Fish products developed from ground rainbow trout and tilapia fillet mixtures: physico-chemical, microbiological and toxicological analyses / Produtos de pescado elaborados a partir de misturas de carne moída de truta-arco-íris (Oncorhynchus mykiss) e tilápia (Oreochromis sp. ): análises físico-químicas, microbiológicas e toxicológicas

Constant fishing industry innovations in response to consumer demands for novel, balanced healthy products exhibiting high nutritional value, as well as easy to prepare and ready to consume, have led to the emergence of new technologies. Among them, the development of products based on ground beef and fish mixtures are noteworthy. In this context, the purpose of this study was to develop products from different ground rainbow trout and tilapia fillet ratios, in the form of meatballs and nuggets, determining their proximate composition and physico-chemical, microbiological and toxicological characteristics. The average dry matter percentage of the developed products was 29.20±2.30%, while mineral content was 1.45±0.55%, lipid pulp content, 3.70±0.30% and crude pulp protein, 16.60±2.04%. Total psychotropic counts remained below 102 CFU/g, for standard counting on aerobic microorganism plates. Histamine and sulfur-based compounds were not detected in any of the prepared samples. The purpose of the work proposal was to develop foods and technologies that can serve as standards, allowing for simple handling products that apply ground meat mixtures from different fish species to maintain adequate nutritional indices after processing.(AU)

A constante inovação da indústria pesqueira, em resposta às demandas dos consumidores por novos produtos saudáveis, com alto valor nutritivo, balanceados, de fácil preparo e prontos para consumo, levou ao surgimento de novas tecnologias, entre elas o desenvolvimento de produtos à base de mistura de carne moída de pescado. Nesse contexto, a proposta deste trabalho é desenvolver produtos a partir de carne moída de pescado de truta-arco-íris e tilápia, confeccionando almôndegas e nuggets em diferentes proporções, com determinação da composição centesimal e de suas características físico-químicas, microbiológicas e toxicológicas. A matéria seca percentual média foi de 29,20±2,30%. Teores de matéria mineral nos produtos estão em 1,45±0,55%. Os teores de lipídeos aferidos na polpa foram de 3,70±0,30%, e os níveis de proteína bruta apresentados na polpa de 16,60±2,04%. A contagem de psicrotróficos totais manteve-se abaixo de 102 CFU/g, para contagem padrão em placas de micro-organismos aeróbicos. Nas amostras, não foram detectados níveis de histamina e de compostos à base de enxofre. A proposta de trabalho objetivou, por fim, desenvolver alimentos e tecnologias que possam servir como padrões, permitindo que produtos de manipulação simples e que utilizem misturas de carne moída de diferentes espécies de pescado mantenham índices nutricionais adequados após o processamento.(AU)

Viabilidade econômica da produção do sucedâneo de caviar de ovas da truta arco-íris / Economic viability to produce caviar substitute using roes of rainbow trout

Roes of rainbow trout (Oncorhynchus mykiss) can be used to produce caviar substitutes, resulting ina high-value added product. This study evaluated the market potential of rainbow trout roe inCampos do Jordão City (São Paulo State, Brazil) (22°44′20″S, 45°35′27″W) and the economic aspectsof its production, considering two scenarios: production in existing processing plants (scenario A)and building a plant for its production (scenario B). To estimate the net present value rates (NPV),we used different asking prices (US$ 8.50, US$ 10.00 and US$ 12.50) and interest rates (11%). Themarket research indicated a great potential for the caviar substitute, perceived as a differential ingastronomy. The results showed economic viability in scenario A, with satisfactory profitabilityand return on venture capital for the short-term at different prices. In scenario B, economic viabilityis attained at the highest price, US$ 12.50; all other simulations require careful consideration fromthe investor.(AU)

Ovas de truta arco-íris podem ser utilizadas para a confecção de sucedâneo de caviar, resultandoem produto de alto valor agregado. Este estudo objetivou avaliar o potencial de mercado doproduto no município de Campos do Jordão SP (22°44′20″S, 45°35′27″W), e os aspectoseconômicos da sua produção nas seguintes simulações: produção em estrutura de processamentopreviamente existente (simulação A), construção de estrutura específica para sua produção(simulação B), diferentes preços de venda (R$ 19,00; R$ 22,50 e R$ 28,00), e taxa de juros de 11%para estimativa do valor presente líquido (VPL). O estudo de mercado indicou grande potencialpara o sucedâneo, visto como diferencial na gastronomia. Os resultados demonstraram aviabilidade do investimento para todas as simulações da condição A, com alta rentabilidade erecuperação do investimento em curto prazo. A condição B apresentou viabilidade apenas para opreço de US$ 12.50; todas as outras simulações para esta condição requerem análise cuidadosa porparte do investidor.(AU)

Viabilidade econômica da produção do sucedâneo de caviar de ovas da truta arco-íris / Economic viability to produce caviar substitute using roes of rainbow trout

Roes of rainbow trout (Oncorhynchus mykiss) can be used to produce caviar substitutes, resulting ina high-value added product. This study evaluated the market potential of rainbow trout roe inCampos do Jordão City (São Paulo State, Brazil) (22°44′20″S, 45°35′27″W) and the economic aspectsof its production, considering two scenarios: production in existing processing plants (scenario A)and building a plant for its production (scenario B). To estimate the net present value rates (NPV),we used different asking prices (US$ 8.50, US$ 10.00 and US$ 12.50) and interest rates (11%). Themarket research indicated a great potential for the caviar substitute, perceived as a differential ingastronomy. The results showed economic viability in scenario A, with satisfactory profitabilityand return on venture capital for the short-term at different prices. In scenario B, economic viabilityis attained at the highest price, US$ 12.50; all other simulations require careful consideration fromthe investor.

Ovas de truta arco-íris podem ser utilizadas para a confecção de sucedâneo de caviar, resultandoem produto de alto valor agregado. Este estudo objetivou avaliar o potencial de mercado doproduto no município de Campos do Jordão – SP (22°44′20″S, 45°35′27″W), e os aspectoseconômicos da sua produção nas seguintes simulações: produção em estrutura de processamentopreviamente existente (simulação A), construção de estrutura específica para sua produção(simulação B), diferentes preços de venda (R$ 19,00; R$ 22,50 e R$ 28,00), e taxa de juros de 11%para estimativa do valor presente líquido (VPL). O estudo de mercado indicou grande potencialpara o sucedâneo, visto como diferencial na gastronomia. Os resultados demonstraram aviabilidade do investimento para todas as simulações da condição A, com alta rentabilidade erecuperação do investimento em curto prazo. A condição B apresentou viabilidade apenas para opreço de US$ 12.50; todas as outras simulações para esta condição requerem análise cuidadosa porparte do investidor.

