Estimation, morphometry and ultrastructure of ovarian preantral follicle population in agouti (Dasyprocta leporina) / Morfometria, estimativa e ultraestrutura da população folicular ovariana preantral de cutia (Dasyprocta leporina)

The aim of this study was to characterize the preantral ovarian follicular population in agoutis (D. leporina) by estimating the number of follicles at each developmental category, and also describe the morphometry and the specific features of the follicle and the oocyte by using light and transmission electron microscopy. The length of each ovary was measured using a caliper rule, longitudinally sectioned into two halves and both were immediately fixed to perform the estimation of follicular population and ultrastructural analysis. The mean (±S.E.M.) population of follicular per pair of ovary was estimated at 4419.8±532.26 and 5397.52±574.91 for right and left ovaries, respectively, but no differences were observed between them. The diameters for follicles, oocyte and nuclei were: 18.62±3.40µm, 12.28±2.37µm and 6.10±0.93µm for primordial, 23.75±5.70µm, 14.22±3.00µm and 6.70±1.24µm for primary and 88.55±17.61µm, 52.85±17.56µm and 22.33±17.61µm for secondary follicles, respectively. The most of the follicles found belonged to the primordial category (86.63%), followed by primary (13.01%) and secondary (0.35%) one. Additionally, polyovular follicles were observed in all the animals and they represented 7.51% of the total follicles counted. The ultrastructural analysis showed that the oocyte presented a central and regular nuclei, displaying a homogenous mass. Among the organelles, the mitochondria were the most abundant and the oocyte Golgi apparatus was rarely observed. In conclusion, this work shows for the first time the characterization of the population of preantral follicles in the ovary of Dasyprocta leporina. Those information will be useful for further development and adaptation of biotechniques such as germplasm cryopreservation and in vitro gametes manipulation.(AU)

O objetivo deste trabalho foi caracterizar a população folicular ovariana pré-antral em cutias (D. leporina) estimando o número de folículos em cada categoria de desenvolvimento, e também descrever a morfometria e as características específicas do folículo e oócito usando microscopia de luz e eletrônica de transmissão. O comprimento de cada ovário foi medido utilizando um paquímetro, seccionados longitudinalmente em duas metades e ambos foram imediatamente fixados para realizar a estimativa da população folicular e análise ultraestrutural. A média (±S.E.M.) da população folicular por par de ovário foi estimada em 4419,8±532,26 e 5397,52±574,91 nos ovários direito e esquerdo, respectivamente, mas não foram observadas diferenças entre eles. Os diâmetros dos folículos, oócito e núcleos, respectivamente, foram: 18,62±3,40µm, 12,28±2.37µm e 6,10±0,93µm para primordial, 23,75±5,70µm, 14,22±3,00µm e 6,70±1,24µm para primário e 88,55±17,61µm, 52,85±17,56µm e 22,33±17,61µm de folículos secundários. A maioria dos folículos encontrados pertencia à categoria primordial (86,63%), seguido pelo primário (13,01%) e um secundário (0,35%). Adicionalmente, os folículos poliovulares foram observados em todos os animais e representavam 7,51% do total de folículos contados. A análise ultra-estrutural mostrou que o oócito apresentou núcleos centrais e regulares, exibindo uma massa homogênea. Dentre as organelas, as mitocôndrias foram as mais abundantes e o aparelho de Golgi do oócito foi raramente observado. Em conclusão, este trabalho mostra pela primeira vez a caracterização da população de folículos pré-antrais do ovário da Dasyprocta leporina. Essas informações serão úteis para o desenvolvimento e adaptação de biotécnicas, como a criopreservação de germoplasma e manipulação de gametas in vitro.(AU)

Ultrastructure of bovine placenta during all gestational period / Ultraestrutura de placenta bovina durante todo o período gestacional

Placentas from pregnant cows with different gestation periods were used. Placental fragments of all groups were processed and evaluated by transmission electron microscopy. After fragment analysis, bovine placenta was observed to be epitheliochorial type in early pregnancy, becoming progressively sinepiteliocorial at the beginning of the second trimester. There are no ultrastructural evidences of inflammation in the region of caruncles throughout gestation, despite the invasion of caruncle proper lamina by trophoblast cells. However, throughout pregnancy and especially at the end, there were evident signs of cell degeneration in both trophoblast and the uterine epithelium. The active trophoblast cells intensely phagocytize cellular debris. There are complex interdigitations between the surface of the trophoblast and the uterine epithelium, which is related to the increase of the exchange surface between mother and fetus. At the end of pregnancy, interdigitations disappear, favoring the detachment and expulsion of the placenta after birth.(AU)

Foram utilizadas placentas de vacas abatidas em frigorífico com diversos tempos gestacionais. Fragmentos de placentomo de todos os grupos foram processados e avaliados em microscopia eletrônica de transmissão. Após análise dos fragmentos, observou-se que a placenta bovina é do tipo epiteliocorial no início da gestação, tornando-se sinepiteliocorial progressivamente a partir do início do segundo mês de gestação. Não existem evidências ultraestruturais de inflamação na região das carúnculas durante toda a gestação, apesar da invasão da lâmina própria caruncular por células trofoblásticas. No entanto, durante toda a gestação e em especial ao seu final, foram observados sinais evidentes de degeneração celular, tanto do trofoblasto como do epitélio uterino. As células trofoblásticas ativas fagocitam intensamente os debris celulares originados dessas degenerações. Existem complexas interdigitações entre a superfície do trofoblasto e do epitélio uterino, o que estaria relacionado com o aumento da superfície de troca entre mãe e feto. Ao final da gestação, praticamente desaparecem essas interdigitações, favorecendo o descolamento e a expulsão da placenta após o parto.(AU)

