• 대한수의학회지
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• 제31권4호
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• pp.523-527
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• 1991

The effects of ethylene glycol, DMSO and glycerol as cryoprotectants and the effect of concentrations(0, 0.25, 0.5 and 1.0M) of sucrose in the diluent on the viability of the aggregated morulae frozen rapidly in liquid nitrogen$(LN_2)$ vapour were examined. The morulae were produced by aggregation of ICR and CBA mice embryos at 8-cell stage. Before freezing the embryos were equilibrated in 1.5M cryoprotectants+0.25M sucrose in oae-step or in 3.0M cryoprotectants+0.25M sucrose in two-steps. The embryos were pipetted into the freezing medium fraction of 0.25ml plastic straws. The straws were frozeu by directly transfer into $LN_2$ vapour(about lcm above $LN_2$) for 2 minutes, and then plunged into $LN_2$. After thawing the cryoprotectants were diluted with 0, 0.25, 0.5 or 1.0M sucrose solution. The post-thawed in vitro viability of the aggregated embryos was significantly dependent on the type and concentration of cryoprotectants in the freezing medium and also on the concentration of sucrose in the diluent. When the aggregated embryos were equilibrated in 1.5M cryoprotectants +0.25M sucrose in one-step and diluted with 0.5M sucrose after thawing, the survival rate of the embryo5 was significantly(p<0.05) higher in DMSO(62.5%) or ethylene glycol(52.2%) than in glycerol(33.3 %). In the case that the concentration of the cryoprotectants was raised to 3.0M in two-steps, thc higher survival rate of the embryos was obtained in ethylene glycol or glycerol than in DMSO followed by diluting them with 0.5 or 1.0M sucrose after thawing(p<0.01).

• 대한수의학회지
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• 제29권1호
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• pp.61-68
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• 1989

This study was carried out to determine suitable selection factors for recipients and embryos which could improve pregnancy rates following bovine embryo transfer. The experiment included 52 surgical transfers from February, 1985 through June, 1986 performed on Kyungpook Breeding Center in southern Korea. The pregnancy rate was highest when recipients were in estrus within 6 hours before the donor to 12 hours after the donor (78.3% versus 50% for recipients in estrus earlier or later). Pregnancy rates were acceptable following culture under field conditions for up to 17 hours. More recipients over 15 months of age (76.1%) remained pregnant than those under 15 months (66.7%). Embryos transferred during the months from February to July resulted in higher pregnancy rates than those transferred during the remaining 6 months (77.3% versus 57.1%). Transferrable embryos were classified A (best) to C (worst); those graded A or B resulted in significantly higher pregnancy rates than those graded C (81.8% and 73.3% versus 25.0%, p<.05). Pregnancy rates among recipients of the Korean native breed tended to be higher than among Holstein recipients (100% versus 71.1%). Similarly, when the embryo was transferred to the right uterine horn, pregnancy rates tended to be higher than when it was transferred to the left (81.3% versus 65%). Pregnancy rates did not differ according to the stage of development of the embryo; they were for morulae, tight morulae, blastocysts, and advanced blastocysts, respectively: 75.0%, 66.7%, 75.0%, and 77.4%.

• 한국수정란이식학회지
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• 제18권3호
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• pp.269-274
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• 2003

본 연구는 미성숙 돼지난자가 체외성숙$.$배양액인 TCM-199과 NCSU-23배지에서의 배양상태를 비교하였고, 체외성숙용 TCM-199배지에 Earle'ssalt와 Hank's salt의 첨가에 의한 세포분화와 배발달 상태를 각각 조사하였다. 1. TCM-199와 NCSU-23 배양액에 각각의 supplement를 첨가하여 5% $CO_2$조건하에서 체외성숙을 유기한 결과, TCM-199 배양액에서 유의성(p<0.05)이 검증되었다. 2. 체외성숙용 TCM-199배지의 Earle's salt와 Hank's salt의 조성 성분에 따른 미성숙 돼지난포란의 배발달률은 Earle's가 Hank's보다 약간 높았으나 유의적인 차이는 나타나지 않았다. 3. 총1,455개의 미성숙 난포란 중에서 999(68.6%)개가 세포분화를 나타내었고, 그중 blastocyst의 62개(6.2%)를 포함한 617개(61.8%)의 mo-rulae와 blastocyst의 배발달을 보였다.

