• 한국수정란이식학회지
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• 제23권2호
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• pp.87-91
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• 2008

This study was carried out to investigate the effects of the supplementation of glutamine, glucosamine and glutathione on the porcine oocytes on IVM rates. Cocs were incubated in NCSU-23 supplemented with at $2.0{\sim}10.0\;mM$ glucosamine, $0.5{\sim}4.0\;mM$ glutamine and $0.1{\sim}1.0\;mM$ glutathione for 48 hrs. Oocytes were transferred to 50 ul drops of maturation medium covered with mineral oil and cultured in a $CO_2$ incubator ($38^{\circ}C$, 5% $CO_2$, 95% air). The IVM rates of oocytes cultured in NCSU-23 supplemented with 0.5, 1.0, 2.0 and 4.0 mM glutamine for 48 hrs were $46.0{\pm}4.5%$, $52.0{\pm}4.8%$, $50.0{\pm}4.2%$ and $44.0{\pm}4.5%$, respectively. The IVM rates of oocytes cultured in NCSU-23 supplement with 2.0, 5.0, 7.0, 10.0 mM glucosamine for 48 hrs were $44.0{\pm}4.5%$, $42.0{\pm}4.5%$, $38.0{\pm}4.6%$ and $24.0{\pm}4.8%$, respectively. The IVM rates of oocytes cultured in NCSU-23 supplemented with glucosamine were no significantly increased compare to the control ($42.5{\pm}4.0%$). The IVM rate of oocytes cultured in NCSU-23 supplemented with 3.0, 5.0, 7.0, 10.0 mM glutathione for 48 hrs were $40.0{\pm}3.2%$, $54.0{\pm}4.2%$, $48.0{\pm}4.5%$, $44.0{\pm}4.8%$, respectively. The IVM rate of oocytes cultured in NCSU-23 supplemented with glutamine and glutathione were significantly increased co~pared to those control ($42.5{\pm}4.0%$). Glucosamine did not affect the IVM rates of oocytes. IVM rates of oocytes cultured in NCSU-23 medium for 48 hrs were significantly increased compared to the cultured for 40 hrs.

• 한국가축번식학회지
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• 제27권1호
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• pp.35-44
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• 2003

The present study was performed to investigate the first polar body(PB) extrusion during in-vitro maturation(IVM) and to examine the effect of different maturation time on the embryo development of Korean Native Cows(KNC) with regard to blastocyst(BL) cell numbers and pregnancy rates. PB extrusion did not take place for the first 12 hours(hr) of IVM, and most of KNC oocytes extruded PB from 14 to 20 hr after the onset of maturation. There was no significant difference in cleavage and 8-cell stage rates among the treatment groups, but BL and BL/8-cell rates were significantly higher(P<0.05) in 18 hr maturation group(31.0$\pm$5.7 and 82.0$\pm$5.1%) than 22 and 24 hr maturation group. The proportion of BL formed on day 7 and 8 was significantly higher(P<0.05) in 18 hr maturation group(85%) than in 24 hr maturation group(55%). There was a significant difference(P<0.05) in inner cell mass, trophectoderm and total cell number between day 7 BL produced by in-vivo and IVM 18 hr and day 8 BL produced by IVM 18 hr and 24 hr. Pregnancy rates are also significantly higher(P<0.05) in in-vivo(56.3%) and IVM 18 hr day 7(50.0%) group than day 8 treatment groups(18 hr: 16.7%, 24 hr: 10.5%). These results suggest that KNC oocytes achieve developmental competency within 20 hr of IVM, and "short" IVM (18 hr) is more effective than "long" IVM(24 hr) in embryo development rates, BL cell numbers and pregnancy rates.

• Clinical and Experimental Reproductive Medicine
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• 제46권4호
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• pp.166-172
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• 2019

Objective: In vitro maturation (IVM) of immature oocytes can be useful for some infertile patients. In IVM programs, the rates of embryo formation and pregnancy are low. Therefore, it is essential to recognize the main factors involved in regulating oocyte maturation in vitro. The purpose of this study was to investigate the effects of growth differentiation factor 9 (GDF9) and cumulus cell (CC) supplementation in IVM medium on the rates of embryo formation and viability of human blastocysts. Methods: A total of 80 germinal vesicle oocytes from stimulated cycles underwent an IVM program. The oocytes were divided into four groups, where group I consisted of IVM media only and served as the control, group II consisted of IVM+CCs, group III consisted of IVM+GDF9 (200 ng/mL), and group IV consisted of IVM+CCs+GDF9 (200 ng/mL). Intracytoplasmic sperm injection was performed on the IVM oocytes, and the cleavage embryos that were generated were vitrified. Following thawing, the embryos were cultured for 3 additional days, and the viability rates of the developed blastocysts were determined. Results: The maturation rate of the oocytes did not differ significantly across the four groups. The fertilization rate in group II was significantly higher than that in the control group (76.5% vs. 46.2%). Embryo formation was significantly more frequent in all experimental groups than in the control group, while blastocyst formation did not show significant differences in the three experimental groups compared to the control. The mean viability rates in groups II, III, and IV were 58.16%, 55.91%, and 55.95%, respectively, versus 37.78% in the control group (p< 0.05). Conclusion: Supplementation of IVM culture media with GDF9 and CCs enhanced the fertilization, embryo formation, and viability rates of blastocysts generated from vitrified cleavage embryos.