Hiperplasia em túnicas de artérias coronárias de beijupirás criados em sistema offshore / Hyperplasia in tunics of coronary arteries in beijupirá farmed in offshore system

As lesões coronarianas já foram relatadas em espécies de peixes anádromos, com características semelhantes às observadas em aterosclerose humana, mas não em peixes cultivados sem interferência de padrões reprodutivos. Objetivou-se descrever lesões coronarianas em beijupirá (Rachycentron canadum) criado em sistema offshore, em 54 exemplares coletados ao longo do cultivo de oito meses. Amostras de tecido cardíaco de alevinos e juvenis foram submetidas à análise histopatológica e à microscopia eletrônica de transmissão (MET), para visualização de lesões coronarianas, sendo as lâminas histológicas coradas pela técnica de hematoxilina-eosina, ácido periódico de Schiff, alcian blue e reticulina de Gomori. Exames realizados pela fotomicroscopia de luz evidenciaram lesões arteriais caracterizadas por hiperplasia das túnicas íntima e média de artérias coronárias em, respectivamente, 29,63% e 79,63% dos animais, com redução do lúmen arterial. Na MET, foram observadas alterações na estrutura das coronárias com espessamento do endotélio e membrana basal, proliferação de fibroblastos e fibras colágenas, com acúmulo de material lipídico subendotelial, resíduos celulares aderidos à membrana basal e vesículas pinocitóticas com presença de lisossomos isolados. Constatou-se que os peixes da espécie Rachycentron canadum criados em cativeiro desenvolvem lesão arterial do tipo crônica degenerativa inflamatória.

The coronary lesions have been reported in species of anadromous fish with similar characteristics as those observed in human atherosclerosis, but not in farmed fish without interference of reproductive patterns. This study aimed to describe coronary lesions in beijupirá (Rachycentron canadum) farmed in an offshore system, in 54 specimens collected along an eight month cultivation period. Samples of heart tissue of fry and juveniles were subjected to histopathological analysis and transmission electron microscopy (TEM) for visualization of coronary lesions, the histology slides were stained using hematoxylin-eosin, periodic acid Schiffh, alcian blue, and reticulin of Gomori. Examinations performed by light microscopy showed arterial lesions characterized by hyperplasia of the intima and media tunics, respectively in 29.63% and 79.63 % of the animals, with reduced lumen. In TEM changes in the structure of the coronary endothelium and thickening of the basement membrane, proliferation of fibroblasts and collagen fibers, with subendothelial accumulation of lipid material, cellular debris adhering to the basement membrane and presence of pinocytotics vesicles and isolated lysosomes were observed. It has been found that the Rachycentron canadum fish species farmed in captivity develop arterial lesion of the chronic inflammatory degenerative type.

Tracking viral particles in the intestinal contents of the American bullfrog, Lithobates catesbeianus, by Transmission Electron Microscopy / Pesquisa de partículas virais em conteúdo intestinal de rãs-touro americanas, Lithobates catesbeianus, por meio da microscopia eletrônica de transmissão

Feces are an important viral agent elimination route for infected carrier animals and in aquatic organisms these pathogenic agents can very rapidly propagate due to the habitation environment. The objective of this work is to track viral particles in the intestinal contents of bullfrogs (Lithobates catesbeianus) from five commercial frog farms in the region of Vale do Paraíba, in the State of São Paulo, Brazil, using negative contrast transmission electron microscopy (TEM). The Coronaviridae, Paramyxoviridae, Parvoviridae and Herpesviridae families were observed and photographed in specimens. This work emphasizes the importance of adopting sanitary measures in commercial farms and confirms that observing feces by TEM is an efficient and rapid diagnostic tool for detecting viral agents...

Sabendo-se que as fezes são uma importante via de eliminação de agentes virais pelos animais portadores e que, por estarem na água, os agentes patogênicos podem se propagar mais rapidamente, objetivou-se a pesquisa de vírus em conteúdo intestinal de rãs-touro (Lithobates catesbeianus) de cinco ranários comerciais na região do Vale do Paraíba, no estado de São Paulo, pela técnica de microscopia eletrônica de transmissão. As famílias Coronaviridae, Paramixoviridae, Parvoviridae e Herpesviridae foram observadas e fotografadas. Este trabalho ressalta a importância da adoção de medidas sanitárias nas criações, além da confirmação de que a observação de fezes pela microscopia eletrônica de transmissão é uma eficiente ferramenta de diagnóstico rápido para agentes virais...

Expression of E-cadherin in pig kidney

E-cadherin is a cell adhesion molecule that plays an important role in maintaining renal epithelial polarity and integrity. The purpose of this study was to determine the exact cellular localization of E-cadherin in pig kidney. Kidney tissues from pigs were processed for light and electron microscopy immunocytochemistry, and immunoblot analysis. E-cadhedrin bands of the same size were detected by immunoblot of samples from rat and pig kidneys. In pig kidney, strong E-cadherin expression was observed in the basolateral plasma membrane of the tubular epithelial cells. E-cadherin immunolabeling was not detected in glomeruli or blood vessels of pig kidney. Double-labeling results demonstrated that E-cadherin was expressed in the calbindin D28k-positive distal convoluted tubule and H(+)-ATPase-positive collecting duct, but not in the aquaporin 1-positive, N-cadherin-positive proximal tubule. In contrast to rat, E-cadherin immunoreactivity was not expressed at detectable levels in the Tamm-Horsfall protein-positive thick ascending limb of pig kidney. Immunoelectron microscopy confirmed that E-cadherin was localized in both the lateral membranes and basal infoldings of the collecting duct. These results suggest that E-cadherin may be a critical adhesion molecule in the distal convoluted tubule and collecting duct cells of pig kidney.

