This study was conducted to establish the optimal culture conditions for in vitro production of bovine embryos derived from slaughter house ovaries. Cumulus-oocyte- complexes (COCs) collected by aspiration from follicles of 2~7 mm in diameter were matured in Ham's F-10 medium supplemented with 0.01 /m1 epidermal growth factor (EGF) at 39 in a humidified atmosphere of 5% in air. After 24 hrs of culture, the oocytes were co-cultured with epididymal sperm selected off by Percoll-density gradient in TALP medium for 24 hrs. The presumptive zygotes were cultured in HECM-6 medium for 3 d post-insemination, and followed by cultured in TCM199 medium until 7 to 10d post-insemination. The cultures were compared of their cleavage and development into later stage in culture medium by additions of different protein sources (PVA, BSA and BCS) and by different embryo density. The rates of cleavage and development rates into blastocyst were not significantly (P<0.05) different among the culture media containing with BSA (75.0% and 40.5%), BCS (76.7% and 38.0%) and PVA (72.5% and 42.2%), respectively. Significantly (P<0.05) higher blastocysts rates were obtained in culturing of 30 and 40 embryos in each 50l droplets of culture medium than in 5, 10 and 20 embryos. These results indicate that the optimal density of embryos is 30~40 embryos in a 50l droplet of culture medium. Furthermore there is no effect of different protein sources on early embryonic development.

the present study was carried out to develop a completely defined culture system and determine if high NaCl concentrations in defined (PVA added) or semi-defined (BSA added) medium is toxic to bovine embryos. Oocytes from slaughterhouse ovaries were matured and fertilized in vitro. After 30 h of insemination, only 2-cell stage embryos were selected and cultured for this experiment. The culture media used were as follows : TLP(114 mM of NaCl) + BSA (3 mg/ml), TLP + PVA (1 mg/ml), mTLP(96 mM of NaCl) + BSA, mTLP + PVA. Six to ten embryos were placed into a 301 drop of each medium and the embryos were examined at 10 day post-insemination without medium renewal. The experiment was replicated 4 times. All data were analyzed by chi-square. There were no significant differences among TLP-BSA, mTLP-BSA and mTLP-PVA in blastocyst development (21.6, 17.2 and 20.2%), respectively. Also, no differences were obtained in hatching rates (11.7, 9.9 and 12.2%), respecitively. However, there were significant differences between TLP-PVA (1.7% and 0.6%) and other group in blastocyst formation and hatching rates, respectively (p<0.01). Development of in vitro produced embryos cultured in BSA containing medium was not affected by high NaCl concentration, but in the completely defined medium, embryonic development was highly affected by NaCl. This study shows that reduced NaCl concentration in completely defined medium is beneficial for development of bovine pre-implantation embryos in vitro.

This study was conducted to establish the optimal conditions for in vitro embryo production using oocytes derived from follicles of slaughter-house ovaries. The ovaries of Hanwoo were obtained from a local slaughter-house. The oocytes were aspirated from visible follicles of 2~7mm in diameter. The recovered oocytes which were completely surrounded by at least 2 layers of cumulus cells and a homogeneous cytoplasmic pigmentation were used. The selected oocytes were washed 3 or 4 times with D-PBS containing 10% bovine calf serum (BCS) and matured in vitor (IVM) in Ham's F-10 supplemented with 10% BCS or 0.01 /ml epidermal growth factor(EGF) at 39 under 5% CO2 in air for 24 hours. They were fertilizqed in vitro (IVF) with fresh sperm separated by Percoll density gradient or swim-up in TALP media. The zygotes were cultrued with or without bovine oviductal epitherial cells(BOEC) in media(HECM-6 supplemented with 11 amino acid and / or TCM-199 supplemented with 10% BCS) for 7 to 10 days. The results obtained were as follow: The cleavage rate and developmental rate to blastocyst after maturation and IVF were not significantly different between Ham's F-10 with EGF(76.0% vs. 44.0%) and BCS(75.9% vs. 43.6%)(P<0.05). The cleavage rate and development rate to blastocyst after fertilizing by swim-up or Percoll method were not signifciantly(P<0.05) different between swim-up (80.2% vs. 29.2%) and Percoll(81.9% vs. 26.5%) (P<0.05). The cleavage rate in TCM 199(80.5) was signficiantly higher than that in HECM-6 (72.0%) (P<0.05). However, developmental rate to blastocyst using TCM 199 following HECM-6 for 3 or 4 days (42.2%) was significantly higher than that in TCM-199 alone(26.7%)(P<0.05). The cleavage rate and development rate of embryos produced in vitro by exchange timing for HECM-6 media were not significantly different between in day 3(78.6% vs. 45.5%) and day 4(75.0% vs. 43.2%)(P<0.05). The cleavage rate and developmental rate to blastocysts according to co-culture system were not significantly different between with (74.2% vs. 41.4%) and without BOEC(73.95 vs. 43.5%) (P<0.05). The number of blastomere in blastocyst stage after co-culture with or without BOEC was not significantly different (106.75.1 and 96.64.0). In conclusion, the most transferable IVP embryos could be produced from Ham's F-10 medium for IVM, Percoll density gradient method for IVF sperm separation and in vitro culture in HECM-6 until day 3 or day 4, and then transferred into TCM-199 until day 9 within adequate embryo density in culture droplets after insemination.

