Comparison between concentration and type of intracellular cryoprotectants and the presence of sucrose for cryobanks of somatic cells derived from captive Pumas.

The loss of wild biodiversity has prompted the development of cryobanks, such as those of somatic cells. This is the reality of Pumas, wild felids of ecological importance that suffer from anthropogenic actions, population decline, and subsequent loss of genetic diversity. Somatic cell banks are a strategy for conserving population diversity. We compared different concentrations and types of intracellular cryoprotectants (dimethyl sulfoxide, DMSO; ethylene glycol, EG) associated with 0.2 M of sucrose (SUC) in the cryopreservation of the somatic cells of captive Pumas. The cells were cryopreserved by slow freezing with different solutions containing Dulbecco's modified Eagle's medium with 10% fetal bovine serum and varying concentrations of DMSO and EG in the absence or presence of SUC. The cells were analyzed for morphological characteristics, viability, proliferative activity, metabolic activity, and apoptosis levels. Cells maintained similar fusiform morphology before and after cryopreservation. There was no difference in viability, regardless of the reduction in the concentration and type of intracellular cryoprotectants and sucrose. Similarly, proliferative activity, metabolic activity, and apoptosis levels were not altered by the composition of the cryoprotectants. In summary, we demonstrate that reducing the concentration of DMSO or EG ensures adequate cryopreservation of Puma somatic cells, regardless of the presence of SUC.

Comparative effect of cryoprotectant combinations on the conservation of somatic cells derived from jaguar, Panthera onca, towards the formation of biologic banks.

Due to the reduction of the jaguar population, the formation of somatic cell cryobanks represents an interesting tool for its conservation. Nevertheless, the success of these cryobanks depends on the cryoprotectants used in cryopreservation. We evaluated the effects of the intracellular cryoprotectants (10% dimethyl sulfoxide, DMSO; 10% ethylene glycol, EG) in the absence or presence of an extracellular cryoprotectant (0.2 M sucrose, SUC) on the morphology, confluence, viability, and metabolism of somatic cells derived from five jaguars belonging to Brazilian zoos. The morphology was presented in a descriptive manner, while the confluence, viability and metabolic activity were presented as means and compared using statistical tests. Non-cryopreserved cells were used as control and compared to frozen/thawed cells using cryoprotectants. No difference was observed for the morphology and confluence among non-cryopreserved and cryopreserved cells, regardless of the cryoprotectants. Only cryopreserved cells in EG (45.8%±12.9) had a reduction in their viability when compared to non-cryopreserved cells (97.8%±1.1). Only cryopreserved cells in DMSO with SUC (76.0%±2.7) or absence of SUC (77.0%±3.7) maintained their metabolic activity after thawing, when compared to non-cryopreserved cells (100.0%±6.7). Therefore, combinations of DMSO in the absence and presence of SUC were efficient in the cryopreservation of somatic cells of jaguars.

Evaluation of the damage caused by in vitro culture and cryopreservation to dermal fibroblasts derived from jaguars: An approach to conservation through biobanks.

Ex-situ conservation strategies such as the formation of somatic cell banks are valuable tools for the conservation of jaguars, whose population has been declining in recent years. Once properly established, these cells can be successfully leveraged for future applications. We aimed to assess the effects of in vitro culture and cryopreservation on the establishment of fibroblasts derived from jaguars. Initially, we identified five dermal fibroblastic lines using morphology and immunophenotyping assays; these lines were then subjected to two experiments. In the first experiment, the viability, metabolism, and proliferative activity of cells at different passages (first, third, and tenth) were evaluated. In the second experiment, the cells were cryopreserved and the levels of reactive oxygen species (ROS), mitochondrial membrane potential (ΔΨm) and apoptosis were evaluated after one, three, and ten passages. Noncryopreserved cells were used as controls. The in vitro culture after first, third, and tenth passages and cryopreservation conditions did not affect the proliferative activity and viability. However, cells cultured until tenth passage and frozen/thawed cells showed reduced metabolism. In addition, cryopreserved cells showed higher levels of intracellular ROS and altered ΔΨm when compared with those of noncryopreserved cells. Finally, frozen/thawed cells cultured after ten passages showed reduced proliferative activity and number of viable cells than did frozen/thawed cells cultured after one and three passages. In summary, we have shown that viable fibroblasts can be established from jaguar skin and that although these cells do not show altered viability and proliferative activity, they do undergo damage during extended culture and cryopreservation.

