• Reproductive and Developmental Biology
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• v.36 no.2
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• pp.115-120
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• 2012

This study was conducted to examine the optimal concentration and treatment time of antioxidants for inhibition of the ROS generation in bovine somatic cell nuclear transfer (SCNT) embryos. Bovine oocytes were activated parthenogenetically, during which oocytes were treated with various antioxidants to determine the optimal concentrations and kind of antioxidants. Determined antioxidants were applied to oocytes during in vitro maturation (IVM) and/or SCNT procedures. Finally, antioxidant-treated SCNT embryos were compared with in vitro fertilized (IVF) embryos. $H_2O_2$ levels were analyzed in embryos at 20 h of activation, fusion or insemination by staining of embryos in $10{\mu}M$ 2'7'-dichlorodihydrofluorescein diacetate (H2DCFDA) dye, followed by fluorescence microscopy. $H_2O_2$ levels of parthenogenetic embryos were significantly lower in $25{\mu}M$ ${\beta}$-mercaptoethanol (${\beta}$-ME), $50{\mu}M$ L-ascorbic acid (Vit. C), and $50{\mu}M$ L-glutathione (GSH) treatment groups than each control group ($24.0{\pm}1.5$ vs $39.0{\pm}1.1$, $29.7{\pm}1.0$ vs $37.0{\pm}1.2$, and $32.9{\pm}0.8$ vs $36.3{\pm}0.8$ pixels/embryo, p<0.05). There were no differences among above concentration of antioxidants in direct comparison ($33.6{\pm}0.9{\sim}35.2{\pm}1.1$ pixels/embryo). Thus, an antioxidant of $50{\mu}M$ Vit. C was selected for SCNT. $H_2O_2$ levels of bovine SCNT embryos were significantly lower in embryos treated with Vit. C during only SCNT procedure ($26.4{\pm}1.1$ pixels/embryo, p<0.05) than the treatment group during IVM ($29.9{\pm}1.1$ pixels/embryo) and non-treated control ($34.3{\pm}1.0$ pixels/embryo). Moreover, $H_2O_2$ level of SCNT embryos treated with Vit. C during SCNT procedure was similar to that of IVF embryos. These results suggest that the antioxidant treatment during SCNT procedures can reduce the ROS generation level of SCNT bovine embryos.

• Asian-Australasian Journal of Animal Sciences
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• v.22 no.4
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• pp.500-506
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• 2009

A modified Tris-buffered medium (mTBM) has been widely used as an insemination medium for porcine in vitro fertilization (IVF). We examined whether mTBM could be used for bovine IVF. Bovine cumulus-oocyte complexes (COCs) were cultured in a serum-free medium containing 30 ng/ml EGF for 22 h. After culture, COCs were inseminated with spermatozoa for 12 h in mTBM containing 5 mM caffeine and 10 g/ml heparin. The penetration of oocytes increased significantly (p<0.05) as the sperm concentration increased from 0.1 (30%) to 1-10 $(87-100%){\times}10^6$ cells/ml. This was significantly different from values obtained at 1 (87%) and 10 $(100%){\times}10^6$ cells/ml. However, when COCs were inseminated with spermatozoa from different bulls, the proportions (62-100%) of oocytes penetrated varied according to the bull. The proportion (18%) of oocytes penetrated was significantly (p<0.05) lower in a fertilization medium without caffeine and heparin but increased with the addition of caffeine and/or heparin to the medium, and the proportion (93-96%) of oocytes penetrated increased significantly (p<0.05) when the medium was supplemented with heparin and caffeine. In this medium, sperm penetration was first observed at 3 h after insemination. Irrespective of the presence of glucose in the fertilization medium, the proportion (93-97%) of oocytes penetrated and the proportion (83-84%) of embryos at the ${\geq}2$-cell stage cultured in a chemically defined medium were not significantly different. However, the proportion of embryos developing to the blastocyst stage was significantly (p<0.05) higher in the presence (11%) of glucose in the fertilization medium than in its absence (2%). In conclusion, the present study demonstrated that bovine oocytes penetrated in vitro in mTBM can develop to the blastocyst stage and mTBM may be used for the in vitro production of bovine embryos.

