• Journal of Veterinary Clinics
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• v.27 no.2
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• pp.154-158
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• 2010

Embryo Transfer (ET) is one of the assisted reproductive technologies and a useful tool for improving herds. The purpose of this study is to produce the calves using frozen embryos which were produced in the top one percent Holstein in Canada by ET. One hundred seventeen recipients were used for surrogate mothers and seventy cows were diagnosed to be pregnant. Fifty seven calves were born successfully and thirteen out of them failed to produce viable calves (abortion: 4, stillbirth: 9). Their gestational length, birth body weight and sex ratio for all the viable calves(n = 57) were $278.1{\pm}3.6$ days (range: from 271 to 286 days), $44.0{\pm}3.0\;kg$ (range: from 37 to 49 kg) and 57.9 vs. 42.1 % (male 33 and female 24), respectively. Microsatellite analysis confirmed that they were derived from frozen embryos. In conclusion, this study demonstrated that viable calves derived from frozen-thawed embryos from Canada were born by ET.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 1997.05a
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• pp.27-34
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• 1997

Bovine oocytes surrounded with compact cumulus cells were cultured for 20 to 22 hours($38^{\circ}C$, 5% $CO_2$) in modified TCM-199 medium supplemented with 5% superovulated cow serum(SCS) and inseminated by in vitro capacitated spermatozoa. Day 7 to 8 embryos were equilibrated for 10 minutes in 1.3M methyl cellosolve(MC) <1.1M diethylene glycol(DEG), 1.8M ethylene glycol(EG), 1.6M propylene glycol(PG) and 1.1 M 1,3-butylene glycol(BG) solutions. They were then loaded into 0.25ml straws, placed into an alcohol bath freezer at $0^{\circ}C$, cooled from $0^{\circ}C$ to $-6^{\circ}C$ at $-1^{\circ}C$/minute, seeded, held for 10 minutes, and stored in liquid nitrogen. After thawing in $30^{\circ}C$ water, the embryos wee rehydrated in TCM-199 medium and then cultured for 48 hours in TCM-199 plus 5% SCS. Embryos were considered viable if they progressed to later developmental stages with a good morphology. Some of the embryos frozen in each cryoprotectant were thawed and transferred non-surgically without removing the cryoprotectant. Hatched embryos survived freezing and one-step dilution as follows : EG(50.0%), MC(53.6%), DEG(56.9%), PG(58.0%) and BG(11.5%). The survival rate of embryos cooled at -0.3^{\circ}C$ vs. $-0.5^{\circ}C$/minute was not significantly different(P<0.05), however, blastocysts hatched most often (P<0.01) in vitro when cooled at a rate of $0.3^{\circ}C$/minute(64.6%), 31/48) than at $-0.5^{\circ}C$/minute(22.6%, 12/53). Pregnancy rates resulting from embryos frozen in the different cryoprotectants were as follows : MC(48%, 10/21); DEG(30%, 3/10); EG(74%, 20/27); and PG(40%, 4/10). These results indicate that MC, DEG, EG and PG have utility as cryoprotectants for the freezing and thawing of IVF Bovine embryos.

• Clinical and Experimental Reproductive Medicine
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• v.24 no.1
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• pp.111-118
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• 1997

