Conservação de tecido somático de catetos (pecari tajacu linnaeus, 1758) submetido a diferentes condições de armazenamento

• Autor(a) principal: Queiroz Neta, Luiza Bento de
• Data de Publicação: 2018
• Tipo de documento: Dissertação
• Idioma: por
• Título da fonte: Repositório Digital da Universidade Federal Rural do Semi-Árido (RDU)
• Texto Completo: https://repositorio.ufersa.edu.br/handle/prefix/967
• topic: Mamíferos silvestres; Recuperação post-mortem; Células somáticas; Wild mammals; Postmortem recovery; Somatic cells; CNPQ::CIENCIAS AGRARIAS::MEDICINA VETERINARIA

Cloning by somatic cell nuclear transfer is an attractive tool for biodiversity conservation and its efficiency depends on the obtaining and selection of nucleus donor cells derived from the skin of individuals of interest. Specifically for collared peccaries, wild mammals found sometimes in regions difficult to access or far from specialized laboratories, the storage at 4–6°C of skin tissues would be an alternative for the conservation of the genetic material of these animals. Therefore, the aim was to evaluate different periods of storage and the presence of nutrient medium on the recovery of somatic cells derived from skin of collared peccaries. Thus, 476 ear explants (9.0 mm3) recovered from eleven adult animals from the Centro de Multiplicação de Animais Silvestres (CEMAS/UFERSA) were distributed in seven groups: samples not refrigerated (control) and refrigerated in absence or presence of Dulbecco’s Modified Eagle’s Medium supplemented with 2.2 g/L sodium bicarbonate, 10% fetal bovine serum and 2% antibiotic–antimycotic for 10, 30 and 50 days of storage. For the histological analyzes, samples were fixed in 4% paraformaldehyde, processed and stained with hematoxylin-eosin for evaluation of epidermal and dermal thickness, number of fibroblasts and halos. Moreover, fragments were evaluated for the quantification of argyrophilic nucleolar organizer region (AgNORs) and metabolic activity by 3-[4,5-dimethylthiazol- 2-yl] -2,5-diphenyltetrazolium bromide (MTT). For in vitro culture analyzes, explants were cultured and evaluated for cell morphology, culture quality, cell viability by trypan blue, proliferative activity by determination of the growth curve and doubling time of the cell population and metabolic activity by MTT assay. Comparisons between the refrigerated and not refrigerated fragments were analyzed using the GraphPad Prisma software. As for the tissue thickness, only the fragments stored for 50 days in the absence of nutrient medium showed a reduction and increase, respectively, in the thickness of the epidermis (55.8 ± 3.2 μm vs. 64.8 ± 2.1 μm) and dermis (172.7 ± 2.5 μm vs. 147.6 ± 2.5 μm) when compared to control (P < 0.05). Moreover, the storage period, regardless of the presence of medium, promoted a reduction in the number of fibroblasts and increase in the number of halos. Also, the number of AgNORs indicating proliferative activity, and metabolic activity of the explants, decreased with the storage period. After the in vitro culture of the explants, only the fragments stored without medium for 50 days were not able to obtain somatic cells. Likewise, cells from explants in the presence of medium for 10 days showed similar characteristics to the cells of not refrigerated explants, especially regarding the duration of culture, doubling time of the cell population, day of all attached explants and subconfluence total time. Thus, viable cells derived from collared peccaries can be recovered from tissue fragments stored for up to 50 days in the presence of nutrient medium; nevertheless, refrigerated somatic tissues up to 10 days in the presence of medium resulted in more viable cells