Evaluation of alternative ingredients as fish meal replacers in on-growing diets for red porgy (Pagrus pagrus)

ªAD DE LAS PALMAS DE GRAN CANARIA Anexo 1 lm t i(�Hl�!yn IYE't�I tan o Sanidad Animal Scgvrid,1d Aliml'nl,inJ Dª MARÍA SORAYA DÉNIZ SUÁREZ, SECRETARIA DEL INSTITUTO UNIVERSITARIO DE SANIDAD ANIMAL Y SEGURIDAD ALIMENTARIA DE LA UNIVERSIDAD DE LAS PALMAS DE GRAN CANARIA. CERTIFICA Que el Consejo de Doctores del Departamento en su sesión de fecha 21 de diciembre de 2011 tomó el acuerdo de dar el consentimiento para su tramitación, a la tesis doctoral europea titulada: "Evaluación de ingredientes alternativos a la harina de pescado en dietas de engorde para bocinegro (pagrus pagrus)", presentada por la doctoranda Dª Josefa García Romero, dirigida por la Dra. Dª. Lidia Robaina Robaina y Dr. D. Rafael Ginés Ruiz. Y para que así conste, y a efectos de lo previsto en el Artº 73.2 del reglamento de Estudios de Doctorado de esta Universidad, firmo la presente en Las Palmas de Gran Canaria, a veintidós de diciembr� de dos mil once. Anexo II Anexo IIAnexo II Anexo II UNIVERSIDAD DE LAS PALMAS DE GRAN CANARIA UNIVERSIDAD DE LAS PALMAS DE GRAN CANARIAUNIVERSIDAD DE LAS PALMAS DE GRAN CANARIA UNIVERSIDAD DE LAS PALMAS DE GRAN CANARIA Departamento: Instituto Universitario de Sanidad Animal y Seguridad Alimentaria Programa de Doctorado: Acuicultura Título de la Tesis Título de la TesisTítulo de la Tesis Título de la Tesis “Evaluation of alternative ingredients as fish meal replacers in on-growing diets for red porgy ( Pagrus pagrus )” Tesis Doctoral presentada por Josefa García Romero Josefa García RomeroJosefa García Romero Josefa García Romero Dirigida por la Doctora Lidia Esther Robaina R Doctora Lidia Esther Robaina R Doctora Lidia Esther Robaina R Doctora Lidia Esther Robaina Robaina obaina obaina obaina y el Doctor Doctor Doctor Doctor Rafael Ginés Ruiz Rafael Ginés RuizRafael Ginés Ruiz Rafael Ginés Ruiz La Directora La Directora La Directora La Directora El Director El Director El Director El Director La La La La Doctorando Doctorando Doctorando Doctorando Las Palmas de Gran Canar Las Palmas de Gran CanarLas Palmas de Gran Canar Las Palmas de Gran Canari ii ia, a 16 de Diciembre de 2011 a, a 16 de Diciembre de 2011a, a 16 de Diciembre de 2011 a, a 16 de Diciembre de 2011 LIDIA ESTHER ROBAINA ROBAINA, PROFESORA LIDIA ESTHER ROBAINA ROBAINA, PROFESORA LIDIA ESTHER ROBAINA ROBAINA, PROFESORA LIDIA ESTHER ROBAINA ROBAINA, PROFESORA EN EL ÁREA DE EN EL ÁREA DE EN EL ÁREA DE EN EL ÁREA DE CONOCIMIENTO DE ZOOLOGÍA CONOCIMIENTO DE ZOOLOGÍA CONOCIMIENTO DE ZOOLOGÍA CONOCIMIENTO DE ZOOLOGÍA DEL DEPARTAMENTO DE BIOLOGÍA DEL DEPARTAMENTO DE BIOLOGÍA DEL DEPARTAMENTO DE BIOLOGÍA DEL DEPARTAMENTO DE BIOLOGÍA DE LA UNIVERSIDAD DE LAS PALMAS DE GRAN CANARIA DE LA UNIVERSIDAD DE LAS PALMAS DE GRAN CANARIADE LA UNIVERSIDAD DE LAS PALMAS DE GRAN CANARIA DE LA UNIVERSIDAD DE LAS PALMAS DE GRAN CANARIA. .. . INFORMA: INFORMA:INFORMA: INFORMA: Que Doña Jos Doña JosDoña Jos Doña Josefa García Romero efa García Romeroefa García Romero efa García Romero, Licenciada en Ciencias de Mar, ha realizado bajo mi dirección y asesoramiento el presente trabajo titulado: “Evaluation of alternative ingredients as fish meal replacers in on “Evaluation of alternative ingredients as fish meal replacers in on“Evaluation of alternative ingredients as fish meal replacers in on “Evaluation of alternative ingredients as fish meal replacers in on- -- -growing growing growing growing diets for red porgy ( diets for red porgy (diets for red porgy ( diets for red porgy ( Pagrus pagrus Pagrus pagrusPagrus pagrus Pagrus pagrus )”. (Evaluación de )”. (Evaluación de )”. (Evaluación de )”. (Evaluación de ingredientes alte ingredientes alteingredientes alte ingredientes alternativos rnativos rnativos rnativos como sustitutos como sustitutoscomo sustitutos como sustitutos de la harina de pescado en dietas de engorde para el de la harina de pescado en dietas de engorde para el de la harina de pescado en dietas de engorde para el de la harina de pescado en dietas de engorde para el bocinegro ( bocinegro (bocinegro ( bocinegro ( Pagrus pagrus Pagrus pagrusPagrus pagrus Pagrus pagrus )”) )”))”) )”), ,, , el cual considero reúne las condiciones y calidad científica para optar al Grado de Doctor. FDO.: LIDIA ESTHER ROBAINA ROBAIN FDO.: LIDIA ESTHER ROBAINA ROBAINFDO.: LIDIA ESTHER ROBAINA ROBAIN FDO.: LIDIA ESTHER ROBAINA ROBAINA AA A RAFAEL GINÉS RUIZ, PROFESOR RAFAEL GINÉS RUIZ, PROFESORRAFAEL GINÉS RUIZ, PROFESOR RAFAEL GINÉS RUIZ, PROFESOR TITULAR DE UNIVERSIDAD EN EL TITULAR DE UNIVERSIDAD EN EL TITULAR DE UNIVERSIDAD EN EL TITULAR DE UNIVERSIDAD EN EL ÁREA DE CONOCIMIENTO DE PRODUCCIÓN ANIMAL ÁREA DE CONOCIMIENTO DE PRODUCCIÓN ANIMAL ÁREA DE CONOCIMIENTO DE PRODUCCIÓN ANIMAL ÁREA DE CONOCIMIENTO DE PRODUCCIÓN ANIMAL DE DE DE DE LA LA LA LA FACULTAD DE VETERINARIA FACULTAD DE VETERINARIA FACULTAD DE VETERINARIA FACULTAD DE VETERINARIA DE LA UNIVERSIDAD DE LAS PALMAS DE LA UNIVERSIDAD DE LAS PALMAS DE LA UNIVERSIDAD DE LAS PALMAS DE LA UNIVERSIDAD DE LAS PALMAS DE GRAN CANARIA DE GRAN CANARIADE GRAN CANARIA DE GRAN CANARIA. .. . INFORMA: INFORMA:INFORMA: INFORMA: Que Doña Josefa García Romero Doña Josefa García RomeroDoña Josefa García Romero Doña Josefa García Romero, Licenciada en Ciencias de Mar, ha realizado bajo mi dirección y asesoramiento el presente trabajo titulado: “Evaluation of alternative ingredients as fish meal replacers in on “Evaluation of alternative ingredients as fish meal replacers in on“Evaluation of alternative ingredients as fish meal replacers in on “Evaluation of alternative ingredients as fish meal replacers in on- -- -growing growing growing growing diets for red porgy ( diets for red porgy (diets for red porgy ( diets for red porgy ( Pagrus pagrus Pagrus pagrusPagrus pagrus Pagrus pagrus )”. (Evaluación de ingredientes )”. (Evaluación de ingredientes)”. (Evaluación de ingredientes )”. (Evaluación de ingredientes alternativos alternativos alternativos alternativos como como como como s ss sustitutos ustitutosustitutos ustitutos de de de de la harina de pescado en dietas de engorde para el la harina de pescado en dietas de engorde para el la harina de pescado en dietas de engorde para el la harina de pescado en dietas de engorde para el bocinegro ( bocinegro (bocinegro ( bocinegro ( Pagrus pagrus Pagrus pagrusPagrus pagrus Pagrus pagrus )”) )”))”) )”), ,, , el cual considero reúne las condiciones y calidad científica para optar al Grado de Doctor. FDO.: FDO.: FDO.: FDO.: RAFAEL GINÉS RUIZ RAFAEL GINÉS RUIZRAFAEL GINÉS RUIZ RAFAEL GINÉS RUIZ E EE E E EE E E EE Ev vv v v vv v v vv va aa a a aa a a aa al ll l l ll l l ll lu uu u u uu u u uu ua aa a a aa a a aa at tt t t tt t t tt ti ii i i ii i i ii io oo o o oo o o oo on nn n n nn n n nn n o oo o o oo o o oo of ff f f ff f f ff f a aa a a aa a a aa al ll l l ll l l ll lt tt t t tt t t tt te ee e e ee e e ee er rr r r rr r r rr rn nn n n nn n n nn na aa a a aa a a aa at tt t t tt t t tt ti ii i i ii i i ii iv vv v v vv v v vv ve ee e e ee e e ee e i ii i i ii i i ii in nn n n nn n n nn ng gg g g gg g g gg gr rr r r rr r r rr re ee e e ee e e ee ed dd d d dd d d dd di ii i i ii i i ii ie ee e e ee e e ee en nn n n nn n n nn nt tt t t tt t t tt ts ss s s ss s s ss s a aa a a aa a a aa as ss s s ss s s ss s f ff f f ff f f ff fi ii i i ii i i ii is ss s s ss s s ss sh hh h h hh h h hh h m mm m m mm m m mm me ee e e ee e e ee ea aa a a aa a a aa al ll l l ll l l ll l r rr r r rr r r rr re ee e e ee e e ee ep pp p p pp p p pp pl ll l l ll l l ll la aa a a aa a a aa ac cc c c cc c c cc ce ee e e ee e e ee er rr r r rr r r rr rs ss s s ss s s ss s i ii i i ii i i ii in nn n n nn n n nn n o oo o o oo o o oo on nn n n nn n n nn n- - -g gg g g gg g g gg gr rr r r rr r r rr ro oo o o oo o o oo ow ww w w ww w w ww wi ii i i ii i i ii in nn n n nn n n nn ng gg g g gg g g gg g d dd d d dd d d dd di ii i i ii i i ii ie ee e e ee e e ee et tt t t tt t t tt ts ss s s ss s s ss s f ff f f ff f f ff fo oo o o oo o o oo or rr r r rr r r rr r r rr r r rr r r rr re ee e e ee e e ee ed dd d d dd d d dd d p pp p p pp p p pp po oo o o oo o o oo or rr r r rr r r rr rg gg g g gg g g gg gy yy y y yy y y yy y ( (( ( ( (( ( ( (( ( P PP P P P P P P P P Pa a a a a a a a a a a ag g g g g g g g g g g gr r r r r r r r r r r ru u u u u u u u u u u us s s s s s s s s s s s p p p p p p p p p p p pa a a a a a a a a a a ag g g g g g g g g g g gr r r r r r r r r r r ru u u u u u u u u u u us s s s s s s s s s s s ) )) ) ) )) ) ) )) ) J JJ J J JJ J J JJ Jo oo o o oo o o oo os ss s s ss s s ss se ee e e ee e e ee ef ff f f ff f f ff fa aa a a aa a a aa a G GG G G GG G G GG Ga aa a a aa a a aa ar rr r r rr r r rr rc cc c c cc c c cc cí íí í í íí í í íí ía aa a a aa a a aa a R RR R R RR R R RR Ro oo o o oo o o oo om mm m m mm m m mm me ee e e ee e e ee er rr r r rr r r rr ro oo o o oo o o oo o Grupo de Investigación en Acuicultura (GIA) Instituto Canario de Ciencias Marinas (ICCM) Instituto Universitario de Sanidad Animal y Seguridad Alimentaria (ULPGC) Thesis for the degree of Doctor of Phylosophy University of Las Palmas de Gran Canaria, 2011 Directors: Dra. Lidia Esther Robaina Robaina & Dr. Rafael Ginés Ruiz A mi familia, A mi familia,A mi familia, A mi familia, CONTENTS CONTENTSCONTENTS CONTENTS List of tables List of tablesList of tables List of tables List of figures List of figuresList of figures List of figures Abbreviations AbbreviationsAbbreviations Abbreviations Acknowledgments AcknowledgmentsAcknowledgments Acknowledgments 1 11 1. .. .GENERAL INTRODUCTION GENERAL INTRODUCTIONGENERAL INTRODUCTION GENERAL INTRODUCTION 1 1.1 1.11.1 1.1. .. . AQUACULTURE PRODUCTION……………………………………… AQUACULTURE PRODUCTION………………………………………AQUACULTURE PRODUCTION……………………………………… AQUACULTURE PRODUCTION…………………………………………… ………… ……… …… … 1 1.2 1.21.2 1.2. .. . PROTEIN IN DIETS FOR FISH SPECIES………………………………... PROTEIN IN DIETS FOR FISH SPECIES………………………………...PROTEIN IN DIETS FOR FISH SPECIES………………………………... PROTEIN IN DIETS FOR FISH SPECIES……………………………….... .. ... .... .. 2 1.2.1. Importance of protein in compounds feed for farming finfish……………… 3 1.2.2. Fish meal as the main protein source in fish diets………………………...... 4 1.2.3. Fish meal current world production and situation………………………...... 5 1.3 1.31.3 1.3. .. . ALTERNATIVE PROTEIN ALTERNATIVE PROTEIN ALTERNATIVE PROTEIN ALTERNATIVE PROTEIN INGREDIENT INGREDIENTINGREDIENT INGREDIENTS SS S TO FISH MEAL IN FIS TO FISH MEAL IN FIS TO FISH MEAL IN FIS TO FISH MEAL IN FISH H H H DIETS……………………………… DIETS………………………………DIETS……………………………… DIETS…………………………………………………………………………… ………………………………………………………………………………………… ……………………………………………... ...... ..... .... .. 6 1.3.1. Alternative protein sources from terrestrial origin………………………….. 6 1.3.2. Product and by-products from marine sources as novel ingredients in aquaculture feed…………………………………………………………………... 8 1.3.2.1. Crab products and by-products………………………………………………..... 11 1.3.2.1.1. Chitin long-change polysaccharide as potential bio-component from crustacean meals………………………………………………………………….. 1 4 1.3.2.1.2. Dietary crab meal and fish flesh quality…………………………………….... 15 1.3.2.2. Other potential ingredients from marine sources: by-products processing from echinoderm industry……………………………………………………….. 1 7 1.4 1.41.4 1.4. .. . THE THE THE THE RED PORGY ( RED PORGY (RED PORGY ( RED PORGY ( Pagrus p Pagrus pPagrus p Pagrus pagrus agrusagrus agrus ; Linnaeus, 1758)……. ; Linnaeus, 1758)…….; Linnaeus, 1758)……. ; Linnaeus, 1758)…….……………… ……………………………… ……………….. .... ... .. .. .. . 17 1.4.1. External appearance, geographical distribution, habitat and reproduction……………………………………………………………………...... 17 1.4.2. Natural diet of fish red porgy………………………………………………….... 18 1.4.3. Red porgy culture…………………………………………………………………. 19 1.4.3.1. Dietary protein and carotenoid pigments requirements……………….…... 20 2 22 2. .. .OJECTIVES OJECTIVESOJECTIVES OJECTIVES 25 3 33 3. .. .GENERAL MATERIALS AND METHODS GENERAL MATERIALS AND METHODSGENERAL MATERIALS AND METHODS GENERAL MATERIALS AND METHODS 29 3.1 3.13.1 3.1. .. . ANIMALS………………………………………………………………………… ANIMALS…………………………………………………………………………ANIMALS………………………………………………………………………… ANIMALS………………………………………………………………………….. .... .. 29 3.2 3.23.2 3.2. .. . CULTURE CULTURE CULTURE CULTURE CONDITIONS…………………………………………… CONDITIONS……………………………………………CONDITIONS…………………………………………… CONDITIONS……………………………………………… …… …………. ……….………. ………. 30 3.2.1. Facilities……………………………………………………………………………. 30 3.2.2. Experimental tanks………………………………………………………………. 30 3.2.3. Experimental conditions…………………………………………………………. 30 3.3 3.33.3 3.3. .. . EXPERIMENTAL FEED INGREDIE EXPERIMENTAL FEED INGREDIEEXPERIMENTAL FEED INGREDIE EXPERIMENTAL FEED INGREDIENTS……… NTS………NTS……… NTS………………………………… …………………………………………………… …………………………… …… … 31 3.3.1. River crab, Procambarus clarkii , meal………………………………………… 31 3.3.2. Marine crab, Chaceon affinis and Paramola cuvieri , meals………………... 32 3.3.3. Sea urchin, Diadema antillarum , meal………………………………………... 34 3.3.4. Other feed ingredients……………………………………………………………. 35 3.4 3.43.4 3.4. .. . FEED PROCESSING… FEED PROCESSING…FEED PROCESSING… FEED PROCESSING………………………………………………………… ……………………………………………………………………………………………………………… …………………………………………………………. ….…. …. 35 3.4.1. Formulation………………………………………………………………………... 35 3.4.2. Diets elaboration………………………………………………………………...... 35 CONTENTS CONTENTSCONTENTS CONTENTS 3.5 3.53.5 3.5. .. . BIOLOGICAL AND FEED UTILIZATION PARAMETERS…………… BIOLOGICAL AND FEED UTILIZATION PARAMETERS……………BIOLOGICAL AND FEED UTILIZATION PARAMETERS…………… BIOLOGICAL AND FEED UTILIZATION PARAMETERS………………. ….…. …. 37 3.6 3.63.6 3.6. .. . INSTRUMENTAL COLOUR DETERMINATION………………………… INSTRUMENTAL COLOUR DETERMINATION…………………………INSTRUMENTAL COLOUR DETERMINATION………………………… INSTRUMENTAL COLOUR DETERMINATION………………………….. .... ... .. . 38 3.7 3.73.7 3.7. .. . ANALYSI ANALYSIANALYSI ANALYSIS O S OS O S OF P F PF P F PR RR ROXIMATE OXIMATE OXIMATE OXIMATE AND AND AND AND FAT FATFAT FATTY ACIDS COMPOSITION TY ACIDS COMPOSITIONTY ACIDS COMPOSITION TY ACIDS COMPOSITION…… ………… ……. .. . 39 3.8 3.83.8 3.8. .. . CAROTENOIDS ANALYSIS……….… CAROTENOIDS ANALYSIS……….…CAROTENOIDS ANALYSIS……….… CAROTENOIDS ANALYSIS……….………………………………………... ……………………………………...……………………………………... ……………………………………...... ...... .... .. . 40 3.8.1. Carotenoids extraction………………………………………………………....... 40 3.8.2. Carotenoids separation and relative quantification (%) by thin layer chromatography (TLC)…………………………………………………………… 41 3.9 3.93.9 3.9. .. . AMMONIA AMMONIAAMMONIA AMMONIA EXCRETION EXCRETIONEXCRETION EXCRETION ASSESSMENT………………………………… ASSESSMENT………………………………… ASSESSMENT………………………………… ASSESSMENT……………………………………. ….…. …. 42 3.9.1. Water sampling for ammonia-N determination……………………………… 42 3.9.2. Analytical method………………………………………………………………… 42 3.10 3.103.10 3.10. .. . DIGESTIBILITY DETERMINATION……………………………………… DIGESTIBILITY DETERMINATION………………………………………DIGESTIBILITY DETERMINATION……………………………………… DIGESTIBILITY DETERMINATION………………………………………… …… …. .. . 44 3.10.1. Experimental tanks and faecal samples collection…………………………... 44 3.10.2. Chromium (III) oxide (Cr2O3) determination…………………………………. 44 3.10.3. Apparent digestibility coefficients determination (ADCs)……………….. 44 3.11 3.113.11 3.11. .. . FLESH TEXTURE PROFILE ANALYSIS………………………………… FLESH TEXTURE PROFILE ANALYSIS…………………………………FLESH TEXTURE PROFILE ANALYSIS………………………………… FLESH TEXTURE PROFILE ANALYSIS……………………………………. ….…. …. 45 3.12 3.123.12 3.12. .. . SENSORY ANALYSIS………………………………………………………… SENSORY ANALYSIS…………………………………………………………SENSORY ANALYSIS………………………………………………………… SENSORY ANALYSIS…………………………………………………………… …… … 46 3.13 3.133.13 3.13. .. . FLESH FLESHFLESH FLESH LIPID OXIDATION…… LIPID OXIDATION…… LIPID OXIDATION…… LIPID OXIDATION…………………………… ……………………………………………… ………………………………… …………………… …………… …… …………….. …………..………….. ………….. 47 3.14 3.143.14 3.14. .. . STATISTICAL ANALYSI STATISTICAL ANALYSISTATISTICAL ANALYSI STATISTICAL ANALYSIS…………………………………………………… S……………………………………………………S…………………………………………………… S……………………………………………………... ...... .... .. . 47 4 44 4. .. . STUDY I: STUDY I:STUDY I: STUDY I: Marine and freshwater crab meals in diets for red Marine and freshwater crab meals in diets for red Marine and freshwater crab meals in diets for red Marine and freshwater crab meals in diets for red porgy ( porgy (porgy ( porgy ( Pagrus pagrus Pagrus pagrusPagrus pagrus Pagrus pagrus ): effect on growth, fish composition and ): effect on growth, fish composition and ): effect on growth, fish composition and ): effect on growth, fish composition and skin colour skin colourskin colour skin colour 51 4.1. Introduction………………………………………………………………………... 53 4.2. Materials and methods…………………………………………………………… 55 4.3. Results……………………………………………………………………………… 59 4.4. Discussion………………………………………………………………………...... 64 4.5. Conclusions………………………………………………………………………... 68 5 55 5. .. . STUDY II STUDY IISTUDY II STUDY II: Marine and freshwater crab me Marine and freshwater crab meMarine and freshwater crab me Marine and freshwater crab meals in diets for red als in diets for red als in diets for red als in diets for red porgy ( porgy (porgy ( porgy ( Pagrus pagrus Pagrus pagrusPagrus pagrus Pagrus pagrus ): ): ): ): digestibility digestibilitydigestibility digestibility, ammonia excretion and , ammonia excretion and, ammonia excretion and , ammonia excretion and phospho phosphophospho phosphorous and calcium retention rous and calcium retentionrous and calcium retention rous and calcium retention. .. . 69 5.1. Introduction………………………………………………………………………... 70 5.2. Materials and methods…………………………………………………………… 72 5.3. Results……………………………………………………………………………… 77 5.4. Discussion………………………………………………………………………...... 82 5.5. Conclusions……………………………………………………………………….... 86 6. 6.6. 6. STUDY III STUDY IIISTUDY III STUDY III: Marine and freshwater crab meals in diets for red Marine and freshwater crab meals in diets for red Marine and freshwater crab meals in diets for red Marine and freshwater crab meals in diets for red porgy ( porgy (porgy ( porgy ( Pagrus pagrus Pagrus pagrusPagrus pagrus Pagrus pagrus ): effect o ): effect o): effect o ): effect on fillet fatty acid profile and flesh n fillet fatty acid profile and flesh n fillet fatty acid profile and flesh n fillet fatty acid profile and flesh quality parameters quality parametersquality parameters quality parameters 87 6.1. Introduction………………………………………………………………………... 88 6.2. Materials and methods…………………………………………………………… 90 6.3. Results……………………………………………………………………………… 95 6.4. Discussion………………………………………………………………………...... 101 6.5. Conclusions……………………………………………………………………….... 105 CONTENTS CONTENTSCONTENTS CONTENTS 7. 7.7. 7. STUDY IV: STUDY IV:STUDY IV: STUDY IV: Meals of spider marine crab ( Meals of spider marine crab (Meals of spider marine crab ( Meals of spider marine crab ( Paramola cuvieri Paramola cuvieriParamola cuvieri Paramola cuvieri ) and ) and ) and ) and sea urchin ( sea urchin (sea urchin ( sea urchin ( Diadema antillarum Diadema antillarumDiadema antillarum Diadema antillarum ) in diets for red porgy ( ) in diets for red porgy () in diets for red porgy ( ) in diets for red porgy ( Pagrus PagrusPagrus Pagrus pagrus pagruspagrus pagrus ): e ): e): e ): effect on growth performance, ammonia ffect on growth performance, ammonia ffect on growth performance, ammonia ffect on growth performance, ammonia excretion, skin excretion, skin excretion, skin excretion, skin colour and colour and colour and colour and flesh quality flesh quality flesh quality flesh quality 107 7.1. Introduction………………………………………………………………………... 108 7.2. Materials and methods…………………………………………………………… 110 7.3. Results……………………………………………………………………………… 117 7.4. Discussion………………………………………………………………………...... 125 7.5. Conclusions……………………………………………………………………….... 132 8 88 8. .. . CONCLUSIONS CONCLUSIONSCONCLUSIONS CONCLUSIONS 135 9 99 9. .. . RESUMEN EN ESPAÑOL RESUMEN EN ESPAÑOLRESUMEN EN ESPAÑOL RESUMEN EN ESPAÑOL 137 9.1. Introducción………………………………………………………………………... 139 9.2 Objetivos 159 9.3. Materiales y métodos……………………………………………………………... 160 9.4. Resumenes de los experimentos………………………………………………… 180 9.5. Conclusiones……………………………………………………………………...... 185 10 1010 10. .. . REFERENCES…………………………………………………………... REFERENCES…………………………………………………………...REFERENCES…………………………………………………………... REFERENCES…………………………………………………………..... .... .. 187 Acknowledges AcknowledgesAcknowledges Acknowledges VI hecho y terminado nunca……por tu confianza y sacrificio de entender que era lo que tenia que hacer…..por decirme todos los días y a pesar de las dificultades por las que hemos pasado de que estabas orgulloso de mí…que momento mas ideal para expresarte mi amor profundo y que quede grabado, decirte que no olvidaré nunca tu ayuda, apoyo y tu amor. 1. GENERAL INTRODUCTION 1. GENERAL INTRODUCTION1. GENERAL INTRODUCTION 1. GENERAL INTRODUCTION INTRODUCTION INTRODUCTIONINTRODUCTION INTRODUCTION - 1 - 1.1 1.1 1.1 1.1 AQUACULTURE PRODUCTION AQUACULTURE PRODUCTIONAQUACULTURE PRODUCTION AQUACULTURE PRODUCTION The accessibility of people to sufficient, safe and nutritious food to maintain a healthy and active life is expressed by the term of “Food Safety”. Improving food security constitutes a necessary precursor for the global intention towards reduction of hunger and poverty (Seligman et al ., 2010). Fish has long been recognized as a relative cheap source of high-quality protein, essential vitamins and minerals, playing an important role in the food safety and the nutrition of the developing countries (FAO, 2003). Fish supply has been depending of fishing wild captures for many years. However, global capture fisheries has almost stopped growing since the mid1980s, especially marine fisheries resources which have been exploited to their maximum or beyond the level of sustainability, whereas the worldwide demand for fish is still increasing (FAO, 2010). Aquaculture is perceived as the faster growing food-producing sector with a great potential to meet the increasing worldwide demand of sea food. Thus, in order to alleviate the pressure on fish stocks the aquaculture appears as the most feasible complement to fisheries. According to FAO (2010), world aquaculture output has increased substantially over the last 50 years from a global food fish production of 3.9% in early 1950s to 47% in 2008 (excluding aquatic plants), being estimated this production bring off more than 50% of global food fish consumption by 2012. In contrast to world capture fisheries production, which is almost stopped, the aquaculture sector maintained a worldwide annual growth rate of 8.3 % between 1970 and 2008, with a per capita supply from aquaculture increasing by ten times from 0.7 kg to 7.8 kg in the same period (Fig. 1.1). World aquaculture production from seawater represents a 36% of the global aquaculture production and produces many high value finfish, crustaceans and molluscs. Marine fish represents a 3.4% (1.8 million of tonnes) of the total aquaculture production in 2008 (FAO, 2010). INTRODUCTION INTRODUCTIONINTRODUCTION INTRODUCTION - 2 - Capture Aquaculture 0 5 10 15 1970 1980 1990 2000 2008 Fishery food supply (kg/capita) Capture Aquaculture 0 5 10 15 1970 1980 1990 2000 2008 Fishery food supply (kg/capita) Figure 1. Figure 1.Figure 1. Figure 1.1 11 1 Relative contribution of aquaculture and capture fisheries to food fish consumption from 1970 to 2008 (adapted from FAO, 2010). 