• 한국가축번식학회지
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• 제21권3호
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• pp.287-292
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• 1997

This study was carried out to investigate the effects of concentration and kinds of cryoprotectants, equilibraction time, thawing temperature and time, sucrose concentration on the survival rates of frozen bovine demi-embryos. The bovine demi-embryos following dehydration by cryoprotectants a various concentration of sucrose were freezed by cell freezer and thawed in 3$0^{\circ}C$ water bath. Survival and in vitro developmental rates was defined as development rates on in vitro culture or FDA-test. The results are summarized as follows : 1. The high survival rates of demi-embryos after frozen-thawing in freezing medium was attained 2.0M glycerol. The high survival rates of demi-embryos after frozen-thawing in freezing medium was obtained using single cryoprotectant(25.0~30.0%) than mixed cryoprotectants(16.7~19.0%). 2. The survival rates of demi-embryos after frozen-thawing in freezing medium added 1.5M, 2.0M glycerol＋0.25M sucrose(37.5~33.3%) were higher survival rates than those of sucrose concentration of 0.50, 0.75M(12.5~26.7%). 3. The equilibration time on the survival rates of demi-embryos was attained after short period of time(30.0~35.0%) in the freezing medium higher than long period of time(21.1%). 4. The thawing temperature on the survival rates of demi-embryos was attained at 3$0^{\circ}C$ of thawing temperature(26.7~40.0%) higher than $25^{\circ}C$ or 37$^{\circ}C$ of thawing temperature(13.3~20.0%). 5. The thawing time on the survival rates of demi-embryos was attained at 1~5 minutes of thawing time(26.7~33.3%) in the freezing medium higher than 10 minutes of thawing time(13.3~18.8%).

• Asian-Australasian Journal of Animal Sciences
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• 제18권9호
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• pp.1242-1248
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• 2005

This study was designed to investigate the in vitro developmental ability and apoptosis of bovine embryos nucleartransferred (NT) with frozen-thawed or cooled donor cells. Cultured adult bovine ear cells were used as donor cells after sub-culturing to confluence (CC), cooling to 4$^{\circ}C$ for 48 h, or freezing-thawing (FT). Apoptotic cells in blastocysts were evaluated for apoptosis by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) method. Fusion, cleavage and blastocyst rates were 69.0 (167/242), 68.8 (115/167), and 29.9 (50/167) with CC cells, 70.4 (88/125), 69.3 (61/88), and 29.6 (26/88) with cooled cells and 66.1 (117/177), 70.1 (82/117), and 13.7 (16/117) with FT cells, respectively. Blastocyst rates of NT embryos derived from FT cells were significantly lower than those from CC or cooled cells (p<0.05). In addition, NT blastocysts produced by using FT cells showed significantly higher apoptosis rates (6.4${\pm}$4.0%) than those produced by CC (2.8${\pm}$1.7%) or cooled (2.3${\pm}$1.3%) cells. However, cooling of donor cells had no significant adverse effect on blastocyst rate as well as apoptosis rate. Therefore, our results suggest that cooled cells may be used as an alternative to freshly cultured confluent culture cells, as donor cells, for the production of Somatic nuclear cloned cattle.

• 한국수정란이식학회지
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• 제9권1호
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• pp.65-71
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• 1994

This study were carried out to investigate the effective concentration of cryoprotectant agents and sucrose by one-step straw method, and to determine the optimum thawing temperature and equilibration time of frozen bovine embryos. The bovine embryos following dehydration by cryoprotective agents and a various concentration of sucrose were directly plunged into liquid nitrogen and thawed in 3O$^{\circ}C$ water. Survival rate was defined by FDA test. The results are summarized as follows : 1. The survival rate of bovine embryos thawed after rapid freezing in the freezing medium containing a various kinds of cryoprotective agents added 0.25M and O.50M sucrose were 28.6% and 25.0%, 35.1% and 31.6%, 32.4% and 24.4%, 34.2% and 28.2%, 18.9% and 17.6%, 14.7.% and 21.6%, respectively. 2. The survival rate of bovine embryos thawed after rapid freezing in the freezing medium containing a various concentration of sucrose added 1.5M and 2.OM glycerol, i.5M and 2.OM DMSO and 1.5M and 2.OM propanediol were 22.9~37.8%, 2O.7~31. 3%, 19.2~30.0% and 17.2~25.0%, respectively. 3. The temperature thawed at 2$0^{\circ}C$ after rapid freezing of bovine embryos resulted in a significantly higher embryos survival rate than did at 3$0^{\circ}C$ and 35$^{\circ}C$. 4. The equilibration time on the survival rate of bovine embryos was attained after short period of time(2.5~5 min.) in the freezing medium higher than long period of time (1O~20 min.).

