Inhibition of Na+, K+ -ATPase with ouabain is detrimental to equine blastocysts

Although equine blastocysts ≤ 300 µm in diameter can be successfully vitrified, larger equine blastocysts are not good candidates for cryopreservation. As Na+, K+-ATPase is involved in maintaining blastocyst expansion, perhaps inhibition of this enzyme would be a viable method of reducing blastocyst diameter prior to cryopreservation. Objectives were to evaluate effects of ouabain-induced inhibition of Na+, K+-ATPase in equine blastocysts. Sixteen mares were ultrasonographically monitored, given deslorelin acetate to induce ovulation, and inseminated. Embryos (D7 and D9) were harvested and Na+, K+-ATPase inhibited for 1 or 6 h by exposure to 10-6 M ouabain, either natural ouabain or conjugated to fluorescein (OuabainFL), during incubation at 37° C. Evaluations included morphometric characteristics (bright field microscopy) and viability (Hoescht 33342 + propidium iodide). Blastocysts incubated for 6 h in Holding medium + ouabain (n=3) had, on average, a 45.7% reduction in diameter, with adverse morphologic features and no re-expansion after subsequent incubation in Holding medium for 12 h. In subsequent studies, even a 1-h exposure to Ouabain or OuabainFL, caused similar reductions, namely 38.7 ± 6.7% (n=5) and 33.6 ± 3.3% (n=7) for D7 and D9 blastocysts, respectively. Ouabain binding was confirmed after OuabainFL exposition and all embryos (n=12) lost viability. We concluded that Na+, K+-ATPase inhibition with ouabain caused death of equine blastocysts and therefore was not a viable method of reducing blastocyst size prior to cryopreservation.

Inhibition of Na+, K+ -ATPase with ouabain is detrimental to equine blastocysts

Although equine blastocysts ≤ 300 µm in diameter can be successfully vitrified, larger equine blastocysts are not good candidates for cryopreservation. As Na+, K+-ATPase is involved in maintaining blastocyst expansion, perhaps inhibition of this enzyme would be a viable method of reducing blastocyst diameter prior to cryopreservation. Objectives were to evaluate effects of ouabain-induced inhibition of Na+, K+-ATPase in equine blastocysts. Sixteen mares were ultrasonographically monitored, given deslorelin acetate to induce ovulation, and inseminated. Embryos (D7 and D9) were harvested and Na+, K+-ATPase inhibited for 1 or 6 h by exposure to 10-6 M ouabain, either natural ouabain or conjugated to fluorescein (OuabainFL), during incubation at 37° C. Evaluations included morphometric characteristics (bright field microscopy) and viability (Hoescht 33342 + propidium iodide). Blastocysts incubated for 6 h in Holding medium + ouabain (n=3) had, on average, a 45.7% reduction in diameter, with adverse morphologic features and no re-expansion after subsequent incubation in Holding medium for 12 h. In subsequent studies, even a 1-h exposure to Ouabain or OuabainFL, caused similar reductions, namely 38.7 ± 6.7% (n=5) and 33.6 ± 3.3% (n=7) for D7 and D9 blastocysts, respectively. Ouabain binding was confirmed after OuabainFL exposition and all embryos (n=12) lost viability. We concluded that Na+, K+-ATPase inhibition with ouabain caused death of equine blastocysts and therefore was not a viable method of reducing blastocyst size prior to cryopreservation.(AU)

Sodium iodide: an alternative treatment option for feline sporotrichosis? / Iodeto de sódio: uma alternativa de tratamento para a esporotricose felina?

Background: Sporotrichosis is caused by pathogenic fungi of the genus Sporothrix. The clinically relevant species are S. schenckii, S. globosa and S. brasiliensis. In Brazil, S. brasiliensis is the most prevalent etiological agent among humans and cats. In cats with sporotrichosis, skin lesions are mainly characterized by nodules and ulcers, usually located in the head, nasal region and limbs. The presence of respiratory signs concomitantly with cutaneous lesions is frequent, especially sneezing, and may be associated with lesions located in the nasal mucosa. Ketoconazole (KTZ), itraconazole (ITZ), potassium iodide (KI), sodium iodide (NaI), terbinafine (TRB), fluconazole (FLZ) and amphotericin B (AMB) are the drugs currently available for treating feline sporotrichosis. ITZ remains the drug of choice. ITZ combined with KI has been successfully used in the treatment of naïve cats (especially cases with lesions in the nasal region), cases of recurrence and refractory to ITZ. Clinical cure with NaI has been described in some cases, but its use has been limited by adverse reactions. The conventional formulation is the saturated solution and the recommended dose in the treatment of feline sporotrichosis is 10 mg/kg every 12 h. Cats are sensitive to iodide preparations and should be carefully monitored for clinical evidence of iodism, such as apathy, anorexia, vomiting, diarrhea, hypothermia, hyperthermia, cardiomyopathy, hyperexcitability, muscular spasms and ptyalism. The purpose of this study was to evaluate the therapeutic response of NaI capsules in feline sporotrichosis.Materials, Methods & Results: An observational cohort study was conducted in cats with sporotrichosis at the Laboratory of Clinical Research in Dermatozoonoses in Domestic Animals (Lapclin-Dermzoo), Evandro Chagas National Institute of Infectious Diseases (INI)/Oswaldo Cruz Foundation (Fiocruz), Rio de Janeiro, Brazil.[...]

Sodium iodide: an alternative treatment option for feline sporotrichosis? / Iodeto de sódio: uma alternativa de tratamento para a esporotricose felina?

Background: Sporotrichosis is caused by pathogenic fungi of the genus Sporothrix. The clinically relevant species are S. schenckii, S. globosa and S. brasiliensis. In Brazil, S. brasiliensis is the most prevalent etiological agent among humans and cats. In cats with sporotrichosis, skin lesions are mainly characterized by nodules and ulcers, usually located in the head, nasal region and limbs. The presence of respiratory signs concomitantly with cutaneous lesions is frequent, especially sneezing, and may be associated with lesions located in the nasal mucosa. Ketoconazole (KTZ), itraconazole (ITZ), potassium iodide (KI), sodium iodide (NaI), terbinafine (TRB), fluconazole (FLZ) and amphotericin B (AMB) are the drugs currently available for treating feline sporotrichosis. ITZ remains the drug of choice. ITZ combined with KI has been successfully used in the treatment of naïve cats (especially cases with lesions in the nasal region), cases of recurrence and refractory to ITZ. Clinical cure with NaI has been described in some cases, but its use has been limited by adverse reactions. The conventional formulation is the saturated solution and the recommended dose in the treatment of feline sporotrichosis is 10 mg/kg every 12 h. Cats are sensitive to iodide preparations and should be carefully monitored for clinical evidence of iodism, such as apathy, anorexia, vomiting, diarrhea, hypothermia, hyperthermia, cardiomyopathy, hyperexcitability, muscular spasms and ptyalism. The purpose of this study was to evaluate the therapeutic response of NaI capsules in feline sporotrichosis.Materials, Methods & Results: An observational cohort study was conducted in cats with sporotrichosis at the Laboratory of Clinical Research in Dermatozoonoses in Domestic Animals (Lapclin-Dermzoo), Evandro Chagas National Institute of Infectious Diseases (INI)/Oswaldo Cruz Foundation (Fiocruz), Rio de Janeiro, Brazil.[...](AU)

Comparative study of three different dyes for evaluation of the physical integrity of the plasma membranes of bovine spermatozoa / Estudo comparativo de três diferentes corantes para avaliação da integridade física da membrana plasmática de espermatozoidesbovinos criopreservado

The morpho-functional nature of the plasma membrane assists to transport nutrients into the interior of spermatozoa, which requires the physical integrity of the membrane. This present study aims to compare three different dyes used to determine the integrity of the plasma membrane of bovine spermatozoa after freezing. The dyes tested were eosin 3% (p/v), trypan blue 0.4% (p/v), and propidium iodide (1.5 mM). Two sample groups were formed for the research. In the first group, one ejaculate was collected from each 11 animals, and in the second group, ten ejaculates were obtained from one animal. The fresh semen was evaluated using microscopy. The post-freezing samples were assessed using microscopy and flow cytometry. The results are presented in the form of average viability percentages ± standard deviations. The values membrane integrity, for the dyes eosin, trypan blue, and propidium iodide were 56.8±8.3%; 56.1±7.9%; 54.6±9.1%; respectively. There were no significant differences between the groups (p≥0.001). In comparative analysis of the techniques, flow cytometry enabled a greater number of cell evaluations, compared to phase contrast microscopy, with less variability and faster sample evaluation, hence adding value to the microscopy tests.

