• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2001년도 춘계학술발표대회
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• pp.20-20
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• 2001

It has been reported that cloning cattle is inefficient. One of the problems was placental abnormality, finally resulting in fetal mortality after transfer of nuclear transfer (NT) bovine embryos. This study was focused on the allocations of embryonic cells to the inner cell mass (ICM) or to the trophectoderm(TE) in NT bovine blastocysts. Somatic cells were derived from a Day 45 fetus of gestation, individually transferred into enucleated oocytes and developed to the blastocyst stage in vitro. Differential staining was used to assess the qualify of blastocysts derived from NT, IVF and in vivo. Development rate of NT embryos to blastocysts (25.0%, 41/164) was similar to that of IVF embryos (28.7%, 49/171). The total cell number of NT blastocysts (101.3$\pm$45.9) was not different compared with that of IVF embryos (107.9$\pm$34.2, P>0.05), but was lower than in vivo embryos (122.5$\pm$21.6, P<0.05). Ratio of ICM/total cells was higher in NT embryos (51.6$\pm$ 18.6%) than in IVF and in vivo embryos (42.3$\pm$ 15.3% and 34.9$\pm$8.9%, respectively) (P<0.05). Most IVF (56.8%, 25/44) and in vivo blastocysts(80.8%, 21/26) was distributed in the proportion of ICM/total cells ranging from 20 to 40% group. However, most NT blastocysts was biased in the 40-60%(34.1%, 15/44) and >60% (31.8%, 14/44) groups. Our findings suggest that placental abnormalities or early fetal losses in the present cloning system may be due to aberrant allocation of NT embryos to the ICM cells.

• 한국수정란이식학회:학술대회논문집
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• 한국수정란이식학회 2002년도 국제심포지엄
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• pp.117-117
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• 2002

The oocytes from most of animal species accumulate genetic information and other necessary materials during oogenesis for the later use in the early development. Over the years oocyte maturation has been studied extensively both in vitro and in vivo. Particularly, maturation of follicular oocyte in vitro becomes one of the important tools for the studies of basic cell biology, the in vitro technology of animal production, and in particular, the somatic cell cloning by nuclear transfer. We examined meiotic maturation and cumulus expansion in the presence of translation or transcription inhibitors for varying periods of in viかo maturation (IVM) of pig oocyte. In Experiment 1, the results revealed that translation and transcription inhibitors inhibited cumulus expansion and meiotic maturation during 35h of IVM. However, 50 to 60% of the oocytes underwent nuclear maturation without cumulus expansion during 75h of IVM. The rest of the oocytes were arrested at metaphase I (40-50%) in the presence of the inhibitors. In Experiment II, the OCCs were exposed to the drugs only for 15h to examine translation and transcription inhibitors on cumulus expansion and meiotic maturation. Transcription inhibitors for 15h did not arrest meiotic maturation when the oocytes were cultured for subsequent, necessary period of IVM, whereas cumulus expansion was completely inhibited, suggesting that initial 15h is critical transcription activity far cumulus expansion. Translation inhibitors for 15h exposure did not alter cumulus expansion and meiotic maturation during subsequent culture in the absence of the drugs. In Experiment III, the OCCs were exposed to the drugs only for later 30h to examine the influence of transcription and translation inhibitors on oocyte maturation. Interestingly, all meiotic maturation underwent normally with full expansion of cumulus. Similar results were obtained from Experiment IV where 5h of exposure from 15 to 20h of IVM culture to the drugs was performed and subsequently cultured for same period in fresh medium. Taken there results together, both transcription and translation are necessary for nuclear maturation and cumulus expansion, and first 15h IVM for cumulus expansion is critical. The arrested oocytes by the drugs were still capable of undergoing nuclear maturation, although cumulus expansion was affected.

• 한국임상수의학회지
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• 제31권4호
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• pp.267-271
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• 2014

