• Clinical and Experimental Reproductive Medicine
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• 제29권1호
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• pp.21-28
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• 2002

Objective: To evaluate whether co-culture of oocytes on vero cell monolayers from Day 0 (Day 0 group) after egg retrieval results in an increase in developmental capacity such as fertilization rate, embryo quality, blastulation and clinical pregnancy rate compared with co-culture of oocytes from Day 1 (Day 1 group). Methods: Sperms were treated with Hams F-10 supplemented with 10% human follicular fluid (hFF). Vero cells for co-culture were prepared in TCM-199 with 10% FBS. Oocytes were co-cultured from Day 0 and fertilized oocytes were co-cultured from Day 1 on vero cell monolayers in DMEM with 10% and 20% hFF, respectively after egg retrieval. On day 1, 2 and 5, fertilization rate and grade of embryos and blastocysts were evaluated. Results (fertilization rate, cleavage rate, grade of embryos and blastocysts and pregnancy rate) were considered statistically significant when p value was less than 0.05 using t-test and $x^2$. Results: In sibling oocytes of same cycles, no differences were found in fertilization rate (94.6 vs. 91.4%), cleavage rates (94.6 vs. 91.4%), embryo grade (on day 2 and 3) and blastulation (65.6 vs. 57.0%) and their grade. In different oocytes of different cycles (patients), no differences were found in fertilization (79.8 vs. 78.3%), cleavage rates (77.7 vs. 76.4%) and blastulation (56.0 vs. 45.3%), but pregnancy rate was higher in the Day 0 group than in the Day 1 group (60.0 vs. 42.9%). Conclusions: This study revealed that the embryonic development capacities were not affected by the different co-culture time in the sibling oocytes of same cycles. Although no statistical significance, because of small size of study, there was a trend for higher pregnancy rates in Day 0 group compared to Day 1 group in different oocytes of different cycles.

• Ocean and Polar Research
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• 제40권3호
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• pp.161-167
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• 2018

We examined the effect of salinity and water temperature on hatching and survival rates of fertilized eggs of hybrid grouper (Epinephelus fuscoguttatus ♀ ${\times}$ E. lanceolatus ♂) at different developmental stages, determining optimal conditions for their long-distance transportation. Deformities and hatching rates of fertilized grouper eggs were observed at salinities of 24, 27, 30, 33, 36, and 39 psu. The optimal salinity was determined to be 36 psu, with a survival rate of $70.0{\pm}2.0%$. Transportation experiments at 36 psu were conducted at water temperatures of 21, 24, 27, and $30^{\circ}C$, different developmental stages such as morula, 5-myomere, and tail beating for hatching and survival rates. The optimal water temperature and developmental stage for transporatation were $30^{\circ}C$ and tail beating stage and those hatching rates were $50.6{\pm}1.9%$ and $86.3{\pm}1.3%$, respectively. At $21^{\circ}C$, the survival rate by transportation water temperature was highest ($73.1{\pm}10.6%$), but the hatching rate ($17.1{\pm}3.1%$) was lowest. Therefore, the hybrid grouper fertilized eggs (E. fuscoguttatus ♀ ${\times}$ E. lanceolatus ♂) can be most efficiently produced under long-distance transportation conditions during the tail beating stage and at a water temperature of $30^{\circ}C$.

• 대한수의학회지
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• 제35권1호
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• pp.187-195
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• 1995

The present study carried out to determine the developmental capacity of bovine oocytes matured in epidermal growth factor(EGF)-containing medium, the developmental competence of bovine embryos using synthetic oviduct fluid(SOF) and the effect of glucose on the development of bovine embryos. In experiment 1, oocytes, obtained from abattoir ovaries, were matured in EGF-containing medium for 24 hours, followed by exposure to Korean native cattle spermatozoa for 18 hours and cultured by utilizing co-culture system with bovine oviduct epithelial cells(BOEC) in TCM199. In experiment 2, early bovine embryos were cultured in SOF with or without BOEC and compared with those in TCM199 with BOEC. In experiment 3, bovine embryos were cultured in the presence or absence of glucose. Seven and ten days after in vitro fertilization, developmental competence of embryos were evaluated. The rate of cleavage was significantly(P<0.05) higher in EGF-containing maturation medium(70.0%) than in control(57.7%). The rates of development to morulae and blastocysts were 30.6% and 23.3% there was no significant difference between them. The rates of in vitro fertilized embryos to morulae and blastocysts cultured in SOF with BOEC(30.4%) and in TCM199 with BOEC(38.0%) were significantly(P<0.01) higher than cultured in SOF without BOEC(13.4%) at seven days after in vitro fertilization. The rates of embryos to blastocysts cultured in SOF with BOEC(29.4%) and in TCM199 with BOEC(35.9%) were significantly(P<0.05) higher than cultured in SOF without BOEC(13.4%) at ten days after in vitro fertilization. The rates of early embryos to morulae and blastocysts cultured in the presence or absence of glucose were 12.2% and 17.5% each other, there was no significant difference between them. The results show that bovine oocytes matured in the presence of EGF can cleave better, SOF with BOEC can replace serum containing complex media, TCM199 with BOEC in bovine embryo culture and glucose have little effect on the culture of early bovine embryos.

