Advances in reproduction technologies, such as in vitro maturation, IVF, and in vitro culture, stimulated research for efficient cryopreservation techniques for mammalian oocytes. It is well known that the oocyte is the largest cell of an animal's body and as such, is full of water and, in many species, fat, making it difficult to cryopreserve. The objective of this work was to study the effect of vitrification for cryopreservation of the metaphase II plate (MPII) of sheep oocytes. Ovaries from 20 ewes of Kazakh Arkharo-Merino breed were acquired after slaughter and maintained at 37°C in TCM-199. The maturation medium was TCM-199, containing 1 mM of glutamine, 10% FBS, 5 μg mL–1 FSH, 5 μg mL–1 LH, 1 μg mL–1 oestradiol, 0.3 mM sodium pyruvate, and 100 mM cysteamine. The oocytes were incubated in 400 μL of medium in 4-well dishes covered with mineral oil. The IVM conditions were 5% CO2 in humidified air at 39°C for 24 h. Then they were placed for 10 min in a media with Hoechst 33342 (3 μg mL–1) and cytochalasin B (7 μg mL–1) to facilitate the enucleation of the MPII with a minimum volume of ooplasm. The MPII plates were divided into 2 groups: the vitrification group was exposed to vitrification media containing 1.12 M ethylene glycol (ET) + 0.87 M ME2SO for 5 min and was exposed in vitrification media containing 2.24 M ET + 1.75 M ME2SO for 5 min, and then in vitrification solution containing 4.48 M ET + 40% ME2SO + 0.25 M sucrose for 30 s. Oocytes were loaded into cryoloop and plunged into liquid nitrogen (LN2). Oocytes were thawed in a 25°C water bath and then placed in TCM-199 at 20% fetal bovine serum. After 15 min of incubation the oocytes were activated for extrusion of the second polar body in 1 mg mL–1 Ca ionophore for 5 min and washed for 5 min followed by 4 h in 6-DMAP (0.12 mM) + cycloheximide (0.6 μg mL–1). After activation the MPII were washed and cultured for 20 h. The control group received the same treatment, but they were not vitrified. Differences between the experimental groups were tested using Chi-squared test. Our research showed the expulsion of the second polar body after activation was observed in more than 62.2% of the MPII that were not vitrified (control group), whereas 40.5% of vitrified plates had expulsion of polar bodies (P < 0.05). These preliminary studies showed that it is possible to vitrify MPII plates. On the other hand, the drastic reduction of the volume of the sheep oocytes might make cryopreservation possible with greater efficiency.

Advances in reproduction technologies, such as in vitro maturation, IVF, and in vitro culture, have stimulated research for efficient cryopreservation techniques for mammalian oocytes. It is well known that the oocyte is the largest cell of an animal’s body and as such, is full of water and, in many species, fat, making it difficult to cryopreserve. The objective of this work was to study the effect of vitrification for cryopreservation of the metaphase II plate (MPII) of sheep oocytes. In our experiment, we used the Vit-Master™ (MTG, Bruckberg, Germany). Ovaries from 19 ewes of Kazakh Arkharo-Merino breed were acquired after slaughter and maintained at 37°C in TCM-199. The maturation medium was TCM-199, containing 1 mM of glutamine, 10% fetal bovine serum, 5 μg mL−1 FSH, 5 μg mL−1 LH, 1 μg mL−1 oestradiol, 0.3 mM sodium pyruvate, and 100 mM cysteamine. The oocytes were incubated in 400 μL of medium in 4-well dishes covered with mineral oil. The IVM conditions were 5% CO2 in humidified air at 39°C for 24 h. Then, oocytes were placed for 10 min in medium with Hoechst 33342 (3 μg mL−1) and cytochalasin B (7 μg mL−1) to facilitate enucleation of the MPII with a minimum volume of ooplasm. The MPII plates were divided into 2 groups: the vitrification group was exposed to vitrification media containing 1.12 M ethylene glycol (ET) + 0.87 M ME2SO for 5 min and was exposed in vitrification media containing 2.24 M ET + 1.75 M ME2SO for 5 min, and then in vitrification solution containing 4.48 M ET + 40% ME2SO + 0.25 M sucrose for 30 s. Oocytes were loaded into a cryoloop and using negative pressure of liquid nitrogen in the chamber for freezing with the VIT-Master. Oocytes were thawed in a 25°C water bath and then placed in TCM-199 at 20% fetal bovine serum. After 15 min of incubation, the oocytes were activated for extrusion of the second polar body in 1 mg mL−1 Ca ionophore for 5 min and washed for 5 min followed by 4 h in 6-DMAP (0.12 mM) + cycloheximide (0.6 μg mL−1). After activation, the MPII were washed and cultured for 20 h. The control group received the same treatment but were not vitrified. Differences between the experimental groups were tested using Chi-squared test. Our research showed that expulsion of the second polar body after activation was observed in more than 59.7% of the MPII that were not vitrified (control group), whereas 37.7% of vitrified plates had expulsion of polar bodies (P < 0.05). These preliminary studies showed that it is possible to vitrify MPII plates. On the other hand, the drastic reduction of the volume of the sheep oocytes might make cryopreservation possible with greater efficiency.

