• Clinical and Experimental Reproductive Medicine
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• 제27권4호
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• pp.367-372
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• 2000

Objective: This study was performed to evaluate whether vitrification method could be used for the cryopreservation of human blastocysts derived from IVF program. Methods: Surplus embryos were obtained from consented IVF patients. Controlled ovarian hyperstimulation was done with midluteal GnRH agonist, gonadotropin and hCG. After oocyte retrieval and insemination, fresh embryo transfer was done at $4{\sim}8$ cell stage. The surplus embryos after ET were cultured in blastocyst medium up to 6 days after oocyte retrieval. Obtained blastocysts were cryopreserved with our vitrification method. Blastocysts were exposed to 1.5 Methylene glycol (EG) in phosphate buffered saline (PBS) for 2.5 minutes, followed by 5.5 M EG plus 1 M sucrose for 20 seconds. Then 1 to 3 blastocysts were mounted on electron microscope (EM) grid and the grid was plunged into liquid nitrogen for storage. For thawing, blastocyst-containing EM grids were sequentially transferred in 1.0 M, 0.5 M, 0.25 M, 0.125 M and 0 M sucrose solution at the intervals of2.5 minutes. And blastocysts were cultured for about 6 hours and only re-expanded blastocysts were transferred to uterus of the patients on 4 to 5 days after ovulation in natural cycle or on 18 to 19 day of artificial cycle. Results: From Oct. 1998 to Jul. 1999, 34 patients were agreed to participate in this study. The mean age and duration of infertility of the patients were 31.6 years and 4.1 years, respectively. Among 34 cycles. replacements could be done in 20 cycles (58.8%). A total 93 blastocysts were thawed and 48 (51.6%) of them survived. Thirty-eight blastocysts, mean 1.9 embryos per patient, were transferred, resulting in 5 clinical pregnancies which consisted of 1 triplet, 2 sets of twins and 2 singleton pregnancies. The pregnancy rate per transfer was 25% and implantation rate was 23.6%. Five patients delivered 7 healthy babies including 2 sets of twins at term. Conclusion: Successful pregnancies and deliveries were established after transfer of vitrified human blastocysts. Vitrification using ethylene glycol as cryoprotectant and electron microscope grid is a rapid and simple method that can be effectively applied for the cryopreservation of human blastocysts.

• 한국가축번식학회지
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• 제21권1호
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• pp.47-52
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• 1997

본 연구는 체외생산된 생쥐 1-세포기배의 초자화동결이 체외/체내 발달율과 수정란 이식후 태어난 산자의 염색체에 미치는 영향을 검토하기 위해 실시하였다. 체외수정하여 얻어진 생쥐 1-세포기배는 EFS40 (40% ethlyene glycol, 30% Ficoll, 0.3 M sucrose) 동결액을 이용하여, 상온 ($25^{\circ}C$)에서 30초 동안 노출한후, 액체질소에 침지하여 초자화동결하였다. 동결후 융해된 생쥐 1-세포기 수정란은 M16 배양액에서 4일동안 배반포기까지 배양하였고, 이때 배반포기까지의 체외배 발달율은 71.5%였으며, 배양된 배반포기배는 가임신 3일된 대리모의 한쪽 또는 양쪽 자궁각에 (6~8개/자궁각) 이식하였다. 모든 대리모는 분만을 유기하였으며, 그 결과를 요약하면 다음과 같다. 임신율과 체내 생존율 즉, 산자 생산(80.0, 39.6%)에 있어서 대조군 (77.8, 50.0%)과 유의차가 없었다. 또한 수정란 이식후 태어난 모든 산자의 염색체 (n=40)는 정상이었다. 이상의 결과로 미루어볼 때 본 실험에서 이용된 초자화동결방법은 생쥐 1-세포기배의 동결에 효과적으로 이용될 수 있음을 시사한다.

