Gilthead seabream (Sparus aurata) is one of the most important species of intensively reared fish in the Mediterranean region. Its short history of domestication, along with the need to develop markets, new products and efficiency in the production process, has resulted in an increased interest in the potential genetic improvement of this species. Little work has so far been directed at establishing the procedures for selective breeding in gilthead seabream at a commercial level, although genetic parameters calculated in other studies have indicated that there is a large potential for improvement in certain traits. Selective breeding of commercial gilthead seabream populations is constrained by aspects of the biology that complicate the production of genetic groups and the maintenance of same-age offspring populations. The aim of this thesis was to develop a protocol for the selective breeding of gilthead seabream, specifically to serve a commercial hatchery and on-growing unit in Cyprus, where the fieldwork was carried out. The hatchery broodstock was monitored over a three-year period to identify the rate of sex reversal in introduced fish and to quantify the sex ratio of the stock over time. The analysis of the egg production records was used to evaluate the success of photoperiod manipulation in each group. Size variation in the larval and juvenile stages is a common problem in the rearing of gilthead seabream, leading to cannibalism and labour-intensive sorting operations. Studies on larval populations, from first feeding through to metamorphosis, indicated the origin of size variation was the differences in early feeding ability. The size advantages could be maintained throughout the larval period. During the juvenile stage of the farm production system, a method to standardise the size sorting of populations by grading was developed in order to counter environmental effects of separating groups of fish. Using this method, grading would be suitable to form the first stage of a selection programme for growth rate. The potential gain of selection for growth rate during the on-growing stage was very high, using a simulated criterion and previous estimates of heritability. Other possible quality traits for selection were also examined and quantified in the hatchery populations. Existing and specifically developed microsatellite markers were used for the assignment of offspring to parents from mass spawning of the hatchery broodstock. The effective population size of single spawning events were found to be low and determined by a high variation in contribution to mass spawning. Contribution was found to be significantly linked to body size, which led to the formation of a replacement policy for the broodstock to maximise spawning performance. Survival of individual families through the larval period was also examined. Based on the results of the experimental work, a two-stage selection programme was designed, along with the presentation of specific procedures for each stage of the production system. This project makes recommendations on various strategies that can increase the effective population size within a selection programme and these are discussed as part of the genetic management of hatchery populations. Significant progress has also been made in the use of genetic markers in monitoring the rate of inbreeding and contribution of individual broodfish, which are considered essential in this species.

In the present study the effects of characteristic freezing times and storage time at -22°C on the quality of the adductor muscle of post-rigor scallops (Pecten maximus)and gilthead seabream fillets (Sparusa urata) were studied in regard to the integrity of muscle structure, myofibrillar protein denaturation and aggregation, lipid degradation, texture and sensory changes. This information would be useful for achieving optimal conditions for freezing these species and assessing their quality during frozen storage for commercial purposes. Scallop muscles and gilthead seabream fillets were frozen individually with characteristic freezing times that can be met in commercial practice of freezing seafoods. After freezing, the samples were thawed and their quality was evaluated. Fresh samples were analyzed as controls. Intermediate characteristic freezing times (i. e. 89 and 49 minutes for scallop muscles and 74 minutes for gilthead seabream fillets) caused more damage to cell structure of both species than the shorter and longer characteristic freezing times tested. Short characteristic freezing times (i. e. 19 minutes for scallop muscles, and 2 and 18 minutes for gilthead seabream fillets) reduced the thawing losses of both species compared to the longer characteristic freezing times (. e. 235 to 1000 minutes for scallop muscles, and 640 minutes for gilthead seabream fillets) tested. Freezing at short characteristic freezing times produced raw fillets similar in texture to the fresh fillets. Therefore, short characteristic freezing times (equal to or less than 19 minutes) are beneficial for freezing both species. Scallop muscles and gilthead seabream fillets were kept frozen for up to 301 and 340 days, respectively. Sampling was carried out at regular intervals on fresh and stored frozen samples. Storage time affected the integrity of infra-cellular organelles, reduced the water holding capacity, caused structural changes to myofibrillar proteins and affected the sensory attributes of both species. Frozen scallop muscles were in acceptable eating condition after a storage period of ten months, with most of the changes in bio-chemical and physical properties being pronounced after three months of storage. Based on the changes in taste scores versus storage time, it was assessed that the practical storage life of frozen gilthead seabream fillets was circa 5 to 6 months Cat+-ATPase activities for scallop muscles and a linear model that combines free fatty acids, peroxide values and protein content in centrifugal tissue fluids for gilthead seabream fillets, may be reliable methods for industry to use for assessing their quality during long term storage at -22°C.