Clinical use of a ceramide-based moisturizer for treating dogs with atopic dermatitis

In humans, skin barrier dysfunction is thought to be responsible for enhanced penetration of allergens. Similar to conditions seen in humans, canine atopic dermatitis (CAD) is characterized by derangement of corneocytes and disorganization of intercellular lipids in the stratum corenum (SC) with decreased ceramide levels. This study was designed to evaluate the effects of a moisturizer containing ceramide on dogs with CAD. Dogs (n = 20, 3~8 years old) with mild to moderate clinical signs were recruited and applied a moisturizer containing ceramide for 4 weeks. Transepidermal water loss (TEWL), skin hydration, pruritus index for canine atopic dermatitis (PICAD) scores, and canine atopic dermatitis extent and severity index (CADESI) scores of all dogs were evaluated. Skin samples from five dogs were also examined with transmission electron microscopy (TEM) using ruthenium tetroxide. TEWL, PICAD, and CADESI values decreased (p < 0.05) and skin hydration increased dramatically over time (p < 0.05). Electron micrographs showed that the skin barrier of all five dogs was partially restored (p < 0.05). In conclusion, these results demonstrated that moisturizer containing ceramide was effective for treating skin barrier dysfunction and CAD symptoms.

Histologia e ultraestrutura do rim e rim cefálico do pacu / Histology and ultrastructure of kidney and cephalic kidney in Pacu

O pacu, Piaractus mesopotamicus, é um teleósteo da Família Characidae, intensivamente cultivado no Brasil devido sua rusticidade, crescimento rápido e fácil adaptação. O conhecimento morfológico dos sistemas corpóreos, incluído órgãos linfóide, se faz necessário, para uma melhor produção no cultivo de peixes, fornecendo subsídios na manutenção dos estoques. O objetivo deste estudo foi descrever morfologicamente o rim e rim cefálico de Piaractus mesopotamicus, analisando os perfis celulares de cada órgão com o uso de microscopia de luz e microscopia eletrônica de transmissão. O resultado da análise macroscópica mostrou que a localização do rim e rim cefálico são as mesmas encontradas na maioria dos teleósteos. O rim apresentou uma forma em "H", onde a região média se expandia sobre a bexiga natatória. O rim cefálico se apresentou como uma dilatação na região cranial do rim, mostrando-se bem visível. Na microscopia eletrônica de transmissão também foram observadas similaridades ultraestruturais com outros teleósteos. Observando nossos resultados concluímos que histologicamente e ultraestruturalmente, os órgãos linfóides rim e rim cefálico de Piaractus mesopotamicus são similares aos de outros teleósteos.

The Pacu, Piaractus mesopotamicus, is a teleostei of the Characidae family, intensively cultivated in Brazil due to its rusticity, fast growth and easy adaptation. Morphological knowledge of the body systems including the lymphoid organs is necessary to improve fish production and supply subsidies for the maintenance of stocks. This study aimed to describe morphologically kidney and head kidney of Piaractus mesopotamicus, analyzing the cellular profile of each organ with the use of light microscopy and electronic transmission microscopy. The result of the macroscopic analysis showed that the localization of the kidney and head kidney is the same as found in the majority of the teleostei. The kidney presented a form in "H", where the medium region is expanded on the natatorium bladder. The head kidney presented a dilatation in the cranial region of the kidney. In the transmission electronic microscopy we found ultrastructural similarities with other teleostei fish. It can be concluded that the lymphoid organs (kidney and head kidney) of Piaractus mesopotamicus are histologically and ultra-structurally similar to the other teleostei.

Pathological findings in fetuses of goats and cattle poisoned by Sida carpinifolia (Malvaceae) / Achados patológicos em fetos de caprinos e bovinos intoxicados por Sida carpinifolia (Malvaceae)

In Brazil, the consumption of Sida carpinifolia by livestock has been associated with neurological diseases linked to lysosomal storage disorders. This paper describes the pathological findings in two caprine fetuses from dams that were experimentally poisoned with S. carpinifolia. The goats were orally dosed with 10 and 13g/kg of a paste of green chopped S. carpinifolia for 30 days and were observed for an additional 15 days period after the last dosage with the plant; thereafter they were euthanized and necropsied. The dams showed only slight clinical signs. The study also includes the findings in one bovine fetus from a naturally S. carpinifolia poisoned cow which showed mild incoordination, generalized tremors, staggering, and frequent falls. The cow was euthanized and necropsied. While there were no significant histopathological changes in the goats, in the cow vacuolation of Purkinje neurons of the cerebellum, pancreatic acinar cells, and thyroid follicular cells were observed. The main microscopic changes observed in the caprine and bovine fetuses were vacuolation in the epithelium of renal tubules, thyroid follicular cells, and Purkinje neurons of the cerebellum. Transmission electron microscopy of sections from CNS of the cow and its fetus revealed vacuoles containing fine granular material surrounded by membrane. Lectin-histochemistry of CNS sections from goat fetuses marked lightly to sWGA lectins, WGA, and Con-A.