• 한국작물학회지
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• 제59권4호
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• pp.498-504
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• 2014

Potato virus Y (PVY) and potato leafroll virus (PLRV) are among the most damaging potato viruses and prevalent in most potato growing areas. In this study, cryopreservation was used to eradicate PVY and PLRV using two cryogenic methods. Potato shoot tips proliferated in vitro were cryopreserved through droplet-vitrification and encapsulation-vitrification using plant vitrification solution 2 (PVS2; 30% glycerol + 15% dimethyl sulfoxide + 15.0% ethylene glycol + 13.7% sucrose) and modified PVS2. Both cryogenic procedures produced similar rates of survival and regrowth, which were lower than those from shoot tip culture alone. The health status of plantlets regenerated from shoot tip culture alone and cryopreservation was checked by reverse transcription-polymerase chain reaction. The frequency of virus-free plants regenerated directly from highly proliferating shoot tips reached 42.3% and 48.6% for PVY and PLRV, respectively. In comparison, the frequency of PVY and PLRV eradication after cryopreservation was 91.3~99.7% following shoot-tip culture. The highest cryopreserved shoot tip regeneration rate was observed when shoot tips were 1.0~1.5 mm in length, but virus eradication rates were very similar (96.4~99.7%), regardless of shoot tip size. This efficient cryotherapy protocol developed to eliminate viruses can also be used to prepare potato material for safe long-term preservation and the production of virus-free plants.

• 한국발생생물학회:학술대회논문집
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• 한국발생생물학회 2003년도 제3회 국제심포지움 및 학술대회
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• pp.128-128
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• 2003

The aims of this study are 1) to test oocytes and embryos collected from in-vivo and in-vitro to achieving the valuable protocol by culturing, vitrifying and thawing of oocytes/embryos, and 2) to transfer them to recipient, and finally have resulted in pregnancies from recipient females after surgical or nonsurgical transfer. In vitro maturation and fertilization were performed according to the procedures of Funahashi et al. Fertilized oocytes were cultured in glucose-free NCSU 23 supplemented with 5 mM sodium pyruvate, 0.5 mM sodium lactate and 4 mg/ml bovine serum albumin for 2 days at 39$^{\circ}C$, and 10% fetal bovine serum was added to the culture medium thereafter. Embryos were treated with 7.5$\square$g/ml cytochalasin-B for 30 min, centrifuged at 13,000 ${\times}$ g for 13 min and then exposed sequentially to an ethylene glycol (EG) vitrification solution, aspirated into OPSs, and plunged/thawed into/from liquid nitrogen. In vivo embryos were surgically collected from three donors after Al. Forty-six embryos (18, 9 and 19 embryos, respectively) were washed 3 times in mPBS+10%FBS, followed treatments : cultured, centrifuged, vitrified, recovered and transferred to recipients as in vitro prepared embryos. Three recipients received surgically 34(control), 188 and 184 embryos (derived from abattoir), respectively. Another three recipients were received nonsurgically 150, 100 and 150 embryos, respectively. All recipient sows exhibited delayed returns to estrus. To our knowledge, these results suggest that required an improved techniques, more vigorous embryos preparation and cleaner uterous condition(use gilt).

• 한국수정란이식학회지
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• 제11권1호
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• pp.41-50
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• 1996

This experiment was carried out to investigate the ovarian responses of the ovulation point, ovarian weight and size, the number of ovarian follicles and collected embryos, and to study the effects of the developmental stages (oocytes, 2-4 cell. 8-16 cell and morulae), additional levels of Ficoll (0, 15, 30%) on the survival rate (FDA-test) of rat embryos frozen in vitrification solution (20% glycerol + 10% ethylene glycol + 10% sucrose). Sunanarized results was as follows; 1. The mean ovulation point per head was 7, and the weight of ovaries was 0.03g. The size of ovary was 5.9 mm(L) and 4.6 mm(W), and the number of ovarian follicles over and below 2 mm was 4.7 and 8.7, respectively. The number of the collected embryos per head was 5.5 (79%). 2. 2. The FDA score of embryos frozen in 20 G 10 E 10 S without Ficoll was 2.8 (oocyte), 2.6 (2-4 cell), 3.9 (8-16 cell) and 3.6 (morula), respectively. However, there were no significant differences among treatments. 3. The FDA score of embryos frozen in 20 G 10 E 10 S with 15% Ficoll was 3.4 (oocyte), 4.0 (2-4 cell), 4.7 (8-16 cell) and 4.8 (morulae), respectively (P>0.05). 4. The FDA score of embryos frozen in 20 G 10 E 10 S with 30 % Ficoll was 3.7 (oocyte), 3.2 (2-4 cell), 4.4 (8-16 cell) and 4.4 (morulae), respectively (P>0.05). 5. As shown in the above results, the higher survival rate was obtained in the treatment of 15% Ficoll than that of 30%. And the survival rate (FDA-test)of the oocytes and 2-4 cell stages of the rat embryos was lower than that of 8~16 cell and morulae stages. It was considered that 8-16 cell and morulae could be available for the successful freezing by vitrification of rat embryos with 15% Ficoll except for oocytes.

