Influence of storage time and nutrient medium on recovery of fibroblast-like cells from refrigerated collared peccary (Pecari tajacu Linnaeus, 1758) skin

Abstract

Animal cloning is a promising technology for biodiversity conservation, and its success depends on the recovery of nucleus donor cells. Specifically for collared peccaries, found sometimes in regions that are difficult to access, the storage at 4-6°C of skin tissues would be an alternative for the conservation of genetic material. Therefore, we aimed to evaluate different storage periods and the presence of a nutrient medium at 4-6°C on the recovery of somatic cells from the skin of collared peccaries. To analyze cell recovery rates, ear explants were distributed in non-refrigerated samples and samples refrigerated for 10, 30, and 50 d in the absence or presence of nutrient medium. All explants were analyzed by histologically and cultured. Only the fragments stored for 50 d without medium showed an increase in the total thickness of skin. Moreover, increased storage period, regardless of the presence of medium, increased the halo number and reduced the metabolic activity. After culture, only the fragments stored without medium for 50 d did not yield any somatic cells. Cells recovered from explants stored for 10 d showed similar characteristics to these recovered from non-refrigerated explants, regardless of the presence of medium, including the day at which explants achieved attachment and the total time to reach subconfluence. In conclusion, viable cells can be recovered from somatic tissues of collared peccaries stored for up to 50 d in the presence of medium, and tissues refrigerated for up to 10 d in the presence of medium yielded more viable cells.

Isolation, characterization, and cryopreservation of collared peccary skin-derived fibroblast cell lines

“…In this sense, studies related to the conservation of the collared peccary have been intensified, especially aimed at improving the techniques related to the preservation of somatic samples. Using this study, we established a culture condition for explants derived from the skin of adult collared peccaries (Santos et al, 2016) and developed a protocol for cryopreservation (Borges et al, 2017(Borges et al, , 2018a(Borges et al, , 2018b) and refrigeration of these explants (Queiroz Neta et al, 2018). In order to conduct the cloning experiments on this species by a somatic cell nuclear transfer, as well as to produce induced pluripotent cells, it is necessary to establish properly characterized cell lines.…”

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

“…The samples were fixed in 4% paraformaldehyde and processed for embedding in paraffin as described by Queiroz Neta et al () for morphometric analysis, cell quantification and distribution analysis. Sections of 5.0 µm thickness were stained with haematoxylin‐eosin.…”

Potential role of intraspecific and interspecific cloning in the conservation of wild mammals

“…Karyoplasts have been routinely cryopreserved by slow freezing (Sharma et al , 2018) using a combination of dimethyl sulfoxide (DMSO), FBS, and sucrose as the cryoprotectant, as observed with Iberian lynx ( Lynx pardinus , León-Quinto et al , 2014). Although it is more desirable to use a somatic cell bank after tissue culture, the absence of in vitro culture conditions sometimes makes these banks unfeasible, resulting in the immediate formation of the targets for those somatic tissues (Borges et al , 2017a,b; Queiroz Neta et al , 2018). The three somatic tissue conservation techniques used for wild animals are slow-freezing cryopreservation (Mestre-Citrinovitz et al , 2016), vitrification (Borges et al , 2018a,b), and cooling at 4–6°C (Queiroz Neta et al , 2018).…”

“…Although it is more desirable to use a somatic cell bank after tissue culture, the absence of in vitro culture conditions sometimes makes these banks unfeasible, resulting in the immediate formation of the targets for those somatic tissues (Borges et al , 2017a,b; Queiroz Neta et al , 2018). The three somatic tissue conservation techniques used for wild animals are slow-freezing cryopreservation (Mestre-Citrinovitz et al , 2016), vitrification (Borges et al , 2018a,b), and cooling at 4–6°C (Queiroz Neta et al , 2018). In collared peccaries, we compared two techniques of vitrification and we observed that solid-surface vitrification was found to be a more efficient method for vitrifying skin tissue when compared with direct vitrification in cryovials, probably due to tissues not being involved in large amounts of cryoprotectants before passing through a drastic change in temperature during the solid-surface vitrification (Borges et al , 2017b).…”