• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.11-11
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• 2001

In this study, we examined the developmental potential of reconstructed bovine embryos and the fate of donor mitochondria during their preimplantation development after nuclear transfer. Isolated cumulus cells were used as donor cells in nuclear transfer. Cumulus cells labelled with MitoTracker Green FM fluorochrome were injected into enucleated bovine MII oocytes and cultured in vitro. MitoTracker labelling on donor cells did not have a detrimental effect on blastocyst formation following nuclear transfer. Cleavage rate was about 69%(56/81) and blastocyst formation rate was 6.2% (5/81) at 7 days after nuclear transfer. The labelled mitochondria dispersed to the cytoplasm and became distributed among blastomeres and could be identified up to the 8- to 15-cell stages. Small patches of mitochondria were detected in some 8- to 15-cell stage embryos (5/20). However, donor mitochondria were not detected in embryos at the 16-cell stage and subsequent developmental stages. In the control group, mitochondria could be identified in arrested 1-cell embryos up to 7 days after nuclear transfer These results suggest that donor mitochondria disappear from recipient cytoplasm before 16-cell stage following nuclear transfer in reconstructed bovine embryos.

• Journal of Embryo Transfer
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• v.16 no.1
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• pp.29-34
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• 2001

To investigate the effect of co-transfer of trophoblastic vesicle (TV) with frozen-thawed in vitro Produced (IVP) bovine embryo on pregnancy rate, IVP blastocysts were transferred to synchronized recipients. Elongated blastocysts were recovered at Day 13 to 15, and dissected more than 4 pieces to removed the embryonic disc. Throphoblastic fragments were cultured for 48 hours to make throphoblastic vesicles (TVs). TVs were cryopreserved in ethylene glycol or vitrification solution and frozen-thawed TVs were co-transferred to recipients with frozen-thawed IVP embryos. 1 The recovery rate of elongated blastocyst on Day 13 to 15 was 22.5% (18/80) and the size of recovered elongated blastocysts was 0.2∼5.0mm. 2. Eighteen elongated blastocysts were dissected into 88 pieces and 61.4% of those pieces were formed to TV (54/88) 3. The viability of frozen-thawed TV in ethylene glycol was higher than in vitrified solution (92.8% vs. 68.8%) 4. The pregnancy rate in co-transfer with frozen-thawed TV and IVP blastocyst was better than transfer only IVP blastocysts (50.0% vs. 23.1%).

• Reproductive and Developmental Biology
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• v.28 no.4
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• pp.257-260
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• 2004

Efficiency of somatic cell nuclear transfer was investigated in mice. First, oocyte activation was induced by SrCl₂, and the rate of development was compared with embryos from normal fertilization. Although more than one half of SrCl₂-treated oocytes developed to blastocysts (146/262, 55.7%), the rate of blastocyst formation was significantly lower than normal fertilization controls (59/79, 74.6%). Second, enucleation of oocytes was performed using Polscope that enables non-invasive visualization of metaphase spindles. Such approach could not only avoid damage of oocytes during an exposure to UV light often employed in conventional enucleation procedures, but could also assure the removal of nuclei from all oocytes operated because of monitoring the location of spindles during an entire process of enucleation. Morphologically normal blastocysts were obtained from the transfer of cumulus cell nuclei into enucleated oocytes. However, the rate of development into the blastocyst stage was still low (4/93, 4.3%). This reflects that the nuclear transfer procedure used in this study was not sufficiently optimized, and other factors may also impact greatly the efficiency of nuclear transfer. Including an induction of oocyte activation and method of enucleation tested in this study, a lot more elements are remained to be optimized to improve the efficiency of somatic cell nuclear transfer in mice.

