• 한국수정란이식학회지
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• 제15권2호
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• pp.191-196
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• 2000

the present study was carried out to develop a completely defined culture system and determine if high NaCl concentrations in defined (PVA added) or semi-defined (BSA added) medium is toxic to bovine embryos. Oocytes from slaughterhouse ovaries were matured and fertilized in vitro. After 30 h of insemination, only 2-cell stage embryos were selected and cultured for this experiment. The culture media used were as follows : TLP(114 mM of NaCl) + BSA (3 mg/ml), TLP + PVA (1 mg/ml), mTLP(96 mM of NaCl) + BSA, mTLP + PVA. Six to ten embryos were placed into a 30$\mu$1 drop of each medium and the embryos were examined at 10 day post-insemination without medium renewal. The experiment was replicated 4 times. All data were analyzed by chi-square. There were no significant differences among TLP-BSA, mTLP-BSA and mTLP-PVA in blastocyst development (21.6, 17.2 and 20.2%), respectively. Also, no differences were obtained in hatching rates (11.7, 9.9 and 12.2%), respecitively. However, there were significant differences between TLP-PVA (1.7% and 0.6%) and other group in blastocyst formation and hatching rates, respectively (p<0.01). Development of in vitro produced embryos cultured in BSA containing medium was not affected by high NaCl concentration, but in the completely defined medium, embryonic development was highly affected by NaCl. This study shows that reduced NaCl concentration in completely defined medium is beneficial for development of bovine pre-implantation embryos in vitro.

• Reproductive and Developmental Biology
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• 제38권4호
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• pp.147-158
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• 2014

Differentiated nuclei can experimentally be returned to an undifferentiated embryonic status after nuclear transfer (NT) to unfertilized metaphase II (MII) oocytes. Nuclear reprogramming is triggered immediately after somatic cell nucleus transfer (SCNT) into recipient cytoplasm and this period is regarded as a key stage for optimizing reprogramming. In a recent study (Dai et al., 2010), use of m-carboxycinnamic acid bishydroxamide (CBHA) as a histone deacetylase inhibitor during the in vitro early culture of murine cloned embryos modifies the acetylation status of somatic nuclei and increases the developmental competence of SCNT embryos. Thus, we examined the effects of CBHA treatment on the in vitro preimplantation development of porcine SCNT embryos and on the acetylated status of histone H3K9 on cloned embryos at the zygote stage. We performed the three groups SCNT: SCNT (NT), CBHA treatment at the porcine fetus fibroblast cells (PFFs) used as donor cells prior to SCNT (CBHA-C) and CBHA treatment at the porcine SCNT embryos during the in vitro early culture after oocyte activation (CBHA-Z). The PFFs were treated with a $15{\mu}M$ of CBHA (8 h) for the early culture and the porcine cloned embryos were treated with a $100{\mu}M$ concentration of CBHA during the in vitro early culture (10 h). Cleavage rates and development to the blastocyst stage were assessed. No significant difference was observed the cleavage rate among the groups (82.6%, 76.4% and 82.2%, respectively). However, the development competence to the blastocyst stage was significantly increased in CBHA-Z embryos (22.7%) as compared to SCNT and CBHA-C embryos (8.6% and 4.1%)(p<0.05). Total cell numbers and viable cell numbers at the blastocyst stage of porcine SCNT embryos were increased in CBHA-Z embryos as compared to those in CBHA-C embryos (p<0.05). Signal level of histone acetylation (H3K9ac) at the zygote stage of SCNT was increased in CBHA-Z embryos as compared to SCNT and CBHA-C embryos. The results of the present study suggested that treatment with CBHA during the in vitro early culture (10 h) had significantly increased the developmental competence and histone acetylation level at the zygote stage.

• 한국수정란이식학회지
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• 제12권2호
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• pp.161-169
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• 1997

This study was conducted to compare the insemination time of bovine oocytes and determine the effects of glucose(1.5 mM) on the development of bovine embryos at early cleavage stage. Oocytes were matured for 24 h, followed by exposure to sperm and cultured in modified Tyrode's media drops or with bovine oviduct epithelial cell monolayer prepared in TCM199(BOECM). Insemination time and culture system were varied in each experiment. In experiment 1, to investigate the developmental capacity of bovine embryos after different time of exposure to sperm, bovine ova and sperm were co-incubated for 18, 30 or 54 h, respectively. The development to blastocysts of 30 and 54 h insemination groups were significantly higher(P<0.05) than 18 h group, and in case of blastocysts of cleaved embryos, 30 h group were significantly higher(P<0.05) than other groups. In experiment 2, we investigated the effect of glucose on early bovine embryos. After 18 h insemination, in vitro fertilized oocytes were separated following 3 groups ; G+0, C+24 and C+48. Oocytes of G+0 group were cultured in glucose added Tyrode's medium after fertilization, oocytes in C+24 and C+48 groups were cultured in glucose free Tyrode's medium after fertilization. After 24 h culture, G+24 group was moved to glucose added medium. All oocytes of 3 groups were moved to BOECM after 48 h culture. The rates of cleavage and development to blastocysts in G+0 group were significantly lower than other groups. In experiment 3, we determined the effects of glucose exposure from 8 to 20 h after insemination on the cleavage and development of oocytes. The oocytes in glucose added group had high capacity of cleavage and further development. This study shows that in bovine oocytes, the optimal exposure to sperm is 30 h and glucose exposure to bovine one-cell embryos is detrimental to their first cleavage and further development in vitro but there has no evidence of detrimental effect of glucose(1.5 mM) exposure to bovine embryos over the two-cell stage in vitro.

