Orientador: Marco Antonio Belo / Banca: Annelise Carla Camplesi dos Santos / Banca: Wilson Gomez Manrique / Banca: Luciano Melo souza / Banca: Silvia Patrícia Carraschi de Oliveira / Resumo: Com o objetivo de estudar a fisiopatologia da reação inflamatória em tilápias, este estudo avaliou a participação do tratamento exógeno com cetoconazol sobre o efeito glicocorticoide de tilápias do Nilo, Oreochromis niloticus, durante aerocistite aguda induzida por Aeromonas hydrophila. Para tanto, foram utilizadas 91 tilápias, constituindo cinco tratamentos: Não tratado e não inoculado (Padrão fisiológico); Não tratado e inoculado (controle infectado); Tratado com dexametasona (DEXA) e inoculado; Tratado com cetoconazol (CETO) e inoculado; Tratado com DEXA + CETO e inoculado. Após anestesia profunda nos períodos experimentais de 6, 24 e 48 horas pós-inoculação (HPI) coletou-se o exsudato para contagem celular e sangue para determinação do hemograma completo, proteinograma, bioquímico sérico. Na fase inicial da infecção (6HPI), o tratamento subcutâneo das tilápias com 200 UI-kg-1 de cetoconazol resultou em atividade pró-inflamatória caracterizada pelo aumento do acúmulo de granulócitos e diminuição de trombócitos no exsudato, associado à neutrofilia e aumento dos níveis séricos de haptoglobina e ceruloplasmina. No quadro eritrocitário inicial de tilápias tratadas com cetoconazol, observou-se diminuição do número de hemácias com diminuição do hematócrito e do volume corpuscular médio, caracterizando alterações microcíticas comuns em deficiências por ferro, corroborando os aumentos observados nos níveis séricos das proteínas de fase aguda haptoglobina e ceruloplasmina. No pico ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: In order to study the pathophysiology of the inflammatory reaction in tilapias, this study evaluated the participation of the exogenous treatment with ketoconazole on the glucocorticoid effect of Nile tilapia, Oreochromis niloticus, during acute aerocysteine induced by Aeromonas hydrophila. For this, 91 tilapias were used, constituting five treatments: Untreated and uninoculated (Physiological pattern); Untreated and inoculated (infected control); Treated with dexamethasone (DEXA) and inoculated; Treated with ketoconazole (CETO) and inoculated; Treated with DEXA + CETO and inoculated. After deep anesthesia in the experimental periods of 6, 24 and 48 hours post-inoculation (HPI), the exudate was collected for cell count and blood for determination of the complete blood count, proteinogram, serum biochemistry. In the initial phase of infection (6HPI), the subcutaneous treatment of tilapia with 200 IU kg -1 of ketoconazole resulted in proinflammatory activity characterized by increased granulocyte accumulation and decreased thrombocytes in the exudate associated with neutrophilia and increased levels of serum levels of haptoglobin and ceruloplasmin. In the initial erythrocyte framework of ketoconazole-treated tilapia, a decrease in the number of red cells with decreased hematocrit and mean corpuscular volume was observed, characterizing common microcytic changes in iron deficiencies, corroborating the observed increases in serum levels of acute phase proteins haptoglobin and ceruloplasmin. At the peak of cellular exudation to the inflamed focus 24 HPI, tilapia treated with ketoconazole showed increased thrombocyte accumulation associated with thrombocytopenia and increased serum levels of transferrin. In the late phase of infection (48 HPI), subcutaneous treatment of tilapia with 200 IU kg-1 of ketoconazole resulted in proinflammatory activity characterized... (Complete abstract click electronic access below) / Doutor

