Orientador: Luiz Edivaldo Pezzato / Coorientador: Márcia M. Pereira Sartori / Banca: Elyara Maria Pereira-da-Silva / Banca: Ademir Fernandes Calvo Junior / Banca: Ana Lúcia Salaro / Banca: Hamilton Hisano / Resumo: A hierarquia de dominância e o confronto agonístico causam estresse aos animais. Essa pesquisa objetivou quantificar o efeito do estresse após o estabelecimento da hierarquia social avaliando as respostas metabólicas entre juvenis de tilápia-do-Nilo. Foram distribuídos 20 peixes machos em 10 aquários com capacidade de 110 litros, adotado um delineamento inteiramente cazualizado totalizando cinco tratamentos com quatro repetições. Os animais foram alimentados com ração prática contendo 28,0% de proteína bruta e 3000 kcal de energia digestível. Foi realizada a divisão hierárquica dos indivíduos em dominantes e submissos segundo Falter (1983). Para o tratamento de comunicação visual, os aquários foram divididos com placas de vidro translúcidas, onde os animais não tiveram contato "físico" não havendo nenhum tipo de contato coma água do coespecífico; para o tratamento de comunicação química havia um sistema de bombeamento que homegeneizava a água dos animais, não havendo nenhum tipo de contato visual com o coespecífico. O animal isolado não recebeu água de nenhum outro peixe e nem teve contato visual. As avaliações do efeito do estresse hierárquico foram medidas por meio de três estudos: a) digestibilidade aparente, b) tempo de trânsito gastrintestinal e c) morfometria do epitélio intestinal dos peixes. O dados de tempo de trânsito gastrointestinal foram representados em forma de gráficos e os dados morfométricos foram avaliados pela análise de variância multivariada, considerando o par altura de vilo (AV) e profundidade de cripta (PC). Com base nos resultados obtidos pode-se concluir que o estresse hierárquico tem efeito significativo no trânsito gastrointestinal, aumentando a velocidade de trânsito da ingesta em até duas horas em animais submissos e que a porção proximal do intestino é a mais adequada para avaliar o efeito do estresse hierárquico por meio ... / Abstract: The dominance hierarchy and agonistic confrontation cause stress to the animals. This study aimed to quantify the effect of stress after the establishment of social hierarchy evaluating metabolic responses between the juvenile Nile tilapia. 20 male fish were distributed in 10 tanks with a capacity of 110 liters, adopted an outline fully cazualizado total of five treatments with four replications. The animals were fed a practical diet containing 28.0% crude protein and 3000 kcal of digestible energy. The hierarchical division of the individuals were in dominant and submissive second Falter (1983). For the treatment of visual communication, aquaria were divided plates with translucent glass, where the animals had no "physical" contact not having any type of contact water conspecific eat; for the treatment of chemical communication had a pumping system that homegeneizava water animals, without any eye contact with the conspecific. The isolated animal received no water no other fish and even had eye contact. The effect of the hierarchical stress ratings were measured by means of three studies: a) digestibility, b) and c gastrointestinal transit time) morphology of the intestinal epithelium of the fish. The data gastrointestinal transit time were represented as graphs and morphometric data were evaluated by multivariate analysis of variance, considering the pair villi height (AV) and crypt depth (PC). Based on the results obtained it can be concluded that the hierarchical stress has significant effect on the gastrointestinal transit, increasing the speed of transit of food intake within two hours in submissive animals and the proximal portion of the intestine is the most appropriate to evaluate the effect of hierarchical stress by pair (AV: PC). / Doutor

Orientador: Edmir Daniel Carvalho / Banca: Newton Castagnolli / Banca: Ricardo Pereira Ribeiro / Resumo: A técnica de criação de peixes em tanques-rede no Brasil é uma técnica que está sendo bem difundida principalmente nas regiões sudeste e nordeste do país e é uma alternativa para o aproveitamento racional dos corpos d'água. A tilápia é uma das principais espécies selecionada para este modelo de produção, devido suas características biológicas e ecológicas, tendo retorno comercial garantido e disponibilidade de incentivos governamentais. Esta prática está abrindo grandes perspectivas para uma rápida expansão da piscicultura industrial no Brasil podendo resultar em ganhos para o país como, geração de renda e redução da dependência da importação do pescado, assim contribuindo para o equilíbrio da balança comercial. O objetivo desse estudo foi avaliar o desempenho zootécnico da tilápia monossexo, Oreochromis niloticus, linhagem tailandesa, no sistema de produção com 10 tanques-rede com um volume útil de 6m3 com uma densidade de aproximadamente 500peixes/m3 para juvenis e 20 tanques com volume útil de 18m3 com uma densidade de aproximadamente 160peixes/m3 para engorda, na represa de Jurumirim, Alto do Rio Paranapanema, SP. Foram realizadas biometrias mensais com uma média de 60 peixes por tanque-rede e concomitantemente mensuração das variáveis físico-quimicas ao redor dos tanques. Para o desempenho zootécnico foi avaliado o fator de condição específico (K); a biomassa total (BT); o ganho de peso diário (GP); a conversão alimentar aparente (CAA); o crescimento específico (CE); a taxa de sobrevivência ao final do cultivo (TS); a relação peso total/comprimento padrão (Wt/Ls); a curva de crescimento em comprimento padrão; a curva de crescimento em peso total e para as variáveis físicoquímicas da água foram analisados... