• 한국수정란이식학회지
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• 제31권1호
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• pp.27-32
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• 2016

This study was conducted to evaluate the effects of different volume ($100{\mu}l$ vs. 2 ml) of microdrop culture on B6D2F1 mice oogenesis. In the present study, B6D2F1/CrljOri $F_1$ mice were utilized in order to maximize oogenesis. Also we used TCM-199, Dulbecco's medified Eagle's medium (DMEM), embryo culture medium (Fertilization medium, Cleavage medium, Blastocyst medium), G series medium and One step medium. Blastulation rate was not different between groups ($58.4{\pm}2.9%$ vs. $61.2{\pm}4.8%$). Zona hatched rate ($38{\pm}15.4%$ vs. $27{\pm}3.4%$) and attached rate ($55{\pm}13.9%$ vs. $46{\pm}3.9%$) did not differ by the volume of culture media. Total cell numbers ($59.8{\pm}9.7$ vs. $70.3{\pm}8.7$), ICM cell numbers ($15.8{\pm}0.6$ vs. $16.8{\pm}1.5$), TE cell numbers ($44.0{\pm}9.7$ vs. $53.6{\pm}7.3$), % ICM ($26.4{\pm}2.9%$ vs. $23.8{\pm}3.3%$) and ICM:TE ratio ($1:2.8{\pm}0.4$ vs. $1:3.2{\pm}0.6$) were not different between groups (i.e., $100{\mu}l$ vs. 2 ml). These results show that the capacity of the culture medium did not effect the cell numbers of B6D2F1 mice blastocysts. In summary, these results can provide fundamental data to maximize culture condition for in vitro fertilization on B6D2F1 mice.

• 공업화학
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• 제8권5호
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• pp.763-770
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• 1997

(O/W)/O 에멀젼형 액막을 이용하여 비점이 비슷한 벤젠-싸이클로헥산 혼합물을 분리하였다. 액막 제조 조건이 선택도, 회수율 및 에멀젼 액적의 크기에 주는 영향을 고찰하였으며, 다단 맥동 추출탑을 이용한 연속 추출 실험을 통하여 비정상상태와 정상상태에서의 추출거동을 검토하였다. 추출실험결과 유화 교반 속도 4000rpm, Tween 80 농도 0.4%, 내유상에 대한 막상의 부피비 0.75, 연속상에 대한 분산상의 부피비 0.5, 투과시간10 분에서 선택도 및 벤젠의 회수율이 가장 좋았다. 에멀젼 액적의 크기 분포 측정 결과, 교반속도가 커질수록 또는 분산상의 microdrop holdup이 작을수록 분산상 액적의 평균크기가 작았으며, 분산상 microdrop holdup의 영향을 고려한 modified Calabrese의 식을 적용한 결과 turbulence damping constant의 값은 2.28이었다. 연속 추출 실험 결과 탑내 교반속도 300 rpm, 맥동 횟수 2 times/sec, 연속상 유속 30ml/min, 에멀젼상 유속 12.0ml/min에서 가장 좋은 추출 효과를 얻을 수 있었다.

• Journal of Animal Science and Technology
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• 제44권6호
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• pp.701-710
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• 2002