Tesis por compendio / Aunque el uso de altos niveles de fuentes de proteína vegetal en piensos para doradas de engorde se ha alcanzado con éxito en cuanto al crecimiento, estas dietas todavía están asociadas a efectos negativos en la eficiencia nutricional y en la capacidad inmunitaria. El intestino es el órgano donde se produce la primera interacción entre el pez, los nutrientes y las bacterias del medio, y desarrolla un papel crucial en la digestión de los nutrientes y la respuesta infamatoria e inmune. Esta tesis doctoral se centra en el impacto de distintas dietas con altos niveles de proteína vegetal, y especialmente, en la evaluación del estatus intestinal de las doradas de engorde alimentadas con altos niveles de sustitución de la harina de pescado durante un periodo largo de tiempo. Los cambios observados en el intestino se caracterizaron mediante el uso de distintas estrategias, como el análisis de la digestibilidad y la retención de amino ácidos, de la excreción de amonio, de la actividad de enzimas digestivos, de los cambios histológico o de la expresión de genes relacionados con la función y el mantenimiento de la arquitectura intestinal, así como técnicas ómicas para el análisis del proteoma y de la microbiota intestinal. Se ensayaron distintos niveles de sustitución de harina de pescado, pero el impacto de las dietas con una sustitución completa, bien complementada con subproductos de origen marino o suplementada con aminoácidos libres sintéticos, recibió mayor atención. La sustitución completa de la harina de pescado provocó una reducción, aunque ligera, del crecimiento y de la eficiencia digestiva y nutritiva de la dorada de engorde, aunque el impacto sobre el crecimiento era mayor cuando los peces eran alimentados desde la época de juveniles con estas dietas. La digestibilidad y el nivel de síntesis de proteína se vio alterada, aunque no se observaron diferencias significativas en la actividad enzimática digestiva. No obstante, el impacto de las fuentes vegetales cuando no había fuentes de proteína marina en la dieta era especialmente crítico para la supervivencia de los peces. En el intestino de estos peces solo se observaron diferencias menores relacionadas con la inflamación a nivel histológico, pero también se observó una disminución en la expresión génica de genes involucrados en la inflamación y la respuesta inmune. El análisis de la microbiota intestinal reveló cambios significativos en la composición de su composición, especialmente en el intestino posterior, sugiriendo una posible falta de capacidad de regular la respuesta inmune y de modular la colonización de bacterias patógenas tras un largo periodo de alimentación con esta dieta. Por otro lado, el análisis del proteoma de la mucosa intestinal también mostró un claro impacto sobre distintos procesos biológicos relacionados con el mantenimiento del homeostasis intestinal y de la integridad epitelial. Por el contrario, no se observó un impacto de la sustitución de la harina de pescado a nivel de expresión génica o del proteoma cuando se incorporaba a la dieta una fuente de proteína marina complementaria, aunque sí que se observaron algunos signos menores de inflamación. Por último, se desarrolló un sistema ex vivo para estudiar la respuesta inflamatoria e inmune de la mucosa intestinal a la presencia de distintas bacterias, y se realizó un ensayo preliminar en dorada para evaluar el efecto de la dieta sobre esta respuesta. En resumen, en este trabajo se ha realizado una evaluación extensa y detallada de los efectos a nivel intestinal de la inclusión de altos niveles de proteína vegetal en la dieta para doradas de engorde. Los resultados indican que las alteraciones en la capacidad inmune, la homeostasis y la microbiota intestinal aparecían solo cuando la proteína procedía exclusivamente de fuentes vegetales, y podrían explicar la mayor mortalidad registrada con esta dieta. / Malgrat que la utilització d'alts nivells de proteïna vegetal en pinsos per a dorades en la fase d'engreixament s'ha aconseguit amb èxit en quan al creixement, aquestes dietes encara s'associen amb freqüència amb efectes negatius en l'eficiència nutricional i la capacitat immunitària. L'intestí és l'òrgan on es produeix la primera interacció entre el peix, els nutrients de la dieta i les bactèries de l'ambient, i juga un paper fonamental en la digestió dels nutrients i en la resposta inflamatòria i immune. Aquesta tesi doctoral es centra en l'impacte de diferents dietes experimentals amb un alt nivell de proteïna vegetal, i especialment, en l'avaluació de l'estat de l'intestí de les dorades d'engreixament alimentades durant un llarg període amb alts nivells de substitució de farina de peix. Els distints canvis observats a nivell intestinal es van descriure mitjançant l'ús de distintes estratègies, com l'anàlisi de la digestibilitat i la retenció dels aminoàcids, de l'excreció d'amoni i de l'activitat enzimàtica, dels canvis histològic o de l'expressió de gens relacionats amb la funció i el manteniment de l'estructura intestinal, així com tècniques òmiques per a l'anàlisi del proteoma i de la microbiota intestinal. Es van assatjar diferents nivells de substitució de farina de peix, però l'impacte de les dietes amb substitució completa, bé complementada amb subproductes d'origen marí o suplementada amb aminoàcids lliures sintètics, va rebre major atenció. La substitució completa de la farina de peix va tenir un efecte lleugerament negatiu sobre el creixement i l'eficiència digestiva i nutritiva de la dorada d'engreixament, encara que l'impacte era major quan els peixos eren alimentats des de la fase de juvenils amb aquesta dieta. La digestibilitat i el nivell de síntesis de proteïna es va veure alterada, encara que no s'observaren diferències significatives en l'activitat dels enzims digestius. No obstant, l'impacte de les fonts vegetals quan s'eliminaven per complet les fonts de proteïna marina de la dieta era especialment crític en la supervivència dels peixos. En l'intestí d'aquests peixos sols s'observaren xicotets indicis d'inflamació a nivell histològic, però també es va observar una disminució l'expressió de gens involucrats amb el procés inflamatori i la resposta immune. L'estudi de la microbiota intestinal va revelar canvis significatius en la composició, especialment a l'intestí posterior, suggerint una possible falta de capacitat de regular la resposta immunitària i de modular la colonització per part de patògens després d'un llarg període d'alimentació amb aquesta dieta. D'altra banda, l'anàlisi del proteoma de la mucosa intestinal també va mostrar un impacte clar sobre diferents processos biològics relacionats amb el manteniment de l'homeòstasi intestinal i de la integritat de l'epiteli. Per contra, no