• Clinical and Experimental Reproductive Medicine
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• 제19권2호
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• pp.163-168
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• 1992

This study was carried out to set up the ovum bank for ovum donation and to determine the best freezing method for human immature oocytes. Human immature follicular oocytes were cryopreserved by slow freezing and rapid thawing method. Immature follicular oocytes were treated by propanediol(PROH) solution by 2 and 4 step method in protocols A & B, respectively. In protocol C, immature oocytes were exposed to sucrose prior to treatment of PROH by 4 step method. We compared survival rate, maturation rate, and fertilization rate of immature oocytes among three protocols. Results were as follows. 1. Oocytes treated by the protocol C showed the highest survival rate( 70.3 %) and maturation rate(34.6%) after thawing. 2. Survival rate of oocytes treated by the protocol C was significantly higher than that of the protocol B after thawing(p<0.05). In conclusion, treatment of oocytes with sucrose prior to expose PROH was the best freezing method. Sucrose may have reduced the toxic effect of cryoprotectant to oocytes. We failed to induce fertilization of oocytes, which were treated by any protocols, by conventional insemination method, but obtained 28.8% fertilization rate by using partial zona dissection(PZD) method. This result suggests that micromanipulation(PZD) of the thawed oocytes before insemination will improve the fertilization rate.

• 한국가축번식학회지
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• 제21권2호
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• pp.95-102
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• 1997

The studies on the carried out to investigate the effective concentration of cryoprotectant agents and sucrose by one-step straw method of 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 concentrations 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 devellpmental rate on in vitro culture or FDA-test. The results are smmarized as followes : 1. The high in vitro developmental rates of bovine frozen embryos after rapidly thawed in freezing medium was attained 2.0M glycerol, 2.0M DMSO, 1M or 2.0M propanediol. 2. The high in vitro developmental rates of bovine frozen embryos after rapidly thawed in freezing medium was obtained single cryoprotectant(6.7~17.4%) than mixed cryoprotectants(6.7~16.7%). 3. In vitro developmental rate of bovine embryos after rapid frozen-thawing in the freezing medium added 0.25M and 0.50M sucrose were higher cleavage rate than those of sucrose concentration of 0.75M and 1.00M. 4. The freezing methods on in vitro developemental rates of bovine embryos was attained slow freezing method(9.70~15.6%) higher than rapid freezing method(9.4~13.3%).

• 한국수정란이식학회지
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• 제14권1호
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• pp.31-37
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• 1999

The objective of this study was to optimize the selection of sperm sources, optimal culture systems and vitrification method depends on sperm sources. The oocytes were inseminated with either KPN 105, 114, 191, SNU 101, 102, 103 or epididymis and then embryos inseminated were cultured in oviductal cell co-culture or HECM-6 as defined me dium. The blastocysts produced were pooled according to sperm sources as KPN, SNU or epididymis and then vitrified by OPP vitrification method. The results obtained were as follows: 1. The cleavage(86.2 or 84.7%) and development rates to blastocyst (30.6 or 32.0%) were not significantly different between oviductal cell co-culture or HECM-6 culture systems(P<0.05). 2. To determine the optimal sperm sources for using IVF in this system, cleavage rates in KPN 191 and SNU 101 (74.2, 55.8%) were significantly lower rather than those in KPN 105, 114, SNU 102, 103 or epididymis (86.7, 85.1, 89.8, 85.5 or 81.2%), but development rates to blastocyst in KPN 114, SNU 103 or epididymis sperm (30.0, 33.0 or 28.6%) were significantly higher rater than those in KPN 105, 191, SNU 101, 102(21.4, 15.4, 14.9 or 25.4%), respectively (P<0.05). 3. The blastocysts produced were pooled according to sperm sources as KPN, SNU or epididymis and then vitrified by OPP vitrification method. The survival rates were not significantly different among sperm sources (89.6%: 43/48 ; 90.1%: 46/51 ; 83.3% : 20/24). These results obtained indicate that the defined medium, HECM-6, could be use to produce of IVP bovine embryos. Since the frozen semen must be required to maintain of unvariation data in IVP embryo production system, KPN 114 and SNU 103 produced in our laboratory were useful for this purpose. The blastocysts produced by different sperm sources as KPN, SNU or epididymis were vitrified by OPP vitrification method and survived very high rates. The OPP vitrification method could be susceptibility to use of IVP bovine blastocyst embryos.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2001년도 춘계학술발표대회
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• pp.5-5
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• 2001

