• Clinical and Experimental Reproductive Medicine
• /
• 제43권3호
• /
• pp.152-156
• /
• 2016

Objective: We studied the effect of the site of laser zona opening on the complete hatching of mouse blastocysts and the cell numbers of the completely hatched blastocysts. Methods: Mouse blastocysts were randomly allocated to the inner cell mass (ICM) group (zona opening performed at the site of the ICM, n=125), the trophectoderm (TE) group (zona opening performed opposite to the ICM, n=125) and the control group (no zona opening, n=125). Results: The rate of complete hatching of the blastocysts was not significantly different in the ICM and the TE group (84.8% vs 80.8%, respectively; p=0.402), but was significantly lower in the control group (51.2%, p<0.001). The cell numbers in the completely hatched blastocysts were comparable in the control group, the ICM group, and the TE group ($69{\pm}19.3$, $74{\pm}15.7$, and $71{\pm}16.8$, respectively; p=0.680). Conclusion: These findings indicate that the site of laser zona opening did not influence the rate of complete hatching of mouse blastocysts or their cell numbers.

• Clinical and Experimental Reproductive Medicine
• /
• 제27권4호
• /
• pp.367-372
• /
• 2000

Objective: This study was performed to evaluate whether vitrification method could be used for the cryopreservation of human blastocysts derived from IVF program. Methods: Surplus embryos were obtained from consented IVF patients. Controlled ovarian hyperstimulation was done with midluteal GnRH agonist, gonadotropin and hCG. After oocyte retrieval and insemination, fresh embryo transfer was done at $4{\sim}8$ cell stage. The surplus embryos after ET were cultured in blastocyst medium up to 6 days after oocyte retrieval. Obtained blastocysts were cryopreserved with our vitrification method. Blastocysts were exposed to 1.5 Methylene glycol (EG) in phosphate buffered saline (PBS) for 2.5 minutes, followed by 5.5 M EG plus 1 M sucrose for 20 seconds. Then 1 to 3 blastocysts were mounted on electron microscope (EM) grid and the grid was plunged into liquid nitrogen for storage. For thawing, blastocyst-containing EM grids were sequentially transferred in 1.0 M, 0.5 M, 0.25 M, 0.125 M and 0 M sucrose solution at the intervals of2.5 minutes. And blastocysts were cultured for about 6 hours and only re-expanded blastocysts were transferred to uterus of the patients on 4 to 5 days after ovulation in natural cycle or on 18 to 19 day of artificial cycle. Results: From Oct. 1998 to Jul. 1999, 34 patients were agreed to participate in this study. The mean age and duration of infertility of the patients were 31.6 years and 4.1 years, respectively. Among 34 cycles. replacements could be done in 20 cycles (58.8%). A total 93 blastocysts were thawed and 48 (51.6%) of them survived. Thirty-eight blastocysts, mean 1.9 embryos per patient, were transferred, resulting in 5 clinical pregnancies which consisted of 1 triplet, 2 sets of twins and 2 singleton pregnancies. The pregnancy rate per transfer was 25% and implantation rate was 23.6%. Five patients delivered 7 healthy babies including 2 sets of twins at term. Conclusion: Successful pregnancies and deliveries were established after transfer of vitrified human blastocysts. Vitrification using ethylene glycol as cryoprotectant and electron microscope grid is a rapid and simple method that can be effectively applied for the cryopreservation of human blastocysts.

• 한국가축번식학회지
• /
• 제22권2호
• /
• pp.177-186
• /
• 1998

While tubal pregnancy is frequently observed in human, it has been reported to rarely occur in other mammals. To investigate the reason of the absence of tubal pregnancy in other mammals, the ability of bovine tubal(oviductal) fluid to su, pp.rt the outgrowth of mouse embryos waw examined by using an in vitro model system wherein the trophoblast cells of hatched mouse blastocysts attach to and outgrow on tissue culture plates coated with FBS. When mouse blastocysts grwon in vitro from 2-cell embryos were cultrued in the dishes coated with FBS, human follicular fluid(hFF) and bovine follicular fluid(bFF), respectively, underwent outgrowth by spreading onto the plastic dishes during 48 hr. In contrast, none of the embryos cultured in the dishes coated with BSA or bovine obiductal fluid(bOF) did outgrow but remained as late blastocysts. Since addition of bOF at 5mg/ml or higher conc. to the culture medium resulted in degeneration of all embryos during 48 hr culture, 10mM conc. of glutathione(GSH) was added to the bOF-containing medium to circumvent the toxicity of bOF. In addition, bOF was heated $65^{\circ}C$ for 30 min(hbOF) to get rid of its precipitating properties and then added to the culture medium. When blastocysts were cultured in the presence of both hbOF and GSH 45.4% of embryos attached to the culture dishes. However, none of these embryos underwent outgrowth. Fially embryos were cultured in the presence of both hbOF and GSH but in the dishes coated with FBS. When they were examined after 48 hr, all of the blastocysts exhibited well-developed outgrwoth. Based upon these results, it is concluded that bovine oviductal fluid is capable of su, pp.rting the attchment of mouse blastocysts onto the culture plaste whereas it cannot promote the outgrwoth of mouse blastocysts in vitro, probably due to the lack of outgrwoth factor.

