• 한국가축번식학회지
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• 제17권3호
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• pp.201-207
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• 1993

We determined whether the ultrarapid freezing method is applicable to micromanipulated mouse embryos. One-cell mouse embryos were microinjected with MThGH gene. Nuclei from one-cell embryos of F1(C57BL$\times$CBA) mice were transplanted into enucleated one-cell embryos of ICR mice. The injected and nucleated embryos that developed to 2-cell stage were cryopreserved by ultrarapidfreezing. The embryos equilibrated in freezing medium(3 M DMSO＋0.25 M sucrose＋2% FBS in PBS) were directly immersed into liquid nitrogen and then thawed in 37$^{\circ}C$ water. Development rates of the microinjected and nuclear-transplanted embryos to blastocyst stage after ultrarapidly freezing and thawing were 31% and 55%, respectively. The frozen-thawed embryos were transferred to pseudopregnant recipient, which then gave birth to 17 offsprings. Twelve(14% of the transferred embryos) and five(20%) offsprings were derived from microinjected and nuclear-transplanted embryos, respectively. The results indicate that the DNA injected and nuclear-transplanted mouse embryos are cryopreservable at 2-cell stage by ultrarapid freezing method.

• 한국수정란이식학회지
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• 제16권1호
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• pp.29-34
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• 2001

To investigate the effect of co-transfer of trophoblastic vesicle (TV) with frozen-thawed in vitro Produced (IVP) bovine embryo on pregnancy rate, IVP blastocysts were transferred to synchronized recipients. Elongated blastocysts were recovered at Day 13 to 15, and dissected more than 4 pieces to removed the embryonic disc. Throphoblastic fragments were cultured for 48 hours to make throphoblastic vesicles (TVs). TVs were cryopreserved in ethylene glycol or vitrification solution and frozen-thawed TVs were co-transferred to recipients with frozen-thawed IVP embryos. 1 The recovery rate of elongated blastocyst on Day 13 to 15 was 22.5% (18/80) and the size of recovered elongated blastocysts was 0.2∼5.0mm. 2. Eighteen elongated blastocysts were dissected into 88 pieces and 61.4% of those pieces were formed to TV (54/88) 3. The viability of frozen-thawed TV in ethylene glycol was higher than in vitrified solution (92.8% vs. 68.8%) 4. The pregnancy rate in co-transfer with frozen-thawed TV and IVP blastocyst was better than transfer only IVP blastocysts (50.0% vs. 23.1%).

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2004년도 춘계학술발표대회
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• pp.218-218
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• 2004

This study was designed to investigate the in vitro developmental ability and apoptosis of embryos nuclear transferred (NT) with frozen-thawed (FT) or cooled donor cells in bovine. Cultured adult bovine ear cells were used as donor cells at confluent condition (CC), after cooling at 4℃ for 48 hour, or after FT. (omitted)

• 대한수의학회지
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• 제27권2호
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• pp.331-334
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• 1987

The effects of ethylene glycol as a cryoprotective agent on the survival of frozen-thawed mouse embryos were examined. The effects of the stage of development of ethylene glycol were also examined. Eight-cell embryos, morulae, early blastocysts and mid-blastocysts were recovered from superovulated immature ddY mice. Ethylene glycol was added to the embryos in 5 equal increments 5 minutes apart, giving a final concentration of 1.5M. The embryos were cooled to ${-6^{\circ}C}$ at ${1^{\circ}C/min}$ and seeding was induced at ${-6^{\circ}C}$. After being held for a further 5minutes at the seeding temperature, the samples were cooled to ${-35^{\circ}C}$ at ${0.3^{\circ}C/min}$ and then transferred to liquid nitrogen. Rapid thawing was done by placing the straws in ${37^{\circ}C}$ water. The thawed embryos were diluted in PBS of same time and manners as adding procedures. Survival of 8-cell embryos and morulae were assessed as a normal development of the embryos to the blastocyst stage and expanding blastocyst after 54 hours and 48 hours of in vitro culture, respectively. While those of the early and mid-blastocysts were assessed to the expanding blastocyst stage after 24 hours of in vitro culture. The survival rates of 8-cell embryos, morulae, early blastocysts and mid-blastocysts were 73.8%, 74.3%, 87.8% and 77.4%, respectively. Significant difference on the survival rate among the four stages of development was not observed.

• 한국수정란이식학회지
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• 제29권1호
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• pp.13-19
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• 2014

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 $38.5^{\circ}C$, 5% $CO_2$ 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 $-7^{\circ}C$, and the straw was seeded during 8 minutes-holding time, and was cooled to $-35^{\circ}C$ at the cooling rate of $0.3^{\circ}C/min$, 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 ($56.86{\pm}26.53%$) were slightly higher than those by the slow-rate freezing method ($55.07{\pm}26.43%$) 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 $72.65{\pm}18.3%$ and $79.06{\pm}17.8%$, 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 $66.22{\pm}18.8%$ and $45.76{\pm}12.8%$, 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).