• 한국자원식물학회지
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• 제17권2호
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• pp.75-81
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• 2004

본 연구는 산림 수목 종자를 대상으로 다양한 초저온 동결 조건을 적용하고, 초저온 저장 후에 나타나는 종자의 발아 특성 및 유묘의 생장 특성 조사하기 위해 수행되었다. 유리화 과정을 기초로 한 초저온 저장은 다릅나무 종자의 발아율을 크게 감소시켰으나, 초저온 저장 전 동결보호제 처리와 초저온 저장 후의 빠른 해빙은 종자의 발아율 감소를 완화시켰다. 초저온 저장 전 동결보호제 노출 시간은 종자의 발아율에 영향을 주었다. 즉 동결보호제 노출시간이 길어짐에 따라 종자의 발아율은 감소하였다. 그러나 초저온 저장 기간은 종자의 발아율에 영향을 주지 않았다. 또한 동결보호제의 노출 시간과 초저온 저장 기간은 유묘의 생장 특성에 영향을 주지 않았다. 따라서 수목 종자의 초저온 저장은 조직의 손상이나 변형이 없이 장기간 저장할 수 있는 현지 외 보존 기술로 적절하다고 판단된다. 그러나 산림 종자의 보다 효율적인 초저온 저장을 위해서는 각 수종의 종자 특성에 맞는 동결보호제 및 처리 기술이 개발되어야 할 것으로 판단된다.

• 한국자원식물학회지
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• 제33권6호
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• pp.675-681
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• 2020

This study describes an efficient and stable droplet vitrification following cryopreservation of strawberry shoot tip (Fragaria × ananassa Duch.) accessions 'Massey' and 'MDUS3816'. The shoot tips were precultured in Murashige and Skoog (MS) liquid medium supplemented with sucrose (0.3-0.7M). Precultured explants were osmoprotected with loading solution (LS, C4) containing 17.5% glycerol and 17.5% sucrose for 40 min and exposed to dehydration solution (B1) containing 50% glycerol and 50% sucrose for 40 min at 25oC. Subsequently, the explants were transferred onto droplets containing 2.5 µL PVS3 on sterilized aluminum foils (4 cm× 0.5 cm) prior to direct immersion in liquid nitrogen (LN) for 1 h. The highest regrowth rate (%) in both the cultivars was obtained when the shoot tips were precultured with 0.3M sucrose for 30 h + 0.5M sucrose for 16 h at 25oC. The cryopreserved shoots tips exhibited 57.8 % recovery rate by culturing in NH₄NO₃-free MS medium supplemented with 3% sucrose, 1.0 g/L casein, 1.0mg/L GA₃, and 0.5 mg/L BA for 5 weeks and in MS medium supplemented with 0.5 mg/L GA₃ for 8 weeks. Variation was not observed in both of ploidy analysis and morphological investigation on plantlets of two accessions cryopreserved under variable preculture conditions.

• 한국산림과학회지
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• 제107권3호
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• pp.258-265
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• 2018

본 연구는 희귀자생식물인 솜양지꽃의 효율적인 초저온 보존 조건을 탐색하고자 행하였다. 종자의 활력은 PVS2와 PVS3 용액 처리구에서는 약 80% 이상으로 대조구보다 훨씬 높은 활력을 보였다. 종자의 활력은 PVS3가 PVS2 처리보다 높게 나타났다. 종자의 활력은 sucrose처리에도 불구하고 대조구보다 낮았다. PVS2의 60분 처리구와 PVS3의 30분 처리구에서 95%의 발아율을 보였으나 그 외 처리구에서는 발아율이 낮았다. PVS2와 PVS3 처리구에서 배양된 유묘의 생장은 PVS2와 PVS3 용액 30분 처리구를 제외하고는 대조구에 비해 생장이 좋지 않았다. 두 가지 초저온동결보존법 간 유묘 생장에서는 encapsulation법과 vitrification법을 비교한 결과 미세한 차이를 보였으나 통계적으로는 유의성이 없었다. 본 연구는 솜양지꽃의 보존에 도움이 될 것으로 판단된다.