O aspecto morfo-funcional da membrana plasmática facilita o transporte de nutrientes para o interior do espermatozoide, o que está ligado à integridade física da membrana plasmática. O presente trabalho objetivou comparar três diferentes corantes na avaliação da integridade física da membrana plasmática do espermatozoide bovino, pós-congelamento. Utilizando os corantes eosina 3% (p/v), azul de tripan 0,4% (p/v) e iodeto de propídio (1,5mM). Foram formados dois grupos para pesquisa. O primeiro, um ejaculado de cada 11 animais e um segundo grupo de dez ejaculados de um único animal. Foi realizada avaliação microscópica do sêmen fresco (motilidade total, motilidade progressiva, vigor e integridade da membrana plasmática). Das amostras congeladas foram avaliados todos os parâmetros descritos anteriormente, além da técnica de eosina, azul de tripan, e iodeto de propídio. Os resultados estão apresentados como média de porcentagem ± desvio padrão. Os valores para a integridade de membrana, utilizando os corantes eosina, azul de tripan, e iodeto de propídio, foram 56.8±8.3%; 56.1±7.9%; 54.6±9.1%, respectivamente. Não há diferença significativa entre os grupos (p≥0.001). Na análise comparativa das técnicas podemos observar que a citometria de fluxo, possibilita um número maior de avaliação celular, quando comparado à técnica de microscopia de campo claro, possibilitando uma menor variabilidade e maior rapidez na avaliação da amostra.

Comparative study of three different dyes for evaluation of the physical integrity of the plasma membranes of bovine spermatozoa / Estudo comparativo de três diferentes corantes para avaliação da integridade física da membrana plasmática de espermatozoidesbovinos criopreservado

The morpho-functional nature of the plasma membrane assists to transport nutrients into the interior of spermatozoa, which requires the physical integrity of the membrane. This present study aims to compare three different dyes used to determine the integrity of the plasma membrane of bovine spermatozoa after freezing. The dyes tested were eosin 3% (p/v), trypan blue 0.4% (p/v), and propidium iodide (1.5 mM). Two sample groups were formed for the research. In the first group, one ejaculate was collected from each 11 animals, and in the second group, ten ejaculates were obtained from one animal. The fresh semen was evaluated using microscopy. The post-freezing samples were assessed using microscopy and flow cytometry. The results are presented in the form of average viability percentages ± standard deviations. The values membrane integrity, for the dyes eosin, trypan blue, and propidium iodide were 56.8±8.3%; 56.1±7.9%; 54.6±9.1%; respectively. There were no significant differences between the groups (p≥0.001). In comparative analysis of the techniques, flow cytometry enabled a greater number of cell evaluations, compared to phase contrast microscopy, with less variability and faster sample evaluation, hence adding value to the microscopy tests.(AU)

O aspecto morfo-funcional da membrana plasmática facilita o transporte de nutrientes para o interior do espermatozoide, o que está ligado à integridade física da membrana plasmática. O presente trabalho objetivou comparar três diferentes corantes na avaliação da integridade física da membrana plasmática do espermatozoide bovino, pós-congelamento. Utilizando os corantes eosina 3% (p/v), azul de tripan 0,4% (p/v) e iodeto de propídio (1,5mM). Foram formados dois grupos para pesquisa. O primeiro, um ejaculado de cada 11 animais e um segundo grupo de dez ejaculados de um único animal. Foi realizada avaliação microscópica do sêmen fresco (motilidade total, motilidade progressiva, vigor e integridade da membrana plasmática). Das amostras congeladas foram avaliados todos os parâmetros descritos anteriormente, além da técnica de eosina, azul de tripan, e iodeto de propídio. Os resultados estão apresentados como média de porcentagem ± desvio padrão. Os valores para a integridade de membrana, utilizando os corantes eosina, azul de tripan, e iodeto de propídio, foram 56.8±8.3%; 56.1±7.9%; 54.6±9.1%, respectivamente. Não há diferença significativa entre os grupos (p≥0.001). Na análise comparativa das técnicas podemos observar que a citometria de fluxo, possibilita um número maior de avaliação celular, quando comparado à técnica de microscopia de campo claro, possibilitando uma menor variabilidade e maior rapidez na avaliação da amostra.(AU)

Equine Sporotrichosis and iatrogenic hypothyroidism / Esporotricose equina e hipotireoidismo iatrogênico

Background: Sporotrichosis is a subacute or chronic granulomatous fungal infection that affects the dermis and subcutaneous tissue. Its etiology is related to a complex of Sporothrix spp. species of worldwide distribution. Usually, sporotrichosis results from traumatic inoculation of Sporothrix spp. into the dermis, affecting humans and other mammals, but a greater number of cases have been reported in felines, a fact increasingly important in the zoonotic transmission of diseased animals to man. In horses, there may be multiple ulcerated wounds covered with scabs and slightly elevated plaques or nodules that occur along lymphatic chains in limbs and can be unique and located. Iodides are considered drugs of choice for the treatment of sporotrichosis in horses. The main side effect of prolonged use of these substances is iodism and thyroid dysfunction. The aim of this study was to report a clinical case of sporotrichosis in an equine treated in the state of Paraná, southern Brazil, which showed secondary iatrogenic hypothyroidism with prolonged use of sodium and potassium iodide. Case: A female, 3-year-old, Quarter Horse, with 400 kg of liveweight presented at clinical examination of the cutaneous tissue elevated lesions of nodular aspect, with 2 to 4 cm in diameter, absence of itching and some nodules presenting alopecia areas in the chest and abdominal region, and in the [...](AU)

Equine Sporotrichosis and iatrogenic hypothyroidism / Esporotricose equina e hipotireoidismo iatrogênico

Background: Sporotrichosis is a subacute or chronic granulomatous fungal infection that affects the dermis and subcutaneous tissue. Its etiology is related to a complex of Sporothrix spp. species of worldwide distribution. Usually, sporotrichosis results from traumatic inoculation of Sporothrix spp. into the dermis, affecting humans and other mammals, but a greater number of cases have been reported in felines, a fact increasingly important in the zoonotic transmission of diseased animals to man. In horses, there may be multiple ulcerated wounds covered with scabs and slightly elevated plaques or nodules that occur along lymphatic chains in limbs and can be unique and located. Iodides are considered drugs of choice for the treatment of sporotrichosis in horses. The main side effect of prolonged use of these substances is iodism and thyroid dysfunction. The aim of this study was to report a clinical case of sporotrichosis in an equine treated in the state of Paraná, southern Brazil, which showed secondary iatrogenic hypothyroidism with prolonged use of sodium and potassium iodide. Case: A female, 3-year-old, Quarter Horse, with 400 kg of liveweight presented at clinical examination of the cutaneous tissue elevated lesions of nodular aspect, with 2 to 4 cm in diameter, absence of itching and some nodules presenting alopecia areas in the chest and abdominal region, and in the [...]

