• Korean Journal of Poultry Science
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• v.28 no.2
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• pp.125-133
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• 2001

The goal of this paper was to examine the qualify zygote-acquiring method for in-vitro culture and the in-vitro culture method of the acquired zygote from a technological perspective. We have reported the results on the introduction of foreign DNAs using the described culturing method. After performing in-vitro and surrogate eggshell culture on a zygote acquired from the abdomen of a hen, 25.8% hatchability was acquired. After microinjecting foreign DNAs into the acquired zygote and performing in-vitro and surrogate eggshell culture using the same method, 13.1∼11.7% hatchability was acquired. Having compared the developments of the control subjects and the experimental subjects, the viability of the experimental subjects on the 4∼5th day of culturing was much lower compared to that of the control subjects. This is a result that shows that the microinjection process of foreign DNAs might have a negative effect on the existence of the embryo; therefore, various technical attempts should be made to minimize such negative effects. Having microinjected foreign DNAs into the zygote of a hen to produce transgenic chickens, 3 transgenic founders were Produced and 70 G1 progeny were produced as a result of the progeny test that had been performed to the present.

• Journal of Embryo Transfer
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• v.27 no.2
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• pp.71-80
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• 2012

Biotechnologies for cloning animals and in vitro embryo production have the potential to produce biomedical models for various researches. Especially, pigs are a suitable model for xenotransplantation, transgenic production and various areas of reproductive research due to its physiological similarities to human. However, utilization of in vitro-produced embryos for transfer remains limited. Despite improvement over past few decades, obstacles associated with the production of good quality embryos in vitro still exist which limit the efficiency of cloning. One of major problems includes improper in vitro maturation (IVM) and culture (IVC). Oxidative stress caused from in vitro culture conditions contributes to inadequate IVM and IVC which leads to poor developmental competence of oocytes, failure of fertilization and embryo development. To reduce the oxidative stress, various antioxidants have been used to IVM and IVC system. However, limited information is available on the effects of resveratrol on livestock reproductions. Resveratrol is a polyphenolic natural product and well known as an antioxidant in foods and beverages (e.g. in grapes and red wine). Resveratrol is known to be cardioprotective, anticarcinogenic, anti-inflammatory, antioxidant and antiapoptotic. This paper will review the effects of resveratrol on in vitro maturation of oocytes and embryo development.

• Reproductive and Developmental Biology
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• v.38 no.1
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• pp.41-52
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• 2014

Embryonic genome activation (EGA) is a highly complex phenomenon that is controlled at various levels. New studies have ascertained some molecular mechanisms that control EGA in several species; it is apparent that these same mechanisms regulate EGA in all species. Protein phosphorylation, DNA methylation and histone modification regulate transcriptional activities, and mechanisms such as ubiquitination, SUMOylation and microRNAs post-transcriptionally regulate development. Each of these regulations is highly dynamic in the early embryo. A better understanding of these regulatory strategies can provide the possibility to improve the reproductive properties in mammals such as pigs, to develop methods of generating high-quality embryos in vitro, and to find markers for selecting developmentally competent embryos.

• Proceedings of the Botanical Society of Korea Conference
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• 1999.07a
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• pp.75-78
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• 1999

To obtain direct evidence for thed involvement of C-AB13, a carrot (Daucus carota L.) homolog of VPI/Ab13, seed-specific transcription factor, in the acquisition of desiccation tolerance carrot non-embryogenic cells (NC) in which the C-AB13 gene was expressed ectopically was prepared. Non-transgenic NC, in which expression of C-AB13 was not detected, did not exhibit desiccation tolerance even after treatment with abscisic acid (ABA). In transgenic NC that expressed C-AB13, embryo-specific ABA-inducible genes (ECP genes) were expressed upon ABA-treatment. Furthermore, the transgenic NC became desiccation-tolerant upon ABA-treatment, but not tolerate desiccation without ABA-treatment. These results provide direct evidence for the involvement of C-AB13 in the ABA-induced acquisition of desiccation tolerance.

