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Background: There are few studies on stem cell isolation in wild animals that provide isolation and culture protocols of these cells in vitro. Among the wild species studied, we present the collared peccary (Tayassu tajacu) as a model with potential to obtain and use MSC in preclinical studies. These animals are phylogenetically close to the domestic pig, popularly known as peccaries and found naturally in South America, Central America and the South of the United States. The aim of the present study was to establish a protocol for the isolation, in vitro cell expansion, differentiation and assessment of the stromal MSC growth curve before and after thawing.Materials, Methods & Results: Mesenchymal stem cells (MSC) from collared peccary bone marrow (Tayassu tajacu) were isolated and expanded by centrifuge in Ficoll® solution and cultured in DMEM® High Glucose medium. The culture was assessed by assays of colony forming units CFU-F and growth curve by saturation (GCS). Cultures in the third passage, with 70% confluence, were replicated at 105 cells/mL concentration in the culture media to induce osteogenic cell differentiation and adipogenic cell differentiation, respectively. The MSC were frozen in nitrogen for 40 days, thawed and re-assessed for cell viability and GCS.Discussion: The bone marrow collected presented high mononuclear cellularity, with a mean variability of 94.5% and 60.83 ± 4.27 UFC were identified in the samples and cells with fibroblast-like-cell morphology were observed. When they were expanded, the mean cell viability was 95%, the mean cell concentration obtained was 233.31 ± 20.04 cells per 25cm2 bottle and the culture reached the growth plateau in GCS between the 13th and 16th day. The osteoblastic cell differentiation assay showed after 18 days, morphology similar to osteoblasts, with irregular cytoplasm limits, cell prolongation formation and flattened appearance. After staining with Alizarin Red, the nucleus presented a wine red coloring and the cytoplasm, more basophilic and well-defined, with calcium deposits inside the cells. The cultures submitted to adipogenic differentiation were large, hexagonal, irregular and presented birrefringent cytoplasm granules after the third week of culture. When stained with Oil Red it was observed that the cytoplasm granules were scattered small fat vacuoles and stained maroon. The viability after thawing was 78% and the mean cell concentration obtained in GCS was 199.71 ± 14.72 cells per 25 cm2 bottle. The curves reached the saturation plateau early, on the eighth day of observation. From then onwards the cultures entered became exhausted and the cell concentration of the samples decreased progressively until minimum values. These results showed the presence of a well-defined MSC population in the collared peccary bone marrow with a high rate of replication in vitro and potential for differentiation confirmed by the adipogenic and osteogenic lines. The cryopreservation technique adopted presented satisfactory results, but indicated a significant cell stress after thawing that justifies investigation of the apoptosis rates induced post thawing in the species. Furthermore, the bone marrow collection did not harm the animals and the facility of stromal MSC isolation and culture qualifies the collared peccary as a viable alternative model to obtain MSC and for studies in the area of cell therapy.

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Pesq. Vet. Bras. 33(Supl.1):8-14, dezembro 2013 8 RESUMO.-[Avaliação do eletrocardiograma em cutias (Dasyprocta primnolopha, Wagler 1831) não-anestesiadas clinicamente saudáveis.] A cutia é uma das espé-cies mais intensamente caçados em toda a Amazônia e as regiões semi-áridas do nordeste do Brasil. Considerando--se a tendência atual no manejo de animais silvestres em cativeiro, o objetivo deste estudo foi determinar os valores de referência para o coração cutia criadas em cativeiro, com base em avaliações do eletrocardiograma (ECG). Foram selecionadas cutias adultas e sem sinais clínicos de doença cardíaca (n=30). Os animais foram contidos fisicamente e, em seguida, o ECG foi realizado. Medições padronizadas foram tomadas para estabelecer a análise estatística dos dados. Análise do complexo QRS apresentou valores compatíveis com os relatórios pregressos em animais animais de companhia, assim como para os poucos dados disponí-veis para outras espécies selvagens e exóticas, com exceção da onda T, que mostrou amplitude semelhante à onda R em The agouti is one of the most intensely hunted species throughout the Amazon and the semiarid regions of north-eastern Brazil. Considering the current tendency of wild animal management in captivity, the objective of this study was to determine heart reference values for agouti raised in captivity, based on electrocardiographic assessments (ECG). Adult agouti were selected without clinical signs of heart disease (n=30). The animals were restrained physically and then the ECG was performed. Standardized measurements were taken to establish the statistical analysis of the data. Analysis of the QRS complex showed values compatible with previous reports in peer animals and the limited data available for other wild and exotic species, except for the T wave that showed similar amplitude to the R wave in all the animals studied. The data obtained provided the first reference values for ECG tracings in agouti, contributing to a better understanding of heart electrophysiology in identifying myocardial pathology in these animals. Electrocardiogram assessment in non-anaesthetized clinically healthy agouti (Dasyprocta primnolopha, Wagler 1831) todos os animais estudados. Os dados obtidos permitiram a aquisição dos primeiros valores de referência para os traçados de ECG em cutias, contribuindo para uma melhor compreensão eletrofisiologia cardíaca, na identificação de miocardiopatia nesses animais.

