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The number of wild animals threatened or at risk of extinction constantly increases and, as a result, biodiversity decreases. Therefore, it is necessary to set in motion proceedings to preserve the genetic variability and to overcome factors leading to this situation. Banks of cryopreserved cells, such as skin fibroblasts, from wild animals could be used both for genetic studies and as a cell source for technologies able to reproduce individuals. In this study we report that a single basic protocol provides a method for obtaining, culturing, and cryopreserving skin fibroblasts from a wide range of wild animals. Skin biopsies were taken in Cantabria (Spain) from live, anesthetized brown bear (Ursus arctos, n = 4), and in Asturias (Spain) from carcasses of accidentally killed individuals from the following species: grey wolf (Canis lupus, n = 1), red fox (Vulpes vulpes, n = 1), eurasian badger (Meles meles, n = 1), ermine (Mustela erminea, n = 1), fallow deer (Dama dama, n = 7), pyrenean chamois (Rupicapra pyrenaica, n = 1), western roe deer (Capreolus capreolus, n = 1), wild boar (Sus scrofa, n = 1), striped dolphin (Stenella coeruleoalba, n = 1) and one avian species, capercaillie (Tetrao urogallus, n = 1). Once obtained and transported to the laboratory, all samples were processed identically. Tissue samples were manually cut into small pieces and enzymatically digested with 0.05% trypsin-EDTA (Sigma-Aldrich, Madrid, Spain) for 30 min, and then with collagenase (300 units/mL) (Sigma) for 14 h at 38°C. Disaggregated cells were centrifuged at 1600 rpm for 10 min and the pellet was diluted with D-MEM (Sigma) containing 10% FBS. Cells were counted and a minimum of 3 × 105 cells were placed in a 25 cm2 flask for culture under 5% CO2 in air and high humidity at 37°C (red fox, badger, ermine, western roe deer) or 38°C (brown bear, grey wolf, fallow deer, pyrenean chamois, wild boar, striped dolphin, capercaillie). Confluent fibroblast monolayers were obtained after 5–7 days in culture except for the striped dolphin whose sample was contaminated. Two to three passages were performed using 75 cm2 flasks before freezing fibroblasts in D-MEM containing 10% DMSO (Sigma) and 10% FBS. Total cell yield from a confluent flask was 5–11 × 106 cells, depending on the animal species. Fibroblasts recovered from all thawed samples grew to confluence, and karyotypes were performed at this step. It was possible to obtain, culture, freeze, and thaw skin fibroblasts successfully from all the above-mentioned land species. Although more research is needed, preliminary data suggest that the above-described procedure can be applied to mammals and birds, thus helping to preserve genetic material of wild animals. Grant Support: Consejería de Medio Ambiente, Ordenación del Territorios e Infraestructuras, Asturias, SPAIN. The authors acknowledge CEPESMA, Luarca, Asturias, SPAIN.

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Background: This work summarizes the mortality cases of twenty-five free-ranging Eurasian wild brown bears (Ursus arctos arctos) from the Cantabrian mountain range submitted for necropsy in Asturias and Castilla y León (northwestern Spain) from “1998 to 2018”. Results: Causes of death were classified both based on (i) infectious or non infectious etiology and (ii) caused by “human intervention” or “natural causes”. In four cases (16%) it was not possible to determine the cause of death due to the inadequate preservation of collected specimens or insufficient tissue availability. Some bears died from a combination of human activities or natural trauma and secondary bacterial infection (i.e. clostridiosis). Based on infectious or non infectious etiology nine out of the 21 (42.9%) brown bears in which the cause of death could be determined died due to infectious diseases, namely gangrenous myositis, infectious canine hepatitis or septicemia. The remaining twelve (57.1%) animals died due to non infectious causes, which included handling, traumatic lesions such as shooting, snare, fighting or infanticide, strychnine poisoning, mushroom poisoning or neoplasia. Exertional myopathy was additionally observed in the handled animal and in one bear found in a snare. Mortality data was also stratified by deaths caused by “human intervention” or “natural causes”. Seven out of the 21 (33.3%) brown bears died as a consequence of “human intervention” due to illegal hunting (shooting or snare), handling or strychnine poisoning. In contrast, fourteen (66.7%) brown bears died by “natural causes” due to traumatic lesions (fights, traumas, or infanticide), infectious canine hepatitis, neoplasia or mushroom poisoning. In four cases traumatic lesions were complicated with clostridiosis which finally caused the death of those animals. Two additional bears showed septicemia and gangrenous myositis secondary to fighting. Another two animals developed exertional myopathy.Conclusions: In a non-hunted population of Eurasian brown bear from the Cantabrian mountain range, main cause of death is attributed to “natural causes” mostly due to traumatic lesions and infectious diseases (primary or secondary developed) which is in contrast to previously reported data for other bear populations. These data are valuable and may help in the conservation and management of this recovering population.

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