Biochemical effects of encapsulated radiovaccine via alginate nanoparticles as useful strategy for booster in immunized rainbow trout against Ichthyophytirius multifiliis

Background: The Ichthyophthirius multifiliis is one of the most important protozoan pathogens of farmed and wild fish populations. The vaccination against I. multifiliis can be considered as an alternative to chemical treatments. There are three routes of vaccination; the injection, immersion and oral administration. Oral immunization would be a good candidate since handling fish is not required, it is a not stressful method and it needs less time and labor. In this study a nanoparticle system for protection of radiovaccine and efficient oral vaccine delivery to the intestinal tract against I. multifiliis was tested in rainbow trout.Materials, Methods & Results: Fifty rainbow trout (40 - 50 g) were infected with I. multifiliis via live trophont, trophont were collected with 200-mesh sieve. After collection, dose of gamma ray (170 Gray) was used for irradiation of parasite. Commercial Ergosan was suspended in sterile 0.15 M (pH 7.2) and sonicated. Remaining powder was irradiated by gamma irradiator. The alginate nanoparticle encapsulated radiovaccine was achieved by the addition of PBS (1 mL) to the alginate nanaoparticle solution (4 mL) plus radiovaccine (37˚C, pH 7.5). Fish were distributed equally into 5 groups (60 fish/aquarium). In this study, elevated levels of WBC was found in fish immunized with non-capsulated and encapsulated radiovaccine at 20 days after challenge (P < 0.05). No significant differences in haematocrit and RBC were found among all of groups (P < 0.05). Also, plasma total protein level was found to decrease in vaccinated fish with non-capsulated radiovaccine at 7 and 20 days after challenge. Significant decrease in glucose level was shown in immunized rainbow trout with encapsulated radiovaccine (P < 0.05). A significant increase in plasma glucose level was shown in rainbow trout challenged with live trophonts at 7 days after challenge (P < 0.05).[...](AU)

Biochemical effects of encapsulated radiovaccine via alginate nanoparticles as useful strategy for booster in immunized rainbow trout against Ichthyophytirius multifiliis

Background: The Ichthyophthirius multifiliis is one of the most important protozoan pathogens of farmed and wild fish populations. The vaccination against I. multifiliis can be considered as an alternative to chemical treatments. There are three routes of vaccination; the injection, immersion and oral administration. Oral immunization would be a good candidate since handling fish is not required, it is a not stressful method and it needs less time and labor. In this study a nanoparticle system for protection of radiovaccine and efficient oral vaccine delivery to the intestinal tract against I. multifiliis was tested in rainbow trout.Materials, Methods & Results: Fifty rainbow trout (40 - 50 g) were infected with I. multifiliis via live trophont, trophont were collected with 200-mesh sieve. After collection, dose of gamma ray (170 Gray) was used for irradiation of parasite. Commercial Ergosan was suspended in sterile 0.15 M (pH 7.2) and sonicated. Remaining powder was irradiated by gamma irradiator. The alginate nanoparticle encapsulated radiovaccine was achieved by the addition of PBS (1 mL) to the alginate nanaoparticle solution (4 mL) plus radiovaccine (37˚C, pH 7.5). Fish were distributed equally into 5 groups (60 fish/aquarium). In this study, elevated levels of WBC was found in fish immunized with non-capsulated and encapsulated radiovaccine at 20 days after challenge (P < 0.05). No significant differences in haematocrit and RBC were found among all of groups (P < 0.05). Also, plasma total protein level was found to decrease in vaccinated fish with non-capsulated radiovaccine at 7 and 20 days after challenge. Significant decrease in glucose level was shown in immunized rainbow trout with encapsulated radiovaccine (P < 0.05). A significant increase in plasma glucose level was shown in rainbow trout challenged with live trophonts at 7 days after challenge (P < 0.05).[...]

HIDROLISADOS PROTEICOS DE VÍSCERA RESIDUAL DE SARDINHA NA ALIMENTAÇÃO DE TRUTAS ARCO-ÍRIS

Gonçalves, André Fernando Nascimento. Hidrolisados proteicos de víscera residual de sardinha na alimentação de trutas arco-íris 2019. 111 f. Tese (Doutorado em Ciência Animal) Universidade do Estado de Santa Catarina. Centro de Ciências Agroveterinárias (CAV/UDESC), Lages, SC, 2019. Os hidrolisados proteicos são produtos obtidos da ação de enzimas proteolíticas através da quebra das proteínas. Pode ocorrer um aumento da digestibilidade, resultando em peptídeos de diferentes tamanhos e aminoácidos livres. A escolha da protease hidrolítica depende do custo da enzima e do resultado final desejado após a hidrólise. As enzimas têm diferentes especificidades para clivar as ligações peptídicas, alterando o perfil de peptídeos e aminoácidos, interferindo na qualidade e eficiência do hidrolisado obtido. O presente trabalho é composto por três estudos, com o propósito de desenvolver e caracterizar hidrolisados proteicos de vísceras de sardinha (Sardinella sp.) e sua influência na alimentação de trutas arco-íris (Oncorhynchus mykiss). O primeiro estudo consistiu em desenvolver e caracterizar um hidrolisado proteico de vísceras de sardinha produzido com as enzimas do próprio trato digestivo do animal. Foram testados diferentes pHs, temperaturas e tempo de hidrólise. As características adequadas para a produção de hidrolisado que economizam tempo, reagente e trabalho são pH 8,4, incubação de 4 horas com uma temperatura de 42 °C. O segundo estudo objetivou caracterizar e comparar o hidrolisado produzido com enzima endógena de sardinha com hidrolisados produzidos com as enzimas comerciais Protamex® e pepsina suína. Foi observado que a caracterização dos hidrolisados foi influenciada pelo tipo da enzima utilizada. A quantidade de nutrientes e o perfil de aminoácidos dos três hidrolisados indicam que eles podem ser utilizados na aquicultura. As enzimas endógenas produziram um hidrolisado com maiores quantidades de aminoácidos hidrofóbicos livres e peptídeos de baixo peso molecular. A enzima Protamex® produziu um hidrolisado com um perfil de peptídeos mais equilibrado. Nas condições produzidas, os hidrolisados inibiram o crescimento de bactérias láticas. O terceiro estudo teve como intuito utilizar estes três hidrolisados proteicos de vísceras de sardinha na alimentação de trutas arco-íris. As ferramentas de avaliação foram o desempenho produtivo, a dinâmica das reservas energéticas teciduais, o perfil de metabólitos no sangue, a atividade das vias metabólicas hepáticas e as enzimas digestivas. Foram utilizados 300 juvenis de trutas arco-íris, divididos em 20 caixas de polietileno com capacidade para 100 L. de água, resultando em 15 juvenis de truta arco-íris por unidade experimental. O ensaio teve a duração de 56 dias. O delineamento foi inteiramente casualizado com quatro tratamentos e cinco repetições, sendo realizado ANOVA e as médias comparadas pelo teste de Tukey (5%). As dietas contendo hidrolisado produzido com enzima Protamex® e as dietas contendo hidrolisado produzido com pepsina suína, não diferiram (P>0,05) da dieta controle, proporcionando os melhores resultados de ganho de peso. A dieta controle foi a mais consumida pelas trutas. Foi observado que o hidrolisado produzido com enzimas endógenas de sardinha é pouco palatável, tendo prejudicado o crescimento dos animais. As diferenças relacionadas às reservas energéticas, parâmetros sanguíneos e síntese de aminoácidos, podem ter sido influenciados pelo consumo. A enzima Protamex® e a pepsina suína produziram hidrolisados que estimularam as proteases alcalinas totais. Foi constatado que as vísceras de sardinha hidrolisadas por enzimas comerciais ou pelas enzimas endógenas produzem ingredientes com características promissoras. Porém, estudos para retirada do amargor dos hidrolisados necessitam ser investigados.

