• 대한수의학회지
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• 제33권1호
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• pp.179-188
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• 1993

The developmental capacity of bovine oocytes under three different culture systems was investigated in this experiment ; One was culture in TCM199 with bovine oviductal epithelial cells(BOEC) for in vitro culture, another was culture in TCM199 with BOEC for 2 days and then transfer of 4~8cell embryos to rabbit oviduct(RO) and the other was transfer of 1 or 2cell embryos to RO for in vivo culture. And the other concern of this experiment was to investigate the effect of culture period and transfer site on recovery. Immature bovine oocytes were cultured in TCM199 with granulosa cells for 22-24hrs and then fertilized in vitro using frozen-thawed semen treated with BO-caffine and BO-BSA. Fifteen to 18hrs after in vitro fertilization oocytes were cultured in TCM199 with BOEC or transferred to RO for 5 days. The rate of development to the morula or blastocyst was higher in transfer of 1 or 2cell embryos to RO(23.1%) than culture in TCM199 with BOEC(11.7%). But, there was no difference between transfer of 1 or 2cell embryos and transfer of 4~8cell embryos to RO(12.8%). Recovery under different culture periods in RO was significantly higher in 90~95hrs(70.1%) than 122~125hrs(50.9%, p<0.05) and recovery significantly increased when oocytes were transferred deeper in RO(2.5cm>, 47.7% ; 2.5~4.5cm, 63.9% ; 4.5cm<, 77.3%, p<0.05). The results show that transfer of 1 or 2cell embryos to RO is an effective means of supporting the further development of in vitro matured and fertilized bovine oocytes than culture in TCM199 with BOEC or transfer of 4~8cell embryos to RO, and recovery from RO increases when oocytes are transferred deeper and incubated shorter in RO.

• Reproductive and Developmental Biology
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• 제37권3호
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• pp.155-159
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• 2013

본 연구는 제주흑우의 정액의 동결과 보존기술 개발을 위해 AndroMed와 Trialdyl를 사용하여 보다 우수한 희석제와 우수 종모우 선정을 위해 실시되었다. 종모우 1번부터 4번까지 개체 정자의 성상비교에서 수정능 획득과 첨체반응의 비율은 36.8%, 26.8%, 37.8% 그리고 38%의 결과를 나타내었다. 2번 개체 정자에서 수정능 획득과 첨체반응의 비율이 유의적으로(p<0.05) 낮은 결과를 보였다. 제주흑우 정액을 동결하기 전 정자의 생존율은 약 $93.27{\pm}1.62$, 동결 융해 후 생존율은 $73.34{\pm}3.27%$로 나타나 동결전 정액의 생존율이 유의적으로(p<0.05) 높은 결과를 나타내었으나, 사멸 정자의 비율은 약 $7.35{\pm}2.63%$와 $13.71{\pm}2.85%$의 결과를 나타내었다. 그리고 $Triladyl^{(R)}$ 동해 방지제를 사용하였을 때, 정자의 운동성은 $72.86{\pm}2.83%$, $AndorMed^{(R)}$를 사용하였을 때 운동성은 $81.47{\pm}2.48%$로 $AndorMed^{(R)}$ 동해방지제를 사용하였을 때 운동성이 다소 높은 경향을 보였으나, 유의적인 차이는 나타나지 않았다. 마찬가지로 정자의 사멸율에서도 $Triladyl^{(R)}$ 동해 방지제는 $18.41{\pm}3.42%$, $AndorMed^{(R)}$ 동해방지제는 $17.26{\pm}4.25%$의 결과를 보여 AndroMed를 사용한 동해방지제가 정자의 생존율을 향상시키는 것으로 나타났다. 결론적으로 제주흑우 정액의 동결 보존을 위해 보다 향상된 동결보호제 선정과 동결보존 기술 개발이 필요할 것으로 사료된다.

• Clinical and Experimental Reproductive Medicine
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• 제42권3호
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• pp.94-100
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• 2015

Objective: The goal of this study was to ascertain optimal assisted hatching (AH) method in frozen embryo transfer. We compared the effect of depending on whether mechanical or laser-AH was performed before or after the vitrification of embryo development rate and blastocyst cell numbers. Methods: In order to induce superovulation, pregnant mare's serum gonadotropin followed by human chorionic gonadotropin were injected into 4- to 5-week-old female mice. 2-cell embryos were then collected by flushing out the oviducts. The Expanded blastocysts were recovered after the collected embryos were incubated for 48 hours, and were then subjected to artificial shrinkage (AS) and cross-mechanical AH (cMAH) or quarter-laser zona thinning-AH (qLZT-AH) were carried out using the expanded blastocysts before or after vitrification. After 48 hours of incubation, followed by vitrification and thawing (V-T), and blastocysts were fluorescence stained and observed. Results: The rate of formation of hatched blastocysts after 24 and 72 hours of incubation was significantly higher in the AS/qLZT-AH/V-T group than in the other groups (p<0.05). The cell number of the inner cell mass was higher in AS/V-T/non-AH and AS/V-T/cMAH groups than those of others (p<0.05). In the control group, the number of trophectoderm and the total cell number were higher than in the AS-AH group (p<0.05). Conclusion: The above results suggest that AS and AH in vitrification of expanded blastocysts lead to the more efficient formation of hatched blastocysts in mice.

