el cultivo larvario de especies de peces marinos (Scott y Baynes,1979; Hernández-Cruz et al., 1990; Koven et al., 1990, Salvesen et al., 2000). Los efectos beneficiosos de la presencia de fitoplancton en el tanque se pueden resumir en que contribuye a una mayor oxigenación del agua del tanque; ayuda a la eliminación de sustancias de desecho (amonio y nitritos) producidos por las larvas y presas presentes en el tanque; controla la flora bacteriana, ya que la mayoría de microalgas presentan actividad antibacteriana a través de la producción de exudados orgánicos y metabolitos tóxicos para las bacterias oportunistas. En el caso particular de Nannochloropsis sp. la especie utilizada en este estudio, la principal flora bateriana esta compuesta por Alfaproteobacterias y el grupo de bacterias tipo Cytophaga-Flavobacterium, las cuales actúan como herramienta de biocontrol de otras comunidades bacterianas que se desarrollan en los tanques de cultivo larvario (Nakase y Eguchi, 2007). A su vez, el incremento de la carga bacteriana en el agua de cultivo, como consecuencia de la repetida adición de fitoplancton al tanque, puede contribuir al desarrollo de flora intestinal inicial de las larvas (Skjermo y Vadstein 1993). Además, la presencia de fitoplancton en el tanque de cultivo, ayuda al mantenimiento del valor nutritivo de las presas vivas. En rotíferos enriquecidos con emulsiones la pérdida de lípidos se estima en un 20% diario en sistemas de cultivo en agua verde, mientras que en los sistemas de cultivo en agua clara hay un descenso más acentuado de la calidad nutritiva de las presas (Reitan et al., 1993; Planas y Cunha., 1999). Se ha observado también, que las larvas cultivadas en agua verde mantienen las reservas vitelinas 2-3 días, tienen una tasa de crecimiento mayor y sufren de 2 a 3 veces menor mortalidad larvaria que cuando se cultivan en agua clara (Papandroulakis et al, 2001; Van der Meeren et al, 2007). Finalmente, se puede decir que el fitoplancton actúa como un filtro lumínico, ya que las microalgas reducen el efecto de la reflexión de la luz en las paredes de tanque, que junto con el uso de tanques con paredes oscuras (Hinshaw,1985), contribuye a una mejora de la actividad alimenticia de la larva, al favorecer un mayor contraste de las presas, (Naas et al 1992,1996, Ramos y Kobayashi, 1985; Muller-Feuga et al, 2003). Actualmente existe una tendencia al uso de concentrados, pastas, liofilizados y biomasa congelada de diferentes microalgas. Hay diferentes estudios comparativos entre el uso de este tipo de productos y las microalgas vivas, así Cañavate y Fernández-Díaz, (2001) no encuentran diferencias en el Spanish summary 237 crecimiento y supervivencia en la larva de dorada (Sparus aurata) con el uso de biomasa congelada frente al alga viva. Sin embargo, estos autores mencionan un deterioro en la calidad de agua del tanque, por el incremento de los valores de amonio, con el uso de biomasa inerte en los sistemas que trabajan en circuito cerrado. Por otro lado, el uso de este tipo de productos limita los efectos beneficiosos exclusivos de la biomasa viva, como son el efecto bacteriostático o la colonización de la flora intestinal de la larva y tanque larvario. 2.4.1.5.-Uso de antibióticos El exceso de carga bacteriana en los tanques de cultivo, como consecuencia de la introducción de presas vivas y el uso de sistemas con limitada o nula renovación, hace que haya sido frecuente el uso de antibióticos para su control. El uso de estas sustancias en el cultivo larvario ha sido un amplio tema de discusión entre diferentes autores y trabajos con diferentes especies. Así, Hernández-Cruz et al.,1994 muestran que el uso de ácido oxolínico tiene resultados adversos en la supervivencia larvaria, es más los rotíferos enriquecidos sometidos a tratamientos con antibiótico muestran un menor contenido en n-3 HUFA que los no tratados. Sin embargo, otros trabajos, correlacionan el uso de la oxitetraciclina, con un incremento de la supervivencia y crecimiento de las larvas de trompetero australiano (Latris lineata) (Bataglene et al.,2006). 2.4.2.-Parámetros físico-químicos Entre el elevado número de parámetros físico químicos que, potencialmente, pueden afectar o condicionar el cultivo larvario (temperatura, salinidad, iluminación, productos de excreción, gases disueltos, pH etc.) se han seleccionado aquellos considerados como más determinantes del crecimiento, la supervivencia y la calidad de las larvas. 2.4.2.1.-Temperatura Dentro de los parámetros físico-químicos uno de los que tienen mayor importancia en el cultivo larvario es la temperatura. Su efecto se puede separar en dos etapas: durante el desarrollo del embrión y durante el desarrollo de la larva. Temperatura de incubación El desarrollo embrionario y larvario de los peces está altamente influenciado por la temperatura de incubación del embrión, así las temperaturas elevadas aceleran el desarrollo embrionario, mientras que las bajas temperaturas lo disminuyen (Blaxter, 1988). Los trabajos de autores como Spanish summary 238 Polo et al. (1991) señalan, que la dorada europea admite un amplio rango de temperatura de incubación que va, desde 14 a 28 ºC, siendo el óptimo en torno a 19º C. Otros autores han relacionado la temperatura óptima de incubación con las condiciones naturales asociadas a las latitudes de los stocks de reproductores considerados. Se indica sin embargo que, por debajo de 16 ºC y por encima de 22º C las tasas de eclosión se reducen y los porcentajes de larvas con deformidades se incrementan notablemente. Resultados similares han sido descritos para otras especies de espáridos como el pargo japonés (Pagrus major), (Mihelakakis y Yoshimatsu, 1998). En el caso de la lubina (Dicentrarchus labrax) se ha señalado que un ligero incremento de la temperatura durante la incubación tiene un efecto positivo en el crecimiento muscular en etapas posteriores (López-Albors et al., 2003). Temperatura de cultivo larvario Tandler y Sherman, (1981), determinaron que la supervivencia larvaria de la dorada (Sparus aurata) no se ve afectada por el efecto de la temperatura de cultivo larvario empleada que puede ir desde 17º a 23 ºC, aunque la mayor supervivencia se obtuvo a 17ºC, asociando un incremento de la temperatura con un mayor crecimiento de las larvas. De la misma manera, Polo et al., (1991) determinan el rango de 16-22 º C como idóneo para el cultivo de esta especie, situando el óptimo en 19ºC y correlacionando las temperaturas fuera del mismo con un aumento en la mortalidad y un incremento de la incidencia de anormalidades en las larvas. Otros estudios demuestran que la temperatura puede tener efectos negativos sobre la supervivencia larvaria así, Tandler et al, (1989), correlacionan el efecto de la temperatura de cultivo con la estructura de tallas de la población, señalando que a temperaturas más elevadas el efecto de dispersión de la talla de la población puede ser mayor lo que contribuye a un aumento de la mortalidad por el efecto del canibalismo a edades tempranas. 2.4.2.2.-Salinidad A pesar de que suele ser un parámetro que se mantiene relativamente estable, algunos criaderos presentan ocasionalmente problemas de bajadas de salinidad a consecuencia de las escorrentías en épocas de lluvias. Se ha visto que produce efectos sobre el cultivo larvario condicionando las tasas de eclosión del huevo e incluso incidiendo en el desarrollo de deformidades esqueléticas. Tal es el caso del “Southern black bream” (Acanthopagrus butcheri) donde la reducción de la salinidad por debajo de 15‰ se refleja con una reducción en la eclosión y un incremento del porcentaje de larvas con deformidades de columna, que puede afectar hasta al 100% de la Spanish summary 239 población a una salinidad de 5‰ y hasta un 20%, a salinidades de 20‰ (Haddy y Pankhurst, 2000). Resultados similares se han evidenciado en el caso de la dorada, donde una bajada brusca de salinidad de 37 a 32‰ durante los primeros días de vida de la larva se refleja en un incremento significativo del porcentaje de anomalías operculares respecto al control mantenido a salinidad constante en la misma etapa (datos propios, no publicados). Por el contario en especies como la lubina (Dicentrarchus labrax) la bajada de salinidad a 15‰ resulta en un aumento de la supervivencia y una mejora en el porcentaje de larvas con vejiga natatoria, resultados similares fueron obtenidos en el caso de la lisa pardete (Mugil cephalus) (Harel et al., 1998). Por otro lado, la combinación de los dos parámetros anteriormente mencionados temperatura y salinidad tiene una importancia significativa en la obtención de larvas viables. Así valores extremos de 28ºC y 12‰ de salinidad aumentan el porcentaje de larvas que presentan deformidades en el pargo japonés Pagrus major (Mihelakakis y Yoshimatsu, 1998). 2.4.2.3.-Productos de excreción nitrogenados El amonio es el principal producto de excreción de los peces, como resultado del catabolismo proteico, siendo especialmente tóxico, en el caso de las larvas. La producción de amonio y detritos orgánicos por el alimento no consumido como pueden ser las microalgas y presas muertas, contribuyen al incremento de la cantidad de amonio en los tanques larvarios. Parra y Yúfera, (1999), sitúan en 20ppm las concentraciones letales de amonio en cultivos de larvas de dorada (Sparus aurata) de 12 días, siendo la forma más tóxica el amonio no ionizado (N-NH3) con una concentración letal para el 50% de la población de 0,24 ppm. En el caso del nitrito (NO2- ) la mortalidad del 100% de la población se observa a 4.500 ppm, y el 24h LC50 en 1.997 ppm. Sin embargo, estas concentraciones son dependientes de factores como el pH, salinidad ó temperatura, así como de la especie, ya que en el caso de las larvas de lenguado senegalés (Solea senegalensis) de 7 días estos mismo autores citan una concentración letal para el N-NH3 de 80 ppm y la 24h-Lc 50 de 1,32 ppm. En el caso del NO2la mortalidad del 20% de la población se observa a concentraciones de 5.000 ppm. Cabe mencionar además la relación que se ha encontrado entre los niveles de amonio de (0,05-0,015 ppm de NH3-H) y la aparición de daños en las estructuras cartilaginosas de las larvas de pargo japonés (Mihelakakis y Yoshimatsu, 1998). Spanish summary 240 2.4.2.4.