1.2 PROTEIN IN DIETS FOR FISH SPECIES 1.2 PROTEIN IN DIETS FOR FISH SPECIES1.2 PROTEIN IN DIETS FOR FISH SPECIES 1.2 PROTEIN IN DIETS FOR FISH SPECIES In the context of animal feeding, protein mainly refers to crude protein content that is commonly determined as nitrogen content x 6.25. This definition is based on the assumption that the nitrogen content of most protein found in animal is about 16% (NCR, 1993). Amino acids are the building block of protein. Thus, protein are composed of up to 20 α-amino acids linked into chains by peptide bonds and cross-linked between chains by sulfhydryl bonds, hydrogen bonds and Van der Waals forces. Fish consume protein to obtain free amino acid, certain of them are essential components as fish can not synthesize them de novo and must be supplied in the diet (Laird and Needham, 1988). Amino acids, after being absorbed by the gastrointestinal tract, are transported to different tissues and organs to be used for fish growth (protein and/or synthesis of several other components or even to produce energy through catabolic processes) (NRC, 1993). Therefore, fish have a dietary protein requirement which means the minimum amount of protein needed to meet requirements of amino acids for achieving INTRODUCTION INTRODUCTIONINTRODUCTION INTRODUCTION - 3 - optimum growth and metabolic necessary process. Quantitative dietary protein requirements has been established in many fish species mainly by the doseresponse method, in which graded amounts o high-quality protein were proved in a reference diet (NCR, 1993). Carnivorous species requirements vary between 3555% of the diet, being appreciably higher than those in diets of terrestrial warmblooded animals (Merzt, 1972; Smith, 1989). One of the main factors affecting nitrogen excretion is the quantity and quality of protein in the diet (Lied and Braaten, 1984; Liu et al ., 2009). Thus, protein is one of the key nutrients in the diets of aquatic species, not only as a nutrient but also as a factor involved in water quality. 1.2.1 1.2.11.2.1 1.2.1 Importance ImportanceImportance Importance of protein of proteinof protein of protein in compounds feed in compounds feedin compounds feed in compounds feed for farming finfish for farming finfishfor farming finfish for farming finfish Formulated feeds are obviously the first step to develop intensified aquaculture systems. Feeds is still the most expensive operating cost in most fish farming (Deutsch et al ., 2007), often ranging from 30% to 60% of the total variable expenses, where proteins are the most expensive components of formulated feed (Tacon and Metian, 2008; FAO, 2010). The production of aquafeed compounds for aquaculture in 2006 was around 22.73 million tonnes (Tacon and Metian, 2008). With the increase of the aquaculture growth rate, is estimated that this production reached 29.3 million tonnes in 2009 (Fig. 1.2). Chinese carp constitutes the mayor group in compound feed production with 11.40 millions tonnes or 47% of the total production. On a global basis, it is estimated that 3.72 million tonnes of fish meal, or the equivalent of 16.6 million tonnes of pelagic forage, was used in the production of 22.73 million tonnes of compound fed in 2006 (Tacon and Metian, 2008; Hardy, 2010). It is therefore important to consider these protein needs in the context of sustainability of aquaculture development. INTRODUCTION INTRODUCTIONINTRODUCTION INTRODUCTION - 4 - Tilapia 7% Marine finfish 7% Trout 3% Salmon 8% Catfish 6% Chinese carps 42% Freshwater crustacean 4% Otros 6% Shrimp 17% 11.40 1.00 2.08 2.15 5.03 2.34 1.84 3.76 Tilapia 7% Marine finfish 7% Trout 3% Salmon 8% Catfish 6% Chinese carps 42% Freshwater crustacean 4% Otros 6% Shrimp 17% 11.40 1.00 2.08 2.15 5.03 2.34 1.84 3.76 Figure 1.2 Figure 1.2 Figure 1.2 Figure 1.2 Total estimated compound feed production (millions tonnes) for the main farmed aquaculture species in 2009 (adapted from FAO 2004-2009). 1.2.2 1.2.21.2.2 1.2.2 Fish meal as the main protein source in fish diets Fish meal as the main protein source in fish diets Fish meal as the main protein source in fish diets Fish meal as the main protein source in fish diets Due to its elevated nutritional quality, including high protein content, good balance of amino and fatty acids, low concentrations of anti-nutrients and being highly digestible, fishmeal (FM) has long been used as the main protein source for compound feed in farms, not only from aquaculture but also from terrestrial origin. Standard fishmeal typically has a crude protein ranges from 64% to 67%, although special high quality product contents between 68% and 72%, with up to 12% fat. In addition, freshness of the used raw material is reflected by a low biogenic amine concentration (e.g. maximum 500 ppm histamine), which is an important parameter to define the final quality of meals. Traditionally, demand sectors for fish meal was to feeding poultry, pigs and ruminants. However, the recent expansion development of aquaculture has been an important factor for increasing global demand for this product. For aquaculture, the demand of fish meal since 1980 has increased by almost 50% in 2008, and according to New (2002) it may estimate to increase by 70% in 2012. Contrary, in other sectors of animal feed the use of fish meal has considerably INTRODUCTION INTRODUCTIONINTRODUCTION INTRODUCTION - 5 - declined (IFFO, 2009) (Fig. 1.3). The biggest aquaculture consumers group of fishmeal in 2006 was marine shrimp followed by marine fish and other farming fish species such as salmon, Chinese carp, trout, eel, catfish and tilapia (Hardy, 2010). Figure 1.3 Figure 1.3Figure 1.3 Figure 1.3 Fish meal used in different farmed animals sectors from 1980 to 2008. 1.2.3 1.2.31.2.3 1.2.3 Fish meal current world production and situation Fish meal current world production and situation Fish meal current world production and situation Fish meal current world production and situation Since 1950 increased demand of fish meal, mainly for using in animal feed, has made this a global high value commodity. However, anchovy ( Engraulis ringens ) stocks, main species used to obtain fish meal are very unstable as their populations decline notably when the climate phenomenon “ el Niño ” occurs. Moreover, there is a strong administrative scheme controlling closed seasons for fishing sustainable and preserving the health of stocks. Because of this the production of fishmeal in recent years has suffered a gradual decline in global production passing from 2.3 million tons in 2000 to 1.34 million tonnes in 2009, which represents a reduction of 58% in their production (FAO-Fish Globe, 2010). These lower yields are due mainly as a result of lower Peruvian catches, which were 70% less than those achieved in 2000 (FAO-Fish Globe, 2010). The main country producing and exporting fishmeal, mostly coming from anchovy species is Peru followed by Chile, while the main consumer market is China, followed by Japan, Taiwan and the European Union (FAO-Fish Globe, Fish meal used in 1980 Pigs 36% Rest 4% Aquaculture 10% Poultry 50% Fish meal used in 2008 Pigs 22% Poultry 8% Rest 8% Aquaculture 62% Fish meal used in 1980 Pigs 36% Rest 4% Aquaculture 10% Poultry 50% Fish meal used in 2008 Pigs 22% Poultry 8% Rest 8% Aquaculture 62% INTRODUCTION INTRODUCTIONINTRODUCTION INTRODUCTION - 6 - 2010). However, Chinese stocks of fish meal have been one of the lowest levels providing a substantial price increase. Respect to Chile, it increased its production to 2009 due mainly to the demand of high quality meal from China. Nonetheless, the earthquake occurred in February of 2010 seriously damaged the Chilean fish meal factories causing a cut of 200,000 tons in the production and export. On the other hand, the possible reduction of Peru's fishing quota for the following years along with the impact of the climatic phenomenon " el Niño " would lead to reduced even more the availability of fishmeal in the market (FAO-Fish Globe 2010). Based on this scenario, the predictions for the production of fishmeal could not be more pessimistic and uncertainty. Moreover, if the demand was already strong this will cause an even more rise in fish meal prices making it as a finite commodity. The reliance on fish meal makes the aquaculture industry less sustainable and because of this, predictions for fish meal consumption in marine fish feed is estimated to be reduced in a 31.3% from 2006 to 2020 (Gatlin et al ., 2007; Tacon et al ., 2006), being needed replacing it with other protein sources that are readily available and at lower prices. 1.3 1.3 1.3 1.3 ALTERNATIVE PROTEIN ALTERNATIVE PROTEIN ALTERNATIVE PROTEIN ALTERNATIVE PROTEIN INGREDIENT INGREDIENTINGREDIENT INGREDIENTS SS S TO FISH MEAL IN FIS TO FISH MEAL IN FIS TO FISH MEAL IN FIS TO FISH MEAL IN FISH DIETS H DIETS H DIETS H DIETS 1.3.1 Alternative protein sources from 1.3.1 Alternative protein sources from1.3.1 Alternative protein sources from 1.3.1 Alternative protein sources from terrestrial origin terrestrial origin terrestrial origin terrestrial origin Recently, a large number of studies have been focused on the replacement of fish meal with alternative protein ingredients, especially those from plant origin and terrestrial animal by-products, in compound aquaculture feeds to alleviate problems of supply and high price of fish meal. In this sense, the effect of partial or total replacement of fish meal by plant protein sources, which are generally less expensive and more ready available, has been accomplished in many bred fish species with different success degree in growth performance, feed utilization and fish quality (Moyano et al ., 1992; Gomes et al ., 1995; Robaina et al ., 1997, 1999; Hardy, 1996; Kaushik et al ., 2004; Francesco et al ., 2007; Shafaeipour et al ., 2008). Soybean is so far the plant source INTRODUCTION INTRODUCTIONINTRODUCTION INTRODUCTION - 7 - widely used instead of fish meal (Robaina et al ., 1997, 1999; Mambrini et al ., 1999; Refstie et a l., 2000; Wang et al ., 2006; Venou et al ., 2006; Overland et al ., 2009); other sources as those from peas (Hardy et al ., 1996; Gouveia et al ., 2000; Borlongan et al ., 2003; Sánchez-lozano et al ., 2009; Davies et al ., 2010), different lupine species (Robaina et al ., 1995; De la Higuera et al ., 1988; Glencross et al ., 2003; Serrano et al ., 2011) and other plant meals (Watanabe et al , 1997; Davies et al ., 1997; Mente et al ., 2003; Palmegiano et al ., 2006; Sánchez-lozano et al ., 2007; Sánchez-Lozano et al ., 2009). However, it is well recognized that utilization of high dietary levels of several plant protein sources in carnivorous species could induce problems linked to the deficiency in the essential amino acids, presence of many specific antinutritional factor, palatability, poor digestibility and absence of n-3 highly unsaturated fatty acids (HUFA) (Francis et al ., 2001). In addition, specific gastrointestinal damage has also being associated with high dietary levels of plant sources (Caballero et al ., 2003; Penn et al ., 2011). On the other hand, omnivorous species have shown greater tolerance with higher dietary inclusion level (Kaushik et al ., 1993; Robinson and Li, 1994; El-Saidy and Gaber, 2002). Although improvements in the raw materials and feed processing technologies have enabled promising results in overcoming plant protein sources, problems such as presence of anti-nutrients can not be avoided (Drew et al ., 2007). Some author such as Tacon and Metian (2008) argued that while trying to make costeffective diets by using cheaper ingredients, the use of plant protein sources even may increase the costs since it may be necessary to treat anti nutritional factors, and to add certain amino-acids in order to improve the nutritional profile of the diets. Regarding to by-product meals from animal protein, these appear to have the greatest potential as cost-effective replacements for fish meal especially in carnivorous fish species (Bureau et al ., 2000). However, the use of this type of protein sources there may be to produce variable growth results and reduction in INTRODUCTION INTRODUCTIONINTRODUCTION INTRODUCTION - 14 - 1.3.2. 1.3.2.1.3.2. 1.3.2.1.1 Chitin long 1.1 Chitin long1.1 Chitin long 1.1 Chitin long- -- -change polysaccharide as potential bio change polysaccharide as potential biochange polysaccharide as potential bio change polysaccharide as potential bio- -- -components from components from components from components from crustacean meals crustacean mealscrustacean meals crustacean meals Marine crustacean shell wastes have long been the primary source of the polysaccharide known as chitin (Healy et al ., 2003). Chemically, the structure of chitin consist in units of N-acetyl-2-amino-2-deoxy-D glucopyranose (C8H15O6N) bound by b (1→4) glycosidic linkages (Díaz-Rojas et al ., 2006). Chitin is obtained from crustacean shell waste such as shrimp, prawn, krill, crab and lobster, with shrimp carcass contenting the higher amount of chitin (30-40%) followed by crab (15-30%) (Qin and Aboh, 1997). Commercially, it is mostly used as a raw material to produce chitosan oligosaccharides and glucosamine in a worldwide amount of 37,000 tons/year (Ferraro et al ., 2010). Chitosan is obtained mainly from chitin isolated where the industrial extraction process used can be tentatively divided into three major step: 1) shells grinding, 2) decalcified process which consist in an elimination of inorganic matter (metals, salts and calcium carbonate) through treating shell with dilute hydrochloric acid in acidic medium, and 3) de-proteinization with the extraction of protein matter in alkaline medium or by proteases derived from bacteria (DiazRojas et al ., 2006). In aquaculture, chitin and chitosan has been proved in some cultured species as immunostimulant showing its protective effect against bacterial diseases (Anderson and Siwicki, 1994; Esteban et al ., 2000; Ortuno et al ., 2000; Esteban et al ., 2001; Cuesta et al ., 2003; Wang and Chen, 2005; Gopalakannan and Venkatesan, 2006). Regarding to fish growth parameters, the dietary effects of chitin and chitosan have been yielded different results. For instance, Shiau and Yu (1999) observed depressed growth in tilapia ( Oreochromis niloticus × O. auratus ) after feeding chitin and chitosan at the 2%, 5% and 10% inclusion level, suggesting an interference of chitosan and chitin in the absorption of the nutrients. Later on, in common carp Cyprinus carpio , lower dietary chitosan content (1%) has proved to INTRODUCTION INTRODUCTIONINTRODUCTION INTRODUCTION - 15 - enhance growth performance (Gopalakannan and Venkatesan, 2006), explained by the crucial role of chitosan to enhance the digestion and absorption of nutrients at low dietary inclusion levels. However, in the same study, fish fed on chitin (1%) showed depressed growth after 30 days of feeding due to the fish developing intolerance to chitin. Contrary, diet containing higher dosage (up to 10%) of chitin or chitosan did not affect fish growth when feeding to red sea bream Pagrus major , Japanese eel Anguila japonica and yellow tail Seriola quiqueradiata (Kono et al ., 1987). A recent research was done to study the presence of chitinolytic enzymes system and the apparent digestibility of chitinous waste meal in cobia fish (Fines and Holt, 2010). Results showed that fish stomach chitinase and chitobiase activities were high, even when antibiotic were supplemented suggesting that chytinolytic bacteria are not significant. In addition, the apparent digestibility of chitin was high either for crab meal (67%) or shrimp meal (78%), and the crude protein, lipid and gross energy digestion for crab and shrimp meals were not significantly different respect to fish meal. With a caloric content for the chitin of 17.1 kJ g-1 (Gutoska et al ., 2004), it could constitute a great percentage of the total energy intake in species which natural feeding are based on crustacean and therefore chitin intake are considerable. In this respect, chitinous waste material can be utilized to replace carbohydrates and protein from fish meal if we consider the available energy from the chitin (Fines and Holt, 2010). Moreover, same authors pointed out the indeed to realise similar feeding experiments in other species with similar feeding behaviour to known more about the potential use of these kind of ingredients in aquaculture diets. 1.3.2.1.2 Dietary crab meal and fish flesh quality 1.3.2.1.2 Dietary crab meal and fish flesh quality1.3.2.1.2 Dietary crab meal and fish flesh quality 1.3.2.1.2 Dietary crab meal and fish flesh quality Fish quality involves different concepts depending on fish farmer, the processing industry or consumer understanding. Thus, organoleptic properties, nutritional value and flesh self life, are sets of characteristic that contribute to fish quality as perceived by the consumer. All mentioned characteristic are INTRODUCTION INTRODUCTIONINTRODUCTION INTRODUCTION - 16 - strongly related with fish chemical composition, which depends on many quality aspects, among them diet composition is highly important (Lie et al ., 2001; Grigorakis, 2007). It seems that all acquired quality attributes of fish fillet are well correlated with fat content and lipid class in tissue (Waagbo et al ., 1993; Guillou et al ., 1995; Morris et al ., 1995). Lipid metabolism and accumulation in fish as response to dietary protein sources has been showed (Lupatsch et al ., 2002; Francesco et al ., 2007), affecting to flesh quality parameters such as sensory characteristic (Williams et al ., 2003 b; Karlsen et al ., 2006; Suontama et al ., 2007b) and the fillet texture of the farmed fish (Hernández et al ., 2007). Few studies exist about the effect of dietary crustacean meal on final flesh quality with most of them being realized to examine crustacean krill meal. Fernández (2006) reported a significant increase of ω-3/ω-6 ratio in muscle of blackspot Pagellus bogaraveo fed on a 30% dietary inclusion level of crustacean meal (10% krill meal and 20% shell shrimp meal) related to the high dietary ω3/ω-6 relation in the crustacean meals. Suontama et al . (2007a) showed that a substitution of 20% the fish meal protein by protein from krill Thysanoessa inermes did not modified fillet fatty acid profile in Atlantic salmon and halibut Hippoglossua hipoglossus , while in the same work higher substitution of the fish meal, either for krill meal (60%) or amphipodo Themso libellula meal (40%), produced significant changes in fillet fatty acid content. Furthermore, substituting fish meal by krill meal did not appear to influence muscle sensory evaluation or texture to any major extent in Atlantic salmon (Karlsen et al ., 2006; Suoantama et al ., 2007b). Regarding to crab meal and flesh quality, Gonzalez et al . (2006) studied the effect of crab meal-supplemented diets (5%) on southern flounder Paralichthys lethostigma muscle quality. Results indicated that flounder fed on crab meal significantly increased ARA (20:4n-6), adrenic (22:4n-6) and DHA (22:6n-3) muscle fatty acids content with an overall higher n-3/n-6 ratio, improving the INTRODUCTION INTRODUCTIONINTRODUCTION INTRODUCTION - 17 - nutritional value of fish fillet. This clearly suggests that crab meal finishing diets could be utilized to enhance final product quality. 1.3.2.2 Other potential ingredients from marine sources: by 1.3.2.2 Other potential ingredients from marine sources: by1.3.2.2 Other potential ingredients from marine sources: by 1.3.2.2 Other potential ingredients from marine sources: by- -- -products processing products processing products processing products processing from echinoderm industry from echinoderm industryfrom echinoderm industry from echinoderm industry Some research has been done in order to explore the possible uses of other marine products and by-products such as those from echinoderm species. From green sea urchin only gonads and for cucumber only muscle bands and body wall are desirable, while the remaining tissue together with the unwanted edible material, that not meet commercial requirement of quality and are discarded. In case of by-product from Atlantic cucumber Cucumaria frondosa and green sea urchin Strongylocentrotus droebachiensis estimated discard amount may reach up to 50% and 80% respectively (Mamelona et al ., 2010). Nutritional composition reveals that these undesirable raw materials are rich in many valuable nutrients as proteins (4.5-14.5% wet weight) and lipids (1.9-4.6%) together with a 35% of essential amino acids of the total ones and also rich in polyunsaturated fatty acids (44% of total fatty acid), with high EPA (17%) but low DHA (0.2%). In terms of vitamins, they are mainly rich in alpha-tocopherol or vitamin E (Zhong et al ., 2007; Mamelona et al ., 2010a; b). 1.4 1.4 1.4 1.4 THE THE THE THE RED PORGY ( RED PORGY (RED PORGY ( RED PORGY ( Pagrus Pagrus Pagrus PagrusPagrus Pagrus Pagrus Pagrus ; Linnaeus, 1758) ; Linnaeus, 1758); Linnaeus, 1758) ; Linnaeus, 1758) 1.4.1 External appearance, geographical distribution, habitat and r 1.4.1 External appearance, geographical distribution, habitat and r1.4.1 External appearance, geographical distribution, habitat and r 1.4.1 External appearance, geographical distribution, habitat and reproduction eproductioneproduction eproduction Red porgy (Fig. 1.4) is a sparid marine fish with a wide geographical distribution extends along the Western Atlantic coasts, from North Carolina to Argentina, and Eastern, from British Island to South of Senegal including the archipelagos of Madeira, Canaries and Cape Verde. It is also present in the Mediterranean and the Adriatic seas (Manooch and Hassler, 1976; Bauchot et al ., 1981; Pajuelo et al ., 1996). INTRODUCTION INTRODUCTIONINTRODUCTION INTRODUCTION - 18 - Phylum: Chordata Superclass: Gnathostomata Class: Osteichtyes Orden Perciformes Suborden: Percoidei Family Sparadae Genus: Pagrus Species: Pagrus pagrus Phylum: Chordata Superclass: Gnathostomata Class: Osteichtyes Orden Perciformes Suborden: Percoidei Family Sparadae Genus: Pagrus Species: Pagrus pagrus Figure 1.4 Figure 1.4Figure 1.4 Figure 1.4 Taxonomic classification of red porgy. Red porgy is a bottom fish that generally lives in warm temperatures and subtropical habitats (Afonso et al ., 2008). Juveniles specimens are often found on sandy bottoms, at 20-50 m depth, while higher fish size are frequently found on rocky bottoms in a depth around 50 m. According to Manooch (1976) and Alekseev (1983), red porgy is a hermaphrodite protogynous reproductive specie, where individual are females at smaller sizes and males at larger sizes. The change of sex occurs in fish sizes between 32.5 to 42.5 cm, and gonads maturation occurs at 3-4 years of age (Manoocher and Alekseev, 1990); although this species undergoes a first sexual maturity of females when reach about 250-300 grams whole weight during their second and third year of life. The spawning season appears between January and April, with water temperatures between 16 °C and 21 °C. 1.4.2 Natural diet of the 1.4.2 Natural diet of the1.4.2 Natural diet of the 1.4.2 Natural diet of the red porgy red porgy red porgy red porgy Similar feeding habit have been observed for individuals of both the Eastern Mediterranean (Labropoulou et al ., 1999) and the western Atlantic (Manooch, 1977), as well as in Tunisian coast (Chakroun-Marzouk and Kartas, 1987). Detailed studies about feeding habits of juvenile red porgy such as INTRODUCTION INTRODUCTIONINTRODUCTION INTRODUCTION - 19 - Castriota et al . (2006) reveals that from the 78 mostly epibenthic identified species consumed, crustacean are the dominant group followed by molluscs and fish, with echinoderms sea urchin also consumed in a lower proportion. Furthermore, despite the large number of taxa found, few species accounted for most of the diets, suggesting specialized feeding. The individual feeding specialization was based on hermit crabs, brachyuran crabs and prosobranch gastropods (Castriota et al ., 2006). The knowledge of red porgy natural diet and feeding habits helps to understand the nutritional mechanisms involved in their feeding biology, which are necessary for commercial cultured red porgy diet improvement. 