Essential oil of Aloysia citriodora Paláu and citral: sedative and anesthetic efficacy and safety in Rhamdia quelen and Ctenopharyngodon idella.

OBJECTIVE: To verify the efficacy of citral in inducing sedation and anesthesia in silver catfish (Rhamdia quelen) and grass carp (Ctenopharyngodon idella) and to assess the safety of essential oil (EO) of Aloysia citriodora and citral in inducing and maintaining anesthesia in silver catfish. STUDY DESIGN: Clinical study, randomized, parallel, multi-arm with control group in target species. ANIMALS: A total of 96 juvenile and 72 adult silver catfish and 80 juvenile grass carp were used. METHODS: Silver catfish and grass carp were exposed to different concentrations of citral, 15-675 and 15-600 µL L-1, respectively, during the maximum period of 30 minutes to verify sedation and anesthesia induction and recovery times. In addition, for anesthetic induction, silver catfish were exposed to the EO of A. citriodora and citral at 225 µL L-1 for 3.5 minutes. Then, fish were transferred to an anesthesia maintenance solution at 50 µL L-1 for 10 minutes to assess hematologic and biochemical variables at 60 minutes, 2 and 6 days after treatment. RESULTS: Citral only induced sedation from 15, 25 and 40 µLL-1 in both species. Anesthesia without mortality was induced in silver catfish at 50-600 µL L-1 and grass carp at 75-450 µL L-1. At 675 and 600 µL L-1, mortality was recorded in silver catfish and grass carp, respectively. The EO of A. citriodora and citral were safe in inducing and maintaining anesthesia in silver catfish, with mean corpuscular hemoglobin concentration being the only variable that varied in relation to time and treatments. CONCLUSIONS AND CLINICAL RELEVANCE: Citral was effective in inducing sedation and anesthesia in both species. In addition, A. citriodora EO and citral were safe in inducing and maintaining anesthesia in silver catfish. Both agents are promising substances for the development of new drugs for fish.

Diphenyl diselenide dietary supplementation protects against fumonisin B1-induced oxidative stress in brains of the silver catfish Rhamdia quelen.

The trend toward using plant-based ingredients in aquafeeds has raised important concerns for aquaculture owing to the negative impacts of mycotoxins on fish health; with emphasis for contamination by fumonisin B1 (FB1). The brain is an important target of FB1; however, study of the pathways linked to brain damage is limited to an analysis of histopathological alterations. Reports have demonstrated the protective effects of dietary supplementation with diphenyl diselenide (Ph2Se2) in the brains of fish subjected to several environmental insults; nevertheless, its neuroprotective effects in fish fed with diets contaminated with FB1 remain unknown. Therefore, the aim of this study was to evaluate whether oxidative damage may be a pathway associated with FB1-induced neurotoxicity, as well as to evaluate whether dietary supplementation with Ph2Se2 prevents or reduces FB1-mediated brain oxidative damage in silver catfish. Brain reactive oxygen species (ROS), lipid peroxidation (LOOH) and protein carbonylation increased on day 30 post-feeding in animals that received FB1-contaminated diets compared to the control group, while brain antioxidant capacity against peroxyl radicals (ACAP) levels and catalase (CAT), glutathione peroxidase (GPx) and glutathione S-transferase (GST) activities were lower. Diphenyl diselenide dietary supplementation avoid increases in brain ROS levels, as well minimizing the augmentation of LOOH levels. Furthermore, Ph2Se2 prevented impairment of brain ACAP levels, as well as GPx and GST activities elicited by FB1-contaminated diets. These data suggest that dietary supplementation with 3 mg/kg Ph2Se2 prevented FB1-induced brain damage in silver catfish, and this protective effect occurred through avoided of excessive ROS production, as well as via prevention of brain lipid damage. Furthermore, Ph2Se2 exerted its neuroprotective effects via ameliorative effects on the enzymatic and non-enzymatic antioxidant defense systems, and may be an approach to prevent FB1-induced brain oxidative stress; however, is not an alternative to prevent the impairment on performance caused by FB1.

