• 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.

• 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.

• 한국가축번식학회지
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• 제16권1호
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• pp.7-14
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• 1992

These experiments were designed to investigate the effects of glycosaminoglycans(GAGs), which maybe produced by cumulus cells, on in vitro maturation process of bovine follicular oocytes. The rates of in vitro maturation of bovine oocytes were examined after incubating with the various concentrations of a hyaluronic acid, chondroitin sulfate, or heparin for 26 hours. The results obatained in these experiments were summarized as follows : 1. When the cumulus or removed oocytes treated with hyaluronic acid(200, 400, 800 and 1, 600$\mu\textrm{g}$/ml), the maturation rates of cumulus-removed oocytes were 48.6, 59.4, 68.4 and 61.3% respectively. Especially, at a concentration of 800$\mu\textrm{g}$/ml, the maturation rates(68.8%) of cumulus-removed oocytes were slightly lower than that(78.3%) of cumulus-enclosed oocytes. However, the treatment of hyaluronic acid showed significantly higher maturation rates compared to that(48.4%) of control group of cumulus-removed oocytes(p<0.05). Therefore, these results suggest thate hyaluronic acid had a beneficial effect on maturation of bovine follicular oocytes. 2. The maturation rate of cumulus-free oocytes treated with chondroitin sulfate(200, 400, 800 and 1, 600$\mu\textrm{g}$/ml) was 52.3, 56.1, 55.9 and 52.2% respectively. These rates were not different from those(52.9%) of control group. However, these rates in cumulus-free oocytes were significantly lower than that(79.0%) in cumulus-enclosed oocytes(p<0.01). Therefore, these results suggest that chondroitin sulfate do not affect on the maturation of oocytes. 3. In cumulus-free oocytes cultured with different heparin concentration, the maturation rates were ranged from 48.5 to 52.1%, showing no differencies from that(50.7%) of control group. However, these rates were significantly lower than 80.0% in cumulus-enlosed oocytes(p<0.01). 4. The nuclear maturaton of oocytes was increased by treatment of each of three glycosaminoglycans(hyaluronic acid, chondroitin sulfate, and heparin). In addition, the treatment of mixed together showed the significant additive effect. Hyaluronic acid was more effective than chondroitin sulfate and heparin were.

• 한국수정란이식학회지
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• 제24권2호
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• pp.121-125
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• 2009

In the present study, effects of concentration of cryoprotectant solutions on the nuclear maturation of vitrifiedthawed bovine oocytes were examined. Also, the developmental capacity of vitrified-thawed immature oocytes following ICSI was investigated. Oocytes were cultured in TCM-199 medium supplemented with 5% FBS at $38^{\circ}$C in 5% $CO_2$ and air. The in vitro maturation rate of vitrified oocytes was 24.5 ${\pm}$ 4.2%. The in vitro maturation rate of vitrified oocytes was lower than that of the control (72.0 ${\pm}$ 3.5%, p<0.05). The in vitro maturation rate of vitrified${\sim}$thawed oocytes incubated in TCM-199 medium supplemented with 1.0${\sim}$5.0 ug CB were 26.7 ${\pm}$ 3.2%, 35.7 ${\pm}$ 3.2%, 54.0 ${\pm}$ 3.0%, 42.5 ${\pm}$ 3.6%, respectively. The in vitro maturation rate (57.0 ${\pm}$ 3.0%) of the vitrified-thawed oocytes treated with 3.0 ${\mu}$g CB for 20 min was the highest of all vitrification groups, although the maturation rate were significantly (p<0.05) lower than those of fresh oocytes. The in vitro maturation rates of the vitrified-thawed (with EDS and EDT) oocytes were 53.8 ${\pm}$ 3.4%, 51.1 ${\pm}$ 3.5%, respectively. This results were lower than the control group (72.0 ${\pm}$ 3.0%). The in vitro developmental rates of the vitrified-thawed oocytes following ICSI were 28.6 ${\pm}$ 4.5%, 25.6 ${\pm}$ 4.3%, respectively. This results were lower than the control group (40.0 ${\pm}$ 4.0%).

