• 한국가축번식학회지
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• 제17권2호
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• pp.103-111
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• 1993

These experiments were undertaken to establish the optimal culture systems for in vitro maturation, fertilization and subsequently embryonic development of porcine immature follicular oocytes isolated from the ovary of slaughtered pigs. Porcine ovaries were brought to the laboratory from local slaughter house within 1 hour after slaughtering and cumulus oocytes complexes were recovered from antral follicles (3~5mm) with 23 gauge needle. To maturate follicular oocytes, cumulus oocytes complexes were washed three times with TCM-199 containing 25mM HEPES and incubated (39$^{\circ}C$, 5% CO2 in air) for 42hrs. Ejaculated and liquid storaged boar spermatozoa capacitated with different sperm capacitation methods and media were prepared forfertilizaing of matured follicular oocytes in vitro. Fertilization was performed by adding 5~10${mu}ell$ of capacitated spermatozoa containing 1~5$\times$105 sperm/ml to droplets. Eighteen to twenty-eight hours after sperm insemination, fertilized eggs were washed three times with culture media and transferred to the culture media. The fertilization rates of in vitro matured follicular oocytes cultured in B. O., TCM-HEPES, m-KRB, and TALP-II media were 61.3%, 83.0%, 88.9% and 89.2%, respectively. In addition, the polyspermy rates were 60.7%, 66.5%, 53.8%, and 43.9%, respectively. These data indicated that the highest of fertilization and the lowest of polyspermy rate was shown in TALP-II medium. Spermatozoa capacitated by caffeine, heparin, and percoll density gradient treatment in the 4 different media, the fertilization rates were 33.0~57.2%, 39.9~90.2%, and 52.6~92.8%, respectively, showing the lowest rate in caffeine treatment. The development rate of follicular oocytes, fertilized with the spermatozoa capacitated by caffeine, heparin, and percoll gradient in the TALP-II medium, upto 2 to 4-cell stages were 32.6%, 74.5% and 70.9%, respectively. Finally, fertilization rates of follicular oocytes cultured with follicular fluid containing medium from 10 to 100% were 61.2~94.1% and the rates (90~94%) with 10~20% follicular fluids were significantly higher than those (85.3%) of cultured in the media without follicular fluid. In addition, the rates of pronucleus formation were also higher in follicular fluid treated group (73.1~83.0%) than those (64.7%) of oocytes cultured without follicular fluid. The highest fertilization and pronucleus formation rates was found in oocytes cultured with 10% follicular fluid. These results suggest that the addition of heparin or percoll density gradient method is better capacitation method. Furthermore, the addition of porcine follicular fluid to the fertilization medium may improve the fertilization rates and formation of pronucleus.

• Reproductive and Developmental Biology
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• 제28권2호
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• pp.95-99
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• 2004

본 연구는 미성숙돼지 난모세포의 체외 성숙에 있어서 myo-inositol의 영향을 알아보기 위하여 실시하였다. 미성숙 돼지 난모세포을 myo-inositol을 포함하는 또는 포함하지 않은 체외 성숙배지에서 44시간 체외 성숙을 유도하였을 때 성숙율은 myo-inositol을 첨가한 성숙배지에서 성숙시킨 실험구에 유의하게 높았다(P<0.05). Myo-inositol 에 의한 성숙율 향상에 있어서 난구세포의 영향을 알아보기 위하여 난구세포의 치밀도에 따라 분류하여 미성숙 난모세포을 myo-inositol을 포함하는 체외 성숙배지에서 배양하였을 때 난구세포가 치밀한 미성숙 난모세포에서가 더 높은 성숙율을 나타내었다(P<0.05). 그러나 난구세포가 치밀하지 않은 미성숙 난모세포도 myo-inositol을 포함하는 성숙배지에서 배양하면 성숙율은 대조구에 비하여 유의하게 높았다(P<0.05). 이러한 결과는 돼지 난모세포의 체외 성숙배지에 myo-inositol의 첨가는 체외 성숙율을 향상시킬 수 있음을 나타내고 있다.

