• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.73-73
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• 2001

This study was to examine whether the vitrified, one-step diluted and direct transferred Hanwoo IVM/IVF/IVC blastocysts can be successfully survived in vivo and they were succeeded into the live birth. For vitrification, blastocysts were serially exposed in glycerol (G) or/and ethylene glycol (EG) mixtures [10% (v/v) G for 5 min, 10% G plus 20% EG (v/v) for 5 min, and 25% G plus 25% EG (v/v) for 30 sect] which is diluted in 10% FBS added D-PBS. Thawing of straw was carried out in air for 10 sec and then in water bath of $25^{\circ}C$ for 20 sec. One-step dilution within the straw was done in water bath of $25^{\circ}C$ for 1 min. Vitrified and one-step diluted embryos were directly transferred into 36 (natural or hormone induced synchronized) recipient cows in 6 areas of Kyungsang Buk-Do. Pregnancies were confirmed at first when recipient cows did not return to the subsequent estrus cycle, and later by manual palpation per rectum on day 45, 90 and then living calves were derived into parturition. Overall pregnancy was 33.3%(12/36), However, higher pregnancy was obtained when the recipients exhibited estrus one day earlier than the age of transferred embryos (53.3 vs 25.0-27.3%), irrespective of synchronization methods. Also, parous recipients became pregnant higher than nulliparous heifers, And, there were not different in pregnancy rates by the aspect of corpus luteum (CL) quality of recipients (good, 29.4; fair, 37.5; poor, 33.3%). One hundred eight of frozen-thawed Hanwoo blastocysts were directly transferred into 36 recipient cows. In 12 of pregnant cows, 3 cows were aborted and 9 cows were calved [single, 66.7% (6/9): twin, 33.3% (3/9)]. Total embryo implantation rate was 11.1% (12/108). However, 9 Hanwoo calves were lived. Therefore, these results demonstrate that direct transfer technique of vitrified and one-step diluted bovine blastocysts can be applied easily and effectively with the higher pregnancy rate on field trial without the equipment and embryological skills.

• Clinical and Experimental Reproductive Medicine
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• v.45 no.1
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• pp.52-55
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• 2018

This study retrospectively assessed whether time-lapse data relating to developmental timing and morphology were associated with clinical outcomes, with the eventual goal of using morphokinetic variables to select embryos prospectively for cryopreservation. In this study, we examined the clinical outcomes of single vitrified-warmed blastocyst transfer cycles that were cultured in a time-lapse incubation system. The morphokinetic variables included uneven pronuclei, an uneven blastomere, multinucleation, and direct, rapid, and irregular division. A total of 164 single vitrified-warmed blastocyst transfer cycles were analyzed (102 cycles of regularly developed blastocysts and 62 cycles of blastocysts with morphokinetic variables). No significant differences in the age of females or the standard blastocyst morphology were found between these two groups. The regularly developed blastocysts showed significantly higher implantation and clinical pregnancy rates than the blastocysts exhibiting morphokinetic variables (30.4% vs. 9.7% and 37.3% vs. 14.5%, respectively; p< 0.01). The blastocysts that exhibited morphokinetic variables showed different mean development times compared with the regularly developed blastocysts. Although morphokinetic variables are known to have fatal impacts on embryonic development, a considerable number of embryos developed to the blastocyst stage. Morphokinetic variables had negative effects on the implantation and clinical pregnancy rates in vitrified-warmed blastocyst transfer cycles. These findings suggest that blastocysts cultured in a time-lapse incubation system should be considered for selective cryopreservation according to morphokinetic variables.

• Korean Journal of Animal Reproduction
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• v.18 no.1
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• pp.55-62
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• 1994

