• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2004.10a
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• pp.72-72
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• 2004

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2004.10a
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• pp.97-97
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• 2004

• Korean Journal of Animal Reproduction
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• v.24 no.3
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• pp.311-317
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• 2000

This study was carried out to compare the temperature and time of heat shock, and the effect of heat shock on development of embryos after in vitro maturation and fertilization in bovine oocytes. The results obtained were as follows. 1. The optimum temperature and time of heat shock were 41$^{\circ}C$ and 30sec on in vitro development of embryos from 4~8 cell to blastocyst. 2. The rates of cleavage on zygotes produced on in vitro were significantly increased by heat shock after IVM than before IVM(P<0.05). 3. When the oocytes were treated heat shock after IVM and 5 days cultured, developmental rates to blastocyst were increased than other experimental treatments.

• Korean Journal of Animal Reproduction
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• v.22 no.2
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• pp.163-169
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• 1998

본 실험은 체외 생산된 소 배반포기배에 대한 laser drilling 처리가 배의 부화율에 미치는 영향을 조사하고자 실시하였다. 그 결과는 다음과 같다. 소 수정란의 체외 발달율을 조사하였던 바, 82.3%의 난할율( 2-세포기)과 체외수정 후 배양 7일째에 32.6%의 배반포 발달율을 나타내었다. 이렇게 생산된 배반포기배에 laser drilling 효과를 조사하였던 바, 처리 후 24시간째의 부화진행율(90.0%)은 대조군(44.4%)보다 유의하게 높게 나타났다(p<0.0001). 또한, 처리 후 48시간째의 부화율(68.0%)도 대조군(33.3%)보다 유의하게 높게 나타났다. 이러한 결과는 laser drilling이 체외 생산된 소 배반포기배의 부화진행율과 부화율을 유의하게 증가시킬 수 있다는 것을 알 수 있었다(p<0.001).

• Korean Journal of Animal Reproduction
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• v.23 no.2
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• pp.133-139
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• 1999

This study was carried out to investigate in vitro/in vivo development of vitrified-thawed immature mouse oocytes. Immature mouse oocytes were vitrified with EFS40 (40% ethylene glycol, 18% ficoll and 0.5 M sucrose). Thawed oocytes were matured for 16 hr in vitro. Matured oocytes with the first polar body were fertilized with the concentration of 1~2$\times$10$^{6}$ $m\ell$ of epididymal sperm. After fertilization, cleavage ($\geq$ 2-cell) and in vitro/in vivo development rates were examined. $\pi$ Ie results were summarized as follows: in vitro maturation rate of immature mouse oocytes in vitrified-thawed group was similar to that in exposed group (67.5%) and control (66.3%), but cleavage rate of vitrified-thawed oocytes (64.9 %) and blastocyst formation rate (59.0%) were significantly different compared to those of exposed group (83.7 and 74.7%) and control (90.7 and 83.7%) (p＜0.05). However, when the blastocysts derived from immature mouse oocytes vitrified-thawed were transferred to pseudopregnant mouse, total implantation (31.3%) was slightly lower than that in control (40.8%), but live fetus formation rate (66.7%)was slightly higher than that in control (58.1%), there was not significantly different. Therefore, when the blastocyts produced in vitro were transferred into recipients, although the development in vitro of oocytes vitrified-thawed was decreased, live fetus formation rate was similar to that of control group. The present results indicate that immature mouse oocytes can be frozen successfully by vitrification with EFS40.

• Proceedings of the KSAR Conference
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• 2003.06a
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• pp.58-58
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• 2003

포유동물 체외 수정란의 체외 발육율을 향상시키기 위해서는 배양액내에 생성되는 free radical을 제거하는 것이 한가지 요인으로 지적되고 있다. 체외 배양액내에 생성되는 free radical을 제거하기 위하여 여러 종류의 항산화제 첨가 배양이 많이 이용되어 체외발육성적을 향상시키고 있다. 포유동물의 번식에 있어서 Melatonin은 광주기 조절 작용에 기여하는 호르몬으로서 혈중 Melatonin의 농도는 번식 작용에 중요한 역할을 하며, 또한 수정란의 세포분열과 세포주기에 영향을 미치며 배양액내의 free radical을 제거하여 수정과 초기 수정란 발달에 영향을 미쳐 항산화 기능이 있는 것으로 보고되고 있다.

