• Asian-Australasian Journal of Animal Sciences
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• v.20 no.4
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• pp.487-495
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• 2007

The objectives of the present study were to initiate cloning of Korean native goat by somatic cell nuclear transfer (NT) and to examine whether unovulated (follicular) oocytes can support the same developmental ability of NT embryos as ovulated (oviductal) oocytes after hCG injection in stimulated cycles of the goat. The in vivo-matured and immature oocytes were collected from the oviducts and follicles of superovulated does, respectively, and the immature oocytes were maturated in vitro. Ear skin fibroblasts derived from a 3-yr-old female Korean native goat were used as the donors of nuclei or karyoplasts. Following fusion, activation and in vitro culture to a 2- to 4-cell stage, 49 in vitro-derived and 105 in vivo-derived embryos were transferred to 6 and 17 recipient does, respectively. One doe and three does of the respective groups were identified as pregnant by ultrasonography on day 30 after embryo transfer. However, only one doe, which had received in vivo-derived embryos, delivered a normal female kid of 1.9 kg on d 149. The cloned kid gained more weight than her age-matched females as much as 87% during the first 4 mo after birth (17.7 vs. $9.4{\pm}0.8$ kg) and reached puberty at 6-mo age a few months earlier than normal female does. The telomere length of the kid, which was similar to that of the donor fibroblast at 2-mo age, decreased 8% between 2- and 7-mo ages. Moreover, at 7-mo age, she had 21% shorter telomere than her age-matched goats. To our knowledge, this is the first case in which a cloned animal born with a normal weight exhibited accelerated growth and development. The unusually rapid growth and development of the cloned goat may have resulted from SCNT-associated epigenetic reprogramming involving telomere shortening.

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.66-66
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• 2001

Since the first birth of pig derived from embryonic cells by nuclear transfer, many researches to produce cloned pig have been carried out. Recently, two reports about the birth of somatic cell cloned pigs using in vivo oocytes and also Betthauser et al. (2000) reported the birth of somatic cell cloned pigs using in vitro oocytes. So here we investigated the effect of activation method and culture medium on in vitro development of porcine nuclear transfer embryo using fetal fibroblast. Oocytes derived from slaughter house obtained ovaries were matured for 42 to 44 h in TCM 199. Matured oocytes were denuded using 0.1% hyaluronidase and then Oocytes with the first polar body were used for enucleation by aspirating the first polar body and adjacent cytoplasm in TCM 199 supplemented with 7.5 $\mu\textrm{g}$ cytochalasin B. Petal fibroblast cells were prepared from 35 days old fetus. To be used as donor cells, fetal fibroblast cells were serum starved for 3 to 5 days and then isolated into single co:1 by trypsinization. Nuclear transfer embryos were fused using 2 times 1.25㎸ for 30$mutextrm{s}$. Fused NT embryos were activated with calcium ionophore (CI) and 6-dimethyl-aminopurine (6-DMAP). Activated oocytes were cultured in NCSU 23 or BECM 3 for 6 days. There was no significant difference between chemical activation and no chemical activation for blastocyst development rate(11.6 vs. 14.8%). However, cell number was significantly higher when NT embryos were activated with CI and 6-DMAP (31.2 vs. 22.6). When NT embryos were cultured in NCSU 23 or BECM 3, blastocyst development rate was 16.4 and 13.2%, respectively, and cell number was 31.5 and 24.1, respectively. These results suggest that chemical activation after fusion and culture in NCSU 23 could increase cell number of porcine NT embryos.

• Proceedings of the KSAR Conference
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• 2003.06a
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• pp.28-28
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• 2003

Haploid parthenotes have been shown to be developmentally delayed compared with diploid parthenogenetic embryos in the mouse and pig. These developmental defects have been hypothesized to rusult from insufficient parthenogenetic activation, suboptimal in vitro culture conditions, or genemic imprinting. In the present study we compared the incidence of apoptosis and apoptosis related gene expression in pig haploid, diploid parthenotes and fertilized embryos. In vitro matured porcine oocytes were activated by electrical stimulation. Haploid activated oocytes with two polar bodies under stereomicroscopy were defined haploid parthenotes, oocytes with one polar body were defined as diploid parthenotes after 3h cycloheximide teatment. The morphological analysis of apoptosis in embryos was carried out using propidium iodide staining and terminal deoxynucleotidyl transferase mediated dUTP nick end labeling. The expression of Bcl-xL, Bak and P53 in haploid, diploid and in vivo fertilized blastocysts was determined using RT-PCR. Lower number of the haploid pig parthenotes developed to the morulae and blastocysts compared to the diploid parthnotes. Number of cells significantly lower in the haploid-derived blastocysts than diploid-derived it. Developmentally retarded haploid parthenotes exibited apoptosis at a significantly higher frequency than did diploid parthenotes and fertilized embryos. Level of Bcl-xL expression, diploid parthenotes similar to in vivo-derived it was higher than haploid parthenotes. However, Bak and P53 mRNA expression were not different among haploid, diploid, and fertilized embryos. This result suggested that parthenogenetic activation and parthenogenesis themselves do not cause apoptosis, but haploid increases the incidence of apoptosis in preimplantation embryos. Apoptosis may be due to decrease expression of Bcl-xL in haploid parthenotes developing in vitro.

