• Journal of Animal Science and Technology
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• v.61 no.2
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• pp.61-68
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• 2019

The hG-CSF (human Granulocyte Colony-Stimulating Factor) is a growth and stimulation factor capable of inducing the proliferation of bone marrow cells, several types of leukocytes, among other hematopoietic tissue cells. hG-CSF is used in used to treat anomalies that reder a small number of circulating white blood cells, which may compromise the immune defenses of the affected person. For these reasons, the production of hG-CSF in a bioreactor system using the mammary gland of genetic modified animals is a possibility of adding value to the bovine genetic material and reducing the costs of hG-CSF production in pharmaceutical industry. In this study, we aimed the production of transgenic hG-CSF bovine through the lipofection of bovine primary fibroblasts with an hG-CSF expression cassette and cloning these fibroblasts by the somatic cell nuclear transfer (SCNT) technique. The bovine fibroblasts transfected with the hG-CSF cassette presented a stable insertion of this construct into their genome and were efficiently synchronized to G0/G1 cell cycle stage. The transgenic fibroblasts were cloned by SCNT and produced 103 transferred embryos and 2 pregnancies, one of which reached 7 months of gestation.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.82-82
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• 2003

Offspring have been produced from somatic cells in a number of species. This biotechnology introduced a new phenomenon in reprogramming and differentiation of somatic cell, namely totipotency. However, birth of oversized calves and perinatal abnormalities such as increased gestation length, lack of spontaneous parturition, higher incidence of dystocia, and reduced perinatal viability of offspring are frequently observed in pregnancies of cloned bovine fetuses. Disturbance of feto-placenta has been proposed as likely causes for abnomal growth. However. Little is known the mechanism responsible for the perinatal problems. Therefore, we focused on gestation length in somatic cell nuclear recipient cows. To solve this issues, placental tissues of control and cloned bovine were obtained by a cesarean section (C-section) before 5 days of paturition. Total RNA from control and cloned bovine placenta was extractd by TRIzol reagent. GeneFishing DEG kits (Seegene) were used to identify differentially expression genes. Total RNA (3 ug) were synthesized by M-MLV reverse transcriptase (200 u/ul) with 10 uM dT-annealing control primer (ACP1) at 42C for 90 min. Then, first-strand cDNA (50 ng) was amplified using the 5 uM arbitary ACP (1-20) and 10 uM dT-ACP2 primers. Some specific expression genes were amplified, Now, we are cloning and sequencing. These finding strongly can be support to solve the problems for parturition delay in nuclear transfer cows, suggest that placenta specific proteins are key indicators for the aberration of gestation and placental function in cows.

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.18-18
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• 2001

Despite of importance of integrated events of nucleus and microtubule remodeling in nuclear transferred embryos with somatic cells, little information is available on this subject. In this study we configured chromatin and microtubule organization following somatic cell nuclear transfer in pre- and non-activated bovine oocytes in order to clearify nuclear remodeling process and to demonstrate centrosome inheritance during nuclear transfer. The cumulus-oocyte complexes were collected from slaughterhouse and were matured in vitro for 20 h in TCM 199 supplemented hormone. Matured bovine oocytes were enucleated by aspirating the frist polar body and metaphase chromatin using a beveled pipette. Bovine fibroblast cells were fused into enucleated oocyte by electrical stimulation. Reconstructed oocytes were activated with ionomycine and 6-dimethylaminopurin, and then cultured in CRlaa medium. The organization of nuclear and microtubules were observed using laser-scanning confocal microscopy. At 1 hour after fusion, microtubule aster was seen near the transferred nucleus in most oocytes regardless activation condition. While most of fibroblast nuclei remodeled to premature chromosome condensation (PCC) and to the two masses of chromosome in non-activated oocytes, a few number of fibloblasts went to PCC and multiple pronuclear like structures in activated oocytes. Microtubular spindle was seen around condensed chromosome. Gamma-tubulin was detected in the vicinity of condensed chromosome, suggesting this is a transient spindle. The spindle seperated nucleus into two masses of chromatin which developed to the pronuclear like structures. Two pronuclear like structures were than apposed by microtubular aster and formed one syngamy like nuclear structure at 15 h following nuclear transfer. At 17 to 18 h after fusion, two centrosomes were seen near the nucleus, which nucleates micrtubules for two cell cleavage. While 31% of reconstructed oocytes in non-activated condition developed to morulae and blastocysts, a few reconstructed oocytes in pre-activated condition developed to the blastocyst. These results suggested introduction of foreign centrosome during nuclear transfer, which appeared to give an important role for somatic cell nuclear reprogramming.

