• 한국수정란이식학회지
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• 제6권1호
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• pp.25-32
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• 1991

The present study was performed to investigate the effects of co-culture with granulosa cells on in vitro fertilization and cleavage of early bovine embryo development. Bovine oocytes were matured for 20-24 hrs in vitro with granulosa cells or without and then fertilized in vitro using frozen-thawed spermatozoa treated with BO-caffeine, BO-BSA(2OmM heparin added). At l8hrs after insemination, oocytes were fixed and examined or further cultured in TCM 199 for 48hrs. The fertilization rates between the control(70.4%) and the groups of co-cultured with granulosa cell(2.5$\times$106 cells/ml; 71.6%, 5.0$\times$ 106/ml; 71.9%, l.0$\times$ 107/ml; 71.1%) did not differ significantly. The cleavage rates in the groups co-cultured with granulosa cell(2.5$\times$ 106 cells/mi; 43.6%, 5.0$\times$ 106/ml; 46.8%. l.0$\times$ 107/ml; 45.0%)were significantly higher than that of without granulosa cell, respectively(P<0.05). However there were no significant differences between the groups co-cultured with granulosa cells. The result indicated that co-culture with granulosa cell was effective means to cleavage of bovine follicular oocytes but did not affect the in vitro fertilization.

• 한국수정란이식학회지
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• 제17권3호
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• pp.211-218
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• 2002

본 연구는 체외에서 소 난포란유래의 배반포 생산에 있어서 배지 내에 첨가하는 외인성 고정질소 원으로써 아미노산과 FBS의 첨가효과를 검토하였다. 소 난포란의 체외성숙은 TCM-l99용액, 체외 수정은 Fer-TALP용액으로 행하였으며, 체외수정후 24시간째(day 1)의 수정난자를 체외배양에 제공하였다. 체외배양용 기초배지는 YS용액, 기초 배양법은 25개 난자/10 ${\mu}\ell$ 배지의 단순.미소적배양법을 이용하였다. 본 연구의 결과를 요약하면 다음과 같다. 1. 체외 수정란의 배양에 있어서 비필수 아미노산(MEM 유래) 첨가가 무첨가군보다 높은 배반포 발달율을 나타냈다. 2. 체외 수정란의 배양에 있어서 필수 아미노산(RPMI 1640 유래) 첨가가 무첨가군보다 유의하게 높은 배반포 발달율을 나타냈다. (P<0.05) 3. Day 1에 비 필수 아미노산, day 5에 필수 아미노산을 첨가하였을 경우 부화 배반포로의 발생율 및 배반포로의 부화율을 향상시켰다. 4. Day 1에 비필수.필수 아미노산을 첨가한 후 day 3, day 4, day 5에 각각 필수 아미노산만을 배지로부터 제거 시 배반포의 부화율은 현저하게 낮았다. 5. FBS의 첨가시기는 배양 후기(day 5)에 첨가할 수록 배반포율 또는 부화 배반포율이 유의하게 상승하였다(p<0.05). 이상의 결과에서 소 난포란 유래 배반포의 체외생산에 있어서 배지에 첨가하는 외인성 고정 질소 원들의 첨가에 있어서 첨가시기 및 농도를 조절함으로써 양질의 배반포 생산을 향상시킬 수 있는 것으로 사료된다.

• 한국수정란이식학회지
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• 제12권1호
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• pp.27-36
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• 1997