The black-rumped agouti ( Dasyprocta prymnolopha , Wagler 1831) is currently under intense ecologic pressure, which has resulted in its disappearance from some regions of Brazil. Echocardiography is widely used in veterinary medicine but it is not yet part of the clinical routine for wild animals. The objective of the present study was to assess the applicability of the echocardiographic exam in nonanesthetized agouti and to establish normal reference values for echocardiographic measurements in bidimensional mode (2D), M-mode, and Doppler for this species, and a lead II electrocardiogram was simultaneously recorded. Twenty agouti were used in this study. All the echocardiographic measurements were positively correlated with weight (P < 0.05), and there were no significant differences between sexes (P > 0.05). Blood flow velocities in the pulmonary and aortic artery ranged from 67.32-71.28 cm/sec and 79.22-101.84 cm/sec, respectively. The isovolumic relaxation time was assessed in all the animals and ranged from 38.5 to 56.6 ms. The maximum value for the nonfused E and A waves and the Et and At waves was 158 beats/min for both. The results obtained for the morphologic and heart hemodynamic measurements can guide future studies and help in the clinical management of these animals in captivity.

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Resumo: As biotécnicas da reprodução são importantes ferramentas para conservação de espécies domésticas e silvestres, pois permitem a recuperação e uso futuro de material reprodutivo. O objetivo deste estudo foi avaliar parâmetros morfológicos de CCOs de cutias, obtidos pela técnica de fatiamento do ovário para utilização em protocolos de maturação e fecundação na produção in vitro de embriões. Foram utilizadas dezessete cutias fêmeas do NEPAS, CCA- UFPI, com idade e peso médios de 3,9 anos e 2,16Kg, respectivamente, que foram submetidas à ovariossalpingohisterectomia. Os ovários após dissecados e pesados em balança de precisão, foram fatiados individualmente. Procedeu-se a busca e seleção dos CCOs em estereomicroscópio, os quais foram identificados e quantificados por cada ovário, além de classificados quanto a sua morfologia segundo a quantidade de camadas de células do cumulus e ao citoplasma em quatro graus. Verificou-se que a técnica de fatiamento do ovário possibilitou a obtenção de CCOs de cutias, com recuperação de grande quantidade e de variados graus de qualidade. Não houve correlações entre a idade dos animais e o peso dos ovários; a idade e o número de CCOs obtidos; entre o peso das cutias e o peso dos ovários e o número de CCOs obtidos.

Stem cells are present in the adult tissues of most diverse species. Bone marrow is recognized to be the most exploited site to obtain stem cells and cell progenitors. The objective of the present study was to characterize hematopoietic progenitor (HP) morphology and analyze the performance of adherent cell progenitors (ACPs) cultivated in vitro from black-rumped agouti bone marrow (Dasyprocta prymnolopha). Bone marrow aspirates were obtained from tibia crest and used to prepare histological slides and identify cell morphology. Cells were also scattered on culture plates for later isolation, expansion, and quantification. Smears obtained from bone marrow demonstrated HPs at different stages of maturity. In culture, these cells showed fibroblastoid morphology and a strong tendency to form colonies, demonstrated by the presence of cell aggregates, cytoplasmic elongations lying side by side. An 80% cell confluence was observed at 18 days in culture and progressive reduction in the percentage of nonadherent mononuclear cells. After eight passes, a mean cell viability of 96.07% was observed, from a pool of 1.6 × 10(7) cells (ACP). Thirteen 25-cm(2) culture bottles were trypsinized, resuspended in freezing medium, stored in 14 criotubes at a concentration of 1 × 10(6) cells per milliliter, and placed in liquid nitrogen at -196°C. Agouti bone marrow demonstrated high plasticity, moreover different HP lines, and a population of adherent cells demonstrated morphology similar to mesenchymal stem cells in culture.