Full confluency, serum starvation, and roscovitine for inducing arrest in the G0/G1 phase of the cell cycle in puma skin-derived fibroblast lines

The puma population is constantly decreasing, and cloning by somatic cell nuclear transfer can be used to conserve the species. One of the factors determining the success of the development of cloned embryos is the cell cycle stage of the donor cells. We evaluated the effects of full confluency (~100%), serum starvation (0.5% serum), and roscovitine (15 µM) treatments on the cell cycle synchronization in G0/G1 of puma skin-derived fibroblasts by flow cytometric analysis. Also, we assessed the effects of these synchronization methods on morphology, viability, and apoptosis levels using microscopy tools. The results showed that culturing the cells to confluence for 24 h (84.0%), 48 h (84.6%), and 72 h (84.2%) and serum starvation for 96 h (85.4%) yielded a significantly higher percentage of cells arrested in the G0/G1 (P < 0.05) phase than cells not subjected to any cell cycle synchronization method (73.9%). Nevertheless, while serum starvation reduced the percentage of viable cells, no difference was observed for the full confluence and roscovitine treatments (P > 0.05). Moreover, roscovitine for 12 h (78.6%) and 24 h (82.1%) was unable to synchronize cells in G0/G1 (P > 0.05). In summary, full confluency induces puma fibroblast cell cycle synchronization at the G0/G1 stage without affecting cell viability. These outcomes may be valuable for planning donor cells for somatic cell nuclear transfer in pumas.(AU)

Isolation and amplification of Anaplasma marginale in chicken embryo fibroblast cell culture / Isolamento e amplificação de Anaplasma marginale em cultivos celulares de fibroblastos de embriões de galinhas

Anaplasma marginale (A. marginale) is a worldwide pathogen that infects a variety of ruminants, but mostly cattle. The present study aimed to describe an isolation technique for A. marginale, using chicken embryo Þ broblast (CEF) cell culture. Blood and tick samples were collected from 5 calves from 2 to 3 months old, which were considered to be infected with A.marginale due to anemia, jaundiced mucous membranes, and prostration. DNA extraction and PCR were performed for diagnosis using blood and tick samples. All tick and blood samples tested positive in PCR. Additionally, ticks were crushed with the aid of a blender for inoculation in CEF cell culture. After inoculation, the cultures were kept at 37ºC and 5% CO2 for 15 days. The cell supernatant of cell cultures was again analyzed using PCR and Wright stain method to conÞ rm A. marginale isolation. Cell cultures tested positive in PCR, and the presence of the agent was demonstrated by Wright stain. Therefore, by using CEF cell culture it was possible to isolate and amplify the A. marginale in a concentration of 1.3 x 107.2 bodies per mL. The CEF cells are undemanding and easy to preserve; they are an option for isolation and production of A. marginale under laboratory conditions.(AU)

Anaplasma marginale (A. marginale) é um patógeno mundial que infecta uma variedade de ruminantes, mas principalmente bovinos. O presente estudo teve como objetivo descrever uma técnica de isolamento para A. marginale, utilizando cultivo celular de Þ broblastos de embriões (CFE) de galinhas. Para isso, foram coletadas amostras de sangue e de carrapatos de 5 bezerros, entre 2 e 3 meses de idade, os quais, devido a anemia, icterícia de mucosas e prostração, foram considerados supostamente infectados com A. marginale. Ethics Approval This study was approved by Credenciamento Institucional para Atividades com Animais em Ensino ou Pesquisa (CIAEP: 02.0420.2021). Consent to participate Not applicable Consent to publish Not applicable Data availability Not applicable Para o diagnóstico, realizaram-se extração de DNA e posterior PCR a partir das amostras de sangue e de carrapatos coletados. Todos os carrapatos e amostras de sangue foram positivas para o teste de PCR. Além disso, os carrapatos foram triturados com o auxílio de um liquidiÞ cador para inoculação em CFE. Após a inoculação, as culturas foram mantidas a 37ºC e a 5% de CO2 durante 15 dias. O sobrenadante celular das culturas foi novamente analisado por PCR e pela técnica de coloração de Wright para conÞ rmar o isolamento de Anaplasma marginale. As culturas celulares foram po sitivas por PCR, e a presença do agente foi comprovada por meio da coloração de Wright. Portanto, utilizando CFE, foi possível isolar e ampliÞ car o A. marginale em uma concentração de 1,3x107,2 bactérias por ml. As células da CEF são pouco exigentes, de fácil manutenção e uma boa opção para isolamento e produção de A. marginale em condição laboratorial.(AU)

Walnut ointment promotes full-thickness burning wound healing: role of linoleic acid

