• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2001년도 춘계학술발표대회
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• pp.31-31
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• 2001

This study was to establish in uitro culture system of mouse preantral follicles and to obtain higher in vitro development rates and production of live young. Preantral follicles were obtained from 12-day-old FI mouse (C57BL $\times$ CBA) by enzymatical methods. Oocyte-granulosa cell complexes (OGCs) of preantral follicles were loaded on Transwell-COL insert and cultured in $\alpha$MEM supplemented with 5% FBS, 100 mIU/$m\ell$ FSH and 100 mIU/$m\ell$ hMG for IVG. IVM was performed in $\alpha$MEM supplemented 1.5 IU/$m\ell$ hCG for 18 hrs and IVF was carried out in Ml6 medium. Embryos were cultured in modified Ml6 medium supplemented 10% FBS for 4 days. The effect of the OGCs size on the nuclear/cytoplasmic maturation was significantly higher in 120-150 ${\mu}{\textrm}{m}$ (MII: 33.0%, $\geq$2-cell: 36.7%, $\geq$morula: 20.9%) than in 70-110 ${\mu}{\textrm}{m}$ (MII: 12.2%, $\geq$2-cell: 10.2%, $\geq$morula: 4.8%) (p<0.001). In period of the IVG days, the rate of $\geq$2-cell was significantly higher in 10 days(38.2%) than in 12 days (20.0%) (p<0.01). In period of IVF time, 9 hrs ($\geq$2-cell: 31.5%, $\geq$ morula: 14.3%) indicated significantly higher cytoplasmic maturation rate than 4 hrs ($\geq$2-cell: 17.5%, This study was to establish in vitro culture system of mouse preantral follicles and to obtain higher in vitro development rates and production of live young. Preantral follicles were obtained from 12-day-old FI mouse (C57BL $\times$ CBA) by enzymatical methods. Oocyte-granulosa cell complexes (OGCs) of preantral follicles were loaded on Transwell-COL insert and cultured in $\alpha$MEM supplemented with 5% FBS, 100 mIU/$m\ell$ FSH and 100 mIU/$m\ell$ hMG for IVG. IVM was performed in $\alpha$MEM supplemented 1.5 IU/$m\ell$ hCG for 18 hrs and IVF was carried out in Ml6 medium. Embryos were cultured in modified Ml6 medium supplemented 10% FBS for 4 days. The effect of the OGCs size on the nuclear/cytoplasmic maturation was significantly higher in 120-150 ${\mu}{\textrm}{m}$ (MII: 33.0%, $\geq$2-cell: 36.7%, $\geq$morula: 20.9%) than in 70-110 ${\mu}{\textrm}{m}$ (MII: 12.2%, $\geq$2-cell: 10.2%, $\geq$morula: 4.8%) (p<0.001). In period of the IVG days, the rate of $\geq$2-cell was significantly higher in 10 days(38.2%) than in 12 days (20.0%) (p<0.01). In period of IVF time, 9 hrs ($\geq$2-cell: 31.5%, $\geq$ morula: 14.3%) indicated significantly higher cytoplasmic maturation rate than 4 hrs ($\geq$2-cell: 17.5%, $\geq$morula: 4.8%) and 7 hrs ($\geq$2-cell: 20.4%, $\geq$morula: 6.1%) (p<0.01). However, there was no difference in cytoplasmic maturation between co-cultured preantral follicle ( $\geq$morula: 17.4%) and preantral follicle cultured in Ml6 ( $\geq$morula: 17.4%). 22 morula and blastocysts produced in above optimal condition were transferred to uterus of 2 pseudopregnant recipients, 1 recipient was pregnant and then born 1 live young. This result demonstrates that in vitro culture system of preantral follicles can be used efficiently as another method to supply mouse oocyte.morula: 4.8%) and 7 hrs (2-cell: 20.4%, $\geq$morula: 6.1%) (p<0.01). However, there was no difference in cytoplasmic maturation between co-cultured preantral follicle ( $\geq$morula: 17.4%) and preantral follicle cultured in Ml6 ( $\geq$morula: 17.4%). 22 morula and blastocysts produced in above optimal condition were transferred to uterus of 2 pseudopregnant recipients, 1 recipient was pregnant and then born 1 live young. This result demonstrates that in vitro culture system of preantral follicles can be used efficiently as another method to supply mouse oocyte.

