• Clinical and Experimental Reproductive Medicine
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• 제29권4호
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• pp.317-322
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• 2002

Objective: These experiments were conducted to investigate the optimal expose length of propidium iodide (PI) and bisbenzimide on differential staining of mouse blastocysts. Materials and methods: A total 964 blastocysts (early${\sim}$hatched) was exposed to PI (n=831) (group I: $\leq$ 10; II: $11{\sim}15$; III: $16{\sim}20$; IV: $\geq$21 sec) and bisbenzimide (n=133) (group A: $\leq$1; B: $1{\sim}$3; C: $\geq$ 4 hr) in several periods for differential staining. Statistical analysis was performed using t-test with SigmaPlot-2001. P-values < 0.05 were accepted as statistically significant. Results: In case of PI exposure, differential staining rates were significantly higher (p<0.05) in group I (89.8%) than in any others (group II: 77.6%; III: 29.6%; IV: 22.2%) and higher (p<0.05) in group II than in group III and IV. In case of bisbenzimide exposure, differential staining rates were not statistically differences in three groups (group A: 97.4%; B: 87.8%; C: 93.3%). Conclusion: The differential staining rates of mouse blastocysts are not affected by the exposure length of bisbenzimide. However, blastocysts were exposed to PI with period of shorter than 15 sec show best outcomes of differential staining rates.

• 한국발생생물학회:학술대회논문집
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• 한국발생생물학회 2003년도 제3회 국제심포지움 및 학술대회
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• pp.108-108
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• 2003

The purpose of this is to investigate the effects of vitrification in open pulled straws (OPS) on in vitro survival of porcine embryos. Blastocysts were produced by in vitro fertilization of slaughterhouse-derived, in vitro matured oocytes with frozen-thawed boar semen, and subsequent culture on granulosa cell monolayer. After frozen-thawing, embryos were culture in NCSU-23 medium with 5 mM hypotaurine, 4 mg/$m\ell$ BSA and 10 ng/$m\ell$ for 48 hrs to survival tests. When blastocysts were frozen-thawed by OPS methods, the embryos with normal morphology were 32.1, 34.5 and 38.9 % in early blastocyst, blastocyst and expanded blastocyat stages. The rates of partial damaged embryos were significantly (P<0.05) higher in early biastocysts than expanded blastocysts. In another experiment, the embryos frozen by OPS methods were cultured for 48 hrs for survival and developmental rates in vitro. The proportions of embryos hatched were 11.8, 20.2 and 33.3% in embryos frozen-thawed at stages of early blastocyst, blastocyst and expanded embryos. On the other hand, The proportions of embryo with normal morphology after culture were 23.5, 25.0 and 33.3% in embryos frozen-thawed at stages of early blastocyst, blastocyst and expanded embryos. These finding indicate the possible broader application for OPS methods that this procedure described is relatively harmless, that it can be used for blastocysts of different developmental stages.

• Journal of Animal Science and Technology
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• 제58권3호
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• pp.11.1-11.6
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• 2016

Background: This study was investigated the effects of culture media conditions on production of eggs fertilized in vitro of embryos from ovaries of high grade Korean native cow, Hanwoo. Methods: The IVMD 101 and IVF 100 were used for in vitro maturation of selected Hanwoo oocytes and In vitro embryo culture after in vitro fertilization, respectively. The IVMD 101 and IVD 101 were used for in vitro culture and completely free of serum. Results: The cleavage rates of 2-cell embryos in reference to Hanwoo oocytes were 86.7, 92.9, and 90.1 % in the control group, IVDM101 medium and IVD101 medium, respectively which indicates that the IVDM101 medium and IVD101 medium may result favorable outcomes. The in vitro development rates of blastocysts were 12.4, 38.4 and 32.4 % in the control group, serum free IVMD101 medium and IVD101 medium, respectively. For hatched blastocysts, it was 5.3, 33.9, and 28.6 % in the control group, serum free IVMD101 medium and IVD101 medium, respectively. Hence, more favorable results were expected for the hatched blastocysts in which the IVMD101 medium and IVD101 medium were used than the control group. Average cell numbers of blastocysts were 128.3, 165.7, and 163.6 in the groups of TCM-199 + 10 % FBS medium, IVMD 101 medium, and IVD 101 medium, respectively which clearly show that the IVMD 101 and IVD 101 medium consequence significantly higher cell numbers compared to the control group (i.e., TCM-199 +10 % FBS medium). Pregnancy rate after embryo transfer was 39.6 % when the serum free medium was used which is higher than that of the medium supplemented with serum (32.8 %). In addition, stillbirth rates were 4.9 % in the group of serum free medium whereas it was 13.6 % in the serum supplemented medium (13.6 %). Conclusions: Taken altogether, serum free media, the IVMD 101 and IVD 101 represented more favorable results in the embryo development rate of embryos, cell numbers of blastocyst, and pregnancy rate. Of note, the IVMD 101 medium showed better outcomes hence, it might be a better option for future applications for in vitro culture of bovine embryos.

