• Asian-Australasian Journal of Animal Sciences
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• 제8권6호
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• pp.641-645
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• 1995

This study was conducted to develop a method for production of nuclear transplant bovine embryos using in vitro-matured (IVM) oocytes and to examine the effect of different conditions of electrofusion on fusion rate and developmental capacity of donor nucleus transplanted to enucleated oocytes. Eight- to sixteen-cell embryos derived from oocytes matured and fertilized in vitro used as donor blastomeres and IVM oocytes were used as recipient oocytes. Oocytes were enucleated immediately after 23-24 h IVM and then reconstituted with a donor blastomere in two different micromanipulation media. Fusion rate and subsequent development of the reconstituted oocytes was compared under the different electric stimuli and recipient oocyte ages. Success rate of enucleation was significantly higher in TCM-199 medium containing FCS than in DPBS. The high fusion rate(75-94%) and development (6.4-14.8%) to morulae and blastocyst (M + B) were obtained from 0.6-0.75 kV/cm DC voltage, although total cleavage was not different among the electric pulses. Most optimal condition of electric stimulation for fusion and development was 1 DC voltage of 0.75 kV/cm, in which 80.5% of oocytes were fused, 80.0% and 31.7% of which was cleaved and developed to M + B, respectively. No M + B was obtained from 1.2 kV/cm DC voltage regardless of pulse frequency. Recipint oocyte age at electrofusion greatly affected the cleavage and subsequent development to M + B, showing high rate at 40-41 h oocyte maturation. These results suggest that a suitable condition of electrofusion for donor nuclei derived from IVF may be 1-2 DC pulses of 0.7 kV/cm for $70{\mu}sec$ and that processing of a transplanted nucleus in IVM oocytes may be affected by maturation age of recipient oocytes.

• Reproductive and Developmental Biology
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• 제35권3호
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• pp.313-317
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• 2011

This study was conducted to examine the effect of oocyte donor age and micromanipulation medium on the development of mouse cloned embryos receiving cumulus cells. Mouse oocytes were obtained from 6 to 11 week-old mice BDF1 female mice(experiment 1) and cumulus cells were used as donor cells. Micromanipulation procedures for nuclear transfer(NT) were performed in FHM, M2 or Hepes-buffered TCM199(TCM199) medium(experiment 2). After nuclear transfer, the reconstructed oocytes were activated by 10 mM $SrCl_2$ in Ca-free CZB medium in the presence of 5 II ${\mu}$g/ml cytochalasin B for 5 h and cultured in KSOM medium for 4 days. In experiment 1, the survival rate of oocytes after injection of cumulus cells were significantly(p<0.05) lower in oocytes from 6~7 week-old mice(53.3%) than in oocytes from 8~9(80.9%) and 10~11 week-old mice(77.1%). In experiment 2, the survival rate of oocytes after cell injection were significantly(p<0.05) higher in FHM and M2 medium(71.7% and 76.9%) than in TCM199 medium(51.2%). The activation rates of cloned embryos were not different among the micromanipulation media. However, the embryos developed to blastocyst stage were significantly(p<0.05) higher in FHM medium(13.9%) than in M2 and TCM199 medium(0.0% and 0.0%). In conclusion, the present study suggest that oocytes from above 8 week-old mice are superior to oocytes from 6~7 week-old mice as a source of recipient cytoplasm and FHM is superior to M2 and TCM199 as a micromanipulation medium for mouse somatic cell cloning.

• Clinical and Experimental Reproductive Medicine
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• 제24권2호
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• pp.167-177
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• 1997