Densimetric fractionation of organic matter in an agricultural chronosequence in no-till areas in the Cerrado region, Brazil / Fracionamento densimétrico da matéria orgânica em uma cronossequência de agricultura em áreas de plantio direto no Cerrado

Mineralization of organic residues deposited on the soil surface in a no-till system (NT) maintains a continuous flow of different forms of carbon (C), which might interfere with densimetric fractions of soil organic matter (SOM). Currently, there are few studies on variations in these fractions in NT areas with different deployment times in the Cerrado region. Thus, the objective of this study was to evaluate the total C, nitrogen (N), and 13C in the soil, as well as to quantify the mass and contents of C, N, and 13C in the light and heavy fractions of SOM extracted using different solutions (water, sodium iodide [NaI], and sodium polytungstate [SPT]) in areas of Cerrado, pastures, and NT with different deployment times with Distroferric Red Latosol soil. The study areas are located in Montividiu, Goiás (GO), Brazil: (1) pasture of Brachiaria decumbens (PA); (2) NT with three years of deployment with soybean in summer and fallow instead of a second crop (NT3); (3) NT with 15 years of deployment with soybean in the summer and maize/sorghum as the second crop (NT15); and (4) NT with 20 years of deployment with soybean in the summer and maize as the second crop (NT20). These areas were compared to an area of native Cerrado sensu stricto (CE). The NT according to the deployment time showed an increase in the contents of C (0-0.05 m) and N (0-0.2 m) in the soil. The origin of C in the soil of the NT areas is associated with C4 plants; however, in up to 0.2 m of the soil profile, the 13C contents reduced according to NT deployment years. Extraction of light-fraction organic matter (LFOM) with SPT better represents this SOM fraction quantitatively and qualitatively than extraction of LFOM with water and NaI. This pattern is evident because of a greater consistency in the mass, C, N, and 13C contents of the LFOM extracted with SPT among the evaluated areas and the lower C content in residual particulate organic matter among the extractants.(AU)

A mineralização dos resíduos orgânicos depositados na superfície do solo no sistema de plantio direto (SPD) mantém um fluxo contínuo de diferentes formas de carbono, podendo interferir nas frações densimétricas da matéria orgânica do solo (MOS). Atualmente, são escassos resultados de pesquisa que avaliem às variações destas frações em áreas de SPD com diferentes anos de implantação na região do cerrado. Dessa forma, o objetivo deste trabalho foi avaliar o C, N e 13C total do solo, bem como, quantificar a massa, C, N e 13C das frações leves e pesadas da MOS extraídas com diferentes soluções (água, iodeto de sódio NaI e politungstato de sódio PTS) em áreas de Cerrado, pastagem e SPD com diferentes anos de implantação sob um Latossolo Vermelho Distroférrico. As áreas de estudo encontram-se localizadas em Montividiu (GO), e apresentam as seguintes características: 1) pastagem de Brachiaria decumbens (PA); 2) SPD com 3 anos de implantação com cultivo de soja no verão e pousio na safrinha (SPD3); 3) SPD com 15 anos de implantação com rotação soja no verão milho/sorgo safrinha (SPD15) e 4) SPD com 20 anos de implantação com rotação soja no verão milho safrinha (SPD20); sendo estas áreas comparadas a uma área de cerrado nativo stricto sensu (CE). O SPD em função do tempo de implantação aumentou os teores de C (0,0-0,05 m) e N (0,0-0,20 m) do solo. A origem da carbono do solo nas áreas de SPD é referente à plantas do ciclo fotossintético C4, no entanto, verifica-se que até os 0,20 m do perfil do solo, os resultados de 13C estão reduzindo em função dos anos de adoção do SPD. A extração da matéria orgânica leve (MOL) com PTS melhor representa quantitativamente e qualitativamente essa fração da MOS, quando comparada à extraída com água e NaI.[...](AU)

Pravastatin induces cell cycle arrest and decreased production of VEGF and bFGF in multiple myeloma cell line / Pravastatina induz parada no ciclo celular e diminuição na produção de VEGF e bFGF em linhagem de Mieloma Multiplo

Abstract Multiple myeloma (MM) is a B cell bone marrow neoplasia characterized by inflammation with an intense secretion of growth factors that promote tumor growth, cell survival, migration and invasion. The aim of this study was to evaluate the effects of pravastatin, a drug used to reduce cholesterol, in a MM cell line.Cell cycle and viability were determinate by Trypan Blue and Propidium Iodide. IL6, VEGF, bFGF and TGF were quantified by ELISA and qRT-PCR including here de HMG CoA reductase. It was observed reduction of cell viability, increase of cells in G0/G1 phase of the cell cycle and reducing the factors VEGF and bFGF without influence on 3-Methyl-Glutaryl Coenzyme A reductase expression.The results demonstrated that pravastatin induces cell cycle arrest in G0/G1 and decreased production of growth factors in Multiple Myeloma cell line.(AU)

Resumo O Mieloma Múltiplo é uma neoplasia de linfócitos B da medula óssea, caracterizada por inflamação com uma intensa secreção de fatores de crescimento que promovem o aumento do volume do tumor, sobrevivência celular, migração e invasão. O objetivo deste estudo foi avaliar os efeitos da pravastatina, uma droga usada para reduzir o colesterol, em um linhagem de MM. O ciclo celular e viabilidade foram determinadas por Trypan Blue e iodeto de propídio. IL6, VEGF, bFGF e TGF foram quantificadas por ELISA e qRT-PCR, incluindo aqui de HMG CoA redutase. Observou-se a redução da viabilidade das células, aumento de células na fase G0/G1 do ciclo celular e redução no VEGF e bFGF, sem influência na expressão da enzima 3-Metil-Glutaril Coenzima A redutase. Os resultados demonstraram que a pravastatina induz parada no ciclo celular em G0/G1 e diminuição da produção de fatores de crescimento em várias linhas de células de Mieloma.(AU)

Fracionamento físico da matéria orgânica e índice de manejo de carbono de um Argissolo submetido a sistemas conservacionistas de manejo / Physical fractionation of organic matter and carbon management index of an Alfisol subjected to conservation management systems

O fracionamento físico da matéria orgânica (MO) do solo é uma alternativa para a obtenção de frações lábeis utilizadas no cálculo do índice de manejo de carbono (IMC). O objetivo deste trabalho foi avaliar a eficiência do fracionamento físico granulométrico (53µm) e densimétrico, com soluções de iodeto de sódio 1,8Mg m-3 (NaI) ou politungstato de sódio 2,0Mg m-3 (PTS), na separação das frações lábeis (particulada e leve, respectivamente) da MO e o seu uso na estimativa do IMC. Amostras da camada de 0-20cm de um Argissolo Vermelho sob preparo convencional (PC) e plantio direto (PD), combinados com dois sistemas de culturas (aveia/milho e aveia+ervilhaca/milho+caupi), foram avaliadas quanto ao estoque de carbono orgânico total (COT), e nas frações particulada (COP) e leve (C-FL) da MO. No fracionamento físico densimétrico, com a utilização de NaI, foi recuperado menos C-FL em relação ao uso de PTS. A recuperação de COP no fracionamento granulométrico foi intermediária entre a recuperação de FL-NaI e de FL-PTS. O IMC com os dados do fracionamento granulométrico apresentou elevada correlação com o IMC dos dados de fracionamento densimétrico (NaI e PTS), havendo porém uma subestimação dos resultados com o uso de NaI. Os sistemas de manejo sem revolvimento do solo e com maior aporte de resíduos vegetais resultaram em maior IMC, tanto pelo aumento do índice de labilidade quanto do índice de estoque de carbono. O IMC a partir de frações físicas densimétricas da MO demonstra ser um índice eficiente na discriminação de sistemas de manejo, podendo ser utilizado na avaliação das práticas de manejo do solo.

