There are diverse methods of cryopreservation of mammalian embryos with variable degrees of success. Although cryopreservation technique of mammalian embryos has been advanced, freezing stress affect to cellular event such as apoptosis and autophage in embryos. The objective of the study is to investigate the affection of to survival, development, live offspring, apoptosis and autophagy on embryo. Mouse embryos were vitrified and thawed using normal straw and modified cut standard straw (M-CSS), then in vitro cultured until blastocyst stage and transferred to recipient. Recovery rates (100 vs 99.2%), survival rates (99.2 vs 78.6%), developmental rates (18.4 vs 10.7%), total cell numbers (45 vs 37), preganacy rates (34.5 vs 25%) and offspring numbers (10.1 vs 4.9 %) of M-CSS group are significantly higher than those of normal straw vitrified group. Also, rate of apoptosis in blastocysts developed using M-CSS (1.9%) was significantly lower than using normal straw vitrification (2.7%). Apoptosis-related gene, caspase 3, was expressed at the highest level in blastocysts derived from normal straw group. However, no differences of autophagy related gene, Atg6 and expression of LC3 between normal straw and M-CSS groups were observed. In conclusion, the standard vitrification procedure induces mitochondrial apoptosis in zygotes in an autophagy-independent manner, whereas the novel M-CSS procedure may improve embryo vitrification.

One-step dilution and direct transfer would be a practical technique for the field application of frozen embryo. This study was to examine whether Jeju Black Cattle (JBC, Korean Cattle) can be successfully cloned from vitrified and one-tep diluted somatic cell nuclear transfer (SCNT) blastocyst after direct transfer. For vitrification, JBC-SCNT blastocysts were serially exposed in glycerol (G) and ethylene glycol (EG) mixtures〔10% (v/v) G for 5 min., 10% G plus 20% EG (v/v) for 5 min., and 25% G plus 25% EG (v/v) for 30 sec.〕which is diluted in 10% FBS added D-PBS. And then SCNT blastocysts were loaded in 0.25 ml mini straw, placed in cold nitrogen vapor for 3 min. and then plunged into LN2. One-step dilution in straw was done in 25℃ water for 1 min, by placing vertically in the state of plugged- end up and down for 0.5 min, respectively. When in vitro developmental capacity of vitrified SCNT blastocyst was examined at 48 h after one-step dilution, hatched rate (56.4%) was slightly lower than that of control group (62.5%). In field trial, when the vitrified-thawed SCNT blastocysts were transferred into uterus of synchronized 5 recipients, a cloned female JBC was delivered by natural birth on day 299 and healthy at present. In addition, when the short tandem repeat marker analysis of the cloned JBC was evaluated, microsatellite loci of 11 numbers was perfectly matched genotype with donor cell (BK94-14). This study suggested that our developed vitrification and one-step dilution technique can be applied effectively on field trial for cloned animal production, which is even no longer in existence.

In vitro-grown axillary buds of Melia aredarach were successfully cryopreserved by vitrification. On the MS medium supplemented with BA 1 mg/L, multiple shoots were developed within 4~5 weeks. Plantlets of Melia azedarach were cold-hardened at 10℃ for a 16-hr photo-period for 6 weeks. Excised axillary shoot-tips from hardened plantlets were precultured on a solidified Murashige & Skoog agar medium (MS) supplemented with 0.7 M sucrose for 1 day at 25℃. Axillary shoot-tip meristems wert dehydrated using a highly concentrated vitrification solution (PVS2) for 60 min at 0℃ prior to a direct plunge into liquid nitrogen (LN). The PVS2 vitrification solution consisted of 30% glycerol (w/v), 15% ethylene glycol (w/v), 15% DMSO (w/v) in MS medium containing 0.4M sucrose. After short-term warming in a water bath at 40℃, the meristems were transferred into 2 ml of MS medium containing 1.2M sucrose for 15 min and then planted on solidified MS culture medium. Successfully vitrified and warmed meristems resumed growth within 2 weeks and directly developed shoots without intermediary callus formation. The survival rate of cold-hardened plantlets for 3 and 4 weeks was 90%. We did not find any difference in PCR-band patterns between control and cryopreserved plants. This method appears to be a promising technique for cryopreserving axillary shoot-tips from in vitro-grown plantlets of Medicinal plants.