• 한국가축번식학회지
• /
• 제11권3호
• /
• pp.218-222
• /
• 1987

This study was conducted using inbred ICR mice to investigate the sex-ratio of preimplantation mouse embryos. For the investigation of sex-ratio of mouse embryos, the karyotype of embryos collected at 70-72, 74-76, 78-80 and 82-84 hr after HCG injection was analyzed by chromosomal analysis. Eight-cell embryos were cultrued up to blastocyst stage, then divided them into three groups(fast-, intermediate- and slow-) according to the blastocoel formation. The sex-ratio was also investigated by chromosomal analysis. 1. The highest apperance of eight-cell and morula was observed at the embryos collected respectively at 66-68 hr(84.6%) and 82-84 hr(79.3%) compared to any other group. 2. The successful rate of embryos sexing at 4-, 8-cell and morula stage were 23.1% (3/13), 42.1%(138/328) and 32.6%(47/141), respectively. The respective sex ratios (female vs male) of 4-, 8-cell and morula were 66.7:33.3, 49.3:50.7 and 39.5:60.5. 3. Of the 476 eight-cell embryos cultured in vitro, 427(89.7%) embryos were developed to the blastocysts and the number of fast-, intermediate- and show-developing embryos were 139, 144 and 144, respectively. 4. Female to male ratios fo fast-, intermediate- and slow-developing group were 23.0:77.0, 55.2:44.8 and 73.8:26.2, respectively. Significantly higher (P<0.05) number of female (48/65;73.8%) was observed in the group of slow-developing embryo than that out of total number of embryos(82/188;43.6%).

• 한국가축번식학회지
• /
• 제12권1호
• /
• pp.31-36
• /
• 1988

There studies were conducted using inbred ICR mice to examine the sex of preimplantation mouse embryo. The morphological normality of mice embryos treated with the culture medium containing rat H-Y antiserum(10%, v/v) plus complement(20%,v/v) was observed and also the sexing of embryos was investigated by chromosomal analysis. The results obtained were summarized as follows: 1. The viability of preimplantation mouse embryos, which were incubated in vitro with different media condition, was scored 68.9-85.5% in control group. However, 151 embryos normally developed up to blastocyst and 160 embryos were retarded growth or destroyed out of total 311 embryos treated in the medium containing H-Y antiserum(10%, v/v) plus complement(20%,v/v). 2. H-Y antiserum was prepared from inb red rats (Wistar and Donryu strain) with different immunization times (4, 5 and 6th) to examine the specific titer of embryos by the number of immunization. Precentage of normally developed embryos incubated either in the medium containing the antiserum of Wistar plus complement or Donryu plus complement was revealed 50.9, 47.4 and 50.0% (4, 5 and 6th immunization and 47.8, 41.2 and 48.7%, respectively. 3. Twenty two females and five males were identified out of fourty-eight normally developed embryos incubated in the medium containing H-Y antiserum plus complement by chromosomal analysis.

• Interdisciplinary Bio Central
• /
• 제2권4호
• /
• pp.16.1-16.4
• /
• 2010

Cryopreservation of mouse embryos and spermatozoa has become the foremost technique for preserving large numbers of different strains of mice with induced mutations. In 1998, our mouse bank was established in the Center for Animal Resources and Development (CARD), Institute of Resource Development and Analysis, Kumamoto University, Japan, based on the Preservation, supply and development of genetically engineered animals report published by the Ministry of Education, Culture, Sports, Science and Technology. We cryopreserve mouse embryos and sperm, supply these resources, organize training courses to educate people and form part of a domestic and international network of both mutagenesis and resource centers. We currently have over 1,500 mouse strains, 842,000 frozen embryos and 26,000 straws containing frozen sperm. Moreover, we disclose information about 1,300 deposited strains. Furthermore, over 400 strains of frozen embryos or mice produced from frozen embryos and sperm are being supplied to the requesters both domestically and internationally. Additionally we hold training courses on the cryopreservation of mouse germplasm 2~3 times a year, both domestically and internationally. In the course, we teach basic reproductive engineering techniques to trainees on a man-to-man basis. We have already held 28 training courses on the cryopreservation of mouse germplasm at our center and at other institutes.

