• Asian-Australasian Journal of Animal Sciences
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• 제14권6호
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• pp.754-758
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• 2001

Bovine oocytes with compact and complete cumulus cells were cultured for up to 24h in TCM199 buffered with 25mmol/l hepes and supplemented with 10% FBS (fetal bovine serum), 1mg/ml $17{\beta}$-estradiol, 20IU/ml hCG(human chorionic gonadotropin). All of the oocytes were divided into at 6 groups depending upon incubation times (control, 0 hour, 6 hours, 12 hours, 16 hours, 18 hours). To all experimental media, $200{\mu}g/ml$ puromycin was added at different incubation times mentioned above. Following these culture times, in vitro insemination was conducted with frozen-thawed bovine spermatozoa in medium BO (Brackett and Oliphant medium for in vitro insemination) with $10{\mu}g/ml$ BSA(bovine serum albumin) and 10 mg/ml heparin added. After 22h culture, the oocytes were fixed with acetic alcohol solution and stained with orcein acetic solution to evaluate sperm nuclear progression. Addition of puromycin after 0, 6 and 12 h of culture resulted in near of oocyte maturation at the M1 stage. Contrariwise, puromycin addition after 12 h of culture led to restoration of nuclear progression to M2 stage. On the one hand, puromycin affected the synthesis of Cyclin B protein that may be involved in the oocyte maturation and sperm capacitation for in vitro fertilization. The present study suggests the participation of protein synthesis, cyclin B, in the oocyte development from M1 to M2 stages in vitro.

• 한국자원식물학회:학술대회논문집
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• 한국자원식물학회 2002년도 제9차 국제심포지움 및 추계정기학술발표회
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• pp.6-7
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• 2002

The goal of this research was to develop the effectiveness of in vitro culture method for A. formosanus and study the environment in vitro conditions affecting on growth. The first series of experiments were examined to investigate the response of three different basal media, MS (Murashige and Skoog, 1962), Knudson (KC; Knudson, 1946) and modified hyponex on growth and multiplication during in vitro culture. Multiple shoot proliferation was induced in shoot tip explants on Hyponex (H3) media supplemented with BA (1 mg1$^{-1}$) or TDZ (1-2 mg1$^{-1}$). Addition of activated charcoal (1%) to the TDZ containing medium promoted rapid shoot tip proliferation (11.1 shoots per explant) but the same medium had an opposite effect resulting in poor proliferation in the nodal explants. However, the regenerated shoots had slow growth rate and failed to elongate. This problem was overcome by transferring the shoot clumps to a hormone free H3 media supplemented with 2% sucrose and 0.5% activated charcoal. Using bioreactor culture for scaling up was also shown the best way for multiple shoot induction and growth of this plant.(중략)

• Journal of Plant Biotechnology
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• 제42권2호
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• pp.111-116
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• 2015

Interest and great demand for blueberry (Vaccinium corymbosum) have increased, as V. corymbosum is now one of the most economically important crops in Korea. It is expected that blueberry production and the area planted for cultivation will increase consistently in the years ahead because of high profitability and the consumer's demand for healthy ingredients. Effective mass production of blueberry is urgently needed for commercial cultivation establishment, but a main limitation is lack of a propagation system that produces a disease-free plant material for commercial plantation. A large amount of research has focused entirely on developing tissue culture techniques for blueberry propagation. However, controlling fungal and bacterial contamination of woody plant material is extremely difficult. Our study was conducted to investigate the effect of biocide addition during the in vitro culture of blueberry on plantlet growth and contamination occurrence. Four biocides, including Plant Preservative Mixture ($PPM^{TM}$), vancomycin, nystatin and penicillin G, were used in varying concentrations during the in vitro propagation of blueberry. When nystatin was added into the medium at low concentrations, the overall growth of blueberry plantlets was retarded. Addition of vancomycin and penicillin G in high concentrations decreased contamination but induced plantlet mortality. On the other hand, when 1ml/L $PPM^{TM}$ was added, the growth characteristics of blueberry plantlets did not significantly differ from non-treatment (control), and the contamination occurrence rate was very low. From these results, we found that the addition of the appropriate biocide could provide an effective method to reduce contamination in the culture process, thereby raising in vitro production efficiency.