1.4.3 Red porgy culture 1.4.3 Red porgy culture1.4.3 Red porgy culture 1.4.3 Red porgy culture The diversification of aquaculture through the production of new species is one of the most important initiatives towards stabilization of the productive sector, currently characterized by high production of a small number of species. In selecting a new candidate species for aquaculture, some requirements or aspects of the species are considered important such as good growth rate, high market value and a large demand for human consumption. In addition, biological and technical aspects of the culture conditions must also be overcame by the new species (Suquet et al. , 2002). In this sense, red porgy has a high meat quality prized, making it a species of interest to both commercial fisheries and recreational anglers. Fillets are characterised by delicate flavour, firm flesh with high protein and low fat content. Red porgy has an important market demand on the Mediterranean and Atlantic coast (Kokokiris et al ., 2006). Unfortunately, owing to prolonged exploitation, some red porgy populations have become over fished (Haimovici, 1998: Vaughan et al., 2002; Afonso et al ., 2008), and consequently wild catches of this species are very limited or insufficient to meet the current market demand. INTRODUCTION INTRODUCTIONINTRODUCTION INTRODUCTION - 20 - Preliminary studies carried out with this species under culture conditions have shown good adaptability to growing conditions, spontaneous spawning, high growth rates and no serious problems of disease or mortality (Kentouri et al , 1994; Kokokiris, 1998; Cejas et al ., 1999). These facts make red porgy one of the priority marine species for aquaculture diversification of the Spanish Atlantic coast or Mediterranean Sea since the nineties of the last century (Kentouri et al ., 1995; Hernandez-Cruz et al ., 1999). 1.4.3.1 1.4.3.1 1.4.3.1 1.4.3.1 Dietary DietaryDietary Dietary protein protein protein protein and carotenoids pigments and carotenoids pigments and carotenoids pigments and carotenoids pigments requirements. requirements.requirements. requirements. One of the main constraints in the culture of the red porgy is the loss of its characteristic red-pink skin colour under cultured conditions (Kentouri et al ., 1995; Klios et al ., 1997; Basurco and Abellán, 1999). The preservation of the natural colour and the similarity in external appearance towards the wild specimens has a great importance from a commercial point of view, associated with the acceptance or rejection of the product by consumers (Shahidi et al ., 1998; TECAM, 1999). Fish are unable to bio synthesize carotenoids de novo (Goodwin, 1984), hence, carotenoids must be provided in the red porgy diet. Thus, most nutritional studies realized on this species have principally focussed on different carotenoid sources and inclusion levels about the effect on skin coloration. From the different carotenoids tested, only astaxanthin, in its esterified form, has given desirable results (Cejas et al ., 2003; Chatzifotis et al ., 2005; Kalinowski et al ., 2005; Tejera et al., 2007). In wild specimens, the astaxanthin carotenoid is provided by small crustaceans that constitute the natural feed of this species (Labropoulou et al ., 1999; Castriota et al ., 2005). Therefore several studies have used crustacean meals as a source of astaxanthin in order to overcome the red porgy skin pigmentation problem, given interesting results (Cejas et al ., 2003; Kalinowski et al ., 2005; 2007). INTRODUCTION INTRODUCTIONINTRODUCTION INTRODUCTION - 21 - Another key constriction in cultured red porgy is their high protein dietary requirement, around 50% dry weight, in artificial diets (Schuchart et al ., 2008), where fish meal has currently been used as protein source. Some works has been realized to study the partial replacement of fish meal protein by protein from hydrolyzed krill meal in red porgy diet (Schchardt, 2005). In general, good feed utilization, growth performance as well as skin colour enhanced results were obtained in a diet with a 20% inclusion of krill meal and low dietary lipid level (15%). In the same study, a reduction of the dietary protein level by increasing the dietary fish oil, clearly resulted in a reduction on fish growth when fat levels where above to 15%. Red porgy is considered a lean fish (Ruedas et al ., 1997). In this line, both dietary protein quantity and quality are considered important in these animals compared to other types of less expensive energy ingredients such as fats. Consequently, for red porgy being competitive in the market remains that not only return their natural skin pigmentation is necessary, but also replacing the expensive fish meal in their diet is important as this would reduce feed cost. In this sense, development of new supplies of protein and natural pigments, especially from marine sources, in on-growing red porgy diets would assist in their economical rearing to reach a marketable size. The present study is focused on identifies potential fish meal alternative ingredients and natural pigment, especially from marine origin, for red porgy diets. The results from this work could give valuable answers to reduce the inclusion level of the expensive fish meal in red porgy diets. Moreover would greatly improve basic nutritional and metabolic knowledge of the red porgy and their effects by dietary changes. All studied aspects will contribute to producing diets for farmed red porgy that more closely resembles their natural diet, thus promoting fish growth and colour towards the wild specimens. 2. OBJECTIVES 2. OBJECTIVES2. OBJECTIVES 2. OBJECTIVES MATERIALS AND METHODS MATERIALS AND METHODSMATERIALS AND METHODS MATERIALS AND METHODS - 30 - phenoxyethanol (0.1 mL L-1), and then randomly distributed into the circular fibreglass tanks. 3.2 3.23.2 3.2 CULTURE CULTURE CULTURE CULTURE CONDITIONS CONDITIONSCONDITIONS CONDITIONS 3.2.1 3.2.13.2.1 3.2.1 Facilities Facilities Facilities Facilities The feeding trials described in the present work were performed at the Marine Culture facilities of the Canary Institute of Marine Sciences (ICCM), belonging to the Canary Island Government (Agencia Canaria de Investigación, Innovación y Sociedad de la Información del Gobierno de Canarias). 3.2.2 3.2.23.2.2 3.2.2 Experimental tanks Experimental tanks Experimental tanks Experimental tanks Tanks system were composed by a group of cylinder conical tanks with a total volume of 500 litres and disposed with a central drainage channel. Each tank had dimensions of 1.5 m of diameter and 1.0 m of depth in the central part and was continuously supplied with natural sea water under experimental conditions. All tanks were individually covered to prevent the fish jumping out the tanks. 3.2.3 3.2.33.2.3 3.2.3 Experimental conditions Experimental conditions Experimental conditions Experimental conditions Experiences were realised with tanks held indoors and natural light condition with a natural photoperiod (latitude 28º, 10’ North). Fish were fed until apparent satiation 2 time a day and six day per week. Daily mortality was recorded as well as the supplied and waste feed in order to determine the daily feed intake. Water dissolved oxygen and temperature were weekly measurement with the oximeter YSI 95 Dissolved Oxygen, 95/10 FT CE model. MATERIALS AND METHODS MATERIALS AND METHODSMATERIALS AND METHODS MATERIALS AND METHODS - 31 - 3.3 3.33.3 3.3 EXPERIMENTAL FEED INGREDIE EXPERIMENTAL FEED INGREDIE EXPERIMENTAL FEED INGREDIE EXPERIMENTAL FEED INGREDIEN NN NTS TS TS TS 3.3.1 3.3.13.3.1 3.3.1 R R R River crab iver crabiver crab iver crab, ,, , Procamba ProcambaProcamba Procambarus rus rus rus clarkii clarkiiclarkii clarkii , meal , meal, meal , meal Phylum: Arthropoda Subphylum: Crustacea Class: Malacostraca Order: Decapada Suborder: Astacidea Family: Cambride Genus: Procamburus Specie: Procambrus clarkii Phylum: Arthropoda Subphylum: Crustacea Class: Malacostraca Order: Decapada Suborder: Astacidea Family: Cambride Genus: Procamburus Specie: Procambrus clarkii Figure Figure Figure Figure 3. 3.3. 3.2 22 2 Taxonomic classification of the river crab Procambarus clarkii . The crab Procambarus clarkii (Fig. 3.2) is a freshwater crayfish specie native to North-eastern of Mexico and the South-eastern of United States. However, it can be found in a wide range of places due to the deliberate introduction of this species in many countries. In Spain, this crayfish was introduced in 1974 for commercial purposes. The first introduction occurred in the Guadalquivir River at the South of Spain (Hadsburgo-Lorena, 1979; Algarin, 1980, Ocete and López, 1983). Nowaday, Spain is among the top three countries that most contribute to the world market of this river crab (Laurent, 1990). The red river crab meal, of the present work was provided by a company located in the southern of Spain (Seafood Sevilla S.L.). This company processes this river crab for human consumption. The meal was generated from the crab byproducts and processing waste mainly consisting of shells, legs, viscera, deformities animal and out market size discards, which all still has a considerable nutritional value. The raw material was processed at high temperature by the company towards to obtain the river crab meal used in the present work (Fig. 3.5). MATERIALS AND METHODS MATERIALS AND METHODSMATERIALS AND METHODS MATERIALS AND METHODS - 32 - 3.3.2 Marine 3.3.2 Marine3.3.2 Marine 3.3.2 Marine crab crab crab crab, ,, , Chaceon affinis Chaceon affinisChaceon affinis Chaceon affinis and and and and Paramola cuvieri Paramola cuvieriParamola cuvieri Paramola cuvieri , meals , meals, meals , meals Phylum: Arthropoda Subphylum: Crustacea Class: Malacostraca Order: Decapada Infraorder: Brachyura Family: Geryonide Genus: Chaceon Specie: Chaceon affinis Phylum: Arthropoda Subphylum: Crustacea Class: Malacostraca Order: Decapada Infraorder: Brachyura Family: Geryonide Genus: Chaceon Specie: Chaceon affinis Figure Figure Figure Figure 3 33 3. .. .3 33 3 Taxonomic classification of the marine crab Chaceon affinis . The red crab Chaceon affinis (Fig. 3.3) is the largest epibenthic brachyuran which inhabit oceanic water throughout the eastern Atlantic Ocean, Iceland to Senegal and around all the Macaronesian Islands (López Abellán et al ., 2002). In the Canary Islands, this species was first collected during a scientific survey in July 1985 (Lozano et al ., 1992). It is has been caught at depths ranging from 550 to 1200 m by scientific surveys and by fishermen (Pinho et al ., 2001; López et al ., 2002). In spite of the stock having an economic potential and the presence of some local fisheries of this specie in north-western Spain (Galicia Bank), there is no still commercial market development of this species for human consumption. The red crab Paramola cuvieri (Fig. 3.4) it is widely distributed throughout the eastern Atlantic, from southern Iceland (63 º N) and Norway to South Africa (36 º S), including the Azores, Madeira, Canaries and Cape Verde, also in some areas of the Mediterranean. In the Canaries, it is occasionally captured between 120 and 860 m depth and until now, there is not a local market for this species and constitutes a discard by-catch in the local fisheries (Sanchez et al ., 2004). It is caught with bottom traps as a secondary species in fisheries, especially shrimp ( Plesionika edwardsii ) between 250 and 350 m and with the red crab ( Chaceon affinis ) between 600 and 900 m. MATERIALS AND METHODS MATERIALS AND METHODSMATERIALS AND METHODS MATERIALS AND METHODS - 33 - Phylum: Arthropoda Subphylum: Crustacea Class: Malacostraca Order: Decapada Infraorder: Brachyura Family: Homolidae Genus: Paramola Specie: Paramola cuvieri Phylum: Arthropoda Subphylum: Crustacea Class: Malacostraca Order: Decapada Infraorder: Brachyura Family: Homolidae Genus: Paramola Specie: Paramola cuvieri Figure 3.4 Figure 3.4Figure 3.4 Figure 3.4 Taxonomic classification of the marine crab Paramola cuvieri . 3.3.2.1 Preparation of the two marine crab meals. The two marine crabs, C. affinis , and P. cuvieri , were caught close to the Canaries’ Coast in Spain by local fishermen. Both meals were prepared locally at our laboratory facilities from the whole crabs individuals using a process adapted from Sudaryono et al ., (1996). First, animals were individually cleaned and autoclaved at high pressure for 15 min, then oven-dried for 12 hours at 55ºC and finally ground in a hammer mill through a 0.5 mm mesh and stored in vacuum plastic bags at 4ºC. The subsequent batch meals were finally mixed until obtained an only homogenous batch (Fig. 3.5). Figure 3.5 Figure 3.5Figure 3.5 Figure 3.5 River and marine crabs meals used in the experimental diets. River crab meal Marine crab meal River crab meal Marine crab meal MATERIALS AND METHODS MATERIALS AND METHODSMATERIALS AND METHODS MATERIALS AND METHODS - 34 - 3.3.3 3.3.3 3.3.3 3.3.3 Sea Sea Sea Sea urchin urchinurchin urchin, ,, , Diadema antillarum Diadema antillarumDiadema antillarum Diadema antillarum , meal , meal, meal , meal Figure Figure Figure Figure 3.6 3.63.6 3.6 Taxonomic classification of the long-spined sea urchin Diadema antillarum . The long-spined black sea urchin Diadema antillarum (Philippi) (Fig. 3.6), is an amphi-Atlantic warm species that plays a key-role in benthic communities as the main herbivorous across the temperate warm-waters of the central-east Atlantic (26-36 º N) (Alves et al ., 2003; Tuya et al ., 2004). In this sense, the longspined black sea urchin is directly involved in the transformation of large rockyreefs covered by erect fleshy algae to overgrazed, deforested, stable substrates dominated by encrusting organisms. These so-called “urchin-grazed barrens” are a global phenomenon; generally support less biodiversity and productivity that nearby vegetated area (Pinnegar et al ., 2000). Individuals of the sea urchin were caught from the seafloor Canaries’ coast and meal was prepared locally from whole animals, previously removing part of the surrounding whole body spines, and using the same adapted process to obtain the marine crab meals (Sudaryono et al ., 1996). After drying, the material was also grounded in a hammer mill through a 0.5 mm mesh and stored in vacuum plastic bags at 4ºC. The different batch meals were mixed until obtained an only homogenous batch. Phylum: Echinodermata Class: Echinoidea Order: Diadematoida Genus: Diadema Species: Diadema antillarum Phylum: Echinodermata Class: Echinoidea Order: Diadematoida Genus: Diadema Species: Diadema antillarum MATERIALS AND METHODS MATERIALS AND METHODSMATERIALS AND METHODS MATERIALS AND METHODS - 35 - 3.3.3 Other feed ingredients 3.3.3 Other feed ingredients3.3.3 Other feed ingredients 3.3.3 Other feed ingredients Fish meal and fish oil used in all experiments were from Peruvian origin and provided by the company Proaqua Nutrición S.A, España. The used pregelatinized starch “Merigel 100” and the vitamin C “Stay C” were donated by Spanish companies Especialidades Puma, S.A. (Amylum Group) and DMSNutrición S.A, respectively. 3.4 FEED 3.4 FEED3.4 FEED 3.4 FEED PROCESSING PROCESSINGPROCESSING PROCESSING 3.4.1 3.4.1 3.4.1 3.4.1 Formulation FormulationFormulation Formulation Proximate composition and total carotenoids content of all used feed ingredients were analysed prior to diet formulation. In all experiences, diets were formulated to be isocaloric and isoproteic with 12% lipid and 50% protein, in dry weight basis, to meet the specie requirements as previously reported by Schuchardt et al. (2008). A diet based on high quality fish meal (FM) was used as a control diet. The treatments were formulated by replacing the desirable % of FM protein in the control diet with protein from the alternative tested meals. Fish oil was the main lipid source in all experimental diets. Dietary starch content varied between diets and was used as a filler to keep the same protein and lipid levels in all experimental formulation. 3.4.2 Diets elaboration 3.4.2 Diets elaboration3.4.2 Diets elaboration 3.4.2 Diets elaboration A mix of minerals and hydro-soluble vitamin (Table 3.1) were prepared and stored at 4ºC until diets elaboration. In both mixtures α-cellulose was used as a carrier component. The other mix was the fat-soluble vitamins (Table 3.1), which was prepared at the time of diets elaboration, using ethoxyquin as antioxidant. To prepare the diets, all dry ingredients were added and mixed sequentially, from the lower to the higher amount. Fat-soluble vitamins were dissolved in the fish oil and added to the whole dry ingredients mixture, being gently mixed to obtain a homogenous mass. Vitamin C was introduced at the end MATERIALS AND METHODS MATERIALS AND METHODSMATERIALS AND METHODS MATERIALS AND METHODS - 36 - Vitamins (g/ kg diet) Minerals (g/ kg diet) Hydro-soluble Cianocobalamine (B12) 0.5 (H 2 PO 4 )Ca 1.605 Biotin (B7) 0.001 CaCO 3 4.0 Folic acid (B9) 0.01 FeSO 4 .7H 2 O 1.5 Pyridoxine (B6) 0.04 MgSO 4 .7H 2 O 1.605 Riboflavine (B2) 0.05 K 2 HPO 4 2.8 Thiamine (B1) 0.04 Na 2 PO 4 H 2 O 1 Choline 2.7 Al(SO 4 ) 3 .6H 2 O0.02 Pantotenic acid 0.12 ZnSO 4 .7H 2 O 0.24 Niacin (B3) 0.2 CuSO 4 .5H 2 O 0.12 Ascorbic acid (C) 0.7 KI 0.02 Mio-inositol 2 CoSO 4 .7H 2 O0.08 Lipid-soluble MnSO 4 H 2 O 0.08 ∝-tocopherol (E) 0.25 Retinol acetate (A) 0.025 Medianone (K3) 0.02 Cholecalciferol (D3) 0.005 of the process to minimize losses due to possible oxidation. The last component added was the water, about 10%, in which the choline chloride vitamin was dissolved. Finally, the resultant mixture diet was pelleted with a CMP (Mod CL3, USA) pellet machine, throughout 5mm die diameter to obtain an adequate feed pellet according to the fish size. The obtained pellets were disposed into different trays and dried in an aerated oven at a temperature of about 38ºC for 12 hours for then being disposed in closed plastic bags and stored at 4ºC. Table Table Table Table 3. 3.3. 3.1 11 1 Vitamins and minerals premixes used in all experimental diets. MATERIALS AND METHODS MATERIALS AND METHODSMATERIALS AND METHODS MATERIALS AND METHODS - 37 - 3.5 BIOLOGICAL AND FEED UTILIZA 3.5 BIOLOGICAL AND FEED UTILIZA3.5 BIOLOGICAL AND FEED UTILIZA 3.5 BIOLOGICAL AND FEED UTILIZATION PARAMETERS TION PARAMETERSTION PARAMETERS TION PARAMETERS The following equations were used to study the effect of the experimental diets on growth performance and feed utilization. 3.5.1 Relative growth 3.5.1 Relative growth 3.5.1 Relative growth 3.5.1 Relative growth Represent the relation between the biomass (g) increase and initial weight. Growth (%) = [(final weight (g) – initial weight (g))/ initial weight (g)] x 100 3.5.2 Condition factor (K) 3.5.2 Condition factor (K)3.5.2 Condition factor (K) 3.5.2 Condition factor (K) Represent the relation between the total animal weight and length. K = weight (g)/ (length (cm)) 3 3.5.3 Specific grow 3.5.3 Specific grow3.5.3 Specific grow 3.5.3 Specific growth rate (SGR) th rate (SGR)th rate (SGR) th rate (SGR) Is the percentage of daily growth increase. SGR= [(Ln final weight -Ln initial weight)/nº experiment days)] x 100 3.5.4 Feed conversion ratio (FCR) 3.5.4 Feed conversion ratio (FCR)3.5.4 Feed conversion ratio (FCR) 3.5.4 Feed conversion ratio (FCR) Represent the relation between feed intake and weight gain. FCR= feed intake (g)/weight gain (g) 3.5.5 Hepatosomatic index (H 3.5.5 Hepatosomatic index (H3.5.5 Hepatosomatic index (H 3.5.5 Hepatosomatic index (HS SS SI II I) )) ) Represent the relation between the liver weight and total animal weight. HSI= [liver weight (g)/ fish weight (g)] x 100 3.5.6 Visceral index (VSI) 3.5.6 Visceral index (VSI)3.5.6 Visceral index (VSI) 3.5.6 Visceral index (VSI) Represent the relation between total viscera weight and fish weight. VSI= [viscera weight (g)/ fish weight (g)] x 100 3.5.7 Protein utilization ratio (PER) 3.5.7 Protein utilization ratio (PER) 3.5.7 Protein utilization ratio (PER) 3.5.7 Protein utilization ratio (PER) This index relates the fish weight with total protein intake, and constitutes a measurement of dietary protein utilization. PER= weight gain (g)/ protein intake (g) MATERIALS AND METHODS MATERIALS AND METHODSMATERIALS AND METHODS MATERIALS AND METHODS - 38 - 3 33 3.6 INSTRUMENTAL COLOUR DETERMINATION .6 INSTRUMENTAL COLOUR DETERMINATION.6 INSTRUMENTAL COLOUR DETERMINATION .6 INSTRUMENTAL COLOUR DETERMINATION For skin colour measurements, a portable colorimeter (Hunter Lab MiniScanTM XE, USA) was used in all experiments. Measurements were taken directly from the left front lateral skin zone (Kalinowski et al ., 2005). The colour parameters obtained were L* , a* and b* from the Hunter system (Fig. 3.7). Figure 3.7 Figure 3.7Figure 3.7 Figure 3.7 Colour measurements taken with the portable colorimeter. L* , is the lightness and ranges from 0 for black to 100 for white. The parameter a* represents chromaticity ranging from green (negative values) to red (positive values), while b* represents chromaticity ranging from blue (negative values) to yellow (positive values), according with the recommendations of the international Commission on Illumination, CIE (1976). From a* and b* values, Hue (Hab) and chroma (Cab) values were calculates by the following equations: Hab= arctan (a*/ b*) , (Hunt, 1977). Cab= ((a*) 2+ (b*) 2)1/2 , (Hunt, 1977). Hue is an angular variable and represents the observable colour. Values of 0º, 90º, 180º and 270º indicate a red, yellow, green and blue hue respectively. Chroma parameter expresses the saturation or intensity of the observable colour. Hunter Lab MiniScanTM XE MATERIALS AND METHODS MATERIALS AND METHODSMATERIALS AND METHODS MATERIALS AND METHODS - 39 - 3.7 ANALYSI 3.7 ANALYSI3.7 ANALYSI 3.7 ANALYSIS OF P S OF PS OF P S OF PR RR ROXIMATE AND FAT OXIMATE AND FATOXIMATE AND FAT OXIMATE AND FATT TT TY ACIDS Y ACIDSY ACIDS Y ACIDS COMPOSITION COMPOSITION COMPOSITION COMPOSITION. .. . All samples were properly collected and stored at -80ºC until analysis. 3.7.1 Moisture 3.7.1 Moisture3.7.1 Moisture 3.7.1 Moisture Moisture was determined by drying the samples at 105ºC until a constant weight was reached (AOAC, 1995). 3.7.2 Ash 3.7.2 Ash3.7.2 Ash 3.7.2 Ash For determining ash content, sample was dried in an oven at a temperature of 405ºC until a constant weight was attained (AOAC, 1995). 3.7.3 Crude Protein 3.7.3 Crude Protein3.7.3 Crude Protein 3.7.3 Crude Protein Protein sample content was determined following the kjeldhal method (AOAC, 1995). Sample was previously digested with concentrated sulphuric acid at a temperature of 420ºC. 3.7 3.73.7 3.7.4 Crude Lipids .4 Crude Lipids.4 Crude Lipids .4 Crude Lipids Total lipid was extracted from the samples by the method of Folch et al . (1957), using a mixture of chloroform:methanol (2:1, v/v) containing 0.01% of BHT. After lipid extraction, solvent was dried under nitrogen atmosphere and then weighted. 3.7.5 Fatty acid profile 3.7.5 Fatty acid profile3.7.5 Fatty acid profile 3.7.5 Fatty acid profile Fatty acid profile from lipid was obtained as described by Christie (1982). Thus, the extracted total lipids were trans-esterified by adding to the samples a solution of Toluene with BHT and another with methanol and sulphuric acid at 1%. Later, samples were incubated for 16 hours at 50ºC. Afterwards, pure distilled water and hexane: dyetil ether (1:1) with BHT at 0.01% was added to the sample. The obtained fatty acid methyl esters (FAMES) were evaporated to dryness under nitrogen atmosphere, weighed and diluted with hexane at 20mg/ml concentration. For the identification and quantification of FAMES, a gas MATERIALS AND METHODS MATERIALS AND METHODSMATERIALS AND METHODS MATERIALS AND METHODS - 46 - T TT Table 3.2 able 3.2able 3.2 able 3.2 Parameters description of the texture profile analysis. 