es van observar un impacte de la substitució de la farina de peix a nivell d'expressió gènica o proteoma quan s'incloïa a la dieta una font complementària de proteïna d'origen marí, encara que sí que s'observaven alguns signes d'inflamació. Per últim, es va desenvolupar un sistema ex vivo per avaluar la resposta inflamatòria i immune de la mucosa intestinal davant la presència de diferents bactèries, i es va realitzar un assaig preliminar per determinar l'efecte de la dieta sobre aquesta resposta. En resum, en aquest treball s'ha realitzat una avaluació extensa i detallada dels efectes a nivell intestinal de la inclusió d'alts nivells de fonts de proteïna vegetal a les dieta per a les dorades d'engreixament. Els resultats indiquen que les alteracions en la capacitat immunitària, l'homeòstasi i la microbiota intestinal eren observades solament quan la proteïna era exclusivament obtinguda de fonts vegetals, i podrien explicar la major mortalitat observada amb aquesta dieta. / Although the inclusion of plant protein sources at high levels in aquafeeds for on-growing gilthead seabream has been successfully achieved on gilthead seabream in terms of growth, these diets are still associated to detrimental effects in feed efficiency and immune capacity. The intestine is the organ where takes place the first interaction of the host with dietary antigen or environmental bacteria, and plays a major role in the digestion of nutrients and the inflammatory and the immune response. The present PhD thesis focus on the impact of classical formulated high plant protein diets on fish performance, but especially, on evaluation of the intestinal status in on-growing fish long-term fed with high levels of fishmeal replacement. Changes at intestinal level were characterized by using different approaches, including protein and amino acid digestibility and retention and ammonia excretion, digestive enzyme activity, histology, expression of genes related with inflammation, immunity, structure and digestion, but also using whole tissue-level techniques for the analysis of the impact on proteome and gut microbiota. Different levels of fishmeal replacement were assayed, although the impact of diets with total replacement, complemented by inclusion of alternative marine by-products or supplemented by free amino acids, received greater attention. Total fish replacement produced a negative but minor impact on the growth and nutritive and digestive performance of on-growing gilthead seabream. Nevertheless, when fish were fed from juvenile stage with plant protein based diets, a higher negative impact in growth terms was noticed. Digestibility and metabolic use of amino acids was altered, but no differences were observed in the digestive enzyme activities. Nonetheless, feeding fish with total dietary fishmeal replacement by plant protein without any marine protein source was especially critical for survival rate. In these fish, gut histological assessment only revealed minor alterations related with an inflammatory response, but gene expression assay showed a down-regulation of several genes involved in the inflammatory and immune response. Moreover, a drastic change in the microbiota composition was observed, especially at the hindgut, revealing a possible lack of capacity to regulate a defensive response and to face with pathogen colonisation after a long-term coupling with these diet. Likewise, gut mucosa proteome analysis also suggests an impact on biological processes related with the maintenance of gut homeostasis and the epithelial integrity. In contrast, total fishmeal replacement did not induce alterations at transcript or proteomic level when diet was complemented with marine ingredients, although some minor inflammatory signs were reported. On the other hand, an ex vivo system to study the inflammatory and immune response of the gut mucosa to the presence of different bacteria was developed, and a preliminary assay evaluating the impact of the diet on this response was performed. To sum up, present works represents a wide assessment at intestinal level of the effects of including plant protein sources at high levels in aqua feeds for on-growing gilthead seabream. Results indicate that alterations in the immune capacity, the gut homeostasis and the microbiota were observed when protein was exclusively provided by plant sources, and could explain the higher mortality reported with this diet. / Estruch Cucarella, G. (2018). ASSESSMENT OF THE LONG-TERM IMPACT OF HIGH PLANT PROTEIN DIETS ON THE INTESTINAL STATUS OF THE ON-GROWING GILTHEAD SEA BREAM (Sparus aurata, L.) [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/113063 / Compendio

The current general economic and food crises have generated an unsettled future for food and feed production and prices in general. Increasing demand, prices and fluctuations in supply in world markets for fishmeal, fish oil, soybean meal, maize and wheat meal emphasize the need to reduce the dependence of the fish feed industry on these ingredients by increasing choices among a wider range of raw materials. Legume seed such as peas, chickpeas and faba beans are promising ingredients for aquafeeds due to their high protein compared to cereals but also for their energy content. The objective of the present study was to investigate the use of these legumes in both high and low inclusion level in diets for the two main species farmed in the Mediterranean countries namely, European seabass (Dicentrarchus labrax L.) and gilthead seabream (Sparus aurata L.). In Chapter 3 the effects of different processing conditions were examined on whole seed flours of tested ingredients with respect to both nutritional and antinutritional factors and physical characteristics of the experimental diet pellets including high and low levels of each legume. Extrusion of raw material resulted in a clear reduction in trypsin inhibitors (TI) with chickpeas showing a decrease of up to 90% and complete inactivation for wheat flour, while for peas and faba beans reduction was less than 50% in most cases. Extrusion processing was less effective in the reduction of phytic acid and total tannins, occasionally reaching 22% and 18% respectively. Oligosaccharides and total NSP were not drastically affected by processing, however faba bean NSP showed greater reduction. A redistribution of soluble and insoluble NSP fractions was noted for chickpea and pea flours after extrusion. Physical characteristics of the pellets were not negatively affected for any of the tested diets. In Experiment I the effects of total or partial wheat substitution by legumes were investigated on nutrient digestibility, gastrointestinal evacuation rate and serum glucose response of European seabass. Use of