• 한국수정란이식학회:학술대회논문집
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• 한국수정란이식학회 2002년도 국제심포지엄
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• pp.79-79
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• 2002

본 연구에서는 in vitro에서 성숙된 난자의 핵성숙(Polar Body extrusion)에 소요되는 시간과 배반포 단계로의 발달능력 사이의 관계를 비교하여 조기에 발달능력을 가진 embryo를 선발할 수 있는 IVP 체계를 개발하고자 하였으며 in vitro maturation(IVM)에 따른 first polar body(PB) 형성, IVM과 IVF 시간이 oocyte의 발달에 미치는 영향과 생산된 배반포의 세포수를 평가하였다. IVM은 TCM199 배양액을 사용하였고 in vitro fertilization(IVF)은 Fer -TALP용액을 사용하였으며 in vitro culture(IVC)는 CRlaa 배양액을 사용하여 2일까지는 0.3% BSA를 3일 부터는 10%FBS와 bovine oviduct epithelial cell을 첨가하여 배양하였다. IVM 시간에 따른 PB의 출현율은 0hr(0%), 6hr(0%), 12hr(0%), 14hr(8.7%), 16hr(40.5%), 18hr(48.0%), 20hr(65%), 22(68%) 그리고 24hr(74.5%)을 보였으며 IVM 시간에 따른 cleavage 및 8cell 발달율 사이에는 유의적인 차이가 없었으나 배반포(BL) 및 8cell에서 배반포로 발달률은 18시간(BL 31$\pm$6, BL/8cell 82 $\pm$5%)에서 가장 높게 나타났으며 24시간(BL 17$\pm$2, BL/8cell 60$\pm$8%)과 유의적인 차이를 보였다(P<0.05). IVC 7일째 배반포의 총세포수와 trophoblast(TE) 세포수는 IVM 18시간(mean$\pm$S.E.; total: 131.1$\pm$34.0, TE: 97.6$\pm$29.6)에서 24시간(total: 112.2$\pm$17.5, TE: 80.1$\pm$15.6)보다 유의하게 많은 것으로 나왔으나(P<0.05) 7일째의 inner cell mass(ICM) 숫자(18hr 33.5$\pm$12.8 vs 24hr 32.1$\pm$12.0)와 8일째 ICM, TE 그리고 총 세포수에는 유의성 있는 차이가 없었다. IVM 18시간에서 PB 형성과 8cell 발달률 사이에 높은 상관성을 보였고 배반포 및 8cell에서 배반포 단계로 높은 발달률을 보였으며 생산된 배반포의 TE 숫자와 총 세포수가 유의하게 많은 것으로 나타났다. 따라서 IVM 18시간 실시하였을 경우 보다 많은 세포수를 가진 배반포 발달 가능성이 높은 embryo를 조기에 선발 가능할 것으로 사료된다.

• 한국수정란이식학회지
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• 제15권2호
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• pp.167-173
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• 2000

The principal objective of this study was to clone transgenic embryos in order to improve the efficiency of transgenic animal production by the combination of microinjection and nuclear transplantation techniques. Mature female New Zealand White rabbits were superovulated by eCG and hCG treatments, fllowed by natural mating. Zygotes were collected from the oviducts at 18∼22 h after hCG injection by flushing with D-PBS containing 5% fetal calf serum(FCS). Two to three picoliters of green fluorescent protein(GFP) gene wa microinjected into male pronucleus. The foreign gene-injected zygotes were cultured in TCM-199 or RD medium containing 10% FCS with a monolayer of rabbit oviductal epithelial cells in a 5% CO2 incubator. The morulae expressing GFP gene were selected and their blastomeres were separated for the use of nuclear donor. Following nuclear transplantation of fluorescence-positive morula stage blastomeres, 13 (21.3%) out of 61 fused oocytes developed to blastocyst stage and all of the cloned blastocysts expressed GFP. The results indicate that the screening of transgene in rabbit embryos by GFP detection could be a promisible method for the preselection of transgenic embryos. Also the cloning of preselected transgenic embryos by nuclear transplantatin could be efficiently applied to the multiple production of transgenic animals.

