• Korean Journal of Animal Reproduction
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• v.16 no.4
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• pp.341-346
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• 1993

The development of isolated blastomeres from mammalian preimplantation embryos has been basically studied for the multiplication of embryos from superior animals. Therefore, this study was investigated the effect of co-culture with mouse fetal fibroblast cells(MFFC) on in vitro development of blastomeres from mouse preimplantation embryos. Mature female ICR mice were treated with hormone to induce superovulation and embryos were collected at each 2, 4, and 8-cell stage. Then, after removing zona pellucida with protease, blastomeres were isolated by micropipetting, or reconstituted with different stage blastomere, and incubated for 72 hrs either in T6 or TCM199 or on the monolayer of MFFC, which was prepared with fibroblast cells from 14∼14 day mouse fetus. After incubation, we examined their development rates every day and the nuclei numbers of each blastocyst by Hoechst-33342 staining. In the development rates of blastomeres, there were no significant differences between media but the higher rateswere found in the monolayer of MFFC, regardless of reconsititution. In addition, blastomeres cultured with MFFC had slightly greater number of nuclei than those cultured in single media. Generally, the higher development rates of blastomeres were found from earlier stage embryos than the later ones, regardless of culture conditions. Reconsitituted blastomeres had more nuclei but did not show the higher development rates, compared to the single blastomeres. Taken together, our results suggest that co-culture with MFFC have a beneficial effect on the in vitro development of blastomeres from mouse embryos.

• Korean Journal of Animal Reproduction
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• v.17 no.4
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• pp.287-294
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• 1994

This study was to investigate the effects of electrofusion, activation and developmental stage of donor embryos on in vitro development of nuclear transplant bovine embryos. A single blastomere nucleus from 8-cell to morula stage embryos produced by in vitro fertilization(IVF) was transferred into a recipient oocyte enucleated at 23∼25 h after in vitro maturation(IVM) or into a recipient oocyte enucleated and cultured for 14∼15 h. In one experiment the nuclear transplant embryos were subjected to additional activation treatments. Fusion rate of nuclear transplant eggs was high at direct current(D.C) voltages of 1.0 and 1.5 kV/cm 991.5 and 93.3%, respectively), but decreased at 2.0kV/cm (81.8%). Additional activation treatments by electric pulases or 7% ethanol did not affect the cleavage and development of nuclear transplant embryos. Development of nuclear transplant embryos slightly increased by delayed nuclear transfer and fusion (42∼43 h after IVM). With this system, blastocysts were obtained from transfer of 8-cell to morula stage donor nuclei (9.6%∼2.4%). The result of this study suggests that nucleo-cytoplasmic interactins, expecially activation of ooplast are very important for the development of nuclear transplant embryos, and donor cell stage does not affect the development of nuclear transplant embryos.

• Korean Journal of Animal Reproduction
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• v.8 no.2
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• pp.110-115
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• 1984

These experiments were carried out to obtain the information about the optimal pH osmolality affecting in vitro fertilization of the mouse eggs, to elucidate the 2-cell block to development in vitro and to find out the method of controlling the subsequent embryo development in vitro. pH and osomlality was adjusted by adding NaCl or NaHCO3 to the basic salt solution. In vitro fertilization were carried out by inroducting the cumulus masses to the suspension of epididymal spermatozoa at each pH, osmolality, and 10${\mu}$M-EDTA medium. The results obtained in these experiments were summarized as follows: 1. The fertilization rates in vitro at each medium of 235, 252, 269, 286, 306, 323, 345, 368, 393 mosmol were 15.6, 38.2, 65.7, 75.6, 80.9, 74.3, 58.1, 35.1, 24.3, 11.1%, respectively. 2. The fertilization rates in vitro at each medium of pH 6.1, 6.4, 6.7, 7.0, 7.3, 7.6, 7.9, 8.1 were 11.8, 17.9, 32.4, 61.9, 79.5, 76.7, 53.5, 13.6%, respectively. 3. In case of ICR female x ICR male embryos, the development rate of 2-cell embryos to 4-8 cell embryos was 16.2% at normal medium, but the rate was increased to 49.3% in medium containging 10 ${\mu}$M-DETA; In case of C3H female x ICR male embryos, the development rate was 41.0% at normal medium, but the rate was increased to 71.7% at 10 ${\mu}$M-EDTA-medium.

