• Korean Journal of Agricultural Science
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• v.43 no.4
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• pp.575-580
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• 2016

This study was performed to determine whether supplementation of tauroursodeoxycholic acid (TUDCA), an endoplasmic reticulum (ER) stress inhibitor, during vitrified cryopreservation enhances the development of frozen mouse embryos. Mouse 8-cell stage embryos were collected and exposed to a cryoprotectant solution containing TUDCA or TM (tunicamycin, an ER stress inhibitor) at room temperature and stored in liquid nitrogen following vitrification. The final concentration of TUDCA or TM was $50{\mu}M$. The survival and development rates of mouse 8-cell stage embryos exposed to TUDCA- or TM-containing solutions at room temperature or stored in liquid nitrogen following vitrification were measured. There were no significant differences in survival rate and blastocyst formation rate among control, TUDCA, and TM groups after embryos were exposed to vitrification solutions at RT. When mouse 8-cell stage embryos were treated with TUDCA or TM and then stored in liquid nitrogen, the survival rates of control and TUDCA groups were significantly higher than for the TM group. Blastocyst formation rate of the TUDCA group following in vitro culture was significantly higher than that in control or TM groups. The TM group showed a lower (p < 0.05) blastocyst formation rate than the other two groups. Our results indicate that TUDCA supplementation during cryopreservation of mouse embryos could enhance their development capacity.

• Journal of Veterinary Clinics
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• v.16 no.1
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• pp.163-176
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• 1999

The nuclear transplantation technique is known as the most potential and efficient method for producing large numbers of genetically identical animals from a single embryo and somatic cells. After Dolly was introduced in 1997, many scientists were amazed. A possibility came to a reality that live offspring could be produced with differentiated somatic cells from an adult animal. On the other side, many in the press and the sensationalists focused on the socially, ethically and scientifically unacceptable sides of the technology. In this article, the history, current status and prospects of the technological development of nuclear transplantation in mammals and its application to the production of cloned animals are described. For the efficient and successful production of cloned embryos by nuclear transplantation, the right selection, preactivation and micromanipulation of oocytes as capacious recipient cytoplasm, the adequate and benefitial preparation of multiple totipotent embryonic and somatic cells as donor nuclei, fusion of them and in vitro production of cloned embryos are very critical. Recently the overall efficiency of production of cloned embryos and offspring in livestock has been much improved. Cloning will also be a more efficient, faster and useful way of creating transgenic fetuses for gene therapies, gene pharming, organs for xenotransplantation by preselection and mass production of transgenic embryos and consequently improving the production efficiency in transgenic animals. Further technical development of nuclear transplantation will enable large-scale production of cloned livestock and in near future the commercial cloning of animals will become a reality.

• Journal of Embryo Transfer
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• v.10 no.1
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• pp.65-72
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• 1995

The purposes of this study were to produce cloned rabbit embryos and offsprings by nuclear transplantation(NT) using in vitro matured oocytes as nuclear recipient cytoplasm and to determine the effect of frozen nuclei donor embryos on the production efficiency of cloned embryos. The 8cell embryos were collected from the mated does by flushing oviducts with Dulbecco's phosphate buffered saline containing 10% fetal calf serum(FCS) at 40 hours after hGG injection. A portion of collected embryos were preserved at 4$^{\circ}C$ for 24 hours and a portion of them were frozen by vitrification method. The embryos used for donor nuclei were synchronized in the phase of Gi /S transition. The in vitro matured oocytes were used as recipient cytoplasm following removing the nucleus and the first polar body. The synchronized blastomeres from fresh, cooled or frozen embryos were injected into the enucleated oocytes by micromanipulation and were electrofused by electrical stimulation of three pulses for 60 $\mu$sec at 1.0 W /cm in 0.28 M mannitol solution. The fused oocytes were co-cultured with a monolayer of rabbit oviductal epithelial cells in M-199 solution containing 10% FCS for 120 hours at 39$^{\circ}C$ in a 5% $CO_2$incubator. Following in vitro culture of the NT embryos to blastocyst stage, they were stained with Hoechst 33342 dye for counting the number of blastomeres by fluorescence microscopy. The nuclear transplant embryos developed in vitro to 2- to 4-cell stage were transferred into the oviducts of synchronized recipient does. The results obtained were summarized as follows: 1. The fusion rates of the blastomeres from fresh, cooled and frozen embryos with the in vitro matured and enucleated oocytes were 100, 95.8 and 64, 3%, respectively. 2. Development in vitro to blastocyst was significantly(p<0.05) different between the cloned embryos with the blastomeres from fresh, cooled or frozen embryos as 39.0, 20. 9 and 15.7%, respectively. 3. The mean numbers of cell cycle per day during in vitro culture of cloned embryos blastomeres from fresh, cooled or frozen embryos was 1.31, 1.29 and 1.16, respectively. 4. A total of 77 nuclear transplant embryos were transferred into 6 recipient does, of which two offsprings were produced from a foster mother 31 days after embryo transfer.