No Brasil, o consumo de Sida carpinifolia por animais de produção tem sido associada a doenças neurológicas relacionadas com doença de depósito lisossômico. Este trabalho descreve os achados patológicos observados em dois fetos caprinos de mães que foram experimentalmente intoxicadas por S. carpinifolia. As cabras foram intoxicadas experimentalmente com S. carpinifolia nas doses de 10 e 13g/kg durante 30 dias e foram acompanhadas durante 15 dias após o consumo da planta. Após este período foram eutanasiadas e necropsiadas. O estudo também inclui os achados patológicos encontrados em um feto de uma fêmea bovina intoxicada naturalmente pela planta, que mostrou leve incoordenação, tremores generalizados, andar desequilibrado e quedas frequentes. A vaca foi eutanasiada e necropsiada. Embora não houvesse alterações histológicas significativas nas cabras, vacuolização dos neurônios de Purkinje do cerebelo, das células acinares do pâncreas e nas células foliculares da tireoide foram observadas na vaca. As principais alterações histológicas observadas nos fetos caprinos e no feto bovino foram vacuolização no epitélio dos túbulos renais, nas células foliculares da tireoide e nos neurônios de Purkinje do cerebelo. Na microscopia eletrônica de transmissão do sistema nervoso central da vaca e de seu feto revelaram-se vacúolos contendo material finamente granulado e delimitado por membrana. Na técnica de lectina-histoquímica dos fetos caprinos houve marcação leve no SNC para as lectinas sWGA, WGA e para Con-A.

Experimental poisoning of goats by Ipomoea carnea subsp. fistulosa in Argentina: a clinic and pathological correlation with special consideration on the central nervous system / Intoxicação experimental por Ipomoea carnea subsp. fistulosa em caprinos da Argentina: correlação clínico-patológica, com especial atenção ao sistema nervoso central

Ipomoea carnea subsp. fistulosa, aguapei or mandiyura, is responsible for lysosomal storage in goats. The shrub contains several alkaloids, mainly swansonine which inhibits lysosomal α-mannosidase and Golgi mannosidase II. Poisoning occurs by inhibition of these hydrolases. There is neuronal vacuolation, endocrine dysfunction, cardiovascular and gastrointestinal injury, and immune disorders. Clinical signs and pathology of the experimental poisoning of goats by Ipomoea carnea in Argentina are here described. Five goats received fresh leaves and stems of Ipomoea. At the beginning, the goats did not consume the plant, but later, it was preferred over any other forage. High dose induced rapid intoxication, whereas with low doses, the course of the toxicosis was more protracted. The goats were euthanized when they were recumbent. Cerebrum, cerebellum, medulla oblongata, pons and colliculi, were routinely processed for histology. In nine days, the following clinical signs developed: abnormal fascies, dilated nostrils and abnormal postures of the head, cephalic tremors and nystagmus, difficulty in standing. Subsequently, the goats had a tendency to fall, always to the left, with spastic convulsions. There was lack in coordination of voluntary movements due to Purkinje and deep nuclei neurons damage. The cochlear reflex originated hyperreflexia, abnormal posture, head movements and tremors. The withdrawal reflex produced flexor muscles hypersensitivity at the four legs, later depression and stupor. Abnormal responses to sounds were related to collicular lesions. Thalamic damage altered the withdrawal reflex, showing incomplete reaction. The observed cervical hair bristling was attributed to a thalamic regulated nociceptive response. Depression may be associated with agonists of lysergic acid contained in Ipomoea. These clinical signs were correlated with lesions in different parts of the CNS.

Ipomoea carnea subsp. fistulosa, aguapeí ou mandiyura, causa uma doença de depósito lisossomal em caprinos. A planta contém vários alcalóides, principalmente swansonine, que inibe uma α-mannosidase lisossomal e uma mannosidase II do Golgi. A intoxicação ocorre pela inibição dessas hidrolases. Há vacuolização neuronal, disfunção endócrina, lesões cardiovasculares e gastrointestinais e distúrbios imunológicos. No presente trabalho é descrita a intoxicação experimental por Ipomoea carnea subsp. fistulosa em caprinos da Argentina. Cinco cabras receberam folhas frescas e ramos de Ipomoea. No começo eles não consumiram a planta, mas depois ela foi preferida a qualquer outras forrageiras. Altas doses induziram uma intoxicação rápida, enquanto que com doses baixas demora mais tempo. As cabras foram sacrificadas quando ficavam em decúbito. Cérebro, cerebelo, medula oblonga, ponte e colículos foram processados rotineiramente para histologia. Aos nove dias, encontramos: fascie anormal, narinas dilatadas, posturas anormais da cabeça, tremores cefálicos e nistagmo, dificuldade para permanecer na estação. Posteriormente, presentearam a tendência de queda, sempre à esquerda, com convulsões espásticas. Os neurônios de Purkinje e os os núcleos profundos foram danificados. Como conseqüência, emergiu a falta de coordenação de movimentos voluntários. A hiper-reflexo coclear originou postura anormal, movimentos de cabeça e tremores. A retirada produz reflexos hipersensibilidade nos flexores dos quatro membros, depois depressão e letargia. As respostas anormais estão ligadas a lesões do colículo. Injurias no tálamo alteram o reflexo de retirada, mostrando uma reação incompleta. Os pelos eriçados da região cervical podem representar uma resposta nociceptiva também regulada pelo tálamo. A depressão pode estar associada com agonistas do ácido lisérgico presentes na Ipomoea. Esses sintomas estão relacionados com lesões em diferentes partes do SNC.