3.12 SENSORY ANALYSIS 3.12 SENSORY ANALYSIS3.12 SENSORY ANALYSIS 3.12 SENSORY ANALYSIS The sensory profile analysis was determined on the cooked fillets of red porgy fed with the different experimental diets. A preliminary training session was aimed before to select the profile attributes, to establish the sample evaluation procedures and to define the score sheet. The dorsal half part of each left fillet was divided into three portions of approximately 3x3 cm and cooked in aluminium boxes, previously identified with codes, for 10 min in a steam oven at 150ºC. Immediately after cooking, fillets were offered to a panel of selected trained judges (ISO, 1985; 1993). Judges were randomly offered closed food boxes labelled with codes containing the fillets. Attributes of odour (marine, oily and atypical), appearance/aspect (whiteness, shininess and cohesiveness), texture (juiciness, firmness and adhesiveness), flavour (marine, oily and atypical) and residual taste (permanence, marine and earthy) were tested for samples of fish fed the experimental diets, and classified by the judge in a continuous scale from 0 to 100 for each parameter. The sensory analysis was carried out in two sessions, Parameters Description (units) Hardness Mechanical required force to compress the sample to certain level Force (N)= (kg × m × s -2 ) Fracturability Required force to produce the rupture of the sample piece Force (N)= (kg × m × s -2 ) Cohesiveness Maximum extent of the sample before breaking ratio wit hout unit Springiness The full or partial recovery of the sample, after stopping the compression force, to its original shape Distance (m) Adhesiveness Required work to separate the sample from the compression cylinder Work (J)= (kg × m 2 × s -2 ) Gumminess Is the product of hardness and cohesiveness. Force (N)= (kg × m × s -2 ) Chewiness The required energy to break up the sample Work (J)= (kg × m 2 × s -2 ) MATERIALS AND METHODS MATERIALS AND METHODSMATERIALS AND METHODS MATERIALS AND METHODS - 47 - and to reduce the panellists’ effect an analysis of repeated measurements was done. 3.13 FISH FIL 3.13 FISH FIL3.13 FISH FIL 3.13 FISH FILL LL LET LIPID OXIDATION ET LIPID OXIDATION ET LIPID OXIDATION ET LIPID OXIDATION The lipid oxidation of fish muscle was determined by measuring the 2thiobarbituric acid reactive substances (TBARS), according to the methodology of Shahidi and Hong (1991). Firstly, fish muscle sample was mixed with a volume (2 ×grams of sample) of 10% trichloroacetic acid (w/v) in a test tube and homogenized (Ultraturrax T25, Jane and Kukel GmbH, Geermany) for 1 minute. The process was carried out at refrigeration temperature, keeping the tubes cooled with ice. Then the samples were centrifuged at 4,000 rpm and 4ºC for 30 minutes. After that, 2 ml of the final extract, previously filtrated, were added to 2 ml of 0.02 M aqueous TBARS in a test tube. The test tubes were heated at 100ºC for 1 hour; after this time, samples were refrigerated under a continuous flow of water and read at 532 nm in a spectrophotometer (Genesys UV10, Thermo Fisher Scientific Inc. Waltham, USA). The results were obtained using a standard curve with 1,1,3,3 tetrametoxipropano (TMP) and expressed as tribarbitúrico acid reactive substances (TBARS) in mg of malonaldehyde per kilogram of sample. 3.14 3.143.14 3.14 STATISTICAL ANALYSI STATISTICAL ANALYSI STATISTICAL ANALYSI STATISTICAL ANALYSIS SS S All scalar data type was tested for normality of distribution and homogeneity of variance (Sokal and Rohlf, 1995). To compare treatments, parametric and homoscedastic data were analysed by one-way ANOVA followed by Tukey’s test for multiple comparisons. For non-normal data and/or data not showing homogeneity of variance, the Kruskal-Wallis multiple range test was employed. When two treatments were established, data were compared statistically by means of T-Student test (Sokal and Rohlf, 1995). In case of considering two factors and their interaction, data were submitted to a two-way ANOVA by using linear model procedure. All statistical analyses were performed MATERIALS AND METHODS MATERIALS AND METHODSMATERIALS AND METHODS MATERIALS AND METHODS - 48 - using the SPSS v. 13.1 statistical package. Significance was set at P <0.05. The angular colour parameter, hue, was analysed for statistical differences following circular statistical procedures. The estimation of the mean value and standard deviation was performed with descriptive statistics for circular distributions. The Rayleigh test was applied to check the uniformity of circular distribution (Zar, 1999). As sampled populations was not unimodal, differences in the hue variables between and experimental groups were tested using the non parametric Watson’s U 2 test and accepted at P <0.05 (Zar, 1999). All tests were performed with Oriana, version 3, statistical software (Kovach Computing Services, Pentraeth, Wales, UK). STUDIES STUDIESSTUDIES STUDIES STUDY I: Marine and freshwater crab meals in diets for red porgy STUDY I: Marine and freshwater crab meals in diets for red porgy STUDY I: Marine and freshwater crab meals in diets for red porgy STUDY I: Marine and freshwater crab meals in diets for red porgy ( (( ( Pagrus pagrus Pagrus pagrusPagrus pagrus Pagrus pagrus ): effect on growth, fish composition and skin ): effect on growth, fish composition and skin ): effect on growth, fish composition and skin ): effect on growth, fish composition and skin colour (Aquaculture Research 41 (2010), 1759 colour (Aquaculture Research 41 (2010), 1759colour (Aquaculture Research 41 (2010), 1759 colour (Aquaculture Research 41 (2010), 1759- -- -1769) 1769)1769) 1769) STUDY II: Marine and freshwater crab me STUDY II: Marine and freshwater crab meSTUDY II: Marine and freshwater crab me STUDY II: Marine and freshwater crab meals in diets for red porgy als in diets for red porgy als in diets for red porgy als in diets for red porgy ( (( ( Pagrus pagrus Pagrus pagrusPagrus pagrus Pagrus pagrus ): ):): ): digestibility, ammonia excretion, phosphorous digestibility, ammonia excretion, phosphorous digestibility, ammonia excretion, phosphorous digestibility, ammonia excretion, phosphorous and calcium retention and calcium retentionand calcium retention and calcium retention STUDY III: STUDY III: STUDY III: STUDY III: Marine and freshwater crab meals in diets for red porgy Marine and freshwater crab meals in diets for red porgy Marine and freshwater crab meals in diets for red porgy Marine and freshwater crab meals in diets for red porgy ( (( ( Pagrus pagrus Pagrus pagrusPagrus pagrus Pagrus pagrus ): effect in fillet fatty acid profile and flesh ): effect in fillet fatty acid profile and flesh ): effect in fillet fatty acid profile and flesh ): effect in fillet fatty acid profile and flesh quality param quality paramquality param quality parameters eterseters eters STUDY IV: STUDY IV:STUDY IV: STUDY IV: Meals of spider marine crab ( Meals of spider marine crab ( Meals of spider marine crab ( Meals of spider marine crab ( Paramola cuvieri Paramola cuvieriParamola cuvieri Paramola cuvieri ) and sea ) and sea ) and sea ) and sea urchin ( urchin (urchin ( urchin ( Diadema antillarum Diadema antillarumDiadema antillarum Diadema antillarum ) in diets for red porgy ( ) in diets for red porgy () in diets for red porgy ( ) in diets for red porgy ( Pagrus Pagrus Pagrus Pagrus pagrus pagruspagrus pagrus ): e ): e): e ): effect on growth performance, ammonia excretion, skin ffect on growth performance, ammonia excretion, skin ffect on growth performance, ammonia excretion, skin ffect on growth performance, ammonia excretion, skin colour and flesh quality colour and flesh qualitycolour and flesh quality colour and flesh quality STUDY I STUDY ISTUDY I STUDY I -51 - Marine and freshwater crab meal Marine and freshwater crab mealMarine and freshwater crab meal Marine and freshwater crab meals in diets for red porgy ( s in diets for red porgy (s in diets for red porgy ( s in diets for red porgy ( Pagrus Pagrus Pagrus Pagrus pagrus pagruspagrus pagrus ): effect on growth, fish composition and skin colour ): effect on growth, fish composition and skin colour ): effect on growth, fish composition and skin colour ): effect on growth, fish composition and skin colour (Aquaculture Research 41 (2010), 1759 (Aquaculture Research 41 (2010), 1759(Aquaculture Research 41 (2010), 1759 (Aquaculture Research 41 (2010), 1759- -- -1769) 1769)1769) 1769) Abstract AbstractAbstract Abstract River crab (RC) meal ( Procambarus clarkii ) and marine crab (MC) meal ( Chaceon affinis ) were tested as a partial replacement for fish meal in diets for red porgy ( Pagrus pagrus ), and their effect on growth performance, fish proximate composition and skin coloration were evaluated. Red porgy were fed during 165 days with five diets. High-quality fish meal diet was used as a control diet (CD). Protein of fish meal in the control was replaced by increasing the dietary levels of protein derived from RC and MC by up to 10% and 20% of each of them (RC10, RC20, MC10 and MC20). Fish fed on MC20 showed the highest values in feed intake, weight gain and growth (%). No differences were found in FCR and protein efficiency ratio (PER) among treatments. Inclusion of both crab meals in diets significantly decreased the lipid content in whole fish compared to control animals. Feeding both crab meals resulted in colour improvement compared to that of the control fish, with better hue values for the RC meal group than those for the MC meal group. The crab meals tested in the present study are suitable as a partial replacement for fish meal in diets for the red porgy, with the MC meal improving growth and both crab meals improving skin colour, with further improvements in skin colour produced in fish-fed diets containing the RC meal. Keywords: Keywords: Keywords: Keywords: carotenoids, crab meal, fish composition, Pagrus pagrus , skin colour STUDY I STUDY ISTUDY I STUDY I - 52 - 4. 4.4. 4.1 11 1 Introduction Introduction Introduction Introduction Red porgy, Pagrus pagrus , is a marine fish species with an important market demand and greatly appreciated by consumers on the Mediterranean and Atlantic coasts (Kokokiris et al ., 2006); therefore, it is considered, in this region, to be a priority species for aquaculture diversification. Preliminary studies carried out with this species under culture conditions have shown good adaptability together with efficient growth rate (Divanach et al ., 1999). However, one of the main constraints in the culture of this marine species is the loss of its characteristic red-pink skin colour, which is a huge problem from a commercial point of view, associated with the acceptance or rejection of the product by consumers (TECAM, 1999; Cejas et al ., 2003). One of the key factors responsible for the discoloration of cultured red porgy skin is the fact that fish are unable to biosynthesize carotenoids de novo (Goodwin, 1984); hence, under aquaculture conditions, carotenoids must be provided in the diet. Thus, most recent nutritional studies done on this species have principally focussed on different carotenoid sources and levels and their effect on skin coloration. In fact, the inclusion of dietary astaxanthin, mainly in its esterified form, has been shown to be an effective carotenoid for farmed red porgy skin to acquire the characteristic redpink coloration (Cejas et al ., 2003; Chatzifotis et al ., 2005; Pavlidis et al ., 2006; Tejera et al ., 2007). Another important limitation in farmed red porgy is the high protein requirement (around 50%) for this species. To date, only one study has focused on determining the protein requirement for this species at the on-growing stage, utilizing a high-quality fish meal tested as a sole protein source (Schuchardt et al ., 2008). Due to the global fish meal production situation (Tacon and Metian, 2008) there is a great need for studying alternative protein sources for this species, ideally by-products from different industries. By-products from the crab industry have an attractive potential not only as a protein source, but also as a carotenoid source (Shahidi and Synowiecki, 1991; STUDY I STUDY ISTUDY I STUDY I - 53 - Cremades et al ., 2003). The partial replacement of fishmeal by crab meal from Pleuroncodes planipes and Cancer pagurus in white shrimp ( Litopenaeus vannamei ) and Atlantic cod ( Gadus morhua ) diets, respectively, has yielded successful growth results in both species (Goytortúa-Bores et al ., 2006; Toppe et al ., 2006; Villarreal et al ., 2006). Also, the inclusion of crab meal from P. planipes has been shown to be an effective source of astaxanthin for rainbow trout (CoralHinostroza et al ., 1997; 1998). Regarding freshwater crabs, in Spain the crayfish Procambarus clarkii is an introduced crustacean from North America but nowadays Spain is one of the main producers in the global crayfish market. Factories that process this animal for human consumption produce large quantities of waste products that are utilized as supplementary animal feed ingredients, constituting a good source of protein, astaxanthin and chitin (Lee, 1990; Negro and Garrido-Fernandez, 2000; Cremades et al ., 2003; Pérez-Gálvez et al ., 2008). Together with crustacean species, there are others with great potential such as the marine crab Chaceon affiniss. This species is distributed throughout the Eastern Atlantic Ocean, from Iceland to Senegal and around all the Macaronesian Islands. It is a deep-sea crab caught at depths ranging from 550 to 1200 m by scientific surveys and by fishermen (Pinho et al ., 2001; López-Abellán et al ., 2002). In spite of the stock having economic potential and the presence of some local fisheries, there is no commercial market development of this species for human consumption. Therefore it is an interesting source of protein and carotenoids for fish diets. The aim of the present study is to partially replace dietary fishmeal protein with proteins from crab meal of P. clarkii and C. affiniss species and to evaluate their effect on red porgy growth performance, skin coloration and whole proximate composition. STUDY I STUDY ISTUDY I STUDY I - 54 - 4. 4.4. 4.2. 2. 2. 2. Materials and methods Materials and methodsMaterials and methods Materials and methods 4. 4.4. 4.2.1 2.1 2.1 2.1 Crab meals Crab mealsCrab meals Crab meals Two different crab meals were used for fish meal protein replacement in the diets. The first was a MC meal from the benthonic deep-sea crab, C. affinis, commonly caught close to the Canaries’ coast in Spain. The meal was prepared locally from whole crabs using a process adapted from Sudaryono (1996). First, individuals were autoclaved at high pressure for 15 min, then oven-dried for 12 hours at 55ºC and finally ground in a hammer mill through a 0.5 mm mesh and stored in vacuum plastic bags at 4ºC. The second used in the present trial, river crab meal (RC), was provided by a company located in the South of Spain (Seafood Sevilla S.L.) and obtained by processing the red river crab P. clarkii . The proximate composition and total carotenoid content of both crab meals are shown in Table 1.4 Table Table Table Table 4 44 4.1 .1.1 .1 Proximate composition (% dry wt) and total carotenoid content (mg kg-1) of fish (FM), river (RC) and marine (MC) crab meals used in experimental diets FM FMFM FM RC RCRC RC MC MCMC MC Crude protein 67.29 42.24 45.85 Crude lipids 11.61 6.05 5.85 Ash 15.34 30.69 28.48 Moisture 13.96 8.87 8.17 Carotenoid 3.37 15.74 5.90 4.2.2 4.2.24.2.2 4.2.2 Fish and culture conditions Fish and culture conditions Fish and culture conditions Fish and culture conditions Red porgy with initial mean weight of 233 ± 36 g [(mean ± standard deviation (SD)] were distributed in circular fibreglass tanks, of 500 L, at an initial density of 5.59 kg m-3. Tanks were provided with natural seawater with 17-19 ºC temperature and 7-10 mg L-1 dissolved oxygen range. Fish were carefully fed until apparent satiation 2 times per days (9:00 and 15:00), 6 days per week, with daily feed intake recorded. STUDY I STUDY ISTUDY I STUDY I - 55 - 4.2.3 4.2.34.2.3 4.2.3 Experimental diets Experimental diets Experimental diets Experimental diets Five isocaloric (12%) and isoproteic (50%) diets were formulated (Table 4.2) and tested in triplicate groups of fish for 165 days. A control diet consisted of high quality fish meal and fish oil (CD) and four diets were formulated where 10% and 20% of fish meal protein in CD was replaced by either marine crab meal protein (diets MC10 and MC20) or river crab meal protein (diets RC10 and RC20). Diets were prepared by mixing ingredients in a horizontal ribbon mixer and pelleting them (CPM, CL3 Pellet mill model. USA) through a 5-mmdiameter die. Table Table Table Table 4. 4.4. 4.2 22 2 Ingredients (g kg-1) and proximate analyses of the experimental diets a a a a Fish meal. b b b b River crab Procambarus clarkii meal. c c c c Marine crab Chaceon affinis meal. d d d d Merigel 100 Amylum Group. e e e e Carbohydrate = 100 – protein – lipid – ash. f f f f Gross Energy = (23.6 MJ kg-1 x % protein + 39.8 MJ kg-1 x % lipid + 17.2 MJ kg-1 x % carbohydrate)/100. C C C CD DD D RC10 RC10RC10 RC10 RC20 RC20RC20 RC20 MC10 MC10MC10 MC10 MC20 MC20MC20 MC20 Ingredients FM a aa a 672 608 543 608 543 RC b bb b - 105 215 - - MC c cc c - - - 98 213 Fish oil 70 70 70 71 70 Gelatinized starch d dd d 213 172 127 178 128 Vitamin premix 20 20 20 20 20 Mineral premix 20 20 20 20 20 CMC 0.5 0.5 0.5 0.5 0.5 Proximate composition (% dry wt) Crude protein 46.68 46.75 46.64 46.30 47.08 Crude lipids 11.59 10.68 11.61 11.42 11.53 Ash 11.21 14.61 16.74 14.73 17.32 Carbohydrate e ee e 30.52 27.96 25.01 27.55 24.07 Moisture 8.33 6.61 7.8 6.20 5.83 Gross Energy (MJ kg-1) f ff f 20.73 20.10 19.93 20.21 19.84 Total carotenoid (mg kg1 ) 2.86 3.36 5.56 3.02 3.58 STUDY I STUDY ISTUDY I STUDY I - 62 - 4. 4.4. 4.3.4 Skin carotenoid concentration and TLC analysis 3.4 Skin carotenoid concentration and TLC analysis3.4 Skin carotenoid concentration and TLC analysis 3.4 Skin carotenoid concentration and TLC analysis Skin carotenoid concentration was enhanced by the addition of both crab meals. The highest skin carotenoid concentration was observed in fish fed with RC20, followed by RC10 and MC20 diets, which did not differ from one another, only from fish fed on CD and MC10 diets (Table 4.5). Skin total carotenoid separation carried out by thin-layer chromatography revealed two fractions: a red and a yellow fraction (Table 4.6). Including crab meal in the diets resulted in lower yellow pigment proportions while increasing the proportion compared to the control fish. Red porgy fed on RC10 and RC20 presented a higher percentage of red, hence a lower percentage of the yellow fraction, in comparison to the rest of the treatment groups. Comparing results obtained from cultured individuals against wild red porgy, fish fed with RC meals had values closest to wild red porgy. Table Table Table Table 4. 4.4. 4.6 6 6 6 Red and yellow pigments fractions, separated on TLC silica gel plates, as a percentage of the total pigments (red+yellow) from fish skin carotenoid extracts Red pigment (%) Red pigment (%)Red pigment (%) Red pigment (%) Yellow pigment (%) Yellow pigment (%)Yellow pigment (%) Yellow pigment (%) CD CDCD CD 31.65±6.69 c 68.35±6.97 a RC10 RC10RC10 RC10 48.45±3.66 ab 51.55±4.35 bc RC20 RC20RC20 RC20 54.20±3.66 a 45.80±3.66 c MC10 MC10MC10 MC10 40.53±0.26 cb 59.47±0.26 ab MC20 MC20MC20 MC20 39.33±1.38 cb 60.67±1.38 ab Column means with common letter denote no significant differences. STUDY I STUDY ISTUDY I STUDY I - 63 - 4. 4.4. 4.4 44 4 Discussion Discussion Discussion Discussion 4. 4.4. 4.4.1 Growth performance 4.1 Growth performance 4.1 Growth performance 4.1 Growth performance From the beginning of the experiment all RC and MC meal-based diets were well accepted and utilized by cultured red porgy. This was clearly reflected in growth performance results that were similar to control animals. Nevertheless, fish from MC20 treatment group, presented higher growth performance compared to the rest of the experimental groups. Previous work in smaller-size red porgy reported no improvement in growth parameters by feeding fish with a combination of commercial pellets (Trouw Spain, 16% lipid) and defrosted shrimp, Pleisonika sp. (88% and 12%, respectively), with FCR values around 2 (Cejas et al ., 2003). In earlier work carried out on the same species, Kalinowski et al . (2007) reported a similar SGR (0.42–0.48%) but higher FCR (2.1–2.5) rates when 16% of fish meal was substituted with shrimp shell meal over a longer feeding period (180 days) and smaller initial fish size. Moreover, growth enhancement and better protein utilization measured by PER were also observed. In the present study, the PER values in all experimental groups were statistically similar suggesting that the replacement of fish meal protein by either crab meal did not significantly affect dietary protein utilization. Since the PER value from fish fed on MC diets did not differ from the rest of treatments, the better growth found in MC-fed fish cannot be explained by a better protein utilization from MC meal. The reason for a higher growth in MC-fed fish could be related to the higher feed intake also presented for this group. The increase of feed intake is often in line with a better appetence of the diet but also with a reduction in dietary energy, as a consequence of poorer digestibility in diets containing high ash levels. Therefore the fish compensates reduced dietary energy by eating more. Meals prepared from crustaceans and their by-products are usually high in ash content (about 20% or higher), which adversely affects fish feeds digestibility (NRC, 1993; Robaina et al ., 1997). STUDY I STUDY ISTUDY I STUDY I - 64 - Few studies have presented data on crab meal digestibility in fish, showing species-specific abilities; thus lower crab meal digestibility values have been found in haddock Melanogrammus aeglefinus (82%) (Tibbetts et al ., 2004), and higher in Atlantic cod Gadus morhua (89%) and halibut Hippoglossus hippoglossus (88%) (Tibbetts et al ., 2006). In the latter study, although comparable protein digestibility coefficients were found for the fish meal (92.8%) and crab meal (89.4%) was found in diets for cod, a medium range energy digestibility coefficient for crab meal (82.4%) in comparison to the fish meal (86.4%) was obtained, which could affect fish feed intake. Toppe et al . (2006) also reported a negative correlation between the dry matter digestibility and the dietary ash in an experiment with cod fed dietary inclusion levels of crab meal from to 54 to 176 g kg-1. Moreover, a linear increase in feed intake was found with increasing dietary ash content, resulting in an improvement in growth performance. In the present study, together with MC diets, RC diets also presented high ash content and red porgy fed on these diets did not show higher feed intake; so, from this point of view, the higher feed intake observed for MC meal diets cannot be easily explained by a lower digestibility as a result of high ash content in these diets. It is also important to mention that crustacean shells contain 50–80% chitin, a complex amino polysaccharide [(poly-β-(1→4)-N-acetyl-glucosamine)], which is normally poorly digested. The natural diet of red porgy includes a high proportion of chitin-rich crustaceans (Labropouluo, 1999; Castriota et al ., 2005), thus suggesting, as in Atlantic cod, a high activity of the digestive enzyme chitinase in this species (Danulat and Kausch 1984; Danulat, 1986), supporting the idea that crustacean products are identified as good candidates to replace fish meal in diets for this species (Toppe et al ., 2006). Apart from this, the increase of feed intake due to the greater appetence for marine crab meal should be considered. Prey attraction in fish is mostly a consequence of visual, olfactory and gustatory stimulus that detects soluble STUDY I STUDY ISTUDY I STUDY I - 65 - compounds present in the surrounding environment. These soluble compounds are often low molecular weight substances containing nitrogen (Jones, 1992; Carr et al ., 1996; Yacoob and Browman, 2007). Crustacean meals are used as dietary attractants and stimulants in many aquatic species, due to the presence of certain amino acids that stimulate the reception and palatability (smell and taste) of animal and feeding behaviour (Shimizu et al ., 1990; Smith et al ., 2005; GoytortúaBores et al ., 2006). Therefore, in the present study the higher feed intake observed in CM fed fish is probably related more to the attractant and stimulant power of marine crab meal. Also, CM diets had a lighter colour compared to the other diets and could therefore have affected attractability and feed intake as has been suggested for cod (Toppe et al . 