• 한국수정란이식학회지
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• 제27권4호
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• pp.259-264
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• 2012

Solution of glycerol, ethylene glycol, sucrose, dextrose (GESD) and cryotop methods were carried out to investigate the survivability on vitrification of embryos. Embryos cultured in vitro were vitrified by GESD of 10 or 8 step and cryotop methods of 6 step, from cryopreservation step to frozen-thawed and culture step. Survival rate and ICM, TE cells of embryos were investigated after frozen-thawed 24 h. As a results, cryotop method was significantly (p<0.05) higher ($85.76{\pm}5.3$ vs. $66.71{\pm}2.4$, $44.80{\pm}2.1%$) than GESD 10 or 8 step methods on survivability. Also, In ICM cell number, cryotop method was significantly (p<0.05) higher to $45.67{\pm}4.7$ cells than GESD 8 step method. TE cell number was significantly (p<0.05) highest to $111.00{\pm}11.0$ cells in cryotop method. On the other hand, survival rate, TE and total cell number were all the significantly (p<0.05) high, except ICM in GESD 10 step method between GESD 10 step method and GESD 8 step method. In conclusion cryotop method was to be most effective, but it is considered necessary to study vitrification method for step-by-step freezing and thawing process.

• 한국수정란이식학회:학술대회논문집
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• 한국수정란이식학회 2003년도 제3회 발생공학 국제심포지움 및 학술대회
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• pp.80-80
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• 2003

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2001년도 춘계학술발표대회
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• pp.5-5
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• 2001

This study was to test whether Hanwoo in vitro matured oocytes can be successfully cryopreserved by a new vitrification procedure using MVC method. For the vitrification, oocytes were pretreated in 10% ethylene glycol (EG10) for 5-10 min, exposed in EG30 for 30 sec, each oocytes were individually put on the inner wall of 0.25 $m\ell$ straw, and then straws were directly plunged into L$N_2$. Thawing was taken by 4-step procedures [1.0 Msucrose (MS), 0.5 MS, 0.25 MS, and 0.125 MS] at 37$^{\circ}C$. In vitro developmental capacity (survival, cleavage ($\geq$2-cell) and blastocyst rates) in vitrified group was no significant difference compared to that in other treatment groups (exposed; 100.0, 74.4, 32.3% and control; 100.0, 78.3, 36.3%): high mean percentage of oocytes (91.2%) was survived, 69.4% of them were cleaved and 27.9% of cleaved embryos were developed to blastocyst. Especially, after transfer of in vitro developed embryos in vitrified group, four of six recipient animals were found to pregnant and three of them were ongoing pregnant by manual palpation at 250 days after transfer. However, among them, two healthy female calves (23 and 25kg) were born. This result demonstrates that MVC method is very appropriate freezing method for the Hanwoo in vitro matured oocytes and that ovum bank can be maintained efficiently by MVC cryopreservation method.

• 한국가축번식학회지
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• 제22권2호
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• pp.171-176
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• 1998

본 연구는 체외에서 배양된 생쥐 완전탈출 배반포기배를 동결보존액 EFS35를 이용하여 초자화 동결하였을 때 체내발달의 적합성 여부를 조사하기 위해 실시하였다. 공시된 완전탈출 배반포기배($\theta$ 130$\mu\textrm{m}$)는 체내에서 생산된 전핵기 수정란을 5일 동안 체외배양하여 얻었으며, 10% ethylene glycol(EG)에 5분 노출한 후 EFS35(35% EG, 18% Ficoll, 0.3 M sucrose)에 30초 동안 노출하거나, 초자화 동결하였다. 융해 후, 재팽창이 이루어진 완전탈출 배반포기배는 가임신 3일된 대리모의 한쪽 또는 양쪽 자궁각(4~6개/자궁각)에 이식하였다. 대리모의 임신율과 착상율은 임신 15일재 외과적 해부로 판정하였다. 그 결과를 요약하면 다음과 같다. 융해 30분 후, 완전탈출 배반포기배의 체외생존율은 노출군(65.5%)과 동결군(54.5%)간에 유의한 차이는 없었다. 또한, 체내발달율을 조사하였던 바, 착상율에 있어서 동결군(41.0%)과 대조군(58.5%)간에 유의한 차는 없었지만, 정상산자율에서는 동결군(24.0%)의 결과가 대조군(58.3%)보다 유의하게 낮게 나타났다(p<0.05). 이러한 결과는 EFS35를 이용한 완전탈출 배반포기배의 초자화 동결은 정상산자율은 감소하였지만, 완전탈출 배반포기배의 이용 효율성을 넓히는데 이용될 수 있다는 것을 알 수 있었다.