• 대한수의학회지
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• 제39권1호
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• pp.220-225
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• 1999

The aim of these experiments was to investigate the effects of bovine oviduct epithelial cells (OEC) derived from different segments to bind sperm binding and maintain their motility in vitro. In experiment 1, the number of sperm attached to OEC derived from isthmus or ampulla, the motility of unattached sperm during co-culture and fertilizing ability were assessed. In experiment 2, heparin treated sperm (hsp) or no treated sperm (nsp) were used to evaluate OEC binding ability of capacitated sperm. In experiment 1, regardless of their origin, approximately 65% of the sperm were attached to OEC within 2h. From 6h of co-culture, the numbers of unattached sperm on ampullary OEC were significantly higher than those on isthmic OEC (p<0.005). From 12h of co-culture, the motility of unattached sperm on isthmic OEC were significantly higher than those on ampullary OEC(p<0.05). The cleavage rate of oocytes inseminated on OEC derived from isthmic segment was also significantly higher than those from ampullary segment (p<0.01). In experiment 2, the numbers of unattached hsp on OEC were significantly higher than those of controls (p<0.01), between 2-24h examination. From 12h of co-culture, the motility of unattached nsp were significantly greater than those of hsp (p<0.01). These results show that bovine OEC derived from the isthmus play more important role(s) for sperm binding, maintaining motility and fertilization in vitro than those from the ampulla, and heparin induced capacitation may change sperm binding ability on OEC in vitro.

• 한국농업기계학회:학술대회논문집
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• 한국농업기계학회 1996년도 International Conference on Agricultural Machinery Engineering Proceedings
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• pp.1045-1054
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• 1996

Tissue culture, or micropropagation , is being used for the vegetative multiplication of several hundred millions of superior plants annually for horticulture and forestry. It is often more expensive than other forms of propagation using cuttings or seeds, because it is labor intensive and more specialized . The aim of automation is to reduce the cost per plantlet by reducing labor input, and finally, to yield profit, as business activity . Labor usually account for 70-80% of th ein vitro and ex vitro cost. This paper aspects of tissue culture automization , such as technical and economical approaches in view of automization.

• 한국발생생물학회지:발생과생식
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• 제18권3호
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• pp.153-160
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• 2014

Adaptive development of early stage embryo is well established and recently it is explored that the mammalian embryos also have adaptive ability to the stressful environment. However, the mechanisms are largely unknown. In this study, to evaluate the possible role of aquaporin in early embryo developmental adaptation, the expression of aquaporin (AQP) 5 gene which is detected during early development were examined by the environmental condition. To compare expression patterns between in vivo and in vitro, we conducted quantitative RT-PCR and analyzed localization of the AQP5 by whole mount immunofluorescence. At in vivo condition, Aqp5 expressed in oocyte and in all the stages of preimplantation embryo. It showed peak at 2-cell stage and decreased continuously until morula stage. At in vitro condition, Aqp5 expression pattern was similar with in vivo embryos. It expressed both at embryonic genome activation phase and second mid-preimplantation gene activation phase, but the fold changes were modified between in vivo embryos and in vitro embryos. During in vivo development, AQP5 was mainly localized in apical membrane of blastomeres of 4-cell and 8-cell stage embryos, and then it was localized in cytoplasm. However, the main localization area of AQP5 was dramatically shifted after 8-cell stage from cytoplasm to nucleus by in vitro development. Those results explore the modification of Aqp5 expression levels and location of its final products by in vitro culture. It suggests that expression of Aqp5 and the roles of AQP5 in homeostasis can be modulated by in vitro culture, and that early stage embryos can develop successfully by themselves adapting to their condition through modulation of the specific gene expression and localization.