The physical fractionation of soil organic matter (SOM) is an alternative in assessing the amount of labile fraction that is used to calculate the carbon management index (CMI). The objective this research was to assess the efficiency of particle-size physical fractionation, (53-µm mesh), and density physical fractionation, with sodium iodide 1.8Mg m-3 (NaI) or sodium polytungstate 2.0Mg m-3 (PTS) solutions, at recovering SOM labile fractions (particulate and light, respectively) and at estimating the CMI. Soil samples of the 0-20cm layer of a sandy clay loam Acrisol under conventional tillage (CT) and no-tillage (NT) combined with two cropping systems (oat/maize and oat+vetch/maize+cowpea) were analyzed for stocks of total organic carbon (TOC), particulate organic carbon fraction (POC) and light fraction carbon (LF-C). In density physical fractionation, with the use of NaI was recovered less LF-C than PTS. The recovery of POC by particle-size fractionation was intermediate between the recovery of NaI-LF and PTS-LF. The CMI calculated after particle-size fractionation showed high correlation with CMI after density fractionation with NaI or PTS, although results were rather underestimated with NaI. Conservation management systems without soil disturbance and with higher crop residue addition enhanced the CMI, because of increments of both lability index and carbon pool index. The CMI based on SOM physical densimetrical fraction shows evidence of being and efficient index to discriminate management systems that can be used to assess soil management practices.

Fracionamento físico da matéria orgânica e índice de manejo de carbono de um Argissolo submetido a sistemas conservacionistas de manejo / Physical fractionation of organic matter and carbon management index of an Alfisol subjected to conservation management systems

O fracionamento físico da matéria orgânica (MO) do solo é uma alternativa para a obtenção de frações lábeis utilizadas no cálculo do índice de manejo de carbono (IMC). O objetivo deste trabalho foi avaliar a eficiência do fracionamento físico granulométrico (53µm) e densimétrico, com soluções de iodeto de sódio 1,8Mg m-3 (NaI) ou politungstato de sódio 2,0Mg m-3 (PTS), na separação das frações lábeis (particulada e leve, respectivamente) da MO e o seu uso na estimativa do IMC. Amostras da camada de 0-20cm de um Argissolo Vermelho sob preparo convencional (PC) e plantio direto (PD), combinados com dois sistemas de culturas (aveia/milho e aveia+ervilhaca/milho+caupi), foram avaliadas quanto ao estoque de carbono orgânico total (COT), e nas frações particulada (COP) e leve (C-FL) da MO. No fracionamento físico densimétrico, com a utilização de NaI, foi recuperado menos C-FL em relação ao uso de PTS. A recuperação de COP no fracionamento granulométrico foi intermediária entre a recuperação de FL-NaI e de FL-PTS. O IMC com os dados do fracionamento granulométrico apresentou elevada correlação com o IMC dos dados de fracionamento densimétrico (NaI e PTS), havendo porém uma subestimação dos resultados com o uso de NaI. Os sistemas de manejo sem revolvimento do solo e com maior aporte de resíduos vegetais resultaram em maior IMC, tanto pelo aumento do índice de labilidade quanto do índice de estoque de carbono. O IMC a partir de frações físicas densimétricas da MO demonstra ser um índice eficiente na discriminação de sistemas de manejo, podendo ser utilizado na avaliação das práticas de manejo do solo.(AU)

The physical fractionation of soil organic matter (SOM) is an alternative in assessing the amount of labile fraction that is used to calculate the carbon management index (CMI). The objective this research was to assess the efficiency of particle-size physical fractionation, (53-µm mesh), and density physical fractionation, with sodium iodide 1.8Mg m-3 (NaI) or sodium polytungstate 2.0Mg m-3 (PTS) solutions, at recovering SOM labile fractions (particulate and light, respectively) and at estimating the CMI. Soil samples of the 0-20cm layer of a sandy clay loam Acrisol under conventional tillage (CT) and no-tillage (NT) combined with two cropping systems (oat/maize and oat+vetch/maize+cowpea) were analyzed for stocks of total organic carbon (TOC), particulate organic carbon fraction (POC) and light fraction carbon (LF-C). In density physical fractionation, with the use of NaI was recovered less LF-C than PTS. The recovery of POC by particle-size fractionation was intermediate between the recovery of NaI-LF and PTS-LF. The CMI calculated after particle-size fractionation showed high correlation with CMI after density fractionation with NaI or PTS, although results were rather underestimated with NaI. Conservation management systems without soil disturbance and with higher crop residue addition enhanced the CMI, because of increments of both lability index and carbon pool index. The CMI based on SOM physical densimetrical fraction shows evidence of being and efficient index to discriminate management systems that can be used to assess soil management practices.(AU)

Influência do bloqueio da Na+, K+-ATPase em blastocistos equinos

Atualmente, um dos desafios na reprodução equina é a baixa taxa de sobrevivência de embriões criopreservados com diâmetro > 300 m, usando métodos de vitrificação ou clássicos. Pesquisas sobre a influência da bomba de sódio e potássio (Na+, K+-ATPase) no desenvolvimento embrionário de equinos têm mais de 30 anos e hoje se sabe que participa da cavitação embrionária, modifica o gradiente iônico das células trofoblásticas, além de influenciar a formação, distribuição e permeabilidade das tight junctions (TJ) entre células trofoblásticas. Devido aos diversos tipos de isoformas da Na +, K+-ATPase, a depender das subunidades que a compõe, e a peculiaridades do desenvolvimento embrionário nas diferentes espécies, esse trabalho é caracterizado por uma revisão de literatura, para demonstrar os resultados dos principais estudos na área. Apresenta os resultados de um estudo que foi desenvolvido com o objetivo de buscar um meio capaz de reduzir o diâmetro do embrião equino a campo, para apoiar a vitrificação do blastocisto. Embriões equinos foram recuperados e submetidos a inibição da Na+, K+-ATPase por 1 ou 6 h, utilizando ouabaína isolada ou conjugada à sonda fluorescente (OuabaínaFL), durante incubação a 37° C. Os embriões foram avaliados quanto a características morfométricas (microscopia em campo claro) e de viabilidade (Hoechst 33342 + iodeto de propídeo). Acredita-se que este tenha sido o primeiro estudo que objetivou avaliar primariamente os efeitos da inibição da Na+, K+-ATPase com ouabaína, um inibidor específico, sobre embriões equinos. Observou-se que a inibição da Na+, K+-ATPase com ouabaína é capaz de reduzir o diâmetro dos blastocistos equinos, independetemente do dia de desenvolvimento (D7 ou D9), tendo sua confirmada a capacidade de ligação aos embriões a partir de estudos com ouabaína conjugada a sonda fluorescente. Todavia, a inibição da Na+, K+-ATPase compromente a viabilidade do embrião, não sendo recomendada a sua adoção com estratégia para redução do diâmetro de blastocistos equinos.

