• 한국동물생명공학회지
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• 제35권1호
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• pp.94-101
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• 2020

In the early development of parthenogenetic embryo, cytoplasm and nucleic acid fragmentation may be a cause of lower embryo development. The purpose of this study was to evaluate whether embryonic development and apoptosis factors can be reduced by controlling the in-vitro culture environment by the addition of hormones, pregnancy serum and uterine milk. Our study showed that the activity of Casp-3 increased within the cytoplasm when artificially used hormones to induce the incubation environment, and PCNA's manifestation was low. However, the addition of pregnant serum appeared to lower the Casp-3 activity compared to the other groups. In addition, MMP-9 activity was increased and early embryo development and cytoplasmic fidelity were also increased. Therefore, the results of the present study showed that the use of gestational serum in the development of parthenogenetic embryo inhibit apoptosis and increases cytoplasmic reorganization by natural environmental control in in vitro culture.

• 한국수정란이식학회지
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• 제5권2호
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• pp.11-22
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• 1990

Remarkable progress has recently been made in embryo transfer technology, resulting in the birth of IVF and nuclear transfer offsprings in swine. However, further progress of the technology to (I) make a safe, effective and economic estrual-cycle synchronization compound, (2) regulate each step of sperm capacitation (3) induce monospermic fertilization, (4) in vitro grow and mature oocytes, (5) fertilize the oocytes efficently, (6) culture the oocytes to the blastocyst stage in defined media, (7) produce multiply copies of embryos with superior genetic merit, (8) preselect the sex of these superior offsprings, and (9) preserve embryos by freezing and storage in liquid nitrogen is required before this promising technology is applied routinely to swine for practical use.

• 한국가축번식학회지
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• 제21권4호
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• pp.389-396
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• 1997

Arrest in embryo development during in vitro culture has been reported in various mammals. Although some cause of the arrest have been suggested, little is known of the way that can overcome the arrest using in vitro culture system. The antioxidant, $\beta$-mercaptoethanol($\beta$-ME), has been shown to play an important role in embryo development. This study was designed to examine the effect of $\beta$-ME on the developing boving embryos produced in vitro by IVM and IVF. To select a, pp.opriate concentration of $\beta$-ME during whole culture period (7 days), various concentrations (10, 50 and 100$\mu$M) of $\beta$-ME were added to the CZB medium and their effects was significantly higher in 100$\mu$M of $\beta$-ME. The effects on development of embryos cultured with and without somatic cells to blastocyst stage were greater in FCS treatment (56.6 and 29.3%) than in BSA treatment(25.5 and 12.8%). We also evaluated the effects of $\beta$-ME addition on the blastocyst formation when embryos at different stages were exposed to 100$\mu$M $\beta$-ME. $\beta$-ME promoted increased development of embryo to blastocyst stage and the effect was greater in 6-cell to morula embryos than in embryos fewer than 4-cells at the initiation of treatment. The results suggested that $\beta$-ME can improve bovine embryo development by overcoming the arrest in early development.

• Clinical and Experimental Reproductive Medicine
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• 제44권1호
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• pp.40-46
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• 2017

Objective: To describe in vitro development of human embryos derived from an individual with a homozygous pathogenic variant in NLRP7 (19q13.42) and recurrent hydatidiform mole (HM), an autosomal recessive condition thought to occur secondary to an oocyte defect. Methods: A patient with five consecutive HM pregnancies was genomically evaluated via next generation sequencing followed by controlled ovarian hyperstimulation, in vitro fertilization (IVF) with intracytoplasmic sperm injection, embryo culture, and preimplantation genetic screening. Findings in NLRP7 were recorded and embryo culture and biopsy data were tabulated as a function of parental origin for any identified ploidy error. Results: The patient was found to have a pathogenic variant in NLRP7 (c.2810+2T>G) in a homozygous state. Fifteen oocytes were retrieved and 10 embryos were available after fertilization via intracytoplasmic sperm injection. Developmental arrest was noted for all 10 embryos after 144 hours in culture, thus no transfer was possible. These non-viable embryos were evaluated by karyomapping and all were diploid biparental; two were euploid and eight had various aneuploidies all of maternal origin. Conclusion: This is the first report of early human embryo development from a patient with any NLRP7 mutation. The pathogenic variant identified here resulted in global developmental arrest at or before blastocyst stage. Standard IVF should therefore be discouraged for such patients, who instead need to consider oocyte (or embryo) donation with IVF as preferred clinical methods to treat infertility.