Oocyte donation program developed to reach the pregnancy in those patients suffering from premature ovarian failure or surgery induced menopause, particularly in their reproductive age. With technical advances and popularity of ART (assisted reproductive technology), the indication of oocyte donation program extended to low responders, and even to naturally menopaused patients that has led them quite successfully to getting in pregnancy. The purpose of this study was to evaluate which one is involved in the decline of fertility between the oocyte and uterine factor. One hundred five cycles of oocyte donation program were performed in 84 patients from Jan., 1993 to Dec., 1996. Oocytes were donated from healthy, young, fertile anonymous donors or relatives or infertile patients with supernumerary oocytes. The study population was divided into 3 groups according to the age of recipients. Group 1 was less than 35 years old, Group 2 was between 35 to 39 years old, and Group 3 was more than 39 years old. The results were as follows: The mean age of oocyte donor was $31.5{\pm}3.3$ (range; 25-36). The mean concentration of basal serum FSH and peak serum estradiol were not different among groups. The mean number of oocytes retrieved from donors, embryos transferred to recipients, and fertilization rate were not different among groups. The clinical pregnancy rate was 37.3% in Group 1, 31.6% in Group 2, and 31.6% in Group 3, respectively. The spontaneous abortion rate was 16.0% in Group 1, 16.7% in Group 2, and 16.7 in Group 3, respectively. The multiple pregnancy rate was 20.0% in Group 1, 16.7% in Group 2, 16,7% in Group 3, respectively, The implantation rate was 11.3% in Group 1, 10.3% in Group 2 and 10.0% in Group 3, respectively. All of the pregnancy outcomes were not different statistically among groups. In conclusion, endometrial receptivity does not seem to be impaired as age increases with transfer of good quality embryos and adequate endometrial preparation.

• 한국수정란이식학회지
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• 제10권1호
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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.

• Clinical and Experimental Reproductive Medicine
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• 제47권2호
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• pp.94-100
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• 2020

The inverse correlation between maternal age and pregnancy rate represents a major challenge for reproductive endocrinology. The high embryo ploidy error rate in failed in vitro fertilization (IVF) cycles reflects genetic misfires accumulated by older oocytes over time. Despite the application of different follicular recruitment protocols during IVF, gonadotropin modifications are generally futile in addressing such damage. Even when additional oocytes are retrieved, quality is frequently poor. Older oocytes with serious cytoplasmic and/or chromosomal errors are often harvested from poorly perfused follicles, and ovarian vascularity and follicular oxygenation impact embryonic chromosomal competency. Because stimulation regimens exert their effects briefly and immediately before ovulation, gonadotropins alone are an ineffective antidote to long-term hypoxic pathology. In contrast, the tissue repair properties (and particularly the angiogenic effects) of platelet-rich plasma (PRP) are well known, with applications in other clinical contexts. Injection of conventional PRP and/or its components (e.g., isolated platelet-derived growth factors as a cell-free substrate) into ovarian tissue prior to IVF has been reported to improve reproductive outcomes. Any derivative neovascularity may modulate oocyte competence by increasing cellular oxygenation and/or lowering concentrations of intraovarian reactive oxygen species. We propose a mechanism to support intrastromal angiogenesis, improved follicular perfusion, and, crucially, embryo ploidy rescue. This last effect may be explained by mRNA upregulation coordinated by PRP-associated molecular signaling, as in other tissue systems. Additionally, we outline an intraovarian injection technique for platelet-derived growth factors and present this method to help minimize reliance on donor oocytes and conventional hormone replacement therapy.

• 한국수정란이식학회지
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• 제10권2호
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• pp.105-113
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• 1995