개 복제는 성숙된 중기의 난자를 수술적으로 회수하여 바로 사용해야 하기 때문에 다른 번식 보조술에 비하여 체내 난자 성숙의 정확한 예측이 특히 더 중요하다. 따라서 본 연구는 개에서 체내 성숙 난자 회수 시 방사면역분석기법(RIA)과 비교하여 화학발광효소면역분석기법(CLEIA)의 신뢰성을 평가하고, 참고값을 설정하기 위해 실시되었다. 배란일(Day 0) 결정을 위하여 발정전기와 발정기의 혈청 프로게스테론 농도가 RIA와 CLEIA 방법으로 분석되었다. Day 3에 수술적인 방법으로 체내 난자를 회수하였고, bisbenzimidazole로 핵을 염색한 뒤 성숙을 현미경으로 평가하였다. 평균 호르몬 농도는 CLEIA 값 ($7.64{\pm}0.06ng/ml$)이 RIA 값 ($6.46{\pm}0.04ng/ml$, P < 0.0001)보다 유의적으로 높았다. Day 0일 때 CLEIA 값 ($10.01{\pm}0.34ng/ml$)은 RIA 값 ($7.91{\pm}0.14ng/ml$)과 다르지 않았으나, Day -1과 Day 1일 때는 CLIEA ($6.41{\pm}0.15$ and $14.25{\pm}0.44ng/ml$)가 RIA($4.95{\pm}0.10$ and $11.29{\pm}0.34ng/ml$)보다 유의적으로 더 높은 값을 나타내었다. 그러나 두 가지 방법 모두 Day -1에서 Day 2까지 프로게스테론 농도가 유의적으로 점차 증가하였다. 그러므로 CLEIA 방법으로 난자 성숙을 결정하기 위해서는 더 넓은 범위와 높은 참고 값이 고려되어야만 한다.

• 한국동물생명공학회지
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• 제38권2호
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• pp.47-53
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• 2023

Despite numerous advances in in-vitro embryo production (IVP), many documented factors have been shown to influence the development of mammalian preimplantation embryos and the success of IVP. In this sense, elevated levels of reactive oxygen species (ROS) correlate with poor outcomes in assisted reproductive technologies (ART) due to oxidative stress (OS), which results from an imbalance between ROS production and neutralization. Indeed, excessive production of ROS compromises the structural and functional integrity of gametes and embryos both in vivo and in vitro. In particular, OS damages proteins, lipids, and DNA and accelerates cell apoptosis. Several in-vivo and in-vitro studies report an improvement in qualityrelevant parameters after the use of various antioxidants. In this review, we focus on OS and the source of free radicals and their effects on oocytes, sperm, and the embryo during IVP. In addition, antioxidants and their important role in IVP, supplementation during oocyte in vitro maturation (IVM), in vitro culture (IVC), and semen extenders were discussed. Nevertheless, various methods for determining the level of ROS in germ cells have been briefly described. Still, it is crucial to develop standardized antioxidant supplement systems to improve overall IVP success. Further studies should explore the safety, efficacy, mechanism of action, and combination of different antioxidants to improve IVP outcomes.

• 한국수정란이식학회지
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• 제30권4호
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• pp.283-287
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• 2015

The aim of the present study was to investigate the ovulation rate and its relationship to fertilization ability in Landrace, Durock and Crossbred pigs. Gilts were natural mated at a body weight of at least 120 kg under the same hormone treatment. Embryos were surgically collected 1 day after natural mating (Day 0). Embryos derived from in vivo-fertilized oocytes were cultured in medium PZM-3. The ovaries were examined and the pathological findings were recorded. The number of corpus hemorrhagicum was counted, and was assumed to equal the ovulation rate. There was no difference in the number of corpus hemorrhagicum (20.4, 28.8 and 23.2) and ovulation (13.5, 26.8 and 17.2) in the Landrace, Durock and Crossbred pigs. The two pronucleus formation was 76.0, 80.0 and 86.9%. The Day-7 embryos had blastocyst rates of 68.0, 75.0 and 73.9%. There was no difference in the number of total cells and apoptotic cells. In the future, more studies require determining relationships between ovulation and fertilization rate in different species of pigs.

• Animal Bioscience
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• 제34권4호
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• pp.546-557
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• 2021