-Iluminación La mayoría de larvas de peces marinos son predadores visuales (Blaxter y Staines., 1970). Por ejemplo, se ha demostrado que la larva de dorada es un predador visual que necesita luz para ser efectiva en sus ataques (Tandler y Mason 1983). Así, Tandler y Helps (1985) demostraron que durante los primeros 12 días de vida de la larva hay una correlación positiva entre el aumento del foto período de 12 a 24 h de luz con la supervivencia en larvas de dorada europea, lo mismo encuentra Barahona-Fernándes, (1979) en larvas de lubina. También se correlaciona un aumento de la intensidad luminosa de 205 a 1370 lux con un mayor crecimiento y supervivencia larvaria, con óptimos que se sitúan entre 1370-5140 lux cuando se combina luz artificial con luz natural en fotoperiodo continuo y sistemas de cultivo en agua verde (Tandler y Mason, 1984). A partir del día 20 de vida no se encuentran diferencias significativas con el cambio de foto período de 12 a 24 horas lo que está relacionado con el desarrollo del sistema visual de la larva (Roo et al., 1999, 2001). Las larvas de espáridos como dorada (Sparus aurata) y bocinegro (Pagrus pagrus) nacen con un sistema visual simple únicamente conformado por fotoreceptores denominados conos, los cuales les capacitan para ver solo en condiciones de alta intensidad de luz. Sin embargo, el sistema visual continúa desarrollándose y aumentando su complejidad, y así al día 18-20 de vida aparecen los precursores de los fotoreceptores de tipo bastones, que mejoran la agudeza visual de la larva permitiéndole predar en condiciones de baja intensidad de luz. Este sistema dual compuesto por conos y bastones no se completa hasta el día 25-30 de vida según la temperatura de cultivo de la larva. Para el periodo comprendido entre los 50-60 días Tandler y Helps 1985 obtienen los mejores crecimientos con una reducción del foto período en el caso de la dorada europea. Otro de los factores que hay que tener en cuenta y que se relacionan con la iluminación en los tanques larvarios es la coloración y reflexión de las paredes del tanque, Naas et al., (1996) y Hinshaw, (1985) describen que los tanques de paredes negras son los que mejor asemejan las condiciones de iluminación natural cuando están provistos de una iluminación adecuada. Finalmente, cabe mencionar la relación encontrada entre la iluminación y el desarrollo esquelético de las larvas, en casos particulares, como el “silver pearfish” (Carapus homei), las larvas de esta especie necesitan pasar por un periodo de oscuridad para completar su metamorfosis, este cambio lo realizan en el interior del pepino de mar ocelado (Bohadschia argus) en el interior del cual tiene lugar una reducción natural del número de vertebras así como una compresión de las mismas para adoptar su forma definitiva de alevín (Parmentier et al., 2004). Spanish summary 241 2.4.3.-Nutrición larvaria Un elemento crítico en el cultivo larvario de peces marinos es la primera alimentación. Las larvas de peces marinos generalmente eclosionan con un cantidad muy limitada de reservas endógenas, por lo que su supervivencia va a estar enormemente condicionada a la alimentación suministrada. El reducido tamaño de la boca de la mayor parte de las larvas de peces marinos, unido a un limitado desarrollo del sistema digestivo, hacen del comienzo de la alimentación exógena una etapa crítica para el éxito del cultivo larvario. 2.4.3.1.-Cantidad de presas Tandler y Sherman, (1981), determinaron que la cantidad de presas óptimas para obtener buenas supervivencias larvarias se encuentran entre 10-15 rot/ml para cultivos intensivos de dorada (Sparus aurata) a una densidad larvaria de 100indv.l-1. Resultados similares han sido citados por Giménez y Estévez (2008) quienes apuntan una densidad de 5-10rot.ml-1, para el cultivo larvario de dentón (Dentex dentex) a una densidad de entre 10 a 40indv.l-1 . Autores como Houde, (1977) y O´Connell y Raymond (1970) indican las concentraciones para el uso de otras presas como copépodos. Así densidades superiores a 1nauplio.ml-1 son necesarias para el cultivo de la anchoa de caleta (Anchoa mitchilli) y el lenguado americano (Achirus lineatus) mientras que concentraciónes de 4 nauplios.ml-1 se asocia con las mejores tasas de supervivencia (68%), en larvas de anchoa del pacífico (Engraulix mordax), lo que da idea a su vez de las diferencias interespecificas para este parámetro. 2.4.3.2.-Secuencia alimentaria-tipos de presas-co-alimentación La secuencia de presas vivas a utilizar es uno de los elementos críticos para obtener el éxito en el cultivo larvario de nuevas especies de peces marinos. Generalmente, la elección de la secuencia alimentaria adecuada, vendrá determinada fundamentalmente por el nivel de desarrollo del organismo a cultivar, que puede variar según las características intrínsecas de las diferentes especies. Generalmente, será el tamaño de la boca y la disponibilidad de distintos tipos de presas que puedan ser producidas de una manera masiva y sostenible, uno de los principales limitantes. Así, en el caso de la mayoría de los espáridos es frecuente el uso de una presa inicial de menor tamaño, como es el rotífero eurihalino del genero Brachionus, que puede ser de diferentes especies o morfo tipos basados en el tamaño de la lorica; Brachionus plicatilis, tipo L (grande), B. ibericus o tipo S (Pequeño) y B.rotundiformis ó SS(Super pequeño) (Hagiwara et al., 2007). Al rotífero le sigue un periodo corto de transición a nauplios de Artemia, para continuar con Spanish summary 248 Así, dentro del proceso industrial de cría de alevines, las empresas se ven obligadas a implantar procesos de criba para eliminar los individuos que presentan algún tipo de anormalidad no comercial, ya que de manera generalizada las granjas de engorde no aceptan lotes con más de un 5% de peces deformes en valoración visual. Este proceso añade unos costes asociados que se estima, repercuten en 0,01€ en el coste de producción de un alevín. Hay criaderos que niegan la existencia de deformidades en sus lotes, que por una estrategia de marketing necesariamente va acompañada de un elevado grado de selección de los animales antes de su venta. Figura 11: Criba manual de individuos deformes (fotografía cortesía de Tinamenor S.A). Las diferentes anomalías que afectan a la calidad de los peces, en mayor o menor medida, pueden ser agrupadas principalmente en dos grupos: de pigmentación y de esqueleto. 2.5.2.1.-Anomalías de pigmentación Las anomalías de pigmentación consisten en una alteración parcial o total de la coloración del cuerpo. Es en peces planos, donde han sido ampliamente descritas por su importancia económica (Gavai et al., 2002; Sæle et al., 2003; Aritaki y Seikai., 2004; Lewis y Lall., 2006). La pigmentación anormal consiste en hipomelanosis o pseudoalbinismo cuando afecta a la cara cara dorsal e hipermelanosis o ambicoloración si afecta a la ventral (Venizelos y Benetti 1999) (Figura 12). Las anomalías de pigmentación son consecuencia de una distribución diferencial y o diferencias fisiológicas celulares de los cromatóforos (Kelhs 2004; Burton 2005) que han sido asociadas a factores como las condiciones intensivas de cultivo, densidad, iluminación, el Spanish summary 249 sustrato o la dieta (Seikai et al. 1987; Estévez et al. 1999, 2001; Benetti 1997). En poblaciones naturales suceden tanto el pseudoalbinismo (Venizelos y Benetti 1999) como la ambicolaración (Astarloa 1995), con inferior incidencia que en poblaciones de cultivo (Bolker y Hill 2000). Figura 12. Ejemplares de lenguado senegales (Solea senegalensis) con pigmentación normal y con pseudoalbinismo. 2.5.2.2.-Anomalías de esqueleto Existen diferentes estudios acerca del desarrollo osteológico y la aparición de anomalias esqueléticas en espáridos, como dorada (Sparus aurata) (Faustino, 2002; Boglione et al., 2001; Koumondouros et al., 1997a), el pargo japonés (Pagrus major) (Moteki, 2002; Kihara et al., 2002; Matsuoka, 2003), dentón (Dentex dentex) (Koumoundouros et al., 2001a), breca (Pagellus erythinus) (Boglione et al., 2003; Sfakianakis et al., 2004), sargo picudo (Diplodus puntazo) (Boglione et al., 2003; Favaloro and Mazzola, 2003) y sargo Diploduss sargus (Koumoundouros et al., 2001b; Sfakianakis et al., 2003). De manera general, las anomalías de esqueleto más importantes en peces marinos son las que afectan al neurocráneo, espina dorsal y esqueleto apendicular ya que alteran de manera severa la morfología de estos y tienen una importante repercusión económica en las empresas. Las deformidades asociadas al neurocráneo afectan fundamentalmente al complejo opercular y mandíbulas y son encontradas frecuentemente en los criaderos comerciales. Las anomalías del complejo opercular pueden estar presentes a uno o ambos lados del pez y consistir en un plegamiento o una formación incompleta del complejo opercular (Koumoundourous, et al., Spanish summary 250 1997b) a menudo esta anomalía está asociada con malformaciones de los arcos branquiales (Sadler et al., 2001) (Figura 13). Estas deformidades suceden desde edades muy tempranas en el desarrollo, sin asociación con un lado corporal concreto, y pudiendo alcanzar incidencias de hasta el 98,3% de la población (Beraldo et al., 2003). Figura 13. Ejemplares juveniles de pargo o bocinegro (Pagrus pagrus) y corvina (Argyrosomus regious) con anomalía del complejo opercular (a,c); b) detalle del plegamiento hacia el interior. Las deformidades de las mandíbulas, consisten en torsiones de la mandíbula inferior y superior o prolongación de éstas en diferente magnitud (Cobcroft et al., 2001), que en ocasiones han sido asociadas a efectos letales (Barahona-Fernandes, 1982) (Figura 8). Figura 14. Deformidades mandibulares en:a,b) pargo o bocinegro, c) dorada. Spanish summary 251 En general, el desarrollo ontogénico del neurocráneo y de las aletas es previo a la eclosión, siendo completado a etapas más avanzadas del desarrollo. Las deformidades de las aletas se caracterizan por torsión, falta parcial o total de las mismas y puede alcanzar incidencias de hasta un 65% en larvas de dorada (Sparus aurata) (Koumoundouros et al.,1997b) aunque tienen una relevancia menor en su repercusión comercial (Figura 14). Figura 15. Anomalías en los radios de las aletas pectorales en; a) dorada, b) pargo ó bocinegro. Las anormalidades de la columna vertebral son una de las más relevantes en piscicultura no sólo por su severo efecto sobre la morfología del pez sino también por la influencia que ejercen sobre caracteres productivos como el crecimiento (Gjerde et al. 2005; Kause et al. 2005). Las principales deformidades de columna son la escoliosis, lordosis, cifosis y fusiones vertebrales, que en ocasiones es posible encontrar en un mismo pez de forma combinada (Afonso et al., 2000) (Figura 16). Spanish summary 252 Figura 16. Anomalías de columna en bocinegro (Pagrus pagrus). Las anormalidades de la columna han sido documentadas en especies de aguas dulce (Akiyama et al., 1985; Madsen y Dalsgaard 1999) y en especies marinas (Paperna, 1978; Takashima, 1978; Barahona-Fernandes, 1982; Taniguchi et al., 1984; Daoulas et al.,1991; Andrades et al., 1996; Koumoundouros et al.,1997b, 2001a,b, 2002; Faustino y Power, 1998, 2001; Afonso et al., 2000; Boglione et al., 2001; Faustino, 2002; Gavaia et al., 2002; Kihara et al., 2002; Moteki, 2002; Favaloro y Mazzola, 2003; Boglione et al., 2003; Matsuoka, 2003; Sfakianakis et al., 2004). 