• Korean Journal of Veterinary Research
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• v.39 no.1
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• pp.189-203
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• 1999

To determine sex of bovine embryos using hamster histocompatibility Y(H-Y) antibodies, bovine compact morulae were incubated for 6 hours in TCM199 supplemented with 10% hamster H-Y antiserum and the embryos with developmental arrest were diagnosed as male embryos, while the embryos showing development during the incubation as female embryos. This presumptive embryo sexing was confirmed by polymerase chain reaction(PCR)method. 1. In the result of hamster sperm cytotoxicity test to measure H-Y antibody titer, the rate of dead sperm was considerably lower in H-Y antiserum absorbed with hamster male splenocytes than in H-Y antiserum absorbed with hamster female splenocytes or H-Y antiserum unabsorbed with splenocytes(p<0.01). 2. The rate of oocytes fertilized in vitro and the rate of blastocysts of the fertilized oocytes were 58.5% and 32.4%, respectively. The rate of blastocysts on day 8 was 15.9%, denoting the highest rate during whole culture period posterior to in vitro fertilization (IVF). 3. The bovine 16 cell and compact morulae embryos incubated in the medium supplemented with hamster H-Y antibodies showed 37.1% and 48.9% of developmental arrest which were diagnosed as male, respectively, and rates of redeveloped embryos from the arrested were 24.1% in 16 cell and 44.3% in compact morulae embryos, respectively, denoting higher rate of sex determination and rate of redevelopment in compact morulae than 16 cell embryos. 4. Bovine compact morulae of Korean cattle and Holstein were treated with hamster H-Y antibodies for sex determination and the rates of developmental arrest(diagnosed as male) were 48.4% for Korean cattle and 47.9% for Holstein, respectively. The rates of redeveloped embryos to blastocyst after treatment were 42.6% for Korean cattle and 41.8% for Holstein, respectively, showing no significant differences of sex determination and redevelopment between both breed. 5. The sex determination of bovine embryos(Korean cattle and Holstein) using hamster H-Y antibodies was diagnosed by PCR for confirmation, denoting the rates of 86.1% for Korean cattle and 85.9% for Holstein male embryos, respectively, and the rates of 91.9% for Korean cattle and 90.1% for Holstein female embryos, respectively, with no significant differences of sex determination between both breed. These results indicated that hamster H-Y antibodies can be usable for sex determination of bovine embryos of Korean cattle and Holstein, the viability of bovine embryos was sustained while being cultured in the medium supplemented with hamster H-Y antibodies of appropriate titer and sex determination of bovine embryos by PCR can be feasible for confirmation.

• Journal of Embryo Transfer
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• v.11 no.3
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• pp.283-289
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• 1996

This study was carried out to determine the sex of genomic and embryonic DNA using polymerase chain reaction(PCR). Bovine specific(216bp) and Y chromosome speicific DNA primers(l4lbp) were synthesized and tested for sexing. Bovine embryos used in this study were produced by in vitro fertilization. Few blastomeres for PCR were bisected by nicromanipulator and demi -embryos were cultured in TCM 199 medium containing 0.1% of solcoseryl. The results obtained were as follows; 1. Average optical density of genomic DNA extracted from blood of Hanwoo was 1.79$\pm$ 0.14. 2. 2. The ratio of the demi-embryos developed to blastocyst was 62.1 and 81.9% in morula and blastocyst, respectively. 3. When DNA of 2~4, 5~10 and more than 11 blastomeres was amplified with Y chromosome specific DNA primer by PCR, appreance rate of Y specific DNA band was 16.7, 46.2 and 40.0%, respectively. At least 5 to 10 blastomeres were required to determine the sex of embryos. 4. The rate of demi-embryos developed to blastocyst was 73.3% in TCM 199 medium supplemented with 0.1% solcoceryl. but 55.6% in control.

