The purpose of this study was to assess follicular viability and competence through in vitro culture of preantral follicles isolated from vitrified mouse whole ovaries. Mouse preantral follicles were enzymatically isolated from vitrified-warmed and fresh ovaries and cultured for 10 days followed by in vitro oocyte maturation. In vitro matured oocytes were fertilized and cultured to the blastocyst stage. Five minutes pre-exposure to vitrification solution of whole ovaries had significantly higher (p<0.05) oocyte survival and maturation rates than between 10 min exposure groups. Oocyte diameter was significantly smaller (p<0.05) in the 5 and 10 min exposure groups (69.4±2.8 and 67.8±3.1) when compared to that of control group (71.7±2.1). There was no statistical significant difference in blastocyst development rates between vitrification group (8.6%) and the fresh control group (12.0%). The mean number of cells per blastocyst was significantly lower (p<0.05) in the vitrification group (41.9±20.2) than in the fresh control group (55.1±22.5). The results show that mouse oocytes within preantral follicles isolated from the vitrified whole ovaries can achieve full maturation, normal fertilization and embryo development.

원전에서 발생되는 방사성폐기물에 대한 고화처리 방법 중 하나인 유리화기술이 일부 가연성폐기물에 대해 적용되고 있다. 국내외적으로 중저준위 방사성폐기물의 효과적인 감용과 안정적인 처분을 위해 다양한 폐기물에 대한 유리화기술 적용방안이 확대 연구되고 있으며, 최근에는 가연성폐기물 뿐만 아니라 알루미늄 금속과 같은 비가연성폐기물에도 유리화 연구가 활발하게 진행되고 있다. 공기조화계통 (HVAC)에는 주로 필터가 이용되고 있으며, 사용 후 필터는 여과재 (유리섬유 및 알루미늄)를 이용하여 배기체를 흡착하기 때문에 방사성폐기물로 처리가 되어야 한다. 본 연구는 필터에 대한 처리기술 연구를 위해 유도가열식 저온용융로 (Cold Crucible Induction Melter: CCIM)를 이용한 유리화 타당성 연구를 수행하였다. 사용후 필터에 대한 유리화 (Vitrification)는 먼저 유리섬유 및 알루미늄 함량을 고려한 최적의 유리조성을 개발 하였으며, 개발된 유리조성을 이용하여 최적의 폐기물 저감을 위한 용융변수와 최종 생성된 유리고화체의 특성을 분석하였다. 사용후 필터 유리화용 조성유리는 주로 SiO2와 B2O3로 구성되어 있다. 전기로를 이용한 용융물 특성시험에서는 폐기물 투입률 및 최종 생성물인 유리고화체의 특성이 검토되었다. 본 연구에서는 알루미늄 금속과 유리섬유로 구성된 필터에 대한 유리조성 개발과 이를 통해 생성된 유리고화체의 물리화학적 특성을 검토하고 유리화 타당성을 확인하였다.

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.

The objective of this study was to investigate the effectiveness of cryopreservation methods for the effect of various vitrification containers, such as EM-grid, OPS, or cryo-loop on the survival and developmental rate of vitrified mouse pronuclear embryos, and mouse cleavage embryo, at 21, 24, 27 and 30 hr after hCG injection. Post-thaw cleavage was similar among treatments, while the developmental rates of mouse blastocyst and hatched blastocyst were higher ( <0.05) in 27 hr and 30 hr than 21 hr. The developmental rate of hatched blastocyst at vitrified cleavage mouse embryos in cryo-loop was significantly higher than vitrified pronuclear embryos of control group as well as EM-grid and OPS ( <0.05). The developmental rate using cryo-loop was higher than EM-grid, but in case of OPS at vitrified cleavage and mouse pronuclear embryos, no significant difference was noticed. These results of our study show that the developmental rates of mouse embryos were unaffected by various vitrification containers, but in case of mouse embryos and hatched blastocysts at late vitrified pronuclear embryos the developmental rates were higher than early vitrified pronuclear embryos. Moreover, the developmental rate of hatched blastocyst at vitrified cleavage mouse embryos was significantly higher than vitrified pronuclear embryos. For better execution of this study, it will be mandatory to include improvement of vitrification containers, cryopreservation methods and conditions, higher survival rate, safe preservation, contamination and embryo loss.

