Made available in DSpace on 2014-06-11T19:31:05Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-10-10Bitstream added on 2014-06-13T20:41:17Z : No. of bitstreams: 1 paiva_kc_dr_arafcf.pdf: 853846 bytes, checksum: 260b6535d470ef7f6e656a8ba1f4e0f3 (MD5) / Universidade Estadual Paulista (UNESP) / O presente trabalho teve como objetivo estudar se os diferentes níveis de substituição do milho pelo sorgo com baixo teor de tanino em rações para Tilápias do Nilo afetam a composição corporal, qualidade dos filés, parâmetros bioquímicos do sangue e a histomorfometria do fígado e intestino. A amostra foi constituída por 250 Tilápias do Nilo (Oreochromis niloticcus) machos com peso médio de 20g as quais receberam alimentação durante o período experimental com rações isocalóricas e isoprotéicas constituindo-se o grupo controle com ração a base de milho (T1) e os grupos experimentais com os seguintes níveis de substituição de milho por sorgo: 25% (T2); 50% (T3); 75% (T4) e 100% (T5). Foram estudados os seguintes parâmetros coletados no momento do abate: bioquímicos (hematologia, colesterol total plasmático (CTP) e triacilgliceróis plasmáticos (TGP)); histomorfometria de fígado e porções intestinais e composição centesimal do peixe inteiro. Os peixes foram filetados e armazenados sob congelamento a -20oC por um período total de 10 meses. Amostras de filés foram coletados nos tempos 0 (imediatamente após o abate, antes do congelamento – Tempo I), após 5 meses de congelamento (Tempo II) e após 10 meses de congelamento (Tempo III) e em cada um dos tempos foram realizadas as seguintes análises: composição centesimal, NNP (Nitrogênio Não Protéico), BNVT (Bases Nitrogenadas Voláteis Totais), TBARS (Substância Reativa ao Ácido Tiobarbitúrico), pH, textura por força de cisalhamento (FC), colesterol total do músculo (CT) e analise sensorial. Os resultados encontrados demonstraram que a substituição de milho por sorgo na ração não influenciou negativamente o desempenho dos animais, sendo peso (em média 150g) e comprimento (em média 20cm) percebidos sem diferenças estatísticas; os pesos do fígado e deposição de gordura visceral aumentaram... / The purpose of this work is to study whether the different levels of substitution of corn by sorghum with low tannin content in diets for Nile tilapia affect body composition, quality of steaks, blood biochemical parameters and histomorphometry of liver and intestine. The sample consisted of 250 male Nile tilapia (Oreochromis niloticcus) with average weight of 20 g which were fed during the experimental period with isocaloric and isoproteic diets constituting the control group with diets based on corn (T1) and the experimental groups with the following levels of substitution of corn for sorghum: 25% (T2), 50% (T3), 75% (T4) and 100% (T5). The following parameters collected at slaughter were studied: biochemicals (hematology, total plasma cholesterol and plasma triglycerides), histomorphometry of liver and intestinal portions and proximate composition of whole fish. The fish were filleted and stored under freezingzer at -20oC for a total period of 10 months. Fillet samples were collected at time 0 (immediately after slaughter, before freezing – Time I), after 5 months of freezing (Time II) and after 10 months of freezing (Time III) and each time the following analyses were done: proximate composition, NNP (Nonprotein Nitrogen), BNVT (Total Volatile Base Nitrogen), TBARS (Thiobarbituric acid reactive substance), pH, texture by shear force (FC), total cholesterol of muscle (CT) and sensory analysis. The results demonstrated that the substitution of corn by sorghum in the diet did not affect negatively the performance of the animals, being weight (150 g on average) and length (20 cm on average) noticed without statistical differences, the liver weight and visceral fat deposition increased in the treatment with sorghum (T5 showing 2.42 g and 1.58 g respectively). Hematology showed a slight raise in VCM (Mean Corpuscular Volume) 166.8fL and 164.03fL in T4 and T5 respectively, against... (Complete abstract click electronic access below)

