Age and sex effects on histological features and in vitro culturing of Antillean manatee (Trichechus manatus manatus, Linnaeus 1758) dermis.

The biobanks from dermal biopsies represent an interesting strategy for biodiversity conservation. Nevertheless, the morphological and cellular patterns of the dermis can be influenced by the age and sex of the individual. Therefore, evaluating these factors is interesting for forming biobanks of Antillean manatees. These animals, representatives of marine fauna, have had their population reduced, and biobanks are essential for their conservation. Then, we evaluated the effects of age (3.5 years vs. 3.6-16 years vs. 23.6 years) and sex (males vs. females) on morphological and cellular parameters using histological and in vitro culture techniques. Regardless of age, no differences were observed for dermal thickness, collagen fibres, tissue proliferative activity and viable cell recovery. Nonetheless, fibroblast reduction was observed in groups aged 23.6 years compared to other animals (p < 0.05). Additionally, cells from animals aged 3.6-16 years showed more significant mitochondrial damage than the other groups (p < 0.05). Regardless of sex, no differences were observed for dermal thickness, collagen fibres, tissue proliferative activity and viable cell recovery; however, females had fewer fibroblasts than males (p < 0.05). Cells from females showed lower mitochondrial damage when compared to cells from males. In summary, although age and sex do not influence dermal thickness and cell recovery, variations in the number of fibroblasts and mitochondrial characteristics were observed among the groups. These differences may be significant for understanding the dermis aspects to be correlated to biobank systems.

The initial steps toward the formation of somatic tissue banks and cell cultures derived from captive Antillean manatee (Trichechus manatus manatus) skin biopsies.

The declining population of the Antillean manatee caused by ecosystem degradation and rising pollution has prompted interest in developing conservation strategies for this species. Given this scenario, somatic tissue banks are important tools for acquiring knowledge about the species, as well as for obtaining somatic cells for biotechnological and ecotoxicological applications. Therefore, we aimed to assess the effects of slow freezing (SF) and solid-surface vitrification (SSV) of the dermis of captive Antillean manatees on the histology and ultrastructure of the tissue and cell viability in culture. While the SSV did not change the dermis thickness, the SF maintained the tissue proliferative potential, assessed by the nucleolar organizer region area, similar to noncryopreserved tissues. Moreover, both techniques reduced the number of fibroblasts and increased the percentage of collagen fibers. Nevertheless, only tissues cryopreserved with SF and noncryopreserved tissues were able to produce cells after in vitro culture. Although SF did not alter cell viability and proliferative activity, cells derived from cryopreserved tissues showed decreased metabolism, altered apoptosis, increased levels of reactive oxygen species, and mitochondrial membrane potential compared to cells from noncryopreserved tissues. In summary, we demonstrated for the first time that Antillean manatee somatic tissues can be cryopreserved by SF, and cells can be obtained after in vitro culture. Improvements in cryopreservation conditions, especially vitrification, of somatic samples are needed to increase the quality of somatic tissue banks in this species.

Influence of Intracellular Cryoprotectants on the Conservation of Dermal Somatic Tissues Derived from Antillean Manatees (Trichechus manatus manatus Linnaeus, 1758).

Cryopreservation of somatic tissue has been studied as a tool for the knowledge and conservation of endangered species, such as Antillean manatees. The use of vitrification protocols is an important step in the establishment of biological banks. To decrease the damage caused by this technique, a reduction in the concentration of cryoprotectants has been proposed. Therefore, we aimed to evaluate combinations and concentrations of intracellular cryoprotectants for the conservation of somatic tissues derived from Antillean manatees. Dulbecco's modified Eagle's medium, F-12 composed of 10% fetal bovine serum and 0.25 M sucrose, was supplemented with 3.0 M ethylene glycol (EG) plus 3.0 M dimethyl sulfoxide (DMSO), or 1.5 M EG plus 1.5 M DMSO or 3.0 M EG or 3.0 M DMSO, to produce four solutions for solid-surface vitrification. Noncryopreserved tissues were used as the controls. After warming, tissues derived from four Antillean manatees were evaluated for ultrastructure, histology, and in vitro culture. No differences were observed among the cryopreserved and noncryopreserved tissues in terms of ultrastructure. The dermis thickness of the cryopreserved fragments in solutions containing 3.0 M EG plus 3.0 M DMSO, 3.0 M EG, and 3.0 DMSO was similar to that of the control. Moreover, cryopreservation with 3.0 M EG plus 3.0 M DMSO maintained tissue proliferative capacity potential evaluated by quantification of nucleolar organizing regions. Nevertheless, none of the cryopreserved fragments were able to maintain the number of fibroblasts and the collagen percentage as compared with that of the noncryopreserved fragments. Also, none of the cryopreserved fragments in the different solutions were able to produce cells in vitro. In summary, even reducing the concentration of intracellular cryoprotectants as well as their association did not guarantee the maintenance of cells after in vitro culture. Further studies are needed to optimize the cryopreservation protocols in Antillean manatee somatic tissues.

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.

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.