• 한국자동차공학회논문집
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• 제10권3호
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• pp.237-244
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• 2002

Analyzing acoustic-structural systems such as automobiles and aircraft, the FRF-based substructuring (FBS) method is one of the most powerful tools. In this paper, a general procedure for the parametric sensitivity analysis of vibro-acoustic problems has been presented using the multi-domain FRF-based substructuring formulation. For an acoustic-structural system sub-structured by multiple domains, the substructuring formulation gives the reaction farces on the interface boundaries. The design sensitivity formula is obtained from the direct differentiation of the reaction force expression with respect to the design vector. As a practical application, the proposed design sensitivity formula is applied to an engine mount system of passenger car. An objective of the problem is to identify the most effective engine mounts and bushes in minimizing the interior noise over the concerned rpm range. The comparison of the sensitivity results with those of the finite difference method shows excellent agreement. In addition, stiffness modifications of the mounts and bushes identified through the design sensitivity analysis lead to a successful decrease of the interior noise. This results show usefulness of the present method very well.

• 미생물학회지
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• 제46권1호
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• pp.86-92
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• 2010

양계장 부산물 시료로부터 우모 분해세균 31균주를 분리하였다. 수집된 우모 분해세균에 대해 16S rRNA 염기서열을 해석하여 계통학적 특성을 검토한 결과, Firmicutes (21균주), ${\gamma}$-proteobacteria (4균주), Actinobacteria (4균주) 그리고 Bacteroidetes (2균주) 계통군에 속하는 다양한 세균이 확보되었다. 우모 분해세균 중 우모 분해율이 75-90% 이상인 우수균주 Chryseobacterium sp. FBF-7, Stenotrophomonas maltophilia FBS-4 그리고 Lysinibacillus sp. FBW-3를 최종 선발하였다. 이들 균주를 이용한 생물학적 분해법과 Ca(OH)2를 이용한 화학적 분해법에 의해 추출된 아미노산의 특성을 비교한 결과, Chryseobacterium sp. FBF-7에 의해 추출된 총 아미노산 함량이 1661.6 ${\mu}mol$/ml로 선발 균주 중 가장 높게 나타났으며, 화학적 분해법에 의해 추출된 총 아미노산 함량과 유사하였다. Chryseobacterium sp. FBF-7에 의해 생성된 필수 아미노산 함유량은 619.3 ${\mu}mol$/ml로 총 아미노산의 37%를 함유하였으며, 화학적 분해법에 의한 경우, 596.9 ${\mu}mol$/ml의 필수 아미노산을 생산하여 총 아미노산 함유량의 32%를 차지하는 것으로 나타났다. Chryseobacterium sp. FBF-7에 의해 추출된 아미노산의 조성을 검토한 결과, valine, glutamic acid, aspartic acid, glycine 그리고 proline이 주요 아미노산이었으며, 특히 aspartic acid, threonine, serine, cysteine 그리고 tyrosine이 화학적 추출법보다 더 높게 추출되는 특징을 나타내었다.

• 한국해양공학회지
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• 제15권4호
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• pp.1-7
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• 2001

Mainly, Floating Breakwaters (FBs) have been constructed in many coastal regions due to the advantages of the coastal environment and construction cost. In general, the FB becomes fixed or its width broadened because the movement of the FB comes to be large and its the wave control function lower for the long period incident waves. This study discusses the wave control function of two-rowed Fixed Floating Breakwater (FFBs) that have narrower width than that of the one-rowed FFB by using numerical approach. Boundary Element Method (BEM) based on the Green formula and Eigenfunction Expansion Method (EEM) are applied to evaluate the three-dimensional wave transformation near the wave fields of two-rowed FFBs. The validity of the present study is confirmed by comparing it with the results of Ijima et al. (1974) and Yoshida et al. (1992) for the one-rowed Fixed Floating Structure. It is revealed that the wave control function of two-rowed FFBs is more effective than that of the one-rowed FFB.