• 한국가축번식학회지
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• 제23권3호
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• pp.239-245
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• 1999

본 연구는 돼지난자의 체외성숙시 난구세포와 cat-alase의 영향을 검토하고저 수행되었다. 체외성숙율은 catalase의 첨가유무에 관계없이 유의적인 차이는 인정되지 않았으나, 난구세포가 부착된 난자가 제거된 난자에 비해 유의적으로 높은 성숙율을 나타냈다 (P＜0.05). 또한 48시간 (57%) 동안 난구세포가 부착된 경우 단지 처음 24시간 (42%) 동안만 난구세포가 부착된 경우에 비하여 유의적으로 높은 성숙율을 나타냈다 (P＜0.05). 한편, 난구세포가 부착 또는 제거된 난자의 성숙시 처음 24시간 동안보다는 후반기 24시간 동안 catalase를 첨가한 경우 유의적으로 높은 성숙율을 나타냈으며 (P＜0.05), 난구세포를 제거한 경우 성숙배양 24시간에서 M-II기로 성숙된 난자가 관찰되었다. 그러나 난구세포가 부착된 난자를 72시간 성숙배양 했을 때, catalase 무첨가 (65%)보다는 첨가시(79%) 유의적으로 높은 성숙율을 나타냈다. 본 연구의 결과로부터 난구세포는 돼지난자의 체외성숙시 필수적인 것으로 인정되며, catalase는 첨가시기에 따라 난자의 성숙에 효과적으로 작용하였으며, 체외성숙시 난자의 노화를 방지할 수 가능성이 있는 것으로 추측된다.

• 한국수정란이식학회지
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• 제5권1호
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• pp.21-27
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• 1990

The technique for maturation of follicular oocyte has been devised to provide such a low cost and in ptentifut number supply of bovine embryo. Some of problems concerning production of bovine embtyo in vitro were discussed in this paper. Bovine follicular oocytes cultured in vitro achieved normal fertilization but cleavage rates to blastocyst were low compared to the oocyte matured in vivo. It has been concluded that a deficient cytoplasmic maturation occurs in the oocytes matured in vitro. These results indicate that the studies for maturation of bovine follicular oocytes in vitro need improvement of culture conditions and to define the characteristics that might be indicative of healthy oocyte.

• 한국가축번식학회지
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• 제21권2호
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• pp.111-116
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• 1997

We determined the effects of follicular fluid fractions in the maturation medium on bovine oocyte maturation, fertilization and subsequent development, as well as on number of cells in blastocysts following culture. Follicular fluid and oocytes from bovine follicles less than 5 mm in diameter were collected from the ovaries of slaughtered cows. Follicular fluid was separated into different molecular weight fractions by untrafiltration through a membrane using a centrifuge at 500$\times$g, for 2h. For the maturation medium, follicular fluid fractions (30%, v/v), whole fluid (30%) or PVP(3mg/ml) were added to TCM 199(0.1$\mu\textrm{g}$/ml estradiol-17$\beta$, 100IU hCG). After maturation for 24h, oocytes were fertilized in vitro with bull frozen-thawed spermatozoa and cultured on a monolayer of granulosa cells for 9 days after fertilization. There were no differences in maturation rates or fertilization rates among any maturation conditions. The rates of development to >2-cell stage of the oocytes were significantly decreased when fraction of follicular fluid below 10,000 MW were added into maturation medium, compared with control and fraction above 10,000 MW(26.0% vs 40.8% to 64.0%, respectveily. p<0.01). Likewise, the rates of development to blastocysts of fertilized oocytes were significantly decreased in maturation medium containing fraction of follicular fluid (<10,000 MW). The average cell number of blastocysts derived from oocytes that matured in the fraction(>10,000 MW) of follicular fluid was 154.7$\pm$13.7. These embryos contained more cells than those matured in whole follicular fluid, or the fraction(<10, 000 MW) of follicular fluid or control(107.0$\pm$8.4, 91.8$\pm$11.8 and 95.8$\pm$6.2, respectively). In conclusion, we found that fractions of follicular fluid contained factors stimulating or inhibiting oocyte cytoplasmic matruation. These suggest that a factor(s) inducing cytoplasmic maturation of oocytes may exist in >10,000 MW fraction of follicular fluid.