Purinergic signaling and gene expression of purinoceptors in the head kidney of the silver catfish Rhamdia quelen experimentally infected by Flavobacterium columnare.

The head kidney is a lymphoid immune organ that plays a key role in the immune and inflammatory responses of teleost fish. It is associated with immunoglobulin G production and differentiation of B cells. The presence of a multi-enzymatic complex found anchored in the plasma membrane makes the head kidney an important purinergic-dependent tissue. Purinergic signaling has been associated with these responses under pathological conditions via regulation of extracellular adenosine triphosphate (ATP), the main damage molecular associated pattern agent released during bacterial infections. The aim of this study was to determine whether purinergic signaling is a pathway associated with impairment of immune responses in silver catfish (Rhamdia quelen) experimentally infected by Flavobacterium columnare, as well as to evaluate the role of P2 purine receptors in this response. Triphosphate diphosphohydrolase (NTPDase) activity in the head kidney was significantly lower in silver catfish experimentally-infected F. columnare 72 h post-infection (hpi) than in the control group, while no significant difference was observed with respect NTPDase activity on adenosine diphosphate, as well as on 5'-nucleotidase and adenosine deaminase activities. Extracellular ATP levels were significantly higher in the head kidney of experimentally-infected fish than in the control group at 72 hpi. Finally, p2ry11 and p2rx3 purine receptor levels were significantly higher in experimentally-infected fish than in the control group at 72 hpi. We conclude that purinergic signaling in the head kidney of silver catfish infected by F. columnare creates a pro-inflammatory profile that may contribute to impairment of immune and inflammatory responses via reduction of ATP hydrolysis and its accumulation in the extracellular milieu, accompanied by upregulation of p2ry11 and p2rx3 purine receptors, leading to pro-inflammatory status.

Branchial bioenergetics dysfunction as a relevant pathophysiological mechanism in freshwater silver catfish (Rhamdia quelen) experimentally infected with Flavobacterium columnare.

Flavobacterium columnare, the causative agent of columnaris disease, is a serious bacterial disease responsible for causing devastating mortality rates in several species of freshwater fish, leading to severe economic losses in the aquaculture industry. Notwithstanding the enormous impacts this disease can have, very little is known regarding the interaction between the host and bacterium in terms of the mortality rate of silver catfish (Rhamdia quelen), as well its linkage to gill energetic homeostasis. Therefore, we conducted independent experiments to evaluate the mortality rates caused by F. columnare in silver catfish, as well as whether columnaris disease impairs the enzymes of the phosphoryl transfer network in gills of silver catfish and the pathways involved in this inhibition. Experiment I revealed that clinical signs started to appear 72 h post-infection (hpi), manifesting as lethargy, skin necrosis, fin erosion and gill discoloration. Silver catfish began to die at 96 hpi, and 100% mortality was observed at 120 hpi. Experiment II revealed that creatine kinase (CK, cytosolic and mitochondrial) and pyruvate kinase (PK) activities were inhibited in silver catfish experimentally infected with F. columnare, while no significant difference was observed between experimental and control groups with respect to adenylate kinase activity. Activity of the branchial sodium-potassium pump (Na+, K+-ATPase) was inhibited while reactive oxygen species (ROS) and lipid peroxidation levels were higher in silver catfish experimentally infected with F. columnare than in the control group at 72 hpi. Based on these data, the impairment of CK activity elicited by F. columnare caused a disruption in branchial energetic balance, possibly reducing ATP availability in the gills and provoking impairment of Na+, K +ATPase activity. The inhibition of CK and PK activities appears to be mediated by ROS overproduction and lipid peroxidation, both of which contribute to disease pathogenesis associated with branchial tissue.

Aloysia triphylla in the zebrafish food: effects on physiology, behavior, and growth performance.