• 농업과학연구
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• 제22권1호
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• pp.62-68
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• 1995

본 연구는 돼지 수정란의 2, 4, 8세포배 및 상실배로부터 분리된 분할배의 작성율, 발생능 및 급속동결에 미치는 영향을 구명하고자 실시되었으며, 그 결과는 다음과 같다. l. Pronase 처리로 분할배를 작성했을 때, 성공율은 2세포배에서 85.7% 이었고, 평균은 68.0% 이었다. Manipulator를 이용했을 경우에는 2세포배 76.6% 및 4세포배 74.3%의 성공율을 나타냈다. 2. 분할배를 체외배양했을 때 배반포기까지의 발달율은 2, 4, 8세포배 및 상실배에서 pronase 처리의 경우 각각 24.1%, 20.4%, 25.5% 및 26.2%이었고 manipulator를 이용하였을 경우 각각 36.4%, 39.5%, 36.1% 및 4l.9% 이었다. 3. 분할배를 동결 융해했을 때 배반포기까지의 발달율은 2세포배에서는 glycerol(16.l%), 4세포배에서는 DMSO(16.7%) 및 상실배에서는 ethyleneglycol(27.6%)의 내동제를 이용한 경우에서 각각 가장 좋은 성적을 나타냈다.

• 대한수의학회지
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• 제37권3호
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• pp.647-659
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• 1997

To overcome the difficulties of collecting and culture of primary cell from genital tract on embryonic development, the present study was carried out to investigate the critical effect of cell lines, such as BRL and Vero cell and its conditioned medium on the development of early Korean native cattle embryos in vitro. Oocytes collected from slaughterhouse ovaries were matured in TCM199 containing FSH, estradiol-$17{\beta}$ and FBS with granulosa cell monolayer for 24 hours and then fertilized in vitro using frozen-thawed, heparin-treated spermatozoa in TALP for 30 hours. And then early embryos (1-2cell) were cultured in TCM199 containing 10% FBS with BOEC, Granulosa, BRL, Vera cell monolayers and conditioned medium for 2~3 days. Development to morulae and blastocysts were recorded, also examined the number of blastomeres presented a valuable parameter for the evaluation of embryonic development. The early cleavage rates of in vitro fertilized embryos co-cultured, there was no differences between primary cell and cell lines(p<0.05). The rate of development to the later stage, coculture of BRL cell was significantly higher than that of the primary cell(p<0.05). The rates of development to morula and blastocyst were significantly higher in vero cell than BRL, Granulosa, Oviduct epithelial cell conditioned medium. In the result of effect of serum on development of early bovine embryos, the use of media containing serum were significantly higher than the use of not containing one on development of early and later stage of embryos. The result of number of blastomeres in blastocysts, there is no differences between primary cell and cell lines. The blastocysts from coculture were higher than from conditioned medium in blastomere cells. In summary, these experments have proved that the culture system in TCM199 with BRL, Vero cell monolayers is effective on in vitro development of early bovine embryos, In addition, it is effective to development of bovine embryos that containing serum in conditioned medium, or in co-culture rather than in conditioned medium alone. The use of cell lines opponent to primary cells is effective in bovine embryo culture.

• 한국가축번식학회지
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• 제17권1호
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• pp.57-68
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• 1993

This stduy was carried out to find a reliable method for the production of in vitro fertilized embryos having more excellent development capacity and freezability in the rabbit and cattle. The greatest number of rabbit oocytes was recovered 6hrs after HCG injection(P<0.05). The maturation rate in vitro was slightly higher in the oocytes(6-h-oocytes) from 6h than those (8-h-oocytes)from 8 hrs after HCG injection and the beneficial effect of FSH during oocyte maturation was significantly great in the oocytes from large follicles. The cleavage rate into 2-to-6-cell stage was not differ between the 6-h-oocytes and 8h-oocytes, but the cleavage of these oocytes was greatly promoted by FSH addition to maturation medium and the cleavge of 8-h-oocytes matured without FSH was significantly low. The embryo development into 16-cell to morula was not promoted by the co-culture with rabbit oviduct epithelial cells. The freezability by embryo stages was ovidusly high at 4-cell and morula stage in 6-h-oocytes and the viability of 16-cell embryos from 8h-oocytes was similar to that of morula stage. The implantation sites after surgical tranfer of fresh rabbit embryos were not implanted. In bovine experiment, the in vitro development into 16-cell and morula after in vitro maturation and fertilization in the follicular oocytes was slightly improved by the co-culture with granulosa cells compared to that with oviduct epithelial cells and the frozen-thawed viability rate of these embryos ranged from 14 to 40%. The excellent fresh embryos were transferred nonsurgically to 6 recipients, but were not pregnant.

