Use of antioxidants and time of cold storage: effects over viability parameters and enzymatic levels in semen of rainbow trout (Oncorhynchus mykiss, Walbaum, 1792) / Uso de antioxidantes e tempo de armazenamento a frio: efeitos sobre parâmetros de viabilidade e níveis enzimáticos no sêmen da truta arco-íris (Oncorhynchus mykiss, Walbaum, 1792)

The cold storage of milt implies potentials alterations in its quality because the storage generates as main process, free radicals that produce spermatozoa membrane lipids damage with the consequent motility and fertilising capacity disruptions. To decrease the damage generated by free radicals the cells have antioxidant defences (proteins, enzymes, and low molecular weight substances). The objective of the present study evaluated the time storage effect and different antioxidants prepared in spermatic diluents on sperm viability of O. mykiss milt stored at 4°C. The two-way ANOVA denoted that the time storage and antioxidant influence have significant effects separated or combined on viability parameters (sperm motility and viability, proteins concentrations and superoxide dismutase enzymatic activity in seminal plasma). In contrast, only the storage time affected the fertilising capacity and catalase enzymatic activity in seminal plasma. The resulting analysis can conclude that the antioxidant presence improves the viability of cold stored milt, especially the transport conditions and the antioxidants allow the fecundity despite motility decrease.

O armazenamento a frio de leite implica potenciais alterações em sua qualidade, pois gera como processo principal radicais livres que provocam danos aos lipídios da membrana dos espermatozoides, com as consequentes alterações na motilidade e na capacidade de fertilização. Para diminuir os danos causados pelos radicais livres, as células têm defesas antioxidantes (proteínas, enzimas e substâncias de baixo peso molecular). O presente estudo avaliou o efeito do tempo de armazenamento e diferentes antioxidantes preparados em diluentes espermáticos no armazenamento de viabilidade de O. mykiss milt a 4°C. A ANOVA de duas vias denotou que o armazenamento no tempo e a influência antioxidante têm efeitos significativos separados ou combinados nos parâmetros de viabilidade (motilidade espermática, viabilidade espermática, concentrações de proteínas e atividade enzimática da superóxido dismutase no plasma seminal), enquanto apenas o tempo de armazenamento afetou a capacidade de fertilização e atividade enzimática da catalase no plasma seminal. A análise resultante pode concluir que a presença de antioxidante melhora a viabilidade do leite frio, especialmente as condições de transporte, e os antioxidantes permitem a fecundidade apesar da diminuição da motilidade.

Use of antioxidants and time of cold storage: effects over viability parameters and enzymatic levels in semen of rainbow trout (Oncorhynchus mykiss, Walbaum, 1792)

Abstract The cold storage of milt implies potentials alterations in its quality because the storage generates as main process, free radicals that produce spermatozoa membrane lipids damage with the consequent motility and fertilising capacity disruptions. To decrease the damage generated by free radicals the cells have antioxidant defences (proteins, enzymes, and low molecular weight substances). The objective of the present study evaluated the time storage effect and different antioxidants prepared in spermatic diluents on sperm viability of O. mykiss milt stored at 4°C. The two-way ANOVA denoted that the time storage and antioxidant influence have significant effects separated or combined on viability parameters (sperm motility and viability, proteins concentrations and superoxide dismutase enzymatic activity in seminal plasma). In contrast, only the storage time affected the fertilising capacity and catalase enzymatic activity in seminal plasma. The resulting analysis can conclude that the antioxidant presence improves the viability of cold stored milt, especially the transport conditions and the antioxidants allow the fecundity despite motility decrease.

Resumo O armazenamento a frio de leite implica potenciais alterações em sua qualidade, pois gera como processo principal radicais livres que provocam danos aos lipídios da membrana dos espermatozoides, com as consequentes alterações na motilidade e na capacidade de fertilização. Para diminuir os danos causados pelos radicais livres, as células têm defesas antioxidantes (proteínas, enzimas e substâncias de baixo peso molecular). O presente estudo avaliou o efeito do tempo de armazenamento e diferentes antioxidantes preparados em diluentes espermáticos no armazenamento de viabilidade de O. mykiss milt a 4°C. A ANOVA de duas vias denotou que o armazenamento no tempo e a influência antioxidante têm efeitos significativos separados ou combinados nos parâmetros de viabilidade (motilidade espermática, viabilidade espermática, concentrações de proteínas e atividade enzimática da superóxido dismutase no plasma seminal), enquanto apenas o tempo de armazenamento afetou a capacidade de fertilização e atividade enzimática da catalase no plasma seminal. A análise resultante pode concluir que a presença de antioxidante melhora a viabilidade do leite frio, especialmente as condições de transporte, e os antioxidantes permitem a fecundidade apesar da diminuição da motilidade.

Use of antioxidants and time of cold storage: effects over viability parameters and enzymatic levels in semen of rainbow trout (Oncorhynchus mykiss, Walbaum, 1792) / Uso de antioxidantes e tempo de armazenamento a frio: efeitos sobre parâmetros de viabilidade e níveis enzimáticos no sêmen da truta arco-íris (Oncorhynchus mykiss, Walbaum, 1792)

Abstract The cold storage of milt implies potentials alterations in its quality because the storage generates as main process, free radicals that produce spermatozoa membrane lipids damage with the consequent motility and fertilising capacity disruptions. To decrease the damage generated by free radicals the cells have antioxidant defences (proteins, enzymes, and low molecular weight substances). The objective of the present study evaluated the time storage effect and different antioxidants prepared in spermatic diluents on sperm viability of O. mykiss milt stored at 4°C. The two-way ANOVA denoted that the time storage and antioxidant influence have significant effects separated or combined on viability parameters (sperm motility and viability, proteins concentrations and superoxide dismutase enzymatic activity in seminal plasma). In contrast, only the storage time affected the fertilising capacity and catalase enzymatic activity in seminal plasma. The resulting analysis can conclude that the antioxidant presence improves the viability of cold stored milt, especially the transport conditions and the antioxidants allow the fecundity despite motility decrease.

