Genotoxicity and mutagenicity of water contaminated with tannery effluents, as evaluated by the micronucleus test and comet assay using the fish Oreochromis niloticus and chromosome aberrations in onion root-tips

Cytotoxicity of metals is important because some metals are potential mutagens able to induce tumors in humans and experimental animals. Chromium can damage DNA in several ways, including DNA double strand breaks (DSBs) which generate chromosomal aberrations, micronucleus formation, sister chromatid exchange, formation of DNA adducts and alterations in DNA replication and transcription. In our study, water samples from three sites in the Córrego dos Bagres stream in the Franca municipality of the Brazilian state of São Paulo were subjected to the comet assay and micronucleus test using erythrocytes from the fish Oreochromis niloticus. Nuclear abnormalities of the erythrocytes included blebbed, notched and lobed nuclei, probably due to genotoxic chromium compounds. The greatest comet assay damage occurred with water from a chromium-containing tannery effluent discharge site, supporting the hypothesis that chromium residues can be genotoxic. The mutagenicity of the water samples was assessed using the onion root-tip cell assay, the most frequent chromosomal abnormalities observed being: c-metaphases, stick chromosome, chromosome breaks and losses, bridged anaphases, multipolar anaphases, and micronucleated and binucleated cells. Onion root-tip cell mutagenicity was highest for water samples containing the highest levels of chromium.

Partial molecular characterization of the Nile tilapia (Oreochromis niloticus) alpha-cardiac muscle actin gene and its relationship to actin isoforms of other fish species

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Respostas da Tilápia do Nilo (Oreochromis niloticus) à atratividade e palatabilidade de ingredientes utilizados na alimentação de peixes

Avaliaram-se as respostas da tilápia do Nilo à atratividade e palatabilidade de 14 ingredientes. O método utilizado foi de dupla escolha, comparando-se cada ingrediente peletizado à uma ração controle. Foram empregados quatro aquários (750 litros) contendo, cada um, três alevinos e dois comedouros instalados nos cantos direito e esquerdo, sendo registrados tempo decorrido entre colocação do ingrediente e chegada dos animais aos comedouros, freqüência de visitas aos comedouros, número de grânulos ingeridos e freqüência de ejeções. Os tratamentos foram comparados pela prova não-paramétrica de Kruskal-Wallis e a comparação dos ingredientes para o conjunto de variáveis, por intermédio da Análise de Agrupamento e Análise de Componentes Principais. Os ingredientes foram assim classificados: a) baixa atrato-palatabilidade = farelos de trigo, soja e algodão, farinha e raspa de mandioca, farinha de girassol e fubá de milho, b) média atrato-palatabilidade = levedura de cana-de-açúcar e glúten de milho e c) alta atrato-palatabilidade = ovo integral liofilizado, farinhas de crisálidas, peixes, carne e camarão. Ingestão de grânulos (manhã e tarde) e freqüência de visitas aos comedouros (tarde) foram consideradas as variáveis mais discriminatórias e freqüência de ejeção de grânulos e tempo gasto para aproximação dos comedouros (manhã e tarde), as menos discriminatórias. As respostas comportamentais dos peixes variaram de acordo com o ingrediente apresentado. A avaliação do grau de atrato-palatabilidade deve ser realizada considerando-se uma combinação de parâmetros.

Digestibilidade aparente e trânsito gastrintestinal em tilápia do Nilo (Oreochromis niloticus), em função da fibra bruta da dieta