legumes in seabass and seabream diets resulted in an overall increase in gastrointestinal evacuation time and a delay in glucose load. Specifically, gastric evacuation time was greatly delayed when seabass was fed a diet with high levels (30%) of chickpeas, while foregut evacuation time was mostly delayed by the diet including a high level (30%) of faba beans. In addition, glucose levels in seabass serum were also affected by the type of carbohydrates ingested with wheat starch showing more rapid increase and decrease of glucose compared to fish fed pea and chickpea diets, while faba bean starch resulted in a lower glucose peak. In Experiment II growth, digestibility, hematological parameters, histological effects and fillet organoleptic characteristics and the interaction between starch inclusion level (8% and 16% or 17% and 35% of legumes respectively) and legume type were estimated when tested legumes replaced wheat in European seabass diets. Digestibility coefficients were satisfactory for all nutrients (>93%) while legume diets at a low level had beneficial effects on growth parameters when compared to the control diet, with chickpeas showing a significant improvement in SGR (P<0.05). High level legume diets did not result in any negative effect on growth. HSI was increased with increasing starch/legume inclusion in the feed and serum glucose also increased for fish fed high levels of faba beans and chickpeas. Carcass proximate composition was not affected by replacement of wheat in the diets, excluding the increase of fat content in fish fed chickpeas. Sensory analysis showed no differences between fish fed the control and high legume inclusion diets. Lastly in Experiment III growth, hematological parameters, histological effects and the interaction between starch inclusion level (low and high) and legume type were evaluated when tested legumes were included in gilthead seabream diets. Decreased, but not significantly so, growth was observed for all diets including legumes compared to the control. Poorer SGR were observed for pea and faba bean diets when these legumes were included at high levels. Liver glycogen increased with increasing starch level, but HSI did not differ significantly for any of the diet treatments. Histological examination of hindgut did not show pathological effects, such as enteritis, for in either species or for any of the diets. Increased absorptive vacuoles were found for control and pea diets (high level) only for seabass. The findings of this thesis showed that the two important species cultivated in Mediterranean countries responded differently to the same raw materials used at high levels in the diets. Overall legumes had a strong effect on gastrointestinal evacuation reducing the rate of feed or digesta passage. Peas, chickpeas and faba beans successfully replaced wheat in seabass diets resulting in improved growth coefficients. However, when the same legumes included in seabream diets growth performance was not improved compared to the wheat based diet.

La daurade royale (Sparus aurata, L.) est une espèce migratrice, connue pour coloniser les lagunes côtières, fortement anthropisées, du printemps à l'automne. Identifier les lagunes clés pour l'accomplissement du cycle de vie doit donc permettre de mieux gérer et protéger les populations de daurades du Golfe du Lion. Dans ce cadre, la microchimie de l'otolithe est un outil précieux. Au cours de sa croissance, cette concrétion de l'oreille interne incorpore des éléments chimiques prélevés dans le milieu. Piégés dans le réseau cristallin, ils sont une image des conditions environnementales rencontrées par le poisson à une date donnée ce qui rend possible, en théorie, de reconstituer ses migrations. Néanmoins, pour cela, il faut (i) connaître précisément la croissance du poisson et de ses otolithes, (ii) mettre en évidence des différences chimiques entre les zones clés pour le cycle de vie en identifiant les outils statistiques appropriés pour la discrimination des habitats. Ainsi, (i) la comparaison objective de modèles de croissance, associée à l'analyse d'un spécimen exceptionnellement âgé a permis de remettre en cause les données classiquement acceptées sur la croissance de la daurade et le schéma de croissance de ses otolithes a été établi ; (ii) la comparaison de 4 méthodes statistiques de discrimination a permis de sélectionner la méthode du random forest pour différencier les signatures chimiques des otolithes de S.aurata de différentes lagunes. Ces avancées ont permis de confirmer le schéma migratoire précédemment décrit, tout en révélant une variabilité des comportements entre les individus et au cours de la vie du poisson. Les juvéniles semblent préférer les lagunes dessalées, suggérant un rôle de nurserie pour ces habitats, alors que les adultes préfèrent des lagunes profondes comme l'étang de Thau. Le maintien de la diversité des caractéristiques des différentes lagunes est donc nécessaire, afin d'assurer la pérennité de la population de S.aurata dans le Golfe du Lion. / The Gilthead seabream (Sparus aurata L.) is a migratory species, known to colonize coastal lagoons with high anthropogenic load, from spring to autumn. It is necessary to identify key lagoons for the seabream life cycle to allow better management and protection of gilthead seabream populations in the Gulf of Lions. For this, otolith microchemistry is a powerful tool. As it grows, this inner ear concretion absorbs chemical elements taken in its surrounding. Traped in the crystal net, these elements are an image of the environmental conditions met by the fish individual. It is then possible to rebuild fish past migrations. For this it is necessary to (i) precisely know the fish and otoliths growth calendars and (ii) identify chemical differences between the key habitats for the life cycle by choosing statistical methods for habitat discrimination.(i) The objective comparison of many growth models, combined to the analysis of an exceptionally old specimen allowed to propose a new scheme for the gilthead seabream growth and its otolith growth pattern was established ; (ii) the comparison of 4 statistical discrimination methods allowed to select the random forest method to differentiate the otolith chemical signatures of gilthead seabreams from various lagoons. These insights confirmed the already described migration pattern and emphasized a behavioural variability between individuals and between periods of life. The juveniles seem to prefer brackish lagoons, suggesting a nursery rôle for these habitats, whereas the adults prefer deeper lagoons as the Thau lagoon. The maintenance of the lagoons diversity is thus necessary for the preservation of the Gilthead seabream population in the Gulf of Lions.