• Clinical and Experimental Reproductive Medicine
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• 제26권3호
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• pp.363-368
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• 1999

연구목적: 본 연구는 동해방지제인 EFS40을 이용한 초자화동결이 생쥐 미성숙란의 cytoskeleton과 염색체의 성상에 미치는 영향을 indirect immunocytochemistry방법과 염색체 분석법으로 알아보고자 실시하였다. 연구재료 및 방법: 본 실험은 생쥐 미성숙란을 EFS40 (40% ethylene glycol, 18% ficou과 0.5 M sucrose가 들어있는 M2배양액)으로 초자화 동결하여 융해한 후 16시간동안 체외 성숙을 유도하여, 제 1극체가 나타난 성숙된 난자를 기준으로 동해제노출군 또는 대조군과 비교 조사하였다. 결과: 초자화동결된 미성숙란의 응해 후 생존율과 체외성숙율은 90.3%과 64.7%로써, 동해제노출군 (86.7%, 69.2%)과 대조군 (100%, 58.3%)에 유사하였다. 초자화동결이 미성숙란의 microtubule과 microfilament에 미치는 영향을 조사하였던 바, 동결군의 microtubule과 micro-filament의 정상적인 형성율 (93.9%, 100.0%)은 동해 제노출군 (94.4%, 100.0%)과 대조군 (100.0%, 100.0%)의 성적과 유사하게 나타났다. 또한, 초자화동결군에서 정상적인 염색체수를 가진 난자의 비율도 65.8%로써, 대조군(79.6%)과 노출군 (69.0%)의 결과와 유의한 차이가 없었다. 결론: 생쥐 미성숙란을 EFS40에 노출하고 동결하는 것이 미성숙란의 cytoskeleton과 염색체성상에 영향을 미치지 않으며, 본 연구에서 사용된 EFS40을 이용한 초자화동결법은 생쥐 미성숙란 동결에 적합하다는 것을 알 수 있었다.

• Reproductive and Developmental Biology
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• 제35권2호
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• pp.137-141
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• 2011

These study was to investigate the in vitro fertilization and viability of fresh and vitrified oocytes. Also, the developmental capacity of IVF and intracytoplasmic sperm injection (ICSI) oocytes were investigated. Then vitrification was performed with the use of 20% ethylene glycol + 20% DMSO + 0.5 M sucrose + 10% FCS + TCM-199 medium. Vitrification immature oocytes are cultured in vitrification solution for 10 min afterwards transferred to expose at room temperature for 5 min. and transferred to the ice water for 5 min. The oocytes were sealed in a 1.0 mm straw and placed in a $LN_2$ container. Frozen oocytes were rapidly thawed in a water bath at $30{\sim}35^{\circ}C$, and then placed in TCM-199 medium containing 0.5 M sucrose for 5 min each, respectively, at $38^{\circ}C$. After being washed for 2~3 times, using fresh medium the oocytes were cultured in TCM-l99 medium supplemented with 5% FCS at $38^{\circ}C$ in 5% $CO_2$ and air. The normal morphology of fresh and vitrified-thawed oocytes were $87.1{\pm}2.1%$ and $54.8{\pm}2.5%$, respectively. The viability rates of fresh and vitrified-thawed oocytes were $70.0{\pm}2.2%$ and $41.9{\pm}2.6%$, respectively. Viability rates of vitrified-thawed oocytes were lower than that of fresh follicular oocytes (p<0.05). The in vitro maturation rates of fresh and vitrified oocytes were $45.1{\pm}3.6%$ and $28.9{\pm}4.4%$, respectively. The IVF rates of fresh follicular and vitrified-thawed oocytes were 34.00.2% and $20.2{\pm}2.6%$, respectively. The in vitro maturation and fertilization rates of vitrified-thawed oocytes were lower than those of the fresh follicular oocytes (p<0.05). A total of 350 oocytes were fixed and stained after co-incubation with spermatozoa, of which 88 had identifiable nuclear material. After IVF for 20 hrs, $25.1{\pm}3.4%$ of the oocytes found to have been penetrated by spermatozoas. Oocytes were fixed and stained after ICSI, and 105 oocytes contained identifiable nuclear material. After IVF and ICSI for 20 hrs, $34.3{\pm}3.4%$ and $59.0{\pm}2.0%$ of the oocytes were found to have been penetrated by spermatozoas. The developmental rates upon ICSI were significantly higher than those of the IVF method (p<0.05).