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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 38C. 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 37C (red fox, badger, ermine, western roe deer) or 38C (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: Consejera de Medio Ambiente, Ordenacin del Territorios e Infraestructuras, Asturias, SPAIN. The authors acknowledge CEPESMA, Luarca, Asturias, SPAIN.

Production of embryos in vitro with improved inner cell mass (ICM) and high ICM per total cell rate is a major objective in reproductive biotechnology. Exogenous all-trans retinoic acid (ATRA), a vitamin A metabolite, and endogenous retinoid regulate development and differentiation during bovine morula to blastocyst transition in vitro. ATRA binds to retinoic acid-receptor (RAR), and the ATRA isomere 9-cis-retinoic acid (9-cis-RA) binds to both RAR and the retinoid X receptor (RXR). The unspecific binding of 9-cis-RA to receptors makes it difficult to study RXR transactivation. Therefore, in this work we studied blastocyst development and cell counts by using a specific synthetic RXR agonist [LG100268 LG; a gift of Ligand Laboratories] as opossed to the effect exerted by ATRA upon RAR binding. Cumulus-oocyte complexes from slaughterhouse ovaries were matured and fertilized in vitro. Presumptive zygotes were cultured in B2 medium with Vero cells until 139 h post-insemination (Day 6), the time at which embryos [morulae (e90%) + early blastocysts] underwent treatments for 48 h in 400 L of SOFaaci + 5% FCS. Data (5 replicates per experiment) were analyzed by CATMOD for effects, processed by GLM and Duncan's test, and expressed as LSM SE (a,b,c P d 0.05). After a LG dose-response experiment (n = 480 morulae), blastocysts rates from LG 1 M on Day 7 were higher than LG 10 M, LG 0.1 M, and LG 0 M (Day 7: 42.8 4.1 vs. 34.4 3.7, 36.8 3.7, and 32.4 3.7, respectively). On Day 8, LG 1 M also yielded more blastocysts than LG 0.1 M (50 4.2 vs. 44.4 3.7, respectively). By differential cell counting (n = 113 blastocysts), hatched blastocysts with LG 10 M showed proliferation in the ICM, while trophectoderm (TE) cells decreased conversely to LG concentration. These effects were not obvious in expanded blastocysts. In a subsequent experiment (n = 340 morulae), ATRA led to blastocysts rates on Day 8 that were higher than negative, untreated controls, but not different from LG 1 M (42.4 2.4 vs. 33.1 2.0 and 36.0 2.4, respectively). ATRA and LG 1 increased TE in expanded blastocysts (n = 42) (102 13.2 and 96.23 13.2, respectively vs. 72.8 10.9 in the untreated group) but not in their hatched counterparts (n = 44). There were no differences in the ICM; but percentages of ICM per total cells were higher in hatched blastocysts cultured with ATRA than in expanded LG 1 M blastocysts and expanded controls (39.5 5.5 vs. 24.2 5.7, and 20.9 4.7, respectively). Manipulation of retinoid receptor-specific pathways make it possible to control blastocyst development and differentiation, leading to embryos of improved quality and viability. Work is in progress to analyze gene expression in these blastocysts.This work was supported by grant MCYT, project AGL-2005-04479.

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 38C. 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 37C (red fox, badger, ermine, western roe deer) or 38C (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: Consejera de Medio Ambiente, Ordenacin del Territorios e Infraestructuras, Asturias, SPAIN. The authors acknowledge CEPESMA, Luarca, Asturias, SPAIN.