La fraîcheur est un paramètre clé de la qualité du poisson. Les méthodes actuelles appliquées en routine pour déterminer la fraîcheur du poisson ne sont pas applicables à toutes les espèces et reflètent davantage un début d'altération du produit. Ainsi, la recherche d'indicateurs précoces de fraîcheur du poisson représente encore un défi majeur et d'actualité dans l'industrie de la pêche. Le but de ces travaux de thèse était de démontrer que les fonctions et l'intégrité mitochondriales étaient susceptibles de constituer des indicateurs précoces de la fraîcheur de filets de poisson. En effet, la mitochondrie est la "centrale" énergétique de la cellule eucaryote et joue un rôle clef dans les mécanismes de mort cellulaire tels que l'apoptose et la nécrose. Les fonctions et l'intégrité mitochondriales de cellules musculaires de filets de poisson ont été étudiées à différents temps de conservation post mortem à 4°C. Le modèle d'étude était la daurade royale (Sparus aurata) (lignée cellulaire de fibroblastes (SAF-1) et muscles de poisson). Dans un premier temps, la structure des mitochondries de poisson a été étudiée par microscopie électronique à transmission. De nombreuses dégradations de la structure des mitochondries ont été observées dans les filets à partir de 72 heures (J3) de conservation à 4°C. Ces altérations se sont accentuées à J4 et J6. La fonctionnalité des mitochondries a ensuite été évaluée selon deux approches : la respiration mitochondriale (oxygraphie) et le potentiel membranaire mitochondrial (ΔΨₘ) estimé avec la sonde fluorescente Rhodamine 123. A partir de 96 heures de conservation à 4°C (J4), ces deux paramètres ont été significativement impactés témoignant d'une altération des fonctions et de l'intégrité mitochondriales.Ces résultats sont ainsi en corrélation avec l'altération structurale observée par microscopie. En parallèle, une méthode d'évaluation du potentiel membranaire a été développée avec un fluorimètre à microvolume à partir d'un modèle bactérien puis de mitochondries isolées. Ces travaux de thèse ont démontré que l'étude des fonctionnalités mitochondriales constitue un marqueur fiable et précoce de la fraîcheur des filets de poisson. Des connaissances supplémentaires sur les mécanismes cellulaires post mortem ont également été apportées. Ces résultats constituent ainsi le point de départ pour le développement d'un kit d'évaluation de la fraîcheur et ouvrent la voie pour la recherche de marqueurs de fraîcheur et de congélation/décongélation basés sur les fonctionnalités et intégrité mitochondriales. / Freshness is a key parameter of fish quality. Current routine techniques to determine fish freshness are not applicable to all species and reflect a late stage of alteration. Thus, research on early indicators of fish freshness still represents a major and topical challenge in fishing industry. This PhD research project aimed to demonstrate that mitochondrial functions and integrity constitute early indicators of fish fillet freshness. Mitochondria are the powerhouse of the cell and play a central role in cell death mecanisms such as apoptosis and necrosis. Mitochondrial function and integrity in fish filet muscle cells were studied at different times of storage post mortem at 4°C. The species studied as a model was the gilthead seabream (Sparus aurata) (gilthead seabream fibroblast cell line (SAF-1) and fish fillets). Firstly, the structure of fish mitochondria was studied by transmission electron microscopy. Numerous mitochondrial structural alterations have been observed in fish fillet from 72 hours (D3) of storage at 4°C. These alterations were more pronounced at D4 and D6. Then, mitochondrial functionality was assessed with two approaches: mitochondrial respiration (oxygraphy) and mitochondrial membrane potential (ΔΨₘ) estimated with the fluorescent probe Rh123. From 96 hours of storage at 4°C (D4), these two parameters were significantly disrupted demonstrating the alteration of mitochondrial function and integrity. The results are in correlation with the mitochondrial structural alterations described by microscopy. In parallel, a method of mitochondrial membrane potential evaluation has been developed with a micro-volume fluorimeter, first using bacteria and then isolated mitochondria. This work demonstrated that the mitochondrial functionality study constitutes a reliable and early fish filet freshness indicator. Additional knowledge on cell mechanisms in post mortem condition has been brought. These results constitute the starting point for the development of a fish freshness assay kit and pave the way to research on others freshness and freeze-thawing indicators based on mitochondrial integrity and functionality.