This study was to test whether Hanwoo in vitro matured oocytes can be successfully cryopreserved by a new vitrification procedure using MVC method. For the vitrification, oocytes were pretreated in 10% ethylene glycol (EG10) for 5-10 min, exposed in EG30 for 30 sec, each oocytes were individually put on the inner wall of 0.25 $m\ell$ straw, and then straws were directly plunged into L$N_2$. Thawing was taken by 4-step procedures [1.0 Msucrose (MS), 0.5 MS, 0.25 MS, and 0.125 MS] at 37$^{\circ}C$. In vitro developmental capacity (survival, cleavage ($\geq$2-cell) and blastocyst rates) in vitrified group was no significant difference compared to that in other treatment groups (exposed; 100.0, 74.4, 32.3% and control; 100.0, 78.3, 36.3%): high mean percentage of oocytes (91.2%) was survived, 69.4% of them were cleaved and 27.9% of cleaved embryos were developed to blastocyst. Especially, after transfer of in vitro developed embryos in vitrified group, four of six recipient animals were found to pregnant and three of them were ongoing pregnant by manual palpation at 250 days after transfer. However, among them, two healthy female calves (23 and 25kg) were born. This result demonstrates that MVC method is very appropriate freezing method for the Hanwoo in vitro matured oocytes and that ovum bank can be maintained efficiently by MVC cryopreservation method.

• Clinical and Experimental Reproductive Medicine
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• 제45권3호
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• pp.110-115
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• 2018

Objective: To compare our in-house method of embryo freezing with Cryotop vitrification in terms of immediate survival, subsequent cleavage and blastocyst formation, and cell numbers in blastocysts. Methods: Two-cell mouse embryos were randomly allocated into three groups: a non-frozen control group (group 1, n = 300), a group that underwent Cryotop vitrification (group 2, n = 300), and a group that underwent our in-house freezing method (group 3, n = 300). Results: There were no significant differences between groups 2 and 3 in the immediate survival rate (96.3% vs. 98.6%, respectively; p= 0.085), the further cleavage rate (91.7% vs. 95.0%, respectively; p= 0.099), or the blastocyst formation rate (80.7% vs. 78.6%, respectively; p= 0.437). The cell numbers in the blastocysts from groups 1, 2, and 3 were comparable ($88.99{\pm}10.44$, $88.29{\pm}14.79$, and $86.42{\pm}15.23$, respectively; p= 0.228). However, the percentage of good-quality blastocysts in the Cryotop vitrification group was significantly higher than in the group in which our in-house method was performed, but was lower than in the control group (58.0%, 37.0%, and 82.7%, respectively; p< 0.001). Conclusion: At present, our method is inferior to the commercial Cryotop vitrification system. However, with further improvements, it has the potential to be useful in routine practice, as it is easier to perform than the current vitrification system.

• 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.

• Reproductive and Developmental Biology
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• 제34권3호
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• pp.271-274
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• 2010

