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The modulation of antioxidant responses and onset of oxidative stress is due to several factors, including temperature, diets, and hypoxia followed by re-oxygenation in several aerobic organisms including rotifers. In this context, the aims of this study were (1) to establish a protocol for evaluating in vivo ROS (reactive oxygen species) concentration in rotifer Brachionus plicatilis and (2) to determine the effects on population growth and oxidative stress responses in rotifers fed with different diets: commercial baker’s yeast (Saccharomyces cerevisiae) or concentrated microalgae Nannochloropsis oculata. The rotifers were stocked in five densities (200, 400, 660, 800, and 1000 rotifers mL−1) and exposed to four concentrations of dichlorofluorescein diacetate (H2DCF-DA) (4.4, 8.7, 17.5, and 35 nmol L−1) to optimize a protocol for in vivo ROS determination. The methodology was standardized using 8.7 nmol H2DCF-DA L−1 and it is effective when rotifer density lies within 400 and 1000 rotifers mL−1. In the second experiment, rotifers were fed with 55 × 109 microalgae cells or 0.6 g yeast per million rotifers for 4 days in quadruplicate. Rotifers fed with yeast showed poorer oxidative condition as shown by (a) a higher in vivo ROS concentration; (b) a higher GST (glutathione S-transferase) activity; and (3) a reduced total antioxidant capacity. In conclusion, the methodology to measure in vivo ROS concentration using the H2DCF-DA was efficiently standardized for the first time to rotifers B. plicatilis. Although the yeast-fed rotifers showed higher population growth comparing with this concentration of microalgae, the rotifers fed with N. oculata faced a safer pro-oxidant scenario and presented a higher percentage of egg-carrying females throughout the experiment indicating a better physiological condition.

8 pages, 2 figures, The nitrification process in recirculating aquaculture systems can reduce water pH. Fish can also be exposed to water acidification during transport, an important feature in the aquarium industry, as live fish can be kept in a closed environment for more than 24 h during overseas aerial transportation. Therefore, it is important to study the responses of fish to acidic environments. We investigated the impacts of acute exposure to decreasing pH levels in orange clownfish Amphiprion percula juveniles on their survival and oxidative stress status. Fish were exposed to pH 5, 6, 7, and 8 for 96 h. We observed a significant reduction in survival (85%) and protein damage as measured by P-SH (protein thiol) for fish maintained at pH 5. Despite no effects on survival or oxidative damage, fish exposed to pH 6 showed an increase in their antioxidant defense systems, demonstrating this pH level could not be suitable for them as well. Furthermore, there were no negative effects for fish kept at pH 7, compared to those maintained at pH 8 during this short-term evaluation, The authors are grateful to CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) for research fellowships provided for L.A. Sampaio (# 307445/2016-0), and to CAPES (Coordenacão de Aperfeiçoamento de Pessoal de Nível Superior, Brazil) for a research grant provided for M.D.D. Carneiro.

The nitrification process in recirculating aquaculture systems can reduce water pH. Fish can also be exposed to water acidification during transport, an important feature in the aquarium industry, as live fish can be kept in a closed environment for more than 24 h during overseas aerial transportation. Therefore, it is important to study the responses of fish to acidic environments. We investigated the impacts of acute exposure to decreasing pH levels in orange clownfish Amphiprion percula juveniles on their survival and oxidative stress status. Fish were exposed to pH 5, 6, 7, and 8 for 96 h. We observed a significant reduction in survival (85%) and protein damage as measured by P-SH (protein thiol) for fish maintained at pH 5. Despite no effects on survival or oxidative damage, fish exposed to pH 6 showed an increase in their antioxidant defense systems, demonstrating this pH level could not be suitable for them as well. Furthermore, there were no negative effects for fish kept at pH 7, compared to those maintained at pH 8 during this short-term evaluation.