Dietary supplements are commonly used by animals and humans and play key roles in diverse systems, such as the immune and reproductive systems, and in metabolism. Essential oils (EOs), which are natural substances, have potential for use in food supplementation; however, their effects on organisms remain to be elucidated. Here, we examine the effects of dietary Aloysia triphylla EO supplementation on zebrafish behavior, metabolism, stress response, and growth performance. We show that fish fed diets containing A. triphylla EO presented an anxiolytic response, with reduced exploratory activity and oxygen consumption; no changes were observed in neuroendocrine stress axis functioning and growth was not impaired. Taken together, these results suggest that the A. triphylla EO supplementation is a strong candidate for use in feed, since it ensures fish welfare (anxiolytic behavior) with decreased oxygen consumption. This makes it suitable for use in high-density production systems without causing damage to the neuroendocrine stress axis and without growth performance being impaired.

Aloysia triphylla essential oil as additive in silver catfish diet: Blood response and resistance against Aeromonas hydrophila infection.

The essential oil of Aloysia triphylla (EOAT) is a promising product with potential use in aquaculture systems. This study evaluated hematological/biochemical responses and survival of silver catfish (Rhamdia quelen) fed a diet containing EOAT and infected by Aeromonas hydrophila. After 21 days of feeding trial, fish were infected with A. hydrophila following a 10-day period of observation. Blood collection was performed before and after the bacterial challenge. Dietary EOAT by itself seems to affect some blood parameters, decreasing total leukocyte, lymphocyte, and neutrophil counts and increasing total protein values. However, 2.0 mL EOAT/kg diet showed a possible potential protective effect after A. hydrophila infection, maintaining the evaluated parameters similar to basal values (from healthy fish before the feeding trial) and promoting survival of silver catfish.

Anesthesia and anesthetic action mechanism of essential oils of Aloysia triphylla and Cymbopogon flexuosus in silver catfish (Rhamdia quelen).

OBJECTIVES: To document the time for anesthesia induction and recovery using different concentrations of essential oils (EOs) of Cymbopogon flexuosus and Aloysia triphylla in silver catfish (Rhamdia quelen), and to determine whether the mechanism of action of either EO involves the benzodiazepine (BDZ) site of the GABAA receptor. STUDY DESIGN: Experimental study. ANIMALS: A total of 144 silver catfish, length 7.5 ± 1.1 cm, weighing 3.95 ± 0.85 g. METHODS: Essential oils were evaluated at concentrations of 25, 150 and 300 µL L-1, and also ethanol alone (seven groups, n = 6 per group). Induction of sedation or anesthesia and recovery were assessed. In a further six groups (n = 6 per group), fish were exposed to both EOs (25, 150 or 300 µL L-1) with diazepam 150 µm, and also diazepam (10 µm) alone. Flumazenil (5 or 10 µm) was added to the recovery water of fish exposed to diazepam (150 µm) or both EOs (150 and 300 µL L-1) (total of 10 groups = 60 fish). RESULTS: Both EOs induced anesthesia at concentrations of 150 and 300 µL L-1, and sedation at 25 µL L-1. There was no significant difference between EOs for reaching deep anesthesia; there was a significantly longer recovery time for the EO of C. flexuosus. The addition of diazepam (150 µm) resulted in faster induction of anesthesia with both EOs, with no significant change in recovery times. Flumazenil (10 µm) reversed the diazepam-induced anesthesia, but not the anesthesia induced by EOs. CONCLUSIONS AND CLINICAL RELEVANCE: The EO of C. flexuosus induced effective sedation (25 µL L-1) and anesthesia (150 and 300 µL L-1) without short-term mortality. The modulation of the BDZ site of the GABAA receptor in the anesthetic action mechanism of both EOs was not demonstrated.

Pre-sedation and transport of Rhamdia quelen in water containing essential oil of Lippia alba: metabolic and physiological responses.

