M.Sc. (Zoology) / The catchment area of the Olifants River is being subjected to increased agricultural and mining activities, industrial development and urbanisation. As a result of this, the water quality 'of the Olifants River and some of its tributaries (e.g. Selati River) has been deteriorating since 1983. This causes reason for concern as one of the downstream water users in the Olifants River catchment is the Kruger National Park, which requires of good quality to sustain its terrestrial and aquatic ecosystems It was therefore necessary to determine what extent activity midstream-or--tlie I Olifants River, especial y in the Phalaborwa area, influenced the water quality of the Olifants River. In this study, an evaluation was done of the water quality (physically and chemically of the Lower Olifants River inside of the Kruger National Park, as well as the Lower Selati River, a tributary of the Olifants River which flows through the Phalaborwa area. Special attention was paid to the metal concentrations in the water, sediment and fish. Water and sediment were sampled every alternate month from April 1990 to February 1992 at six sampling sites along the Lower Olifants River and one in the Selati River. Standard methods were used to determine the physical and chemical characteristics (e.g. pH, TDS, etc.) of the water. The fish species Barbus marequensis was also sampled every alternative month, but only at three sampling sites along the Olifants River and one in the Selati River. Organs and tissues that were dissected, included the gills, fat, liver, gonads, gut, muscle, skin, blood, vertebrae, kidney and bile, as well as the gut contents. In February 1992, additional sampling was performed at Pioneer Dam, a natural reference point used in this study. Atomic absorption spectrophotometry was used in the laboratory to determine the Cr, Cu, Fe, Mn, Ni, Pb, Sr and Zn concentrations in the water, sediment and fish samples. (The water quality of the Selati River was found to be stressful to aquatic life, especially .) with regard to the sodium, fluoride, chloride, sulphate, potassium, total ~dissolved salts 1 and metal concentrations (except strontium). The Selati River also influenced the ': water quality of the Olifants River after the Selati-Olifants confluence. Most of the :. time the water quality of the Lower Olifants River in the KNP did, however, comply With the recommended guideline limits, except for the metal concentrations. The high metal concentrations detected in the water (in some cases sub-lethal levels) indicated some degree of metal pollution, but, due to the hardness of the Olifants River water, conditions were not necessarily toxic to the aquatic life. The accumulated metals in the organs and tissues of marequensis gave a good indication of the metal levels to which the fish were exposed. More metals were taken up by the fish from April 1990 to February 1991 than from April 1991 to February 1992. The various metals were . distributed differently to the tissues of B. marequensis, with the highest zinc concentrations being accumulated by the skin and ovaries; the highest copper and iron by the liver, kidney and gut; the highest chromium and nickel by the blood; and the highest manganese, lead and strontium by the vertebrae and gills. These results afforded guidelines as to the types of tissue which should be sampled as the most suitable for the analysis of the various metals in polluted waters.

Three investigations were conducted to assess the effects of selected bio-active feed additives on tilapia (Oreochromis niloticus) growth performance, feed utilisation, haemato-immunological status, intestinal morphology and microbiology. The first experiment was conducted to evaluate the effects of feeding tilapia semi-practical diets supplemented with exogenous enzymes (phytase, protease or carbohydrase). After six weeks of the dietary supplementation, tilapia fed diets supplemented with exogenous enzymes (phytase and carbohydrase) showed enhanced growth performance and higher microvilli density when compared to the control group. Additionally, the intestinal bacterial community profile of tilapia fed the carbohydrase supplemented diet was significantly altered in contrast to those fed the control diet. In the second experiment, tilapia were fed with practical diets supplemented with exogenous enzymes (phytase, protease or xylanase) for eight weeks. Tilapia fed the xylanase supplemented diet demonstrated significantly higher final body weight (FBW), improved feed conversion ratio (FCR) and better protein efficiency ratio (PER) when compared to the control group. Apparent digestibility coefficients of protein, ash, energy, phosphorus, calcium and sodium were highest in tilapia fed a diet supplemented with phytase. The third experiment was carried out to assess the combined effects of dietary exogenous enzymes (phytase, protease and xylanase) and probiotics (Bacillus subtilis, B. licheniformis and B. pumilus) on tilapia growth performance and health. After seven weeks of feeding, tilapia fed the diet supplemented with both exogenous enzymes and probiotics showed significantly higher FBW, specific growth rate, improved FCR and better PER. The serum lysozyme activity was observed to be significantly higher in tilapia fed the probiotic supplemented diet when compared to the control group. The dietary supplementation with combined exogenous enzymes and probiotics increased intestinal perimeter ratio, microvilli count (density), diameter and subsequently resulted in higher enterocyte absorptive area in tilapia. This study demonstrates that feeding tilapia with dietary exogenous enzymes can enhance growth performance and modulate microbial community profile. In addition, supplementation with both exogenous enzymes and probiotics is capable of improving tilapia growth performance, immune parameters and intestinal morphology.