Purpose: To investigate the active ingredients of walnut ointment (WO) and its mechanism in repairing wounds. Methods: The ingredients of WO were detected by gas chromatography­mass spectrometry. The effect of linoleic acid (LA) was tested by in vitro Alamar Blue (AB) reagent. Image J software, histological and immunohistochemical analysis were used to confirm the healing effect of LA in the porcine skin model. The animals were euthanized after the experiment by injection of pentobarbital sodium. Results: LA, 24% in WO, promotes keratinocytes and fibroblasts proliferation, which were 50.09% and 15.07% respectively higher than control (p < 0.05). The healing rate of the LA group (96.02% ± 2%, 98.58% ± 0.78%) was higher than the saline group (82.11% ± 3.37%, 88.72% ± 1.73%) at week 3 and week 4 (p < 0.05). The epidermal thickness of the LA was 0.16 ± 0.04 mm greater and the expression of the P63 and CK10 proteins was stronger in the LA group than the control (p < 0.05). Conclusions: LA, which is the main components in WO can promote full-thickness burning wounds (FBWs) by stimulating cell proliferation and differentiation.

Viability of fibroblasts from "Curraleiro Pé Duro" cattle after different cryopreservation protocols / Viabilidade de fibroblastos de bovinos da raça Curraleiro Pé Duro submetidos a diferentes protocolos de criopreservação

A raça tropicalmente adaptada Curraleiro Pé Duro (CPD) possui grande rusticidade e capacidade de produção. A técnica de criopreservação de células somáticas permite estocar, por tempo indeterminado, o material genético. O objetivo deste trabalho é avaliar a viabilidade de fibroblastos, pós-criopreservação, em protocolos diferentes. Foi utilizada uma biópsia auricular de um bovino CPD, que passou por antissepsia e anestesia local. Posteriormente o material foi processado e incubado para ser observado quanto à confluência e à morfologia. Em seguida, os fibroblastos foram criopreservados em três tratamentos (T1, T2 e T3). Após serem criopreservados, foram descongelados e analisados quanto à viabilidade celular, à capacidade de crescimento e à morfologia. Na análise de variância e das médias, foram comparados pelo teste de Tukey com significância de 5%. Não foram observadas, nos protocolos, diferenças estatísticas entre a viabilidade celular (T1 = 67,98%, T2 = 71,42% e T3 = 69,93%), a capacidade de confluência (T1 = 80%, T2 = 90%, T3 = 85%) ou a passagem de origem das células. A criopreservação de fibroblastos auriculares de bovinos CPD não mostrou diferença entre os três métodos, sugerindo até que o método que demanda equipamentos menos especializados (T1) é tão eficiente quanto os demais.

Cytotoxicity and the bioconversion strategy of Aristolochia spp

Aristolochia plants are notable from an ethnopharmacological viewpoint, but the relevance of these species for medicinal purposes has been debated because of their inherent toxicity. The convergence of these contrasting realities can be readily achieved using bioconversion methods, which have been shown to be useful tools for numerous applications, including the detoxification of biomass. In this context, methanolic extracts of leaves from Aristolochia triangularis and Aristolochia gibertii, as well as the feces of Battus polydamas larvae fed with leaves from these plants, were prepared, and their cytotoxic activities were evaluated on a human fibroblast cell line (GM07492). The leaf extracts were found to be cytotoxic, leading to reductions of 42.1 and 33.8% on cell viability, respectively, while the fecal extracts were considered inactive. In addition to evidencing the cytotoxicity of A. triangularis and A. gibertii, these findings demonstrated a potential bioconversion strategy for obtaining aristolochiaceous extracts with reduced toxicity using the larvae of a specialist phytophagous insect, thus renewing expectations in relation to the pharmacological importance of Aristolochia spp. The results were also ecologically relevant, as B. polydamas larvae were found to be able to detoxify compounds from host plants.(AU)

Cytotoxicity and the bioconversion strategy of Aristolochia spp

Aristolochia plants are notable from an ethnopharmacological viewpoint, but the relevance of these species for medicinal purposes has been debated because of their inherent toxicity. The convergence of these contrasting realities can be readily achieved using bioconversion methods, which have been shown to be useful tools for numerous applications, including the detoxification of biomass. In this context, methanolic extracts of leaves from Aristolochia triangularis and Aristolochia gibertii, as well as the feces of Battus polydamas larvae fed with leaves from these plants, were prepared, and their cytotoxic activities were evaluated on a human fibroblast cell line (GM07492). The leaf extracts were found to be cytotoxic, leading to reductions of 42.1 and 33.8% on cell viability, respectively, while the fecal extracts were considered inactive. In addition to evidencing the cytotoxicity of A. triangularis and A. gibertii, these findings demonstrated a potential bioconversion strategy for obtaining aristolochiaceous extracts with reduced toxicity using the larvae of a specialist phytophagous insect, thus renewing expectations in relation to the pharmacological importance of Aristolochia spp. The results were also ecologically relevant, as B. polydamas larvae were found to be able to detoxify compounds from host plants.(AU)