Orientador: Pedro de Magalhães Padilha / Banca: Luiz Edivaldo Pezzato / Banca: Lincoln Carlos Silva de Oliveira / Resumo: O trabalho avaliou o perfil metaloproteômico de amostras de tecido muscular de tilápia do Nilo (Oreochromis niloticus). Métodos eletroforéticos em segunda dimensão (2D PAGE) foram utilizados no fracionamento das proteínas e na identificação por análise de imagens obtidas nos géis de poliacrilamida. Foi feito mapeamento de cálcio, cobre, ferro, manganês e zinco nos spots proteicos por Absorção Atômica usando o módulo chama (FAAS) e forno de grafite (GFAAS). As proteínas dos spots nos quais foram identificados a presença de Ca, Cu, Fe, Mn e Zn foram caracterizadas por Espectrometria de Massas (ESI MS MS). Em média foram detectados 620 spots por gel, com desvio padrão de 11,3 e correlação média de 73% entre os géis. As determinaçoes por FAAS/GFAAS indicaram a presença de cálcio em todos os spots, cobre em 2 spots, ferro em um único spot, manganês em 6 spots, e zinco em 3 spots. Dos 18 spots preoteicos nos quais foram identificados a presença dos íons metálicos, 11 foram caracterizados por ESI MS MS, proteínas podem ser candidatas a biomarcadoras de Ca, Cu, Fe, Mn e Zn / Abstract: The study evaluated the metalon protemic profile samples of muscle tissue of Nile tilapia (Oreochromis niloticus). Methods for second dimension electrophoresis (2D PAGE) has been used in the fractionation of proteins and identification by analysis of images obtained in polyacrylamide gels. Mapping of calcium, copper, iron, manganese and zinc in protein spots was done by Atomic Absorption using the module calls flame (FAAS) and graphite furnace (GFAAS). Were characterized by mass spectrometry (ESI MS MS) the protein spots were identified in which the presence of Ca, Cu, Fe, Mn and Zn. On average 620 spots per gel, with a standard deviation of 11.3 and average correlation of 73 % between the gels were detected. Determination by FAAS / GFAAS indicated the presence of all the spots calcium, copper in two spots of a single spot iron, manganese in 6 spots in 3 spots zinc. The 18 spots preoteicos in which the presence of metal ions have been identified, 11 were characterized by ESI MS MS proteins may be candidates for biomarkers Ca , Cu, Fe , Mn and Zn / Mestre

Made available in DSpace on 2014-06-11T19:28:24Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-02-27Bitstream added on 2014-06-13T20:37:17Z : No. of bitstreams: 1 000740068.pdf: 1647648 bytes, checksum: b1d214f874d365863fcad053a496ee8f (MD5) / O trabalho avaliou o perfil metaloproteômico de amostras de tecido muscular de tilápia do Nilo (Oreochromis niloticus). Métodos eletroforéticos em segunda dimensão (2D PAGE) foram utilizados no fracionamento das proteínas e na identificação por análise de imagens obtidas nos géis de poliacrilamida. Foi feito mapeamento de cálcio, cobre, ferro, manganês e zinco nos spots proteicos por Absorção Atômica usando o módulo chama (FAAS) e forno de grafite (GFAAS). As proteínas dos spots nos quais foram identificados a presença de Ca, Cu, Fe, Mn e Zn foram caracterizadas por Espectrometria de Massas (ESI MS MS). Em média foram detectados 620 spots por gel, com desvio padrão de 11,3 e correlação média de 73% entre os géis. As determinaçoes por FAAS/GFAAS indicaram a presença de cálcio em todos os spots, cobre em 2 spots, ferro em um único spot, manganês em 6 spots, e zinco em 3 spots. Dos 18 spots preoteicos nos quais foram identificados a presença dos íons metálicos, 11 foram caracterizados por ESI MS MS, proteínas podem ser candidatas a biomarcadoras de Ca, Cu, Fe, Mn e Zn / The study evaluated the metalon protemic profile samples of muscle tissue of Nile tilapia (Oreochromis niloticus). Methods for second dimension electrophoresis (2D PAGE) has been used in the fractionation of proteins and identification by analysis of images obtained in polyacrylamide gels. Mapping of calcium, copper, iron, manganese and zinc in protein spots was done by Atomic Absorption using the module calls flame (FAAS) and graphite furnace (GFAAS). Were characterized by mass spectrometry (ESI MS MS) the protein spots were identified in which the presence of Ca, Cu, Fe, Mn and Zn. On average 620 spots per gel, with a standard deviation of 11.3 and average correlation of 73 % between the gels were detected. Determination by FAAS / GFAAS indicated the presence of all the spots calcium, copper in two spots of a single spot iron, manganese in 6 spots in 3 spots zinc. The 18 spots preoteicos in which the presence of metal ions have been identified, 11 were characterized by ESI MS MS proteins may be candidates for biomarkers Ca , Cu, Fe , Mn and Zn