• Asian-Australasian Journal of Animal Sciences
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• 제8권6호
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• pp.599-605
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• 1995

The ultrastructures of in vitro-derived bovine blastocysts have been compared with those of blastocysts obtained from a superovulated cow. In vivo blastocysts obtained from the uterus showed well-differentiated features, while in vitro-derived embryos, which were developed from in vitro fertilized ovum, showed insufficient cellular organizations. In vitro-derived embryos contained many undefined cellular organizations in the perivitelline spaces compared with in vivo-derived blastocysts. Other features of in vivo and in vitro blastocysts were characterized by differential development of microvilli projection into blastocoele from the surface of the trophoblast cells. The conceivable reason for the difference between in vivo and in vitro developments of bovine embryos is that it is likely that in vitro culture system adopted in the present experiment may not be sufficient for better embryonic development.

• 한국수정란이식학회지
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• 제8권1호
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• pp.13-19
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• 1993

These experiments were carried out to determine the effect of pregnancy in bisected embryo. The embryos of ICR mouse were microsurgically bisected at morula and blastocyst stage using microsurgical blade attached a micromanipulator. These bisected embryos without zona pellucida were cultured up to blastocyst stage and cell count and diameter of stained blastomere, and transferred pseudopregnant mice. And the development of these bisected embryos was compared with the results of production of young of the corresponding intact embryos or cell stage. When the bisected mouse embryos were cultured in vitro for 20 to 24 hours in morula stage(77.2%) or 3 to 6 hours in blastocyst stage(84.1%), them were developed to the expanded blastocyst stage. There were no significant(P<0.05) differences in the development rate of bisected embryos between in morula and blastocyst stages. The embryo size of blastocyst developed in vitro from bisected embryo was small(P<0.05)than intact embryo. However, the number of blastomeres with bisected embryo (24.7+1.3and 21.5+1.2 respectively) were significantly(P<0.05) reduced, compared with that of intacted embryos(36.3+1.1 and 41.4+1.2 respectively). When compared with the result of pregnancy rate(63.6%) after surgical transfer of bisected morulae, a similar result(65.4%) was obtained with bisected blastocyst stage(P< 0.05). However, production of youngs (38.8%) after transfer of bisected morula, a similar result (38.1%) was obtained with bisected blastocyst stage (P<0.05).

• 한국수정란이식학회지
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• 제6권1호
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• pp.33-40
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• 1991

In vitro developmental ability of early preimplantation monse embryos was shown to be depend on the embryonic stages, media and snpplements and their interaction(Experiment 1). The development of I-cell embryos were more promoted in the complex medinm(Ham's Fl0) than in the simple one(m-KRB), but that of 2-cell embryos showed the reverse effect. The bovine serum albumin(BSA) as a medium snpplement more promoted the development of I- and 2-cell embryos, compared with human fetal cord serum(HCS). On the other hand, the harmful effect of HCS was especially shown on the early cleavage in the embryonic development of the two stages. The effect of serum, in the respect of interaction between media and snpplements. was also more significantly appeared in m-KRB than Ham's Fl0. In the experiment 2, when the harmful effect of HCS was compared with that of fetal bovine serum(FBS), the former more promoted the development of l - and 2-cell embryos than the latter. The effect of HCS was more significantly shown in the development of I-cell than that of 2-cell embryos. Conclusively, as I- and 2-cell embryos were different in the requirements for the in vitro development. the optimal medium and supplement have to be selected for each embryonic stage. It is also respected to the better result if it take into consideration into the kinds of sera when serum is used for culture of early preimplantation embryos.

• Reproductive and Developmental Biology
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• 제29권3호
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• pp.181-186
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• 2005

본 연구에서는 배양 체계(혈청 첨가 TCM199, TALP, CRlaa 및 혈청 미첨가; IVMD101, IVF100, IVMD101)가 체외수정 또는 미세주입된 수정란의 체외 발달에 미치는 효과를 검토하였다. 또한 미세주입에 사용하는 GFP유전자의 양 및 미세주입 수정란에서 형광발현 양상을 검토하였다. 체외수정된 수정란의 $\geq$ 2세포기, 8세포기 및 배반포 도달율이 미세주입 수정란에 비하여 유의하게 높았다. 혈청 미첨가 배지에서의 8세포기 발달율이 수정란의 종류에 관계없이 유의하게 높았으나(p<0.05; $ 3.3\%\;vs.\;15.5\%$ 및 $21.4\%\;vs.\;39.4\%$, respectively), 배반포 도달율은 유사한 경향이었다($2.7\%\;vs.\;2.3\%$ 및 $23.0\%\;vs.\;23.6\%$, respectively). 한편, 2ng/uL 유전자를 미세주입한 수정란의 $\geq$ 2세포기, 8세포기 및 배반포 도달율이 4 및 8ng/uL의 것에 비하여 높은 경향이었다. 미세주입 수정란의 형광발현율은 1세포기가2및 8세포기에 비하여 유의하게 높았으나(p<0.05), 4세포기 및 배반포 단계와는 차이가 없었다.