Escherichia coli vacuolating factor, involved in avian cellulitis, induces actin contraction and binds to cytoskeleton proteins in fibroblasts

Avian pathogenic Escherichia coli (APEC) isolated from avian cellulitis lesions produces a toxin, named Escherichia coli vacuolating factor (ECVF), that causes cell vacuolization and induces inflammatory response in broiler chicken. Methods We investigated the intracellular activities of ECVF in avian fibroblasts using fluorescence staining, electron microscopy, MTT and LDH measurements. As ECVF act specifically in avian cells, we performed blotting assay followed by mass spectrometry to better understand its initial intracellular protein recognition. Results ECVF induced actin contraction, mitochondrial damage and membrane permeability alterations. Ultrastructural analysis showed intracellular alterations, as nuclear lobulation and the presence of degraded structures inside the vacuoles. Moreover, ECVF induced cell death in fibroblasts. ECVF-biotin associates to at least two proteins only in avian cell lysates: alpha-actinin 4 and vinculin, both involved in cytoskeleton structure. Conclusion These findings demonstrated that ECVF plays an important role in avian cellulitis, markedly in initial steps of infection. Taken together, the results place this toxin as a target for drug and/or vaccine development, instead of the use of large amounts antibiotics.(AU)

Escherichia coli vacuolating factor, involved in avian cellulitis, induces actin contraction and binds to cytoskeleton proteins in fibroblasts

Avian pathogenic Escherichia coli (APEC) isolated from avian cellulitis lesions produces a toxin, named Escherichia coli vacuolating factor (ECVF), that causes cell vacuolization and induces inflammatory response in broiler chicken. Methods We investigated the intracellular activities of ECVF in avian fibroblasts using fluorescence staining, electron microscopy, MTT and LDH measurements. As ECVF act specifically in avian cells, we performed blotting assay followed by mass spectrometry to better understand its initial intracellular protein recognition. Results ECVF induced actin contraction, mitochondrial damage and membrane permeability alterations. Ultrastructural analysis showed intracellular alterations, as nuclear lobulation and the presence of degraded structures inside the vacuoles. Moreover, ECVF induced cell death in fibroblasts. ECVF-biotin associates to at least two proteins only in avian cell lysates: alpha-actinin 4 and vinculin, both involved in cytoskeleton structure. Conclusion These findings demonstrated that ECVF plays an important role in avian cellulitis, markedly in initial steps of infection. Taken together, the results place this toxin as a target for drug and/or vaccine development, instead of the use of large amounts antibiotics.(AU)

Desafios para a reprodução assistida em animais de vida livre / Challenges for assisted reproduction in free-living animals

A reprodução assistida se faz necessária em programas de conservação de espécies ameaçadas de extinção, sendo um facilitador de transporte e troca de material genético. Neste contexto, o acesso ao material de animais de vida livre é essencial para incrementar o banco genético da espécie em questão, no entanto adaptar os métodos possíveis à realidade do campo torna essa área de pesquisa desafiadora. Ainda hoje os espermatozoides são os gametas mais acessados em animais de vida livre, porém com pouco uso efetivo para criopreservação e produção de filhotes. É pungente a necessidade de mais pesquisas nesta área, uma vez que há centenas de espécies brasileiras ameaçadas, com especificidades fisiológicas e que habitam habitats variados, o que demanda adaptações espécie-específicas e hábitat específicas.

Assisted reproduction is necessary for conservation programs for endangered species, facilitating transport and exchange of genetic material. In this context, access to material from free-living animals is essential to increase the genetic bank of the species in question. However, adapting the possible methods to the reality of the fieldwork makes this area of research a challenge. Even today, sperm are the most accessed gametes in free-living animals, but with little effective use for cryopreservation and production of offspring. The need for more research in this area is acute, as there are hundreds of Brazilian species under threat, with physiological specificities, and that inhabit varied habitats, which demand species-specific adaptations and specific habitats.

Estabelecimento de linhagens celulares em felídeos silvestres Uma revisão dos efeitos das condições de cultivo e criopreservação sobre a qualidade celular / Establishment of cell lines in wild felids A review of the effects of culture and cryopreservation conditions on cell quality

Linhagens celulares, principalmente fibroblastos derivados da pele, podem ser ferramentas interessantes para a conservação e multiplicação de felídeos silvestres ameaçados de extinção. Esses animais em virtude de seu quantitativo reduzido têm nos bancos de células somáticas uma alternativa para a conservação de material biológico derivado de pequenas populações, garantindo assim o armazenamento da diversidade genética de diferentes grupos de indivíduos. Isso porque tais linhagens, quando apropriadamente estabelecidas por meio das técnicas de cultivo in vitro e criopreservação, permitem seu emprego na obtenção de embriões por clonagem por transferência nuclear de célula somática e produção de células induzidas à pluripotência. Assim, uma das etapas essenciais para o uso dessas linhagens de maneira eficiente consiste na avaliação dos efeitos das condições de cultivo in vitro e criopreservação das células, visando reconhecer os danos gerados pelas manipulações e identificar os ajustes necessários aos protocolos empregados. Portanto, o objetivo desta revisão consiste em reunir e explorar informações úteis sobre as condições de cultivo in vitro e criopreservação de células derivadas de felídeos silvestres, visando o estabelecimento de linhagens celulares na conservação e multiplicação destas espécies.(AU)