This study aimed to assess whether the addition of calcium carbonate (CaCO3) to increase Calcium levels reduces the long-term nitrite (NO2−) toxicity in pacu Piaractus mesopotamicus juveniles. The fish were exposed for 60 days to six treatments comprised by the interaction of three NO2− concentrations (0, 15 and 30 NO2−-N mg L−1) and two Calcium levels (60 and 150 CaCO3 mg L−1), all in triplicate. The zootechnical indices, as well as blood and oxidative stress parameters were evaluated at the end of experimental period. The results showed that at lower hardness, NO2− exposure led to enhanced lipid peroxidation (LPO) levels of muscle and gills and compromised the food conversion rate. Otherwise, the LPO levels were kept at basal condition and no changes were found in zootechnical parameters at higher hardness treatments. Thus, the increase in Calcium levels up to 150 CaCO3 mg L−1 using CaCO3 may reduce the NO2− toxic levels in pacu juveniles, and may be an interesting strategy of water quality management, especially in intensive production systems.

This study investigated the secondary stress responses of Paralichthys orbignyanus exposed to ammonia and nitrite and after recovery. Fish were exposed to 0.12, 0.28, and 0.57 mg NH₃‐N/L, or 5.72, 10.43, and 15.27 mg NO₂‐N/L for 10 d followed by the same time length for recovery. Ammonia‐ and nitrite‐free water was used as a control treatment. Blood samples were collected after 1, 5, and 10 d of exposure and after recovery. Fish exposed to ammonia presented lower and higher glucose levels after 10 d of exposure and recovery, respectively. Ammonia induced initial and transient ionic disturbances and metabolic alkalosis. Nitrite exposure caused hyperglycemia, increased plasma K⁺ levels, and respiratory alkalosis, whereas metabolic acidosis was observed after recovery. Increased proportion of monocytes and/or granulocytes and reduced number of lymphocytes were demonstrated in fish exposed to 0.28 mg NH₃‐N/L (Day 1) and 10.43 mg NO₂‐N/L (Day 5) and after recovery in the 0.28 and 0.57 mg NH₃‐N/L treatments. Exposure to ammonia decreased the proportion of granulocytes on Day 5. In conclusion, exposure to concentrations at 0.12 mg NH₃‐N/L and 5.72 mg NO₂‐N/L provoked physiological disorders in Brazilian flounder. Nonetheless, fish exposed to 5.72 mg NO₂‐N/L following a 10‐d recovery period showed complete resumption of homeostasis.

Summary The aim of this study was to evaluate the anaesthetic efficacy and the effect of sedation with tricaine (TMS®), also known as MS-222, on secondary and oxidative stress parameters in juvenile tambaqui, Colossoma macropomum transported in hyperoxia for 2, 6 and 10 hr. Juveniles were placed in aquaria containing six different concentrations of buffered tricaine (150, 180, 210, 240, 270 and 300 mg/L) and the times for anaesthesia induction and recovery determined. Fish transported in hyperoxic conditions were investigated for glycemia, ionic concentration (K+, Ca++, Na+) and osmolality, haematocrit (Ht) and haemoglobin concentration (Hb), partial pressure of gases (pCO2 and pO2), pH and bicarbonate concentration (HCO3−) in whole blood collected from caudal vasculature. Total antioxidant capacity against peroxyl radicals (ACAP), glutathione-S-transferase (GST) activity and lipid peroxidation levels (TBARS) were investigated in gills, brain and liver. All concentrations of TMS® induced deep anaesthesia in juvenile tambaqui in this study. A concentration of 240 mg/L of TMS® was sufficient to induce rapid anaesthesia (

The aim of this study was to evaluate the anaesthetic activities of the extractives of Nectandra grandiflora and Spilanthes acmella in juvenile tambaqui fish, Colossoma macropomum as well as to investigate their effects on oxidative stress status of fish submitted to transport in hyperoxia. Fish were placed in aquaria containing five different concentrations of essential oil (EO) of N. grandiflora (25; 50; 100; 200; and 300 μL L − 1 ) or waxy extract of S. acmella (5; 10; 15; 20; and 25 mg L − 1 ), and the times of anaesthetic induction and recovery were determined by concentration-response trials. Fish transported inside plastic bags in hyperoxic conditions for 2, 6 and 10 h with or without anaesthetics were investigated for the occurrence of oxidative stress through the assessment of total antioxidant capacity against peroxyl radicals (ACAP), glutathione S-transferase (GST) activity and lipid peroxidation levels (TBARS) in muscle, gills, liver and brain. Both extractives of S. acmella and N. grandiflora induced deep anaesthesia in juvenile C. macropomum . Although transient changes occurred in ACAP and GST activity after transport, irrespective of transport time or anaesthetic used, antioxidant components in these extractives reduced or maintained lipid peroxidation within basal levels in the assessed organs. The use of 10 mg L − 1 of extract of S. acmella was suitable to induce fast anaesthesia ( − 1 and 30 μL L − 1 of the extractives of S. acmella and N. grandiflora respectively, enhanced protection against oxidative damage mainly in muscle and gills of juvenile tambaqui and therefore have the potential to be used as chemoprotectants for transport purposes. Statement of relevance This study presents novel products to be used as anaesthetics/sedatives for fish.