• Clinical and Experimental Reproductive Medicine
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• 제32권2호
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• pp.177-185
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• 2005

Objective: This study was conducted to find an optimal condition for the vitrification of mouse morulae and expanded blastocysts. Materials and Methods: Mouse embryos were obtained at 2-cell stage and cultured to morula and expanded blastocyst stage in Human Tubal Fluid (HTF) medium supplemented with 10% Serum Substitute Supplement (SSS). The vitrification solutions used were EFS30, EFS35 and EFS40 that contains 30%, 35% and 40% ethylene glycol, respectively, with 18% ficoll and 0.5 M sucrose diluted in Dulbecco's phosphate-buffered saline (DPBS) medium supplemented with 10% SSS. The vitrification procedure was performed in EFS solution with three steps, followed by thawing in 6 steps with 0.5 M sucrose, and then survival and hatching-hatched rate per embryos recovered were compared among six groups. Results: After 24 h culture in different vitrification and thawing solution, the survival rate of morula embryos was 94.1%, 85.4% and 59.7% for EFS30, EFS35 and EFS40 group, respectively. Hatching rate of morula embryos after 72 h culture was 30.6%, 25% and 11.3% for EFS30, EFS35 and EFS40 group, respectively. The survival rate of expanded blastocyst embryos after 24 h culture was 90.4%, 98.5% and 100% for EFS30, EFS35 and EFS40 group, respectively. Hatching rate of expanded blastocyst embryos after 48 h culture was 46.2%, 57.6% and 64.3% for EFS30, EFS35 and EFS40 group, respectively. Conclusion: The EFS30 solution was the best for vitrification of mouse morulae. The EFS40 solution was the best for vitrification of mouse expanded blastocysts. The mouse expanded blastocyst was better than mouse morula for vitrification of mouse embryos.

• Reproductive and Developmental Biology
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• 제28권3호
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• pp.161-166
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• 2004

체외수정 후 체외배양48시간에 난구세포의 제거 유ㆍ무에 따라 CR/sub 1aa/ 배양액에 homoglobin을 0, 1, 5 ㎍/㎖를 첨가한 구의 상실배기 이상 체외발육성 적은 난구세포를 제거한 구에서 23.8%, 33.3% 및 26.8% 였으며, 난구세포를 제거하지 않은 구에서는 각각 39.5%, 54.8% 및 48.8%로서 난구세포를 제거하지 않은 구에 Hb를 1 ㎍/㎖를 첨가한 구가 여타구보다 통계적으로 높은 결과를 얻었다(P<0.05). 체외수정 후 체외배양 96시간 후 난구세포를 제거 유ㆍ무에 따라 Hb를 0, 1, 5 ㎍/㎖를 첨가하였을 때, 상실배기 이상 체외발육성적은 난구세포를 제거한 구에서 각각 28.6%, 46.2% 및 39.1%였으며, 난구세포를 제거하지 않은 구에서는 각각 33.9%, 67.2% 및 46.0%로서, 난구세포를 제거하지 않은 구에 Hb를 1 ㎍/㎖를 첨가구가 여타구에 비해 통계적으로 유의하게 높게 나타났다(P<0.05). CR/sub 1aa/ 배양액에 L-NAME를 0, 10, 50 및 100 mM을 첨가한 구에서 상실배 이상 발육된 체외발육성적은 각각 55.6%, 64.9%, 58.8% 및 66.7%로써 L-NAME 첨가구가 무첨가구에 비해 커다란 차이가 나타나지 않았다(P>0.05). 모든 처리구에 배반포까지 발육된 체외수정란의 세포수에는 커다란 차이가 인정되지 않았다.

• 한국가축번식학회지
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• 제26권1호
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• pp.61-68
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• 2002