본 연구는 소 난자의 초자화 동결 방법을 설정하기 위하여 체외성숙 소 난자에서 난구세포가 부착된 상태 또는 제거한 상태로 microdrop (MD) 방법과 straw (Straw) 방법을 이용하여 초자화 동결하여 생존율을 검사하였다. 동결 융해난자는 a) 단위발생을 유도하였고 b) 체외수정 후 전핵 형성을 관찰하였으며 c)체외수정 후 수정란 발달을 검사하였다. 초자화 동결 난자의 생존율은 MD 방법을 이용하였을 때가 Straw를 이용하였을 때 보다 높았다 (92.50 vs. 74.19%, p<0.05). MD 방법을 이용하였을 때 대부분의 난자가 생존을 하였다. 단위발생을 유도하였을 때 난할율과 배반포 발달율은 MD (45.05%, 10.81%, p<0.05)가 Straw 방법 보다 높았다 (27.17%, 6.52%, p<0.05). 난구세포의 부착 유무에 따라 동결 융해 후 체외수정 하여 자웅 전핵 형성을 각각 검사하였다. 난구세포 제거 난자에서는 MD와 Straw 방법으로 동결 융해하였을 때 차이가 없었다(80.36% vs. 67.31 %, p<0.05). 정상 수정율 (2PN)에서는 세 처리간에 차이가 없었다 (Fresh; 54.55% vs. MD; 42.22% vs. Straw; 37.14%, p>0.05). 그러나 미수정란 (<1PN)은 Fresh 난자가 동결융해 난자보다 유의적으로 낮았다 (Fresh; 32.47% vs. MD; 57.78% and Straw 62.86%, p<0.05). 다정자 침입은 (3PN) 신선란 (12,99%)에서 발생하였으나 동결 융해 난자에서는 발생하지 않았다. 난구세포가 제거된 난자에서, 정상 수정율 (2PN)은 Fresh와 동결 융해 난자간에 유의적인 차이를 보였다 (Fresh; 59.38% vs. MD; 17.31% and Straw; 30.43%, p<0.05). 또한 미수정율 (<1PN)에 있어서도 신선란과 동결 융해난은 유의적인 차이를 보였다 (Fresh; 23.44% vs. MD; 73.08% and Straw 58.70%, p<0.05). 다정자 침입 (3PN, >4PN)은 신선란과 동결 융해란 모두에서 나타났다. 체외수정 후, 난구세포가 부착된 난자의 2세포기 발달율은 신선란에 비하여 MD 또는 Straw 처리구에서 유의적으로 낮았다 (Fresh; 81.76% vs. MD; 22.22% and Straw; 11.36%, p<0.05). 동결융해난자는 배반포 발달율에 있어서도 신선란에 비하여 유의적으로 낮았다 (Fresh; 28.38 vs. MD; 1.71% and Straw 0%, p<0.05). 난구세포가 제거된 난자에서 2세포기 발달율은 신선란과 MD에서 차이가 없었다 (27.59% vs. 19.25%, p<0.05), 그러나 배반포 발달율에 있어서는 신선란이 MD 또는 Straw 처리구보다 유의적으로 높았다 (4.31% vs. 0.62% and 0%, respectively; p<0.05). 이상의 결과에서, 초자화 동결융해한 소 난자는 체외수정 후 배반포로 발달이 가능함을 나타내주고 있다.

• 한국수정란이식학회지
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• 제12권3호
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• pp.293-300
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• 1997

Supplementation of maturation medium with additional granulosa cells has beneficial effect on in vitro maturation of bovine follicular oocytes and their subsequent cleavage and development in vitro. However, maturation system using granulosa cells have some disadvantages that collection of granulosa cells is cumbersome and metabolic activity of the cells is variable according to ovarian cycle or follicular size. We hypothesized that bovine immsture oocytes matured without granulosa cell coculture can fertilize and develop normally if the medium volume per oocyte is reduced during in vitro maturation. Immature oocytes were matured for 24 hours in a TCM199 containing 10% fetal calf serum, anterior pitultary hormone (0.02 AU /ml Antrinⓡ) and estradiol with or without granulosa cells in vitro. In Group 1, 35 to 40 oocytes were matured in a well of 4-well plastic dish containing 500 $\mu$l of maturation medium and granulosa cells, and 9 to 10 oocytes were matured in a 50-$\mu$l drop of maturation medium without granulosa cells in Group 2. After maturation, oocytes were coincubated with sperm for 30 hours in a modified Tyrode's medium (IVF). Inseminated oocytes were cultured in a microdrop (30 $\mu$l) of a synthetic oviduct fluld medium (SOFM) containing BSA, Minimum Essential Medium essential and non-essential amino acids for 9 days. As a preliminary experiment, we investigated the beneficial effect of granulosa cells during maturation on subsequent cleavage and development using the same type of culturedishes (4-well dish). Granulosa cells could not increase embryo cleavage after fertilization but significantly improved (p<0.05) embryo development to expanding blastocyst (Table1 and 2). In Group 1, 68 and 80% of inseminated oocytes have cleaved at 30 hours and 2 days after IVF, respectively, which is similar (p>0.05) to the result of Group 2 (69% at 30 hours and 78% at 2 days after IVF). The oocytes in Group 2 showed 21 and 11% of developmental rates to expanding and hatching blastocysts, respectively, which was not significantly different (p>0.05) from those (20 and 10%, respectively) of oocytes in Group 1. In conclusion, it has been clarified that a microdrop culture system without granulosa cells for in vitro maturation can support bovine embryonic development to blastocyst in vitro as readily as a granulosa cell coculture system.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2004년도 춘계학술발표대회
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• pp.258-258
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• 2004

Successful cryopreservation of mammalian oocytes would provide a source of materials for in vitro embryo production. This study was conducted to determine vitrification conditions for bovine immature oocytes using micro-drop method and, to examine maturation, fertilization and development of vitrified bovine immature oocytes. (omitted)

• 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.