• 한국수정란이식학회지
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• 제23권1호
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• pp.19-24
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• 2008

The purpose of this study was to compare the efficiency of slow freezing with that of vitrification method for the cryopreservation of human embryos. Human embryos were derived from in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) and the mixed solution of propanedial (1.5, 1.0, 0.5M PROH) and sucrose (0.1M), ethylene glycol (7.5, 15%), dimethyl sulfoxide (7.5, 15% DMSO), sucrose (0.5, 1.0M) and SPS (Serum Protein Substitute) was used for a cryoprotectant for slow freezing and vitrification solution, respectively. Rates of recovery after thawing, morphological normality, post-thaw viability, arrest, morphological abnormality and preimplantation development were compared between two protocols. After freezing-thawing, recovery and survial rate of slow freezing was (88.6% and 73.4%), whereas vitrification was (99.2% and 96.2%) (p<0.05). The arrest rate of slow freezing was significantly lower compared with those of vitrification(8.7% vs 29.9%) (p<0.05). Preimplantation development to the 2-cell (83.8% vs 67.7%), 4-cell (69.0% vs 47.2%) and 8-cell (62.4% vs 37.8%) stages 24, 48 and 72 h after thawing, respectively, were higher in the slow freezing than the vitrification. After slow freezing and vitrification of human embryo at 2-8cell stage, the rate of recovery rate, survival rate and partial damage rate were 92.0% vs 100%, 80.4% vs 96.2% and 52.2% vs 19.0%, respectively. And partial damage rate was significantly lower than those of slow freezing method (p<0.05). These results demonstrate that a slow freezing using PROH is more efficient than a vitrification for cryopreserving the human zygotes, although the vitrification yielded better recovery, survival and partial damage of frozen-thawed 2-8 cell stage embryos than slow freezing method.

• 한국수정란이식학회지
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• 제24권3호
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• pp.207-212
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• 2009

The aim of present experiment was to examine commercial synthetic extender(AndroMed) for semen cryopreservation of Korean Black Bull. Semen was collected from a Korean Black Bull using an artificial vagina and transported to the laboratory. The semen was diluted 1:1 by AndroMed. The pellect was diluted to final sperm concentration of $5{\times}10^5/ml$ by doubling in every 10 minutes at $4^{\circ}C$ cold chamber. The semen was equilibrated for 1 hr at cold chamber and packed to 0.5 ml straw. The semen straws were located above 5 cm of liquid nitrogen for 5 minutes, above 5 cm for 10 minutes and above 10 cm for 10 min. And then the frozen straw was plunged to $LN_2$. The presented straws were examined the viability and motility after thawed at $37^{\circ}C$ water bath. Hanwoo semen was used as KPN (Korea Proven Bull Number) in this experiment. The survival rates was significantly higher in fresh semen than frozen semen ($80{\pm}14%\;and\;43{\pm}11%$). However, the motility rates was similar (80.7% and 66.4%). The survival and motility rates were higher in 5cm, 10 min treatment group than the other two groups in straw-located height and duration above $LN_2$ ($50{\pm}14%$ and 70.7% vs, 33.18% and $65{\pm}7%$ vs, 30.14% and 65.7%, respectively). The development rates to cleavage was higher in Black Cow than Hanwoo semen (62.2%, 64.4%), However, The development rates to blastocyst was higher in Hanwoo than Black cow semen (25.9%, 23.0%). In conclusion. The present results that acceptable fertilization and cryopreservation could be obtained by in vitro fertilization with frozen-thawed semen using a synthetic semen extender (AndroMed).