Gonçalves, André Fernando Nascimento. Protein hydrolysates of residual sardine viscera in rainbow trout feeding 2019. 111 s. Thesis (Doctorate in Animal Science) - University of the State of Santa Catarina. Agroveterinary Science Center (UDESC/ CAV), Lages, SC, 2019. Protein hydrolysates are products obtained from the action of proteolytic enzymes through the breakdown of proteins. There may be an increase in digestibility, resulting in peptides of different sizes and free amino acids. The choice of hydrolytic protease depends on the cost of the enzyme and the desired end result after hydrolysis. The enzymes have different specificities to cleave the peptide bonds, altering the profile of peptides and amino acids, interfering in the quality and efficiency of the obtained hydrolyzate. The present work is composed by three studies, with the purpose of developing and characterizing protein hydrolysates of sardine viscera (Sardinella sp.) And its influence on the feeding of rainbow trout (Oncorhynchus mykiss). The first study consisted of developing and characterizing a protein hydrolyzate of sardine viscera produced with the enzymes of the animal 's own digestive tract. Different pHs, temperatures and hydrolysis time were tested. Suitable characteristics for hydrolyzate production that save time, reagent and work are pH 8.4, incubation of 4 hours with a temperature of 42°C. The second study aimed to characterize and compare the hydrolyzate produced with endogenous sardine enzyme with hydrolysates produced with the commercial enzymes Protamex® and porcine pepsin. It was observed that the hydrolyzate characterization was influenced by the type of enzyme used.The amount of nutrients and the amino acid profile of the three hydrolysates indicate that they can be used in aquaculture. Endogenous enzymes produced a hydrolyzate with higher amounts of free hydrophobic amino acids and low molecular weight peptides. The enzyme Protamex® produced a hydrolyzate with a more balanced peptide profile. Under the conditions produced, the hydrolysates inhibited the growth of lactic acid bacteria. The third study aimed to use these three protein hydrolysates of sardine viscera in the feeding of rainbow trout. The evaluation tools were the productive performance, the dynamics of the tissue energy reserves, the profile of metabolites in the blood, the activity of the hepatic metabolic pathways, and the digestive enzymes. 300 juveniles of rainbow trout were used, divided into 20 polyethylene boxes with capacity for 100 L of water, resulting in 15 juveniles of rainbow trout per experimental unit. The assay lasted for 56 days. The experimental design was completely randomized with four treatments and five replications. The analysis of variance (ANOVA) and the means were compared using the tukey test (5%). The diets containing hydrolyzate produced with Protamex® enzyme and the diets containing hydrolyzate produced with porcine pepsin did not differ (P> 0.05) from the control diet, providing the best results of weight gain. The control diet was the most consumed by trout. It was observed that the hydrolyzate produced with endogenous sardine enzymes is poorly palatable, having a detrimental effect on the growth of the animals. Differences related to energy reserves, blood parameters and amino acid synthesis may have been influenced by consumption. The enzyme Protamex and porcine pepsin produced hydrolysates.

Efeitos de imunossupressores sobre o sistema imunológico em transplantes de células germinativas em trutas arco-íris

Resumo YOSHINAGA, T.T. Efeitos de imunossupressores sobre o sistema imunológico em transplantes de células germinativas em trutas arco-íris Os transplantes de células germinativas e enxertos de testículo podem ser aplicados na reprodução de espécies comerciais e na conservação de espécies ameaçadas de extinção. No entanto, a maior limitação dos transplantes está na sua limitada eficiência, devido à baixa porcentagem de hospedeiros capazes de gerar gametas derivados do doador e da rejeição de enxertos de testículo alogênicos após poucas semanas de sua implantação. O sistema imunológico dos hospedeiros podem estar envolvidos na baixa de eficiência dos transplantes e na rejeição dos enxertos. Desta forma, este trabalho buscou verificar se a administração de drogas imunossupressoras como o tacrolimus e ciclosporina em emulsão poderiam ser utilizados para prolongar a viabilidade de enxertos de testículo em trutas arco-íris. Na primeira parte, experimentos in vitro com leucócitos do sangue periférico demonstraram que ambos o tacrolimus e a ciclosporina impedem a proliferação celular mesmo sob estímulo proliferativo da concanavalina. Nos ensaios in vivo, ambas as doses de tacrolimus (0,5 e 1,5 mg/kg) e a mais baixa de ciclosporina (20 mg/kg) inibiram significativamente a expressão de il2 no rim cefálico três dias após a injeção. Já a dose de 40 mg/kg de ciclosporina inibiu a expressão de il2 por até sete dias após a injeção. Na segunda parte, enxertos alogênicos de testículo foram realizados em animais tratados semanalmente com emulsões de tacrolimus. As análises histológicas (coloração H.E.) e as de RT-PCR (vasa e txdnc6) demonstraram a presença de espermatogônias na primeira semana e indicaram a presença de espermátides/espermatócitos na quinta semana, respectivamente, em alguns animais do grupo tratado com a dose de 0,5 mg/kg de tracrolimus. No grupo tratado com a dose mais alta de tacrolimus (1,5 mg/kg) e no grupo controle (sem imunossupressor), células germinativas ou marcadores destas não foram detectados. Estes resultados, portanto, fazem do tacrolimus um imunossupressor promissor para utilização em enxertos alogênicos e também em transplantes de células germinativas de truta arco-íris. A administração concomitante de um outro imunossupressor conjugado ao tacrolimus (na baixa dosagem) para inibir duas ou mais vias de ativação do sistema imunológico, conforme utilizado em transplantes de órgãos em humanos, pode ser uma alternativa para otimizar os efeitos imunossupressivos nos animais receptores. Palavras-Chave Reprodutores substitutos; imunossupressão;transplante de células germinativas;