• Clinical and Experimental Reproductive Medicine
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• v.26 no.3
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• pp.345-353
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• 1999

In the studies on the hatching mechanisms in mammals, many investigators focused on the embryonic intrinsic factor(s) in in vitro culture, but the uterine environment as the extrinsic factor(s) is thought to play an important role in hatching mechanism. Therefore, to evaluate the effect of uterine environment on the hatching event in vivo, the immature(GV) and ovulated(MII) oocytes, and the late 2-cell embryos of mouse were transferred to pseudopregnant foster mother's uterus during peri-implantation period. So it was verified whether there would happen hatching by only uterine environment independently on embryonic stage. The ultrastructural changes of the zona surface of transferred group were compared with those 01 in vivo and vitro group by SEM. 36 hrs after transfer, the immature and ovulated oocytes almost degenerated, and the late 2-cell embryos developed to various embryonic stages. However, the embryos which didn't develop to blastula stage did not hatch. The ultrastructural network of ZP in transferred group seemed to be smoothed uniformly, which was different from in vitro group. In conclusion, it is suggested that the uterine environment during peri-implantation period enhances the embryo hatching by provoking the structural change of ZP.

• Clinical and Experimental Reproductive Medicine
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• v.32 no.4
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• pp.331-336
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• 2005

Objective: To investigate the effects of female age on in vitro maturation and fertilization of immature oocytes from controlled ovarian hyperstimulation (COH) in human IVF-ET program. Method: A total of 96 immature oocytes (GV & metaphase I) obtained from 40 cycles of IVF-ET (29 patients). The mean age of female patients was $31.8{\pm}3.1years$. Ovulation was triggered by urinary or recombinant hCG. Immature oocytes were cultured with YS medium containing 30% of patients' human follicular fluids, LH (1 IU/mL), FSH (1 IU/mL) and EGF (10 ng/mL), and then matured oocytes were fertilized by ICSI. In vitro maturation and fertilization of immature oocytes were analyzed according to age of female (< 34 or ${\geq}34years$). Results: The maturation rate was similar between two groups (68% vs 64%). The fertilization rate of in?vitro-matured oocytes was higher in patients < 34 years old, but there was no statistical significance (64% vs 50%, p=0.347). The fertilization rate of in-vitro-matured oocytes was significantly lower compared with those of in-vivo-matured oocytes in both age groups (64% vs 79%, p=0.035, 50% vs 86%, p=0.007). Conclusion: In older female group, fertilization rate of in-vitro-matured oocytes seems to be decreased. Further investigations should be warranted to increase fertilization potential of in-vitro-matured oocytes.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.128-128
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• 2003

The aims of this study are 1) to test oocytes and embryos collected from in-vivo and in-vitro to achieving the valuable protocol by culturing, vitrifying and thawing of oocytes/embryos, and 2) to transfer them to recipient, and finally have resulted in pregnancies from recipient females after surgical or nonsurgical transfer. In vitro maturation and fertilization were performed according to the procedures of Funahashi et al. Fertilized oocytes were cultured in glucose-free NCSU 23 supplemented with 5 mM sodium pyruvate, 0.5 mM sodium lactate and 4 mg/ml bovine serum albumin for 2 days at 39$^{\circ}C$, and 10% fetal bovine serum was added to the culture medium thereafter. Embryos were treated with 7.5$\square$g/ml cytochalasin-B for 30 min, centrifuged at 13,000 ${\times}$ g for 13 min and then exposed sequentially to an ethylene glycol (EG) vitrification solution, aspirated into OPSs, and plunged/thawed into/from liquid nitrogen. In vivo embryos were surgically collected from three donors after Al. Forty-six embryos (18, 9 and 19 embryos, respectively) were washed 3 times in mPBS+10%FBS, followed treatments : cultured, centrifuged, vitrified, recovered and transferred to recipients as in vitro prepared embryos. Three recipients received surgically 34(control), 188 and 184 embryos (derived from abattoir), respectively. Another three recipients were received nonsurgically 150, 100 and 150 embryos, respectively. All recipient sows exhibited delayed returns to estrus. To our knowledge, these results suggest that required an improved techniques, more vigorous embryos preparation and cleaner uterous condition(use gilt).

