• 한국수정란이식학회지
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• 제22권4호
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• pp.265-269
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• 2007

This study was conducted to investigate the efficacy of vitrification procedure for the cryopreservation of porcine oocytes and the utilization of vitrified oocytes as recipient cytoplasts for somatic cell nuclear transfer (NT), and observed that porcine oocytes are evaluated by pronuclear formation, and parthenogenetic development. Single fetal donor cells were deposited into the perivitelline space of vitrified enucleation oocytes, followed by electrical fusion and activation. NT embryos were cultured in NCSU-23 medium supplemented with 5% FBS, at $38.5^{\circ}C$ in 5% $CO_2$ and air. 1. When the developmental rates of the oocytes after being culture for $0{\sim}10$ hours vitrified with EDS and ETS were 42.0%, 38.0%, respectively. This results were lower than the control group(62.2%). 2. When the developmental rates of the oocytes after being culture for $0{\sim}10$ hours vitrified-thawed with sucrose and glucose, 5% PVP, NCSU-23 supplemented with 10% FBS were 33.3%, 25.9%, respectively. This results were lower than the control group(55.6%). 3. The fusion and development to the blastocyst stage between the NT embryos constructed with the vitrified and non-vitrified oocytes were significant differences. Developmental rate of oocytes and NT embryos constructed with the vitrified or non-vitrified oocytes were $13.0{\pm}2.4%\;and\;23.2{\pm}2.4%$, respectively.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2002년도 춘계학술발표대회 발표논문초록집
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• pp.58-58
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• 2002

Currently, the technique of pronuclear microinjection is the most successful and most widely-used method for producing transgenic animals. Among this technique, surperovulation and embryo transfer are the crucial steps for obtaining a large number of fertilized eggs and birth as much as potential founders from the transferred embryos. (omitted)

• 한국가축번식학회지
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• 제21권4호
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• pp.373-379
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• 1997

This study was carried out to determine the hormone treatment scheme for an efficient superovulation and optimal recovery time for obtaining pronuclear embryos suitable for DNA injection in Korean native goats. For a superovulation, FSH(5.6mg) was given over four days in twice daily injections with (FSH/hCG group) or without(FSH group) hCG(100 IU) co-injection at the time of 7th FSH injection. Estrus cycle was synchronized by norgestomet ear-implantation for 11 days and its removal at the time of 6th FSH injection. Among the treated goats, the percentage of ovulated goats, which were examined at 70 to 76 h following implant removal, was greater in FSH/hCG group than in FSH group (100% vs 36.4%) but there was no significant difference in the mean numbers of ovulation points and fertilization rates between the two groups. To optimize hCG treatment scheme and recovery time, we injected hCG at the time of 7th (FSH/hCGa) or 8th(FSH/hCGb) FSH injection and then examined the developmental stage of the embryos recovered at different times after implant removal. In FSH/hCGa group, significant portions(31 to 44%) were beyond 1-cell stage, which was non-injectable, irrespective of their recovery time. However, in FSH/hCGb group recovered at 70 to 76 h after implant removal, great portions(69%) were fertilized and most of them(96.6%) were injectable 1-cell stage. Considering together the fertilization rate and developmental stage of recovered embryos, it is recommendable to administrate hCG at the time of final 8th FSH injection and collect the embryos at 70 to 76 h after implant removal to obtain injectable embryos as many as possible in Korean native goats.

• Asian-Australasian Journal of Animal Sciences
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• 제19권2호
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• pp.171-175
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• 2006

The aim of this in vitro study was to test the effect of microinjection (Mi) of foreign gene into the rabbit egg pronucleus and epidermal growth factor (EGF) addition on the blastocyst rate, the cell number and the diameter of embryos, and to determine possible relationships between embryo cell number and embryo diameter. Blastocyst rate was significantly decreased in gene- Mi (G-Mi/E0) group (63.1%) comparing to intact ones (83.5%, $p_1$<0.05). The addition of EGF at 20ng/ml (G-Mi/E20) or 200 ng/ml (GMi/ E200) to gene-Mi embryos did not affect blastocyst rate (65.6 and 55.2% resp.). As a control for Mi, the eggs were microinjected with the same volume of phosphate-buffered solution (PBS-Mi) instead of the gene construct solution. Cell numbers and embryo diameters were measured from embryo images obtained on confocal laser scanning microscope. Bonferroni-modified LSD test showed that the embryo cell number in PBS-Mi group was significantly lower ($p_1$<0.05) and in gene-Mi group was tended to decrease compared with intact embryos. Embryo diameter was not different among experimental groups. No effect of EGF given at any doses both on the cell number and embryo diameter was found. A positive correlation between cell number and embryo diameter was observed in all groups of embryos. Since embryo diameter was not changed under the influence of Mi or EGF addition in this study, this seems to be more conservative characteristics of the embryo morphology. These results suggest that the pronuclear microinjection compromises developmental potential of embryos, decreasing blastocyst rate and embryo cell number, whilst embryo diameter is not affected. No effects of EGF on studied parameters were confirmed. Declined quality of Mi-derived embryos is caused by the microinjection procedure itself, rather than by the gene construct used.