Resumo O armazenamento a frio de leite implica potenciais alterações em sua qualidade, pois gera como processo principal radicais livres que provocam danos aos lipídios da membrana dos espermatozoides, com as consequentes alterações na motilidade e na capacidade de fertilização. Para diminuir os danos causados pelos radicais livres, as células têm defesas antioxidantes (proteínas, enzimas e substâncias de baixo peso molecular). O presente estudo avaliou o efeito do tempo de armazenamento e diferentes antioxidantes preparados em diluentes espermáticos no armazenamento de viabilidade de O. mykiss milt a 4°C. A ANOVA de duas vias denotou que o armazenamento no tempo e a influência antioxidante têm efeitos significativos separados ou combinados nos parâmetros de viabilidade (motilidade espermática, viabilidade espermática, concentrações de proteínas e atividade enzimática da superóxido dismutase no plasma seminal), enquanto apenas o tempo de armazenamento afetou a capacidade de fertilização e atividade enzimática da catalase no plasma seminal. A análise resultante pode concluir que a presença de antioxidante melhora a viabilidade do leite frio, especialmente as condições de transporte, e os antioxidantes permitem a fecundidade apesar da diminuição da motilidade.

Evaluación de la mineralización en mandíbula y diente de trucha arcoíris (Oncorhynchus mykiss) mediante microscopía electrónica de barrido de presión variable acoplada a detector de espectroscopía de rayos X de energía dispersiva / Evaluation of mineralization in jaw and teeth of rainbow trout (Oncorhynchus mykiss) using variable pressure scanning electron microscopy coupled to energy-dispersive X-ray spectroscopy detector

RESUMEN: En salmonicultura se utilizan imágenes de rayos X, para evaluar la columna vertebral y las aletas, pero estas no permiten cuantificar los minerales que constituyen el hueso, para esto se utilizan otras técnicas que son destructivas. La Microscopía Electrónica de Barrido de presión variable (VP SEM) acoplada al detector espectroscopia de rayos X de energía dispersiva (EDX) acoplado, nos permite analizar la microestructura y a la vez determinar elementos químicos, porcentaje y distribución presentes en puntos específicos en una muestra. Se utilizaron 5 truchas control y 5 con deformidad mandibular, de agua dulce en etapa de smolt, se analizó la estructura y mineralización del hueso, se eligieron en promedio 6 puntos de cuantificación por región de interés y se analizaron mediante Microscopía Electrónica de Barrido (VP SEM-EDX). Los datos obtenidos muestran porcentaje en masa de Ca promedio en hueso articular de truchas fueron de 8.07 % y de 14.48 % en truchas con deformidad mandibular y control respectivamente y el porcentaje en masa promedio de P es de 4.07 % y 7.60 %, en truchas con deformidad mandibular y control respectivamente. Se identificó además otros elementos presentes en la muestra como Mg, Na, C, O, N, S, F, Zn, Al y Fe, con especial interés en el aumento de carbono en las muestras analizadas con deformidad mandibular y la presencia de aluminio en todas las muestras. La Técnica de VP SEM-EDX, permite evaluar de forma directa, sin destrucción de la muestra y con una preparación mínima de la muestra. En el hueso, la aplicación más frecuente de SEM-EDX es la medición del contenido de Ca y P y la relación que existe entre estos elementos Ca/P, en la muestra. Paralelamente la técnica nos permite la detección de otros microelementos provenientes del agua o de la alimentación y que eventualmente pueden provocar alteraciones en los peces, confirmando la hipótesis que el microanálisis elemental tiene utilidad para la salmonicultura.

SUMMARY: In salmon farming systems, X-ray images are used to evaluate the spine and fins, but these do not allow quantifying the minerals that make up the bone, for different techniques that are destructive are used. Variable Pressure Scanning Electron Microscopy (VP SEM) coupled to an Energy Dispersive X-ray spectroscopy detector (EDX), allows us to analyze the microstructure and at the same time determine chemical elements, percentages, and distribution present at specific points in a sample. Five control and five jaw deformity trout, from freshwater and in the smolt stage were used. The structure and mineralization of the bone were analyzed, an average six quantification points were chosen per region of interest (ROI) and then they were analyzed by Scanning Electron Microscopy (VP SEM-EDX). The data obtained have shown the average mass percentage of calcium in trout joint bone was 8.07 % and 14.48 % in jaw deformity and control trout, respectively; and the average mass percentage of phosphorus is 4.07 % and 7.60 %, in jaw deformity and control trout, respectively. Other elements present in the sample were also identified, such as magnesium, sodium, carbon, oxygen, nitrogen, sulphur, fluorine, zinc, aluminium, and iron, with special interest the increase of carbon in the analyzed samples with mandibular deformity and the presence of aluminum in all samples. The VP SEM-EDX Technique allows direct evaluation, without destruction of the sample and with minimal sample preparation. In bone, the most frequent application of SEM-EDX is the measurement of the content of calcium (Ca) and phosphorus (P) and the relationship that exists between these elements, calcium/phosphorus (Ca/P), in the sample. At the same time, the technique allows us to detect other microelements from water or food that can eventually cause alterations in fish, confirming the hypothesis that elemental microanalysis is useful for salmon farming.

Effects of elderflower extract enriched with polyphenols on antioxidant defense of salmon leukocytes

BACKGROUND: In fish farming, the plant extracts containing antioxidant compounds have been added to the diet for enhancing pathogen resistance. In vitro studies evaluating the antioxidant effect of herbal extracts on fish cell models have focused on ROS production and the respiratory burst mechanism. However, the effects on enzymatic antioxidant defense on salmon leukocytes have not been evaluated. This study aims to evaluate the enzymatic antioxidant defense and ROS-induced cell damage in Salmon Head Kidney-1 (SHK-1) cell line exposed to polyphenol-enriched extract from Sambucus nigra flowers. RESULTS: Firstly, the Total Reactive Antioxidant Power (TRAP) assay of elderflower polyphenol (EP) was evaluated, showing 459 and 489 times more active than gallic acid and butyl hydroxy toluene (BHT), respectively. The toxic effect of EP on salmon cells was not significant at concentrations below 120 mg/ mL and no hemolysis activity was observed between 20 and 400 mg/mL. The treatment of SHK-1 cell line with EP decreased both the lipid peroxidation and protein oxidation induced by H2O2, which could be associated with decreasing oxidative stress in the SHK-1 cells since the GSH/GSSG ratio increased when only EP was added. CONCLUSIONS: These results suggest that plant extracts enriched with polyphenols could improve the enzymatic antioxidant defense of salmon leukocytes and protect the cells against ROS-induced cell damage