Currently, one of the challenges on equine reproduction is the low survival rate of horse embryos cryopreserved with diameter > 300 m, using either vitrification or classic methods. Researches about the influence of sodium pump and potassium (Na+, K+-ATPase) on horse embryo development have more than 30 years and nowadays it is known that participates on embryo cavitation, changes ionic gradient of trophoblastic cells, as well as influences formation, distribution and permeability of tight junctions (TJ) between trophoblastic cells. Due to many Na+, K+-ATPase isoforms, depending on comprising subunits, as well as peculiarities on embryo development depends on species, this work is characterizied by a literature review to demonstrate the main results in the field of research. It is also presented the results of a study that aimed to develop a medium able to reduce horse embryo diameter in the field, to support horse blastocyst vitrification. Horse embryos were hasvested and subjected to Na+, K+-ATPase inhibition for 1 or 6 h, using ouabain isolated or conjugates to fluorescent probe (OuabaínaFL), during incubation at 37 ° C. Embryos were assessed for morphometric characteristics (bright field microscopy) and viability (Hoechst 33342 + propridium iodide). To our knowledge, this is the first study that primarly aimed to evaluate the influence of Na+, K+-ATPase inhibition with ouabain, a specific inhibitor, on the horse embryos. It was observed that Na+, K+-ATPase inhibition with ouabain is able to reduce horse blastocyst diameter, in spite of embryo development (D7 ou D9), and ouabain binding to blastocysts were confirmed after embryo exposition to fluorescent conjugated ouabain. However, Na+, K+-ATPase inhibiton compromised embryo viability and it is not recommended as a strategy to reduce horse blastocyst diameter.

Surto de abscesso mandibular por Pseudomonas aeruginosa em ovinos / Outbreak of mandibular abscess by Pseudomonas aeruginosa in sheep

Descreve-se um surto de abscesso mandibular em ovelhas da raça Bergamácia no município de Botucatu, estado de São Paulo. Do rebanho de 120 animais, 35 apresentaram aumento de volume mandibular com a presença de nódulos únicos, de consistência pétrea, de diferentes tamanhos, fistulados ou não e sem indicativos de inflamação dos tecidos moles adjacentes. Os animais eram criados em pasto de Panicum maximum cv. Tanzânia com água e sal mineral ad libitum e everminados, via oral, com pistolas dosificadoras. O material para diagnóstico microbiológico e antibiograma foi coletado de cinco animais acometidos, por punção e aspiração dos nódulos. Dos 35 animais acometidos, 19 foram submetidos ao exame radiográfico, um ao exame tomográfico e outro à biópsia óssea da região submandibular. O único ovino que morreu, encontrava-se em estado de caquexia provavelmente devido à localização do aumento de volume que afetou a implantação dos dentes molares daquela região impedindo a apreensão e mastigação adequadas levando a perda da condição corporal e morte. Ao exame necroscópico, observaram-se áreas de necrose caseosa na mandíbula direita de onde isolou-se Pseudomonas aeruginosa. O tratamento utilizado foi baseado na aplicação de iodeto de sódio a 10 por cento por via intramuscular e antibioticoterapia segundo antibiograma com enrofloxacina por via intramuscular, porém com pouca eficácia. Diante do quadro clínico, dos dados de anamnese, da localização das lesões no tecido ósseo mandibular, do resultado do cultivo microbiológico, das alterações radiográficas e tomográficas foi feito o diagnóstico de abscesso mandibular causado por Pseudomonas aeruginosa.

We describe an outbreak of mandibular abscess in Bergamácia sheep from the municipality of Botucatu, São Paulo, Brazil. In a herd of 120 sheep, 35 showed an increase of the mandibular volume with presence of a single nodule of stony consistency and different sizes, often fistulated but with no signs of inflammation in the surrounding soft tissues. The sheep were raised on a Panicum maximum cv. Tanzania pasture, with water and mineral salt ad libitum, and were treated orally against parasites with drenching pistols. Material for microbiological diagnosis and antibiogram was collected from the nodules of five affected sheep, through puncture and aspiration. From 35 affected sheep, 19 were submitted to radiographic examination, one was submitted to computer tomography and another to bone biopsy of the submandibular region. The only sheep that died was in a state of cachexia, probably due to the location of the volume increase which affected the molar hindering apprehension and appropriate chewing. Necropsy revealed areas of caseous necrosis in the jaw, from where Pseudomonas aeruginosa was isolated. The sheep were treated with intramuscular application of 10 percent sodium iodide and the antobiotgic enrofloxacin, but with little efficacy. Based on the clinical data from medical records, the location of lesions in the jaw bone, the result of microbiological culture, radiographic and computer tomography, the diagnosis of mandibular abscesses due to Pseudomonas aeruginosa infection was made.

Surto de abscesso mandibular por Pseudomonas aeruginosa em ovinos / Outbreak of mandibular abscess by Pseudomonas aeruginosa in sheep

Descreve-se um surto de abscesso mandibular em ovelhas da raça Bergamácia no município de Botucatu, estado de São Paulo. Do rebanho de 120 animais, 35 apresentaram aumento de volume mandibular com a presença de nódulos únicos, de consistência pétrea, de diferentes tamanhos, fistulados ou não e sem indicativos de inflamação dos tecidos moles adjacentes. Os animais eram criados em pasto de Panicum maximum cv. Tanzânia com água e sal mineral ad libitum e everminados, via oral, com pistolas dosificadoras. O material para diagnóstico microbiológico e antibiograma foi coletado de cinco animais acometidos, por punção e aspiração dos nódulos. Dos 35 animais acometidos, 19 foram submetidos ao exame radiográfico, um ao exame tomográfico e outro à biópsia óssea da região submandibular. O único ovino que morreu, encontrava-se em estado de caquexia provavelmente devido à localização do aumento de volume que afetou a implantação dos dentes molares daquela região impedindo a apreensão e mastigação adequadas levando a perda da condição corporal e morte. Ao exame necroscópico, observaram-se áreas de necrose caseosa na mandíbula direita de onde isolou-se Pseudomonas aeruginosa. O tratamento utilizado foi baseado na aplicação de iodeto de sódio a 10 por cento por via intramuscular e antibioticoterapia segundo antibiograma com enrofloxacina por via intramuscular, porém com pouca eficácia. Diante do quadro clínico, dos dados de anamnese, da localização das lesões no tecido ósseo mandibular, do resultado do cultivo microbiológico, das alterações radiográficas e tomográficas foi feito o diagnóstico de abscesso mandibular causado por Pseudomonas aeruginosa.(AU)

We describe an outbreak of mandibular abscess in Bergamácia sheep from the municipality of Botucatu, São Paulo, Brazil. In a herd of 120 sheep, 35 showed an increase of the mandibular volume with presence of a single nodule of stony consistency and different sizes, often fistulated but with no signs of inflammation in the surrounding soft tissues. The sheep were raised on a Panicum maximum cv. Tanzania pasture, with water and mineral salt ad libitum, and were treated orally against parasites with drenching pistols. Material for microbiological diagnosis and antibiogram was collected from the nodules of five affected sheep, through puncture and aspiration. From 35 affected sheep, 19 were submitted to radiographic examination, one was submitted to computer tomography and another to bone biopsy of the submandibular region. The only sheep that died was in a state of cachexia, probably due to the location of the volume increase which affected the molar hindering apprehension and appropriate chewing. Necropsy revealed areas of caseous necrosis in the jaw, from where Pseudomonas aeruginosa was isolated. The sheep were treated with intramuscular application of 10 percent sodium iodide and the antobiotgic enrofloxacin, but with little efficacy. Based on the clinical data from medical records, the location of lesions in the jaw bone, the result of microbiological culture, radiographic and computer tomography, the diagnosis of mandibular abscesses due to Pseudomonas aeruginosa infection was made.(AU)

Experimental dry socket: microscopic and molecular evaluation of two treatment modalities / Alveolite experimental: análise microscópica e molecular de duas modalidades de tratamento