Abstract YOSHINAGA, T.T Effects of immunosuppressants on immune system in germ cell transplantation in rainbow trout Germ cell transplantation and testis graft can be applied for the reproduction of commercial or endangered species. However, mechanisms of rejection from the host immune system might limit the production of surrogate gametes and progeny after transplantation and induce rejection of testis allografts within few weeks. In this work, we aimed to administer immunosuppressants-containing emulsions in order to verify whether they are capable to prevent immune rejection and promote testis allograft survival in rainbow trout. In the first part of this study, it was demonstrated in vitro that tacrolimus and cyclosporine were able to inhibit leucocyte proliferation even under concanavalin-induced mitotic stimulation. In in vivo experiments, both dosage of tacrolimus (0,5 and 1,5 mg/kg) and a lower of cyclosporine (20 mg/kg) inhibited significantly the expression of il2 in head kidney at three days post-injection. A higher dosage of cyclosporine (40 mg/kg) was able to inhibit il2 expression for up to seven days post-injection. In the second part, testis allografts were conducted in fish treated weekly with tacrolimus-containing emulsions. Histological (H.E. staining) and RT-PCR (vasa and txdnc6) analysis demonstrated the presence of spermatogonias in the first week and indicated the presence of spermatids/spermatocytes in the fifth week, respectively, in some animals treated with 0,5 mg/kg of tacrolimus. In the group treated with the highest tacrolimus dose (1,5 mg/kg) and in the control group (without immunosupressant), no germ cells or their respective markers were detected. These results suggest that tacrolimus comprise a promising immunosuppressant, with applications to testis allografts or germ cell transplantation in rainbow trout. Co-administration combining tacrolimus (at lower dose) with other immunosuppressive drugs for inhibiting more than two activation pathways of the immune system, as done in human organ transplantation, can be an alternative to optimize the immunossupressive effects in host organisms. Keywords Surrogate broodstock; immunosuppression; germ cell transplantation;

Histopathological and Immunohistochemical Diagnostic of Carcinosarcoma of Mandible in a Rainbowtrout (Onchorhyincus mykiss)

Background: The most common health problems described in fishes are noninfectious, infectious, and metabolic disorders. Fishes have neoplasias that are similar to those observed in mammals. Though these neoplasias do not cause economic losses to aquaculture, they have aroused a crescent interest from the comparative pathology’s point of view. Neoplasias of the oral cavity in fishes are usually reported as individual cases, but mandible tumors are not common, and most of them are squamous cells carcinomas. The aim of this study is to report a case of carcinosarcoma of the mandible in a rainbow trout (Oncorhynchus mykiss), obtained from a trout farm in Campos do Jordão, São Paulo, Brazil. Case: The fish underwent necropsy and tissue samples were collected and fixed with a buffer-neutralized formalin solution, which were then embedded in paraffin, sectioned in 5 μm sections and stained with hematoxylin-eosin. The histological sections were stained for immunohistochemical procedures, according to a modified avidin-biotin-peroxidase complex technique. Slides containing tissue were deparaffinized with xylol and rehydrated with alcohol of different grades. The endogenous peroxidase activity was blocked by incubating the slides for 20 min at 0.3% H2O2 in a solution of 5% methanol. After rinsing the slides in water and in a PBS/0.05%-Tween 20 solution, they were incubated in normal serum diluted 1:100 containing 10% bovine albumin in PBS (BSA), at room temperature for 30 min in humid chamber. After incubation, the primary anti-cytokeratin and anti-vimentin antibody was added to a 1:200 dilution in 10% PBS-BSA, and the slides were incubated until the next day at 41oC in humid chamber. The slides were then rinsed in PBS and incubated for 7 min in a 50 mL 30.3-diaminobenzidine solution containing 1% PBS-BSA in 50 mL H2O2. Hematoxylin was used for counterstaining. The immunohistochemical analysis showed a tumor with epithelial and mesenchymatous neoplastic components.(...)

Histopathological and Immunohistochemical Diagnostic of Carcinosarcoma of Mandible in a Rainbowtrout (Onchorhyincus mykiss)

Background: The most common health problems described in fishes are noninfectious, infectious, and metabolic disorders. Fishes have neoplasias that are similar to those observed in mammals. Though these neoplasias do not cause economic losses to aquaculture, they have aroused a crescent interest from the comparative pathologys point of view. Neoplasias of the oral cavity in fishes are usually reported as individual cases, but mandible tumors are not common, and most of them are squamous cells carcinomas. The aim of this study is to report a case of carcinosarcoma of the mandible in a rainbow trout (Oncorhynchus mykiss), obtained from a trout farm in Campos do Jordão, São Paulo, Brazil. Case: The fish underwent necropsy and tissue samples were collected and fixed with a buffer-neutralized formalin solution, which were then embedded in paraffin, sectioned in 5 μm sections and stained with hematoxylin-eosin. The histological sections were stained for immunohistochemical procedures, according to a modified avidin-biotin-peroxidase complex technique. Slides containing tissue were deparaffinized with xylol and rehydrated with alcohol of different grades. The endogenous peroxidase activity was blocked by incubating the slides for 20 min at 0.3% H2O2 in a solution of 5% methanol. After rinsing the slides in water and in a PBS/0.05%-Tween 20 solution, they were incubated in normal serum diluted 1:100 containing 10% bovine albumin in PBS (BSA), at room temperature for 30 min in humid chamber. After incubation, the primary anti-cytokeratin and anti-vimentin antibody was added to a 1:200 dilution in 10% PBS-BSA, and the slides were incubated until the next day at 41oC in humid chamber. The slides were then rinsed in PBS and incubated for 7 min in a 50 mL 30.3-diaminobenzidine solution containing 1% PBS-BSA in 50 mL H2O2. Hematoxylin was used for counterstaining. The immunohistochemical analysis showed a tumor with epithelial and mesenchymatous neoplastic components.(...)(AU)