This study was conducted to investigate the survival rate of frozen-thawed spermatozoa in equine by glycerol concentration and freezing speed Two stallions (1 Thoroughbred-13 year old and 1 Arab-7 year old) bred in Korea Racing authority was examined for 1 times in a couple of weeks. Semen was collected by condom method standing heated mare and were centrifuged 650 g for 15 min. and isolated the seminal plasma. Thick fraction of semen was diluted EDTA-Lactose-egg yolk diluents to 1:1 and contained in 0.5 ml straw as $6{\sim}14{\times}10^7\;cells/ml$. Final concentrations of glycerol were 3, 5 and 7% in cryopreseved diluents and added 4 times for 2 hours equilibration. For the freezing, equilibrated straws were located 3 or 5 em above $LN_2$ gas for 5 or 10 min. Survival rates of pre-frozen sperm were $65.0{\pm}13.2%$, $68.3{\pm}10.4%$, $66.7{\pm}11.5%$ and post-frozen were $53.3{\pm}23.1%$, $45.0{\pm}15.0%$, $50.0{\pm}18.0%$ in 3, 5, 7% glycerol concentration, respectively. There was no difference between glycerol concentrations. Survival rates of frozen-thawed sperm on freezing speed were $36.7{\pm}10.4%$, $40.0{\pm}7.1%$, $30.0{\pm}13.2%$ at 3 cm-5 min and $33.3{\pm}11.5%$, $31.7{\pm}2.9%$, $21.7{\pm}10.4%$ at 3 cm-10 min in 3, 5, 7% glycerol concentration, respectively. Survival rates of frozen-thawed sperm on freezing speed were $43.3{\pm}15.3%$, $32.0{\pm}17.9%$, $22.3{\pm}15.7%$ at 5cm-5 min and were $47.5{\pm}15.0%$, $43.3{\pm}12.6%$, $48.3{\pm}15.3%$ at 5cm-10 min in 3, 5, 7% glycerol concentration, respectively. There were significantly different between groups (p<0.05). These results suggest that glycerol concentration did not affect cryopreservation of stallion semen within 3~7% but freezing speed affects. In our experiment, the best cryopreservation condition was at 5 cm above $LN_2$ gas for 10 min for pre-freezing and 7% of glycerol concentration. These results lead to commercial AI with frozen-thawed stallion semen.

• Journal of Chest Surgery
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• 제31권12호
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• pp.1127-1133
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• 1998

배경: 광범위한 기관의 병변시 이를 절제하고 기관을 대체시킬 수 있는 이상적인 대치물질로 현재 많이 연구되고 있는 기관의 초냉동보관 동종이식편의 상피세포 및 연골조직의 생육성 유지와 항원성의 변화를 토끼 기관을 이용하여 연구하였다. 대상 및 방법: 토끼 45마리를 각각 15마리씩 3개의 군으로 나누어 1군은 자가 이식술을 시행하였고 2군은 초냉동보관하지 않은 동종이식편으로 기관 대치술을 시행하였으며 3군은 영하 196$^{\circ}C$에서 1달간 보관한 초냉동보관 동종이식편으로 경부기관 정위치에 이식수술을 시행하였다. 수술 후 7일, 14일, 30일 후에 각군 당 5마리씩 무작위로 선별하여 기관 이식편의 조직학적 검사를 시행하여 상피세포 및 연골조직의 생육성 및 거부반응 정도를 조사하였다. 결과: 상피세포 재생정도에 있어서 7일째에는 세 군간에 차이가 없었으나 14일과 30일에는 1군에서 2군과 3군에 비해 상피세포 재생정도가 좋았다. 2군과 3군에서는 차이가 없었다. 수술 후 7일째까지 거부반응은 2군과 3군 모두에서 거의 나타나지 않았으나 14일과 30일째에는 7일째에 비해 2군과 3군 모두에서 통계학적으로 유의하게 거부반응이 많이 나타났다(P<0.05). 3군에 있어서 2군에 비해 거부반응이 적었으나 통계학적인 유의성은 없었다. 모든 경우에 연골세포는 생육성을 유지하고 있었으며 거부반응도 없었다. 결론: 1달 동안 초냉동보관 된 기관의 동종이식편은 상피세포와 연골의 생육성을 유지할 수 있으며 이는 초냉동보관 기관 동종이식편이 기관 대체물질로 사용할 수 있는 가능성을 보여준다고 할 수 있다. 그러나 초냉동보관으로 항원성을 완전히 없앨 수는 없으며 따라서 거부반응을 더욱 더 줄일 수 있는 방법에 대한 연구가 계속되어야겠다.