Os efeitos de diferentes níveis de fibra bruta na digestibilidade aparente e velocidade de trânsito gastrintestinal de tilápias do Nilo alimentadas com dietas purificadas fornecidas foram avaliados. Utilizaram-se cinco aquários circulares (250 L) para alimentação, dotados de sistema fechado de filtragem, reabastecimento e aquecimento da água, e cinco aquários de digestibilidade (100 L), dotados de sistema individual de filtragem, reabastecimento e aquecimento. Utilizaram-se 32 peixes por aquário, com peso inicial médio de 30,65 ± 0,50 g. Adotou-se o delineamento inteiramente casualizado caracterizado por cinco níveis de fibra bruta (2,5; 5,0; 7,5; 10,0 e 12,5%) e 5 repetições. Concluiu-se que níveis crescentes de fibra bruta, em dietas purificadas, interferem significativamente na digestibilidade aparente da matéria seca, proteína bruta e do extrato etéreo. Níveis de até 5,00% de fibra bruta não diminuiu a digestibilidade aparente da matéria seca e da proteína bruta e 7,50% de fibra bruta não diminuiu a digestibilidade aparente do extrato etéreo da dieta purificada pela tilápia do Nilo. Entretanto, o aumento do teor de fibra bruta da dieta diminui significativamente o tempo de trânsito gastrintestinal.

Farinha de resíduos da filetagem de tilápias na alimentação de tilápia-do-nilo (Oreochromis niloticus) na fase de reversão sexual

Este trabalho foi realizado com o objetivo de avaliar a inclusão da farinha de resíduos da indústria de filetagem de tilápias (FT) na alimentação da tilápia-do-nilo na fase de reversão sexual. Foram utilizadas 375 larvas com dois a três dias de idade, distribuídas em 25 aquários com capacidade para 30 L, em delineamento inteiramente casualisado, com cinco tratamentos e cinco repetições, sendo a unidade experimental constituída por um aquário com 15 larvas. Foram elaboradas cinco rações isoprotéicas e isoenergéticas com 38,6% de proteína digestível e 3.800 kcal de ED/kg, com níveis de 0, 5, 10, 15 e 20% de inclusão de FT. Os valores de temperatura, oxigênio dissolvido, pH e condutividade elétrica da água dos aquários durante o período experimental foram de 24,53 ± 1,60ºC; 4,81 ± 0,68 mg/L; 7,70 ± 0,25; e 175,97 ± 108,46 µS/cm, respectivamente. Ao final do experimento, não foram observadas diferenças no peso e comprimento final, no fator de condição, na sobrevivência e no índice de reversão sexual das larvas alimentadas com a FT. A inclusão de até 20% de FT em rações para tilápia do Nilo na fase de reversão sexual não causa prejuízo ao desempenho dos peixes.

Digestibilidade aparente de macrófitas aquáticas pela tilápia-do-nilo (Oreochromis niloticus) e qualidade da água em relação às concentrações de nutrientes

Objetivou-se com este estudo determinar os coeficientes de digestibilidade aparente (CDA) da proteína bruta e dos aminoácidos de duas espécies de macrófitas aquáticas flutuantes (Eichhornia crassipes e Pistia stratiotes) pela tilápia-do-nilo (Oreochromis niloticus) e verificar a qualidade da água dos aquários de digestibilidade em relação às concentrações de nitrogênio e fósforo. Foram elaboradas três rações, marcadas com 0,10% de óxido de cromo-III, sendo uma ração-referência (purificada) e as demais contendo 30% de cada uma das macrófitas aquáticas. As tilápias-do-nilo (58,8 + 18,5 g) foram alimentadas até a saciedade aparente e a coleta de fezes foi feita pelo sistema Guelph modificado. Os CDA médios da proteína e dos aminoácidos foram, respectivamente, 93,17 e 93,32% para a ração-referência; 59,23 e 60,35% para E. crassipes; e 52,24 e 57,40% para P. stratiotes. Não foram constatadas diferenças significativas entre os valores de CDA da proteína e dos aminoácidos dos ingredientes vegetais. Os resultados obtidos demonstraram reduzida eficiência da tilápia-do-nilo em assimilar a maioria dos aminoácidos de E. crassipes e P. stratiotes. As excretas das tilápias-do-nilo contribuíram para o aumento das concentrações de nitrogênio e fósforo na água dos aquários, independentemente da ração fornecida.