CARACTERIZAÇÃO NUTRICIONAL E MÉTODOS DE CONSERVAÇÃO EM PESCADO

O pescado é uma fonte nutricional de alta qualidade e o consumo desta matriz vem sendo cada vez mais fomentado para garantia da saúde humana. O Brasil possui ampla variedade de peixes dulcícolas que apresentam potencial para produção de carne. A família Serrasalmidae participa ativamente na economia do país, porém, devido principalmente a falta de informações técnicas e científicas, o potencial aquícola do mesmo é ainda pouco explorado, fortalecendo o incentivo de pesquisas relacionadas às diversas espécies de peixes brasileiros dessa e de outras famílias, principalmente no aspecto nutricional. No entanto, o pescado caracteriza-se como uma matriz altamente perecível e análises mais rápidas e confiáveis, como avaliação da textura, para verificação do frescor e qualidade estão sendo constantemente desenvolvidas. Como estratégia de redução e controle da carga microbiana, tecnologias de conservação, como a radiação UV-C e a EAM, representam importantes ferramentas para a manutenção da qualidade do pescado e têm sido implantadas visando o aumento da validade comercial dos produtos derivados desta matriz. Desta forma, o presente estudo foi dividido em quatro etapas: (1) Analisar o perfil de ácidos graxos e composição centesimal de quatro espécies de peixes dulcícolas brasileiros de maior importância comercial da região Centro-Oeste (Artigo I). Os resultados demonstraram que a espécie Piaractus brachypomus (PB) apresentou maior quantidade de ácidos graxos ômega-3, principalmente EPA e DHA, e exibiu índices de qualidade nutricional superiores em relação a outras espécies, sendo considerada excelente fonte de lipídeos de alta qualidade; (2) Analisar o perfil de ácidos graxos e composição centesimal de seis espécies de peixes dulcícolas brasileiros de diferentes famílias ainda pouco exploradas pela piscicultura continental nacional (Artigo II). Os resultados demonstraram que os peixes Brycon cephalus (BC), Cichla ocellaris (CO) e Hypostomus affinis (HA) apresentaram perfis similares, exibindo altas concentrações de ácidos graxos ômega-3, especialmente DHA, além de excelentes índices de qualidade nutricional. Estas espécies foram consideradas matrizes de relevante qualidade lipídica e desejáveis ao consumo humano; (3) Avaliar a validade comercial de filés de Truta Arco-Íris (Oncorhynchus mykiss) submetidos à radiação UV-C e/ou acondicionadas em embalagens em atmosfera modificada (EAM) (Artigo III). Baseado nos resultados, concluiu-se que o tratamento de EAM (80% CO2/20% N2) aumentou a validade comercial dos filés de truta arco-íris em, no mínimo, duas vezes, retardando o crescimento microbiano, além de postergar as alterações químicas; (4) Correlacionar parâmetros de textura com o perfil de aminoácidos livres de nove espécies de peixes dulcícolas brasileiros (Artigo IV). A realização das análises experimentais e estatísticas encontra-se em andamento e os resultados parciais estão apresentados no documento.

Fish is a high quality nutritional source and the consumption of this matrix has been encouraged to guarantee human health. Brazil has a wide variety of freshwater fish species with great potential of meat production. Serrasalmidae family is participating actively in the economy, however, mainly due to lack of technical and scientific data, the potential of Brazilian aquaculture is underexploited, which strength the incentive of research related to the various species of Brazilian fish, especially in nutritional aspect. However, fish is characterized as a highly perishable matrix and faster and reliable analysis to verify the freshness and fish quality, as texture analyses, are constantly being developed. Among microbial load reduction and control strategy, preservation methods such as EAM and UV-C, represent important tools for maintaining the fish quality and has been implemented in order to increase the commercial validity of the derived products. Thus, this study was divided into four stages: (1) Analyze the fatty acids profile and chemical composition of four largest commercial importance freshwater fish species of the Midwest of Brazil (Paper I). The results showed that the species Piaractus brachypomus (PB) showed a higher amount of omega-3 fatty acids, especially EPA and DHA, and exhibited greater nutritional quality indexes in relation to other species, being considered an excellent source of high lipids quality; (2) Analyze the fatty acids profile and chemical composition of six Brazilian freshwater fish species from different families still underexploited by the national fish farming (Paper II). The results showed that the fish Brycon cephalus (BC), Cichla ocellaris (CO) and Hypostomus affinis (HA) presented similar profiles demonstrating high concentrations of omega-3 fatty acids, particularly DHA, in addition to excellent levels of nutritional quality indexes. These species were considered relevant lipid quality matrices and well desirable for human consumption; (3) Evaluate the commercial validity of Rainbow trout (Oncorhynchus mykiss) fillets submitted to UV-C radiation and/or under modified atmosphere packaging (MAP) (Paper III). Based on the results, it was concluded that the MAP (80% CO2 / 20% N2) increased the commercial validity of rainbow trout fillets in at least twice, retarding microbial growth and delaying chemical changes. (4) Correlate texture parameters with the free amino acids profile of nine Brazilian freshwater fishes species (Paper IV). The experimental and statistical analysis is under progress and partial results are presented in the document.