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The technique of fish breeding in cages is spreading rapidly in southeastern and northeastern Brazil and it is an alternative for the rational exploitation of bodies of water. The (nile) tilapia is one of the main species selected for this model of production because of its biological and ecological characteristics, profitability and availability of official incentives. This practice is opening great perspectives for the fast expansion of the fish breeding industry in Brazil, resulting in profits for the country, generating income and reducing the dependence on imported fish, thus contributing for our trade balance. The objective of this study was to evaluate the performance of the mono-sexed (nile) tilapia, Oreochromis niloticus, in the system of production with 10 cages with volume of 6 m3 with a density of approximately 500fish/m3 for juveniles and 20 tanks with volume of 18m3 with a density of approximately 160fish/m3 for growth, in Jurumirim Dam, in the Paranapanema River. Monthly biomeasurements with an average of 60 fish per cage were carried out along with the measurement of the physichal and chemical variables around the cages. For the performance, the specific condition factor (K), was evaluated, the total biomass (TB), the daily weight gain (WG), the apparent food conversion (AFC), the specific growth (SG), the survival rate (SR) at the end of the culture, the relation total weight/standard (Wt/Ls) length, the growth curve of standard length, the growth curve of total weight were evaluated. For the physical chemical variables of the water, the temperature (oC), pH, the dissolved oxygen (DO), the electric conductivity and the transparency were analyzed. In this system, it was possible to breed fish with commercialization weight (more than 500g), between 90 and 150 days of culture, with bad CAA rates - ranging from 3.13 to 5.43 food/Kg of fish... (Complete abstract click electronic access below) / Mestre

Orientador: Maeli Dal Pai Silva / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-08T01:50:52Z (GMT). No. of bitstreams: 1 Barretto_JeaneMarleneFogacadeAssis_D.pdf: 16620509 bytes, checksum: f55fbc50c521ec1697bb9e56f7c70d08 (MD5) Previous issue date: 2006 / Resumo: o desenvolvimento da musculatura em animais utilizados como alimento tem importância na qualidade e no aumento da massa muscular. Esse estudo teve por objetivo avaliar as características morfológicas e o crescimento da musculatura miotomal em tilápia do Nilo (Oreochromis niloticus) submetida à dieta com suplementação de vitamina C. O experimento foi realizado a partir da primeira alimentação exógena até 60 dias. As larvas foram distribuídas em cinco tratamentos, numa densidade de 10 indivíduos/L onde foram administrados os seguintes níveis de suplementação de vitamina C/ kg de ração balanceada: T1 = sem suplementação, T2 = 250 mg, T3 = 500 mg, T4 =1000 mg, T5 = sem suplementação até o vigésimo dia do tratamento, e com suplementação de 500 mg do vigésimo primeiro ao sexagésimo dia de experimento. Foram realizadas cinco repetições por tratamento com distribuição randômica. Cinco peixes de cada tratamento foram anestesiados com Ms-222 - SIGMA, sacrificados e a biometria foi efetuada após 20, 40 e 60 dias de experimento. Após isso, amostras de músculo foram congeladas em n-hexana resfriada em nitrogênio líquido e cortes histológicos foram submetidos à coloração HE (para análise da morfologia geral da musculatura), à reação mATPase, após pré incubação ácida (para a análise das características da ATPase miofibrilar) e à reação NADH-TR (para análise do metabolismo oxidativo das fibras). Aos 20 e 60 dias do experimento, cinco amostras de cada tratamento foram submetidos às reações imunohistoquímicas MyoD e miogenina (fatores de transcrição miogênica - para avaliar o grau de proliferação e diferenciação das celulas precursoras miogênicas). Também foram coletadas amostras de músculo para fixação em formalina neutra tamponada, processadas para serem embebidos em paraplast e os cortes histológicos foram submetidos à reação imunohistoquímica PCNA (Antígeno Nuclear de Proliferação Celular). A suplementação com os maiores níveis de vitamina C e após o período de restrição promoveu maior ganho de massa corpórea. O crescimento muscular em todos os tratamentos ocorreu principalmente por hiperplasia. Durante o experimento não foram observadas alterações morfológicas e histoquímicas nas fibras musculares. A suplementação de 500mg vit C kg-1da ração após a restrição por 20 dias promoveu a proliferação e a diferenciação das células precursoras miogênicas 500mg vit C kg-1 da ração foi o nível de suplementação apropriado para a miogênese e o desenvolvimento da Tilápia / Abstract: Muscle development in animais utilized for food has importance to muscle quality and mass increase. The aim of this study was evaluate the morphologic and the growth characteristics of the miotomal muscle in Nile tilapia (Oreochromis niloticus) submitted to a vitamin C supplemented diet. The experiment was carried through from the first exogenous feeding up to 60 days. The larvae were distributed into five treatments, in aquaria at 10/L density. The levels of vitamin C supplement/kg of ration were: T1=no supplement, T2=250mg vit C kg-1 of diet, T3=500mg vit C kg-1of diet, T4= 1000mg vit C kg-1 of diet, T5= no supplement until 20 days and 500mg vit C kg-1 of diet on days 20 to 60. There were 5 repetitions for each treatment. After 20, 40 and 60 days, 5 fish from each treatment were anaesthetized with MS-222 - SIGMA, sacrificed and weighed. Muscle fragments were cooled in liquid nitrogen. Histological sections were submitted to the following reactions: Haematoxilin-Eosin (for analysis of the muscle morphology and to determine muscle fibers diameters); Nicotinamide Adenine