• 한국수정란이식학회지
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• 제17권1호
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• pp.61-65
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• 2002

한우 미성숙 난자의 vitrification 동결시 발생단계별 생존성과 체외발생율을 알아보고자 난자를 0, 10, 14 및 20시간 성숙배양시킨 후 vitrification 동결 융해 후의 체외발생율을 조사하였다 본 연구에서 나타난 결과를 요약하면 다음과 같다. 1. 0, 10, 14 및 20시간 성숙배양시킨 난자를 vitrification 동결보존 후 MR 단계로의 발생율은 각각 33.3%, 55.0%, 68.3% 73.3%였으며, diploid로의 발생율은 26.7%, 21.7%, 6.7%, 1.7%로서 대조군의 M II 단계의 78.2%에 비해 낮게 나타났으나 diploid단계의 3.6%에 비해서는 높게 나타났다. 2. 미성숙 난자를 0, 10, 14 및 20시간 성숙배양 시킨 후 vitrification 동결 응해 후의 생존율은 각각 38.0%, 30.0%, 20.0% 및 12.0%로서 비동결 대조군의 48.0%에 비해 낮은 생존율을 나타냈다. 3. 미성숙 난자를 0, 10, 14 및 20시간 성숙배양 시킨 다음 vitrification동결 응해 후 수정하였 때 체외수정율은 64.6%, 61.6%, 54.8%, 32.3% 였으며, 배 반포로의 체 외 발생 율은 각각 32.3%, 21.7%, 14.5%, 4.6%로서 대조춘의 80.0%와 55.0%에 비해 낮은 체외수정율과 체외발생율을 나타냈다.

• 한국발생생물학회:학술대회논문집
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• 한국발생생물학회 2003년도 제3회 국제심포지움 및 학술대회
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• pp.128-128
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• 2003

The aims of this study are 1) to test oocytes and embryos collected from in-vivo and in-vitro to achieving the valuable protocol by culturing, vitrifying and thawing of oocytes/embryos, and 2) to transfer them to recipient, and finally have resulted in pregnancies from recipient females after surgical or nonsurgical transfer. In vitro maturation and fertilization were performed according to the procedures of Funahashi et al. Fertilized oocytes were cultured in glucose-free NCSU 23 supplemented with 5 mM sodium pyruvate, 0.5 mM sodium lactate and 4 mg/ml bovine serum albumin for 2 days at 39$^{\circ}C$, and 10% fetal bovine serum was added to the culture medium thereafter. Embryos were treated with 7.5$\square$g/ml cytochalasin-B for 30 min, centrifuged at 13,000 ${\times}$ g for 13 min and then exposed sequentially to an ethylene glycol (EG) vitrification solution, aspirated into OPSs, and plunged/thawed into/from liquid nitrogen. In vivo embryos were surgically collected from three donors after Al. Forty-six embryos (18, 9 and 19 embryos, respectively) were washed 3 times in mPBS+10%FBS, followed treatments : cultured, centrifuged, vitrified, recovered and transferred to recipients as in vitro prepared embryos. Three recipients received surgically 34(control), 188 and 184 embryos (derived from abattoir), respectively. Another three recipients were received nonsurgically 150, 100 and 150 embryos, respectively. All recipient sows exhibited delayed returns to estrus. To our knowledge, these results suggest that required an improved techniques, more vigorous embryos preparation and cleaner uterous condition(use gilt).