M.Sc. / Developments in the catchment area of the Olifants River, such as poor agricultural practices and overgrazing, caused increased amounts of erosion material to enter the river. This leads to the silting up of the Phalaborwa Barrage, situated in the river, on the border of the Kruger National Park. Because of the occasional flushing of the Barrage, in order to retain storage capacity, the Olifants River inside the Kruger National Park is subjected to very high sediment loads. This has led to poor water quality and subsequent fish mortalities. The objective of this study was therefore to investigate the possible effects of silt loads released from the Barrage on the survival of selected life stages of Oreochromis mossambicus. Silt was collected from the Phalaborwa Barrage, and the silt/fish interaction was invesigated under controlled laboratory conditions. Oreochromis mossambicus was exposed to various silt concentrations for different exposure periods to determine the LC50 and sub-lethal concentration ranges. After determination of sub-lethal values, 0. mossambicus was exposed to these concentrations to determine whether they have any effects on the oxygen consumption and selected haematological parameters of exposed fish. Lethal levels for suspended sediment are interrelated with age differences of the fish, particle size of the suspended sediment and concentration. Increased suspended sediment disrupted activity and respiratory patterns, changed orientation responses and ultimately resulted in the death of the fish. The gills and the opercular cavities of the dead fish were usually clogged with a thick layer of mucus and silt. The laboratory experiments revealed that LC5 0 values for 0. mossambicus ranged from 26,78 to 54,20g silt/1 for larvae, 35,10 to 39,20g silt/1 for juveniles and 32,93 to 35,43g silt/1 for adult fish. The larval stage was generally less tolerant of suspended silt than juveniles and adults. Sub-lethal levels for adult fish ranged from 0 to 27g silt/l, where no mortalities occurred. Exposure of adult fish to sub-lethal concentrations of silt did alter routine oxygen consumption by causing increased rates (9 to 330% from initial) of oxygen consumption. This indicates that sub-lethal levels of suspended silt concentrations result in an increase in oxygen consumption by the fish due to an increase in metabolic rate associated with increased activity and stress. Silt exposure Exposure of adult fish to sub-lethal silt concentrations (<20g silt/l) resulted in increases in haemotocrit, red blood cell counts, white blood cell counts and blood glucose concentrations, but decreases in haemoglobin and mean corpuscular volume when compared to that for the control fish. These differences became more apparent as the silt exposure concentration increased with statistically significant differences (P<0.05) between most of the selected