The present study was performed to investigate the survival and subsequent embryonic developmental rate of immature and mature oocytes after vitrification and pronuclear stage embryos after slow-freezing and vitrification. We have also tried to examine the dependency of concentrations (7.5, 15%) and exposure time (5, 10, 20 min) of ED cryoprotectant on developmental rate of pronuclear stage embryos. The developmental rates of 2-ce1l and blastocyst embryos at mature oocytes were significantly (p<0.05) higher than immature oocytes. After slow freezing, vitrification and thawing of pronuclear stage embryo, the survival and developmental rates of blastocysts and hatched blastocysts were significantly (p<0.05) higher after vitrification than after slow-freezing. On contrary, the developmental rates of 2-cell embryos were significantly (p<0.05) higher after slow freezing than after vitrification. The cryopreservation methods of pronuclear stage embryos vitrified by exposed to 7.5% ED solution for 5 minutes was significantly (p<0.05) higher than other experimental group. The results of our study suggest 1hat the developmental rates of mature oocytes have been more successful than immature oocytes during vitrification. Vitrification was more efficient than slow freezing in case of pronuclear stage embryos. The effective cryopreservation method of pronuclear stage embryos was vitrified by exposed to 7.5% ED solution for 5 minutes.

The in vitro maturation rate of vitrified-thawed canine oocytes was 30.8±3.4%. The in vitro maturation rate of vitrified oocytes was lower than that of the control (52.0±2.5%, p<0.05). The in vitro maturation rate of vitrified-thawed oocytes were significantly (p<0.05) lower than those of fresh oocytes. The in vitro maturation and developmental rates of the vitrified-thawed oocytes were 17.5±2.5% and 8.8±3.4%, respectively. This results were lower than the control group (43.6±3.2% vs 20.0±3.0%). SOD1 gene expression of 1～2 mm of follilce size were higher than those of above 6 mm follicle size. SOD2 gene expression of 1～2 mm of follicle size were significantly higher than those of above 6 mm follicle size (p<0.01). The expression pattern of SOD1, 2 was constantly expressed in both groups but strongly expressed in follicles (1～2 mm) group when compared to the above 6 mm follicles. SOD gene expression between groups the fresh and vitrified oocytes groups were significant differences in rates. However, RGS gene expression between groups the fresh and vitrified oocytes groups were no significant differences in rates.

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.

가압경수로 원전 농축폐액건조설비(CWDS)에서 생성된 농축폐액건조물에 대한 고화 방안이 국내외적 으로 다양하게 연구되어 왔다. 농축폐액의 고형화는 시멘트, 파라핀 및 폴리머와 같은 고화제를 이용하여 수행되어 왔다. 동시에 농축폐액에 대한 감용비 및 운영상의 효과를 극대화하기 위한 농축폐액건조물 전처 리 방안이 연구되었다. 건조된 분말 형태의 폐기물을 유리화 설비에서 직접 처리할 경우 비산에 의한 배기 체 계통 및 폐기물 투입구 막힘 현상을 초래할 수 있으며, 취급 중 비산에 의한 방사성피폭을 초래할 가능 성이 있다. 본 연구는 분말형태의 폐기물을 유리화설비에서 고화하기 위한 전처리방안을 수립하고 이를 통해 설비운영 및 폐기물 운영관리의 안전성을 확보하는데 목적이 있다.

We investigated the cleavage rate and blastocyst yield for each culture condition to enhance tolerance of cryo-preservation of bovine IVF embryo with relatively lower cryo-tolerance compared to in vivo embryo. The cleavage rate and blastocysts yield for CR1aa, IVMD, IVD, CR1aa+10% FBS were 73.2, 69.3, 72.8, 68.5% and 44.1, 30.8, 33.3, 48.0%, respectively. The values did not differ among each treatments without serum. For embryo vitrification, In vivo and In vitro blastocysts were exposed to VS1(10% glycerin, 0.1 M glucose, 0.1 M sucrose, PEG 1%) for 5 min, and VS2 (10% glycerin, 10% EG, 0.2 M glucose, 0.2 M sucrose, PEG 2%) for 5 min and then VS3 (10% glycerin, 30% EG, 0.3 M glucose, 0.3 M sucrose, PEG 3%) for 1 min. The exposed embryos were then loaded into the 0.25 ml plastic straws and then plunged into liquid nitrogen. The straws were held for period of 1 to 2 weeks before thawing. In embryo viability, no differences in blastocyst re-expansion rates were found between in vivo and in vitro embryos. whereas expansion-BL rates was significantly higher for in vivo-derived embryos (72.7%) when compared to in vitro-derived embryos (51.4%), respectively (P<0.05). In conclusion, our results indicate that combined use of CRIaa culture medium with vitrification might enhance tolerance of cryopreservation for bovine IVF embryo production.