• 한국수정란이식학회지
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• 제21권3호
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• pp.255-262
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• 2006

본 연구는 Open Pulled Straw(OPS)방법에 의해 동결-융해한 돼지 수정란의 체외 생존 능력을 검토하기 위하여 수행되었다. 미성숙 난자의 체외 성숙을 위하여, 돼지 난소는 도축장에서 회수하였으며, 난구세포로 쌓여있는 난자는 직경 $2{\sim}6mm$의 난포로부터 난포액과 함께 흡입에 의하여 회수하였다. 회수된 미성숙 난자의 체외 성숙을 위하여 5 mM hypotaurine, 0.57 mM cysteine, 10% 난포액, 10 IU/ml PMSG 및 10 IU/ml hCG가 함유된 NCSU-23 배양액 내에서 $21{\sim}22$ 시간 배양 후, 호르몬 물질을 제거한 성숙 배양액 내에서 $21{\sim}22$ 시간 동안 추가 배양하였다. 한편 체외 수정을 위하여 동결-융해한 정액은 5.56 mM glucose, 0.33 mM na-pyruvate, 100 IU/ml penicillin, $100 {\mu}g/ml$ streptomycin 및 4 mg/ml BSA가 첨가된 D-PBS 액을 가지고 1,500 rpm에서 10분간 2회 원심분리를 실시하여 세척하였다. 체외 수정을 위한 배양액은 pH $7.2{\sim}7.4$에서 2 mM caffeine과 2 mg/ml BSA가 첨가된 mTBM 배양액을 이용하였다. 체외 수정시 정자의 최종 농도는 $2.5{\times}10^6cells/ml$로 조정하였다. 수정 8시간 후, 체외 발육을 위하여 5.0 mM hypo-taurine, 4 mg/ml BSA 및 10 ng/ml EGF가 첨가된 NCSU- 23액으로 옮겨 7일간 배양을 계속하였다. 그 후 배반포의 여러 단계에서 OPS 법에 의해 동결-보존하였다. 그 결과, 동결-융해 후 형태학적으로 정상적인 수정란의 비율은 초기 배반포(28.3%)보다는 확장 배반포(38.9%)단계에서 동결했을 때 유의적으로 높게 나타났다(p<0.05). 한편, 동결-융해 후 상해를 입은 수정란의 비율은 확장 배반포보다는 초기 배반포 단계에서 동결된 수정란에서 유의적으로 높은 성적을 보였다(p<0.05). 또 다른 실험에서, 수정란의 동결-융해 후 형태학적으로 정상인 수정란을 48시간 추가 배양했을 때, Hatching 된 수정란의 비율은 초기 배반포, 배반포 및 확장배반포기 단계에서 동결-융해한 수정란에서 각각 6.7, 20.0 및 33.3%로 발육 단계가 높을수록 동결-융해 후의 생존율도 높게 나타났다. 본 연구의 결과로부터, 돼지에서 수정란의 동결-융해 후 생존성의 향상을 위해서는 발육 단계가 높은 배반포기 단계에서의 동결이 요구되며 본 연구에서 이용된 OPS 동결 방법이 폭넓게 활용될 것으로 사료된다.

• 한국가축번식학회지
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• 제17권3호
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• pp.233-242
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• 1993