Contamination of aquatic ecosystems (for example, dams, lakes, rivers, streams) with heavy metals (according to McDonald and Wood (1993), the metals that are of greatest concern in fresh waters are Hg, Pb, Cd, Cu, Zn, AI, Mn and Cr (approximately in order of decreasing toxicity)) has been receiving increased worldwide attention, and the literature has many publications on the topic. These amplified levels of contamination have become a global problem in both developed and developing countries and the impact of them on aquatic environments is severe due to the inability of water to disperse contaminants. To meet this threat, ecotoxicologists face a difficult challenge on how to assess the long-term impacts of metals which have already been released into the environment and which persist in sediment, water and biota. In recent years, the field of biomarker study has evolved and expanded rapidly to address this void. Biomarkers examine molecular and biochemical responses in exposed individuals in an effort to assess the status of an impacted environment and they can also serve as a basis for a Rapid Risk Assessment of Fish Health (RRAFH) employed by water quality managers. This effort to incorporate biomarkers into RRAFH research will eventually be worthwhile. Moreover, with this multidisciplinary approach, cause-effect relationships may be examined. It was in this context that the present study evaluated three biomarkers (metallothionein (MT), Na+K+ ATPase and heat shock/stress protein 70 (HSP 70)) to determine the integrated physiological responses in Oreochromis mossambicus to one metal (copper (Cu)) at a sub lethal level over the short term (96, 48, 24, 16, 12, 6, 4 and 2 h respectively). The information gained from these tests was valuable in suggesting what the baseline levels of biomarkers in O. mossambicus would be, as studies of metabolic adjustments to stress in fish have been focused on salmonids. The demand for sensitive, specific and rapid biological assays was also addressed by the development of an HSP 70 ELISA. This assay with, with additional refinement, will also prove to be a valuable tool in the management of Cu pollution in this species. Evaluation of the biomarkers in the gills of O. mossambicus indicated that this species has relatively high levels of MT in their gills compared with other species of fish. Exposure to sub lethal Cu concentrations [Cu] caused fluctuating, but not significant, levels in Na+K+ ATPase and HSP 70 during the exposure time, and the actual [Cu] in the gill seems to be the biggest factor when interpreting results. Significant decreases were found in MT levels in the gill tissue at 2 h and 12 h of exposure. HSP 70 results also indicated non-significant decreases to Cu exposure. Data from the gills analysed in the current study suggest that down-regulation in the biomarker response might be a better indicator of Cu exposure or effect in O. mossambicus. These data also suggest that further studies should rethink the amount of Cu that the fish under investigation would find sub-lethal as this metal seems to be very well regulated in this species of fish and seems frequently to non-significantly effect the chosen biomarkers. Evaluation of the biomarkers in the liver of O. mossambicus indicated that MT levels present in the liver most likely fall within the in range of normal baselevels for tilapia living in a Cu-rich environment and that HSP 70 in the liver only responds significantly to increased aqueous [Cu] at 16 h of exposure. This study also confirms current knowledge that the toxicity assessment of any pollutant, including Cu, cannot be exclusively based on one biomarker and that the true value of these biomarkers only becomes evident when they are used not as individual indicators but as integrated responses that influence one another. All these results furnish useful data for future research into the sub lethal effects of a pollutant such as Cu. The next step would be to test the usefulness of these physiological methods for detection of similar responses in natural fish populations inhabiting Cu polluted waters. This will provide a database relating the presence of Cu to biological effects at a molecular level. / Prof. J.H.J. Van Vuuren

