Exogenous ATP in the Cryopreservation of Brycon orbignyanus Spermatozoa.

BACKGROUND: ATP exogenous (ATPe) has been used successfully in improving motility and fertility for many animal species. However this has not yet been tested on Brycon orbignyamus. OBJECTIVE: The objective of this study was to evaluate the use of ATPe for the cryopreservation of sperm from B. orbignyamus. MATERIALS AND METHODS: The ATPe concentrations tested were 1.0 µM, 5.0 µM and 10 µM combined with Beltsville Thawing SolutionTM extender and dimethylformamide at 7.5%. The sperm were frozen in a nitrogen vapour vessel and stored in liquid nitrogen at -196 ºC. The parameters of viability post-thawing were evaluated using CASA, and flow cytometer. RESULTS: The ATPe did not promote improvements in spermatic kinetics, and in the higher concentrations caused a worsening in these parameters. Also there was loss of mitochondrial functionality and greater cellular disruption with the concentration of 10 µM. CONCLUSION: We do not recommend the addition of ATP for cryopreserving B. orbignyamus.

Vitellogenin levels and others biomarkers show evidences of endocrine disruption in fish species from Iguaçu River - Southern Brazil.

The adverse effects of endocrine disrupting chemicals (EDCs) on aquatic wildlife and human health represent a current issue of high public concern. Even so, they are still poorly studied in aquatic environments of South America. The aim of the present study was to investigate the impact of EDCs in five cascading reservoirs from the Iguaçu River, evaluating reproductive endpoints in three native fish species (Astyanax bifasciatus, Chrenicicla iguassuensis and Geophagus brasiliensis). Additionally, a polyclonal antiserum anti-vitellogenin from G. brasiliensis and a capture ELISA assay were developed for detection of estrogenic or anti-estrogenic activities in male and female fish, respectively. Vitellogenin (VTG) levels in male fish from the Iguacu River was observed, as well as decreased levels of vitellogenin and estradiol in the plasma of female fish. These findings were associated with immature gonads and lower gonadosomatic index in G. brasiliensis adult females from the Foz do Areia (FA) Reservoir. Additionally, both endemic species (Astyanax bifasciatus and Chrenicicla iguassuensis) displayed immature gonads and histological changes, such as degeneration of germ cells, in other studied reservoirs. The current results suggest that these reproductive responses may be associated with the bioavailability of EDCs in the Iguaçu River. These impacts are likely related to chemicals released by human activities, especially from sewage and industrial sources and agricultural production, detected in previous studies. Overall, the FA reservoir was potentially the most affected by chemicals with endocrine properties, and further studies are necessary to identify and quantify these chemicals.

Cylindrospermopsin effects on cell viability and redox milieu of Neotropical fish Hoplias malabaricus hepatocytes.

Cylindrospermopsin (CYN) is a cyanotoxin that is cytotoxic to a wide variety of cells, particularly to the hepatocytes. In this study, the toxic effects of purified CYN were investigated in primary cultured hepatocytes of Neotropical fish Hoplias malabaricus. After isolation, attachment, and recovery for 72 h, the cells were exposed for 72 h to 0, 0.1, 1.0, 10, and 100 µg l-1 of CYN. Then, cell viability and a set of oxidative stress biomarker responses were determined. Catalase, superoxide dismutase, glucose-6-phosphate dehydrogenase, and glutathione S-transferase activities were not affected by exposure to CYN. Concentration-dependent decrease of glutathione reductase activity occurred for most CYN-exposed groups, whereas non-protein thiol content increased only for the highest CYN concentration. Lipid peroxidation, protein carbonylation, and DNA damage levels were not altered, but reactive oxygen species levels increased in the cells exposed to the highest concentration of CYN. Cell viability decreased in all the groups exposed to CYN. Thus, CYN may cause a slight change in redox balance, but it is not the main cause of cell death in H. malabaricus hepatocytes.

Effects of realistic concentrations of TiO2 and ZnO nanoparticles in Prochilodus lineatus juvenile fish.

The impact of nanoparticles on fish health is still a matter of debate, since nanotechnology is quite recent. In this study, freshwater benthonic juvenile fish Prochilodus lineatus were exposed through water to three concentrations of TiO2 (0.1, 1, and 10 µg l(-1)) and ZnO (7, 70, and 700 µg l(-1)) nanoparticles, as well as to a mixture of both (TiO2 1 µg l(-1) + ZnO 70 µg l(-1)) for 5 and 30 days. Nanoparticle characterization revealed an increase of aggregate size in the function of concentration, but suspensions were generally stable. Fish mortality was high at subchronic exposure to 70 and 700 µg l(-1) of ZnO. Nanoparticle exposure led to decreased acetylcholinesterase activity either in the muscle or in the brain, depending on particle composition (muscle-TiO2 10 µg l(-1); brain-ZnO 7 and 700 µg l(-1)), and protein oxidative damage increased in the brain (ZnO 70 µg l(-1)) and gills (ZnO 70 µg l(-1) and mixture) but not in the liver. Exposed fish had more frequent alterations in the liver (necrosis, vascular congestion, leukocyte infiltration, and basophilic foci) and gills (hyperplasia and epithelial damages, e.g., epithelial disorganization and epithelial loss) than the control fish. Thus, predicted concentrations of TiO2 and ZnO nanoparticles caused detectable effects on P. lineatus that may have important consequences to fish health. But, these effects are much more subtle than those usually reported in the scientific literature for high concentrations or doses of metal nanoparticles.

