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Improved Production Efficiencies of Various Adeno-Associated Virus (AAV) Serotypes and a Novel Universal AAV Titration Method (다양한 adeno-associated virus (AAV) 혈청형의 효율성 높은 생산법과 새로운 공통적 정량법 개발)

  • Cho, Young-Hwa;Choi, Ye-Jin;Yun, Jung-Hee;Kim, Nam-Hee;Choi, Mi-Ra;Choi, Young-Kook;Kim, Kyung-Hee;Lee, Young-Ill;Lee, Beom-Jun;Park, Kee-Rang
    • Journal of Life Science
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    • v.22 no.6
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    • pp.703-712
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    • 2012
  • Adeno-associated virus (AAV) has been considered to be a very safe and efficient gene delivery system. However, the major obstacles to therapeutic usage of AAV have been to achieve highly efficient and reproducible production processes, and also to develop a reliable quantifying method of various serotypes with a simple protocol. We compared the efficiency of the conventional production protocol of AAV2 and adenovirus (Ad) co-infection to that of a new method containing AAV2 infection followed by pHelper transfection. We tested HEK293 and 293T, and further examined the time-dependent changes of AAV2 production. The new method of AAV2 and pHelper DNA gave about ten times higher production efficiency than that of the conventional protocol. The highest production efficiency in 293T was achieved as $1.61{\times}10^5$ virus genomes (v.g.)/cell by the new method of 10 MOI of AAV2 infection and 5 days post-infection. This protocol of the highest efficiency was then applied to produce various AAV serotypes and showed the efficiencies higher than $10^5$ v.g./cell. Next, we designed the universal PCR primers of highly conserved regions for various AAV serotypes to develop a simple and reliable titration method. The universal primers could amplify all the tested AAV serotypes with similar sensitivities by ten molecular copies. Therefore, this pair of universal primers can be further utilized to detect AAV contaminants in therapeutic adenoviral vectors.

Antioxidative Effects of Pleurotus eryngii and Its By-products (새송이 버섯과 그 부산물의 항산화성)

  • Cho, Hyun-So;Lee, Hyun-Ji;Lee, Soo-Jung;Shin, Jung-Hye;Lee, Hyun-Uk;Sung, Nak-Ju
    • Journal of Life Science
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    • v.18 no.10
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    • pp.1360-1368
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    • 2008
  • Physicochemical characteristics and antioxidative activity were measured to investigate the possibility for functional characteristics of Pleurotus eryngii and its by-products. By-products of Pleurotus eryngii were classified with mushroom, fungal body and fermented mushroom by-product. Moisture was the highest in fermented mushroom by-product and crude protein was 1.72%, in mushroom. Crude fiber content was less than 10% except the fungal body by-product. Mineral content appeared to be the highest in the fermented mushrooom with a value of 3,696.1 mg/100 g, and potassium was a predominant mineral in Pleurotus eryngii as well as its by-products. Amino acid content was the highest in mushroom with a level of 989.59 mg/100 g. DPPH radical scavenging ability of the fermented mushroom was the highest, and its methanol extract and water extract exhibited $64.07{\pm}0.23%$ and $76.27{\pm}1.46%$ of scavenging activity at a concentration of 10 mg/ml. Reducing power was significantly higher in the fermented mushroom in comparison with those of the mushroom, mushroom by-product, and fungal body by-product. The reducing power of the water extract of fermented mushroom was the highest with a value of $2.22{\pm}0.03$. SOD-like activities for the individual samples except the fungal body by-product were higher than 50% at a concentration of 10 mg/ml. The hydroxyl radical scavenging abilities of the individual samples except the fungal body by-product were over 50%. Nitrite scavenging effects were better in pH 2.5 than in pH 4.0. While the nitrite scavenging effects of methanol extracts were $42.93{\pm}1.71{\sim}72.97{\pm}2.18%$, those of the water extracts were $57.66{\pm}1.80{\sim}81.07{\pm}0.81%$. Antioxidative activity of the fermented mushroom appeared to be the highest among the mushroom by-products. Taken together, these results provide an insight into utilization of the mushroom by-products as materials for functional foods and animal feed.