• 한국동물번식학회:학술대회논문집
• /
• 한국동물번식학회 2001년도 춘계학술발표대회
• /
• pp.20-20
• /
• 2001

It has been reported that cloning cattle is inefficient. One of the problems was placental abnormality, finally resulting in fetal mortality after transfer of nuclear transfer (NT) bovine embryos. This study was focused on the allocations of embryonic cells to the inner cell mass (ICM) or to the trophectoderm(TE) in NT bovine blastocysts. Somatic cells were derived from a Day 45 fetus of gestation, individually transferred into enucleated oocytes and developed to the blastocyst stage in vitro. Differential staining was used to assess the qualify of blastocysts derived from NT, IVF and in vivo. Development rate of NT embryos to blastocysts (25.0%, 41/164) was similar to that of IVF embryos (28.7%, 49/171). The total cell number of NT blastocysts (101.3$\pm$45.9) was not different compared with that of IVF embryos (107.9$\pm$34.2, P>0.05), but was lower than in vivo embryos (122.5$\pm$21.6, P<0.05). Ratio of ICM/total cells was higher in NT embryos (51.6$\pm$ 18.6%) than in IVF and in vivo embryos (42.3$\pm$ 15.3% and 34.9$\pm$8.9%, respectively) (P<0.05). Most IVF (56.8%, 25/44) and in vivo blastocysts(80.8%, 21/26) was distributed in the proportion of ICM/total cells ranging from 20 to 40% group. However, most NT blastocysts was biased in the 40-60%(34.1%, 15/44) and >60% (31.8%, 14/44) groups. Our findings suggest that placental abnormalities or early fetal losses in the present cloning system may be due to aberrant allocation of NT embryos to the ICM cells.

• 한국동물번식학회:학술대회논문집
• /
• 한국동물번식학회 2004년도 춘계학술발표대회
• /
• pp.201-201
• /
• 2004

In this study we examined whether vitrified Hanwoo (Korean cattle) IVM/IVF/IVC blastocysts can survive in vitro/in vivo by a quick one-step dilution method and these embryos result in live births. Blastocysts produced in vitro were vitrified by serial exposure to glycerol (G) and/or ethylene glycol (EG) mixtures of 10% (v/v) G for 5 min, 10% G plus 20% EG (v/v) for 5 min, and 25% G plus 25% EG (v/v) for 30 see. (omitted)

• 한국수정란이식학회지
• /
• 제14권1호
• /
• pp.39-46
• /
• 1999

To establish the optimal culture systems for production of transferable embryos in Korean Cattle, pregnancy rates of IVF-derived blastocysts according to different culture media, culture method and culture duration were compared. Development of IVF-derived embryos to blastocysts was most effective in YS medium group co-cultre with cumulus cells. Blastocysts cultured for 6 to 8 d in vitro showed higher hatching rate and good quality. Pregnancy rates after transfer of IVF-derived blastocysts cultured for 7 or 8 d were high. Through our experiments, it is considered that improvement of culture media and culture method is necessary for mass production of blastocysts with excellent of good quality in Korean Cattle.

• 한국가축번식학회지
• /
• 제20권3호
• /
• pp.333-341
• /
• 1996

One of the biggest problems involved in transgenic animal production is lack of appropriate market genes. To overcome this problem, we tested whether the genes of SEAP (secreted alkaline phosphatase) and GFP (green fluorescence protein) on our retrovirus vectors can be applicable to the transgenic animal production. The main advantage of these marker genes over other generally mainpulation can be selected without sacrificing viability. The results obtained in this study are summarized as follows: 1. Removal of zona pellucida from the mouse zygotes did not affect embryo developments to blastocysts. 2. Co-culture of zona-free embryos with virus-producing cells for 6 hours also did not affect embryo developments to blastocysts. 3. Among 58 blastocysts developed from the zona-free zygotes co-cultured with the virus-producing cells, SEAP expression was observed from the 6 blastocysts. 4. Expression of the GFP gene was detected from the virus- producing cells but no embryo expressing the gene was counted among 50 blastocysts developed from the zona-free zygotes co-cultured with the virus-producing cells.