This study was carried out to investigate the effect of co-culture system(bovine oviduct epithelial cells; BOEC) and defined culture system(modified TALP ; mTALP) on the development of IVM-IVF embryos, and survival of in vitro produced blastocysts after freezing and thawing. Occytes from the slaugheterhous ovaries were matured and fertilized using general protocol. The results obtained were as the following: 1. Survival rates of frozen-thawed blastocysts using 10% glycerol as cryoprotectant was higher in day 7 blastocysts than in Day 8 and 9 blastocysts from co-cultrue system, but survival rate of frozen-thawed blastocysts was higher in Day 10 blastocysts than in day 8 and 9 blastocysts from defined culture system. Regardless of their age, survival rate of frozen-thawed blastocysts was significantly higher (p<0.05) in co-culture system than in defined culture system. 2. The cell number of blastocysts was significanlty higher (p<0.05) in Day 7 blasotcysts than in Day 8 and 9 blastocysts from co-cultures, but the cell number of blsstocysts was significantly higher (p<0.05) in Day 10 blastocysts than in Day 8 and 9 blastocysts from defined culture system. Regardless of the culture system, blastocysts with higher cell number showed higher survival rates after freezing and thawing.

효율적인 homozygous 동물을 생산하기 위한 실험의 단계로 염색체가 4배체인 수정란의 이용성을 타진하기 위해 생쥐 수정란과 cytochalasin B를 사용하여 4배체 유도에 관한실험을 수행하였다. 생쥐 2-세포기 수정란을 10/ml 농도의 cytochasin B로 약 20시간 배양하였을 때 모든 수정란은 발육을 거의 멈추었으나, 이 수정란을 cytochalasin B-free medium에 체외배양하였을 때 발육이 재개되어 48시간 후 상실기나

To establish the optimal culture systems for production of transferable embryos in Korean Cattle, pregnancy rates of IVF-derived blastocysts according to different culture media, culture method and culture duration were compared. Development of IVF-derived embryos to blastocysts was most effective in YS medium group co-cultre with cumulus cells. Blastocysts cultured for 6 to 8 d in vitro showed higher hatching rate and good quality. Pregnancy rates after transfer of IVF-derived blastocysts cultured for 7 or 8 d were high. Through our experiments, it is considered that improvement of culture media and culture method is necessary for mass production of blastocysts with excellent of good quality in Korean Cattle.

적정 배양액의 선정, ITS의 첨가와 BSA의 농도조절 및 NaCl 농도의 조절을 통해 소 수정란의 무혈청, 체세포배제 배양체계를 확립하기 위하여 수행한 실험에서 다음과 같은 결론을 얻었다. 1. 배양액으로 CRlaa, TALP 및 SOF를 사용하여 발육률을 검토한 결과, 발육률의 유의적인 차이는 나타나지 않았다. 2. 배양액내의 고분자물질원으로 BSA, FBS 및 PVA를 첨가하여 사용한 결과 BSA 및 FBS 첨가군이 PVA 첨가군보다 유의적으로 높

난관상피세포와 그 배양액에서 분비된 고분자 분획이 소정자의 수정능력에 어떠한 영향을 미치는지 알아보고자 실시한 실험에서 다음과 같은 결론을 얻었다. 1. 난관상피세포배양액으로부터 MW 5 kDa cut-off bucket을 사용하여 탈염 및 농축을 실시, 단백질/고분자분획을 회수하고 이를 체외수정용 배양액에 첨가하고 난관상피세포 monolayer와 공배양을 통해 체외수정을 실시한 결과 고분자분획첨가 및 난관상피세포 공배양(OM+OEC)군이 고분자분획첨가

The objective of this study was to evaluate the embryonic development ability and the appearance of blastocysts of bovine in vitro fertilized oocytes cultured in different culture media, and also to evaluate survival rate after thawing of frozen embryos by using 1.5 or 1.8M ethylene glycol(EG) with sucrose or trehalose. Fertilized oocytes were divided into three groups; i ) monolayer of cumulus /granulosa cell prepared by TGM 199+5% calf serum(TGM199), ii)GRlaa+5% CS, iii)SOF+5% CS, and they were cultured after insemination for 9 days, at 39˚C, under 5% in air, but SOF+5% CS was cultured at 39˚C, under 5% 02, 5% GO2, 99% N2. Blastocysts derived from GRlaa + 5% CS on day 7~8 after insemination were frozen by using 1.5M EG or 1.8M EG with/without 0.2M sucrose or O.1M trehalose. The development rate of blastocysts on day 7 after insemination in SOF+5% CS was significant higher than in TCM199 or CR1aa(P<0.05). The appearance rate of blastocysts on day 7-8 after insemination was higher than in TCM199, when fertilized oocytes were cultured in GRlas or SOF. The survival rate of frozen blastocysts after thawing tended to increase, when blastocysts were frozen by using 1.8M EG with 0.2M sucrose or O.1M trehalose. These results indicated that SOF or CRlaa media with amino acids was superior to TCM199 with monolayer in terms of blastocyst development in culturing of in vitro fertilized bovine nocytes, and sucrose or trehalose was supposed to prevent embryos from the freezing shock.

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 l of maturation medium and granulosa cells, and 9 to 10 oocytes were matured in a 50-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 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.