• Korean Journal of Animal Reproduction
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• v.11 no.2
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• pp.127-131
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• 1987

This study was carried out to obtain the basic information on splitting and culture of mouse and bovine embryos. Two-, four-, eight-, cell and morula mouse embryos were digested with pronase, splitted in vitro by micro-glass needel with hand, and bovine embryos were splitted by micromanipulator. The splitted embryos were cultured under 5% of CO2 gas in air at 37$^{\circ}C$ for 48-72 hours. The results obtained in this study were summarized as follows: 1. The mouse and cattle were superovulated by 5IU of PMS and HCG, and 2500IU of PMS and 25mg of PGF2$\alpha$, respectively. The average number of embryos after superovulation were 32.5$\pm$8.2 and 7.5$\pm$3:1, respectively. 2. Out of total 122 embryos splitted, the successful splitting rate was 75.0%, 66.7%, 68.4% and 71.4% in 2-, 4-, 8- and morula embryos in mouse, respectively. There was no different splitting rate between mouse(71.4%) and bovine embryos(66.7%) in morula. 3. The successful culture rate of splitted embryos was 68.0% and 67.9% in mouse and bovine embryos, respectively.

• Journal of Animal Science and Technology
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• v.65 no.4
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• pp.767-778
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• 2023

The aim of the research is to identify that porcine oocytes can function as recipients for interspecies cloning and have the ability to develop to blastocysts. Furthermore each mitochondrial DNA (mtDNA) in interspecises cloned embryos was analyzed. For the study, mouse-porcine and porcine-porcine cloned embryos were produced with mouse fetal fibroblasts (MFF) and porcine fetal fibroblasts (PFF), respectively, introduced as donor cells into enucleated porcine oocytes. The developmental rate and cell numbers of blastocysts between intraspecies porcine-porcine and interspecies mouse-porcine cloned embryos were compared and real-time polymerase chain reaction (PCR) was performed for the estimate of mouse and porcine mtDNA copy number in mouse-porcine cloned embryos at different stages.There was no significant difference in the developmental rate or total blastocyst number between mouse-porcine cloned embryos and porcine-porcine cloned embryos (11.1 ± 0.9%, 25 ± 3.5 vs. 10.1 ± 1.2%, 24 ± 6.3). In mouse-porcine reconstructed embryos, the copy numbers of mouse somatic cell-derived mtDNA decreased between the 1-cell and blastocyst stages, whereas the copy number of porcine oocyte-derived mtDNA significantly increased during this period, as assessed by real-time PCR analysis. In our real-time PCR analysis, we improved the standard curve construction-based method to analyze the level of mtDNA between mouse donor cells and porcine oocytes using the copy number of mouse beta-actin DNA as a standard. Our findings suggest that mouse-porcine cloned embryos have the ability to develop to blastocysts in vitro and exhibit mitochondrial heteroplasmy from the 1-cell to blastocyst stages and the mouse-derived mitochondria can be gradually replaced with those of the porcine oocyte in the early developmental stages of mouse-porcine cloned embryos.

• Korean Journal of Animal Reproduction
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• v.12 no.1
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• pp.37-41
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• 1988

These studies were carried out to examine the sex of preimplantation mouse embryo. For the investigation of sex-ration of mouse embryos, morula and blastocysts stage embryos treated with H-Y antiserum (10%, v/v) and FITC anti-mouse-IgG were divided into the positive and negative embryos. Positive and negative identified embryos were observed the viability according to the in vitro cultured and the sex ratio was also investigated by chromosomal analysis. The results obtained in these studies were summarized as follows: 1. Two hundred sixty-seven recovered embryos of morula or blastocyst stage were incubated in medium containing H-Y antiserum and FITC anti-mouse-IgG. Positively or negatively identified embryos were 139 and 128. This trend indicated the approximal sex ratio was 1:1. 2. Sex ratio was measured using the embryos treated with indirect immunofluorescence assay to examine the relationship between embryo developmental stage and sex ratio. Sex ratio of morula stage embryos was 45.2% positive and 54.8% negative, on the other hand, the ratio switched to 56.4% positive and 43.6% negative embryo in blastocyst stage. 3. Fourty-seven positive and 57 negative embryos were obtained out of 104 morula stage embryos treated with indirect immunofluorescence assay. Survived positive or negative embryos during in vitro culture were 42 and 49, respectively out of 47 and 57 embryos. 4. The numbers of negative and positive embryos were 171 and 92 out of 163 blastocyst embryos which were incubated in the medium containing H-Y antiserum and FITC anti-mouse-IgG. The result of karyotype test showed the successful rate of sexing embryo is positive and negative embryos was63.0% (58/92) and 62.0% (44/71). The final female to male ratio within 58 positive embryos was 22.7:77.6, and the ratio of the 44 negative embryos was 77.3:22.7.

• Journal of Veterinary Clinics
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• v.8 no.1
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• pp.103-108
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• 1991

It was carried out to investigate the effect of the kind of medium, the concentration of serum added and the temperature of storage on the survival of mouse and bovine embryos preserved In vitro. The survival of frozen-thawed mouse embryos after cooling at 4$^{\circ}C$ was also investigated. It was possible to preserve the embryos of mouse until 4 days and of cattle in a day without significant decrease of the survival rates. The survival rates of frozen-thawed mouse embryos after cooling at 4$^{\circ}C$ over 3 days were below 20%.