Optimum zinc supplementation level in Nile tilapia Oreochromis niloticus juveniles diets

The present study was designed to determine the optimum dietary zinc supplementation to Nile tilapia juveniles (13.3 +/- 1.13 g), by using vegetable-based diets supplemented with increasing levels of zinc from commercial-grade zinc sulfate monohydrate, a previously determined zinc source of higher bioavailability. The basal diet was supplemented with 25, 50, 100, 150, 200, 300, or 400 mg/kg Zn. The experiment was conducted in forty 250-l tanks arranged in a recirculating water system. The experimental period was divided in two phases. For the first 10-week experimental phase, fish were fed satiation diets supplemented with increasing levels of zinc. For the second 5-week experimental phase, fish that were fed diets supplemented with 0-300 mg/kg Zn during the first phase were fed the 400 mg/kg Zn-supplemented diet; fish fed the diet supplemented with 400 mg/kg Zn (first phase) were fed the nonzinc-supplemented diet (second phase). Broken-line analysis showed that the optimum dietary zinc supplementation ((ZnSO4H2O)-H-.) to Nile tilapia juveniles, using weight gain and bone zinc saturation as response criteria, was 44.50 and 79.51 mg/kg Zn, respectively. When challenged by a zinc-deficient diet, tilapia mobilized stored bone zinc to preserve its zinc status. By considering that bone zinc saturation is a more accurate response criterion than weight gain, it was concluded that the optimum dietary zinc supplementation ((ZnSO4H2O)-H-.) in vegetable-based diets to Nile tilapia juveniles is 79.51 mg/kg Zn. (C) 2004 Elsevier B.V. All rights reserved.

Physiological responses of Nile tilapia, Oreochromis niloticus, fed vitamin C- and lipid-supplemented diets and submitted to low-temperature stress

Water temperature alterations can determine harmful physiological modifications in fish, which should be prepared to cope with this, and nutrition strategies seem to be essential. This study evaluated the effects of different levels of vitamin C and lipids on physiological responses of Nile tilapia, Oreochromis niloticus, submitted to temperature stress. There were two phases: Phase I - preparing fish to store vitamin C and lipid at appropriate temperature, and Phase II - evaluating the contributions these reserves make to fish physiology under low-temperature stress. The experiment used a 3 x 2 factorial design with three vitamin C levels (300, 600, and 1200 mg/kg diet) and two lipid levels (8.0 and 12.0%), plus absence of nutrient test and a diet of 6.0% lipids and 125.0 mg/kg vitamin C. In Phase I, 192 fish were kept at 26.0 +/- 1.0 C for 112 d, and in Phase II, 48 fish were kept at 18.0 +/- 0.5 C for 32 d and at 15.0 +/- 0.5 C for 11 d. Fish fed C0L0 diet showed lower erythrocytes values in both phases; higher vitamin C supplement determined higher red blood cell (RBC) number and higher hematocrit (Htc) (Phase II); Htc was significantly lower in Phase II; after temperature stress, fish fed C0L0 diet had higher mean corpuscular volume, lower hemoglobin corpuscular concentration, and significantly lower vitamin C concentration in the liver; and higher supplementation determined a higher concentration in the liver (Phases I and II). Higher plasmatic cortisol concentration was seen in fish fed C0L0 diet. In conclusion, our results show that the absence of vitamin C in diets impairs RBC formation and does not enable fish to cope with stress; excess vitamin C is efficient in mitigating stress and 600 mg/kg diet is economic and physiologically sufficient to prepare fish for coping with low-temperature stress. Lipid supplementation does not determine alterations in stress biochemical parameters.