Effect of water temperature and salinity in oviposition, hatching success and infestation of Aphanoblastella mastigatus (Monogenea, Dactylogyridae) on Rhamdia quelen.

Several environmental parameters may influence biological processes of several aquatic invertebrates, such as the Monogenea. Current analysis investigates oviposition, hatching success and infestation of Aphanoblastella mastigatus, a parasite of the silver catfish Rhamdia quelen at different temperatures (~ 24 and 28 °C) and salinity (by adding sodium chloride to water, at concentrations 0, 5 and 9 g/L) in laboratory. There was no significant difference in oviposition rate and in A. mastigatus infestation success at 24 and 28 °C. On the other hand, the concentration 9 g/L of sodium chloride in the water impaired the parasite's survival and the viability of the eggs. Results show that its usage is efficient as a possible prophylactic treatment. Eclosion rate of A. mastigatus's eggs was significantly higher at 28 °C, although it was significantly less from 5 g/L. Two oviposition peaks (06h15 and 18h15) occurred during a 24-hour period, or rather, during the highest variations in luminosity. Further studies are recommended with greater temperature intervals and more intense experimental infestations to verify the effects of temperature in the life span and infestation success of A. mastigatus.

Effect of water temperature and salinity in oviposition, hatching success and infestation of Aphanoblastella mastigatus (Monogenea, Dactylogyridae) on Rhamdia quelen / Efeito da temperatura da água e salinidade na oviposição, sucesso de eclosão e infestação de Aphanoblastella mastigatus (Monogenea, Dactylogyridae) em Rhamdia quelen

Abstract Several environmental parameters may influence biological processes of several aquatic invertebrates, such as the Monogenea. Current analysis investigates oviposition, hatching success and infestation of Aphanoblastella mastigatus, a parasite of the silver catfish Rhamdia quelen at different temperatures (~ 24 and 28 °C) and salinity (by adding sodium chloride to water, at concentrations 0, 5 and 9 g/L) in laboratory. There was no significant difference in oviposition rate and in A. mastigatus infestation success at 24 and 28 °C. On the other hand, the concentration 9 g/L of sodium chloride in the water impaired the parasite’s survival and the viability of the eggs. Results show that its usage is efficient as a possible prophylactic treatment. Eclosion rate of A. mastigatus’s eggs was significantly higher at 28 °C, although it was significantly less from 5 g/L. Two oviposition peaks (06h15 and 18h15) occurred during a 24-hour period, or rather, during the highest variations in luminosity. Further studies are recommended with greater temperature intervals and more intense experimental infestations to verify the effects of temperature in the life span and infestation success of A. mastigatus.

Resumo Diversos parâmetros ambientais podem afetar os processos biológicos de diversos organismos invertebrados aquáticos, como os Monogenea. Neste estudo, nós investigamos a oviposição, sucesso de eclosão e infestação de Aphanoblastella mastigatus, parasito de jundiá Rhamdia quelen, em diferentes condições de temperatura (~ 24 e 28 °C) e salinidade (por meio da adição de cloreto de sódio na água nas concentrações de 0, 5 e 9 g/L) em laboratório. Não houve diferença significativa na taxa de oviposição nem no sucesso de infestação de A. mastigatus à 24 e 28 °C; por outro lado, a concentração de 9 g/L de cloreto de sódio na água inviabilizou não só a sobrevivência do parasito mas também a viabilidade dos ovos. Esse resultado indica que seu uso é eficiente como potencial tratamento profilático. A taxa de eclosão dos ovos de A. mastigatus foi significativamente maior a 28 °C. A partir de 5 g/L, a mesma foi significativamente menor. Durante um período de 24 horas, dois picos na oviposição ocorreram nos momentos de maior variação da luminosidade (06h15 e 18h15). Futuros estudos são recomendados utilizando intervalos de temperatura e intensidades de infestação experimental maiores para verificar a influência da temperatura na longevidade e sucesso de infestação de A. mastigatus.