Effect of Doenjang with Black Soybean on Cytokine Production and Inhibition of Tumor Metastasis (검정콩 된장의 사이토카인 생성 및 종양전이 억제에 미치는 영향)

  • Lim, Sun-Young;Park, Kun-Young;Bae, Myung-Suk;Kim, Kwang-Hyuk
    • Journal of Life Science
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    • v.19 no.2
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    • pp.264-270
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    • 2009
  • We investigated the effect of black soybean and doenjang with black soybean on production of cytokines including interleukin-2 (IL-2), interleukin-6 (IL-6) and tumor necrosis factor-${\alpha}$ (TNF-${\alpha}$) compared with yellow soybean and doenjang with yellow soybean. We also determined inhibitory effect of two types of soybeans and doenjang on tumor metastasis produced by colon 26-M3.1 carcinoma cells. The cytokine productions of mouse splenocytes increased by the exposure of lipopolysaccharide (LPS, 1 ${\mu}g$/ml) compared to without treatment of LPS. The LPS-induced IL-2 production was highest in the black soybean methanol extract, while the methanol extract from black soybean doenjang fermented for 2 mo showed the higher levels of IL-2 without LPS (p<0.05). In case of LPS-induced IL-6 production, the methanol extracts from control, yellow soybean, black soybean doenjang fermented for 2 and 7 mo showed higher levels of IL-6 compared to those of black soybean, and yellow soybean doenjang fermented for 2 mo (p<0.05). The methanol extract of black soybean doenjang fermented for 2 mo showed significantly higher levels of TNF-${\alpha}$ in both with and without LPS (p<0.05). In experiment of tumor metastasis, the treatment (1 mg/mouse) of methanol extract from black soybean doenjang fermented for 7 mo inhibited tumor metastasis by 50% and had the highest inhibitory effect among other samples (p<0.05). From these results, doenjang manufactured with black soybean modulated the production of cytokine and showed anticancer effect, suggesting that this effect was increased with increased periods of fermentation.

Flavonoid Profiles of Quercus mongolica Fisch. ex Ledeb. and Q. serrata Murray (Fagaceae) in Mt. Seorak, Korea: Taxonomical and Ecological Implications (설악산 신갈나무와 졸참나무의 플라보노이드 조성과 분류학적, 생태학적 의미)

  • Park, Jin Hee
    • Journal of Life Science
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    • v.24 no.10
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    • pp.1092-1101
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    • 2014
  • In this study, the distribution patterns of Quercus mongolica and Q. serrata in Korea were investigated, and the possibility of introgressive hybridization and gene flow between Q. mongolica and Q. serrata in Mt. Seorak was inferred by flavonoid analyses. The most critical factor in the vertical and horizontal distribution patterns of Q. mongolica and Q. serrata was the temperature, in accordance with latitude and altitude. The species showed a zonal distribution, with a Q. mongolica zone in the upper area and a Q. serrata zone in the lower area. In Mt. Seorak, Central Korea, the range of the vertical distribution of Q. mongolica was generally above an altitude of 100 m, whereas that of Q. serrata was an altitude of 0-400 m (-500) and rarely above an altitude of 500 m. However, in Mt. Jiri, Southern Korea, Q. serrata was found up to an altitude of 1,000~1,200 m, whereas the frequency of Q. mongolica was reduced at lower elevations and the species was rare below an altitude of 300 m, although pure stands were found on higher mountain slopes above an altitude of 1,200 m. The altitudinal distribution of the two species overlapped, where the two species occurred together. The leaf flavonoid constituents of thirty-four individuals of Q. mongolica and Q. serrata in Mt. Seorak and Mt. Jiri, Korea were examined. Twenty-four flavonoid compounds were isolated and identified. These were glycosylated derivatives of flavonols kaempferol, quercetin, isorhamnetin, myricetin. Five compounds among the flavonoid compounds were acylated. Kaempferol 3-O-glucoside, quercetin 3-O-glucoside, quercetin 3-O-galactoside, and its acylated compounds were major constituents and present in all individuals. Quercus mongolica is distinguished from Q. serrata by the presence of quercetin 3-O-arabinosylglucoside, a high concentration of three acylated compounds (kaempferol 3-O-glucoside, quercetin 3-O-glucoside, and quercetin 3-O-galactoside), and a relatively low concentration or lack of rhamnosyl flavonol compounds. Intraspecific variations, however, were found in the flavonoid profiles of Q. mongolica and Q. serrata, and the flavonoid profiles of individuals belonging to the two species in a hybrid zone (sympatric zone) tended to be similar, qualitatively and quantitatively. These findings strongly suggest that gene exchange or gene flow occurs through introgressive hybridization between Q. mongolica and Q. serrata in Mt. Seorak.