Several studies have suggested eugenol as a suitable anaesthetic for fish. However, it has also been regarded as a toxic and aversive substance to several aquatic organisms, including fish. This study sought to assess the eugenol-induced behavioural alterations and its seizurogenic potential to fish. Moreover, a distinctive methodology for an in vivo evaluation of the brain activity was also presented. Prior to the evaluation of eugenol-induced responses, fish were exposed to pentylenetetrazole (PTZ), to characterize any seizure-like patterns. Antagonizing responses to PTZ were assessed in fish receiving diazepam (BDZ) and subsequently exposed to PTZ. Tambaqui fish juveniles, Colossoma macropomum (15.8 ± 2.8 g) were used as models and assayed as follows: (i) fish exposed to PTZ (15 mM) and (ii) fish receiving a dose of BDZ (10 mg Kg–1) and later exposed to PTZ (15 mM) (BDZ–PTZ group). Thereafter, fish were evaluated throughout (iii) eugenol exposure at 65 μL L–1 (ethanolic solution) and recovery. Control fish and a vehicle control group (ethanol at 585 μL L–1) were also established. PTZ baths elicited body immobilization preceded by hyperactivity in a stereotyped seizure-like behaviour with increased EEG wave amplitude and frequency. PTZ effects in the brain were attenuated by a pre-administration of BDZ. Upon eugenol exposure, tambaqui had an intense neuronal excitability, showing a clonus-like seizure behaviour, also corroborated by the EEG patterns, which were consistent with a seizure-like response. Responses of eugenol-exposed fish resembled those of the PZT-exposed animals, with epileptiform discharges. EMG was in line with the EEG modulation, showing increased tracing oscillations and higher mean amplitudes in PTZ-exposed fish whereas in BDZ–PTZ group muscle contraction was less frequent and powerful. Fish exposed to eugenol showed initially some muscle activity followed by a loss of muscle tonus over time. In summary, our results showed that upon eugenol exposure, although a time-dependent body immobilization was attained, fish presented an intense neuronal excitability comparable to that evoked by PTZ. Eugenol failed to promote depression of the CNS and therefore may be not suitable to be used for general anaesthesia of C. macropomum. As eugenol could be implicated in seizurogenesis and be potentially toxic to the fish brain, protocols suggesting the broad use of eugenol for short-term anaesthesia or euthanasia of fish should be carefully revised, as it raises important concerns in terms of ethics and fish welfare.

O objetivo desse trabalho foi avaliar o efeito combinado da temperatura e alimentacao sobre a taxa de excrecao de amonia do piaussu. Os peixes (10,3 ± 1,7 g) foram aclimatados em um sistema de recirculacao de agua e submetidos a cinco temperaturas (15, 19, 23, 27 and 31°C), alimentacao (duas vezes ao dia ate saciedade) e regimes de jejum diferentes. Nove peixes por tratamento foram distribuidos individualmente em tanques e a taxa de excrecao de amonia foi estimada ate 24 h, a cada quatro horas, para os tratamentos de animais alimentados e em jejum. Durante esse periodo o consumo de racao foi estimado. Como esperado, a taxa de excrecao dos animais alimentados, em termos gerais, foi maior comparada aos peixes nao alimentados, exceto pelos peixes mantidos a 15oC. Temperaturas mais altas aumentaram a excrecao a partir de 27oC nos tratamentos em jejum. O aumento da temperatura foi diretamente proporcional a um maior consumo de racao, o qual foi o principal responsavel pela variabilidade na taxa de excrecao. Picos de excrecao pos-prandial ocorreram desde as primeiras quatro horas ate 12 h apos a alimentacao e foram seguidos por um retorno aos niveis basais apos 16 h. A taxa de excrecao de amonia do piaussu e influenciada por ambas a temperatura e alimentacao, sendo fortemente influenciada pela ultima. Baseando-se nos dados do presente estudo, a temperatura de criacao para essa especie devera ser mantida em torno de 27oC. Palavras-chave : peixes de agua doce, metabolismo, nitrogenio, sistema de recirculacao, teleosteos.