• Korean Journal of Animal Reproduction
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• v.21 no.2
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• pp.157-165
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• 1997

포유류 배반포배의 epiblast는 내세포괴에 포함되어 있으며, 이 epiblast cells이 배 및 태아의 생식세포와 일반 체세포로 분화된다 (Beddington, 1986; Lawson 등, 1991). 그런데 조기에 발달된 부화배반포기 배가 지연발생된 부화배반포기 배보다도 많은 epiblast cells을 가지고 있다고 한다(Talbot 등, 1995). 그래서 본 연구에서는 체외수정 유래의 배반포배의 발육속도에 따른 내세포괴/영양배엽세포의 비율 및 배아간세포 확립 효율을 비교하여 발달일령 간에 차이가 있는지를 규명하고자 하였다. 공시한 소의 난포란을 TCM-199에 0.5$\mu\textrm{g}$/ml FSH, 5$\mu\textrm{g}$/ml LH, 10% FBS, 100 units/ml penicilin, 및 100$\mu\textrm{g}$/ml streptomycin을 첨가하여 39$^{\circ}C$, 5% CO2 조건하에서 24시간 동안 체외성숙한 후, 5$\mu\textrm{g}$/ml heparin으로 수정능이 획득된 1$\times$106 sperm/ml의 정자로 체외수정을 유도하였다. 체외수정 후 18~20시간에 과립막세포를 vortexing으로 제거하여 얻은 모든 체외수정란을 3mg/ml BSA, 20${\mu}\ell$/ml NEM amino acids, 40${\mu}\ell$/ml BME amino acids, 10mM glycine 및 1mM alanine이 함유된 CR1aa 배양액에서 BRL 단층세포와 공배양을 실시하였다. 수정후 7, 8 및 9일재 (체외수정일 : 0일)에 확장배반포기까지 발달한 수정란을 이중염색 및 배아간세포의 확립 실험에 공시하였다. 체외수정후 24시간에 분할된 총 1,145개의 수정란이 7, 8 및 9일째에 후기 배반포기까지 각각 222(15.6%), 103(7.2%) 및 52(3.6%)로 발달하여 총 377개 (26.4%)가 발달하였다. 내세포괴/영양배엽세포의 비율은 7일 및 8일째 배반포배에서 각각 47.2$\pm$11.9/95.1$\pm$24.4개 (33/67%) 및 40.3$\pm$12.4/83.3$\pm$26.9개 (33/67%)로서 9일째 배반포배의 19.3$\pm$8.1/62.3$\pm$23.1개 (24/76%) 보다 유의적(P<0.05)으로 높았다. ES-like cells을 확립하기 위하여 후기배반포기 배를 mouse embryonic fibroblast 단층 공배양기에 옮긴 후 5일에 내세포괴의 부착 여부를 판정하고, 10일에 배아간세포의 확립 여부를 판정하였다. 그 결과 7, 8 및 9일째의 배반포기배의 각각 47.7% (82/172), 30.9%(22/71) 및 15.6%(5/32)에서 배아간세포가 확립되었다. 이상의 결과에서 배반포기까지의 발육이 빠른 수정란에서 영양배엽에 대한 내세포괴세포의 비율이 높았고 배아간세포의 확립율도 높다는 사실을 입증할 수 있었으며, 이와 같은 결과에서 체외수정란 유래 배반포배의 질을 결정할 수 있을 것으로 생각된다.

