• Clinical and Experimental Reproductive Medicine
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• v.28 no.3
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• pp.235-245
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• 2001

Objectives: The purpose of this present study was to compare mouse embryo development in 3 commercial media and hatching competence of mouse embryo with or without enzymatic treatment. Methods: Collected 375 mouse embryos were divided into three groups, and then cultured in IVF-20 (G2), Medicult IVF (M3), P-1 (blastocyst M), respectively. Three day mouse morulae were cultured in G2 media treated with pronase. The results were analyzed using Chi-square test, and considered statistically significant when p<0.01. Results: The developmental rate of 2 cell mouse embryo after 72 hours was highest in IVF-20 (G2) among conventional 3 media. The hatching rate of mouse morulae was low when clultured in G2 media without pronase during 48 hours. However, it was higher when cultured in media treated with $1{\mu}g/ml$, $2.5{\mu}g/ml$, $5{\mu}g/ml$ pronase, respectively. Conclusions: Using good media and digestion of zona pellucida with enzymatic treatment improve development and hatching rate of embryo. Therefore, implantation and pregnancy rate could be improved.

• Clinical and Experimental Reproductive Medicine
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• v.21 no.3
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• pp.325-330
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• 1994

The study has been carried out in order to evaluate the effects of embryonic stage, and cryopreservation method on the rates of viability and development of the cryopreserved mouse early embryos. The results were as following:In the treatment steps of cryoprotectant, for the fertilized oocyte with pronucleus(PN), 2-step was better than the others. And for the other embryos, 4-step was better than 2- or 3-step. In respect to the embryonic stage, as the embryos developed from fertilized oocytes to 8-cell embryos, the rates of viability and development were increased higher. Therefore, 8-cell embryo was better stage than the others. In respect to the kind of cryoprotectants, PROH was better than DMSO for the fertilized oocyte, as a cryoprotectant. DMSO, for the 2-cell embryos and PROH and DMSO for the 4- and 8-cell embryos were suitable for cryopreservation.

• Clinical and Experimental Reproductive Medicine
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• v.45 no.3
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• pp.110-115
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• 2018

Objective: To compare our in-house method of embryo freezing with Cryotop vitrification in terms of immediate survival, subsequent cleavage and blastocyst formation, and cell numbers in blastocysts. Methods: Two-cell mouse embryos were randomly allocated into three groups: a non-frozen control group (group 1, n = 300), a group that underwent Cryotop vitrification (group 2, n = 300), and a group that underwent our in-house freezing method (group 3, n = 300). Results: There were no significant differences between groups 2 and 3 in the immediate survival rate (96.3% vs. 98.6%, respectively; p= 0.085), the further cleavage rate (91.7% vs. 95.0%, respectively; p= 0.099), or the blastocyst formation rate (80.7% vs. 78.6%, respectively; p= 0.437). The cell numbers in the blastocysts from groups 1, 2, and 3 were comparable ($88.99{\pm}10.44$, $88.29{\pm}14.79$, and $86.42{\pm}15.23$, respectively; p= 0.228). However, the percentage of good-quality blastocysts in the Cryotop vitrification group was significantly higher than in the group in which our in-house method was performed, but was lower than in the control group (58.0%, 37.0%, and 82.7%, respectively; p< 0.001). Conclusion: At present, our method is inferior to the commercial Cryotop vitrification system. However, with further improvements, it has the potential to be useful in routine practice, as it is easier to perform than the current vitrification system.

• Journal of Embryo Transfer
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• v.29 no.2
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• pp.111-117
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• 2014

This study was conducted to optimize the efficiency of cloning and to produce cloned mice. The majority of cloned mammals derived by nuclear transfer (NT) die during gestation and have enlarged and dysfunctional placentas. In this study, the optimized conditions were established to produce clone mice. The parthenogenetic oocytes were activated after 6 h regardless of cytochalasin B (CB) concentration. CB treatment ($2{\mu}g/ml$) was found second polar body. Lower concentration of CB was decreased the activation rate, but the second polar body was the best highly increased during 6 h incubation. The small fragments were exhibited in the $5{\mu}g/ml$ treatment of CB, but it was not found in lower concentration groups (> $2.5{\mu}g/ml$). To examine effects of $SrCl_2$ on the adult cumulus cells, somatic cell NT oocytes were exposed during 0.5, 1 and 6 hrs. The second polar body was significantly greater in 0.5 h exposure group (6.6%) than 1, 6 hrs. Developmental rate from 2-cell to 4-cell was the lowest in 7.5 mM Strontium chloride ($SrCl_2$) groups (84.1% and 64.3%) than 5, 10 m $MSrCl_2$. The implantation rate was not significantly difference among 5, 7.5 and 10 m $MSrCl_2$ group. Three live fetuses were produced by SCNT. SCNT placentas were remarkably heavier than IVF group (8 fetuses) (0.34, 0.34, 0.33 vs 0.14 g) compared with the placenta weight of IVF and SCNT clones.

