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Aquaculture is probably the fastest-growing food-producing sector, accounting for nearly 50 percent of the world’s food fish [...]

Póster presentado en el XX International Symposium on Fish Nutrition and Feeding "Towards Precision Fish Nutrition and Feeding", celebrado en Sorrento (Italia) entre el 5 y el 9 de junio de 2022., The digestive tract houses a consortium of microorganisms that plays critical roles in the nutrition and health of the host. Gut microbiota changes during the daily cycle, however, there is limited knowledge on absolute (vs. relative) temporal microbiota abundance variation (Vandeputte et al., 2021). Water temperature is a crucial factor in fish production, influencing among others feed intake and transit time. The aim of the present study was to assess the effect of rearing temperature and daily feeding rhythms on the fish gut microbiota. The greater amberjack (Seriola dumerili) was used as experimental species, a promising species for the diversification of Mediterranean aquaculture. Fish were randomly distributed in three independent RAS systems set to 18, 22 and 26°C. Juveniles were reared for 12 days and fed ad libitum three times daily with a commercial diet. Then, nine fish per temperature were sampled every 6 h for 24 h for fecal microbiota analysis. Results from 16S rDNA qPCR revealed no effect of temperature and sampling time on bacterial abundance per mg of gut content. By contrast, when focusing on the quantity of active cells (qPCR of 16S rRNA), differences with respect to sampling time but not temperature, were observed. The bacterial activity increased a factor 30 due to feeding but was reduced to prefeeding levels four hours after the last meal. This pattern was also observed in the specific bacterial activity (RNA:DNA ratio), with almost 2 orders of magnitude increase during the period of active feeding. At 18°C a higher dispersion in the results was observed, probably because of the slower gut transit time. Regarding community composition, feeding promoted an increase in the relative and absolute abundance of the order Vibrionales. In conclusion, this is the first study assessing the quantitative and qualitative effects of water temperature and daily feeding rhythm on the gut microbiota in fish. Feeding was the main driver modulating bacterial activity and suggest resource limitation of the gut microbiota during non-fed periods. However, low temperature seems to slow down the process, probably due to a slower digestive process., Project RTI2018-096134-B-I00 (MCIU-AEI, Spain + FEDER)

Trabajo presentado en el XX International Symposium on Fish Nutrition and Feeding "Towards Precision Fish Nutrition and Feeding", celebrado en Sorrento (Italia) entre el 5 y el 9 de junio de 2022.

Trabajo presentado en el Aquaculture Europe 2021, celebrado en Madeira (Portugal), del 4 al 7 de octubre de 2021, • Temperature is one of the most important factors regulating the rate of many biological processes. • The study of fish digestive biochemistry is essential to obtain valuable information on factors affecting food transformation and growth, and therefore Aquaculture profitability. • The greater amberjack, Seriola dumerili, is a fast-growing pelagic teleost with great interest for the diversification of marine fish aquaculture in the Mediterranean region., This research was funded by the project RTI2018-096134-B-I00 from the Spanish Ministry of Science granted to MY. CN-G was supported by the researcher contract DOC_01203 funded by PAIDI 2020.

Trabajo presentado en el 18th International Symposium on Fish Nutrition and Feeding (ISFNF. 40 years of research in fish nutrition), celebrado en Las Palmas de Gran Canaria del 3 al 7 de junio de 2018., Efficient macronutrients digestion depends on the time the feed is available for hydrolysis within the different sections of the digestive tract under the appropriate digestive enzymes. In fish, feed transit rates usually are estimated from gut filling or evacuation after a unique meal. Nevertheless, food residence time within the gut is dependent on the feeding frequency, as well as on the environmental conditions. In this study we have analyzed the transit rate in the stomach and intestine in early juveniles of cobia (Rachycentron canadum) under routine feeding and at two different temperatures. Juveniles (3.7±0.4 g wet BW) were randomly distributed to 6 experimental 200-L tanks (60 fish tank-1) and reared at two temperatures (30 and 34 °C) in recirculation systems for six weeks. Fish were fed twice a day (8:00 and 16:00 h) with a diet containing 47% protein and 10% lipid. The diet included yttrium (Y) as marker only in the morning meal. At the end of the experiment, fish were sampled every 4 hours and Y-content in the stomach and intestine was analyzed by ICP-mass spectrometry. Total gut content at each sampling time was estimated from the weight of the stomach and intestine, and the transit rate from the difference of Y content between successive sampling. In juvenile cobia, mass and volume of the stomach are much higher than in the rest of the intestine. The feed transit rate in the stomach was notably faster at 34 than at 30 °C during the first eight hours post-feeding; afterwards the rate was similar at both temperatures. Contrarily, no differences were found in the intestine, but transit rate declined drastically to residual values 12 h post-feeding. The increased temperature induced a change of filling pattern and a decrease of food residence time in the stomach., Project WISEFEED funded by the European Union's H2020 program (Marie Skłodowska-Curie grant No 691150).