2006). 4. 4.4. 4.4.2 Fish proximate composition 4.2 Fish proximate composition4.2 Fish proximate composition 4.2 Fish proximate composition The addition of both crab meals to the diets significantly reduced the total lipid content in whole fish. This lipid reduction could be due in part to a higher proportion of chitin and chitosan in these diets, which is known to bind lipid in the digestive tract and lower its absorption in humans and rats (Mun et al ., 2006; Zhang et al ., 2008). Although this species should be able to highly digest this complex polysaccharide as mentioned previously, some of them may act in this manner. On the other hand, accumulation of lipids in fish put on the crab meal diet could also be related to a dietary imbalance between saturated and unsaturated fatty acids, as a consequence of the high level of saturated fatty acids normally found in crab meals. In the present work, the proximate composition of the experimental diets was similar, but changes in relative proportions of fatty acids by crab meal inclusion could affect lipid utilization and deposition. The fatty acid analysis of diets and tissues were not determined in the present experiment, so more research is needed to better understand the nutritional value of fatty acids in the crab meals used for the red porgy. Differences in the minerals content of the crab meal diets compared to the control fish meal diet should also be considered regarding growth parameters and body lipid content. Calcium and STUDY I STUDY ISTUDY I STUDY I - 66 - phosphorus content are normally higher in crab meal than fish meal. At the levels of crab meal tested in the present experiment and according to data from Toppe et al . (2006), the final dietary phosphorus content should be higher than that of the control diet. Phosphorus deficiency in fish diets normally results in body fat accumulation, as a result of cellular hypoxia and inhibition of oxidative phosphorylation (Sakamoto and Yone 1980; Sugiura et al ., 2004). Non-polar lipid accumulation in muscle and liver, mainly oleic and palmitic acid, has been found in some species when fed a P-deficient diets (Takeuchi and Nakazoe, 1981). On the contrary, the lower body lipid content in the present experiment could be related to higher dietary phosphorus content in the crab based diets compared to the fish meal diet. Red porgy is a lean fish species, so more research is also needed to elucidate the effect of these changes on fatty acid body lipid content, fish behaviour and fish quality. Moreover, mineral excretion parameters should be considered to finally define the appropriate level of inclusion of these types of ingredients in diets; other aspects such as feed levels and retention of unwanted substances like fluorine, heavy metals and organic pollutants should be considered. 4.3 Skin coloration 4.3 Skin coloration4.3 Skin coloration 4.3 Skin coloration From the different carotenoids tested to return the characteristic skin coloration to cultured red porgy, only dietary astaxanthin has given optimistic results (Cejas et al ., 2003; Chatzifotis et al ., 2005; Kalinowski et al ., 2005; Tejera et al ., 2007), with esterified astaxanthin being better utilized than unesterified astaxanthin by red porgy for skin pigmentation purposes (Tejera et al ., 2007). In several crustacean species, astaxanthin is quantitatively the most prevalent carotenoid, and a major part is often in an esterified form (Matsuno and Hirao, 1989). Therefore several studies have used crustacean meals as a source of astaxanthin in order to overcome the red porgy skin pigmentation problem, given interesting results (Cejas et al ., 2003; Kalinowski et al ., 2005; 2007). In the STUDY I STUDY ISTUDY I STUDY I - 67 - present trial, a positive effect of carotenoids from both crab meals was clearly observed in red porgy skin also. This visual analysis was corroborated with results from colorimetric variables ( a* , b* , hue and chroma). The only skin colour variable not affected positively by crab meals was lightness. Variations of this parameter have often been attributed to different environmental factors such as background colour, light and intensity spectrum and density (Van der Salm et al ., 2006). From the two crab meals used, the RC meal produced better skin colour results, as indicated by both visual and colorimetric evaluation, especially at the highest level of inclusion (RC20). These results were expected since river crab meal and river crab meal based diets presented higher total carotenoid content than marine crab meal and its diets. It is important to mention that results obtained in our laboratory showed a total carotenoid concentration of 15 µg g-1 and 5 µg g-1 for RC meal and MC meal respectively, with a lower concentration of RC meal than reported by other authors due mainly to different batches of raw products and also to processing differences. Together with visual and colorimetric evaluation, skin total carotenoid concentration was also determined and once again red porgy fed on the river crab meal based diets presented the highest concentrations in this tissue. Using the TLC chromatographic technique, two fractions were obtained, red and yellow, tentatively identified as astaxanthin and tunaxanthin respectively. Wild red porgy presents approximately 64% and 36% of red and yellow fractions respectively; therefore the RC20 treatment group presented the closest profile to wild individuals (54 and 46% for the red and yellow fractions respectively). In the study carried out by Tejera et al . (2007), astaxanthin and its esters (ranging from 65 to 84% of total carotenoid) were the major carotenoid present in the skin of the red porgy and tunaxanthin diesters were the second most abundant carotenoid comprising 16 to 35% of the total carotenoid in the astaxanthin-supplemented treatments. In the present study, the low percentage STUDY I STUDY ISTUDY I STUDY I - 68 - of red fraction found in the skin of fish fed on RC10, MC20 and MC10, could be attributed to the lower total carotenoid content present in the respective diets (3 to 3.58 µg g-1) in comparison to the levels tested by Tejera et al . (2007) (ranging from 25 to 50 µg g-1). 4. 4.4. 4.5 55 5 Conclusion Conclusion Conclusion Conclusions ss s In conclusion, the present work clearly shows the good potential of crab meals as a dietary ingredient for red porgy. The partial replacement of fish meal protein by up to 20% from MC meal positively influenced the red porgy growth performance. On the other hand, MC meal at the highest inclusion level tested achieved a more intense red colour than control fish, but RC meal revealed as more efficient pigment source for this species at the levels used in this study. More research is needed to correctly define the appropriate levels of inclusion of these ingredients. STUDY II STUDY IISTUDY II STUDY II - 69 - Marine and freshwater crab meals in diets for red porgy ( Marine and freshwater crab meals in diets for red porgy (Marine and freshwater crab meals in diets for red porgy ( Marine and freshwater crab meals in diets for red porgy ( Pagrus Pagrus Pagrus Pagrus pagrus pagruspagrus pagrus ): ):): ): di didi digestibi gestibigestibi gestibility, lity, lity, lity, ammonia excretion, phosphorous and calcium ammonia excretion, phosphorous and calcium ammonia excretion, phosphorous and calcium ammonia excretion, phosphorous and calcium retention retentionretention retention Abstract AbstractAbstract Abstract Two trials were conducted to evaluate the nutrient digestibility of river crab (RC) ( Procambarus clarkii ) and marine crab (MC) ( Chaceon affinis ) meals in diets for red porgy ( Pagrus pagrus ) and the effect on ammonia-N excretion and P and Ca retention. In trial I, the apparent digestibility coefficient (ADC) of nutrients and energy contents in RC and MC meals was determined. Results showed that, the ADC values for protein, lipids, ash, dry matter and gross energy were significantly higher for MC than RC meal. Both crab meals were efficiently digested despite their high chitin and ash contents. In trial II, red porgy were fed over 6 months with five diets: a control diet (CD) based on high quality fish meal (FM), and four diets where FM protein was replaced at rate of 10% or 20% by protein derived from either RC or MC meals (RC10, RC20, MC10 and MC20). Replacement of the FM protein by RC or MC proteins had no effect both on protein utilization and ammonia-N excretion rates, which ranged from 105 to 119 mg N-NH4+ kg-1 day-1 for all diets. A linear increase was observed in the dietary Ca/P ratio by the increasing of the dietary RC and MC meal inclusion, showing in a reduction in the whole fish body content of P, Ca and ash with interesting similar whole body Ca/P ratio (1.59-1.63) for all treatments. Accordingly, Ca, P and ash retention rates were significantly reduced by increasing the dietary proportion of both crab meals. Moreover, a negative relationship between fish growth and final whole body Ca, P and ash content was obtained. However, no significant differences were observed in total P excretion value (kg P t-1). Keywords: Keywords: Keywords: Keywords: alternative ingredient; crab meal; digestibility; ammonia-N excretion; calcium retention; phosphorus retention; red porgy. STUDY II STUDY IISTUDY II STUDY II - 70 - 5.1 5.15.1 5.1 Introduction IntroductionIntroduction Introduction The red porgy, Pagrus pagrus , is a sparid bottom fish that lives in warm and subtropical waters on both sides of the Atlantic and in the Mediterranean Sea. Its meat quality is highly prized, making it a species of interest to both commercial fisheries and recreational anglers. Unfortunately, owing to prolonged exploitation, some red porgy populations have become overfished (Haimovici, 1998: Vaughan et al ., 2002; Afonso et al ., 2006). However, the positive results obtained in studies on red porgy development under aquaculture conditions (Divanach et al ., 1993; Kentouri et al ., 1994, 1995; Hernández-Cruz et al ., 1999; Cejas et al ., 1999), together with the capacity of red porgy to acquire its characteristic red-pink skin coloration when fed by diets containing carotenoid (Cejas et al ., 2003; Chatzifotis et al. , 2005; Kalinowski et al ., 2005; Pavlidis et al ., 2006; Tejera et al ., 2007), make this species a potential candidate for aquaculture in Mediterranean and Atlantic coastal regions. An important limitation to farmed red porgy is their high dietary protein requirement (around 50%). This, coupled with the low global production of fish meal (FM) (Tacon and Metian, 2008), highlights the need to search for alternative protein sources. In this sense, crabs and crab waste products are a source of protein and pigments (Cremades et al., 2003; Toppe et al ., 2006), with potential use of these products to feed farmed red porgy. A recent study with this species showed that diets including crab meal are highly attractant and palatable promoting better fish growth. In addition, the required skin colour was achieved, and growth rates and feed performance were even better than fish fed a control FM-based diet (Garcia-Romero et al ., Study I). Certainly, crab meals contain chitin and large amount of ash that can adversely affect the digestibility of nutrients (NCR, 1993; Robaina et a l., 1997; Shi-Yen et al ., 1999; Krogdahl et a l., 2005). However, some studies on crab meal digestibility in fish have shown species-specific differences, with digestibility values of 82% in haddock ( Melanogrammus aeglefinus ) (Tibbetts et al ., 2004), 89% STUDY II STUDY IISTUDY II STUDY II - 71 - in cod ( Gadus morhua ) and 88% in Atlantic halibut ( Hippoglossus hippoglossus ) (Tibbetts et al ., 2006). The ability of a species to consume these types of feed with no adverse effect on nutrient digestibility depends on the adequate secretion of gastric acid and appropriate enzyme activity (Wood et al ., 1992; Gutowska et al ., 2004). High chitinase production has been found in fish that feed naturally on crustaceans. The high intake of chitin with such natural diets suggests that chitinolytic enzymes play an important role in digestion (Gutowska et al ., 2004; Fines and Holt, 2010). For cultured species, data about the effects on total ammonia-N (N-NH4+) excretion patterns would be very helpful to validate feed nutrient utilization. The main factors affecting fish total ammonia-N excretion are those that influence the catabolism and deposition (or retention) of protein by fish (Lied and Braaten 1984; Liu et al ., 2009), especially the quantity and quality of protein in the diet. Another crab meal characteristic is the high amount of Ca content, found as mineral way making up the exoskeleton. In fish, bones take up a large proportion of Ca and P through the formation of hydroxyapatite. Both Ca and P have numerous essential biological functions (Lall and Lewis-McCrea, 2007). The dietary Ca requirements of fish depend on the water chemistry, species differences, dietary P levels and the available sources of P. Dietary Ca can, however, inhibit the absorption of other dietary minerals such as P, Mg and Zn (Nakamura, 1982; Hardy and Shearer, 1985; Gatlin and Philips, 1989). The present work was undertaken to determine nitrogen utilization from two different crab meal origin included in diets for red porgy ( Pagrus pagrus ), by measuring postprandial ammonia nitrogen (N-NH4+) excretion patterns. Since crab meals are normally rich in Ca, a further aim was to evaluate the effects of the dietary Ca they provide on whole fish Ca and P contents as well as their retention rates. In a parallel experiment, the apparent digestibility coefficients (ADCs) for nutrients in the two crab meals were determined. STUDY II STUDY IISTUDY II STUDY II - 78 - Table 5. Table 5.Table 5. Table 5.4 44 4 Total digestibility of diets containing fish meal (CD), river crab meal (RCD) and marine crab meal (MCD) used in the trial I. CD CDCD CD RC RCRC RC D DD D MC MCMC MC D DD D Diet ADC (%) Diet ADC (%)Diet ADC (%) Diet ADC (%) 88.09±0.44a 74.04±0.37 b 86.71±0.78 a Column, means ± SD, with different letter denote a significant difference. Table 5. Table 5.Table 5. Table 5.5 5 5 5 Apparent digestibility coefficients (ADCs) of crude protein, crude lipids, ash, dry matter and gross energy in the river crab (RC) and marine crab (MC) meals. ADCs (%) ADCs (%)ADCs (%) ADCs (%) RC RCRC RC MC MCMC MC Protein 70.69 ± 0.23 b 95.21 ± 0.88 a Lipid 75.74 ± 0.88 b 99.74 ± 0.78 a Ash 13.00 ± 3.67 b 27.00 ± 3.54 a Dry matter 69.04 ± 1.37 b 82.75 ± 1.37 a Gross Energy (MJ 74.14 ± 0.86 b 89.73 ± 2.71 a Column, means ± SD, with different letter denote a significant difference. 5. 5.5. 5.3.2 Trial II. 3.2 Trial II.3.2 Trial II. 3.2 Trial II. 5.3.2.1 Biochemical content of the diets The proximate diet composition (Table 5.3) reflected the compositions of the included ingredients. Thus, higher ash values were observed with increasing proportions of any crab meal. Ca levels increased with the dietary ash content, with comparable values for the diets containing RC or MC meal. A slight reduction in the P content of all diets with crab meal respect to the CD was also observed. Thus, the Ca/P ratio increased in RC and MC diets. Table 5.6 shows the crude amino acids profile for the FM, RC meal and MC meal, together with experimental diets. The FM had more essential (EAA) and non-essential amino acids (NEAA) than the RC or MC meals (with similar levels). However, the amino acid profiles of all the crab meal-based diets closely resembled that of the CD. In addition, all diets showed a similar EAA and NEAA profile with a similar ratio of EAA/NEAA, ranging from 0.91 to 0.95. STUDY II STUDY IISTUDY II STUDY II - 79 - Table Table Table Table 5. 5.5. 5.6 66 6 Crude essential (EAA) and non-essential (NEAA) amino acid profile of fish meal (FM), river crab (RC) and marine crab (MC) meals, and the experimental diets (g AA x 100g protein-1) used to fed red porgy in trial II. Meals MealsMeals Meals Diets DietsDiets Diets FM FMFM FM RC RCRC RC MC MCMC MC CD CDCD CD RC10 RC10RC10 RC10 RC20 RC20RC20 RC20 MC10 MC10MC10 MC10 MC20 MC20MC20 MC20 EAA EAAEAA EAA Arginine 5.88 4.43 4.99 2.66 2.60 2.55 2.63 2.59 Histidine 3.46 1.25 1.24 1.57 1.47 1.38 1.47 1.34 Isoleucine 3.79 3.03 2.53 1.71 1.68 1.66 1.66 1.66 Leucine 7.49 5.45 4.69 3.39 3.30 3.23 3.27 3.20 Lysine 9.18 3.93 3.51 4.15 3.93 3.71 3.91 3.65 Methionine 5.05 1.47 1.64 2.29 2.13 1.98 2.14 2.03 Phenylalanine 4.03 3.41 2.77 1.82 1.80 1.78 1.77 1.76 Threonine 4.47 3.05 2.92 2.02 1.96 1.91 1.96 1.93 Tryptophan 1.17 0.76 0.70 0.53 0.52 0.50 0.51 0.48 Valine 4.58 3.39 3.29 2.07 2.02 1.98 2.02 1.99 Sum of EAA 49.12 30.16 28.29 22.21 21.41 20.68 21.34 20.63 NEAA NEAANEAA NEAA Aspartic acid 9.64 7.67 6.19 4.36 4.29 4.22 4.22 4.10 Glutamic acid 14.61 11.25 9.71 6.61 6.48 6.36 6.41 6.20 Alanine 6.78 5.02 3.95 3.06 2.99 2.93 2.95 2.95 Cystine 2.23 2.49 2.31 1.01 1.02 1.04 1.02 1.11 Glycine 6.29 4.45 4.14 2.84 2.77 2.70 2.76 2.70 Proline 4.00 3.05 3.47 1.81 1.77 1.74 1.79 1.77 Serine 4.29 3.08 3.10 1.94 1.89 1.85 1.9 1.84 Tyrosine 3.05 2.70 2.64 1.38 1.37 1.36 1.37 1.39 Hydroxyproline 0.74 0.19 0.14 0.34 0.31 0.29 0.31 0.29 Sum of NEAA 51.63 39.89 35.64 23.35 22.89 22.49 22.73 22.35 EAA/NEAA EAA/NEAA EAA/NEAA EAA/NEAA 0.95 0.77 0.79 0.95 0.93 0.91 0.93 0.92 5. 5.5. 5.3.2.2 3.2.23.2.2 3.2.2 Postprandial ammoni Postprandial ammoni Postprandial ammoni Postprandial ammonia aa a- -- -N excretion patterns N excretion patternsN excretion patterns N excretion patterns and nitrogen retention and nitrogen retentionand nitrogen retention and nitrogen retention The postprandial ammonia-N excretion rates (mg kg1 body weight) showed similar patterns of change in all experimental diet groups (Fig. 5.1). An increasing ammonia-N excretion was detected after feeding, reaching a maximum value at 6 h in all treatments. The inclusion of crab meals at two probed levels had no effect on total ammonia-N excretion (mg kg1 body weight) even when the total excretion rate was expressed as units of N intake percentage. Thus, the total ammonia-N excretion rates (mg kg-1 d-1) obtained were 105, 113, 117, 119 and 112 for the CD, RC10, RC20, MC10 and MC20 diets respectively, while the total ammonia-N excretion per unit of N intake was 14, 16, 15, 13 and 11%. An increase of N retention percentage linked to higher proportions of RC and MC meal was observed (Table 5.7). STUDY II STUDY IISTUDY II STUDY II - 80 - 0 10 20 30 40 50 08:00 08:00-10:00 10:00-12:00 12:00-14:00 14:00-16:00 16:00-20:00 20:00-22:00 Time (hours) CD CR10 CR20 CM10 CM20 Figure 1.5 Figure 1.5Figure 1.5 Figure 1.5 Daily ammonia nitrogen excretion pattern in red porgy after feeding the experimental diets (Means ± SD). Means without letters denote no significant difference between diets at any interval. Table 5. Table 5.Table 5. Table 5.7 77 7 Nitrogen intake (N) and ammonia-N excretion (N-NH4+) of red porgy fed the different diets in trial II. CD CDCD CD RC10 RC10RC10 RC10 RC20 RC20RC20 RC20 MC10 MC10MC10 MC10 MC20 MC20MC20 MC20 N a (mg kg-1 d-1) 780 ± 110 721 ± 180 750 ± 131 979 ± 152 1014 ± 159 N-NH4+ (mg kg-1 d-1)105 ± 12 113 ± 11 117 ± 15 119 ± 14 112 ± 20 N-NH4+/N (%) 14% 16% 15% 13% 11% Nitrogen retention b 21.92±0.19 21.57±1.05 23.22±0.17 22.79±0.86 23.09±0.72 a aa a Total nitrogen intake in the ammonia-N excretion tests of trial I. b bb b Nitrogen retention rate (%)={(Final nutrient content − initial nutrient content) / nutrient intake}x100. Values are means ± standard deviations (n=3) for each treatment. Means with no letters denote a lack of a significant difference. 5. 5.5. 5.3. 3.3. 3.2 22 2.2 .2 .2 .2 Whole body Ca and P content Whole body Ca and P content Whole body Ca and P content Whole body Ca and P content Compared to fish fed control diet, whole body Ca and P contents were reduced in all fish fed RC and MC diets, fish fed CM20 diet showed the lowest values (Table 5.8). The Ca and P and ash contents of the CD fish were the closest to those of wild red porgy. STUDY II STUDY IISTUDY II STUDY II - 81 - Ca retention was high in CD fish but it fell significantly with increasing dietary proportions of crab meals, with the lowest value for the MC20 fish (Table 5.6). All crab meal-based diets also resulted in lower P retention (Table 5.6), with a reduction of around 10% in RC10, RC20 and MC10 compared to CD fish, while the MC20 fish showed a reduction of 24%. The ash content and ash retention values for fish fed any crab meal diets also fell significantly. However, the whole body Ca/P ratio resulted very similar among treatments (1.59-1.63), being those values lower than that showed for wild red porgy (1.85) (Table 5.7). Table Table Table Table 5. 5.5. 