In most tropical river systems there has been a lack of integrated ecological research to investigate the dynamics and impacts of invasive species on recipient river systems. This is in sharp contrast to temperate river systems. This thesis investigated the nature, extent, and impact of Nile tilapia, Oreochromis niloticus (Linnaeus, 1758), on indigenous congenerics within the Limpopo River basin in northern South Africa. An integrated approach was adopted to gain a better understanding of factors that allow Nile tilapia to be a successful invader and also to gain an insight into its invasion rate and conservation implications within South Africa. Morphometric and genetic variation between Nile tilapia, indigenous congenerics and their associated hybrids were determined. Intermediate meristic characters obscured the identification of hybrid specimens from pure morpho-specimens and species identity was only confirmed through mtDNA analysis. Preliminary evidence points to unidirectional hybridization among Oreochromis congeners in the Limpopo River system. The hypothesis that bigger Nile tilapia males may have a competitive advantage over spawning grounds and in female mate choice is proposed. The trophic ecology of Nile tilapia was investigated using both stomach contents and stable isotope analysis. A high similarity in stomach contents was observed but interspecific differences were revealed in the isotopic composition of diets that suggest fine scale patterns of resource partitioning that could be achieved by the ability of fish to selectively feed on what is immediately available and the ability to perceive the dynamics that determine food resource availability. Ecological niche models were used to determine the potential invasive range of Nile tilapia and revealed broad invasive potential over most river systems in southern Africa that overlapped the natural range of endemic congenerics. It was noted that model performance and the degree of niche conservatism varied significantly with variable selection and spatial extent of study area. This implied that the spatial distribution of suitable and unsuitable environmental variables varied between the native and introduced ranges of Nile tilapia and also indicated the ability of Nile tilapia to survive in conditions incongruent with its native range. The extreme hardiness and adaptive life history characteristics of Nile tilapia have probably predisposed it to be a successful invader in novel systems within southern Africa. Lastly, a qualitative risk assessment method was developed as a potential application to determine the risk of establishment and spread of the invasive Nile tilapia. Results showed that in the absence of quantitative data on ecosystem structure and functioning, habitat suitability analysis in terms of known physiological tolerance limits to minimum water temperature, presence or absence of dams, seasonality of river flows and the presence of indigenous fish species of concern could be adequate for identifying vulnerable river systems. The model developed also provides an objective method that is easy to implement, modify and improve on as new data become available. Furthermore, the model can be applied to highlight areas of uncertainty where future research should be directed. / Thesis (PhD)--University of Pretoria, 2012. / Zoology and Entomology / Unrestricted

Numerous food-borne illnesses are associated with fish and shellfish annually due to consumers choosing to eat seafood raw or undercooked and consumers not properly handling and preparing seafood. The 2009 FDA Food Code suggests intact fish and shellfish should be cooked to an internal temperature of 63°C to target Salmonella spp. Selected fish and shellfish were cooked to 64°C ± 1 and 74°C ± 1 and evaluated for consumer acceptability, characteristics of doneness at temperature endpoints, and physical changes of texture (TPA, KSC, and WB), color, and cook loss. Temperature endpoints represented the recommended internal temperature and ten degrees higher to increase lethality of Salmonella spp. Differences (p < 0.05) in texture were determined among the methods and products tested. However, consumer acceptability (n = 50) for fish and shellfish products (salmon: baked, poached; tilapia: baked, pan-fried; and shrimp: boiled) cooked to 64°C ± 1 and 74°C ± 1 were liked equally (p > 0.05), with mean hedonic scores falling between 6 (like slightly) and 7 (like moderately). A trained descriptive panel (n = 7) reviewed visual and non-oral texture indicators of doneness to distinguish 64°C ± 1 and 74°C ± 1. Firmness and shape of shrimp, separation between muscle flakes and fillet edge color of baked tilapia, and firmness and edge color of the fillet for baked salmon were identified as indicators to determine doneness. Overall, 74°C could be recommended as the internal temperature for cooking fish/shellfish such as salmon, tilapia, and shrimp without diminishing eating quality or acceptability. / Master of Science in Life Sciences