Trabajo presentado en el 18th International Symposium on Fish Nutrition and Feeding (ISFNF. 40 years of research in fish nutrition), celebrado en Las Palmas de Gran Canaria del 3 al 7 de junio de 2018., Water temperature is a key factor affecting metabolic rates and therefore fish feeding physiology and behavior. These effects have a special significance in the scenario of the global warming. In this study we have examined effects of an increased water temperature on the daily pattern of gastrointestinal pH, enzymatic proteolytic digestive activity and the food transit time in early juveniles of cobia (Rachycentron canadum), a candidate species for aquaculture in tropical and subtropical waters. Juveniles (3.7±0.4 g wet BW) were randomly distributed to 6 experimental 200-L tanks (60 fish tank-1) and reared at two temperatures (30 and 34 °C) in recirculation systems for six weeks. Fish were fed twice a day (8:00 and 16:00h) with an experimental diet containing 47% protein and 10% lipid. At the end of the experiment, fish were sampled every 4 hours to obtain daily patterns of processes related to digestive activity. Cobia reared at 30oC grew faster and with higher FCR than those at elevated temperature (34oC). The fish reared at high temperature had lower intestinal luminal pH but similar gastric pH. Overall the pepsin activity during the complete daily cycle, but it was significantly different from the fish maintained at 30oC only at 8 hours post-feeding. Similarly, the trypsin activity was not significantly affected by the water temperature but tended to be higher at 34°C during the first hours postfeeding. The transit rate was notably faster 34°C in the stomach but similar in the intestine. These results indicate that the slight increase of proteolytic activity that was observed at 34°C cannot compensate for the faster gut transit rate. The reduced time the dietary proteins are available for hydrolysis can explain the lower growth observed at this temperature., Project WISEFEED funded by the European Union's H2020 program (Marie Skłodowska-Curie grant No 691150).

Trabajo presentado en el 7th Fish & Shellfish Larviculture Symposium (Larvi 2017), celebrado en Gent del 4 al 7 de septiembre de 2017., Larval rearing is the most sensitive phase in marine fish farming due to animal small size, high mortality rates, and imappropriate rearing protocols that later may induce poor performance in post-llarvae and juveniles. The better way to avoid nutritional problems is to adapt th.e feeding protocols to the larval feeding pattems and nutritional requirements. Feeding is a complex process including food uptake, digestion, and processing of the absorbed nutrients, and many metabolic and physiological processes that are rhythmically regulated in order to accomplish the daily energetic demands. Therefore, the objective of the present work was to determine the biological basis that regulates the digestive function throughout the daily cycle during Senega.lese sole larval and post-larval stages in order to improve the digestive efficiency., Acknowledgements: financed by projects RIDIGEST (AGL2011-23722; MINECO; Spain) and SOLEAWIN (310305/FEP/71; Portugal) with FEDER/ERDF contribution and CCMAR/Multi/04326/2013 (Portugal) from FCT. Sofia Engrola was supported by FCT investigator grant IF/00482/2014/CP1217/CT0005 funded by the European Social Found, the Operational Programme Human Potential and the Foundation for Science and Technology oí Portugal (FCT).

Trabajo presentado en el Aquaculture Europe 16 (Food for Thought), celebrado en Edimburgo del 20 al 23 de septiembre de 2016., The Atlantic bluefin Tuna (Thunnus thynnus) is one of the most commercially valuable marine fish species. Therefore, considerable research effort is being done since many years to advance in the farming techniques of this species. A good knowledge of the main events occurring at ontogenetic level is essential to advance in the design of more appropriate feeds and feeding protocols during the larval stage. In a first study we examined the organogenesis and development in general at histological level (Yúfera et al., 2014). In this study we have examined the activity of some digestive enzymes during the first weeks of life. With this aim the tuna larvae have been reared under mesocosm system with copepods as primary food source., This research was financed by Project WEANTUNA (Eurostar E!7994) with FEDER/ERDF contribution.