• Journal of Embryo Transfer
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• v.6 no.2
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• pp.18-29
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• 1991

Nuclear transplantation technique is known as the most potential and efficient method for producing a large number of genetically identical animals from a single embryo. The technical development of nuclear transplantation in mammals and its application to the production of cloned animals are described. For the efficient and successful production of cloned embryos by nuclear transplantation, the right selection and micromanipulation of recipient eggs or embryos as capacious recipient cytoplasm, the adequate and benefitlal preparation of multiple totipotent embryonic cells as donor nuclei, and also the fusion technique are very critical. Recent studies approaching to these critical points are introduced and discussed. Up to date, the overall efficiency of production of cloned embryos and offspring in livestock is estimated to be low. Further technical development of nuclear transplantation will enable large-scale production of cloned livestock and in near future the commercial cloning of animals will become a reality.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2006.10a
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• pp.54-55
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• 2006

• Journal of Embryo Transfer
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• v.25 no.4
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• pp.215-219
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• 2010

The objective of this study was to examine the reproductive characteristics of cloned miniature piglets produced from surrogate domestic pigs. Somatic cell nuclear transfer (SCNT) miniature pig embryos were transferred into domestic pigs. As controls, domestic pigs of the same breed with surrogates for SCNT embryos and miniature pigs of the same breed with the somatic cell donor were bred by artificial insemination and natural mating, respectively. Surrogate domestic pigs that farrowed cloned miniature piglets had a significantly longer gestation length (118.1 days) than conventionally bred domestic (115.4 days) and miniature (115.5 days) pigs. Furthermore, the birth weight of cloned miniature piglets produced from domestic pigs (743 g) was significantly greater than that of miniature piglets produced by natural breeding (623 g). Also, cloned miniature piglets had a significantly lower weaning rate (49.7%) than conventionally produced domestic (91.5%) and miniature (100%) piglets. No differences were observed between female and male cloned piglets in gestation length, litter size, birth weight, or weaning rate. Our results demonstrate that gestation length is extended in domestic pigs that are transferred with SCNT miniature pig embryos and that cloned miniature piglets have increased birth weight and high pre-weaning mortality.

• Journal of Embryo Transfer
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• v.10 no.1
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• pp.73-82
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• 1995

large scale production of cloned embryos requires the technology of multiple generation nuclear transplantation(NT) using NT embryos as the subsequent donor nuclei. The purposes of this study were producing the second generation cloned rabbit embryos, and also to determine the electrofusion rate and in vitro developmental potential comparatively in the cloned embryos of the first and second NT generation. The embryos of 16-cell stage were collected from the mated does by flushing oviducts with Dulbecco's phosphate buffered saline(D-PBS) containing 10% fetal calf serum(FCS) at 47 hours after hCG injection In the first generation NT, the nuclear donor embryos were synchronized in the phase of Gi /S transition of 32-cell stage. The first generation NT embryos which were developed to 8-cell were synchronized in Gi /S transition phase of the following 16-cell stage and used as donor nuclei for second generation Synchronization of the cell cycle of blastomeres was induced, first, using an inhibitor of microtuble polymerization, colcemid for 10 hours to arrest blastomeres in M phase, and secondly, using a DNA synthesis inhibitor, aphidicolin for 1.5 to 2 hours to arrest them in Gi /S transition boundary. The recipient cytoplasms were obtained by removing the nucleus and the first polar body from the oocytes collected at 14 hours after hCG injection. The separated donor blastomeres were injected into the enucleated recipient oocytes by micromanipulation and were electrofused by electrical stimulation of three pulses for 60 $\mu$sec at 1.25 kV /cm in 0.28 M rnannitol solution The fused oocytes were co-cultured with a monolayer of rabbit oviductal epithelial cells in M-199 solution containing 10% FCS for 120 hours at 39$^{\circ}C$ in a 5% $CO_2$ incubator. Following in vitro culture of the first and second generation cloned embryos to blastocyst stage, they were stained with Hoechst 33342 dye for counting the number of blastomeres by fluorescence microscopy. The results obtained were summarized as follows: 1. The electrofusion rate was found to be similar as 79.4 and 91.5% in the first and second generation NT rabbit embryos, respectively. 2. The in vitro developmental potential to blastocyst stage of the second generation NT embryos (23.3%) was found significantly(p<0.05) lower, compared with that of the first generation NT embryos (56.8%). 3. The mean blastomeres counts of embryos developed to blastosyst stage following in vitro culture for 120 hours and also their daily cell cycles during the culture period were decreased significantly (p<0.05) to 104.3 cells and 1.33 cylces in the second NT generation, compoared with 210.4 cells and 1.54 cycles in the first NT generation, respectively.

