• Development and Reproduction
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• v.23 no.1
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• pp.43-54
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• 2019

We examined the effects of endoplasmic reticulum (ER) stress inhibitor treatment during the micromanipulation of porcine somatic cell nuclear transfer (SCNT) on the in vitro development of SCNT embryos. ER stress inhibitors such as salubrinal (200 nM) and tauroursodeoxycholic acid (TUDCA; $100{\mu}M$) were added to the micromanipulation medium and holding medium. The expression of X-box binding protein 1 (Xbp1), ER-stress-associated genes, and apoptotic genes in SCNT embryos was confirmed at the one-cell and blastocyst stages. Levels of Xbp1 splicing and expression of ER-stress-associated genes in SCNT embryos at the one-cell stage decreased significantly with TUDCA treatment (p<0.05). The expression of ER-stress-associated genes also decreased slightly with the addition of both salubrinal and TUDCA (Sal+TUD). The expression levels of caspase-3 and Bcl2-associated X protein (Bax) mRNA were also significantly lower in the TUDCA and Sal+TUD treatments (p<0.05). At the blastocyst stage, there were no differences in levels of Xbp1 splicing, and transcription of ER-stress-associated genes and apoptosis genes between control and treatment groups. However, the blastocyst formation rate (20.2%) and mean blastocyst cell number ($63.0{\pm}7.2$) were significantly higher (p<0.05) for embryos in the TUDCA treatment compared with those for control (12.6% and $41.7{\pm}3.1$, respectively). These results indicate that the addition of ER-stress inhibitors, especially TUDCA, during micromanipulation can inhibit cellular damage and enhance in vitro development of SCNT embryos by reducing stress levels in the ER.

• Reproductive and Developmental Biology
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• v.28 no.1
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• pp.1-6
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• 2004

This study was conducted to investigate the effects of donor cell type, individual, passage number and trypsinization time on the in vitro development of bovine somatic cell nuclear transfer embryos. Three cell types (skin, muscle and cumulus cells) and cells from 3 individuals were used for nuclear transfer. Cell were passaged by 5, 15 or 30 times, and cell were trypsinized for 1 or 3 min before injection. Nuclear transfer were performed by conventional fusion method. Development rates to the blastocyst stage were not significantly different among three cell types (16.5∼23.9%) and individuals (16.4∼19.5%). Blastocyst formation rate of cloned embryos reconstituted with cells at passage 30 (5.8%) was significantly lower than those of embryos reconstituted with 5- and 15-passaged cells (25.3 and 23.5%, respectively, P＜0.05). The rate of embryos developed to the blastocyst stage was higher in embryos reconstituted with cells trypsinized for 1 min (30.7%) compared to embryos reconstituted with cells trypsinized for 3 min (P＜0.05). The result of the present study indicates that different donor cell types and individuals used in this study did not affect the development of cloned bovine embryos. However, passage number and trypsinization time of donor cells affect the in vitro development of cloned bovine embryos.

• Reproductive and Developmental Biology
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• v.32 no.4
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• pp.249-253
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• 2008

Interspecies somatic cell nuclear transfer (iSCNT) is a valuable tool for studying the interactions between an oocyte and somatic nucleus. The object of this study was to investigate the developmental competence of in vitro-matured porcine oocytes after transfer of the somatic cell nuclei of 2 different species (goat and rabbit). Porcine cumulus oocytes were obtained from the follicles of ovaries and matured in TCM-199. The reconstructed embryos were electrically fused with 2 DC pulses of 1.1kV/cm for $30{\mu}s$ 0.3M mannitol medium. The activated cloned embryos were cultured in porcine zygote medium-3 (PZM-3), mSOF or RDH medium for 7 days. The blastocyst formation rate of the embryos reconstructed from goat or rabbit fetal fibroblasts was significantly lower than that of the embryos reconstructed from porcine fetal fibroblast cells. However, a significantly higher number of embryos reconstructed from goat or rabbit fetal fibroblasts cultured in mSOF or RDH, respectively, developed to the morular stage than those cultured in PZM-3. These results suggest that goat and bovine fetal fibroblasts were less efficacious than porcine-porcine cloned embryos and that culture condition could be an important factor in iSCNT. The lower developmental potential of goat-porcine and porcine-bovine cloned embryos may be due to incompatibility between the porcine oocyte cytoplasm and goat and bovine somatic nuclei.