Orientador: Margarida Maria Barros / Resumo: Visando o preparo da tilápia do Nilo, Oreochromis niloticus, para o período de inverno, foram avaliados durante 112 dias diferentes níveis de suplementação de lipídeo e vitamina C, no desempenho produtivo e parâmetros fisiológicos. Foi utilizado um lote de 192 alevinos, com peso médio de 5,57 0,50 g distribuídos aleatoriamente em 32 tanques-rede de 200 L cada, dispostos em oito aquários de 1000 L, dotados de sistema de aquecimento e de recirculação de água com filtragem mecânica e biológica. O experimento foi conduzido num esquema fatorial 2 x 3, com dois níveis de lipídeo (8,0 e 12,0%) e três níveis de vitamina C (300,0; 600,0 e 1200,0 mg de vitamina C/kg da dieta), com quatro repetições, mais dois tratamentos, um ausente de suplementação dos nutrientes testes e outro acrescido de 6,0% de lipídeo e 125,0 mg de vitamina C/kg da dieta. Foram confeccionadas oito rações práticas, isoprotéicas com 32,0% de proteína digestível. O suplemento vitamínico e mineral utilizado foi isento de vitamina C, sendo considerada como fonte somente o adicionado à dieta. A vitamina C utilizada foi a polifosfatada com 35,0% de atividade e a fonte de lipídeo o óleo de soja. Os resultados permitiram concluir que a energia adicional proveniente do lipídeo é depositada na cavidade abdominal na forma de gordura visceral; que a deposição desta gordura como reserva de energia para transpor o período de inverno foi obtida em todos os níveis de suplementação de lipídeo, sendo a suplementação com 8,0% a mais adequada; que a concentração hepática de vitamina C é proporcional a concentração desta na dieta porém, em função da capacidade de reserva do fígado a quantidade de 600,0 mg de vitamina C/kg da dieta mostra-se mais apropriada economicamente e que a ausência de vitamina C prejudica a eritropoiese e a síntese de colágeno. / Abstract: It were evaluated during 112 days different lipid and vitamin C supplementation in practical diets, on growth performance and physiologic parameters of Nile tilapia, aiming the animals preparation for winter time. It was utilized 192 fingerlings, selected from the same spawn, with average weight of 5.57 0.50 g. These were randomly stocked into 32 200-l aquaria, supplied with heat system and recycled water with mechanic and biological filter. The experiment was conducted in factorial design 2 x 3, two levels of lipid (8.0 and 12.0%) and three levels of vitamin C (300.0; 600.0 and 1,200.0 mg of vitamin C/kg diet), with four replicates each treatment, plus two treatments, absence of test nutrients supplementation and supplemented with 6.0% of lipid and 125.0 mg of vitamin C/kg diet. Eight experimental practical diets were formulated to contain 32.0% of digestible protein. Vitamin and mineral mix was absent of vitamin C, considering as a source only the supplemented in diets. The vitamin C utilized was polyphosphate 35.0% activity. Based on the results it was concluded that the additional energy coming from lipid is accumulated in abdominal cavity as visceral fat; fat deposition as a energy reserve for winter time was obtained in all lipid supplementation, however 8.0% supplementation seems to be more appropriate; vitamin C liver concentration is proportional to diet concentration, but because of liver reserve capacity the amount of 600.0 mg of vitamin C/kg diet appears to be more economic appropriate and that vitamin C absence impairment erythropoiesis and collagen syntheses. / Mestre

