• Development and Reproduction
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• v.21 no.2
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• pp.205-213
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• 2017

The aim of this study was to determine the effect of additional alpha-linolenic acid (ALA) supplementation during in vitro maturation (IVM) and culture (IVC) on nucleic maturation and embryo development of pigs. Cumulus-oocyte complexes (COCs) were incubated in IVM medium containing different concentration of ALA (25, 50 and $100{\mu}M$) for 44 h. After in vitro maturation, nuclear maturation of oocytes were evaluated by aceto-orcein stain. Mature oocytes with $50{\mu}M$ ALA were fertilized and cultured in IVC medium with ALA (25, 50 and $100{\mu}M$) during early-embryogenesis (48 hours after fertilization). Then, embryos were cultured with $25{\mu}M$ ALA during early embryogenesis and/or late embryogenesis (120 hours after early-embryogenesis). In results, oocyte maturation were significantly increased by $50{\mu}M$ ALA treatment groups compared with control groups (p<0.05). Treatment of $25{\mu}M$ ALA during early-embryogenesis enhanced cleavage rate of embryo compared with other groups (p<0.05), whereas formation and total cell number of blastocyst had no significant difference. Similarly, cleavage rate of embryos were increased by $25{\mu}M$ ALA supplement during early- or late-embryogenesis than ALA treatment both stage of embryogenesis (p<0.05), but did not influence to blastocyst formation. Interestingly, total cell number of blastocyst were enhanced in ALA treatment group during early-embryogenesis. These findings indicated that ALA supplement enhance the nuclear maturation of oocyte and embryo development, however, excessive ALA could negatively influence. Therefore, we suggest that ALA is used for improvement of in vitro production of mammalian embryo and further study regarding with functional mechanism of ALA is needed.

• Journal of Embryo Transfer
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• v.18 no.2
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• pp.109-113
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• 2003

The study was carried out to investigate the effects of morphology, preservation and reproductive cycle of oocytes in vitro maturation of cats oocytes and development of IVM embryos. The results were summarized as follows : 1. Nuclear status of GV and MI of in vitro cultured(24 h) oocytes with and whithout cumulus cells were 74.3% and 25.7%, 28.6% and 11.4%, 77.1% and 5.7%, respectively, The rate of oocytes with cumulus cells was higher than that of denuded oocytes. 2. Nuclear status of GV and MI of in vitro cultured(24 h) oocytes recovered from ovaries collected at different stages of the reproductive cycle(inactive, follicular and luteal) were 88.6% and 6.5%, 60.0% and 11.4%, 77.1% and 5.7%, respectively. 3. Nuclear status of fresh and salts-stored oocytes with and whithout cumulus cells were 74.3%, 25.7% and 37.1%, 11.4% and 57.1%, 13.3%, 17.1%, 3.3%, respectively. The rate of oocytes with cumulus cells(13.3%∼74.3%) was higher than that of denuded oocytes(3.3%∼57.1%).

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2002.11a
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• pp.73-73
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• 2002

Nuclear transfer (NT) techniques have advanced in the last years, and cloned animals have been produced by using somatic cells in several species including pig. However, it is difficult that the nuclear transfer porcine embryos development to blastocyst stage overcoming the cell block in vitro. Abnormal segregation of chromosomes in nuclear transferred embryos on genome activation stage bring about embryo degeneration, abnormal blastocyst, delayed and low embryo development. Thus, we are evaluated that the correlations of the frequency of embryo developmental rates and chromosome aberration in NT and In viかo fertilization (IVF) derived embryo. We are used for ear-skin-fibroblast cell in NT. If only karyotyping of embryonic cells are chromosomally abnormal, they may difficultly remain undetected. Then, we evaluate the chromosome aberrations, fluorescent in situ hybridization (FISH) with porcine chromosome 1 submetacentric specific DNA probe were excuted. In normal diploid cell nucleus, two hybridization signal was detected. In contrast, abnormal cell figured one or three over signals. The developmental rates of NT and IVF embryos were 55% vs 63%, 32% vs 33% and 13% vs 17% in 2 cell, 8 cell and blastocyst, respectively. When looking at the types of chromosome aberration, the detection of aneuploidy at Day 3 on the embryo culture. The percentage of chromosome aneuploidy of NT and IVF at 4-cell stage 40.0%, 31.3%, respectively. This result indicate that chromosomal abnormalities are associated with low developmental rate in porcine NT embryo. It is also suggest that abnormal porcine embryos produced by NT associated with lower implantation rate, increase abortion rate and production of abnormal fetuses.

