• Journal of Embryo Transfer
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• v.4 no.1
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• pp.35-40
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• 1989

The effects of cryoprotectants (glycerol, DMSO and ethylene glycol) and the concentrations (0, 0, 25, 0.5and 1.0 M) of sucrose in the diluent on the is vitro survival of mouse morulae froaen rapidly in liquid nitrogenvapour were examined. When the embryos were equilibrated in 1.5 M cryoprotectants +0.25 M sucrose in one-step or in 3.0 M cryoprotectants +0.25 sucrose in two-step and diluted with 0, 0.25, 0.5, or 1.0 M sucrose solution after thawing, high survival rates were obtained in ethylene glycol (48.0% to 88.2 %) or in glycerol (35.0 % to 77.8 %). These results show that 1.5 M ethylene glycol is a highly efficient cryoprotective agent for the rapid freezing of mouse morula embryos and 0.5 M sucrose was optimal concentration in the diluent after thawing.

• Journal of Embryo Transfer
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• v.13 no.1
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• pp.37-41
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• 1998

This study was conducted to investigate the survival and hatching rates after refrozen-thawed bovine IVF blastocysts. The survival rates after refrozen-thawed bovine IVF blastocysts produced on day 7, day 8 and day 9, were 66.6%(16/24), 62.5%(15/24) and 65.3%(17/26), respectively. The survival and hatching rates after the first frozen-thawed bovine JVF blastocysts were 90.0%(27 /30) and 70.0%(21 /30), but in refrozen-thawed bovine IVF blastocysts were 66.2%(49 /74) and 45.9%(34 /74), respectively. The results of this study were suggest that refrozen-thawed bovine IVF embryos had survival ability.

• Journal of Embryo Transfer
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• v.24 no.4
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• pp.271-274
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• 2009

To establish a protocol of estrus induction and synchronization in European mouflon, we performed artificial insemination using frozen-thawed semen and exogenous hormones. CIDR was inserted into vaginas of four mouflons for 16 days. A day before removal of CIDR, PG 600 was injected intramuscularly. $PGF_2{\alpha}$ was injected when removing CIDR. Artificial insemination was cervically conducted with injecting LHRH 48 hours after CIDR withdrawal. Even though no pregnancy was confirmed, estrous signs were notified like open cervix, congestion of vaginal wall and discharge of cervical mucus. Further research in the wild sheep would be needed for development of artificial breeding methods and advancing sustainability of domestic zoos.

• Journal of Embryo Transfer
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• v.26 no.3
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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.

• Clinical and Experimental Reproductive Medicine
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• v.45 no.1
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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.

• Journal of Embryo Transfer
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• v.22 no.4
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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.

• Journal of Embryo Transfer
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• v.17 no.1
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• pp.55-59
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• 2002

The objective of this study is to determine the developmental competence of in vitro matured bovine oocytes after intracytoplasmic sperm injection(ICSI) with frozen-thawed epididymal spermatozoa. The ovaries were obtained from slaughtered Korean native cows. Oocytes matured in vitro for 24 hrs were fertilized by ICSI with frozen-thawed epididymal spermatozoa. After ICSI, a group of oocytes was activated with 7% ethanol fur 5 min, and the other group was not activated. The oocytes were cultured in TCM-199 medium containing hormones and 10% FCS for 24~30 hrs in a incubator with 5% $CO_2$ in air at 38.5$^{\circ}C$. The percentage of oocytes reaching M II after 24 hrs and 30 hrs of incubation were significantly higher(p<0.05) after culture with TCM-199 media(80.0% and 88.3%) than M I(8.3% and 6.7%). The rate of cleavaged embryos to blastocyst obtained by ICSI treated activation oocytes was significantly higher(p<0.05) than that of nonactivation oocytes(22/46, 47.8% vs 10/39, 25.6%). The rates of embryos development to blastocyst obtained by ICSI treated sperm of flesh, epididymal and frozen-thawed epididymal were 24/45(53.3%), 15/40(37.5%), 11/43(25.6%), respectively and these values of fresh sperm injection were higher than frozen-thawed epididymal sperm. We also concluded that embryos can be produced with ICSI of in vitro matured oocytes by ICSI using frozen-thawed epididymal semen.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2002.11a
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• pp.78-78
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• 2002