Avaliação da viabilidade de microrganismos probióticos incorporados em ração de truta-arco-íris (Oncorhynchus mykiss) / [Evaluation of the viability of probiotic microorganisms impregnated into rainbow trout (Oncorhynchus mykiss) ration]

A produção aquícola mundial tem demandado estudos que buscam soluções para os problemas produtivos e sanitários apresentados na piscicultura. Nesse contexto, o uso de produtos probióticos demonstra ser uma abordagem promissora. Dessa forma, o presente trabalho teve como objetivo avaliar a viabilidade de um pool de cepas de Lactobacillus incorporados à ração de truta-arco-íris. Para tanto, as respectivas cepas foram previamente cultivadas em caldo MRS e submetidas à criocentrifugação. As células obtidas foram ressuspensas em soro fisiológico acrescentado de 2% de óleo de soja, sendo posteriormente incorporadas à ração por meio de misturador circular. Essa ração foi devidamente armazenada sob refrigeração a 4°C, por 120 dias, sendo retiradas aleatoriamente três amostras, a cada 15 dias, para avaliação da viabilidade das bactérias, por meio da contagem em placas. Os resultados demonstraram que houve uma redução não significativa (P>0,05) na população de células viáveis, correspondente a 2%, após 30 dias de armazenamento. Verificou-se também, após esse período, uma população aproximada equivalente a 3,00 x 108 UFC/g. Dessa forma, demonstrou-se a possibilidade de desenvolvimento de formulações probióticas por meio da incorporação de cepas específicas em rações de trutas, o que poderá contribuir para o desenvolvimento de tecnologias alternativas para produção de peixes.(AU)

Among the exotic species of fish produced in Brazil, the rainbow trout (Oncorhynchus mykiss) has been highlighted during the past few years. The expansion of this sector has been intensifying the need for further studies in order to decrease production and health problems presented in aquaculture, as well as to develop alternative technics to controversial antimicrobial treatments. In this context, the use of probiotics has been shown to be a promising approach to the improvement in trout husbandry. Thus, the present study aimed to contribute to the development of an alternative technology in the rainbow trout husbandry by evaluating the effect of probiotic preparations on the performance of the animals subjected to conventional husbandry conditions or chronic stress. Therefore, in this paper we report the results regarding the evaluation of the viability of a pool of 4 Lactobacillus probiotic strains incorporated into rainbow trout ration using soybean oil as vehicle. The results have demonstrated a high efficacy of cell incorporation into the ration with high viability rate after storage for 120 days at 4°C.(AU)

Assessment and incidence of fish associated bacterial pathogens at hatcheries of Azad Kashmir, Pakistan / Avaliação e incidência de patógenos bacterianos associados a peixes em incubadoras da Caxemira de Azad, Paquistão

Abstract Fish is the most indispensable source of proteins for individuals and have high nutritional value. On the other hand, the fish culturing raised issues of fish health due to close contact between the aquatic environment and the fish pathogens. So, the aim of the current study was to identify the bacterial pathogens and screen the injured Rainbow trout rearing in different trout hatcheries run under fisheries department of the government of Azad Jammu and Kashmir, Pakistan. Seven bacterial pathogens such as Shigella flexneri, Enterobacter amnigenus, Salmonella Typhimurium, Serratia odorifera, Pseudomonas aeruginosa, Streptococcus pyogenes, and Bacillus cereus were isolated and identified. Results revealed that the injury of fish specimens was due to overcrowding. Instead of rainbow coloration, specimens have darker black in color. The water of ponds was not clean and clear and such conditions was because of the greater quantity of feed thrown in the water. It was concluded that poor hygienic water condition and overloading allowed the opportunistic bacterial contaminations to succeed which cause a serious threat to hatcheries.

Resumo O peixe é a fonte mais indispensável de proteínas para os indivíduos e tem alto valor nutricional. Por outro lado, a cultura dos peixes levantou questões sobre a saúde dos peixes devido ao próximo contato entre o ambiente aquático e os agentes patogênicos desses peixes. Assim, o objetivo do presente estudo foi identificar os patógenos bacterianos e rastrear a criação da truta arco-íris que apresentou lesões em diferentes incubadoras de trutas, com supervisão do departamento de pesca do governo de Azad Jammu e Caxemira, Paquistão. Sete patógenos bacterianos foram isolados e identificados, tais como: Shigella flexneri, Enterobacter amnigenus, Salmonella typhimurium, Serratia odorifera, Pseudomonas aeruginosa, Streptococcus pyogenes e Bacillus cereus. Os resultados revelaram que a lesão de espécimes de peixes foi devido à superlotação. Em vez da coloração do arco-íris, os espécimes tiveram uma coloração preta mais escura. A água das lagoas não era limpa e nem clara, e tais condições ocorreram devido a maior quantidade de alimento lançada na água. Concluiu-se que a precária condição higiênica da água e também a sobrecarga permitiram que as contaminações bacterianas oportunistas fossem bem-sucedidas, causando séria ameaça às incubadoras.

Toxicity of Zinc at sublethal exposure to rainbow trout (Oncorhynchus mykiss) / Toxicidad de zinc por exposición subletal en trucha arco iris (Oncorhynchus mykiss) / Toxicidade por exposição ao zinco em trutas arco-íris (Oncorhynchus mykiss)

Abstract Zinc (Zn), can alter different physiological processes in fish such as growth and serum parameters. The objective of this study was to investigate the toxicity of Zn on sublethal exposure to rainbow trout (Oncorhnychus mykiss). Fish exposed to higher levels of Zn grew slower than fish exposed to lower levels of Zn. Weight gain and SGR (Specific Growth Rate) decreased linearly as the Zn level in the water increased. The CF (Condition factor) of fish grown in water with a high concentration of Zn also decreased significantly compared to the low concentration of Zn, whereas the Food Conversion Ratio (FCR) increased with the concentration of Zn in water. Both AST (Aspartate Transaminase) and ALT (Alanine Transaminase) activities were significantly altered by Zn and showed a linear pattern and decreased after 30 days, while ALP (Alkaline Phosphatase) levels showed a remarkable increase from a mean level of control. This study suggests that biochemical parameters of growth and serum could be used as important and sensitive biomarkers to monitor heavy metal toxicity on fish health status.