• Clinical and Experimental Reproductive Medicine
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• 제21권3호
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• pp.315-323
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• 1994

Mammalian oviductal epithelial cells have been known to improve in vitro fertilization and embryonic development. Recently, co-cultured human embryos with the epithelial cells in human genital tract has been reported to improve the pregnancy rate. The purpose of the study was to investigate the effects of the epithelial cells of human genital tract on the development of mouse early embryos and human fertilized oocytes. The epithelial cells of human genital tract were collected from the fallopian tubes which were obtained during hysterectomy in fertile women and from the endometrium during endometrium biopsy. Collected human ampullary cells(HACs) and endometrial cells(HECs) were cultured for 10 days to establish primary monolayer. Second passaged HACs and HECs were obtained by trypsinization were cryopreserved in PBS with 1.5 M DMSO for later use. To investigate the effect when co-cultured with HACs and HECs, we tried to apply strict quality control on mouse embryo, from two cell to blastocyst prior to human trial. The results of quality control were as follows; In Group I (Ham's F10 with 10% FCS), Group IT (co-cultured with HACs) and Group ill (co-cultured with HECs), developmental rates to blastocyst were 63.3%(253/400), 76.0%(304/ 400),74.0%(296/400), respectively. Hatching rates were 36.8%(147/400), 41.80/0(167/400), 38.0%(152/400), respectively(p<0.05). To perform the human IVF, cryopreserved HACs were thawed at 37$^{\circ}C$ waterbath, seeded on the well dish and cultured for 48 hI'S. The pronuclear stage embryos were transferred to the seeded well dish. After 24 hRS, co-cultured embryos were examined and transferred to patient's uterus. The results of human IVF when co-cultured with HACs were that fertilization and developmental rates were 61.8% (256/414), 95.3% (244/256) as compared with 57.2% (279/488) and 94.6%(264/279) in Ham's F10 supplemented with 10% FCS(control). However, 62.9% (161/256) of co-cultured human embryos showed good embryos(no or slight fragmentation) as compared with 53.8 % (150/279) in control(p < 0.05). Pregnancy rate was 40.0% (12/30) when co-cultured with HACs whereas 30.6%(11/36) in control. In conclusions, co-culture system using HACs and HECs improved the developmental and hatching rates of mouse embryo. Also, in human IVF system when co-cultured with HACs, it improved both the quality of human embryos and the pregnancy rate.

• 대한수의학회지
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• 제33권1호
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• pp.151-169
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• 1993

The present study was carried out to investigate the best condition for nuclear-cytoplasm fusion and in vitro culture of nuclear transplanted embryos and to investigate the production of nuclear transplanted offsprings. 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) pulses at output voltage of 1.0, 1.5 and 2.0 kV/cm for 100, 150 and $200{\mu}$ sec to induce cell fusion. 1. The culture of intact or zona cut 2-cell embryos in the medium supplemented with cytochalasin B($5{\mu}g/m{\ell}$) and colcemide($0.1{\mu}g/m{\ell}$)for 30 and 60 minutes did not affect the development to later stage. 2. The in vitro developmental rates of group A(a nucleus from one of the blastomeres was removed) and B(electrofusion of group A) were significantly lower than that of control group(p<0.01). 3. When nuclear transplanted embryos were submitted to electrofusion, the significantly higher fusion rates of 2-cell donor nuclei were achieved at the electric field strength of DC 1.5kV/cm for 100 and $150{\mu}$ sec, DC 2.0 kV/cm for $100{\sim}200{\mu}$ sec than DC 1.0 kV/cm for 100 and $150{\mu}$ sec(p<0.01). The significantly higher fusion rates of 4-cell donor nuclei were achieved at DC 2.0 kV/cm for 100 and $150{\mu}$ sec than DC 1.0kV/cm for $100{\sim}200{\mu}$ sec(p<0.01). These fusion rates in 8-cell donor nuclei were 88.7~99.3%. 4. The developmental potency to blastocyst in 2- and 4-cell donor nuclei was significantly higher in DC 1.0 and 2.0 kV/cm for $100{\sim}200{\mu}$ sec treated group and DC 2.0 kV/cm for 150 and $200{\mu}$ sec treated group (p<0.01). The developmental potency to blastocyst in 8-cell donor nuclei was significantly higher in DC 2.0 kV/cm for $100{\mu}$ sec treated group than in DC 1.0 kV/cm for $100{\mu}$ sec treated group and DC 2.0 kV/cm for 150 and $200{\mu}$ sec treated group(p<001). 5. The developmental potency to blastocyst after nuclear transplantation was significantly higher in 2-cell donor nuclei than in 8-cell donor nuclei(p<0.01). 6. The success rate of nuclear injection into enucleated 2-cell embryos was significantly higher in 2-cell donor nuclei than in 4- or 8-cell donor nuclei(p<0.01). 7. The culture time taken for the nuclear transplanted 2-cell embryos to blastocyst stage was significantly longer in 2-cell donor nuclei than in 8-cell donor nuclei(p<0.01). 8. There was no significant difference in the developmental potency of nuclear transplanted embryos within the concentration of EGF at 0 to 15 ng per $m{\ell}$ of BMOC-3 solution. 9. The production rates of offspring after transfer of nuclear transplanted embryos to recipient mouse were significantly higher in 2-cell donor nuclei than in 8-cell donor nuclei(p<0.01).