DESENVOVIMENTO E OTIMIZAÇÃO DE MÉTODO POR CLAE - EM/EM PARA DETERMINAÇÃO DE RESÍDUOS EM PESCADO

Este trabalho teve como objetivo avaliar a ocorrência de resíduos de antimicrobianos da classe dos anfenicóis em filé de tilápia do Nilo e de truta arco-íris , provenientes de frigoríficos localizados em Minas Gerais, por cromatografia líquida (CL) acoplada à espectrometria de massas (EM). O método CLAE desenvolvido utilizou coluna Aqua C18 (50 x 2,0 mm, 5 um),e fase móveis (A) água + 0,1% de ácido fórmico e (B) metanol + 0,1% de ácido fórmico com um fluxo de 0,4 mL/min e um gradiente de eluição.O espectrômetro de massa operou por ionização electrospray, modo de ionização negativa e as amostras foram analisadas por monitoramento de reações múltiplas. O método forneceu boa resolução entre os picos de tianfenicol, florfenicol e cloranfenicol com 3,9; 5,1 e 6,5 minutos de tempo de retenção, respectivamente. O método de extração otimizado consistiu em única etapa de extração liquido-liquido com recuperações apropriadas. O método atendeu aos parâmetros de validação (EC657/2002), fornecendo valores de CC de 0,019; 54,81 e 54,933 g/kg para cloranfenicol, florfenicol e tianfenicol, respectivamente. Dentre as 117 amostras analisadas, 12 apresentaram resíduos de cloranfenicol em concentração de 0,02 a 0,49 g/kg, das quais três estavam acima do limite mínimo de desempenho requerido (LMDR) estabelecido para o cloranfenicol (0,30 g/kg), com concentrações de 0,37; 0,42 e 0,49 g/kg. Resíduos de tianfenicol foram encontrados em duas das 117 amostras, em concentrações abaixo do limite máximo de resíduo (LMR). O florfenicol foi detectado em todas as amostras, com teores abaixo do LMR. A presença de resíduos de cloranfenicol em alimentos representa um risco à saúde dos consumidores. Apesar dos teores de tianfenicol e florfenicol encontrados estarem dentro dos padrões aceitáveis deve haver constante vigilância no uso desses e de pescados pelas consequências na saúde pública.

This study aimed to evaluate the occurrence of antimicrobial residues of the anfenicóis class in the Nile tilapia fillet and rainbow trout, from refrigerators located in Minas Gerais, for liquid chromatography (LC) coupled with mass spectrometry (MS). The HPLC method developed used Aqua C18 column (50 x 2.0 mm, 5 um), and mobile phase (A) water + 0.1% formic acid and (B) methanol + 0.1% formic acid with a flow 0.4 mL / min and a gradient of eluição.O mass spectrometer operated by electrospray ionization, negative ionization mode and the samples were analyzed by multiple reaction monitoring. The method provided good resolution between peaks thiamphenicol, florfenicol and chloramphenicol with 3.9; 5.1 and 6.5 minutes retention time, respectively. The optimal extraction method consisted of only liquid-liquid extraction step with appropriate recovery. The method met the validation parameters (EC657 / 2002), providing CC values of 0.019; 54.81 and 54.933 mg / kg to chloramphenicol, florfenicol and thiamphenicol, respectively. Among the 117 samples analyzed, 12 showed chloramphenicol residues in concentration from 0.02 to 0.49 mg / kg, three of which were above the minimum required performance limit (MRPL) set for chloramphenicol (0.30 mg / kg ), with concentrations of 0.37; 0.42 and 0.49 ug / kg. Thiamphenicol residues were found in two of 117 samples at concentrations below the maximum residue limit (MRL). Florfenicol was detected in all samples, with levels below the MRL. The presence of chloramphenicol residues in food poses a risk to consumer health. Despite thiamphenicol and florfenicol levels found are within acceptable standards must be constant vigilance in the use of these and other antibiotics in fish production for the consequences on public health.

Influence of rearing temperature changes on thrombocytes aggregation in rainbow trout / Influência de alterações de temperatura na agregação de trombócitos emtruta arco-íris

Fish, as poikilotherms, are subjected to the influence of the environmental temperature. It has been already reported that fish immune system is influenced by temperature; however, no information is available concerning if temperature has any effect on thrombocytes. Thrombocytes from fish reared at 6°C, 10oC and 20oC were assessed for alteration in their aggregation capacity. Thrombocyte percentages were altered by the water temperature at which the fish was reared, decreasing from 19% (at 10°C) to 13% (at 6°C) and increasing to 24% (at 20°C). However, the aggregation capacity was not significantly compromised by these temperature changes in an individual cell basis, which would suggest a capability of this cell, to maintain this indispensable function at different temperatures.(AU)

Os peixes, por serem animais pecilotermos, estão sujeitos à influências da temperatura do ambiente. Vários trabalhos já descreveram que o sistema imune é influenciado pela temperatura, contudo não existem informações concernentes à influencia desta sobre os trombócitos. Trombócitos provenientes de trutas arco-íris (Oncorrhynchus mykiss) mantidas a 6°C, 10ºC e 20ºC foram testados quanto à sua capacidade de agregação. A porcentagem de trombócitos sofreu alteração dependendo da temperatura da água em que os animais foram mantidos, diminuindo de 19% (a 10°C), para 13% (a 6°C) e aumentando para 24% (a 20°C). Entretanto, a capacidade de agregação individual de cada célula não foi significativamente afetada por essas mudanças de temperatura, o que sugere a qualidade dessa célula em manter essa função indispensável independente da temperatura.(AU)

Reproductive behavior of the rainbow trout Salmo irideus Gibbons (Osteichthyes,Salmonidae) under intensive culture / COMPORTAMENTO REPRODUTIVO DE TRUTA ARCO-IRIS Salmo irideus GIBBONS ( OSTEICHTHYES, SALMONIDAE ) EM CULTIVO INTENSIVO

The purpose of this paper is to study some aspects of the rainbow trout (Salmo irideus) reproduction under intensive culture. The structural characteristics and macroscopic variations of the ovaries were described, and based on these observations a maturity scale of six stages was elaborated. The spawning time and type were determined based on the gonadal maturation curve and in the distribution of the monthly relative frequency of stages. It was observed that the spawning type is total and occurred in June and July when the mean monthly values of the water temperature and the photoperiod reached its lowest values. It was also determined that the females and males reached the first sexual maturation when they were, respectively, 21.7 cm and 20.1 cm long and one year old.

O presente trabalho teve o objetivo de estudar alguns aspectos do comportamento reprodutivo de truta arco-íris, Salmo irideus, em cultivo intensivo. As características e variações macroscópicas dos ovários foram descritas, sendo que com estes resultados elaborou-se uma escala de maturidade com seis estádios. Determinaram-se o tipo de desova, definido como total, e a época da desova, que ocorreu nesta espécie em junho e julho, quando o fotoperíodo e a temperatura da água atingiram seus menores valores. O comprimento e a idade da primeira maturação, para fêmeas e machos, foram calculados, sendo, respectivamente, 21,7 cm e 20,1 cm, e a idade, ao redor de doze meses para ambos os sexos.