• 한국수정란이식학회지
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• 제17권3호
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• pp.203-209
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• 2002

본 실험은 원시난포의 체외배양 체계를 확립할 수 있는 가능성을 검토하기 위해 10일과 20일령 마우스의 난소에서 분리한 난포를 체외배양하여 난포의 형태변화, 난자의 성장를 및 난자의 핵성숙단계를 조사하여 비교하였다. 그 결과를 요약하면 다음과 같다. 1. 10일령과 20일령의 마우스에서 분리한 난포는 배양 6일째부터 난포강이 형성되었다. 2. 10일령과 20일령 마우스의 난소에서 분리한 난포의 수는 평균 21.5와 33.3개였고, 배양하기 전에 투명대를 제외한 난자의 직경은 51.85 $mu extrm{m}$와 57.50 $\mu\textrm{m}$였으나, 10 일간 배양한 후 측정한 난자의 직경은 55.95 $\mu\textrm{m}$와 63.11 $\mu\textrm{m}$로 증가하였다. 3. 10일령과 20일령에서 M II까지 핵이 성숙된 난자는 각각 4.3%와 22.1%였고. GVBD기 이상은 각각 14.5%와 61.1%였다.

• Journal of Veterinary Science
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• 제24권1호
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• pp.4.1-4.16
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• 2023

Background: In vitro culture of preantral follicles is a promising technology for fertility preservation. Objectives: This study aims to investigate an optimized three-dimensional (3D) fetal bovine serum (FBS)-free preantral follicle culture system having a simple and easy operation. Methods: The isolated follicles from mouse ovaries were randomly divided in an ultra-low attachment 96-well plates supplement with FBS or bovine serum albumin (BSA) culture or encapsulated with an alginate supplement with FBS or BSA culture. Meanwhile, estradiol (E₂) concentration was assessed through enzyme-linked immunosorbent assay of culture supernatants. The diameter of follicular growth was measured, and the lumen of the follicle was photographed. Spindle microtubules of oocytes were detected via immunofluorescence. The ability of oocytes to fertilize was assessed using in vitro fertilization. Results: The diameters were larger for the growing secondary follicles cultured in ultra-low attachment 96-well plates than in the alginate gel on days 6, 8, and 10 (p < 0.05). Meanwhile, the E2 concentration in the BSA-supplemented medium was significantly higher in the alginate gel than in the other three groups on days 6 and 8 (p < 0.05), and the oocytes in the FBS-free system could complete meiosis and fertilization in vitro. Conclusions: The present study furnishes insights into the mature oocytes obtained from the 3D culture of the preantral follicle by using ultra-low attachment 96-well plate with an FBS-free system in vitro and supports the clinical practices to achieve competent, mature oocytes for in vitro fertilization.

• 한국수정란이식학회지
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• 제12권2호
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• pp.151-159
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• 1997

In vitro fertilization(IVF) derived morula and blastocyst embryos were bisected by a simple method and cultured in vitro without zona pellucida And also bisected embryos were frozen-thawed and cultured in vitro) to evaluate the survival rate. The results obtained were as follows : The average number of grade I or II immature follicular oocytes recovered by slicing method per ovary was 11.9 from 142 ovaries. Following in vitro fertilization, the rates of cleavage and in vitro development to morula and blatocyst were 61.7 and 32.2% respectively. The successful bisection rate of IVE embryos was 67.51%, and the embryos of blastocyst stage were bisected successfully at significantly(P

• 한국가축번식학회지
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• 제14권1호
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• pp.17-22
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• 1990

The present study was carried to investigate the development potential of bovine follicular oocytes matured and fertilized in vitro. Bovine oocytes matured in vitro were fertilized, cultured, and transfered to the rabbit oviduct for in vitro culture to evaluate the development potential. The rates of in vitro maturation, fertilization and polyspermy were 96%(355/369), 90%(320/355) and 15%(17/320), respectively. The percentage of oocytes cleaved after culture for 48 hours, to 2 cell, 3~4 cell, 6~8 cell stage were 17%(60/356), 21%(75/356) and 19%(67/356), respectively and overall cleavage rate was 57%(202/356). The rate of recovered embryos after 5 days in rabbit oviduct was 56%(80/142), and 21%(17/80) of recovered embryos developed over morula stage.