• 한국수정란이식학회:학술대회논문집
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• 한국수정란이식학회 2002년도 국제심포지엄
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• pp.3-7
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• 2002

In vitro embryo culture techniques provide significant contributions not only for a basic research of fertilization and early embryogenesis, but also for a low cost mass production of bovine embryos for transfer, embryo diagnosis, nuclear cloning and the production of transgenic cows. This presentation introduces newly developed serum-free media (IVD101 and IVMD101) that are effective far high yields of transferable embryos of excellent quality from in vitro-matured and fertilized oocytes. Both serum-free media are superior to a conventional serum-containing medium on the increased rates of blastocyst formation, post-thaw embryo viability, and pregnancy after transfer. Furthermore, reduced risks of calf mortality and large calf syndrome are also observed for the serum-free-derived embryos. Serum-derived embryos contain a large number of lipid droplets and immature mitochondria in their cytoplasm that may account for the lower production of transferable embryos and poor embryo quality.

• 한국수정란이식학회지
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• 제13권2호
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• pp.147-157
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• 1998

This study was conducted to investigate the effects of in vitro fertilization, culture and embryo development according to in vitro maturation rate, protectant composition and equilibrium time after frozen /thawing of bovine immature oocytes. This results obtained in studies on the effect of different cryoprotectants on the viability, maturation and development of in vitro bovine oocytes were as follow: 1.The post-thawing of immature oocytes matured to metaphase II during culture time for 0 to 26 h, and those group (62~3%) were low than control group (76.7%). The optimal maturation time of frozen-thawed immature oocytes was at 24 h. 2.The viability of cryopreserved immature oocytes was not affected by sort of cryoprotectants. The developmental competence of frozen4hawed oocytes was not affected by cryoprotectants. These results indicate that an optimal maturation time of frozen /thawed immature oocytes was at 24h. Furthermore the viability of cryopreserved immature oocytes was not affected by sort of cryoprotectants and developmental competence of frozen /thawed oocytes.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2000년도 국제심포지움
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• pp.5-6
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• 2000

Nitric oxide (NO) is a simple combined molecule of oxygen and nitrogen, and has a wide variety of action on the physiological and pathophysiological function of the body. It is a key transducer of the vasodilator message from the endothelium to vascular cells. However, its different roles have been elucidated by numerous researches, which was undertaken in the 80's and 90's. Three types of NO synthase were involved in synthesizing NO and they are identified in different tissues and cells including macrophage, endothelial cells and even tumor cells. In the late 90's, we undertook a number of researches for elucidating the effect of NO on embryo development, since developmentally arrested bovine embryos contained large amount of NO metabolites in their cytoplasm. Subsequently, we found that the addition of a spontaneous NO donor to culture medium markedly inhibited embryo development and that its inhibitory role was independent of embryonic genome activation. Research was focused to find a way to prevent the inhibitory action of NO on embryo development and demonstrated that the addition of hemoglobin, a NO scavenger, to embryo culture medium greatly stimulated in vitro-development of bovine and mouse embryos. Based on these research outcomes, we developed a NO action-free culture system for embryos and other tissues. The efficacy of such system has subsequently been confirmed by achieving the high rates of preimplantation development and blastocyst formation in the NO action-free culture of mouse and bovine embryo. In this article, we briefly introduced the nature of NO and our research outcomes on the role of NO in embryo development.

• 농업과학연구
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• 제43권1호
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• pp.52-60
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• 2016

