• Journal of Embryo Transfer
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• v.24 no.3
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• pp.183-187
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• 2009

In this study, we aimed to determine whether the evaluated markers of cell death could be found at particular developmental stages of normal porcine in vitro fertilization (IVF) embryos. We investigated the characteristics of spontaneous and induced apoptosis during preimplantation development stages of porcine IVF embryos. In experiment 1, to induce apoptosis of porcine IVF embryos, porcine IVF embryos at 22h post insemination were treated at different concentration of actinomycin D (0, 5, 50 and 500 ng/ml in NCSU medium). Treated embryos were incubated at $39^{\circ}C$ in 5% $CO_2$, 5% $O_2$ for 8h, and then washed to NCSU medium and incubated until blastocyst (BL) stage. We examined cleavage rate at 2days and BL development rate at 7days after in vitro culture. A significantly lower rate of cleavage was found in the 500 ng/ml group compared to others (500 ng/ml vs. 0, 5, 50 ng/ml; 27.8 % vs. 50.0%, 41.2%, 35.9%), and BL formation rate in 500 ng/ml was lower than that of others (500 ng/ml vs. 0, 5, 50 ng/ml; 8.0% vs. 12.6%, 11.2%, 12.6%). In experiment 2, to evaluate apoptotic cells, we conducted TUNEL assay based on morphological assessment of nuclei and on detection of specific DNA degradation under fluorescence microscope. This result showed that apoptosis is a normal event during preimplantation development in control group (0 ng/ml actinomycin D). A high number of BL derived control group contained at least one apoptotic cell. Actinomycin D treated BLs responded to the presence of apoptotic inductor by significant decrease in the average number of blastomeres and increase in the incidence of apoptotic cell death. In 500 ng/ml group, the incidence of apoptosis increased at 4-cell stage and later. This result suggested that apoptosis is a process of normal embryonic development and actinomycin D is useful tool for the apoptosis study of porcine preimplantation embryos.

• Journal of Embryo Transfer
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• v.32 no.4
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• pp.337-342
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• 2017

The present study was aimed to determine the effects of green tea extract (GTE) and beta-mercaptoethanol (${\beta}-ME$) supplementation in boar sperm freezing extender on in vitro fertilization (IVF) and reactive oxygen species (ROS) and glutathione (GSH) levels of presumptive zygotes (PZs). Experimental groups were allocated into lactose egg yolk (LEY) without antioxidant (control), GTE (1,000 mg/l in LEY) and ${\beta}-ME$ ($50{\mu}M$ in LEY). In freezing, spermatozoa extended with LEY were cooled to $5^{\circ}C$ for 3 h and then kept at $5^{\circ}C$ for 30 min following dilution with LEY containing 9% glycerol and 1.5% Equex STM. The final sperm concentration was $1{\times}10^8/ml$. Spermatozoa were loaded into straws and frozen in nitrogen vapor for 20 min. For IVF, oocytes were matured in NCSU-23 medium and co-cultured with spermatozoa following thawing at $37^{\circ}C$ for 25 sec. At 12 h following IVF, IVF parameters (sperm penetration and monospermy) were evaluated. In addition, GSH and ROS levels of PZs were determined by Cell Tracker Blue CMF2HC and DCHFDA, respectively. IVF parameters did not show any significant difference among the experimental groups. GSH and ROS levels of PZs were not significantly different between groups. In conclusion, antioxidant supplementation in boar sperm freezing could not influence IVF parameters, ROS and GSH levels of PZs.

• Journal of Embryo Transfer
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• v.5 no.2
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• pp.11-22
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• 1990

Remarkable progress has recently been made in embryo transfer technology, resulting in the birth of IVF and nuclear transfer offsprings in swine. However, further progress of the technology to (I) make a safe, effective and economic estrual-cycle synchronization compound, (2) regulate each step of sperm capacitation (3) induce monospermic fertilization, (4) in vitro grow and mature oocytes, (5) fertilize the oocytes efficently, (6) culture the oocytes to the blastocyst stage in defined media, (7) produce multiply copies of embryos with superior genetic merit, (8) preselect the sex of these superior offsprings, and (9) preserve embryos by freezing and storage in liquid nitrogen is required before this promising technology is applied routinely to swine for practical use.

• Reproductive and Developmental Biology
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• v.29 no.3
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• pp.181-186
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• 2005

This experiment was conducted to investigate effects of the two different in vitro production systems, serumcontaining system (IVM, IVF and IVC; TCM199, TALP and CR1aa) and serum-free system (IVM, IVF and IVC; IVMD101, IVMD100 and IVMD101), on the development of in vitro fertilized or DNA-microiniected embryos. We also examined the effect of DNA dosage and its expression pattern in embryos. The DNA used for microinjection was a green fluorescence protein gene. The development rates to $\geq$ 2cell, 8cell and blastocyst stage were significantly higher in vitro fertilized embryos than those in DNA-microinjected embryos. The development rate to the 8-cell stage was significantly higher in serum-free system than in serum-containing system (p<0.05; $3.3\%\;vs.\;15.5\%\;and\;21.4\%$, respectively). The development rates to the blastocyst stage of in vitro fertilzed or DNA-microinjected embryos between two different culture system ($2.7\%\;vs.\;2.3\%\;and\;23.0\%\;vs.\;23.6\%$, respectively) were not different. The development rates of embryos injected 2 ng/uL DNA was higher. than those of embryos injected 4 or 8 ng/uL DNA. The GFP expression rate of 1-cell embryos was significantly higher than that of 2-cell and 4-cell embryos, whereas the rates were not different between 4-cell and blastocyst-stage embryos.

