• 한국수정란이식학회지
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• 제27권1호
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• pp.63-69
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• 2012

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 ($p$ <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 ($p$ <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.

• 한국수정란이식학회지
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• 제26권3호
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• pp.201-207
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• 2011

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.

• Asian-Australasian Journal of Animal Sciences
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• 제22권2호
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• pp.174-180
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• 2009

This study was carried out to establish an appropriate condition for the efficient cryopreservation of the mouse pronuclear embryo. In vitro cryopreservation of pronuclear embryos was carried out by slow freezing or vitrification methods and development rate of 2-cell, blastocyst and hatched blastocyst was measured as well as survival rate of the thawed pronuclear embryo. After slow freezing, vitrification and thawing of mouse pronuclear embryos, the survival rate and blastocyst development rate for the vitrification group was 97.3 and 53.4%, respectively, which was significantly higher as compared to the slow freezing group with 88.6 and 23.9%, respectively (p<0.05). Blastocyst developmental rate in each experimental group was significantly higher for 21 h in the post-hCG group at 40.5-57.0% than the 24 h post-hCG group at 40.5% (p<0.05). ICM (Inner cell mass) cell numbers of blastocyst-stage embryos during the different stages of mouse pronuclear embryos, slow freezing and vitrification period in the control and vitrification groups were 22.1${\pm}$2.7 and 17.0${\pm}$3.1-22.0${\pm}$3.2, respectively; hence, the slow freezing group (10.2${\pm}$2.0) had significantly higher cell numbers than those of the other two groups (p<0.05). Trophoblast (TE) cell number in the control group, 65.8${\pm}$12.6, was significantly higher than in the slow freezing group, 41.6${\pm}$11.1 (p<0.05). The total cell numbers in the control group and 21 h post hCG group were 87.9${\pm}$13.6 and 81.8${\pm}$14.1, respectively, and were significantly higher than for the slow freezing group (51.8${\pm}$12.6; p<0.05).

• 한국수정란이식학회지
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• 제14권1호
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• pp.9-15
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• 1999

This study was conducted to investigate the effect of $\alpha$-tocopherol and cysteamine with Whitten's medium in supporting the development on in vitro maturation(IVM), in vitro fertilization (IVF) and in culture(IVC) on porcine oocytes. When the immature oocytes were cultured of $\alpha$-tocopherol for 40h, the nuclear maturation rates were 39, 4, 52.5 and 54.1%, respectivley. The nuclear maturation rates of treat groups were signficantly (P<0.05) higher than those of non-treat groups. After matureation, the oocytes were inseminated in vitro in medium 199 with ejaculated spermatoza for examination of sperm penetration, polyspermy, male pronuclear(MPN) formation, and cleavage rate. Sperm penetration rates of treat higher than the control groups(P<0.05), and MPN formation rates were significantly(P<0.05) higher on treated groups (24.3~53.1%) than control groups(14.2~21.4%). After insemination, the cleavage rates at 120hr were groups higher than control groups(P<0.05).

• 한국수정란이식학회:학술대회논문집
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• 한국수정란이식학회 2001년도 추계학술대회 및 정기총회
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• pp.29-29
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• 2001

• 한국수정란이식학회:학술대회논문집
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• 한국수정란이식학회 2001년도 추계학술대회 및 정기총회
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• pp.35-35
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• 2001

• 한국수정란이식학회지
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• 제16권2호
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• pp.139-144
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• 2001

본 연구는 숫소 개체별, 정자의 형태, 정자의 전처리 및 난자의 투명대부착과 미부착별로 현미조작에 의해 난자의 세포질내 단일정자를 주입했을 때 웅성전핵 형성율과 체외발생율을 조사하였다. 1. 숫소 개체별 정자를 이용하여 ICSI를 하였을 때 웅성전핵 형성율은 각각 73.9∼87.0%였고 체외발생율은 33.3∼60.9%를 나타냈다. 2. 신선 및 동결정자와 미부절단 정자와 두부, 미부손상 정자를 이용하여 ICSI를 하였을 때 웅성전핵 형성율은 각각 82.0%, 78.0% 42.2%, 51.1%였고, 체외발생율은 56.0%, 42.0%, 17.8%, 22.2%로서 신선정자를 이용했을 때가 다른 처리군에 비해 높은 전핵형성율과 체외발생율을 나타냈다. 3. 정자를 heparin, BFF, His, Ca Ionophore, I + caffeine 액으로 처리후 ICSI를 하였을 때 전핵 형성율은 66.7∼82.2%였고, 체외발생율은 33.3∼60.6%로서 I + caffeine 처리법이 다른 처리군에 비해 높게 나타났다. 4. 투명대 부착 난자 및 미부착 난자로 ICSI를 하였을 때 전핵형성율은 각각 80.0%, 72.0%였고, 체외발생율은 46.0%, 36.0%로서, 토명대부착 난자가 투명대 미부착 난자에 비해 높은 웅성전핵 형성율과 체외발생율을 나타냈다.