• Clinical and Experimental Reproductive Medicine
• /
• 제30권4호
• /
• pp.269-280
• /
• 2003

Objective: We reported the overcoming effect of $Ni^{2+}$ on the in vitro 2-cell block of mouse embryos. In this study, we aim to investigate whether $Ni^{2+}$ should induce intracellular $Ca^{2+}$ transient in the mouse embryos. Materials and Methods: Embryos were collected at post hCG 32hr from the oviduct of the ICR mouse and cultured in M2 medium omitted phenol red. Intracellular $Ca^{2+}$ was checked by using a confocal laser scanning microscope and fluo-3AM by using various intracellular $Ca^{2+}$ antagonists. Results: In 1mM $Ni^{2+}$ treated medium which contained $Ca^{2+}$(1.71mM), 75.7% of the embryos showed $[Ca^{2+}]i$ transient about 200 sec later. In the $Ca^{2+}$-free medium, 69.8% of the embryos showed $[Ca^{2+}]i$ transient. In U73122, phospholipaseC(PLC) inhibitor (5uM, 10min) pretreated group, 33.3% of the embryos showed $[Ca^{2+}]i$ transient. Heparine, inositol 1, 4, 5-triphosphate receptor(IP3R) antagonist preinjected embryos showed no response with 1mM $Ni^{2+}$. In danthrolene treatment, ryanodine receptor(RyR)-antagonist, 43% embryos showed $[Ca^{2+}]i$ transient but they showed delayed response about 340sec in the presence of $Ca^{2+}$. Conclusions: Summing up the above results, $Ni^{2+}$ seems to induce $Ca^{2+}$-release from the $Ca^{2+}$-store even in the $Ca^{2+}$-free medium. IP3 receptors of the mouse 2-cell embryos might have an essential role for the intracellular $Ca^{2+}$ increase by $Ni^{2+}$.

• 한국수정란이식학회지
• /
• 제12권3호
• /
• pp.247-251
• /
• 1997

This research was conducted to observe developmental capacity of the early embryos aggrigated to phytohemagglutinin-M(PHA-M) in the culture of mouse embryos in vitro. The results showed that the development of blastocyst increased to 2-celT >< 2-cell : 68. 9%, 4-cell $\times$4-cell : 92.5% and 8-cell $\times$8-cell : 97.3% in the aggrigated embryos of ICR mouse, and 2-cell $\times$ 2-cell : 90.0%, 4-cell $\times$4-cell : 93.9% and 8-cell $\times$ 8-cell : 100% in the aggrigated embryos of two different strains (ICR $\times$ CBA/J mouse). (Key words : aggrigated embryos, in vitro 2-cell block, phytohemagglutinin-M, blastocyst)

• 한국가축번식학회지
• /
• 제14권3호
• /
• pp.205-211
• /
• 1990

Single nuclei from two-, four- and eight-cell mouse embryos were transplanted into enucleated two-cell mouse embryos by micromanipulation and sendai virum mediated fusion. no significant difference in successful injectin rate and fusion rate was found between the cell stages of nuclear donor embryos. There nuclear transplant embryos receiving different cell stage nuclei were cultured in vitro for 96 hours. 75.3% of 255 embryos receiving 2-cell nuclei, 68.2% of 236 embryos reciving 4-cell nuceli and 46.9% of 228 embryos receiving 8-cell nuclei were developed to blastocyst, respectively. The number of blastomeres was significantly(P<0.05) reduced in the embryos receiving 8-cell nuclei, compared with the embryos receiving 2-cell, 4-cell nuclei or the intact embryos. Also the size of blastocysts was significantly(P<0.05) smaller in the embryos receiving 8-cell nuclei, compared with the intact or other nuclear transplant embryos. These results suggest that single nuclei introduced into the enucleated two-cell embryos are able to support the in vitro development of the reconstituted embryos to blastocysts. The prominant retardation of blastocoele formation and cell division was shown in nuclear transplant embryos receiving eight-cell nuclei when they were cultured in vitro.

