Currently aquaculture is the largest growing food sector in the world, however, its future growth is limited by its heavy reliance on fishmeal (FM). Overfishing of wild marine fish stocks used for FM is putting too much pressure on the stocks, making FM unsustainable as a dominant protein source as aquaculture continues to grow. Plant proteins (PP) are an ideal alternative to FM because of their wide availability and relatively low cost. Soybean meal (SBM) is the most commonly used PP in aquaculture, but its inclusion in feeds is limited due to reduced digestibility and the presence of antinutritional factors ultimately leading to a reduced growth performance of fish that are fed with the SBM-based diet. Therefore, the goal of this thesis was to mitigate the negative effects of PP and improve the growth of fish fed with a PP-based diet, utilizing SBM as our PP in feeds. Three feeding trials were conducted to test the efficacy of 3 approaches towards improving the use of PP in fish. The first trial (Chapter 2), tested the effect of nutritional programming (NP) on the utilization of SBM in zebrafish (Danio rerio). NP is the theory that an organism can be ‘programmed’ to better utilize a dietary component by being exposed to that component in its early life stages. This study also tested the effect of NP through the broodstock by exposing the broodstock to SBM for 4 weeks prior to breeding. In addition, a combination of both programming techniques was also tested. The results found that neither of the programming techniques had a significant effect on the growth performance of the zebrafish. Among the two groups from the programmed broodstock, the group that also received early stage NP had a significantly higher expression of PepT1, a di- and tri- peptide transporter. Also, the dual programmed group had the highest length-to-width ratio of the distal villi among groups that were fed SBM, which signifies an increase in surface area for nutrient absorption in the intestine. The findings from this study suggest that early stage NP may increase the absorption of nutrient from PP-based feeds within the intestine. The second feeding trial (Chapter 3), utilized the supplementation of health-promoting dipeptides to improve the use of SBM-based feeds for zebrafish. The three dipeptides used in this study were alanyl-glutamine, carnosine, and anserine. The five groups in this study consisted of three groups receiving an SBM-based diet with one of the dipeptides supplemented into it, a (-) Control group receiving a non-supplemented SBM-based diet, and a (+) Control group receiving a FM-based diet. Both the alanyl-glutamine and carnosine supplemented groups experienced a significantly higher weight gain compared to the (-) Control group. In addition, the alanyl-glutamine supplemented group had a significantly higher length-to-width ratio of the intestinal villi and, had a numerically higher expression of both nutrient absorption genes measured, PepT1 and fabp2, compared to the (-) Control group. This finding suggests that the supplementation of alanyl-glutamine into SBM-based diets may improve the intestinal absorptive capacity of the fish fed with the SBM. The results from this study also support the use of both alanyl-glutamine and carnosine supplementation as a means of improving the growth performance of fish fed with a SBM-based diet. The third feeding trial (Chapter 4), was conducted on largemouth bass (LMB) (Micropterus salmoides). This study also focused on NP as a means of improving the utilization of dietary SBM, similar to Chapter 2. In this study, live feed was used as a vector to program the larval LMB to SBM. The programmed group in this study received Artemia nauplii that were enriched with a SBM solution, during the larval stage. The bass were then fed with a FM-diet for 7 weeks, before being reintroduced to SBM for the final 6 weeks of the study (PP-Challenge). The programmed LMB experienced a significantly higher weight gain compared to the non-programmed fish also undergoing the PP-Challenge, and achieved a weight gain similar to that of the LMB that were being fed with an FM-diet. In addition, the programmed LMB had significantly longer distal villi and a higher length-to-width ratio of the villi, compared to the non-programmed group. The findings from this study support the use of live feed as a vector for NP and improving the growth performance of a carnivorous aquaculture species fed with a SBM-based diet. The overall findings from these studies suggest that both NP and dipeptide supplementation are feasible means of improving the utilization of SBM in fish. The mechanism behind NP seems to lie in the intestine. In both zebrafish and LMB, NP was found to reduce the inflammatory impact on the intestine and increase the surface area for absorption of the intestinal villi. The supplementation of alanyl-glutamine had similar effects on the intestine as NP and improved the growth performance of zebrafish. The observations from these studies seem to point to mitigating the negative effects of SBM on the intestine as the key to improving the growth performance of fish fed with a SBM-based diet.