5.8 88 8 Whole body calcium (Ca), phosphorus (P) and ash composition (g kg-1), retention rates (%) and phosphorus excretion (PE) of red porgy fed the experimental diets. Wild Wild Wild Wild CD CDCD CD RC10 RC10RC10 RC10 RC20 RC20RC20 RC20 MC10 MC10MC10 MC10 MC20 MC20MC20 MC20 Ash 37 34.63 31.15 27.94 29.68 26.05 Ca 3.99 3.72 3.02 2.76 2.81 2.56 P 2.15 2.27 1.89 1.71 1.75 1.61 Ca/P 1.85 1.63 1.60 1.62 1.60 1.59 Ca R a aa a - 55.19±0.96 a 33.54±2.16 b 22.10±0.73 c 30.52±2.70 b 15.54±0.77 d P R a aa a - 48.87±0.87 a 43.85±2.70 b 44.11±1.47 b 43.94±1.63 b 36.63±1.78 c Ash R a aa a - 44.97±0.66 a 29.57±1.92 b 24.89±0.80 c 30.23±0.83 b 21.43±1.01 c PE b bb b -12.34±1.88 13.12±2.73 12.92±2.48 13.89±2.61 10.71±3.30 Different letters in the same row denote significant differences among groups. a aa a R: retention (%)={(Final nutrient content − initial nutrient content) / nutrient intake} x100. b bb b PE: P excretion (kg t-1)=[{FCR x nutrient in diet (g) – nutrient retained in fish (g)} /production (t)] x1000 5.4 5.45.4 5.4 Discussion Discussion Discussion Discussion 5.4.1 5.4.1 5.4.1 5.4.1 Digestibility DigestibilityDigestibility Digestibility The ADC of protein (ADCP) was high in MC meal (95%) and medium-high in RC meal (71%). These results, especially those for the MC meal, are similar to those reported by other authors. For example, Tibbetts et al . (2006), who worked STUDY II STUDY IISTUDY II STUDY II - 82 - with cod ( Gadus morhua ), reported ADCP values of 96%, 89% and 67% for krill meal (obtined from whole animals), crab by-product meal and shrimp meal. Tibbetts et al . (2004) also reported an ADCP value of 82% in haddock ( Melanogrammus aeglefinus ) fed crab meal, while Peach (2005) give a value of 88%, also fed with crab meal, in halibut ( Hippoglossus hipoglossus ). The lower ADCP value associated with the RC meal might be a consequence of its higher ash content (307 g/kg; 173 g/kg as part of the entire diet) compared to MC meal (284 g/kg; 167 g/kg as part of the entire diet). Ash has a well known negative effect on the assimilation of nutrients (NRC, 1993; Robaina et al ., 1997). However, in other species, dietary ash contents higher than those of this study (184 g/kg), also from crab meal, appeared to have no effect on the ADCP (Toppe et al ., 2006). Origin and processing of meals can also affect the bioavailability of nutrients. Although MC meal was produced from whole crabs, and the RC meal from crab by-products, their amino acid profiles were very similar. Thus, differences in processing would appear to be responsible for differences in digestibility. Many authors indicate that the high-temperature heating of protein ingredients during processing could negatively affect the nutritional value of the final meal, leading to differences in ADCP associated with changes in the chemical or conformational composition of the proteins (Opstevedt et al ., 1984; Anderson et al ., 1993; Oduguwa et al ., 1998; Ljokjel et al ., 2004; Sorensen et al ., 2005). The present MC meal was made from fresh, whole crabs at low temperature, while the RC meal was produced by a high temperature process. The ADC of lipids (ADCL) was higher for MC meal (99%) than RC meal (75%). Crustaceans generally have high contents of unsaturated fatty acids, triglycerides and phospholipids, the later known to be highly digestible by fish (Colombo-Hixon et al ., 2010). Since no differences were seen in the lipid profiles of the diets in trial I (data not shown), the differences in ADCL would once again appear to be related to the processing. STUDY II STUDY IISTUDY II STUDY II - 83 - The ADC of ash (ADCA) differed strongly between CD, RC meal and MC meal of diets used in the trial I (38%, 13% and 27% respectively). In a similar study to the present work, Toppe et al . (2006) observed a significant reduction in the ADCA of crab meal compared to a control where ash content of the meal was increased, however, the ADCL and ADCP values were not affected. The high calcium content of RC and MC meals is largely responsible for their high ash content. That feature may prevent the assimilation of minerals (Nakamura, 1982; Hardy and Shearer, 1985; Galtin and Philips, 1989), explaining why both RC and MC meals had a lower ADCA than CD in trial I. However, the calcium content of both these meals was practically the same; thus, this cannot explain the differences in ADCA (13% and 27% for RC and MC meal respectively). Sorensen et al . (2005), indicate that the bioavailability of minerals on feed is even more sensitive to thermal processing than other nutrients since they may undergo physico-chemical changes. Such changes might have occurred during the processing of RC meal. The ADC of energy (ADCE) was high in MC meal (90%), in agreement with the results reported in cobia ( Rachycentron canadum ) (90%) (Fines et al ., 2010), and somewhat lower for the RC meal (74%) with respect to values reported for crab meal in haddock (83%) and cod (82%) (Tibbets et al ., 2004; 2006), 5.4.2 5.4.2 5.4.2 5.4.2 Postprandial ammonia Postprandial ammoniaPostprandial ammonia Postprandial ammonia- -- -N excretion patterns N excretion patternsN excretion patterns N excretion patterns The partial substitution of FM by crab meal (at rate of 10 or 20%) had no effect on protein utilization as determined by ammonia-N excretion. This was confirmed by the N retention results (Table 5.7) and other indices such as protein efficiency ratio (PER) (García-Romero et al ., 2010; Study I). This good protein utilization is partly explained by the adequate amino acid profile of the diet, which was not altered by the inclusion of the crab meals (Table 5.6). These results agree with those reported by García (2002), who indicated that red porgy fed a diet of FM with 20% substituted for shrimp meal led to no significant changes in STUDY II STUDY IISTUDY II STUDY II - 84 - ammonia-N excretion. However, in the same work, substitution at the rate of 40% did lead to an increase in ammonia-N excretion perhaps indicating an imbalance in the diet’s amino acid profile and/or the interaction of some components when such ingredients are included at higher rates. Sub-optimal levels of dietary energy from non-protein (lipid and carbohydrate) sources are known to increase the catabolism of proteins leading to the excretion of ammonia-N (Cho and Kaushik, 1990). In the present work, although carbohydrate content of the diet fell from 213 g kg-1 in the CD to 128 g kg-1 and 127 g kg-1 in the RC20 and MC20 diets respectively, no effect on ammonia-N excretion was seen. This might be due to the level of dietary lipids was enough to meet energy demands, or an efficient use of carbohydrates from the crab meals. In agreement with Shuchardt et al . (2008), red porgy shows greater capacity to use carbohydrates than lipids. In cobia, strong chitinase activity has been detected in the stomach, allowing them to use ingested chitin as an energy source (Fines and Holt, 2010). 5.4.3 5.4.3 5.4.3 5.4.3 Calcium and phosphorus retention Calcium and phosphorus retentionCalcium and phosphorus retention Calcium and phosphorus retention Ca and P concentrations in whole fish fell with the inclusion of RC and MC in the diet. Most studies report that the differences between P content and its retention in different tissues are associated to dietary P levels and bioavailability of the P source (Buyukates et al ., 2000; Sathoh et al ., 2003). Dietary Ca, however, can interfere with P assimilation (Nakamura, 1982; Davies et al ., 1993; Peñaflorida, 1999; Cheng et al ., 2006). Lee et al . (2010) reported lower available P concentrations in rainbow trout ( Oncorhynchus mykiss ) fed diets based on seafood processing waste-product meals as they have high calcium concentrations. In the present work, the high Ca concentrations of the crab meals could have inhibited the assimilation of P, leading to a reduction in the whole body P content. In return, this lower quantity of available or assimilatable P may have affected the deposition of Ca, thus reducing the whole body content; several authors have STUDY II STUDY IISTUDY II STUDY II - 85 - shown that the amount of available P in the diet limits the deposition of Ca (Magbenka et al ., 2005; Lee et al ., 2009). The last group of authors also showed that, with respect to the transport of dietary Ca in the blood, supplementation with P is more important than supplementation with Ca since fish have the capacity to absorb calcium from the environment. It has been reported that minerals such as Zn influence bone mineralisation by acting as cofactors to enzymes involved in the process (Gómez et al ., 1999; Ye et al ., 2006). When calcium is present in the diet at over 2% it can interfere with the assimilation of Zn and Mg (Hardy and Shearer, 1985; Hossain and Furuichi, 2000; Apines et al ., 2003; Ye et al ., 2006), a fact reflected in the reduction of whole body ash contents. In this sense, the smaller amounts of ash in fish fed the RC and MC meal diets may not only be due to a reduction in whole body Ca and P, but also to a reduction in other minerals as result to the inhibition of assimilation by dietary Ca. In the present work, it would be interesting to metion that although with same dietary level of Ca and P for the RC10 and RC20 respect to the MC10 and MC20, the poorer ash digestibility for the RC, half of that for MC, should indicate a lower Ca and P bioavailability for the RC diets compared to the MC ones. This fact was not however related with the observed Ca and P content in the whole fish, which was some higher for the RC with respect to the MC diets. On the other hand, a negative relationship (R2=-0.44) between the whole body ash content and the SGR (Study I) of the fish for the different treatments was observed. This suggest that, in addition to the anterior described interaction between Ca and P, an import effect of the fish weight increasing rates by feeding the different diets on the final fish ash ca and P content seems to occurr. Contrary, in spite of the similar growth rates showed for CD, RC10 and RC20 feeding fish (Study I), clear differences in body ash, Ca and P exist among them; in this sense the lower contents in RC fish could be due to the lower ash and dry matter digestibility for this meal. STUDY II STUDY IISTUDY II STUDY II - 86 - Although Ca/P ratios appreciably differed between diets, the same ratio (about 1.6) was seen in all fish, irrespective of diet. Thus, red porgy appear to be able to adjust the levels of Ca in their body to maintain a constant Ca/P ratio as occur in other species (Ogino et al ., 1979; Watanabe et al ., 1980; Lee et al ., 2009). However, the values showed in the present work were higher than those reported in other species (Nordrum et al ., 1997; Lee et al ., 2009). Although the P retention declined with an increasing of crab meal proportion in the diet, no significant differences were seen in the estimate of excreted P (loading phosphorus). Nevertheless, the values obtained are slightly higher than those estimated for rainbow trout (Satoh et al ., 2003), as well as those found in red sea bream ( Pagrus major ) (Sarker et al ., 2007). Fish meal is the usual source of phosphorus, but it is reported to be poorly assimilatable; therefore, it is excreted (Watanabe et al ., 1980). The present results suggest that the amounts and type of available P required by red porgy be further investigated. 5. 5.5. 5.5 55 5 Conclusion ConclusionConclusion Conclusion The present results show that the tested crab meals are suitable for inclusion in the diet of farmed red porgy. The partial substitution of FM protein by up to 20% with protein from RC or MC meal did no affect dietary protein utilization. The RC meal was readily digestible but the MC meal even more so, being related this difference to the meals processing. The Ca content of the crab meals appeared to reduce whole body Ca and P contents. However, compared to the control, no significant differences were seen in terms of estimated total P excretion (kg P t-1) by feeding with crab meals. STUDY III STUDY IIISTUDY III STUDY III - 87 - Marine and freshwater crab meals in diets for red porgy ( Marine and freshwater crab meals in diets for red porgy (Marine and freshwater crab meals in diets for red porgy ( Marine and freshwater crab meals in diets for red porgy ( Pagrus Pagrus Pagrus Pagrus pagrus pagruspagrus pagrus ): effect in fillet fatty acid profile and flesh quality parameters. ): effect in fillet fatty acid profile and flesh quality parameters.): effect in fillet fatty acid profile and flesh quality parameters. ): effect in fillet fatty acid profile and flesh quality parameters. Abstract AbstractAbstract Abstract The present study was conducted to evaluate the effect of using river crab ( Procamburus clarkii ) meal (RC) and marine crab ( Chaceon affinis ) meal (MC) on the red porgy ( Pagrus pagrus ) fillet fatty acid profile and quality. Red porgy were fed during 165 days with five diets: a control diet based on high-quality fish meal (CD); four diets replacing 10% and 20% fish meal protein by protein from both RC or MC meals (RC10, RC20 & MC10, MC20). Fillet fat content resulted significantly higher in MC20 fish than the rest of diets. Feeding with both RC and MC meals slightly increased muscle contents of polyunsaturated (PUFA), 20:4n-6 (ARA), 20:5n-3 (EPA), 22:6n-3 (DHA) and n-3/n-6 ratio as well as decrease of n-9 fatty acids, although difference are only significant for the ARA content. After inclusion of both crab meals, values of atherogenicity index (AI) resulted similar among treatments while thrombogenecity index (TI) were reduced, but not significantly, reflecting the higher n-3 fatty acid content in fillets from crab diets. Sensory analysis showed that red porgy fillets of all treatments were very well appreciated with some differences detected in only MC20 fillets, which had significantly higher scores in the attributes of oily odour and flavour, cohesiveness aspect and firmness texture compared to the rest of diets. RC meal dietary inclusion did not promote any changes in sensory evaluation panel. Instrumental muscle texture did not revealed differences between treatments. Results from TBARS of raw fillets showed that the inclusion of both crab meals clearly delay lipid oxidation compared to a FM based diet, during refrigerated storage at 4ºC. Thus, for 4 and 7 days of storage, fillets of RC or MC diets, attained significantly lower TBARS values than those fed on CD. Keywords: Keywords: Keywords: Keywords: alternative ingredients; crab meal; flesh quality; lipid oxidation; red porgy. STUDY III STUDY IIISTUDY III STUDY III - 94 - using a flat-ended (Type P/100) aluminium compression plate with a test speed 0.8 mm/s and a strain to 80%. During the test, seven mechanical texture parameters were calculated: hardness (N), fracturability (N), adhesiveness (J), springiness (m), cohesiveness, chewiness (J) and gumminess (N). 6. 6.6. 6.2.6 2.62.6 2.6 Determination of Determination ofDetermination of Determination of lipid oxidation in fish fillet lipid oxidation in fish fillet lipid oxidation in fish fillet lipid oxidation in fish fillet To determine the oxidative stability of fish fillet during refrigerated storage, nine fish from each treatment were collected, filleted and kept at 4ºC until analysis. Oxidative changes in samples were monitored at 0, 4 and 7 days of storage. The index of lipid oxidation was determined as thiobarbituric acid reactive substances (TBARS) (Shaidi and Hong, 1991). Fish sample (3 g) was mixed with 6 ml of 10% trichloroacetic acid (w/v) and homogenized for 60 s and centrifuged at 4,000 rpm at 4ºC for 30 minutes. After filtration, 2 ml of the filtrate were added to 2 ml of 0.02 M TBA solution and heated at 100ºC for 1 hour. Then the absorbance was measured at 532 nm by UV/Vis spectrophotometer (Genesys UV10, Thermo Fisher Scientific Inc. Waltham, USA). TBARS value was expressed as mg of malonaldehyde (MDA) per kg of fillet. 6. 6.6. 6.2.7 Statistical analysis 2.7 Statistical analysis2.7 Statistical analysis 2.7 Statistical analysis One-way analysis of variance (ANOVA) was conducted for the all data. The homogeneity of variance was tested using Levene’s test and the normal distribution of data was checked (Sokal and Rohlf, 1995). When the differences were significant at the P < 0.05 level, Tukey’s range test was utilized to compare the mean values among the treatments due to the main effects. For data not displaying normality and/or homogeneity of variance, a non-parametric analysis and multiple range test (Kruskal-Wallis) were used. In case of the thiobarbituric acid-reactive substances data, a general linear model with two fixed factors, time and dietary level inclusion, was used. All statistical analyses were tested at 0.05 significance level using the SPSS (13.1) statistical package. STUDY III STUDY IIISTUDY III STUDY III - 95 - 6.3 6.36.3 6.3 Results Results Results Results 6. 6.6. 6.3.1 Fatty acid composition of the lipid fraction in crabs meals and experimental 3.1 Fatty acid composition of the lipid fraction in crabs meals and experimental 3.1 Fatty acid composition of the lipid fraction in crabs meals and experimental 3.1 Fatty acid composition of the lipid fraction in crabs meals and experimental diets dietsdiets diets Some differences were observed on fatty acid profile (g/100 g fatty acids) in two different crab meals (Table 6.2). Thus, higher proportion of saturated fatty acids (SFA) (22.76%) and lower of monounsaturated fatty acids (MUFA) (34.11%) were obtained for the CM respect to the RC (14.98% and 46.89%, for SFA and MUFA respectively). Similarly, MC contained higher concentrations of total PUFA (polyunsaturated fatty acids), n-3, n-3 HUFA (highly unsaturated fatty acids) and DHA (docosahexaenoic acid; 22:6n-3) (42.87%, 27.18%, 25.52% and 13.47%, respectively), compared to those for RC meal (37.21%, 21.59%, 18.40% and 9.05%, respectively). On the other hand, RC meal showed the higher concentrations of total n-9 fatty acid (29.14%) and linolenic acid (0.91%) s, respect to those for MC meal (20.81% and 0.34%, respectively). ARA (arachidonic acid; 20:4n-6) fatty acid content was similar for both crab meals but 4.5 fold higher than the observed in the fish meal. According to total PUFA content, MC meal profile resulted closer to that of FM. Meanwhile, fatty acid analyses of experimental diets showed similar profile without significant differences among diets (Table 6.2). The main dietary fatty acids were 16:00, 18:1n-9, EPA (eicosapentaenoic acid; 20:5n-3) and DHA. Compared to the control diet, a general increase in the ARA fatty acid content with increase the dietary inclusion of both crab meals were detected, being 48% and 66% higher for RC20 and MC20, respectively. 6. 6.6. 6.3 33 3.2 Proximate fillet composition .2 Proximate fillet composition.2 Proximate fillet composition .2 Proximate fillet composition The proximate composition of fish muscle at the end of the trial is shown in Table 6.3. Results were very similar for protein, ash and moisture content among fish fed with diets containing both crab meals at the two tested levels, and the fish fed with the control diet. Significantly higher lipid content (4.73%) was obtained STUDY III STUDY IIISTUDY III STUDY III - 96 - for the MC20 feeding fish compared with the other treatments (between 3.67% and 3.80%). Table Table Table Table 6. 6.6. 6.3 3 3 3 Muscle proximate composition (%wet wt) of red porgy fed the experimental diets. Different letters in same row denote significant differences among diets (P<0.05). 6. 6.6. 6.3. 3.3. 3.3 Fillet fatty acid composition 3 Fillet fatty acid composition3 Fillet fatty acid composition 3 Fillet fatty acid composition The fatty acid composition (g/100 g fatty acids) of fish fillet at the end of the trial is shown in Table 6.4. Very similar SFA and MUFA content were obtained in all treatments. Replacing FM with both RC and MC meals slightly increased PUFA, n-3, n-3 HUFA, ARA, EPA and DHA as well as decrease n-9, especially oleic acid (18:1n-9). Only ARA content was found significantly different with the lowest value for CD feeding fish (0.86%). Consequently, dietary inclusion of crab meals promote higher fillet overall n-3/n-6 ratio although the differences were not significant yet ( P <0.05). AI index resulted similar between different treatments while TI index showed a trend to reduce with inclusion crab meals (Table 6.4). Fatty acid content was also calculed as percentage of wet tissue (Table 6.5). The results were determined by the different fillet fat content, in fact, the increment of total lipid in fish fed MC20 diet promote differences in the correspondent fatty acid content. Thus, significantly higher content of MUFA, PUFA, n-3, n-3-HUFA, ARA and DHA, (25%, 40%, 47%, 50%, 100% and 40% higher, respectively) respect to fish fed CD diet, were obtained when fish was feeding with CM20 diet. CD CDCD CD RC10 RC10RC10 RC10 RC20 RC20RC20 RC20 MC10 MC10MC10 MC10 MC20 MC20MC20 MC20 Protein 23.00±0.13 22.88±0.18 22.91±0.11 23.05±0.13 22.91±0.22 Lipid 3.67±0.39 b 3.73±0.55 b 3.77±0.24 b 3.80±0.65 b 4.73±0.30 a Ash 1.52±0.03 1.54±0.05 1.59±0.06 1.59±0.05 1.58±0.06 Moisture 73.58±1.16 73.89±1.16 73.33±0.23 73.44±1.02 72.26±1.92 STUDY III STUDY IIISTUDY III STUDY III - 97 - Table Table Table Table 6. 6.6. 6.4 44 4 Muscle fatty acid profile (g/100 g fatty acids) of red porgy fed the experimental diets a aa aAI, atherogenic index; TI, thrombogenecity index. * Letter by rows denotes significant difference between treatments ( P <0.05, ANOVA ) Fatty acids Fatty acidsFatty acids Fatty acids CD CDCD CD RC10 RC10RC10 RC10 RC20 RC20RC20 RC20 CM10 CM10CM10 CM10 CM20 CM20CM20 CM20 14:00 4.63 4.84 4.80 4.86 4.78 15:00 0.88 0.76 0.90 0.97 0.78 16:00 22.40 21.93 22.14 22.57 21.59 17:00 0.87 0.96 0.98 0.94 0.98 18:00 6.97 6.55 6.90 6.95 6.40 20:00 0.23 0.23 0.25 0.22 0.22 ∑ ∑∑ ∑SFA SFASFA SFA 35.98 35.27 35.97 36.51 34.74 14:1n - 7 0.07 0.08 0.08 0.08 0.08 14:1n-5 0.15 0.17 0.19 0.17 0.15 15:1n-5 0.08 0.08 0.10 0.09 0.09 16:1n-5 0.21 0.23 0.25 0.23 0.24 16:1n-7 7.52 7.77 7.76 7.72 7.75 18:1n-5 0.11 0.11 0.11 0.11 0.11 18:1n-7 3.31 3.33 3.37 3.36 3.34 18:1n-9 18.32 16.91 16.39 16.66 16.74 20:1n-5 n.d 0.19 0.20 0.19 0.19 20:1n-7 0.75 0.65 0.49 0.80 0.71 20:1n-9 0.70 0.60 1.00 0.75 0.70 22:1n-9 n.d 0.24 0.24 0.23 0.23 22:1n-11 0.67 0.66 0.62 0.62 0.60 ∑ ∑∑ ∑MUFA MUFAMUFA MUFA 31.89 31.02 30.80 31.01 30.93 16:2n - 6 0.24 0.22 0.12 0.26 0.27 16:2n-4 0.86 0.92 0.92 0.93 0.91 16:3n-4 n.d 0.03 0.04 0.01 0.04 16:3n-3 0.13 0.13 0.12 0.14 0.14 16:3n-1 0.20 0.19 0.23 0.22 0.21 16:4n-3 0.61 0.66 0.66 0.63 0.65 16:4n-1 0.06 0.06 0.08 0.07 0.06 18:2n-9 0.24 0.21 0.15 0.21 0.25 18:2n-6 6.97 6.24 5.70 5.48 6.07 18:2n-4 0.29 0.32 0.31 0.31 0.31 18:3n-6 0.27 0.28 0.28 0.27 0.29 18:3n-4 0.27 0.29 0.30 0.31 0.31 18:3n-3 0.93 0.87 0.84 0.79 0.87 18:4n-3 0.72 0.79 0.77 0.74 0.81 18:4n-1 0.15 0.16 0.15 0.15 0.18 20:2n-9 0.21 0.19 0.17 0.20 0.23 20:2n-6 0.20 0.20 0.21 0.20 0.21 20:3n-6 0.19 0.19 0.20 0.20 0.21 20:3n-3 0.06 0.07 0.07 0.06 0.07 20:4n-6 0.86b 1.11 b 1.32 a 1.27 a 1.32 a 20:4n-3 0.59 0.64 0.62 0.63 0.68 20:5n-3 5.51 6.16 6.16 5.96 6.20 22:4n-6 0.28 0.31 0.30 0.29 0.32 22:5n-6 0.30 0.35 0.37 0.36 0.36 22:5n-3 1.98 1.91 1.92 1.86 2.07 22:6n-3 10.14 11.04 11.12 10.79 11.27 ∑ ∑∑ ∑PUFA PUFAPUFA PUFA 31.76 33.54 33.13 32.34 34.31 ∑ ∑∑ ∑ n nn n - -- - 3 33 3 20.68 23.56 22.29 21.35 22.76 ∑ ∑∑ ∑n nn n- -- -6 66 6 8.45 8.72 8.20 7.84 8.76 ∑ ∑∑ ∑n nn n- -- -9 99 9 19.03 16.59 16.95 17.31 17.44 ∑ ∑∑ ∑n nn n- -- -3 HUFA 3 HUFA3 HUFA 3 HUFA 18.28 21.11 19.90 19.08 20.29 n nn n- -- -3/n 3/n3/n 3/n- -- -6 66 62.44 2.71 2.72 2.72 2.62 ARA/EPA ARA/EPAARA/EPA ARA/EPA 0.15 0.17 0.21 0.21 0.21 EPA/DHA EPA/DHAEPA/DHA EPA/DHA 0.54 0.52 0.56 0.56 0.56 A AA AI II I a a a a 0.67 0.65 0.67 0.68 0.65 T TT TI II I a a a a 0.40 0.35 0.37 0.38 0.36 STUDY III STUDY IIISTUDY III STUDY III - 98 - Table Table Table Table 6. 6.6. 6.5 5 5 5 Muscle fatty acid profile (g / 100 g wet fillet) of red porgy fed the experimental diets. Fatty acid Fatty acidFatty acid Fatty acid s ss s CD CDCD CD RC10 RC10RC10 RC10 RC20 RC20RC20 RC20 MC10 MC10MC10 MC10 MC20 MC20MC20 MC20 ∑SFA 1.31 1.39 1.24 1.15 1.65 ∑MUFA 1.17 b 1.12 b 1.06 b 1.07 b 1.47 a ∑PUFA 1.15 b 1.32 b 1.14 b 1.02 b 1.61 a ∑n-3 0.73 b 0.87 b 0.77 b 0.70 b 1.07 a ∑n-6 0.34 0.35 0.28 0.26 0.41 ∑n-9 0.70 0.65 0.58 0.56 0.82 ∑n-3 HUFA 0.64 b 0.78 b 0.69 b 0.60 b 0.95 a ARA 0.03 b 0.04 b 0.04 b 0.04 b 0.06 a DHA 0.38 b 0.43 b 0.38 b 0.35 b 0.53 b EPA 0.20 0.24 0.21 0.20 0.29 Linolenic 0.03 0.03 0.03 0.02 0.04 Linoleic 0.25 0.24 0.20 0.18 0.28 Oleic 0.67 0.65 0.56 0.54 0.79 Letter by rows denotes significant difference between treatments. 6. 6.6. 6.3.4 Sensory analysis of fillets 3.4 Sensory analysis of fillets3.4 Sensory analysis of fillets 3.4 Sensory analysis of fillets The results of sensory evaluation of cooked red porgy fillets are presented in Table 6.6. Fillets were very well appreciated by the judges, independently of the experimental diets. All differences found in values provided by the sensory analyses were for fish fed on MC meal supplemented diets, specially at the highest level (MC20), whereas the dietary inclusion of RC meal, both RC10 and RC20 level, did no showed appreciable changes respect to CD group. Thus, fish fed on MC meal seemed to produce an oilier perception with higher oily odour and flavour scores, being significantly higher in MC20 fillets than fillets of fish fed CD or the rest of treatments. Also, fillets from fish fed on MC meal based diets appeared to have more cohesiveness aspect, with only significant difference for MC20 diet. Regarding to texture attributes, flesh firmness by feeding on MC meal was significantly higher than those of fish fed CD, RC10 and RC20 diet, and once again different for the MC20 diet. No significant differences were observed in the rest of the assayed attributes. STUDY III STUDY IIISTUDY III STUDY III - 99 - Table Table Table Table 6. 6.6. 6.6 66 6 Sensory analysis of cooked fillets from red porgy fed the experimental diets. Means whit different letter denote significant differences ( P <0.05) 6. 6.6. 