Effect of water temperature and salinity in oviposition, hatching success and infestation of Aphanoblastella mastigatus (Monogenea, Dactylogyridae) on Rhamdia quelen

Abstract Several environmental parameters may influence biological processes of several aquatic invertebrates, such as the Monogenea. Current analysis investigates oviposition, hatching success and infestation of Aphanoblastella mastigatus, a parasite of the silver catfish Rhamdia quelen at different temperatures (~ 24 and 28 °C) and salinity (by adding sodium chloride to water, at concentrations 0, 5 and 9 g/L) in laboratory. There was no significant difference in oviposition rate and in A. mastigatus infestation success at 24 and 28 °C. On the other hand, the concentration 9 g/L of sodium chloride in the water impaired the parasites survival and the viability of the eggs. Results show that its usage is efficient as a possible prophylactic treatment. Eclosion rate of A. mastigatuss eggs was significantly higher at 28 °C, although it was significantly less from 5 g/L. Two oviposition peaks (06h15 and 18h15) occurred during a 24-hour period, or rather, during the highest variations in luminosity. Further studies are recommended with greater temperature intervals and more intense experimental infestations to verify the effects of temperature in the life span and infestation success of A. mastigatus.

Resumo Diversos parâmetros ambientais podem afetar os processos biológicos de diversos organismos invertebrados aquáticos, como os Monogenea. Neste estudo, nós investigamos a oviposição, sucesso de eclosão e infestação de Aphanoblastella mastigatus, parasito de jundiá Rhamdia quelen, em diferentes condições de temperatura (~ 24 e 28 °C) e salinidade (por meio da adição de cloreto de sódio na água nas concentrações de 0, 5 e 9 g/L) em laboratório. Não houve diferença significativa na taxa de oviposição nem no sucesso de infestação de A. mastigatus à 24 e 28 °C; por outro lado, a concentração de 9 g/L de cloreto de sódio na água inviabilizou não só a sobrevivência do parasito mas também a viabilidade dos ovos. Esse resultado indica que seu uso é eficiente como potencial tratamento profilático. A taxa de eclosão dos ovos de A. mastigatus foi significativamente maior a 28 °C. A partir de 5 g/L, a mesma foi significativamente menor. Durante um período de 24 horas, dois picos na oviposição ocorreram nos momentos de maior variação da luminosidade (06h15 e 18h15). Futuros estudos são recomendados utilizando intervalos de temperatura e intensidades de infestação experimental maiores para verificar a influência da temperatura na longevidade e sucesso de infestação de A. mastigatus.

Cellular responses of Prochilodus lineatus hepatocytes after cylindrospermopsin exposure.

Cylindrospermopsin is a potent toxicant for eukaryotic cells produced by several cyanobacteria. Recently, primary hepatocyte cultures of Neotropical fish have been established, demonstrating to be a quite efficient in vitro model for cellular toxicology studies. In the current study, a protocol for culture of Prochilodus lineatus hepatocytes was established and utilized to investigate the cellular responses to purified cylindrospermopsin exposure. Hepatocytes were successfully dissociated with dispase, resulting in a cell yield of 6.36 × 10(7)cells g(-1) of liver, viability of 97% and attachment on uncoated culture flasks. For investigation of cylindrospermopsin effects, hepatocytes were dissociated, cultured during 96 h and exposed to three concentrations of the toxin (0.1, 1.0 or 10 µgl(-1)) for 72 h. Cylindrospermopsin exposure significantly decreased cell viability (0.1 and 1 µgl(-1)) and multixenobiotic resistance mechanism, MXR (all exposed groups), but increased reactive oxygen/nitrogen species levels (all exposed groups) and lipid peroxidation (10 µgl(-1)). On the other hand no significant alterations were observed for other biochemical biomarkers as 2GSH/GSSG ratio, protein carbonyl levels and DNA strand breaks or glutathione S-transferase and glucose 6-phosphate dehydrogenase activities. In conclusion, hepatocytes might be made sensitive to cylindrospermopsin, at least in part, due to reduction of xenobiotics and endobiotics efflux capacity by MXR. Additionally, the toxin exposure suggests important issues regarding hepatocytes survival at the lowest cylindrospermopsin concentrations.

Vitellogenesis and other physiological responses induced by 17-beta-estradiol in males of freshwater fish Rhamdia quelen.

This study investigated the effects of different doses of 17-beta-estradiol (E(2)) in Rhamdia quelen. Groups of males exposed to different doses of E(2) (0.1 mg kg(-)(1), 1 mg kg(-)(1) and 10 mg kg(-)(1)) were compared with non-exposed male and female fish groups. Among the considered biomarkers, no significant differences were observed for micronuclei test, reduced glutathione concentration and lipid peroxidation. All E(2)-treated individuals had decreased glutathione S-transferase activity. Increased catalase and superoxide dismutase activities, increased vitellogenin expression and decreased metallothionein concentration were observed in males treated with the highest dose. Liver of all test groups showed necrotic areas, but cytoplasm vacuolization was again found only in the individuals exposed to highest dose. E(2) causes deleterious hepatic effects to R. quelen, and vitellogenin expression, catalase and superoxide dismutase activity and metallothionein concentration represent appropriate biomarkers for studying E(2) effects. Additionally, the response of some biomarkers was similar in males exposed to E(2) and unexposed females, and therefore exposure to endocrine disruptors may cause consequences for fish populations.