Dinucleotide Tetrazolium Reductase (NADH-TR), for muscle fibre metabolic evaluation (oxidative and glycolitic); and miofibrillar ATPase (mATPase), in pH 9.4, after acid preincubation to study myosin ATPase characteristics. After 20 and 60 days, muscle samples from five fish of each treatment were immersed in n-Hexane cooled in liquid nitrogen and histological sections were submitted to the MyoD and myogenin immunohistochemical reaction to evaluate cell proliferation and differentiation. Another five fish per treatment were embedded in Paraplast and histological sections were submitted to PCNA (Proliferating Cell Nuclear Antigen) immunohistochemical reaction to evaluate the degree of cell proliferation per treatment. During the experiment, ali treatments increased in weight and length. Weight differences were significant only on day 60. Supplementation with high vitamin C levels and after a period of restriction promoted an increase in body mass. Muscle fiber morphology, metabolic and myosin ATPase characteristics were normal and similar in all treatments. Muscle growth in all treatments was predominantly by hyperplasia. Proliferation and differentiation of myogenic precursor cells was higher with 500mg vit C kg-1 of supplemented diets throughout the experiment. Restriction of this supplementation for 20 days did not harm differentiation and still promoted myogenic precursor cell proliferation. The appropriate levei of vitamina C to myogenesis and the development of the Tilapia was 500mg vit C kg-1 of the ration / Doutorado / Biologia Celular / Doutor em Biologia Celular e Estrutural

Orientadores: Fernanda Elizabeth Xidieh Murr, Kil Jin Park / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-08T23:04:25Z (GMT). No. of bitstreams: 1 Simoes_MarciaRegina_D.pdf: 3855399 bytes, checksum: 7d7e8805878319750d638aadcdd2e2de (MD5) Previous issue date: 2007 / Resumo: Foi estudada a influência de dois tratamentos na secagem convectiva de filé de Tilápia do Nilo (Oreochromis niloticus), variedade Tailandesa. No primeiro tratamento foi realizado o estudo do processo de desidratação osmótica utilizando solução binária (água + NaCl) e ternária (água + NaCl + sacarose). Foram verificadas as influências dos fatores: temperatura, concentração da solução osmótica e tempo de imersão, nas respostas, ganho de sólidos, perda de água, GS/PA e atividade de água através da Metodologia de Superfície de Resposta. Para cada solução, foi escolhida uma condição otimizada, que foi determinada pelos menores valores de GS/PA e atividade de água. As condições escolhidas foram: temperatura de 34ºC, concentração de NaCl de 24,6% p/p e tempo de imersão de 230min para a desidratação osmótica com solução de NaCl. Para a desidratação osmótica com solução de NaCl + sacarose as condições escolhidas foram: temperatura de 34ºC, concentração de NaCl de 13% p/p, tempo de 185min e concentração de sacarose de 37% p/p. Para as melhores condições de desidratação osmótica obtida para cada solução, foram realizadas cinéticas de desidratação osmótica. No segundo tratamento foi avaliada a influência da temperatura de secagem e da concentração da fumaça líquida no processo de defumação do filé de tilápia (Oreochromis niloticus). A condição escolhida foi a de 40ºC de temperatura e concentração de fumaça de 15% p/p. As curvas de secagem convectiva para os dois tratametnos foram obtidas a 40ºC, 50ºC e 60ºC de temperatura e 1,5m/s de velocidade do ar de secagem. Para o ajuste dos dados experimentais foram utilizados os modelos difusional e o de PAGE. Os valores obtidos foram da ordem de 10-10m2/s. O modelo de PAGE apresentou melhor ajuste em relação ao modelo difusional. Na análise sensorial todas as amostras obtiveram boa aceitabilidade. A amostra desidratada em NaCl e seca, apresentou maior dureza e gomosidade. Na análise de microestrutura a amostra tratada com NaCl + sacarose mostrou conservar melhor a estrutura do pescado e o processo de defumação conservou a estrutura muscular do pescado defumado e seco / Abstract: This work investigated two procedures for the convective drying of Thai tilapia (Oreochromis niloticus) fillet. The first procedure carried out the osmotic dehydration process using a binary solution (water + NaCl); the second used ternary solutions (NaCl + sucrose and water). The influence of temperature, salt concentration, sugar concentration and immersion time on mass loss, water loss, solid gain, solid gain / water loss ratio (SG/WL) and water activity were evaluated, through the Response Surface Methodology (RMS). For each solution a condition to carry out the convective drying was chosen, wich was determined by the smallest value of the SG/WL ratio and the water activity. The chosen conditions were: 34 °C, salt concentration of 24.6 % and immersion time of 230 min for dehydration using water + NaCl; 34 °C, 13 % of salt, 37 % of sugar concentration and immersion time of 185 min for dehydration using water + NaCl + sucrose. For the best osmotic dehydration conditions achieved for each solution, osmotic dehydration kinetic was obtained. In the second procedure, the influence of drying temperature and concentration of liquid smoke in the smoking process of Thai tilapia fillet was evaluated. Convective drying was carried out in a fixed bed dryer at 40 °C, 50 °C e 60 °C and air velocity of 1.5 m/s. Drying curves were fitted to experimental data using a diffusion model, considering no shrinkage, and also using PAGE¿s model. The obtained diffusion coefficients were in the order of 10-10 m2/s. PAGE¿s model presented better fittings in relation to the diffusion model. Sensorial analysis showed great acceptability for all samples. The sample dehydrated in NaCl and dryed showed the highest hardness and springness. Microstruture analyses indicated the sample treated with NaCl + sucrose better preserved the structure and that the smoking process kept the original structure of the smoking and dryed fish / Doutorado / Doutor em Engenharia de Alimentos