• 한국가축번식학회지
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• 제17권3호
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• pp.193-199
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• 1993

Bovine follicular oocytes were matured in two different conditions, TCM 199＋10% FBS with or without hormones (0.01 unit/ml ovine follicle stimulating hormone, 0.01 unit/ml ovine luteinizing hormone and 1$\mu\textrm{g}$/ml $\beta$-estradiol). There was no significant difference in maturation and fertilization rates of the oocytes between two groups. The result indicates that hormonal treatment does not have beneficial effect on in vitro maturation and fertilization of follicular oocytes. IVF-derived cone-cell bovine embryos were injected with foreign DNA (CChcLf) by microinjection method and then co-cultrued with bovine oviductal epithelial cells. Developmental rate of microinjected embryos to blastocyst stage (21%) was similar to that of non-injected embryos(29%). This result represents that microinjected bovine embryos produced in vitro have a potential of development to normal blastocysts.

• 한국가금학회지
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• 제40권3호
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• pp.197-205
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• 2013

동결 닭 원시생식세포의 생식계열 키메라를 이용한 생체에의 복원을 실용화하기 위해서는, 닭 원시생식세포의 동결보존기술의 향상에 의해 동결 및 융해 후의 많은 생존세포를 확보하는 것이 반드시 필요하다. 닭 원시생식세포는 배양 5.5일령의 닭 원시생식선으로부터 채취하고, MACS 방법에 의해서 순수 닭 원시생식세포를 분리했다. 15% 각각의 EG를 동결보호제로 사용한 처리군이 각 군의 농도에 상관없이 유의적(p<0.05)으로 PG 처리군보다 동결 및 융해 후의 세포의 생존율이 높음을 확인하였다. 특히, 10% EG 처리군에서 85.63%로 동일한 농도의 PG 처리군(66.81%)보다 유의적(p<0.05)으로 가장 높은 생존율을 보였다. 한편, 상업용 닭(한협3호)에서도 오계와 비슷한 경향의 결과를 확인하였다. 이상의 결과들로부터 유리화 동결에 있어서 가장 높은 생존율을 보인 10% EG이 최적의 동결 보호제로서 사용 가능함을 확인하였다.

• 한국수정란이식학회지
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• 제26권4호
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• pp.223-227
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• 2011

The objective of this study was to compare of different isolation method of mouse preantral follicles, and to examine in vitro development of mouse preantral follicles isolated by different method. Preantral follicles were mechanically or enzymatically extracted from mouse ovaries. Mechanical isolation method used fine gauge needles and enzymatic method of isolating follicles used collagenase. The recovered preantral follicles were cultured for 10 days in alpha-minimal essential medium (${\alpha}$-MEM) + 5% FBS + Insulin-Transferrin-Selenium (ITS) + 100 mIU/ml FSH. The collected primary follicles by enzymatic treatment were higher than mechanical method. Others stage preantral follicle by mechanical isolation were higher than enzymatic method. After 10 days of culture, no statistical differences were shown in survival rates of preantral follicle among the 2 culture groups. The metaphase II rates of the oocytes were significantly higher (p<0.05) in mechanical method (17.8%) than in enzymatic method (5.1%). These results suggest that the isolation method of choice depends on the target stage preantral follicles and mechanical isolation is an optimal method of preantral folliclesin a culture of mouse preantral follicle.

• 한국수정란이식학회지
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• 제22권3호
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• pp.179-184
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• 2007

체세포의 배양 방법은 체세포 핵이식에 의한 형질 전환 돼지 생산에 있어서 중요한 요인 중 하나이다. 본 연구에서는 돼지 태아 섬유 아세포의 효율적인 배양 방법을 수려하였다. 돼지 태아 섬유 아세포는 임신 33일째 태아로부터 제조하였으며, 돼지 태아 섬유아세포의 증식을 혈청과 배지 종류별로 분석하였다. 그 결과, 15% ES screened FBS가 포함된 DMEM 배지에서의 배양은 15% FBS보다 세포수의 증가가 훨씬 더 빠르게 나타났다. 또한, 태아 섬유아 세포는 DMEM/F-12와 다르게 ES midified DMEM과 DMEM 배지에서 $7{\sim}8$번째 계대까지 증식이 유지되었다. 이러한 배양 조건에서 PGK-neo 벡터(pKJ2)를 돼지 태아 섬유아 세포에 도입한 다음 12일간 $300\;{\mu}g/ml$ G418이 포함된 배지에서 선별하여 colony를 얻을 수 있었다. 이러한 결과는 본 연구에 이용된 배양 시스템이 transgenic vector를 도입시킨 돼지 체세포의 screening에 이용될 수 있음을 보여주고 있다.