• Archives of Plastic Surgery
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• 제36권2호
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• pp.233-236
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• 2009

Purpose: Spindle cell lipoma(SCL) is an uncommon subcutaneous soft tissue neoplasm that arises in the shoulder and posterior neck of older male patients. The imaging appearance of SCL is not pathognomonic and can display some features overlapping with liposarcoma. We report a case of SCL on the posterior neck. Method: The patient is a 50 - year - old man with a slowly enlarging subcutaneous mass on the right side of posterior neck. Computed tomographic imaging revealed a 7.0 cm sized, well - circumscribed, heterogenous and fatty mass with enhanced solid components. Whole body Fluorine - 18 Fluorodeoxyglucose Positron emission tomogram(FDG PET-CT) showed little increase of FDG uptake on the right posterior neck and there was no distant metastasis. Results: The mass was surgically removed. The resection margin was free of tumor on frozen biopsy. Histopathologic examination indicated spindle cell lipoma consisting of a mixture of mature adipocytes and uniform spindle cells within a matrix of mucinous material. Conclusion: Although CT image of solidtary mass in posterior neck is similar with the one of liposarcoma, we should consider that it may be a spindle cell lipoma if PET-CT and other systemic studies reveal no distant metastasis. And we should perform fine needle aspiration to differentiate SCL from malignant lesions.

• Applied Microscopy
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• 제31권2호
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• pp.185-197
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• 2001

여러 가지 장점이 있음에도 일정성이 유지되지 않는 다는 단점이 있는 마이크로파 고정 효과를 "저에너지 마이크로파"를 사용하여 보완하고자 하였다. 이를 위해 사람 위선암(gastric adenocarcinoma)을 저 에너지 마이크로파(low energy of microwave, LEM)로 고정하여 미세구조와 항원-항체반응 수준에서 그 효과를 관찰하고자 하였다. LEM으로 고정한 시료에서 항원-항체반응은 monoclonal 생쥐-항-사람-p53 (IgG2b, kappa)과 토끼-항-사람-c-erbB-2로 처리하여 결과를 냉동 절편과 비교하였다. 암세포 특이 항원은 LEM으로 처리한 시료에서 발색반응 산물의 확산이 적은 것으로 관찰되었고 보다 쉽게 인지될 수 있었다. LEM으로 고정한 위선암 조직의 미세구조는 보존된 것으로 보였으나 그 고정효과는 2차 원인에 의해 손상되는 것으로 보였으므로 이를 보상하기 위해서 LEM 고정 후 저농도 화학 고정액으로 재차 처리하여 다소의 일정성을 유지할 수 있었으나 위선암 조직은 더 낮은 마이크로파 에너지 요구성이 있는 것으로 생각할 수 있었다. 따라서 암세포가 요구하는 "최소 마이크로파 요구 범위"를 밝히기 위해서 배양 HeLa 세포를 더 낮은 에너지의 마이크로파와 저농도 화학고정액으로 처리한 결과 HeLa세포 미세구조가 보존되는 등 비교적 좋은 효과를 볼 수 있었다. 이 결과로 보면 LEM 조사(照射)로서 생체 저분자 및 수용성 단백질이 crosslink되는 효과가 있는 것으로 보았고 이를 저농도 화학 고정액으로 재차 고정하는 방법으로서 미세구조 및 항원성 보존 효과가 있는 것으로 사료되었으므로 이에 대하여 논의하고자 하였다.

• 한국수산과학회지
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• 제20권3호
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• pp.191-201
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• 1987