Trabajo presentado en Aquaculture Europe (Adding value), celebrado en San Sebastián del 14 al 17 de octubre de 2014., To attain higher growth, and consequently, greater profitability in hatcheries it is essential to understand species feeding behavior and nutritional requirements. It has been reported that larvae do not display constant food ingestion under natural or laboratory conditions but have daily feeding rhythms (Kotani & Fushimi, 2011). Sole is a species with a complex metamorphosis, which implies important changes in food habits and in digestive physiology. In juveniles and adults it was suggested the existence of circadian feeding rhythms (Navarro et al., 2009), nevertheless, the larval daily feeding rhythm and the potential effect on digestive and efficiency remain unknown.The aim of this study was to understand the daily feeding behavior and digestive metabolism of Senegalese sole post-larvae (35 days post hatching-dph) under a 12h light:12h dark photoperiod in order to improve feeding protocols and promote growth., Project AGL2011-23722 (MINECO, Spain, and FEDER/ERDF). S. Engrola acknowledge financial support by Fundação para a Ciência e a Tecnologia, Portugal, throught grant SFRH/BPD/49051/2008.

Trabajo presentado en eñ 16th International Symposium on Fish Nutrition and Feeding (ISFNF 2014), celebrado en Cairns (Australia) del 25 al 30 de mayo de 2014., Both the improvement of current feeding protocols of marine fish larvae based on live prey and the design of suitable inert feeds requires a good knowledge of the complexity of larval feeding behavior and of factors modulating food processing. Some of these factors affecting net efficiency of the digestive process are: the species, developmental stage, and diet composition. On the other hand, daily feeding ration and number of meals per day affect the absorption and assimilation of nutrients, and hence final growth. Senegalese sole (Solea senegalensis) has become a species with high commercial interest for aquaculture. The rapid physiological changes that Senegalese sole larvae undergo throughout development, including a pelagic pre-metamorphic and a benthic post-metamorphosis phase, complicate the determination of their feeding needs during larvae and post-larvae rearing. In this study, the daily feeding behavior and digestive enzymes activity in S. senegalensis were analyzed in pre-metamorphic (3 and 6 days post hatching; dph) and post-metamorphic larvae (20 and 30 dph) during a 24h cycle, with a photoperiod of 12 light:12 dark. Gut content was measured by counting rotifers mastax in pre-metamorphic larvae and by weighing the dissected gut in post-metamorphic larvae. Enzymes activity (trypsin and lipase) was analyzed individually per larvae using fluorogenic substrates. Results showed that Senegalese sole larvae have a diurnal feeding behavior during the pelagic phase, reaching the highest gut content at the end of the light period. On the other hand, metamorphosed larvae showed feeding activity during the whole daily cycle, although the gut content tended to be higher during nocturnal hours. Trypsin activity at pre-metamorphic stages was closely related to the amount of food present in the digestive tract while in post-metamorphic larvae the activity kept almost constant during the whole day, with the exception of first and last hours in the day, when activity levels were lower. Regarding to lipase activity, results point to a continuous production of lipase, although activity levels tended to be higher during dark phase in all stages. These results clearly confirm that moving from pelagic to benthic life involves a change in the processes related to feeding and digestion by sole post-larvae. Future experiments will explore whether this pattern may continue changing beyond the first month of life up to acquiring a definitive juvenile feeding strategy. This knowledge will allow the designof more appropriate feeding protocols during the first weeks for a better nutrient utilization and therefore enhanced growth and development., Funded by Project AGL2011-23722, MICINN, Spain and FEDER/ERDF.

Trabajo presentado en Aquaculture Europe (Adding value), celebrado en San Sebastián del 14 al 17 de octubre de 2014.