The studies were carried out to investigate the effects of co-culture with cumulus cells and oviduct epithelial cells on the in vitro fertilization and cleavage rate of bovine follicular cocytes and to determine the optimum thawing temperature and equilibration time on in vitro developmental rate of frozen bovine embryos. The ovaries were obtained from slaughtered Korean native cows. The follicular oocytes were cultured in TGM-199 medium containing 10 IU /ml의 PM SG, 10 IU /ml의 hCG, ip g/ml의 $\beta$-estradiol and 10% FCS for 24~48 hrs in incubator with 5% $CO_2$ in air at 38.5$^{\circ}C$. The bovine embryos following dehydration by cryoprotective agents and a various concentration of sucrose were directly plunged into liquld nitrogen and thawed in 3$0^{\circ}C$ water. Survival rate was defined as developmental rate on in vitro culture or FDA-test. The results are sunanarized as followes :1. The in vitro fertilization and in vitro developmental rates of bovine oocytes co-cultured with cumulus cells in TCM499 medium were 75.0~76.8% and 17.3~27.6%, respect-ively. And in-vitro fertilization rates of cumulus-enclosed oocytes(55.4%)were significantly(p<0.05) higher than cumulus-denuded oocytes (23.1%). 2. The in vitro fertilization and in vitro developmental rates of bovine oocytes co-cultured with l$\times$ l04cells /ml, 1 x l06cells /ml, lx l08cells /ml and 1 x l015cells /ml oviduct epithelial cells in TCM-199 medium were 74.5~77.8% and 15.7~21.20 respectively.3. The in-vitro fertilization and in vitro developmental rates of bovine oocytes cocultured in '1CM-199 media containing PMSG, hCG, PMSG+hCG. PMSG+$\beta$-estradiol, hCG+$\beta$-estradiol 0 to 40 hrs after insemination were 74.0~77.4% and l8.9~23.l%, re-spectiv ely.4.The survival rates of bovine embryos thawed after rapid freezing in the freezing medium containing a various concentration of sucrose added 1.5M and 2.OM glycerol,DMSO and propanediol were 23.5~31.4% and 20.6~34.l%, respectively. 5. The temperature thawed at 3$0^{\circ}C$ after rapid freezing of bovine embryos resulted in a significantly higher embryos survival rate than did at 2$0^{\circ}C$ and 35$^{\circ}C$.6. The equilibration time on the survival rates of bovine embryos was attained after short period of time(2.5~5 min.) in the freezing medium higher than long period of time (10~20min.). (Key words : bovine embryos, co-culture, freezing, in vitro development)

• 한국가축번식학회지
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• 제14권1호
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• pp.17-22
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• 1990

The present study was carried to investigate the development potential of bovine follicular oocytes matured and fertilized in vitro. Bovine oocytes matured in vitro were fertilized, cultured, and transfered to the rabbit oviduct for in vitro culture to evaluate the development potential. The rates of in vitro maturation, fertilization and polyspermy were 96%(355/369), 90%(320/355) and 15%(17/320), respectively. The percentage of oocytes cleaved after culture for 48 hours, to 2 cell, 3~4 cell, 6~8 cell stage were 17%(60/356), 21%(75/356) and 19%(67/356), respectively and overall cleavage rate was 57%(202/356). The rate of recovered embryos after 5 days in rabbit oviduct was 56%(80/142), and 21%(17/80) of recovered embryos developed over morula stage.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2003년도 생물공학의 동향(XIII)
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• pp.113-117
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• 2003

모낭은 복잡한 상호작용을 통하여 성장 및 주기가 조절되며 다양한 세포들로 구성진 기관이다. 본 연구에서는 조직공학적 방법을 이용하여 모낭을 구성하는 세포들을 분리 및 배양하였고 세포들의 형태와 증식양상을 관찰할 수 있었고, 기관배양의 방법을 통하여 in vitro에서 첨가물에 대한 모발의 성장 및 주기의 변화를 살펴보았다.

• 한국수정란이식학회지
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• 제8권2호
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• pp.143-149
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• 1993

The objective of this study was to produce calves derived from in vitro fertilization of in vitro matured follicular oocytes. Oocytes aspirated from small antral folicles of ovaries obtained at a local slaughter house were matured and fertilized in vitro. At l8hrs after insemination with Korean native cattle semen, oocytes were co-cultured for 6~7 days by utilizing co-culture system with bovine oviduct epithelial cell. After co-culture, good or excellent quality late morulae or early blastocysts were selected by morphological criteria under stereo microscope. Selected embryos were transferred to recipients on day 6 or 7 (estrus = day 0). Recipients were monitored by observation for estrus and rectal palpation after 60 days from embryo transfer. One of them went to term with the birth of a calf. This case is the first production of calf derived from in vitro fertilization in Korea.