This work evaluated methods for goat morulae cryopreservation by using cryoloop: vitrification (V) and super-cooling ultra-rapid vitrification (SCURV). The vitrification method was applied according to the method described by Vajta et al. (1998 Mol. Reprod. Dev. 51, 53–58). Both treatments used a vitrification solution [VS: 20% (3.6 mol L−1), ethylene glycol (EG), 20% (2.4 mol L−1) dimethylsulfoxide (Me2SO)] 0.5 mol L−1 sucrose in DPBS with 10% BSA in both methods. In our experiment, we used the Vit-Master™ (MTG, Bruckberg, Germany). The super-cooled LN facilitates heat transmission between LN and the cryosolution interface and this is efficient for bovine semen and blastocyst cryoconservation (Arav et al. 2002 Mol. Cell. Endocrinol. 187, 77–81). By surgical flushing 25 super stimulated goats, 127 transferable morulae were harvested; 39 morulae were transferred fresh to synchronized recipients (control) and the others were cryopreserved by V (n = 46) or SCURV (n = 42), respectively thawed or warmed, and transferred to recipients. Embryos were vitrified using the cryoloop. They were first incubated in 50% VS for 2 min and then transferred for 30 s into 100% VS. Each embryo was loaded by cryoloop, which was immediately submerged into and stored in liquid nitrogen. Warming was done by placing the narrow end of the cryoloop into DPBS + 0.25 M sucrose for 5 min. Embryos were then transferred into DPBS + 0.125 M sucrose for 3 min and finally to DPBS until transfer. The SCURV morulae were then exposed to 50% and 100% VS at 37°C for 2 min and 30 s, respectively. Embryos after saturation in VS were transferred by cryoloop and using negative pressure of liquid nitrogen in the chamber for freezing with the VIT-Master. Thawing vitrify embryos was accomplished by placing the vitrified embryos in solutions of sucrose 0.25 M and 0.125 M with 2- and 3-min exposures accordingly. After thawing, embryos were transferred. Statistical analysis was done using Student’s test. The kidding rate following transfer of fresh, frozen-thawed vitrification, and SCURV methods were 22, 16, and 16 kids, respectively. No statistical difference was found for the percentage of does kidding following transfer thawed after vitrification (34.7 ± 4.5%a), and SCURV methods (38.1 ± 5.9%b). The survival rate following transfer of fresh embryos (56.4 ± 4.9c) was higher and in line with previous findings using VS. Differences were statistically significant (ac, bc P < 0.05). Importantly, our data suggested that the SCURV method can be used for cryopreservation of goat morulae and has similar success to the vitrification method. While further work on the developmental competence of embryos cryopreserved with the SCURV method is needed, we hypothesise that SCURV, with a faster freeze rate and potentially a lower level of cryoprotectants, may be able to minimize ice crystal formation; SCURV should be further evaluated as a routine mechanism for cryopreserving goat embryos.

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.