• Clinical and Experimental Reproductive Medicine
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• 제44권2호
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• pp.79-84
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• 2017

Objective: Optimizing in vitro maturation (IVM) media to achieve better outcomes has been a matter of interest in recent years. The aim of this prospective clinical trial was to investigate the effects of different media on the IVM outcomes of immature oocytes at the germinal vesicle (GV) stage. Methods: A total of 400 immature oocytes at the GV stage with normal morphology were retrieved from 320 infertile women aged $31{\pm}4.63years$ during stimulated intracytoplasmic sperm injection (ICSI) cycles. They were divided into groups of homemade IVM medium (I, n = 100), cleavage medium (II, n = 100), blastocyst medium (III, n = 100), and Sage IVM medium (IV, n = 100) and cultured for 24 to 48 hours at $37^{\circ}C$. ICSI was performed, and the rates of fertilization and embryo formation were compared across the four groups. Results: In the 400 retrieved GV oocytes, the total maturation rates showed significant differences in groups I to IV (55%, 53%, 78%, and 68%, respectively, p<0.001). However, there were no significant differences in the fertilization, embryo formation, or arrest rates of metaphase II oocytes across these groups. In all groups, GV maturation was mostly completed after 24 hours, with fewer oocytes requiring 48 hours to mature (p<0.01). Moreover, the rate of high-quality embryos was higher in group IV than in the other groups (p=0.01). Conclusion: The quality of the IVM medium was found to affect clinical IVM outcomes. Additionally, blastocyst medium may be a good choice in IVM/ICSI cycles as an alternative IVM medium.

• 한국수정란이식학회지
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• 제22권4호
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• pp.219-222
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• 2007

These study was carried out to investigate the effects of the collection time, culture time and activation of canine oocytes on in vitro maturation rates. The activated oocytes were cultured in 10% FCS+TCM-199 media containing hormonal supplements (10 IU/ml HCG, 10 IU/ml PMSG, 10 ug/ml gonadotropin) at 5% $CO_2$, 95% air, $38^{\circ}C$. 1. IVM rate of in vitro cultured cumulus-attached oocytes recovered from ovaries that collected at follicular and luteal stages of the reproductive cycles were 11.4% and 5.7%, respectively. IVM rate of oocytes recovered from ovaries that collected at follicular stages of the reproductive cycles was significantly higher than that of luteal stage (p<0.05). 2. When IVM was carried out at different periods of 40, 48, and 70 hrs, the IVM rates of oocytes matured in vitro were 2.9%, 8.6%, 5.7%, respectively. These results indicate that the IVM time between $48{\sim}70$ hrs gives the highest maturation rate for the oocytes matured at the different stages. 3. IVM rate of oocytes matured in vitro for 10 hrs after single and combined activation treatment by ET, IP and CH and Ca+DMAP, CH+DMAP, ET+CH were $11.5{\pm}1.2%,\;10.8{\pm}1.0%,\;9.6{\pm}1.2%\;and\;12.4{\pm}1.5%,\;11.8{\pm}1.5%,\;11.2{\pm}1.4%$ respectively. This was higher than that in both single and combined stimulated groups compared to control group ($6.2{\sim}7.2%$).

• 한국수정란이식학회지
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• 제23권2호
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• pp.93-100
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• 2008

This study was conducted to establish an in vitro maturation (IVM) system by selection of efficient macromolecule in the porcine in vitro production (IVP) technology. To choose the efficient macromolecules in the development of porcine embryos, the effects of 3 kinds of macromolecules (porcine serum; PS, porcine follicular fluid; pFF, and polyvinyl alcohol; PVA) supplemented in IVM media on the maturation, cleavage, and development rates to blastocyst of parthenogenetic activation (PA) and in vitro fertilization (IVF) embryos were examined. The maturation rates of porcine oocytes in media supplemented with PS were significantly higher than those with pFF and PVA (92.4% vs. 85.4%, 77.1%; p<0.05). In the cleavage and development to blastocyst rates, supplement with PS or pFF in the IVM media was more effective than PA. However, there were no significant differences in cleavage and development to blastocyst between PS and pFF group. From the results of this study, it was demonstrated that PS was optimal macromolecule in the porcine IVM media.

• 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.