The role of heat shock proteins in shielding organism from environmental stress is illustrated by the large-scale synthesis of these protein by the organism studied to date. However, recent evidence also suggests an important role for heat shock protein in fertilization and early development of mammalian embryos. Effects of elevated in vitro temperature on in vitro produced bovine embryos were analysed in order to determine its impact on the expression of heat shock protein 70 (HSP70) by control and frozen-thawed after in vitro fertilization (IVF) or nuclear transfer (NT). The objective of this study was to assess the developmental potential in vitro produced embryos with using of the various containers and examined expression and localization of heat shock protein 70 after it's frozen -thawed. For the vitrification, in vitro produced embryos at 2 cell, 8 cell and blastocysts stage after IVF and NT were exposed the ethylene glycol 5.5 M freezing solution (EG 5.5) for 30 sec, loaded on each containers such EM grid, straw and cryo-loop and then immediately plunged into liquid nitrogen. Thawed embryos were serially diluted in sucrose solution, each for 1 min, and cultured in CRI-aa medium. Survival rates of the vitrification production were assessed by re-expanded, hatched blastocysts. There were no differences in the survival rates of IVF using EM grid, cryo-loop. However, survival rates by straw were relatively lower than other containers. Only, nuclear transferred embryos survived by using cryo-loop. After IVF or NT, in vitro matured bovine embryos 2 cell, 8 cell and blastocysts subjected to control and thawed conditions were analysed by semiquantitive reverse transcription polymerase chain reaction methods for hsp 70 mRNA expression. Results revealed the expression of hsp 70 mRNA were higher thawed embryos than control embryos. Immunocytochemistry used to localization the hsp70 protein in embryos. Two, 8-cell embryos derived under control condition was evenly distributed in the cytoplasm but appeared as aggregates in some embryos exposed frozen-thawed. However, under control condition, blastocysts displayed aggregate signal while Hsp70 in frozen-thawed blastocysts appeared to be more uniform in distribution.

• Clinical and Experimental Reproductive Medicine
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• v.42 no.4
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• pp.175-180
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• 2015

Objective: Embryo loading (EL) is a major step in embryo transfer (ET) and affect on the success of in vitro fertilization (IVF). This study aimed to compare the effect of two different EL techniques on the rates of pregnancy and delivery in IVF/ET cycles. Methods: 207 fresh ET and 194 Frozen-thawed ET (FET) cycles were included in this retrospective study. Two groups (A and B) were defined based on the EL technique used. In group A, the entire catheter was flushed with Ham's F-10 medium. The embryos were then drawn into the catheter using one air bracket. In group B, $70{\mu}L$ of air was aspirated into the syringe and the catheter was flushed using Ham's F10 medium. The medium, air, embryos, air, and finally another layer of medium were then sequentially drawn into the catheter. The main outcome measures were the pregnancy and delivery rates. Results: The groups did not differ with respect to the etiology of infertility, the source of spermatozoa, the quality of the embryos, the type of EL catheter, and the ease of transfer. The pregnancy rate was similar between two groups. In fresh ET cycles, a higher delivery rate was observed in group B than it group A (78.1% vs. 60%, p=0.1). In FET cycles, the rate of delivery was significantly higher in group B than in group A to a nonsignificant extent (88.9% vs. 58.8%, p=0.06). Conclusion: EL techniques did not have a significant impact on the delivery rate in either fresh or FET cycles.

• Korean Journal of Veterinary Research
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• v.38 no.2
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• pp.394-400
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• 1998

Cryopreservation of embryos by vitrification is a simple method to preserve bovine embryos for subsequent embryo transfer, but embryonic viability after vitrification has been inconsistent and low compared with conventional slow freezing. The aim of the present study is to examine the effect of serum or serum albumin in a vitrification solution and epidermal growth factor(EGF) or fibroblast growth factor(FGF) on in vitro viability of bovine blastocysts frozen by vitrification. Bovine blastocysts were produced by in vitro maturation, fertilization of follicular oocytes and culture of embryos in a synthetic oviduct fluid medium(SOFM) containing BSA and 19 essential and nonessential amino acids. Blastocysts with excellent or good morphology were selected at 7 or 8 days after culture and utilized for vitrification. In experiment 1, blastocysts were vitrified in a solution containing semi-fetal calf serum(SFCS) or BSA(5 or 10mg/ml) and then their subsequent viabilities were examined by culturing thawed embryos in a SOFM containing BSA and 19 amino acids. Effect of EGF or FGF added to a SOFM containing polyvinyl alcohol(PVA) on the viability of vitrified-thawed blastocysts was investigated in experiment 2. BSA added at 5 or 10mg/ml to a vitrification solution showed significantly higher(p < 0.05) developmental rate to expanded and hatching blastocysts than SFCS, but there was no significant difference in the developmental rate to hatched blastocysts after thawing. Supplementation of a culture medium with EGF and/or FGF significantly increased(p < 0.05) embryo development to expanded blastocysts compared with control but showed no beneficial effect on the development to hatching or hatched blastocysts. Coculture of thawed embryos with granulosa cells in a TCM 199 containing 10% fetal calf serum(FCS) showed the highest developmental rate to expanded, hatching and hatched blastocysts among the groups tested. In conclusion, supplementation of a vitrification solution with BSA at 5mg/ml and culture of thawed blastocysts in a medium containing EGF and/or FGF can improve in vitro viability of bovine blastocysts frozen by vitrification.