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2004년도 춘계학술발표대회
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• pp.278-278
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• 2004

The purpose of this study was to establish the convenient freezing method for more cheap and simple. Semen quality was evaluated the motility, viability, abnormality, acrosome intactness and membrane integrity. And there were also examined the developmental rates of IVM/IVF embryos using frozen-thawed boar semen in each treatment group. (omitted)

• 한국수정란이식학회지
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• 제3권1호
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• pp.38-42
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• 1988

These studies were conducted to investigate the effect of repreated superovulation on embryo production, the effect of the frozen-thawed embryos transferred on the developmental stage and grade, and donor-recipient synchrony on pregnancy rate in Korean native cattle. The results obtained in these studies were as follows: 1. Repeated superovulations in Korean Native Catile were not affected on the number of corpus luteum (CL), embryos recovered and embryos cleaved (range: 4.8 $\pm$ 4.21 to 9.5 $\pm$ 6.50, 1.8 $\pm$ 2.53 to 8.2 $\pm$ 8.04 and 1.6 $\pm$ 2.32 to 4.0 $\pm$ 4.59, respectively). 2. Blastocyst embryos (38.5%) showed higher pregnancy rate than morula (31.6%). 3. The pregnancyrates of cattle transferred with good and fair embryos were 33.3% and 40.4%, respectively. 4. The pregnancy rate when the donors exhibited estrus 12 hours earlier than the recipients (62.5%) was higher than when the donors and recipients exhibited estrus at the same time (33.3%) or when the donors exhibited estrus 12 hours later than the recipients (20.0%).

• 한국가축번식학회지
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• 제18권1호
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• pp.25-29
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• 1994

This study was carried out to investigate the in-vitro fertilization rate survival rate of rapidly frozen porcine immature embryos. The porcine embryos following dehydration by cryoprotectants containing sucrose were directly plunged into liquid nitrogen and thawed in 3$0^{\circ}C$ water bath. Survival rate was defined as development rate on vitro culture or FDA test. The results are summarized as follows : 1. The in vitro fertilization rate of all frozen immature oocytes(6.7~26.7%) was very low, 35.0% of unfrozen oocytes and the rate of immature oocytes was very higher than that of mature oocytes. 2. The survival rate of all frozen immature oocytes(10.3~25.0%, 13.3~30.0%) was very low, 45% of unfrozen oocytes and the rate of immature oocytes was slightly higher than that of mature oocytes.

• 한국수정란이식학회지
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• 제5권2호
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• pp.38-44
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• 1990

This study was carried out to produce superior dairy cattle by embryo transfer. Seven dairy cows were superovulated with divided injection of FSH 4Omg for 5 days started on day 9 to 14 of the estrus cycle and injection of PGF$_2$$\alpha$ 45mg on day 4 of FSH injection. Donor cows were flushed to collect embryos on day 7 or 8 of the estrus cycle. Fresh embryos collected were transferred to synchronized dairy recipients or frozen using glycerol 3 step method to he equilibrated. And 35 embryos which were frozen using glycerol 6 step method were imported from U.S.A. After glycerol dilution of frozen embryos was done by reverse density during freezing. frozen-thawed embryos were transferred to synchronized dairy or beef recipients. The results obtained were as follows; 1. Total of 24 embryos were collected from 7 donor cows flushed and transferable embryos were 18 (75.0%). 2. Among 24 embryos. morula, early blastocyst, blastocyst, expanded blastocyst and unfertilized ova were 3 (12.5%), 1 (4.2%), 10 (41.6%), 4 (16.7%) and 6 (25.0%), respectively. 3. Heat inducing rate after 1st and 2nd injections of PGF$_2$$\alpha$ in Holstein and beef cattle was 83.3% and 71.4% and 62.5% and 69.2%, respectively. 4. Among 56 recipients, 23 head were pregnant (41.1%). The pregnancy rate of fresh embryos was 50.0% (1/2 heads) and the pregnancy rate of frozen embryos which were frozen using glycerol 3 step and using glycerol 6 step imported from U.S.A. was 52.6%(l0/19 heads) and 34.3%(12/35 heads), respectively. 5. The pregnancy rate of blastocyst (60.0%) was higher than that of morula (39.0%), early blastocyst (25.0%) and expanded blastocyst (0%). 6. The pregnancy rate of grade I embryos (52.2%) was higher than that of grade 2 (34.6%) and grade 3 (28.6%). 7. The pregnancy rate according to synchrony of recipient with donor was higher in simultaneous recipient (55.0%) and +l2hrs' (53.8%) than -24hrs' (23.5%), -l2hrs' (20.0%) and +24hrs' (0%).

• 한국수정란이식학회:학술대회논문집
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• 한국수정란이식학회 2006년도 The 6th Intermational Symposium on Developmental Biotechnology
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• pp.40-41
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• 2006