The heavy reliance on live feeds is currently restricting the growth and sustainability of the aquaculture industry, therefore, the overall goal of this research was to improve the utilization of formulated dry diets at first feeding of larval fish. This was done with a specific focus on the production and provision of the optimal dietary protein form and composition. Chapter 2 aimed to provide an efficient protein source for larval fish by using same-species muscle and endogenous enzymes to produce hydrolysates and by providing a series of diets with increasing molecular weight protein fragments through larval development. Largemouth Bass (Micropterus salmoides) (LMB) muscle was mixed with the digestive enzymes from adult LMB and hydrolyzed for 1.5, 3, and 6 h, respectively. Five diets were produced, an intact diet containing non-hydrolyzed muscle and four diets with 37% muscle hydrolysate inclusion. The molecular weight profile of those diets were formulated to vary based on the inclusion level of each hydrolysate. To account for gut development, one group of larval LMB was fed a weekly series of diets with an increasing molecular weight profile. The initial inclusion of the hydrolysates significantly improved the total length of the larval LMB; however, neither the hydrolysate inclusion nor the series of dietary molecular weight profiles improved the overall growth of larval LMB. The inclusion of hydrolysates significantly decreased the occurrence of skeletal deformities. The results from this study suggest that the inclusion of same-species hydrolysates can improve the initial growth of first-feeding LMB, but further research is necessary to determine the optimal molecular weight profile, hydrolysate inclusion level, and physical properties of feeds to improve the overall growth performance during the larval stage. Chapter 3 compared the effect of dietary inclusion of a fish muscle hydrolysate produced from species-specific muscle and enzymes to hydrolysates produced from those of a different species, in diets for larval Walleye (Sander vitreus). Four intact and hydrolyzed protein products were produced from each combination of Walleye muscle and endogenous enzymes, and muscle and endogenous enzymes from Nile Tilapia (Oreochromis niloticus). The hydrolyzed products were continuously mixed for 3 h during the hydrolysis, (at 22oC and 28oC for Walleye and Tilapia enzymes, respectively), and the pH was adjusted throughout the process to mimic gastric and intestinal digestion conditions. Four diets were produced with the dietary protein supplied as a 50/50 ratio of the intact and hydrolyzed muscle from the respective muscle/enzyme combination. There was a significant interaction effect between muscle and enzyme source on the growth of larval Walleye. At the conclusion of the study, the larval Walleye that received the diet with muscle hydrolysate produced with Walleye muscle and Walleye endogenous enzymes had a significantly higher average weight than all other groups, and significantly higher postprandial levels of total free amino acids and indispensable amino acids in the muscle. Each hydrolysate-based diet led to a significant reduction in skeletal deformities and survival, compared to a group fed with a commercial diet. The results from this study suggest that species-specific muscle and enzymes produce a more optimal dietary protein source for larval fish than non-species-specific products, but further research should focus on improving the physical properties of the formulated diets to improve survival of fish larvae. Chapter 4 proposed a practical controlled hydrolysis method to utilize the endogenous enzymes within the fish body for the breakdown of tissues proteins, and to produce a species-specific meal that is tailored to the nutritional requirements and absorptive capacity of fish larvae. Four Zebrafish (Danio rerio) meals were produced from whole-body adult Zebrafish, three hydrolysates that were hydrolyzed for 1, 2, and 3 h, respectively, and an unhydrolyzed meal. From these meals, three diets were produced, each defined by their supply of dietary protein. The Unhydro diet was solely based on the unhydrolyzed Zebrafish meal. The 50% Hydro diet was based on 50% Zebrafish hydrolysate mix and 50% unhydrolyzed Zebrafish meal. The 100% Hydro diet was 100% based on the Zebrafish meal hydrolysate. The hydrolysate mix contained equal parts of the 1, 2, and 3 h hydrolysates. Proteomic analysis showed that the proposed hydrolysis method was able to efficiently hydrolyze the protein within Zebrafish body. The feeding trial found no significant differences in the final weight, total length, or survival between the Unhydro, 50% Hydro, and 100% Hydro groups, but the 50% Hydro group did express a significant upregulation of PepT1 at 24 h after feeding, compared to the Unhydro group. The growth results paired with PepT1 gene expression potentially indicate Zebrafish larvae to be adapted to dry feeds at first feeding and able to utilize dietary protein in different molecular forms efficiently for growth. Overall, the proposed hydrolysis method provides a practical and cost-effective approach to producing species-specific fishmeal hydrolysates. Further research is necessary to determine whether the produced hydrolysates can improve the growth of larval fish in other fish models. Further insight into behavioral and physiological responses