Η τσιπούρα είναι ένα είδος με ιδιαίτερο εμπορικό ενδιαφέρον. Στην Ελλάδα η εκτροφή της είναι ιδιαίτερα σημαντική. Παρά τη μεγάλη μελέτη λόγω της εκτροφής του είδους, υπάρχουν ακόμα πολλά αναπάντητα ερωτήματα ειδικά σε θέματα της αναπαραγωγής. Η μελέτη που έγινε δίνει πληροφορίες σχετικά με την πλευστότητα αυγών τσιπούρας εκτροφής, προέλευσης Ατλαντικού και Μεσογείου, φυσικής και όψιμης φωτοπεριόδου, όπως αυτή διαμορφώνεται καθ’ όλη τη διάρκεια της αναπαραγωγικής περιόδου (4,5 μήνες) και σε 7 διαφορετικές αλατότητες (32, 33, 34, 35, 36, 37, 38 ppt). Η αλατότητα ωοτοκίας ήταν 40 ppt. Μετρήθηκαν επίσης οι διάμετροι των αυγών σε δύο από αυτές τις αλατότητες (32 και 35ppt). Τα αποτελέσματα έχουν ιδιαίτερο οικολογικό ενδιαφέρον, καθώς επιτρέπουν τη δόμηση κάποιων υποθέσεων σχετικά με την επίδραση διαφορετικών συνθηκών, στην πιθανότητα επιβίωσης των αυγών και συνεπώς και επανένταξης νέων ατόμων στο φυσικό περιβάλλον. / Gilthead seabream is a species of high commercial interest. It is very important for Greek fish farming. Although, there has been great research on the species, there are still many questions unanswered, especially in the area of reproduction. This study gives information regarding the egg buoyancy of farmed gilthead seabream coming from the Atlantic or the Mediterranean, under natural or late photoperiod, during the whole reproduction period (4,5 months)and under 7 different salinities (32, 33, 34, 35, 36, 37, 38 ppt). Salinity at reproduction was 40 ppt. The diameter of the eggs was also measured (at 32 and 35 ppt). The results have particular ecological interest, as they allow the formation of some hypothesis regarding the influence of different factors in the recruitment of gilthead seabream in nature.

The gilthead seabream (Sparus aurata) and European seabass (Dicentrarchus labrax) require n-3 long-chain polyunsaturated fatty acids (LC-PUFA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), for optimal growth and health. Due to the rapid growth of global aquaculture the quantity of marine oils used in aquafeeds has been limited, yet the overall quantity of oil in an aquafeed has increased by the addition of vegetable oil (VO) to supply dietary energy. For aquaculture to continue to grow more fish must be produced with less marine ingredients, yet EPA and DHA must be maintained at levels above fish requirements. This project set out to re-evaluate the requirement for EPA and DHA in gilthead seabream and European seabass. Two dose-response studies were designed and executed where juvenile seabream and seabass were fed one of six levels of EPA+DHA (0.2 – 3.2 % as fed). Biometric data were collected and analysed to determine new requirement estimates for EPA+DHA for fish of two weight ranges (24 – 80 g and 80 – 200 g). The effects of the dietary LC-PUFA gradient on lipid composition and metabolism were also considered.