2-DEOXYGLUCOSE-INDUCED FOOD-INTAKE BY NILE TILAPIA, OREOCHROMIS-NILOTICUS (L)

The alimentary and glycemic responses to cytoglycopenia were studied in thirty-one Nile tilapia alevins of indeterminate sex and age, measuring on average 10.67 +/- 0.82 cm. The cytoglycopenia was provoked by ip injection of 60 mg/kg 2-deoxy-D-glucose (2-DG, N = 16). The control group (N = 15) was submitted to ip injection of 0.2 ml saline. Blood samples for glucose determination were obtained before and three hours after drug administration by cardiac puncture. Food was then offered ad libitum. One hour later the animals were sacrificed and their stomachs removed. The difference in wet weight between full and empty stomach was utilized to quantify the food intake. Median food intake was 0.3877 g for the fish treated with 2-DG and 0.107 g for the animals injected with saline. This difference was statistically significant by the Mann-Whitney test (P<0.05). The median values of blood glucose levels before drug injection were 46.19 mg/100 ml in the 2-DG-treated fish and 44.54 mg/100 ml in the control group. Three hours after drug administration, the values were 48.64 mg/100 ml in the experimental group and 56.90 mg/100 ml in the control group. The difference between the values of blood glucose before and after the drug was not significant for either group. We conclude that glucoprivation provokes food intake in fish and that the same glucoprivation was not sufficient to provoke hyperglycemia.

Environmental cadmium exposure and metabolic responses of the Nile tilapia, Oreochromis niloticus

The contamination of water by metal compounds is a worldwide environmental problem. This study was undertaken to evaluate the impact of short-term cadmium exposure on metabolic patterns of the freshwater fish Oreochromis niloticus. The fish were exposed to 320, 640, 1280 and 2560 mug/l sublethal concentrations of Cd++ (CdCl2) in water for 7 days. The specific activities of the enzymes phosphofructo kinase (PFK-E.C.2.7.1.11.), lactate dehydrogenase (LDH-E.C.1.1.1.27.) and creatine kinase (CK-E.C.2.7.3.2.) were decreased in white muscle after cadmium treatments, indicating decreases in the capacity of glycolysis in this tissue. Cadmium exposure induced increased glucose concentration in white muscle of fish. on the other hand, cadmium exposure at sublethal concentrations increased phosphofructo kinase and LDH in red muscle of fish. Cadmium significantly decreased total protein concentrations in liver and white muscle regardless of tissue glycogen levels. The data suggest that cadmium acts as a stressor, leading to metabolic alterations similar to those observed in starvation. (C) 2001 Elsevier B.V. Ltd. All rights reserved.

Relação Cálcio/Fósforo Disponível em Rações para Tilápia do Nilo (Oreochromis niloticus)

The objective of this work was to evaluate the effect of Ca: available P (P-a) ratio in the diets on performance and bone mineral depositions of juvenile Nile tilapia (2.5 +/- 0.5g). Seven isoproteic and isoenergetic diets (28% CP and 3100 Kcal of DE/Kg of diet), with similar crude fiber and sulfur aminoacids content, were used. The diets had 0.50 and 0.25; 0.50 and 0.50; 0.50 and 0.75; 0.80 and 0.40; 0.80 and 0.80; and 0.80 and 1.20% of Ca and P-a which corresponded to Ca/P-a ratios of 2:1;1:1 and 1:1.5, respectively. An additional control diet, with low levels of Ca and Pa (0.02 and 0.12%, respectively) was used. A completely randomized design with a 2'3 factorial arrangement (Ca level and Ca:P-a ratio) on treatments, plus the additional control diet and three replications was used. Five fishes per aquaria were randomly allotted to 21 fiberglass aquaria (80L). The temperature of the water was maintained close to 26degreesC, and the oxygen, pH and ammonia levels of the water were monitored. The results showed that it was necessary a minimum dietary level of 0.25% of P-a for a satisfactory bone mineral depositions, and that the best performance results were achieved using diets with Ca: P-a ratios between 1:1 and 1:1.5.