• Asian-Australasian Journal of Animal Sciences
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• 제26권4호
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• pp.501-508
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• 2013

The effects of amino acids and dipeptides on in vitro production of porcine embryos and accumulation of ammonia in culture medium during developmental stages were examined in this study. The maturation, fertilization and development of embryonic cultures were performed in modified Tissue culture medium (mTCM)-199 supplemented with 10% (v/v) porcine follicular fluid, modified Tyrode's albumin lactate pyruvate (mTALP) medium, and modified North Carolina State University (mNCSU)-23 medium, respectively. In addition, amino acids and dipeptides of different concentrations and combinations were used to treat the embryos. The addition of L-alanyl-L-glutamine (AlnGln)+L-glycyl-L-glutamine (GlyGln) significantly (p<0.05) improved oocyte maturation, fertilization and the incorporation and oxidation of 14C(U)-glucose when compared to the control group and other treatment groups. Additionally, 2-4 cell, 8-16 cell, morula and blastocyst development increased significantly (p<0.05) following treatment with AlnGln+GlyGln when compared to the control group and other treatment groups, while this treatment reduced the accumulation of ammonia. Taken together, these findings suggest that treatment with AlnGln+GlyGln may play an important role in increasing the rate of porcine oocyte maturation, fertilization and embryonic development by reducing the level of accumulated ammonia measured in the culture media.

• Reproductive and Developmental Biology
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• 제32권3호
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• pp.147-152
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• 2008

The aim of this study was to investigate what components of porcine epididymal fluid (pEF) influences the nuclear maturation of porcine germinal vesicle oocytes. Porcine cumulus-oocytes complexes from follicles were cultured in TCM 199 containing pEF. After 48 h cultures, oocytes were examined for evidence of GV breakdown, metaphase I, anaphase-telophase I, and metaphase II. Maturation rate of oocytes was significantly increased in media supplemented with 10% pEF during in vitro maturation (IVM) than in those without pEF. When lipid component of pEF was removed by treating n-heptane, no significant difference was observed in maturation of oocytes between n-heptane treatrment and intact pEF group. However, the proportion of oocytes reaching at metaphase II (M II) was significantly (p<0.05) decreased in the oocytes cultured in media containing trypsin-treated pEF compared to those in media with intact pEF. When porcine GV oocytes were matured in the medium supplemented with intact pEF or pEF heated at $56^{\circ}C$ and $97^{\circ}C$, rates of oocytes remained at GV stage were 11.7%, 29.4% and 42.0%, respectively. However, there were no difference in proportion of oocytes reaching at MII stage among intact pEF group and $56^{\circ}C$ group. Present study suggests that 1) pEF contains an enhancing component(s) for nuclear maturation in vitro of oocytes, 2) protein(s) of pEF may be capable to promote nuclear maturation in vitro, and 3) enhancing component for nuclear maturation may consist of two factors, which are responsible for germinal vesicle breakdown (GVBD) and promotion of MII stage.

• 한국수정란이식학회지
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• 제15권1호
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• pp.1-8
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• 2000

The ultrasound-guided oocytes cllection (ovum pick-up ; OPU) has become a substitution for superovlation in cattle. The objective of this study was to examine the effect of OPU frequency on the in vitro production of embryos in Hanwoo cattle. Six cycling Hanwoo cows were distributed into two groups for either once or twice weekly OPU sessions. Oocytes were collected by ultrasound-guided follicle aspiration(SA600) using a 6.5HMz transducer and attached with 18 gauge needle, with vacuum pressure of 40 mmHg. The cumulus-oocyte complexes (COCs) collected from each donor were matured in TCM 199 supplemented with 10% fetal bovine serum at 5% CO2 in air at 38.5$^{\circ}C$ for 22h and in vitro matured oocytes were co-incubated with sperm(separated by Percoll gradient) for 6h. The zygotes were co-cultured on cumulus cell monolayer in 10ul droplets in the same culture medium and conditions used for IVM for 7 days. On Day 7 of culture, development to blastocysts was examined. Although the number of oocytes collected was variable depending on individuals, overall embryo production in the twice per week OPU sessions was better that in the once per week sessions(6~21 vs 2~7 blastocysts produced, respectively). Two cows(E, A) were good oocyte donors and embryo production was superior in cow C ; however, cow F was a poor donor as compared to the others. In conclusion, these results suggest that for embryo production, twice weekly OPU sessions were better than once per week for producing embryos in vitro from Hanwoo cattle.