Genetic Diversity and Relationship of the Walleye Pollock, Theragra chalcogramma Based on Microsatellite Analysis (Microsatellite marker 분석을 이용한 명태(Theragra chalcogramma) 5 집단의 유전적 다양성 및 유연관계 분석)

  • Dong, Chun Mae;Kang, Jung-Ha;Byun, Soon-Gyu;Park, Kie-Young;Park, Jung Youn;Kong, Hee Jeong;An, Cheul Min;Kim, Gun-Do;Kim, Eun-Mi
    • Journal of Life Science
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    • v.26 no.11
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    • pp.1237-1244
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    • 2016
  • A comprehensive analysis of the genetic diversity and relationship of the cold-water fishery walleye pollock (Theragra chalcogramma), the most abundant economically important fishery resource in the East sea of Korea, has not been carried out, despite its importance in Korea. The present study assessed the genetic diversity and relationship between five walleye pollock populations (Korean population, Russian population, USA population, and Japanese populations) of T. chalcogramma using eight microsatellite DNA (msDNA) markers to provide the scientific data for the preservation and management of the Pollock fishery resource. The results of the analysis of 186 individuals of the Pollock revealed a range of 7.13-10.63 numbers of alleles (mean number of alleles=9.05). The means of observed heterozygosity ($H_O$), expected heterozygosity ($H_E$) were 0.732 and 0.698, respectively. The results of genetic distance, Pairwise $F_{ST}$, UPGMA (UPGMA: un-weighted pair-group method with an arithmetical average) (the phylogenetic tree), PCA (PCA: Principal Coordinate analysis) analysis pointed to significant differences between the Korean population, Russian population, USA population, and Japanese populations, although small (p<0.05). These results shed light on the genetic diversity and relationships of T. chalcogramma and can be utilized for research on the evaluation and conservation of Korean T. chalcogramma as genetic resources.

Toll-like Receptor 4-mediated Apoptotic Cell Death in Primary Isolated Human Cervical Cancers (부인과질환 특이적 종양의 TLR4 매개성 apoptosis 유발에 관한 연구)

  • Won, Jinyoung;Hong, Yunkyung;Park, Sookyoung;Kim, Joo-Heon;Hong, Yonggeun
    • Journal of Life Science
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    • v.28 no.6
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    • pp.718-725
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    • 2018
  • Toll-like receptor 4 (TLR4) has been implicated in cell proliferation and apoptosis in several types of cancer. In this study, the impact of TLR4 activation on apoptotic cell death in gynecologic cancers induced by lipopolysaccharide (LPS) was investigated. Cervical cancer cell lines were produced from isolated surgical specimens supplied by Paik Hospital. The primary cultures of normal myometrium and gynecologic cancers, including cervical, endometrial, and ovarian cancers, were used to examine the differences in morphological characteristics between normal and cancerous cells. A reverse transcription polymerase chain reaction analysis was used to determine the relative expression levels of TLR4 gene involved in apoptosis-associated signaling in cervical cancer cells. The cancer cell colonies showed a tendency to reach high levels of confluency compared with normal cells. In addition, an enhanced growth rate and loss of contact inhibition were observed in gynecologic cancer cells compared with normal cells (doubling times of 16.6 hr vs. 26 hr, respectively). The expression level of ITGA5, an alpha-5 integrin marker, was upregulated in normal myometrial cells, but this tendency was not exhibited in cervical cancer cells. Furthermore, p53 tumor suppressor gene expression was upregulated, whereas TLR4 and caspase-3 gene expressions were downregulated in cervical cancer cells. Notably, the expression levels of TLR4 and caspase-3 were increased significantly in LPS-treated cancer cells compared with those in non-LPS-treated cells. These results suggest that the TLR4-mediated caspase-dependent apoptotic signaling pathway could be suggested as a therapeutic target for the treatment of gynecologic cancers, including cervical cancers.