Reproductive behavior of the rainbow trout Salmo irideus Gibbons (Osteichthyes,Salmonidae) under intensive culture. / COMPORTAMENTO REPRODUTIVO DE TRUTA ARCO-IRIS Salmo irideus GIBBONS ( OSTEICHTHYES, SALMONIDAE ) EM CULTIVO INTENSIVO.

The purpose of this paper is to study some aspects of the rainbow trout (Salmo irideus) reproduction under intensive culture. The structural characteristics and macroscopic variations of the ovaries were described, and based on these observations a maturity scale of six stages was elaborated. The spawning time and type were determined based on the gonadal maturation curve and in the distribution of the monthly relative frequency of stages. It was observed that the spawning type is total and occurred in June and July when the mean monthly values of the water temperature and the photoperiod reached its lowest values. It was also determined that the females and males reached the first sexual maturation when they were, respectively, 21.7 cm and 20.1 cm long and one year old.

O presente trabalho teve o objetivo de estudar alguns aspectos do comportamento reprodutivo de truta arco-íris, Salmo irideus, em cultivo intensivo. As características e variações macroscópicas dos ovários foram descritas, sendo que com estes resultados elaborou-se uma escala de maturidade com seis estádios. Determinaram-se o tipo de desova, definido como total, e a época da desova, que ocorreu nesta espécie em junho e julho, quando o fotoperíodo e a temperatura da água atingiram seus menores valores. O comprimento e a idade da primeira maturação, para fêmeas e machos, foram calculados, sendo, respectivamente, 21,7 cm e 20,1 cm, e a idade, ao redor de doze meses para ambos os sexos.

Histopathological and Immunohistochemical Diagnostic of Carcinosarcoma of Mandible ina Rainbowtrout (Onchorhyncus mykiss)

Background:  :  : The most common health problems described in fishes are noninfectious, infectious, and metabolic disorders. Fishes have neoplasias that are similar to those observed in mammals. Though these neoplasias do not cause economic losses to aquaculture, they have aroused a crescent interest from the comparative pathologys point of view. Neoplasias of the oral cavity in fishes are usually reported as individual cases, but mandible tumors are not common, and most of them are squamous cells carcinomas. The aim of this study is to report a case of carcinosarcoma of the mandible in a rainbow trout (Oncorhynchus mykiss), obtained from a trout farm in Campos do Jordão, São Paulo, Brazil. Case: The fish underwent necropsy and tissue samples were collected and fixed with a buffer-neutralized formalin solution, which were then embedded in paraffin, sectioned in 5 ?m sections and stained with hematoxylin-eosin. The histological sections were stained for immunohistochemical procedures, according to a modified avidin-biotin-peroxidase complex technique. Slides containing tissue were deparaffinized with xylol and rehydrated with alcohol of different grades. The endogenous peroxidase activity was blocked by incubating the slides for 20 min at 0.3% H2O2 in a solution of 5% methanol. After rinsing the slides in water and in a PBS/0.05%-Tween 20 solution, they were incubated in norma

Histopathological and Immunohistochemical Diagnostic of Carcinosarcoma of Mandible ina Rainbowtrout (Onchorhyncus mykiss)

Background:  :  : The most common health problems described in fishes are noninfectious, infectious, and metabolic disorders. Fishes have neoplasias that are similar to those observed in mammals. Though these neoplasias do not cause economic losses to aquaculture, they have aroused a crescent interest from the comparative pathologys point of view. Neoplasias of the oral cavity in fishes are usually reported as individual cases, but mandible tumors are not common, and most of them are squamous cells carcinomas. The aim of this study is to report a case of carcinosarcoma of the mandible in a rainbow trout (Oncorhynchus mykiss), obtained from a trout farm in Campos do Jordão, São Paulo, Brazil. Case: The fish underwent necropsy and tissue samples were collected and fixed with a buffer-neutralized formalin solution, which were then embedded in paraffin, sectioned in 5 ?m sections and stained with hematoxylin-eosin. The histological sections were stained for immunohistochemical procedures, according to a modified avidin-biotin-peroxidase complex technique. Slides containing tissue were deparaffinized with xylol and rehydrated with alcohol of different grades. The endogenous peroxidase activity was blocked by incubating the slides for 20 min at 0.3% H2O2 in a solution of 5% methanol. After rinsing the slides in water and in a PBS/0.05%-Tween 20 solution, they were incubated in norma

Production of all-female triploid rainbow trout, Oncorhynchus mykiss. I Embryo survival / PRODUÇÃO DE LOTES MONOSSEXOS FEMININOS TRIPLÓIDES DE TRUTA ARCO-ÍRIS, Oncorhynchus mykiss. I SOBREVIVÊNCIA DOS EMBRIÕES

The survival rate among all-female and mixed-sex groups of diploid and triploid rainbow trout, Oncorhynchus mykiss, during the embryonic development period was compared. AII-female groups were obtained using sperm from masculinized genotypic female with 17-methyltestosterone. Triploidy was induced by heat shocking the eggs at 28°C for 20 minutes, starting 10 minutes post-activation. Triploidization rate was estimated from erythrocyte major axis measurement as 94 and 100%, respectively to all-female and mixed-sex groups. Compared with the diploid control, heat-shocked eggs showed higher mortality primarily after hatching period; groups fertilized with semen from masculinized female showed higher mortality rates than groups fertilized with semen from normal males.

Durante o período de desenvolvimento embrionário, foram comparadas as taxas de sobrevivência de lotes monossexos femininos e de lotes de sexos mistos de truta arco-íris, Oncorhynchus mykiss, diploides e triploides. Os lotes monossexos femininos foram obtidos usando-se sêmen de fêmeas genotípicas masculinizadas com 17 -metiltestosterona e a triploidia foi induzida por choque térmico a 28°C aplicado durante 20 minutos, com início 10 minutos após a ativação dos ovos. As taxas de triploidização, estimadas pela medida do eixo maior dos eritrócitos, foram de 94 e 100%, respectivamente, para os grupos monossexos femininos e de sexos mistos. Os grupos de indivíduos triplóides, resultantes de tratamento com choque térmico, apresentaram maior mortalidade do que os seus respectivos controles diploides, sobretudo na fase eleuteroembrionária e as parcelas que foram fertilizadas com sêmen de fêmeas masculinizadas apresentaram mortalidade superior à daquelas que foram fertilizadas com sêmen de machos normais.