This study was conducted to examine the efficiency of enucleation and blastomere isolation from recipient oocytes and donor embryos, respectively and to determine the effect of oocyte age and electric voltage on the fusion rate and in vitro development of the fused oocytes in rabbit nuclear transplantation. Immature oocytes collected from ovarian follicles were matured in vivo for 12 h in TCM-199 containing FCS and hormones and in vivo matured oocytes were collected 17 to 18 h post-HCG. The fresh and frozen donor embryos of 8- to 16-cell stage were collected from the oviduct of superovulated does. The proportion of successfully enucleated oocytes was greatly lower in in vitro matured oocytes (42.3%) than that (62.7%) in in vivo matured oocytes The level of cytochalasin B for in vivo matured oocytes did not affect the efficiency of enuleation, but 7.5 $\mu$g /mL cytochalasin B for in vitro matured oocytes showed a high enucleation rate significantly. The isolation efficiency of a single blastomere nucleus did not differ between 8- and 16-cell stage embryos. The percentage of single blastomeres isolated from 16-cell stage fresh embryos after 0.5% pronase treatment was greatly higher at 16-min treatment (94.4%) than at 8-min(78. 1%) and the blastomeres(61.5%) isolated from frozen-thawed embryos after 16-min pronase were significantly fewer than those of fresh embryos. The age of recipient oocytes affected nuclear fusion rate. The reconstituted oocytes fused at 24-h age showed slightly higher fusion rate (77.8%) than those (65.0%)fused at 18-h age. The fusion rate of in vitro and in vivo matured oocytes inserted with fresh blastomere did not differ among electric voltages, but the cleavage rate and development to morula-blastocysts of in vitro matured oocytes was more higher under 0.6 kV/cm than under 0.8 to 1.2 kV/cm, while the cleavage rate and development of in vivo matured oocytes was higher under 0.8 to 1.0 kV/cm than under 1.2 kV/cm. The fusion and cleavage rate fol1owing insertion with frozen-thawed blastomere was not different between the in vitro and in vivo matured oocytes and was similar to those from fresh blastomere insertion.

• 한국수정란이식학회지
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• 제12권2호
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• pp.171-179
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• 1997

The objectives of the present study were improvements in the efficiency of developmental rates to morula and blastocyst stages to produce a large number of genetically identical nuclear transplant embryos. The oocytes collected from slaughterhouse ovaries were matured for 24 h and then enucleated and cultured to allow cytoplasmic maturation and gain activation competence. And then the donor embryos were treated for 12 h with 10 $\pi$g /ml nocodazole and 7.5 $\pi$g /ml cytochalasin B to synchronize the cell cycle stage at 26 h after the onset of culture. The blastomeres were transferred into the perivitelline space of the enucleated nocytes and blastomeres and oocytes were fused by electrofusion. The cloned embryos were then cultured in various conditions to allow further development. The age of the recipient(30 vs 40 h) had no significant effect on the fusion rates(82.4 vs 82.1%) and the developmental rates to morula /blastocyst(9.8 vs 11.0%). Effect of Nocodazole treatment on the donor cell cyle synchronization to improve the developmental rates of bovine nuclear transplant embryos was significantly higher than control group(21.4 vs 10.1%, p<0.05). Significant differences were in the percentage of fusion rates(72.9,77.1vs 61.9%) in three types of fusion medium(PBS(+), mannitol and sucrose, p<0.01). The developmental rates of bovine nuclear transplant embryos appeared to be highest in mSOF medium under 5% 0$_2$ condition, but no significant differences were found when compared with TCM199-BOEC and mSOF under two different oxygen ratio(5 and 20%).

• 한국수정란이식학회지
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• 제9권1호
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• pp.23-29
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• 1994

The long term goal of this research is to develop an efficient procedure for large scale production of genetically identical or cloned animals. To improve nuclear transpalntation efficiency in the rabbit, this study evaluated the age of nuclear recipient oocytes on the different steps of nuclear transplantation. The ovulated oocytes in different ages were collected from the superovulated does by flushing oviducts with Dulbecco's phosphate buffered saline(D-PBS) supplemented with 10% fetal calf serum(FCS) from 13 to 15, 17 to 20 and 23 to 26 hours after hCG injection. The denuded oocytes were used as nuclear recipient cytoplasm following enucleation by micromanipulation. The blastomeres separated from the 8-cell embryos were used as nuclear donor. The enucleated oocytes receiving a blastomere in the perivitteline space were fused in the 0.28 M mannitol solution at 1.5 kV/cm, 60 sec for three times. The fused oocytes were co-cultured with the monolayered rabbit oviductal epithelial cells in TGM-199 solution with 10% FCS for 72 hours at 37$^{\circ}C$ in a 5% $CO_2$ incubator. The cultured nuclear transplant embryos and in vivo developed embryos collected at 72 hours after hCG injection were stained with Hoechst 33342 dye. Their cell numbers were counted under a fluorescent microscope. The results obtained were summarized as follows ; 1. The aged oocytes(20 hrs. post hCG) showed significantly(P<0.05) higher fusionrates(70 ~ 90%) than the recently ovulated oocytes(30.8%) 2. The aged oocytes which were electrically activated and fused at 20 hours developed to blastocyst at significantly(P<0.05) high rate, while none of the recently ovulated oocytes developed to blastocyst. 3. Even though the aged oocytes at 23~26 hours showed higher fusion rate(85.7%), not only they were inadequate to manipulate but also their developmental potential to blastocyst was highly impaired. 4. The developmental potential in vitro of nuclear transplant embryos was significantly retarded than in vivo deveolped embryos.