in fish to imbalanced dietary amino acid profiles was provided in Chapter 5. The objective of this study was to determine how stomachless fish respond to diets deficient in the main limiting IDAA (lysine, methionine, and threonine), using Zebrafish as a model species. Six semi-purified diets were formulated for this study. The CG diet contained casein and gelatin as its only protein sources, while FAA50 diet had 50% of is dietary protein supplied with crystalline amino acids. Both were formulated to contain identical, balanced amino acid profiles. The remaining diets were supplied with the same amino acid mix as the FAA50 diet, but with minor adjustments to create deficiencies of the selected IDAA. The (-) Lys, (-) Met, and (-) Thr diets had lysine, methionine, and threonine withheld from the free amino acid (FAA) mix, respectively, and the Def diet was deficient in all three. The fish were fed to apparent satiation three times a day, and each feeding was carefully observed to ensure all feed added to the tanks was consumed. The results showed that although the singular deficiency of the three main limiting amino acids did not induce significant changes in feed intake, the combined deficiency of the three IDAA significantly increased the feed intake of juvenile Zebrafish. This increased feed intake prevented the IDAA deficiencies from significantly reducing growth, however, the feeding efficiency was also reduced. There was also an observed upregulation of neuropeptide Y (NPY), an orexigenic hormone, in the Def group, compared to the FAA50 group. The outcomes of this study provide insight into the behavioral and physiological response to dietary amino acid imbalances of stomachless fish and suggests stomachless fish increase their feed intake when challenged with IDAA-deficient diets, and that the regulation of NPY might play a role in this response. Chapter 6 assessed the postprandial FAA dynamics in the plasma, liver, and muscle of three species; 1) Largemouth Bass – warm-water, stomach-possessing carnivorous species; 2) Walleye – cool-water, stomach-possessing carnivorous species; and 3) Zebrafish– tropical, stomachless omnivorous species. Two diets were formulated for this study, a diet based on intact casein and gelatin (CG), and a diet with 50% of its protein supplied in FAA form (FAA50). Forty-two fish from each species were utilized, with one group of 21 receiving the CG diet, and the other 21 receiving the FAA50 diet. All fish were starved for 24 hours prior to the final feeding before sampling. Three fish were sampled at each time point, with three samples (plasma, liver, and muscle) taken from each fish. Samples were taken prior to feeding (0 h) and then at 0.5, 1, 2, 3, 6, and 12 h after feeding, for all species. A significant three-way interaction was observed between the diet, species, and postprandial time on the total FAA, IDAA, and DAA levels in the plasma, liver, and muscle, indicating that the postprandial FAA patterns were significantly different between species and in response to the different diets. In stomach-possessing species, dietary amino acids from the FAA50 diet were absorbed more rapidly than those from the CG diet, resulting in fewer correlations with the dietary IDAA profiles. The absorption of FAA in cool-water Walleye was more gradual and prolonged than the warm-water LMB, leading to more significant correlations with the dietary IDAA and more sustained peaks. The postprandial peaks of FAA typically occurred at the same time in the stomachless Zebrafish fed with the CG or FAA50 diet. The levels of FAA were noticeably lower after feeding with the FAA50 diet in Zebrafish, compared to the CG diet. These results provide a reference for differences in the FAA dynamic patterns of three species with differing physiological characteristics, when fed diets with intact protein or supplemented with FAA. The findings presented in this dissertation provide support and novel methods for the production and inclusion of species-specific protein hydrolysates as an ideal protein source in formulated diets for first-feeding larval fish. This research contributes to the development of larval diets that can release the limitations of growth placed on the aquaculture industry by the reliance on live feeds, particularly within the hatchery sector. This research also provides further understanding of dietary protein utilization and delivers new fish nutrition knowledge that will benefit the aquaculture industry as a whole.

Submitted by Caroline Falcao (caroline.rfalcao@ufpe.br) on 2017-05-22T17:37:54Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Tese-Aurelyanna- OCEANOGRAFIA.pdf: 849615 bytes, checksum: 58c710c655e4bb0252be1dd111aa04c0 (MD5) / Made available in DSpace on 2017-05-22T17:37:54Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Tese-Aurelyanna- OCEANOGRAFIA.pdf: 849615 bytes, checksum: 58c710c655e4bb0252be1dd111aa04c0 (MD5) Previous issue date: 2014-08-22 / O alimento vivo é um fator fundamental para o desenvolvimento larval de crustáceos decápodes. Copépodos se destacam como alimento vivo para larvas carnívoras devido ao seu excelente valor nutricional, alta digestibilidade e tamanho adequado. Porém, o uso destes organismos em larviculturas ainda depende do aprimoramento das técnicas de cultivo. O presente trabalho foi dividido em três capítulos. O objetivo dos capítulos 1 e 2 foi aprimorar a técnica