• 한국동물학회지
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• 제28권3호
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• pp.179-193
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• 1985

鷄胚의 筋原細胞의 증식과 融合에 미치는 계배 抽出液의 영향을 조사하였다. 1. 배양액내의 계배 추출액의 농도가 높을수록 근원세포의 증식은 촉진되었으며, 반면에 근원세포 융합은 지연되었다. 2. 계배 추출액의 단백질을 Sephadex G-75로 分劃하고, 각 분획을 근원세포의 배양액에 첨가한 결과 分子量 40,000과 22,000 dalton 사이의 분획이 근원세포의 증식과 융합을 촉진시켰다. 3. 계배 추출액의 단백질을 ammonium sulfate로 분획시켜 각 분획을 근원세포의 배양액에 첨가한 결과 70% 이상 포화 용액에서 침전하는 분획이 근원세포의 증식과 융합을 현저히 증가시켰다. 이 유효 분획을 Sephadex G-75로 재차 분획하여 각 분획의 효과를 조사한 결과 근원세포의 증식과 융합을 촉진시키는 분획이 계배 추출액을 Sephadex G-75로 분획하여 얻은 유효 분획과 거의 동일한 효과를 나타내었다.

• 한국수정란이식학회지
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• 제22권4호
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• pp.251-255
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• 2007

This study was carried out to examine on developmental competence of Hanwoo embryos cultured in vitro according to culture conditions and freezing methods. The in vitro developmental competence to blastocyst stage at Day 8 of culture in SOF was significantly (p<0.05) higher than that in CR1aa (30.3% vs. 18.4%). The in vitro developmental rate of morula and blastocysts cultured in group culture was significantly (p<0.05) higher than that in individual culture (41.4% and 36.0% vs. 21.1% and 10.5%, respectively). The cell number of Day 8 blastocysts in group culture was significantly (p<0.05) higher than that in the individual culture ($120.1{\pm}12.8\;vs.\;94.1{\pm}12.1$, respectively). The survival rates of frozen-thawed balstocysts that were exposed in 1.5 M ethylene glycol or 1.5 M ethylene glycol containing 0.1 M sucrose were 77.5% and 78.7%, respectively. The survival rates of blastocysts cultured for 48 h in slow freezing and vitrification was not significantly different (73.3 and 74.0%). In conclusion, in vitro developmental competence of bovine embryos was influenced on the culture medium (SOF) and culture method (Group culture). Survival rate of frozen-thawed of bovine embryos was not influenced on freezing solutions and freezing methods.

• Clinical and Experimental Reproductive Medicine
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• 제27권1호
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• pp.1-7
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• 2000

Objective: Mammalian embryos undergo changes of energy environment for transfer from oviduct to uterus. Also, the human reproductive organ (oviduct, uterus) contains energy sources of different concentration (oviduct - glucose: 0.5 mM, pyruvate: 0.32 mM, lactate: 10.5 mM; uterus - goucose: 3.15 mM, pyruvate: 0.1mM, lactate: 5.87 mM, respectively). This study was conducted to examine the effect of these energy sources added in DMEM with glutamine on the mouse embryo development. Methods: There was used ICR female mouse. Two cell embryos of mouse are collected by method of 'flushing'. Flushing fluid was used Ham's F-10 added to 20% FBS. The collected 2 cell embryos were cultured in media such as Control (only DMEM), group A and B (DMEM supplemented with 0.5 mM and 3.15 mM glucose), and group C and D (DMEM supplemented with 0.1 mM and 0.32 mM pyruvate), and group E and F (DMEM supplemented with 5.87 mM and 10.5 mM lactate). All experimental media supplemented with 20% hFF, respectively. Pattern of embryo development was observed to interval at 24hr during 96hr. Results : The media with glutamine added glucose (group A: 51.0%; group B: 48.4%) was significantly (p<0.05) higher than other experimental group in development into the morula stage after 24 hr in culture, but not significantly different compared with control and the rate of development into the blastocyst was significantly (p<0.05) low in the both of pyruvate (group C: 7.9% group D: 6.8%) and lactate (group E: 7.1%, group F: 7.1%) treatment group after 48 hr in culture. Development into the blastocyst and hatched balstocyst after 72 hr in culture revealed similarly in control (81.9%) and glucose treatment group (group A: 83.3%, group B: 82.8%). However, development into the hatched and attached blastocyst after 96hr in culture revealed significantly (p<0.05) development in the glucose treatment group (group A: 82.3%, group B: 78.5%) than control (63.2%), and its of pyruvate (group C: 34.1%, group D: 34.1%) and lactate (group E: 25.9%, group F: 33.3%) treatment group were significantly (p<0.05) lower than control similar to previous observations. Conclusion : The glucose added to the DMEM with only glutamine, as energy source, was highly to the rate of development compared with control, but the other energy sources were not, synthetically. Above refer to, the human reproductive organ (oviduct, uterus) contains energy sources of different concentration. Thus, further studies are will examine continuously to effects by interaction of different energy sources in the mouse embryo development, and these results will provide to foundation on the human embryo culture.