Intracytoplasmic sperm injection (ICSI) recently has been utilized widely as the most successful technique to overcome the unfertilization problem in cases of severe male infertility in couples who could not be treated by conventional IVF. Recently, indications of ICSI have been extended further and more fertilized oocytes become available. Thus, it is necessary to examine the efficiency of freezing the surplus embryos obtained from ICSI. We compared the survival rate and the future outcome of cryopreserved embryos obtained either after conventional IVF or ICSI during the same period. After ICSI or IVF, five best-quality embryos from each patient were transferred in the stimulation cycle and the surplus pronuclear (PN) stage oocytes or multicellular embryos were cryopreserved by slow freezing protocol with 1,2-propanediol (PROH) as a cryoprotectant. A total of 792 embryos from ICSI trial were thawed and 65.2% (516/792) survived. The survival rates of PN stage oocyte, multicellular embryo and PN + multicellular embryo were 63.5%, 68.2%, 64.0%, respectively. After 111 transfers, 34 pregnancies were achieved, corresponding to a clinical pregnancy rate of 30.6% per transfers. We thawed 1033 embryos from IVF trials and 57.5% (594/1033) survived. In IVF cycle, the survival rates of PN stage oocyte, multicellular embryo and PN + multicellular embryo were 58.2%, 65.2%, 40.2%, respectively. Thirty eight clinical pregnancies were established after 134 transfers, corresponding to a pregnancy rate of 28.4% per transfer. The cleavage rate of thawed PN stage oocytes from ICSI trial (61.3%) was significantly higher than those from conventional IVF (53.4%). The developmental rates of good embryo (${\geqq}$ grade II) in thawed PN stage oocytes obtained from conventional IVF and ICSI were 63% and 65%, respectively. We concluded that PN stage oocytes, multicellular embryos resulting from ICSI procedure can be successfully frozen/thawed with reasonable clinical pregnancy rates comparable to those of IVF.

• Journal of Embryo Transfer
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• v.23 no.1
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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.

• Journal of Embryo Transfer
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• v.27 no.4
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• pp.259-264
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• 2012

Solution of glycerol, ethylene glycol, sucrose, dextrose (GESD) and cryotop methods were carried out to investigate the survivability on vitrification of embryos. Embryos cultured in vitro were vitrified by GESD of 10 or 8 step and cryotop methods of 6 step, from cryopreservation step to frozen-thawed and culture step. Survival rate and ICM, TE cells of embryos were investigated after frozen-thawed 24 h. As a results, cryotop method was significantly (p<0.05) higher ($85.76{\pm}5.3$ vs. $66.71{\pm}2.4$, $44.80{\pm}2.1%$) than GESD 10 or 8 step methods on survivability. Also, In ICM cell number, cryotop method was significantly (p<0.05) higher to $45.67{\pm}4.7$ cells than GESD 8 step method. TE cell number was significantly (p<0.05) highest to $111.00{\pm}11.0$ cells in cryotop method. On the other hand, survival rate, TE and total cell number were all the significantly (p<0.05) high, except ICM in GESD 10 step method between GESD 10 step method and GESD 8 step method. In conclusion cryotop method was to be most effective, but it is considered necessary to study vitrification method for step-by-step freezing and thawing process.

• 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.

• Korean Journal of Animal Reproduction
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• v.15 no.2
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• pp.133-139
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• 1991

This stduy was carried out in order to investigate the effects of concentration and equilibration time of cryoprotective agents on survival rate of slow and ultrarapidly frozen in vitro fertilized bovine embryos. In vitro fertilized bovine embryos, following dehydration by cryoprotective agents and sucrose, were slowly freezed(from 2$0^{\circ}C$ to -7$^{\circ}C$/-1$^{\circ}C$/min., from -7$^{\circ}C$ -35$^{\circ}C$/-0.2$^{\circ}C$/min. from -35$^{\circ}C$ to -38$^{\circ}C$/-0.3$^{\circ}C$/min.) by cell freezer and directly plunged into liquid nitrogen and thawed in 38$^{\circ}C$ water. Survival rate was defined by development rate to the morula and blastocyst stage after in vitro cultured and FDA test. The results are summarized as follows : 1. The survival rates of in vitro fertilized bovine embryos after slow frozen-thawing in the freezing medium of 0.25M sucrose added 2.5M glycerol, 3.0M DMSO, 2.0M propanediol and 2.5M glycerol＋2.0M propanediol were 84.3%, 85.9%, 77.8%, 74.3%, respectively. 2. The survival rates of in vitro fertilized bovine embryos after slow frozen-thawing in the freezing of 0.50M sucrose added 2.5M glycerol, 3.0M DMSO, 2.0M propanediol and 2.5M glycerol＋2.0M propanediol were 83.8%, 85.1%, 71.4%, 74.6%, respectively. 3. The survival rates of in vitro fertilized bovine embryos after ultrarapid frozen-thawing in the freezing of 0.25M sucrose added 2.5M glycerol, 3.0M DMSO, 2.0M propanediol and 2.5M glycerol＋3.0M propanediol were 69.3%, 70.8%, 63.2%, 67.1%, respectively. 4. The survival rates of in vitro fertilized bovine embryos after ultrarapid frozen-thawing in the freezing of 0.25M sucrose added 2.5M glycerol, 3.0M DMSO, 2.0M propanediol and 2.5M glycerol＋2.0M propanediol were 69.4%, 70.1%, 62.3%, 63.5%, respectively. 5. The survival rates of in vitro fertilized bovine embryos after slow and ultrarapid fromthawing in the freezing medium of sucrose added cryoprotective agents were not significant difference between 5min. and 10min. of equilibration time.