Resumen La presente tesis aborda los métodos estadísticos de monitorización y predicción de procesos multivariantes y autocorrelacionados en la industria. La investigación se orienta, por un lado, al estudio de los métodos de monitorización que culmina con el desarrollo de un gráfico de control de procesos; y por otro lado, al análisis de los métodos de predicción, donde se elabora el modelo dinámico sobre estructuras latentes (MDEL). En los dos casos, los modelos de series temporales constituyen una herramienta fundamental para modelar la estructura de autocorrelación en los datos. El estudio del modelo de monitorización sigue un orden lógico de complejidad de los métodos de control de procesos usados en la industria. Se empieza por analizar los gráficos de control univariante; los gráficos multivariantes, y sus limitaciones en el control de procesos autocorrelacionados, con lo cual se introducen los gráficos multivariantes para procesos autocorrelacionados, donde se desarrolla el gráfico de control multivariate autocorrelated and adapted EWMA chart (MAAEWMA). El desempeño del gráfico MAAEWMA fue comparado con el gráfico MEWMA ajustado a ruidos blancos. Los resultados mostraron que el gráfico MAAEWMA es más rápido en detectar cambios en la media del proceso, y es más eficiente para procesos con media y alta autocorrelación. Por otro lado, el método predictivo fue elaborado para modelar los procesos multivariantes y autocorrelacionados. Para ello, se estudiaron los métodos sobre estructuras latentes PCA y PLS, y sus respectivas versiones dinámicas: dynamic PCA (DPCA) y dynamic PLS (DPLS). El desarrollo del modelo dinámico sobre estructuras latentes (MDEL) consistió en la combinación del modelo DPCA y de la función de transferencia (FT). El modelo DPCA fue aplicado a los datos de entrada y de salida del proceso de manera separada para determinar las variables latentes que recogen la máxima variabilidad dinámica en los datos. La estructura dinámica en las variables latentes resultantes fue modelada mediante una función de transferencia para analizar la influencia de los datos de entrada sobre los datos de salida. Los modelos MDEL y DPLS fueron ajustados en condiciones idénticas a un conjunto de datos simulados. La calidad de ajuste de los modelos fue evaluada mediante la variabilidad explicada de las variables latentes (R2), y la capacidad predictiva fue testada mediante validación cruzada, donde fue calculado el mean absulute error (MAE) de los dos modelos. El MDEL presentó mejores resultados comparado al DPLS, con mayor variabilidad explicada porlas variables latentes, y con una capacidad predictiva robusta y estable. Los modelos propuestos fueron aplicados para monitorizar y predecir el proceso de cría de tilapia en Mozambique. El gráfico MAAEWMA señló cambios significativos del ambiente acuático en el proceso, y con el modelo MDEL se analizó el impacto de dichos cambios en el crecimiento de la tilapia. / Resum La present tesi aborda els mètodes estadístics de monitoratge i predicció de processos multivariants i autocorrelacionados en la indústria. La investigació s'orienta, d'una banda, a l'estudi dels mètodes de monitorització que culmina amb el desenvolupament d'un gràfic de control de processos; i d'altra banda, a l'anàlisi dels mètodes de predicció, on s'elabora el model dinàmic sobre estructures latents (MDEL). En els dos casos, els models de s`eries temporals constitueixen una eina fonamental per a modelar l'estructura d'autocorrelació en les dades. L'estudi del model de monitoratge segueix un ordre lògic de complexitat dels mètodes de control de processos usats en la indústria. Es comença per analitzar els gràfics de control univariant;els gràfics multivariants, i les seves limitacions en el control de processos autocorrelacionados, amb la qual cosa s'introdueixen els gràfics multivariants per a processos autocorrelacionados, on es desenvolupa el gràfic de control multivariate autocorrelated and adapted EWMA chart (MAAEWMA). L'acompliment del gràfic MAAEWMA va ser comparat amb el gràfic MEWMA ajustat a sorolls blancs. Els resultats van mostrar que el gràfic MAAEMA és més ràpid en detectar canvis en la mitjana del procés, i és més eficient per a processos amb mitja i alta autocorrelació. D'altra banda, el mètode predictiu va ser elaborat per modelar els processos multivariants i autocorrelacionados. Per a això, es van estudiar els m`etodes sobre estructures latents PCA i PLS, i les seves respectives versions dinàmiques: dynamic PCA (DPCA) i dynamic PLS (DPLS). El desenvolupament del model dinàmic sobre estructures latents (MDEL) va consistir en la combinació del model DPCA i de la funció de transferència (FT). El model DPCA va ser aplicat a les dades d'entrada i de sortida del procés de manera separada per a determinar les variables latents