2.5.2.3.-Factores condicionantes de la aparición de anomalías de esqueleto La aparición de deformidades esqueléticas en peces está relacionada con factores medioambientales, nutricionales y genéticos. Entre los factores ambientales están los abióticos (intensidad de la luz, oxígeno disuelto, temperatura, pH, salinidad, flujo de agua, asociados al cultivo), bióticos (bacterias, virus, hongos y parásitos) y xenobióticos (alguicidas, fungicidas, herbicidas, insecticidas, efluentes industriales y metales pesados). Entre los nutricionales están los relacionados con los ácidos grasos esenciales, fosfolípidos, aminoácidos, proteínas, vitaminas y minerales, mientras que entre los factores genéticos están los unigénicos y los poligénicos. Spanish summary 253 2.5.2.3.1.-Factores medioambientales Dentro de los factores ambientales abióticos se ha descrito la luz, la temperatura o la salinidad como causantes de la aparición de deformidades, en peces marinos (Battaglene and Talbot, 1990; Polo et al.,1991; Mihelakakis y Yoshimatsu, 1998; Cobcroft et al., 2001; Sfakianakis et al., 2004). Otros factores que puede provocar repercusiones negativas en la integridad del esqueletovértebras en larvas son los traumas mecánicos durante el desarrollo embrionario, o durante las primeras etapas de cultivo larvario, consecuencia de la manipulación de huevos y larvas. El desarrollo anómalo de la vejiga natatoria, (Chatain,1989,1990; Andrades, 1993;) y las variaciones en la hidrodinámica del tanque (Chatain, 1994; Divanach et al., 1997; Koumoundouros et al., 1997a,b; Kihara et al., 2002) se ha visto que favorecen la aparición de lordosis. De manera general, el sistema de cultivo ha sido descrito como factor modulador de la aparición de deformidades esqueléticas, fundamentalmente relacionado con la intensificación de las técnicas de cría, asociando el uso de sistemas extensivos y semi-intensivos a la obtención de peces con muy baja incidencia de deformidades esqueléticas más similares a las de los peces salvajes, cuando se comparan con los obtenidos en ejemplares cultivados mediante técnicas intensivas (Divanach y Kentouri, 1983; Divanach et al.,1996; Boglione et al., 2001; Koumoundouros et al., 2001a; Sfakianakis et al., 2004; Roo et al., 2005b; Giménez y Estévez, 2008b) (Figura 7). Los antecedentes de factores medioambientales bióticos como generadores de deformidades esqueléticas son escasos, siendo los parásitos los que tienen una mayor influencia. Parásitos pertenecientes a la familia mixosporea han sido descritos como causantes de deformidades esqueléticas en trucha arcoiris (Oncorhyncus mykiss), seriola coreana (Seriola quinqueradiata) y perca de río (Perca fluviatilis) (Lom et al., 1991). La relación de factores xenobióticos con la aparición de deformidades esqueléticas ha sido señalada para pesticidas (Chun et al. 1981; Thi Hong Lien et al., 1997), herbicidas (Koyama, 1996), hidrocarburos (Grady et al. 1992), compuestos orgánicos y organoclorados (Lindesjöö et al. 1994) y metales (Slominska y Jezierska, 2000). También el exceso de antibióticos en los tratamientos de enfermedades ha sido relacionado con la inducción de deformidades esqueléticas (Toften y Jobling, 1996). Spanish summary 254 2.5.2.3.2.-Factores nutricionales Además de los factores ambientales, son muy numerosos los factores nutricionales que actúan directamente sobre el metabolismo del hueso y el cartílago, como las vitaminas y ácidos grasos, además de otros factores que de manera indirecta condicionan estos procesos como son las hormonas, prostaglandinas, citoquinas y factores de crecimiento (Watkins y Seifert, 2000a). En el caso particular de los peces, se ha visto que los factores nutricionales tales como las deficiencias y excesos de vitaminas y ácidos grasos esenciales durante las primeras etapas de de crecimiento o las dietas de los reproductores pueden alterar el desarrollo osteológico de los embriones y larvas (Kanazawa et al., 1983; Akiyama et al., 1986, Knox et al., 1988; Chatain and Ounais–Guschemann, 1990; Afonso et al., 2000, Cahu et al., 2003, Saele et al., 2003; Hamre et al., 2005). Aunque, en los peces no se ha establecido una relación entre la composición de ácidos grasos de la dieta y la del cartílago y hueso, diferentes estudios con otro vertebrados como pollos y ratas han demostrado que las variaciones en la relación de las series n-3/n-6 PUFA en la dieta alteran la composición de los ácidos grasos del hueso y cartílagos (Xu et al., 1994; Watkins et al., 1991,2000b; Liu et al., 2004). En este sentido, Gapasin y Durai, (2001), han encontrado una relación entre la aparición de deformidades esqueléticas y los ácidos grasos esenciales de la dietas en los peces marinos larvas, juveniles y reproductores. Aunque, los mecanismos por los cuales los ácidos grasos controlan el desarrollo osteológico no son bien conocidos. Sin embargo, su implicación a nivel molecular ha sido demostrada, actuando como moduladores del genoma a través de receptores nucleares específicos, como los receptores activados de proliferación de Peroxisomas (PPAR) que se unen a la molécula de ADN como heterodímero con el receptor X del ácido retinoico (RXR). Este receptor actúa como factor de trascripción ligando-activado (Mangelsdorf et al., 1994) y los genes que lo regulan están involucrados en el desarrollo esquelético durante la ontogénesis, lo que pone de manifiesto que los HUFA pueden regular la expresión de genes del desarrollo y de este modo afectar el desarrollo del esqueleto, esto tiene muchas implicaciones en la salud humana y ha sido muy poco estudiado en el desarrollo de los peces (Cahu et al., 2003). Sin, embargo recientemente Villeneuve et al., 2005, 2006 han reflejado que el exceso de HUFA en las dietas acelera la diferenciación de los osteoblastos a través de una sobre-regulación del Receptor X retinoico α y las proteínas de morfogénesis del hueso (BMP4), lo que se refleja con la aparición de un exceso de vértebras en las larvas de lubina (Dicentrarchus labrax). Spanish summary 255 En cuanto a los aminoácidos esenciales, las deficiencias en triptófano han sido asociadas con la presencia de deformidades (Walton et al., 1984; Akiyama et al., 1985, 1986; Wilson, 1989). La deficiencia de péptidos en la dieta también influye en la incidencia de malformaciones esqueléticas en el desarrollo larvario (Cahu et al., 1999). En cuanto a las vitaminas, ha sido demostrada la influencia de los excesos o defectos las vitaminas A y C en las incidencias de las deformidades esqueléticas en peces (Halver, 1989; Dedi et al., 1995; Takeuchi et al., 1995, 998). En este sentido, es importante resaltar que el ácido retinoico tiene capacidades teratogénicas con influencia en los sucesos que acontecen durante la embriogénesis de animales y humanos, viéndose los efectos de éste, medidos a través de la expresión de los genes Hox y Sonic Hedgehog (shh). Así, un exceso de ácido retinoico en la dieta de las larvas de lenguado del Pacífico (Paralichthys olivaceus) provoca la aparición de compresión de las vértebras (Takeuchi et al., 1998). 2.5.2.3.3.-Factores genéticos Andrades et al. (1996) explican que la mayoría de los juveniles lordóticos observados en una granja de engorde de dorada (Sparus aurata), provenían probablemente de larvas lordóticas supervivientes, sugiriendo que las causas primarias de la aparición de lordosis podrían ser, entre otras, de origen genético que pueden afectar a los huevos durante el desarrollo embrionario. La primera asociación familiar de una deformidad severa en dorada se debe a Afonso et al. (2000), quienes describieron la aparición simultánea de una triple deformidad de columna (LEC; lordosis, escoliosis y cifosis) en todas las réplicas de descendientes de una misma familia. Astorga et al. (2003a,b) estudiaron el efecto de la consanguinidad en la aparición de las deformidades esqueléticas de dorada, mediante el cruce de reproductores normales con distintos niveles de consanguinidad (F=0,125; F=0,25), a las edades de 4, 14 y 35 días post-eclosión y a los 194 días de edad. Spanish summary 257 9.3.