• Clinical and Experimental Reproductive Medicine
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• 제25권2호
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• pp.171-177
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• 1998

The combination of testicular sperm extraction (TESE) with ICSI can achieve normal fertilization and pregnancy rate and is effective method in obstructive and non-obstructive azoospermic patients. But, when pregnancy was not occurred, repeated testicular biopsies are not evitable. Therefore, in this study, we observed the survival rate of testicular spemratozoa and spermatozoa extracted from the seminiferous tubules after cryopreserved-thawed used for next IVF cycle with ICSI. In a total of 23 cases, obstructive azoospermia was 17 cases and non-obstructive azoospermia was 6 cases. In obstructive azoospermia, after thawing, motile spermatozua was observed in 13 cases (76.5%). The fertilization rate with 2PN was 67.6% and 5 pregnancies (29.4%) were achieved. In non-obstructive azoospermia, motile spermatozoa was observed in 2 case (33.3%) after thawing. The fertilization rates with 2PN was 53.7% and 3 pregnancies (50.0%) were achieved. A comparison of the results of motile spermatozoa after thawed testicular spermatozoa and spermatozoa extracted from the thawed seminiferous tubule section were 3 cases (60.0%) and 12 cases (66.6%), respectively. The fertilization and pregnancy rates of thawed testicular spermatozoa and spermatozoa extracted from the thawed seminiferous tubule section were 69.4% and 20.0%, 62.5% and 38.8%, respectively. Conclusively, thawed testicular spermatozoa and spermatozoa extracted from the thawed seminiferous tubule section can achieve normal fertilization and pregnancy and cryopreservation of testicular spermatozoa and seminiferous tubule may avoid repetition of testicular biopsies in azoospermic patients in whom the only source of spermatozoa is the testis.

• Clinical and Experimental Reproductive Medicine
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• 제27권2호
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• pp.191-200
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• 2000

Objective: This study was carried out to compare the effects of the stepwise exposure treatments on the morphological normality, fertilization and blastocyst formation rate of the frozen-thawed mouse mature oocytes by vitrification or ultra-rapid freezing and to use as a fundamental data for the cryopreservation of human oocytes. Materials and Methods: The morphological normality and fertilization rates of the vitrified and ultra-rapid frozen mouse mature oocytes after three-stepwise exposure treatments (1step, 3step and 5step) were observed. After choosing the 3step exposure treatment groups, we observed the morphological normality and fertilization, blastocyst formation rate of the vitrified and ultra-rapid frozen mouse mature oocytes. Results: The morphological normality and fertilization rates of the vitrified mouse mature oocytes after three-stepwise exposure treatments (1step, 3step and 5step) were 75%, 85%, 88% and 58%, 61 %, 54% respectively. There were no significant differences among treatments(p>0.05). The morphological normality and fertilization rate of the control was 92% and 65%. There were no significant differences in fertilization rate among control and treatments (p>0.05). The morphological normality and fertilization rates of the ultra-rapid frozen mouse mature oocytes after three-stepwise exposure treatments (1step, 3step and 5step) were 83%, 83%, 84% and 75%, 63%, 56% respectively. There were no significant differences among treatments (p>0.05). The morphological normality and fertilization rate of the control was 95% and 67%. There were no significant differences among control and treatments (p>0.05). The morphological normality and fertilization rate of the vitrified or ultra-rapid frozen mouse mature oocytes after 3step exposure treatment were 69% and 75%, respectively. The blastocyst formation rate was 60% and 57%. The results did not differ significantly between vitrification and ultra-rapid freezing (p>0.05). Conclusion: As known in the above results, there were no significant differences in the fertilization and blastocyst formation rate of the frozen-thawed mouse mature oocytes by vitrification or ultra-rapid freezing among the control and treatments. It is suggested that vitrification and ultra-rapid freezing method were effective for the cryopreservation of mouse mature oocytes.