Resumo: O estado nutricional dos reprodutores é um dos fatores determinantes para o sucesso da reprodução de tilápia-do-nilo. A qualidade dos ingredientes que compõe uma dieta é fundamental na fisiologia reprodutiva dos peixes. Os lipídios possuem um efeito poupador de custo para uma dieta, como fonte de energia. Baseados nestes estudos, o objetivo deste trabalho foi avaliar o efeito da suplementação alimentar com diferentes fontes lipídicas sobre os parâmetros reprodutivos, de matrizes de tilápia-do-nilo da linhagem GIFT. Para isso, dietas contendo 42% de proteína bruta e 4356 Kcal/kg EB, contendo diferentes fontes lipídicas (óleo de linhaça - TOL, soja - TOS, palma - TOP, peixe - TOPX ou fígado de bacalhau - TOFB, com inclusão de 4%) foram fornecidas para fêmeas de tilápia (peso médio de 150g ±12,6) durante 90 dias. Para o desfio de jejum, três lotes de ovos das fêmeas de cada tratamento foram coletados, misturados e incubados. Após a eclosão dos ovos e absorção do saco vitelínico, 300 larvas foram distribuídas em 15 aquários experimentais, e mantidas em jejum até a mortalidade total. A suplementação de 4% de óleo de palma na dieta dos peixes influenciou positivamente nos índices somáticos (hepatosomático e gordura visceral), nos parâmetros reprodutivos (numero total, fertilidade e fecundidade dos ovos; fecundidade, número total e desafio de jejum nas larvas); taxas de colesterol e triglicerídeos e fator de condição. A adição do óleo de palma não apresentou alterações hepáticas decorrentes da suplementação. Não houve diferenças significativas observadas em relação à leucometria diferencial, eritrograma e nas proteínas plasmáticas totais das matrizes de tilápia. A análise histológica do fígado de matrizes... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The nutritional status of the breeding is one of the determining factors for successful reproduction of the Nile tilapia. The quality of the ingredients that make up a diet is essential in the reproductive physiology of fish. The lipids have a sparing effect of cost for a diet as a source of energy. Based on these studies, the objective of this study was to evaluate the effect of dietary supplementation with different lipid sources on reproductive parameters, arrays of Nile tilapia GIFT strain. To do this diets containing 42% crude protein and 4356kcal/kg EB, containing different lipid sources (linseed oil - TOL, soybean oil - TOS, palm oil - TOP, fish oil - TOPX or cod liver oil - TOFB, with inclusion of 4%) were provided to female tilapia (average weight 150g ±12,6) for 90 days. For the challenge of fasting, three lots of eggs from females of each treatment were collected, mixed and incubated. After the eggs hatch and absorption of the vitelline sac, 300 larvae were distributed in 15 experimental aquaria, and kept fasting until the total mortality. The supplementation of 4% of palm oil in fish diet had a positive effect on somatic rates (hepatic-somatic and visceral fat) in reproductive parameters (total number, fertility and fecundity of eggs, fertility, and total number of larval challenge of fasting); cholesterol and triglycerides and condition factor. The addition of palm oil did not show hepatic alterations due to supplementation. No significant differences were observed in relation to the differential leukocyte counts, and plasma proteins erythrogram total of matrices of tilapia. Histological analysis of liver arrays showed that fish fed with linseed oil showed indicators of a possible liver steatosis. The arrays fed diets containing fish oil and cod liver oil, also presented positive results, however, the... (Complete abstract click electronic access below) / Orientador: Marta Verardino De Stéfani / Coorientador: Teresa Cristina Ribeiro Dias Koberstein / Banca: João Batista Kochenborger Fernandes / Banca: Elisabeth Criscuolo Urbinati / Banca: Newton Castagnolli / Banca: Sergio Fonseca Zaiden / Doutor

With World Health Organization estimation of population increase to over 9 billion by 2050, malnutrition and food insecurity are predicated to be two of the main challenges facing the world in the future. Fish is a source of protein that can have a significant contribution for solving the global food crisis. Fish for human consumption is provided by both the aquaculture industry and the capture fisheries, however, the flat-lined production of seafood from capture fisheries means aquaculture production should double in number to provide enough protein by 2050. Culturing fish at higher densities is being used as a way to increase the global