정어리를 효율적으로 이용하기 위하여 각종 조리재료 및 식품가공용 중간소재로 이용할 수 있는 정어리냉동조미육의 가공조건을 구명하고 저장 중의 품질안정성에 대하여 검토하였다. 정어리냉동조미육을 가공하기 위하여 정어리육을 채육한 다음 육에 대하여 대두유의 유화커어드 $20.6\%$, 식염 $0.5\%$, 설탕 $2.0\%$, sodium bicarbonate $0.4\%$, polyphosphosphate $0.2\%$, MSG $0.1\%$ 및 양파가루 $0.3\%$, 마늘가루 $0.1\%$, 생강가루 $0.1\%$, 대두단백질 $3.0\%$, 그리고 저장 중의 지질산화를 방지 할 목적으로 sodium erythortate를 $0.1\%$ 첨가하여 잘 혼합한 후 $-35^{\circ}C$에서 동결시켜 $-20^{\circ}C$에서 저장하는 것이 좋았다. 정어리냉동조미육의 수분함량은 $67\~70\%$, 조단백질 $14\~16\%$, 조지방 $11\~12\%$였으며, 저장 중 제품의 pH는 다소 감소하고 휘발성염기질소는 약간 증가하였으며 생균수는 거의 변화가 없었다. 동결저장 중 과산화물값, 카르보닐값 및 TBA값을 측정한 결과 sodium erythorbate를 첨가한 제품(B)는 지질산화가 효율적으로 억제되었다. 제품의 정미성분 중 IMP는 저장 중 감소하였오 hypoxanthine은 증가하였다. 제품의 유리아미노산은 histidine과 glutamic acid가 대부분을 차지하였고 저장 중 약간 증가하였다. 정어리냉동조미육의 정미성분의 주체는 양적으로 보아 유리아미노산과 핵산관련물질이었고 저장중 총량은 거의 변화가 없었다. 제품의 주요구성지방산은 18:2, 18:1, 16:0 및 18:3 등이있고 저장 중 22:6의 감소율은 제품(B)에서 현저하게 억제되었다. 저장 중 제품 모두 유리드립과 가압드립은 증가하였고 염용성질소는 감소하였다. 텍스튜어는 경도와 질김성이 다소 증가하였으며 탄성과 응집력은 거의 변화가 없었다. 색조는 L값(명도)은 감소하고 b값(황색도)은 약간 증가하였으며 그 변화폭은 표면이 더욱 컸다. 관능검사 결과 제품 모두 저장 120일 동안 품질이 안정하게 유지되었으며 제품(B)의 품질안정성이 가장 우수하였다.

• 한국수정란이식학회지
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• 제30권1호
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• pp.7-15
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• 2015

The objective of this study was to investigate the efficiency of nicotinic acid on sperm cryosurvival and fertilization ability in frozen-thawed boar semen. Boar semen was collected by glove-hand method and was frozen using freezing solution treated to 0, 5, 10 and 20 mM of nicotinic acid. The frozen sperm for sperm characteristic analysis was thawed such as viability, acrosome reaction, and mitochondrial integrity. The frozen-thawed sperm was estimated by SYBR14/PI double staining for viability, FITC-PNA/PI double staining for acrosome reaction and Rhodamine123/PI double staining for mitochondrial integrity using a flow cytometry. The embryo was estimated in vitro development and DCFDA staining for reactive oxygen species assessment. As results, frozen-thawed sperm viability was significantly higher in 5 and 10 mM ($61.1{\pm}1.5%$,$64.7{\pm}2.0%$) of nicotinic acid than other groups (0 mM, $52.1{\pm}2.3%$; 20 mM, $47.8{\pm}5.1%$, P<0.05). The live sperm with acrosome reaction was significantly higher in 5 and 10 mM of nicotinic acid ($26.1{\pm}1.8%$, $24.9{\pm}1.5%$) than other groups (0 mM, $35.3{\pm}0.8%$; 20 mM, $36.5{\pm}1.9%$, P<0.05). The live sperm with mitochondrial integrity was significantly higher in 5 and 10 mM ($84.2{\pm}3.6%$, $88.4{\pm}2.3%$) of nicotinic acid than other groups (0 mM, $77.3{\pm}4.4%$; 20 mM, $73.3{\pm}3.6%$, P<0.05). Blastocyst rate of in vitro development was significantly higher in 10 mM ($17.0{\pm}1.3%$) of nicotinic acid than other groups (0 mM, $9.4{\pm}0.5%$; 5mM, $12.6{\pm}0.8%$; 20 mM, $5.0{\pm}1.0%$, P<0.05). Moreover, total cell number was higher in 5 and 10 mM ($53.6{\pm}2.9%$, $57.9{\pm}2.8%$) of nicotinic acid than other groups (0 mM, $41.0{\pm}1.4%$; 20 mM, $23.2{\pm}2.8%$, P<0.05). Hydrogen peroxide in embryos was lower in 5 mM nicotinic acid ($0.7{\pm}0.1%$) than other groups (0 mM, $1.0{\pm}0.1%$; 10mM, $0.9{\pm}0.0%$; 20 mM, $1.4{\pm}1.0%$, P<0.05). In conclusion, nicotinic acid-treated semen improves cryosurvival and quality of spermatozoa. Also, the fertilized oocytes with nicotinic acid improve quality of embryo and blastocyst formation.