• 한국잠사곤충학회지
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• 제49권1호
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• pp.28-32
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• 2007

누에체액이 동물세포 배양의 필수첨가물인 FBS를 대체할 수 있는 세포증식 효과가 있는 것으로 확인되었으나, 누에의 배발에 상처를 내어 채혈하는 방법으로는 대량 채취가 어렵다. 본 연구에서는 이러한 문제점을 해결코자 대량의 체액을 한꺼번에 얻을 수 있는 대규모 채혈법을 검토하게 되었다. 1. 누에 머리부분과 꼬리부분(배발 포함)에 상처를 낸 후 원심하여 체액을 채취하는 방법(절피원심법)이 기존의 소규모 채취한 체액과 유사한 세포증식 촉진효과를 보였다. 2. 절피원심한 체액의 원심속도에 따른 세포증식 촉진 효과는 500rpm과 1000rpm에서 양호한 값을 보였다. 3. 원심속도를 500rpm으로 고정한 경우 원심시간을 5분에서 30분까지 달리하여도 세포증식 효과는 유사하였고, 체액 회수율은 원심시간이 길어질수록 증가하였으나 일정시간 이상부터는 증가하지 않았다. 4. 절피원심법으로 대규모 채취한 체액의 액상회수율은 누에(백옥잠)생체중의 19.5%이었고, 분말회수율은 1.4% 이었으며, 분말체액에서도 세포증식 촉진 효과가 인정되었다.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2001년도 춘계학술발표대회
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• pp.76-76
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• 2001

The objective of this study was to optimize the vitrification method of in vitro produced bovine embryos. Thus, in vitro produced embryos at 8 cell, morula and blastocyst stages were vitrified on electron microscope grids (EM grids) or in open pulled straws (OPS) with EG5.5 (5.5 M ethylene glycol, 1.0 M sucrose and 10% FBS in m-DPBS medium) freezing solution and their survival rates after thawing were compared. The embryos on EM grids or in OPS were briefly exposed to EG5.5 freezing solution and plunged directly into liquid nitrogen within 30 to 35 sec. Post-thawed embryos were serially diluted in 0.5, 0.25 and 0.125 M sucrose in m-DPBS, each for 1 min, and then cultured in CRI aa medium supplemented with 10% FBS. Embryonic survival rate was assessed as re-expanded and hatched rates of those embryos after warming. The rates of re-expansion embryos did not significantly different between EM grid (8 cell: 42.10%, morula: 66.66% and blastocyst: 77.08%) and OPS (8 cell: 47.36%, morula: 61.90% and blastocyst: 83.33%) methods. In addition, the hatched rates in EM grid (8 cell: 31.57%, morula: 57.14% and blastocyst: 72.91%) were similar to those in OPS (8 cell. 34.21%, morula: 50.00% and Blastocyst: 77.08%). Interestingly, even at the same blastocyst stage, the in vitro survival of day 7 embryos (EM grid: 79.48 and OPS: 87.18%) was higher than those of day 8 embryos (EM grid: 72.10 and OPS: 82.06%). The total cell number of blastocyst developed in vitro after vitrification was examined with Hoechst 33342 staining to compare the embryo quality among different treatment groups. The total cell number of blastocyst was not significantly different between vitrified groups (EM grid: 162.4$\pm$8.0 and OPS: 158.4$\pm$7.1) and unvitrified control (168.0$\pm$5.6). These results indicate that both vitrification containers can provide the high rate of embryo survival. Moreover, the OPS container may not need a cap to protect the container from floating after immersion in L$N_2$. Therefore, this study suggest that bovine embryos can be cryopreserved easily, effectively and successfully by vitrification method using EM grid or OPS with EG5.5 freezing solution. In the future, the Pregnancy rate would be investigated after transfer of our vitrified embryos into the appropriated recipients.