This study was carried out to test the effects of fluorescein diacetate(FDA 0${mu}ell$/ml, 0.5${mu}ell$/ml, 5${mu}ell$/ml, 10${mu}ell$/ml or 0${mu}ell$/ml, 5${mu}ell$/ml in PBS) treated before culture on the survival of vitrified mouse morulae in vitrification solution(20% glycerol＋glycerol＋10% ethylene glycol＋30% ficoll＋10% sucrose). The results were summarized as follows; 1. The survival rate of FDA-tested fresh mouse morulae after 24 hours culture was over 96%((P4.8) in the control or treatment groups with various levels of FDA. Because the rate of mouse morulae which developed to hatched blastocysts was higher with the various levels of FDA treatment(67%) than control(50%), it was considered that toxicity of FDA did not affect the survival of mouse morulae. 2. When mouse morulae were FDA-tested in FDA 0(control), 0.5, 5, and 10${mu}ell$/ml treatment after vitrification, the development rate to expanded blastocyst were 66, 82, 64 and 76%, and FDA scores were P4.2(84%), P4.7(94%), P4.2(84%) and P3.9(78%), respectively. There were no significant differences between control and FDA treatments, but there were significant difference between 0.5${mu}ell$/ml)94%) and 10${mu}ell$/ml(78%) treatment(P<0.01). 3. The survival rates of cultured mouse morulae according to FDA-scores(P0=non-fluorescence; P1~P5=according to their fluorescence) after vitrification were P5;92%, P4;67%, P3;42% and P2.P0;0%, respectively. 4. Implantation rates of morulae stage embryos cultured into early blastocysts and implanted into uterine hornes vitrification were 14 and 11% embryos treated control(0${mu}ell$/ml) and FDA 5${mu}ell$/ml and the normal fetus development was 2% embryos for both treatments. Results of this percent study indicated that toxicity of FDA does not affect not only the survival of fresh and vitrified mouse morulae but also the development rate and implantation of fetus after transfer as well. The development rates of mouse morulae with the FDA score of P5, P4 and P3 were 92, 67 and 42%, respectively, it was considered that FDA-test was fit for the judge of survival.

• Proceedings of the KSAR Conference
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• 2004.06a
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• pp.201-201
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• 2004

In this study we examined whether vitrified Hanwoo (Korean cattle) IVM/IVF/IVC blastocysts can survive in vitro/in vivo by a quick one-step dilution method and these embryos result in live births. Blastocysts produced in vitro were vitrified by serial exposure to glycerol (G) and/or ethylene glycol (EG) mixtures of 10% (v/v) G for 5 min, 10% G plus 20% EG (v/v) for 5 min, and 25% G plus 25% EG (v/v) for 30 see. (omitted)

• Proceedings of the KSAR Conference
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• 2002.06a
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• pp.41-41
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• 2002

Bovine cumulus-oocyte complexes were recovered from ovaries at a slaughter and then divided into five groups: control group(unvitrified oocytes), 0 hr. group(composed of oocytes vitrified before the onset of maturation) and 10, 14, and 20 hrs groups(vitrified respectively at 10, 14 and 20 hrs after the onset of maturation). The oocytes remained vitrified for 24 hrs, and then were thawed in 30℃ water bath. Survival and cleavage rates were defined as development rate on in vitro culture and stained with aceto-orcein or FDA test.

• 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.

• Journal of Embryo Transfer
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• v.21 no.1
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• pp.53-58
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• 2006

This study was conducted to comparing on vitrification of mouse oocytes and embryos using CPS, conventional straws and CPS by evaluating in morphological survival for oocytes, and embryonic cleavages and blastocyst formation for embryos. The morphological survival in vitro after thawing of vitrified oocytes using CPS (75%) and conventional straws (72%) were significantly higher (p<0.05) than that using OPS (68%). The blastocyst formation rates of vitrified embryos using CPS (48.6%) and unfrozen control embryos (56.0%) were significantly higher (p<0.05) than those of conventional straws (43.4%) and OPS (37.7%). The rates of morula formation were also higher to control, CPS, conventional straws and OPS in orderly. These results show that CPS has the advantages of achieving a high survival and safety preservation.

• Journal of Embryo Transfer
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• v.9 no.1
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• pp.7-14
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• 1994