• Korean Journal of Animal Reproduction
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• v.24 no.3
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• pp.289-297
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• 2000

This study was conducted to investigate whether various protein sources and co-culture affect in vitro development of porcine zygotes derived from In vitro maturation/fertilization (IVM/IVF). These results obtained in these experiments are summarized as follows 1. When porcine oocytes matured and fertilized In vitro were cultured in NCSU 23 medium supplemented with various BSA concentrations (0.4, 0.8 and 3.2%), In vitro developmental rates of porcine zygotes to blastocyst stage were 22.9, 18.4 and 14.6%, respectively. High concentration of BSA (3.2%) showed a smaller nuclei number (36.1$\pm$11.8) of blastocysts than 0.4 and 0.8% BSA groups (53.2$\pm$27.4 and 61.2$\pm$22.5, respectively) (P<0.05). This result indicates that high concentration of BSA is detrimental on preimplantation development of IVF-derived porcine embryos. 2. No differences were detected in the developmental rate and mean nuclei number of porcine embryos between 10 and 20% FBS concentrations in culture medium. 3. IVF-derived porcine embryos co-cultured with mouse or porcine embryonic fibroblast cells showed a lower development to the blastocyst stage than those without co-culture system. Consequently, the present study suggests that high concentration of BSA as a protein source in culture medium suppresses development potential of porcine embryos produced In vitro. In addition, co-culture with somatic cells is not effective on in vitro development of IVF-derived porcine embryos to blastocyst stage.

• Journal of Veterinary Clinics
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• v.13 no.2
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• pp.149-152
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• 1996

This study was carried out to evaluate the efficiency of production of cloned embryos by nuclear translatation (NT) when using 4-cell to compact morula stage embryos as nuclear donor. In micromanitulation and electrofusion of blastomeres from 4-cell to morula stage embryos, the successful injection rate was higher with late stage blastomeres, on the contrary the fusion rate was lower. The in vitro developmental rate of NT embryos was not significantly different between cell-stages of donor blastomeres. Although the overall rate of production of cloned embryos with 4-cell. 8-cell, early and late morula stage embryos was 14.0, 18.0, 15.3 and 14.1%, respectively, the mean number of blastocysts produced with a donor embryo was the most (4.51) with the compact morulae. Therefore, it can be suggested that the embryos at thelate stage is more beneficial for the mulciple production of cloned embryos, If the late stage blastomeres have maintained their totipotency to produce intact offspring.

• Journal of Embryo Transfer
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• v.9 no.1
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• pp.23-29
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• 1994

The long term goal of this research is to develop an efficient procedure for large scale production of genetically identical or cloned animals. To improve nuclear transpalntation efficiency in the rabbit, this study evaluated the age of nuclear recipient oocytes on the different steps of nuclear transplantation. The ovulated oocytes in different ages were collected from the superovulated does by flushing oviducts with Dulbecco's phosphate buffered saline(D-PBS) supplemented with 10% fetal calf serum(FCS) from 13 to 15, 17 to 20 and 23 to 26 hours after hCG injection. The denuded oocytes were used as nuclear recipient cytoplasm following enucleation by micromanipulation. The blastomeres separated from the 8-cell embryos were used as nuclear donor. The enucleated oocytes receiving a blastomere in the perivitteline space were fused in the 0.28 M mannitol solution at 1.5 kV/cm, 60 sec for three times. The fused oocytes were co-cultured with the monolayered rabbit oviductal epithelial cells in TGM-199 solution with 10% FCS for 72 hours at 37$^{\circ}C$ in a 5% $CO_2$ incubator. The cultured nuclear transplant embryos and in vivo developed embryos collected at 72 hours after hCG injection were stained with Hoechst 33342 dye. Their cell numbers were counted under a fluorescent microscope. The results obtained were summarized as follows ; 1. The aged oocytes(20 hrs. post hCG) showed significantly(P<0.05) higher fusionrates(70 ~ 90%) than the recently ovulated oocytes(30.8%) 2. The aged oocytes which were electrically activated and fused at 20 hours developed to blastocyst at significantly(P<0.05) high rate, while none of the recently ovulated oocytes developed to blastocyst. 3. Even though the aged oocytes at 23~26 hours showed higher fusion rate(85.7%), not only they were inadequate to manipulate but also their developmental potential to blastocyst was highly impaired. 4. The developmental potential in vitro of nuclear transplant embryos was significantly retarded than in vivo deveolped embryos.

• Korean Journal of Animal Reproduction
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• v.26 no.4
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• pp.385-394
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• 2002

This study was constructed the correlations of the embryonic developmental rates and the frequency of chromosome aberration using ear-skin-fibroblast cell in nuclear transfer (NT) derived embryos. Karyoplast-oocyte complexes were fused and activated simultaneously, then cultured for seven days to assess development. The developmental rates of NT and in vitro fertilization (IVF) embryos were 55.4% vs 63.5%, 31.7% vs 33% and 13.4% vs 16.8% in 2 cell, 8 cell and blastocyst, respectively. Firstly, the frequency of chromosome aberrations were evaluated using fluorescent in situ hybridization (FISH) technique with porcine chromosome 1 submetacentric specific probe. Chromosome aberration was detected at day 3 on the embryo culture, the percentages of chromosomal aneuploidy in NT and IVF embryos at 4-cell stage were 40%, 31.3%, respectively. Secondly, embryonic fragmentation was evaluated at 4-cell stage embryo. Frequency of embryonic fragmentations was in 51.3% of NT, 61.3% of IVF, 28.9% of parthenogenetic activation at 4-cell stage. The proportion of fragmentation in NT embryos was higher than activation embryos. This result indicates that chromosomal abnormalities and embryonic fragments are associated with low developmental rate in porcine NT embryo. It is also suggest that abnormal porcine embryos produced by NT related with lower implantation rate, increased abortion rate and production of abnormal fetuses.