Ultrastructural and morphometric description of the ear skin and cartilage of two South American wild histricognate rodents (Dasyprocta leporina and Galea spixii) / Descrição ultraestrutural e morfométrica da pele e cartilagem auricular de dois roedores histricognatos silvestres da América do Sul (Dasyprocta leporina e Galea spixii)

Skin and cartilage have been the main source for the recovery of somatic cells to be used in conservation strategies in wild mammals. In this sense, an important step for the cryopreservation of these samples is to recognize the properties of the skin and cartilage. Thus, knowing that the skin may differ among species and aiming to contribute to the establishment of cryobanks, the study examined the differences in the ear skin and cartilage of wild rodents from South America, agouti (Dasyprocta leporina) and spix's yellow-toothed cavy (Galea spixii). Ultrastructural and quantitative methods were used to measure skin and cartilage thickness, density of collagen and elastic fibers, cell type number and distribution, and proliferative activity. Although ultrastructural analysis revealed a similar pattern between species, morphometric analysis of the skin and cartilage showed differences between agoutis and cavies regarding thickness of epidermis layers (corneum: 5.3±2.5μm vs. 3.9±0.6μm; intermediate: 16.4±6.2μm vs. 23.4±8.1μm; basal: 9.9±2.1μm vs. 4.8±0.5μm), dermis (183.1±44.0μm vs. 258.2±22.9μm), total skin (211.8±46.0μm vs. 290.3±23.7μm) and perichondrium (27.6±6.1μm vs. 10.5±1.8μm). A greater number of epidermal cells (61.7±15.2 vs. 24.8±7.6) and chondrocytes (32.7±9.0 vs. 27.5±4.7) were observed in agouti, while the cavy presented a greater number of melanocytes (12.6±4.7 vs. 29.9±6.2), keratinocytes (14.7±4.2 vs. 29.8±7.6), and fibroblasts (103.6±24.7 vs. 112.2±11.3). Moreover, a higher percentage of collagen fibers and proliferative activity was observed in the skin of cavies, when compared to the skin of agoutis. Therefore, there are differences between agouti and cavy for ear skin and cartilage, requiring the establishment of species-specific cryopreservation protocols.(AU)

A pele e cartilagem têm sido uma importante fonte de recuperação de células somáticas a serem utilizadas em estratégias de conservação em mamíferos silvestres. Nesse contexto, uma importante etapa para criopreservação é conhecer, inicialmente, as propriedades que compõem a pele e cartilagem. Sabendo, então, que a pele pode diferir-se entre espécies e com o objetivo de contribuir para o estabelecimento de criobancos, o estudo evidenciou as diferenças da pele e da cartilagem do pavilhão auricular apical de cutias (Dasyprocta leporina) e preás (Galea spixii) que são roedores silvestres presentes na América do Sul. Para tanto, métodos ultraestruturais e quantitativos foram utilizados para mensurar a espessura da pele e da cartilagem, densidade de fibras colágenas e elásticas, número e distribuição dos tipos celulares e atividade proliferativa. Embora as propriedades ultraestruturais em cutias e preás tenham se mostrado semelhantes, avaliações acerca da morfometria da pele e da cartilagem demonstrou diferenças, especialmente nas camadas epidérmicas (córnea: 5,3±2,5μm vs. 3,9±0,6μm; espinhosa: 16,4±6,2μm vs. 23,4±8,1μm; basal: 9,9±2,1μm vs. 4,8±0,5μm), derme (183,1±44,0μm vs. 258,2±22,9μm), pele total (211,8±46,0μm vs. 290,3±23,7μm) e pericôndrio (27,6±6,1μm vs. 10,5±1,8μm). Além disso, um número maior de células epidérmicas (61,7±15,2 vs. 24,8±7,6) e condrócitos (32,7±9,0 vs. 27,5±4,7) foram observados em cutias, enquanto em preás um maior número de melanócitos (12,6±4,7 vs. 29,9±6,2), queratinócitos (14,7±4,2 vs. 29,8±7,6) e fibroblastos (103,6±24,7 vs. 112,2±11,3) foram evidenciados. Ainda, em preás, uma maior porcentagem de fibras colágenas e da atividade proliferativa foram observadas quando comparadas a pele de cutias. Portanto, existem diferenças entre cutias e preás para pele e cartilagem do pavilhão auricular, exigindo desta forma um estabelecimento de protocolos de criopreservação específica para cada uma destas espécies.(AU)

Effects of Vitrification Techniques on the Somatic Tissue Preservation of Agouti (Dasyprocta leporina Linnaeus, 1758).

The cryobanks of agouti somatic tissues represent a promising tool for the conservation of this species and of those that are phylogenetically related and endangered. For these purposes, one strategy to guarantee the quality of samples after warming would be to choose the appropriate tissue vitrification technique. Therefore, we evaluated the effects of two different techniques, direct vitrification in cryovials (DVC) and solid-surface vitrification (SSV), on the preservation of ear somatic tissues derived from agoutis kept in a scientific center of creation. Noncryopreserved somatic tissues were used as controls. Although SSV reduced the thickness of the dermis and cartilage (p < 0.05), the epidermal thickness of these samples was observed to be similar to controls (p > 0.05). Notably, the number of fibroblasts was not altered with either technique. However, both vitrification methods led to an increase in the number of perinuclear halos, with a particularly strong increase observed in DVC-derived fragments (p < 0.05). Compared with the DVC group, SSV showed a larger number of normal chondrocytes and smaller number of degenerate chondrocytes. Furthermore, the number of empty lacunae in SSV-derived fragments remained similar to controls (p > 0.05). In summary, SSV was found to be a more efficient method for vitrifying agouti somatic tissues compared with DVC. These results are important for the proper formation of agouti somatic banks, an essential step in the study of biological resources in this species.