Au, Yee Man Candy. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 128-144). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iii / Acknowledgements --- p.v / List of Tables --- p.x / List of Figures --- p.xi / List of Abbreviations --- p.xiii / Chapter 1. --- Chapter One Introduction / Chapter 1.1 --- Homeostasis and detoxification of metal ions --- p.1 / Chapter 1.2 --- Biochemistry of metallothionein --- p.3 / Chapter 1.2.1 --- Structure of metallothionein --- p.4 / Chapter 1.2.2 --- Isoforms of metallothionein --- p.5 / Chapter 1.2.3 --- Roles of metallothionein --- p.6 / Chapter 1.2.4 --- Structure of metallothionein gene --- p.9 / Chapter 1.2.5 --- Metal responsive element (MRE) --- p.10 / Chapter 1.2.6 --- Regulation of MT gene --- p.11 / Chapter 1.3 --- Metal responsive element-binding transcription factor 1 (MTF-1) --- p.16 / Chapter 1.3.1 --- Structure of MTF-1 --- p.16 / Chapter 1.3.2 --- Target genes of MTF-1 --- p.18 / Chapter 1.4 --- Teleost MT and MTF-1 --- p.20 / Chapter 1.5 --- Tilapia --- p.26 / Chapter 1.6 --- Tilapia MT and MTF-1 --- p.26 / Chapter 1.7 --- Aims of study --- p.30 / Chapter 2. --- Chapter Two Materials and Methods / Chapter 2.1 --- Quantification of MTF-1 isoforms and MT mRNA levels in tilapia and Hepa-Tl cells by real-time PCR --- p.32 / Chapter 2.1.1 --- Heavy metal exposure on tilapia --- p.32 / Chapter 2.1.1.1 --- Animals --- p.32 / Chapter 2.1.1.2 --- Heavy metal exposure --- p.32 / Chapter 2.1.1.3 --- Total RNA extraction --- p.33 / Chapter 2.1.1.4 --- Reverse Transcription --- p.35 / Chapter 2.1.2 --- Heavy metal exposure on Hepa-Tl cells --- p.36 / Chapter 2.1.2.1 --- Cell Culture --- p.36 / Chapter 2.1.2.2 --- Metal treatment on Hepa-Tl cells --- p.37 / Chapter 2.1.3 --- SYBR green --- p.39 / Chapter 2.1.3.1 --- Primer Design --- p.39 / Chapter 2.1.3.2 --- Validation of cycling condition --- p.41 / Chapter 2.1.3.3 --- Determination of relative amount of target gene present in the samples --- p.43 / Chapter 2.1.3.4 --- Statistical analysis --- p.44 / Chapter 2.1.4 --- TaqMan probes --- p.44 / Chapter 2.1.4.1 --- Primer Design --- p.44 / Chapter 2.1.4.2 --- Validation of cycling condition --- p.45 / Chapter 2.2 --- Localization study of MTF-1 isoforms --- p.46 / Chapter 2.2.1 --- Amplification of the full length tilapia MTF-1 isoforms --- p.46 / Chapter 2.2.2 --- Preparation of Escherichia coli competent cells --- p.48 / Chapter 2.2.3 --- Transformation --- p.49 / Chapter 2.2.4 --- Confirmation of the insert of the ligation products --- p.50 / Chapter 2.2.5 --- Cloning of MTF-1-L and MTF-1-S gene into phrGFPII-1 vector --- p.51 / Chapter 2.2.6 --- Transient transfection of plasmids to Hepa-Tl cells --- p.54 / Chapter 2.2.7 --- Staining of the nucleus by Hoechst 33342 --- p.55 / Chapter 2.2.8 --- Metal treatment on Hepa-Tl cells --- p.55 / Chapter 2.3 --- Electrophoretic mobility shift assay (EMSA) --- p.56 / Chapter 2.3.1 --- Preparation of Hepa-Tl whole-cell protein extract --- p.56 / Chapter 2.3.2 --- In vitro transcription/translation of tilapia MTF-1 isoforms --- p.57 / Chapter 2.3.3 --- Annealing of the tiMREg oligonucleotides --- p.58 / Chapter 2.3.4 --- Labeling of the annealed tiMREg oligonucleotides --- p.58 / Chapter 2.3.5 --- Electrophoretic mobility shift assay --- p.59 / Chapter 3. --- Chapter Three Results / Chapter 3.1 --- Quantification of MTF-1 isoforms and MT mRNA levels in tilapia and Hepa-Tl cells by real-time PCR --- p.62 / Chapter 3.1.1 --- Validation of primers for real-time PCR --- p.62 / Chapter 3.1.2 --- Tissue distribution of MTF-1 isoforms in tilapia and Hepa-Tl cell-line --- p.63 / Chapter 3.1.3 --- Effect of metal treatment on MTF-1 isoforms and MT gene expression level in different tissues of tilapia and Hepa-Tl cell-line --- p.68 / Chapter 3.2 --- Localization study of MTF-1 isoforms --- p.82 / Chapter 3.2.1 --- Cloning of MTF-1 isoforms into phrGFPII-1 vector --- p.82 / Chapter 3.2.2 --- Transient transfection of phrGFPII-1 plasmids to Hepa-Tl cells --- p.82 / Chapter 3.3 --- Electrophoretic mobility shift assay (EMSA) --- p.96 / Chapter 4. --- Chapter Four Discussion / Chapter 4.1 --- Tissue distribution of MTF-1 isoforms --- p.104 / Chapter 4.2 --- Effect of metal stress on the mRNA expression level of MT and MTF-1 isoforms --- p.106 / Chapter 4.3 --- In vitro study of the localization of the MTF-1 isoforms --- p.114 / Chapter 4.4 --- DNA binding of MTF-1 synthesized by in vitro transcription/translation method --- p.121 / Chapter 4.5 --- Conclusion --- p.125 / Chapter 5. --- REFERENCES --- p.128

Frequent measures that aim to identify the tolerance of an organism to various environmental conditions rely on the mortality of said organism. However, the effects of sub-lethal stress can be just as important to consider as they may give rise to how an organism may live in such an environment (growth, reproduction, etc.). Coping with changes in environmental conditions can have a high energy cost. Even starting a cellular stress response alone has proven to be costly. It is therefore reasonable that organisms in stressful situations will dedicate energy sources to survival mechanisms, and downregulate non-necessary activities like growth, and reproduction. As a tropical freshwater species, Nile Tilapia are subject to both Winter Stress Syndrome and saltwater intrusion as global climate change progresses, making them an ideal model organism. In order to test the physiological limits of this species, we exposed Nile Tilapia to a variety of treatments (two temperatures: 21°C & 14°C, three salinities: 0ppt, 16ppt, 34ppt) for one hour. By manipulating both temperature and salinity simultaneously the author hopes to shed light on the future of this species as global climate change progresses. Cell cycle arrest can occur at cellular checkpoints such as the ones located at G1 or G2/M. Monitoring the DNA replication process is crucial to cellular activities, and disruptions such a cell size issues or DNA damage can cause this process to stop indicating the presence of sub-lethal stress. There were no significant changes in the proportions of cells in G1, S, or G2 due to an increase of salinity in warm water. In cold water, there was a steady decrease of the percentage of cells in G1 as salinity increased suggesting cell cycle arrest is occurring at a different checkpoint. A significant increase in the number of cells in G2 in response to cold temperature was found, however, this effect was not made greater by the addition of salinity. Increasing number of cells in G2/M suggests that there is cell cycle arrest occurring at the G2/M checkpoint. The concentrations of three proteins involved in the regulation and arrest of the cell cycle were measured in gill tissue by dot blotting. Western blotting was performed to ensure the specificity of the protein antibodies. Three proteins of interest were chosen due to their roles in regulating cell cycle proliferation (Proliferating Cell Nuclear Antigen), growth arrest (Growth Arrest and DNA Damage-45) and programmed cell death (p53). Proliferating Cell Nuclear Antigen (PCNA), Gadd45, p53 all showed significant decreases in concentration in gill tissue exposed to saltwater (34 ppt) in the warm temperature treatment. There was no significant effect of salinity within the cold temperature treatment for any of the proteins tested. The CCAAT/Enhancer-binding proteins (C/EBP) are a class of transcription factors that act upon cellular proliferation and differentiation. C/EBP-𝛿 is the specific protein that is activated in response to stress stimuli. There were no significant differences observed in C/EBP-𝛿 concentrations in gill tissue.