• Reproductive and Developmental Biology
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• v.31 no.2
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• pp.103-108
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• 2007

This study was carried out to investigate the effects of cryoprotectants, warming solution and removal of lipid on open pulled straw vitrification (OPS) method of porcine embryos produced by nuclear transfer (NT) of fetal fibroblasts. All solutions used during vitrification were prepared with holding medium consisting of 25 mM Hepes buffered TCM199 medium containing 20% fetal bovine serum (FBS) at $38.5^{\circ}C$. The blastocysts derived from NT with or without lipid were vitrified in each medium of different concentrations of dimethyl sulfoxide (DMSO) and ethylene glycol (EG). Also, blastocysts after cryopreservation were warmed into different concentrations of sucrose in warming solution. The optimal concentrations of cryoprotectants in vitrification solution were 10% DMSO + 10% EG in vitrification solution 1 (VS1) and 20% DMSO + 20% EG in vitrification solution 2 (VS2). The optimal concentrations of sucrose were 0.3 M sucrose in warming solution 1 (WS1) and 0.15 M sucrose in warming solution 2 (WS2). lipid removal from oocytes before NT enhanced the viability of NT embryos after vitrification. Our results show that use of the OPS method in conjunction with lipid removal provides effective cryopreservation of porcine nuclear transfer embryos.

• Journal of Animal Reproduction and Biotechnology
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• v.35 no.3
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• pp.258-264
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• 2020

This study investigated the effect of variation in the number of somatic-cell-cloned embryos and their developmental stage at transfer on pregnancy, as well as the influence of the estrus status of recipient pigs on in vivo development of cloned porcine embryos after embryo transfer. For somatic cell nuclear transfer (SCNT), fibroblast cells were obtained from a male porcine fetus. Recipient oocytes were collected from prepubertal gilts at a local abattoir and then cultured. After SCNT, reconstructed embryos of different numbers and developmental stages were transferred into recipient pigs. The developmental stage of the cloned embryos and the number of transferred embryos per surrogate showed no significant differences in terms of the resulting cloning efficiency. However, the pregnancy rate improved gradually as the number of transferred cloned embryos was increased from 100-150 or 151-200 to 201-300 per recipient. In pre-, peri-, and post-ovulation stages, pregnancy rates of 28.6%, 41.8%, and 67.6% and 16, 52, and 74 offspring were recorded, respectively. The number of cloned embryos and estrus status of the recipient pig at the time of transfer of the cloned embryo affect the efficiency of pig production; therefore, these variables should be particularly considered in order to increase the efficiency of somatic cell pig cloning.

• Reproductive and Developmental Biology
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• v.33 no.3
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• pp.157-162
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• 2009

This study was performed to comprehend the developmental characteristics of cloned embryos knocked out (KO) of $\alpha$-1,3-galactosyltransferase (GalT) gene. Immature oocytes were collected and cultured for 40 hrs (1-step) or 20hrs (with hormone) + 20hrs (without hormone) (2-step). The embryos transferred with miniature pig ear fibroblast cell were used as control. The reconstructed embryos were cultured in PZM-3 with 5% $CO_2$ in air at $38.5^{\circ}C$ for 6 days. To determine the quality of the blstocysts, TUNEL and quantitative realtime RT-PCR were performed. The embryos were transferred to a surrogate (Landrace) at an earlier stage of the estrus cycle. The maturation rate was significantly higher in 2-step method than that of 1-step (p<0.05). The blastocyst development of GalT KO embryos was significantly lower than that of normal cloned embryos (p<0.05). The total and apoptotic cell number of GalT KO blastocysts was not different statistically from control. The relative abundance of Bax-$\alpha$/Bcl-xl ratio was significantly higher in both cloned blastocysts than that of in vivo blastocysts (p<0.05). Taken together, it can be postulated that the lower developmental potential and higher expression of apoptosis related genes in GalT KO SCNT embryos might be a cause of a low efficiency of GalT KO cloned miniature pig production.