• Asian-Australasian Journal of Animal Sciences
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• v.20 no.5
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• pp.718-724
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• 2007

Considerable attention has been focused on the cloning of mammalian embryos, as a consequence of poor development, in order to enhance the application of genetic engineering. Experiments were conducted to compare the developmental competence of parthenotes and reconstructed (NT) rabbit eggs with fetal fibroblasts (FFs) following various activation regimens. Oocytes and NT eggs were exposed to: electric stimulation (EST, Group 1) and EST followed by 6-dimethylaminopurine (DMAP, Group 2), cycloheximide (CHX, Group 3) or DMAP/CHX (Group 4). Pronuclear (PN) status, cleavage, blastocyst development and the ploidy were assessed. In parthenote groups 1, 2, 3 and 4, the PN formation differed significantly. And, the cleavage and blastocyst rates were 41.7 and 5%, 75.6 and 53.7%, 68 and 36%, 82.1 and 52.6%, respectively, among treatments. Polyploidy was observed in 17.2% of EST plus DMAP and 44.9% of EST plus DMAP/CHX groups. In SCNT groups (Group 1, 2, 3 and 4), the cleavage and blastocyst rates were 28.6 and 7.1%, 58.3 and 29.2%, 56.8 and 24.1%, 64.5 and 27.8%, respectively. The chromosomal composition differed significantly (p<0.05) among treatments. In Group 2 and 3, 53.8% and 81.8% of embryos revealed diploid chromosomal sets, respectively. However, in Group 4, 53.3% of embryos showed abnormal ploidy (mixoploid). Although DMAP or combination with DMAP/CHX resulted in higher in vitro development of rabbit SCNT embryos, higher incidence of chromosomal abnormality may induce problems related to fetal loss of at late stage of development.

• Journal of Embryo Transfer
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• v.29 no.4
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• pp.375-383
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• 2014

Klotho (KL) is a single transmembrane protein composed of KL1 and KL2 repeats possessing ${\beta}$-glucuronidase activity and maintains calcium homeostasis in physiological state. It has been implicated in pigs that calcium is important for the establishment and maintenance of pregnancy, and our previous study has shown that transient receptor potential vanilloid type 6 (TRPV6), a calcium ion transporter, is predominantly expressed in the uterine endometrium during pregnancy in pigs. However, expression and function of KL in the uterine endometrium has not been determined in pigs. Thus, the present study determined expression and regulation of KL in the uterine endometrium during the estrous cycle and pregnancy in pigs. Real-time RT-PCR analysis showed that levels of KL mRNA decreased between Days 12 to 15 of the estrous cycle, and its expression showed a biphasic manner during pregnancy. KL mRNA was expressed in conceptuses and in chorioallantoic tissues during pregnancy. Explant culture study showed that expression levels of KL were not affected by treatment of steroid hormones or interleukin-1beta during the implantation period. Furthermore, levels of KL mRNA in the uterine endometrium from gilts carrying somatic cell nuclear transfer (SCNT)-derived embryos were significantly lower than those from gilts carrying natural mating-derived embryos on Day 12 of pregnancy. These results exhibited that KL was expressed at the maternal-conceptus interface in a pregnancy status- and stage-specific manner, and its expression was affected by SCNT procedure, suggesting that KL may play an important role in the establishment and maintenance of pregnancy in pigs.

• Reproductive and Developmental Biology
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• v.33 no.3
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• pp.157-162
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• 2009

This study was performed to comprehend the developmental characteristics of cloned embryos knocked out (KO) of $\alpha$-1,3-galactosyltransferase (GalT) gene. Immature oocytes were collected and cultured for 40 hrs (1-step) or 20hrs (with hormone) + 20hrs (without hormone) (2-step). The embryos transferred with miniature pig ear fibroblast cell were used as control. The reconstructed embryos were cultured in PZM-3 with 5% $CO_2$ in air at $38.5^{\circ}C$ for 6 days. To determine the quality of the blstocysts, TUNEL and quantitative realtime RT-PCR were performed. The embryos were transferred to a surrogate (Landrace) at an earlier stage of the estrus cycle. The maturation rate was significantly higher in 2-step method than that of 1-step (p<0.05). The blastocyst development of GalT KO embryos was significantly lower than that of normal cloned embryos (p<0.05). The total and apoptotic cell number of GalT KO blastocysts was not different statistically from control. The relative abundance of Bax-$\alpha$/Bcl-xl ratio was significantly higher in both cloned blastocysts than that of in vivo blastocysts (p<0.05). Taken together, it can be postulated that the lower developmental potential and higher expression of apoptosis related genes in GalT KO SCNT embryos might be a cause of a low efficiency of GalT KO cloned miniature pig production.