• 한국가축번식학회지
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• 제17권1호
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• pp.69-74
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• 1993

This study was investigated to examine the effect of conditioned medium from bovine oviductal cell(BOEC) in the co-culture system with BOEC on in vitro development of in vitro produced bovine embryos. Oocyte-cumulus complexes were cultured for 24 hrs in TCM-199 supplemented with 10% fetal calf serum, 1$\mu\textrm{g}$/ml FSH and 21U hCG, 1$\mu\textrm{g}$/ml oestradiol-17$\beta$ at 39$^{\circ}C$ under 5% CO2 in air. In vitro fertilization was performed with epididymal sperm and heparin (10$\mu\textrm{g}$/ml, 15min.) or caffeine(2.5mM)-treated spermatozoa. Oocytes were incubated with 1$\times$106 spermatozoa/ml for 18 hrs and then cultured in various culture system for 7 days. The development rates of 16-cell or blastocyst stages were recorded on 4, 7 days, respectively, after incubating. The proportions ofembryonic development into molulae and blastocysts were higher in cumulus cell co-culture(23.4%) and BOEC co-culture(34.3%) than in M199-FCS(6.1%). Similarily, the development rates into molulae and blastocysts were significantly higher in BOEC-conditioned medium than those in M199-FCS. Therefore, it is suggested that BOEC co-culture and BOEC conditioned medium increase significantly the development of in vitro produced bovine embryos in in vitro system.

• 한국발생생물학회지:발생과생식
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• 제25권1호
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• pp.43-53
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• 2021

We examine the effect of endoplasmic reticulum (ER) stress inhibitor treatment time on the in vitro development of porcine somatic cell nuclear transfer (SCNT) embryos. Porcine SCNT embryos were classified by four groups following treatment time of ER stress inhibitor, tauroursodeoxycholic acid (TUDCA; 100 µM); 1) non-treatment group (control), 2) treatment during micromanipulation process and for 3 h after fusion (NT+3 h group), 3) treatment only during in vitro culture after fusion (IVC group), and 4) treatment during micromanipulation process and in vitro culture (NT+IVC group). SCNT embryos were cultured for six days to examine the X-box binding protein 1 (Xbp1) splicing levels, the expression levels of ER stress-associated genes, oxidative stress-related genes, and apoptosis-related genes in blastocysts, and in vitro development. There was no significant difference in Xbp1 splicing level among all groups. Reduced expression of some ER stress-associated genes was observed in the treatment groups. The oxidative stress and apoptosis-related genes were significantly lower in all treatment groups than control (p<0.05). Although blastocyst development rates were not different among all groups (17.5% to 21.7%), the average cell number in blastocysts increased significantly in NT+3 h (48.5±2.3) and NT+IVC (47.7±2.4) groups compared to those of control and IVC groups (p<0.05). The result of this study suggests that the treatment of ER stress inhibitor on SCNT embryos from the micromanipulation process can improve the reprogramming efficiency of SCNT embryos by inhibiting the ER and oxidative stresses that may occur early in the SCNT process.

• 한국수정란이식학회지
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• 제14권3호
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• pp.219-224
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• 1999

The effect of several potential antioxidants were examined as a means of increasing the in vitro development of in vitro matured and in vitro fertilized oocytes into morulae and blastocysts. Korean native cattle embryos after in vitro fertilization were cultrued for 7 days at 38.5$^{\circ}C$ in CR1aa containing varing concentration of the antioxidants in a gas phases consisting of 5% CO2, 95% humidified air. The results obtained were summarized as follows; The proportion of embryos developed to morulae and blastocysts in CR1aa containing 2.5uM $\alpha$-tocopherol(11.0% and 6.0%) was significantly higher than those of 0, 5.0, and 7.5uM $\alpha$-tocopherol (P<0.05). concentration of 50uM L-ascorbic acid (7.5% blastocysts) did affect the proportion of embryos developing into blastocystes(P>0.05). Addition of 200uM cysteamine was significantly higher than those of 0, 100 and 300uM (P<0.05). When the fertilized oocytes were cultured at 0. 200, 400 and 600uM of selenium for 168 hrs, the morulae rates were 12.2, 5.2, 16.0 and 16.1% respectively, and addition of 200uM selenium was significantly higher than those of 0, 400, 600uM (P<0.05). These results suggested that the addition of $\alpha$-tocopherol, L-ascorbic acid, cysteamine and selenicum can enhanced development to the morulae and blastocysts of in vitro derived fertilized oocytes.