• Journal of Embryo Transfer
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• v.12 no.1
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• pp.91-102
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• 1997

This study was carried out to enhance the efficiency of Korean Native cattle embryos and establish the techniques for producing the twin calves. Bisected embryos without zona pellucida which were divided by simple method not using holding pipette or whole two embryos were transferred to recipients.The pedigrees of monozygotic twin calves produced by transfer of bisected pair embryos were identified. The results obtained were as follows ; The average successful bisection rate was 89.16%. The embryos of blastocyst stage (91.66%) were bisected successfully at significantly (P<0.05) higher rate, compared with the morula stage embryos (86.66%). The average survival rate of bisected embryos following 24 hours culture was 59.02%. The survival rate of morula stage embryos (62.50%) was significantly (P<0.05) higher than that of blastocyst stage embryos (55.5%). For the production of monozygotic twin calves, ten pairs of flesh or frozen demi-em- lymphocytes antigen, the twin calves produced by transfer of bisected pair embryos of Korean Native cattle were identified in pedigrees and confirmed as monozygotes.

• Clinical and Experimental Reproductive Medicine
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• v.43 no.3
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• pp.164-168
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• 2016

Objective: Assisted reproductive technology has been associated with an increase in multiple pregnancies. The most effective strategy for reducing multiple pregnancies is single embryo transfer. Beginning in October 2015, the National Supporting Program for Infertility in South Korea has limited the number of embryos that can be transferred per in vitro fertilization (IVF) cycle depending on the patient's age. However, little is known regarding the effect of age and number of transferred embryos on the clinical outcomes of Korean patients. Thus, this study was performed to evaluate the effect of the number of transferred blastocysts on clinical outcomes. Methods: This study was carried out in the Fertility Center of CHA Gangnam Medical Center from January 2013 to December 2014. The clinical outcomes of 514 women who underwent the transfer of one or two blastocysts on day 5 after IVF and of 721 women who underwent the transfer of one or two vitrified-warmed blastocysts were analyzed retrospectively. Results: For both fresh and vitrified-warmed cycles, the clinical pregnancy rate and live birth or ongoing pregnancy rate were not significantly different between patients who underwent elective single blastocyst transfer (eSBT) and patients who underwent double blastocyst transfer (DBT), regardless of age. However, the multiple pregnancy rate was significantly lower in the eSBT group than in the DBT group. Conclusion: The clinical outcomes of eSBT and DBT were equivalent, but eSBT had a lower risk of multiple pregnancy and is, therefore, the best option.

• Journal of Embryo Transfer
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• v.15 no.1
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• pp.47-56
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• 2000

This study was carried out to improve a technique of embryo transfer for twin calves production in Hanwoo cattle. Blastocysts for the donor of embryo transfer were classified into three criteria by accessment of morphology; early blastocyst, blastocyst and expanded blastocyst. Tow embryos were introduced transcervically into utrerine horn either of Hanwoo or Holstein by ipsilaterally or contralaterally to the corpus luteum. Thiry-six out of 57 recipients cows were inseminated by artificially on the next day of estrus, and followed by transfer of embryos into contralaterally. The pregnance rates of recipients following transfer of bovine embryos of day 7, 8 and 9 was 43.5, 18.2 and 8.3%, respectively. These results appeared that these was a significant (P<0.05) difference between on day-7 embryos and day-9 embryos, but not between on day-8 and day-9 embryos. Although there was not significant(P<0.05) difference in the pregnancy rates between the blastocysts(11/25, 44%) and expanded blastocysts(2/19, 10.5%) and between the blastocysts and early blastocysts(2/13, 15.4%), the embryos at blastocyst stage are more suitable than others for obtaining higher rate of pregnancy. There was no significant difference on pregnancy of the embryos transferred prior to presence(6/21, 29%) or absence (9/36, 25%) of artificial insemination. On pregnancy of Holstein, 2(15.4%) out of 13 recipients were pregnant in heifer. Similar Pregnancy rates were obtained between 1∼2 parities and 3∼4 parities by 30% (6/20) and 27.3%(3/11), respectively. Taken together, there was not significant difference in pregnancy rate due to small number of recipients used for this experiment. Both of Hanwoo and Holstein introduced the embryos by contralsterally to the corpus luteum were slightly higher pregnancy rate compare to by ipsilaterally (12/41, 29.3% vs, 3/16, 18.8%). The ratio of production of twin and single calves in Holstein was 20% (9/45) and 2.2% (1/45), respectively. However, in Hanwoo cows both of production of twin and single were similar as 8%. This result suggests that Holstein as recipients was superior to Hanwoo cows for production of twin calves. Out of all 15 pregnant, 12(80%) were produced a total of 22 normal calves in which the others composed of abnormal, as judging as 2(13.3%) for abortion and 1(6.6%) for stillbirth during the pregnant period.