aquaculture production. Modern aquaculture intensified practices expose the fish to various stressors that can negatively impact their welfare. Good nutrition can improve the health status of aquacultured fish while accumulation of specific nutrient can add value to the fillet. In line with that, the studies described in this dissertation aimed to examine the vital role of fish in human health and evaluate strategies to improve the sustainable aquaculture production of fish using novel aquafeeds. This was completed by conducting a survey of the essential minerals and heavy metals of commercially available catfish, salmon, and tilapia fillets in the United States marketplace and three fish nutritional trials to evaluate the impacts of novel aquafeeds on production and health of fish and the food quality of their respective fillets. Selenium with the established health benefit for human and fish was the trace mineral of interest in this dissertation. This element is the main component of a group of selenium containing proteins called selenoproteins that are antioxidant and maintain oxidative homeostasis of fish and human. Additionally, selenium is an antagonist for many heavy metals such as mercury. The market study revealed that catfish, salmon and tilapia samples available in United States marketplace are good source of selenium, zinc and phosphorous. The concentration of heavy metals in all the samples were lower than safety standards. Nile tilapia (Oreochromis niloticus) and rainbow trout (Oncorhynchus mykiss), which are two economically important aquacultured fish, were used as animal models in the fish nutritional trials. Different forms of selenium supplemented to Nile tilapia diet. The form of Dietary Se form did not have any significant (p>0.05) effect on growth, and plasma and hepatic GPX activity of Nile tilapia. However, organic selenium supplement showed significantly (p<0.05) higher bioavailability and accumulated in the fillets at the significantly higher concentration. In the second fish trial selenoneine, a novel selenium containing compound, was supplemented at various concentrations to rainbow trout diet. Other than growth parameters multiple other health biomarkers (lysozyme, glutathione peroxidase, superoxide dismutase activities) were examined to determine whether selenoneine supplementation had a positive impact on fish health. Selenoneine enriched diet significantly(p<0.05) increased the weight gain, lysozyme and oxidative enzymes activities of rainbow trout. Lastly rainbow trout feed was supplemented with commercially available probiotic (Actisaf®), prebiotic (Safmmann®), and their mixture (synbiotic). These dietary supplements did not have any significant effect on fish production but led to a significant increase in activity of oxidative enzymes in plasma when compared to control diet. These results suggest that feed supplements used in this dissertation can be used to promote the fish health raised in recirculating aquaculture systems and support an economically and environmentally sustainable aquaculture. / PHD / Fish contributes significantly to human’s diet in terms of high quality, easily digestible animal protein that helps fight malnutrition and food insecurity. More than half of the fish for human consumption is produced by the aquaculture industry and the growth of the industry is very critical for meeting the future needs for protein. Other than production of high-quality protein the aquaculture industry has made a great contribution to people’s livelihood, poverty alleviation, income generation, employment and, trade globally. To continue contributing effectively to global food security aquaculture industry should overcome various obstacles such as disease and sustainable production of high-quality feed. In this dissertation, important roles of fish in human diet was examined by measuring the mineral composition of commercially available catfish, salmon and tilapia in the United States market place. Also diets of Nile tilapia and rainbow trout were supplemented with various forms of selenium, prebiotics, and/or probiotics which have positive effect on health of both human and fish. These supplementations led to production of valued-added fish fillets enriched in selenium and improved the fish growth and health.

