Different cryopreservation requirements in foetal versus adult skin cells from an endangered mammal, the Iberian lynx (Lynx pardinus).

Cryobanking somatic foetal cells acquire much relevance in endangered species for biodiversity conservation purposes. Such cells could be later used to reintroduce the lost genes into the breeding pool, by inducing pluripotency and/or nuclear transfer if necessary. Since requirements for preserving foetal cells are not always the same as for adult ones, we evaluated the cryosensitivity of foetal skin cells in comparison with adult ones from the critically endangered Iberian lynx. Responses to cryoinjury were analyzed in both thawed cell types by means of cell viability and functionality (by analyzing their membrane integrity, metabolic activity, glycosaminoglycan content and proliferative activity). Freezing media included the permeating cryoprotectant Me2SO, either alone or along with the non-permeating cryoprotectant sucrose at 0.1 or 0.2M. When Me2SO was the only cryoprotectant, survival rate fell in thawed foetal cells to 54±4% (against 89±6% for thawed adult ones) and both proliferative and metabolic activities remained significantly lower than values for thawed adult cells. However, the combination of sucrose (both 0.1 as 0.2) and Me2SO in foetal cells significantly increased their survival rates (to 71±4% and 73±5%, respectively), proliferative activities (partially at day 7 and completely at day 14 after thawing) and metabolic activities. Our findings clearly show a difference between foetal and adult cells concerning their cryopreservation sensitivity and requirements, as well as their recovery time after thawing. These results are of relevance for the cryopreservation of foetal and adult cells from the Iberian lynx and could be also useful for other mammals.

Developing biological resource banks as a supporting tool for wildlife reproduction and conservation The Iberian lynx bank as a model for other endangered species.

This work presents a Biological Resource Bank generated as a complementary supporting tool for the reproduction and the in situ and ex situ conservation of the Iberian lynx. In its design we prioritized the preservation of a maximum of the current genetic and biological diversity of the population, and the harmless collection of the samples. To provide future reproductive opportunities through any possible technique, we processed and cryopreserved germinal cells and tissues from dead animals, 7 males and 6 females, as well as somatic cells and tissues from 69 different individuals. This somatic cell reserve reflects a very important fraction of the population biodiversity which, furthermore, will allow the development of a wide variety of studies that can be easily extrapolated to the majority of the population. We have developed a new non-destructive method to isolate cells with stem-cell-like properties. If considered convenient in the future, and after proper research, such cells could permit therapeutic applications and perhaps be a good source to be used in somatic cell nuclear transfer. Samples of whole blood and its derivatives, hairs, urine and feces from many different individuals were also preserved. Proper storage of such samples is required to allow epidemiological studies to be performed for the testing of different etiological hypotheses or, in general, to develop any bio-sanitary study to improve conservation strategies within the natural habitat. This work describes the main aspects involved in the practical implementation of the Iberian lynx Biological Resource Bank, as a model that could be useful for the development of similar banks for other endangered species.