• Korean Journal of Animal Reproduction
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• v.18 no.1
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• pp.7-13
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• 1994

To verify in vivo viability of IVF-derived bovine embryos, morula and blastocysts that developed from in vitro matured and fertilized ova were transferred to the uteri of recipient cows and normal calves were produced. To produce IVF-derived bovine morula or blastocysts, ova matured and fertilized in vitro were cultured in culture medium for 7~8 days at 39$^{\circ}C$ under the humicified atmosphere of 5% CO2. Two different culture systems, a co-culture system with TCM-199 and bovine epithelial cells (BOEC) and CR1aa without somatic cell support, were compared. Cleavage rates to 2~8 cell stage and developmental rates of IVF-derived bovine embryos to blastocyst stage were not different between co-culture system (51.3 and 14.0%) and CR1aa medium (60.4 and 22.1%), respectively. Embryos were classified into three grades by embryo quality and then one or two embryos in higher quality(A and B grades) were transferred to the uterus of recipients. In this study Korean Native calf was first born after transfer of IVF-derived embryos. Total four live calves were normally developed to term from IVF-derived bovine blastocysts and one female fetus was still-born approximatedly 8 months of gestation, but there was no pregnancy after transfer of morula. Therefore, normal calves could be produced after transfer of IVF-derived bovine embryos cultured in CR1aa medium without somatic cell support. In addition, our results suggest that in transfer of IVF-derived bovine embryos blastocyst stage is better than morula.

• Korean Journal of Animal Reproduction
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• v.26 no.3
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• pp.223-228
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• 2002

This study was carried out to examine the effects of antioxidants and buthionine sulfoximide(BSO) on the development of porcine in vitro maturation(IVM) and in vitro fertilization(IVF) oocytes. Cumulus cell free embryos derived from porcine IVM/IVF oocytes were cultured NCSU23 medium with antioxidants or BSO under an atmosphere of 5% $CO_2$ and 5% $O_2$at 38.5$^{\circ}C$ for 5~6 days. The embryos cultured in medium with BSO showed a significanthy(P<0.05) lower rates(8.4~15.7%) of the development to the morulae and blastocyst stage than control group(35.9%). When the embryos were cultured with NAC, ebselen, glutathione and BSO, the proportions of embryos beyond morulae and blastocysts were significantly(P<0.05) higher in medium with NAC(40.5%), ebselen(44.2%) and glutathione(36.0%) than BSO(10.9%). In conclusions, these results indicate that NAC, ebselen and glutathione as a antioxidants can increase the proportion of embryos that develop beyond morulae stage. BSO, intracellular glutathione inhibitors, is suppressed the development of porcine embryos derived from IVM/IVF.

• Reproductive and Developmental Biology
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• v.28 no.2
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• pp.113-117
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• 2004

The present study was carried out to examine the effects of $O_2$ concentrations and culture media (North Carolina State University (NCSU)-23, porcine zygote medium(PZM)-3 or PZM-4) on in vitro development of porcine IVM/IVF embryos. Porcine oocyte-cumulus complexes were cultured in BSA-free NCSU-23 medium containing porcine follicular fluid (10%), cysteine (0.9 mM), $\beta$-mercaptoethanol (25 $\mu\textrm{g}$/$m\ell$), epidermal growth factor (10 ng/$m\ell$) and hormonal supplements (PMSG and hCG: 10 IU/$m\ell$) for 20∼22 h. They were then cultured in the same medium but without hormonal supplements for an additional 20∼22 h. After culture, cumulus-free oocyte were coincubated with liquid boar spermatozoa for 5∼6h. Putative zygotes were transferred to NCSU-23, PZM-3 and PZM-4 medium under the condition of 5% $O_2$ or 20% $O_2$ concentrations. At 48 h, no mean differences were found in cleavage rates. However, the rates of blastocyst formation at day 7 after in vitro fertilization were significantly higher in PZM-3 medium under the condition of 5% $O_2$ concentration than other treatments (19.9$\pm$2.4 vs. 11.1$\pm$2.0 to 16.0$\pm$2.5%, P<0.05). The total cell numbers of blastocysts were significantly higher in 5% $O_2$ than in 20% O2 (P<0.05). However, no differences was found among the culture media within each $O_2$ concentrations. In conclusion, the use of PZM-3 medium in 5% $O_2$ concentration was effective on in vitro development of porcine IVM/IVF embryos.