6.3.5 Texture parameters 3.5 Texture parameters3.5 Texture parameters 3.5 Texture parameters Data on texture parameters assayed on raw fillets of red porgy fed the different diets are showed in Table 6.7. Dietary inclusion of both crab meals at the two probed levels, 10% and 20%, had no significant effect on any of the seven texture parameters analysed Table Table Table Table 6. 6.6. 6.7 7 7 7 Texture parameters of raw fillets from red porgy fed the experimental diets Experimental Diets Experimental DietsExperimental Diets Experimental Diets Attributes AttributesAttributes Attributes CD CDCD CD RC10 RC10RC10 RC10 RC20 RC20RC20 RC20 MC10 MC10MC10 MC10 MC20 MC20MC20 MC20 ANOVA ANOVAANOVA ANOVA Marine 46 46 41 47 46 p = 0.97 Oily 5 b 5 b 4 b 6 ab 8 a p = 0.03 Odour OdourOdour Odour Atypical 3 3 3 3 3 p = 0.87 Whiteness 53 55 54 53 49 p = 0.86 Shine 45 50 38 44 42 p = 0.54 Aspect AspectAspect Aspect Cohesivenes 40 b 41 b 39 b 44 b 54 a p = 0.03 Juiciness 39 43 41 42 42 p = 0.76 Firmness 30 b 39 b 34 b 45 ab 51 a p = 0.00 Texture TextureTexture Texture Adhesivenes 39 35 32 39 35 p = 0.76 Marine 44 45 40 45 40 p = 0.74 Oily 4 b 4 b 4 b 5 ab 8 a p = 0.04 Flavour FlavourFlavour Flavour Atypical 3 2 4 4 4 p = 0.53 Persistance 36 41 42 38 35 p = 0.85 Marine 33 43 35 41 38 p = 0.19 Residual Residual Residual Residual flavour flavourflavour flavour Earthy 3 3 3 3 3 p = 0.98 Acceptance AcceptanceAcceptance Acceptance 73 74 73 75 73 p = 0.78 CD CDCD CD RC10 RC10RC10 RC10 RC20 RC20RC20 RC20 MC10 MC10MC10 MC10 MC20 MC20MC20 MC20 Fracture (N) 1.87±0.61 1.90±0.48 1.60±0.38 1.82±0.44 1.79±0.54 Hardness (N) 2.80±0.76 3.07±0.67 2.95±0.75 3.09±0.64 3.00±0.56 Springiness 0.43±0.08 0.42±0.07 0.44±0.08 0.42±0.05 0.47±0.09 Cohesiveness 3.74±0.92 4.29±0.42 4.04±0.40 4.33±0.56 4.07±0.91 Gumminess 11.43±2.98 14.60±2.69 12.54±2.78 12.98±2.65 12.42±2.58 Chewiness (N) 5.01±0.94 6.43±0.93 4.29±0.88 5.72±0.90 5.48±0.82 Adhesiveness -0.03±0.01 -0.03±0.01 -0.03±0.09 -0.03±0.01 -0.03±0.01 STUDY III STUDY IIISTUDY III STUDY III - 100 - 6. 6.6. 6.3.6 Lipid oxidation of red porgy fill 3.6 Lipid oxidation of red porgy fill3.6 Lipid oxidation of red porgy fill 3.6 Lipid oxidation of red porgy fillets etsets ets The effect of dietary treatment on muscle lipid oxidation, measured as changes in TBARS values at 0, 4 and 7 days of storage after slaughtering is illustrated in Figure 6.1. A significant dietary effect on lipid oxidation was observed ( P <0.00) in addition to a significant interaction effect of time and dietary treatments ( P <0.00). No differences in TBARS values between diets were observed at the moment of slaughter (0 days). However, at days 4 and 7 of storage, fish giving diets containing both RC meal and MC meal, at the two inclusion levels, attained significantly lower TBARS values in comparison with red porgy fed with fish meal based diet (CD). A trend to reduce TBARS values by increasing the dietary crab meals was observed. The lowest TBARS value, at days 4 and 7, was observed in fish fed with RC20, followed by MC20, RC10 and MC10 diets, which did not differ from one to another (Fig. 6.1). 0,00 0,04 0,08 0,12 0,16 0 4 7 Days of storage MDA (mg) CD RC10 RC20 MC10 MC20 a c b c bb a b bb 0,00 0,04 0,08 0,12 0,16 0 4 7 Days of storage MDA (mg) CD RC10 RC20 MC10 MC20 a c b c bb a b bb Figure Figure Figure Figure 6. 6.6. 6.1 11 1 Evolution of fillet lipid oxidation during refrigerated storage of fillets from red porgy fed the experimental diets. STUDY III STUDY IIISTUDY III STUDY III - 101 - 6.4 6.46.4 6.4 Discussion Discussion Discussion Discussion Data of fillet chemical composition were in the range (lipids slightly higher) of results reported by Robaina et al . (2005) and Schuchard et al . (2008) for farmed red porgy of 400 g body weight. According to the nutritional classification cited by Nunes et al . (2003), based on the lipid and protein content of 27 consumed fish species, results obtained in the present study indicate that the red porgy even farmed is considered a lean fish specie (lipid < 5%; protein >20% wet weight), showing an excellent protein source with low fat content. Different results are reported in the literature regarding the effect of crustacean meal on muscle composition in reared red porgy and other species. In a previous work, Schuchard (2005), reported an increase of 13% in muscle lipid content in red porgy fed diet with 20% of krill meal. Opposite results were showed by Chebbaki (2001), with a decrease in muscle fat in red porgy fed krill meal. However, no effect on total lipid was observed on Atlantic salmon ( Salmo salar ) and Atlantic halibut ( Hippoglossua hipoglossus. ) fed diets containing different crustacean meal levels (Olsen et al ., 2006; Suontama et al ., 2007). In the present study, the significant higher fat muscle content of fish fed with MC20 diet could be more related to their higher feed intake and weight (see García-Romero et al . Study I), since lipid tissue content in fish tend to increase with these two factors (Martínez et al ., 1992; Shearer, 1994; Ruedas et al ., 1997). As in most fish, the fillet fatty acid composition reflected that of diet (Madsen et al ., 2000; Bell et al ., 2002; Glencross et al ., 2003). Nevertheless, in all treatments, some fillet fatty acids are presented in different proportion relative to diets. For instance, 18:1n-9 fillet content was higher (40-50%) regardless of dietary contents, which was also reported in gilthead sea bream (Arantzamendi, 1999). The level of the essential fatty acid EPA was reduced (40-50%) in muscle compared to diet, whereas DHA concentration was maintained near or slightly above diet value. This fact denote the preference of red porgy to utilize EPA for energy purposes while a selective deposition of DHA, as previously observed both STUDY III STUDY IIISTUDY III STUDY III - 102 - in fatter muscle species (Froyland et al ., 2000; Izquierdo et al ., 2003) and lean fish (Kennedy et al ., 2007; Valente et al ., 2011). Regarding to differences between treatments, total SFA and MUFA muscle content appears to be unaffected by the dietary inclusion of the two different crab meals. The main fatty acid detected in SFA and MUFA were 16:00 (palmitic acid) and 18:1n-9 (oleic acid) respectively, common in other farmed marine fish (Krajnovic-Ozteric et al ., 1994; Glencross et al ., 2003; Izquierdo et al ., 2005; Grigorakis et al ., 2011). On the contrary, feeding red porgy with both RC and MC meal diets, promoted an increase of PUFA, n-3, n-3HUFA, ARA, EPA and DHA along with a lower n-9 fatty acid (mainly 18:1n-9) when compared to CD feeding fish. Yet, only the ARA content resulted statistically higher (P<0.005). Presumably, the level of ARA in muscle showed a linear increase with inclusion level of crab meals in agreement with the ARA meal value (Tables 6.2 and 6.4). Higher ARA content has been found in wild fish respect to farmed one, included red porgy, due to the fed lipids from marine organisms (Ohshima et al ., 1983; Ruedas et al ., 1997; Izquierdo et al ., 2005). Furthermore, the increment of total lipid in CM20 fillets induce to higher level of some fatty acids, included essential fatty acids, even further improving the nutritional value of red porgy flesh for human consumption. Regarding to lipid quality, good values of atherogenicity (AI) and thrombogenicity (TI) indices (Ulbricht and Southgate, 1991; Sidhu, 2003; Ruxton et al ., 2004; Seierstad et al ., 2005) were obtained in all fillets (Table 6.4). In addition, the increase of n-3 fatty acids, and hence the n-3/n-6 ratio, found in fish fed by crab meals diets caused an improvement of TI index. Obtained values in present work for AI and TI are slightly higher and comparable to those reported for wild and farmed red porgy respectively (Rueda et al ., 1997; Miniadis-Meimaroglou et al ., 2007). Regarding to results of sensory evaluation, some differences were showed in CM10 and CM20 fillets. However, no effect on the final fish acceptance was determined with high scores for all treatments (Table 6.6). The differences were STUDY III STUDY IIISTUDY III STUDY III - 103 - found in oily odour and flavour, being CM20 fillets statistically higher. These oily changes were no related with other characteristics as marine o atypical flavour intensity, and would be more related to the higher fat content in these fishes. In other species such as farmed southern flounder ( Paralichthys lethostigma ), cooked flesh acquired a less fish flavour intensity when fed a 5% crab meal supplemented diet (González et al ., 2006). The results for other attributes are however quite not consistent with the above explanations mentioned for MC20 oily differences. Thus, although oily fillets are normally associated to lower fillet firmness (Lie et al ., 2001), in present case higher values for the firmness and cohesiveness were obtained for the MC20 feeding. In other lean farmed species such as cod ( Gadus morhua ), Karsel et al . (2006), showed that firmness texture of cooked flesh increased by increasing krill meal inclusion level. On the other hand, the instrumental texture measurements, performed in raw red porgy fillet, were unaffected by the inclusion of the two different crab meals in diet. This fact is in accordance with those reported by González et al . (2006) in southern flounder fed on crab meal and Karlsen et al . (2006) in cod fed on graded levels of krill (0-100%). Data of texture were similar to results obtained in the same species in a previous work (Robaina et al ., 2005), while texture parameters of cohesiveness, gumminess and chewiness were higher compared to gilthead sea bream ( Sparus auratus ) (Castro, 2010), both studies performed under the same fish sized and instrumental conditions. The data reported about malonaldehyde (MDA) content in red porgy fillets ranged from 0.024-0.034 mg kg-1, at slaughtering day, to 0.054-0.124 mg kg-1 at seventh day of refrigerated (4ºC) storage. Those values were lower than those observed in other marine commonly farmed fish species. Thus, both in European sea bass ( Dicentrachus labrax ) of 250 g (Poli et al ., 2001) and in gilthead sea bream of similar size at the present study (Castro, 2010), muscle MDA content increased from 0.04 to 0.115 mg kg-1 and from 0.160 to 0.260 mg kg-1 respectively, at seventh day of refrigerated (4ºC) storage. STUDY IV STUDY IVSTUDY IV STUDY IV - 110 - Aside from promote fish growth enhancement, a new ingredient is able as suitable ingredient if their inclusion not negatively affect the final product quality as well as environment pollution. Likewise, the present study is focused on evaluate two natural ingredients from marine origin crab meal of P. cuvierri and sea urchin meal of D. antillarum as suitable ingredients for red porgy diet through an overall evaluation about the effect on fish growth performance, skin coloration, ammonia-N excretion, flesh quality and lipid flesh oxidation during refrigerated storage. 7.2 7.27.2 7.2 Materials and methods Materials and methods Materials and methods Materials and methods 7.2.1 7.2.17.2.1 7.2.1 Marine crab Marine crab Marine crab Marine crab and sea urchin meals elaboration and sea urchin meals elaboration and sea urchin meals elaboration and sea urchin meals elaboration Marine crab meal (SMC) was prepared from whole individuals of Paramola cuvieri , which were captured as by-catch in local crab fisheries in the Canaries’ Coast, Spain. While sea urchin meal (SU), was obtained by processing D. antillarum specimens also caught in sea bottom of Canaries’ coast. All animals were provided by fishermen’s, being meals obtained at the laboratory using an adapted process from Sudaryono et al . (1996). First, individuals were autoclaved at high pressure for 15 min, then cold down quickly before oven-dried at 55ºC for 12 hours and finally ground in a hammer mill through a 0.5 mm mesh and stored in vacuum plastic bags at 4ºC. Results for proximate composition, total carotenoid content and fatty acid profile of meals are shown in Tables 7.1 and 7.3. 7.2.2 7.2.27.2.2 7.2.2 Experimental diets Experimental diets Experimental diets Experimental diets Five isocaloric (13%) and isoproteic (48%) diets were formulated (Table 7.2) and tested in triplicate groups of red porgy. The control diet (CD) consisted of Peruvian high quality fish meal and fish oil. Two diets were formulated by replacing 10% and 20% of fish meal protein in CD with SMC meal protein (diets SMC10 and SMC20). Another two diets were formulated by inclusion 8% and 16% of the SU meal (diets SU8 and SU16). All diets were prepared by mixing the ingredients in a horizontal ribbon mixer before pelleting in a California Pellet Mill STUDY IV STUDY IVSTUDY IV STUDY IV - 111 - (CL3 Pellet mill, USA) through out a 5 mm matrix diameter. The chemical composition of diets and fatty acid profile are reported in Tables 7.2 and 7.3. Table 7.1 Table 7.1Table 7.1 Table 7.1 Proximate composition (% dry wt) and total carotenoid content (mg kg-1) of the fish meal (FM), spider marine crab meal (SMC) and sea urchin meal (SU) used in the experimental diets a a a a Spider marine crab Paramola cuvieri meal b b b b Sea Urchin Diadema atillarum meal Table Table Table Table 7. 7.7. 7.2 22 2 Ingredients (g kg-1) and proximate analysis of the experimental diets a a a a Fish meal (Peruvian origin) b b b b Spider marine crab, Paramola cuvieri , meal c c c c Sea Urchin, Diadema Atillarum , meal d d d d Merigel 100 Amylum Group e e e e Carbohydrate = 100 – (protein + lipid + ash) f f f f Gross Energy = (23.6 MJ kg-1 x % protein + 39.8 MJ kg-1 x % lipid + 17.2 MJ kg-1 x % carbohydrate)/100 FM FMFM FM SMC SMCSMC SMC a a a a SU SUSU SU b b b b Crude 68.12 37.59 15.83 Crude 10.11 5.11 5.87 Ash 15.34 31.98 48.13 Moisture 6.81 5.77 3.17 Carotenoid 3.28 11.96 6.50 CD CDCD CD SMC10 SMC10SMC10 SMC10 SMC20 SMC20SMC20 SMC20 SU8 SU8SU8 SU8 SU16 SU16SU16 SU16 Ingredients IngredientsIngredients Ingredients FM a aa a 700 642 571 695 676 SMC b bb b - 124 248 - - SU c cc c - - - 80 160 Fish oil 86 86 86 82 80 Gelatinized starch d dd d 169 103 50 98 39 Vitamin premix 20 20 20 20 20 Mineral premix 20 20 20 20 20 CMC 5 5 5 5 5 Proximate composition (% dry wt) Proximate composition (% dry wt)Proximate composition (% dry wt) Proximate composition (% dry wt) Crude protein 47.23 46.75 46.69 47.08 46.73 Crude lipids 13.39 13.68 13.20 13.42 13.53 Ash 11.27 14.71 15.83 15.01 18.56 Carbohydrate e ee e 28.11 24.86 24.28 24.49 21.18 Gross Energy (MJ kg-1) f ff f 21.31 20.75 20.44 20.66 20.05 Total carotenoid (mg kg-1) 2.62 3.58 5.04 2.79 3.08 STUDY IV STUDY IVSTUDY IV STUDY IV - 112 - Table Table Table Table 7. 7.7. 7.3 33 3 Fatty acid (g/100 g fatty acids) profile of the meals and experimental diets Fatty a Fatty aFatty a Fatty acids cidscids cids FM FMFM FM SMC SMCSMC SMC SU SUSU SU CD CDCD CD SMC10 SMC10SMC10 SMC10 SMC20 SMC20SMC20 SMC20 SU8 SU8SU8 SU8 SU16 SU16SU16 SU16 14:00 0.05 3.1 5 16.83 7.25 6.78 7.28 7.13 7.87 15:00 0.23 0.73 1.24 0.67 0.45 0.56 0.46 0.57 16:00 4.24 17.46 21.64 21.55 18.32 19.82 18.73 20.64 17:00 1.68 1.05 0.44 1.59 1.57 1.64 1.52 1.53 18:00 10.25 5.86 3.93 4.38 3.62 4.15 3.68 4.36 20:00 0.42 0.54 5.32 0.24 0.20 0.26 0.20 0.26 ∑ ∑∑ ∑SFA SFASFA SFA 16.87 28.79 49.41 35.68 30.94 33.71 31.73 35.23 14:1n - 7 10.21 0.06 0.22 0.02 0.05 0.04 0.03 0.05 14:1n-5 0.06 0.19 0.06 0.28 0.16 0.16 0.13 0.16 15:1n-5 0.90 0.02 0.04 0.09 n.d 0.40 n.d n.d 16:1n-5 0.62 0.42 0.20 0.13 0.37 n.d 0.38 0.37 16:1n-7 8.68 6.35 4.86 9.03 8.76 9.07 8.73 8.76 18:1n-5 0.12 0.38 0.40 0.11 0.13 0.14 0.13 0.13 18:1n-7 4.37 4.59 4.65 3.26 3.25 3.47 3.27 3.25 18:1n-9 10.88 23.30 4.77 9.79 9.62 9.70 9.40 9.62 20:1n-5 n.d. 0.75 0.50 0.22 0.26 0.43 0.35 0.26 20:1n-7 0.43 1.85 5.37 0.24 0.68 0.89 0.75 0.68 20:1n-9 0.95 1.85 5.76 0.81 0.68 0.89 0.75 0.68 22:1n-9 0.44 2.39 1.10 0.10 0.21 0.29 0.23 0.21 22:1n-11 0.57 1.30 4.69 0.73 0.67 0.87 0.64 0.67 ∑ ∑∑ ∑MUFA MUFAMUFA MUFA 38.23 43.44 32.61 24.81 24.84 26.33 24.79 24.84 1 6:2n - 6 1.55 0.49 0.12 0.39 0.44 0.45 0.39 0.40 16:2n-4 1.53 0.95 0.57 0.32 0.37 0.37 0.36 0.37 16:3n-4 0.06 0.16 0.13 0.64 0.69 0.68 0.69 0.61 16:3n-3 0.41 0.48 1.27 1.63 1.68 1.62 1.67 1.65 16:3n-1 1.70 1.11 0.90 0.25 0.25 0.27 0.26 0.25 16:4n-3 n.d. 0.10 0.20 1.28 1.39 1.37 1.40 1.39 16:4n-1 n.d. 0.20 0.04 n.d. 0.02 0.02 0.02 0.02 18:2n-9 0.12 0.22 1.75 0.07 0.09 0.08 0.08 0.07 18:2n-6 1.17 1.86 0.50 3.84 3.86 3.89 3.85 3.86 18:2n-4 0.54 0.19 0.30 0.38 0.21 0.21 0.21 0.21 18:3n-6 0.42 0.09 0.63 0.31 0.30 0.33 0.29 0.30 18:3n-4 0.22 0.24 0.28 0.25 0.33 0.29 0.26 0.28 18:3n-3 0.48 0.37 1.51 0.99 1.06 1.05 1.05 1.06 18:4n-3 0.21 0.34 1.44 1.69 1.70 1.70 1.65 1.70 18:4n-1 n.d. 0.05 0.21 0.22 0.24 0.20 0.23 0.24 20:2n-9 0.07 0.14 0.57 0.09 0.12 0.14 0.13 0.12 20:2n-6 0.20 1.08 1.34 0.19 0.16 0.22 0.20 0.16 20:3n-6 0.10 0.13 0.06 0.10 0.12 0.11 0.12 0.12 20:3n-3 n.d. 0.20 0.64 n.d. 0.09 0.10 0.09 0.12 20:4n-6 1.90 7.18 8.07 1.10 1.10 1.33 1.14 1.61 20:4n-3 0.54 0.32 0.42 0.78 0.87 0.83 0.84 0.87 20:5n-3 14.02 1.04 0.80 12.45 12.04 12.20 12.87 12.04 22:4n-6 0.44 0.18 0.20 0.59 0.66 0.55 0.65 0.66 22:5n-6 n.d. n.d n.d 0.32 0.33 0.34 0.30 0.33 22:5n-3 2.19 1.04 1.10 2.87 2.71 2.21 2.54 2.71 22:6n-3 15.18 12.71 0.77 10.31 10.06 10.48 10.95 10.06 ∑ ∑∑ ∑PUFA PUFAPUFA PUFA 43.05 36.41 23.82 41.06 40.89 41.04 42.24 41.21 ∑ ∑∑ ∑ n nn n - -- - 3 33 3 33.04 23.59 7.38 32.00 31.60 31.56 33.06 31.60 ∑ ∑∑ ∑n nn n- -- -6 66 6 5.78 9.44 10.92 6.84 6.97 7.22 6.94 7.44 ∑ ∑∑ ∑n nn n- -- -9 99 9 12.47 29.74 13.96 10.86 10.72 11.10 10.59 10.70 ∑ ∑∑ ∑n nn n- -- -3 HUFA 3 HUFA3 HUFA 3 HUFA 31.94 22.31 3.73 26.41 25.77 25.82 27.29 25.80 AA/EPA AA/EPAAA/EPA AA/EPA 0.14 6.90 7.34 0.09 0.09 0.11 0.09 0.13 EPA/DHA EPA/DHAEPA/DHA EPA/DHA 0.92 0.08 1.43 1.21 1.20 1.16 1.18 1.20 n nn n- -- -3/n 3/n3/n 3/n- -- -6 66 6 5.72 2.49 0.68 4.68 4.53 4.37 4.76 4.25 STUDY IV STUDY IVSTUDY IV STUDY IV - 113 - 7.2.3 7.2.37.2.3 7.2.3 Fish and culture conditions Fish and culture conditions Fish and culture conditions Fish and culture conditions Red porgy with initial mean weight of 205 ± 39 g (mean ± SD) were distributed in 15 fibreglass circular tanks of 500 l, at an initial density of 6.3 kg m-3. Tanks were provided with natural seawater with 17-18 ºC and 7-9.7 mg L-1 of temperature and dissolved oxygen respectively. Fish were carefully hand feeding to apparent satiation twice per day (8:00 and 15:00), six days per week for 180 days. Uneaten feed was daily recovered for feed intake correction. 7.2.4 7.2.47.2.4 7.2.4 Sampling and colour evaluation Sampling and colour evaluation Sampling and colour evaluation Sampling and colour evaluation Skin colour parameters, body weight and total length were taken in all fish previously anesthetised with 2-phenoxyethanol (0.1 mL L-1) at the beginning of the experiment and at 45, 90, 135, 180 feeding days. For proximate analysis of muscle and liver, nine individuals per treatment were slaughtered. Nine fish per treatment were also sampled for skin carotenoids concentration at the end of the triall. All samples were stored at -80ºC until analysis. 2.5. Samples recollection and ammonia nitrogen concentration 2.5. Samples recollection and ammonia nitrogen concentration2.5. Samples recollection and ammonia nitrogen concentration 2.5. Samples recollection and ammonia nitrogen concentration Ammonia-N determinations were carried out over 24 h on days 145, 154 and 163. Feeding was performed to apparent satiation at 08:00 am. Inlet and outlet water samples (50 mL) were then taken from each tank every 2 h between 08:30 am and 08:30 pm to analyse the ammonia-N excretion of the fish fed the different diets. Samples of water were kept in dark recipients and stored at 4ºC until analysis. A tank without fish and another with unfed fish were also sampled to determine the blank-corrected value. 7. 7.7. 7.2.6 2.62.6 2.6 Colour measurements Colour measurements Colour measurements Colour measurements Skin colour parameters were lightness ( L* ), redness ( a* ) and yellowness ( b* ), in accordance with the recommendations of the International Commission on Illumination, CIE (1976). Colour variables were measured from the left front lateral zone (Kalinowski et al ., 2005) using a portable colorimeter (Hunter STUDY IV STUDY IVSTUDY IV STUDY IV - 114 - MiniScanTM XE plus). Hue [ Hab=arctan(b*/a* )] and Chroma [ Cab=(a*2+b*2 )1/2], were calculated according to Hunt (1997). 7. 7.7. 7.2.7 2.72.7 2.7 Carotenoids analysis Carotenoids analysis Carotenoids analysis Carotenoids analysis Carotenoids of meals and diets were extracted according to Barua et al . (1993), while for skin, due to the high water content, according to Schiedt and Liaaen-Jensen (1995). Total carotenoid concentration was calculated spectrophotometrically at λ=470nm using E 1%,1 cm= 2100, when carotenoids were expressed as astaxanthin equivalent, by the following equation: µg g-1 sample = 10000*V*A/W*E 1%,1 cm where V (mL) is the volume of the extract, W (g) the weight of sample, A the absorbance and E 1%,1 cm the extinction coefficient. For quantification of red and yellow pigments, final n-hexane solutions, containing total skin carotenoids, were analyzed by TLC using pre-coated plates of silica gel (G60 Merck glass plates, 20 x 20 cm). Hexane/diethylether 20:1 was used as a developing system. Separated skin pigments were tentatively identified on the basis of relative motility on a TLC plate. For quantification, each separated component was scraped off the silica plate and washed several times with acetone until no colour was observed. Thereafter, the acetone volume was filtered evaporated to dryness and dissolved in hexane for further quantification by UV/Vis spectroscopy, reading at λ maxima and using an extinction coefficient (E1%,1cm) of 2500 for unknown compounds (Britton et al ., 1995). Unpublished colour variables of wild red porgy skin from our research group were used as referential values. 7. 7.7. 7.2.8 2.82.8 2.8 Proximate analysis and fatty acid profile Proximate analysis and fatty acid profile Proximate analysis and fatty acid profile Proximate analysis and fatty acid profile Proximal analysis and fatty acid profile was performed for meals and diets in triplicate. For muscle samples three fish from each experimental tank were STUDY IV STUDY IVSTUDY IV STUDY IV - 115 - used. Moisture, crude protein and ash content were determined according to AOAC (1995). Lipids were extracted with chloroform-methanol (2:1, v/v), as described by Folch et al . (1957). The fatty acid methyl esters were obtained by transesterification with 1% sulphuric acid in methanol (Christie, 1982), purified by absorption chromatography on NH2 Sep-pak cartriges (Waters, S.A., Milford, MA, USA) and separated and quantified by gas-liquid chromatography as described by Izquierdo et al . (1992). Fatty acids were identified by comparison to external standards. Data were expressed as percentage of total identified fatty acids. The index of atherogenicity and thrombogenecity related to effects of different fatty acids on human’s health were calculated in concordance to Ulbricht and Southgate (1991): Index of atherogenicity (AI) AI= [(12:00) + (4 x 14:00) + (16:00)] x [(PUFA n-6 and n-3) + MUFA]-1 Index of thrombogenicity (TI) TI= [(14:00) + (16:00) + (18:00)] x [(0.5 x MUFA) + (0.5 x n-6) +(3 x n-3) + (n-3/n6)]-1 7. 7.7. 7.2.9 2.92.9 2.9 Texture analysi Texture analysi Texture analysi Texture analysis ss s After a starving period of 24 h, fish were slaughtered in a small tank with ice and seawater. Nine fish from each treatment were randomly sampled and kept with ice in polyethylene boxes at 4ºC for a day. The texture analysis was conducted in a Stable Micro System texture analyser (TA.XT2, Surrey, England) equipped with a 5 kg load cell. All tests were carried out at refrigeration temperatures, keeping the fillets on ice. After removal of the skin, three blocks (2×2×1.2 cm) were obtained from each fillet above the lateral line. Fish fillets were compressed up to breaking. Compression test was defined as the force exerted to deform a block fillet using a flat-ended (Type P/100) aluminium compression plate with a speed to 0.8 mm/s and strain to 80% penetration. The STUDY IV STUDY IVSTUDY IV STUDY IV - 116 - total force, F1 (N), needed to compress the fillet was recorded. 