• Journal of Embryo Transfer
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• v.33 no.3
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• pp.119-127
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• 2018

The purpose of this study is to examined the electrofusion and activation conditions for the production of porcine somatic cell nuclear transfer (SCNT) embryos. In this study, immature oocytes were cultured in TCM-199 with and without hormones for 22 hours. Skin fibroblasts cells of porcine were transferred into the perivitelline space of enucleated in vitro matured oocytes. Cell fusion was performed with two different pulses that each one pulse (DC) of 1.1 kV/cm or 1.5 kV/cm for $30{\mu}sec$. After fusion subsequent activation were divided into three groups; non-treatment (control) and treatment with 2 mM 6-DMAP or $7.5{\mu}g/ml$ cytochalasin B for 4 hours. Transferred embryos were cultured in PZM-3 (Porcine Zygote Medium-3) in $5%\;CO_2$ and 95% air at $39^{\circ}C$ for 7 day. Apoptosis-related genes (Caspase-3, BCL-2, mTOR, and MMP-2) were analyzed by immunofluorescence staining. There was no significant difference between two different electrofusion stimuli in the cleavage rate; $64.9{\pm}4.8%$ in 1.1 kV/cm and $62.7{\pm}4.0%$ in 1.5 kV/cm. However, blastocyst formation rate (%) was significantly different among three different activation groups (no treatment, 2 mM 6-DMAP or $7.5{\mu}g/ml$ cytochalasin B) combined with electrofusion of 1.1 kV/cm. The blastocyst formation rate was $12.6{\pm}2.5$, $20.0{\pm}5.0$, and $34.9{\pm}4.3%$ in control, 2 mM 6-DMAP, and $7.5{\mu}g/ml$ cytochalasin B, respectively. Immunofluorescence data showed that expression levels of caspase-3 in SCNT embryos undeveloped to blastocyst stage were higher than those in the blastocyst stage embryos. Expression levels of Bcl-2 in blastocyst stage embryos were higher than those in the arrested SCNT embryos. These results showed that the combination of an electric pulse (1.1 kV/cm for $30{\mu}sec$) and $7.5{\mu}g/ml$ cytochalasin B treatment was effective for production of the porcine SCNT embryos.

• Journal of Embryo Transfer
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• v.33 no.4
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• pp.221-228
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• 2018

The osmolarity of a medium that is commonly used for in vitro culture (IVC) of oocytes and embryos is lower than that of oviductal fluid in pigs. In vivo oocytes and embryos can resist high osmolarities to some extent due to the presence of organic osmolytes such as glycine and alanine. These amino acids act as a protective shield to maintain the shape and viability in high osmotic environments. The aim of this study was to determine the effects of glycine or/and alanine in medium with two different osmolarities (280 and 320 mOsm) during IVC on embryonic development after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT) in pigs. To this end, IVC was divided into two stages; the 0-2 and 3-7 days of IVC. In each stage, embryos were cultured in medium with 280, 320, or 360 mOsm and their combinations with or without glycine or/and alanine according to the experimental design. Treatment groups were termed as, for example, "T(osmolarity of a medium used in 0-2 days of IVC)-(osmolarity of a medium used in 3-7 days of IVC)" T280-280 was served as control. When PA embryos were cultured in medium with various osmolarities, T320-280 showed a significantly higher blastocyst formation (29.0%) than control (22.2%) and T360-360 groups (6.9%). Glycine treatment in T320-280 significantly increased blastocyst formation (50.4%) compared to T320-280 only (36.5%) while no synergistic was observed after treatment with glycine and alanine together in T320-280 (45.7%). In contrast to PA embryonic development, the stimulating effect by the culture in T320-280 was not observed in SCNT blastocyst development (27.6% and 23.7% in T280-280 and T320-280, respectively) whereas the number of inner cell mass cells was significantly increased in T320-280 (6.1 cells vs. 9.6 cells). Glycine treatment significantly improved blastocyst formation of SCNT embryos in both T280-280 (27.6% vs. 38.0%) and T320-280 (23.7% vs. 35.3%). Our results demonstrate that IVC in T320-280 and treatment with glycine improves blastocyst formation of PA and SCNT embryos in pigs.

