• Proceedings of the KSAR Conference
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• 2003.06a
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• pp.57-57
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• 2003

수정란의 체외생산(IVP)기술은 동물생산기술의 적용과 생리학이나 세포생물학의 기본 연구에 새로운 biotchnologies의 발생에 있어 매우 중요하고 흥미로운 사건으로서 여러 종류의 포유류에 적용되고 있다. 이러한 기술은 3가지의 절차를 밟아야 하는데 In vitro maturation(IVM), In vitro fertilization(IVF) 및 In vitro development(culture)(IVD)가 그것이다. 그런데 돼지는 다태동물로서 난소의 난포가 성장할 때 난포간 meiotic competence가 난자에 의하여 진행되므로 난포내의 난포액에 의하여 난자의 발육이 좌우된다고 보고 있다 돼지WP에 사용되는 난자는 난포의 직경이 3~5mm에서 수집하고 cumulus-oocyte complexes(COCs)의 형태에 따라 선택하는 것이 보편화되어 있다. 실험목적은 돼지 난소의 antral follicles이 성장할 때 크기에 의하여 oocyte meiotic competence의 방출에 어떤 영향을 주는지를 난자의 체외성숙과 체외수정 및 체외발육 비율을 각각 조사하여 미성숙 난자의 체외배양 기술을 개선할 목적이었다. 수행내용은 도축돈 난소의 난포크기별 3mm<, 3~5mm, 5mm)로 나누어, 다시 말해서 preantral stage, antral stage, postantral stage으로 구분하여 채취한 COCs를 IVM, IVF, IVC 상태에서의 COCs 형태, cell cleaved rate, developmental rate 등을 조사하였다.

• Journal of Embryo Transfer
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• v.29 no.2
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• pp.133-139
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• 2014

$K^+$ channels are involved in the regulation of a variety of physiological functions, including proliferation, apoptosis and differentiation, in mammalian cells. Our previous study demonstrated that the blockage of $K^+$ channels inhibits mouse early embryonic development. This study was designed to identify the effect of $K^+$ channels during bovine embryonic development. $K^+$ channel blockers (tetraethylammonium (TEA), $BaCl_2$, quinine, ruthenium red and fluoxetine) were added to the culture medium during in vitro fertilization (IVF) for 6 h to first identify the short-term effect of these chemicals. Among $K^+$ channel blockers, fluoxetine, which is used as a selective serotonin reuptake inhibitor, significantly increased the blastocyst formation rate by approximately 6% when compared to control. During the in vitro maturation (IVM) of immature oocytes and the in vitro culture (IVC) of embryos, the oocytes and embryos were exposed to fluoxetine for either a short-term (6 h) or a long-term (24 h) to compare the embryonic development in response to exposure time. The 6 h exposure to fluoxetine during IVM did not affect the blastocyst formation rate, but the rate of blastocyst formation was reduced after the 24 h exposure. On the other hand, embryonic development increased approximately 10% in both groups of embryos exposed to fluoxetine for 6 and 24 h during IVC. Taken together, fluoxetine treatment during IVF and IVC, but not IVM, enhances bovine embryonic development. These results suggest that fluoxetine-modulated signals in oocytes and embryos could be an important factor towards enhancing bovine embryonic development.

• Clinical and Experimental Reproductive Medicine
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• v.47 no.2
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• pp.130-134
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• 2020

Objective: The aim of this study was to compare the effects of conventional insemination (in vitro fertilization [IVF]) and intracytoplasmic sperm injection (ICSI) on the fertilization, developmental competence, implantation potential, and clinical pregnancy rate of embryos derived from in vitro matured oocytes of patients with polycystic ovary syndrome (PCOS). Methods: A prospective study was carried out among 38 PCOS patients who had undergone In vitro maturation (IVM) treatment. In total, 828 immature oocytes were collected from 42 cycles and randomly assigned for insemination by IVF (416 oocytes) or ICSI (412 oocytes). After fertilization, the embryos were cultured until the blastocyst stage and single embryos were transferred after endometrial preparation and under ultrasound guidance. Results: No significant differences were found in the maturation rate (78.1% vs. 72.6% for IVF and ICSI insemination, respectively; p= 0.076), fertilization rate (59.4% vs. 66.9% for IVF and ICSI insemination, respectively; p= 0.063), or the formation of good-quality blastocysts (40.9% vs. 46.5% for IVF and ICSI insemination, respectively; p= 0.314). Implantation and clinical pregnancy also did not show significant differences. Conclusion: There was a comparable yield of in vitro matured oocytes derived from PCOS patients in terms of fertilization, blastocyst formation, implantation rate, and clinical pregnancy between IVF and ICSI insemination. These findings provide valuable insights for choosing assisted reproductive treatment in women with PCOS, as IVM offers promising outcomes and is less invasive and less costly.

