• Clinical and Experimental Reproductive Medicine
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• 제33권4호
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• pp.265-272
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• 2006

목 적: 본 연구는 생쥐 포배기 배아로부터 내세포괴를 분리하는 방법과 지지세포의 종류와 mitomycin C 처리 시간이 내세포괴 colony 형성률에 미치는 영향을 관찰하기 위해 시행되었다. 연구방법: 일반적인 면역절제술, 주사바늘을 이용한 부분 영양막세포 절개법, 포배기 배아 공배양법으로 내세포괴를 분리한 후, 상업적으로 구입이 가능한 STO 또는 직접 제조한 생쥐 배아섬유아세포 (pMEF)를 지지세포로 이용하여 배양하였다. 또한, mitomycin C를 1, 2, 3시간 동안 처리한 각각의 지지세포에서 7일 동안 배양한 후, 내세포괴 colony 형성률을 살펴보았다. 결 과: STO 지지세포에서는 부분 영양막세포 절개법을 사용한 경우 (52%)가 면역절제술 (12%)이나 포배기 배아 공배양법 (16%)을 사용한 경우보다 내세포괴 colony 형성률이 유의하게 높았다 (p<0.05). pMEF 지지세포에서의 형성률은 부분 영양막세포 절개법을 사용한 경우 (88%)와 포배기 배아 공배양법 (82%)을 사용한 경우가 면역절제술 (16%)을 사용한 경우보다 높았다 (p<0.05). STO와 pMEF 모두에서, 2시간 mitomycin C 처리군 (52%, 88%)이 1시간 처리군 (9%, 42%)과 3시간 처리군 (18%, 76%)보다 높은 내세포괴 colony 형성률을 보여주었다 (p<0.05). 결 론: 이상의 결과는 부분 영양막세포 절개법이 생쥐 포배기 배아로부터 내세포괴를 분리하는 가장 효과적인 방법이며, 가장 적절한 mitomycin C 처리 시간은 2시간이라는 것을 보여준다. 그러나 이와 같은 부분 영양막세포 절개법의 효용성을 보다 명확하게 확인하기 위해서는 분리한 내세포괴를 계대배양하여 줄기세포주로서의 특성을 확인하는 실험이 추가적으로 필요할 것으로 생각된다.

• 한국수정란이식학회지
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• 제31권4호
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• pp.367-374
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• 2016

This study was conducted to evaluate the effect of transfer temperature of epididymis on survival rate of semen and development ability of B6D2F1 mice embryos. No significant differences were noted in the survival rate of semen ($59.0%{\pm}0.1$ vs. $47.6%{\pm}0.1$), in vitro fertilization rate ($90.7%{\pm}0.1$ vs. $90.7%{\pm}0.1$), developmental rate ($90.0%{\pm}0.1$ vs. $90.0%{\pm}0.1$), and blastocysts formation rate ($53.1%{\pm}0.2$ vs. $52.3%{\pm}0.2$) between groups. (NS; P>0.05). However, the zona hatched rate was significantly higher in the $4^{\circ}C$ group compared to those of the $37^{\circ}C$ group ($47.8%{\pm}0.1$ vs. $25.6%{\pm}0.2$; p<0.05). When it comes to cell numbers of blastocysts, the % ICM (/total cells) was significantly higher in the group of $4^{\circ}C$ compared to the $37^{\circ}C$ ($27.0%{\pm}0.1$ vs. $18.3%{\pm}0.1$; p<0.05). However there were no differences in total cell numbers ($72.7{\pm}31.6$ vs. $62.0{\pm}36.6$), ICM cell numbers ($17.0{\pm}7.8$ vs. $14.6{\pm}8.6$), TE cell numbers ($55.8{\pm}29.8$ vs. $64.0{\pm}24.4$), the ratio of ICM:TE ($1:4.2{\pm}4.1$ vs. $1:6.4{\pm}7.2$) between two groups (NS; P>0.05). Taken altogether, it is expected to achieve the best developmental ability of B6D2F1 mice embryos in the transfer temperature of epididymis. Also these results can provide fundamental data to maximize culture condition for in vitro fertilization on B6D2F1 mice. In future, therefore, it is expected that results herein might be applied for in vitro culture of human embryos.

