• 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.

• 한국수정란이식학회지
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• 제9권2호
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• pp.173-180
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• 1994

The present experirnents on cryopreservation were carried out to investigate effect of solution toxicity, equilibration time and cell stages on the post-thaw survival of mouse morulae and blastocyst embryos cryopreserved by vitrification in EFS solution. The mouse embryos were exposed to the EFS solution in one step at room temperature, kept in the EFS solution during different period for toxicity test, vitrified in liquid nitrogen and thawed rapidly. After the mouse morulae embryos were exposed to EFS solution for 2 and 5 ruin. at room temperature and then they were washed in 0.5 M sucrose solution and basal mediurn(D-PBS + 10% FCS), they were cultured to examined cryoprotectant toxicity induced injury during exposure, most of embryos developed to expanded blastocysts(100 and 90.0%). However, when the exposure time was extended to 10 and 20 min, these development rates dropped dramatically in 10 ruin. (75.0%) and 20 ruin. (4.5%), respectively. When the compacted morulae were vitrified in EFS solution after equilibration for 2 and 5 min, the embryos have developed to normal blastocyst following thawing, washing and culture processes was 89.3 and 89.6%. However, when the exposure time was expanded to 10 ruin, this survival rate dropped to 68.8%. When the blastocyst were vitrified in EFS solution after equilibration for 2, 5 and 10 minutes, the survival rate of embryos which developed to normal blastocyst following thawing and culture processing were 58.5, 46.7 and 22.4%, respectively. The optimal time of equilibration of mouse morula and blastocysts in EFS solution seemed o be 2 and 5 ruin.

• Clinical and Experimental Reproductive Medicine
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• 제27권3호
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• pp.283-289
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• 2000

Objective: The study was performed to compare the survival rate and the development of day 2 mouse embryos which had freezing procedures done. Methods: We used three different vitrification solutions (EFS, VS14, DPS) and a ultrarapid freezing solution (UFS) for cryopreservation of day 2 mouse embryo. Results: We tested toxicity by exposing embryos to vitirification solutions and a ultrarapid freezing solution. The survival rates are 100%, 97.8%, 95.6% and 100% (EFS, VS14, DPS and UFS). After cultured for 96 hours, hatching rates of each group are 93.5% (no freezing), 95.6% (EFS), 86.4% (VS14), 93.0% (DPS), and 93.0% (UFS). There is no significant differences among groups. The survival rates after thawing cryopreserved embryos are 80.2%, 91.7%, 69.5%, 0% and 91.8% (slow freezing, EFS, VS14, DPS and UFS). Also cultured for 96 hours, the hatching rates are 93.5% (no freezing), 84.1% (slow freezing), 93.9% (EFS), 48.5% (VS14) and 70.1% (UFS). Conclusion: The survival rates of vitrification in EFS solution and ultrarapid freezing are higher than slow freezing (p<0.05). The hatching rate of vitrification in EFS solution cultured for 96 hours is highest, so vitrification of day 2 mouse embryos in EFS solution considered as more effective for cryopreservation.

• Reproductive and Developmental Biology
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• 제28권3호
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• pp.173-179
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• 2004

본 연구는 돼지 미성숙 난포란의 유리화 동결액에 노출시 독성과 삼투압 스트레스가 가장 적은 유리화 동결액을 조사하기 위하여 실시하였으며, 그 결과를 요약하면 다음과 같다. 미성숙 난포란을 EFS(40% ethylene glycol +18% ficoll +0.3 M sucrose) 용액, ES(5.5 M ethylene glycol +1.0 M sucrose) 용액, GE(10% glycerol +20% ethylene glycol) 용액에 각각 노출하고 대조구로 아무 처리하지 않은 난포란을 체외성숙하여 관찰한 체외성숙율은 ES 용액과 대조구 에서 EFS 용액과 GE 용액보다 유의적으로 높았으며(P<0.05), 유리화 동결액의 독성에 의한 난포란의 퇴화율은 대조구, ES 용액, EFS 용액, GE 용액 순으로 유의적으로 낮은 퇴화율을 나타내었다(P<0.05). 미성숙 난포란을 EFS 용액, ES용액, GE용액에 각각 노출하고 대조구로 아무 처리하지 않은 난포란을 체외성숙, 체외수정하여 관찰한 정상 수정율은 대조구가 세 종류의 유리화 동결액보다 유의적으로 높았고(P<0.05), 유리화 동결액 간에는 유의적인 차이가 없었다. 다정자 침입율과 난자당 침입한 평균 정자수는 모든 처리구간에 유의적인 차이가 없었다. 미성숙 난포란을 EFS 용액, ES 용액, GE 용액에 각각 노출하고 대조구로 아무 처리하지 않은 난포란을 체외성숙, 체외수정, 체외 배양하여 관찰한 수정 3 일째의 분할율은 ES 용액과 대조구가 EFS 용액과 GE 용액보다 유의적으로 높았으며(P<0.05), 수정 7일째의 배반포 형성율은 모든 처리구간에 유의적인 차이가 없었다. 배양기간 동안 나타난 퇴화율은 대조구가 세 가지 유리화 동결액에 비해 유의적으로 낮았고(P<0.05), ES 용액은 EFS 용액과 GE 용액보다 유의적으로 낮은 퇴화율을 나타내었다(P<0.05).