In animal reproduction, the quality of oocytes and embryos has been evaluated by the expression of specific molecules. Follistatin (FST), which was isolated from follicular fluid, binds and bio-neutralizes the TGF-${\beta}$ superfamily members. Previous studies using the bovine model showed FST could be an important molecular determinant of embryo developmental competence. However, the effect of FST treatment on porcine embryo developmental competence has not been established. In this study, the effect of exogenous FST on porcine embryo developmental competence was investigated during in vitro culture. FST (10 ng/ml) treatment induced a significant decrease in the rate of cell arrest at the 4-cell stage. The expression levels of DNA-methyltransferase 1 (DNMT1), histone deacetylase 1 (HDAC1), and histone deacetylase 2 (HDAC2) were decreased in 4-cell stage embryos. FST treatment also resulted in significant improvements in developmental competence of embryos in terms of blastocyst formation rate and OCT-4 mRNA levels, the latter being related to pluripotency. In conclusion, during in vitro culture, FST treatment significantly ameliorated 4-cell block during embryonic development and improved embryo developmental competence. Therefore, FST treatment may potentially have a functional role in porcine embryogenesis that is broadly applicable to enhance in vitro embryo development.

• 한국수정란이식학회지
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• 제15권3호
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• pp.271-277
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• 2000

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 $\mu\textrm{g}$/m1 epidermal growth factor (EGF) at 39$^{\circ}C$ in a humidified atmosphere of 5% $CO_2$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 50$\mu$l 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 50$\mu$l droplet of culture medium. Furthermore there is no effect of different protein sources on early embryonic development.

• 한국수정란이식학회지
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• 제12권2호
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• pp.181-188
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• 1997

This study was carried out to determine the effect of bovine follicular fluid(bFF), hormones, and fetal bovine serum(FBS) supplemented in the medium on the in vitro fertilization and development of bovine embryos. The ovaries were obtained from a local abattoir and placed in physiological saline kept at 30~32˚C and brought to the laboratory within 3~4 hours. The oocytes and follicular fluid were collected by aspiration from visible follicles, and the oocytes of grades I on the basis of the morphology of cumulus cells attached and the homogeneity of cytoplasmic granules were selected and used for maturation. The basal media used for oocyte maturation, fertilization and embryo development in vitro were Ham' F-10, TALP and TCM-199, respectively. The hormones supplemented in maturation medium were consisted of 35 pg /ml FSH, 10 pg /ml LH and 1 pg/mi estradiol-l7$\beta$. The bFF collected from 5~9 mm follicles was centrifuged, filtered and inactivated by heat-treatment at 56˚C for 30 min. FBS also was inactivated with the same method and kept at -20˚C until use. The embryos were co-cultured with the monolayer of bovine oviductal epithelial cells at 39˚C under 5% $CO_2$ in air for 9 days. The results obtained were summarized as follows: The fertilization rate of oocytes was found 87.4% from 10% FBS and hormones treatment for IVM, and 37.1% of these TVF embryos were developed to blastocyst stage in 10% FBS groups. Compared with this control system, the fertilization rate was decreased significantly(P<0.05) in the maturation without either FBS or hormones. These IVF embryos were developed to morula stage at the similar rate, but to blastocyst at significantly(P<0.05) lower rate in the embryo culture with or without FBS supplementation. The fertilization rate(82.9%) in hormones and 10% inactivated bFF was similar with 10% FBS and hormone groups(87.4%), but decreased significantly(P<0.05) in 20 or 30% bFF (61.0 or 66.0%), respectively. In vitro developmental competence to blastocyst stage in 10% FBS and 20% inactivated bFF(37.1% and 31.4%) was higher than in 10 or 30% inactivated bFF(20.0 or 19.2%) or 10, 20 and 30% fresh bFF(19.1, 21.0 and 17.5%) The results indicated that the in vitro fertillzation and development rate of the embryos should be improved in 10% FBS or 20% inactivated culture system and 20% inactivated bFF might be available economically for bovine oocyte maturation and embryo culture instead of fetal bovine serum.