• 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.

• International Journal of Oral Biology
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• 제35권1호
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• pp.1-5
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• 2010

Human fibroblasts that maintain the structural integrity of connective tissues by secreting precursors of the extracellular matrix are typically cultured with serum. However, there are potential disadvantages of the use of serum including unnatural interactions between the cells and the potential for exposure to animal pathogens. To prevent the possible influence of serum on fibroblast cultures, we devised a serum-free growth method and present in vitro data that demonstrate its suitability for growing porcine fetal fibroblasts. These cells were grown under four different culture conditions: no serum (negative control), 10% fetal bovine serum (FBS, positive control), 10% knockout serum replacement (KSR) and 20% KSR in the medium. The proliferation rates and viabilities of the cells were investigated by counting the number of cells and trypan blue staining, respectively. The 10% FBS group showed the largest increase in the total number of cells ($1.09\;{\times}\;10^5\;cells/ml$). In terms of the rate of viable cells, the results from the KSR supplementation groups (20% KSR:64.7%; 10% KSR: 80.6%) were similar to those from the 10% FBS group (68.5%). Moreover, supplementation with either 10% ($3.0\;{\times}\;10^4\;cells/ml$) or 20% KSR ($4.8\;{\times}\;10^4\;cells/ml$) produced similar cell growth rates. In conclusion, although KSR supplementation produces a lower cell proliferation rate than FBS, this growth condition is more effective for obtaining an appropriate number of viable porcine fetal fibroblasts in culture. Using KSR in fibroblast culture medium is thus a viable alternative to FBS.

• Reproductive and Developmental Biology
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• 제39권3호
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• pp.49-57
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• 2015

In order to achieve successful in vitro production of embryo, it is necessary to establish intrauterine environment during in vitro culture. Thus, this study was investigated to establish embryo culture system using co-incubated collagen matrix gel (CM) with endometrial epithelial cells (EC). Endometrial epithelial cells were isolated from porcine endometrium at follicular phase, the cells seeded in insert dish for co-incubation with CM-coated culture dish. Then, culture media treated with/without 2.0 IU/ml hCG or 10 ng/ml $IL-1{\beta}$. After incubation for 24 h, the co-incubated insert dishes were removed from CM-coated culture dish before embryo culture. Embryos at 48 h after in vitro fertilization (IVF) were cultured on the dish for 120 h with porcine zygote medium. We determined PTGS-2 expression in the ECs, VEGF protein in co-incubated CM with EC and observed cleavage rate and blastocyst development of embryos at 168 h after IVF. In result, expression of PTGS-2 was higher at co-incubated EC with hCG and $IL-1{\beta}$ groups than EC without hCG and $IL-1{\beta}$. The VEGF protein was detected at co-incubated CM with EC, EC treated with hCG and $IL-1{\beta}$ groups higher than CM group. Also, cleavage rate was no significantly difference among all group, however, blastocyst development was significantly higher in co-incubated CM with EC treated with hCG group than un-treated groups (p<0.05). Therefore, we suggest that novel embryo culture system using co-incubated collagen matrix gel with endometrial epithelial cells treated with $IL-1{\beta}$ is beneficial and useful for enhancing the production of porcine blastocysts in vitro.