• Clinical and Experimental Reproductive Medicine
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• 제27권4호
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• pp.359-366
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• 2000

The present studt was performed to investigate the effect of treatment and samples of human follicular fluid (hFF) on the development in vitro of mouse embryos. The two cell stage embryos collected at 40 h post-hCG injection were cultured in the modified human tubal fluid (m-RTF) containing 15% synthetic serum substitute (SSS) or human tubal fluid (hFF) for up to 3 days at $37^{\circ}C$ in 5% $CO_2$ incubator. Also the composition of hormone, total protein and protein pattern of hFF samples were analyzed. The developmental rate of mouse embryos developed to blastocyst were not significant difference in the m-RTF containing 15% hFF filtered with 0.22 or 0.8 ${\mu}m$ syringe filter, however, the embryos cultured in the m-RTF containing inactivated hFF were significantly (p<0.05) developed at the high rate to blastocyst than those containing fresh hFF and SSS. The in vitro developmental rate to blastocyst and hatched blastocyst in the m-RTF containing 15% hFF sample A (90.5 and 85.4%, respectively) and SSS (79.4 and 75.3, respectively) were significantly (p<0.05) increased, compared with hFF sample B (64.2 and 54.1 %, respectively). The hFF sample A tended to be higher concentration of LH, FSR, total protein and the ratio of progesterone/$E_2$ and lower concentration of $E_2$ and progesterone than the hFF sample B, but there were no differences in the protein pattern between the two hFF samples. The results of these study suggest that the addition of hFF to the culture medium enhances the development in vitro to blastocyst and hatched blastocyst, but the in vitro developmental rate of mouse embryos is different between hFF samples.

• Reproductive and Developmental Biology
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• 제31권1호
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• pp.7-13
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• 2007

The present study investigated the effects of follicle stimulating hormone (FSH) and human chorionic gona-dotrophin (hCG) on the nuclear maturation of canine oocytes. Oocytes were recovered from mongrel female ovaries in various reproductive states; follicular, luteal or anestrous stage. Oocytes were cultured in sserum-free tissue culture medium (TCM)-199 supplemented with various concentrations of FSH (Exp. 1: 0, 0.5, 1.0 or 10 IU) or hCG (Exp.2:0, 0.5, 1.0 or 10 IU) or both (Exp. 3:1 IU FSH +1 IU hCG) for 72 hr to determine the effective concentration of these hormones, and to examine their combined effect. After maturation culture, oocytes were denuded in PBS containing 0.1% (w/v) hyaluronidase by gentle pipetting. The denuded oocytes were stained with $1.9\;{\mu}M$. Hoechst 33342 in glycerol and the nuclear state of oocytes was evaluated under UV light. More (p<0.05) oocytes matured to MII stage when follicular stage oocytes were supplemented with 1 IU FSH (6.2%) compared with the control, 0.1 or 10.0 IU FSH (0 to 1.2%). Significantly higher (p<0.05) maturation rate to MII stage was observed in follicular stage oocytes supplemented with 1.0 IU hCG (7.2%) compared with the control or other hCG supplemented groups (0 to 1.5%). However, the combination of FSH and hCG did not improve the nuclear maturation rate of canine oocyte (2.4 %) compared with FSH (6.2%) and hCG alone (7.2%). In conclusion, FSH or hCG alone significantly increased the maturation of canine oocytes to MII stage.