Trabajo Fin de Máster.-- Universidade do Algarve. Faculdade de Ciências e Tecnologia. Mestrado em Biologia Marinha., [EN] The greater amberjack, Seriola dumerili, a fast growing pelagic fish, was one of the species selected to increase the farmed fish variety in Europe, due to its adaptation to captivity conditions, good flesh quality and high market demand. Inadequate feeding protocol has been suggested as one of the causes behind mortalities during the larval stage. Aiming to advance feeding optimisation during the early stage of this species, this study examines the ontogeny of digestive function using molecular and biochemical approaches. Gene expression of digestive enzyme precursors and the enzyme activity has been determined during the first 51 days post hatching (dph) and the diurnal period of 19 dph larvae, reared in semi-intensive conditions. The expression of pancreatic proteases precursors (try3, ctra and ctrb1) increased from first-feeding, while gastric chitinase (chia1), gastric protease (pga3) and proton pump (atp4a2) after 10 dph. The precursor genes of pancreatic lipases (cel1, cel2 and cel3) peaked between 6 and 10 dph, phospholipase A2 (pla2g1b) rose only after 25 pdh, while α-amylase (amy2a) increased mainly from 20 dph. The trypsin activity was more evident from first-feeding to 25 dph and chymotrypsin activity from this day onwards. Acidic chitinase and pepsin activity appeared, respectively, at 16 and 30. The activity of 7C-like lipase was evident from first-feeding but, similarly to 4C-like lipase significantly increased from 15 dph, while amylase peaked from 6 to 22 dph. Aminopeptidase and alkaline phosphatase activities started at 20 dph indicating the functional maturation of brush border of the enterocytes. The daily pattern analysis showed a food anticipatory strategy in the expression of proteases related genes. Lipases activity was more evident during the morning hours, followed by amylase, and, in the afternoon, by proteases. Results, also, suggested an alternation in the activity of chymotrypsin and trypsin. These ontogenetic patterns are concordant with a carnivorous marine teleost., [PT] A limitação principal no setor da aquicultura europeia é a baixa diferenciação de espécies em cultivo. Ao longo da última década, os investimentos em investigação no domínio da aquicultura procuraram aumentar a pouca variedade de peixes de cultura, com sucesso. Uma das espécies visadas é a Seriola dumerili, um peixe carnívoro, de crescimento rápido e pelágico. Este é selecionado pela sua adaptação às condições de cativeiro, boa qualidade da carne, elevado valor de mercado e procura. Contudo, o potencial industrial desta espécie é ainda limitado pela baixa produção em massa de juvenis de alta qualidade. Uma elevada taxa de mortalidade, sobretudo durante a transição da alimentação exógena-endógena e desmame limitam a sua produção, além das doenças e do canibalismo. Um importante avanço no cultivo larvar foi verificado com a implementação da tecnologia de mesocosmos e com a inclusão da Artemia e de rotíferos na dieta. Apesar disso, vários estudos sugerem que estes problemas ainda não estão totalmente resolvidos, devido a uma lacuna de conhecimento dos requerimentos nutricionais e fisiologia digestiva durante o crescimento das larvas. Na verdade, é referida a falta de sincronia entre a composição do alimento e o desenvolvimento do sistema digestivo. Foi avaliada a atividade de apenas algumas enzimas nesta espécie mas a expressão genética dos seus precursores e os padrões enzimáticos e genéticos diários nunca foram tratados na Seriola ssp. Com esse objetivo, este estudo pretende obter pela primeira vez um conhecimento mais aprofundado sobre o desenvolvimento da capacidade máxima digestiva em larvas e juvenis precoces do lírio (Seriola dumerili). As larvas foram criadas em condições intensivas, num sistema de aquicultura com recirculação de água (RAS). Com abordagens de biologia molecular e bioquimica, foram avaliadas a expressão dos genes de precursores de enzimas digestivas e a atividade das mesmas enzimas. As amostras incluíram larvas desde os primeiros 30 (expressão) e 51 (enzimas) dias pós-eclosão (dpe). Simultaneamente, foram determinados padrões diários de expressão e atividade enzimática em larvas com 19 dpe, com amostragens periódicas das 7:30h às 21:00h. Se realizou a extração do RNA das larvas e, por transcrição reversa, obteve-se cDNA. A seguir, mediante primers específicos e inclusão de marcadores multiplicaram os genes de interesse e, finalmente, foi avaliada a sua expressão. Por outro lado, para a atividade enzimática, as larvas foram homogeneizadas, isolando o extrato de enzimas pancreáticas e o extrato das células da borda em escova. A