• Korean Journal of Animal Reproduction
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• v.17 no.2
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• pp.113-120
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• 1993

To provide the optimal culture conditions for the developm,ent of in vit개 produced embryos, we have been investigated various culture media as well as co-cultrue systems using porcine cumulus cells or mouse fetal fibroblast cells. 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) in various maturation media for 42 hrs. Ejaculated and liquid storaged boar spermatozoa capacitated with different sperm capacitation methods and media were rpepared for fertilizing of matured follicular oocytes in vitro. Fertilization was performed by adding 5~10${\mu}\ell$ fo 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 various culture media, to the culture media with a monolayer of somatic cells. The in vitro development rates of 1-cell embryos cultured with three times with culture media and transferred to the various culture media, to the culture media with a monolayer of somatic cells. The in vitro development rates of 1-cell embryos cultured with three different media, m-KRB, BECM and TCM-HEPES were 0~1.0%, showing extremely lower rates. Especially, most of embryos were observed to arrest the development beyond 4-cell stages. The rates of embryos developed to 2-, 4-, 8-, 16-, 32-cell and morula or blastocyst stage in co-culture with porcine cumulus cells and mouse fetal fibroblast cells were 61.1~67.0%, 59.0~58.0%, 42.5~43.1%, 28.4~30.2% and 20.4~21.0%, respectively. These development rates upto morula or blastocyst stages were significantly higher than those of the embryos cultured in the basic culture medium(P<0.01). These findings suggest that co-culture of in vitro fertilized eggs with porcine cumulus cells or mouse fetal fibroblast cells enhance the development of fertilized eggs to morula or blastocyst stage in vitro.

• Korean Journal of Animal Reproduction
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• v.26 no.2
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• pp.173-182
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• 2002

The mechanisms underlying of the visual assessment and resulting in optimum embryonic development following in vitro maturation, fertilization, and culture are unclear, It was known that in vitro produced embryos show more frequent occurrence of fragmentation, which resulted in poor developmental potential and decreased implantation rate. The objective of this study was to investigate the apoptotic rates in bovine blastocyst derived from in vitro fertilization (IVF) and nuclear transfer (NT). In addition, the expression levels of Bcl-2 and Bax gene were investigated in the blastocyst to confirm their potential roles in the regulation of apoptosis during preimplantation embryonic development. Analysis of apoptosis was carried out by using terminal deoxynucleotidyl transferase mediate dUTP nick end labeling (TUNEL) method. The levels of Bcl-2 and Bax gene in the blastocyst derived from IVF and NT were determined by RT-PCR. The proportion of TUNEL positive signal in blastocyst derived from NT was significantly higher than that in blastocyst derived from IVF (p<0.001). Bcl-2 expression level of blastocyst derived from IVF was higher than that of blstocyst derived from NT. However, high expression level of Bax was observed in the blastocyst derived from NT. These results indicates that apoptosis is more responsible for fiagmentation in bovine blastocyst derived from NT than IVF. These results suggested that the increase of developmental failure followed by NT could be caused by nuclear fragmentation as apoptosis.

• Reproductive and Developmental Biology
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• v.29 no.3
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• pp.201-205
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• 2005

The objective of this study was to investigative the effects of amino acids supplementation on maturation, fertilization and embryo development of pig oocytes. Essential amino acids (EA), non-essential amino acids (NA) or both amino acids (EA + NA) were supple-mented to North Carolina State University (NCSU) 23 medium containing porcine follicular fluid (pFF). When the amino acids were supplemented to the maturation medium, the maturation rates were higher (p<0.05) in the NA group than control ($83.3{\pm}0.04\%\;versus\;70.0{\pm}0.05\%$, but the subsequent cleavage rates and development to morula and blstocyst stage between aminoacid supplement groups and control were not different. The developmental rates to morula and blastocysts stage were not significantly different regardless of amino acid supplementation to culture medium. In addition, supplementation of amino acids did not significantly affect the rate of fertilization and polyspermy. When the amino acids were supplement to culture medium, the number of trophectodermal (TE) cells was significantly (p<0.05) higher in amino acid supplement group than that of control ($18.6{\pm}0.5\;versus\;16.1{\pm}0.6$), whereas the numbers of inner cell mass (ICM) cells were not different among the treaonent groups and control ($29.0{\pm}0.9\~31.5{\pm}1.2$). Total cell number was also significantly (p<0.05) higher in EANA group ($50.0{\pm}1.0$) than that of control group ($44.2{\pm}1.1$). These results indicate that the amino acid supplementation to maturation and culture medium may not significantly stimulate early embryo development, but may improve the TE cell number of blastocyst stage in the pig.