Using the METHONTOLOGY Approach to a Graduation Screen Ontology Development: An Experiential Investigation of the METHONTOLOGY Framework

  • Park, Jin-Soo;Sung, Ki-Moon;Moon, Se-Won
    • Asia pacific journal of information systems
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    • v.20 no.2
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    • pp.125-155
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    • 2010
  • Ontologies have been adopted in various business and scientific communities as a key component of the Semantic Web. Despite the increasing importance of ontologies, ontology developers still perceive construction tasks as a challenge. A clearly defined and well-structured methodology can reduce the time required to develop an ontology and increase the probability of success of a project. However, no reliable knowledge-engineering methodology for ontology development currently exists; every methodology has been tailored toward the development of a particular ontology. In this study, we developed a Graduation Screen Ontology (GSO). The graduation screen domain was chosen for the several reasons. First, the graduation screen process is a complicated task requiring a complex reasoning process. Second, GSO may be reused for other universities because the graduation screen process is similar for most universities. Finally, GSO can be built within a given period because the size of the selected domain is reasonable. No standard ontology development methodology exists; thus, one of the existing ontology development methodologies had to be chosen. The most important considerations for selecting the ontology development methodology of GSO included whether it can be applied to a new domain; whether it covers a broader set of development tasks; and whether it gives sufficient explanation of each development task. We evaluated various ontology development methodologies based on the evaluation framework proposed by G$\acute{o}$mez-P$\acute{e}$rez et al. We concluded that METHONTOLOGY was the most applicable to the building of GSO for this study. METHONTOLOGY was derived from the experience of developing Chemical Ontology at the Polytechnic University of Madrid by Fern$\acute{a}$ndez-L$\acute{o}$pez et al. and is regarded as the most mature ontology development methodology. METHONTOLOGY describes a very detailed approach for building an ontology under a centralized development environment at the conceptual level. This methodology consists of three broad processes, with each process containing specific sub-processes: management (scheduling, control, and quality assurance); development (specification, conceptualization, formalization, implementation, and maintenance); and support process (knowledge acquisition, evaluation, documentation, configuration management, and integration). An ontology development language and ontology development tool for GSO construction also had to be selected. We adopted OWL-DL as the ontology development language. OWL was selected because of its computational quality of consistency in checking and classification, which is crucial in developing coherent and useful ontological models for very complex domains. In addition, Protege-OWL was chosen for an ontology development tool because it is supported by METHONTOLOGY and is widely used because of its platform-independent characteristics. Based on the GSO development experience of the researchers, some issues relating to the METHONTOLOGY, OWL-DL, and Prot$\acute{e}$g$\acute{e}$-OWL were identified. We focused on presenting drawbacks of METHONTOLOGY and discussing how each weakness could be addressed. First, METHONTOLOGY insists that domain experts who do not have ontology construction experience can easily build ontologies. However, it is still difficult for these domain experts to develop a sophisticated ontology, especially if they have insufficient background knowledge related to the ontology. Second, METHONTOLOGY does not include a development stage called the "feasibility study." This pre-development stage helps developers ensure not only that a planned ontology is necessary and sufficiently valuable to begin an ontology building project, but also to determine whether the project will be successful. Third, METHONTOLOGY excludes an explanation on the use and integration of existing ontologies. If an additional stage for considering reuse is introduced, developers might share benefits of reuse. Fourth, METHONTOLOGY fails to address the importance of collaboration. This methodology needs to explain the allocation of specific tasks to different developer groups, and how to combine these tasks once specific given jobs are completed. Fifth, METHONTOLOGY fails to suggest the methods and techniques applied in the conceptualization stage sufficiently. Introducing methods of concept extraction from multiple informal sources or methods of identifying relations may enhance the quality of ontologies. Sixth, METHONTOLOGY does not provide an evaluation process to confirm whether WebODE perfectly transforms a conceptual ontology into a formal ontology. It also does not guarantee whether the outcomes of the conceptualization stage are completely reflected in the implementation stage. Seventh, METHONTOLOGY needs to add criteria for user evaluation of the actual use of the constructed ontology under user environments. Eighth, although METHONTOLOGY allows continual knowledge acquisition while working on the ontology development process, consistent updates can be difficult for developers. Ninth, METHONTOLOGY demands that developers complete various documents during the conceptualization stage; thus, it can be considered a heavy methodology. Adopting an agile methodology will result in reinforcing active communication among developers and reducing the burden of documentation completion. Finally, this study concludes with contributions and practical implications. No previous research has addressed issues related to METHONTOLOGY from empirical experiences; this study is an initial attempt. In addition, several lessons learned from the development experience are discussed. This study also affords some insights for ontology methodology researchers who want to design a more advanced ontology development methodology.