The effects of transporting silver catfish (Rhamdia quelen) for 6 h in plastic bags containing 0 (control), 30 or 40 µL/L of essential oil (EO) from Lippia alba leaves were investigated. Prior to transport, the fish in the two experimental groups were sedated with 200 µL/L of EO for 3 min. After transport, dissolved oxygen, carbon dioxide, alkalinity, water hardness, pH, temperature and un-ionized ammonia levels in the transport water did not differ significantly among the groups. However, total ammonia nitrogen levels and net Na(+), Cl(-) and K(+) effluxes were significantly lower in the groups transported with EO of L. alba than those in the control group. PvO2, PvCO2 and HCO3(-) were higher after transporting fish in 40 µL/L of EO of L. alba, but there were no significant differences between groups regarding blood pH or hematocrit. Cortisol levels were significantly higher in fish transported in 30 µL/L of EO of L. alba compared to those of the control group. The metabolic parameters (glycogen, lactate, total amino acid, total ammonia and total protein) showed different responses after adding EO to the transport water. In conclusion, while the EO of L. alba is recommended for fish transport in the conditions tested in the present study because it was effective in reducing waterborne total ammonia levels and net ion loss, the higher hepatic oxidative stress in this species with the same EO concentrations reported by a previous study led us to conclude that the 10-20 µL/L concentration range of EO and lack of pre-sedation before transport are more effective.

Rhamdia quelen (Quoy & Gaimard, 1824), submitted to a stressful condition: effect of dietary addition of the essential oil of Lippia alba on metabolism, osmoregulation and endocrinology

The aim of this study was to evaluate the effect of the essential oil of Lippia alba (EOLA) as a feed additive on ionoregulatory and metabolic parameters and pituitary hormones expression in silver catfish, Rhamdia quelen , submitted to a stressful condition (stocking density of 10.6 kg m-3 and limited space). Fish were fed with different concentrations of EOLA (0.0 - control, 0.25 and 0.50 mL kg food-1) for 20 days. Metabolic parameters were not affected by the diet, with the exception of alanine aminotransferase, which was higher in the liver of fish fed 0.50 mL EOLA kg food-1. Plasma ions and activity of H+-ATPase did not change, but fish fed 0.25 mL EOLA kg food-1 presented higher Na+/K+-ATPase activity. Somatolactin expression in the pituitary was higher in the fish fed 0.25 mL EOLA kg food -1, but the expression of growth hormone and prolactin did not change. Therefore, dietary EOLA does not exert a protective effect in R. quelen submitted to a stressful situation because it did not alter most measured parameters. The use of 0.25 mL EOLA kg food-1 seems to be more suitable than 0.50 mL EOLA kg food-1 since the latter may be related to liver damage.

O objetivo deste estudo foi avaliar o efeito do óleo essencial de Lippiaalba (OELA) como aditivo em rações na ionoregulação, parâmetros metabólicos e expressão de hormônios hipofisários em jundiás, Rhamdiaquelen, submetidos a uma situação estressante (densidade de estocagem de 10,6 kg m-3 e espaço limitado). Os peixes foram alimentados com diferentes concentrações de OELA (0,0 - controle, 0,25 e 0,50 mL kg de ração-1) durante 20 dias. Parâmetros metabólicos não foram afetados pela dieta, com a exceção da alanina aminotransferase, que foi mais elevada no fígado dos peixes alimentados com 0,50 mL de OELA kg de ração-1. Íons plasmáticos e a atividade da H+-ATPase não apresentaram nenhuma alteração, mas os peixes alimentados com 0,25 mL OELA kg de ração-1 apresentaram maior atividade da Na+/K+-ATPase. A expressão da somatolactina na hipófise de peixes alimentados com 0,25 mL OELA kg de ração-1 aumentou, porém a expressão do hormônio de crescimento e da prolactina não mudou. Portanto, a adição do OELA na ração não tem um efeito protetor em jundiás submetidos a uma situação estressante, pois não influiu na maioria dos parâmetros medidos. O uso de 0,25 mL OELA kg de ração-1 parece ser mais adequado que 0,50 mL OELA kg de ração-1, uma vez que este nível de inclusão pode estar relacionado a danos hepáticos.