• Clinical and Experimental Reproductive Medicine
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• 제46권4호
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• pp.178-188
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• 2019

Objective: To determine the clinical pregnancy (CP) and live birth (LB) rates arising from frozen embryo transfers (FETs) that had been generated under the influence of in vitro fertilization (IVF) adjuvants given to women categorized as poor-prognosis. Methods: A registered, single-center, retrospective study. A total of 1,119 patients with first FETs cycle include 310 patients with poor prognosis (109 treated with growth hormone [GH], (+)GH group vs. 201 treated with dehydroepiandrosterone, (-)GH group) and 809 patients with good prognosis (as control, (-)Adj (Good) group). Results: The poor-prognosis women were significantly older, with a lower ovarian reserve than the (-)Adj (Good) group, and demonstrated lower chances of CP (p< 0.005) and LB (p< 0.005). After adjusting for confounders, the chances of both CP and LB in the (+)GH group were not significantly different from those in the (-)Adj (Good) group, indicating that the poor-prognosis patients given GH had similar outcomes to those with a good prognosis. Furthermore, the likelihood of LB was significantly higher for poor-prognosis women given GH than for those who did not receive GH (p< 0.028). This was further confirmed in age-matched analyses. Conclusion: The embryos cryopreserved from fresh IVF cycles in which adjuvant GH had been administered to women classified as poor-prognosis showed a significant 2.7-fold higher LB rate in subsequent FET cycles than a matched poor-prognosis group. The women with a poor prognosis who were treated with GH had LB outcomes equivalent to those with a good prognosis. We therefore postulate that GH improves some aspect of oocyte quality that confers improved competency for implantation.

• 한국가축번식학회지
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• 제21권2호
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• pp.131-137
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• 1997

본 연구는 초자화 동결된 생쥐 팽창, 탈출, 완전탈출 배반포기배의 체내 발달율을 조사하기 위해 실시하였다. 체외수정하여 얻어진 생쥐 배반포기배는 EFS40(40% ethylene glycol, 30% Ficoll, 0.3M sucrose)으로 초자화 동결하였다. 팽창, 탈출 배반포기배는 20% ethylene glycol에 5분동안 평형시킨 다음, EFS40 용액에 1분간 노출후 액체질소에 침지하여 초자화 동결하였다. 완전탈출 배반포기배는 0.4% BSA가 첨가된 m-CR1 배양액에서 5일동안 배양하여 얻었으며, 10% EG에 5분, EFS40에 30초동안 노출하여 초자화 동결시켰다. 융해후 재팽창이 이루어진 배반포기배는 가임신 3일된 대리모의 한쪽 또는 양쪽 자궁각에(6∼8개/자궁각) 이식하였다. 대리모의 임신율과 착상율은 임신 15일째 외과적 해부로 판정하였다. 그 결과를 요약하면 다음과 같다. 1) 임신율과 정상 산자율은 초자화 동결된 팽창 배반포기배의 경우 77.8과 25.0%이었고, 탈출 배반포기배의 경우는 77.8과 26.4%로서 각각의 대조군에 있어서 66.7과 42.9%, 83.3과 40.4%에 비해 유의차가 없었다. 2) 완전탈출 배반포기배의 체외 발달율은 34.0%였고, 3) 체내 발달율은 33.3%였다. 이러한 결과는 본 실험에 사용된 EFS40 동결액을 이용한 초자화 동결방법이 생쥐 팽창, 탈출 배반포기배의 초자화 동결은 물론, 완전탈출 배반포기의 초자화 동결에도 유용하게 이용될 수 있다는 가능성을 시사하였다.

• 한국수정란이식학회지
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• 제11권3호
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• pp.271-281
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• 1996