• 한국가축번식학회지
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• 제22권1호
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• pp.35-42
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• 1998

This experiment was carried out to improve in vitro development of rabbit one-cell embryos to the blastocyst stage. One-cell rabbit embryos were collected at 19\ulcorner20hr after superovulation induction and incubated at 39\ulcorner in 5% CO2 for 72hr. In order to find optimum conditions in medium that affects the rabbit embryo's development in vitro, RDH medium which mixed with RPMI1640, DMEM and Ham's F10 was compared with the previously reported mediums (Ham's F10 and RD) for embryo development and cell numbers. Three additives (BSA, taurine and glucose) were tested for the development of rabbit one-cell embryos in vitro. When the embryos were cultured in RDH medium, their development was markedly promoted as compared with Ham's F-10 or RD alone. Glucose exhibited no significant effects on embryo development and cell numbers. BSA a, pp.ared to promote transition from morula to blastocyst stage and taurine increased cell numbers of cultured embryos markedly regardless of medium. BSA and taurine together in RDH medium showed the additive effects on embryos development and cell number.

• 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.

• 한국산학기술학회논문지
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• 제20권9호
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• pp.510-516
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• 2019

본 연구는 염소 수정란의 체외배양용 배지에 FBS, gBS 및 PVA 첨가시 배발달 및 apoptosis 발생률을 분석하여 체외배양시 각 첨가물의 효과를 조사하였다. 체외성숙 후 체외수정을 실시하여 체외배양 배지에 10% FBS, 10% gBS 및 10% PVA를 첨가하고 배발달 효율과 배반포의 품질을 확인하기 위해 TUNEL assay를 통해 apoptosis 발생 비율을 조사한 결과, 난할률 및 배반포 형성률에서 처리군 모두 대조군(무혈청) 보다 유의적으로 높은 결과를 보였다. 특히 gBS와 PVA 처리군에서 각각 31.95, 35.29%로 유의적으로 가장 높은 배반포 형성률을 보였다. 또한 TUNEL assay의 결과에서도 배발달 비교실험에서와 같이 대조군에서 가장 적은 총세포수와 가장 높은 apoptosis 비율을 보였으며, gBS와 PVA 처리군에서 가장 많은 총세포수 및 가장 적은 apoptosis 비율을 보였다. 염소의 체외배양 효율 향상을 위한 본 연구의 결과, 무혈청 배지나 FBS첨가 보다는 gBS나 PVA의 첨가가 배발달 효율뿐만 아니라 배반포 품질에도 긍정적임을 알 수 있었으나, gBS내 확인되지 않는 물질들의 위험성을 감안했을 때 PVA가 보다 안전하고 효율적일 것으로 생각된다.

• 한국수정란이식학회지
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• 제18권3호
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• pp.237-242
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• 2003

본 연구에서는 소에 있어서 수정란 이식을 다각적으로 분석하고 개선하기 위해 이식한 결과를 비교 검토하였고 그 결과는 다음과 같다. 1. 채란된 한우 난포란을 이용하여 생산된 체외수정란을 서로 다른 지역별로 이식한 바 B지역(경주)에서는 106의 수란우에 이식하여 51두(48.1％)가 수태하였고, A지역(김천) 수태율(33.8％)과 D 지역(경산) 수태율(35.3％)보다 유의하게 높았지만, C 지역(탑리)에서는 유의차가 존재하지 않았다. 따라서, 수정란이식에 있어서 이식 지역과 사육 환경, 시술자의 숙련도에 따라 수태율이 달라질 수 있다는 것을 알 수 있었다. 2. 수란우의 산차에 따른 수태율은, 미경산우 42.9％로서 경산우의 36.6％에 비해 다소 높은 수태율을 보였으나 유의적인 차이는 인정되지 않았다(P<0.05). 3. 이식 수정란의 발육단계는 중기 배반포 또는 후기 배반포로서 구분하여 수란우에 이식하였다. 중기 배반포가 45.5％, 후기 배반포 41.0％로서 중기 배반포가 후기 배반포보다 높은 수태율을 나타내었지만 유의한 차이는 인정되지 않았다. 배반포기가 생성된 날짜에 따른 수란우의 수태율은 7일째 생성된 배반포 이식시 수태율은 43.8％, 8일째 생성된 배반포 이식시 수태율은 32.9％, 9일째 생성된 배반포 이식시 수태율은 20.0％로서 7일째 배반포의 수태율 이 다른 두 처리군보다 수태율은 높았지만, 유의차는 인정되지 않았다.