utilização de kits apropriados com protocolos específicos permitiu determinar a atividade enzimática, através de medições de absorção e fluorescência. A atividade de proteases, lipases e carboidrases e os respetivos padrões de expressão dos precursores foram medidos e comparados, para ajudar a esclarecer os mecanismos de regulação. A expressão dos precursores de proteases alcalinas (tripsina, quimotripsina) e de lipases ativadas por sais biliares foi registada antes e próximo da abertura da boca, respeitivamente. Após a transição para alimentação exógena, observou-se expressão dos carregadores de peptídeos e da quitinase ácida, mas só após o dia 20 registou-se expressão do mRNA de protease ácida, amilase e fosfolipase. Em particular, a pepsina e os precursores atp4ase mostram uma forte sincronia com o desenvolvimento do estômago. Uma atividade precoce da 7C-like lipase parece ter um papel na absorção dos nutrientes do saco vitelino antes da abertura da boca. Durante os primeiros dias de alimentação exógena, o metabolismo das proteínas e dos hidratos de carbono parece ser já relevante. Uma vez que o teor de hidratos de carbono na dieta é baixo, o papel da amilase é ainda desconhecido. Calcula-se que participe na digestão de rotíferos. Do 10 ao 22 dpe, o metabolismo dos lípidos ganha importância, especificamente com a 7C-like lipase. Estes resultados podem evidenciar algumas deficiências na assimilação de proteínas durante o estado larvar, embora outros estudos os considerem comuns. Com o início da alimentação com ração, o metabolismo das proteínas volta a ser altamente relevante. Com efeito, a quimotripsina, a aminopeptidase e a fosfatase alcalina começaram subitamente a manifestar a sua atividade. Por estes motivos, o desmame poderá ter influência na produção da atividade enzimática. Contudo, os valores destas três enzimas diminuem rapidamente, na presença da produção de pepsina, que é um dos fatores indicadores da mudança para digestão adulta. Surpreendentemente, a pepsina não coincide com a atividade da quitinase ácida, que aparece no 19 dpe. A sua síntese deveria ser simultânea, dado que estão relacionadas com o funcionamento do estômago. Uma única larva usada como amostra, em vez de um pool, e a resistência da quitinase alcalina em pH baixo podem justificar estes resultados. Ao mesmo tempo, também se coloca a hipótese de uma falta de sincronia entre o desenvolvimento do estômago e a produção de pepsina. As 4C e 7C-like lipases mostram uma diminuição após o dia 30, sugerindo uma mudança para lípidos mais complexos. A análise dos padrões diários mostrou que a expressão génica se mantém mais constante comparada com a atividade. Entre todos os genes, apenas as transcrições de mRNA relativo ás proteases apresentaram um padrão diário inconstante. Com efeito, os carregadores de tripsina, quimotripsina e peptídeos mostram padrões mais elevados durante a manhã. Como relatado sobre outras espécies, considera-se que uma adaptação aos ritmos de alimentação estará relacionada com a programação genética. Contrariamente às proteases, as outras enzimas não mostram variações durante o período de estudo. Na análise enzimática, são verificadas mais flutuações. Por um lado, as larvas parecem reagir à ingestão de alimento com um aumento de atividade de proteases e amilase. Em particular, a quimiotripsina cresce em poucas horas, enquanto a tripsina leva mais tempo, atingindo o pico no final da tarde. Uma elevada atividade de tripsina à noite poderá explicar os baixos valores obtidos na análise ontogenética. Por outro lado, as larvas concentraram atividade de lipase nas primeiras horas da manhã, imediatamente antes da alimentação. De forma semelhante à expressão das proteases, as lipases parecem estar envolvidas num mecanismo de antecipação, que limita a perda de energias. Este fator, de acordo com os resultados ontogenéticos, mostra uma vez mais a importância do metabolismo dos lípidos nesta fase larvar. Tal como verificado em outros estudos, muitas vezes os padrões de expressão não correspondem aos padrões enzimáticos, sugerindo um importante papel e impacto dos mecanismos de regulação. A este propósito, é sabido que muitas respostas metabólicas são determinadas por genes induzidos por fatores ambientais, cujos mecanismos são ainda desconhecidos. Os resultados de este estudo aumentam o conhecimento da ontogenia, fisiologia e metabolismo do trato digestivo em larvas de Seriola dumerili, apontando uma direção para mais estudos. Novas noções deverão melhorar a otimização dos protocolos de alimentação e favorecer a sustentabilidade das culturas. Como resultado, o aumento das taxas de sobrevivência e crescimento irá melhorar os rendimentos dos produtores e os objetivos de sustentabilidade.