Orientadores: Felix Guillermo Reyes Reyes, Marili Villa Nova Rodrigues / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-19T20:15:42Z (GMT). No. of bitstreams: 1 Branco_LucianadaConceicaoCastello_M.pdf: 696133 bytes, checksum: 0c1ae932e7cdd4cd2fea5388f7a0bb5a (MD5) Previous issue date: 2012 / Resumo: Os antimicrobianos são aplicados mundialmente na tilapicultura para o tratamento de bacterioses, principais doenças que acometem esta espécie. Entre os antibióticos, a enrofloxacina (ENR), pertencente à família das quinolonas, é utilizada internacionalmente e tem demonstrado ser eficaz no tratamento de infecções bacterianas. No Brasil, seu uso na criação de peixes ainda não foi aprovado, mas o Ministério da Agricultura, Pecuária e Abastecimento (MAPA) incluiu, em 2010, este fármaco no Plano Nacional de Controle de Resíduos e Contaminantes em Peixes (PNCRC). A ENR é biotransformada pelos animais à ciprofloxacina (CIP), composto este que apresenta atividade antimicrobiana e é utilizado na medicina humana. O processamento de alimentos tem mostrado ser um fator positivo para a segurança dos alimentos, devido à possível degradação de resíduos de antimicrobianos que poderiam causar o desenvolvimento de resistência em micro-organismos ou induzir efeitos adversos na saúde de seres humanos. Assim, é de grande importância, do ponto de vista da saúde pública e de conhecimento científico, conhecer o comportamento de resíduos de medicamentos veterinários nos alimentos quando submetidos ao processamento. O fishbúrguer, preparado com carne de tilápia, têm demonstrado elevada aceitação pelos consumidores. Isto reflete o novo estilo da vida em que os requisitos para a compra de um alimento incluem os benefícios nutricionais e também a conveniência de preparação. Para avaliar a diminuição dos níveis de resíduo de ENR e CIP presentes em filé de tilápia, durante processo de produção e fritura dos mesmos, torna-se necessário o desenvolvimento e validação de metodologia analítica que apresentem precisão, exatidão e sensibilidade para determinar este composto e seu metabólito. Portanto, os objetivos deste trabalho foram em filé de tilápia, bem como em um produto derivado, fishbúrguer, para avaliar a estabilidade dessas substâncias durante o processo de produção e fritura. Para este fim, um método simples, rápido e de baixo custo, utilizando LC-MS, foi desenvolvido e validado para a determinação simultânea de ENF e CIP. O preparo de amostra foi baseado na precipitação de proteínas com ácido tricloroacético, extração com acetonitrila e limpeza do extrato com hexano. A separação cromatográfica foi realizada em uma coluna C18. Para ambos os compostos, as curvas de calibração na matriz demonstraram linearidade superior a 0,99. O método foi seletivo, exato (91-100% de recuperação) e preciso (coeficientes de variação abaixo de 5%). Limites de detecção e quantificação foram, respectivamente, 3,5 e 10,4 µg/kg para ambos os compostos. Os valores calculados CCa e CCß foram, respectivamente, 107 e 114 µg/kg para ENR e 105 e 110 µg/kg para CIP (LMR estabelecido pela Comissão da UE é de 100 µg/kg para a soma de ENR e CIP). O método foi empregado para a determinação de resíduos de ENR e CIP em amostras de filés de tilápia obtidos a partir de peixes expostos a ENR (dose única de 40 mg/kg de peso corpóreo - administrado via ração). Os animais foram sacrificados após 4, 8 e 12 h de exposição. Resíduos acima do LMR foram encontrados mesmo após 12 h de administração do fármaco. A fim de avaliar a influência do processo de produção e fritura sobre os níveis residuais de ENR e CIP, fishbúrgueres foram preparados com filé de tilápias expostas a ENR. O método analítico para determinação da ENR e CIP no filé de tilápia foi adaptado e validado para a análise de fishbúrgueres antes e após fritura. Os métodos adaptados foram validados e mostraram-se linear, com coeficiente de correlação maior que 0,99, precisos (coeficiente de variação abaixo de 6%) e exatos (recuperação média de 90 a 106%). Foram analisadas amostras de fishbúrgueres (antes e após fritura) utilizando os métodos validados. Não foi verificada redução significativa dos níveis residuais da soma de ENR e CIP durante o preparo dos fishbúrgueres. No entanto, redução (p<0,05) de, aproximadamente, 10% foi verificada como consequência da fritura. Porém, essa redução pode ser considerada baixa em termos de segurança dos consumidores / Abstract: Antimicrobials are used worldwide for the treatment of tilapicultura bacteriosis, main diseases that affect this species. Among the antibiotics, enrofloxacin (ENR), belonging to the quinolone family, is used internationally and has been shown to be effective in treating bacterial infections in fish. In Brazil, its use in fish farm is not yet approved, but the Ministry of Agriculture, Livestock and Supply (MAPA) included, in 2010, this drug in the National Plan for Control of Residues and Contaminants in Fish (PNCRC). ENR is biotransformed in the body of animals being converted to ciprofloxacin (CIP), an active compound used in human medicine. Food processing has shown to be a positive factor for the food safety, due to the possible degradation of antimicrobial agents that could potentially cause development of resistance in microorganisms or induce adverse health effects in humans. Thus, it is of great importance, from the standpoint of public health and scientific knowledge, to know the behavior of veterinary drugs in foods when subjected to processing for consumption. The fishburger, prepared with meat from tilapia, have shown high