El presente trabajo de investigación busca describir las dificultades que ha ocasionado el protocolo técnico de registro sanitario de importación de productos pesqueros y acuícolas congelados impuesto por la autoridad sanitaria competente del Perú respecto a la importación y comercialización del filete de tilapia congelado proveniente de un nuevo productor chino a partir de la experiencia de la empresa Umi Foods S.A.C., por lo tanto nuestro estudio se divide en cuatro (4) capítulos, los cuales son detallados a continuación. El primer capítulo se centra en el plan de investigación, por medio del cual planteamos la pregunta, el problema y los objetivos, tanto principales como específicos y la formulación de la hipótesis inicial de la investigación. El segundo capítulo da a conocer los antecedentes de la investigación, tanto nacionales como internacionales y el marco teórico del estudio, así como las restricciones para la importación y los requisitos para la comercialización del filete de tilapia congelado. El tercer capítulo se centra en la metodología de la investigación, donde detallamos el tipo, propósito, categorías, delimitación, diseño y tamaño de muestra de la investigación. Asimismo, en este capítulo desarrollamos las entrevistas de profundidad, las cuales serán de utilidad para el recojo de información. Finalmente, el cuarto capítulo se enfoca en el respectivo análisis y discusión de resultados de nuestra investigación para así encontrar los hallazgos y realizar tanto las conclusiones como las recomendaciones finales por medio del análisis de documentos y de la información obtenida mediante las entrevistas de profundidad desarrolladas en nuestro estudio. / The present research seeks to describe the difficulties caused by the technical protocol of import sanitary registration for frozen fishery and aquiculture products from the sanitary authority of Peru regarding the import and commercialization of frozen tilapia fillet that comes from a new Chinese producer and according to the experience of the company Umi Foods S.A.C., therefore our study is divided into four chapters that are detailed as below. The first chapter focuses on the planning research, which is about proposing the research question, the main and specific problems and objectives. In this chapter we also make known about the starting hypothesis of our working research. In the second chapter we present the national and international backgrounds and the theory framework of the research as well as the import restrictions and the commercialization requirements of the frozen tilapia fillet. The third chapter explains the methodology research, which we detail the type, purpose, categories, delimitation, design and sampling size of the research. Also, in this chapter we develop depth interviews that will be very useful for the research’s collecting information. Finally, the fourth chapter is about the respective analysis and discussion of the research´s results in order to get the findings of the study and make the final conclusions and recommendations according to the documents’ analysis and to the obtained information from held depth interviews in our research. / Tesis

The potential for commercial tilapia aquaculture to be developed taking an economic-focused approach was investigated in Mexico. The research examined various issues related to production, marketing and the business environment of the industry. Findings revealed that farmed tilapia products in Mexico can be produced competitively and profitably in large quantities, not only due to its suitability for culture in most of the country; but also due to the availability of more profitable markets (i.e. supermarkets), increasing demand for high quality tilapia products (e.g. fresh, large sizes and more value-added products) and implementation of more efficient business strategies (e.g. economies of scale and partnerships) and newer technologies (i.e. husbandry and equipment). Public/private sector partnerships proved to be the most feasible way to promote and develop tilapia farming in Mexico, particularly in the case of small and medium enterprises (SMEs). Through either economical, technological or consumables support from development bodies; and integration with other agri-business (e.g. agriculture and livestock) or within the industry (i.e. horizontally and/or vertically). In which economies of scale were promoted, efficiency was improved, dealing power was increased, and costs and risks were reduced. In which larger businesses reported production costs 50% lower (around MX 11 kg⁻¹) than SMEs, allowing them to compete against larger sources (i.e. fisheries and imports).Additionally, a strong and fast moving domestic market influenced by the decline outputs (22% between 1990 and 2003) from the main source (i.e. catching sector) and the availability of more value-added products (e.g. fillets in various presentations) have promoted its expansion into more profitable markets (i.e. supermarkets and exports) and in sustained and/or increased prices within the past decade (compared to other seafood commodities, e.g. shrimp and salmon).However, concerns arise about the long-term sustainability of tilapia farming due to the high production costs (overall median value MX$ 19 kg⁻¹), small and inconsistent outputs (85% of the farms interviewed produced less than 100 t year⁻¹), lack of knowledge of proper farming techniques and marketing strategies, unlawful competition from imported products (labelling and taxes), poor law enforcement and monitoring from regulatory institutions, and poor institutional support and inadequate extension services, all of which have affected the sustainable development of tilapia farmers and associated groups. Further research is required for the development and promotion of more efficient and economically viable strategies for tilapia farming businesses to target key internal markets. Similarly, improved and more rigorous monitoring of development and support programs performance is required.