M.Sc. / Please refer to full text to view abstract

M.Sc. (Zoology) / Tilapia sparrmanii (Smith) obtained from the Provincial Fishery in Lydenburg was used for toxicological studies. The haematology of T. sparrmanii was investigated after exposure to sub-lethal doses of atrazine, zinc and iron for 72 hours in a continuous flow and static system, as well as prolonged (4 weeks) exposure in the continuous flow system. After exposure the blood of the experimental fish were sampled. Mean values and standard errors were obtained for several haematological parameters such as number of red and white blood cells, haematocrit, haemoglobin, mean corpuscular volume, pH, plasma glucose-, protein-, lipid- and lactate concentrations, as well as plasma Na+, K+, eland Ca+. Statistically significant differences were found between the values of haematological parameters of experimental and control fish. A tendency towards haemoconcentration was observed when fish were exposed to the low pollutant concentrations, while signs of haemodilution was found at higher concentrations. The results in this study demonstrate that, after prolonged exposure, T. sparrmanii develops a tolerance and partially recovers from the stress condition. The bioconcentration of atrazine in the blood of T. sparrmanii after exposure were determined by liquid chromatography and zinc and iron by atomic absorption spectrophotometry. During this study it was found that atrazine, zinc and iron concentrated in the blood of T. sparrmanii after short- and prolonged exposure to the pollutants. The routine oxygen consumption of "!'. sparrmanii exposed to atrazine, zinc and iron were determined in a continuous flow apparatus over 72 hours. The oxygen consumption of the experimental fish fluctuated for the first 8-12 hours during the acclimation period. The influence of atrazine on the routine oxygen consumption was not so drastic as that of zinc and iron. Zinc caused a decrease in the routine oxygen consumption of the experimental fish over 72 hours, while iron increased the routine oxygen consumption...

M.Sc. (Zoology) / Tilapia sparrmanii (Smith) was exposed to sub-lethal doses of chromium, manganese, zinc and iron for 96 hours at an acidic pH (5), a physiological pH (7,4) and an alkaline pH (9), as well as a prolonged exposure (2 - 4 weeks) at an uncontrolled pH. In the laboratory fish were kept in aquaria which were supplied with continuously flowing borehole water. Controlled laboratory conditions existed during experimentation. Blood and liver samples of the experimental fish were sampled after exposure. The bioconcentration and the effect of the selected metals at the mentioned conditions, were thereby determined on bloodcoagulation, total and differential leucocyte counts, and liver enzYmes to investigate the possible damage to the liver. As metals have the ability to enter and concentrate in the body, the bioconcentration of each metal was determined by atomic absorption sPectrophotometry in the blood and liver. Chromium showed an increase in concentration in both the blood and liver, with an increase in pH after short term (96h) exposure. The statistical significant increases of manganese concentration in the blood over a short term were not reflected in the liver. It may be indicative of homeostatic control. A similar phenomenon was found with .zd.nc , The concentration of zinc in the liver did not reflect the progressive decrease in the blood wi th a increase in pH. Iron revealed a statistical significant increase in bioconcentration in the liver with, an increase in pH, which is indicative of the activity of the liver. The long term exposure of fish to chromium and manganese caused a significant increase in the concentration of the metals in the blood and liver. The only significant increase in concentration after exposure to iron, occured . in the Iiver. Exposure to zinc however caused a statistical significant decrease in concentration in both the blood and liver. Differential leucocyte counts reflected lymphocytosis, eosinophilia, monocytopenia and neutropenia, after short term exposure. These conditions nearly always resulted in leucopenia. Long term exposure to manganese and zinc reflected both eosinophilia and neutropenia. Chromium reflected eosinophilia, and iron, neutropenia. The only statistically significant change in the number of leucocytes after long term exposure, was caused by zinc and resulted in leucopenia. According to the photokymographic observation of blood coagulation by the thrombelastograph, exposure to the selected heavy metals lead to a prolonged clotting time and thrombocytopenia. Long term exposure (4 weeks) to manganese caused another bleeding disease, called hemophilia. It was thus evident that the exposure to heavy metals led to clotting defects, which caused bleeding.