• Korean Journal of Animal Reproduction
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• v.20 no.4
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• pp.459-465
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• 1997

The present study was undertaken to determine the effect of NT time on the rate of fusion a and suhseguent development In vitro and determine the optimal strength and duration of DC pulse for electrofusion of IVF donor embryo nuclei and IVM recipient oocytes. The recipient oocytes were enucleated 25 ~ 2Sh after IVM and further cultured for 18 ~ 20h prior to fusion for oocyte aging. IVF embryos as donor nuclei were C co cultured with BOEC for 16- to 32-cell stage development. The transfer time of donor bIas tomeres was 1~3h post-enucleation in early NT group and 1 ~ 18h post-enucleation in late NT group, respectively and fusion was performed 43~4Sh post-IVM. The fusion rate did not differ between the early NT and late NT group, but the rate of cleavage and 8- to 16-cell stage embryos in the late NT group was more higher than that in the early NT group. The fusion, cleavage and M+B development was high from O.7SkV /cm DC than from 1.0kV /cm DC voltage, resulting in 17.6% M+B from 0.75kV /cm DC voltage. No difference in fusion rate was among pulse durations, but 50 and 70 usec pulse duration showed slight high cleavage and M + B d development. The results indicate that the best NT time of IVF donor blastomeres into the enucleated oocytes was 42~44 post-IVM and the most suitable condition for electrofusion was a single 0.7SkV /cm DC voltage for SO~70$\mu$sec.

• Clinical and Experimental Reproductive Medicine
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• v.39 no.4
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• pp.166-171
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• 2012

Objective: We compared the assisted reproductive technology (ART) outcomes among infertile women with polycystic ovary syndrome (PCOS) treated with IVM, conventional IVF, GnRH agonist, and GnRH antagonist cycles. Methods: The prospective study included a total of 67 cycles in 61 infertile women with PCOS. The women with PCOS were randomized into three IVF protocols: IVM/IVF with FSH and hCG priming with immature oocyte retrieval 38 hours later (group A, 14 cycles), GnRH agonist long protocol (group B, 14 cycles), and GnRH antagonist multi-dose flexible protocol (group C, 39 cycles). IVF outcomes, such as clinical pregnancy rate (CPR), implantation rate (IR), miscarriage rate (MR), and live birth rate (LBR), were compared among the three groups. Results: Age, BMI, and basal FSH and LH levels did not differ among the three groups. The number of retrieved oocytes and 2 pronucleus embryos was significantly lower in group A compared with groups B and C. The CPR, IR, MR, and LBR per embryo transfer showed no differences among the three groups. There was no incidence of ovarian hyperstimulation syndrome in group A. Conclusion: The IR, MR, and LBR in the IVM cycles were comparable to those of the GnRH agonist and GnRH antagonist cycles. The IVM protocol, FSH and hCG priming with oocyte retrieval 38 hours later, is an effective ART option that is comparable with conventional IVF for infertile women with PCOS.

• Korean Journal of Animal Reproduction
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• v.21 no.3
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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.

• Journal of Embryo Transfer
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• v.31 no.3
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• pp.191-197
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• 2016

Historically, Korea old cattle had been consisted with various lines of coat color brindle, black and white-brown breeds or more. The two rare lines of black and white coat color are maintained for animal resources and preserved critically. The present study was carried out to evaluate potential usage of cysteamine supplementation during in vitro matration (IVM) and in vitro culture/production of embryo (IVP) by transvaginal ultrasound-guided follicle aspiration (Ovum Pick-Up: OPU) for the establishment of cryo-banking system. Immature slaughterhouse-derived cumulus-oocyte complexes (SL-COCs) were matured in IVM medium supplemented with 0, 0.1, 0.3 or 0.9 mM cysteamine, and then cultured in mSOF-BAS for 8 days after in vitro fertilization. The treatment of 0.1 mM cysteamine on SL-COCs showed higher rate of blastocyst, so OPU-derived COCs from rare breeds were matured in TCM media supplemented with or without 0.1 mM cysteamine, FSH and 5% FBS. The embryos were evaluated their developmental stages on day 8. During IVM, cysteamine treatment significantly increased the embryo production rate of slaughterhouse-derived COCs (19.6% vs. 30.5%). The presence of cysteamine during IVM of OPU-derived COCs from rare Korean cattle breeds (albino white and black line) also increased embryo production rates than those from SL-COCs (27.4% vs. 41.9% and 36.4%). With these results, cysteamine treatment during IVM is one of key factors IVP of blastocysts to establish banking system of endangered rare Koarean cattle with OPU derived transferable blastocysts.