Despite teleosts being the largest and most diverse group of vertebrates, fish models currently used to study photoperiodic effects on fish physiology have been limited to a few species, most of which are temperate seasonal breeders. The overall aim of this work was to expand our knowledge on circadian biology and environmental physiological effects in Nile tilapia (Oreochromis niloticus niloticus), a continuous breeding species of tropical-subtropical origin. The circadian light axis of Nile tilapia is described with regards to melatonin production. Circadian melatonin profiles of fish under 12L:12D photoperiods were observed to be low at day and high at night, suggesting melatonin to be an entraining signal as observed in all other vertebrates. When constant light (LL) was used, such day and night fluctuations where abolished. However when fish where exposed to constant darkness (DD) a strong robust endogenous melatonin rhythm was found, suggesting the presence of circadian oscillators in this species. Importantly, this endogenous rhythm was observed to be maintained for at least three weeks under darkness and proved to be circadian in nature. Moreover, although the melatonin system was able to produce day and night melatonin rhythms when exposed to a different (6L:6D) photocycle, the oscillator appeared to not be entrainable to such a short photo cycle when exposed to DD, as melatonin levels remained high. When comparing the circadian organization of different teleost species including Nile tilapia, preliminary studies showed at least three divergent circadian light organizations in teleosts. Nile tilapia was characterised by a pineal gland far less sensitive than in other fish species as demonstrated through in vitro studies. Furthermore, pineal melatonin production was clearly dependent on the light perceived by the eyes as ophthalmectomy resulted in basal plasma melatonin levels during the dark period. These findings are the first to be reported in a teleost and could be comparable to the circadian light organization of higher vertebrates such as mammals. The onset of puberty of Nile tilapia was studied with regards to the newly discovered Kiss1/GPR54 system. Such a system has recently been discovered in mammals and found to be the primary switch of the brain-pituitary-gonadal (BPG) axis. The results of this study not only suggest a link between the Kiss1/GPR54 system and the onset of III puberty in this tropical batch spawning teleost, that would be a highly conserved feature across vertebrates, but also that the transcriptional mechanisms regulating GPR54 expression could be directly or indirectly influenced by light. Finally, a study was conducted on the effects of different intensities of continuous light (LL) on the growth and sexual development of Nile tilapia up to first maturation. The results showed a significant growth response of fish in all LL treatments compared to control fish. Importantly, this confirmed that LL enhances growth in this species and suggests that it is the light regime more than the intensity which is having an effect. This work thus provides important basic knowledge of the light entrainment pathway and circadian melatonin rhythms in Nile tilapia. Of special importance is the discovery of a strong endogenous melatonin oscillator and a novel circadian organization in fish which would seem to be homologous to that observed in higher vertebrates. Moreover, this work provides evidence that the newly discovered Kiss1/GPR54 system has a similar role in fish as has been found in mammals and that such a system could be directly or indirectly regulated by light. If so, Nile tilapia and other fish species could become important models in the chronobiology and reproduction fields. Finally, this work not only increases our basic and applied knowledge of this species, but also broadens our understanding of the circadian light axis in teleosts and its mediatory effects on reproduction.

In order to combine previous observations from different sources on sex determination, and to identify sex chromosomes including the major sex determination locus in Nile tilapia, physical and genetic maps based on sex-linked markers and genes (such as sex-linked AFLPs, microsatellites, ovarian aromatase and DMO genes) were integrated and anchored. An accurate physical map using FISH techniques on mitotic cells was developed based on a previous map and 23 tilapia BAC clones previously assigned to linkage groups (LGs) 1, 3, 6, 7, 10 and 12; and on meiotic cells, 2 BAC clones containing the SLAM OniY227 and the dmrt4 gene were mapped. The six linkage groups were then assigned to different chromosomes, but surprisingly, the putative sex LG1 was located to a small submetacentric chromosome and not to the larger subtelocentric chromosome 1, where LG3 was assigned instead. The other LGs were assigned to different chromosomes and oriented with respect to the centromeres. A detailed comparison of the physical distribution of markers on chromosome 1 with respect to LG3 revealed a suppression in recombination in the subtelomeric region of the q arm between the marker GM354 (0 cM) and clcn5 (29 cM) and an abrupt increment of recombination between clcn5 (29 cM) and GM128 (77 cM) close to the centromere (Flpter=0.2). The unpairing region (20% of the total length) observed on the larger bivalents of XY fish during early pachytene in meiotic cells has been confirmed by DAPI staining and FISH to be at the terminal part of the q arm, opposite to the centromere. Comparison with six other tilapia species (2n=44) revealed a well conserved karyological distribution of the suspected LGs associated with sex determination (1 and 3). Besides, in O. karongae (2n=38) it was shown by SATA and UNH995/UNH104 marker hybridisation that LG1 has been re-arranged into the subtelomeric chromosome 2 as a result of a telomere-telomere fusion. A pool of 15 tilapia BAC clones previously localised on chromosome 1 and containing sex-linked AFLPs, dmrt1, dmrt4 and several SINEs were screened for new microsatellites; BACs were digested with SAU3AI and TC, GT, ATCT and CTGT probes radio-labelled with 32P. The high abundance of repetitive sequences in the BACs used led to only one useful polymorphic and co-dominant marker being obtained, associated to a BAC clone containing a copy of the dmrt1 gene on chromosome 1 (Flpter=0.85). Four linkage maps were constructed from an XY male, XY neofemale, XX neomale and XX female, mapping 4 and 8 markers on LG1 and LG3 (including the dmrt1 associated microsatellite) respectively. A specific sex-determination locus was identified on LG1 clearly linked with UNH995. However there appeared to be different allelic strengths for this sex determination locus, as shown by different sex ratios associated with different UNH995 genotypes. Additionally, one of the two XX fish mapped, showed the location of the recessive black blotching trait on LG3 (chromosome 1) between the markers GM128 and GM526, close to the centromere (Flpter=0.14). The results presented suggest a nascent Y chromosome in early stage of differentiation in Nile tilapia and with a functional master gene on LG1 close to the marker UNH995 (Flpter=0.67) located on the q arm of a small submetacentric chromosome. The potential influences of the autosomal LG3 (chromosome 1) in sex differentiation are also discussed.