• Reproductive and Developmental Biology
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• v.31 no.4
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• pp.261-265
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• 2007

Insulin, transferrin and selenium (ITS) complex is reported to improve in vitro development of oocytes and embryos. This study was carried out to investigate the effects of ITS during in vitro culture (IVC) of porcine parthenogenetic and nuclear transfer (NT) embryos on subsequent developmental capacity in vitro. The electrically activated oocytes were cultured in Porcine Zygote Medium (PZM-3) with various concentrations (0, 0.1, 0.5, and 1.0%) of ITS for 7 days. Also, the electrically activated reconstructed embryos were cultured in PZM-3 with various concentrations (0, 0.1, 0.5, and 1.0%) of ITS for 6 days. Addition of ITS to culture medium did not affect development of porcine parthenogenetic embryos in vitro. To test the effect of ITS on the in vitro development of porcine NT embryos, factorial experiments were also performed for in vitro maturation (IVM) medium (TCM-199) with or without 1% ITS and culture medium (PZM-3) with or without 0.5% ITS. Addition of 0.5% ITS to culture medium increased (p<0.05) the proportion of NT blastocysts compared with non-treated group. In contrast, addition of 1% ITS to culture medium was ineffective or had a detrimental effect. Also, addition of ITS only to maturation medium increased (p<0.05) the percentage of NT blastocysts formation compared with the control group. In conclusion, addition of ITS to IVM or IVC medium could improve subsequent blastocyst development of porcine NT embryos.

• Korean Journal of Poultry Science
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• v.42 no.3
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• pp.239-245
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• 2015

Transgenic animals have been widely used for developmental biology studies, as disease models, and even in industry such as transgenic bioreactor animals. For transgenic birds, quail has the great advantages of small body size, short generation time, and frequent egg production. To date, retroviral or lentiviral transduction has been used to generate transgenic quail for various purposes. However, the efficiency of transgenic offspring production with these methods is relatively low and viral vector usage has safety issues. Unfortunately, non-viral transgenesis has not been established in quail due to a deficiency of stem cell and germ cell culture systems. In this study, we established a direct in ovo lipofection method that could be used to create transgenic quail without germline-competent cells or viruses. To optimize the injection stage during embryo development, the liposome complex (containing piggyBacCMV-GFP and transposase plasmids) was introduced into an embryonic blood vessel at 50 hr, 55 hr or 60 hr. GFP expression was detected in various tissues (heart, kidney, liver and stomach) on day 12 of incubation under a fluorescence microscope. Additionally, GFP-positive cells were detected in the recipient embryonic gonads. In conclusion, the direct in ovo lipofection method with the piggyBac transposon could be an efficient and useful tool for generating transgenic quail.

• Asian-Australasian Journal of Animal Sciences
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• v.8 no.6
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• pp.557-562
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• 1995

In this study, characteristics of chick primordial germ cells (PGCs), which is the founder cell of the germline, and gonadal development of the chick embryo between 12hrs and 6 day of incubation were investigated by transverse serial sections of chick embryos under the light microscopic observation. In embryo stage 20 (3 day of incubation), there are a lot of PGCs at the mesenchym, which were moving to the thickened epithelium (gonadal ridge). The PGCs arrive at both right and left gonad primordial in equal number prior to stage 24 (4 day of incubation), but in the following stages, the distribution of the PGCs became asymmetrical. More PGCs colonized the left than the right gonad, but the reason for the unequal distribution of PGCs is uncertain. The PGCs have mostly settled in the gonadal ridge (GR) at 6 day embryo. This study was conducted to investigate characteristics of the PGC migration and gonadal formation and observe the best condition for PGC isolation, culture and to attempt the possibility of the production for transgenic germline chimeras with manipulated PGCs.