• Animal Bioscience
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• v.35 no.2
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• pp.177-183
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• 2022

Objective: The present study evaluated the efficiency of embryo development and pregnancy of somatic cell nuclear transfer (SCNT) embryos using different source-matured oocytes in Camelus dromedarius. Methods: Camelus dromedarius embryos were produced by SCNT using in vivo- and in vitro- matured oocytes. In vitro embryo developmental capacity of reconstructed embryos was evaluated. To confirm the efficiency of pregnancy and live birth rates, a total of 72 blastocysts using in vitro- matured oocytes transferred into 45 surrogates and 95 blastocysts using in vivo- matured oocytes were transferred into 62 surrogates by transvaginal method. Results: The collected oocytes derived from ovum pick up showed higher maturation potential into metaphase II oocytes than oocytes from the slaughterhouse. The competence of cleavage, and blastocyst were also significantly higher in in vivo- matured oocytes than in vitro- matured oocytes. After embryo transfer, 11 pregnant and 10 live births were confirmed in in vivo- matured oocytes group, and 2 pregnant and 1 live birth were confirmed in in vitro- matured oocytes group. Furthermore, blastocysts produced by in vivo-matured oocytes resulted in significantly higher early pregnancy and live birth rates than in vitro-matured oocytes. Conclusion: In this study, SCNT embryos using in vivo- and in vitro-matured camel oocytes were successfully developed, and pregnancy was established in recipient camels. We also confirmed that in vivo-matured oocytes improved the development of embryos and the pregnancy capacity using the blastocyst embryo transfer method.

• Korean Journal of Animal Reproduction
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• v.18 no.1
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• pp.39-46
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• 1994

This experiment was carried out to develop an in vitro culture system for rabbit embryos. The zygotes or 2-cell embryos were collected from the oviducts of the superovulated and mated does with D-PBS/10% FCS at 24 hours after hCG injection. The in vitro developmental rate of blastocyst formation and the number of nuclei in the embryos were examined under the following treatments; 1) TCM-199 with 10% FCS, 2) EBSS with 10% FCS, 3) rabbit vitreous humor(VH), 4) TCM-199 with 10% FCS＋BOEC, 5) TCM-199 with 10% FCS＋ROEC, 6) EBSS with 10% FCS＋BOEC and 7) EBSS with 10% FCS＋ROEC. For a comparative study of in vivo and in vitro development, the fresh blastocysts, which were developed in vivo for 96 hours after hCG injection, were collected from the uterus and their numbers of nuclei were counted. 1. The zygotes or 2-cell embryos developed to the blastocyst stage in TCM-199, EBSS and VH at the rates of 93, 92 and 89%, respectively. 2. The higher developmental rates 95~98% of blastocyst formation was achieved when the embryos were co-cultured with a monolayer of bovine or rabbit oviductal epithelial cells in TCM-199 or EBSS. No significant difference in developmental rates was shown between bovine and rabbit oviductal epithelial cells. 3. In a comparative study of in vivo and in vitro development, the total numbers of nuclei were significantly less in the in vitro cultured embryos(104~224) than the in vivo developed embryos(1, 0090 at 96 hours after hCG injectin. 4. The mean cell cycle numbers in the embryos cultured for 72 hours in TCM-199 with 10% FCS, EBSS with 10% FCS, TCM-199 with 10% FCS＋BOEC, TCM-199 with 10% FCS＋ROEC, EBSS with 10% FCS＋BOEC and in vivo was 7.38, 6.63, 7.76, 7.69, 7.01 and 9.92, respectively. From these results, it can be suggested the optimal culture system for in vitro culture of rabbit embryos is a co-culture system with bovine or rabbit oviductal epithelial cells in TCM-199 with 10% FCS. Considering the significant reduction in total numbers of nuclei in the in vitro cultured embryos, the advanced research on development of in vitro culture system for rabbit embryos is expected.