-Objetivos El objetivo principal de este trabajo fue “la mejora de la tecnología de producción de larvas y alevines de bocinegro”. Los objetivos específicos se dividen en dos etapas consecutivas que se complementan, una primera parte del trabajo en la que se describen aspectos específicos sobre la biología y desarrollo de las primeras etapas de vida de esta especie como son el desarrollo del sistemas visual y digestivo, el desarrollo osteológico o la aparición de deformidades esqueléticas en relación al sistema de cultivo empleado y al valor nutricional de las presas vivas. A continuación en la segunda parte, se ha aplicado el conocimiento adquirido para la mejora del protocolo de cría de esta especie. Los objetivos planteados se han logrado mediante la realización de 4 estudios que se incluyen en esta trabajo de tesis doctoral. Estudio I. Desarrollo del sistema visual de las larvas de bocinegro Pagrus pagrus (Linnaeus, 1758) en relación a los cambios del sistema digestivo y hábitos alimenticio. En este estudio se pretende describir la formación del sistema visual de las larvas de bocinegro, y su correlación con el desarrollo del sistema digestivo. Este objetivo se plantea con el fin de obtener información básica, como herramienta para la búsqueda de las condiciones de cultivo generales para esta especie: como son la necesidad de cambios de iluminación, uso de agua verde y determinación de la secuencia alimentaria idónea. Se realizó una experiencia de cultivo para lograr este objetivo. Estudio II. Desarrollo osteológico y aparición de deformidades esqueléticas en las larva de de bocinegro Pagrus pagrus (Linnaeus, 1758) bajo diferentes técnicas de cultivo. En este estudio se pretende describir el efecto de la intensificación de las técnicas de cría larvaria en relación al desarrollo osteológico de esta especie y la incidencia de malformaciones esqueléticas. Este objetivo se plantea con el fin de obtener información básica a cerca del patrón de desarrollo osteológico y la incidencia de las anomalías esqueléticas de esta especie, que permitirá determinar la calidad de los alevines producidos e idoneidad de las técnicas de cría aplicadas. Se realizaron dos experiencias de cultivo para lograr este objetivo. Spanish summary 258 Estudio III. Efecto del contenido de DHA en los rotíferos sobre la incidencia de deformidades esqueléticas del bocinegro Pagrus pagrus (Linnaeus, 1758). En este estudio se pretende describir el efecto ciertos factores nutricionales sobre el crecimiento, supervivencia e incidencia de las malformaciones esqueléticas en los primeros estadios de desarrollo de esta especie. Este objetivo se plantea con el fin de identificar factores nutricionales que afectan al desarrollo larvario y mejorar los productos enriquecedores de presas vivas para esta especie. Se realizaron dos experiencias de cultivo para lograr este objetivo. Estudio IV. Avances en las técnicas de cultivo de bocinegro Pagrus pagrus (Linnaeus, 1758): Comparación de sistemas de cultivo larvario intensivos y semi-intensivos. En este estudio se pretende mejorar los protocolos de producción larvaria de esta especie: Para alcanzar este objetivo se realizaron tres experiencias centradas en el estudio de los cambios en los regimenes de luz, la densidad de presas y el protocolo de co-alimentación, comparando a su vez diferentes sistemas de cultivo. Spanish summary 265 Figure 26: Sección longitudinal de los tanques de alevinaje. Figura 27. a) Vista longitudinal, planta y fotografía de tanques de alevinaje. -=---, D m_)1 ------ B \--"===~~ Spanish summary 266 4.3.-Especie objeto de estudio El bocinegro o pargo (Pagrus pagrus), es un teleósteo, que pertenece a la familia Sparidae, su clasificación taxonómica detallada se especifica a continuación: Phyllum: Chordata Superclase: Gnathostomata Clase: Osteichthyes Orden: Perciformes Suborden: Percoidei Familia: Sparidae Género: Pagrus Especie: Pagrus pagrus Figura 28. Ejemplar reproductor, huevos, larvas y alevines de bocinegro e instalaciones comerciales de engorde en tanques donde se realizaron experiencias piloto de engorde de esta especie. 4.3.1 Habitat Se trata de una especie euriterma que se adapta a un amplio rango de temperaturas y a una gran variedad de hábitats, generalmente se encuentra en fondos rocosos y pedregales en una batimetría media 50 m de profundidad, los ejemplares jóvenes (denominados palletes en Canarias) es frecuente encontrarlos a menor profundidad en un rango de 10-30m. En el medio natural, es un Spanish summary 267 pez que se alimenta de moluscos y crustáceos principalmente, gracias a su potente mandíbula, mientras que en condiciones de cultivo se adapta con facilidad al alimento seco. Se trata de una especie hermafrodita proterogínia (hembra en su primea etapa de vida y macho posteriormente) y la época de puesta, en el medio natural, oscila entre los meses de diciembre y enero en el Atlántico occidental (Ciechomski y Weiss, 1973), y en el caso de Canarias se puede prolongar hasta comienzo de primavera (Pajuelo y Lorenzo, 1996). En esta misma localización y en condiciones de cultivo la época de puesta da comienzo a principios de marzo pudiéndose prolongar hasta finales mayo sin manipulación foto-térmica (Cejas et al., 1997). 4.3.2 Distribución geográfica1.3.- Distribución Esta especie ha sido citada en diferentes regiones, pudiéndose encontrar en el Mar Mediterráneo y Adriático; en el Atlántico este desde las Islas Británicas hasta el sur de Angola, incluyendo las Islas Canarias, Azores y Madeira; y en el Atlántico oeste se puede localizar desde Nueva York, hasta el sur de Argentina (Manooch y Hassler, 1978) (Figura 29). Figura 29. Distribución geográfica de la especie Pagrus pagrus (Fish Base, 2008). 4.3.3 Perspectivas de cultivo1.3.- Distribución El bocinegro (Pagrus pagrus) desde hace algunos años, es uno de los peces marinos que han sido propuestos como candidato potencial para la diversificación de la acuicultura marina (Kentouri et al., 1995; Hernández-cruz et al., 1999). Tiene un alto precio y buena aceptación en el mercado, lo que es una condición indispensable para la introducción de una nueva especie ya que la rentabilidad del cultivo comercial es la última finalidad de la investigación en acuicultura. Spanish summary 268 El engorde comercial de bocinegro, todavía no es una realidad, si bien esta estadísticamente señalada una primera producción en Grecia en el año 1999 con 100 toneladas (FEAP, 2006), además durante el desarrollo de este trabajo se han realizado diferentes experiencias de engorde a escala piloto, tanto en jaulas como en tanques, con empresas locales con producciones muy reducidas. Aunque, las condiciones para el transporte de alevines han sido descritas por Pavlidis et al, (2003), los datos de producción de alevines son escasos y se puede resaltar la producción de 1,1 millones de alevines en Grecia en el año 2000 y la reciente publicación de datos correspondientes a los años 2006, 2007 y 2008 señalan una producción de alrededor de 2 millones de alevines anuales en este país (FEAP, 2008), sin embargo no se han encontrado datos a cerca de las producciones obtenidas con estos alevines. A escala experimental se han de destacar las producciones obtenidas en Canarias que se han incrementado en los últimos años, pasando de 12.000 unidades en el año 2002 hasta los cerca de 100.000 alevines que se han producido de manera regular en los años 2005 y 2006. Los estudios de cultivo larvario de esta especie muestran resultados dispares según las condiciones de cultivo empleadas (Papandroulakis et al., 2004; Kentouri et al., 1995; HernándezCruz et al., 1999; Michelakakis et al., 2001; Papandroulakis et al., 2004). Y diferentes estudios de investigación básica han descrito en detalle la ontogenia de diferentes tejidos y órganos (Socorro et al., 2001, Roo et al., 1999, Darias et al., 2005, 2007) así mismo la descripción detallada del desarrollo osteológico del bocinegro ha sido recientemente publicada por Socorro (2006). 4.4.-Condiciones experimentales 4.4.1.-Cultivos auxiliares Para el desarrollo de las diferentes experiencias de cultivo larvario fue necesario la producción de “alimento vivo”, término que define al conjunto de organismos que se utilizan como dieta de las larvas de peces marinos en sus etapas más tempranas, cuando todavía no es viable la alimentación con dietas inertes, y que por sus características biológicas necesitan ser producidos en condiciones específicas dentro de las diferentes unidades de la instalación. 4.4.1.1 Cultivo de fitoplancton. En todas las experiencias de cultivo larvario se utilizó la microalga Eustigmatoficea, Nannochloropsis sp. Las principales características de esta especie han sido descritas por Maruyama et al., (1989) y se presenta en la siguiente tabla III. Spanish summary 269 Tabla III. Características biológicas de Nannochlropsis sp (Maruyama et al., 1989). Dimensiones: 2-4 μm. Forma celular: circular a ovalada. Forma del cloroplasto: copa u ovalada. Propagación: fisión binaria. Retículo endoplásmico en el cloroplasto: presente. Disposición de los tilacoides: 3-tilacoides. Pigmentos predominantes: clorofila a, caroteno, violaxantina, éster de vauqueriaxantina. Figura 30: Esquema de una célula de Nannocloropsis sp. y visión microscópica en cámara Neubauer. El sistema de cultivo empleado para la producción masiva de fitoplancton, fue de tipo “Bach” o sistema cerrado. Este se realizó en la unidad de producción de alimento vivo, en volúmenes crecientes de 50, 230 y 460 litros, utilizando bolsas de polietileno transparentes (Figura 31). En el cultivo se utilizó, agua de mar filtrada mecánicamente a través de un filtro de polyester reforzado y fibra de vidrio (Mod.00689; Astral pool, Barcelona, España) relleno con un lecho de arena de diversa granulometría. Seguidamente, el agua fue esterilizada por radiación UV a una longitud de onda de 254 nm con un equipo de desinfección industrial (Mod. M-3PE-300; Wedeco AG, ;tJ Cloroplasto -__ Tilacoides ,_'o. ¡ R.E del Cloroplasto _,. . Nucleo l M.tocondria Spanish summary 270 Herford, Alemania). La salinidad natural (37‰) se redujo, para el proceso de cultivo mediante la adición de la cantidad necesaria de agua dulce para alcanzar una salinidad final de 25‰, que se comprobó mediante un refractómetro portátil (Mod. SZJ-S, Madrid, España). A cada bolsa de cultivo se le introdujo uno o dos difusores de cerámica según el tamaño de la misma, ubicados a una distancia de 15cm del fondo, a través de los cuales se suministró aireación para favorecer la mezcla del medio y evitar la sedimentación de las células. Los cultivos, se mantuvieron con fotoperiodo continuo, combinando la iluminación natural con luz artificial, mediante luminarias fluorescentes (Mod. TLD 58W/54-765, Philips, Francia), que mantenían un intensidad mínima durante la noche de 9.500 lux medida con un luxómetro digital (Mod. HT170N; Italia). Figura 31. Bolsas de cultivo de fitoplancton de 50, 230 y 460 litros de volumen. Independientemente del volumen de cultivo, el protocolo consistió en la introducción