Lippia alba essential oil promotes survival of silver catfish (Rhamdia quelen) infected with Aeromonas sp.

In vitro and in vivo activity of the Lippia alba essential oil (EO) against Aeromonas sp. was evaluated. In the in vitro assay the minimum inhibitory concentration (MIC) and a minimum bactericidal concentration (MBC) of EO for Aeromonas cells were determined using the microdilution method. Twenty five strains of Aeromonas sp. isolated from infected fish obtained from local fish farms were used. MIC and MBC values were 2862 and 5998 µg mL-1 for L. alba EO and 0.5 and 1.2 µg mL-1 for gentamicin, respectively. In the in vivo assay silver catfish juveniles (Rhamdia quelen) (7.50 ± 1.85 g and 10.0 ± 1.0 cm) with typical injuries associated to Aeromonas infection were divided into four treatments (in triplicate n=10): untreated fish (negative control), 10 mg L-1 of gentamicin, and 20 or 50 µL L-1 of EO. Fish were maintained in aerated 20 L plastic boxes. After 10 days survival of silver catfish infected with Aermonas sp. and treated with essential oil (50 µL L-1) was greater than 90%.

Using the essential oil of Aloysia triphylla (L'Her.) Britton to sedate silver catfish (Rhamdia quelen) during transport improved the chemical and sensory qualities of the fish during storage in ice.

UNLABELLED: Exposure of silver catfish to 40 µL/L of the essential oil of Aloysia triphylla (AT) during in vivo transport delayed the onset and resolution of rigor mortis as well as the degradation of IMP into HxR compared to the control. The fish that were treated with 30 or 40 µL/L of AT received lower sensory demerit scores after 10 d of storage in ice compared to the control, and the fish that were treated with 40 µL/L of AT had a longer sensory shelf life than did the control. These results indicated that using AT as a sedative in the water in which the silver catfish were transported extended their freshness and increased their shelf life during refrigerated storage. PRACTICAL APPLICATION: Interest in natural anesthetics, such as Aloysia triphylla, has increased in the field of commercial aquaculture because they reduce the number of fish lesions acquired during capture, handling, and transportation. Fish sedated with the essential oil of A. triphylla at 40 µL/L during transport before slaughter exhibited a delay in the loss of freshness and an increased shelf life in ice. In addition to improving animal welfare before slaughter, the essential oil appears to be a promising product for improving fish conservation in the food industry.

Anesthetic activity of the essential oil of Aloysia triphylla and effectiveness in reducing stress during transport of albino and gray strains of silver catfish, Rhamdia quelen.

This study investigated the efficacy of the essential oil (EO) of Aloysia triphylla as an anesthetic for albino and gray strains of silver catfish, Rhamdia quelen. Juveniles were exposed to concentrations between 20 and 800 µL L(-1) EO of A. triphylla to evaluate time of induction and recovery from anesthesia. In another experiment, both strains were divided into four groups such as 0 (control), 30, 40, or 50 µL L(-1) EO and transported for 5 h. The longest time for anesthetic induction and recovery was observed in the albinos. Both strains reached anesthesia in the 100-800 µL L(-1) (11.1-1.24 min) range, without mortality, being 200 µL L(-1) the best response considering time to anesthesia (5.35 min). Albinos transported with all EO concentrations showed higher values of carbon dioxide in the water of transport, but lower levels were observed in grays transported with 40 and 50 µL L(-1) EO when compared to control fish. The same concentrations did not prevent significant whole-body cortisol rise at the end of transport in the albino strain. Juveniles of both strains transported with EO presented lower ion loss to the water compared to control fish. The EO of A. triphylla is an effective anesthetic for albino and gray silver catfish. This EO increases whole-body cortisol levels in the albino strain, but as it reduces net ion loss as in the gray strain, it can be also recommended for transport.

The use of nitazoxanide against the pathogens Ichthyophthirius multifiliis and Aeromonas hydrophila in silver catfish (Rhamdia quelen).