Objective: If fertilization does not occur within a specific period, the quality of unfertilized oocytes in the oviduct (in vivo aging) or in culture (in vitro aging) will deteriorate over time. Icariin (ICA), found in all species of Epimedium herbs, has strong antioxidant activity, and is thought to exert anti-aging effects in vitro. We asked whether ICA protects oocytes against age-related changes in vitro. Methods: We analyzed the reactive oxygen species (ROS) levels and expression of antioxidant, maternal, and estrogen receptor genes, and along with spindle morphology, and the developmental competence and quality of embryos in the presence and absence of ICA. Results: Treatment with 5 μM ICA (ICA-5) led to a significant reduction in ROS activity, but increased mRNA expression of glutathione and antioxidant genes (superoxide dismutase 1 [SOD1], SOD2, peroxiredoxin 5, and nuclear factor erythroid 2-like 2), during aging in vitro. In addition, ICA-5 prevented defects in spindle formation and chromosomal alignment, and increased mRNA expression of cytoplasmic maturation factor genes (bone morphogenetic protein 15, cyclin B1, MOS proto-oncogene, serine/threonine kinase, and growth differentiation factor-9). It also prevented apoptosis, increased mRNA expression of antiapoptotic genes (BCL2-like 1 and baculoviral IAP repeat-containing 5), and reduced mRNA expression of pro-apoptotic genes (BCL2 antagonist/killer 1 and activation of caspase-3). Although the maturation and cleavage rates were similar in all groups, the total cell number per blastocyst and the percentage of apoptotic cells at the blastocyst stage were higher and lower, respectively, in the control and ICA-5 groups than in the aging group. Conclusion: ICA protects oocytes against damage during aging in vitro; therefore, it can be used to improve assisted reproductive technologies.

• 한국가축번식학회지
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• 제23권1호
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• pp.53-61
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• 1999

본 연구는 생쥐 preantral follicle 의 체외성장 및 성숙에 있어서 gonadotrophins 인 FSH 와 LH 의 효과를 조사하기 위하여 실시하였으며, 그 결과는 다음과 같다. 1. FSH 첨가군들은 대조군에 비하여 유의하게 높은 생존율과 성숙율을 나타냈으며, 100$m\ell$D/$m\ell$ 의 FSH 농도가 preantral follicle 의 체외배양에 적정한 농도인 것으로 나타났다. 2. HMG 첨가군은 FSH 첨가군보다, 통계적 유의차는 인정되지 않았지만, 높은 생존율과 성숙율을 나타냈다. 3. FSH 와 LH 의 첨가비율이 100$m\ell$U/$m\ell$ 대 10$m\ell$D/$m\ell$(10:1) 에서 가장 높은 생존율과 성숙율을 나타냈다. 4. FSH 혹은 HMG 첨가시, 정상적인 oestradiol 과 progesterone 분비양상을 나타냈으며, HMG 첨가군에서 유의하게 높은 농도의 oestradiol 과 progesterone 을 분비하였다. 이상의 결과를 종합해 볼 때, gonadotrophins 은 preantral follicle 의 체외성장 및 성숙뿐만 아니라 steroidogenesis 에서 중요한 역할을 수행한다는 것을 확인할 수 있었다.

• 한국수정란이식학회지
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• 제19권2호
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• pp.81-87
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• 2004

본 연구는 재래산양에 있어서 과배란 처리시 공란 산양의 반복사용에 따른 oocyte의 회수방법과 회수란의 질적 개선방법을 확립하기 위하여 실시하였으며, 성선자극호르몬의 1회 처리와 반복처리, 호르몬제의 종류에 따른 oocyte의 회수율과 등급을 조사하였다. 공시동물은 체중 15~25kg 전${\cdot}$후의 성숙한 미경산 재래산양으로서 발정 동기화를 위하여 CIDR를 10일간 질내에 삽입하고 과배란 처리는 70 mg의 FSH 와 1,000 IU의 PMSG를 투여하여 과배란을 유기하였다. ${PGF_{2\alpha}$ 는 8일째에 투여하여 과배란을 유기하였으며, CIDR는 10일째 제거와 동시에 hCG 400 IU를 투여하였다. 난자의 회수는 hCG 투여 후 35~40시간째에 외과적인 방법으로 실시하였으며, 회수한 난자는 난구세포와 세포질의 부착정도에 따라 4등급으로 분류하여 회수율을 조사하였다. FSH와 PMSG를 투여하여 공란 산양으로 처음 사용하였을 때 49두에서 324(6.63${\pm}$0.63) 개와 27두에서 305(11.30${\pm}$1.29)개의 배란점을 확인하였으며, 이 중 43.8%(142/324)와 69.2%(211/305)가 회수되어 두당 회수율은 2.92${\pm}$0.52 개와 7.81${\pm}$1.14개로서 FSH와 PMSG간에 전체 회수율과 두당 회수율은 PMSG 처리구가 유의적(P<0.05)으로 높았다. 반복 사용하였을 때 FSH와 PMSG 처리구의 두당 배란수는 2.88${\pm}$0.69 개와 7.56${\pm}$1.11 개였으며, 회수율은 각각 17.4%(4/23) 및 44.9%(61/136) 로 PMSG 처리구가 유의적(P<0.05)으로 높았다. 두당 회수율은 0.50${\pm}$0.27개 및 3.39${\pm}$0.86개로서 PMSG 처리구가 유의적으로 높았다. 회수한 난포란의 1 및 2등급에 있어서 공란 산양으로 처음 사용하였을 때 FSH 투여구는 각각 20.6%(GI) 및 14.9%(GII)였으며, PMSG 투여구는 12.9%(GI) 및 14.1%(GII)로 나타났다. 반복 사용하였을 때, 회수한 난포란의 등급은 FSH 투여구는 각각 31.3%(GI) 및 28.1%(GII)였으며, PMSG 투여구는 36.4%(GI), 5.1%(GII)로 나타났다. 이상의 결과로 볼 때 배란된 난자의 회수율은 PMSG 가 FSH보다 높았으며, 난포내 난자의 회수율은 투여 호르몬 및 반복사용 여부에 따른 차이는 없었다.