• 한국수정란이식학회지
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• 제33권4호
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• pp.205-210
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• 2018

This study was performed to investigate the effect of types of oil (OVOIL vs. OIL) on B6D2F1 mice oogenesis. In this study, B6D2F1 F1 mice were used in order to maximize oogenesis. The expansion rate of cumulus cells ($82.0%{\pm}0.2$ vs. $78.0%{\pm}0.1$), in vitro fertilization rate ($92.0%{\pm}0.1$ vs. $88.0%{\pm}0.1$), developmental rate ($91.0%{\pm}0.1$ vs. $87.0%{\pm}0.2$), blastocysts formation rate ($56.0%{\pm}0.1$ vs. $57.0%{\pm}0.1$), and zona hatched rate($41.4%{\pm}0.2$ vs. $24.0%{\pm}0.1$) were not different between groups (NS; P>0.05). However, there was a significant difference in maturation rate; the OVOIL group showed higher maturation rate compared to that of the OIL group ($96.0%{\pm}0.1$ vs. $87.0%{\pm}0.1$; P<0.05). In the blastocysts cell numbers, the total cell numbers ($83.9{\pm}26.1$ vs. $56.9{\pm}23.9$), ICM cell numbers ($15.7{\pm}8.8$ vs. $6.3{\pm}3.5$), TE cell numbers ($68.3{\pm}25.7$ vs. $50.7{\pm}24.1$), % ICM ($21.6%{\pm}0.1$ vs. $12.7%{\pm}0.1$), and the ratio of ICM:TE ($1:6.2{\pm}6.5$ vs. $1:10.3{\pm}7.0$) were significantly higher in the OVOIL group than the OIL group (P<0.05). These results suggested that it is expected to achieve the more developmental ability of B6D2F1 mice depending on the type of oil (OVOIL vs. OIL). In addition, the results can provide essential information for culture condition on B6D2F1 mice. Henceforth, thus, it is expected that these results herein might be used for in vitro culture of human embryos.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2001년도 춘계학술발표대회
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• pp.68-68
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• 2001

Blastocyst formation, consisting of the inner cell mass (ICM) and trophectoderm (TE), is the first differentiation process during embryonic development in mammals. It has been hypothesized that the proportion of ICM to TE in the blastocyst may be crucial for subsequent developmental competence of early embryos, which it may be expressed as a sensitive indicator for evaluating in vitro systems. In this study ICM/total cell ratio of nuclear transfer (NT) embryos was compared with IVF-derived and in vivo embryos. Somatic cell nuclei obtained from a fetus at Day 40 of gestation were transferred into the enucleated oocyte and then cultured in NCSU 23 medium for 6 days as previously described (Koo et al., Biol. Reprod. 2000; 63:986-992). ICM and TE cells of blastocysts were determined by using a differential staining method (Han et al., Biol. Reprod. 1999; 60:1110-1113). Development rate (9.8$\pm$2.5%, 23/225) to the blastocyst stage of NT embryos was lower than IVF embryos (23.8$\pm$2.7%, 53/223). Thus, a difference was detected in the in vitro developmental rate to blastocyst stage between NT and IVF-derived embryos (P<0.05). In the next experiment, we investigated ICM and TE nuclei to assess the quality of blastocysts that produced by NT, IVF and in vivo, respectively. NT blastocysts (27.6$\pm$8.3) showed a smaller total cell number than IVF-derived (42.6$\pm$17.4) and in vivo embryos (283.9$\pm$103.5) (P<0.05). Ratios of ICM/total cells in NT, IVF and in vivo blastocysts were 15.1$\pm$ 18.6% (n=56), 12.3$\pm$9.2% (n=57) and 30.4$\pm$6.8% (n=40), respectively. Individual blastocysts for the ratio of ICM/total cells were assigned to 3 groups (I; <20%, II; 20 to 40% and III;>40%). As the results, most in vivo blastocysts (97.5%, 39/40) were distributed into group II while most NT (78.6%, 44/56) and IVF-derived blastocysts (82.5%, 47/57) were allocated to group I. Thus, our data show that NT or IVF-derived embryos have aberrant morphology during early development in vitro systems, suggesting that these anomalies may result in developmental failures of the NT embryos to term.