• Reproductive and Developmental Biology
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• 제33권3호
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• pp.125-131
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• 2009

The aim of this study is to investigate the effect of porcine epididymal fluid (pEF) on in vitro-maturation and subsequent fertilization of porcine follicular oocytes. Porcine cumulus-oocytes complexes retrieved from antral follicles were cultured in tissue culture medium (TCM)-l99 supplemented with pEF of different concentrations. At 48 h after culture, development of oocytes to germinal vesicle (GV) breakdown, metaphase I, anaphase-telophase I, and metaphase II were examined Significant (p<0.05) increase in the proportion of oocytes developed to MII stage was observed in oocytes cultured in pEF-containing TCM-l99 than in oocytes cultured in pEF-free TCM-199 (46.2% vs 16.7%), which was a dose-dependent manner. Subsequently, the proportion of monospermic fertilization were significantly (p<0.05) increased in oocytes cultured in the TCM supplemented with pEF than those cultured in pEF-free TCM-199 (51.0% vs 24.1%). In the second series of experiment, the percentage of MII oocytes was significantly (p<0.05) increased after exposure of oocytes to pEF during the first 22 h period of culture than after exposure of oocytes to pEF during the next 24 h of culture, while no significant difference in the percentage of monospermy was observed. The results of this study demonstrate that pEF contains at least enhancing component(s) for nuclear maturation.

• 한국가축번식학회지
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• 제21권1호
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• pp.53-59
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• 1997

This study was conducted to investigate the effect of hormones on in vitro maturation of porcine follicular oocytes. The results obtained were as follows : 1. The maturation rates of oocytes cultured in medium containing FSH 1, 10, 50 and 100$\mu\textrm{g}$/ml were 44.6, 58.5, 42.6 and 37.9%, respectively. And those were no difference to maturation rates among the medium containing FSH. However, the maturation rate(13.7%) of oocytes cultured without FSH was significantly(P<0.05) lower than those of oocytes cultured with FSH. 2. The maturation rates of oocytes cultured in medium containing hCG 0, 1, 10, 50 and 100IU/ml were 12.2, 11.9, 17.9, 21.9 and 45.6%, respectively. The maturation rate of oocytes cultured with 100IU/ml hCG was significantly(P<0.05) higher than those of oocytes cultured with 0~50IU/ml hCG concentrations. 3. The maturation rates of oocytes cultured in medium containing E2 0, 1, 10, 50 and 100$\mu\textrm{g}$/ml were 13.7, 10.5, 14.9, 11.4 and 5.9%, respectively. There were no differences to maturation rates among the E2 concentrations. 4. The FSH＋hCG treatment was the highest maturation rate in medium containing different combination of FSH, hCG and E2.

• International Journal of Oral Biology
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• 제30권4호
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• pp.131-134
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• 2005

In the present study, performances of several in vitro maturation (IVM) systems for porcine follicular oocytes were evaluated, and an efficient chemically defined IVM system for porcine oocytes was proposed. The proposed one-step culture system supplemented with polyvinylalcohol (PVA) gave competitive efficiencies in terms of oocyte maturation and blastocyst development after parthenogenetic activation and in vitro culture, compared with the conventional two-step culture system by a supplementation of porcine follicular fluid (pFF). Additionally, it is identified that the proposed chemically defined one-step culture system yielded the comparable level of blastocyst production to the conventional maturation system in porcine somatic cell nuclear transfer (SCNT). Therefore, one can eliminate un-expected effects accompanied by supplementation of pFF. No medium replacement during whole maturation period is an additional benefit by applying this new system. Thus, these data support that the developed PVA supplemented chemically defined one-step IVM system for porcine follicular oocyte might be used in porcine SCNT program.

• Asian-Australasian Journal of Animal Sciences
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• 제14권10호
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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.