Resumen TZinc (Zn), puede alterar diferentes procesos fisiológicos en peces, tales como, el crecimiento y los parámetros séricos. El objetivo de este estudio fue investigar la toxicidad de Zn en la exposición subletal de la trucha arco iris (Oncorhnychus mykiss). Los peces expuestos a niveles más altos de Zn crecieron más lentamente que los peces expuestos a niveles más bajos de Zn. El aumento de peso y la SGR (tasa de crecimiento específica) disminuyeron linealmente a medida que aumentó el nivel de Zn en el agua. El CF (factor de condición) de los peces cultivados en agua con una alta concentración de Zn, también disminuyó significativamente en comparación con la baja concentración de Zn, mientras que el índice de conversión de alimentos (FCR) aumentó con la concentración de Zn en el agua. Las actividades AST (Aspartate Transaminase) y ALT (Alanine Transaminase) fueron significativamente alteradas por el Zn y mostraron un patrón lineal y disminuyeron después de 30 días, mientras que los niveles de ALP (Fosfatasa Alcalina) mostraron un aumento notable desde un nivel medio de control. Este estudio sugiere que los parámetros bioquímicos de crecimiento y suero podrían usarse como biomarcadores importantes y sensibles para controlar la toxicidad de metales pesados en el estado de salud de los peces.

Resumo O TZinc (Zn) pode alterar diferentes processos fisiológicos em peixes, como parámetros de crescimento e soro. O objetivo deste estudo foi investigar a toxicidade do Zn na exposição subletal à truta arco-íris (Oncorhnychus mykiss). Os peixes expostos a níveis mais altos de Zn cresceram mais lentamente do que os peixes expostos a níveis mais baixos de Zn. O ganho de peso e a SGR (Taxa de Crescimento Específico) diminuíram linearmente à medida que o nível de Zn na água aumentou. O CF (fator de condição) de peixes cultivados em água com alta concentração de Zn também diminuiu significativamente em comparação com a baixa concentração de Zn, enquanto a tax on de conversão alimentar (FCR) aumentou com a concentração de Zn na água. As atividades de AST (Aspartate Transaminase) e ALT (Alanine Transaminase) foram significativamente alteradas pelo Zn e mostraram um padrão linear e diminuíram após 30 dias, enquanto os níveis de ALP (Fosfatase Alcalina) mostraram um aumento notável em relação ao nível médio de controle. Este estudo sugere que parâmetros bioquímicos de crescimento e soro podem ser usados como biomarcadores importantes e sensíveis para monitorar a toxicidade de metais pesados no estado de saúde dos peixes.

Proximate and Nutritional Content of Rainbow Trout (Oncorhynchus mykiss) Flesh Cultured in a Tropical Highland Area

Abstract The present study was performed to assess the proximate and nutrient content of rainbow trout flesh, cultured in the Doi Inthanon Fisheries Research Unit, Chiang Mai Inland Fisheries Research and Development Center, Thailand. 240 fish were randomly distributed across 12 cages with 20 fish cage-1. Sixteen individual fish from each cage were randomly collected at different ages of 10, 12 and 24 months. Body composition, pH, water-holding capacity, shear force, collagen content analysis, sensory, lipid oxidation, and fatty acids profile were evaluated. The results indicated that body composition and carcass length were significantly higher in fish aged 24 months, except for carcass and viscero-somatic index percentages (P ≤ 0.05). Fish at 24 months showed significantly higher pH, moisture, fat, shear force, thiobarbituric acid reactive substances, and total collagen content values than fish at 10 and 12 months (P ≤ 0.05). However, protein percentage, sensory measurement and water-holding capacity were significantly higher in younger fish at 10 and 12 months. The average lipid content and n-6/n-3 ratios were significantly greater in fish at 12 months and in ventral fillets (P ≤ 0.05). However, polyunsaturated fatty acids: saturated fatty acid ratio was higher in fish at 24 months and in dorsal fillets. In conclusion, rainbow trout cultured in sub-tropical, montane conditions can be valuable sources of protein, unsaturated fatty acids, eicosapentaenoic acids, and docosahexaenoic acids.