Production of all-female triploid rainbow trout, Oncorhynchus mykiss. I Embryo survival / PRODUÇÃO DE LOTES MONOSSEXOS FEMININOS TRIPLÓIDES DE TRUTA ARCO-ÍRIS, Oncorhynchus mykiss. I SOBREVIVÊNCIA DOS EMBRIÕES

The survival rate among all-female and mixed-sex groups of diploid and triploid rainbow trout, Oncorhynchus mykiss, during the embryonic development period was compared. AII-female groups were obtained using sperm from masculinized genotypic female with 17-methyltestosterone. Triploidy was induced by heat shocking the eggs at 28°C for 20 minutes, starting 10 minutes post-activation. Triploidization rate was estimated from erythrocyte major axis measurement as 94 and 100%, respectively to all-female and mixed-sex groups. Compared with the diploid control, heat-shocked eggs showed higher mortality primarily after hatching period; groups fertilized with semen from masculinized female showed higher mortality rates than groups fertilized with semen from normal males.

Durante o período de desenvolvimento embrionário, foram comparadas as taxas de sobrevivência de lotes monossexos femininos e de lotes de sexos mistos de truta arco-íris, Oncorhynchus mykiss, diploides e triploides. Os lotes monossexos femininos foram obtidos usando-se sêmen de fêmeas genotípicas masculinizadas com 17 -metiltestosterona e a triploidia foi induzida por choque térmico a 28°C aplicado durante 20 minutos, com início 10 minutos após a ativação dos ovos. As taxas de triploidização, estimadas pela medida do eixo maior dos eritrócitos, foram de 94 e 100%, respectivamente, para os grupos monossexos femininos e de sexos mistos. Os grupos de indivíduos triplóides, resultantes de tratamento com choque térmico, apresentaram maior mortalidade do que os seus respectivos controles diploides, sobretudo na fase eleuteroembrionária e as parcelas que foram fertilizadas com sêmen de fêmeas masculinizadas apresentaram mortalidade superior à daquelas que foram fertilizadas com sêmen de machos normais.

Macroscopic and microscopic evaluation of rainbow trout, sêmen, Salmo irideus Gibbons / AVALIAÇÃO MACRO E MICROSCÓPICA DO SÊMEN DA TRUTA ARCO-ÍRIS, Salmo irideus GIBBONS

In order to improve the artificial reproduction in freshwater fishes of economical value, a study of the seminal characteristics of 79 males of rainbow trout, Salmo irideus Gibbons, two years old, was carried out at Estação Experimental de Salmonicultura de Campos do Jordão, during de reproductive season of 1980, 1981 and 1983. The seminal characteristics examined and the values obtained when estimated by confidence interval (95%) were: semen volume : 5.01 to 12.36 ml; sperm concentration: 15.07 to 20.99 x 106/mm³; percentage of sperm motility: 55.86 to 72.94 and percentage of live sperm by differential staining method, 54.13 to 75.75. There was no statistically significant difference between the percentage of sperm motility by microscopic examination and percentage of live sperm obtained by differential staining method, when the data of productive season of 1981 and 1983 were analyzed by the X² (chi-square) test (p 0.05).

Visando ao aperfeiçoamento da reprodução artificial de peixes de água doce de valor econômico, estudou-se o perfil espermático de 79 (setenta e nove) reprodutores de truta arco-íris, Salmo irideus Gibbons, com dois anos de idade, pertencentes à Estação Experimental de Salmonicultura de Campos do Jordão, São Paulo, durante os períodos reprodutivos de 1980, 1981 e 1983. As características seminais examinadas e os valores obtidos, quando estimados pelo intervalo de confiança de 95%, foram: volume seminal: 5,01 a 12,36 ml; concentração de espermatozoides x 106/mm3: 15,07 a 20,99; estimativa porcentual da motilidade espermática: 55,86 a 72,94; porcentagem de espermatozoides vivos avaliados pela coloração diferencial nos anos de 1981 a 1983, respectivamente, 54,13 a 75,75. Não se evidenciou diferença significativa entre a estimativa porcentual de elementos vivos por meio da motilidade espermática por exame direto subjetivo e aquela de espermatozoides vivos, não corados, pelo método de coloração diferencial, quando foram analisados os dados dos períodos reprodutivos de 1981 e 1983, pelo teste do qui-quadrado (X²), ao nível de 5% de significância.

Macroscopic and microscopic evaluation of rainbow trout, sêmen, Salmo irideus Gibbons / AVALIAÇÃO MACRO E MICROSCÓPICA DO SÊMEN DA TRUTA ARCO-ÍRIS, Salmo irideus GIBBONS.

In order to improve the artificial reproduction in freshwater fishes of economical value, a study of the seminal characteristics of 79 males of rainbow trout, Salmo irideus Gibbons, two years old, was carried out at Estação Experimental de Salmonicultura de Campos do Jordão, during de reproductive season of 1980, 1981 and 1983. The seminal characteristics examined and the values obtained when estimated by confidence interval (95%) were: semen volume : 5.01 to 12.36 ml; sperm concentration: 15.07 to 20.99 x 106/mm³; percentage of sperm motility: 55.86 to 72.94 and percentage of live sperm by differential staining method, 54.13 to 75.75. There was no statistically significant difference between the percentage of sperm motility by microscopic examination and percentage of live sperm obtained by differential staining method, when the data of productive season of 1981 and 1983 were analyzed by the X² (chi-square) test (p 0.05).

Visando ao aperfeiçoamento da reprodução artificial de peixes de água doce de valor econômico, estudou-se o perfil espermático de 79 (setenta e nove) reprodutores de truta arco-íris, Salmo irideus Gibbons, com dois anos de idade, pertencentes à Estação Experimental de Salmonicultura de Campos do Jordão, São Paulo, durante os períodos reprodutivos de 1980, 1981 e 1983. As características seminais examinadas e os valores obtidos, quando estimados pelo intervalo de confiança de 95%, foram: volume seminal: 5,01 a 12,36 ml; concentração de espermatozoides x 106/mm3: 15,07 a 20,99; estimativa porcentual da motilidade espermática: 55,86 a 72,94; porcentagem de espermatozoides vivos avaliados pela coloração diferencial nos anos de 1981 a 1983, respectivamente, 54,13 a 75,75. Não se evidenciou diferença significativa entre a estimativa porcentual de elementos vivos por meio da motilidade espermática por exame direto subjetivo e aquela de espermatozoides vivos, não corados, pelo método de coloração diferencial, quando foram analisados os dados dos períodos reprodutivos de 1981 e 1983, pelo teste do qui-quadrado (X²), ao nível de 5% de significância.