acceptance by the consumers. This follows the new style of life in which the requirements for the purchase of a food include the nutritional benefits and the convenience of preparation. To evaluate the degradation of ENR present in meat processed as tilapia fishburger, it becomes necessary to develop and validate analytical methods that present precision, accuracy and sensitivity to determine this compound and CIP as well. Therefore, the objectives of this study were to develop and validate analytical method for the determination of ENR and CIP in tilapia fillet, as well as in a product derived from tilapia meat (fishburger) and to evaluate the stability of these substances during the frying of fishburgers prepared from tilapias exposed to ENR. For this purpose, a simple, fast and low cost LC-MS method for the simultaneous determination of ENR and CIP residues in tilapia fillet and fishburgers was developed and validated. Sample preparation was done by protein precipitation with trichloroacetic acid, acetonitrile extraction and extract clean up with hexane. The chromatographic separation was performed on a C18 column. For both compounds, matrix matched calibration curves showed linearity higher than 0.99. The method was selective, accurate (91-100% recovery) and precise (coefficients of variation below 5%). Limits of detection and quantification were, respectively, 3.5 and 10.4 µg/kg for both compounds. The calculated CCa and CCß values were, respectively, 107 and 114 µg/kg for ENR and 105 and 110 µg/kg for CIP (MRL established by the EU Commission is 100 µg/kg for the sum of ENR and CIP). The method was employed for the determination of ENR and CIP residues in samples of tilapia fillets obtained from fishes exposed to ENR (single dose of 40 mg/kg b.w incorporated in the feed). The animals were killed after 4, 8 and 12 h of exposure. Sum of residues of ENR and CIP above the MRL were found, even after 12 h of ENR administration. In order to evaluate the influence of cooking (frying) on the residual levels of ENR and CIP, fishburgers were formulated with fillet of tilapias exposed to ENR. The analytical method for the determination of ENR and CIP in tilapia fillet was adapted and validated for the analysis of the fishburgers before and after frying. Adapted and validated methods showed linearity with a correlation coefficient greater than 0.99, selectivity, precision (coefficient of variation below 6%) and accuracy (average recovery of 90 to 106%). Samples of fishburgers (before and after frying) were analyzed using the validated methods. A small reduction (approximately 10%) in the residual levels of ENR and CIP as a consequence of frying was verified, indicating that both compounds are stable to cooking conditions / Mestrado / Ciência de Alimentos / Mestre em Ciência de Alimentos

Reproductive containment in farmed fish is highly desired for sustainable aquaculture to prevent genetic introgression with wild conspecifics and enhance productivity by suppressing sexual maturation. A number of strategies have already been implemented or have been tested in commercially important fish (e.g. triploidy, monosexing, hormonal therapies); however, they either do not result in 100% containment, or they cannot be applied to all species. One promising new approach consists in disrupting primordial germ cells (PGCs), at the origin of germline cells, to induce sterility. The work carried out in this doctoral thesis aimed to investigate the genes involved in the survival of germ cells and subsequently conduct a functional analysis of candidate genes using CRISPR/Cas9 gene editing system to ultimately provide the basis for the development of a novel sterilisation technique. Nile tilapia was chosen as the experimental animal as it is a major aquaculture species worldwide and the control of reproduction plays a critical role in the farming productivity in this species. In addition, the species has clear advantages as its whole genome sequence is accessible, the generation time is relatively short and zygotes can be available all year round. Initially, a panel of 11 candidate genes with reported roles in survival of PGCs was investigated during the ontogenic development which led to the selection of piwi-like (piwil) gene as a target for genome editing. Then, high temperature was tested as a means to induce germ cell loss to better understand the mechanism underlying germ cell survival and apoptosis, and this study confirmed the functional importance of piwil genes in relation to germ cell loss and proliferation. In addition, the study suggested potential subfunctionalisation within the Bcl-2 gene family which requires further investigation. The next step aimed to optimise the CRISPR/Cas9 gene editing method by improving the microinjection system and testing different concentrations of sgRNAs. Over 95% of injected embryos showed on-target mutation in piwil2 via zygote injection of CRISPR/Cas9 reagents and complete KO larvae were shown in half of the mutants, producing putative sterile fish. However, there was no clear association between the phenotypes in PGCs and the mutation rate. Further comparative studies of mutant screening methods including T7E1, RGEN, HRMA, fragment analysis and NGS revealed that the genotypes of F0 are highly mosaic, suggesting that deep sequencing is recommended for accurate and high throughput F0 screening and further improvement for predictable genome editing is required for a reliable gene functional analysis in F0. In summary, the current thesis provided new scientific knowledge and supporting evidence for the use of the CRISPR/Cas9 gene editing platform to study gene function associated with sterility, with the ultimate goal to develop an alternative sterilisation method in fish.