• Journal of Animal Science and Technology
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• v.66 no.5
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• pp.905-919
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• 2024

Porcine oocytes undergo in vitro maturation (IVM) for 42-44 h. During this period, most oocytes proceed to metaphase and then to pro-metaphase if the nucleus has sufficiently matured. Forty-four hours is sufficient for oocyte nuclear maturation but not for full maturation of the oocyte cytoplasm. This study investigated the influences of extension of the IVM duration with rapamycin treatment on molecular maturation factors. The phospho-p44/42 mitogen-activated protein kinase (MAPK) level was enhanced in comparison with the total p44/42 MAPK level after 52 h of IVM. Oocytes were treated with and without 10 µM rapamycin (10 R and 0 R, respectively) and examined after 52 h of IVM, whereas control oocytes were examined after 44 h of IVM. Phospho-p44/42 MAPK activity was upregulated the 10 R and 0 R oocytes than in control oocytes. The expression levels of maternal genes were highest in 10 R oocytes and were higher in 0 R oocytes than in control oocytes. Reactive oxygen species (ROS) activity was dramatically increased in 0 R oocytes but was similar in 10 R and control oocytes. The 10 R group exhibited an increased embryo development rate, a higher total cell number per blastocyst, and decreased DNA fragmentation. The mRNA level of development-related (POU5F1 and NANOG) mRNA, oocyte-apoptotic (BCL2L1) genes were highest in 10 R blastocysts. These results suggest that prolonged IVM duration with rapamycin treatment represses ROS production and increases expression of molecular maturation factors. Therefore, this is a good strategy to enhance the developmental capacity in porcine oocytes.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.7-8
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• 2003

Since it was first reported in 1997, somatic cell cloning has been demonstrated in several other mammalian species. On the mouse, it can be cloned from embryonic stem (ES) cells, fetus-derived cells, and adult-derived cells, both male and female. While cloning efficiencies range from 0 to 20%, rates of just 1-2% are typical (i.e. one or two live offspring per one hundred initial embryos). Recently, abnormalities in mice cloned from somatic cells have been reported, such as abnormal gene expression in embryo (Boiani et al., 2001, Bortvin et al., 2003), abnormal placenta (Wakayama and Yanagimachi 1999), obesity (Tamashiro et ai, 2000, 2002) or early death (Ogonuki et al., 2002). Such abnormalities notwithstanding, success in generating cloned offspring has opened new avenues of investigation and provides a valuable tool that basic research scientists have employed to study complex processes such as genomic reprogramming, imprinting and embryonic development. On the other hand, mouse ES cell lines can also be generated from adult somatic cells via nuclear transfer. These 'ntES cells' are capable of differentiation into an extensive variety of cell types in vitro, as well assperm and oocytes in vivo. Interestingly, the establish rate of ntES cell line from cloned blastocyst is much higher than the success rate of cloned mouse. It is also possible to make cloned mice from ntES cell nuclei as donor, but this serial nuclear transfer method could not improved the cloning efficiency. Might be ntES cell has both character between ES cell and somatic cell. A number of potential agricultural and clinical applications are also are being explored, including the reproductive cloning of farm animals and therapeutic cloning for human cell, tissue, and organ replacement. This talk seeks to describe both the relationship between nucleus donor cell type and cloning success rate, and methods for establishing ntES cell lines. (중략)

• Korean Journal of Agricultural Science
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• v.22 no.1
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• pp.62-68
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• 1995

This study was carried out to examine splitting, developmental capacity and rapid freezing of blastomeres separated from 2-, 4-, 8-cell and morula from porcine embryos. The results obtained in this study were summerized as follows : 1. The successful splitting rate by pronase was 85.7% in 2-cell embryos(average splitting rate, 68.0%), and by manipulator was 76.6% and 74.3% in 2- and 4-cell embryos. 2. The developmental capacity rates of splitted embryos by the pronase treatment were 24.1%, 20.4%. 25.5% and 26.6% in 2-, 4-, 8-cell and morula, and by manipulator were 36.4%, 39.5%, 36.1% and 41.9%, respectively. 3. The successful results of in vitro culture after frozen-thawed of splitted embryos were 16.1%(glycerol) in 2-cell, 16.7%(DMSO) in 4-cell and 27.6%(ethyleneglycol) in morula, respectively.