• Asian-Australasian Journal of Animal Sciences
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• 제20권5호
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• pp.693-700
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• 2007

The present study was conducted to examine the effect of different levels of essential and nonessential amino acid in NCSU-23 medium on the in vitro-produced porcine embryo as it develops from the zygote to the blastocyst stage. Four experiments were performed, each with a completely randomized design involving 5 to 8 replications of treatments. In order to know the effect of nonessential amino acids in NCSU-23 medium, 0, 5, 10 and $20{\mu}/ml$ MEM were supplemented there to, (Exp. 1) and the medium was supplemented with same level of essential amino acids (Exp. 2). The combined effect of nonessential (0, 5, 10 and $20{\mu}/ml$ MEM) and essential amino acids (0, 5, 10 and $10{\mu}/ml$ MEM) in NCSU-23 medium (Exp. 3), first 72 h with non-essential amino acids (at 0, 5, 10 and $20{\mu}/ml$ MEM), and last 4 d with essential amino acids with the same level as NEAA (Exp. 4) were examined. The embryo development was monitored and the quality of blastocysts was evaluated by counting the number of total cells and determining the ratio of inner cell mass (ICM) to trophoectoderm (TE) cells. When Eagle's nonessential amino acids (MEM) added to NCSU-23 medium, it significantly increased the likelihood of development to the 2- to 4-cell stage and subsequent blastocyst development. Supplementation of different levels of essential amino acids in the NCSU-23 medium decreased cleavage rate, rate of morula and blastocyst development and the number of ICMs. In the case of the combined effect of essential and nonessential amino acids, better and significant results were found for blastocysts, hatching blastocysts and for ICM numbers which were also dose dependent. With respect to the biphasic effect of nonessential and essential amino acids, nonessential amino acids increased cleavage whereas essential amino acids increased the total cell number. Neither the nonessential nor the essential group of amino acids, on their own, affected blastocyst cell number or the differentiation of cells in the blastocyst. In conclusion, this study determined the role of nonessential and essential amino acids in the culture of the porcine embryo and showed that the embryo requires different levels of amino acids as it develops from the zygote to the blastocyst stage.

• Clinical and Experimental Reproductive Medicine
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• 제24권1호
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• pp.21-26
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• 1997

본 연구는 체외생산된 생쥐 배반포기배의 ICM 세포에서 EGF-R 발현유무를 immunosurgery와 indirect immunofluorescence (간접 면역 형광방법) 을 이용하여 조사하고자 실시하였다. 본 실험에 사용된 ICM 세포는 체외수정 후 96시간째에 회수된 생쥐 배반포기배를 immunosurgery 방법을 이용하여 얻어졌으며, 회수된 ICM세포는 생사유무와 EGF-R 발현유무 조사에 공시 되어졌다. 그 결과를 요약하면 다음과 같다. ICM세포의 회수율은 rabbit anti-mouse serum (antiserum) 과 guinea pig serum (complement) 에 각각 15-30 분과 15-60분 동안 처리 했을 경우 8.0-84.2% 였으며, 또한 처리시간이 각각 30분과 60분일 때 가장 높은 회수율 (84.2%) 을 얻었다. Immunosurgery 후 얻어진 ICM세포의 생존 유무를 조사하기 위해 live/dead염색 방법을 이용하였던 바, 처리된 ICM세포중 93.8-100%의 생존율을 나타내어 회수된 ICM세포는 유해한 영향을 받지 않았다는 것을 알 수 있었다. 또한, 간접면역 형광방법을 이용하여 ICM세포에서 EGF-R가 발현되는 것을 확인 하였다. 따라서, ICM세포에서의 EGF-R의 발현은 인위적으로 첨가된 EGF의 이용가능성을 높임으로서 체외에서의 착상전 배 발달을 증진시킬 수 있을 것으로 사료된다.