Tesis por compendio / [ES] Los ácidos grasos de cadena muy larga (VLC-FA; >C24), aunque presentes en pequeñas cantidades, juegan un importante papel para el correcto desarrollo y funcionalidad de los tejidos neurales en vertebrados, especialmente durante su desarrollo temprano. Sin embargo, a pesar de su aparente importancia, los estudios sobre estos compuestos en peces son escasos. La biosíntesis de los VLC-FA se lleva a cabo mediante las denominadas proteínas de elongación de los ácidos grasos de cadena muy larga 4 (Elovl4) y, en consecuencia, la dotación y la función de estas enzimas determinan la capacidad endógena que una determinada especie tiene para satisfacer las demandas fisiológicas de VLC-FA, especialmente durante su desarrollo temprano. Además, esta producción endógena de los ácidos grasos poliinsaturados de cadena muy larga (VLC-PUFA) es dependiente de los sustratos. Así, para su biosíntesis se requiere de ácidos grasos más cortos, es decir ácidos grasos poliinsaturados de cadena larga (LC-PUFA; C20-24) que actúen como precursores, los cuales son incorporados principalmente a través de la dieta. Teniendo esto en mente, el presente trabajo de investigación tuvo como objetivos caracterizar los genes elovl4 en dorada (Sparus aurata) y en lenguado senegalés (Solea senegalensis), determinar la función de sus correspondientes proteínas codificadas, así como analizar el patrón de expresión tisular de elovl4. Además, se ha investigado la regulación nutricional de los genes implicados en la biosíntesis de VLC-PUFA (elovl4a, elovl4b) y LC-PUFA (fads2, elovl5) durante las primeras etapas del ciclo de vida de ambas especies (larvas y poslarvas), mediante el uso de dietas adaptadas a cada etapa del desarrollo. Los resultados confirmaron que ambas especies de peces poseen dos genes elovl4 distintos, denominados elovl4a y elovl4b según su homología con sus ortólogos de pez cebra. Asimismo, los ensayos funcionales de sus correspondientes proteínas, llevados a cabo en levaduras, indicaron que tanto Elovl4a como Elovl4b tienen la capacidad de elongar los ácidos grasos precursores (C20-24) hasta VLC-FA en ambas especies. Sin embargo, Elovl4b mostró mayor actividad que Elovl4a para elongar todos los ácidos grasos poliinsaturados a productos de cadena más larga, especialmente de la serie n-3. Además, los resultados de expresión génica indicaron que, aunque fueron detectados transcritos de elovl4 en la mayoría de los tejidos analizados, ambos genes elovl4 se expresaron más intensamente en los tejidos neurales de ambas especies, como el cerebro y los ojos, que mostraron los niveles de expresión más altos de elovl4a y elovl4b, respectivamente. Además, los resultados procedentes de los ensayos de regulación nutricional indicaron que los genes fads2, elovl5, elovl4a y elovl4b pueden ser regulados a través del contenido en LC-PUFA presente en la dieta. Es importante destacar que elovl4a y elovl4b fueron regulados de manera distinta según las hipotéticas necesidades de VLC-PUFA asociadas, de manera específica, con cada etapa del desarrollo temprano y dependiendo de disponibilidad dietaria de LC-PUFA. Estos hallazgos pueden contribuir a alcanzar una mejor comprensión de la vía biosintética de los VLC-FA en los teleósteos marinos, resaltando así el papel crucial que los productos de Elovl4 desempeñan para el correcto desarrollo y mantenimiento de las funciones neurofisiológicas durante las primeras etapas del desarrollo de los peces. Asimismo, estos resultados pueden ayudar a esclarecer el mecanismo molecular que controla la biosíntesis de VLC-PUFA, así como a establecer sus requerimientos específicos a lo largo del desarrollo de los teleosteos marinos en función de la especie. De esta manera, se abre la posibilidad de incorporar con éxito fuentes lipídicas alternativas a través de una programación nutricional temprana que estimule la biosíntesis de los VLC-PUFA durante las primeras etapas de alimentación exógena. / [CA] Els àcids grassos de cadena molt llarga (VLC-FA; >C24), encara que presents en petites quantitats, juguen un important paper per al correcte desenvolupament i funcionalitat dels teixits neurals en vertebrats, especialment durant el seu desenvolupament inicial. No obstant, malgrat la seua aparent importància, els estudis sobre aquests compostos en peixos son escassos. La biosíntesi dels VLC-FA es porta a terme mitjançant les denominades proteïnes d'elongació dels àcids grassos de cadena molt llarga 4 (Elovl4) i, en conseqüència, la dotació i la funció d'aquests enzims determinen la capacitat endògena que una determinada espècie té per a satisfer les demandes fisiològiques de VLC-FA, especialment durant el seu desenvolupament inicial. A més, aquesta producció endògena dels àcids grassos poliinsaturats de cadena molt llarga (VLC-PUFA) és depenent dels substrats. Així, per a la seua biosíntesi es requereix d'àcids grassos més curts, és a dir àcids grassos poliinsaturats de cadena llarga (LC-PUFA; C20-24) que actuen com a precursors, els quals són incorporats principalment a través de la dieta. Tenint això en compte, el present treball de recerca va tindre com a objectius caracteritzar els gens elovl4 en orada (Sparus aurata) i llenguado (Solea senegalensis), determinar la funció de les seues corresponents proteïnes codificades, així com analitzar el patró d'expressió tissular de elovl4. A més, es va investigar la regulació nutricional dels gens implicats en la biosíntesi de VLC-PUFA (elovl4a, elovl4b) i LC-PUFA (fads2, elovl5), durant les primeres etapes del cicle de vida d'ambdues espècies (larves i poslarves), mitjançant l'ús de dietes adaptades a cada etapa del desenvolupament. Els resultats van confirmar que totes dues espècies de peixos posseeixen dos gens elovl4 diferents, denominats elovl4a i elovl4b segons la seua homologia amb els seus ortòlegs en peix zebra. Així mateix, els assajos funcionals de les corresponents proteïnes, duts a terme en llevats, van indicar que tant Elovl4a com Elovl4b tenen la capacitat d'elongar els àcids grassos precursors (C20-24) fins a VLC-FA en totes dues espècies. No obstant això, Elovl4b va mostrar