All-male fry are preferred to prevent uncontrolled reproduction before harvest in intensive Nile tilapia (Oreochromis niloticus) aquaculture. Males also grow faster than females. An alternative approach to direct hormonal masculinisation of tilapia fry is to produce fry that are genetically male. However, sex determination system in tilapia is fairly complex. Recent developments have resulted in a linkage map and genetic markers that can be used to analyse the sex determination system. To analyse the genetic sex determination mechanism and to develop marker-assisted selection in the Stirling Nile tilapia population, a fully inbred line of clonal females (XX) was verified using test crosses and DNA markers (mostly microsatellites) to use as a standard reference line in sex determination studies. A series of crosses were performed involving this line of females and a range of males. Three groups of crosses were selected (each group consisted of three families) from progeny sex ratio distributions, and designated as type ‘A’ (normal XY males x clonal XX females), type ‘B’ (putative YY males x clonal XX females) and type ‘C’ (unknown groups of males x clonal XX females), for sex linkage study. For type ‘A’, inheritance of DNA markers and phenotypic sex was investigated using screened markers from tilapia linkage group 1 (LG1) to confirm the LG1-associated pattern of inheritance of phenotypic sex and the structure of LG1. Screened markers from LG1, LG3 and LG23 were used to investigate the association of markers with sex in families of type ‘B’ and ‘C’. In addition, a genome-wide scan of markers from the other 21 LGs was performed to investigate any association between markers and sex, in only families of cross type ‘B’. LG1 associated pattern of inheritance of phenotypic sex was confirmed by genotype and QTL analyses in families of cross type ‘A’. Analyses of genotypes in families of type ‘B’ and ‘C’ showed strong association with LG1 markers but no association with LG3 and LG23 markers. Genome wide scan of markers from all other LGs did not show any significant association between any markers and the sex. The allelic inheritance of two tightly linked LG1 markers (UNH995 and UNH104) in families of type ‘B’ and ‘C’ identified polymorphism in the sex determining locus: one of the alleles was associated mostly with male offspring whereas another allele was associated with both progeny (mostly males in type ‘B’ families, and approximately equal numbers in type ‘C’ families). This knowledge was used to identify and separate supermales (‘YY’ males) that should sire higher proportions of male progeny, reared to become sexually mature for use as broodstock. Two of them were crossed with XX females (one clonal and one outbred) to observe the phenotypic expression of the strongest male-associated allele in progeny sex. The observations of 98% male (99 males out of 101 progeny) and 100% male (N=75) from these two crosses respectively, suggest that a marker-assisted selection (MAS) programme for genetically male Nile tilapia production could be practical. This study also suggests that the departures from the sex ratios predicted using a “simple” XX/XY model (i.e., YY x XX should give all-male progeny) were strongly associated with the XX/XY system, due to multiple alleles, rather than being associated with loci in other LGs (e.g., LG3, LG23). This study also tentatively names the allele(s) giving intermediate sex ratios as “ambivalent” and emphasizes that the presence and actions of such allele(s) at the same sex-determining locus could explain departures from predicted sex ratios observed in some earlier studies in Nile tilapia.