Comparisons of Popularity- and Expert-Based News Recommendations: Similarities and Importance (인기도 기반의 온라인 추천 뉴스 기사와 전문 편집인 기반의 지면 뉴스 기사의 유사성과 중요도 비교)

  • Suh, Kil-Soo;Lee, Seongwon;Suh, Eung-Kyo;Kang, Hyebin;Lee, Seungwon;Lee, Un-Kon
    • Asia pacific journal of information systems
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    • v.24 no.2
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    • pp.191-210
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    • 2014
  • As mobile devices that can be connected to the Internet have spread and networking has become possible whenever/wherever, the Internet has become central in the dissemination and consumption of news. Accordingly, the ways news is gathered, disseminated, and consumed have changed greatly. In the traditional news media such as magazines and newspapers, expert editors determined what events were worthy of deploying their staffs or freelancers to cover and what stories from newswires or other sources would be printed. Furthermore, they determined how these stories would be displayed in their publications in terms of page placement, space allocation, type sizes, photographs, and other graphic elements. In turn, readers-news consumers-judged the importance of news not only by its subject and content, but also through subsidiary information such as its location and how it was displayed. Their judgments reflected their acceptance of an assumption that these expert editors had the knowledge and ability not only to serve as gatekeepers in determining what news was valuable and important but also how to rank its value and importance. As such, news assembled, dispensed, and consumed in this manner can be said to be expert-based recommended news. However, in the era of Internet news, the role of expert editors as gatekeepers has been greatly diminished. Many Internet news sites offer a huge volume of news on diverse topics from many media companies, thereby eliminating in many cases the gatekeeper role of expert editors. One result has been to turn news users from passive receptacles into activists who search for news that reflects their interests or tastes. To solve the problem of an overload of information and enhance the efficiency of news users' searches, Internet news sites have introduced numerous recommendation techniques. Recommendations based on popularity constitute one of the most frequently used of these techniques. This popularity-based approach shows a list of those news items that have been read and shared by many people, based on users' behavior such as clicks, evaluations, and sharing. "most-viewed list," "most-replied list," and "real-time issue" found on news sites belong to this system. Given that collective intelligence serves as the premise of these popularity-based recommendations, popularity-based news recommendations would be considered highly important because stories that have been read and shared by many people are presumably more likely to be better than those preferred by only a few people. However, these recommendations may reflect a popularity bias because stories judged likely to be more popular have been placed where they will be most noticeable. As a result, such stories are more likely to be continuously exposed and included in popularity-based recommended news lists. Popular news stories cannot be said to be necessarily those that are most important to readers. Given that many people use popularity-based recommended news and that the popularity-based recommendation approach greatly affects patterns of news use, a review of whether popularity-based news recommendations actually reflect important news can be said to be an indispensable procedure. Therefore, in this study, popularity-based news recommendations of an Internet news portal was compared with top placements of news in printed newspapers, and news users' judgments of which stories were personally and socially important were analyzed. The study was conducted in two stages. In the first stage, content analyses were used to compare the content of the popularity-based news recommendations of an Internet news site with those of the expert-based news recommendations of printed newspapers. Five days of news stories were collected. "most-viewed list" of the Naver portal site were used as the popularity-based recommendations; the expert-based recommendations were represented by the top pieces of news from five major daily newspapers-the Chosun Ilbo, the JoongAng Ilbo, the Dong-A Daily News, the Hankyoreh Shinmun, and the Kyunghyang Shinmun. In the second stage, along with the news stories collected in the first stage, some Internet news stories and some news stories from printed newspapers that the Internet and the newspapers did not have in common were randomly extracted and used in online questionnaire surveys that asked the importance of these selected news stories. According to our analysis, only 10.81% of the popularity-based news recommendations were similar in content with the expert-based news judgments. Therefore, the content of popularity-based news recommendations appears to be quite different from the content of expert-based recommendations. The differences in importance between these two groups of news stories were analyzed, and the results indicated that whereas the two groups did not differ significantly in their recommendations of stories of personal importance, the expert-based recommendations ranked higher in social importance. This study has importance for theory in its examination of popularity-based news recommendations from the two theoretical viewpoints of collective intelligence and popularity bias and by its use of both qualitative (content analysis) and quantitative methods (questionnaires). It also sheds light on the differences in the role of media channels that fulfill an agenda-setting function and Internet news sites that treat news from the viewpoint of markets.