major activitat que Elovl4a per a elongar tots els àcids grassos poliinsaturats (substrats) fins a productes de cadena més llarga, especialment de la sèrie n-3. A més, els resultats d'expressió gènica van indicar que, encara que van ser detectats trànscrits de elovl4 en la majoria dels teixits analitzats, tots dos gens elovl4 es van expressar més en teixits neurals de totes dues espècies, com el cervell i els ulls, que van mostrar els nivells d'expressió més alts d'elovl4a i elovl4b, respectivament. A més, els resultats procedents dels assajos de regulació nutricional van indicar que els gens fads2, elovl5, elovl4a i elovl4b poden ser regulats a través del contingut en LC-PUFA present en la dieta. És important destacar que elovl4a i elovl4b van ser regulats de manera diferent segons les hipotètiques necessitats de VLC-PUFA associades, de manera específica, amb cada etapa del desenvolupament inicial i depenent de la disponibilitat dietaria de LC-PUFA. Aquestes troballes poden contribuir a tenir una millor comprensió de la via biosintètica dels VLC-FA en els teleostis marins, ressaltant així el paper crucial que els productes d'Elovl4 exerceixen en el correcte desenvolupament i manteniment de les funcions neurofisiològiques durant les primeres etapes del desenvolupament dels peixos. De la mateixa forma, aquests resultats poden ajudar a esclarir el mecanisme molecular que controla la síntesi endògena de VLC-PUFA, així com a establir els requisits específics de cada espècie al llarg del desenvolupament dels teleostis marins. D'aquesta manera, s'obri la possibilitat d'incorporar amb èxit fonts lipídiques alternatives a través d'una programació nutricional inicial que estimuli la biosíntesi de VLC-PUFA durant les primeres etapes d'alimentació exògena. / [EN] Very long-chain fatty acids (VLC-FA; >C24), although present in small amounts, play important roles for the correct development and functionality of neural tissues, especially during early development of vertebrates. However, despite their putative importance, their study in fish is scarce. Biosynthesis of VLC-FA is carried out by the so-called elongation of very long-chain fatty acid 4 (Elovl4) proteins and, consequently, the complement and function of these enzymes determine the endogenous capacity that a given species has for satisfying the physiological demands for VLC-FA, especially during its early development. Moreover, this endogenous production of very long-chain polyunsaturated fatty acid (VLC-PUFA) is substrate-dependent. Therefore, shorter fatty acid precursors, i.e. long-chain polyunsaturated fatty acids (LC-PUFA; C20-24), are required. These nutrients are mostly incorporated by the diet and their bioavailability can influence the capacity of Elovl4 for satisfying the physiological VLC-PUFA demands in marine fish. Thus, nutritional regulation of elovl4, as well as other elongase and desaturase genes involved in LC-PUFA biosynthesis (elovl5, fads2) has been proposed as a strategy to enhance endogenous production of LC-PUFA and VLC-PUFA in fish farming. The present thesis aimed to characterize elovl4 genes from the marine teleosts Sparus aurata and Solea senegalensis, to determine the function of the corresponding encoded proteins, and to analyze the tissue expression pattern of these genes. Moreover, we investigated the nutritional regulation of genes involved in the biosynthesis of the VLC-PUFA (elovl4a, elovl4b) and the LC-PUFA (fads2, elovl5) in early life-cycle stages (larvae and post-larvae) of both fish species fed diets adapted to each development stage. Live preys were supplied to larvae (early and late larvae) and microdiets for post-larvae, with a variable content in VLC-PUFA precursors, i.e. LC-PUFA. The results confirmed that both fish species possess two distinct elovl4 genes termed as elovl4a and elovl4b based on their homology to the zebrafish orthologs. Functional assays of the corresponding proteins in yeast denoted that Elovl4a and Elovl4b from both species have the capability to elongate C20-24 fatty acid precursors to VLC-FA products. However, Elovl4b appeared to have a higher activity than Elovl4a elongating all the polyunsaturated fatty acid substrates assayed to longer chain polyunsaturated products, especially those of the n-3 series. Moreover, gene expression results indicated that, although elovl4 transcripts were detected in most of the tissues analyzed, elovl4 genes were more strongly expressed in the neural tissues of both species, such as brain and eyes, which showed the highest expression levels of elovl4a and elovl4b, respectively. Furthermore, the results from nutritional regulation assays denoted that fads2, elovl5, elovl4a and elovl4b genes could be regulated by the dietary LC-PUFA content. It is important to highlight that elovl4a and elovl4b genes were differently regulated according to the species-specific VLC-PUFA putative needs, associated with each early life-stage and the LC-PUFA dietary availability. Importantly, these findings can contribute to a better understanding of the VLC-FA biosynthetic pathway in marine teleosts, highlighting the crucial role that the Elovl4 products carry out for the correct development and maintenance of neurophysiological functions during early stages of the fish development. Therefore, these results can help to elucidate the molecular mechanism controlling the VLC-PUFA biosynthesis and their species-specific requirements along the marine fish development, opening the possibility to incorporate successfully alternative lipid sources, through an early nutritional programming that stimulates the VLC-PUFA biosynthesis during the first exogenous feeding stages. / Para desarrollarel trabajo de investigación descrito en esta memoria, Miguel Torres Rodríguez recibió una beca predoctoral de la Excma. Diputación de Castellón. / Torres Rodríguez, M. (2021). Estudio de los patrones de expresión de genes implicados en la síntesis de ácidos grasos de cadena muy larga durante el desarrollo de la dorada y el lenguado, y su regulación nutricional [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/166619 / Compendio