• Clinical and Experimental Reproductive Medicine
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• 제50권4호
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• pp.244-252
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• 2023

Objective: We evaluated the efficacy of the newly developed optimized in vitro culture (OIVC) dish for cultivating preimplantation mouse embryos. This dish minimizes the need for mineral oil and incorporates microwells, providing a stable culture environment and enabling independent monitoring of individual embryos. Methods: Mouse pronuclear (PN) zygotes and two-cell-stage embryos were collected at 18 and 46 hours after human chorionic gonadotropin injection, respectively. These were cultured for 120 hours using potassium simplex optimized medium (KSOM) to reach the blastocyst stage. The embryos were randomly allocated into three groups, each cultured in one of three dishes: a 60-mm culture dish, a microdrop dish, and an OIVC dish that we developed. Results: The OIVC dish effectively maintained the osmolarity of the KSOM culture medium over a 5-day period using only 2 mL of mineral oil. This contrasts with the significant osmolarity increase observed in the 60-mm culture dish. Additionally, the OIVC dish exhibited higher blastulation rates from two-cell embryos (100%) relative to the other dish types. Moreover, blastocysts derived from both PN zygotes and two-cell embryos in the OIVC dish group demonstrated significantly elevated mean cell numbers. Conclusion: Use of the OIVC dish markedly increased the number of cells in blastocysts derived from the in vitro culture of preimplantation mouse embryos. The capacity of this dish to maintain medium osmolarity with minimal mineral oil usage represents a breakthrough that may advance embryo culture techniques for various mammals, including human in vitro fertilization and embryo transfer programs.

• Reproductive and Developmental Biology
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• 제32권1호
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• pp.15-20
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• 2008

Pronuclear DNA microinjection has been the most universal method in transgenic animal production but its success rate of transgenesis in mammals are extremely low. To address this long-standing problem, we used retrovirus- and lentivirus-based vectors carrying the enhanced green fluorescent protein (EGFP) gene under the control of ubiquitously active cytomegalovirus (CMV) promoter to deliver transgenes to bovine embryos. The rate of transgenesis was evaluated by counting EGFP positive blastocysts after injection of concentrated virus stock into the perivitelline space of the bovine oocytes in metaphase II. Among two different types of lentivirus vectors derived from FIV (feline immunodeficiency virus) and HIV (human immunodeficiency virus), the former scored the higher gene transfer efficiency; almost 100% of the blastocysts developed from the oocytes infected with FIV-based vector were EGFP positive. As for the vectors derived Com HIV lentivirus, the transgenesis rate of the blastocysts was reduced to 39%.

• Asian-Australasian Journal of Animal Sciences
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• 제21권3호
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• pp.358-363
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• 2008

The intracytoplasmic sperm injection (ICSI) procedure has recently been utilized to produce transgenic animals and may serve as an alternative to the conventional pronuclear microinjection in species such as pigs whose ooplasm is opaque and pronuclei are often invisible. In this study, the effects of sperm membrane disruption and electrical activation of oocytes on in vitro development and expression of transgene green fluorescent protein (GFP) in ICSI embryos were tested to refine this recently developed procedure. Prior to ICSI, sperm heads were treated with Triton X-100+NaCl or Triton X-100+NaCl+NaOH, to disrupt membrane to be permeable to exogenous DNA, and incubated with linearized pEGFP-N1 vector. To induce activation of oocytes, a single DC pulse of 1.3 kV/cm was applied to oocytes for $30{\mu}sec$. After ICSI was performed with the aid of a micromanipulator, in vitro development of embryos and GFP expression were monitored. The chemical treatment to disrupt sperm membrane did not affect the developmental competence of embryos. 40 to 60% of oocytes were cleaved after injection of sperm heads with disrupted membrane, whereas 48.6% (34/70) were cleaved without chemical treatment. Regardless of electrical stimulation to induce activation, oocytes were cleaved after ICSI, reflecting that, despite sperm membrane disruption, the perinuclear soluble sperm factor known to mediate oocyte activation remained intact. After development to the 4-cell stage, 11.8 (2/17, Triton X-100+NaCl+NaOH) to 58.8% (10/17, Triton X-100+NaCl) of embryos expressed GFP. The expression of GFP beyond the stage of embryonic genome activation (4-cell stage in the pig) indicates that the exogenous DNA might have been integrated into the porcine genome. When sperm heads were co-incubated with exogenous DNA following the treatment of Triton X-100+NaCl, GFP expression was observed in high percentage (58.8%) of embryos, suggesting that transgenic pigs may efficiently be produced using ICSI.