• 한국수정란이식학회지
• /
• 제32권2호
• /
• pp.47-52
• /
• 2017

As a part of the effort to improve post-transfer survival rate of embryos in Korean black goats, a technique for laparoscopic uterine transfer of blastocysts was carried out. A total of 26 transferrable embryos (morula to expanded blastocysts) were transferred to 13 recipient goats via transabdominal laparoscopic method. In consequence of our hormone protocol, 65% of the recipients (13/20) were found to have synchronized estrus. After confirmation of corpus luteum in each recipient goat, a Babcock laparoscopic forceps was inserted into the lower abdominal cavity to hold a uterine horn and fasten it near the peritoneum without causing injury. Then 7.5cm long 16G IV catheter was inserted directly into the uterine lumen through the abdominal wall. After removal of the stylet of the IV catheter, the embryo transfer tube (identical in size to the stylet and loaded with blastocysts) was inserted into the uterine lumen through the catheter to unload the embryos. Of the 13 estrus synchronized recipients, 9 were transferred blastocysts and 4 were transferred molurae (2 embryos in each recipient) in uterine ipsilateral to the ovary with corpus luteum. Four of the 9 recipients which blastocysts were transferred using this method has been confirmed pregnant (44.4% pregnancy rate).

• Asian-Australasian Journal of Animal Sciences
• /
• 제20권12호
• /
• pp.1820-1826
• /
• 2007

In this study, we conducted various experiments in order to develop enhanced cultural conditions for in vitro-produced porcine embryos. All embryos were produced by in vitro maturation (IVM) and fertilization (IVF) of immature oocytes from abattoir-derived ovaries. In experiment 1, we cultured IVF embryos in 4 different groups, namely, 0% bovine serum albumin (BSA), 3% BSA, 0.05% Polyvinyl alcohol (PVA), and 0.5% Polyvinylpyrrolidone (PVP) added to the basal fluid cultural medium, Porcine zygote medium 3 (PZM-3). The rates of embryo development were higher in the group where the PZM-3 media had been supplemented with 3% BSA than the other groups. While not statistically significant, the percent of blastocysts and hatched blastocytes were 6.9% and 25.0% in the 3% BSA group vs. 1.2-6.4% and 0-16.7% in the other groups, respectively. In experiment 2, we added 10% fetal bovine serum (FBS) to PZM-3 on day 0 of culture and observed the development rate of blastocysts per day of culture from days 0 to 5. The development rate of blastocysts was higher at 15.6% on day 4 than on any other day, and was significantly higher than on day 0 or day 1 (p<0.05). The development rate of hatched blastocysts was 26.7% on day 4, and was higher than on any other day. In experiment 3, we cultured IVF embryos with different fluid culture media, grouped as 1) PZM-3+0.3% BSA (day0-day7); 2) PZM-3+0.3% BSA${\rightarrow}$day-4) PZM-3+10% FBS; 3) PZM-3+0.3% BSA${\rightarrow}$PZM-3+0.3% BSA+(day-4) FBS 10%; and 4) PZM-3+0.3% BSA+10% FBS (day0-day7). The development rates of blastocysts and hatched blastocysts were 21.5% and 53.1% in group 3, respectively, which was significantly higher than group 4 with respect to blastocyst development (5.2%, p<0.05) but not hatched blastocysts (14.3%). The total cell number (TCN) of blastocysts in group 3 was higher at $37.8{\pm}16.1$ than the other groups at $16.8{\pm}4.4$ - $30.1{\pm}10.9$; however, this was not significantly different. The results of this study showed that PZM-3 containing 0.3% BSA and supplemented with FBS during the later stage of culture on day 4 resulted in better TCNs and an increased rate of hatched blastocysts.

• 한국발생생물학회지:발생과생식
• /
• 제3권1호
• /
• pp.45-51
• /
• 1999

소 배반포로부터 배아주 (embryonic stem, ES) 유사세포를 분리하기 위해서는 영양외배엽 (trophectoderm, TE) 세포를 제거하는 것이 효과적이다. 따라서 본 실험은 효과적으로 TE를 제거하기 위한 calcium ionophore A23187 (CIPA) 처리조건을 확립하고, 분리해낸 ES 유사세포의 in vitro 다능성 (pluripotency)을 검증하고자 수행하였다. CIPA 농도 및 처리시간을 달리 하였을 때 50 $\mu$M에서 25분간 처리한 군이 colony 형성율 (51%)및 10 passage 까지의 배양성적 (4.8%)에서 가장 좋은 결과를 나타내었다. 또한 CIPA를 처리하지 않은 군과의 비교에서도 약 5배의 높은 결과를 보임으로서 본 실험에서 확립된 CIPA 처리조건은 가시적인 toxicity 없이 ES 유사세포의 확립에 이용될 수 있음을 시사하였다. 확립된 ES 유사세포는 heterogeneous한 alkaline phosphatase (AP) 활성을 보여 소 ES 유사세포에 대한 타 보고들과 유사한 결과를 보였다. In vitro 부양배양 (suspension culture)에서는 embryoid body로 분화가 가능하였으며, 약 70% 정도의 euploidism을 보였다. 따라서 본 실험에서 확립된 CIPA의 처리조건이 소 배반포로부터 ES 유사세포를 확립하는데 효과적으로 이용될 수 있음을 확인할 수 있었다.