• Journal of Embryo Transfer
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• v.10 no.1
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• pp.83-90
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• 1995

Production of calves after transfer of nuclear transplant embryos is the latest technology to be applied in commercial livestock breeding. The objective of this study was to establish an efficient procedure to produce offsprings from nuclear transplant embryos. The fusion rates (72.7% vs. 80.8%), cleavage rates (62.5% vs. 71.4%) and rates of development in vitro (12.0% vs. 15.2%) of nuclear transplant embryos were not significantly different between 30 and 40h maturation age of cytoplast. The in vivo and in vitro-derived embryos as nuclei donor were used in this system of bovine nuclear transplantation. Fusion rates of nuclear transplant embryos were not significantly different between in vivo and in vitro-derived embryos (73.0 and 79.2%, respectively). The percentage of embryos reaching the morulae or blastocysts were 21.8% for in vivo-derived embryos and 11.9% for in vitro-derived embryos (p<0.01). Pregnancy rates after embryo transfer of nuclear transplant embryos were not significantly different between in vivo and in vitro-derived embryos (45.9 and 40.5%, respectively). However, calving rates after embryo transfer of nuclear transplant embryos were significantly higher in the in vivo-derived embryos than in vitro (p<0.01). Further research for age of cytoplast and use of in vitro-derived embryos as nuclei donor is required in this system. In conclusion, these results clearly show that the use of in vitro-derived oocytes as recipient cytoplast can improve the nuclear transplant system for genetic progress in cattle.

• Reproductive and Developmental Biology
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• v.41 no.1
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• pp.17-23
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• 2017

The oocyte undergoes various events during in vitro maturation (IVM) and subsequence development. One of the events is production of reactive oxygen species (ROS) that is a normal process of cell metabolism. But imbalances between ROS production and antioxidant systems induce oxidative stress that negatively affect to mammalian reproductive process. In vitro environments, in vitro matured oocytes have many problems, such as excessive production of ROS and imperfect cytoplasmic maturation. Therefore, in vitro matured oocytes still have lower maturation rates and developmental competence than in vivo matured oocytes. In order to improve the IVM and in vitro culture (IVC) system, antioxidants, vitamins were added to the IVM, IVC medium. Antioxidant supplementation was effective in controlling the production of ROS and it continues to be explored as a potential strategy to overcome mammalian reproductive disorders. Based on these studies, we expect that the use of antioxidants in porcine oocytes could improved maturation and development rates.

• Clinical and Experimental Reproductive Medicine
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• v.38 no.4
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• pp.234-237
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• 2011

Objective: During stimulated IVF cycles, up to 15% of oocytes are recovered as immature. The purpose of this study was to investigate the trend of oocyte maturity in repeated ovarian stimulation for IVF. Methods: One hundred forty-eight patients were selected who underwent two consecutive IVF cycles using same stimulation protocol during 2008 to 2010. Ovarian stimulation was performed with FSH and human menopausal gonadotropin and flexible GnRH antagonist protocol in both cycles. Oocyte maturity was assessed according to presence of germinal vesicle (GV) and the first polar body. Immature oocyte was defined as GV stage or metaphase I oocyte (GV breakdown with no visible polar body) and cultured up to 48 hours. If matured, they were fertilized with ICSI. Results: Percentages of immature oocytes were 30.8% and 32.9% ($p$=0.466) and IVM rates of immature oocytes were 36.2% and 25.7% ($p$=0.077), respectively. A significant correlation was noted between percentage of immature oocytes in the two cycles (R=0.178, $p$=0.03). Women with >40% immaturity in both cycles (n=21) showed lower fertilization rate of $in$ $vivo$ matured oocytes (56.4% vs. 72.0%, $p$=0.005) and lower pregnancy rate (19.0% vs. 27.1%, $p$=0.454) after the second cycle when compared with women with <40% immaturity (n=70). In both groups, female age, number of total retrieved oocyte and embryos transferred were similar. Conclusion: In repeated ovarian stimulation cycles for IVF, the immature oocyte tended to be retrieved repetitively in consecutive IVF cycles.

• Proceedings of the KSAR Conference
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• 2001.10a
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• pp.44-46
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• 2001

In vitro produced (IVP) embryos produced by in vitro fertilization (IVF) often exhibit wide variations in developmental competence and viability, considerably more than are exhibited by embryos that develop in vivo. These anomalies in IVP embryos may be due to heterogeneity of oocyte quality, suboptimal culture conditions, disturbances in gene expression, or most likely a combination of these factors (Ho et al., 1994; Roth et al., 1994; McKiernan and Bavister, 1998; Hasler, 1998; Schramm and Bavister, 1999; Doherty et al., 2000; Hyttel et al., 2000; Niemann and Wrenzycki, 2000; Wrenzycki et al., 2001). In research studies or in clinical applications with domesticated animals, cats, non-human primates and humans, oocytes used for IVF are usually collected from a heterogeneous cohort of ovarian follicles that include oocytes which normally might not be ovulated and/or are deficient in developmental competence. (omitted)