Sex determination studies in two species of teleost fish, Oreochromis niloticus and Leporinus elongatus

Genetic analyses of sex determination have identified sex chromosomes in many teleost fish species. However, there are several cases for which sex ratios do not fit perfectly with the expectations of heterogametic systems, suggesting the influence of either minor sex determining genes or environmental influences on the process of sex differentiation. The frequent absence of sex chromosome markers makes the identification of minor sex-determining genes very difficult. It is easier to test first the hypothesis of environmental sex determination (ESD) by studying the temperature effect, since temperature-dependent sex determination has been demonstrated to occur in several vertebrate groups including 1 fish species. To contribute to a better understanding of fish sex determination, we have tested the effects of high temperatures on sex ratios of Oreochromis niloticus, and have attempted to isolate sex chromosome molecular markers in Leporinus elongatus. Treatments of O. niloticus fry at 36 degrees C applied for 10 days and more, and starting 1 week after fertilization markedly increased the proportion of males, and progeny-testing these males confirmed that some of them are sex-reversed genetic females. Two non-coding sequences of L. elongatus Z and W chromosomes were cloned by genomic subtraction. They cross-hybridized with the genome of a close species without providing sex-specific patterns. A collection of L. elongates individuals was subjected to gonadal and chromosomal sexing, and DNA hybridization with both sequences. These analyses revealed 3 individuals having atypical W chromosomes. Interestingly, 2 of these were males having a ZW karyotype. We assume that these atypical sex chromosome arise by exchanges between Z and W chromosomes, and that a transition between female and male heterogamety is underway in this species.

Use of ideal protein concept for precision formulation of amino acid levels in fish-meal-free diets for juvenile Nile tilapia (Oreochromis niloticus L.)

This study was undertaken in a closed system with Nile tilapia (Oreochromis niloticus) to examine the effects of total replacement of fish meal (FM) by soybean meal. Nile tilapia fingerlings with an average weight of 5.34+/-0.08 g were hand-fed one of the five isoenergetic (approximate to13.5 MJ digestible energy kg(-1)) and isoproteic (approximate to31% of digestible protein) experimental diets to satiation, six times a day during 85 days in eight replicate fibreglass tanks (six fish per tank). The control diet containing FM was substituted by soybean meal, with and without essential amino acids (lysine, methionine and threonine) or dicalcium phosphate supplementation. The supplemental amino acids were added at levels to simulate the reference amino acid profile of Nile tilapia carcass protein, based on the ideal protein concept. The results showed that soybean meal diet supplemented only with dicalcium phosphate was inferior to the control diet with FM and soybean meal diets supplemented with dicalcium phosphate and essential amino acids. Multiple essential amino acids and dicalcium phosphate incorporation in soybean meal diets was associated with performance, whole-body composition and carcass yield equal to that of the fish fed with the control diet containing FM. These data suggest that a diet with all plant protein source, supplemented with essential amino acids, based on tissue amino acid profile, can totally replace FM in a diet for Nile tilapia, without adverse effects on the growth performance, carcass yield and composition.

SYNAPTONEMAL COMPLEX-ANALYSIS IN SPERMATOCYTES OF TILAPIA, OREOCHROMIS-NILOTICUS (PISCES, CICHLIDAE)

Some adaptations of the synaptonemal complex (SC) whole-mounting technique first used in plants permitted its application to meiotic studies in tilapia, Oreochromis niloticus. Direct observation of the chromosome pairing process and bivalent structure during the meiotic prophase of this fish species by light and electron microscopy permitted the analysis of SCs in autosomes and the possible identification of sex chromosomes. The analysis of SCs in spermatocytes of 0. niloticus revealed that all 22 bivalent chromosomes completely paired, except for the occurrence of a size heteromorphism in the terminal region of the largest bivalent associated with the presence of an incompletely paired segment during the synapsis process, which may be the cytological visualization of an XX/XY sex chromosome system in this species.