• Journal of Embryo Transfer
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• v.31 no.1
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• pp.89-95
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• 2016

The present study was conduct to examine the $H_2O_2$ expression level and apoptosis-related gene expression levels inporcineskin-derived stem cell-like cells (pSSCs) after adipogenic, chondrogenic, and osteogenic differentiation induction. The pSSCs were obtained by digestion of porcine ear skin biopsy and cultured in each induction medium for 21 to 26 days to induce adipogenic, chondrogenic, and osteogenic differentiation, respectively. The $H_2O_2$ levels of pSSCs after induction culture were evaluated by staining with 2'7'-dichlorodihydrofluorescein diacetate ($H_2DCFDA$). The apoptotic gene expression of pSSCs after induction culture was also estimated by RT-PCR. The pSSCs have a potential to differentiate into three mesodermal cell types (adipocytes, chondrocytes, and osteoblasts). Non-induced control and chondrogenic-induced cells were showed higher $H_2DCFDA$ intensity (P<0.05) than adipogenic- and osteogenic-induced cells. The relative expression of Bax/Bcl-2 level was significantly low (P<0.05) in adipogenic- and osteogenic-induced cells compared to non-induced control. However, there was no difference in the relative expression of Bax/Bcl-2 level among differentiation induction groups. The result of the present study shows that the apoptosis of pSSCs is not detrimentally increased by differentiation induction culture, although chondrogenic-induced pSSCs showed high ROS generation level and apoptotic index similarly to those of non-induced cells.

• Journal of Embryo Transfer
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• v.29 no.1
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• pp.67-71
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• 2014

In porcine embryo culture, one of reactive oxygen species (ROS) is harmful factors that are made during in vitro culture. To decrease the detrimental effect of ROS on embryo development, superoxide dismutase, catalase and glutathione peroxidase could be used in the embryo culture. Out of these antioxidants, 7,8-dihydroxyflavone (7,8-DHF) was reported its antioxidant effects to prevent the glutamine-triggered apoptosis. Therefore, this study was performed to investigate the most appropriate concentration of 7,8-DHF in porcine embryonic development. For that, 5 different concentration (0, 0.1, 0.5, 1, $2{\mu}m$) of 7,8-DHF was supplemented in the porcine IVM media and then maturation and blastocyst formation rates were compared among 5 groups. In maturation rates of porcine oocytes, significant higher maturation rates was shown in the $1.0{\mu}m$ group compared with another 4 groups ($83.3{\pm}2.1$ vs. $80.7{\pm}1.4$, $79.8{\pm}1.4$, $78.3{\pm}1.2$, $79.4{\pm}1.6$), respectively (P<0.05). In the embryo culture, $1.0{\mu}m$ group also showed the significant higher cleavage rates ($76.8{\pm}3.1$ vs. $62.1{\pm}5.0$, $65.7{\pm}4.0$, $68.6{\pm}3.7$, $64.6{\pm}4.0%$) and blastocyst formation rates - ($39.6{\pm}4.0%$ vs. $28.6{\pm}3.3$, $31.1{\pm}3.9$, $29.3{\pm}2.5$, $39.6{\pm}4.0$, $26.4{\pm}3.2%$), respectively (P<0.05). There was no significant difference among 5 groups in the cell number of blastocyst (P<0.05). In conclusion, supplement of $1.0{\mu}m$ of 7,8-DHF was effective to increase the porcine embryonic development competence as antioxidant to ROS.

• Journal of Embryo Transfer
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• v.25 no.3
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• pp.165-169
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• 2010

This study was carried out to assess the effect of vitamin E against the reactive oxygen species (ROS) on chemical activation of in vitro matured oocytes. Bovine oocytes were aspirated from slaughtered ovaries and transferred to maturation medium with or without vitamin E ($100\;{\mu}M$). After 22 hours of culture, oocytes with polar bodies were selected and submitted to activation treatments with or without vitamin E. After activation, oocytes were cultured in mSOF medium and rate of development was monitored. For ROS ($H_2O_2$) detection, in vitro matured and activated oocytes were selected and stained with DCFDA and observed under fluorescence microscope. The ROS contents were not significant differences in IVM rate, activation process and embryonic development to blastocysts with or without vitamin E. The cell number of blastocyst showed significant difference (p<0.05) in embryos matured and activated with vitamin E. The results of the present study demonstrated that the exposure of vitamin E in IVM and activation process improved the quality of embryos evaluated by the cell number of blastocysts.

• Journal of Embryo Transfer
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• v.16 no.3
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• pp.239-243
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• 2001

Selection of oocyte cryopreservation method is a prerequisite factor for developing an effective bank system. Compared with slow freezing method, the vitrification has various advantages such as avoiding intracellular ice crustal formation. In our previous, we attempted to employ a vitrification method using ethylene glycol and an electron microscope grid for cryopreservation of mouse oocytes. However, A high incidence of spindle and chromosome abnormalities was detected in thawed oocytes after vitrification. We examined whether the addition of a cystoskeleton stabilizer Taxol $^{TM}$, to the vitrification solution could promote the post-thawed survival and subsequent development of stored oocytes. More oocytes developed to the 4-cell (44.7% vs. 69.7%), 8-cell (31.8% vs. 64.2%), morula (24.7% vs. 54.3%), and blastocyst (20.3% vs. 49.2%) stages after the addition of Taxol$^{TM}$ to the cryoprotectant than after no addition. 21 and 26 mouse pups were born after transfer of blastocyst derived from oocytes vitrified without and with Taxol. The addition of Taxol to vitrification solution greatly promoted post-thaw preimplantation development of ICR morose oocytes.tes.

• Animal cells and systems
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• v.5 no.3
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• pp.225-229
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• 2001

Nek2 is a mammalian protein kinase that is structurally homologous to NIMA, a mitotic regulator in Aspergillus nidulans. To understand cellular processes in which Nek2 participates during mammalian development, we investigated the expression and subcellular localization of Nek2 in vivo. The Nek2 protein was detected in spermatocytes and in a fraction of actively dividing ovarian follicle cells and of embryonic tissues. We also observed that Nek2 was localized in both the nucleus and centrosome in embryonic cells. Such localization pattern supports the proposal that Nek2 is a mitotic regulator that is involved in multiple cell cycle events during mammalian development.

• Journal of Animal Science and Technology
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• v.58 no.3
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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.