가압경수로 원전의 농축폐액건조설비에서 발생된 농축폐액 건조물을 유리화 하는 방안이 연구되어 왔다. 중저준위 방사성폐기물을 유리화할 경우 최종 생성물은 내구성이 우수하고 현저한 부피저감 효과의 장점을 가지고 있다. 붕산농축폐액에 대한 유리화 타당성 연구는 분말시료의 전처리 방법 개발, 유리조성 프로그램을 이용한 유리개발 및 실증시험으로 수행되었다. 분말시료에 대한 전처리 방안으로는 유리화설비에 투입하기 전에 고형성을 갖도록 펠렛화하는 것이다. 농축폐액 성분중 Na와 B의 함량 분포는 유리속에 용융되는 정도와 설비로부터의 폐기물 배출·처리에 영향을 주기 때문에 이를 고려하여 유리조성이 개발되어야 한다. 실증시험에서는 폐기물 투입률, 배기체 특성 및 최종 생성물인 유리고화체의 특성이 검토되었다. 본 연구는 붕산농축폐액에 대한 유리고화체의 물리화학적 특성을 검토하고 유리화 타당성을 확인하는데 목적이 있다.

The purpose of this study was to determine the effects of Taxol pre-treatment to in vitro matured bovine oocytes, and sucrose and trehalose added to vitrification solution on spindle morphology and embryonic development following cryopreservation. Bovine oocytes were collected from ovaries and matured in tissue culture medium 199 (TCM 199) supplemented with 10% Fetal Bovine Serum (FBS), 0.05ng/ml epidermal growth factor, 0.01 IU/ml luteinizing hormone and estradiol for 22h in , 5% , TCM 199-HEPES containing 20% FBS was used as basic medium (BM) to prepare vitrification solution. Oocytes were pre-treated with Taxol in maturation medium for 15 min prior to vitrification. Oocytes were exposed to 1.6 M ethylene glycol (EG) and 1.3M dimethyl sulfoxide (DMSO) in BM and then were exposed to 3.2 M EG, 2.6 M DMSO and 0.5 M sucrose in BM or 3.2 M EG, 2.6 M DMSO and 0.5 M trehalose in BM. Oocytes with cumulus cells and oocytes without cumulus cells were considered as control 1 and control 2, respectively and held in TCM 199-HEPES at . Oocytes were frozen using modified solid surface vitrification and were stored in cryotubes in liquid nitrogen for more than 1 week. Frozen oocytes were thawed in TCM 199-HEPES containing 0.5 M, 0.25 M and 0.1 M sucrose in BM for 2 min, respectively or 0.5 M, 0.25 M and 0.1 M trehalose in BM for 2 min, respectively. Immunoflurorescence staining of oocytes was performed to assess spindle morphology and chromosome configuration of oocytes. The rates of cleavage and blastocyst were examined following in vitro fertilization. Normal spindle morphology rate of oocytes pre-treated with Taxol prior to vitrification was not higher than that of other vitrified groups. Taxol pre-treatment did not increase cleavage and blastocyst formation rates, although control groups showed significantly higher rates (p<0.05). Percentages of normal spindle and embryonic development were not significantly different among vitrified groups regardless of type of sugar. In conclusion, Taxol pre-treatment of oocytes before cryopreservation did not reduce the damage induced by vitrification and subsequently did not improve embryonic development following vitrification. Trehalose may be used as an alternative non-permeating cryoprotectant in vitrification solution.

The purpose of this study was to compare the efficiency of slow freezing with that of vitrification method for the cryopreservation of human embryos. Human embryos were derived from in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) and the mixed solution of propanedial (1.5, 1.0, 0.5M PROH) and sucrose (0.1M), ethylene glycol (7.5, 15%), dimethyl sulfoxide (7.5, 15% DMSO), sucrose (0.5, 1.0M) and SPS (Serum Protein Substitute) was used for a cryoprotectant for slow freezing and vitrification solution, respectively. Rates of recovery after thawing, morphological normality, post-thaw viability, arrest, morphological abnormality and preimplantation development were compared between two protocols. After freezing-thawing, recovery and survial rate of slow freezing was (88.6% and 73.4%), whereas vitrification was (99.2% and 96.2%) (p<0.05). The arrest rate of slow freezing was significantly lower compared with those of vitrification(8.7% vs 29.9%) (p<0.05). Preimplantation development to the 2-cell (83.8% vs 67.7%), 4-cell (69.0% vs 47.2%) and 8-cell (62.4% vs 37.8%) stages 24, 48 and 72 h after thawing, respectively, were higher in the slow freezing than the vitrification. After slow freezing and vitrification of human embryo at 2-8cell stage, the rate of recovery rate, survival rate and partial damage rate were 92.0% vs 100%, 80.4% vs 96.2% and 52.2% vs 19.0%, respectively. And partial damage rate was significantly lower than those of slow freezing method (p<0.05). These results demonstrate that a slow freezing using PROH is more efficient than a vitrification for cryopreserving the human zygotes, although the vitrification yielded better recovery, survival and partial damage of frozen-thawed 2-8 cell stage embryos than slow freezing method.