Cell lines, mainly fibroblasts derived from the skin, can be interesting tools for the conservation and multiplication of endangered wild felids. These animals, due to their reduced quantity, have somatic cell banks as an alternative for the conservation of biological material derived from small populations, thus guaranteeing the storage of the genetic diversity of different groups of individuals. This is because such lines, when properly established through in vitro culture and cryopreservation techniques, allow their use in obtaining embryos by cloning by somatic cell nuclear transfer and production of cells induced to pluripotency. Thus, one of the essential steps for the use of these lines in an efficient way consists of the evaluation of the effects of the conditions of in vitro culture and cryopreservation of the cells, aiming to recognize the damages generated by the manipulations and to identify the necessary adjustments to the employed protocols. Therefore, the aim of this review is to gather and explore useful information about in vitro culture and cryopreservation conditions of cells derived from wild felids, aiming at the establishment of cell lines in the conservation and multiplication of these species.(AU)

Estabelecimento de linhagens celulares em felídeos silvestres – Uma revisão dos efeitos das condições de cultivo e criopreservação sobre a qualidade celular / Establishment of cell lines in wild felids – A review of the effects of culture and cryopreservation conditions on cell quality

Linhagens celulares, principalmente fibroblastos derivados da pele, podem ser ferramentas interessantes para a conservação e multiplicação de felídeos silvestres ameaçados de extinção. Esses animais em virtude de seu quantitativo reduzido têm nos bancos de células somáticas uma alternativa para a conservação de material biológico derivado de pequenas populações, garantindo assim o armazenamento da diversidade genética de diferentes grupos de indivíduos. Isso porque tais linhagens, quando apropriadamente estabelecidas por meio das técnicas de cultivo in vitro e criopreservação, permitem seu emprego na obtenção de embriões por clonagem por transferência nuclear de célula somática e produção de células induzidas à pluripotência. Assim, uma das etapas essenciais para o uso dessas linhagens de maneira eficiente consiste na avaliação dos efeitos das condições de cultivo in vitro e criopreservação das células, visando reconhecer os danos gerados pelas manipulações e identificar os ajustes necessários aos protocolos empregados. Portanto, o objetivo desta revisão consiste em reunir e explorar informações úteis sobre as condições de cultivo in vitro e criopreservação de células derivadas de felídeos silvestres, visando o estabelecimento de linhagens celulares na conservação e multiplicação destas espécies.

Cell lines, mainly fibroblasts derived from the skin, can be interesting tools for the conservation and multiplication of endangered wild felids. These animals, due to their reduced quantity, have somatic cell banks as an alternative for the conservation of biological material derived from small populations, thus guaranteeing the storage of the genetic diversity of different groups of individuals. This is because such lines, when properly established through in vitro culture and cryopreservation techniques, allow their use in obtaining embryos by cloning by somatic cell nuclear transfer and production of cells induced to pluripotency. Thus, one of the essential steps for the use of these lines in an efficient way consists of the evaluation of the effects of the conditions of in vitro culture and cryopreservation of the cells, aiming to recognize the damages generated by the manipulations and to identify the necessary adjustments to the employed protocols. Therefore, the aim of this review is to gather and explore useful information about in vitro culture and cryopreservation conditions of cells derived from wild felids, aiming at the establishment of cell lines in the conservation and multiplication of these species.

Efeito do extrato de Bidens pilosa L, Mel e pomadas homeopática e alopática na cicatrização de feridas cutâneas de ratos Wistar / [Effect of Bidens pilosa L, Honey extract and homeopathic and allopathic ointments on the healing of skin wounds of Wistar rats]