• Journal of Embryo Transfer
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• v.29 no.2
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• pp.133-139
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• 2014

$K^+$ channels are involved in the regulation of a variety of physiological functions, including proliferation, apoptosis and differentiation, in mammalian cells. Our previous study demonstrated that the blockage of $K^+$ channels inhibits mouse early embryonic development. This study was designed to identify the effect of $K^+$ channels during bovine embryonic development. $K^+$ channel blockers (tetraethylammonium (TEA), $BaCl_2$, quinine, ruthenium red and fluoxetine) were added to the culture medium during in vitro fertilization (IVF) for 6 h to first identify the short-term effect of these chemicals. Among $K^+$ channel blockers, fluoxetine, which is used as a selective serotonin reuptake inhibitor, significantly increased the blastocyst formation rate by approximately 6% when compared to control. During the in vitro maturation (IVM) of immature oocytes and the in vitro culture (IVC) of embryos, the oocytes and embryos were exposed to fluoxetine for either a short-term (6 h) or a long-term (24 h) to compare the embryonic development in response to exposure time. The 6 h exposure to fluoxetine during IVM did not affect the blastocyst formation rate, but the rate of blastocyst formation was reduced after the 24 h exposure. On the other hand, embryonic development increased approximately 10% in both groups of embryos exposed to fluoxetine for 6 and 24 h during IVC. Taken together, fluoxetine treatment during IVF and IVC, but not IVM, enhances bovine embryonic development. These results suggest that fluoxetine-modulated signals in oocytes and embryos could be an important factor towards enhancing bovine embryonic development.

• Reproductive and Developmental Biology
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• v.29 no.4
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• pp.235-239
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• 2005

This study was conducted to test whether the transgenic cattle pass the transgene to their progeny through germ cells, and whether the transgene is expressed in the mammary gland of ransgenic cows. Two male ransgenic calves were born from IVF-derived embryos injected with bovine $\beta-casein/human$ lactoferrin fusion gene and then grew up to be reproducible. Semen was collected from a transgenic bull after 18 mon of age and then frozen. Bovine oocytes matured in vitro were fertilized with spermatozoa of the transgenic bull and cultured in $50\;{\mu}L$ drops of CRlaa medium supplemented with 3 mg/mL BSA. After 48 h of culture, cleaved embryos were determined for the presence of transgenes by DNA polymerase chain reaction (PCR). Proportion of transgene positives among bovine embryos fertilized with sperm of the transgenic bull was $20.9\%$ (28/134). One of transgenic bulls did not produce transgenic sperm. Out of 34 calves produced from recipient heifers inseminated with semen of the other bull, 3 $(8.8\%)$ were transgenic animals (2 females and 1 male). Thus, one transgenic bull showed a low transmission frequency below Mendelian levels in both the IVF-derived embryos and his progeny. It was demonstrated by Southern blot that copy numbers of the transgene in the transgenic progeny enhanced about 1.8 times as compared to those of the founder bull The results demonstrate that the transgenic bull carrying human lactoferrin gene could pass his transgene to the progeny through germ cells, although he is a germ-line mosaic.

• Journal of Embryo Transfer
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• v.28 no.3
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• pp.185-191
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• 2013

The purpose of this study is to improve production efficiency of vitrified-thawed transgenic bovine embryos. Transgenic bovine embryos were produced by injection of FIV-GFP lentiviral vector into perivitelline space of in vitro matured MII stage oocytes, and then in vitro fertilization. EGFP-expressing transgenic bovine blastocysts were cultured in serum-containing and serum-free medium. These blsatocysts were vitrified by pull and cut (PNC) container made with 0.25 cm plastic straw. Results indicate that total developmental rates of normal IVF embryo cultured in serum-containing and-free medium into blastocyst were not significantly different (22.3 vs 21.5%) and those of GFP-expressing transgenic bovine embryo into blastocyst showed no significant difference between serum-containing (13.9%) and-free medium (13.1%). However, developmental rate of GFP transgenic embryo was significantly (P<0.05) lower than its of normal IVF embryos. In additional study, we vitrified GFP transgenic normal bovine blastocysts using PNC vitrification method. Survival rate of vitrified-thawed GFP transgenic blastocyst (23.1%) was significantly (P<0.05) lower than its of normal blastocysts (68.9%). Although, survival rate of vitrified-thawed GFP transgenic blastocyst was lower than its of normal blastocyst, our result may suggested that PNC vitrification method is feasible to cryopreserve transgenic embryos. Our next plan will be the production of GFP express transgenic bovine derived from vitrified-thawed embryos using PNC method.