• Animal Bioscience
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• v.36 no.3
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• pp.441-450
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• 2023

Objective: Serum amyloid A3 (SAA3), an acute phase response protein, plays important roles in opsonization, antimicrobial activity, chemotactic activity, and immunomodulation, but its expression, regulation, and function at the maternal-conceptus interface in pigs are not fully understood. Therefore, we determined the expression of SAA3 in the endometrium throughout the estrous cycle and at the maternal-conceptus interface during pregnancy. Methods: Endometrial tissues from pigs at various stages of the estrous cycle and pregnancy and with conceptuses derived from somatic cell nuclear transfer (SCNT), conceptus tissues during early pregnancy, and chorioallantoic tissues during mid- to late pregnancy were obtained and the expression of SAA3 was analyzed. The effects of the steroid hormones, interleukin-1β (IL1B), and interferon-γ (IFNG) on the expression of SAA3 were determined in endometrial explant cultures. Results: SAA3 was expressed in the endometrium during the estrous cycle and pregnancy, with the highest level on day 12 of pregnancy. The expression of SAA3 in the endometrium was significantly higher on day 12 of pregnancy than during the estrous cycle. Early-stage conceptuses and chorioallantoic tissues during mid to late pregnancy also expressed SAA3. The expression of SAA3 was primarily localized to luminal epithelial cells in the endometrium. In endometrial explant cultures, the expression of SAA3 was induced by increasing doses of IL1B and IFNG. Furthermore, the expression of SAA3 decreased significantly in the endometria of pigs carrying conceptuses derived from SCNT on day 12 of pregnancy. Conclusion: These results suggest that the expression of SAA3 in the endometrium during the implantation period increases in response to conceptus-derived IL1B and IFNG. The failure of those appropriate interactions between the implanting conceptus and the endometrium leads to dysregulation of endometrial SAA3 expression, which could result in pregnancy failure. In addition, SAA3 could be a specific endometrial epithelial marker for conceptus implantation in pigs.

• Animal Bioscience
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• v.35 no.9
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• pp.1360-1366
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• 2022

Objective: The present study analyzed the influence of co-transferring embryos with high and low cloning efficiencies produced via somatic cell nuclear transfer (SCNT) on pregnancy outcomes in dogs. Methods: Cloned dogs were produced by SCNT using donor cells derived from a Tibetan Mastiff (TM) and Toy Poodle (TP). The in vivo developmental capacity of cloned embryos was evaluated. The pregnancy and parturition rates were determined following single transfer of 284 fused oocytes into 21 surrogates and co-transfer of 47 fused oocytes into four surrogates. Results: When cloned embryos produced using a single type of donor cell were transferred into surrogates, the pregnancy and live birth rates were significantly higher following transfer of embryos produced using TP donor cells than following transfer of embryos produced using TM donor cells. Next, pregnancy and live birth rates were compared following single and co-transfer of these cloned embryos. The pregnancy and live birth rates were similar upon co-transfer of embryos and single transfer of embryos produced using TP donor cells but were significantly lower upon single transfer of embryos produced using TM donor cells. Furthermore, the parturition rate for TM dogs and the percentage of these dogs that remained alive until weaning was significantly higher upon co-transfer than upon single transfer of embryos. However, there was no difference between the two embryo transfer methods for TP dogs. The mean birth weight of cloned TM dogs was significantly higher upon single transfer than upon co-transfer of embryos. However, the body weight of TM dogs did not significantly differ between the two embryo transfer methods after day 5. Conclusion: For cloned embryos with a lower developmental competence, the parturition rate and percentage of dogs that remain alive until weaning are increased when they are co-transferred with cloned embryos with a greater developmental competence.

• Journal of Embryo Transfer
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• v.32 no.3
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• pp.131-138
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• 2017

In most mammals, metaphase II (MII) oocytes having high maturation promoting factor (MPF) activity have been considered as good oocytes and then used for assisted reproductive technologies including somatic cell nuclear transfer (SCNT). Caffeine increases MPF activity in mammalian oocytes by inhibiting p34cdc2 phosphorylation. The objective of this study was to investigate the effects of caffeine treatment during in vitro maturation (IVM) on oocyte maturation and embryonic development after SCNT in pigs. To this end, morphologically good (MGCOCs) and poor oocytes (MPCOCs) based on the thickness of cumulus cell layer were untreated or treated with 2.5 mM caffeine during 22-42, 34-42, or 38-42 h of IVM according to the experimental design. Caffeine treatment for 20 h during 22-42 h of IVM significantly inhibited nuclear maturation compared to no treatment. Blastocyst formation of SCNT embryos was not influenced by the caffeine treatment during 38-42 h of IVM in MGCOCs (41.1-42.1%) but was significantly improved in MPCOCs compared to no treatment (43.4 vs. 30.1%, P<0.05). No significant effects of caffeine treatment was observed in embryo cleavage (78.7-88.0%) and mean cell number in blastocyst (38.7-43.5 cells). The MPF activity of MII oocytes in terms of p34cdc2 kinase activity was not influenced by the caffeine treatment in MGCOCs (160.4 vs. 194.3 pg/ml) but significantly increased in MPCOCs (133.9 vs. 204.8 pg/ml). Our results demonstrate that caffeine treatment during 38-42 h of IVM improves developmental competence of SCNT embryos derived from MPCOCs by influencing cytoplasmic maturation including increased MPF activity in IVM oocytes in pigs.