Platelet activation: ultrastructure and morphometry in platelet-rich plasma of horses / Ativação plaquetária: ultraestrutura e morfometria no plasma rico em plaquetas de equinos

This study was conducted to investigate the activation ability of the platelet-rich plasma (PRP) by pharmacological agents, as well as to verify the need or not of this activation for therapeutic use. The PRP was obtained from four healthy crossbred geldings aged 13 to 16 years (15±1years), and was processed for observation and quantification of the platelet morphology by using the transmission electron microscopy. All PRP samples were activated with 10 percent calcium chloride (CaCl2) solution, pure bovine thrombin or associated with CaCl2. The control (pure PRP) was not pharmacologically activated. In the pure PRP samples, 49 percent of the platelets were classified as state of activation uncertain, 41 percent as resting, 9 percent as fully activated and 1 percent as irreversibly damaged. Treatment with 10 percent CaCl2 provided a distribution of 54 percent platelets in state of activation uncertain, 24 percent as fully activated, 20 percent as resting, and 2 percent as irreversibly damaged. The platelet morphology of the bovine thrombin treated samples did not fit into classification adopted, as showing irregular shape with emission of large filamentous pseudopods, appearance of ruptured and whole granules in the remaining cytoplasm and extracellular environment. There was effect of the treatment on the platelet morphology (P=0.03). The 10 percent CaCl2 is an adequate platelet-activating agent. However, in cases the use of PRP under its liquid form is necessary, the use of pure PRP is recommended, since besides presenting an adequate percentage of fully activated platelets it also has significant amount of the resting type, which can be activated by substances found in the injured tissue.

O objetivo desse estudo foi investigar a capacidade de ativação do plasma rico em plaquetas (PRP) por substâncias farmacológicas, assim como verificar a necessidade ou não dessa ativação para uso terapêutico. O PRP foi obtido de quatro equinos mestiços hígidos, machos castrados, com 13 a 16 anos (15±1anos) de idade, e processado para observação e quantificação da morfologia plaquetária mediante a utilização da microscopia eletrônica de transmissão. Todas as amostras de PRP foram ativadas com cloreto de cálcio (CaCl2) a 10 por cento, trombina bovina pura ou associada a CaCl2. O controle (PRP puro) não foi ativado farmacologicamente. Nas amostras de PRP puro, 49 por cento das plaquetas foram classificadas como ativação incerta, 41 por cento em repouso, 9 por cento totalmente ativada e 1 por cento com dano irreversível. O tratamento com CaCl2 a 10 por cento proporcionou uma distribuição de 54 por cento de plaquetas com ativação incerta, 24 por cento totalmente ativada, 20 por cento em repouso, e 2 por cento como com dano irreversível. Amostras tratadas com trombina bovina apresentaram morfologia plaquetária que não se enquadraram na classificação adotada, apresentando forma irregular com emissão de grandes pseudópodes filamentosos, aspecto de rompimento e grânulos inteiros no citoplasma remanescente e meio extracelular. Houve efeito do tratamento sobre a morfologia plaquetária (P=0,03). O CaCl2 a 10 por cento é um adequado agente ativador de plaquetas. Entretanto, nos casos onde se faz necessário o uso de PRP na forma mais líquida, recomenda-se o uso do PRP puro, que além de apresentar uma adequada porcentagem de plaquetas totalmente ativadas, também possui importante quantidade do tipo em repouso, que pode ser ativado por substâncias presentes no tecido lesionado.

Markiana nigripinnis (Perugia, 1891) as a putative member of the subfamily Stevardiinae (Characiformes: Characidae): spermatic evidence

The genus Markiana was until recently recognized as incertae sedis in the family Characidae, even though alternative placements for this genus have been advanced since its original description. More recently, it was hypothesized that Markiana nigripinnis is part of a clade informally named the Astyanax clade, indicating the putative close relationship of Markiana with the genus Astyanax. Examination of sperm ultrastructure of representatives of Astyanax and M. nigripinnis shows no evidence for this hypothesized close relationship. Rather, the spermatozoa of M. nigripinnis share characters found in spermatozoa of the non-inseminating members of the subfamily Stevardiinae, such as an angle of nuclear rotation equal to 85º resulting in a lateral position of the double nuclear fossa and flagellum. As with the non-inseminating Stevardiinae, sperm nuclei are also slightly elongate toward the flagellum, the proximal centriole is partially inside the nuclear fossa and anterior and oblique to the distal centriole, and the midpiece is short and strongly asymmetric. Additionally, M. nigripinnis shares with the other members of the Stevardiinae the presence of only four teeth in the inner row of the premaxillary and a short triangular ectopterygoid, which is never more than twice the length of the palatine.