• Proceedings of the KSAR Conference
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• 2004.06a
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• pp.170-170
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• 2004

First of all, in vitro production (IVP) of porcine embryos is an important as initial step to improve bio-technical applications such as transgenesis and cloning for xenotransplantation. In recent years, considerable progress has been achieved in the IVP embryos using advanced methods for in vitro maturation (IVM) and fertilization (IVF). (omitted)

• Journal of Embryo Transfer
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• v.4 no.1
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• pp.28-34
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• 1989

These experiments were carried out to determine the effect of cell stage in embryo bisection on the sub-Sequent in vitro and in vivo development in mouse. The embryos of ICR mouse were microsurgicaily bisected at 2-cell, 4-cell, 8-cell, morula and blastocyst stage using a microsurgical blade attached a micromanipulator. These demi-embryos without zona pellucida were cultured up to blastocyst stage and transferred to pseudopregnant mice, and the development of these demi-embryos was compared with the results of intact embryos of the corresponding cell stage. The successful rate of mouse embryo bisection at 4-cell stage (59.0%) was significantly (p <0.05) lower than those at 8-cell (75.6%), 2ce11 (80.7%) or morula stage (84.8%), and highest at blastocyst stage (95.7%). When the bisected embryos without any damage from microsurgery were cultured in vitro up to blastocyst,the in vitro de'velopment of demi-embroys bisected at morula to blastocyst was 91.6 to 95.3%, which was similar to the culture result of intact embryos of corresponding stage. However, the in vitro development of demi-em-bryos bisected at 2- to 8-cell stage was signiflcantiy (p <0.05) lower.The post-transfer implantation rate of demi-embryos developed in vitro to eu-blastocyst were 19.6 and 25.4% in demi-embryos bisected at morula and blastocyst stage,respectively and not significantly (P <0.05)different from the result of intact embryos of the same stage. However, the implantation rates of demi-embryos bisected at 2- or 8-cell stage were significantly (P <0.05) lower than the result from the intact embryos of the corresponding stage.

• Korean Journal of Animal Reproduction
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• v.21 no.3
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• pp.293-302
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• 1997

Follicular oocytes of Grade I and II were collected from 2~6 mm ovarian follicles and matured in vitro (IVM) for 24 hrs in TCM-199 su, pp.emented with 35$\mu\textrm{g}$/ml FSH, 10$\mu\textrm{g}$/ml LH, and 1$\mu\textrm{g}$/ml estradiol-17$\beta$ at 39$^{\circ}C$ under 5% CO2 in air. They were fretilized in vitro (IVF) by epididymal spermatozoa capacitated with heparin for 12 hrs. The zygotes were then co-cultured in vitro with bovine oviducted epithelial cells (BOEC) for 7 to 9 days. The optimal time for IVM, the successful enucleation of IVM oocytes by micromanipulation at different oocyte ages after IVM, and the ideal culture system for IVM for effective IVF and in vitro development of IVM-IVF embryos was examined for in vitro production of nuclear recipient oocytes and nuclear donor embryos. To improve the efficiency of nuclear transplantation (NT) of IVF embryo into IVM follicular oocytes, this study evaluated the optimal electric condition and oocytes age for activation of IVM oocytes and in vitro development of NT embryos. In vitro development of NT embryos with preactivation or non-preactivation in enucleation oocytes, cell number of IVN-IVF embryos, and NT embryos wre also examined. The results obtained were as follows; 1. The most suitable enucleation time was at 24 hpm (83.3%) rather than that of 28 hpm(69.6%) and 32 hpm(50.0%). 2. There was no difference among the fusion rates of NT embryos at the voltages of 0.75, 1.0 and 1.5 kV/cm, but the in vitro development rates to morule and blastocyst were significantly (P<0.05) higher at the voltage of 0.75(12.5%) and 1.0kV/cm (12.6%) compared to 1.5kV/cm(0%). 3. No significant difference in activation rates were seen in NT embryos stimulated for 30, 60 and 120 $\mu$sec (71.7, 85.2 and 71.9%, respectively), but the in vitro development rates to morulae and blastocyst were significantly (P<0.05) higher in the oocytes stimulated for 30 $\mu$sec (11.6%) and 60 $\mu$sec(10.7%) than 120 $\mu$sec(0.0%). 4. The fusion rates (71.0 and 87.3%) and the in vitro development rates (9.1 and 12.7%) to morula and blastocyst were seen in the NT embryos stimulated at 28 and 32 hpm under the condition of 1.0 kV/ml, 60 $\mu$sec. However, at 24 hpm the fusion rates were 64.8% and the in vitro development to morula and blastocyst were not seen. 5. The fusion rates between the 8~12, 13~17 and 18~22-cell stage of IVM-IVF embryos were not significantly different. The in vitro development rates of the fused embryos to morula and blastocyst which were received from a blastomere of 8~12, 13~17 and 18~22-cell stages of IVM-IVF embryos were 14.9, 8.3 and 6.5%, respectively. 6. The in vitro development rate of the enucleated recipient oocytes with preactivation (24.2%) to morula and blastocyst was significantly (P<0.05) higher than that of non-preactivation (12.8%). 7. The cell numbers of NT blastocyst and IVM-IVF blastocyst cultured during 7~9 days were 63$\pm$11 and 119$\pm$23, and then their the mean cell cycle number were 5.98 and 6.89, respectively.