• 한국가축번식학회지
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• 제25권4호
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• pp.339-347
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• 2001

정자를 매개체로 한 유전자 전이는 형질 전환 동물의 생산을 위한 가능성 있는 간단한 방법이다. 또한 세포질내 정자 주입법에 의한 외래 유전자의 전이에 의한 형질 전환 동물의 생산이 최근에 보고되었다. 본 연구에서는 정자를 EGFP유전자와 공배양한 후 이를 난모세포내에 미세 주입한 다음, 수정란의 발달과 EGFP유전자의 발현을 조사하였다. 돼지 난자에 정자, 세포질막이 파괴된 정자 또는 정자를 주입하였다. 주입 후 수정된 난자는 NCSU23 배양액에서 배반포까지 배양하였으며, 배발생율과 EGFP 유전자의 발현을 연구하였다. 정자를 미세 주입한 결과 난할율은 67.0%로 정자두부를 미세주입한 난할율인 59.7%보다 높았고, 수정란의 EGFP 유전자 발현율은 각각 42.1와 20.0%로서 전자가 유의하게 높았다. 세포질막을 파괴하기 위해 다른 방법들로 정자를 처리하여 주입하였을 시 구정란의 배발생율에 영향을 주지 않았다 정자, 세포막이 파괴된 정자를 주입하여 배반포 발생율을 조사한 결과, 15.0과 14.2%로서 유의차가 없었다. 동결융해하거나 Triton X-100으로 처리한 정자를 주입하여, EGFP 발현율을 조사한 결과, 동결융해 정자를 사용했을 때의 성적이 38.4%로 타군의 성적인 22.4%에 비해 유의하게 높았다. 미세 주입에 앞서 정자는 EGFP 유전자의 0.01 ng/${\mu}\ell$ 부터 1 ng/${\mu}\ell$까지의 여러 농도로 배양되었다. 그러나 난할율을 조사한 결과 EGFP 유전자의 농도 차이에 따른 유의차가 없었다. 정자 배양액에 첨가된 EGFP 유전자의 농도가 0.1 ng/$m\ell$일 때 EGFP 발현율은 37.4%로서 가장 높은 결과를 보였다. 따라서 본 연구의 결과에 의하면 세포질막을 제거한 정자에 외래 유전자가 묻게 되며, 이 정자는 외래유전자를 수정란내로 옮겨 매개체로서의 역할이 가능할 것으로 생각된다.

• 한국가축번식학회지
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• 제18권1호
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• pp.31-37
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• 1994

This study carried out to investigate the effects of cryoprotectants in the medium on the survival rate of rapidly frozen porcine bisected demi-embryos. The porcine bisected demi-embryos following dehydration by cryoprotectants containing sucrose were directly plunged into liquid nitrogen and thawed in 3$0^{\circ}C$ water bath. Survival rate was defined as development rate on in vitro culture or FDA-test. The results are summarized as follows : 1. The survival rates of without-zona pellucida embryos and 2 blastomeres porcine embryos were 10.0 and 7.1%, respectively. The rate of unfrozen blastomeres (20.00%) was significantly higher than that of non-frozen oocytes. 2. The survival rates of with and without-zona pellucida of bisected porcine embryos by micromanipulator were 20.0 and 14.3%, respectively. 3. The developmental rates of with and without-zona pellucida of bisected poricine embryos by micromanipulator were 13.3 and 7.1%, respectively.