Criteria for Safe Use of Plant Ingredients in Diets for Aquacultured Fish

The Norwegian Food Safety Authority (Mattilsynet) asked the Norwegian Scientific Committee for Food Safety (Vitenskapskomiteen for mattrygghet) to assess if the criteria for safe use of plant ingredients in diets for aquacultured fish fulfil the Feed regulative §7 to “not induce negative health effects in the animal”, and in this context aquacultured fish. The use of feed ingredients of both plant and animal origin is set by the regulation “Forskrift 7. November 2002 nr 1290”, and amendments. The objective of the regulation is to protect animals, consumers and the environment. For animals, the feed shall not pose a risk, or danger, to their health. Aspects to be assessed were whether the changes in fish diet ingredient composition seen in recent years with high levels of plant ingredients, plus additions of immunostimulants, would in any manner challenge fish health and if any ingredient should be limited due to its negative effect, or induce any long-term negative effect. “Long-term” here extends beyond normal production time for consumption, e.g. when substances that might affect fish health are included in broodstock diets. Atlantic salmon (Salmo salar), rainbow trout (Onchorhyncus mykiss), Atlantic halibut (Hippoglossus hippoglossus) and Atlantic cod (Gadus morhua) should especially be addressed. However, since all life stages should be included, especially broodstock, and also possible long-term effects, and literature on these for the requested species is scarce, the assessment mentions studies on other species when relevant. With the exception of full-fat and extracted soybean meal for salmonids, substituting at least part of the fishmeal fraction of aquafeeds with individual plant ingredients is promising, at least in the short to medium term. Indeed in some cases, diets containing up to 20% inclusion level of high-quality plant protein sources have resulted in better nutrient digestibility and growth parameters than the fishmeal-based control diets. When substituting fishmeal with plant ingredients, however, it is necessary to balance the diets regarding limiting amino acids and minerals. Adding plant proteins to fish diets result in the introduction of anti-nutritional factors. There is an urgent need to investigate consequences of various anti-nutritional factors, individually and in combinations, to nutrient digestibility, utilization and metabolism as well as to intestinal function, structure, defence mechanisms and microbiota. Long-term effects also merit investigation. This will aid in the ability to predict how a newly introduced plant ingredient as well as combinations of plant ingredients may affect the fish and identify steps needed to avoid adverse health effects. As many of the potential disadvantages of using plant oils in salmonid diets are related to either very high levels of n-6 PUFA (most available oils) or very high levels of linseed oil, it would be recommended that mixtures of plant oils should be used as feed inclusions. By adjusting the ratio of n-6 and n-3 the level of eicosanoids can be controlled. By including palm oil, potential problems in lipid digestibility and transport can be controlled. A standard inclusion of soybean lecithin may also be advisory. These and other variants of mixtures of oil sources have been explored in recent years with some success in salmonid fish. Such mixtures do not seem to be necessary for marine fish. Modern finfish aquaculture faces problems such as bone and skeletal deformities, cataracts, heart disorders, unspecific ulceration and various digestive disorders including intestinal colic in Atlantic cod, gastric dilatation (bloat) in rainbow trout, and intestinal tumours, at low incidence, in Atlantic salmon broodstock. Most of the mentioned problems have been related to malnutrition, feed, intensive growth and/or unfavourable environmental conditions. The disorders are often not lethal, but may imply a fish welfare problem and increase the susceptibility to secondary disorders and infectious diseases. Major changes in feed composition and feed ingredients may increase the risk for such production-related disorders in intensive fish farming. Care should be taken when choosing plant alternatives, both types and qualities, to prevent nutrition-related diseases such as skeletal deformities, cataracts, heart conditions, and other, unspecific symptoms. The change from marine- to plant-based diet ingredients, results in changed profile and content of undesirable substances. The list of undesirable substances included in the feed legislation is, in general, sufficient, but it should be considered to include pesticides in use today and more of the mycotoxins. Currently only aflatoxin B1 is included, while only recommendations exist for other mycotoxins. Studies of dietary exposure to undesirable substances, e.g. pesticides and mycotoxins, and their toxic effects and toxicokinetics in fish are scarce. To date, the application of pre- and probiotics for the improvement of aquatic environmental quality and for disease control in aquaculture seems promising; however, the information is limited and sometimes contradictory. Currently there are numerous gaps in existing knowledge about exogenous nucleotide application to fish including various aspects of digestion, absorption, metabolism, and influences on various physiological responses, especially expression of immunogenes and modulation of immunoglobulin production. As limited information is available about the effect of immunostimulants, prebiotics and nucleotides on gut morphology, this topic should be given high priority in future studies. Heat processing of raw materials and of the complete fish diets may potentially alter nutritional properties of plant materials. However, the negative effects appear to be modest under practical conditions.

Assessment and incidence of fish associated bacterial pathogens at hatcheries of Azad Kashmir, Pakistan

Abstract Fish is the most indispensable source of proteins for individuals and have high nutritional value. On the other hand, the fish culturing raised issues of fish health due to close contact between the aquatic environment and the fish pathogens. So, the aim of the current study was to identify the bacterial pathogens and screen the injured Rainbow trout rearing in different trout hatcheries run under fisheries department of the government of Azad Jammu and Kashmir, Pakistan. Seven bacterial pathogens such as Shigella flexneri, Enterobacter amnigenus, Salmonella Typhimurium, Serratia odorifera, Pseudomonas aeruginosa, Streptococcus pyogenes, and Bacillus cereus were isolated and identified. Results revealed that the injury of fish specimens was due to overcrowding. Instead of rainbow coloration, specimens have darker black in color. The water of ponds was not clean and clear and such conditions was because of the greater quantity of feed thrown in the water. It was concluded that poor hygienic water condition and overloading allowed the opportunistic bacterial contaminations to succeed which cause a serious threat to hatcheries.

Resumo O peixe é a fonte mais indispensável de proteínas para os indivíduos e tem alto valor nutricional. Por outro lado, a cultura dos peixes levantou questões sobre a saúde dos peixes devido ao próximo contato entre o ambiente aquático e os agentes patogênicos desses peixes. Assim, o objetivo do presente estudo foi identificar os patógenos bacterianos e rastrear a criação da truta arco-íris que apresentou lesões em diferentes incubadoras de trutas, com supervisão do departamento de pesca do governo de Azad Jammu e Caxemira, Paquistão. Sete patógenos bacterianos foram isolados e identificados, tais como: Shigella flexneri, Enterobacter amnigenus, Salmonella typhimurium, Serratia odorifera, Pseudomonas aeruginosa, Streptococcus pyogenes e Bacillus cereus. Os resultados revelaram que a lesão de espécimes de peixes foi devido à superlotação. Em vez da coloração do arco-íris, os espécimes tiveram uma coloração preta mais escura. A água das lagoas não era limpa e nem clara, e tais condições ocorreram devido a maior quantidade de alimento lançada na água. Concluiu-se que a precária condição higiênica da água e também a sobrecarga permitiram que as contaminações bacterianas oportunistas fossem bem-sucedidas, causando séria ameaça às incubadoras.

Expressão da enzima indoleamina-2, 3-dioxigenase em truta arco-íris (Oncorhynchus mykiss) / Expression of the enzyme indoleamine-2, 3-dioxygenase in Rainbow Trout (Oncorhynchus mykiss

A indoleamina 2,3-dioxigenase (IDO) é uma enzima que cataboliza o aminoácido triptofano, levando à inibição da proliferação de linfócitos T, seja pela exaustão desse aminoácido no ambiente, ou pela indução via catabólitos induzindo-os a apoptose. Em mamíferos, esta enzima atua em diversas condições do organismo como a gestação, infecções, inflamações crônicas, transplantes e tumores, atuando na regulação imunológica. Estudos recentes identificaram a presença de moléculas homólogas a IDO em espécies filogeneticamente inferiores, cuja função parece estar restrita ao metabolismo do triptofano como fonte de energia. Este estudo teve por objetivo averiguar a expressão da IDO em células sanguíneas e órgãos hematopoiéticos de truta arco-íris pela imuno-histoquímica, buscando evidências de que a mesma poderia, nesta espécie, estar relacionada ao sistema imune. A expressão de IDO foi observada nos órgãos hematopoiéticos estudados incluindo o rim cefálico que apresentou marcação em células interrenais e leucócitos; baço, na qual a marcação restringiu à alguns leucócitos; no fígado a marcação ficou limitada à apenas algumas células dentro dos vasos sanguíneos e nas extensões sanguíneas pode-se visualizar a marcação de alguns leucócitos como os monócitos, linfócitos e neutrófilos. A predominância da marcação da IDO nesses tecidos pode constituir uma evidência de que a IDO identificada na O. mykiss esteja relacionada ao sistema imunológico nessa espécie...