본 연구는 aesculetin과 $O_2$농도가 체외에서 성숙 수정된 한우 초기배의 체외발육에 미치는 영향을 검토하였다. 실험을 위하여 체외수정 후 40~44시간에서 2~8세포로 발육한 초기배는 6일간 배양하였다. 그 결과, 실험 1에서 상실배와 배반포기배의 발육은 산소농도 20% 보다는 5%에서 유의 적으로 높게 나타났으며 (P＜0.05), 1 $\mu\textrm{g}$/$m\ell$의 aesculetin 첨가시 0, 5, 및 10 $\mu\textrm{g}$/$m\ell$ 첨가에 비하여 유의적 (P＜0.05)으로 높은 발육율을 나타냈다 실험 2에서, 배반포기 및 상실배 이상 발육한 초기배의 비율은 산소농도 20%보다는 5%에서 역시 유의적으로 더 높았으며 (P＜0.05), 1$\mu\textrm{g}$/$m\ell$의 aesculetin 또는 2.5mM taurine 첨가시 배반포기 및 상실배 이상 발육한 초기배의 비율은 다른 실험구에 비하여 유의적으로 높게 나타났다 (P＜0.05). 실험 3에서, aesculetin과 PDGF 동시 첨가 및 taurine과 EGF를 동시에 첨가한 경우 다른 실험구에 비하여 유의적으로 높은 발육율을 얻었으며 (P＜0.05), aesculetin 무첨가 보다는 첨가시 배반포기배의 세포수가 높게 관찰되었으나 배양조건에 관계없이 유의적인 차이는 인정되지 않았다. 한편, 실험 4에서 자연에서 추출한 것과 상품화된 aesculetin이 초기배의 발육에 미치는 영향을 검토한 결과 무첨가에 비해 유의적으로 높은 발육율을 나타냈으나 (P＜0.05), 자연에서 추출한 것 (71%)과 상품화 (70%)된 aescuietin 사이에 유의적인 차이는 인정되지 않았다. 또한 자연에서 추출한 것과 상품화된 aesculetin 첨가시 무첨가에 비해 배반포기배의 세포수가 높게 관찰되었으나 이들 사이에 의한 유의적인 차이는 없었다. 본 연구의 결과로부터, 5%의 산소농도하에서 aesculetin은 항산화제와의 공동첨가에 의해 한우 초기배의 체외발육을 증진시키는 것으로 나타났다.

• 대한수의학회지
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• 제31권2호
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• pp.229-234
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• 1991

Thc chimeric morulae were produced following aggregation of the half embryos which were microsurgically bisected at 8-cell and early morula stage. Different phenotypic embryos were obtained by mating ICR female mice with ICR or CBA male mice. The early morula stage was thc desirable stage for the aggregation of mouse embryos after bisection. The post-thawed survival rates of bisected-aggregated embryos that developed into normal blastocyst after conventional freezing in DMSO and ethylene glycol were 30.5 and 32.896, respectively. One offspring was produced by transferring the 67 frozen-thawed bisected-aggregated embryos.

• 한국임상수의학회지
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• 제13권2호
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• pp.149-152
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• 1996

This study was carried out to evaluate the efficiency of production of cloned embryos by nuclear translatation (NT) when using 4-cell to compact morula stage embryos as nuclear donor. In micromanitulation and electrofusion of blastomeres from 4-cell to morula stage embryos, the successful injection rate was higher with late stage blastomeres, on the contrary the fusion rate was lower. The in vitro developmental rate of NT embryos was not significantly different between cell-stages of donor blastomeres. Although the overall rate of production of cloned embryos with 4-cell. 8-cell, early and late morula stage embryos was 14.0, 18.0, 15.3 and 14.1%, respectively, the mean number of blastocysts produced with a donor embryo was the most (4.51) with the compact morulae. Therefore, it can be suggested that the embryos at thelate stage is more beneficial for the mulciple production of cloned embryos, If the late stage blastomeres have maintained their totipotency to produce intact offspring.

• 한국가축번식학회지
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• 제19권3호
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• pp.161-169
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• 1995

To investigate the metabolism of various substrates in preimplantation bovine embryos, uptake of glucose and pyruvate, and lactate production were measured in single IVF-derived bovine embryos by a non-invasive method. When the embryos were incubated for 5 h in culture medium supplemented with 1 mM glucose and 0.4mM pyruvate as substrates at each developmental stage, glucose uptake was increased with more advanced developmental stages while pyruvate uptake was decreased. Total lactate producton of 2-cell embryos was significantly higher than that of blastocysts (p<0.05). Both of glucose uptake and lactate production in normal morulae produced in vitro was significantly high compared to the degenerated embryos(p<0.05). The results obtained in the study suggest that pyruvate as an exogenous substrate may be support in bovine embryos until 8-cell stage, whereas glucose may be effective as an energy source after morula stage. In addition, it was proven thatlactate was not effective as an energy source in preimplantation development of IVF-derived bovine embryos.

• 한국수정란이식학회지
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• 제8권2호
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• pp.143-149
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• 1993

The objective of this study was to produce calves derived from in vitro fertilization of in vitro matured follicular oocytes. Oocytes aspirated from small antral folicles of ovaries obtained at a local slaughter house were matured and fertilized in vitro. At l8hrs after insemination with Korean native cattle semen, oocytes were co-cultured for 6~7 days by utilizing co-culture system with bovine oviduct epithelial cell. After co-culture, good or excellent quality late morulae or early blastocysts were selected by morphological criteria under stereo microscope. Selected embryos were transferred to recipients on day 6 or 7 (estrus = day 0). Recipients were monitored by observation for estrus and rectal palpation after 60 days from embryo transfer. One of them went to term with the birth of a calf. This case is the first production of calf derived from in vitro fertilization in Korea.