Nile tilapia, Oreochromis niloticus, is one of the most important farmed fish globally. One of the most serious bacterial diseases constraining global tilapia production is Francisellosis caused by Francisella noatunensis subsp. orientalis (Fno). Although outbreaks of Fno are increasing worldwide, there are no licenced commercial vaccines to prevent the disease for use on tilapia farms. Thus, the current treatment of choice is the use of antibiotics combined with increasing water temperature up to 30°C. Studies investigating the diversity of circulating Fno isolates and the immune response of tilapia elicited by vaccination against piscine francisellosis are lacking. In addition, the current conventional and molecular tools used for detection of Fno have many drawbacks, making detection of Fno a challenging process. In this study, five clinical isolates of Fno from diverse geographical locations (UK, Costa Rica, Mexico, Japan and Austria), previously characterised by morphology, genotype, antimicrobial susceptibility and virulence, were used in a proteomic study. The whole proteomic cell profile of the five isolates were homogenous by one-dimension sodium dodecyl polyacrylamide gel electrophoresis (1D-SDS-PAGE), while minor differences in the intensity of 15 proteins between the strains were observed by two-dimension SDS-PAGE (2DE), including some important virulence related proteins. The UK isolate was the most significantly different isolate when compared to the other Fno isolates in the current study. The Fno UK isolate had significantly higher abundance of 10/15 of the significantly expressed proteins including four of the essential pathogenicity and virulence related proteins (IglC, GroEL, DnaK, ClpB) compared to the other used Fno isolates. The antigenic profiles of the five Fno isolates were studied by 1D western blotting using tilapia hyper immune sera which recognised an immunodominant band of a molecular weight of ~ 17-28 kDa in all tested Fno isolates. Liquid chromatography-electrospray ionization-tandem mass spectrometry (LC/ESI/MS/MS) identified 47 proteins in this antigenic band. Some of the identified proteins are associated with Fno pathogenicity. 2D western blot analysis of the vaccine isolate (Fno UK) revealed differential antigen recognition between sera from vaccinated and non-vaccinated fish following experimental challenge (26 antigenic spots recognised by sera from vaccinated fish; 31 antigenic spots recognised by sera from vaccinated and challenged fish and 30 antigenic spots recognised by non-vaccinated and challenged fish). The identity of these proteins was determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and some of them are known Francisella virulence related proteins. Bioinformatics analyses revealed diverse categories of proteins with high biological functions, however the vast majority of these proteins are involved in energy production and metabolic pathways of the bacteria. This detailed analysis will facilitate the development of cross-strain protective subunit Fno vaccines and antigen-targeted Fno diagnostics. The outer membrane proteins (OMPs) of the same five Fno isolates were extracted using the ionic detergent sarkosyl. The OMP fraction of the different isolates were separated via 1D-SDS PAGE and the digested peptides of the UK isolate were analysed by LC/ESI/MS/MS. High degree of similarity was observed in the OMP profile of the five Fno isolates with an abundant protein band at 17-28 kDa, which was found to be antigenic by 1D western blot using convalescent tilapia sera. LC/ESI/MS/MS analysis of the OMPs of the Fno UK isolate identified 239 proteins, including 44 proteins in the antigenic band (17-28 kDa). Comparison between the proteins identified in the immunogenic band of whole cell lysate and OMP fraction of the Fno UK isolate showed 30 common proteins between the two preparations, 17 proteins were identified only in the whole cell extract and 14 were identified only in OMP fraction. Outer membrane proteins (e.g. Omp-A), virulence related proteins such (e.g. IglC) and other stress related proteins (e.g. AhpC/TSA family peroxiredoxin) were more abundant in the OMP fraction than the whole cell lysate. In silico analysis enabled prediction of the function and location of the OMPs identified by Mass-spectrometry. The findings of this study provide preliminary data on bacterial surface proteins that exist in direct contact with the host immune defence during infection and offering an insight into their potential role as novel targets for Fno diagnostics and vaccine development. The efficacy of an injectable whole cell oil-adjuvanted vaccine was evaluated against challenge with heterologous Fno isolates in Nile tilapia, Oreochromis niloticus. Three duplicate groups of 130 healthy Nile tilapia (~15 g) were intraperitoneally (i.p.) injected with the vaccine, adjuvant-alone or PBS followed by an i.p. challenge with three Fno isolates from geographically distinct locations. The vaccine provided significant protection to all