que recullen la màxima variabilitat dinàmica en les dades. L'estructura din`amica en les variables latents resultants va ser modelada mitjançant una funci¿o de transferència per analitzar la influència de les dades d'entrada sobre les dades de sortida. Els models mdel i DPLS van ser ajustats en condicions idèntiques a un conjunt de dades simulades. La qualitat d'ajust dels models va ser avaluada mitjançant la variabilitat explicada de les variables latents (R2), i la capacitat predictiva va ser testada mitjançant validació creuada, on va ser calculat el pixen absulute error (MAE) de els dos models. L'mdel presentar millors resultats comparat al DPLS, amb major variabilitat explicada per les variables latents, i amb una capacitat predictiva robusta i estable. Els models proposats van ser aplicats per monitoritzar i predir el proc¿es de cria de tilapia a Moçambic. El gràfic MAAEWMA va assenyalar canvis significatius de l'ambient aquàtic en el procés, i amb el model mdel es va analitzar l'impacte d'aquests canvis en el creixement de la tilàpia. / Abstract This thesis is based on statistical methods to monitor and predict the multivariate and autocorrelated industrial processes. The research is oriented on the monitoring methods that culminate with the development of a process control chart and analysis of the prediction methods, where the dynamic model on latent structures (MDEL) was developed. In both cases, the time series models were used to model the autocorrelation structure in the data. The analysis of the monitoring models follows a logical order of complexity of the process control methods used in the industry. The univariate and multivariate charts were analysed, and their limitations in the control of autocorrelated processes were considered. Therefore, the multivariate charts for autocorrelated processes were introduced and the multivariate, autocorrelated and adapted EWMA chart (MAAEWMA) was developed. The performance of the MAAEWMA chart was compared with the MEWMA chart adjusted to white noise. The results showed that the MAAEWMA chart was faster in detecting changes in the process mean and was more efficient for processes with medium and high autocorrelation. On the other hand, the predictive method was developed to model the multivariate and autocorrelated processes. To this end, the methods on latent structures PCA and PLS and their respective dynamic versions: dynamic PCA (DPCA) and dynamic PLS (DPLS) were studied. The dynamic model on latent structures (MDEL) consists of the combination of DPCA model and the transfer function (FT). The DPCA model was applied separately to the input and output data of the process. This strategy was applied to guarantee the maximum dynamic variability in the latent varia- bles. The dynamic structure in the latent variables was modelled by a transfer function to analyse the influence of the input on the output data. The MDEL and DPLS models were adjusted in identical conditions to a set of simulated data. The goodness of fit was evaluated by the explained variability of the latent variables (R2) and the predictive capacity of the models was tested through cross validation, where the mean absolute error (MAE) was calculated. The MDEL presented better results than DPLS model, with greater variability explained by the latent variables and a robust and stable predictive capacity. The proposed models were applied to monitor and predict the tilapia crop process in Mozambique. The MAAEWMA chart indicated significant changes in the aquatic environmentand and with the MDEL was analysed the impact of these changes on the growth of tilapia. / Mulema, SA. (2018). Monitorización y Predicción de Procesos Multivariantes y Autocorrelacionados. Aplicación a la producción de Tilapia en Mozambique [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/113070

Consumers regularly decide to consume fish and shellfish raw or undercooked, which can cause foodborne illness due to product contamination or unsafe handling by the consumer.  In order to be considered safe for consumption, intact fish and shellfish should be prepared to an internal temperature of 63"C, according to the 2009 FDA Food Code, with Salmonella spp. as the target organism.  Focus groups (5 groups, 32 participants) were conducted to determine consumer beliefs and concerns regarding fish and shellfish safety and preparation.  Transcripts of focus groups where coded for themes, which were then grouped into categories.  Nine categories emerged including:  experience, trust, confidence, quality of product, motivation, concerns, cooking procedures, cooking instructions, and knowledge.  Emerging themes were used to help develop educational materials to increase consumer ability to properly prepare fish and shellfish.  In a separate experiment, participants (n=6) cooked salmon (baked, broiled), tilapia (baked, broiled), and shrimp (broiled, boiled) according to cookbook-based directions.    Internal temperatures of products were recorded, with 33.3% of products cooked to a temperature less than 63"C.  A group training session was held, during which participants received additional visual and non-oral cues to determine when products were prepared to 63"C and safe food handling practices.  After training, participants prepared the same products.  Participants demonstrated improved food safety behaviors and were more successful at cooking products to temperatures "63"C (94.4% of products).  Improved cooking instruction and educational materials may reduce the risk of foodborne illness from undercooked fish and shellfish. / Master of Science in Life Sciences