Evaluation of different skin regions derived from a postmortem jaguar, Panthera onca (Linnaeus, 1758), after vitrification for development of cryobanks from captive animals.

Biological resource banks represent valuable tools for the conservation of species vulnerable to extinction, such as the jaguar. Cryobanks of skins have the potential to safeguard rare genotypes, allowing the potential exploitation of biological samples in animal multiplication technologies and the study of genetic variability. Determination of the most suitable skin regions for tissue conservation can help increase the efficiency of cryobanks and the storage of biological samples. To this end, we evaluated the effects of vitrification of skin tissues from the ear, caudal, and femoral regions of a post-mortem jaguar belonging to a zoo in Brazil. Non-vitrified and vitrified samples were evaluated and compared using quantitative methods, focusing on skin thickness, cell quantification, number of perinuclear halos, collagen and elastic density, and proliferative activity. No differences were observed in skin thickness, number of perinuclear halos, elastic density, and proliferative activity between non-vitrified and vitrified tissues in skin from any region. However, vitrified tissues derived from femoral skin showed a reduction in the number of fibroblasts, epidermal cells and collagen density compared to non-vitrified tissues. In summary, the ear and caudal regions provided the best conservation of somatic tissues derived from jaguars, and skin samples from these regions are therefore the most suitable for the formation of cryobanks.

Morphological, Ultrastructural, and Immunocytochemical Characterization and Assessment of Puma (Puma concolor Linnaeus, 1771) Cell Lines After Extended Culture and Cryopreservation.

Cell lines are valuable tools to safeguard genetic material from species threatened with extinction that is mainly due to human action. In this scenario, the puma constitutes a species whose population is being rapidly reduced in the ecosystems it inhabits. For the first time, we characterized puma skin-derived cell lines and assessed these cells after extended culture (experiment 1) and cryopreservation (experiment 2). Initially, we identified and characterized four dermal fibroblast lines using morphology, ultrastructure, and immunofluorescence assays. Moreover, we evaluated the effects of culture time (1st, 3rd, and 10th passages) and cryopreservation on their morphology, ultrastructure, viability, metabolism, proliferative activity, reactive oxygen species (ROS) levels, mitochondrial membrane potential (ΔΨm), and apoptosis. The cells showed a typical spindle-shaped morphology with centrally located oval nuclei. The cells were identified as fibroblasts by staining for vimentin. In vitro culture after the 1st, 3rd, and 10th passages did not alter most of the evaluated parameters. Cells in the 3rd and 10th passages showed a reduction in ROS levels (p < 0.05). The ultrastructure revealed morphological damage in the prolongments, and nuclei of cells derived from the 3rd and 10th passages. Moreover, cryopreservation resulted in a reduction in ΔΨm compared with that of noncryopreserved cells, suggesting that the optimization of cryopreservation methods for puma fibroblasts is essential. In conclusion, we found that viable fibroblasts could be obtained from puma skin, with slight changes after the 10th passage in in vitro culture and cryopreservation. This is the first report on the development of cell lines derived from pumas.

Quantitative and descriptive histological aspects of jaguar (Panthera onca Linnaeus, 1758) ear skin as a step towards formation of biobanks.

Skin of mammals vulnerable to extinction, such as the jaguar, is used as a source of material in conservation strategies. The composition of skin is not uniform among species, and the ability to distinguish similarities in skin morphology in animal groups is fundamental in the application of skin tissue for use in biobanks. The aim of our study was to evaluate the structure, composition and capacity for culture of ear skin from the yellow and black jaguars. Both qualitative and quantitative methods were used, focusing on skin thickness, cell quantification and distribution, collagen density, proliferative activity and viability. Histomorphometrical study of the skin showed a total thickness of 273.2 and 274.6 µm for the yellow and black jaguars, respectively. Melanocytes and fibroblasts were, respectively, 9.7 and 23.0 for the yellow jaguar and 11.3 and 26.8 for the black jaguar. A collagen density of 67.0% and 49.0% was observed for yellow and black jaguars, respectively. Both animals presented a proliferative activity varying between 1.20 and 1.30. All tissues could promote cellular detachment, reaching subconfluence in 10-15 days. This kind of information from histomorphometrical features and cell cultures can be essential for a more targeted application of ear skin cryopreservation in this species, as such information will enable understanding the action of substances on tissues during the conservation process.