• 대한수의학회지
• /
• 제33권1호
• /
• pp.171-178
• /
• 1993

The present study was carried out to investigate the developmental potency to blastocyst after freezing and thawing of nuclear transplanted 2-cell embryos. The nuclei from 2-, 4- and 8-cell mouse embryos were transferred into enucleated 2-cell embryos, and the reconstituted embryos were submitted to direct current(DC) pulse at output voltage of 2.0 kV/cm for $100{\mu}$ sec to induce cell fusion. The recovery rate and developmental potency to blastocyst after freezing and thawing of nuclear transplanted 2-cell embryos was investigated. 1. The recovery rate of nuclear transplanted 2-cell embryos in normal morphology after freezing and thawing was significantly higher in rapid freezing(DMSO 4.5M) than in slow cooling(p<0.01). 2. When the recovered embryos in normal morphology were cultured in vitro, there were no significant differences in the developmental potency to blastocyst between the freezing methods and the concentrations of cryoprotectant. In summary, these experiments have proved that rapid freezing method(DMSO 4.5M) is effective in nuclear transplanted 2-cell mouse embryos. If improved micromanipulation techniques and freezing are combined, nuclear transplantation technique will contribute to the improvement of productivity in livestock animals.

• 대한수의학회지
• /
• 제29권2호
• /
• pp.123-128
• /
• 1989

Demi-embryos were successfully produced by bisection of ICR mouse embryos at preimplantation stages. They were microsurgically bisected using a microsurgical blade attached to a micromanipulator after pretreatment with 0.5% pronase in PBS for two minutes or not. Embryos with softened zona pellucida were more easily bisected and less damaged than intact embryos. The highest success rate in bisection has been achieved by selecting blastocysts(94.1% in success rate with intact blastocysts and 100% in success rate with zona softened blastocysts). Demi-embryos without zona pellucida were cultured in D-PBS or M-16 medium at $37^{\circ}C$, 5% $CO_2$ in air for 72 hours for 2-cell stage embryos, 48 hours for 4-to 8-cell stage embryos, 24 hours for morula stage embryos and 6~12 hours for blastocyst stage embryos. For the in vitro culture of 2-cell stage embryos, $100{\mu}M$ 2Na-EDTA was added to the media. M-16 medium was better for the in vitro development of mouse embryos than PBS, and PBS is not considered to be suitable for long-term culture of embryos, especially at early stage of cleavage. In M-16 medium, developing rate of demi-embryos of which pair underwent development to form eublastocysts was 15.8% at 2-cell stage, 16.8% at 4-cell stage, 38% at 8-cell stage, 89.6% at morula stage and 94.4% at blastocyst stage, respectively. The more rapid and efficient production of demi-embryos and higher viability after bisection can be expected by softening zona pellucida with pronase and by selecting morulae or blastocysts rather than embryos at early stage of cleavage.

• 한국가축번식학회지
• /
• 제16권3호
• /
• pp.217-224
• /
• 1992

These studies were carried out ot investigate on the transferred embryo development following ultrarapid frozen for 8-cell and morula of in vitro fertilization mouse embryos. The post-thaw embryo survival was evaluated and compared by cell stage of embryos and by equilibration time before ultrarapid freezing. The results obatined were summerized as follows: 1. The effects of equilibration time of 3 vs. 6 minutes before ultrarapid freezing and after thawing on the morphological survival and the viability of 8-cell and morulas embryos were not significant. 2. When the ultrarapid frozen-thawed 32 eight-cell and 33 morula embryos, and 30 fresh blastocysts were transferred to pseudopregnant recipient mice, the number of normal offsprings produced were 9(28.1%), 14(42.4%) and 18(60.0%), respectively. From the above resutls, it was concluded that the optimal conditions of pH osmolality of the media for mouse IVF and embryo culture, and the period of sperm preincubation might be 7.1, 310 mOsm and 120 min., respectively,a nd somewhat high conception rate might be resulted from transfer of frozen embryos of morula stage and fresh embryos of blastocyst stage.

• 한국수정란이식학회지
• /
• 제6권2호
• /
• pp.47-51
• /
• 1991

This study investigated full-term development potential of ultrarap idly frozen and thawed mouse 2-cell embryos. Mouse 2-cell embryos, dehydrated by exposure to freezing medium, were directly immersed into liquid nitrogen and thawed in 37$^{\circ}C$ water. The embryos that were frozen and thawed were cultured in uitro and transferred to foster mothers to examine there developmental potential. As a result, the frozen-thawed 2-cell embryos developed to blastocysts in vitro as a similar rate as control 2-cell embryos did(in vitro 2-cell, 86.4%; in vivo 2-cell, 90.9%; solution control, 89.9%; control, 89.7%). Normal live young were obtained from transfer of frozen-thawed embryos to the oviduct and uterus of pseudopregnant recipients (3l.4~56.7%).