• Clinical and Experimental Reproductive Medicine
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• 제39권4호
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• pp.153-160
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• 2012

Objective: The aim of this study was to compare vitrification optimization of mouse embryos using electron microscopy (EM) grid, cryotop, and thin plastic strip (TPS) containers by evaluating developmental competence and apoptosis rates. Methods: Mouse embryos were obtained from superovulated mice. Mouse cleavage-stage, expanded, hatching-stage, and hatched-stage embryos were cryopreserved in EM grid, cryotop, and TPS containers by vitrification in 15% ethylene glycol, 15% dimethylsulfoxide, 10 ${\mu}g/mL$ Ficoll, and 0.65 M sucrose, and 20% serum substitute supplement (SSS) with basal medium, respectively. For the three groups in which the embryos were thawed in the EM grid, cryotop, and TPS containers, the thawing solution consisted of 0.25 M sucrose, 0.125 M sucrose, and 20% SSS with basal medium, respectively. Rates of survival, re-expansion, reaching the hatched stage, and apoptosis after thawing were compared among the three groups. Results: Developmental competence after thawing of vitrified expanded and hatching-stage blastocysts using cryotop and TPS methods were significantly higher than survival using the EM grid (p<0.05). Also, apoptosis positive nuclei rates after thawing of vitrified expanded blastocysts using cryotop and TPS were significantly lower than when using the EM grid (p<0.05). Conclusion: The TPS vitrification method has the advantages of achieving a high developmental ability and effective preservation.

• Clinical and Experimental Reproductive Medicine
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• 제43권2호
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• pp.106-111
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• 2016

Objective: To study the clinical outcomes of single frozen-thawed blastocyst transfer cycles according to the hatching status of frozen-thawed blastocysts. Methods: Frozen-thawed blastocysts were divided into three groups according to their hatching status as follows: less-than-expanded blastocyst (${\leq}EdB$), hatching blastocyst (HgB), and hatched blastocyst (HdB). The female age and infertility factors of each group were evaluated. The quality of the single frozen-thawed blastocyst was also graded as grade A, tightly packed inner cell mass (ICM) and many cells organized in the trophectoderm epithelium (TE); grade B, several and loose ICM and TE; and grade C, very few ICM and a few cells in the TE. The clinical pregnancy and implantation rate were compared between each group. The data were analyzed by either t-test or chi-square analysis. Results: There were no statistically significant differences in average female ages, infertility factors, or the distribution of blastocyst grades A, B, and C in each group. There was no significant difference in the clinical pregnancy and implantation rate of each group according to their blastocyst grade. However, there was a significant difference in the clinical pregnancy and implantation rate between each group. In the HdB group, the clinical pregnancy and implantation rate were similar regardless of the blastocyst quality. Conclusion: There was an effect on the clinical outcomes depending on whether the blastocyst hatched during single frozen-thawed blastocyst transfer. When performing single frozen-thawed blastocyst transfer, the hatching status of the frozen-thawed blastocyst may be a more important parameter for clinical outcomes than the quality of the frozen-thawed blastocyst.

• 한국수정란이식학회지
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• 제27권1호
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• pp.63-69
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• 2012

The objective of this study was to investigate the effectiveness of cryopreservation methods for the effect of various vitrification containers, such as EM-grid, OPS, or cryo-loop on the survival and developmental rate of vitrified mouse pronuclear embryos, and mouse cleavage embryo, at 21, 24, 27 and 30 hr after hCG injection. Post-thaw cleavage was similar among treatments, while the developmental rates of mouse blastocyst and hatched blastocyst were higher ($p$ <0.05) in 27 hr and 30 hr than 21 hr. The developmental rate of hatched blastocyst at vitrified cleavage mouse embryos in cryo-loop was significantly higher than vitrified pronuclear embryos of control group as well as EM-grid and OPS ($p$ <0.05). The developmental rate using cryo-loop was higher than EM-grid, but in case of OPS at vitrified cleavage and mouse pronuclear embryos, no significant difference was noticed. These results of our study show that the developmental rates of mouse embryos were unaffected by various vitrification containers, but in case of mouse embryos and hatched blastocysts at late vitrified pronuclear embryos the developmental rates were higher than early vitrified pronuclear embryos. Moreover, the developmental rate of hatched blastocyst at vitrified cleavage mouse embryos was significantly higher than vitrified pronuclear embryos. For better execution of this study, it will be mandatory to include improvement of vitrification containers, cryopreservation methods and conditions, higher survival rate, safe preservation, contamination and embryo loss.