• Asian-Australasian Journal of Animal Sciences
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• v.17 no.8
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• pp.1076-1079
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• 2004

Antibiotics are common additives in culture media during in vitro embryo development, but their effects on oocyte maturation in vitro have not been tested. The effects of penicillin, streptomycin and gentamicin on the maturational competence and subsequent development potential of goat follicular oocytes were examined after parthenogenetic activation in vitro. Maturation rates at 24 h after in vitro maturation, and parthenogenetic development at 48 h after activation, were evaluated by observing the protruding first polar body and the 4 cell stage cleavage, respectively. When streptomycin was present in the maturation medium, the percentages of matured oocytes 24 h after activation were significantly (p<0.01) lower than those from the other groups (42.5-45.7% vs. 69.1-73.8%). Penicillin and gentamicin treatment did not affect the maturation rates or the percentages reaching the 4 cell stage 48 h after activation. There was no significant difference in cleavage rates among the different antibiotic treatments 48 h after activation. Therefore, streptomycin suppresses the in vitro maturation of immature goat oocytes, but does not influence their subsequent development.

• Asian-Australasian Journal of Animal Sciences
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• v.13 no.6
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• pp.856-860
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• 2000

Cloning technology continues to capture widespread attention by the international news media and biomedical and agricultural industries. The future uses of this technology could potentially contribute to major advances in biomedical and agricultural sciences. Cloned transgenic dairy cattle possessing milk promoters directing transgenes will produce pharmaceutical proteins in their milk faster, more efficiently and less expensively than transgenic cattle created using microinjection techniques. Additionally, cloned transgenic fetuses and animals may become a source of cells, tissue and organs for xenotransplantation. Lastly, but maybe most importantly, enhanced production traits and disease resistance may be realized in animal agriculture by utilizing these new technologies. The recent advances in the cattle cloning technology are important but there are still major obstacles preventing widespread commercial use of this technology. The type of donor nucleus, recipient cytoplasm, and cloning procedures used will impact the potential number of clones produced and the uses of the technology. In addition, the new advances in cloning methodology have not improved the relatively low pregnancy rates or reduced the incidence of health problems observed in cloned offspring. These problems may require novel techniques to decipher their cause and new methods of preventing and/or diagnosing them in the preimplantation embryo. The commercial potential is enormous for cloning technology; however, little has been done to improve the efficiencies of the procedure. Improving procedural efficiencies is a critical developmental milestone especially for potential uses of cloning technology in animal agriculture.

• Journal of Embryo Transfer
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• v.13 no.2
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• pp.97-106
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• 1998

The pronuclear injection of metallothionein-human growth hormone (MT-hGH) gene into rabbit zygotes was performed to establish in vitro developmental system and to detect the presence of the injected gene by nested PCR. Mature female New Zealand White rabbits were superovulated by eGG and hCG treatments. The rabbits were mated and the zygotes were collected from the oviducts 18-22 h after hCG injection by flushing with D-PBS. Two to three picoliters of MT-hGH gene was microinjected into male pronuclei. The foreign gene-injected zygotes were cultured in TCM-199 or RD mediurn containing 10% FCS with a monolayer of rabbit oviductal epithelial cefls in a 5% $CO_2$ incubator. The presence of injected DNA in rabbit embryos or blastomeres at different developmental stages .vas detected by a nested PCR analysis. The results are summarized as follows ; 1.The developmental rate of the MT-hGH gene-injected zygotes to blastocyst was significantly higher in TCM-199 medium (68.1%) than in RD medium (42.9%). 2.The gene injection into pronuclei at 18 or 22 hours post hCG treatment during pronuclear stage did not much affect on the in vitro development of the rabbit embryos. 3.The rate of gene-positive embryos detected by the nested PCR analysis was significantly decreased when they developed to blastocysts. The results indicate that the screening of transgene in rabbit embryos by nested PCR analysis could be a prornisible method for the preselection of transgenic embryos. Furthermore, the preselection of transgenic embryos would greatly reduce hoth the cost and effort of production of transgenic animals.