In recent times, diminishing freshwater resources, due to the rapidly increasing drain of urban, industrial and agricultural activities in combination with the impact of climate change, has led to an urgent need to manage marine and brackish water environments more efficiently. Therefore the diversification of aquacultural practices, either by the introduction of new candidate species or by the adaptation of culture methods for existing species, is vital at a time when innovation and adaptability of the aquaculture industry is fundamental in order to maintain its sustainability. The Nile tilapia (Oreochromis niloticus, Linnaeus, 1758), which has now been spread well beyond its natural range, dominates tilapia aquaculture because of its adaptability and fast growth rate. Although not considered to be amongst the most salt tolerant of the cultured tilapia species, the Nile tilapia still offers considerable potential for culture in low-salinity water. An increase in knowledge of the limits and basis of salinity tolerance of Nile tilapia during the sensitive early life stages and the ability to predict responses of critical life-history stages to environmental change could prove invaluable in improving larval rearing techniques and extend the scope of this globally important fish species. The capability of early life stages of the Nile tilapia to withstand variations in salinity is due to their ability to osmoregulate, therefore the ontogeny of osmoregulation in the Nile tilapia was studied from spawning to yolk-sac absorption after exposure to different experimental conditions ranging from freshwater to 25 ppt. Eggs were able to withstand elevated rearing salinities up to 20 ppt, but transfer to 25 ppt induced 100% mortality by 48 h post-fertilisation. At all stages embryos and larvae hyper-regulated at lower salinities and hypo-regulated at higher salinities, relative to the salinity of the external media. Osmoregulatory capacity increased during development and from 2 days post-hatch onwards remained constant until yolk-sac absorption. Adjustments to larval osmolality, following abrupt transfer from freshwater to experimental salinities (12.5 and 20 ppt), appeared to follow a pattern of crisis and regulation, with whole-body osmolality for larvae stabilising at c. 48 h post-transfer for all treatments, regardless of age at time of transfer. Age at transfer to experimental salinities (7.5 – 20 ppt) had a significant positive effect on larval ability to osmoregulate; larvae transferred at 8 dph maintained a more constant range of whole body osmolality over the experimental salinities tested than larvae at hatch. Concomitantly, survival following transfer to experimental salinities increased with age. There was a significant effect (GLM; p < 0.05) of salinity of incubation and rearing media on the incidence of gross larval malformation that was seen to decline over the developmental period studied. It is well established that salinity exerts a strong influence on development and growth in early life stages of fishes therefore the effects of varying low salinities (0 - 25 ppt) on hatchability, survival, growth and energetic parameters were examined in the Nile tilapia during early life stages. Salinity up to 20 ppt was tolerable, although reduced hatching rates at 15 and 20 ppt suggest that these salinites may be less than optimal. Optimum timing of transfer of eggs from freshwater to elevated salinities was 3-4 h post-fertilisation, following manual stripping and fertilisation of eggs, however increasing incubation salinity lengthened the time taken to hatch. Salinity was related to dry body weight, with larvae in salinities greater than 15 ppt displaying, at hatch, a significantly (GLM: p < 0.05) lower body weight but containing greater yolk reserves than those in freshwater or lower salinities. Survival at yolk-sac absorption displayed a significant (GLM; p < 0.05) inverse relationship with increasing salinity and mortalities were particularly heavy in the higher salinities of 15, 20 and 25 ppt. Mortalities occurred primarily during early yolk-sac development yet stabilised from 5 dph onwards. Salinity had a negative effect on yolk absorption efficiency (YAE). Salinity-related differences in oxygen consumption rates were not detectable until 3 days post-hatch; oxygen consumption rates of larvae in freshwater between days 3 – 6 post-hatch were always significantly higher (GLM p < 0.05) than those in 7.5, 15, 20 and 25 ppt, however, on day 9 post-hatch this pattern was reversed and freshwater larvae had a significantly lower QO2 than those in elevated salinities. Salinity had a significant inverse effect on larval standard length, with elevated salinities producing shorter larvae from hatch until 6 dph, after which time there was no significant differences between treatments. Salinity had a significant effect on whole larval dry weight, with heavier larvae in elevated salinities throughout the yolk-sac period (GLM; p < 0.05). The ability of the Nile tilapia to withstand elevated salinity during early life stages is due to morphological and ultrastructural modifications of extrabranchial mitochondria-rich cells (MRCs) that confer an osmoregulatory capacity before the development of the adult osmoregulatory system. A