Aquacultured products assist the human demands for seafood so that foodfish supplies can remain sustainable and consistent. Although the fish-farming industry has seen dramatic growth and intensification in recent years, the latter has led to an increase in bacterial diseases and fish health management problems, resulting in major economic losses around the world. In addition to the lack of understanding of fish physiology, these complications are exacerbated by the inappropriate and controversial use of antibiotics. This work addressed these issues in striped catfish (Pangasius hypophthalmus) and Nile tilapia (Oreochromis niloticus), two economically important foodfish, by investigating alternative, more cost-effective options to promote fish health. The first two studies established reference intervals for immunology, hematology and plasma chemistry analytes in striped catfish in a recirculating aquaculture system (RAS). In a third study, the immunomodulatory effects after directly feeding probiotic strains of Bacillus subtilis NZ86 and O14VRQ in Nile tilapia were ascertained. This last study revealed that supplementation with both of the probiotic strains for 51 days stimulated several local and systemic innate immune responses of tilapia. When these transient probiotic bacteria were present in the gut, a pro-inflammatory environment was developed as evidenced by the localized higher expression of the cytokines tumor necrosis factor (TNF) – α and interleukin (IL) – 1β. Significant increases (p < 0.05) were noted differentially by both probiotic strains throughout the trial in plasma lysozyme content, alternative complement activity, and in the peripheral blood leukocyte profiles. Additionally, there were trends for increased levels of phagocytosis and respiratory burst in leukocytes of the anterior kidney and spleen at the end of the trial, suggesting the potential use of these probiotic strains for improved immune-competence. These findings help to understand and clarify the potential mechanism of action associated with the increased disease resistance recorded in preliminary studies with the same probiotic strains. Implementation of the tools established and validated in this work could be useful in evaluating fish welfare situations involving striped catfish grown in RAS conditions, and also show promise for a healthier foodfish supply where antibiotic applications practices could be minimized. / Ph. D. / Aquaculture, or fish farming, is one of the most prosperous production sectors of animal-derived food. Despite the success story of aquaculture, the fish industry is heavily plagued by bacterial diseases, which cause losses in billions of dollars annually around the world, and directly contribute to increases in human food insecurity. Since the options to cost-effectively address diseases are limited, I explored alternative ways to more safely monitor and also ensure optimal health in striped catfish and tilapia, two globally important aquaculture fishes. I investigated the values of different cellular and chemical components of the blood to monitor the health of striped catfish when grown in indoor recirculating conditions, in order to understand normal catfish physiology. The values of these blood components were comparable to those of other freshwater fishes. As part of another study, I supplemented probiotics in the diet of the tilapia for 51 days, and assessed the effects of these on the immune system of the fish. Dietary supplementation of the probiotics resulted in the presence of the probiotics in the gut of the fish. Furthermore, the presence of these microbes was tightly linked to elevated values of numerous functions of the immune system. These functions included levels of lysozyme, alternative complement, and percentage of neutrophils, which are all related with a state of heightened immunity in the animal host. The tools that I established and validated in this study are promising alternatives to optimize the health of these two important foodfish. Moreover, they could be useful for the fish farmer because of their greater cost-effectiveness, and can potentially lead to a safer foodfish supply by decreasing the reliance on antibiotics.