de una cantidad de inóculo (concentración inicial de 1.5*106 cells.ml-1 ) en las bolsas previamente llenas con el agua de mar preparada (25‰) y la adición de fertilizante comercial (Nutri-Phyt; Fitoplancton marino S.L, Puerto Santa María, España) únicamente el primer día de cultivo. Los ciclos de cultivo tuvieron una duración media de 8 a 10 días tras los cuales se cosechó el volumen total de la bolsa, que alcanzaba unas concentraciones de 35-80*106cells.ml-1 según el volumen de cultivo, siendo generalmente menor (35*106cells.ml-1) en los volúmenes mayores. Diariamente se comprobó el estado del cultivo mediante una valoración fotométrica de la turbidez con un fotómetro portátil (Mod. PF-11; Macherey-Nagel; Durew, Alemania) y contaje con hemocitómetro (Mod. Neubauer, Alemania) de muestras de diferentes bolsas al azar, que permitió determinar la concentración y la presencia de contaminantes biológicos en el medio (Figura 32). Spanish summary 271 Figura 32. Curva de crecimiento tipo y correlación entre medidas de concentración y turbidez del cultivo. Ocasionalmente se encontraron contaminantes como cianobacterias y ciliados tipo Euplotes sp. (Figura 33), en el caso de este último, la adición de formaldehido (38%) (Panreac, España) a una dosis de 0.05 ml.l-1 de cultivo dio excelentes resultados eliminando el 100% de los contaminantes sin comprometer la viabilidad del cultivo de fitoplancton. Figura 33. Organismo contaminante (Euplotes sp).encontrado en los cultivos de Nannochloropsis sp. El fitoplancton producido se utiliza para diferentes acciones dentro del proceso de cría de larvas Concentración (Cells.ml-1) 0 20x10640x10660x10680x106100x106 Turbidez (FAU) 0 200 400 600 800 1000 1200 1400 Concentración vs Turbidez Regresión Días de cultivo Siembra123456789 Concentración ( Cells.ml-1) 0 20x106 40x106 60x106 80x106 100x106 Nannochloropsis sp. Spanish summary 272 de peces. Así, las bolsas de 50 l se utilizaron como inóculo para el escalado del cultivo a 230 l. El procedente de las bolsas de 230 se utilizó a su vez, como inóculo de las bolsas de 460 l y como medio de cultivo para el mantenimiento de las cepas de rotíferos. Finalmente, el fitoplancton producido en las bolsas de 460 l se utiliza en el proceso de cultivo de larvas para la aplicación de las técnicas de agua verde y ocasionalmente para la alimentación de rotíferos. 4.4.1.2 Cultivo de rotíferos. El rotífero utilizado en las experiencias de cultivo larvario fue Brachionus plicatilis. cepa tipo L con una longitud total media de los individuos adultos de 240 µm. Figura 34. Rotíferos (Brachionus plicatilis) utilizados en los cultivos de larvas. El proceso del cultivo consta de varias fases (mantenimiento de la cepa, pre-cultivo y cultivo masivo) lo que permite, por un lado, autonomía en la producción y por otro, la posibilidad de recomenzar el cultivo de forma rápida en caso de fallo o accidente. 4.4.1.2.1.-Mantenimiento de la cepa y pre-cultivo de rotíferos. Generalmente la cepa se mantuvo en recipientes de 2 a 5 l de capacidad que se llenaron con Nannochloropsis sp. a una concentración de 20-25*106 cells.ml-1, al que se añadió la cantidad de rotíferos necesario, para obtener una concentración inicial de 2-5 rot.ml-1. El cultivo se mantuvo con aireación suave a temperatura ambiente y fotoperiodo continuo de 24h. Una vez que el cultivo alcanzó una concentración de 75-100 rot.ml-1, alrededor del 5º-7º día, se cosechó el volumen total y se pasó a un volumen mayor, normalmente, botellas de 20 l repitiendo Spanish summary 273 el mismo protocolo descrito anteriormente. Figura 35. Recipientes para el pre-cultivo de rotíferos. 4.4.1.2.2.-Cultivo masivo. En el proceso de producción masiva de rotíferos se utilizaron tanques cilindro-cónicos de fibra de vidrio, con una capacidad de 1700 l (Figura 36). Las condiciones generales de cultivo se muestran en la tabla IV. Tabla IV. Condiciones generales de la producción de rotíferos en las instalaciones experimentales del ICCM. Agua: Esterilizada con UV Temperatura: 20-25ºC Salinidad: 25 ppt Iluminación: Fotoperiodo natural, sin iluminación directa. Aireación: >2 l/min con un único difusor central, de manguera porosa, situado al 80% de altura del tanque. Oxígeno: Inyección de O2 puro, a través de difusor de manguera porosa, cuando el nivel era inferior a 3,5ppm. Los ciclos de producción (Tabla V) fueron de 8 días de cultivo, iniciándose el proceso con una densidad aproximada de 265 rot.ml-1, a partir del 4º día de cultivo se cosechó en días alternos 400 l de volumen del tanque que fueron repuestos con agua previamente mezclada para alcanzar la salinidad establecida (25ppt). Al 8ºdía se procedió a la cosecha del volumen total y reinicio de un Spanish summary 274 nuevo ciclo. Como medida rutinaria, tras la cosecha, los rotíferos eran sumergidos durante 1 minuto en agua dulce para eliminar posibles contaminantes. Diariamente se determinó la densidad media y el porcentaje de hembras ovígeras en tres contajes individuales de 0,5ml con una Micropieta (Mod. Eppendorf Research 100-1000µl; Hamburgo, Alemania), tomados de una muestra recogida en la zona central del tanque de cultivo, para asegurar una buena homogeneización de la muestra en cada medida. Se anotó la actividad de los individuos, con una estimación visual de la movilidad de los mismos identificándola como (alta-media-baja). Se anotó también la presencia de posibles contaminantes, como ciliados y copépodos, con una estimación visual de concentración (alta-media-baja), y se determinó la calidad del medio de cultivo por la presencia de flóculos o partículas en suspensión y el aumento de viscosidad del mismo, con una estimación visual (Limpio-Medio-Sucio). De la misma manera, a diario se tomaron medidas de temperatura y oxígeno disuelto a las 9:00 y a las 15:00 con una sonda portatil (Mod. Handy Polaris, OxyGuard; Birkerød, Dinamarca). En cuanto a la alimentación, de manera general se utilizó levadura de panificación (Saccharomyces cerevisiae) añadiendo 0,4g/106rotíferos. Únicamente el día de inicio del ciclo se suplementó la levadura con fitoplancton liofilizado, en una concentración de 0,1g/ 106rotíferos. La alimentación se distribuyó manualmente a las 09:00 y a las 15:00 horas y mediante un distribuidor automático a las 21:00 y 03:00 horas. Figura 36. Tanques de producción masiva de rotíferos y sistema de distribución automática del alimento. Spanish summary 281 Tabla VII. Protocolo de descapsulación de Artemia y conservación en salmuera de la planta de producción de alevines Protocolo Descapsulación-Conservación en salmuera de Artemia Hidratación: 45´- 1h 30´. 10-12 litros de agua por kg de ciste seco. Descapsulación Artemia (Kg) Hipoclorito(g) Lejía(l) NaOH (g) Agua de mar( l) Vol total Lejías varias 10 l 0,25 87,5 2,19 16,5 1,31 3,5 Indice de Refracción: 1,352 0,50 175 4,38 33 2,6 7 Conc. Gram/l 40 1,0 350 8,75 66 5,3 14 1,5 525 13,13 99 7,9 21 2,0 700 17,50 132 10,5 28 2,5 875 21,88 165 13,1 35 3,0 1.050 26,25 198 15,8 42 4,0 1.400 35,00 264 21,0 56 5,0 1.750 43,75 330 26,3 70 6,0 2.100 52,50 396 31,5 84 7,0 2.450 61,25 462 36,8 98 7,5 2.625 65,63 495 39,4 105 Notas Lejía: 0.35 gramos de hipoclorito por gramo de cistes secos(INVE). Sosa: 66g de NaOH por kilogramo de cistes secos. Antiespumante. Opc: 250-500ppm, 0.25-0.5 g/l. Agua de mar: La necesaria para alcanzar una concentración final de 14 l/kg de ciste secos. Tiempo:5-8min, según cambio de color (pardo-naranja). Neutralización :Lavar con agua de mar abundante durante 20 min, tb puede utilizarse 50g Tiosulfato/kg ciste. Se puede comprobar la presencia de restos de Cl con ortotolidina u otro detector de cloro. Spanish summary 282 Salmuera Artemia (Kg) Sal (kg) Agua (l) Vol final(l) Conc. Final 0,25 0,29 1,00 1,79 139,4 0,50 0,59 2,00 3,59 139,4 1,0 1,17 4,00 7,17 139,4 1,5 1,76 6,00 10,76 139,4 2,0 2,34 8,00 14,34 139,4 2,5 2,93 10,0 17,93 139,4 3,0 3,52 12,0 21,52 139,4 4,0 4,69 16,0 28,69 139,4 5,0 5,86 20,0 35,86 139,4 6,0 7,03 24,0 43,03 139,4 7,0 8,20 28,0 50,20 139,4 7,5 8,79 30,0 53,79 139,4 Notas Agua: 4l agua de mar( 37% ) por kg de cistes secos. Sal:330 g sal/litro - g de sal en agua de mar. Cistes: VT= Agua + sal + Cistes(2*peso en seco) Concentración: Kg descapsulado/VT Producción teórica: HE* gramos sembrados Conservar en ausencia de luz en lugar fresco(<10º). Elaborado por: Fecha Modif. J.Roo 02/02/2003 Spanish summary 283 4.4.1.3.4.-Eclosión Según las necesidades de alimentación de las larvas se determinó el volumen de cistes en salmuera a eclosionar. El procedimiento se iniciaba con la desinfección del tanque de eclosión, para posteriormente proceder a su llenado con agua del mar filtrada y esterilizada. Se utilizaron tanques cilíndro-cónicos de fibra de vidrio, de 1700 l de capacidad, con aireación central fuerte, iluminación durante 24 horas con una intensidad de 2000 lux. Estos tanques estaban equipados con un intercambiador de calor que permitía mantener la temperatura del cultivo en torno a 2829ºC. El día anterior a la siembra de los cistes en salmuera, se llenaba el tanque y se introducía un sensor de temperatura (Mod. OxyGuard; Birkerød, Dinamarca) que unido a un sistema automático de regulación de temperatura (Mod. AKO Electrómecanica, Barcelona, España ) permitía que el nuevo tanque alcanzase y mantuviese la temperatura requerida para la eclosión. La densidad de siembra se fijó en 1.8g.l-1, el 95% de los huevos eclosionaban en la siguientes 24h, obteniéndose nauplius de Artemia con un tamaño de 450-650μm, dependiendo del tipo de Artemia, y que era el adecuado para la primera fase de transición de alimento vivo de las larvas, paso de rotífero a Artemia (Figura 41). Figura 41. a) Siembra de salmuera; b) Tanques de eclosión; c) Nauplio de Artemia salina recién eclosionado. 4.4.1.3.5.