• Journal of Embryo Transfer
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• v.18 no.3
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• pp.237-242
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• 2003

These studies were carried out to establish an effective in vitro embryo transfer methods by analyzing several factors. The base media were TCM-199 solution for in vitro maturation(IVM) of bovine follicular oocytes and Fer-TALP solution for in vitro fertilization(IVF) and CRlaa medium for in vitro culture(IVC). IVC used the fertilized oocytes of 24-hr culture (day 1)after IVF. Embryos were cultured in drop-culture that contained 25 embryos per 10${mu}ell$. Blastocysts cultured for 7 to 9 in vitro were transferred to recipients. The results obtained were as follows: 1. The pregnancy rate according to different region of embryo transfer were 33.8％, 48.1％, 45.0％ and 35.3％ respectively. 2. The pregnancy rate according to the parity of recipient when embryos were transferred to nulliparous (42.9％) was higher than that of 1∼3nd parlous(36.9％), however there were not show significant difference each other. 3. According to the stage of blastocyst, the pregnancy rate when middle blastocysts (MB) (45.5％) were transferred to recipients were higher than that of late blastocysts (LB) (41.0％). 4. When IVF-derived blastocysts cultured for 7 to 9 day were transferred to recipients, the pregnancy rate was higher 7 day of blastocyst than that of 8 day or 9 day of blastocyst. The results of embryo transfer according to the regions, the parity of recipient and the development stage showed that blastocyst formed for 7day transferred to nulliparou were higher pregnancy rate than others.

• Korean Journal of Animal Reproduction
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• v.18 no.4
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• pp.257-263
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• 1995

소 체외수정란의 체외배양 체계는 현재 완전히 확립되지는 않은 상태로서 8∼16 세포기 발육억제현상, 수정란의 파편하 현상, 성장지연 등 여러 가지 문제점들이 현 배양체계에서 나타난다. 그러나 hepler cell들과의 공배양에 의해 이러한 문제점들은 상당히 극복되며 또한 체외발생의 촉진 및 공배양된 수정란의 이식에 의해 임신율도 향상된다. 현재 소 체외수정란의 공배양에는 난관상피세포가 가장 널리 이용되나 이러한 시스템은 몇가지 문제점이 있다. 즉, primary culture를 확보하기 위하여 신선한 조직을 주기적으로 채취해야 하며 따라서 난관채취에 시간이 소요되고, 불편하며 또한 공배양에 이용되는 세포들이 균일하지 않은바 난관상피세포에 따라 배 발생 촉진작용에 변이를 나타내기도 한다. 그러나 확립된 cell line을 이용할 수 있다면 이러한 난관상피세포의 이용에서 나타나는 문제점들이 해결될 수 있다. Buffalo Rat Liver cell은 이러한 목적에 이용될 수 있는 cell line 중의 하나로서 이들은 여러 가지 성장인자(growth factor)를 분비하는 것으로 알려져 있다. 따라서 본고에서는 소 체외수정란의 공배양을 위한 BRL(Buffalo Rat Liver)cell의 이용성, 이용방법 및 이용에 있어서 고려하여야 할 요인들에 대하여 살펴보고자 한다.

• Journal of Embryo Transfer
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• v.11 no.3
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• pp.201-210
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• 1996

This experiment was carried out to evaluate the effect of early mouse embryonic development in vitro by co-culture with bovine and porcine oviductal epithelial cells (BOEC and POEC). The 2-cell embryos were collected from the oviducts of the superovulated and mated cultured in D-PBS /15% FCS at 48 hours after hCG injection. The in vitro developmental rate of blastocyst formation in the embryos were examined under the fllowing treatments; 1) TCM 199 added 15% HCS, 2) Ham's F-10 added 15% HCS, 3) MediCult IVF medium, 4) TCM 199 added 15% HCS + BOEC, 5) TCM 199 added 15% HCS + POEC, 6) Ham's F40 added 15% HCS + BOEC, 7) Ham's F-10 added 15% HCS + POEC,8) MediCult IVF medium + BOEC, 9) MediCult IVF medium + POEC. For a comparative study of in vitro development for 96 hours after hCG injection, were cultured with oviductal epithelial cell and media only. The obtained results were 2-cell embryos developed to the blastocyst stage in TCM 199, Ham's F-10 and MediCult IVF medium at the rates of 84.4,83.2 and 81.6%. respectively. The higher developmental rates(91~97%) of blastocyst formation was appeared when the embryos were co-cultured with a monolayer of bovine or porcine oviductal epithelial cells in TCM 199 or Ham's F-10 and MediCult IVF media. No significant difference in developmental rates was shown between bovine and porcine oviductal epithelial cells but significant difference in co-culture system in comparison between media only system and co-cultures. In conclusions, oviductal epithelial cells, BOEC and POEC, when co-culture with mouse early embryos improved the rates of development, blastocyst and hatching. Therefore, it is suggested that co-culture system using oviductal epithelial cells improve early embryonic developtnent in mouse.