Tilapia are freshwater fish that have become important in aquaculture and as a stable global source of seafood due to their ability to thrive in different environments. However, tilapia are sometimes considered nutritionally undesirable due to their high n-6 to n-3 fatty acid ratios. A market study was conducted first to determine fatty acid compositions in tilapia fillets in different US markets. Then a research was conducted to enhance nutritional value of tilapia by improving the n-3 and oleic acid contents in fish fillets without compromising fish growth or feed conversion ratios. Feeds were formulated with combinations of high and low n-6, n-3, and oleic acid levels using soybean oil, fish oil, algae oil, and high-oleic sunflower oil. Then 12 diets, including a commercial diet, were assigned to 24 tanks, each with 25 tilapia per tank. A Recirculating Aquaculture System (RAS) was used to grow the fish for 8 weeks. Fatty acid compositions of tilapia fillets were determined and samples were vacuum packed and stored at -10oC and -20oC to test oxidative degradation and fatty acid compositional changes. The market survey data showed that there were significant differences in fatty acid composition, lipid content, and n-6:n-3 fatty acid ratios depending on the country of origin. Samples from USA had ideal n-6:n-3 ratios (1.3 ±0.85) while samples from Southeast Asia had higher n-6:n-3 fatty acids ratio (6.6 ±0.54). Algae oil incorporation significantly increased DHA level while fish oil incorporation significantly increased both EPA and DPA. High-oleic sunflower oil based diets improved oleic acid levels and reduced linoleic acid compared to the soybean oil based diets. Sensory evaluation indicated that lipid source did not significantly impact preference or overall fillet quality, including texture. Interestingly, a survey showed people were interested in value-added tilapia, and would pay up to 30% more for nutritionally enhanced fish compared to the $5.00/lb fresh fillet price currently available in supermarkets. There was no observable oxidation during long term frozen storage. The oxidation study proved that value-addition would not be compromised during the long term storage conditions, even under temperature abuse. It is possible to improve tilapia nutritional quality through diet to provide consumers with value-added products that maintain quality during frozen storage. / Ph. D. / Tilapia are high quality food with high levels of protein. However, many consumers are skeptical about tilapia due to their undesirable lipid composition containing high levels of omega 6. Therefore initially, the market study was developed to identify the lipid composition of tilapia US consumers were exposed to. Then the experiment was designed to create high omega 3 tilapia by improving the diets using mixture of fish oil, algae oil, high-oleic sunflower oil, and soybean oil. The 8 week tilapia growth trial was conducted with 12 experimental diets. Then the fish fillets were collected and analyzed for their fatty acid composition using GC/MS. Also fillet’s shelf-life study was conducted in 2 frozen storage conditions up to 9 months. The market study proved that the omega 6 to omega 3 ratio was favorable especially in USA sourced tilapia. However it also showed wide variation in omega 6 to omega 3 ratio depending on the country of origin. Tilapia from USA had an ideal omega 6:omega 3 ratio (1.3 ±0.85) while tilapia from Southeast Asia had higher omega 6:omega 3 ratio (6.6 ±0.54). The experimental study showed that the algae oil addition significantly improved DHA level while fish oil addition improved EPA and DPA. Higholeic sunflower oil addition improved omega 9 compared to the soybean oil based diets. Sensory evaluation indicated that different lipid sources had no impact on consumer preference. Interestingly, the survey showed that the people were interested in healthier tilapia, and were willing pay up to 30% more to the $5.00/lb of supermarket fresh fillet price. There was no observable degradation during long term frozen storage. The shelf life study proved that valueaddition would not degrade during the long term storage conditions. Therefore, development of high quality tilapia through diet manipulation is possible that does not degrade over 9 months of frozen storage.