PURPOSE: To evaluate two treatment modalities of dry socket in rats and to discuss the first findings of the molecular analysis in this experimental model. METHODS: 84 rats underwent a tooth extraction were divided in 4 groups: I-uninfected socket (control), II-infected socket without any treatment, III-infected socket treated with irrigation of 2% sodium iodide and 3% hydrogen peroxide solution, IV-infected socket submitted to curettage, irrigation with physiological saline solution and fulfilled with metronidazole paste as base. The groups were subdivided in postoperative sacrifice periods: 6/15/28 days. A quantitative and a qualitative microscopic analysis was performed. Also, a quantitative analysis was performed using a RealTimePCR to evaluate the genes expression in the wound healing: Collagen Type I/COL-I, vascular endothelial growth factor/VEGF, osteocalcin/OCN, alkaline phosphatase/ALP, runt-related transcription factor 2/RUNX2 and tumor necrosis factor alpha/TNF-α. RESULTS: The group I showed higher bone formation, followed by groups IV, III, II respectively. The group II presented higher inflammatory infiltrate and the wound healing was delayed compared with other groups. It was obtained a significant positive correlation between bone neoformation and the expression of OCN and RUNX2, inflammatory infiltrate with TNF-α and a negative correlation between bone neoformation and TNF-α. CONCLUSION: No significant difference was found between the treatments.(AU)

OBJETIVO: Avaliar duas modalidades de tratamento da alveolite em ratos e discutir os primeiros achados de uma análise molecular neste modelo experimental. MÉTODOS: 84 ratos foram submetidos a uma extração dentária e foram divididos em quatro grupos: I- alvéolo não infectado (controle), II- alvéolo infectado sem tratamento, III- alvéolo infectado tratado com irrigação de iodeto de sódio a 2% e solução de peróxido de hidrogênio a 3%, IV- alvéolo infectado submetido à curetagem, irrigação com solução salina fisiológica e preenchimento com pasta a base de metronidazol. Os grupos foram subdivididos em períodos de sacrifício pós-operatório: 6/15/28 dias. Uma análise quantitativa e qualitativa microscópica foi realizada. Além disso, uma análise quantitativa foi realizada utilizando RealTimePCR para avaliar a expressão de genes no reparo alveolar: o colágeno tipo I / COL-I, o fator de crescimento endotelial vascular / VEGF, osteocalcina / OCN, fosfatase alcalina / ALP, fator de transcrição runt relacionados 2 / RUNX2 e fator de necrose tumoral alfa / TNF-α. RESULTADOS: O grupo I mostrou maior formação óssea, seguido pelos grupos IV, III, II, respectivamente. O grupo II apresentou maior infiltrado inflamatório e a cicatrização foi atrasada em comparação com outros grupos. Foi obtida uma correlação positiva entre a neoformação óssea e a expressão de OCN e RUNX2, infiltrado inflamatório com TNF-α e uma correlação negativa entre a neoformação óssea e TNF-α. CONCLUSÃO: Nenhuma diferença significativa foi encontrada entre os tratamentos.(AU)

Evaluation of castor oil-based polyurethane membranes in rat bone-marrow cell culturey / Avaliação de membranas à base de óleo de polímero de mamona em culturas de células de medula óssea de ratos

PURPOSE: To evaluate three methods to isolate rats MSCs and to analyze the potential of a castor oil polyurethane base membrane as a scaffold for MSCs. METHODS: Four male Wistar rats, aged 20-30 days were used. Bone marrow aspirates from femur and tibia were harvested using DMEM high glucose and heparin. The cell culture was performed in three different ways: direct culture and two types of density gradients. After 15 days, was made the 1st passage and analyzed cell viability with markers Hoerscht 33342 and propidium iodide. The MSCs were characterized by surface markers with the aid of flow cytometry. After this, three types of castor oil polyurethane membranes associated with the MSCs were kept on the 6-well plate for 5 days and were analyzed by optical microscopy to confirm cell aggregation and growth. RESULTS: Separation procedures 1 and 2 allowed adequate isolation of MSCs and favored cell growth with the passage being carried out at 70% confluence after 15 days in culture. The cells could not be isolated using procedure 3. When the 3 castor oil polyurethane membrane types were compared it was possible to observe that the growth of MSCs was around 80% in membrane type 3, 20% in type 2, and 10% in type 1. CONCLUSION: Both Ficoll-Hypaque densities allow isolation of rat MSCs, and especially castor oil-based membrane type 3 may be used as a scaffold for MSCs.(AU)

OBJETIVO: Avaliar três formas de cultivo de células-tronco mesenquimais de ratos; e analisar o potencial do polímero de mamona na forma de membrana como arcabouço para CTMs. MÉTODOS: Foram utilizados quatro ratos machos Wistar, de 20 a 30 dias de idade. Aspirados da medula óssea do fêmur e da tíbia foram colhidos com DMEM alta glicose e heparina. As células foram isoladas de três formas diferentes: cultivo direto e com dois tipos de gradientes de densidade. Após 15 dias, foi feita a 1ª passagem e analisada a viabilidade celular com os marcadores Hoerscht 33342 e Iodeto de Propídio. As CTMs foram então caracterizadas por marcadores de superfície, com o auxílio de citômetro de fluxo. Após, três tipos de membrana à base de óleo de polímero de mamona associadas com as CTMS foram mantidas em cultivo por cinco dias, e analisados por microscópio ótico para confirmar o crescimento e a adesão celular. RESULTADOS: Após 15 dias, Os procedimentos que utilizaram gradientes de densidade permitiram o isolamento das CTMs e favoreceram o crescimento celular com a passagem, sendo obtido 70% de confluência após 15 dias em cultura. O procedimento direto não se mostrou eficaz para o isolamento das células. O crescimento das CTMs foi aproximadamente 80% sobre a membrana tipo 3, 20% na tipo 2 e 10% na membrana tipo 1. CONCLUSÃO: Os dois gradientes de concentração Ficoll-Hypaque permitem isolar CTMs de ratos; e especialmente a membrana de polímero de mamona tipo 3 pode ser usada como um bom arcabouço para as CTMs.(AU)