clearly defined temporal staging of the appearance of these adaptive mechanisms, conferring ability to cope with varying environmental conditions during early development, was evident. Ontogenetic changes in MRC location, 2-dimensional surface area, density and general morphological changes were investigated in larvae incubated and reared in freshwater and brackish water (15 ppt) from hatch until yolk-sac absorption using Na+/K+-ATPase immunohistochemistry with a combination of microscope techniques. The pattern of MRC distribution was seen to change during development under both treatments, with cell density decreasing significantly on the body from hatch to 7 days post-hatch, but appearing on the inner opercular area at 3 days post-hatch and increasing significantly (GLM; p < 0.05) thereafter. Mitochondria-rich cells were always significantly (GLM; p < 0.05) denser in freshwater than in brackish water maintained larvae. In both freshwater and brackish water, MRCs located on the outer operculum and tail showed a marked increase in size with age, however, cells located on the abdominal epithelium of the yolk-sac and the inner operculum showed a significant decrease in size (GLM; p < 0.05) over time. Mitochondria-rich cells from brackish water maintained larvae from 1 day post-hatch onwards were always significantly larger (GLM; p < 0.05) than those maintained in freshwater. Preliminary scanning electron microscopy studies revealed structural differences in chloride cell morphology that varied according to environmental conditions. Mitochondria-rich cell morphology and function are intricately related and the plasticity or adaptive response of this cell to environmental changes is vital in preserving physiological homeostasis and contributes to fishes’ ability to inhabit diverse environments. Yolk-sac larvae were transferred from freshwater at 3 days post-hatch to 12.5 and 20 ppt and sampled at 24 and 48 h post-transfer. The use of scanning electron microscopy allowed a quantification of MRC, based on the appearance and surface area of their apical crypts, resulting in a reclassification of ‘sub-types’ i.e. Type I or absorptive, degenerating form (surface area range 5.2 – 19.6 μm2), Type II or active absorptive form (surface area range 1.1 – 15.7 μm2), Type III or differentiating form (surface area range 0.08 – 4.6 μm2) and Type IV or active secreting form (surface area range 4.1 – 11.7 μm2). In addition, the crypts of mucous cells were discriminated from those of MRCs based on the presence of globular extensions and similarly quantified.

Lack of affordable feeds is one of the major constraints facing small-scale fish farmers in Tanzania. This study evaluated the suitability of moringa leaf meal (MLM), cassava leaf meal (CLM) and cassava root meal (CRM) as novel ingredients in Nile tilapia, Oreochromis niloticus diets. Each of the ingredients was processed in an attempt to remove the most significant antinutritional factor. A series of five experiments was conducted in a recirculation system using juvenile O. niloticus. The fish were fed isonitrogenous (30g 100g-1), isolipidic (10g 100g-1) and isoenergetic (18 kJ g-1) diets containing graded levels of the processed ingredients to their apparent appetite but not exceeding 10% of their body weight for a period of 8 weeks. Processing led to the removal of 0.3% of saponin from MLM and 60% and 90% of hydrogen cyanide from CLM and CRM respectively. The contents of other inherent antinutritional factors such as phenols, tannins and phytic acid were little affected. Processed MLM, CLM and CRM had 31.1/29.0/1.5g 100g-1 of crude protein, 5.9/10.2/2.4g 100g-1 of crude fibre and 20.1/19.7/15.8k Jg-1 of gross energy. The content of sulphur amino acids was higher in CLM (0.47%) than in MLM (0.23%). Digestibe protein and digestible energy was higher in MLM (25.71g 100g-1/15.44kJ g-1) than in CLM (12.71g 100-1/9.16kJ g-1). CRM had a digestible energy content of 13.5kJ g-1. Inclusion of either of the leaf meals, even at the lowest level of 15g 100g-1 of total dietary protein, led to a significant reduction in feed intake, growth and feed utilisation. Liver and small intestine did not show any histopathological changes which could be linked to dietary treatment. Conversely, cassava root meal could replace up to 75% of wheat meal in the diet without significantly affecting performance. The performance of leaf meals was marginally improved by a combination of blending and feeding stimulants, whereby a blend containing 1 part MLM and 2 parts CLM could provide up to 20g 100g-1 of dietary protein without significantly reducing performance. Biological and economic performance of practical diets containing 30-50g 100g-1 of dietary protein from moringa and cassava blends (LMB) with feeding stimulants was significantly lower than a fishmeal-meal based diet (FM) but comparable to a soybean meal-based diet (SBM). The suitability of MLM and CLM as novel protein sources in O. niloticus diets will depend on 1) improving reduction/removal of inherent antinutritional factors in MLM and CLM as well as improving digestibility of CLM. On the other hand, the suitability of CRM as a carbohydrate energy source will depend on the availability of cost effective protein sources due to its low protein content.