Les invasions biologiques sont reconnues comme un facteur évolutif important sur une échelle de temps courte. Elles affectent notamment la structure génétique des populations, les patrons d’évolution phénotypique et la richesse des faunes de parasites associées aux populations envahissantes. Cette étude se propose de quantifier les conséquences d’une invasion biologique suivant ces trois niveaux (génétique, phénotypique et parasitologique) en prenant pour exemple le cas du tilapia du Mozambique Oreochromis mossambicus. Ce cichlidé africain présente un statut remarquable en biologie de la conservation puisqu’il est à la fois (i) l’une des espèces les plus envahissantes au monde car dispersée à l’échelle globale au cours du XXème siècle et (ii) une espèce « quasi-menacée » (UICN) sur son aire native (partie du sud-est de l’Afrique) du fait de son hybridation massive avec d’autres Oreochromis sp. introduits. La démarche générale employée ici est de décrire l’histoire récente des populations à l’aide de marqueurs nucléaires (AFLP) et des séquences de l’ADN mitochondrial (ADNmt), puis de mettre en relation ces résultats génétiques avec la diversité morphologique et la parasitologie des populations. Deux systèmes différents ont été étudiés : Au sein de l’aire native, l’étude se focalise sur le Limpopo inférieur et le sous-bassin de la Changane (Mozambique). Des patrons d’introgression incluant trois espèces en présence sont détectés, mais les hybrides sont peu fréquents et leur expansion limitée. Ces résultats sont de plutôt bonne augure pour la conservation d’O. mossambicus et ils permettent d’identifier deux zones de conservation prioritaires. L’étude des parasites indique une plus grande diversité parasitaire mais de faibles prévalences dans les sites de moindre valeur en conservation, ce qui pourrait favoriser le succès des espèces introduites et de leurs hybrides. Parmi les territoires envahis, les AFLP et l’ADNmt soutiennent une homogénéité générale et une diversité génétique faible, qui sont interprétées comme le résultat d’un fort goulot d’étranglement précédant l’expansion à l’échelle mondiale. Une structure des populations en lien avec la géographie à large échelle (Nouvelle-Calédonie, Guadeloupe, Jamaïque) est cependant détectée. La variation de la forme du corps est également structurée à large échelle géographique, ce en dépit des fortes variations environnementales enregistrées à l’échelle locale. Cela suggère un effet des contraintes génétiques sur la diversification morphologique contemporaine. L’absence de parasites monogènes sur les populations introduites en Nouvelle-Calédonie peut être mise en relation avec un évènement fondateur, et est proposé comme l’un des facteurs ayant pu favoriser le succès de l’espèce. En conclusion, une faible diversité génétique ne contraint vraisemblablement pas un potentiel envahissant élevé et une diversification rapide chez les tilapias. / Biological invasions are recognized as a significant evolutionary factor over short time scales. In particular, their effect is well recorded on the genetic structure of populations, the patterns of phenotypic evolution and the richness of parasite fauna associated to invasive populations. This study aims at quantifying the consequences of a biological invasion according to these three levels (genetical, phenotypical and parasitological) taking as example the Mozambique tilapia Oreochromis mossambicus. This African cichlid is characterized by an unusual conservation status since it is both (i) ranked among the world’s worst invasive species due to its global dispersion during the 20th century and (ii) sorted as “near-threatened” (IUCN) over its native range (a part of south-east Africa) because of massive hybridization with alien introduced Oreochromis species. The approach used in this study imply to describe the recent history of populations using nuclear (AFLP) and mitochondrial DNA (mtDNA) markers, and then to compare this genetic background to results describing the morphological and parasitological diversity of populations. Two different biological systems were studied: 1) Within the native range, the study focuses on the Lower Limpopo and the Changane sub-drainage (Mozambique). Introgression patterns involving the three co-occurring species were detected, but the frequency of hybrid is low and their geographic expansion is limited. These results provided rather good auspices for the conservation of O. mossambicus, and they allowed to identify two zones of high conservation priorities. The parasitological survey reveals high parasite richness and low prevalences among sites of low conservation values. This last pattern could favour the success of alien introduced species and their hybrids. 2) Among the invasive range of O. mossambicus, both AFLP and mtDNA support a strong genetic homogeneity and a low genetic diversity, a pattern interpreted as resulting from a strong population bottleneck preceding the events of global dispersion. A pattern of population structure related to large scale geography (New Caledonia, Guadeloupe, Jamaica) is nevertheless detected. Body shape variation is also primarily structured at large geographical scale, suggesting a role for genetic constrains on contemporary morphological diversification. The total absence of monogenean parasites in the populations of New Caledonia could result from a founding event and is suggested as a potential factor that could have favoured the O. mossambicus’ success. In conclusion, a low genetic diversity does not likely constraint a strong invasive potential and a rapid phenotypic diversification in tilapias.