The antiparasitic and antibacterial activities of nitazoxanide (NTZ) against Ichthyophthirius multifiliis (Ich) and Aeromonas hydrophila, respectively, were evaluated in silver catfish juveniles (Rhamdia quelen). In the first experiment, fish naturally infected by Ich were treated with different concentrations of NTZ. Fish mortality and the number of trophonts/juveniles were evaluated. In the second experiment, the antimicrobial potential in vitro (MIC test) and preventive potential in vivo against A. hydrophila was evaluated. The treatment with 1.5mgL(-1) NTZ showed the best result against Ich, with a significant reduction of trophonts and fish mortality of 10%. NTZ did not show in vitro activity against A. hydrophila at concentrations tested, but showed a probable preventive activity in vivo. However, these results are preliminary and more studies should be conducted.

Effects of subchronic manganese chloride exposure on tambaqui (Colossoma macropomum) tissues: oxidative stress and antioxidant defenses.

This study aimed to evaluate oxidative stress parameters in juvenile tambaqui (Colossoma macropomum) exposed to 3.88 mg l(-1) Mn(2+) for 96 hours. Biomarkers of oxidative stress, such as thiobarbituric acid reactive substances (TBARS), superoxide dismutase (SOD), catalase (CAT), and glutathione-S-transferase (GST) activities, as well as content of reduced glutathione (GSH), were analyzed in gill, liver, brain, and kidney. The presence of Mn(2+) in the water corresponded to increased levels of Mn(2+) accumulation according to the following sequence: gill > kidney > brain > liver. There was a significant increase in TBARS levels (40 %) and SOD activity (80 %) in addition to a significant decrease in GSH content (41 %) in gills of fish exposed to waterborne Mn(2+). In hepatic tissue of the exposed animals, TBARS levels decreased significantly (35 %), whereas SOD (82 %) and GST activities (51 %) as well as GSH content (43 %) increased significantly. In brain of exposed juvenile fish, only significant decreases in SOD (32 %) and CAT activities (65 %) were observed. Moreover, the kidney of exposed fish showed a significant increase in TBARS levels (53 %) and a significant decrease in SOD activity (41 %) compared with the control. Thus, the changes in biomarkers of oxidative stress were different in the tissues, showing a specific toxicity of this metal to each organ.

The anesthetic efficacy of eugenol and the essential oils of Lippia alba and Aloysia triphylla in post-larvae and sub-adults of Litopenaeus vannamei (Crustacea, Penaeidae).

The aim of this study was to evaluate the anesthesia induction and recovery times of sub-adult and post-larvae white shrimp (Litopenaeus vannamei) that were treated with eugenol and the essential oils (EOs) from Lippia alba and Aloysia triphylla. Oxidative stress parameters in the hemolymph of this species were also analyzed. The concentrations of eugenol, A. triphylla EO and L. alba EO recommended for anesthesia were 200, 300 and 750 µL L(-1) for sub-adults and 175, 300 and 500 µL L(-1) for post-larvae, respectively. The concentrations studied during the transport of sub-adults were between 20 and 50 µL L(-1) eugenol, 20-30 µL L(-1)A. triphylla EO and 50 µL L(-1)L. alba EO. For post-larvae, the optimal concentrations for transport were 20 µL L(-1) eugenol and between 20 and 50 µL L(-1)A. triphylla EO. The white shrimp sub-adults that were exposed to A. triphylla EO (20 µL L(-1)) showed increases in their total antioxidant capacities (150%), catalase (70%) and glutathione-S-transferase (615%) activity after 6 h. L. alba EO (50 µL L(-1)) and eugenol (20 µL L(-1)) also increased GST activity (1292 and 1315%) after 6 h, and eugenol (20 µL L(-1)) decreased the total antioxidant capacity (100%). Moreover, concentrations above 30 µL L(-1) for the EOs of A. triphylla and L. alba and 20 µL L(-1) eugenol were effective at inducing anesthesia and improving the antioxidant system against reactive oxygen species (ROS) after 6 h.