Indoleamine 2,3-dioxygenase (IDO) is an enzyme that catabolizes the amino acid tryptophan, leading to inhibition of T lymphocyte proliferation, whether by depletion of this amino acid in the environment, or by induction via the catabolites inducing apoptosis. In mammals, this enzyme acts on various conditions of the body such as pregnancy, infections, chronic inflammation, transplantation and tumors, acting in immune regulation. Recent studies have identified the presence of homologous molecules IDO lower phylogenetically related species, whose function appears to be confined to the tryptophan metabolism as an energy source. This study aimed to investigate the expression of IDO in blood cells and hematopoietic organs of rainbow trout by immunohistochemistry, seeking evidence that it could, this species is related to the immune system. The expression of IDO was observed in hematopoietic organs studied including head kidney that show labeling in interrenal cells and leukocytes; spleen, in which the marking restricted to a few leukocytes in the liver;, labeling was restricted to only certain cells within the blood vessels and the blood extensions can view the marking of some leukocytes including monocytes, lymphocytes and neutrophils. The predominance of IDO marking these tissues may constitute evidence that IDO identified in O. mykiss is related to the immune system in this species...

Effect of temperature on the expression of IFN-1 (α), STAT-1 and Mx-1 genes in Oncorhynchus mykiss (Salmoniformes: Salmonidae) exposed with the virus of the infectious pancreatic necrosis (IPNV) / Efecto de la temperatura sobre la expresión de genes IFN-1(α), STAT-1 y Mx-1 en alevines de trucha arcoíris Oncorhynchus mykiss (Salmoniformes: Salmonidae) expuestos al el virus de la necrosis pancreática infecciosa (IPNV)

The infectious pancreatic necrosis (IPNV) is the causative agent of an acute illness well characterized in salmonids worldwide. Clinical signs and mortality rates are dependent on several factors such as the viral dose, the age of the fish, the water temperature, among others. An experimental study was conducted to measure the effect of temperature on the gene expression profile of IFN-1(α), STAT-1 and Mx-1 in rainbow trout fry, exposed to IPNV. Fry (n=198) were exposed at 8, 12 and 16°C, and samples were taken for 21 days to determine the virus titer and gene expression. In the first 11 days the greatest viral titer was recorded at 8°C compared with the values obtained at 12 and 16°C. At 8°C, there was a significant increase on day 4 of mRNA Mx-1 (t-test, p<0.05), time in which the viral titer began to decrease. Furthermore, as the viral titer increased, STAT-1 and Mx-1 (r=0.91) and (r=0.96) increased, respectively. The animals were able to recover from day 4 from some of the symptoms of IPN. Clinical disease was developed only in fish exposed to 12°C and all died between days 6 and 14, despite the highly significant increase shown in the average expression level of Mx-1, compared with the values recorded at 8°C and 16°C (Tukey, p<0.0001). Additionally, the expression profiles of IFN-1(α) and STAT-1 decreased completely (~0.016) and (~0.020 times) on day 7. The highest expression level of IFN-1(α), occurred at 16°C (Tukey, p<0.0005). Fry exposed at 16°C were normal during the experiment. IFN-1(α) possibly generated a protector effect from day 2 when they showed a significant expression increase compared with the results at 8°C and 12°C (t-student, p<0.0001); however, STAT-1 was not significantly affected by temperature, although the highest average expression value was recorded at 16°C. Our research supports the expression of relevant anti-viral response genes as IFN-1(α), STAT-1 and Mx-1 are physiologically modulated by the water temperature, directly influencing the development of the IPN disease in rainbow trout.

El virus de la necrosis pancreática infecciosa (IPNV) es el agente etiológico de una enfermedad aguda bien caracterizada en salmónidos alrededor del mundo. Los signos clínicos y la tasa de mortalidad dependen de varios factores tales como la dosis viral, la edad del pez y la temperatura del agua, entre otros. Un estudio experimental se llevó a cabo para medir el efecto de la temperatura sobre el perfil de expresión génica de IFN-1(α), STAT-1 y Mx-1 en alevines de trucha arcoíris expuestos con IPNV. Los alevines (n=198) fueron expuestos a 8, 12 y 16°C, y se tomaron muestras durante 21 días para determinar el título viral y la expresión génica. En los primeros 11 días el mayor titulo viral se registró a 8ºC en comparación con 12 y 16. A 8°C, existió un incremento significativo en el día 4 del ARNm de Mx-1 (t-test, p<0.05), momento en que el título viral empezó a disminuir. Además conforme el título viral aumentaba, también STAT-1 y Mx-1 aumentaron (r=0.91) y (r=0.96) respectivamente. Los animales fueron capaces de recuperarse desde el día 4 de algunos de los síntomas de IPN. La enfermedad clínica se desarrolló únicamente en peces expuestos a 12°C y todos murieron entre el día 6 y 14, a pesar del incremento altamente significativo mostrado en el nivel promedio de expresión de Mx-1 a 12°C, comparados con los valores registrados a 8 y 16°C (Tukey, p<0.0001). Además los perfiles de expresión de IFN-1(α) y STAT-1 decrecieron el día 7 completamente (~0.016) y (~0.020) veces, respectivamente. El nivel de expresión promedio más alto de IFN-1(α) se registró a 16°C (Tukey, p<0.0005). Los alevines expuestos a 16°C se mostraron normales durante el experimento. IFN-1(α) posiblemente generó un efecto protector desde el día 2 cuando mostró un aumento significativo en comparación con los resultados a 8 y 12°C (t-student, p<0.0001); sin embargo, STAT-1 no fue afectado de manera significativa por la temperatura, aunque el más alto valor de expresión promedio se registró a 16°C. Nuestra investigación confirma que la expresión de genes relevantes de respuesta antiviral como IFN-1(α), STAT-1 y Mx-1 son fisiológicamente modulados por la temperatura del agua, influyendo directamente en el desarrollo de la enfermedad de IPN en trucha arcoíris.