• 한국수정란이식학회지
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• 제32권3호
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• pp.105-109
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• 2017

Autophagy is an intracellular degradation and recycling system. Oocyte maturation is dynamic process, in which various proteins should be synthesized and degraded. In our previous study, we reported the loci of autophagosome and dynamics of autophagic activity in porcine oocytes during in vitro maturation. In this study, we verified loci of autophagosome in porcine follicular cumulus-oocyte complex by detection of microtubule-associated protein 1A/1B-light chain 3 (LC3) which is the reliable marker of autophagosome. Porcine ovary including various sizes of follicles was fixed within 1 hour after collection from slaughterhouse. After fixation, immunohistochemistry was conducted on sliced ovary tissue containing various sizes of follicles by using LC3 antibody. As a result, LC3 signal was clearly detected in both cumulus and oocytes of various sizes of follicles. We also found ring shaped signal which represent autophagosome near oocyte membrane. Most of the signals in oocytes were localized nearby cellular membrane while evenly dispersed in cumulus cells. Therefore, this result suggests that autophagy occurs in porcine COCs (cumulus-oocyte complexes) at follicular stage.

• 한국가축번식학회지
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• 제22권3호
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• pp.253-264
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• 1998

Porcine follicular oocyte, collected from antral follicles (2~5 mm in diameter) of gilt ovaries were matured in vitro porcine follicular fluid (pFF) with PMSG (pFF-PMSG) buffer with at 37$^{\circ}C$ under 5% CO2 in air their ability of maturation promoting factor (MPF), of GV and GVBD formation was examined followed during time after in vitro culture. Formation of second metaphase was observed in 57.6% and 71.2% of matured in with pFF-PMSG buffer to 45 and 50 hours after invitro. Porcine oocytes cultured in pFF-PMSG for various periods of up to 30 hours were stained with Hoechst-33342 and classified according to maturation before assaying. Histone H1 kinase (H1K) activity was assayed during meiotic maturation in porcine oocytes matured in pFF-PMSG buffer in vitro. In oocytes matured in pFF-PMSG, H1K activity was at the 30 hours after culture and increased about 15 fold than at the germinal vesicle stage with before at the cultured in vitro. This pattern is similar to those reported in non-mammalian species and su, pp.rts the concepts that H1K is ubiquitous in eukaryotes and controls the meiotic cell cycle in mammals. These results suggest that the maturation pFF-PMSG buffer used influences the fluctuation pattern of H1K activity and biological characteristics of porcine oocytes cultured in vitro.

• 한국가축번식학회지
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• 제21권4호
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• pp.381-387
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• 1997

This study was conducted to investigate the effects of fetal bovine serum(FBS), calf serum(CS) and human serum(HS) on in vitro maturation of porcine follicular oocytes. The results obtained were summarized as follows : 1. The maturation rates of oocytes cultured in medium containing FBS 5, 10, 20 and 30% were 47.0, 63.5, 48.4 and 43.2%, respectively. There were significantly higher than those of non-treated group(25.3%). And the highest maturation rate was the 10% treatment. 2. The maturation rates of oocytes cultured in medium containing CS 5, 10, 20 and 30% were 55.2, 56.6, 59.4 and 46.5%, respectively. There were significantly higher than those of non-treated group(25.3%). And the highest maturation rate was the 20% treatment. 3. The maturation rates of oocytes cultured in medium containing HS 5, 10, 20 and 30% were 74.5, 78.2, 73.1 and 68.6%, respectively. There were significantly higher than those of non-treated group(29.6%). And the highest maturation rate was the 10% treatment.

• Clinical and Experimental Reproductive Medicine
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• 제8권1호
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• pp.1-12
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• 1981

면역학적인 방법을 사용하여 가돈 여포액내의 특이단백질의 존재여부와 이들을 추출하여 난자성숙에 미치는 영향을 조사한 결과 다음과 같았다. 1. 가돈 혈청 및 혈장에는 존재하지 않는 면역학적으로 특이한 두 단백질이 가돈 여포액내에서 검출되었다. 2. 이들 두 단백질은 전기영동적으로 각각 fast alpha-I globulin 과 beta-globulin 의 이동성을 나타냈다. 3. 이들 두 단백질은 생쥐 여포난자의 성숙을 억제시켰다. 본 실험의 결과로 미루어 가돈 여포액내에는 난자성숙을 억제시키는 특이단백질이 존재하는 것으로 사료된다.