O objetivo do presente estudo foi avaliar os efeitos da exposição a ní­­veis subletais de amônia nos parí­¢metros sanguí­­neos e qualidade da carne de juvenis de pacu (Piaractus mesopotamicus). Além disso, também foi realizada a avaliação desses parí­¢metros pós-recuperação em água livre de amônia. Os peixes (27,1 ± 5,4 g) foram expostos a 0,0 (controle); 0,5 e 1,0 mg N-NH3 L-1 por 10 dias seguidos pelo mesmo tempo de recuperação em água livre de amônia. Ao final dos 10 dias de exposição e após o perí­­odo de recuperação foram coletadas amostras de sangue para determinação dos ní­­veis de glicose, lactato e dos valores de hematócrito. Para avaliação dos ní­­veis de peroxidação lipí­­dica, da composição proximal e realização da análise sensorial, amostras de músculo/filé também foram coletadas. A exposição í­Â amônia ocasionou alterações nos parí­¢metros sanguí­­neos e afetou negativamente a análise sensorial dos filés de pacu. Entretanto, a composição proximal e o grau de peroxidação lipí­­dica não foram alterados. Concluindo, a exposição a ní­­veis subletais de amônia induziu respostas secundárias de estresse em juvenis de pacu e alterou caracterí­­sticas organolépticas do filé. Um perí­­odo de recuperação de 10 dias foi suficiente para restaurar a homeostase e a qualidade do filé.

The objective of this study was to investigate the effects of waxy extract of Spilanthes acmella and essential oil (EO) of Nectandra grandiflora on secondary stress responses of tambaqui Colossoma macropomum transported for up to 10 h. Fish were packed in plastic bags and shipped for 2, 6 and 10 h with or without anaesthetics added to the transport water. Whole blood was collected from the caudal vasculature before and after transport and several haematological variables were measured. No mortalities were observed after transport. Attenuation of secondary stress responses was observed with the use of N. grandiflora EO after 6 h transport based on lower glycaemia and no alteration of blood Na+ concentrations after transport compared to reference values of non-transported fish. Furthermore, dissolved oxygen levels were still maintained in the water after transport for 10 h with N. grandiflora EO compared to water of non-transported fish. While no clear advantages were observed with the use of S. acmella extract at 2 mg L−1 after transport, the use of N. grandiflora EO at 30 μL L−1 should be considered for the transport of C. macropomum for it seems to provide a sparing effect on the water dissolved oxygen levels and an improved physiological condition after transport.

This study aimed to evaluate the effect of different water pH levels on the growth performance and oxidative stress responses of pacu juveniles. Pacu were subjected to four different water pH levels (5.5, 6.5, 7.5 and 8.5) for 45 days in recirculation aquaculture systems. The best growth performance occurred at a water pH of 8.5. At a water pH of 8.5, in general, alkalinity, nitrite and un-ionised ammonia were higher and plasma ions Na+ and K+ were lower than at other water pH values. Total ammonia was higher at a water pH of 5.5. Juveniles exposed to acidic water pH (5.5 or 6.5) showed a lower muscle antioxidant capacity against peroxyl radicals (ACAP) and glutathione S-transferase (GST) activity. Water pH did not affect survival, blood glucose and pH, haematocrit, plasma Cl−, and the muscle content of protein thiols (PSH) and thiobarbituric acid reactive substances (TBARS). In conclusion, a water pH of 8.5 is recommended for pacu production systems, since fish presented higher growth and antioxidant statuses at this pH.