Orientador: Fabiana Pilarski / Banca: Margarida Maria Barros / Banca: Róberson Sakabe / Resumo: Essa revisão aborda os efeitos da suplementação com arginina na saúde e nos aspectos produtivos em peixes. A utilização de arginina na dieta mostrou influência na resposta fisiológica dos peixes de acordo com a dose empregada. Os aspectos estudados pelos autores foram agrupados em dez temas principais: efeitos da arginina no sistema digestório e na absorção de nutrientes; digestibilidade e arginina circulante no plasma; analitos e enzimas relacionadas à dieta; índices hematológicos; expressão gênica e secreção de hormônios; função hepática; resposta imune e oxidativa; índices corporais; composição bromatológica e desempenho produtivo. A arginina exerce um efeito significativo no desempenho produtivo e nas variáveis relacionadas à retenção de nutrientes. Sua associação com o aumento da excreção de nitrogênio pelos peixes é evidente, contudo, a interação entre lisina e arginina ainda precisa ser mais investigada pois não está clara a existência de um antagonismo entre esses aminoácidos. Em sanidade, não está elucidada a resposta das variáveis em consequência à suplementação de arginina. A realização de desafios, seja por manejo ou por infecção bacteriana, mostrou-se uma excelente ferramenta para avaliar o efeito imunoestimulante desse aminoácido. Devido sua liberação por células fagocíticas e sua ação bactericida, a síntese de óxido nítrico pela arginina é de extrema importância. Dessa forma, a arginina exibe elevado potencial como aditivo dietário / Abstract: This review explores the effects of arginine supplementation in health and productive aspects of fish. The use of arginine in diet showed influence on fish physiological response according to the dose applied. The aspects studied by the authors were grouped into ten main themes: effects of arginine in the digestive system and absorption of nutrients; digestibility and circulating arginine in plasma; analytes and enzymes related to diet; hematological indices; gene expression and hormone secretion; liver function; and oxidative immune response; body indexes; chemical composition and productive performance. Arginine has a significant effect on growth performance and on variables related to nutrients retention. Is evident its association with increased nitrogen excretion by fish, however, interaction between lysine and arginine needs to be further investigated since it is not clear the presence of antagonism between these amino acids. In health, is not elucidated variables response due to arginine supplementation. The challenges performances, either by management or by bacterial infection, have proved being an excellent tool to assess the effect of this amino acid immunostimulant. Due to its release by phagocytic cells and its bactericidal action, nitric oxide synthesis by arginine is extremely important. Thus, arginine displays high potential as dietary additive / Mestre

O objetivo deste estudo foi identificar SNPs na região promotora e no intron I do gene GH (região alvo) e verificar sua possível associação com o crescimento de O. niloticus, o que foi executado em duas etapas: (1) prospecção de SNPs; (2) associação de SNPs com crescimento das variedades Red-Stirling e Chitralada. As análises de associação foram realizadas por meio de metodologia estatística baseada em análise univariada de modelo linear misto considerando-se efeitos fixos e aleatórios. Nove SNPs foram identificados no promotor (GHP1 a GHP9) e um na região 5 UTR (GHP10), os quais formaram 10 blocos genotípicos (A a J). Na população de associação seis novos blocos foram identificados (K a P). Os blocos B, P, K, L e M foram associados aos melhores pesos e os SNPs GHP6 a GHP10 demonstraram associação significativa (P < 0,05) como o crescimento. Portanto, foi possível estimar um conjunto de genótipos com maior efeito genético aditivo sobre o crescimento, o qual poderia ser utilizado em futuros programas de melhoramento genético assistidos por marcadores moleculares. / The present study aimed to identify SNPs in the proximal promoter region and in the first intron of GH gene and to evaluate if there is association of SNPs variation with the O. niloticus growth rate. Firstly, SNP searching in the two targeted regions was carried out in four strains. Then, two strains, Red-Stirling and Chitralada were used in grow-out testing in cages. Association between SNPs and growth rate were statistically estimated by univariate linear mixed model taking into account fixed and random effects. Nine SNPs were found in the proximal promoter region and one in the 5 UTR region, which formed 10 genotype blocks (A to J). Five of these genotype blocks (F to J) were not found in the grow-out individuals. However, six new genotype blocks (K to P) were identified. Genotype blocks B, P, K, L and M were statistically associated to the best weights, and the SNPs GHP6 to GHP10 individually showed significant association (P < 0,05) with growth. These findings found herein may potentially be used as Marked-Assisted Selection in tilapia breeding programs.

No description available.