The present experiments on cryopreservation were designed to examine the effects of solution toxicity, equilibration time and cell stages on the post-thaw survival of bovine IVF embryos. The oocytes were matured in vitro(IVM) for 24 hrs. in TCM-199 supplemented with 35 $\mu$g /ml FSH, 10 $\mu$g /ml LH, 1 $\mu$g /ml estradiol-17$\beta$ and granulosa cells at 39$^{\circ}C$ under 5% $CO_2$ in air. They were fertilized in vitro(IVF) by epididymal spermatozoa treated with heparin for 24 hrs., and then the zygotes were co-cultured in vitro(IVC) with bovine oviductal epithelial cells for 7 to 9 days. The bovine IVF embryos were exposed to the EFS solution in one step at room temperature, kept in the EFS solution during different period for toxicity test, vitrified in liquid nitrogen, and thawed rapidly. 1. after the bovine blastocysts were exposed to EFS solution for 2 min. at room temperature and then they were washed in 0.5 M sucrose solution and TCM-199, they were cultured to examined cryoprotectant induced injury during exposure, Most of the embryos(95.0%) developed to reexpanded blastocoels. However, when the exposure time was extended to 5 and 10 min, these development rates dropped dramatically in 5 min. (69.5%) and 10 min. (47.4%), respectively, 2. When the bovine IVF embryos were vitrified in EFS solution after the equilibration for 1 and 2 min. exposure, The embryos to have reexpanded blastocoels following thawing, washing and culture processes were found to he 82.6 and 73.9%, respectively. However, when the exposure time was extended to 3 min, this survival rate dropped to 18.2%. The optimal time for equilibration of bovine IVF blastocysts in EFS solution seemed to he 1~2 min. 3. When the bovine IVF embryos were equilibrated for 1 min. the significantly (P<0. 05) higher post-thaw survival rates were obtained from the embryos of blastocyst stage(81.3%) than morulae stage(5. 1%). The optimal cell stage for viterification with EFS solution proven to he blastocyst stage in bovine IVF embryos. 4. The number of blastomeres of blastocyst stage was examined with nuclear staining with Hoechst 33342 during 7 to 9 days post-insemination. The cell counts of frozen bovine IVF embryos were found significantly(P$\geq$7.5 and those of the fresh embryos 76.6$\geq$7. 1, which were cultured in the sarne period and conditions as frozen embryos.

• Clinical and Experimental Reproductive Medicine
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• v.24 no.3
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• pp.347-353
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• 1997

This study was to test whether in vitro/in vivo survival of vitrified mouse blastocysts was influenced by culture conditions and ET method. Mouse blastocysts were obtained from in vitro fertilization and cultured for 4 days in M16 medium, and they were vitrified in EFS40 which contained 40% ethlyene glycol, 18% Ficoll and 0.5 mol sucrose in PBS. In experiment I, in vitro and in vivo survival rate of these embryos were evaluated in different culture condition after thawing. When thawed embryos were cultured in M16 medium as a control, m-CR1 medium contained 20 amino acids (2% BME amino acis and 1% MEM non-essential amino acids solution) and 4 mg/ml BSA and cumulus monolayer cell co-cultured condition in mCR1 medium (10% FBS), their in vitro survival at 24 hr after thawing was not affected by culture condition (75.6, 83.1, 82.4%). However, in vivo survival rates of implantation in m-CR1 medium (80.4%) were significantly higher than those of M16 medium (51.2%), co-culture (57.1%) condition, although there was no difference in live fetuses rates on day 15 gestation (39.0, 49.0, 38.1%). In experiment II, the in vivo development potential of embryos by ET methods was examined. When blastocysts were transferred to the day 2, 3 pseudopregnant recipient without culture soon after thawing, no pregnant recipient was obtained on the day 2 pseudopregnancy, and 50% of pregnancy rates and 15.4% of live fetus rates were obtained on the day 3 pseudopregnant recipients. These results were significantly lower than those of transferred group (day 3 pseudopregnant recipients) after culture for 16 hr post thawing (73.5, 57.1%) (p<0.05). In experiment III, to elevate usability of delayed embryos in vitro/in vivo survival of vitrified embryos (day 4 early, day 5 early and expanding blastocyst) were examined. in vivo survival rates (live fetus, total implantation) were higher in day 4 early blastocysts (33.3, 66.7%) than in day 5 expanding blastocysts (29.0, 38.7%), although the highest in vitro survival rates were obtained in the day 5 expanding brastocysts (78.3%). Therefore, these results suggest that the in vitro/in vivo survival rates of vitrified embryos could be improve by the culture condition and ET method and that the in vivo development rates of delayed embryos were decreased with longer culture duration in vitro. It means that more effective cryopreservation was obtained in day 4 early blastocysts than in day 5 expanding blastocysts.

• Journal of Embryo Transfer
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• v.8 no.1
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• pp.31-36
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• 1993

The post-thaw survival of mouse embryos of the various developmental stages was determined after cryopreservation by vitrification in a solution containing ethylene glycol, Ficoll and sucrose (EFS). All the embryos were equilibrated for 2 minutes just prior to freezing. The number of blastomeres during in vitro development was counted by nuclei higher rates of post-thaw survival were obtained from the embryos of 2-cell(92.2%), 8-cell(77.2%) or morula stage(90.0%) than those of blastocyst stage(62.7%). The number of blastomeres per embryo following in vitro culture for 24 hours was significantly(P<0.05) smaller as 66.0f22.3 in vitrified and thawed morulae than fresh morulae(91.7$\pm$12.2).