-Enriquecimiento Para mejorar el valor nutritivo de la Artemia, se procedió a su enriquecimiento, al igual que en proceso de eclosión se utilizaron tanques cilindro-cónicos de fibra de vidrio, de 1700l de capacidad, con aireación central fuerte, iluminación durante 24 horas, con una intensidad de 2000 lux, equipado con un intercambiador de calor que permitía mantener la temperatura de cultivo en Spanish summary 284 torno a 25-26ºC. Los nauplios de Artemia recién cosechados, se introducían en el tanque de enriquecimiento, lleno con la cantidad necesaria de agua del mar filtrada y esterilizada para mantener una concentración de 250.000-300.000 nauplios.l-1 . De modo general el enriquecedor utilizado fue Easy DHA Selco (Inve, Dendermonde, Bélgica), producto que se presenta en forma de emulsión lipídica. El tiempo de enriquecimiento de los nauplios fue de 18-24 horas a una concentración de 0.6gr.l-1, y añadido en una única dosis al inicio del enriquecimiento (h=0). Una vez enriquecidos los metanauplius de 18-24horas, se filtraron en una bolsa de 125µm de luz de malla, se lavaron con agua de mar abundante para eliminar los posibles restos de emulsión y se concentraron en un cubo de 20l procediendo a su contaje y comprobando visualmente su correcto enriquecimiento por la presencia de gotas lipídicas en el tracto de la Artemia (Figura 42). Los metanauplius enriquecidos se añadían manualmente y mediante distribuidores automáticos de acuerdo a las necesidades de los cultivos larvarios. Figura 42. a) Tanque de enriquecimiento; b) Mantenimiento de Artemia en frío; c) Comprobación del correcto enriquecimiento de los metanauplios. Spanish summary 285 4.4.2.-Cultivo larvario 4.4.2.1.-Huevos Por lo general, los huevos de peces marinos son un estadío biológico fácil de manipular y transportar. Sin embargo, unas condiciones inadecuadas de manipulación pueden dar lugar a una elevada mortalidad de los mismos, y a efectos negativos posteriores tales como bajas tasas de eclosión, elevada mortalidad larvaria durante los primeros días de cultivo o aparición de diferentes anomalías morfo-anatómicas. En consecuencia, el manejo de los huevos de peces debe ser cuidadoso para evitar mortalidades y obtener larvas de calidad. En las distintas experiencias de cultivo larvario, que se presentan en este documento se utilizaron huevos procedentes de puestas naturales de stocks de reproductores pertenecientes al ICCM y al Instituto Español de Oceanografía-Centro Costero de Canarias (IEO-COC). En ambas instalaciones, los ejemplares reproductores fueron alimentados dos veces por semana con dietas comerciales, que se complementaron una vez a la semana con pescado fresco y moluscos como sepia, calamar y mejillón (Figura 43). Figura 43. Ejemplares reproductores de bocinegro (Pagrus pagrus). Independientemente del origen, la manipulación de las puestas siguió una secuencia similar. Los huevos se recogieron en un colector de malla de 500µm dispuesto en la salida superior de agua de los tanques de cultivo, los huevos fertilizados presentan flotabilidad positiva y caen por rebose en dicho colector (Figura 44a). Una vez recogida la puesta se realizó la separación de huevos en un cono de decantación, diferenciándose, la fracción flotante como huevos mayoritariamente viables y la no flotante como mayoritariamente no viables (Figura 44b). Cuando el origen de los huevos Spanish summary 286 no fue del stock del ICCM, estos se trasladaron por avión o barco, en el interior de cubitainers de plástico reforzado, de 20 litros de capacidad, que se mantuvieron en el interior de cajas individuales de poliestireno expandido, que protegen de los golpes y mantienen unas condiciones de temperatura estables durante el transporte (Figura 44c). En cada cubitainer, se introducen una media de 250.000 huevos a una concentración a 25.000 huevos.l-1, llenando únicamente el 50% del volumen del recipiente con agua de mar. Tanto el agua como el 50% restante del volumen del cubitainer se saturaron con oxígeno gas (Figura 44d). Figura 44. a) Colector de huevos, b) Embudo de decantación para separación de fracción de puesta no viable; c,d) Llenado de cubitainer y cajas para transporte de huevos. Una vez en las instalaciones, se procedió a la aclimatación de los huevos a los parámetros físicoquímicos existentes y se hizo una nueva decantación para eliminar los huevos muertos o dañados durante el transporte. Se realizó un recuento de los huevos viables y se procedió a su siembra, por volumetría, en los tanques de cultivo (Figura 45). Figura 45. a) Conteo de huevos; b) Huevos viables para siembra; c) Siembra de huevos en tanques de cultivo. Spanish summary 287 Paralelamente se sembraron seis pequeños recipientes cilíndricos provistos de intercambio de agua y aire para calcular los índices de eclosión y de supervivencia larvaria, al tercer día de vida, justo antes de la apertura de la boca (Figura 46). Figura 46. a) Recipientes usados para determinar porcentajes de eclosión y supervivencia larvaria; b) Huevos muertos y larvas recién eclosionadas no viables; c) Larvas viables. 4.4.2.2.-Cultivo larvario semi-intensivo Las experiencias de cultivo larvario, con tecnología semi-intensiva (Mesocosmos), se realizaron en los tanques de 40.000 litros de capacidad, anteriormente descritos. En todas las experiencias se sembraron huevos fertilizados de 12 a 24h de vida, a una densidad que osciló entre 5-6,5 huevos.l-1. En todos los casos, durante la fase de cultivo larvario se utilizó agua de mar previamente filtrada y esterilizada como se describe en el apartado de cultivos auxiliares. La renovación de agua se incrementó desde un 10% del volumen diario del tanque en las primeras etapas hasta un 25%.h-1 a partir de los 30 días post eclosión (dpe). La salinidad del agua de mar se mantuvo constante durante todas las experiencias (37‰). El oxígeno disuelto y la temperatura se determinaron diariamente, fluctuando entre los diferentes experimentos. De manera general, la iluminación, consistió en una combinación de luz natural y luz artificial con un fotoperiodo 24h horas (el fotoperiodo se varió en experiencias concretas de cultivo larvario como se detalla en las diferentes experiencias), pasando posteriormente a fotoperiodo natural. Los tanques están equipados con 4 luminarias fluorescentes (Mod TLD 36W/54, Philips, Francia) que en combinación con luz natural que penetra a través del techo traslucido mantenían una intensidad de entre 1.000-3.500 Lux en la superficie del agua. En cuanto a la secuencia alimentaria, la tecnología de mesocosmos, incluye el uso de agua verde, Spanish summary 288 por lo que desde el día 2 dpe, se añadió diariamente fitoplancton vivo (Nannochloropsis sp.) para mantener una concentración media de 250-500.000 células.ml-1. Desde el día 2 hasta el 25dpe, la alimentación consistió en rotíferos (Brachionus plicatilis) alimentados con levadura de panificación (Saccharomyces cerevisiae) y posteriormente enriquecidos con diferentes productos comerciales o experimentales según el experimento, manteniendo una concentración 4-5 rotíferos.ml-1 en el tanque larvario y ajustado dos veces al día (08:00;15:00). Desde el día 13 hasta el 20 una vez al día (11:00), se añadieron 175 nauplios.l-1 de Artemia (Tipo AF, INVE Aquaculture, Dendermonde, Bélgica). Desde el día 15 hasta el día 50 se añadieron metanauplios de Artemia (Tipo EG, INVE Aquaculture, Dendermonde, Bélgica), enriquecidos con Easy DHA Selco (INVE Aquaculture, Dendermonde, Bélgica) a una concentración que se incrementó de 250 a 1000 metanauplios.l-1 ) tres veces al día (09:00;15:00 y 20:00) siendo la última toma de Artemia distribuida mediante un distribuidor automático. La concentración de presas en el tanque (rotíferos y Artemia) se evaluó mediante la toma de muestras, dos veces al día (08:00;14:00), de 3-5 puntos del tanque de cultivo, antes de la adición de presas nuevas para restablecer la concentración predeterminada. A partir del 5dpe y hasta los 25dpe se limpio superficie de los tanques mediante el uso de un limpiador de superficie, para la eliminación de la película lipídica que se genera con la adición de presas vivas enriquecidas. Generalmente, el destete dio comienzo el día 20 utilizándose micro dietas de la gama Genma Micro (Skretting, Francia) diseñadas para peces marinos y se dio por finalizado a los 45-50dpe. La alimentación con microdietas se realizó inicialmente de forma manual y posteriormente, cuando se observó una buena aceptación de las mismas, se distribuyeron de forma automática mediante alimentadores automáticos (T-Drum feeders, Arvotec, Noruega), inicialmente cada hora y finalmente cada 15min, durante 24h al día. (Tabla VII). A los 50 días de edad los alevines fueron transferidos a tanques de 10.000 litros en el área de alevinaje, donde se mantuvieron en las mismas condiciones de cultivo (1-10 ind.l-1) en circuito abierto hasta 95 días, cuando los peces fueron contados y se llevó a cabo la caracterización esquelética. 4.4.2.3.-Cultivo larvario intensivo Las experiencias de cultivo larvario, con tecnología intensiva, se realizaron en los tanques de 2.000 litros de capacidad, descritos previamente. Este tipo de tecnología es ampliamente utilizada en los criaderos comerciales, con diferentes variantes en cuanto a las condiciones generales del cultivo como pueden ser el tamaño y forma de los tanques, flujos de agua, posición y número de aireadores o la secuencia alimentaria y productos comerciales y dietas empleadas para el enriquecimiento de presas y destete de las larvas. De manera general, esta técnica se basa en el Spanish summary 289 uso de una densidad larvaria elevada que generalmente oscila entre 50 y 150 larvas.l-1. En el presente trabajo, en todas las experiencias se utilizó una densidad de siembra que osciló entre 100-125 huevos.l-1. Se utilizó agua de mar previamente filtrada y esterilizada, al igual que en el técnica semi-intensiva. La renovación de agua, se redujo de un 25%.h-1 durante la eclosión, a un 10% del volumen del tanque diario en las primeras etapas, incrementándose gradualmente hasta un 25%.h-1. A partir de los 30 días post eclosión. La salinidad del agua de mar se mantuvo constante durante todas las experiencias (37‰) y el oxígeno disuelto y la temperatura se determinaron diariamente, fluctuando entre los diferentes experimentos. De manera general, la iluminación, consistió en una combinación de luz natural y luz artificial, con un fotoperiodo 24h horas (el fotoperiodo se varió en experiencias concretas de cultivo larvario como se detalla en las diferentes experiencias), pasando posteriormente a foto periodo natural. Los tanques están equipados con