• 한국가축번식학회지
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• 제21권2호
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• pp.103-109
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• 1997

The studies on the carried out to investigate to determine the optimum thawing temperature and equilibration time and 1 step straw method of frozen bovine embryos. The follicular oocytes were cultured in TCM-199 medium containing 10 IU/ml PMSG(Sigma, USA), 10 IU/ml hCG(Sigma, USA), 1 $\mu\textrm{g}$/ml $\beta$-estradiol(Sigma, USA) and 10% FCS for 24~48 hrs in incubator with 5% CO2 in air at 38.5$^{\circ}C$ and then matured oocytes were again cultured for 12~18 hrs with motile capacitated sperm by preincubation of heparin. The bovine embryos following dehydration by cryoprotective agents and various concentration of sucrose were directly plunged into liquid nitrogen and thawed in 3$0^{\circ}C$ water. Survival and in vitro developmental rate was defined as developmental rate on in vitro culture or FDA-test. The results are summarized as followes : 1. The equilibration time on in vitro developmental rates of bovine embryos was attained after short period of time(2.5~5 min.) in the freezing medium higher than long period of time (10~20 min.). 2. The temperature thawed at 3$0^{\circ}C$ after rapid freezing of bovine embryos resulted in a significantly higher in vitro developmental rate than did at 2$0^{\circ}C$ and 35$^{\circ}C$. 3. The thawing time on in vitro developmental rates of bovine embryos was attained after short period of time(1~5 min.) in the freezing mediuim higher than long period of time(10min.). 4. The in vitro developmental rates of bovine embryos after rapid frozen-thawing by 1 step straw method in the freezing medium added 1.5M, 2.0M glycerol, DMSO, propanediol and 0.25M, 0.50M, 0.75M, 1.00M sucrose were 12.5~19.4%, 10.0~15.6%, 9.1~13.8% and 6.7~12.9%, respectively.

• 한국가축번식학회지
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• 제24권3호
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• pp.263-268
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• 2000

본 연구는 2-, 4- 또는 8-세포기 생쥐 수정란으로부터 분리된 단일 할구의 발달 및 완만동결 후 생존능력을 조사하기 위하여 실시하였다. 2-, 4-또는 8-세포기 생쥐 수정란에서 각각 223개, 60개, 188개의 할구를 분리하여 96시간 배양하였는데 이중 2-세포기 수정란으로부터 분리된 할구는 111개(49.8%)가 배반포기까지 발육하였으며, 4-세포기와 8-세포기 수정란으로부터 분리된 할구는 각각 12개(20.0%)와 31개(16.5%)가 배반포기까지 발육하였다. 분리된 할구를 완만 동결한 후 융해하여 배양했을 때 배반포기까지의 발육율은 2-세포기 할구는 27.1%(16/50), 4-세포기 할구는 36.4%(4/11) 그리고 8-세포기 할구는 17.6%(3/17)였으며, 융해 후 할구 회수율은 각각 54.2%(65/120), 46.4%(13/28), 24.3%(17/70)을 나타냈다. 2-세포기 할구를 동결 융해한 후 동기화시킨 생쥐 자궁에 이식하여 정상적으로 발달한 태아를 생산하였다. 본 실험의 결과로 완만동결방법이 생쥐의 할구를 동결 보존하는데 이용될 수 있음을 확인하였다.

• Journal of Yeungnam Medical Science
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• 제37권1호
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• pp.47-53
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• 2020

Background: This study was conducted to analyze clinical factors that can affect pregnancy rates in normal responders undergoing the freeze-all policy in in vitro fertilization. Methods: We evaluated 153 embryo transfer cycles in 89 infertile women with normal response to controlled ovarian stimulation (COS). After COS, all embryos were cultured to the blastocyst stage, and good quality blastocysts were vitrified for elective frozen-thawed embryo transfer (FET). Clinical variables associated with COS and the results of COS and culture, including the number of retrieved oocytes, fertilized oocytes, and frozen blastocysts were compared between the pregnant group and the non-pregnant group. Results: After a single cycle of COS for each patient, 52 patients became pregnant while 37 did not. Significant differences were observed in the number of matured oocytes, fertilized oocytes, frozen blastocysts, and transferred embryos. The number of frozen blastocysts in the pregnant group was almost twice that in the non-pregnant group (5.6±3.1 vs. 2.8±1.9, p<0.001). The area under the receiver operating characteristic curve for the 4 frozen blastocysts was 0.801 in the pregnant group. Conclusion: In the freeze-all policy, the number of matured oocytes, number of fertilized oocytes, and number of frozen blastocysts might be predictive factors for pregnancy.