• Asian-Australasian Journal of Animal Sciences
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• 제12권6호
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• pp.966-975
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• 1999

Technologies on preimplantation porcine embryos have been developed quickly and significantly. Successful development of systems for culture of porcine zygotes to the blastocyst stage has made it possible to utilize follicular oocytes for in vitro production of embryos and thus stimulated research on various embryo technologies. Recent technological development of embryo cryopreservation, separation of X- and Y-bearing spermatozoa and non-surgical embryo transfer has also made it easy to utilize in vivo- and in vitro-produced embryos for artificial manipulation to produce clones and transgenic pigs. Further progress in overcoming various problems associated with each embryo technology will result in acceptable efficiency to utilize porcine embryos with a high or increased quality. Combining these technologies will accelerate further expansion of the swine industry not only for meat production but also for the production of therapeutic recombinant proteins and xonografts.

• 한국수정란이식학회지
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• 제19권2호
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• pp.113-119
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• 2004

본 연구는 재래산양에 있어서 과 배란 처리에 의한 oocyte의 회수방법과 양질의 oocyte 회수 체계를 확립하기 위하여 과 배란 처리 후 회수시간이 난포의 발달과 난자의 회수율에 미치는 영향을 조사하였다. 공시동물은 체중 15~25 kg 전${\cdot}$후의 성숙한 미경산 재래산양으로서 발정동기화를 위하여 CIDR를 10일간 질내에 삽입하고 과배란 처리는 FSH를 CIDR 삽입 8, 9, 10일째에 12시간 간격으로 70 mg을 감량 투여하였다. $PGF_{2\alpha}$는 FSH와 함께 8일째에 투여하였으며, CIDR는 10일째에 제거와 동시에 hCG 400 IU를 투여하였다. 난자의 회수는 hCG 투여 후 29~50시간째에 시간대별로 외과적인 방법으로 실시하였다. hCG 투여 후 회수시간에 따른 in vivo란의 회수에 있어서 각각 5.93${\pm}$2.88(29~34시간), 6.82${\pm}$0.95(35~40시간) 및 7.33${\pm}$1.54개(41~50시간)로서 차이가 없었다. 회수율에 있어서는 35~40시간째에 회수하였을 때가 49.7%로서 29~34시간 및 41~50시간째에 38.2% 및 29.5% 보다 유의적(P<0.05)으로 높았다. 두당 회수 난자 수는 각각 2.27${\pm}$0.76, 3.39${\pm}$0.75 및 2.17${\pm}$1.40개로서 차이가 없었다. hCG 투여 후 29~34, 35~40, 및 41~50시간에 관찰된 난포 수는 두당 11.75${\pm}$2.45, 11.87${\pm}$1.34 및 9.20${\pm}$1.50개였다. 또한 배란된 성숙난자의 채란 율은 각각 70.2, 74.7 및 54.3%로서 41~50시간째에 회수하였을 때가 가장 낮았다. 두당 회수율에 있어서도 8.25${\pm}$1.34, 8.87${\pm}$1.10 및 5.00${\pm}$1.30개로서 회수시간에 따른 유의적인 차이는 없었다. 회수한 난포내 미성숙 난자의 등급에 있어서 회수시간대별 1등급은 각각 24.2, 19.5 및 12.0%였으며, 2등급의 경우는 41~50시간이 4.0%로서 29~34시간과 35~40시간의 14.4% 및 16.2%보다 유의적(P<0.05)으로 낮았다.