• Clinical and Experimental Reproductive Medicine
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• 제24권3호
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• pp.325-333
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• 1997

The use of hormonal stimulation in human in vitro fertilization and embryo transfer (IVF-ET) leads to increased production of embryos for ET. So to avoid high pregnancies and to allow conception in future, unstimulated cycles, cryopreservation of spare embryos is desirable. One of the improvement of cryopreservation methods is vitrification. We cryopreserved mouse day 3 embryos by vitrification using the three different vitrification solution (EFS40, VS11 and VS3a). EFS40 solution is consisted of 40% (v/v) ethylene glycol, Ficol170 30% (w/v) and 0.5M sucrose and VS11 is 6.0M ethylene glycol and 1.8M glycerol. And VS3a is 6.5M glycerol and 6% (w/v) BSA (bovine serum albumin). First we tested the toxicity of three vitrification solution by exposure to these solution during 3 min. After washing by thawing solution, the survival rates of each groups are 95.5%, 90.9% and 84.4% (EFS40, VS11 and VS3a). High percentages of them developed to expanded blastocyst and hatching embryos in culture 48hrs 94.2%, 97.7%, 100% and 97.4% (no treatment group, EFS40, VS11 and VS3a). So there is no significant differences among the each group. Second, after thawing of vitirfied embryos, the survival rates of each groups are 96.8% (slow freeze), 94.1% (EFS40), 85.5% (VS11) and 80.0% (VS3a, P vs. no freeze or EFS40 is 0.01). Vitrified embryos exhibited a high rate of development in vitro after 48hrs culture. The percentages of each group to blastocyst and hatching embryos are 88.7% (no freeze), 91.8% (slow freeze), 93.4% (EFS40), 87.7% (VS11) and 73.0% (VS3a, P vs. other group is 0.01). The results suggest that there is no significant differences in exposure of various vitrification solution and day 3 mouse embryos can be vitrified in solution EFS40 and VS11 by simple procedure.

• 한국수정란이식학회지
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• 제14권3호
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• pp.145-153
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• 1999

This study was conducted to investigate the effects of vitrification solution and developmental stage on the survival rate of vitrified-thawed human blastocyst embryos. Human blastocyst embryos were cryopreserved by vitrification using EFS and GE solution, and their survival rates were examined after thawing and further culture. EFS solution was consisted of 40% ethylene glycol, 18% Ficoll 70 and 0.3M sucrose. GE solution was consisted of 25% glycerol and 25% ethylene glycol. Embryos were exposed to EFS and GE solution by 2 steps and 3 steps, respectively, and plunged into liquid nitrogen after loading into 0.25ml plastic straws. Blastocysts were classified into 4 groups in accordance with their developmental stage: into 1) EEB, 2) MEB and 3) EdB, of blastocysts developed on day 5, and 4) 6d-Bla(the blastocysts which formed on day 6). The blastocysts at each stage were vitrified by GE solution and cryopreserved in LN2. After thawing them, we examined their survival rates, respectively. The resulted of this study were as follows: 1. The survival rate of blastocysts vitrified by GE solution was 64.4%, significantly higher than that (5.7%) vitrified by EFS solution (P<0.001). 2. When the blastocysts were vitrified by GE solution according to each developmental stage, the survival rates of EEB, MEB, EdB and 6d-Bla were 65.9%, 65.9%, 73.2% and 58.1%, respectively. In conclusion, the cryopreservation of human blastocysts by vitrification is likely to have a marked advantage in terms of cost, work and time as compared to the conventional slow freezing in IVF-ET programs.

• 한국발생생물학회:학술대회논문집
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• 한국발생생물학회 2003년도 제3회 국제심포지움 및 학술대회
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• pp.113-113
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• 2003

This study was conducted to investigate the effect of vitrification solution(VS) on in vitro developmental competence of immature porcine oocytes. The immature porcine oocytes were exposed to the following vitrification solution, at RT. 1) EFS sol. : 20% ethylene glycol (EG) 3 min, 40% EG + 18%(w/v) Ficoll(MV70, 000) + 0.3 M sucrose 30 sec, 2) GE sol. : 10% glycerol 5 min, 10% G + 20% EG 5 min, 25% G +25% EG 30 sec, 3) EG sol : 1.5M EG 2.5 min, 5.5 M EG + 1.0 M sucrose 30 sec. Oocytes were immediately transferred into 1.0 M, 0.5 M, 0.25 M, 0125 M, 0 M sucrose solution for 2.5 min each at RT. After removal of VS, immature oocytes were matured in vitro and subsequently all oocytes were subjected to IVF followed in vitro culture for 7 days. Maturation rates of oocytes were 38.8%, 44.5%, 22.4% and 57.6%, in EFS, EG, GE and Control, respectively, maturation rates of oocytes in EG and Control was significantly higher than EFS and GE(P<0.01). Fertilization rates of oocytes in Control was significantly higher than other treated groups(P<0.05), but no difference were observed among treated groups. Polyspermic rates were no significant difference among four groups. Cleavage rates of oocytes were 21.9%, 47.1%, 19.0% and 65.9%, in EFS, EG, GE and Control, respectively, cleavage rates of oocytes in EG and Control was significantly higher than EFS and GE(P<0.05), but blastocyst formation rates were no significant difference among four groups. These results suggested that the use of EG solution could be a great challenge for reaching a successful vitrification of immature porcine oocytes.