una única luminaria incandescentes (Mod TLD 36W/54, Philips, Francia) que aportaban una intensidad de entre 1500-3500 Lux en la superficie del agua. Esta tecnología incluyó también el uso de agua verde, por lo que desde el día 2 dpe, se añadió diariamente fitoplancton vivo (Nannochloropsis sp.) para mantener una concentración media de 250-500.000 cells.ml-1. Desde el día 2 hasta el 25dpe, la alimentación consistió en rotíferos (Brachionus plicatilis) alimentados con levadura de panificación (Saccharomyces cerevisiae) y posteriormente enriquecidos, con diferentes productos comerciales o experimentales según las experiencias, manteniendo una concentración 5-10 rotíferos.ml-1 en el tanque larvario y ajustada dos veces al día (08:00;15:00). Desde el día 13 hasta el 20, una vez al día (11:00), se añadieron 250 A0.l-1 Nauplios de Artemia (Tipo AF, INVE Aquaculture, Dendermonde, Bélgica), posteriormente desde el día 15 hasta el día 50, se añadieron metanauplios de Artemia (Tipo EG, INVE Aquaculture, Dendermonde, Bélgica), enriquecidos con el producto A1 DHA Selco (INVE Aquaculture, Dendermonde, Bélgica) a una concentración que se incrementó de 250 a 2500 metanauplios.l-1 ) tres veces al día (09:00;15:00 y 20:00) siendo la última toma de Artemia distribuida mediante un distribuidor automático. La concentración de presas en el tanque, se evaluó mediante la toma de muestras, dos veces al día (08:00;14:00), de 3 puntos del volumen del tanque de cultivo, antes de la adición de presas nuevas al tanque para restablecer la concentración de presas indicada. A partir del 5dpe y hasta los 20dpe se procedió a la limpieza de la superficie de los tanques mediante el uso de un limpiador de superficie, para la eliminación de la película lipídica que se genera con la adición de presas vivas enriquecidas. Generalmente, el destete dio comienzo el día 20 utilizándose microdietas de la gama Genma Micro (Skretting, Francia) diseñadas para peces marinos y se dio por finalizado a los 45-50dpe. La alimentación con microdietas se realizó inicialmente de forma manual y posteriormente cuando se observó una Spanish summary 290 buena aceptación de las mismas, se distribuyó de forma automática mediante alimentadores automáticos (Mod. T-Drum feeders; Arvotec, Noruega), inicialmente cada hora y finalmente cada 15min, durante 24h al día. (Tabla VII). A los 50 días de edad los alevines fueron transferidos a tanques de 10.000 litros en el área de alevinaje, donde se mantuvieron en las mismas condiciones de cultivo (1-10 ind.l-1), en circuito abierto hasta 95 días, cuando los peces fueron contados y se llevo a cabo la caracterización esquelética. . Spanish summary 297 Martoja-Pierson (1970) y de García del Moral (1993) modificadas por Socorro (2006) (Tablas IX, X). Tabla IX. Protocolo de tinción hematoxilina–eosina y con ácido peryódico-reactivo de Shiff-Hx (PAS-Hx) Paso 5a: Tinción con Hematoxilina-eosina (H&E) Paso 5b:Tinción con ácido peryódico-reactivo de Shiff-Hx (PAS-Hx) Producto Tiempo Producto Tiempo Hematoxilina de Harris 15-20 min Acido HIO4 (peryódico) 0,5% 5 min Alcohol ácido 3 baños cortos Agua 5 min Agua Lavado Reactivo de SCHIFF 20 min Agua amoniacal 20seg Agua 5 min Agua en continuo 5 min Hematoxilina 15 min Eosina de Puttis 3-4min Agua 20 min Agua Lavado Una vez finalizada la tinción, se procedió a la deshidratación y clarificado de las muestras (Tabla X). Tabla X. Protocolo de deshidratación y clarificado Paso 6: Deshidratación y clarificado Producto Tiempo Alcohol 96% 5 min Alcohol 100% 10 min Alcohol 100% 5 min Xilol 5 min Xilol 5 min Finalizado el proceso de tinción, las preparaciones fueron observadas y fotografiadas en un fotomicroscopio (Mod. DMBE, Leica, Nussloch, Alemania) para la realización de los estudios descriptivos de las diferentes estructuras. A su vez, cuando fue necesaria la realización de mediciones y contaje de estructuras particulares, se utilizó un programa informático de análisis de imagen (Image-pro Plus versión 2.0; Media Cybernetics, Inc., Buckinghamshire, Inglaterra). Spanish summary 298 4.5.5.-Estudio osteológico Para estudiar el desarrollo osteológico de las larvas desde el día 0 al 50dpe se cogieron 25 individuos, por tanque de cada tratamiento cada 5 días. Estos se fijaron en una solución de formol tamponado al 10% y posteriormente se sometieron al proceso de tinción de estructuras de cartílago-hueso siguiendo la metodología de Taylor y Dyke (1985) y (Dingerkus y Uhler (1977) modificadas por Socorro (2006). Las larvas fijadas en formol se extraen del recipiente de conservación y se introducen en un recipiente de fijación de acuerdo con siguiente procedimiento: Tabla XI. Protocolo de tinción cartílago-hueso Una vez terminado el proceso, las larvas presentan las estructuras esqueléticas teñidas (azul estructuras cartilaginosas y rojo las estructuras óseas) y fueron estudiadas individualmente bajo la Paso 1: Tinción de cartílago Paso 2: Hidratación Paso 3: Clarificación Tiempo Tiempo Tiempo Tinción con Azul Alcian 120 min Alcohol 96% 60 min Proceso de clarificación 60 min Alcohol 96% 60 min Alcohol 96% 60 min Preparacion de Solución de azul Alcian 10mg azul alcián BGX (c.i. 16230) 80ml de alcohol 96% 20ml ácido acético glacial Alcohol 75% 60 min Preparación de la solución de tripsina 90 mg tripsina 30 ml de Na2B4O710H2O (solución saturada) 70 ml agua destilada Alcohol 40% 60min Alcohol 15% 60 min Agua destilada 60min Paso 4: Tinción de hueso Paso 5: Clarificacion y conservación Tiempo Relación Tiempo Tinción con Rojo Alizarina 60min KOH 0,5% - Peróxido 3:1 60min KOH 0,5% - Glicerina 3:1 12-24 horas KOH 0,5% - Glicerina 1:1 12-24 horas KOH 0,5% - Glicerina 1:3 12-24 horas Glicerina + Timol 100% Conservación Preparacion de Solución de rojo alizarina KOH 0,5% en agua. Rojo de alizarina 1 g / litro Preparación de la solución de Glicerina-Peróxido de hidrógeno KOH 0,5% Glicerina (3:1) H2O2 1,2 % Spanish summary 299 lupa (Mod. Olympus, casa pais) para describir su desarrollo osteológico, tomando fotografías digitales,en los momentos más importantes del desarrollo. Figura 51. a) Detalle de estructura craneal de una larva de bocinegro de 8dpe presentado únicamente estructuras cartilaginosas teñidas con azul alcian; d) Larva de 27 días con estructuras calcificadas teñidas con rojo alizarina. 4.5.6.-Caracterización de deformidades Para la caracterización de las deformidades esqueléticas, se tomaron muestras de manera rutinaria a diferentes edades, en todas las experiencias de cultivo, así al finalizar el destete 50dpe, se llevó a cabo una valoración visual de 500 larvas por tanque y por tratamiento. En etapas posteriores, además de la valoración visual, se realizaron valoraciones de placas radiológicas de baja intensidad realizadas con un mamógrafo digital (Mod. Senographer-DHR, General electrics, USA) de las deformidades morfo-anatómicas que aparecían en 100 individuos de cada tratamiento. Los anomalías detectadas se clasificaron de acuerdo con Divanach et al., (1996). Spanish summary 300 Figura 52. Evaluación visual de deformidades, preparación de placas y mamografías resultantes para caracterización en detalle. 4.5.7.-Determinaciones merísticas En determinadas experiencias se realizaron valoraciones merísticas del nº total de vertebras incluyendo el urostilo y nº de costillas pleurales en 250 ejemplares de 95dpe, siguiendo la metodología descrita por Matsuoka (2003). Así, las vertebras con dos espinas neurales y /o con dos espinas hemales se consideraron compuestas por la fusión de dos vertebras. Figura 53. Identificación de vertebras y costillas. Spanish summary 301 4.6.-Analisis bioquímicos Durante el transcurso de las diferentes experiencias, se tomaron muestras de los productos utilizados para la producción y enriquecimiento de las presas vivas (rotíferos y Artemia), piensos y de las propias presas vivas utilizadas, antes y después de su enriquecimiento. Así mismo, se recogieron muestras de larvas de diferentes edades, de cada uno de los tratamientos ensayados. Una vez recogidas, las muestras de presas vivas y larvas, se colocaron sobre una malla de 63µm, se lavaron con agua dulce y posteriormente con agua destilada. A continuación se eliminaba la mayor cantidad de agua posible con papel secante y se procedia a su congelación a -80ºC en bolsas herméticas bajo atmósfera de nitrógeno, para su posterior análisis. Los análisis bioquímicos se realizaron en el laboratorio del Instituto Universitario de Sanidad Animal y Seguridad Alimentaria (IUSA). Se hicieron determinaciones del contenido en humedad, cenizas, proteínas, lípidos totales y ácidos grasos. Todas las determinaciones, se realizaron al menos por triplicado. 4.6.1.-Determinación de la humedad Se determinó siguiendo el método de la American Oficial de Analis Chemisty (AOAC,1995). El procedimiento consiste en secar en una estufa a 110ºC una cantidad de muestra conocida (Pi) hasta peso constante (Pf). Posteriormente se saca la muestra, se introduce en un desecador 30 min y se realiza la pesada. El porcentaje de humedad de la muestra se obtiene con la expresión: 4.6.2.-Determinación de las cenizas El contenido de cenizas se determinó, por medio de la incineración de una cantidad conocida de muestra (Pm) en un horno mufla, a una temperatura de 450ºC durante 24 horas, pesando posteriormente la cantidad de cenizas remanente (Pc) hasta peso constante según la AOAC (1995) y aplicando la siguiente expresión. 4.6.3.-Determinación de las proteínas El contenido proteico se calculó a partir del contenido de nitrógeno total de las muestras, determinado por la técnica de Kjeldhal. Según el método de la AOAC (1995), la técnica consiste en la digestión de las muestras con acido sulfúrico a 420ºC con presencia de un catalizador de cobre durante una hora, seguido de una destilación con Na(OH) al 40% utilizando acido bórico saturado como sustancia receptora en una unidad destiladora (Mod. Foss Tecator, 1002, Höganäs, %H = ((Pi - Pf) × 100)/Pi %Cenizas = (100 x Pm)/Pc Spanish summary 302 Suecia). Finalmente se realiza una valoración con HCl 0,1 M. Para calcular el porcentaje de proteína se aplicó la siguiente expresión. Siendo: V = Volumen de HCl usado en la valoración en ml P = Media de la valoración de los patrones en ml N = Normalidad del HCl Pm= Peso molecular del nitrógeno que es 14,007 F = Factor de conversión empírico que tiene un valor de 6,25 M = Peso de la muestra en mg 4.6.4.