• 한국수정란이식학회지
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• 제16권2호
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• pp.99-106
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• 2001

본 연구에서 도축된 한우 난소를 aspiration법과 면도날 장착으로 제작된 기구를 이용해서 slicing 법으로 난포란을 회수하여 그에 따른 난포란의 회수율과, 난포란을 체외에서 22시간 성숙시킨 후 난포란의 핵 성숙을 및 체외수정 후 수정율과 발달율을 요약하면 다음과 같다. 1. 난포란의 회수율에 있어서 각 난소당 회수는 aspiration법이 6.7개. slicing법이 15.1개의 결과를 나타내어 난소에 대한 난포란의 회수율은 slicing법이 유의적 (P<0.05)으로 높은 결과를 나타내었다. 2 Aspiration법과 slicing법으로 채란된 난자를 체외에서 22시간 성숙시킨 후, 제 2감수분열 중기까지의 핵 성숙율은 각각 83%와 62%로 유의적인(P<0.05) 차이를 보였으나, 난소 한 개당 제 2감수분열 중기까지 성숙된 난포란수는 aspiration과 slicing법이 각자 5.6개와 9.4개로 slicing법에 의해서 유의적 (P<0.05)으로 많았다. 3. Aspiration법과 slicing법으로 채란된 난포란의 발달을 조사결과를 살펴보면 분할율과 배반포기까지의 발달율에서 aspiration법이 유의적 (P<0.07)으로 높게 나타났으나, 이와 상반되게 난소 한 개당 생산된 배반포기 수정란의 수는 slicing법에 있어서 2.8개로 aspiration법의 2.1개보다 유의적인 (P<0.05) 증가를 보였다. 이상의 결과에 따라 많은 난자를 이용하기 위한 목적과 혹은 이식 가능한 다수의 수정란 생산을 위해서는 본 연구에서 고안 제작된 기구를 이용하여 slicing법으로 난포란을 채란하는 것이 효과적인 방법이 될 것으로 사료된다.

• 한국수정란이식학회지
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• 제12권3호
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• pp.293-300
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• 1997

Supplementation of maturation medium with additional granulosa cells has beneficial effect on in vitro maturation of bovine follicular oocytes and their subsequent cleavage and development in vitro. However, maturation system using granulosa cells have some disadvantages that collection of granulosa cells is cumbersome and metabolic activity of the cells is variable according to ovarian cycle or follicular size. We hypothesized that bovine immsture oocytes matured without granulosa cell coculture can fertilize and develop normally if the medium volume per oocyte is reduced during in vitro maturation. Immature oocytes were matured for 24 hours in a TCM199 containing 10% fetal calf serum, anterior pitultary hormone (0.02 AU /ml Antrinⓡ) and estradiol with or without granulosa cells in vitro. In Group 1, 35 to 40 oocytes were matured in a well of 4-well plastic dish containing 500 $\mu$l of maturation medium and granulosa cells, and 9 to 10 oocytes were matured in a 50-$\mu$l drop of maturation medium without granulosa cells in Group 2. After maturation, oocytes were coincubated with sperm for 30 hours in a modified Tyrode's medium (IVF). Inseminated oocytes were cultured in a microdrop (30 $\mu$l) of a synthetic oviduct fluld medium (SOFM) containing BSA, Minimum Essential Medium essential and non-essential amino acids for 9 days. As a preliminary experiment, we investigated the beneficial effect of granulosa cells during maturation on subsequent cleavage and development using the same type of culturedishes (4-well dish). Granulosa cells could not increase embryo cleavage after fertilization but significantly improved (p<0.05) embryo development to expanding blastocyst (Table1 and 2). In Group 1, 68 and 80% of inseminated oocytes have cleaved at 30 hours and 2 days after IVF, respectively, which is similar (p>0.05) to the result of Group 2 (69% at 30 hours and 78% at 2 days after IVF). The oocytes in Group 2 showed 21 and 11% of developmental rates to expanding and hatching blastocysts, respectively, which was not significantly different (p>0.05) from those (20 and 10%, respectively) of oocytes in Group 1. In conclusion, it has been clarified that a microdrop culture system without granulosa cells for in vitro maturation can support bovine embryonic development to blastocyst in vitro as readily as a granulosa cell coculture system.