• Reproductive and Developmental Biology
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• v.28 no.2
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• pp.101-105
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• 2004

This study was carried out to evaluate the effects of glucose and sodium phosphate on in vitro development of porcine oocytes matured and fertilized in vitro. When the culture medium was supplemented with various concentrations of glucose, the higher proportions (23 and 26%) of oocytes developed to morular or blastocyst stages were at the concentrations of 2.78 and 5.56 mM than 0 (9%; P<0.05) and 11.12 mM (18%). In experiment to evaluate effect of sodium phosphate during in vitro development of porcine oocytes, a significantly (P<0.05) higher proportions of embryos developed to morular or blastocyst stages was obtained with sodium phosphateof 0.28 (25%) and 0.53 (27％) mM than 0 (15%), 1.05 (19%) and 2.10 (10%) mM. On the other hand, when oocytes were cultured in medium with (0.53 mM) sodium phosphate, the proportions of developed embryos were significantly (P<0.05) higher in medium without (29%) that than with (14%) 5.56 mM glucose. However, a higher proportion of embryos developed to morular or blastocyst stages were obtained in medium with (23%) that than without (8%) glucose (P<0.05). The minimum essential medium (MEM) added to the culture medium were higher regardless of presence of sodium phosphate and glucose on the development of embryos. Although sodium phosphate and glucose could support morular and blastocyst development to a limited extend (10∼24%), significantly higher proportion (36%) at morular or blastocyst stages was obtained by MEM adding in the medium with sodium phosphate and glucose. These results suggest that the early development of in vitro fertilized porcine oocytes can be maintained efficiently by glucose and sodium phosphate when they were cultured in medium with MEM.

• Reproductive and Developmental Biology
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• v.30 no.4
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• pp.307-311
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• 2006

The purpose of this study was to determine effects of oxytocin and $interleukin-1{\alpha}$ on in vitro development of bovine embryo cultured with endometrial epithelial and stromal cells isolated from bovine uterus. The expressions of COX-2 mRNA in bovine endometrium were also studied. When embryos were cultured with epithelial cells, the rate of blastocysts was significantly (p<0.05) higher in embryos treated with oxytocin than that of control group. The rate of hatched blastocysts was also significantly (p<0.05) higher in embryos treated with oxytocin than those of two control groups. On the other hand, when the embryos were cultured with stromal cells, the rate of blastocysts were significantly (p<0.05) higher than those of groups treated with $IL-1{\alpha}$, oxytocin and control with stromal cells than that of control group without stromal cells. The rate of blastocysts hatched were also significantly (p<0.05) higher in group treated with $IL-1{\alpha}$ than those of control group without stromal cells and oxytocin group. In another experiment, COX-2 gene was expressed in embryo group treated with oxytocin during the co-culture of embryos with epithelial cells. In contrast, COX-2 mRNA was expressed in group treated with $IL-1{\alpha}$ when the embryos were cultured with stromal cell. This result shows that oxytocin and $IL-1{\alpha}$ were stimulate embryo development in vitro when embryos were cultured with epithelial and stromal cells, and can affect the development of bovine embryos in the uterus.