• 한국수정란이식학회지
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• 제23권1호
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• pp.51-57
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• 2008

The present study investigated the efficient methods to produce in vitro Hanwoo embryos, and to improve the pregnancy rate. The developmental rate, total cell number and ICM ratio of in vitro embryos were compared amongst different culture media. Comparisons were also made on the status of recipients, pregnancy rate along with day of transfer after the estrus. Development of embryos into blastocyst stage in IVMD101 supplemented with 5% fetal bovine serum (FBS) group was significantly higher (34.2%) than that of TCM-199 supplemented with 5% FBS (26.8%) and IVMD101 without FBS (25.9%) (p<0.05). The development rate to blastocyst stage was significantly faster in IVMD101(5% FBS) than that of other groups ($0.2{\sim}2.3%$) (p<0.05). The average number of inner cell mass and trophectoderm were similar among treatment groups, which were $36.0{\sim}44.7$ and $83.3{\sim}106.7$. However, total cell number in IVMD101(5% FBS and 0% FBS) was significantly higher than that of TCM199(5% FBS). There were no differences in the pregnancy rate among treatment groups (32.0%, 33.9% and 28.6%, respectively). However, the pregnancy rate of Day 6 embryos cultured in IVMD101(5% FBS) was significantly (p<0.01) higher than IVMD101 without FBS and TCM-199 + 5% FBS (38.0% vs. 17.2% and 32.4%, respectively). No significant difference was observed for the pregnancy rate between heifer and cow transferred with Day 6 embryos cultured in IVMD 101(5% FBS) (42.7% and 39.3%, respectively). However, there was a significant difference of pregnancy rate (p<0.05) in heifer between one and two embryos transferred (31.4% and 41.9%). There was no difference of pregnancy rate among transfer days after estrus between heifer and cow, but the pregnancy rate of transfer to heifer with day 6 after estrus was significantly higher (p<0.05) than that of day 7 and 8 (22.2% vs. 49.0% and 38.7% respectively). Based on the above findings, there is a possibility to produce in vitro produced embryos cultured in IVMD101(5% FBS) showed higher blastocyst rate and the increased cell number. In terms of the pregnancy rate of in vitro produced embryos, the highest pregnancy rate was observed when two embryos were cultured in IVMD101(5% FBS) and transferred.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2001년도 발생공학 국제심포지움 및 학술대회 발표자료집
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• pp.37-43
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• 2001

1. About fifty thousand of cattle embryos were transferred and 16000 ET-calves were born in 1999. Eighty percents of embryos were collected from Japanese Black beef donors and transferred to dairy Holstein heifers and cows. Since 1985, we have achieved in bovine in vitro fertilization using immature oocytes Collected from ovaries of slaughterhouse. Now over 8000 embryos fertilized by Japanese Black bull, as Kitaguni 7 -8 or Mitsufuku, famousbulls as high marbling score of progeny tests were sold to dairy farmers and transferred to their dairy cattle every year. 2. Embryo splitting for identical twins is demonstrated an useful tool to supply a bull for semen collection and a steer for beef performance test. According to the data of Dr.Hashiyada (2001), 296 pairs of split-half-embryos were transferred to recipients and 98 gave births of 112 calves (23 pairs of identical twins and 66 singletons). 3. A blastomere-nuclear-transferred cloned calf was born in 1990 by a joint research with Drs.Tsunoda, National Institute of Animal Industry (NIAI) and Ushijima, Chiba Prefectural Farm Animal Center. The fruits of this technology were applied to the production of a calf from a cell of long-term-cultured inner cell mass (1998, Itoh et al, ZEN-NOH Central Research Institute for Feed and Livestock) and a cloned calf from three-successive-cloning (1997, Tsunoda et al.). According to the survey of MAFF of Japan, over 500 calves were born until this year and a half of them were already brought to the market for beef. 4. After the report of "Dolly", in February 1997, the first somatic cell clone female calves were born in July 1998 as the fruits of the joint research organized by Dr. Tsunoda in Kinki University (Kato et al, 2000). The male calves were born in August and September 1998 by the collaboration with NIAI and Kagoshima Prefecture. Then 244 calves, four pigs and a kid of goat were now born in 36 institutes of Japan. 5. Somatic cell cloning in farm animal production will bring us an effective reproductive method of elite-dairy- cows, super-cows and excellent bulls. The effect of making copy farm animal is also related to the reservation of genetic resources and re-creation of a male bull from a castrated steer of excellent marbling beef. Cloning of genetically modified animals is most promising to making pig organs transplant to people and providing protein drugs in milk of pig, goat and cattle.