7. 7.7. 7.2.1 2.12.1 2.10 00 0 Measurement of Thiobarbituric acid Measurement of Thiobarbituric acid Measurement of Thiobarbituric acid Measurement of Thiobarbituric acid- -- -reactive substances (TBARS) reactive substances (TBARS)reactive substances (TBARS) reactive substances (TBARS) Nine fish from each treatment were collected for the 2-thiobarbituric acidreactive substances (TBARS) analysis (Shaidi and Hong, 1991). At days 0, 4 and 7 of refrigerated storage, a fillet sample of 3 g was mixed with 6ml of 10% trichloroacetic acid (w/v) and homogenized for 60 s. After filtration, 2 ml of the filtrate were mixed with 2 ml of a TBARS solution (0.02 M) and heated at 100ºC for 1 hour. Then, the absorbance was measured at 532 nm by UV/Vis spectrophotometer (Thermo Scientific, Evolution 300 model, Chicago, USA). TBARS values were expressed as mg of malonaldehyde (MDA) per kg of fillet. 7. 7.7. 7.2.11 2.112.11 2.11 Stati StatiStati Statistical analysis stical analysisstical analysis stical analysis Scalar data was tested for normality of distribution and homogeneity of variance (Sokal and Rohlf, 1995). To compare treatments, parametric and homoscedastic data were used by one-way ANOVA followed by Tukey’s test for multiple comparisons. For non-normal data and/or data showing homogeneity of variance, the Kruskal-Wallis multiple range test was used. In case of the thiobarbituric acid-reactive substances data, a general linear model with two fixed factors, time and dietary level inclusion, was used. All statistical analyses were tested at 0.05 significance level using the SPSS (13.1) statistical package. The angular colour parameter, Hue, was analysed by circular statistical test. The estimation of the mean and standard deviation was performed with descriptive statistics for circular distributions. The Rayleigh test was applied to check the uniformity of circular distribution (Zar 1999). As sampled populations was not unimodal, differences in the Hue values were tested using the non parametric Watson’s U 2 test and accepted at P <0.05 (Zar 1999). All tests were performed with Oriana, version 3, statistical software (Kovach Computing Services, Pentraeth, Wales, UK). STUDY IV STUDY IVSTUDY IV STUDY IV - 117 - 7.3 7.3 7.3 7.3 Results ResultsResults Results 7.3.1 7.3.17.3.1 7.3.1 Growth and feed utilization Growth and feed utilization Growth and feed utilization Growth and feed utilization All diets were well accepted from the start of the trial, with higher feed appetence observed in fish fed both SMC and SU meals diets. This fact resulted in higher feed intake and a positive effect on growth performance, although only the highest inclusion level of the two meals (SMC20 and SU16 diets) caused significantly greater feed consumption compared to CD diet (Table 7.4). Better growth response was observed for the inclusion of SU than for SMC meal. Thus, final fish weight, absolute weight gain and specific growth rate (SGR) values were significantly higher both to group with the highest inclusion of spider marine crab meal (SMC20), and groups with sea urchin meal (SU8 and SU16) compared to fish fed CD and SMC10, which did not differed among them. Results for feed conversion ratio (FCR) and the efficiency of dietary protein utilization (PER) were positively affected by increasing dietary levels of SU meal, with significant lower FCR values for both inclusion levels (SU8 and SU16) and significant higher PER values for the highest inclusion level (SU16). Comparing between the two meal groups (SMC and SU), SU16 showed significantly better FCR and PER results than those for SMC10 and SMC20, while SU8 has similar values respect to SMC20 and better compared to SMC10 (Table 7.4). Fish body appearance measured by condition factor, K, was better affected by feeding SU diets, showing significantly lower values respect to CD and SMC fish. Results for HSI had similar trend, with lowering indexes by increasing dietary levels of both meals. No effect was found for visceral index (VSI) between diets (Table 7.4). STUDY I STUDY ISTUDY I STUDY IV VV V - 118 - Table Table Table Table 7. 7.7. 7.4 44 4 Growth performance and feed utilization parameters of red porgy fed the experimental diets (mean ± SD) Significant differences between treatments are indicated wth different letter. a aa a Weight gain Weight gain Weight gain Weight gain = (final weightinitial weight) b b b b Feed intake (g) Feed intake (g)Feed intake (g) Feed intake (g) per fish for 165 day experiment c c c c Specific growth rate= Specific growth rate= Specific growth rate= Specific growth rate= 100x(ln final weight-ln initial weight)/nº days d dd d Feed conversion rat Feed conversion ratFeed conversion rat Feed conversion ratio= io= io= io= feed intake (g)/weight gain (g) e e e e Protein efficiency ratio= Protein efficiency ratio= Protein efficiency ratio= Protein efficiency ratio= weight gain (g)/protein intake (g) (dry matter). f ff f Condition factor (%)= Condition factor (%)= Condition factor (%)= Condition factor (%)= 100x(final weight /(final length) 3 ) g gg g Hepatosomatic index (%)= Hepatosomatic index (%)= Hepatosomatic index (%)= Hepatosomatic index (%)= 100x(liver weight/final weight) h hh h Visceral index (%)= Visceral index (%)= Visceral index (%)= Visceral index (%)= 100x(( (((( ((final weight-final eviscerated fish weight)/final weight) CD CDCD CD SMC10 SMC10SMC10 SMC10 SMC20 SMC20SMC20 SMC20 SU8 SU8SU8 SU8 SU16 SU16SU16 SU16 Initial weight (g) 201.61±22.11 204.60±25.09 201.10±22.89 202.92±24.09 200.68±21.55 Final weight (g) 421.91±43.57 b 433.42±44.03 b 463.78±45.32 a 476.54±46.24 a 479.24±52.18 a Weight gain (g) a aa a 221.02±35.47 b 229.44±45.51 b 253.95±34.87 a 273.92±41.88 a 279.27±43.14 a FI (g fish -1 ) b bb b 379.1±1.59 b 407.00±23.35 ab 432.51±1.26 a 414.96±26.26 ab 444.57±26.61 a SGR c cc c 0.37±0.05 b 0.38±0.07 b 0.42±0.04 a 0.44±0.06 a 0.45±0.05 a FCR d dd d 1.73±0.05 a 1.84±0.07 a 1.70±0.09 ab 1.53±0.03 bc 1.48±0.05 c PER e ee e 1.22±0.04 bc 1.10±0.04 c 1.25±0.07 bc 1.34±0.09 ab 1.44±0.05 a K f ff f 3.10±0.28 a 3.02±0.37 a 2.88±0.30 ab 2.80±0.39 b 2.83±0.29 b HSI g gg g 1.44±0.29 a 1.31±0.20 a 1.11±0.30 b 1.09±0.25 cb 1.01±0.23 c VSI h hh h 5.99±1.42 5.75±1.09 4.98±0.68 5.93±0.85 5.53±0.92 STUDY IV STUDY IVSTUDY IV STUDY IV - 119 - 7.3.2 7.3.27.3.2 7.3.2 Postprandial ammonia nitrogen excretion Postprandial ammonia nitrogen excretion Postprandial ammonia nitrogen excretion Postprandial ammonia nitrogen excretion The postprandial ammonia-N excretion concentration (mg kg1 body weight) showed similar patterns in all feeding groups, with increasing ammonia-N excretion after feeding and reaching maximum values at 4 h in all treatments. Another peak, with less amplitude, was observed at 10 hours after feeding (Fig 7.1). The inclusion of SMC up to 20% level did not significantly affected the total daily ammonia-N excretion (mg N-NH4+ kg-1 day-1) respect to the CD, although a lower value in SMC20 fish was observed (Table 7.5). On the other hand, fish fed on SU meal diets showed lower ammonia-N excretion, only significant for the highest inclusion level (SU16). Per unit of nitrogen intake, total ammonia-N excretion reached 34%, 42%, 32%, 31% and 22% for fish fed on CD, SMC10, SMC20, SU8 and SU16 diets, respectively (Table 7.5). 0 20 40 60 80 8:00 08:00-10:00 10:00-12:00 12:00-14:00 14:00-16:00 16:00-18:00 18:00-20:00 20:00-22:00 Time (hours) mgN-NH 4 + /Kg BW CD SCM10 SCM20 SU8 SU16 Figure Figure Figure Figure 7. 7.7. 7.1 11 1 Daily ammonia nitrogen excretion patterns in red porgy after feeding the experimental diets. Means ± SD, (n=3) for each treatment. Means without letters denote no significant differences. STUDY IV STUDY IVSTUDY IV STUDY IV - 126 - 7. 7.7. 7.3.7 3.73.7 3.7 Flesh texture Flesh texture Flesh texture Flesh texture No significant differences were obtained for texture between CD and the rest of the fish diets, although a reduction trend was observed by increasing SU inclusion. It was only different the value of SU16 fish, lower than that for SMC10 fish (Table 7.10). Table Table Table Table 7. 7.7. 7.10 1010 10 Force, F1 (N), required to compress the raw fillet of red porgy fed the experimental diets. CD CDCD CD SMC10 SMC10SMC10 SMC10 SMC20 SMC20SMC20 SMC20 SU8 SU8SU8 SU8 SU16 SU16SU16 SU16 F 1 1.83±0.70 ab 1 .94±0.75 a 1.76±0.63 ab 1.65±0.73 ab 1.40±0.62 b Means with different letter denote significant difference 7. 7.7. 7.3.8 3.83.8 3.8 Muscle lipid oxidation Muscle lipid oxidation Muscle lipid oxidation Muscle lipid oxidation The effect of dietary treatment on lipid oxidation measured as changes in TBARS values at 0, 4 and 7 days of storage after slaughtering is illustrated in Figure 7.5. A significant dietary influence ( p <0.00) with an effect interaction with time storage ( p <0.00) was observed. Thus, no differences in TBARS values were observed at 0 days while at days 4 and 7 of storage, fish fed SMC diets at the two inclusion level attained significantly lower TBARS values in comparison with CD and SU diets (Fig. 7.5). Figure 7.5 Figure 7.5Figure 7.5 Figure 7.5 Evolution of fillet lipid oxidation during refrigerated storage of fillets from red porgy fed the experimental diets. Significant differences between treatments are indicated with different letter. 0,00 0,04 0,08 0,12 0,16 0,20 047 Days of storage CD SMC10 SMC20 SU8 SU16 aa ab aa a b bb 0,00 0,04 0,08 0,12 0,16 0,20 047 Days of storage CD SMC10 SMC20 SU8 SU16 aa ab aa a b bb STUDY IV STUDY IVSTUDY IV STUDY IV - 127 - 7.4 7.47.4 7.4 Discussion Discussion Discussion Discussion 7.4.1 7.4.1 7.4.1 7.4.1 Growth performance Growth performanceGrowth performance Growth performance In present study, the inclusion of both SMC and SU meals in diets positively affected on the final growth performance. As observed in a previous experiment (García-Romero et al ., 2010; study I) conducted also with red porgy fed on different crab meals origin, river ( Procambarus clarkii ) or marine ( Chaceon affinis ), dietary inclusion of marine origin ingredients highly induced the fish appetite response, clearly reflected in a significantly higher feed intake respect to the control diet together with a positive impact on fish growth. Results obtained are further encouraged for fish fed on SU meal, which attained significantly better feed efficiency (FCR) and protein utilization (PER) than SMC meal diets. Crustacean meals have been used as alternative protein sources in several species showing successful results on diets appetence and growth performance (Smith et al ., 2005; Goytotúa-Bores et al ., 2006; Tibbetts et al ., 2006; Kalinowski et al ., 2007). Feed intake is regulated by the amount of digestible energy in diets but also by the palatability and attractant diet effect associated with the presence of certain stimulant soluble nutrients (Jonnes 1992; Yacoob and Browman, 2007). Digestible energy of SMC and SU meal based diets may be expected to be lower than that of CD, due to some nutrient limiting factors such as the greater ash and chitin content. Nevertheless, since a good digestibility (Study II) has been found in diet containing high amount of ash (from marine crab Chaceon affinis meal), the higher feed consumption observed may be also explained by attractant and good palatability properties conferred by certain components presented in both marine meals. It is important to mention that this fact was supported by the observed fish feeding behaviour of SU and SMC diets, showing a faster initial pellet intake response together with more feed competitiveness and longer sustained appetence. The same feeding behaviour was recently described by Tibbetts et al . (2010), where feeding with krill meal on cod ( Gadus morhua ) and halibut ( Hippoglossus hipoglossus ) significantly increased feed intake, even at STUDY IV STUDY IVSTUDY IV STUDY IV - 128 - higher levels of digestible energy and protein, with increment on growth, FCR and PER rates enhancement krill, as a crustacean, has the role of meal attractant attributed to the soluble protein fraction content, preserved in meals with the adequate processing. In agreement with these authors, feed attractant properties should be highly potential as could affect in double growth in fish species. Both SU and SMC meals were gently processed at low drying temperature, which may preserve certain nutritional attractants components in meals and better general quality. Feeding with SMC diets resulted in a tendency toward to decrease Ammonia-N excretion (AE) respect to those fed the control diet, while diets with SU meal significantly reduced AE by increasing the inclusion level. It is well known that AE is influenced by the fish size (Zhang et al ., 2004; Liu et al ., 2009), however, since SU8 and SU16 fish displayed similar K value condition, the lower AE value in SU16 diet may likely due to a better dietary protein utilization, or even to a “protein-sparing effect” of any non-protein component of SU meal. Furthermore, the lower AE along with the increase of PER value suggests that growth of animals fed SU diets was not only due to the higher feed intake but also better protein utilization of diets containing SU meal. More research should be performed to elucidate possible growth stimulating compounds in these ingredients. The lesser amplitude second peak excretion at about 12 hours after morning feeding may be related with the rule of feeding the fish twice a day, while only once during ammonia experience trials. The presence of two peaks of excretion is widely reported to have two meals (Echavarria and Col, 1993; Engine and Carter, 2001), indicating that the second peak could be a result of metabolism as reflecting residual to these fish have been accustomed to two meals. STUDY IV STUDY IVSTUDY IV STUDY IV - 129 - 7.4.2 7.4.2 7.4.2 7.4.2 Skin colour performance Skin colour performanceSkin colour performance Skin colour performance Several crustacean species are rich in astaxanthin carotenoid, which is often presented in esterified form (Matsuno and Hirao, 1989; Coral-Hinostroza and Bjerkeng, 2002). In the present trial, the carotenoids content from the spider marine crab meal, specially at the highest level of inclusion (SCM20), was an effective dietary ingredient for enhancing skin colour in red porgy based on a* , b* , Hue, Chroma values and skin carotenoids concentration. This observation is in line with the previous work (García-Romero et al ., 2010; study I) where the dietary inclusion crab meals positively affected skin colour, showing crab meals as a good source of esterified astaxanthin, which has proved to be more efficiently utilized than the unesterified form to improve skin colour in red porgy (Kalinowski et al ., 2005; Tejera et al ., 2007). From the two marine crabs species used in the different finding ( C. affinis . in study I and P. cuvieri in present study IV), superior pigmentation efficacy from the P. cuvieri diets over the C. affinis crab diets (MC) were obtained since total carotenoids concentration in SMC meal (11.96 mg kg-1) was higher respect to the MC meal (5.90 mg kg-1). Wild red porgy presents a red-pink skin coloration a blend of red (astaxanthin) and yellow (tunaxanthin) pigments (Tejera et al ., 2007). Thus, the adequate relative proportion between red and yellow pigments in the skin of farmed red porgy promotes the adequate overall colour appearance. In present study fish fed on SMC20 diet had similar pigments relative proportion (60%/ 40%, red/yellow) to wild origin (64%/36%, red/yellow). On the other hand, red porgy fed SU diets did not increase redness parameter colour ( a* ) or deposit high amount of red pigment as we expected, since SU meal contain mainly yellow pigments and red sparids have shown limited ability to convert them (B-carotene, zeaxanthin and lutein) into astaxanthin (Nakazoe et al ., 1984; Chatzifotis et al ., 2005; Chatzifotis et al ., 2009). Although, SU meal was tested mainly under growth performance purposes, it was observed an increase of yellowness ( b* ) supported with a significant major proportion of yellow pigment fraction (88%-91%) in skin STUDY IV STUDY IVSTUDY IV STUDY IV - 130 - fish fed on SU diets. The echinenone (4-keto-β-carotene) is a yellow pigment wellknown as major carotenoid found in gonads of sea urchins (Griffiths, 1966; Tsushima et al ., 1993b; Havardsson and imsland, 1999; Matsuno and Tsushima, 2001). This induces that the yellow pigment from SU was well utilized by the red porgy and deposited in the skin as tunaxanthin. Moreover, it has been proved that red porgy has the ability to oxidize dietary asthaxanthin into tunathanxin, so other precursors of tunaxanthin may be considered (Kalinoswski et al ., 2005). Results showed SU meal pigments content led to deposition of yellow carotenoid in fish skin contributing to a yellowish coloration and it could offer an alternative as yellow pigment source for enhance tunaxanthin content. Skin lightness (L*) was not significantly affected by diets. As occurred in other species, a variation in this parameter seems mainly related with different environmental factors (Pavlidis et al ., 2006). 7.4.3 7.4.3 7.4.3 7.4.3 Composition and fish flesh q Composition and fish flesh qComposition and fish flesh q Composition and fish flesh quality ualityuality uality As shown by numerous species and previous works with red porgy, the fillet fatty acid composition reflects that from the diet (Madsen et al ., 2000; Bell et al ., 2002; Glencross et al ., 2003). In present study, there was an increase of PUFA, total n-3, n-3 HUFA, DHA (22:6n-6), EPA (20:5n-3) and ARA (20:4n-6) fatty acid content, as well as a decrease in total n-6 and saturated fatty acid concomitant with the inclusion of marine crab meal in diets. Similar results were found where feeding southern flounder ( Paralichthys lethostigma ) with crab meal supplemented diets (5%) also improved the muscle nutritional quality with higher n-3/n-6 ratio respect to fish fed on a commercial diet (Gonzalez et al ., 2006). The same trend also occurred by feeding with SU diets although lower accused, with only 10% increase in n-3/n-6 ratio respect to the control group in comparison with SMC10 and SCM20 (15% and 20% respectively). ARA content in diets increased in dose-dependent manner as the amount of crab meal or sea urchin meal increased (up to 13.3 g kg-1 in SMC20 and 16.1 g kg-1 in SU16). Effect of higher feed intake STUDY IV STUDY IVSTUDY IV STUDY IV - 131 - with a feed from marine origin could have an effect on red porgy nutritional fillet enhancement. The TI and AI index was also favourable for SMC fillets as result of the healthier combination on fillet fatty acid profile respect to the control diet. Regarding to SU fillet, those indexes were unaffected respect to the CD fish mainly due to the similar SFA content in fillets. Regarding fillet texture, the reduction of the required force to compress fillets of fish fed on SCM20, SU8 and SU16 respect to the control diet could be related to the slightly increase of fillet fat content found in these diets (table 7.4). Fish tend to increased lipid muscle concentration with growth so differences in muscle hardness between dietary groups may be partially explained by the significantly higher size, and hence higher muscle lipid content, presented in fish fed on SCM20, SU8 and SU16 respect to the control group, as reported the same effect in other cultured fish species (Izquierdo et al ., 2005; Suontama et al ., 2007). According to fillet shelf-life (lipid oxidation) results, the inclusion of crab meal in diet delayed the lipid oxidation in muscle stored at 4ºC respect to fish fed on CD diet. The rate of lipid oxidation in post mortem flesh has been proved to vary with factors such as multi type and level of proand antioxidant presented in fillet (Undeland et al ., 1999; Bell et al ., 2000; Hamre et al ., 2004). Exogenous antioxidants are provided by the diet and retained in fish tissues especially during long-time supplementation period. Among them, carotenoids have been showed to be potential antioxidants (Bell et al ., 2002). In present study SMC10 diets and SU16 diets have similar carotenoids concentration (3.58 mg kg-1 and 3.08 mg kg-1 respectively) but fillet of fish fed on SU16 diet did not show lower oxidation. This suggest that against-oxidation capacity of crab meal may be associated not only with the amount of carotenoids but also probably with the whole action of various constitutes with antioxidant activity. Moreover, astaxanthin has longer showed to present stronger antioxidant functions than other carotenoids (Miki et al ., 1991; Bell et al ., 2002; Guerin et al ., 2003; Wang et STUDY IV STUDY IVSTUDY IV STUDY IV - 132 - al ., 2006). Crab meal was demonstrated to be richer in astaxanthin respect to FM and SU meals. However, this is speculation since antioxidants were not measured in that particular study. From the promote results more experiments are required to evaluate and identify the antioxidant capacity of the different contain pigments, active lipid or protein constituents of the crab meals toward the adequate analytical methodology. 7.5 7.57.5 7.5 Conclusion Conclusion Conclusion Conclusion Results confirm the possibility to use SMC and SU meals as potential feed ingredients in on-growing diets for red porgy, improving feed appetite and growth performance. Furthermore, the inclusion of SMC meal has a positive effect on fish skin colour and especially on the extension of self life, delaying lipid oxidation during storage time. Meanwhile, SU meal promotes yellow skin coloration in red porgy. 8 88 8 8 88 8. .. . . .. .C CC C C CC CO OO O O OO ON NN N N NN NC CC C C CC CL LL L L LL LU UU U U UU US SS S S SS SI II I I II IO OO O O OO ON NN N N NN NS SS S S SS S CONCLUSIONS CONCLUSIONSCONCLUSIONS CONCLUSIONS - 135 - 8. CONCLUSIONS 8. CONCLUSIONS8. CONCLUSIONS 8. CONCLUSIONS 1. 1.1. 1. All tested ingredients from marine origin, meals of Paramola cuvieri crab, Chaceon affinis crab and Diadema antillarum sea urchin, presented a great attractant potential, higher respect to the fish meal. Thus, a better fish feeding response and feed consumption were observed. 2. 2.2. 2. The partial replacement of fish meal by both tested marine crabs, Chaceon affinis and Paramola cuvieri, positively influenced on the red porgy growth performance. On the contrary, the same inclusion levels of the river crab Procambarus clarkii meal did not improved fish growth. 3. 3.3. 3. The inclusion of sea urchin Diadema antilarum meal up to 16% positively influenced red porgy growth performance and significantly enhanced feed conversion and protein efficient rates. 4 44 4. .. . The inclusion of tested crab meals, Procambarus clarkii , Chaceon affinis and Paramola cuvieri, increased the PUFA content, n-3/n-6 ratio and significantly increased the ARA content of red porgy muscle, positively affecting its nutritional value for human consumption. While, the dietary inclusión of sea urchin Diadema antilarum meal, both at the 8% and 16% inclusion level produced the highest ARA content in muscle. 5 55 5. .. . Marine crab Chaceon affinis meal at 20% of dietary inclusion improved the red skin colour compared to that of the control fish. While the river crab Procambarus clarkii meal and the crab Paramola cuvieri meal revealed to be more efficient as pigment source for this species at the two levels used in this study. 6 66 6. .. . Dietary inclusion of sea urchin Diadema antillarum meal significantly increased yellow pigments content in red porgy skin.