Refrigeração do epidídimo canino a 4ºC e recuperação dos espermatozoides epididimários utilizando ACP-106c / Cooling of the canine epididymis at 4ºC and recovery of epididymal sperm using ACP-106c

Objetivou-se avaliar a qualidade dos espermatozoides recuperados da cauda do epidídimo após a refrigeração do complexo testículo-epidídimo (CTE) de cães usando o diluidor ACP-106c. Foram utilizados 60 cães machos adultos, com peso de 10-20 kg. Após a eutanásia, removeu-se o CTE que foi imerso em solução fisiológica 0,9% e transportado em caixa térmica ao laboratório a 30ºC. Para a refrigeração e recuperação dos espermatozoides epididimários, os 60 pares do CTE foram divididos em 4 grupos, de acordo com o tempo de refrigeração do CTE e posterior recuperação espermática: G0h, G6h, G12h e G18h, em que cada par do CTE permaneceu por zero, seis, doze ou dezoito horas a 4ºC, respectivamente. A recuperação dos espermatozoides da cauda do epidídimo foi realizada pela técnica de flutuação utilizando-se o diluidor ACP-106c ou Tris. Para cada epidídimo foi adicionado 1,0 mL de um dos dois diluidores, pré-aquecidos a 37ºC por 5 minutos. Em seguida foram centrifugados a 800g/5 minutos para remoção dos resíduos celulares. Avaliou-se a morfologia, funcionalidade e motilidade espermática total e progressiva, além de parâmetros obtidos pelo CASA. Os dados foram submetidos à ANOVA seguido do teste de Turkey (P < 0,05). Em todos os parâmetros avaliados, não houve diferença entre os diluidores testados (P>0,05). Os valores de motilidade total nos grupos G0h, G6h, G12h, e G18h para o ACP-106c foram 84,4±7,7; 81,6±11,6; 88,3±6,5 e 69,5±16,9, respectivamente, e para o Tris 85,2±8,7; 77,4±14,3; 79,0±17,8 e 65,4±17,9, respectivamente. Um decréscimo na qualidade espermática foi observado após 18 horas de refrigeração em ambos os diluidores. Dessa forma pode-se concluir que o ACP-106c pode ser utilizado para recuperar os espermatozoides epididimários refrigerados e podem ser viáveis por até 12h de refrigeração.

The study aimed to assess the quality of sperm recovered from the epididymal cauda after cooling the complex testis-epididymis (CTE) of dogs using ACP-106c extender. Sixty adult male dogs, weighing 10-20 kg were used. After euthanasia, CTE was removed and it was immersed in 0.9% saline and transported to the laboratory in cooler at 30ºC. For cooling and recovery of epididymal spermatozoa, the 60 pairs of CTE were divided into four groups, according to the refrigeration time of the CTE and subsequent sperm recovery: G0h, G6h, G12h and G18h, wherein each pair of CTE remained zero, six, 12 or 18 hours at 4ºC respectively. The recovery of sperm epididymal tail was conducted by flotation technique using ACP-106c or Tris extender. For each epididymis, it was added 1.0 mL of either extenders, preheated to 37ºC for 5 minutes. They were then centrifuged at 800g/5 minutes to remove the cell debris. Morphology, functionality and total and progressive motility, and parameters obtained by CASA were evaluated. Data were analyzed by ANOVA followed by Turkey test (P <0.05). In all parameters assessed, there was no difference between the extenders used (P>0.05). The values of total motility groups G0h, G6H, G12H, and G18h for ACP-106c were 84.4±7.7, 81.6±11.6, 88.3±6.5 and 69.5±16.9 respectively, and for Tris 85.2±8.7, 77.4±14.3, 79.0±17.8 and 65.4±17.9 respectively. A decrease in sperm quality was observed after 18 hours of cooling in both extenders. Thus it can be concluded that the ACP-106c may be used to recover the epididymal spermatozoa chilled and may be viable for up to 12h cooling.