• Journal of Veterinary Clinics
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• v.24 no.3
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• pp.295-299
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• 2007

The detrimental effects of gene transfection on embryo development and the molecular mechanism behind the differential expression of genes related to early embryo development were assessed in the production of transgenic cow embryos through somatic cell nuclear transfer (NT). Parthenogenetic, IVF, and transgenic NT embryos derived from ${\alpha}_1$-antitrypsin transfected ear fibroblast cells was produced. To investigate the molecular mechanism behind lower developmental competence of transgenic NT embryos, the differential mRNA expression of three genes ($IFN-{\tau}$, Oct4, Fgf4) in the 3 types of embryo (Parthenogenetic, IVF, transgenic NT) was examined. RNA was extracted from ten blastocysts derived from 3 types of embryos and reverse-transcripted for synthesis of the first cDNA. The quantification of 3 gene transcripts ($IFN-{\tau}$, Oct4, and Fgf4) was carried out in three replicate by quantitative real-time reverse transcriptase PCR. Expression level of $IFN-{\tau}$ mRNA was significantly higher in transgenic NT embryos than parthenogenetic and IVF embryos (P<0.05). However, expression level of Oct4 and Fgf4 of transgenic NT embryos was significantly lower than IVF embryos (P<0.05). Altered levels of these three mRNA transcripts may explain some of the embryonic/fetal/neonatal abnormalities observed in offspring from transgenic NT embryos.

• Clinical and Experimental Reproductive Medicine
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• v.16 no.2
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• pp.161-171
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• 1989

The development of 2-cell mouse embryos to the blastocyst stage in vitro has been used as quality control for the culture media and supplements employed for human in vitro fertilization and embryo transfer(IVF-ET). 2-cell mouse embryos were cultured to the blastocyst stage in SECM, Medium 199-Earle's, Ham's F-10 I , Ham's F-10 II , Hoppe & Pitts, MEM and $HT_6$. The protein supplements contained in media were bovine serum albumine, fetal bovine serum and human fetal cord serum. The results were as follows; 1. The successful development was 81.3% in Medium 199-Earle’s, 91.9% in Ham’s F-10 I and 97.1% in $HT_6$. 2. 2-cell mouse embryos developed properly in all supplements but the best development was particularly noted in $HT_6$ media when HFCS was supplied as protein supplement.

• Korean Journal of Animal Reproduction
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• v.23 no.4
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• pp.337-345
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• 1999

The present study was conducted to investigate the effects of various co-culture systems and supplemented protein sources on the in vitro development of bovine IVF embryos. Bovine cumulus oocyte complexes (COCs) were matured and fertilized in vitro. Presumptive zygotes with cumulus cells were transferred to TCM-199 or CRlaa containing 10% FBS or 3mg/$m\ell$ BSA, and cultured for 36~40 hr. After primary culture, cleaved embryos were co-cultured with cumulus cells(CC), bovine oviduct epithelial cells(BOEC) or Buffalo rat liver cells (BRLC) in TCM-199 or CRlaa supplemented with FBS or BSA respectively, for further 6 days. Cleavage rate increased with BSA(P＜0.01) in the both TCM-199(79%) or CRlaa(74%) When embryos were co-cultured with CC or BOEC in TCM-199, blastocyst development was enhanced with BSA(40% and 43%) compared to FBS (22% and 29%) , whereas in CRlaa no difference observed between BSA(40% and 39%) and FBS (40% and 42%). When embryos were co-cultured with BRLC monolayer, FBS enhanced the blastocyst development (P＜0.05) compared to BSA in both TCM-199(41% vs 31%) and CRlaa (44% vs 37%). The result of the present study showed that the cleavage rate of bovine IVF embryos increased with BSA, The result also showed that BSA can enhance the development of IVF embryos in co-culture with CC or BOEC in TCM-199, suggesting the in vitro development is affected by the medium and supplemented protein sources in co-culture with somatic cells.