The effect of superovulation (PMSG, FSH) on the ovarian response of matured cows were tested. The survival rates of bovine embryos and ovarian oocytes frozen by slow, rapid freezing and vitrification were investigated. A total of 15 heads of cow were devided into 3 groups by injection dose of GTH (PSMG, FSH). Each group was superrovulated with injections of 2, 500, 3, OOOJU PSMG and 40mg FSH followed by injection of 30mg PGF2a. Embryos were non-surgically recovered from superovulated cows 6~7days after estrus. The recovered embryos were frozen in 10% glycerol + 10% sucrose by slow and rapid freezing. Ovarian oncytes were frozen in 20% g]ycerol+l0% ethylene glycerol + 30% Ficol + 10% sucrose by vitrification and the survival of frozen embryos and ovarian oncytes were judged by FDA-test. The results are summarized as follows; 1. Estrus after the injection of 2500, 3000 I.U. PMSG and 4Omg FSH were 32.8, 35.0 and 43.4 and the duration of estrus were 18.6, 18.8 and 22.4 hours respectively. 2. The average sizes of the left ovaries were 5.4cm (2, 500 IU PMSG), 5.1cm (3, OOOIU PMSG) and 6.4cm (FSH), and the right were 6.2cm (2, 5001U PMSG), 5.7cm (3, OOOIU PMSG) and 7.&m (FSH) respectively. There were significant differences in the right overies among treatments (P<0.05). 3. The average number of ovarian follicles in the left ovaries were 4.8 (2, 500 IU PMSG), 5.2(3, 000 IU PMSG) and 7.8 (FSH) respectively. There were significant difference in the right ovaries among treatments (P<0.05). 4. In the average numbers of ovulation points in the left ovaries were 3.0 (2, 5001U PMSG), 3.2 (3, OOOIU PMSG) and 4.4 (FSH) respectively, and the right were 7.2 (2, 5001U PMSG), 7.8(3, 000IU PMSG) and 11.4 (FSH). There were significant differences in the right ovaries among treatments (P<0.05). 5. The numbers of the recovered embryos were 20 (2, 5001U PMSG), 19 (3, 000 IU PMSG) and 21 (FSH) respectively, and oncytes and degenerted oncytes were 6.5 and 11.0 Estrus periods of post parturation were 52.4days (2, 5001U PMSG), 69.8days (3, OOOIU PMSG) and 62.4days (FSH) respectively. 6. The FDA score of cow morulae frozen by slow freezing, sernirapid frezing and vitrified freezing was higher in slow (3.1) and vitrified freezing (3.0) than that in semirapid freezing (1.28). The FDA-scores of cow, pig and rabbit ovarian oocytes frozen in 20% glycerol + 10% ethylene glycol + 30% Ficoll + 10% sucrose by vitrification were higher in cows (3.3) than both in pigs (2.6) and rabbits (2.3).

• 한국가축번식학회지
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• 제24권1호
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• pp.89-95
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• 2000

본 실험은 초자화동결된 소 배반포기배를 실험현장에서 효율적으로 융해할 수 있는 기술을 찾고자 실시하였다. 초자화동결은 glycerol (G)과 ethylene glycol (EG) 그리고 10% FBS가 들어있는 m-DPBS를 이용하였으며, 배반포기배는 3단계로 초자화동결 되었는데, 10% G에 5분간 평형, 10% G와 20% EG에 5분간 평형, 그리고 25% G와 25% EG에 30초간 노출하였다. 질소 증기를 3분간 씌고 액화질소에 침지하였다. 융해는 straw 를 공기 중에서 10초간 노출시키고, $25^{\circ}C$ 물에서 빙정이 없어질 때까지 녹인 후 $25^{\circ}C$ 와 36$^{\circ}C$ 에 각각 시간차에 따라 처리군을 나누었다. 초자화동결된 배반포기배를 융해시 시간차에 따라 체외생존능은 융해 24시간과 48시간 후 재팽창과 완전탈출 배반포기배로 평가하였다. 그 결과를 요약하면 다음과 같다. 1) 초자화 동결된 배반포기를 융해시 시간차에 따라 체외생존농을 보았을 때, 1분으로 융해한 군이(86.6, 56.6%) 다른 처리군들보다 (2분 : 93.5, 35.4% ; 2.5분 : 76.9, 30.7% ; 3분 : 88.8, 36.1%; 3.5분 : 83.7, 8.1%) 체외생존능이 높게 나타났다. 2) 1분 융해방법으로 배반포기배의 발달단계에 따라 생존능을 조사하였을 때, 융해 48시간 후 빠르게 발달된 배반포기배의 부화율 (팽윤 : 93.8, 56.3% : 부화초기 : 86.2, 58.6%)은 느리게 발달하는 난자군의 부화율 (초기 : 83.3, 36.6%) 보다 높은 체외생존능을 나타내었다. 3) 또한, 1분 융해방법으로 배반포기배가 생산된 나이에 따라 체외생존능을 조사하였을 때, 융해 48시간 후, 7일 (66.6%) 과 8일 (60.0%)에 생산된 배반포기배가 9일 (22.7%)에 생산된 완전탈출 배반포기배율 보다 유의하게 높은 체외생존율을 나타내었다 (P＜0.05). 그러므로 초자화동결된 배반포기배를 1분 융해방법으로 융해하였을 때 빠르고 효율적으로 체외생존능을 얻을 수 있음을 알 수 있었다.