immunised tilapia groups with a significantly higher relative percent survival (RPS) of 82.3% against homologous challenge, compared to 69.8% and 65.9% after heterologous challenge. Protection correlated with significantly elevated specific antibody responses and western blot analysis demonstrated cross-isolate antigenicity with sera from fish post-vaccination and post-challenge. Moreover, a significantly lower bacterial burden was detected by quantitative real-time polymerase chain reaction (qPCR) in conjunction with significantly greater expression of IgM, IL-1β, TNF-a and MHCII 72 hours post-vaccination (hpv) in spleen samples from vaccinated tilapia compared to those of adjuvant-alone and control fish. The latter results suggested stimulation of protective immune responses following vaccination. In addition, a whole cell formalin killed autogenous immersion vaccine against Fno was developed using the same isolate used for the injectable vaccine. Duplicate tanks of 35 tilapia fry were immersed in the vaccine or in sterile Modified Muller Hinton broth (MMHB) diluted in tank water (1:10 dilution) for 30 s and at 30 days post-vaccination (dpv), all fish groups were immersion challenged with the homologous Fno isolate and monitored for 21 days. A moderate RPS of 43.7% was provided by the vaccine. Serum IgM levels were below the threshold in 30 % of the vaccinated fry 30 dpv. Also, the IgM levels of the vaccinated fry were not significantly different from control fry 21 days-post challenge. A recombinase polymerase amplification (RPA) assay was developed and validated for rapid detection of Fno. The RPA reaction was performed at a constant temperature of 42°C for 20 min. The RPA assay was performed using a quantitative plasmid standard containing a unique Fno gene sequence. Validation of the assay was performed not only by using DNA from Fno, closely related Francisella species and other common bacterial pathogens in fish farms, but also by screening 78 Nile tilapia and 5 water samples collected from UK and Thailand. All results were compared with those obtained by previously established real-time qPCR. The developed RPA showed high specificity in detection of Fno with no cross-detection of either the closely related Francisella spp. or the other species of bacteria tested. The Fno-RPA performance was highly comparable to the published qPCR with detection limits at 15 and 11 DNA molecules detected, respectively. The Fno-RPA was rapid, giving results in approximately 6 min in contrast to the qPCR that required approximately 90 min to reach the same detection limits. Moreover, the RPA was more tolerant to reaction inhibitors than qPCR when tested with field samples. The fast reaction, simplicity, cost-effectiveness, sensitivity and specificity make the RPA an attractive diagnostic tool that will contribute to control the infection through prompt on-site detection of Fno. The overall results of this study indicated that Fno isolates from different origins share a high degree of homology in their proteomic and antigenic profile. Proteomic characterisation data of Fno isolates has contributed to understanding the diversity of Fno isolates and assisted in identifying suitable candidates for developing an effective Fno vaccine. / Moreover, this study has proven the efficacy of a cross protective Fno injection vaccine in tilapia fingerlings, with further optimisation needed for immersion vaccination of fry, and given insights into the immune response of tilapia to vaccination against francisellosis. In addition, it provided a rapid, sensitive, specific and robust molecular tool for detection of Fno that can assist surveillance and control of piscine francisellosis on tilapia farms.

Thesis (MPhil (Animal Sciences. Aquaculture))--University of Stellenbosch, 2005. / This paper contains literature on tilapia culture, feeding and nutritional factors of prime consideration to survival and growth of Oreochromis mossambicus. Results are presented for a three-phase laboratory experiment on survival of O. mossambicus fry in an attempt to evaluate the use of live Spirulina platensis. The experiment was conducted on fry in a closed system in an effort to maximize the use of live Spirulina and also optimize growth and production. Fry were tested for tolerance levels of salinities, 0-35 g/lt, and showed favourable survival rates up to 15 g /lt salinity without being fed. Manipulation of input in freshwater turned high fry mortalities with increasing rates without difference for physical form of Spirulina. Growth was not significantly affected by types of input. However fry grew well at 0-40% rates with considerable survival performance. It is noted good quality of water that allow improved survival and growth of fry in a closed system may be assured with rates of input up to 5 or 10% of bodyweight. These input rates can guide use of live Spirulina in saline water tilapia culture if Spirulina proves good productivity at the consistency of fry tolerance to the salinity levels established in this paper.