• Reproductive and Developmental Biology
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• 제35권4호
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• pp.469-473
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• 2011

These study was carried out to investigate the effects of the supplementation with sodium nitroprusside (SN) and nitric oxide (NO) of canine oocytes on IVM rates. Oocytes were incubated in TCM-199 supplement with at 0.03~0.10 mM SN and 0.3~1.0 mM NO for 48 hrs. Oocytes were transferred to 50 ul drops of maturation medium covered mineral oil and cultured in a $CO_2$ incubator (5% $CO_2$, 95% air, $38^{\circ}C$). The in vitro maturation rate of oocytes cultured for 48 hrs in TCM-199 medium supplement with 0.03, 0.05, 0.07, 0.10 mM SN were $25.9{\pm}3.5%$, $36.4{\pm}3.2%$, $33.3{\pm}3.5%$, $28.8{\m}3.2%$, respectively. The in vitro maturation rate of oocytes cultured for 48 hrs in TCM-199 medium supplement with 0.03~0.07 mM SN were significantly increased compare to the control ($26.0{\pm}2.2%$). The in vitro maturation rates of oocytes cultured for 48 hrs in TCM-199 medium supplement with 0.3, 0.5, 0.7, 1.0 mM NO were $28.0{\pm}4.2%$, $36.5{\pm}3.6%$, $30.0{\pm}3.8%$, $19.2{\pm}3.5%$, respectively. The in vitro maturation rate of oocytes in TCM-199 medium supplemented with 0.3 and 0.5 mM NO were significantly increased compare to the control ($26.0{\pm}2.2%$). The in vitro maturation rates of oocytes cultured for 12~48 hrs in TCM-199 medium supplement with 0.05 mM SN were $26.0{\pm}3.2%$, $28.0{\pm}3.4%$, $38.0{\pm}3.2%$, respectively. The in vitro maturation rate of oocytes cultured for 12~48 hrs in TCM-199 medium supplement with 0.5 mM NO were $22.0{\pm}3.0%$, $30.0{\pm}3.8%$, $36.0{\pm}4.2%$, respectively. These result was significantly increased compare to the control.

• 한국가축번식학회지
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• 제20권3호
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• pp.289-297
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• 1996

This study was conducted to investigate the effects of maturaton medium and porcine follicular fluid on in vitro maturation of porcine follicular oocytes. The results obtained are as follows; 1. When the oocytes were cultured for 42 hours, the maturation rate was significantly (P<0.05) higher in TCM-HEPES(75.5%) than Ham's F-10(60.9%) and mKRB(60.7%) medium. The optimal medium for the maturation of porcine oocytes in vitro was the TCM-HEPES medium. 2. When the oocytes were cultured for 42 hours in TCM-HEPES medium with 10, 20 and 30% of porcine follicular fluid(pFF), the maturation rates of porcine oocytes were 69.1, 65.6 and 63.3%, respectively. The maturation rate(51.4%) of oocytes cultured without pFF was significantly(P<0.05) lower than that of oocytes cultured with pFF. 3. The maturation rates of porcine oocytes cultured for 42 hours in TCM-HEPEs medium with 3 different porcine follicular fluid treatments were 68.6%(centrifused), 72.3%(filtered) and 73.1%(heat treated), respectively. The maturation rate(49.4%) of control group without pFF treatment was significantly(P<0.05) lower than that of oocytes cultured with pFF treatment.

• 한국임상수의학회지
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• 제7권1호
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• pp.419-427
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• 1990

Immature bovine oocytes were cultured to investigate whether the addition of FCS(10% or 20% ), CS (10%or 20% ) or BSA(5mg/ml) to culture medium with or without HEPES and co-culture with granulosa cells affect the frequency of in vitro maturation of extrafollicular bovine oocytes. After culture, the maturation rates were examined by the presence of 1st polar body and nuclear configuration. The maturation rate when FCS and CS as protein supplement were added to culture medium with or without HEPES was significantly higher than when BSA was added, and the maturation rate of extrafollicular bovine oocytes co-cultured with granulosa cells was higher than that cultured without granulosa cells, but there was no significant difference. FCS and CS were shown to be superior protein supplement when compared to BSA, and serum concentration, HEPES and co-culture with granulosa cells did not affect the in vitro-maturation of extrafollicular bovine oocytes.