This study investigated several, previously little-known, aspects of reproductive physiology and endocrinology in the substrate-spawning cÌchlid Tilapia zillii; a tilapia that is becoming increasingly popular in world aquaculture. Studies were undertaken in controlled laboratory aquaria, thereby reducing the potential influence of environmental variation evident in many previous field studies of this species. Analysis involved two strains of T. zillii: strain 'A' (T. zillii) and strain 'B' (formerly known as Tilapia tholloni). Spawning periodicity and total fecundity generally increased with fish size. Egg size varied within a narrow window and did not generally increase with fish size though fish weighing 100 - 200g tended to produce the largest eggs. The best estimate of spawning periodicity was considered to be 'mean days elapsed/spawn' as this figure was based upon both spawning and non-spawning fish in an experimental group. Mean days elapsed/spawn increased with increasing fish size and averaged 61.4 days and 37.5 days in strains 'A' and 'B' respectively. The shortest reproductive cycles observed were just 7 days and 6 days for strains 'A' and 'B' respectively. Total fecundity ranged from 461 - 11640 eggs/clutch. Mean total fecundity was 3606+/-280 in strain 'A' and 3560+/-243 in strain 'B'. Mean egg diameter was 1.5+/-0.04mm and 1.4+/-0.08mm in strains 'A' and 'B' respectively. Fecundity and egg size also varied over successive spawns in serial-spawning females but these variations did not appear to be related to spawning periodicity. Regression analysis revealed strong relationships between fish size (weight and length) and total fecundity, relative fecundity and total egg volume. Relationships between fish size and egg size were generally much weaker. Fecundity and egg size were related to the length of the preceding inter-spawn-interval (ISI) in fish of certain weight categories but not others, providing limited evidence that length of ISI may in par, control fecundity and egg size in this species. Ovarian recrudescence was classified into ten distinct developmental stages based upon oocyte size, biochemical properties and structure. This classification scheme was comparable to classification schemes developed for other teleosts but represents the first detailed description of oocyte growth in a substrate-spawning tilapia. Radioimmunoassay and stereological analysis provided valuable and novel data concerning the dynamics of ovarian development in this species. Levels of 17ßoestradiol (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 8, ovaries were dominated by large late-vitellogenic/maturing oocytes (stages 6 & 7) occupying 60 - 70% of the ovary. Gonadosomatic index (GSI) reached maximal levels by day 14. Since the proportion of stage 6/7 oocytes exhibited little change from day 8 onwards, it is suggested that pre-vitellogenic oocytes are recruited into vitellogenic growth immediately after spawning and complete vitellogenesis as early as day 8 postspawning. Analysis of serial-spawning fish found that initial post-spawn E2 and T peaks (on days 2 - 6) were much lower after the second spawning. Sex steroid levels were also found to be suppressed in confined T. zillii (i.e. where stocking densities were > lOkg/m3). Confined females failed to spawn but displayed a marked tendency to do so after transfer to individual aquaria. Serum E2 and T were suppressed during confinement but increased rapidly following transfer to individual aquaria (coincident with resumed spawning activity). It is suggested that levels of E2 and T under confinement are not sufficient to allow completion of vitellogenic growth and are most probably suppressed via a pheromonal mechanism. Finally, the present study investigated the effect of prolonged food restriction on various aspects of reproduction. T. zillii were rationed from first feeding and throughout the following 17 months. Despite very large differences in fish size, no significant differences were detected in total fecundity, egg diameter nor total egg volume once data had been adjusted for differences in fish size. These data suggest that despite very large differences in food availability throughout the periods of sexual differentiation and on-growing, investment in reproduction remained relatively consistent. It appeared that during food restriction, T. zillii sacrificed body weight and growth so as to maintain reproductive investment. In summary, this study provides valuable and novel information regarding the reproductive physiology and endocrinology of female T. zillii and suggests that this species may be a suitable 'model' species for future work on fecundity and ovarian development.