Cicatrização de ferida é um processo dinâmico, que tem por objetivo restaurar a continuidade do tecido lesionado. No entanto, em alguns casos, é necessário favorecer condições adequadas para viabilizar o processo fisiológico. Neste estudo foram utilizados ratos Wistar, divididos aleatoriamente entre cinco grupos, com 12 animais cada, sendo eles: grupo P (Bidens pilosa L.), grupo mel, grupo Co1 (pomada comercial alopática), grupo Co2 (pomada comercial homeopática) e grupo CT (controle). As lesões foram geradas por incisão com punch de 8mm, sendo tratadas diariamente de forma tópica. Foram eutanasiados quatro animais por grupo, no terceiro, sétimo e 14º dias do experimento, e o material coletado foi armazenado em formalina 10% e encaminhado para processamento histológico. Posteriormente, realizou-se a contagem de leucócitos mononucleares, fibroblastos e neovasos e avaliou-se a arquitetura de fibras colágenas. Os resultados da contagem foram analisados pela ANOVA, seguida pelo teste de Tukey (P<0,05). O modelo experimental proposto neste estudo demonstrou que todos os tratamentos apresentaram potencial cicatrizante, com exceção do mel. A aplicação tópica do creme do extrato de Bidens pilosa L. a 10% apresentou melhor perfil anti-inflamatório; a pomada alopática apresentou boa aderência à superfície da lesão e a pomada homeopática, grande potencial angiogênico, com menor tempo de cicatrização.(AU)

Wound healing is a dynamic process that aims to restore the continuity of injured tissue. However, in some cases it is necessary to favor adequate conditions to enable the physiological process. Wistar rats were randomly divided into 5 groups with 12 animals each, namely: group P (Bidens pilosa L.), group honey, group Co1 (commercial allopathic ointment), group Co2 (commercial homeopathic ointment) and group CT (control). The lesions were generated by an 8mm punch incision and were treated topically daily. Four animals per group were euthanized on the 3rd, 7th and 14th day of the experiment and the collected material was stored in 10% formalin and sent for histological processing, after which mononuclear, fibroblasts and neovascular leukocytes were counted and collagen fiber architecture was evaluated. Counting results were analyzed by ANOVA, followed by Tukey test (p <0.05). The experimental model proposed in this study showed that all treatments had healing potential, except honey. The topical application of 10% Bidens pilosa L. extract cream showed the best anti-inflammatory profile; Allopathic ointment showed good adhesion to the surface of the lesion and homeopathic ointment showed great angiogenic potential with shorter healing time.(AU)

Efeito do extrato de Bidens pilosa L, mel e pomadas homeopática e alopática na cicatrização de feridas cutâneas de ratos Wistar / [Effect of Bidens pilosa L, honey extract and homeopathic and allopathic ointments on the healing of skin wounds of Wistar rats]

Cicatrização de ferida é um processo dinâmico, que tem por objetivo restaurar a continuidade do tecido lesionado. No entanto, em alguns casos, é necessário favorecer condições adequadas para viabilizar o processo fisiológico. Neste estudo foram utilizados ratos Wistar, divididos aleatoriamente entre cinco grupos, com 12 animais cada, sendo eles: grupo P (Bidens pilosa L.), grupo mel, grupo Co1 (pomada comercial alopática), grupo Co2 (pomada comercial homeopática) e grupo CT (controle). As lesões foram geradas por incisão com punch de 8mm, sendo tratadas diariamente de forma tópica. Foram eutanasiados quatro animais por grupo, no terceiro, sétimo e 14º dias do experimento, e o material coletado foi armazenado em formalina 10% e encaminhado para processamento histológico. Posteriormente, realizou-se a contagem de leucócitos mononucleares, fibroblastos e neovasos e avaliou-se a arquitetura de fibras colágenas. Os resultados da contagem foram analisados pela ANOVA, seguida pelo teste de Tukey (P<0,05). O modelo experimental proposto neste estudo demonstrou que todos os tratamentos apresentaram potencial cicatrizante, com exceção do mel. A aplicação tópica do creme do extrato de Bidens pilosa L. a 10% apresentou melhor perfil anti-inflamatório; a pomada alopática apresentou boa aderência à superfície da lesão e a pomada homeopática, grande potencial angiogênico, com menor tempo de cicatrização.(AU)

Wound healing is a dynamic process that aims to restore the continuity of injured tissue. However, in some cases it is necessary to favor adequate conditions to enable the physiological process. Wistar rats were randomly divided into 5 groups with 12 animals each, namely: group P (Bidens pilosa L.), group honey, group Co1 (commercial allopathic ointment), group Co2 (commercial homeopathic ointment) and group CT (control). The lesions were generated by an 8mm punch incision and were treated topically daily. Four animals per group were euthanized on the 3rd, 7th and 14th day of the experiment and the collected material was stored in 10% formalin and sent for histological processing, after which mononuclear, fibroblasts and neovascular leukocytes were counted and collagen fiber architecture was evaluated. Counting results were analyzed by ANOVA, followed by Tukey test (p <0.05). The experimental model proposed in this study showed that all treatments had healing potential, except honey. The topical application of 10% Bidens pilosa L. extract cream showed the best anti-inflammatory profile; Allopathic ointment showed good adhesion to the surface of the lesion and homeopathic ointment showed great angiogenic potential with shorter healing time.(AU)

Primary fibroblast cell cycle synchronization and effects on handmade cloned (hmc) bovine embryos / Sincronização do ciclo celular de fibroblastos primários e efeitos na produção de embriões bovinos clonados por handmade (hmc)