The effects of in vitro maturation and sperm treatment condition on the in vitro fertilization (IVF) and developmental capacity of bovine oocytes were investigated and the development of embryos was compared under the 2 different co-culture system, with GC or BOEC. The cultured embryo to 16 cell or morula wre transferred into recipients or frozen by 2 different freezing method. The results obtained were summarized as follows; 1. In vitro maturation rates of vovine follicular oocytes cultrued in TCM199 with 10% FCS or ECS were 64.0% and 72.7%, but the case of addition of 10% FCS or ECS to TCM199 co-cultured with granulosa cells were 81.3% and 84.0%, respectively. IVM rate of three TCM199 added to granulosa cells was higher than that of media without granulosa cells. 2. When bovine follicular oocytes were matured in TCM199 with 10% FCS and GC and then fertilized in vitro by sperm treated with caffeine, embryo developments of bovine oocytes co-cultured with BOEC were 38.4% and 51.4%, respectively. But those of bovine oocytes co-cultured with GC were 52.2% by sperm treated with caffeine-heparin. 3. Cleavage rates of bovine oocytes cultured with 10% FCS alone and fertilized in vitro by sperm treated with caffeine-heparin was 33.0%. 4. When bovine follicular oocytes were matured in TCM199 with 10% FCS and GC, embryo developments of bovine ooctyes co-cultured with BOEC of GC were 46.0% and 50.2%, respectively. 5. When bovine follicular oocytes were matured in TCM199 with 10% ECS and GC, embryo developments co-cultured with BOEC or GC were 45.2% and 51.4%, respectively. 6. When Korean Native cow's follicular oocytes matured in TCM199 with 10% FCS and GC, embryo developed co-cultured with BOEC or GC were 45.2% and 51.4%, respectively. 6. When Korean Native cow's follicular oocytes matured in TCM199 with 10% FCS and GC, embryo developments of the bovine oocyte co-cultured with BOEC and GC were 41.8% and 60.1%. But with FCS 10% those of the bovine oocytes co-cultured with BOEC and GC were 42.0% and 48.4%, respectively. 7. When Holstein's follicular oocytes were matured in TCM199 with 10% ECS and GC, embryo developments fo the bovine oocytes co-cultured with BOEC and GC were 50.0% and 57.7%, but with ECS 10% those of the bovine oocytes co-cultured with BOEC and GC were 52.2% and 56.5%, respectively. 8. The viability of frozen-thawed embryos ranged from 60~80% and those of frozen-thawed embryos from vitrification was lower than that from conventional metiod. 9. The selected fresh embryos were transferred nonsurgically to 7 recipients but did not result in pregnancy.

• 한국수정란이식학회지
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• 제27권3호
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• pp.155-162
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• 2012

This study was designed to determine whether low-density lipoporoteins (LDL) extracted from egg yolk in extender improve the function of Korean Jeju Black Bull semen. The semen was cryopreserved with 5% ethylene glycol (EG) or 7% glycerol (G) extenders containing 10% egg yolk (EY), 4% LDL and 5% EY or 8% LDL. Frozen-thawed sperm were evaluated sperm motility, viability, membrane integrity and acrosome integrity. Post-thawed sperm motility has been significantly higher (p<0.05) in 4% LDL + 5% EY ($69.00%{\pm}4.18$; EG and $63.00%{\pm}9.75$; 7% G) than 8% LDL ($57.00%{\pm}5.70$; EG and $52.00%{\pm}4.47$;G). Treatment of 4% LDL + 5% EY-EG ($66.85%{\pm}5.06$) has been significantly improved sperm viability compared to other treatments except 10% EY - EG. Moreover, in membrane integrity, swollen sperm ratio has been only significantly increased (p<0.05) in 4% LDL + 5% EY - EG ($64.65%{\pm}6.10$) among all treatments. In assess to detect acrosome integrity, especially, AR pattern ratio has been significantly decreased (p<0.05) in 4% LDL + 5% EY - EG among all treatments. In sperm viability as time passes, between 4% LDL + 5% EY and 10% EY, there was no significant difference, but 8% LDL was significantly decreased sperm viability in EG (1 and 2 hrs) and G (30 min, 1, 2, 5 and 12 hrs) extender. However, there were no significant differences among all treatments except 8% LDL-G in sperm membrane integrity. 8% LDL-G has been significantly decreased swollen sperm ratio at 5 hrs after thawed. It is concluded from these results that 4% LDL + 5% EY to the freezing extender showed more positive effect on the frozen-thawed spermatozoa in Korean Jeju Black bull.

• 한국수정란이식학회지
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• 제30권3호
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• pp.143-147
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• 2015

Cryopreservation of boar semen is continually researched in reproductive technologies and genetic resource banking in breed conservation. For evaluating the boar semen quality, sperm motility (MOT) is an important parameter because the movement of spermatozoa indicates active metabolism, membrane integrity and fertilizing capacity. Various researches have been trying to improve the quality of semen Post-thawed in boar. Recently, polymorphism (g. 35756 T>C) of Estrogen Receptor 1 (ESR1) gene reported to be significant association with MOT. This study was conducted to evaluate the ESR1 gene as a positional controlling for motility and kinematic characteristics of post-thawed boar semen. To results, The g.35756 T>C SNP of ESR1 was significantly associated with frozen semen motility and kinematic characteristics. The g.35756 T>C SNP was high significantly associated with MOT, VCL, VSL and VAP (p<0.001). The SNP was also significantly associated with ALH (P<0.05). Therefore, we suggest that the g. 35756 T>C polymorphism in the intron 1 region of the porcine ESR1 gene could potentially be applied in frozen semen programs to improve MOT trait, but only after validation in other populations.