• 한국수정란이식학회지
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• 제9권1호
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• pp.7-14
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• 1994

The present experiments on cryopreservation were designed to examine the effects of solution toxicity, equilibration time and cell stages on the post-thaw survival of bovine IVF embryos. The oocytes were matured in vitro(IVM) for 24 hrs. in TCM-199 supplemented with 35 $\mu$g /ml FSH, 10 $\mu$g /ml LH, 1 $\mu$g /ml estradiol-17$\beta$ and granulosa cells at 39$^{\circ}C$ under 5% $CO_2$ in air. They were fertilized in vitro(IVF) by epididymal spermatozoa treated with heparin for 24 hrs., and then the zygotes were co-cultured in vitro(IVC) with bovine oviductal epithelial cells for 7 to 9 days. The bovine IVF embryos were exposed to the EFS solution in one step at room temperature, kept in the EFS solution during different period for toxicity test, vitrified in liquid nitrogen, and thawed rapidly. 1. after the bovine blastocysts were exposed to EFS solution for 2 min. at room temperature and then they were washed in 0.5 M sucrose solution and TCM-199, they were cultured to examined cryoprotectant induced injury during exposure, Most of the embryos(95.0%) developed to reexpanded blastocoels. However, when the exposure time was extended to 5 and 10 min, these development rates dropped dramatically in 5 min. (69.5%) and 10 min. (47.4%), respectively, 2. When the bovine IVF embryos were vitrified in EFS solution after the equilibration for 1 and 2 min. exposure, The embryos to have reexpanded blastocoels following thawing, washing and culture processes were found to he 82.6 and 73.9%, respectively. However, when the exposure time was extended to 3 min, this survival rate dropped to 18.2%. The optimal time for equilibration of bovine IVF blastocysts in EFS solution seemed to he 1~2 min. 3. When the bovine IVF embryos were equilibrated for 1 min. the significantly (P<0. 05) higher post-thaw survival rates were obtained from the embryos of blastocyst stage(81.3%) than morulae stage(5. 1%). The optimal cell stage for viterification with EFS solution proven to he blastocyst stage in bovine IVF embryos. 4. The number of blastomeres of blastocyst stage was examined with nuclear staining with Hoechst 33342 during 7 to 9 days post-insemination. The cell counts of frozen bovine IVF embryos were found significantly(P$\geq$7.5 and those of the fresh embryos 76.6$\geq$7. 1, which were cultured in the sarne period and conditions as frozen embryos.

• 한국가축번식학회지
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• 제15권3호
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• pp.173-177
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• 1991

In vitro survival of the mouse morulae frozen by vitrification method(Kasai et al., 1990) was investigated in the present study. The embryos were plunged into LN2 directly after exposure to the vitrification solutions(EFS, GFS and DFS). The results were obtained as follows. The viability of morulae after freezing and thawing was high in EFS(96.7∼100.0%) and GFS vitrification solution(93.3∼96.7%), and the lowest in DFS vitrification solution(0.00∼0.03%).

• The Korean Journal of Physiology and Pharmacology
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• 제2권2호
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• pp.185-192
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• 1998

A role of endogenous somatostatin in pancreatic exocrine secretion induced by intrapancreatic cholinergic activation was studied in the isolated rat pancreas perfused with modified Krebs-Henseleit solution. Intrapancreatic neurons were activated by electrical field stimulation (EFS: 15 V, 2 msec and 8 Hz). Pancreatic exocrine secretion, including volume flow and amylase output, and release of somatostatin from the pancreas were respectively determined. Somatostatin cells in the islet were stained with an immunoperoxidase method. EFS significantly increased pancreatic volume flow and amylase output, which were reduced by atropine by 59% and 78%, respectively. Intraarterial infusion of either pertussis toxin or a somatostatin antagonist resulted in a further increase in the EFS-evoked pancreatic secretion. EFS also further elevated exocrine secretion in the pancreas treated with cysteamine, which was completely restored by intraarterial infusion of somatostatin. EFS significantly increased not only the number of immunoreactive somatostatin cells in the islet but also the concentration of immunoreactive somatostatin in portal effluent. It is concluded from the above results that intrapancreatic cholinergic activation elevates pancreatic exocrine secretion as well as release of endogenous somatostatin. Endogenous somatostatin exerts an inhibitory influence on exocrine secretion induced by intrapancreatic cholinergic activation via the islet-acinar portal system in the isolated pancreas of the rat.