Spatial and temporal synchrony and compatibility between the receptor oocyte and the donor cell nucleus are necessary for the process of embryo cloning to allow nuclear reprogramming and early embryonic development. The objective of the present study was to evaluate three cell cycle synchronization methods on a primary bovine fibroblast culture for 24, 48, or 72 h. These fibroblasts were used as nuclear donors to evaluate their in vitro developmental potential and the quality of the embryos produced through handmade cloning (HMC). No differences were found between the methods used for fibroblast synchronization in G0/G1 (p > 0.05). Production of clones from fibroblasts in four groups- no treatment at 0 h and using serum restriction SR, high culture confluence HCC, and SR+HCC at 24 h- resulted in high cleavage rates that were not different. Embryo production rates were 37.9%, 29.5%, and 30.9% in the 0h, SR24h, and SR+HHC24h groups, respectively, and 19.3% in the HCC group, which was significantly different from the other three (p < 0.05). There were no differences in the quality parameter among the clones produced with fibroblasts subjected to the different synchronization. Finally, when overall clone production was compared versus parthenotes and IVF embryos, the only difference was between clones and parthenogenetic embryos with zona pellucida (30.2% vs 38.6%). The number of blastomeres from the blastocytes produced through IVF was significantly greater than those from embryos activated parthenogenetically and from clones (117, 80, 75.9, and 67.1, respectively). The evaluation of three synchronization methods at different time points did not demonstrate an increase in the percentage of fibroblasts in the G0/G1 phases of the cell cycle; however, good quality and high cloning rates were obtained, suggesting that it is not always necessary to subject the cells to any synchronization treatments, as they would yield equally good cloning results.

A sincronia espacial e temporal e a compatibilidade entre o oócito receptor e o núcleo celular doador são necessárias para o processo de clonagem de embriões a fim de permitir a reprogramação nuclear e o desenvolvimento embrionário precoce. O objetivo do presente estudo foi avaliar três métodos de sincronização do ciclo celular em uma cultura primária de fibroblastos bovinos durante 24, 48 ou 72 h. Estes fibroblastos foram utilizados como doadores nucleares para avaliar o seu potencial de desenvolvimento in vitro e a qualidade dos embriões produzidos por meio da técnica de Handmade cloning (HMC). Não foram encontradas diferenças entre os métodos utilizados para a sincronização de fibroblastos em G0 / G1 (p> 0,05). Produção de clones de fibroblastos nos quatro grupos - sem tratamento a 0 h e com restrição de soro RS, alta confluência celular ACC e RS + ACC às 24 h - resultou em altas taxas de clivagem que não foram diferentes. As taxas de produção de embriões foram de 37,9%, 29,5% e 30,9% nos grupos 0h, RS24h e RS + ACC24h, respectivamente, e 19,3% no grupo ACC, que foi significativamente diferente dos outros três (p <0,05). Não houve diferenças no parâmetro de qualidade entre os clones produzidos com fibroblastos submetidos à sincronização diferente. Finalmente, quando a produção geral de clones foi comparada versus partenotos e embriões de FIV, a única diferença foi entre clones e embriões partenogênicos com zona pelúcida (30,2% vs 38,6%). O número de blastômeros dos blastocitos produzidos através da FIV foi significativamente maior do que os de embriões ativados partenogeneticamente e de clones (117, 80, 75,9 e 67,1, respectivamente).

Fibroblastic osteosarcoma of scapula in a dog Scottish Terrier: case report / Osteossarcoma fibroblástico de escápula em cão Scottish Terrier: relato de caso

Osteosarcoma (OSA) is the most primary bone tumor observed in dogs. It a malignant neoplasias that affects the bone formation being with aggressive clinical-histological behavior a fast growth and high metastatic potential. Is observed in middle aged or older dogs with large or giant breed and restricts to the metaphysis of long bones of appendicular skeleton. In routine there was a Scottish Terrier dog attended, male, ten years old, that arrived intermittent claudication of the left forelimb, sensitivity to palpation and edema of firm consistency in the cervicothoracic region, with no previous historic of fractures or trauma. The association of clinical signs with the radiographic cytological and histopathological aspects resulted in the definitive diagnosis of fibroblastic OSA in scapula. The treatment was the amputation of LTM, promoting pain relief with little or none loss of quality of life.