MEMBRANA PLASMÁTICA: CRIOPRESERVAÇÃO E CAPACITAÇÃO DE ESPERMATOZÓIDES EQUINOS

Esta tese compreende quatro capítulos. Todos os estudos aqui relatados foram executados na Colorado State University (CSU), em Fort Collins-CO/USA. Foram utilizados sêmen de seis garanhões com idade entre 15 e 21 anos, das raças Puro-Sangue Inglês, Quarto de Milha e Árabe; estabulados no Laboratório de Reprodução Equina da CSU. Capítulo 1: dividido em três experimentos. Experimento 1: objetivou-se avaliar efeito do tratamento com 2 mg de ciclodextrina carregada com colesterol e estigmastanol (CCC , CCE); 1, 2 e 3 mg de ciclodextrina carregada com óleo de salmão (S1, S2 e S3) e com óleo de semente de linho (L1, L2 e L3) nas motilidades total (MT) e progressiva (MP) dos espermatozóides após descongelamento. Não foram observadas diferenças entre os tratamentos (p>0,05). Experimento 2: objetivou-se avaliar MT e MP do sêmen tratado com diferentes fontes de lipídeos sonicados em meio Whittens Modificado (MW), após congelamento convencional e congelamento precedido por choque pelo frio; e avaliar efeito do tratamento com 2 mg de CCC e CCE sobre motilidades após choque pelo frio. Os tratamentos usando lipídeos foram: 3 e 6 mg de óleo de salmão (S3 e S6), de óleo de semente de linho (L3 e L6), de gema de ovo de galinha (G3 e G6), de óleo fígado de bacalhau (B3 e B6) e de óleo de açafrão (A3 e A6). Nenhum dos tratamentos utilizando lipídeos melhorou motilidade após congelamento convencional (p>0,05). Para o sêmen submetido a 0 ºC por 30 minutos, os tratamentos G6, CCC e CCE melhoraram MT e MP (p<0,05). Experimento 3: utilizou-se cromatografia gasosa para avaliar eficiência dos tratamentos em alterar o perfil de ácidos graxos da membrana espermática. Os tratamentos foram: 6 g de óleo de semente de linho diluído em etanol (L1); 2 mg de ciclodextrina carregada com óleo de semente de linho (L2) e 6 mg de óleo de semente de linho sonicado em MW (L3). Nenhum dos tratamentos foi capaz de alterar o perfil de ácidos graxos da membrana espermática (p>0,05). Capítulo 2:objetivou-se mensurar quantidade de colesterol e estabelecer a relação colesterol:fosfolipídeos na membrana plasmática de células espermáticas tratadas com diferentes quantidades de CCC (0, 2, 4, 6 e 8 mg). Foram utilizados kit comercial (Cholesterol Liquicolor, Stanbio Laboratory, Boerne, TX, USA) para mensuração do colesterol e ensaio de ferrotiocianato de amônio para mensuração dos fosfolipídeos totais. A quantidade de colesterol encontrada nas células tratadas com 0, 2, 4, 6 e 8 mg de CCC foram respectivamente 0,22; 0,58; 0,77; 0,91 e 1,07 g/106 de espermatozóides, e a relação colesterol:fosfolipídeos para os mesmos tratamentos foram 0,39; 0,72; 1,0; 1,16 e 1,36. A quantidade de colesterol na membrana foi maior a partir do uso de 2 mg de CCC (p<0,05), e a relação colesterol:fosfolipídeos a partir do tratamento com 4 mg de CCC (p<0,05). Capítulo 3: neste estudo o objetivo foi avaliar o comportamento do sêmen sem tratamento (CT) ou tratado com 20% de plasma seminal (PS) e 40 M de PC-12 (PC) em eventos ligados à capacitação espermática durante incubação por 30 minutos (avaliações foram feitas no tempo 0 T0, 5 T5, 15 T15 e 30 minutos T30). Para isso cada um dos tratamentos foi submetido a quatro diferentes corantes: 1- CoroNa Green, que quantifica sódio intracelular (CG); 2- Fluo-3, que quantifica cálcio intracelular (Fluo); 3- FITC-PNA, que quantifica reação acrossômica (FP) e 4- Merocianina, que caracteriza fluidez/desorganização da membrana plasmática (Mero). Yo-Pro-1 ou iodeto de propídeo foram utilizados para avaliar integridade de membrana plasmática. As amostras foram lidas em citometria de fluxo. O tratamento com PC-12 aumentou o percentual de células com acrossoma reagido, detectada na avaliação aos 30 minutos. Este percentual, de 33%, foi maior que o dos demais tratamentos no mesmo tempo (CT: 13% e PS: 4%) e superior aos percentuais para o próprio PC-12 nos tempos 0 (7%), 5 (9%) e 15 minutos (16%). Com relação à fluidez da membrana plasmática não houve diferença no percentual de células com merocianina elevada entre os diferentes tratamentos, nem entre um mesmo tratamento ao longo do tempo (p>0,05). O estudo revelou efluxo de sódio das células espermáticas equinas viáveis após 15 minutos de incubação, sendo que nos tempos 15 e 30 minutos o percentual de celulas positivas para CG foram menores para tratamento com plasma seminal (8,8 e 4,8%) e maiores para tratamento com PC-12 (36 e 15,4%), em relação ao controle (23,6 e 11,8%, p<0,05). Após 15 minutos de incubação o sêmen dos três tratamentos apresentou percentual de células positivas para Fluo superior ao tempo 0, porém o plasma seminal retardou a entrada deste íon nas células, visto que, no T15 percentual de células Fluo+ foi menor que nos outros tratamentos (CT: 87%; PS: 59% e PC: 95%, p<0,05), voltando a se igualar no tempo 30 minutos (CT: 98%%; PS: 85% e PC: 97%, p>0,05). O meio MW utilizado foi capaz de induzir efluxo de sódio e influxo de cálcio, alterações que caracterizam capacitação espermática. Capítulo 4: os objetivos aqui foram avaliar eficiência de diluidores a base de gema de ovo (Lactose-EDTA) ou gema de ovo associada a leite em pó (FR5 modificado FR5M) e eficiência do glicerol (GLI) ou da combinação deste com metilformamida (MF) em manter MT e MP dos espermatozóides após congelamento usando diferentes protocolos (0: congelamento sem resfriamento; 20: resfriamento em palhetas por 20 minutos a 5 ºC antes do congelamento; 120T: resfriamento do sêmen em tubos por 120 minutos a 5 ºC; 120P: resfriamento do sêmen em palhetas por 120 minutos a 5 ºC). O FR5M preservou melhor a MP de células espermáticas em todos os protocolos de congelamento utilizados, mostrando benefício da combinação da gema de ovo com leite em pó no diluente de congelamento (15%, 21%, 26% e 25% para 0, 20, 120T e 120P, respectivamente, vs. 9%, 14%, 19% e 17%, para lactose-EDTA, p<0,05). A utilização de Lactose-EDTA 5%GLI apresentou resultados superiores para MT e MP se comparado ao mesmo diluidor com 3%MF + 2%GLI, o que mostra que o glicerol é um crioprotetor que pode ser utilizado em diferentes protocolos de congelamento (MT para Lactose-EDTA 5%GLI = 33%, 45%, 56% e 53% para 0, 20, 120T e 120P vs. 20%, 30% 35% e 36% para Lactose-EDTA 3%MF+ 2%GLI, p<0,05; MP para Lactose-EDTA 5%GLI = 19% e 17% para 120T e 120P vs. 11% e 10% para Lactose-EDTA 3%MF+ 2%GLI, p<0,05). O resfriamento do sêmen antes do congelamento pode ser realizado tanto em tubos quanto em palhetas de 0,5 mL, sem prejuízos às motilidades total e progressiva do sêmen após congelamento.