by Yan Hiu Mei, Carol. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 114-126). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iii / Acknowledgements --- p.iv / Table of Contents --- p.v / List of Tables --- p.viii / List of Figures --- p.ix / Abbreviations --- p.xii / Chapter CHAPTER 1 --- TRANSGENIC TECHNOLOGY --- p.1 / Chapter 1.1 --- Transgenesis in animals --- p.1 / Chapter 1.2 --- Transgenic fish in toxicology --- p.4 / Chapter 1.2.1 --- Aquatic metal toxicity --- p.4 / Chapter 1.2.2 --- Environmental monitoring of aquatic metal toxicity --- p.5 / Chapter 1.2.3 --- Biomarkers --- p.6 / Chapter 1.3 --- Transgenics in aquaculture --- p.9 / Chapter 1.3.1 --- Revolution is needed in aquaculture --- p.9 / Chapter 1.3.2 --- Aquaculture potential of tilapia in China --- p.10 / Chapter 1.3.3 --- Endocrinology for fish growth --- p.12 / Chapter 1.3.4 --- Growth promotion by exogenous growth hormone in tilapia --- p.14 / Chapter 1.3.5 --- Accelerated growth in transgenic fish --- p.15 / Chapter 1.4 --- General principle in transgenic fish production --- p.16 / Chapter 1.5 --- Project aim --- p.22 / Chapter CHAPTER 2 --- ISOLATION AND CHARACTERIZATION OF ZEBRAFISH METALLOTHIONEIN GENE PROMOTER --- p.23 / Chapter 2.1 --- Introduction --- p.23 / Chapter 2.1.1 --- Metallothionein --- p.23 / Chapter 2.1.2 --- Biological functions --- p.24 / Chapter 2.1.3 --- Metallothionein gene regulations --- p.25 / Chapter 2.1.4 --- Metallothionein as biomarker for metal pollution --- p.26 / Chapter 2.2 --- Materials and methods --- p.28 / Chapter 2.2.1 --- General molecular biology techniques --- p.28 / Chapter 2.2.2 --- Sequences of PCR primers used --- p.31 / Chapter 2.2.3 --- Cloning zebrafish MT gene 5-flanking region --- p.31 / Chapter 2.2.4 --- Cloning zebrafish MT gene --- p.32 / Chapter 2.2.5 --- Cloning full length zMT gene --- p.33 / Chapter 2.2.6 --- Cell culture --- p.35 / Chapter 2.2.7 --- Transient transfection assay --- p.37 / Chapter 2.2.8 --- Electrophoretic mobility shift assay --- p.39 / Chapter 2.3 --- Results --- p.42 / Chapter 2.3.1 --- Zebrafish metallothionein gene --- p.42 / Chapter 2.3.2 --- Deletion analysis of zMT promoter by transient transfection assay --- p.48 / Chapter 2.3.3 --- Functional characterization of zebrafish metallothionein promoter --- p.57 / Chapter 2.4 --- Discussions --- p.61 / Chapter 2.4.1 --- Zebrafish MT gene --- p.61 / Chapter 2.4.2 --- Functional characterization of zebrafish MT promoter --- p.61 / Chapter CHAPTER 3 --- PREPARATION OF GENE CONSTRUCTS FOR TRANSFER IN ZEBRAFISH --- p.65 / Chapter 3.1 --- Introduction --- p.65 / Chapter 3.1.1 --- Zebrafish as model in toxicological studies --- p.65 / Chapter 3.1.2 --- Reporter gene system --- p.66 / Chapter 3.1.3 --- Transgenic reporter fish --- p.68 / Chapter 3.1.4 --- Gene transfer by electroporation in zebrafish --- p.68 / Chapter 3.1.5 --- Objective --- p.69 / Chapter 3.2 --- Materials and methods --- p.70 / Chapter 3.2.1 --- Design of gene constructs for ectopic expression in zebrafish --- p.70 / Chapter 3.2.2 --- Testing electroporation conditions for zebrafish --- p.72 / Chapter 3.3 --- Results --- p.73 / Chapter 3.4 --- Discussions --- p.76 / Chapter 3.4.1 --- Engineering gene constructs --- p.76 / Chapter 3.4.2 --- Applications of transgenic zebrafish --- p.79 / Chapter CHAPTER 4 --- GENE TRANSFER EXPERIMENTS ON TILAPIA --- p.82 / Chapter 4.1 --- Introduction --- p.82 / Chapter 4.2 --- Materials and methods --- p.85 / Chapter 4.2.1 --- Isolation of O. aureus growth hormone --- p.85 / Chapter 4.2.2 --- Engineering gene constructs for ectopic expression in tilapia --- p.86 / Chapter 4.2.3 --- Gene transfer in tilapia --- p.87 / Chapter 4.2.4 --- Screening transgenic tilapia --- p.89 / Chapter 4.3 --- Results --- p.91 / Chapter 4.3.1 --- Tilapia growth hormone --- p.91 / Chapter 4.3.2 --- Gene constructs for ectopic expression in tilapia --- p.94 / Chapter 4.3.3 --- Testing electroporation conditions --- p.96 / Chapter 4.3.4 --- PCR screening for transgenic fish --- p.97 / Chapter 4.4 --- Discussions --- p.101 / Chapter 4.4.1 --- Tilapia growth hormone --- p.101 / Chapter 4.4.2 --- Electroporation experiments on of tilapia eggs --- p.101 / Chapter 4.4.3 --- Improvements on gene construct design for tilapia --- p.104 / Chapter 4.4.4 --- Ethical and safety considerations --- p.106 / Chapter CHAPTER 5 --- REFERENCES --- p.114 / APPENDIX --- p.127