Osteossarcoma (OSA) é o tumor ósseo primário mais observado em cães. Trata-se de uma neoplasia maligna que afeta a formação do osso, com comportamento clínico-biológico agressivo, crescimento rápido e altamente metastático. É mais observado em cães de meia idade, com porte grande ou gigante e, em geral, restringe-se à metáfise dos ossos longos do esqueleto apendicular. Foi atendido um cão Scottish Terrier macho de dez anos de idade que chegou com claudicação intermitente do membro torácico esquerdo (MTE), sensibilidade à palpação e edema de consistência firme na região cervicotorácica, sem nenhum histórico prévio de fraturas ou traumas. O diagnóstico definitivo de OSA fibroblástico escapular foi realizado com base na associação entre os achados clínicos, radiográficos, citológicos e histopatológicos. O tratamento utilizado foi a amputação do MTE, promovendo alívio da dor com pouca ou nenhuma perda da qualidade de vida.

Effects of the application of the amniotic membrane in the healing process of skin wounds in rats

Purpose: To evaluate the efficacy of the application of the human amniotic membrane (HAM) on the inflammatory process, fibroblast proliferation, formation of collagenand reduction of skin wound areas in rats.Methods: Thirty six rats were submitted to a surgical injury induction and divided into two groups (n = 18): group C (control) and T (treated with the HAM). The macroscopic evolution in the wound area and the histological characteristics of the skin samples were evaluated.Results: The regression of the wound area was greater in group T. The histological analysis revealed a significant reduction (p < 0.05) in the inflammatory infiltrate in group T at all experimental periods compared with that in the control group. Furthermore, the group T presented a significant increase in the proliferation of fibroblasts at 14 and 21 days compared with group C (p < 0.05). Regarding the deposition of mature collagen fibers, there was an increase in the replacement of type III collagen by type I collagen in group T (p < 0.05).Conclusion: Treatment with the HAM reduced the healing time as well as the inflammatory responses, increased the proliferation of fibroblasts, and induced a higher concentration of mature collagen fibers.(AU)

Fibroblastic osteosarcoma of scapula in a dog Scottish Terrier: case report / Osteossarcoma fibroblástico de escápula em cão Scottish Terrier: relato de caso

Osteosarcoma (OSA) is the most primary bone tumor observed in dogs. It a malignant neoplasias that affects the bone formation being with aggressive clinical-histological behavior a fast growth and high metastatic potential. Is observed in middle aged or older dogs with large or giant breed and restricts to the metaphysis of long bones of appendicular skeleton. In routine there was a Scottish Terrier dog attended, male, ten years old, that arrived intermittent claudication of the left forelimb, sensitivity to palpation and edema of firm consistency in the cervicothoracic region, with no previous historic of fractures or trauma. The association of clinical signs with the radiographic cytological and histopathological aspects resulted in the definitive diagnosis of fibroblastic OSA in scapula. The treatment was the amputation of LTM, promoting pain relief with little or none loss of quality of life.(AU)

Osteossarcoma (OSA) é o tumor ósseo primário mais observado em cães. Trata-se de uma neoplasia maligna que afeta a formação do osso, com comportamento clínico-biológico agressivo, crescimento rápido e altamente metastático. É mais observado em cães de meia idade, com porte grande ou gigante e, em geral, restringe-se à metáfise dos ossos longos do esqueleto apendicular. Foi atendido um cão Scottish Terrier macho de dez anos de idade que chegou com claudicação intermitente do membro torácico esquerdo (MTE), sensibilidade à palpação e edema de consistência firme na região cervicotorácica, sem nenhum histórico prévio de fraturas ou traumas. O diagnóstico definitivo de OSA fibroblástico escapular foi realizado com base na associação entre os achados clínicos, radiográficos, citológicos e histopatológicos. O tratamento utilizado foi a amputação do MTE, promovendo alívio da dor com pouca ou nenhuma perda da qualidade de vida.(AU)

Primary fibroblast cell cycle synchronization and effects on handmade cloned (hmc) bovine embryos / Sincronização do ciclo celular de fibroblastos primários e efeitos na produção de embriões bovinos clonados por handmade (hmc)

Spatial and temporal synchrony and compatibility between the receptor oocyte and the donor cell nucleus are necessary for the process of embryo cloning to allow nuclear reprogramming and early embryonic development. The objective of the present study was to evaluate three cell cycle synchronization methods on a primary bovine fibroblast culture for 24, 48, or 72 h. These fibroblasts were used as nuclear donors to evaluate their in vitro developmental potential and the quality of the embryos produced through handmade cloning (HMC). No differences were found between the methods used for fibroblast synchronization in G0/G1 (p > 0.05). Production of clones from fibroblasts in four groups- no treatment at 0 h and using serum restriction SR, high culture confluence HCC, and SR+HCC at 24 h- resulted in high cleavage rates that were not different. Embryo production rates were 37.9%, 29.5%, and 30.9% in the 0h, SR24h, and SR+HHC24h groups, respectively, and 19.3% in the HCC group, which was significantly different from the other three (p < 0.05). There were no differences in the quality parameter among the clones produced with fibroblasts subjected to the different synchronization. Finally, when overall clone production was compared versus parthenotes and IVF embryos, the only difference was between clones and parthenogenetic embryos with zona pellucida (30.2% vs 38.6%). The number of blastomeres from the blastocytes produced through IVF was significantly greater than those from embryos activated parthenogenetically and from clones (117, 80, 75.9, and 67.1, respectively). The evaluation of three synchronization methods at different time points did not demonstrate an increase in the percentage of fibroblasts in the G0/G1 phases of the cell cycle; however, good quality and high cloning rates were obtained, suggesting that it is not always necessary to subject the cells to any synchronization treatments, as they would yield equally good cloning results.(AU)