O gênero Markiana até recentemente foi reconhecido como incertae sedis na família Characidae, apesar da localização alternativa para este gênero desde sua descrição original. Mais recentemente, surgiu a hipótese de que Markiana nigripinnis faz parte de um clado chamado informalmente de "Astyanax clade", indicando a suposta relação de Markiana com o gênero Astyanax. A análise da ultraestrutura dos espermatozoides de representantes do gênero Astyanax e M. nigripinnis não mostra nenhuma evidência de estreita relação. Pelo contrário, os espermatozóides de M. nigripinnis compartilham o padrão encontrado nos espermatozoides dos membros não-inseminadores da subfamília Stevardiinae, tais como, um ângulo de rotação nuclear igual a 85º resultando em uma posição de lateral da fossa dupla nuclear e do flagelo. Assim como nos Stevardiinae não inseminadores, os núcleos dos espermatozoides também são ligeiramente alongados em direção ao flagelo, o centríolo proximal é anterior e oblíquo em relação ao centríolo distal e parcialmente inserido na fossa nuclear, e a peça intermediária é curta e fortemente assimétrica. Além disso, M. nigripinnis compartilha com os outros membros da Stevardiinae a presença de apenas quatro dentes na série interna da pré-maxila e um osso ectopterigoide curto e triangular, que nunca ultrapassa o dobro do comprimento do osso palatino.

Transmission electron microscopy for characterization of acrosomal damage after Percoll gradient centrifugation of cryopreserved bovine spermatozoa

The objective of this study was to characterize acrosomal ultrastructure following discontinuous Percoll gradient centrifugation of cryopreserved bovine sperm. Semen was collected from six bulls of different breeds and three ejaculates per bull were evaluated. Frozen semen samples were thawed and the acrosomal region of sperm cells was evaluated by transmission electron microscopy (TEM) before (n = 18) and after (n = 18) Percoll centrifugation. The evaluation of 20 sperm heads from each of the 36 samples analyzed ensured that a large number of cells were investigated. The data were subjected to analysis of variance at a level of significance of 5%. Percoll centrifugation reduced the percentage of sperm exhibiting normal acrosomes (from 61.77 to 30.24%), reduced the percentage of sperm presenting atypical acrosome reactions (from 28.38 to 4.84%) and increased the percentage of sperm exhibiting damage in the acrosome (from 6.14 to 64.26%). The percentage of sperm with typical acrosome reactions was not significantly different before (3.70%) and after (0.67%) centrifugation. TEM distinguished four different types of acrosomal status and enabled ultrastructural characterization of acrosomal injuries. The percentage of sperm exhibiting normal acrosomes decreased and damage in the acrosome was the most frequent acrosomal injury with the Percoll gradient centrifugation protocol utilized.

Doença do armazenamento lisossomal induzida pelo consumo de Sida carpinifolia em bovinos do Rio Grande do Sul / Lysosomal storage disease caused by Sida carpinifolia in cattle in Rio Grande do Sul

This paper reports the natural poisoning by Sida carpinifolia (guanxuma, chá-da-índia) in cattle in Rio Grande do Sul, Brazil. Five cattle were affected in the period 2001-2008. Clinical signs included weight loss, incoordination, walking difficulty, generalized tremors, frequent falls, and death. Microscopically, the main changes were vacuolation of Purkinje neurons in the cerebellum, pancreatic acinar cells, and thyroid follicular cells. Transmission electron microscopy revealed vacuoles bordered by membrane containing finely granular material. Lectin histochemistry showed positive staining in neurons with the lectins Concanavalia ensiformis (Con-A), Triticum vulgaris (WGA), and Succinyl Triticum vulgaris (sWGA).

Relata-se a intoxicação natural por Sida carpinifolia (guanxuma, chá-da-índia) em bovinos no Rio Grande do Sul. Foram afetados cinco bovinos no período 2001-2008. O quadro clínico foi caracterizado por emagrecimento, incoordenação, dificuldade de locomoção, tremores generalizados, quedas frequentes e morte. Microscopicamente, as principais alterações foram vacuolização dos neurônios de Purkinje do cerebelo, das células acinares do pâncreas e das células foliculares da tireoide. A microscopia eletrônica evidenciou vacúolos com conteúdo finamente granulado e delimitado por membrana. Na lectina-histoquímica, observou-se marcação em neurônios com as lectinas Concanavalia ensiformis (Con-A), Triticum vulgaris (WGA) e Succinyl Triticum vulgaris (sWGA).

Carbonated water and necro-apoptotic cell death in chicken embrio low limb / Agua carbonatada y la muerte celular por necro-apoptosis en miembro inferior de embrión de pollo

Carbonated water is a fundamental part of many drinks and its effects have been studied in many pathological situations. However, cells and tissue damage as a consequence of carbonated water has not been the subject of extensive research. We assessed the short-term effects of soda on in vitro Hanging-drop culture of myoblasts and ex vivo lower limb of 8-day-old chicken embryo skeletal muscle tissue. Several groups weren designed: a) Control (Con-tyr), b) Carbonated water (Car), c) Coffe (Caf), and d) Cola beverage (Glu). The samples were observed with light microscopy and digital imaging analysis was performed. The ultra-structure of control and treated tissue were observed with electron microscopy. Immunohistochemistry techniques, such as terminal deoxynucleotidyl transferase-mediated dUTP nick- end labeling (TUNEL), TACTS Blue Label (TdT Kits) of R&D Systems were used. The myoblasts monolayers treated with soda showed plenty of eosinophilics elements. The eosinophily corresponds to higher percentage of cell death. The muscular tissue of the low limb treated with carbonated water (Car) showed calcium phosphate and collagen decreases, 53,86% and 82,95% respectively and enlarged nuclei of a higher size, with an evident loss of the parallel arrangement and fragmented nuclei. Compared to control samples, the muscular disorganization was accompanied by a positive reaction of the apoptotic bodies on TACS, also a positive reaction to ApopTacg and another positive reaction for the metalloproteases in the inter fibrillar cartilage matrix. These changes were not significant in Tyrode's solution controls, Coffee and Cola beverage groups. The morphological outcome can be apoptosis, necrosis or a mixed phenotype, suggesting that the carbonated water toxic effect might be related to these cell death processes. Further research, exploring biochemical factors will be required to elucidate necro-apoptotic cell death induced by carbonated water.