Comparison of fatty acid content of fresh and frozen fillets of rainbow trout (Oncorhynchus mykiss) Walbaum

The aim of this study was to evaluate and compare the percentage content of fatty acids (FA) of 54 rainbow trout fresh and frozen fillets. Frozen fillets were stored at -15ºC for 45 and 90 days. Saturated (SFA), monounsaturated (MUFA), polyunsaturated (PUFA), omega-6 (n-6) and omega-3 (n-3) fatty acids were determined by gas chromatograph coupled to mass spectrometry. The results indicated that during frozen storage, SFA and MUFA content increased by 32.63 and 9.25%, respectively, while PUFA content decreased by 25.3%, n-6 by 12.4% and n-3 by 32.55%. These changes were more significant (P ≤ 0.05) during the first 45 d of storage. It was concluded that the frozen storage had a negative impact on meat quality of rainbow trout due to the reduction of PUFA, n-3/n-6, polyene index and PUFA/ SFA and the increase of the SFA, and atherogenic and thrombogenic indexes, which meant a substantial loss of nutritional value in the fillets of rainbow trout.

Anti-peptide antibodies: a tool for detecting IL-8 in salmonids

Background: Interleukin 8 is a chemokine that is produced by several types of cells, like macrophages and has chemotactic activity in particular on neutrophils, playing a key role during the inflammatory process. It has been demonstrated at the molecular level that this molecule is present and conserved in several vertebrate groups, pointing its importance. Analysis of the amino acid sequence of IL-8, projected from cDNA of Salmo salar, presents homology with the sequences of mammals, poultry and lamprey, indicating the presence of a homologous molecule in higher fish. However, there is no information at protein level, which allows characterizing the regulatory role of this molecule during the immune response in fish. Results: In this work, we designed and synthesized an epitope peptide of 10 residues with a purity of 95 percent and mass of 1158.7 kDa, which showed a random coil structure. From this peptide it was able to generate a polyclonal mono-specific antibody which was capable of detecting the whole molecule of IL-8 in tissue and cellular model of salmonids. Conclusions: The resulting antibody is a versatile tool for detecting IL-8 by different immune techniques such as ELISA, dot blot, western blotting and immunocytofluorescence. Analysis of IL-8 at proteomic level is a useful method for characterizing immune properties of this molecule in fish.

Desarrollo de una formulación de paté a base de descartes de pulpa de trucha arcoiris (Oncorhynchus mykiss) / Development of a paté formulation basis on rainbow trout discards

Se desarrolló una formulación optimizada de paté utilizando descartes de pulpa de trucha arcoiris utilizando la metodología Taguchi teniendo en cuenta que sus beneficios sobre la salud lo convierte en una alternativa viable interesante como un producto con características funcionales para ayudar a los consumidores a tener un estilo de vida saludable. El proceso de optimización se llevó a cabo en dos etapas. Primero se ocupó el arreglo ortogonal L(8)2(7) con 7 variables independientes para seleccionar los factores de control que tuvieran un efecto significativo sobre la calidad sensorial (C.S.) quedando finalmente seleccionadas: ají merken, cloruro de sodio, manteca vegetal junto con el arreglo ortogonal L(9)3(4) .Las respuestas de C.S. fueron sometidas a un análisis estadístico de diferencias de magnitud de promedios y análisis de varianza (ANOVA) determinándose que los factores que influyeron significativamente (p2/Kg. grasa respectivamente, ambos inferiores a los máximos permitidos por la reglamentación chilena. Con respecto a la aceptabilidad del producto fue de 91% y el 87% de los encuestados estaría dispuesto a comprarlo.

An optimized formulation of pate was developed using rainbow trout discard with the employment of Taguchi methodology, taking into account that the health-promoting benefits of this resource makes it a viable alternative as a functional foods to help consumers to get a healthy lifestyle. The optimization process utilizing Taguchi methodology was carried out in two phases. First, an orthogonal array L(8)2(7) with seven independent variables was chosen to select the control factors with a significant effect on the sensory quality (SQ). As a result, the following independent variables were selected: merkén pepper, sodium chloride, vegetal lard and margarine. In the second stage the L(9)3(4) orthogonal array was used. Data were analysed using differences between average values of factors according working levels, and also ANOVA, summing up that merken pepper, sodium chloride and margarine showed a significant effect (p>0.05) on the SQ. Best combination turned to be: merkén 0.7%, sodium chloride 1.3%, vegetal lard 5.2% and margarine 5.2%. Among the chemical characteristics highlighted protein 13.8%, lipid 10.21%, caloric density 175 Kcal/100g and colesterol 46mg/100g. Shelf life study during a period of time of 6 weeks at 5ºC expressed as mesophyl aerobic count (MAC) and peroxide index were 1.6E+0.4 ufc/g and 8.44 meq O2/Kg respectively, both characteristics lower than the maximum limits allowed for chilean regulations. Concerning to the acceptability of the optimized product the hedonic test showed 91% approval and also 87% of consumers would be well disposed to buy this product.

Analysis of the association between spawning time QTL markers and the biannual spawning behavior in rainbow trout (Oncorhynchus mykiss)

The rainbow trout is a salmonid fish that occasionally exhibits broodstocks with biannual spawning behavior, a phenomenon known as a double annual reproductive cycle (DARC). Spawning time quantitative trait loci (SPT-QTLs) affect the time of the year that female rainbow trout spawn and may influence expression of the DARC trait. In this study, microsatellite markers linked and unlinked to SPT-QTLs were genotyped to investigate the underlying genetics of this trait. SPT-QTLs influenced the DARC trait since in two case-control comparisons three linked markers (OmyFGT12TUF, One3ASC and One19ASC) had significant levels of allelic frequency differentiation and marker-character association. Furthermore, alleles of One3ASC and One19ASC had significantly higher frequencies in populations that carried the DARC trait.