• 한국환경보건학회지
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• 제35권6호
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• pp.486-490
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• 2009

In January 2009, the water quality of offshore around the Incheon coast was evaluated by bioassay using early development systems of a sea urchin species, Hemicentrotus pulcherrimus. The results of performing biological evaluations on seawater samples from total of thirteen sites, showed that the formation rates of normal pluteus larva varied from 18% to 71%. In site 5 the seawater sample led to an averaage formation rate of normal larva of 18%, the highest abnormal formation rate hindering the early embryo development of the experimental animal, while that of site 3 averaged 71%, the highest formation rate of normal larva. Seawater samples from site 1, 2, 4, 7, 9, 10, 11 and 12, resulted in average formation rates of normal larva from 33% to 56%, which indicates the developmental damage of early embryos is not severe. Seawater samples from site 5, 6, 8 and 13, resulted in average formation rates of normal larva from 18% to 21% which there was strong damage to the development of early embryos.

• 한국가축번식학회지
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• 제21권3호
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• pp.287-292
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• 1997

This study was carried out to investigate the effects of concentration and kinds of cryoprotectants, equilibraction time, thawing temperature and time, sucrose concentration on the survival rates of frozen bovine demi-embryos. The bovine demi-embryos following dehydration by cryoprotectants a various concentration of sucrose were freezed by cell freezer and thawed in 3$0^{\circ}C$ water bath. Survival and in vitro developmental rates was defined as development rates on in vitro culture or FDA-test. The results are summarized as follows : 1. The high survival rates of demi-embryos after frozen-thawing in freezing medium was attained 2.0M glycerol. The high survival rates of demi-embryos after frozen-thawing in freezing medium was obtained using single cryoprotectant(25.0~30.0%) than mixed cryoprotectants(16.7~19.0%). 2. The survival rates of demi-embryos after frozen-thawing in freezing medium added 1.5M, 2.0M glycerol＋0.25M sucrose(37.5~33.3%) were higher survival rates than those of sucrose concentration of 0.50, 0.75M(12.5~26.7%). 3. The equilibration time on the survival rates of demi-embryos was attained after short period of time(30.0~35.0%) in the freezing medium higher than long period of time(21.1%). 4. The thawing temperature on the survival rates of demi-embryos was attained at 3$0^{\circ}C$ of thawing temperature(26.7~40.0%) higher than $25^{\circ}C$ or 37$^{\circ}C$ of thawing temperature(13.3~20.0%). 5. The thawing time on the survival rates of demi-embryos was attained at 1~5 minutes of thawing time(26.7~33.3%) in the freezing medium higher than 10 minutes of thawing time(13.3~18.8%).

• Asian-Australasian Journal of Animal Sciences
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• 제16권1호
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• pp.15-22
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• 2003

Experiments were conducted to evaluate the effect of blastomere diameters and cell cycle stages on the subsequent development of nuclear transplant rabbit embryos (NT-embryos) using nuclei derived from the 16- or 32-cell stage embryos. All blastomeres and NT-embryos were cultured individually in modified Ham's F-10 medium supplemented with 10% rabbit serum (RS) at $38^{\circ}C$ and 5% $CO_2$ in air. The diameter of blastomeres from 16-cell stage embryos was found twice of those from 32-cell stage (51 vs 27 ${\mu}m$). Significant differences were observed in cleavage rates ($\geq$3 divisions) in the isolated single blastomeres (54 vs 48 for 16-cell; 28 vs 14 for 32-cell, p<0.05), but the fusion rates of oocytes with transferred nuclei were similar between small and large single blastomeres derived from either 16-cell or 32-cell stage embryos. When 16-cell stage blastomeres were used as nuclear donors, cleavage rates ($\geq$3 divisions) of the NT-embryos were greater in the small nuclear donors than in the large donors (73 vs 55%, p<0.05). On the contrary, significantly higher cleavage (43 vs 6%, p<0.05) and developmental rates (14 vs 0%, p<0.05) were observed in the large blastomere nuclear donor group of the 32-cell stage embryos. When the cell cycle stages were controlled by a microtubule polymerization inhibitor (Demicolcine, DEM) or the combined treatment of DEM and Aphidicolin (APH), a DNA polymerase inhibitor, fusion rates were 88-96% for the 16-cell donor group (without DEM treatment), which were greater than the 32-cell donor group (54-58%). Cleavage rates were also greater in the transplants derived from G1 nuclear donor group (93-95%) than those from the DEM and APH combined treatment (73%) for the 16-cell donor group (p<0.05). No significant difference was detected in the morula/blastocyst rates in either donor cell stage (p>0.05). In conclusion, it appeared that no difference in the developmental competence between large and small isolated blastomeres was observed. When smaller 16-cell stage blastomeres were used as nuclear donor, the cleavage rate or development of NT-embryos was improved and was compromised when 32-cell stage blastomeres were used. Therefore, control nuclear stage of the donor cell at $G_1$ phase in preactivated nuclear recipients seemed to be beneficial for the cleavage rate of the reconstructed embryo in the 16-cell transplant, but not for subsequent morula or blastocyst development.