This thesis is compounded by four chapters. All work reported here were performed at Colorado State University (CSU), located in Fort Collins, CO / USA. Six stallions between 15 and 21 years old (Thoroughbred, Quarter Horse and Arabian breeds) and stabled at Equine Reproduction Laboratory at CSU were used in all experiments. Chapter 1: comprised by three experiments. Experiment 1: aimed to evaluate the effects on total motility (TM) and progressive motility (PM) after semen thawing from treatments with 2 mg of cyclodextrin-loaded-cholesterol and 2 mg of cyclodextrin-loaded-stigmastanol (CLC, CLS); 1, 2 and 3 mg of cyclodextrin-loaded-salmon oil (S1, S2 and S3); and 1, 2 and 3 mg of cyclodextrin-loaded-flax seed oil (F1, F2, and F3). No differences among treatments were detected (p>0.05). Experiment 2: aimed to evaluate TM and PM of semen treated with different sonicated lipids into Modified Whittens Medium (MW), after conventional freezing and freezing preceded by cold shock, and assess the effect of treatment with 2 mg of CLC and CLS on motility after cold shock. Treatments using lipids were: 3 and 6 mg of salmon oil (S3 and S6); 3 and 6 mg of flax seed oil (F3, F6); 3 and 6 mg of egg yolk (E3 and E6); 3 and 6 mg of cod liver oil (C3 and C6), 3 and 6 mg of safflower oil (SF3 and SF6). None of the lipid treatments improved motility after conventional freezing (p>0.05). For semen subjected to 0 °C for 30 minutes, TM and PM increased in semen treated with E6, CLC and CLS (p<0.05). Experiment 3: gas chromatography was used to evaluate effectiveness of treatments in altering the sperm membrane fatty acid profile. The treatments were: 6 g of flax seed oil diluted with ethanol (F1); 2 mg of cyclodextrin-loaded- flax seed oil (F2) and 6 mg of flax seed oil sonicated in MW (F3). None of the treatments was able to change fatty acid profile in the sperm membrane (p>0.05). Chapter 2: aimed to measure the amount of cholesterol and to establish the cholesterol:phospholipids ratio in plasma membrane of fresh equine sperm cells treated with different amounts of CLC (0, 2, 4, 6 and 8 mg). A commercial kit was used to measure cholesterol (Cholesterol Liquicolor, Stanbio Laboratory, Boerne, TX, USA) and an ammonium ferrothiocyanate assay for total phospholipids measurement. Amount of cholesterol found for cells treated with 0, 2, 4, 6 and 8 mg CLC were, respectively, 0.22; 0.58; 0.77; 0.91 and 1.07 micrograms/106 sperm, and cholesterol:phospholipids ratios to the same treatments were 0.39; 0.72; 1.0; 1.16 and 1.36, respectively. Increase in cholesterol amount was observed when 2 mg of CLC was used (p<0.05), and cholesterol:phospholipids ratio was higher with 4 mg of CLC (p<0.05). Chapter 3: in this study the objective was to evaluate the response of fresh equine semen without treatment (CT) or treated with 20% of seminal plasma (SP) and 40 M PC-12 (PC) in events related to sperm capacitation during incubation for 30 minutes (evaluations made at time 0 - T0, 5 - T5, 15 T15 and 30 minutes - T30). Each treatment was subjected to four different dyes: 1- CoroNa Green, which measures intracellular sodium (CG); 2- Fluo-3, which measures intracellular calcium (Fluo); 3- FITC-PNA, which measures acrosome reaction (FP) and 4- Merocyanine, which characterizes plasma membrane fluidity/destabilization (Mero). Yo-Pro-1 or propidium iodide were used to assess cell membrane integrity. Samples were read in flow cytometry. An increased percentage of cells with reacted acrosome was observed in semen treated with PC-12 at T30. This proportion (33%) was higher than the other treatments at the same time (CT: 13% and SP: 4%) and higher than the proportion for PC-12 at T0 (7%), T5 (9%) and T15 (16%). There was no difference in the percentage of cells with high merocyanine between different treatments or between the same treatment over time (p>0.05). The study showed sodium efflux after 15 minutes incubation, and at times 15 and 30 minutes the percentage of positive cells for GC were lower in SP treatment (8.8 and 4.8%) and higher for PC-12 (36 and 15.4%) compared to the CT (23.6 and 11.8%, p <0.05). At T15 the semen of all treatments showed higher proportion of Fluo positive cells than T0, but seminal plasma delayed entry of the ion into the cells, since at T15 Fluo proportion of positive cells was lower than the other treatments (CT: 87%; SP: 59% and PC: 95%, p<0.05) and became similar at T30 (CT = 98%, SP= 85% and PC=97%, p> 0.05). The semen extender used was able to inducing sodium efflux and calcium influx, changes characterizing sperm capacitation. Chapter 4: the objectives were to evaluate efficiency of an egg yolkbased medium (lactose-EDTA) or egg yolk associated with powdered milk (modified FR5 - MFR5), and efficiency of glycerol (GLY) or a combination between GLY and dimethylformamide (MF) to maintain TM and PM sperm after freezing using different protocols (0: no cooling; 20: cooling straws for 20 minutes at 5 °C before freezing; 120T: cooling in tubes for 120 minutes at 5 °C; and 120P: cooling in straws for 120 minutes at 5 °C). MFR5 preserved PM in all freezing protocols, showing the benefit of the combination of egg yolk with powdered milk for freezing diluent (15%, 21%, 26% and 25% for 0, 20, 120T and 120P, respectively, vs. 9%, 14%, 19% and 17% for lactose-EDTA, p<0.05). The use of lactose-EDTA 5%GLY showed superior results for TM and PM if compared to the same medium with 3%MF + 2%GLY, indicating that glycerol is a cryoprotectant that can be used in several different freezing protocols (TM for Lactose-EDTA 5%GLY = 33%, 45%, 56% and 53% for 0, 20, 120T and 120P vs. 20% 30% 35% and 36% for LactoseEDTA 3%MF + 2%GLY, p <0.05; MP for Lactose-EDTA 5%GLY = 19% and 17% at 120T and 120P vs. 11% and 10% for Lactose-EDTA 3%MF + 2%GLY, p<0.05). The cooling before freezing can be performed either in tubes as in 0.5 mL straws for 120 minutes, without damage to total and progressive motility of thawed semen.

The acariasis in the air sacs of canaries (Serinus canarius L.) and Australian parakeets (Melopsittacus undulatus, Shaw, 1805) by Cytodites nudus (Vizioli, 1868) / Acarinose dos sacos aéreos de canários (Serinus canarius L.) e periquitos australianos (Melopsittacus undulatus, Shaw 1805) por Cytodites nudus (Vizioli, 1868)

In this paper the authors describes, in a group of canaries, a bronchitis caused by an infestation of the air sacs, by Cytodites nudus (Vizioli, 1868). They further described the observed symptoms, mainly those which are due to the disturbance of the breathing system (intense dyspnea, cough, sneezing, noisy breathing and congestion of the larynx) and those of exciting of the neural system (convulsions with tonic and clonic contractions). The more frequent lesions are: inflammation of the air sacs, with the presence of liquids, mucus and blood coagulum in the cavities. Pericarditis and adherences of the pericardium to the thorax walls. The treatment was based, on fluidifiers and expectorants of secretions, containing either potassium iodide or sodium benzoate. There were often found ectoparasites among the breedings, from which came our cases, for which treatment the Sodium fluoride was utilized. The clinics diagnosis was confirmed by necroscopy performed on the patients or on other birds of the same breedings, with similar symptoms. The authors also describes the incidence of Cytodites nudus in the air sacs of Australian Parakeets (Melopsittacus undulatus Shaw, 1805).

O artigo apresenta resumo em inglês.

The acariasis in the air sacs of canaries (Serinus canarius L.) and Australian parakeets (Melopsittacus undulatus, Shaw, 1805) by Cytodites nudus (Vizioli, 1868) / Acarinose dos sacos aéreos de canários (Serinus canarius L.) e periquitos australianos (Melopsittacus undulatus, Shaw 1805) por Cytodites nudus (Vizioli, 1868)

In this paper the authors describes, in a group of canaries, a bronchitis caused by an infestation of the air sacs, by Cytodites nudus (Vizioli, 1868). They further described the observed symptoms, mainly those which are due to the disturbance of the breathing system (intense dyspnea, cough, sneezing, noisy breathing and congestion of the larynx) and those of exciting of the neural system (convulsions with tonic and clonic contractions). The more frequent lesions are: inflammation of the air sacs, with the presence of liquids, mucus and blood coagulum in the cavities. Pericarditis and adherences of the pericardium to the thorax walls. The treatment was based, on fluidifiers and expectorants of secretions, containing either potassium iodide or sodium benzoate. There were often found ectoparasites among the breedings, from which came our cases, for which treatment the Sodium fluoride was utilized. The clinics diagnosis was confirmed by necroscopy performed on the patients or on other birds of the same breedings, with similar symptoms. The authors also describes the incidence of Cytodites nudus in the air sacs of Australian Parakeets (Melopsittacus undulatus Shaw, 1805).

O artigo apresenta resumo em inglês.