본 실험은 국화의 바이로이드 제거에 이용되는 초저온처리 시 국화 품종 'White ND'을 적합한 처리조건을 확립하기 위해 초저온처리의 단계별 요인을 실험하였다. 그 결과 생장점의 크기는 1 mm(엽원기 2~3매 포함)에서 높은 생존율과 신초 재생율을 나타내었고, vitirification 처리시 PVS3가 효과적이었으며, 처리 시간은 60분 처리 하였을 때 높은 생존율 및 정 상 신초 재생율을 보였다. 또한 vitrification을 위한 전처리 조건은 sucrose 농도를 88 mM 24시간, sucrose 0.3 M 16시간, sucrose 0.5 M 6시간, sucrose 0.7 M 3시간으로 처리하는 것이 초저온 처리 후 생존율 및 신초 재생율을 높이는데 효과적이었으며, 재생된 정상 식물체는 모본과 비교하여 ploidy level이 동일한 것으로 보아 식물체의 유전적 변이가 일어나지 않았다.

This study was carried out to study the survival rate of thawed Hanwoo embryos frozen by the slow-rate freezing or the cryotop vitrification method. Hanwoo cumulus-oocyte complexes were recovered from ovaries at a slaughter house, matured for 20~22 hours, fertilized with Hanwoo semen for 5~6 hours, and cultured for 7~9 days in 38.5℃, 5% CO2 incubator. For freezing, Day 7∼9 blastocysts were collected. Embryos for the slow-rate freezing were equilibrated in 1.8 M ethylene glycol (EG) with Dulbecco's phosphate-buffered saline (D-PBS). Programmable cell freezer was precooled down to —7℃, and the straw was seeded during 8 minutes-holding time, and was cooled to —35℃ at the cooling rate of 0.3℃/min, and then was plunged and stored in liquid nitrogen. Embryos for the cryotop vitrification were treated in TCM199 with 0.5 M sucrose, 16% EG, 16% dimethylsulfoxide (DMSO). Embryos were then loaded individually onto cryotop and plunged directly into liquid nitrogen. The survival rates of embryos frozen by these two freezing methods were evaluated at 12 to 24h post-thawing. The survival rates of frozen/thawed Hanwoo embryos by the cryotop vitrification method (56.86 ± 26.53%) were slightly higher than those by the slow-rate freezing method (55.07 ± 26.43%) with no significant difference. Using the cryotop vitrification and the slow-rate freezing of Hanwoo blastocysts on Day 7 following in-vitro fertilization (IVF) treatment, the survival rates of frozen/thawed Hanwoo embryos were 72.65 ± 18.3% and 79.06 ± 17.8%, respectively. The survival rates by the cryotop vitrification were higher than those by the slow-rate freezing on both Day 8 and 9 with significantly higher survival rate on Day 9 (p<0.05). Using the cryotop vitrification and the slow-rate freezing of Hanwoo embryos to compare between three different blastocyst stages, the survival rates of the blastocyst stage embryos were 66.22 ± 18.8% and 45.76 ± 12.8%, respectively with higher survival rate by the vitrification method (p<0.05). And the survival rate of expanded blastocysts was higher than those of early blastocysts and blastocysts in two freezing methods with significantly higher survival rate by the slow-rate freezing method (p<0.05).

파이로그린공정의 염폐기물처리과정에서 발생되는 주요 산화물 형태의 폐기물에는 희토류폐기물이 있으며 주요 구성 핵종은 Y, La, Ce, Pr, Nd, Sm, Eu, Gd 등 8종이다. 최종적인 희토류폐기물의 형태는 산화물 형태로 발생된다. 본 연구에서는 붕규산 유리계 내에서 희토류 산화물의 유리화 타당성을 평가 하기 위하여 6종의 유리조성을 개발하였다. 희토류 8핵종 혼합에 대한 solubility는 1,200℃에서 25wt% 미만, 1,300℃에서 30wt% 미만 waste loading으로 온도 상승에 따라 증가하는 것으로 나타났으며 liquidus temperature는 균질한 유리가 형성된 20wt% waste loading에서 950℃ 이하로 평가되었다. 희토류 산화물의 유리매질 내 solubility 이상에서는 희토류-oxide-silicate 결정이 생성된 유리세라믹을 이차상으로 형성하였으며 20~25wt% waste loading의 표면균질성이 양호한 유리는 용융온도 1,200~1,300℃ 범위에서 점도 100 poise 이하, 전기전도도 1 S/cm 이상으로 유도가열식 저온용융로설비에서의 운전 용이성이 매우 양호한 것으로 평가되었다. 개발된 유리조성에 대한 기타 물리·화학적 특성 평가를 위한 실험들이 향후 수행될 예정이다.

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.