Effect of Glucose Exposure on the Development of the Mouse Preimplantation Embryo In Vitro (착상전 생쥐배아의 Glucose에 대한 노출이 체외 배발생에 미치는 영향)

  • Kim, Seon-Ui;Eom, Sang-Jun;Yun, San-Hyeon;Im, Jin-Ho;Jeong, Gil-Saeng
    • Korean Journal of Animal Reproduction
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    • v.19 no.3
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    • pp.227-234
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    • 1995
  • This study was carried out to investigate the effects according to the time course of glucose exposure on the development of one-cell embryos beyond morula in CR$_laa$ medium. One-cell zygotes from B6CBA F$_1$ mice were recovered at 24 ~ 25h after hCG and cumulus cells were removed with 0.1% hyaluronidase. The embryos were pooled and subsequently divided into each groups and cultured in CR$_laa$ at 37$^{\circ}C$ in 5% CO$_2$ in aIr. The embryos were either, placed in CR$_laa$ containing various concentration (5.5, 16.5, 27.5 and 38.5 mM) of glucose for 1 min. and subsequently returned to the fresh culture medium (without glucose), or were transferred to the same media containing glucose at 72 h post hCG. The results obtained in these experiments were summarized as follows: 1. The development rates of zygotes, recovered from the oviducts in M2 and cultured in CR$_laa$ with 3mg/Im FAF-BSA, to expanded blastocysts (25.7%) and hatching bIastocysts (17.6%) were significantly higher than those of zygotes recovered in TL Hepes (0% and 0%, respectively). 2. The development rates of one-cell embryos exposed to 27.5 mM glucose at 72 h post hCG for 1 min, were 68.8% (CR$_1$+BSA) a and 77.1% (CR$_1$+FBS) of expanded blastocyst stage, but there were no significant differences between the embryos exposed for 1 min. or transferred at 72 h. 3. Regardless of glucose concentration (5.5, 16.5, 27.5 & 38.5mM), 45.7~61.5% of embryos developed to the blastocyst stage. There were no significant differences between any of the treatments on the devel-opment of one-cell embryos. Therefore, the detrimental effect of highly concentration was not appeared.

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Systems for Production of Calves from Hanwoo(Korean Native Cattle) IVM/IVF/IVC Blastocyst I. Hanwoo IVM/IVF /IVC Blastocyst Cryopreserved by Vitrification (체외생산된 한우 배반포기배로부터 송아지 생산을 위한 체계 I. 체외생산된 한우 배반포기배의 초자화 동결보존)

  • Park, S. P.;Kim, E. Y.;Kim, D. I.;Park, N. H.;Y. S. Won;S. H. Yoon;K. S. Chung;J. H. Lim
    • Korean Journal of Animal Reproduction
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    • v.22 no.4
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    • pp.349-357
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    • 1998
  • This study was to investigate whether the viability of Hanwoo IVM/IVF/IVC blastocysts was maintained after vitrification and thawing. In vitro produced Hanwoo blastocysts were vitrified by two-step method: equilibrated in EG20 for 3 min, and then exposed in EFS40 [40% ethylene glycol (EG), 18% ficoll and 10.26% sucrose in mDPBS containing 10% FBS ]and vitrified in L$N_2$for 30 - 45 sec. After thawing, in vitro survival was assessed as the re-expanded and hatched rates at 24 hand 48 h, respectively. The results obtained in these experiments were summarized as follows: From the 12 replicates, 52.5% of Hanwoo blastocysts were produced in vitro at day 7 after IVF. When the effects of freezing solution to the embryo survival were examined, there is no significant toxicity in exposure (100.0, 73.8%) compared to that af control group (100.0, 87.0%). However, when embryos were vitrified, high survival (86.2, 55.4%) was obtained although it was significantly lower than those of exposure and control group (p<0.05). When the in vitro survival of vitrified embryos according to developmental stage and culture day were examined, it showed that more advanced embryo stage exhibited a significantly higher survival rate irrespective of culture day (p<0.05). Also, even in the same development stage, the in vitro survival of day 7 embryos (re-expanded: 75.0~87.5%, hatched: 21.4~66.7%) was higher than those of day 8 embryos(re-expanded: 58.6~78.3%, hatched: 10.3~52.2%). Therefore, these results suggested that in vitro produced Hanwoo blastocysts can be successfully cryopreserved by simple two-step vitrification method using EFS40 freezing solution, particularly at the expanded and early hatching blastocyst stage regardless of embryo culture duration (day 7 or day 8 after IVF).

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