Traditionally, fish oil (FO) has been used extensively in aquafeeds. The stagnation in global fish oil production coupled with an increased demand for its use in aquaculture feeds, especially salmonid feeds, has greatly inflated fish oil prices. Therefore, in order to sustain the rapid growth of the tilapia industry, the dependence on these commodities in feeds should be reduced through use of cheaper and more sustainable sources of dietary lipids, such as palm oil. This study therefore investigated several, previously poorly understood, effects of palm oil on reproductive performance of the commercial tilapia species, Oreochromis niloticus; which currently ranks as second most popular species in world aquaculture. In the present study broodstock were fed on experimental diets at full and half ration regimes throughout their entire life cycle from exogenous feeding. Studies were conducted in standardised and controlled hatchery conditions, thereby reducing the potential influence of environmental variations. First feeding O. niloticus fry were fed on four diets, cod liver oil (D 1), palm oil (D 2), mixed palm and cod liver oil (D 3) (9:1 ration) and a commercial trout diet as control (D 4) (Skretting, U.K.) on a reducing ration based on fish size. The present study investigated the effect of dietary lipid sources on (1) growth performance, (2) biochemical composition of eggs (total lipid and fatty acid composition), (3) morphological parameters of eggs (total and relative fecundity, egg size, egg weight and EW:BW), (4) larval quality (larval length and weight) and (5) oocyte recruitment and its associated sex steroid hormones. Experimental diets and feeding ration significantly influenced (p<0.05) the growth performance over a period of 120 days. Total lipid and fatty acid composition of eggs originating from broodstock fed on palm oil, mixed palm and cod liver oil (9:1) or a control diet were not significantly different (P>0.05) when fed at either full (3% BWday-1) or half ration (1.5% BWday-1). The present study, however, confirmed that fatty acid composition of fish eggs reflected the fatty acid composition of the diet, although specific fatty acids were selectively utilized or retained in the eggs. The mean inter-spawning interval (ISI) increased with increasing fish size and averaged 14, 19 and 24 days for fish fed on palm oil, mixed palm and cod liver oil or control diets, respectively. The shortest ISI observed was 7 days for fish fed a palm oil diet. Total fecundity ranged from 660 - 820 eggs/clutch. Mean total fecundity was 750, 820 and 660 eggs/clutch for fish fed a palm, mixed palm and cod liver oil or a control diet, respectively, but these differences were not significant (P>0.05). However, relative fecundity and egg weight to body weight rates as a percentage (EW: BW) were found significantly differ (p<0.05) between fish fed the control diet and experimental diets. Mean egg diameter (2.2 mm) was not significantly influenced (p>0.05) by experimental diets. The egg volume, egg dry and wet weight, fertilisation and hatching rate were also not significantly different between fish fed the experimental diets. Oocyte development was classified into distinct stages based upon oocyte size, biochemical properties and structure. The recrudescence to these stages was not significantly influenced by broodstock fed experimental diets either at full or half ration. Steroid hormones and histological analyses provided valuable data concerning the oocyte development and recruitment in this species. Levels of 17ß-oestradiol (E2) and testosterone (T) peaked within 6 days of spawning, suggesting that vitellogenesis began as early as day 2 or 3 post-spawning. By day 6, ovaries were dominated by large late-vitellogenic/maturing oocytes (stages 6 & 7) occupying about 70% of the ovary. Gonadosomatic index (GSI) reached maximal levels by day 6. It is suggested that pre-vitellogenic oocytes are recruited into vitellogenic growth immediately after spawning and complete vitellogenesis on day 6 post-spawning. Finally, the present study investigated the effect of food restriction at two rations (full and half) on broodstock reproductive performance. Oreochromis niloticus were rationed from first feeding and throughout their life-cycle. The dietary regime, full ration (3%) and half ration (1.5%), influenced fish size but despite this variation no significant differences (p>0.05) were detected in total lipid and fatty acid composition in the eggs, total fecundity, egg diameter, total egg volume and larval size. These results suggested that despite large differences in food availability throughout their life cycle, investment in reproduction had remained remarkably consistent. It appeared that during food restriction, O. niloticus sacrificed body weight and growth so as to maintain reproductive investment. In summary, this study provides valuable information using a novel experimental design on the effects of dietary lipid sources on reproductive performance of female O. niloticus. Substituting palm oil for fish oil as the dietary lipid source and reducing ration by half (1.5% BWday-1) had no significant effect on reproductive performance. Therefore it is suggested that under controlled conditions, lipids of non-marine origin, such as palm oil, can be successfully substituted for broodstock diets. Halving feed requirement should also increase profitability of seed production. KEYWORDS: Tilapia; O. niloticus; palm oil; diet; fecundity; spawning periodicity; oocyte recruitment; reproductive performance.

Anti-müllerian hormone (AMH) is expressed in male embryos and represses development of müllerian ducts during testis differentiation in mammals, birds and reptiles. Amh orthologues have been identified in teleosts despite them lacking müllerian ducts. Previously we found sexually dimorphic aromatase activity in tilapia brains before ovarian differentiation. This prompted us to search for further dimorphisms in tilapia brains during sex differentiation and see whether amh is expressed. We cloned the tilapia amh gene and found that it contains 7 exons but no spliced forms. The putative protein presents highest homologies with Amh proteins of pejerrey and medaka as compared to other Perciformes. We analysed amh expression in adult tissues and found elevated levels in testes, ovary and brain. Amh expression was dimorphic with higher levels in XY male brains at 10–15 dpf, when the gonads were still undifferentiated and gonadal amh was not dimorphic. Male brains had 2.7-fold higher amh expression than gonads. Thereafter, amh levels decreased in the brain while they were up-regulated in differentiating testes. Our study indicates that amh is transcribed in male brains already at 10 dpf, suggesting that sexual differentiation may be occurring earlier in tilapia brain than in gonads. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.