• Asian-Australasian Journal of Animal Sciences
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• 제20권5호
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• pp.718-724
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• 2007

Considerable attention has been focused on the cloning of mammalian embryos, as a consequence of poor development, in order to enhance the application of genetic engineering. Experiments were conducted to compare the developmental competence of parthenotes and reconstructed (NT) rabbit eggs with fetal fibroblasts (FFs) following various activation regimens. Oocytes and NT eggs were exposed to: electric stimulation (EST, Group 1) and EST followed by 6-dimethylaminopurine (DMAP, Group 2), cycloheximide (CHX, Group 3) or DMAP/CHX (Group 4). Pronuclear (PN) status, cleavage, blastocyst development and the ploidy were assessed. In parthenote groups 1, 2, 3 and 4, the PN formation differed significantly. And, the cleavage and blastocyst rates were 41.7 and 5%, 75.6 and 53.7%, 68 and 36%, 82.1 and 52.6%, respectively, among treatments. Polyploidy was observed in 17.2% of EST plus DMAP and 44.9% of EST plus DMAP/CHX groups. In SCNT groups (Group 1, 2, 3 and 4), the cleavage and blastocyst rates were 28.6 and 7.1%, 58.3 and 29.2%, 56.8 and 24.1%, 64.5 and 27.8%, respectively. The chromosomal composition differed significantly (p<0.05) among treatments. In Group 2 and 3, 53.8% and 81.8% of embryos revealed diploid chromosomal sets, respectively. However, in Group 4, 53.3% of embryos showed abnormal ploidy (mixoploid). Although DMAP or combination with DMAP/CHX resulted in higher in vitro development of rabbit SCNT embryos, higher incidence of chromosomal abnormality may induce problems related to fetal loss of at late stage of development.

• 한국가축번식학회지
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• 제22권4호
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• pp.419-424
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• 1998

토끼 수정란의 전핵에 MT-hGH 유전자를 주입하고 핵이식 기법으로 형질전환 복제수정란의 생산효율과 PCR 검색으로 복제수정란에서 유전자존재 여부를 조사한 바 다음과 같은 결론을 얻었다. 1. MT-hGH 유전자를 주입하여 8- 및 16- 세포기로 자란 수정란을 공핵란으로 사용하여 핵이식을 실시하였던 바, 세포융합률은 각각 60.0%, 62.8% 로 비슷하였으나 정상수정란을 공급핵으로 사용한 80.4% 보다 유의적으로 낮은 융합률을 보였다. 그러나 이들 복제수정란의 체외발달률은 처리군간에 유의적인 차이는 인정되지 않았다. 2. 유전자 주입 후 8- 및 16- 세포기로 자란 수정란의 할구를 이용하여 핵이식으로 복제하고 체외에서 배반포까지 자란 수정란을 PCR -screening으로 유전자를 검출한 결과, 각각 23% 와 33% 의 유전자 양성 수정란을 감별하였다.

• Clinical and Experimental Reproductive Medicine
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• 제39권3호
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• pp.114-117
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• 2012

Objective: It is well known that fresh blastocyst transfer results in better pregnancy outcomes with a smaller number of transferred embryos compared with cleavage stage embryo transfer. However, in terms of frozen-thawed blastocyst transfer, only a few studies are available. We aimed to evaluate clinical outcomes of frozen-thawed embryo transfer (FET) with blastocysts. Methods: Retrospective analysis of FET cycles with blastocysts (B-FET) between Jan 2007 and June 2009 was performed. Age-matched FET cycles with cleavage stage embryos (C-FET) during the same period were collected as controls. A total of 58 B-FET cycles were compared with 172 C-FET cycles and also compared with those of post-thaw extended culture blastocysts from frozen pronuclear stage embryos (22 cycles). Results: There was no difference in the patient characteristics of each group. The embryos' survival rates after thawing were comparable (>90%) and there was no difference in the implantation rate or clinical and ongoing pregnancy rate among the three groups. Conclusion: In FET, blastocyst transfers may not present better pregnancy outcomes than cleavage stage embryo transfers. A further large-scale prospective study is needed.