• Reproductive and Developmental Biology
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• v.37 no.2
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• pp.57-64
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• 2013

Developmental potential of cloned embryos is related closely to epigenetic modification of somatic cell genome. The present study was to investigate the effects of applying histone deacetylation inhibitor, trichostatin A (TSA) to activated porcine embryos on subsequent development of porcine parthenogenetic and nuclear transfer embryos. Electrically activated oocytes were treated with 5 nM TSA for different exposure times (0, 1, 2 and 4 hr) and then the activated embryos were cultured for 7 days. The reconstructed embryos were treated with different concentrations of 0, 5, 10 and 25 nM TSA for 1 hr. Also 5 nM TSA was tested with different exposure times of 0, 0.5, 1, 2 and 4 hr. And fetal fibroblast cells were treated with 50 nM TSA for 1, 2 or 4 hr and with 5 nM TSA for 1 hr. Cumulus-free oocytes were enucleated and reconstructed by TSA-treated donor cells and electrically fused and cultured for 6 days. In parthenogenetic activation experiments, 5 nM TSA treatment for 1 hr significantly improved the percentage of blastocyst developmental rates than the other groups. Total cell number of blastocysts in 1 hr group was significantly higher than other groups or control. Similarly, blastocyst developmental rates of porcine NT embryos following 5 nM TSA treatment for 1 hr were highest. And the reconstructed embryos from donor cells treated by 50 nM TSA for 1 hr improved the percentage of blastocyst developmental rates than the control group. In conclusion, TSA treatment could improve the subsequent blastocyst development of porcine parthenogenetic and nuclear transfer embryos.

• Journal of Embryo Transfer
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• v.12 no.3
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• pp.307-313
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• 1997

Experiments were conducted to assess the effect of quality and viability of bovine blastocysts derived from in-vitro culture(IVC) of in vitro matured and fertilized(IVM-IVF) oocytes during their transport 2 hours. Follicular oocytes were collected form ovaries obtained at a slaughterhouse and were cultured for 24 hours in TCM-199. The IVM oocytes were fertilized in vitro with caudal epididymis spermatozoa. Fertilized oocytes were cultured for 7 to 9 days, and embryos that developed to the blastocyst stage were used for the experiment. The blastocysts, packed in straws with storage medium that consisted TCM-199 with HEPES equilibratd in air and supplemented with 10% FCS were transported at 39~(2.0 h). The quality of blastocysts was assessed and ranked as A(excel-lent), B(Good), fair or poor after transportation. The percentages of A and B grade blastocysts after transport duration for < 1 hours(97.7%) were similar to the result from transport duration for 1~2 hours (92.9%) and 2~3 hours(89.6%), but significantly(P<0.05) higher than transpot duration for 3~4 hours(76.3%). The percentages of A and B grade blastocysts after transport duration for two hours from developed blastocyst at 7day(100%) and 8day(85.0%) were higher 9day(96.6%) and >9day (40.0%). And early to expanded blastocyst produced in vitro were transferred to recipient cow by additional embryos at 7 and 8th day after AI. Three of them were pregnant to term and produced four twin calves, and two calves was premature birth. The gestation lengths of male to female and female to female twin were 282 and 281 days, respectively. And birth weight of twin calves were male to female(22.Skg) and female to female twin(20.3Okg), respectively.