El agua carbonatada constituye una parte fundamental en muchas bebidas y su efecto ha sido estudiado en muchas situaciones patológicas. Sin embargo, el daño celular y de tejido como consecuencia del agua carbonatada no ha sido claramente investigado. El presente trabajo evalúa el efecto agudo in vitro de la soda sobre mioblastos obtenidos por cultivos en gota pendiente y el efecto sobre tejido muscular esquelético in vivo del miembro inferior de pollo de 8 días de desarrollo. Cuatro grupos de embriones fueron seleccionados al azar: a) Control (Con-tyr), b) Agua Carbonatada o Soda Club (Car), c) Café (Caf) y d) Bebida de cola (Glu). Las muestras fueron observadas por microscopía de luz. El análisis de imágenes digitales fue realizado. La ultraestructura del tejido control y tratado fue observada con Microscopía Electrónica de Transmisión (MET). La determinación de apoptosis fue realizada a través de TUNEL y l TACS Blue Label. La actividad de metaloproteasa MMP-1 fue ensayada. La población de mioblastos tratados con Soda Club mostró un elevado número de elementos eosinofílicos interpretado como un elevado número de células muertas, a diferencia del control y el grupo tratado con cafeína y bebida de Cola. En el tejido muscular se determinó una reducción de fosfatos de calcio y fibras de colágeno en una proporción de 53,86% y 82,95% respectivamente, acompañada por un desarreglo de las fibras y núcleos fragmentados, con reacción positiva para cuerpos apoptóticos y metaloproteasas en la matriz interfibrilar. Los resultados sugieren que el efecto tóxico del agua carbonatada sobre células y tejido pudiera estar vinculado con procesos combinados de muerte celular como necro-apoptosis. Se sugiere una mayor exploración de los eventos moleculares para dilucidar la combinación de los procesos de muerte celular sugerida en el presente trabajo.

Effects of ischemia/reperfusion on beta cells of pancreas and protective effects of melatonin treatment / Efectos de la isquemia/reperfusión sobre células beta del páncreas y efectos protectores del tratamiento de melatonina

Oxygen free radicals are considered to be important components involved in the pathophysiological tissue alterations observed during ischemia-reperfusion (I/R). In this study, we investigated the putative protective effects of melatonin treatment on pancreatic I/R injury. Sprague Dawley male rats were subjected to 30 min of pancreatic pedicle occlusion followed by 90 min reperfusion. Melatonin (10 mg/kg. s.c) was administrated 30 min prior to ischemia or I/R application. At the end of the reperfusion periods, rats were decapitated. Pancreatic samples were taken for transmission electron microscopy. The results indicated that ischemia created b cell damage as evidenced by dilatation between the nucleus inner and outer membrane and degeneration on islets of Langerhans cells, was reversed by melatonin treatment. As melatonin administration reversed these microscopic damage, it seems likely that melatonin protects pancreatic tissue against oxidative damage.

Los radicales libres del oxígeno son considerados como uno de los componentes más importantes que participan en las alteraciones fisiopatológicas del tejido durante la isquemia-reperfusión (I/R). En este estudio, se investigó el supuesto efecto protector del tratamiento de melatonina sobre la lesión pancreática I/R. Ratas Sprague Dawley machos fueron sometidas a 30 minutos de oclusión del pedículo pancreático seguido de 90 minutos de reperfusión. La melatonina (10 mg/kg) fue administrada 30 minutos antes de la isquemia o de la aplicación I/R. Al finalizar los periodos de reperfusión, las ratas fueron decapitadas. Fueron tomadas muestras pancreáticas para el análisis en microscopía electrónica de transmisión. Los resultados indicaron que la isquemia ocasionó daño en las células beta demostrado por la dilatación entre el núcleo interior y la membrana exterior y la degeneración de los islotes de células pancreáticas, los que fueron revertidos por el tratamiento de melatonina. Como la administración de melatonina revirtió estos daños microscópicos, parece probable que ella proteja al tejido pancreático contra el daño oxidativo.

Ultrastructural study of poxvirus causing myxomatosis in rabbits, in São Paulo and Santa Catarina, Brazil / Estudio ultraestructural del poxvirus causal de mixomatosis en conejos, en São Paulo y Santa Catarina, Brasil

The myxomatosis is a contagious worldwide disease caused by poxvirus which infects domestic and wild rabbits. In the present study we present two distinct outbreaks of myxomatosis when raising rabbits, one for commercial purpose of production of meat and skins and, another one for the commercialization of ornamental rabbits. The observed signs were ocular, auricular, nasal, testis lesions and many times scattered throughout the body of the animals. The lesions were characterized by formation of nodules that by palpation disclosed gummy or gelatinous aspect. At the transmission electron microscopy, all the skin and crust samples were analyzed by negative staining technique. A great number of particles with morphology similar to the poxvirus, some enveloped in a brick-shaped and irregular disposition of tubules on the external membrane, measuring 300x240 nm on the average were visualized. Ultra thin sections revealed the presence of intracytoplasmic inclusion bodies surrounded by membrane containing oval particles, measuring 270 x 130 nm, containing nucleus or an internal biconcave (dumbbell-shaped) core. Immature particles (empty), surrounded by membrane were also observed. In addition, intracytoplasmic electron dense inclusion bodies containing viral particles budding of dense amorphous material and intranuclear fibrillar or "digital" inclusions showing a regular striation and arranged in groups were found in the middle of granular material. The nuclei were deformed with densely condensed chromatin forming amorphous and electron dense inclusion bodies. In the immunocytochemistry technique, the antigen-antibody reaction was strongly marked by the particles of colloidal gold, emphasizing the viral particles. The techniques used in this study were important in the diagnosis of the affected animals.