• 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.

• Clinical and Experimental Reproductive Medicine
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• 제18권2호
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• pp.173-179
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• 1991

The purpose of this study was to evaluate the in vitro developmental promoting effect of human amniotic fluid (AF) on pronucleate 1-cell mouse embryos. The AF was obtained from five patients undergoing amniocentesis for the routine diagnosis of fetal abnormality. The supernatant was filtered ($0.22{\mu}m$) after centrifugation and stored at $-20^{\circ}C$. One-cell embryos were cultured in four study groups (10% AF, 0.4% BSA, 10% AF+0.4% BSA & no-supplement in Ham's F10) for 6 days (EXP. 1). Significantly more embryos hatched in 10% AF (P<0.0l), although no difference was found among other three groups. The embryos were also cultured in varous concentration of AF (0, 10, 50 & 100%) for 7 days (EXP. 2). The rate of hatched blastocysts was significantly higher in 10%- and 50% group than in 0% and 100%- one at day 6 (P<0.05) and day 7 (P<0.005), although no difference was found between 10% and 50%- group.

• Asian-Australasian Journal of Animal Sciences
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• 제17권5호
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• pp.617-620
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• 2004

The present study compares development of rabbit embryos generated using different oocyte activation protocols and reconstructed with embryonic or cumulus cells as nuclear donor. In vivo matured oocytes were collected from New Zealand White rabbits at 16 h after ovulation treatment and were activated at18 h of post-ovulation treatment. The following schemes of oocytes activation were tested: 1) single electric pulse (EP, 3.2 kV/cm, 3${\times}$20 $\mu$s, 0.3 M mannitol)+5 min culture in the presence of 5 mM Ionomycin, 2) single electric pulse (EP, 3.2 kV/cm, (${\times}$20 $\mu$s, 0.3 M mannitol)+1 h culture in the presence of 2 mM 6-DMAP, and 3) three electric pulses 30 min apart. Cleavage rate, percentage of expanded and hatched blastocysts as well as total cell number of blastomeres of parthenogenetic embryos were significantly higher using either EP+6-DMAP or 3${\times}$EP schemes, comparing with EP+Ionomycin. Development rate up to hatched blastocyst stage of cloned rabbit embryos using the EP+6-DMAP for activation of nuclei were 19% for embryonic cell nuclei and 36% for cumulus cell nuclei. The best activation protocol optimalized in this study was the combined treatment "P+6-DMAP" which may be potentially used for nuclear transfer protocol.

• 한국수정란이식학회지
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• 제15권3호
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• pp.219-230
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• 2000

This study was carried out to determine the factors on achieving good viability of embryos biopsied fur sexing, to investigate pregnancy rate following embryo transfer(ET) with sexed embryos, and to confirm the accuracy for the calves bort following ET with sexed embryos by polymerase chain reaction(PCR). To investigate viability of Hanwoo embryos after biopsy for sexing, fresh and frozen/thawed embryos were biopsied according to different developmental day of blastocysts, different stage of blastocysts, and different biopsy grade and the embryos themselves were incubated for 2 hours in TCM199 after microsection to be evaluated morphologically for recovery as blastocyst. The results obtained were as follows : 1. The rate of oocytes cleaved in vitro and the rate of blastocyst of the cleaved oocytes were 52.5% and 21.6%, respectively. The rate of blastocyst on day 8 was 11.2%, denoting the highest rate during whole culture period posterior to in vitro fertilization(IVF) 2. After biopsy for sexing, the viability rate of blastocyst on day 7, 8 and 9 was 75.0%, 88.4%, and 100.0%, respectively and the viability of early, mid, and expanded blastocyst after biopsy was 75.0%, 88.9%, and 91.1%, respectively The viability rate of fresh and frozen/thawed embryos was 89.9%, 71.4%, respectively. And the viability of expanded, hatching, and hatched blastocyst of frozen/thawed embryos was : 75.0%, 75.0%, and 50.0%, respectively. The viability of embryos according to biopsy grade of 10∼20%, 21∼30%, and 31∼40% was 85.7%, 91.5%, and 71.4%, respectively. 3. Pregnancy rate after transfer with biopsied embryo between flesh and frozen/thawed embryos was 22.6% and 20.0%, respectively. 4. In comparison between sex by PCR method and sex of calves born after embryo transfer, the accuracy of sex deterimination was 92.3% (12/13).