• Journal of Embryo Transfer
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• v.21 no.3
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• pp.183-190
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• 2006

These studies were conducted to monitor developmental competence of follicular oocytes collected from the carcass of the high meat quality in Korean native cattle using each individual protocol of IVM, IVF and IVC. The follicular oocytes that were collected from the ovaries of the cow yielded 1, $1^+\;and\;1^{++}$ meat quality were matured, fertilized and cultured using each individual protocol of IVM, IVF and IVC. As results, the number of follicular oocytes collected from individual fundamentally-registered cows yielded 1, $1^+\;and\;1^{++}$ meat grade were 28.9, 28.8 and 29.6 per head, respectively. The rates of blastocyst formation after IVM, IVF and IVC were 27.2, 28.7 and 32.9% in the cows yielded 1, $1^+\;and\;1^{++}$ meat quality, respectively. The rate of blastocyst formation was 8.4 per head. The number of follicular oocytes collected from pedigree registered cows yielded 1, $1^+\;and\;1^{++}$ meat quality were 25.8, 27.1 and 27.0 per head, respectively. The rates of blastocyst formation were 23.0, 33.7 and 42.6% in the meat quality of 1, $1^+\;and\;1^{++}$ after in vitro-manipulation, respectively (p<0.05). The rate of blastocyst formation was 8.5 per head. In conclusion, these results suggest that in vitro embryo production system using individual culture system including IVM, IVF and IVC can make good use of the gene from the carcass of the high meat quality in Korean native cattle.

• Journal of Animal Reproduction and Biotechnology
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• v.34 no.3
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• pp.212-221
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• 2019

Sestrin-2 (SESN2) as a stress-metabolic protein is known for its anti-oxidative effects as a downstream factor of PERK pathways in mammalian cells. However, the expression patterns of SESN2 in conjunction with the UPR signaling against to ER stress on porcine oocyte maturation in vitro, have not been reported. Therefore, we confirmed the expression pattern of SESN2 protein, for which to examine the relationship between PERK signaling and SESN2 in porcine oocyte during IVM. We investigated the SESN2 expression patterns using Western blot analysis in denuded oocytes (DOs), cumulus cells (CCs), and cumulus-oocyte complexes (COCs) at 22 and 44 h of IVM. As expected, the SESN2 protein level significantly increased (p < 0.01) in porcine COCs during 44 h of IVM. We investigated the meiotic maturation after applying ER stress inhibitor in various concentration (50, 100 and 200 μM) of tauroursodeoxycholic acid (TUDCA). We confirmed significant increase (p < 0.05) of meiotic maturation rate in TUDCA 200 μM treated COCs for 44 h of IVM. Finally, we confirmed the protein level of SESN2 and meiotic maturation via regulating ER-stress by only tunicamycin (Tm), only TUDCA, and Tm + TUDCA treatment in porcine COCs. As a result, treatment of the TUDCA following Tm pre-treatment reduced SESN2 protein level in porcine COCs. In addition, SESN2 protein level significantly reduced in only TUDCA treated porcine COCs. Our results suggest that the SESN2 expression is related to the stress mediator response to ER stress through the PERK signaling pathways in porcine oocyte maturation.

• Asian-Australasian Journal of Animal Sciences
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• v.14 no.10
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• pp.1360-1366
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• 2001

The aims of this study were first to evaluate the effects of different levels (20, 40 and 100%) and sources (follicular size: large, >7 mm; medium, >5-7 mm; small, 3-5 mm) of porcine follicular fluid (pFF) on the in vitro maturation (IVM) of porcine oocytes, and the effects of fertilization treatments and different culture conditions on development of fertilized oocytes were also investigated. No differences in the maturation (63.6-76.6%) and cleavage (24.8-34.3%) rates were observed among the 20,40 and 100% pFF groups (p>0.05). The cleavage rates of oocytes cultured and fertilized in 40% and 100% pFF maturation media were significantly higher than those fertilized in m199-NBCS (51.0-61.2% vs. 12.8-31.8%. p<0.05), regardless of sources of the pFF. When oocytes were fertilized in m199-NBCS followed by culture in rabbit oviducts for 4 days, the cleavage rate in 40% pFF group was better than that in 100% pFF group (46.9% vs. 32.5%, p<0.05). Two oocytes recovered from the oviducts in the 40% pFF group developed to blastocysts after IVC. However, none developed to blastocysts when fertilized in the IVM medium after being transferred to rabbit oviducts. In conclusion, addition of pFF accompanied with gonadotropins (FSH, LH) in IVM medium enhanced maturation and cleavage rates of porcine oocytes. Direct addition of sperm suspension to IVM medium may be an alternative to simplify the fertilization procedures and to reduce the mechanical lesion during manipulation. Furthermore, rabbit oviducts provide a better environment for the in vitro fertilized oocyte developing to the morula and blastocyst stages.