La mixomatosis es una enfermedad contagiosa de distribución mundial, causada por poxvirus que infecta conejos domésticos y salvajes. En este estudio presentamos dos distintos surtos por mixomatosis que ocurrieron en producciones de conejos, una para fines comerciales de producción de carne y pieles y otra para el comercio de conejos domésticos. Las señales observadas fueron afecciones oculares, nasales, testiculares y, a veces, también distribuida por todo el cuerpo de los animales. Estas se caracterizaban por formación de nódulos que a la palpación tenían un aspecto gelatinoso o gomoso. En la microscopía electrónica de transmisión, por la técnica de contrastación negativa, se pudo observar en todas las muestras examinadas de piel y de costras, un gran número de partículas típicas de poxvirus, con envoltura y forma de ladrillo, mostrando disposición irregular de los túbulos sobre la membrana externa, midiendo 300 x 240 nm en el promedio. Cortes ultrafinos de fragmentos de piel y de costras revelaron la presencia de cuerpos de inclusión intracitoplasmáticas, envueltos por membrana y conteniendo partículas ovales, midiendo 270 x 130 nm, conteniendo núcleo o centro interno bicóncavo (forma de mancuernas). Partículas inmaduras (vacías) envueltas por membrana fueron observadas. También fueron analizados cuerpos de inclusión intracitoplasmáticos, electrodensos, conteniendo partículas virales brotando del material denso y amorfo. Fueron observadas inclusiones intranucleares fibrilares o "digitales" mostrando una estriación periódica y disposición en grupos en medio del material granular. Los núcleos estaban deformados con cromatina densamente condensada formando cuerpos de inclusiones electrodensas y amorfas. En la técnica de imunocitoquímica la reacción antígeno-anticuerpo fue intensamente marcada por las partículas de oro coloidal realzando fuertemente las partículas virales.

Erythropoiesis and erythrophagocytosis in bovine haemal nodes / Eritropoyesis y eritrofagocitosis en nodos linfáticos hemales de bovino

Whether bovine haemal nodes are involved in turnover of red blood cells has been a subject of some controversy In this study, fluorescent and conventional optical microscopy of conventionally or immunohistochemically stained node sections, together with transmission electron microscopy, showed the presence of erythrocyte precursors and megakaryocytes, and evidence of active involvement in the destruction and replacement of old or degenerate red cells and the platelets.

Si nodos linfáticos hemales bovinos están involucrados en la cantidad de volumen de glóbulos rojos ha sido objeto de cierta controversia. En este estudio, secciones de nodos linfáticos teñidas convencionalmente o inmunohistoquimicamente fueron analizadas con microscopía óptica fluorescente y convencional, junto con microscopio electrónico de transmisión, los que revelaron la presencia de precursores eritrocíticos y megacariocitos, y la evidencia de participación activa en la destrucción y sustitución de glóbulos rojos viejos o degenerados y plaquetas.

Immunolocalization of anion exchanger 1 (Band 3) in the renal collecting duct of the common marmoset

The purpose of this study was to determine the expression and distribution of band 3 in the collecting duct and connecting tubules of the kidney of the marmoset monkey (Callithrix jacchus), and to establish whether band 3 is expressed in type A intercalated cells. The intracellular localization of band 3 in the different populations of intercalated cells was determined by double-labeling immunohistochemistry. Immunohistochemical microscopy demonstrated that band 3 is located in the basolateral plasma membranes of all type A intercalated cells in the connecting tubule (CNT), cortical collecting duct (CCD), and outer medullary collecting duct (OMCD) of the marmoset. However, type B intercalated cells and non-A/ non-B intercalated cells did not show band 3 labeling. Electron microscopy of the CNT, CCD and OMCD confirmed the light microscopic observation of the basolateral plasma membrane staining for band 3 in a subpopulation of interacted cells. Basolateral staining was seen on the plasma membrane and small coated vesicles in the perinuclear structure, some of which were located in the Golgi region. In addition, there was no labeling of band 3 in the mitochondria of the CNT, CCD and in OMCD cells. The intensity of the immunostaining of the basolateral membrane was less in the CNT than in the CCD and OMCD. In contrast, band 3 immunoreactivity was greater in the intracellular vesicles of the CNT. From these results, we suggest that the basolateral Cl-/HCO3- exchanger in the monkey kidney is in a more active state in the collecting duct than in the CNT.

Hematology, cytochemistry and ultrastructure of blood cells in fishing cat (Felis viverrina)

Hematological, cytochemical and ultrastructural features of blood cells in fishing cat (Felis viverrina) were evaluated using complete blood cell counts with routine and cytochemical blood stains, and scanning and transmission electron microscopy. No statistically significant difference was found in different genders of this animal. Unique features of blood cells in this animal were identified in hematological, cytochemical and ultrastructural studies. This study contributes to broaden hematological resources in wildlife animals and provides a guideline for identification of blood cells in the fishing cat.