• Clinical and Experimental Reproductive Medicine
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• 제45권1호
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• pp.25-30
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• 2018

Objective: This study conducted a preliminary examination of the effects of three-area laser-assisted zona thinning (LAZT) during the cleavage stage of embryo development on the hatching process in human in vitro fertilization-embryo transfer (IVF-ET) with subjects of advanced female age or frozen-thawed (FT) embryos. Methods: Eight-cell stage embryos were treated with LAZT in three areas of the zona pellucida at $120^{\circ}$ intervals. The control group was embryos without LAZT. Of the 72 consecutive fresh cycles and the 28 FT embryo transfer cycles, the patients in 55 fresh cycles and 17 FT cycles declined LAZT, and those cycles were defined as the control group. Results: In the fresh cycles, the pregnancy rates were similar in the LAZT and control groups. However, in the FT cycles, the pregnancy rate was significantly higher in the LAZT group than in the control group (45.5% in the LAZT group vs. 23.5% in the control group, p< 0.05). Conclusion: These results show that multi-area LAZT resulted in significantly improved pregnancy outcomes in human 8-cell embryos compared to controls.

• 한국수정란이식학회지
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• 제9권1호
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• pp.43-48
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• 1994

This study was carried out to produce monozygotic twin calves by transfer of bisected embryos. Four Korean native cattle donors were superovulated with FSH and flushed to collect embryos on day 6 or 7 of the estrus cycle. Morula and early blastocyst embryos showed 1 or 2 grade were bisected with microblade and each set of demi-embryos without zona pellucida were transferred nonsurgically to 10 recipients respectively. The results obtained were as follows; 1. Twenty four demi-embryos (92.3%) were separated from 13 original embryos and among them 20 demi-embryos (83.3%) had normal appearance without severe damage. 2. Four sets of fresh demi-embryos were transferred to 4 recipients and one recipient was twin pregnant 3. Six sets of frozen-thawed demi-embryos were transferred to 6 recipients. Two recipients were pregnant, one of them twin.

• 한국수정란이식학회지
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• 제10권3호
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• pp.193-202
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• 1995

In order to improve the cryopreservatory techniques of livestock embryos, the quick freezing method which is directly plunged in liquid nitrogen via prefreezing procedure without freezing machine was carried out for mouse embryos treated with permeable and nonpermeable cryoprotectants. The viability of frozen-thawed embryos were evaluated by FDA vital dye test. The results obtained was summaried as follows: 1. A total of 720 embryos were recovered from frozen embryos for viability test. Evalution of the fluorescein diacetate(FDA) vital dye test with mice embryos were resulted of 2.3 total mean score - evaluted in orderly higher mean grade of P3 453 (63%), P2 133(18%), P1 51(7%) and P0 83(12%). 2. An all-round evalution of these combination, the highest viability was showed in 3M ethylene glycol + 0. 25M trehalose treated with the copper prefreezing. 3. Effects of permeable and nonpermeable cryoprotectants combination were evaluated by means FDA score. 3M ethylene glycol + 0.25M trehalose showed the highest survival rates of 2.8 mean FDA score. 4. Effects of permeable cryoprotectants were evaluated by mean FDA score but the results were not significantly different each other. 5. In evalution of the nonpermeable cryoprotectants, 0. 25M trehalose obtalned higher mean FDA score than of 0.25M sucrose and it was significantly different(P<0.05). 6. There was no significantly difference between copper and stainless-steel in prefreezing procedures.

• Clinical and Experimental Reproductive Medicine
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• 제44권1호
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• pp.52-55
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• 2017

The aim of this study was to report a successful pregnancy using completely immotile frozen-thawed spermatozoa selected by laser. A single laser shot was used to detect the presence of viable immotile spermatozoa in fresh and frozen-thawed testicular spermatozoa. The viability rate was 55.8% after the laser detection, and cryopreservation was carried out immediately. The thawing test was performed on the day of oocyte pick-up, and no motile sperm were observed after extending the culture for another 4 hours, while a survival rate of 39.8% was detected using the laser. In all, five mature oocytes were injected, resulting in four cases of normal fertilization (80%) on day 1. Further, two high-quality day 3 embryos were transferred, which resulted in a singleton pregnancy. Our study demonstrates that completely immotile spermatozoa are worth cryopreserving for further intracytoplasmic sperm injection, which provides a new insight into male fertility preservation in cases of completely immotile spermatozoa.