• 한국수정란이식학회지
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• 제13권3호
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• pp.245-249
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• 1998

본 연구는 성장인자 EGF, IGF-1, EGF+IGF-1 이 소 난포란의 체외성숙에 미치는 영향을 규명하기 위하여 실시하였다. 소 난포란의 체외성숙 시 EGF를 농도별 (10, 50 및 100ng/m1)로 첨가하여 실험한 결과 통계학적 유의성은 인정되지 않았으나 24시간 배양 후 성숙율은 대조군에 비하여 높은 경향을 나타내었다. 소난포란의 체외성숙 시 EGF와 IGF-1을 병용 처리하여 실험한 결과, EGF 또는 IGF-1 단독처리 군에 비하여 병용처리군이 유의적으로 높은 체외성숙율을 나타내었다(p〈0.05). 따라서 소난포란의 체외성숙율은 EGF와 IGF-1에 의하여 영향을 받으며 특히 EGF와 IGF-1의 상호작용에 의하여 더 유효한 효과를 나타내는 것으로 사료된다.

• 한국가축번식학회지
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• 제17권4호
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• pp.271-278
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• 1994

The aim of this study was to compare the two culture systems 1) co-culture with cumulus cells and 2) chemically defined medium supplemented with amino acids (CR1aa) and fetal calf serum (FCS) of in vitro produced bovine embryos from follicular oocytes in vitro. Bovine follicular oocytes were collected from ovaries of slaughtered cows and matured in TCM199 supplemented with 10% FCS and hormones (1$\mu\textrm{g}$/ml FSH-P and 1$\mu\textrm{g}$/ml oestradiol-17$\beta$)24 hours at 39$^{\circ}C$ under 5% CO2 in air. The capacitation of spermatozoa from ejaculated or frozen bull semen was induced by centrifugation through Percoll density gradient (45%, 90%). Then capacitated spermatozoa (1$\times$106/ml) were inseminated into 50${mu}ell$ droplet containing matured follicular oocytes and incubated for 40~42 hours. Cleaved embryos of 2~4cell stage were transferred to the co-culture with cumulus cells and/or CR1aa medium supplemented with FCS. In semen source, the developmental rates to the blastocyst and the hatched blastocyst stages were higher in ejaculated semen(27.6% and 14.9%) than those of frozen-thawed semen(18.3% and 11.8%), respectively. In two culture systems, the proportions of embryonic development upto the blastocysts and the hatched blastocysts were higher of CR1aa medium (22.1% and 12.1%) than those of cumulus cell co-culture (16.8% and 5.1%), respectively. The number of cells in exapnded blastocysts was slightly higher in cumulus cells co-culture (122.6$\pm$8.5) than that in CR1aa medium (117.9$\pm$5.9). The present results indicated that the early development of in vitro produced bovine embryos can be maintained efficiently in CR1aa medium as well as in co-culture with cumulus cells.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2001년도 발생공학 국제심포지움 및 학술대회 발표자료집
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• pp.58-58
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• 2001

This study was carried out to investigate the optimal activation condition for parthenogenetic development. In order to activate oocytes at 24 hrs post onset of maturation, the oocytes were cultured 3 - 13 μM Ca for 5 min., 5-8 ㎍/㎖ cytoclacin(CH) for 6 hrs, 0.5-2.0 mM 6-dimethylaminopurine(DMAP) for 3 hrs alone or combination. The activated oocytes were cultured in TCM-199 media at 5% CO₂, 95% N₂, 38℃. 1. The cleavage rate after 48 hrs culture of oocytes treated with 3-13 μM Ca for 5 min. were 9.6%-20.0% and 3.8-7.3%, respectively. When oocyte were treated with 10 μM Ca, the blastocyst formation rate was significantly higher than other group. 2. The cleavage rate after 48 hrs culture of oocytes treated with 5-8 ㎍/㎖ cytoclacin(CH) for 6 hrs, were 9.4%-21.8% and 0.0-7.3%, respectively. When oocyte were treated with 10㎍/㎖ CH, the blastocyst formation rate was significantly higher than other group. 3. The cleavage rate after 48 hrs culture of oocytes treated with 0.5-2.0 mM 6-dimethylaminopurine(DMAP) for 3 hrs were 9.1%-21.8% and 0.0-7.3%, respectively. When oocyte were treated with 2.0mM DMAP, the blastocyst formation rate was significantly higher than other group. 4. The cleavage rate after 48 hrs culture of oocytes treated with Ca＋CH, Ca＋DMAP, CH＋DMAP were 75.9%-93.5% and 9.7 -13.3%, respectively. When oocytes were treated with Ca followed by DMAP, the blastocyst formation rate was significantly higher than other group(p〈0.05). 5. When necleus transferred embryos co-cultured with BSA, EGF and CS, the developmental rate to blastocyst were higher than control group.