Prophylactic effect of levamisole on rainbow trout (Oncorhynchus mykiss) against Yersinia ruckeri

Alteration in the relative percentage of survival (RPS) rate of rainbow trout (Oncorhynchus mykiss) exposed to 5, 10 and 25�g ml-1 levamisole for 2 h against Yersinia ruckeri was investigated. The average weight of the 120 fish used in this study was 6.3g. Upon challenge with a virulent strain, the relative survival percentage of respectively 83.3 percent, 86.7 percent and 76.6 percent was recorded. The results suggest that the application of levamisole in fish farms could increase resistance to infection of fish and offer economic benefits.

Estandarización del manejo y la criopreservación de semen de hembras masculinizadas de trucha arco iris (Oncorhynchus mykiss) / Padronizar a gestão ea criopreservação de sêmen de fêmeas de truta arco-íris (Oncorhynchus mykiss) sob masculinização / Standardization of handling and freezing sperm from masculinized females of rainbow trout (Oncorhynchus mykiss)

La demanda de líneas monosexo hembras en la producción de trucha ha incrementado significativamente en los últimos años, por lo que se han desarrollado tecnologías para estandarizar este proceso como el uso de semen de hembras genéticas sometidas a reversión sexual. El objetivo de la presente investigación fue estandarizar la maduración in vitro y la criopreservación de semen de hembras masculinizadas (neomachos XX) de trucha arco iris (Oncorhynchus mykiss) como estrategia para producir descendencias 100% hembras de reproductores colombianos. Para la obtención del semen los neomachos fueron sacrificados y el semen recuperado fue sometido a proceso de maduración con plasma seminal de machos normales o plasma seminal artificial. Para la criopreservación del semen se probaron los crioprotectores dimetilsulfóxido 10% y metanol 10%. En el experimento se evaluron la movilidad post maduración y post descongelación y la fertilidad del semen. El proceso de maduración tuvo un efecto significativo sobre el porcentaje de movilidad (p<0.05), al obtener los mejores valores con el plasma seminal artificial (55 ± 10.4% espermatozoides móviles). Los mayores valores de movilidad postdescongelación (29.9 ± 13.3%) al igual que la mejor fertilidad (26.33 ± 7.53%), se registraron con el protocolo de dimetilsulfóxido al 10%.

The demand of monosex female stocks in production of trout has significantly increased during the past years, which has led to develop new technologies to standardize this process. The usage of semen of genetic females submitted to sexual reversion is a good choice. The objective of this research was to develop a methodology to mature in vitro and cryopreserved semen of sex-reversed rainbow trout (Oncorhynchus mykiss) females as strategy to produce lineage 100% Colombian trout female. The semen was directly obtained from the gonads after its surgical extraction of the slaughtered individuals, later it was submitted to maturation process implementing seminal plasma of normal males and artificial plasma. The semen was cryopreserved in two extender dimetyhyl sulfoxide 10% and methanol 10%. Postmaturation, postcriopreservation movility and sperm fertility were evaluated. �Maturation process had a significative effect on movility, the highest movility was obtained with artificial seminal plasma (55 ± 10.4 %). Highest post criopreservation movility (29.9 ± 13.3%) and highest fertility rates (26.33 ± 7.53 %) were obtained with dimetyhyl sulfoxide 10%.

A procura de linhas monosexo fêmeas na produção de trutas tem aumentado significativamente nos últimos anos, de modo tecnologias foram desenvolvidas com a finalidade de padronizar este processo como o uso do esperma de genética feminina submetido a reversão sexual. O objectivo do presente inquérito foi para uniformizar a maturação in vitro e criopreservação de sêmen masculinização de fêmeas (neomachos XX) trutas arco-íris (Oncorhynchus mykiss) como uma estratégia para produzir descendentes de 100% do sexo feminino dos jogadores colombianos. Para a obtenção do esperma neomachos foram mortas e sêmen foi recuperado submetida a maturação processo normal de plasma seminal plasma seminal masculina ou artificiais. Para a criopreservação de sêmen foi testado crioprotectores dimethylsulphoxide 10% e 10% de metanol. O experimento foi evaluron mobilidade pós maturação e pós descongelamento e fertilidade do sêmen. O processo de maturação teve um efeito significativo sobre a porcentagem de mobilidade (p<0.05), para obter os melhores valores com artificial seminal plasma (55 ± 10.4% esperma). Os maiores valores de mobilidade postdescongelación (29.9 ± 13.3%), bem como a melhor fertilidade (26.33 ± 7.53%), foram registrados com o protocolo de dimethylsulphoxide a 10%.

Non-specific Immune Response of Rainbow Trout (Oncorhynchus Mykiss) by Dietary Heat-inactivated Potential Probiotics

BACKGROUND: The effects of the dietary administration of two heat-inactivated whole bacteria from the Vibrionaceae family, singly or combined, on innate immune response of the rainbow trout were studied. The two bacteria (Pdp11 and 51M6), which were obtained from the skin of rainbow trout, showed in vitro characteristics that suggested they could be considered as potential fish probiotics. METHODS: The fish were fed four different diets: control (non-supplemented), or diets supplemented with heat-inactivated bacteria at 10(8) cfu/g Pdp11, 10(8) cfu/g 51M6 or with 0.5x10(8) cfu/g Pdp11 plus 0.5x10(8) cfu/g 51M6 for 4 weeks. Six fish were sampled at weeks 1, 2, 3 and 4, and then the main humoral (natural haemolytic complement activity and serum peroxidase content) and cellular innate immune responses (leucocyte peroxidase content, phagocytosis, respiratory burst and cytotoxicity) were evaluated. RESULTS: The serum peroxidase content and the natural haemolytic complement activity increased with time, reaching the highest values in the third and fourth weeks of feeding, respectively. The phagocytic ability of specimens fed the mixture of the two inactivated bacteria was significantly higher than in the controls after 2 and 3 weeks of treatment. The same activity increased significantly in rainbow trout fed the Pdp11 diet for 2 weeks or the 51M6 diet for 3 weeks. Respiratory burst activity was unaffected by all the experimental diets at all times assayed. Cytotoxic activity had significantly increased after 3 weeks in fish fed the 51M6 diet. CONCLUSION: Our results demonstrated the usefulness of incorporating inactivated probiotic bacteria into fish diets.