• 한국수정란이식학회:학술대회논문집
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• 한국수정란이식학회 2004년도 제4회 발생공학 국제심포지움 및 학술대회
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• pp.55-56
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• 2004

• Clinical and Experimental Reproductive Medicine
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• 제34권2호
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• pp.117-124
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• 2007

목 적: 본 연구는 생쥐 전핵시기 배아를 완만동결법과 유리화동결법으로 동결-융해 후 배아의 생존율과 성장률을 비교하고자 시행하였다. 연구방법: 과배란을 유도한 생쥐로부터 전핵시기 배아를 획득하여 10% SSS가 첨가된 HTF 배양액으로 약 1시간 동안 배양한 후 두 개의 전핵이 관찰되는 정상적인 형태의 배아만 선별하여 동결하였다. 동결방법으로는 1.5 M PROH에 0.1 M sucrose가 함유된 완만동결법과 40% ethylene glycol, 18% Ficoll, 0.5 M sucrose가 혼합된 EFS40 용액과 EM grid를 이용하여 이용한 유리화동결법을 실시하였다. 동결-융해 후 전핵시기 배아의 회수율, 생존을 및 부화 포배기로의 성장률과 부화율을 비교하였다. 결 과: 각각의 방법으로 동결-융해 후 24시간 동안 배양하였을 때 2-세포기까지의 성장률은 완만동결군이 59.1%이었고 유리화동결군이 77.0%로 두 군간에 유의한 차이를 보였고 (p<0.003), 48시간 동안의 배양에서도 완만동결군이 53.3%이고 유리동결군이 72.6%로 유의하게 유리화동결군에서 높은 수세포기까지의 성장률을 보였으며 (p<0.003), 72시간 배양하였을 때의 상실배로의 성장률 역시 완만동결군이 46.7%이고 유리화동결군이 67.3%로 유리화동결군에서 유의하게 높은 성장률을 보였다 (p<0.001). 융해 후 144시간 동안 배양하였을 때의 부화포배기로의 성장률은 완만동결군이 26.3%이고 유리화동결군이 43.4%로 유리화동결군에서 유의하게 높은 성장률을 보였다 (p<0.005). 결 론: 생쥐 전핵시기 배아의 동결보존에서 유리화동결법은 완만동결법 보다 시간이 단축되고 비싼 장비가 필요없어 경제적이고 간단했을 뿐 아니라 동결-응해 후 전반적으로 높은 생존율과 성장률을 나타내었다.

• 생명과학회지
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• 제16권4호
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• pp.566-570
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• 2006

현재, 유전자의 in vivo 기능을 연구하기 위해 가장 많이 이용되고 있는 transgenic mice를 생산하기 위한 기본적으로 이용되고 있는 방법이 one cell-stage embryo에 pronuclear microinjection (PMI)이다. 그러나, 이 PMI 후에 one cell-stage embryo들의 생존율은 현저히 감소 (65.4%)할 뿐만 아니라 PMI 후의 embryo의 출생률(26.4%)이 PMI 처리를 하지 않은 것 (41.9%) 보다 현저히 낮다. 더욱이, PMI 방법에 의해 태어난 transgenic founder들의 간 조직에 병리학적 변화가 14.8% 정도에 대해서 같은 한배의 새끼 non-transgenic founder들의 경우도 간 조직에 병리학적 변화가 14.3%로 나타났다. 결론적으로, 이 PMI 방법에 의한 염색체에 물리적 손상은 형질전환 마우스의 생산 및 표현형에 영향을 미치는 잠재적 요소로 생각된다.