-Determinación de los lípidos totales La extracción de los lípidos se realizó según el método de Folck et al. (1957), adaptado por Izquierdo y Gil (1998). El método consistió en tomar una cantidad de muestra entre 50-200 mg, que se homogeneizó en un Ultra Turrax (IKA-Werke, T25 Basic, Germany, Staufen) a 11.000 rpm durante 5 min en una solución de 5 ml de Cloroformo: Metanol (2:1) con 0.01% de BHT. A continuación, la solución resultante, se filtró a presión reducida a través de lana de vidrio y añadiendo KCl al 0.88%, para aumentar la polaridad de la fase acuosa. Por decantación y tras un centrifugado a 2000 rpm durante 5 min se separaron las fases acuosa y orgánica. Una vez retirada la fase acuosa se evaporó a sequedad con una corriente de N2 y se determinó el contenido de lípidos totales de la muestra por gravimetría. 4.6.5.-Determinación de ácidos grasos Los lípidos totales extraídos, se transesterificaron según el método de Christie,(1982). En este procedimiento se añadió Tolueno con BHT y una solución de Metanol:Sulfúrico al 1%. La mezcla se agitó fuertemente para favorecer la disolución de los lípidos, llenando el recipiente con N2 y sellándolo posteriormente. Se dejó incubando 16 horas a 50ºC en agitación. Transcurrido este tiempo, se dejó enfriar la muestra y se le añadió agua destilada ultra pura y Hexano:Dietil eter 1:1 con BHT al 0.01%. Los FAMES purificados se evaporaron a sequedad con N2 y se pesaron. Finalmente, se diluyeron a una concentración de 20mg de FAMES por ml de Hexano, pasándolos a microviales, que se congelaron a -80ºC hasta el momento de su identificación y cuantificación en el cromatógrafo de gases, (Mod. Shimadzu GC-14A; Analytical instrument division, Kyoto, Japon), equipado con un detector de ionización de llama y un integrador Shimazu (CR-5A). Las características de la columna son las siguientes: columna capilar de sílice fundida, de 30m x %Proteina =(V-P) x N x Pm x F/M Spanish summary 303 0.32mm D.I. con supelco-10 como fase estacionaria, (Supelco, Inc., Bellefonte, EE.UU). Actuando como Gas portador:helio. La presión de los gases: He 1 Kg.cm-2, H2 0.5 Kg.cm-2, N2 1 Kg.cm-2, aire 0.5 Kg.cm-2. Y la temperatura: en el inyector 250 ºC, columna según rampa con las siguientes características: temperatura inicial 180ºC durante 10min, tasa de incremento de temperatura 2.5ºC.min-1, temperatura final 215ºC durante 10min. La identificación de los ácidos grasos se llevó a cabo mediante la utilización de EPA 28 como aceite estándar. Comparando los tiempos de retención y las distancias de los picos del análisis de las muestras con el estándar. 4.7.-Análisis estadístico Los resultados obtenidos se han expresado siempre como media ± desviación estándar de la media. Los análisis estadísticos se realizaron con el programa SPSS Versión 14.0 (SPSS Chicago, Illinois, 1999). Los datos de cada experimento, se compararon estadísticamente mediante test de la T-Student (Sokal and Rolf, 1995), cuando había solo dos tratamientos o con un análisis de varianza (ANOVA) si era mayor el número de tratamientos ensayado. Como criterio general se tomó el 5 % como nivel de significación. Una vez habían sido detectadas diferencias estadísticamente significativas con el ANOVA, las diferencias entre medias fueron puestas de manifiesto mediante el test de comparación múltiple de Tukey. Cuando las varianzas eran heterogéneas y/o los datos no se distribuían normalmente se intenta hacerlas homocedásticas y/o que los datos se distribuyeran normalmente transformándolas variables en sus logaritmos o bien con la función arco seno. Si la heterogeneidad o la no distribución normal de los datos persistían, se empleaba el test no paramétrico de Kolmogorov-Smirnov, cuando solo había dos replicados, o si era mayor el nº de tratamientos el test Games-Howell. Finalmente, para el estudio de calidad de los alevines se aplicó un análisis log lineal, con la χi cuadrado de Pearson (Sokal and Rolf., 1995). 4.8.-Nomenclatura de especies citadas Los nombres vulgares de las especies, utilizados en este trabajo fueron tomados del “Diccionario multilingüe de especies marinas para el mundo hispano” de Vera, (1992). En caso de no figurar, la especie, en dicho diccionario se utilizó la denominación FAO en español de la base de datos “Fishbase” y en el caso de no existir el nombre en español, se utilizó la denominación FAO en inglés de esa base de datos. Spanish summary 305 9.5.-Conclusiones Estudio I. Desarrollo del sistema visual de las larvas de bocinegro Pagrus pagrus (Linnaeus, 1758) en relación a los cambios del sitema digestivo y hábitos alimenticios. 1. Las larvas de bocinegro son predadores visuales, que eclosionan con un sistema visual y digestivo incompleto y no funcional. 2. Entre el tercer y cuarto día post eclosión se producen los mayores cambios en el sistema visual y digestivo, como la pigmentación de los fotoreceptores primarios (conos) que coincide con la apertura de la boca y la detección de acividad digestiva en en el intestino medio, lo que es un indicativo de que las larvas están preparadas para comenzar la alimentación exógena. 3. El segundo hito mas importante en el desarrollo del sistema visual se detecta en torno a los 20 dpe, (7,0-7,5mm LT) cuando aparecen los núcleos de los segundo fotoreceptores (bastones). Desde este momento, las larvas de bocinegro presentan una retina con doble sistema de fororreceptores (conos y bastones). 4. La pigmentación de los bastones coincide con la aparición de las primeras células gástricas y la progresiva migración de las larvas desde las capas superficiales a una mayor profundidad en el tanque de cultivo, lo que sugiere cambios en los hábitos alimenticios de las condiciones de cultivo a partir de esta etapa. Estudio II. Desarrollo osteológico y aparición de deformidades esqueléticas en las larva de de bocinegro Pagrus pagrus (Linnaeus, 1758) bajo diferentes técnicas de cultivo. 5. El patrón general de desarrollo osteológico del bocinegro, no se ve afectado por el sistema de cultivo. Sin embargo, el momento de aparición de las diferentes estructuras esqueléticas, que se encuentra mas ligado al crecimiento en talla que a la edad de las larvas, si se vio afectado por el sistema de cultivo empleado. Así, la osificación se completa antes en los sistemas semi-intensivos, donde se obtiene una mayor tasa de crecimiento. Spanish summary 306 6. Independientemente del sistema de cultivo empleado se observó una elevada incidencia de deformidades morfoanatomicas en esta especie. Siendo las aparición de lorsosis y fusiones vertebrales las anomalías más frecuentes. Se registró una baja incidencia de anomalías operculares, lo cual se asoció a la presencia de unas largas espinas operculares desde etapas tempranas de su desarrollo que previenen la curvatura del opérculo hacia el interior de la cámara branquial. La lordosis, fue localizada mayoritariamente entre la 8ª y 12ª vertebra, región de la columna que soporta una mayor presión muscular durante la natación. 7. El sistema de cultivo, tiene efecto sobre la localización de las fusiones vertebrales. Así, en las larvas cultivadas en sistemas intensivos, las fusiones se localizan a lo largo de toda la columna y particularmente en la zona caudal, mientras que en el sistema semi-intensivo se localizan mayoritariament en la zona pre-hemal. 8. La intensificación del sistema de cultivo se reflejo en la aparición de un mayor número de individuos con una vertebra extra, una mayor incidencia de cifosis y anomalías craneales principalmente registradas como acortamiento de la mandibula superior y aparición de mandibulas cruzadas. Estudio III. Efecto del contenido de DHA en los rotíferos sobre la incidencia de deformidades esqueléticas del bocinegro Pagrus pagrus (Linnaeus, 1758). 9. Los requerimientos de DHA para obtener un buen crecimiento en las larvas de bocinegro fueron inferiore as 1.9%. El DPA (22:5n-6) se acumula fácilmente en los tejidos de las larvas de bocinegro cuando estas se alimentan con rotíferos con un elevado contenido en este ácido graso, registrandose una baja supervivencias de las larvas, lo que puede estar relacionado con la elevada incorporación de DPA en el tejido nervioso y visual, afectando a funciones de las biomembranas, como se ha descrito en otros vertebrados. 10. La suplementacion de DHA en la dieta, fue asociada a una mejora en la supervivencia y una reducción en la incidencia de deformidades esqueléticas en general, lo que indica la importancia de este ácido graso en el desarrollo osteológico. Spanish summary 307 Estudio IV. Avances en las técnicas de cultivo de bocinegro Pagrus pagrus (Linnaeus, 1758): Comparación de sistemas de cultivo larvario intensivos y semi-intensivos. 11. Los ácidos grasos más abundantes en las larvas del bocinegro son el 22:6n-3, 16:0, 18:1n9, 18:0, 18:2n-6, 20:5n-3, 16:1n-7,18:1n-7, 18:3n-3 y 20:4n-6. 12. El DHA es acumulado selectivamente en los tejidos de las larvas de bocinegro, en niveles superiores a los aportados en las presas vivas, lo que sugiere la importancia de este ácido graso para esta especie. Los niveles de ácido eicosapentaenoico refleja los cambios en la dieta durante la ontogenia de las larvas de bocinegro mientras que el ácido araquidónico permanece casi constante a lo largo del desarrollo larvario. 13. Durante el desarrollo larvario del bocinegro, se han identificado tres estadios de crecimiento: una primera etapa (5-15 dpe) de elevado crecimiento y periodo muy sensible en la supervivencia larvaria; una segunda etapa (15-30dpe), que se correlaciona con la finalización de la metamorfosis, con un bajo crecimiento y un mejor control de la mortalidad; y finalmente, un tercer perido post-metamorfosis (30-50dpe) con un ligero incremento del crecimiento y baja mortalidad. 14. Durante la primera etapa (5-15 dpe), el incremento en la densidad de presas en el sistema de cultivo semi-intensivo de 1-2 rot.ml-1 a 4-5 rot.ml-1 disminuye la mortalidad larvaria e incrementa las tasas de crecimiento. 15. 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