• Journal of Embryo Transfer
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• v.21 no.2
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• pp.147-156
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• 2006

The objective of this study was to supply excellent genetic resources to livestock farms by transferring embryos produced by genetically superior Korean cows (Hanwoo). Eighty Hanwoo donors were superovulated with gonadotropin ($Folltrpin^(R)\;or\;Antorin^(R)$) for 4 days combined with or without progesterone releasing intravaginal device (CIDR) insertion. The collected fresh or frozen-thawed embryos were transferred to 226 farm recipients. In this study, the effect of CIDR insertion in combination with gonadotropin ($Folltrpin^(R)$) treatments initiated at the random stage of estrous cycle on embryo production was evaluated and compared to conventional superovulation protocol. Moreover, the effect of gonadotropin ($Antorin^(R)$) dose in CIDR-treated Hanwoo donors on the embryo yield was determined. In addition, the effects of embryos (fresh vs. frozen-thawed), embryo transfer person, seasons and farms on the pregnancy rate were evaluated. In Hanwoo donors, CIDR insertion in combination with $Folltrpin^(R)$ treatments regardless of estrous detection resulted in increased numbers of total ova (6.5 vs. 5.8) and transferable embryos (3.9 vs. 3.2) compared to the conventional superovulation protocol (p<0.01). In CIDR-treated Hanwoo donors, the higher dose of $Antorin^(R)$ (36 vs. 28 mg) resulted in the increased number of transferable embryos (8.3 vs. 5.4, p<0.05). The embryos (fresh 43.9% vs. frozen-thawed 23.1%) and embryo transfer person (53.9 vs. $0{\sim}16.7%$) significantly affected the pregnancy rate after embryo transfer (p<0.01). These results suggest that CIDR-based superovulation protocol may be effectively used for production of superior Hanwoo embryos and, multiple ovulation and embryo transfer in Hanwoo might be effectively applied for livestock improvement if pregmancy rate with frozen-thawed embryos and embryo transfer skill would be improved.

• Korean Journal of Animal Reproduction
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• v.11 no.2
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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.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.134-134
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• 2003

Bovine embryos produced by in vitro maturation, feretilization and development was examined for presevation and transfer. The fertilization medium used BO medium with 5 mM/$m\ell$ caffeine and 10$\mu\textrm{g}$/$m\ell$ heparin and adjusted to a pH of 7.2 to 7.4. The final concentration of spermatozoa was adjusted to 1$\times$$10^{6}$ cells/$m\ell$ motile sperm during fertilization in vitro. At 8~10 hrs after insemination, the oocytes were transferred into CR1aa medium and cultured for 7 days. Embryos were preserved by vitrification method for transfer. When the embryos of early, blastocyst and expanded blastocyst stages were frozen-thawed, the proportions of embryos with normal morphology 83.6, 88.1 and 85.2%. (중략)