The effects of ammonia exposure and recovery on oxidative stress parameters and histology of juvenile Brazilian flounder Paralichthys orbignyanus were evaluated. The fish were exposed to 0.12, 0.28 and 0.57 mg NH3-N L−1, plus a control, for 10 days followed by the same recovery time in ammonia-free water. Gill, liver and muscle samples (n = 9) were collected after 1, 5 and 10 days of exposure and after recovery for oxidative stress analysis (antioxidant capacity against peroxyl radicals (ACAP); glutathione S-transferase (GST) activity; lipoperoxidation levels measured through thiobarbituric acid reactive substances (TBARS) content). For histological assessment, gill, liver and brain samples were collected. Exposure to all NH3-N concentrations induced different time- and dose-dependent changes in oxidative stress parameters. Reduced antioxidant capacity of the liver and muscle and enhanced TBARS levels in the gills and liver were demonstrated. Differently, a high ammonia concentration elicited lower hepatic TBARS levels. Enhanced GST activity in all organs and increased antioxidant capacity of the gills were also observed. No ammonia-induced histopathological effects were demonstrated. After recovery, most parameters (liver ACAP, GST activity in the muscle and liver and TBARS in the gills) returned to baseline levels. However, liver TBARS and gill GST activity remained altered 0.57 mg NH3-N L−1 treatment. The recovery period also led to a decrease in gill antioxidant capacity and an increase in muscle antioxidant capacity. In conclusion, a concentration of 0.12 mg NH3-N L−1 induces oxidative stress and antioxidant responses in juvenile Brazilian flounder. Moreover, a 10-day recovery period is not sufficient to restore fish homeostasis.

O presente trabalho tem como objetivo avaliar o impacto da atividade de dragagem de aprofundamento do Porto de Aratu sobre a estrutura da comunidade ictioplanctônica. As coletas foram realizadas em 6 estações de amostragem, tanto na maré enchente, quanto na vazante, antes, durante e após a dragagem. O ictioplâncton foi coletado utilizando-se rede bongo, malha 500 μm, em arrastos superficiais e os parâmetros da qualidade da água (temperatura, salinidade, pH, oxigênio dissolvido, turbidez, amônia e fósforo total) também foram determinados. Foram verificadas diferenças estatísticas significativa para a variabilidade temporal de todos os parâmetros de qualidade da água, exceto a turbidez, e nos índices estruturais da comunidade ictioplanctônica. O período da dragagem apresentou as menores densidades de ovos e larvas, além de menor riqueza e diversidade de táxons, com posterior recuperação no período da pós-dragagem. A análise multivariada de ordenação demonstrou uma separação bastante clara entre as amostragens, demonstrando haver uma variabilidade temporal na composição e abundância do ictioplâncton na área portuária de Aratu, em resposta as características oceanográficas da massa de água e alterações na qualidade, possivelmente associadas à atividade de dragagem.

The present study aimed to evaluate the effects of benzocaine and tricaine methanesulfonate on oxidative stress parameters of juvenile tambaqui tissues. Fish (n=80) were anesthetized with benzocaine (100 mg L-1) or tricaine (240 mg L-1) and two control groups were used (non-anesthetized fish and fish exposed to ethanol-only). After anesthetic induction 10 fish/anesthetic were euthanized after 3, 12 and 24 hours post-anesthesia and tissue samplings (gills, liver and brain) were performed. Samples were submitted to analyses of enzyme activity glutathione-S-transferase (GST), cellular lipid peroxidation (TBARS) and total antioxidant capacity (ACAP). ACAP increased in gills of benzocaine treatment after 12 hours. The liver showed a reduction in ACAP of tricaine treatment after 12 hours. Both anesthetic treatments showed an increase of ACAP at 24 hours compared to control group. The activity of the GST enzyme increased in the gills for treatments benzocaine and tricaine after 3 and 12 hours. Liver showed increased GST activity (benzocaine after 24 hours and tricaine after 3 and 24 hours). Lipid damage decreased in gills (both anesthetics) and brain (tricaine) after 24 hours. The results demonstrate that benzocaine and tricaine did not cause oxidative damage in juvenile tambaqui under the experimental conditions herein established.