• Journal of Embryo Transfer
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• v.30 no.4
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• pp.305-313
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• 2015

Xenotransplantation of pig islet regarded as a good alternative to allotransplantation. However, cellular death mediated by hypoxia-reoxygenation injury after transplantation disturb success of this technique. In the present study, we produce transgenic pig expressing human heme oxygenase 1 (HO1) genes to overcome cellular death for improving efficiency of islet xenotransplantation. Particularly, Korean miniature pig breed, Micro-Pig, was used in the present study. Somatic cell nuclear transfer (SCNT) technique was used to produce the HO1 transgenic pig. Six alive transgenic piglets were produced and all the transgenic pigs were founded to have transgene in their genomic DNA and the gene was expressed in all tested organs. Also, in vitro cultured fibroblasts derived from the HO1 transgenic pig showed low reactive oxygen species level, improved cell viability and reduced apoptosis level.

• Journal of Embryo Transfer
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• v.24 no.4
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• pp.249-251
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• 2009

Polar body was usually used as a determinant of oocyte's maturation. Polar body morphology could reflect the embryo quality and implantation competence. This review only focuses on morphology of the first polar body and embryo developmental rate in the presence or absence of polar body. However, it is very difficult to describe whether polar body has any effects on embryo development in vitro or in vivo. Further intensive research is needed to determine its function on embryo development.

• Journal of Embryo Transfer
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• v.31 no.1
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• pp.97-107
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• 2016

Although many diseases could be treated by the development of modern medicine, there are some incurable diseases including brain cancer, Alzheimer disease, etc. To study human brain cancer, various animal models were reported. Among these animal models, mouse models are valuable tools for understanding brain cancer characteristics. In spite of many mouse brain cancer models, it has been difficult to find a new target molecule for the treatment of brain cancer. One of the reasons is absence of large animal model which makes conducting preclinical trials. In this article, we review a recent study of molecular characteristics of human brain cancer, their genetic mutation and comparative analysis of the mouse brain cancer model. Finally, we suggest the need for development of large animal models using somatic cell nuclear transfer in translational research.

• Journal of Embryo Transfer
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• v.20 no.2
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• pp.105-112
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• 2005

The present study was conducted to examine some factors affecting in vitro development of oocytes from somatic cell nuclear transfer (SCNT) in Korean native goats. Recipient oocytes were surgically collected after superovulation by using CIDR and FSH, PMSG, hCG and estrous synchronization in Korean Native goats. For nuclear transfer, the fibroblasts from caprine ear cells and fetal fibroblasts were surgically harvested and were cultured in vitro until cell confluency in serum-starvation condition (TCM-199 + $0.5\%$ FBS) for 3 to 5 days. The zona pellucidae of matured oocytes were partially drilled by laser irradiation. A single somatic cell was individually transferred into each enucleated oocyte. The reconstructed oocytes were then electrically fused and activated. Activated NT embryos were cultured in mSOF medium supplemented with $0.8\%\;BSA\;6\~7\;day\;at\;39^{\circ}C,\;5\%\;CO_2,\;5\%\;O_2,\;90\%\;N_2$ in air. There were no significant difference in the number of embryos cleaved and 4-cell development between the fibroblast nuclei from mature ear cells and fetal cells, but the rate of 8-cell development was higher (P<0.05) in ear cells $(40.5\%)$ than in fetal cells $(55.5\%)$. However, the embryo development to morula or blastocyst was not significantly different between both the groups$(6.7\%\;vs\;16.0\%)$, respectively. The number of embryo cleaved $(79.0\%)$ were higher (P<0.05) in the oocytes activated with ionomycin+6-DMAP than in the oocytes activated electrically $(9.5\%)$. The development of fused embryos to morula or blastocyst was found $15.6\%$ in ionomycin+6-DMAP, but no morula or blastocysts were developed in electrical stimulation. The development rate of SCNT embryos to morula or blastocyst was love. (P<0.05) in SCNT embryos $(19.0\%\;vs\;0.0\%)$ than that in parthenotes $(66.1\%\;vs\;59.1\%)$. In the parthenotes, the cleavage rate and development to morula or blastocyst were significantly higher (P<0.05) as $86.8\%\;and\;50.0\%$ in ovulated oocytes than in follicular oocytes $(69.0\%\;vs\;23.6\%)$, respectively. These results suggest that some factors Including superovulation treatment, oocyte source, maturation of follicular oocytes, activation method and culture condition may affect in vitro developmental capability of embryos produced by somatic cell nuclear transfer in Korean Native goats, and the fusion rate be greatly low compared with other species.