Both tissue and cell cryopreservation can be applied for biodiversity conservation. The proper preservation of tissues and cells from a wide range of animals of different species is of paramount importance because these cell samples could be used to reintroduce lost genes back into the breeding pool by somatic cell cloning. The aim of this work was to investigate the effect of vitrification on viability of vitrified sheep fibroblasts for conservation of biodiversity so that it might be used in the future to provide nuclear donors. Skin samples collected from 10 adult sheep were cut into small pieces (1×1mm), placed into culture Petri dishes containing DMEM supplemented with 20% (vol/vol) fetal bovine serum, and covered with coverslips followed by incubation at 5% CO2, 95% RH, and 37°C. During culture, fibroblasts left skin samples and proliferated. Culture medium was changed every 4 days. After 21 to 22 days of incubation, a fibroblast monolayer was observed, culture medium was removed, and cells were incubated for 7 to 10min in the presence of Dulbecco’s PBS+0.25% trypsin. Dissociated fibroblasts were washed with DMEM by centrifugation at 300×g for 10min. For vitrification, fibroblast samples were then diluted at a concentration of 2×106cellsmL−1 in DMEM+ 20% ethylene glycol, 20% dimethylsulfoxide, and 0.5molL−1 of sucrose. The fibroblasts were then exposed to 50 and 100% vitrification solution (VS) at 37°C for 5min and 30s, respectively. Fibroblasts after saturation in VS were transferred and placed into 0.25-mL plastic straws. Straws were sealed with modelling clay and plunged into LN. Viability of frozen-thawed fibroblast samples was detected using the Trypan Blue staining method (frozen-thawed: 53.0±2.6%; control (fresh): 98.5±1.2%). The values obtained are expressed as mean±standard error of the mean. Statistical analysis was done using Student’s t-test. Results indicated that there was a significant difference in viability between fresh and cryopreserved fibroblasts. Importantly, our data suggest that the use of vitrification reduced the toxic elements contained in the cryopreservation solution while maintaining a similar ability to produce viable fibroblasts after cryopreservation. Although further work on the viability of sheep skin fibroblast with the vitrification method is needed, these data suggest that with vitrification a faster cooling rate and high level of cryoprotectants are able to minimize ice crystal formation and should be further evaluated as a routine mechanism for cryopreserving sheep fibroblasts.

Wildlife conservation requires innovative preservation methods in order to preserve gene and species biodiversity. Nuclear transfer has the potential to preserve genes from critically endangered wildlife species where few or no oocytes are available from the endangered species, and where cryopreserved cell lines have been conserved in cryobanks. The purpose of this study was to investigate the developmental ability of embryos reconstructed with transfer of cryopreserved somatic cells from the Kazakh argali (Ovis ammon collium) to enucleated domestic sheep (Ovies aries) oocytes. Frozen-thawed fibroblasts were diluted with DMEM (1:5) and centrifuged at 300g for 7 to 10 min. Supernatants were removed, and cells were diluted with DMEM at a concentration of 2 × 106 cells mL−1. Fibroblasts were placed into culture Petri dishes containing DMEM supplemented with 20% (v/v) fetal bovine serum (FBS), and incubated at 5% CO2, 95% relative humidity, and 37°C. After 21 to 22 days of incubation, a fibroblast monolayer was observed, culture medium was removed, and cells were incubated for 7 to 10 min in presence of Dulbecco’s PBS + 0.25% trypsin. Dissociated fibroblasts were washed with DMEM by centrifugation at 300 × g for 10 min. Cumulus-oocyte complexes were aspirated from slaughterhouse ovaries. Subsequently, the cumulus cells were removed by pipetting in 1 mg mL−1 hyaluronidase in HEPES-buffered TCM-199; zonae pellucidae were removed by incubation in 2 mg mL−1 pronase in HEPES-buffered TCM-199 supplemented with 2% cattle serum (T2) for 1 min. Bisection was performed by hand under a stereomicroscope using a microblade in 5 μg mL−1 cytochalasin B in TCM-199 supplemented with 20% cattle serum (T20). Fusions were performed 24 to 28 h after the start of maturation. One cytoplast was attached to one fibroblast in 500 μg mL−1 phytohemagglutinin dissolved in T2. In the fusion chamber, covered with fusion medium (0.3 M mannitol, 0.1 mM MgSO4, 0.05 mM CaCl2, and 0.01% polyvinyl alcohol), one cytoplast-fibroblast pair was fused with one cytoplast in a single step. The fusions were performed with a single DC pulse of 100V, each pulse for 9 μs. Successfully fused embryos were activated 1 h after the end of fusion by incubation in 2 μM calcium ionophore (Sigma, St. Louis, MO, USA) in T20 for 5 min followed by 3-h incubation in microdrops of culture medium containing 2 mM 6-DMAP. After successful reconstruction, 79 nuclear transferred and activated embryos were cultured in well-of-the-wells in trigas (5% O2, 5% CO2, 90% N2) in Submarine incubation system for 7 days. All except 15 embryos cleaved; 35 (44.3%) developed to compacted morula, and 15 (18.9%) to the blastocyst stage. In conclusion, argali embryos developed from reconstruction using their frozen–thawed fibroblasts combined with domestic sheep cytoplasts; however, in vitro developmental ability to the blastocyst stage was limited. Additional research that establishes the early embryo development with optimising nuclear transfer techniques may have a potential role in the conservation of critically endangered wildlife species.