• 한국가축번식학회지
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• 제24권3호
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• pp.311-317
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• 2000

본 연구는 체외에서 한우 난포란의 성숙과 수정후 배의 체외발생에 있어서 온도충격의 영향이 처리온도 및 처리시간에 미치는 효과를 검토하였다. 1. 체외에서 생산된 4~8 세포기 배의 배반포로의 배발달율에 있어서 온도충격의 적정온도는 41$^{\circ}C$, 처리시간은 30초였다. 2. 체외성숙 후의 온도충격이 체외성숙 전의 온도충격보다 수정란의 분할율을 유의하게 상승시켰다(p<0.05). 3. 체외성숙 후와 배양 5일째 배에 각각 온도처리를 하였을 경우 비처리군보다 배반포의 배발달율이 향상되었다.

• 한국수정란이식학회지
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• 제31권3호
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• pp.153-159
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• 2016

(-)-Epicatechin gallate (ECG) is a polyphenol compound of green tea exhibiting biological activities, such as antioxidant and anticancer effects. To examine the effect of ECG on porcine oocytes during in vitro maturation (IVM), oocytes were treated with 0-, 5-, 15-, and $25{\mu}M$ ECG. After maturation, we investigated nuclear maturation, intracellular glutathione (GSH) and reactive oxygen species (ROS) levels and subsequent embryonic development after parthenogenetic activation (PA) and in vitro fertilization (IVF). After 42 hours of IVM, the $5{\mu}M$ group exhibited significantly increased (p< 0.05) nuclear maturation (89.8%) compared with the control group (86.1%). However, the $25{\mu}M$ group observed significantly decreased (p< 0.05) nuclear maturation (83.5%). In intracellular maturation assessment the 5-, 15-, and $25{\mu}M$ groups had significantly increased (p< 0.05) GSH levels and decreased ROS levels compared with the controls. The 5- and $15{\mu}M$ group showed significantly increased (p< 0.05) embryo formation rates and total cell number of blastocysts after PA (18% and 68.9, 15% and 85.1 vs. 12% and 59.5, respectively) compared with controls. Although the $25{\mu}M$ group observed significantly lower blastocyst formation rates after PA (27.6% vs. 23.2%) than control group, the $5{\mu}M$ group showed significantly increased blastocyst formation rates after PA (37.2% vs. 23.2%) compared to the control group. Furthermore, the $5{\mu}M$ group measured significantly increased blastocyst formation rates (20.7% vs. 8.6%) and total cell number after IVF ($88.3{\pm}1.5$ vs. $58.0{\pm}3.6$) compared to the control group. The treatment of $5{\mu}M$ ECG during IVM affectively improved the porcine embryonic developmental competence by regulating intracellular oxidative stress during IVM.

• 한국수정란이식학회지
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• 제22권4호
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• pp.223-227
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• 2007

This study was carried out to investigate the effect of morphology of oocytes, kinds of media, cysteine and myo-inositol supplementation on IVM rate of porcine oocytes. Cumulus- enclosed oocytes were incubated in maturation NCSU-23 and TCM-199 medium with supplementation with 3, 5, 10, 20 mM myo-inositol and 0.05, 0.1, 0.5, 1.0 mM cysteine. 1. When classified by morphology, excellent, good and fair of cumulus-enclosed oocytes were incubated for 48 hrs and the IVM rate were $14.2{\pm}3.7%{\sim}58.7{\pm}4.0%$, respectively. The rate were greater in oocytes with excellent cumulus cells than those without cumulus cells. 2. The IVM rate of oocytes cultured in TCM-199 and NCSU- 23 medium supplementation or non-supplementation with 1.0 mM myo-inositol were $7.5{\pm}4.5%,\;45.0{\pm}4.8%\;and\;4.4%,\;42.5{\pm}4.2%,\;18.0{\pm}5.2%$, respectively. Supplementation with myo-inositol significantly increased the IVM rate of oocytes. 3. The IVM rate of oocytes cultured in NCSU-23 medium supplementation of 3, 5, 10, 20 mM myo-inositol for 48 hrs were $47.5{\pm}4.5%,\;57.5{\pm}4.2%,\;62.5{\pm}4.9%,\;50.0{\pm}5.2%$, respectively. The IVM rate of oocytes in NCSU-23 medium supplemented with 10 mM myo-inositol were significantly increased compared to control ($42.5{\pm}4.0%$). 4. The IVM rate of oocytes cultured for 48 hrs in NCSU-23 media supplement with 0.3, 0.5, 1.0, 2.0 mM myo-inositol were $50.0{\pm}4.5%,\;62.5{\pm}4.2%,\;52.5{\pm}4.9%,\;45.0{\pm}4.2%$, respectively. The IVM rate of oocytes in NCSU-23 medium supplemented with 10 mM cysteine were significantly increased compared to control ($42.5{\pm}4.0%$).