• 한국수정란이식학회지
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• 제31권1호
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• pp.19-25
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• 2016

This study was conducted to evaluate the effects of type of culture media (BM, G2, OS, TCM, and MEM) on B6D2F1 mice oogenesis. In the present study, B6D2F1/CrljOri $F_1$ mice were utilized in order to maximize oogenesis. Also we used TCM-199, Dulbecco's medified Eagle's medium (DMEM), embryo culture medium (Fertilization medium, Cleavage medium, Blastocyst medium), G series medium and One step medium. In vitro maturation was highest in BM followed by the order of OS, MEM, TCM and G2 ($90{\pm}2.8%>88{\pm}3.2%>85{\pm}4.9%>78{\pm}10.2%>64{\pm}7.7%$, respectively). To note, the G2 group was statistically different compared to other groups (p<0.05). On the other hand the fertilization rate was highest in the G2 group followed by BM, OS, TCM, and MEM ($87{\pm}7.2%>85{\pm}6.9%>74{\pm}14.0%>71{\pm}13.8%>2{\pm}1.4%$, respectively). The MEM group was significantly lower compared to other groups (p<0.05). The developmental rate was highest in the OS group followed by the G2 group and the BM group albeit no statistical significance was noted ($73{\pm}11.6%>71{\pm}9.2%>66{\pm}10.4%$). Of note, all cells of the TCM and MEM groups were died during embryonic development. The zona hatched rate ($51{\pm}9.8%$ vs. $50{\pm}9.1%$ vs. $47{\pm}7.2%$ for BM, G2, and OS respectively) and attached rate ($45{\pm}12.3%$ vs. $38{\pm}16.1%$ vs. $37{\pm}11.5%$ for BM, G2, and OS respectively) were not different amongst groups. No difference was found in total cell numbers ($74{\pm}13.9$ vs. $64{\pm}9.2$ vs. $76{\pm}6.7$ for BM, G2, and OS respectively), ICM cell numbers ($20{\pm}1.9$ vs. $14{\pm}1.8$ vs. $15{\pm}2.1$), TE cell numbers ($55{\pm}12.5$ vs. $49{\pm}10.7$ vs. $61{\pm}5.9$), % ICM ($30{\pm}2.8%$ vs. $24{\pm}7.0%$ vs. $22.8{\pm}2.2%$) and ICM:TE ratio ($1:2{\pm}0.5$ vs. $1:3.1{\pm}0.8$ vs. $1:3.1{\pm}0.5$) amongst groups. In summary, these results can provide fundamental data to maximize culture condition for in vitro fertilization on B6D2F1 mice.

• 한국수정란이식학회지
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• 제33권4호
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• pp.221-228
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• 2018

The osmolarity of a medium that is commonly used for in vitro culture (IVC) of oocytes and embryos is lower than that of oviductal fluid in pigs. In vivo oocytes and embryos can resist high osmolarities to some extent due to the presence of organic osmolytes such as glycine and alanine. These amino acids act as a protective shield to maintain the shape and viability in high osmotic environments. The aim of this study was to determine the effects of glycine or/and alanine in medium with two different osmolarities (280 and 320 mOsm) during IVC on embryonic development after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT) in pigs. To this end, IVC was divided into two stages; the 0-2 and 3-7 days of IVC. In each stage, embryos were cultured in medium with 280, 320, or 360 mOsm and their combinations with or without glycine or/and alanine according to the experimental design. Treatment groups were termed as, for example, "T(osmolarity of a medium used in 0-2 days of IVC)-(osmolarity of a medium used in 3-7 days of IVC)" T280-280 was served as control. When PA embryos were cultured in medium with various osmolarities, T320-280 showed a significantly higher blastocyst formation (29.0%) than control (22.2%) and T360-360 groups (6.9%). Glycine treatment in T320-280 significantly increased blastocyst formation (50.4%) compared to T320-280 only (36.5%) while no synergistic was observed after treatment with glycine and alanine together in T320-280 (45.7%). In contrast to PA embryonic development, the stimulating effect by the culture in T320-280 was not observed in SCNT blastocyst development (27.6% and 23.7% in T280-280 and T320-280, respectively) whereas the number of inner cell mass cells was significantly increased in T320-280 (6.1 cells vs. 9.6 cells). Glycine treatment significantly improved blastocyst formation of SCNT embryos in both T280-280 (27.6% vs. 38.0%) and T320-280 (23.7% vs. 35.3%). Our results demonstrate that IVC in T320-280 and treatment with glycine improves blastocyst formation of PA and SCNT embryos in pigs.