de cultivo do copépodo harpacticóide Tisbe biminiensis e testar novas dietas. O cultivo dos copépodos foi realizado em volume de 15L em bandejas plásticas de 0,37m2 adaptadas com drenos para facilitar a troca de água e coleta da prole. No Capítulo 1 foram testadas as rações Alcon Basic® Dieta Controle (RC), ração experimental para peixes Op0 (RE), ração comercial para peixes Nutripeixe AL55® (RP) e ração comercial para camarão marinho Camaronina CR1® (RCM). Apesar da boa aceitabilidade de todas as rações, diferiram de RC a ração RE, apresentando valores inferiores de produção de prole do início dos experimentos até o primeiro pico de produção e a ração RCM, que apresentou resultados inferiores na fase de estabilização da população total e no pico de produção de prole. A produção diária por caixa de cultivo foi de 9.000 ind. L-1 ou 364.864 ind m2 . No Capítulo 2 a dieta controle (RC) foi comparada a dieta teste (RT) ração comercial para peixes marinhos, NRD 5/8, INVE. Durante o período experimental, a produção de prole obtida com a RT não diferiu da produção obtida com a RC, sendo em média 7.400 ind. L-1 . dia -1 ou 300.000 ind. m2 . No Capítulo 3 foi avaliado o desempenho da prole de T. biminiensis como substituta dos náuplios recém-nascidos de Artemia, na alimentação das pós- larvas (PL1 a PL10) do camarão marinho Litopenaeus vannamei. Três tratamentos foram testados: T1 – Controle Artemia, T2 – Mix: Artemia + T. biminiensis e T3 – T. biminiensis. A sobrevivência no T1 foi significativamente maior em comparação com as demais (T1 100% > T2 52% = T3 51%). O comprimento larval foi significativamente maior no tratamento T2 (T2 7.9 mm > T1 7.1 mm = T3 6.8 mm) e não houve diferenças no peso seco. As adaptações realizadas nos sistemas de cultivo reduziram o esforço de coleta e a boa aceitação de T. biminiensis aos diversos tipos de ração reduziram os custos de produção. A quantidade produzida ainda é baixa, mas pode satisfazer a necessidade de setores como a aquicultura ornamental. Os bons resultados alcançados em termos de crescimento e peso no T2 indicam que a combinação de alimentos melhora a qualidade das pós-larvas. A menor sobrevivência das pós-larvas de L. vannamei nos tratamentos T2 e T3 podem estar relacionada a predação, pelos copépodos, de pós-larvas debilitadas. A presença de bactérias patogênicas provindas dos cultivos de copépodos deve ser investigada no futuro. Os resultados indicam que na ausência ou restrição de cistos de Artemia no mercado o cultivo de copépodos poderia vir a representar uma alternativa para as larviculturas desta espécie. / The live food is essential for optimal larval development of decapod crustaceans. Copepods offer excellent nutritional quality, a high degree of digestibility and an adequately small size. However, the use of these organisms in hatcheries still depends on the improvement of farming techniques. This work was divided into three chapters. The aim of Chapters 1 and 2 was to improve the technique of cultivation of harpacticoida copepod Tisbe biminiensis and test new diets. The cultures were performed in plastic trays (0.37 m2 ) in volumes of 15 L with drains to facilitate the water exchange and collection of the offspring. In chapter 1, were tested control diet Alcon Basic® (CD) an experimental diet (ED) for Rachycentron canadum juveniles Op0; a commercial diet Nutripeixe AL55® (FD) and a commercial diet for marine shrimp Camaronina CR1® (MSD). In comparison to the CD treatment, the ED led to significantly lower offspring production from the beginning of the experiment through to peak production and the MSD achieved significantly poorer results regarding the stabilization phase of the overall population and peak offspring production. The results of the daily production per cultivation unit was 9000 ind. L-1 or 364,864 ind m2 . In Chapter 2 the control diet (CD) was compared to test diet (TD) commercial feed for marine fish, NRD 5/8, INVE. During the experimental period, the production of offspring obtained with TD did not differ in yield with the RC, averaging 7,400 ind. L-1 . ind day -1 or 300,000. m2 . In Chapter 3 was evaluated the performance of offspring of the T. biminiensis like substitute of nauplii of Artemia in feeding post-larvae (PL1 to PL10) of the marine shrimp Litopenaeus vannamei. Three treatments were tested: T1 - Artemia Control, T2 - Mix: T. biminiensis + Artemia and T3 - T. biminiensis. The Survival in T1 was significantly higher compared with the other treatments (T1 100% > T2 52% = T3 51%). Larval length was significantly higher in T2 (T2 7.9 mm > T1 7.1 mm = T3 6.8 mm) and no differences in dry weight. The adaptations carried in plastic trays reduced the harvest effort and the. The good acceptance of T. biminiensis the different types of diet reduce production costs. The number of offspring produced is still low, but it can satisfy the sectors such as ornamental aquaculture. The good results achieved in T2 (growth and weight) indicate that the combination of foods improves the quality of post- larvae. The lower survival of post-larvae in treatments T2 and T3 may be related to predation of the weakened post-larvae by copepods. The presence of pathogenic bacteria in cultures of copepods should be investigated in the future. The results indicate that in the absence or restriction of Artemia cysts, the copepods cultures may represent an alternative to the hatcheries.