• Clinical and Experimental Reproductive Medicine
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• v.19 no.2
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• pp.125-131
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• 1992

The possible effect of human follicular fluid(hFF) on the growth and development of fertilized oocytes and embryos is important because the fallopian tubes are exposed to FF after follicular rupture and the processes of fertilization and embryo cleavage occur inside the fallopian tubes. Previously, it was suggested that human FF might adversely affect on the development of early mouse embryos. In order to investigate the effect of hFF on the development of embryos, early mouse embryos were cultured in media containing various protein sources as bovine serum albumin(BSA), fetal cord serum(FCS) and FF. And we evaluated the development of early mouse embryos in terms of the morphology, cleavage rate, and cell count of blastcysts. There were no significant differences in the morula and blstocyst formation rates of 2-cell mouse embryos cultured in the media containg three different protein sources and three different concentrations of FF. The blastocyst formation rate of 1-cell mouse embryo cultured in FF group was significantly higher than that cultured in BSA group(P<0.05). The morula and blastocyst formation rates of 2-cell mouse embryos of the group cultured in the media containing FF were comparable with those of other two groups, in addition, the cell count of blastocysts of FF group in the 2-cell embryo culture was higher than those of BSA group and HCS group(P<0.01), and this finding was also noted in 1-cell embryo culture. There was no difference in the morula and blastocyst formation rates of the 2-cell mouse embryos cultured in the media containing different concentrations of FF. These results suggest that mature human follicular fluid has no inhibitory activity on the development of early mouse embryos even in high concentration and may be a good protein source which is positively associated with the development of mouse embryos in vitro especially in 1 cell embryo culture.

• Korean Journal of Veterinary Research
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• v.30 no.2
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• pp.231-241
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• 1990

The present study was carried out to investigate the viability of frozen-thawed aggregated mouse embryos and to produce the chimeras. Different phenotypic embryos were obtained by mating ICR female mice with ICR or CBA male mice. The aggregated morulae were produced following aggregation of embryos at 4-, 8- and 12- to 16-cell stage. The desirable stage for the aggregation of the mouse embryos was 8- to 16-cell stage. The post-thawed in vitro survival rates of aggregated embryos in glycerol, DMSO and ethylene glycerol were 51.5, 78.6 and 69.4%, respectively. Although the higher survival was obtained in DMSO, there were no significant differences in the survival rates among the three cryoprotectants. A total of 155 frozen-thawed aggregated embryos were transferred to 11 recipient mice, 3 out of 7 offsprings were born to overt chimera. These experiments have proven that mouse chimeras can be obtained from frozen-thawed aggregated embryos.

• Korean Journal of Animal Reproduction
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• v.17 no.3
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• pp.201-207
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• 1993

We determined whether the ultrarapid freezing method is applicable to micromanipulated mouse embryos. One-cell mouse embryos were microinjected with MThGH gene. Nuclei from one-cell embryos of F1(C57BL$\times$CBA) mice were transplanted into enucleated one-cell embryos of ICR mice. The injected and nucleated embryos that developed to 2-cell stage were cryopreserved by ultrarapidfreezing. The embryos equilibrated in freezing medium(3 M DMSO＋0.25 M sucrose＋2% FBS in PBS) were directly immersed into liquid nitrogen and then thawed in 37$^{\circ}C$ water. Development rates of the microinjected and nuclear-transplanted embryos to blastocyst stage after ultrarapidly freezing and thawing were 31% and 55%, respectively. The frozen-thawed embryos were transferred to pseudopregnant recipient, which then gave birth to 17 offsprings. Twelve(14% of the transferred embryos) and five(20%) offsprings were derived from microinjected and nuclear-transplanted embryos, respectively. The results indicate that the DNA injected and nuclear-transplanted mouse embryos are cryopreservable at 2-cell stage by ultrarapid freezing method.

• Journal of Embryo Transfer
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• v.27 no.1
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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.

• Journal of Embryo Transfer
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• v.11 no.1
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• pp.35-40
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• 1996

This study was carried out to select the best cryoprotectant and to establish optimal concentration of the cryoprotectant in ultrarapid freezing of mouse 4-cell embryos and morulae, respectively. We investigated survival of ultrarapid frozen embryos according to various cryoprotectants such as glycerol, ethylene glycol, propylene glycol and dimethyl sulfoxide (DMSO). Suvival of the embryos frozen at different concentrations (3.0, 4.0 and 5.0 M) of indivisual cryoprotectant was also tested. Preimplantation developmental rate (96.3%, 83/86) of 4-cell mouse embryos treated with 4.0 M ethylene glycol after ultrarapid freezing and thawing was higher than those of other cryoprotectants (glycerol, propylene glycol and DMSO). In the ultrarapid freezing of mouse morulae, the highest developmental rate (98.8%, 89 /90) of the embryos to blastocysts was shown in the group of 5.0 M glycerol. Thus, these results demonstrate that 4.0 M ethylene glycol and 5.0 M glycerol are optimal for ultrarapid freezing of 4-cell mouse embryos and morulae, respectively.

• Korean Journal of Animal Reproduction
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• v.13 no.2
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• pp.63-69
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• 1989

Eight-cell mouse embryos equilibrated with vitrification solution(VS3), consisting of glycerol and polyethlene glycol, were plunged directly into liquid nitrogen. The embryos were cryopreserved by vitrification without intra- and extracellular ice formation. After the vitrified embryos were warmed at 4$^{\circ}C$, sucrose dilution procedures were examined and the survival embryos were transferred to recipients after 48h incubation in vitro. The results were obtained as follows. 1. Mouse embryos equilibrated with VS3 were diluted with 1.5m sucrose-HB 1 solution, 0.5M sucrose-HB1 solution for 5min at room temperature, respectively. The proportion of vitrified-warmed embryos developed to blastocyst(85.7%) was as high as that of the embryos diluted with 1.04M sucrose-HB1 solution at 4$^{\circ}C$(85.5%). 2. Normal live youngs were obtained in 53.9%(55/102) of the vitrified-warmed embryos after tranfer to pseudopregnant recipients.