• Asian-Australasian Journal of Animal Sciences
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• 제20권4호
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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.

• 한국가축번식학회지
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• 제21권3호
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• pp.293-302
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• 1997

Follicular oocytes of Grade I and II were collected from 2~6 mm ovarian follicles and matured in vitro (IVM) for 24 hrs in TCM-199 su, pp.emented with 35$\mu\textrm{g}$/ml FSH, 10$\mu\textrm{g}$/ml LH, and 1$\mu\textrm{g}$/ml estradiol-17$\beta$ at 39$^{\circ}C$ under 5% CO2 in air. They were fretilized in vitro (IVF) by epididymal spermatozoa capacitated with heparin for 12 hrs. The zygotes were then co-cultured in vitro with bovine oviducted epithelial cells (BOEC) for 7 to 9 days. The optimal time for IVM, the successful enucleation of IVM oocytes by micromanipulation at different oocyte ages after IVM, and the ideal culture system for IVM for effective IVF and in vitro development of IVM-IVF embryos was examined for in vitro production of nuclear recipient oocytes and nuclear donor embryos. To improve the efficiency of nuclear transplantation (NT) of IVF embryo into IVM follicular oocytes, this study evaluated the optimal electric condition and oocytes age for activation of IVM oocytes and in vitro development of NT embryos. In vitro development of NT embryos with preactivation or non-preactivation in enucleation oocytes, cell number of IVN-IVF embryos, and NT embryos wre also examined. The results obtained were as follows; 1. The most suitable enucleation time was at 24 hpm (83.3%) rather than that of 28 hpm(69.6%) and 32 hpm(50.0%). 2. There was no difference among the fusion rates of NT embryos at the voltages of 0.75, 1.0 and 1.5 kV/cm, but the in vitro development rates to morule and blastocyst were significantly (P<0.05) higher at the voltage of 0.75(12.5%) and 1.0kV/cm (12.6%) compared to 1.5kV/cm(0%). 3. No significant difference in activation rates were seen in NT embryos stimulated for 30, 60 and 120 $\mu$sec (71.7, 85.2 and 71.9%, respectively), but the in vitro development rates to morulae and blastocyst were significantly (P<0.05) higher in the oocytes stimulated for 30 $\mu$sec (11.6%) and 60 $\mu$sec(10.7%) than 120 $\mu$sec(0.0%). 4. The fusion rates (71.0 and 87.3%) and the in vitro development rates (9.1 and 12.7%) to morula and blastocyst were seen in the NT embryos stimulated at 28 and 32 hpm under the condition of 1.0 kV/ml, 60 $\mu$sec. However, at 24 hpm the fusion rates were 64.8% and the in vitro development to morula and blastocyst were not seen. 5. The fusion rates between the 8~12, 13~17 and 18~22-cell stage of IVM-IVF embryos were not significantly different. The in vitro development rates of the fused embryos to morula and blastocyst which were received from a blastomere of 8~12, 13~17 and 18~22-cell stages of IVM-IVF embryos were 14.9, 8.3 and 6.5%, respectively. 6. The in vitro development rate of the enucleated recipient oocytes with preactivation (24.2%) to morula and blastocyst was significantly (P<0.05) higher than that of non-preactivation (12.8%). 7. The cell numbers of NT blastocyst and IVM-IVF blastocyst cultured during 7~9 days were 63$\pm$11 and 119$\pm$23, and then their the mean cell cycle number were 5.98 and 6.89, respectively.