• 한국수정란이식학회지
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• 제26권3호
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• pp.187-193
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• 2011

This study was carried out to establish most suitable freezing condition, to evaluate the different glycerol concentration of freezing and thawing rates on motility, viability, membrane integrity and acrosome intecrity of frozen Korean Jeju Black Bull spermatozoa, Semen was collected from a Korean Jeju Black Bull using an artificial vagina and transported to the laboratory. The semen was extended gradually 1:5 then cooled slowly for 2 hrs to 4$^{\circ}C$. The semen was diluted 1:1 with cryoprotectant extenders (3%, 5% and 7% glycerol) and equilibrated for 2 hrs at cold chamber and packed to 0.5 ml straws. The semen straws were located above 3 cm of liquid nitrogen for 5 minutes, above 5 cm for 10 min and above 8 cm for 10 min. And then the frozen straw was plunged into LN$_2$. The presented straws were examined the viability and motility after thawed at 37$^{\circ}C$ water bath. The viability and membrane integrity immediately post-thawing were significantly higher in samples frozen in 7% glycerol than 3% and 5% glycerol (p<0.05). After CTC staining to assess acrosome integrity, F pattern was significantly increased, but B pattern was significantly decreased in 7% glycerol (p<0.05). Freezing distance of 5 cm from liquid nitrogen and pre-cooling for 10 min yield better survival and membrane integrity, but not significant difference. However, AR pattern according to CTC staining was significantly decreased in 3 cm for 5 min.

• 한국가축번식학회지
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• 제11권2호
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• pp.122-126
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• 1987

This study was carried out to investigate the effects of the glycerol equilibration methods and embryo grades before freezing on the survival rate after thawing in mouse embryos. The results obtained from this study were as follows: 1. The number of embryos of grade I(Excellent), II(Good) and III(Fair) before freezing in this study was 97(27.4%), 160(45.2%) and 97(27.4%), respectively. 2. The average survival rate of frozen-thawed embryos in 3 and 5 steps glycerol equilibration was 66.7% and 64.1%, and the rate of transfererable embryos after culture was 68.9% and 69.0%, respectively. 3. Out of embryo grade I and II before freezing, the transferable rate after thawing was 75.2% and 48.1%, respectively, and grade I embryos before freezing was higher transferable rate than that of grade II.

• 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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• 제22권4호
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• pp.251-255
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• 2007

This study was carried out to examine on developmental competence of Hanwoo embryos cultured in vitro according to culture conditions and freezing methods. The in vitro developmental competence to blastocyst stage at Day 8 of culture in SOF was significantly (p<0.05) higher than that in CR1aa (30.3% vs. 18.4%). The in vitro developmental rate of morula and blastocysts cultured in group culture was significantly (p<0.05) higher than that in individual culture (41.4% and 36.0% vs. 21.1% and 10.5%, respectively). The cell number of Day 8 blastocysts in group culture was significantly (p<0.05) higher than that in the individual culture ($120.1{\pm}12.8\;vs.\;94.1{\pm}12.1$, respectively). The survival rates of frozen-thawed balstocysts that were exposed in 1.5 M ethylene glycol or 1.5 M ethylene glycol containing 0.1 M sucrose were 77.5% and 78.7%, respectively. The survival rates of blastocysts cultured for 48 h in slow freezing and vitrification was not significantly different (73.3 and 74.0%). In conclusion, in vitro developmental competence of bovine embryos was influenced on the culture medium (SOF) and culture method (Group culture). Survival rate of frozen-thawed of bovine embryos was not influenced on freezing solutions and freezing methods.