One of the major problems facing aquaculture in Ghana is the non-availability of quality and affordable fish feeds. The present study investigated the nutritional suitability and cost-effectiveness of some Ghanaian oilseed by-products, soybean meal (Glycine spp), cottonseed meal (Gossypium spp), groundnut cake (Arachis hypogaea L.) and groundnut husk, as alternative protein sources to fishmeal (FM) in the diet of Nile tilapia (Oreochromis niloticus L.). The oilseed meals were used individually, as mixtures, as mixtures enriched with methionine and mixtures detoxified by heat processing (autoclaving) and/or addition of supplements (viz. phytase and ferrous sulphate) intended to reduce levels of the most important antinutritional factors (ANFs). Diets, containing the oilseed meals at inclusion levels from 25% to 75% dietary protein, were formulated to be isonitrogenous (320 g.kg-1), isolipidic (100 g.kg-1) and isoenergetic (18 KJ.g-1) and fed to juvenile Nile tilapia at 4-10% of their body weight for a period of eight weeks. Proximate analysis showed that soybean meal (SBM), cottonseed meal (CSM), groundnut cake (GNC) and groundnut husk (GNH) had 500.3, 441.4, 430.5 and 205.6 g.kg-1 crude protein, 38.2, 89.5, 12.8 and 89.2 g.kg-1 crude fibre and 20.19, 19.61, 23.17 and 22.18 kJ.g-1 gross energy respectively. Generally the oilseed meals had good essential amino acid (EAA) profiles with the exception of GNH. The EAA profile of SBM compared very well with FM but methionine and threonine were low (0.73 and 1.50 % of protein respectively) and the same was true for CSM and GNC with even lower levels. Analyzed ANFs in SBM, CSM, GNC and GNH were 17.54, 31.64, 14.86 and 3.99 g.kg-1 phytic acid, 14.09, 1.24 and 2.34 g.kg-1 trypsin inhibitors and 5.80, 6.50, 8.01 and 10.08 g.kg-1 saponin respectively and in CSM 5.6 g.kg-1 gossypol. Nutrient digestibility of these oilseed proteins suggested that Nile tilapia may be able to utilize SBM, CSM and GNC efficiently as dietary protein sources due to high apparent protein digestibility of 94.50%, 84.93% and 90.01% respectively. However, GNH may not be suitable because of very low apparent protein digestibility (27.67%). These protein sources when used individually were shown to cause depressed growth and feed efficiency when substituting more than 50% of the FM protein in diets. This may be attributed to high levels of ANFs, high fibre content and poor EAA profile. However, the use of mixtures of these meals was found to be marginally more effective than that of single sources. This may have been as a result of lower levels of ANFs and improvement in essential amino acid profile due to mixing. Supplementing the mixtures with methionine led to improvement in feed utilization but without significantly improving the nutritive value compared with FM. Heat processing was effective in reducing heat labile trypsin inhibitors in SBM, CSM and GNC by almost 80%, but not phytic acid and saponins, which remained virtually unaffected. Use of meals detoxified by heat processing with/without supplements at 50% inclusion improved growth and feed utilization compared to the unprocessed meals and performance was generally not significantly different from FM. Cost effectiveness analysis revealed that diets containing single feedstuffs or mixtures, particularly those containing equal proportions of oilseed meals and higher proportion of CSM replacing between 50% - 75% FM protein, were more profitable than FM diet. Similarly, the use of heat processed meals at 50% replacement of FM protein yielded greater profit than all other diets including the FM diet. However, essential amino acid supplementation of the meals was less profitable compared to the control. Generally, fish fed diets with oilseed meals would take longer to attain harvest size compared with FM and this could lead to an increase in production costs or a decrease in the number of production cycles which could be achieved within a year. It can be concluded that there is nutritional and economic justification for using SBM, CSM and GNC as partial replacement for FM in diets of Nile tilapia. Based on growth performance, nutrient utilization and economic benefits the diet with heat processed oilseed meal mixtures (containing equal proportions of 16.67% each) at 50% inclusion has the best prospects for replacing FM protein in diets of O. niloticus.

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