• Journal of Sericultural and Entomological Science
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• v.43 no.1
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• pp.53-54
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• 2001

The chromosome number of Morus bombycis Koidz. and Morus monogolica C.K.Schn. growing wild in the Korea Peninsula is diploid (2n=28) and that of Morus tiliaefolia Makino is hecxaploid (2n=84). The somatic cell division of each species is nomal.

• Reproductive and Developmental Biology
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• v.33 no.4
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• pp.223-228
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• 2009

We attempted to control the maturation promoting factor (MPF) activity and investigated the subsequent reprogramming of bovine somatic cell nuclear transfer (SCNT) embryos. Serum-starved adult skin fibroblasts were fused to enucleated oocytes treated with 2.5 mM caffeine or $150\;{\mu}M$ roscovitine. The MPF activity, nuclear remodeling patterns, chromosome constitutions and development of SCNT embryos were evaluated. Methylated DNA of embryos was detected at various developmental stages. The MPF activity was increased by caffeine treatment or reduced by roscovitine treatment (p<0.05). Blastocyst development was higher in the caffeine-treated groups (27.6%) than that of the roscovitine-treated group (8.3%, p<0.05). There was no difference in the apoptotic cell index among the three groups. However, the mean cell number of blastocysts was increased in the caffeine-treated group (p<0.05). Higher methylation levels were observed in the Day 3 embryos of the roscovitine-treated group (50.8%), whereas lower methylation levels were noted at Day 5 in the caffeine-treated group (12.5%, p<0.05). These results reveal that the increase in MPF activity via a caffeine-treatment creates a more suitable condition for nuclear reprogramming after SCNT.

• Journal of Animal Reproduction and Biotechnology
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• v.35 no.3
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• pp.215-222
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• 2020

Mesenchymal stem cells (MSCs) have been widely used as donor cells for somatic cell nuclear transfer (SCNT) to increase the efficiency of embryo cloning. Since replicative senescence reduces the efficiency of embryo cloning in MSCs during in vitro expansion, transfection of telomerase reverse transcriptase (TERT) into MSCs has been used to suppress the replicative senescence. Here, TERT-transfected MSCs in comparison with early passage MSCs (eMSCs) and sham-transfected MSCs (sMSCs) were used to evaluate the effects of embryo cloning with SCNT in a porcine model. Cloned embryos from tMSC, eMSC, and sMSC groups were indistinguishable in their fusion rate, cleavage rate, total cell number, and gene expression levels of OCT4, SOX2 and NANOG during the blastocyst stage. The blastocyst formation rates of tMSC and sMSC groups were comparable but significantly lower than that of the eMSC group (p < 0.05). In contrast, tMSC and eMSC groups demonstrated significantly reduced apoptotic incidence (p < 0.05), and decreased BAX but increased BCL2 expression in the blastocyst stage compared to the sMSC group (p < 0.05). Therefore, MSCs transfected with telomerase reverse transcriptase do not affect the overall development of the cloned embryos in porcine SCNT, but enables to maintain embryo quality, similar to apoptotic events in SCNT embryos typically achieved by an early passage MSC. This finding offers a bioengineering strategy in improving the porcine cloned embryo quality.

• Journal of Embryo Transfer
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• v.28 no.2
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• pp.133-139
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• 2013