Thesis (MPhil)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: Tilapia is presumed to have a well-defined genetic mechanism of sex determination, but not all sex ratios are compatible with a monofactorial sex determination model. A theory of autosomal gene influence, as well as temperature sex determination (TSD) has been proposed in order to explain large variations in sex ratios. This study assessed the variation in progeny sex ratio in O. mossambicus as a basis for the application of YY male technology in the production of all-male progeny groups. Three populations of O. mossambicus were sampled that are representative of the geographical distribution of the species in Southern Africa. Progeny groups were produced from randomly selected parents and maintained at constant temperature during the labile period of sex differentiation. Variation in sex ratios between different families of the same strain as well as between different strains was calculated. No significant differences were observed in sex ratio between strains, though a significant intra-group variation was identified. This study identified both male and female-biased sex ratios. The data in general conform to a monofactorial sex determination model. Male-biased sex ratio observed in one strain can possibly be ascribed to temperature sex determination (TSD). This strain of O. mossambicus originated from an area with different annual temperature patterns and the possibility of TSD having an adaptive advantage is discussed. This thesis also presents the results of a program to produce monosex male tilapia through the application of the YY male technology in O. mossambicus. Viable XY female and YY male genotypes were produced. XY females sired progenies ranging from 68-94% male ofspring, while YY males sired a mean progeny of 94% male phenotypes. From these results it is concluded that YY male technology provide a viable method for the production of all-male progeny in O. mossambicus. Once available on a commercial scale, the technology can be made more reliable through the application of the appropriate selection methods. / AFRIKAANSE OPSOMMING: Tilapia spesies is oorwegend onderworpe aan ‘n goed gedefinieerde enkelfaktor model van genetiese geslagsbepaling, hoewel afwykings van sodanige geslagsbepalingsmodel soms waargeneem word. Die invloed van outosomale gene, sowel as omgewings temperatuur word voorgehou as verklaring van die waargenome variasies in geslagsverhoudings. Hierdie studie behels die evaluasie van variasie in geslagsdifferensiasie van O. mossambicus as basis vir die implementering van die YY manlike tegnologie vir die produksie van slegs-manlike nageslag groepe. Monsters is bekom van drie populasies van O. mossambicus verteenwoordigend van die geografiese verspreiding van die spesie in Suider Afrika. Nageslag groepe is geproduseer vanaf ewekansig gekose ouerpare en by ‘n konstante temperatuur gehuisves tydens die sensitiewe tydperk van geslagsdifferentiasie. Variasie in geslagsverhouding tussen verskillende families binne dieselfde subpopulasie sowel as binne verskillende populasies is bepaal. Geen betekenisvolle verskille is waargeneem in die geslagsverhouding tussen die verskillende populasies nie terwyl betekenisvolle verskille tussen families binne ‘n populasie waargeneem is. Die data voldoen aan ‘n enkelfaktor genetiese geslagsbepalingsmodel, met die uitsondering van een populasies waar ‘n moontlike omgewingsverwante geslagsbepalingseffek waargeneem is. Die tesis sluit ook in die resultate in van ’n program om YY-tegnologie in O. mossambicus te vestig ten einde manlike nageslag groepe te produseer. Funksionele vroulike XY- en manlike YY genotipes is ontwikkel. Die vroulike XY-genotipes het manlike nageslag groepe opgelewer wat wissel van 68%-94% manlik, terwyl manlike YY-genotipes nageslag groepe gelewer het wat gemiddeld 94% manlik was. Die resultate bevestig die lewensvatbaarheid van YY-tegnologie vir die kommersiële produksie van manlike nageslaggroepe in O. mossambicus.