A sincronia espacial e temporal e a compatibilidade entre o oócito receptor e o núcleo celular doador são necessárias para o processo de clonagem de embriões a fim de permitir a reprogramação nuclear e o desenvolvimento embrionário precoce. O objetivo do presente estudo foi avaliar três métodos de sincronização do ciclo celular em uma cultura primária de fibroblastos bovinos durante 24, 48 ou 72 h. Estes fibroblastos foram utilizados como doadores nucleares para avaliar o seu potencial de desenvolvimento in vitro e a qualidade dos embriões produzidos por meio da técnica de Handmade cloning (HMC). Não foram encontradas diferenças entre os métodos utilizados para a sincronização de fibroblastos em G0 / G1 (p> 0,05). Produção de clones de fibroblastos nos quatro grupos - sem tratamento a 0 h e com restrição de soro RS, alta confluência celular ACC e RS + ACC às 24 h - resultou em altas taxas de clivagem que não foram diferentes. As taxas de produção de embriões foram de 37,9%, 29,5% e 30,9% nos grupos 0h, RS24h e RS + ACC24h, respectivamente, e 19,3% no grupo ACC, que foi significativamente diferente dos outros três (p <0,05). Não houve diferenças no parâmetro de qualidade entre os clones produzidos com fibroblastos submetidos à sincronização diferente. Finalmente, quando a produção geral de clones foi comparada versus partenotos e embriões de FIV, a única diferença foi entre clones e embriões partenogênicos com zona pelúcida (30,2% vs 38,6%). O número de blastômeros dos blastocitos produzidos através da FIV foi significativamente maior do que os de embriões ativados partenogeneticamente e de clones (117, 80, 75,9 e 67,1, respectivamente).(AU)

The Effect of High Temperature on Viability, Proliferation, Apoptosis and Anti-oxidant Status of Chicken Embryonic Fibroblast Cells

The effects of oxidative stress induced by high temperature on the cell viability, proliferation, apoptosis and oxidative status of chicken embryonic fibroblasts (CEF) were analyzed. The viability, proliferation, apoptotic and anti-oxidative status were measured after incubating CEF at the temperatures of 37ºC (control) and 40-44ºC (experimental groups) for 6,12 and 24 hours. The results showed that at high temperature (42-43ºC), the viability of CEF cells decreased after 6, 12 and 24 h of incubation, but the difference was significant only at 43ºC. Cell proliferation was significantly reduced at 44oC/6h. The apoptotic rate of CEF cells was increased following heat treatments in a time-dependent manner. ROS formation increased with increasing temperature, but the difference was only significant at 44ºC/6,12h. Heat stress did not significantly affect the superoxide dismutase (SOD) activity. CAT activity was significantly decreased at 43ºC/24h and 44ºC/12 and 24h. Malondialdehyde (MDA) formation was significantly increased at 43ºC/12h and 44ºC/12 and 24h. In conclusion, heat stress induced the oxidative stress, decreasing the viability, proliferation and anti-oxidative response of CEF cells.(AU)

The Effect of High Temperature on Viability, Proliferation, Apoptosis and Anti-oxidant Status of Chicken Embryonic Fibroblast Cells

The effects of oxidative stress induced by high temperature on the cell viability, proliferation, apoptosis and oxidative status of chicken embryonic fibroblasts (CEF) were analyzed. The viability, proliferation, apoptotic and anti-oxidative status were measured after incubating CEF at the temperatures of 37ºC (control) and 40-44ºC (experimental groups) for 6,12 and 24 hours. The results showed that at high temperature (42-43ºC), the viability of CEF cells decreased after 6, 12 and 24 h of incubation, but the difference was significant only at 43ºC. Cell proliferation was significantly reduced at 44oC/6h. The apoptotic rate of CEF cells was increased following heat treatments in a time-dependent manner. ROS formation increased with increasing temperature, but the difference was only significant at 44ºC/6,12h. Heat stress did not significantly affect the superoxide dismutase (SOD) activity. CAT activity was significantly decreased at 43ºC/24h and 44ºC/12 and 24h. Malondialdehyde (MDA) formation was significantly increased at 43ºC/12h and 44ºC/12 and 24h. In conclusion, heat stress induced the oxidative stress, decreasing the viability, proliferation and anti-oxidative response of CEF cells.