• 한국수정란이식학회지
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• 제13권1호
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• pp.1-9
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• 1998

In order to improve the cryopreservation by vitrification or slow freezing of nuclear transplant rabbit embryos, the effects of factors affecting embryo cryopreservation such as cryoprotectants, equilibration, cooling rate and post-thaw dilution on post-thaw survial and development were determined using intact embryos of morular stage. And the post-thaw development of nuclear transplanted embryos cryopreserved under the optimal conditions examined was compared between vitrification and slow freezing. The cryoprotectant solution used was ethyleneglycol-ficoll-sucrose (EFS) or ethyleneglycol-poly-vinylpyrrolidone-galactose- I (EPG- I ) for vitrification, and EPG- II for slow freezing. To examine the viability of frozen-thawed embryos, the nuclear transplanted embryos were co-cultured in TCM-199 plus 10% FBS with bovine oviduct epithelial cells(BOEC) for 24 hrs and the intact morulae were co-cultured with BOEC for 5 days and 3 days to hatching blastocyst stage in 39 ˚C 5% $CO_2$ incubator. The results obtained were as follows: Following vitrification with EFS, the post-thaw development of rabbit morulae to hatching blastocyst was significantly(P<0.05) higher in compacted stage(82.4%) than in early morular stage(60.0%). The post-thaw development of compacted morulae to hatching blastocyst was similarly high in vitrification with EFS(82.4%), EPG- I (85.0%) and in slow freezing with EPG- II (83.3%). Following vitrification with EPG- I, the post-thaw development of intact rabbit morulae to hatching blastocyst was similar as 78.0% and 85.0% in 1-step and 2-step post-thaw dilution, respectively. The post-thaw development of nuclear transplanted rabbit embryos of compacted morulae stage to hatching blastocyst was similarly 43.6% and 40.0% in vitrification with EPG- Iand slow freezing with EPG- II, respectively. These results indicated that the rabbit nuclear transplant and intact embryos of morulae stage could be well cryopreserved with either vitrification or slow freezing procedure.

• 한국수정란이식학회지
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• 제11권1호
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• pp.7-14
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• 1996

The object of this study was to evaluate the effect of uterine epithelial cells on development of Korean native cattle(KNC) oocytes fertilized in vitro. Qocytes were collected from ovaries of slaughtered Korean Native Cows and matured in TCM199 with granulosa cells supplemented with 10% FBS, 5$\mu$g/ml FSH, 10 JU/ml hCG, and 1$\mu$g/ml estradiol-17$\beta$ for 24 hrs. For co-culture of in vitro development of fertilized ova, oviductal epithelial cells (l$\times$l0˚cells /ml) obtained from slaughtered cow and uterine epithelial cells(1$\times$10˚cells /ml) flushed from the superovulated holstein on Day 7 were incubated in 39$^{\circ}C$, 5% $CO_2$, 95% air. Frozen-thawed KNC sperm was capacitated with BO(Brackett & Oliphant, 1975) medium supplemented with 10mM, 5mM-caffein. Matured oocytes were inseminated for 20 hrs. And then fertilized oocytes were washed with culture medium and transferred to oviductal epithelial cells for in vitro development and three days later a portion of embryos were transferred to uterine epithelial cells. Stastical methods of developmental rates on KNC-IVF oocytes was ANOVA-test. Developmental rates of KNC-IVF oocytes was significant higher(P<0.01) when co-cul-tured with uterine epithelial cells(25.2%) than oviductal epithelial cells. Blatocyst cul-tured for 7 to 9 days were frozen by automatic freezer with 1.4M glycerol-PBS. Survival rates of blastocyst was 40.0%. Fourteen frozen-thawed blastocysts were transferred to five holstein heifers on day 7 after natural estrus. Three recipients were observed twin and one recipient was single by ultra-sound systems on days 45 after embryo transfer.