The objective of this study was to determine the effect of post-activation treatment with cytoskeletal regulators in combination with or without 6-dimethylaminopurine (DMAP) on embryonic development of pig oocytes after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT). PA and SCNT oocytes were produced by using in vitro-matured pig oocytes and treated for 4 h after electric activation with $0.5{\mu}M$ latrunculin A (LA), $10.4{\mu}M$ cytochalasins B (CB), and $4.9{\mu}M$ cytochalasins D (CD) together with none or 2 mM DMAP. Post-activation treatment of PA oocytes with LA, CB, and CD did not alter embryo cleavage (85.8~88.6%), blastocyst formation (30.7~ 32.4%), and mean cell number of blastocysts (33.5~33.8 cells/blastocyst). When PA oocytes were treated with LA, CB, and CD in combination with DMAP, blastocyst formation was significantly (P<0.05) improved by CB+DMAP (42.5%) compared to LA+DMAP (28.0%) and CD+DMAP (25.1%), but no significant differences were found in embryo cleavage (77.5~78.0%) and mean blastocyst cell number (33.6~35.0 cells) among the three groups. In SCNT, blastocyst formation was significantly (P<0.05) increased by post-activation treatment with LA+DMAP (32.9%) and CD+DMAP (35.0%) compared to CB+DMAP (22.0%) while embryo cleavage (85.5~85.7%) and blastocyst cell number (41.1~43.8 cells) were not influenced. All three treatments (LA, CB, and CD with DMAP) effectively inhibited pseudo-polar body extrusion in SCNT oocytes. The proportions of oocytes showing single pronucleus formation were 89.6%, 83.9%, and 93.3%, respectively with the increased tendency (P<0.1) by LA+DMAP and CD+ DMAP compared to CB+DMAP. Our results demonstrate that post-activation treatment with LA or CD in combination with DMAP improves pre-implantation development of SCNT embryos and the stimulating effect of cytoskeletal modifiers on embryonic development is differentially shown depending on the origin (PA or SCNT) of embryos in pigs.

• Journal of Plant Biology
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• v.28 no.2
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• pp.141-148
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• 1985

The effects of sizes and densities of cells cultured, conditioned medium, and media pH on the somatic embryogenesis of carrot (Daucus carota L.) were examined. A large number of globular embryoids was formed after 4 days in cell culture, and later globular embryoids developed into heart and torpedo shape. High cell density resulted in higher number and better growth of embryos, especially on conditioned medium than Murashige-Skoog medium. The fresh weight and number of embryoids formed increased with the decrease in cell size. The significant reduction in fresh weight and number of embryoids was obtained when culturing cells with diameter of over 90 ${\mu}{\textrm}{m}$. Dry weight and number of embryoids were markedly reduced with medium pH of 4 or 7, but promoted with pH 6.0.

• Korean Journal of Veterinary Service
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• v.24 no.1
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• pp.89-94
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• 2001

In order to investigate the relationship between milk hygienic quality and some environmental factors such as the herd size and types of milking machines, we sampled and examined the level of total bacterial count, coliforms, Staphyococcus aureus, somatic cell counts(SCC) and fat rates in raw milk. of the 84 dairy farms, the prevalence of level on number of standard plate count over 100,000cfu/$m\ell$ and coliforms over 1,000cfu/$m\ell$ in bulk milk were 25.0% and 15.6%, respectively. Also, 2 farms(2.4%) were exceed the level on number of 500cfu/$m\ell$ S aureus in raw milk. The prevalence of dairy herd with first grade of total bacterial count(TBC) according to bucket, pipe line and parlour milking system was 40.0%, 74.0% and 84.0%, respectively. The prevalence of dairy herd with first grade of TBC according to grade 1, 2 and 3 by SCC was 77.8%, 83.2%. and 69.2%, respectively. Therefore, the relationships between hygienic quality in raw milk and the herd size, types of milking machines, were significant. In conclusion, this study could be overemphasized the importance of herd management condition for milk hygienic qualify.

• Journal of Embryo Transfer
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• v.30 no.4
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• pp.345-350
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• 2015

In the last 10 years, porcine somatic cell nuclear transfer to generate transgenic pig has been performed tremendous development with introduction and knockout of many genes. However, efficiency of porcine somatic cell nuclear transfer is still low and embryo transfer (ET) is one of important step for production efficiency. In porcine ET for production of transgenic cloned pig, we can consider many of points to increase production rates. In respect of seasonality and weather, porcine ET usually is not performed in summer and winter. Cloned transgenic embryos must be transferred into reproductive tracts of recipients where embryos are located after natural fertilization with similar estrous cycle. If cloned embryos with 2~4 cell stage are transferred, they must be transferred into oviducts in periovulatory stage. Number and deposition sites of transferred cloned embryos are important. And we must compare the methods of ET between surgical and non-surgical ones in respect of production efficiency. Sow recipients after natural estrus is most preferred recipients however its cost is must be considered. Here we will review many of current studies about porcine embryo transfer to increase production efficiency of transgenic pigs and strategies for further studies.