Thesis (MScAgric)--University of Stellenbosch, 2002. / ENGLISH ABSTRACT: Forming part of a conservation programme, this study was concerned with two species of Cichlid fish (Oreochromis mossambicus and O. ni/oticus), which were brought into contact with each other by unnatural ways. They are now hybridizing to some extent and there is also evidence that the foreign O. ni/oticus may out compete the native O. mossambicus. To cast light on what the current distribution is of both these species and the hybrids in Southern Africa, it is important to identify specimens very accurately. In attempting to find genetic markers to distinguish between two species of Cichlids we tested 20 microsatellite dinucleotide (CAn) repeats during a preliminary study and found five of these promising to exhibit little intra-specific genetic diversity but large genetic variation between species. We amplified these five loci in 145 individuals from 10 populations, which included the two species and their hybrids. Exact sizes of the fragments were determined using an automated DNA sequencer. Between the two species, allele sizes were overlapping, but when data were analyzed by statistical models, the differences could be seen for populations, however on individual level there was overlap between the species. The hybrids were found to be intermediate positioned between the two pure species. Our attempt to assign individuals to populations provided doubtful results. Thus, using this set of markers, populations can be ascribed to one of these species, but not individuals by themselves. / AFRIKAANSE OPSOMMING: As deel van 'n natuurbewarings program, word daar in hierdie studie twee spesies van vis ondersoek was in kontak met mekaar gekom het op onnatuurlike wyse. Hierdie twee visspesies vanuit die CICHLIDAEfamilie (Oreochromis mossambicus en 0. ni/oticus) kan hibridiseer wanneer hul saam voorkom, maar dit is ook bekend dat die uitheemse O. ni/oticus die inheemse O. mossambicus kan bedreig in terme van leefruimte, kos en broeispasie. Om die voorkoms van hibriede tussen die twee spesies te ondersoek in Suider Afrika se varswater opvangsgebiede, is dit baie belangrik om individue baie akkuraat te identifiseer. In hierdie poging om genetiese merkers te vind wat die twee spesies van mekaar onderskei, het ons 20 mikrosateliet di-nulkleotied (CAn) herhalende volgordes op verskillende loci ondersoek. Vyf daarvan het belowend voorgekom om as spesie spesifieke merkers te dien. Die fragmente op die vyf loci is ge-amplifiseer in 145 individue vanuit 10 populasies. Presiese groottes van die fragmente is bepaal met behulp van 'n ge-outomatiseerde DNA volgorde bepaler waarna genotiepes vir elke individu toegeken is. Tussen die twee spesies het alleel groottes oorvleuel, maar wanneer data geanaliseer word met behulp van statistiese metodes, was verskille tussen die spesies duidelik op populasie vlak. Die hibriede het intemediêr tussen die twee spesies voorgekom. Dus met behulp van hierdie stel merkers kan onderskei word tussen die twee spesies op populasie vlak, hoewel individue nie op sig self identifiseer kan word nie.

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.