• Asian-Australasian Journal of Animal Sciences
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• v.16 no.6
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• pp.889-893
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• 2003

Immunomodulatory and therapeutic potential of Enrofloxacin was studied in bovine sub clinical mastitis (SCM). The therapeutic efficacy was adjudged by Somatic cell count and Total bacterial count of the milk, whereas, the immuno modulatory potential of the drug was assessed by measuring myeloperoxidase (MPO) and acid phosphates (ACP) enzyme level in the milk leukocytes. Forty-five cows were divided into three equal groups. Gr I consisting 15 cows served as healthy control, whereas, 30 cows (SCM), Gr II and Gr III, selected on the basis of California Mastitis Test (CMT) positive reaction. Gr II cows received 150 mg of Enrofloxacin, once a day for three days and Gr.III received sterile 5 ml PBS (pH 7.4) for 7days, both the treatment were given by intramammary route. The observation was made up to 30 days post-treatment (PT). The CMT of the healthy milk was negative (0), whereas, it ranged between 1 point score and 2 point score in SCM. The Somatic cell count (SCC) and Total bacterial count (TBC) decreased significantly (p<0.05) on day 3 PT in GrII cows in Enrofloxacin treated group, however, such changes were insignificant in PBS treated group. Traces of MPO and ACP enzyme were found in the healthy milk. The mean ACP level enhanced by 70% on day 3 PT in GrII and only 18.7% in Gr. III cows. The mean MPO level enhanced to 32% in Gr. II and 18 % in Gr. III cows on day 3 PT. Concomitant use of Enrofloxacin in SCM at sub optimal dose was found to reduce the bacterial load by increasing the bactericidal enzyme level in the milk polymorphonuclear cells (PMNs) in bovine SCM, which indicates its immunomodulatory potential in mastitis.

• Reproductive and Developmental Biology
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• v.33 no.2
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• pp.93-98
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• 2009

In this study, bovine Dnmt1 cDNA was sequenced and detected Dnmt1 mRNA level in bovine tissues by northern blot, methylation pattern of genome by southern blot, specific localization of Dnmt1 in mouse and bovine preimplantation embryos by immunocytostaining and Dnmt1 protein level in ovary and testis by western blot. Bovine Dnmt1 cDNA sequence showed more homology with that of human than mouse and rat. The RNA level of Dnmt1 was 10 times higher expression in placenta than other tissues. This indicates that placenta was hypermethylated compared to others organs. The genomic DNA could not be cut by a specific restriction enzyme (HpaII) in placenta, lung and liver of bovine. It suggests that Dnmt1 in some somatic cells was already methylated. Dnmt1, which has the antibody epitope 1316~1616, was distributed in nucleus and cytoplasm including the stage of pronuclear stage and maturation of oocyte and gradually weaken to blastocyst stage compare to negative. In addition, Dnmt1 was strongly expressed in tetraploid embryo and cloned 8-cell than IVF 8-cell. An aberrant pattern of DNA methylation in cloned embryo may be abnormal development of fetus, embryonic lethality and placenta dysfunction. The somatic specific band (190kDa) was appeared in ovary and testis, but oocyte specific band (175kDa) was not. Further investigations are necessary to understand the complex links between the methyltransferases and the transcriptional activity of genes in the cloned bovine tissues.

• Korean Journal of Animal Reproduction
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• v.21 no.4
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• pp.345-353
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• 1997

Antioxidants and antioxidants with somatic cell co-culture, bovine oviduct epithelial cells(BOEC) and buffalo rat liver cells(BRLC), were studied as a mean of increasing the development and intracellular glutathione(GSH) concnetrations of bovine embryos derived from in vitro matured(IVM) and in vitro fertilized(IVF) oocytes. Cell numbers and intracellular GSH concentrations of blastocysts were also counted. The developmental rate beyond morula stages in CRlaa containing taurine(2.5mM), superoxide dismutase(SOD, 600U) and catalase(250U) were 1%, 75.0%, 64.8% and 62.3%, respectively. The developmental rate in antioxidant groups was significantly higher than in control(P<0.05). The intracellular GSH concentrations of blastocysts cultured in 0, 2.5mM taurine, 600U SOD and 250U catalase were 33.8pM, 39.3pM, 42.3pM and 54.8pM, respectively. This result indicated that the developmental rates and intracellular GSH concentrations of catalase group was significantly higher than any other groups(P<0.05). The developmental capacity in CRlaa plus various antioxidants co-cultured with BOEC were 55.3%(control), 74.1%(2.5mM taurine), 66.7%(600U SOD) and 70.7%(250U catalase) and in CRlaa plus various antioxidants co-cultured with BRLC in control, 2.5mM taurine, 600U SOD and 250U catalase were 63.8%, 75.5%, 71.0% and 73.5%, respectveily, the intracellular GSH concentrations of blastocyst embryos co-cultured with BOEC and BRLC in CRlaa with 0.25mM taurine, 600U SOD and 250U catalase were 73.4pM and 64.4pM, 79.9pM and 67.5pM, 82.3pM and 71.7pM, and 83.0pM and 80.0pM, respectively. Cell numbers of blastocysts were not difference in all experimental groups. These studies indicate that andtioxidants and antioxidant with somatic cell co-culture can increase the proportion of embryo that developed into morula and blastocysts, and the intracellular GSH concentrations of blastocyst embryos.

• Korean Journal of Animal Reproduction
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• v.13 no.2
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• pp.98-104
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• 1989

Successful techniques of in vitro fertilization(IVF) are valuable for studying the process of fertilization and for developing economical procedures for gene and nuclear transfer in farm animals. To date, bovine IVF system has been developed with oocytes in vitro or vitro, but the resulting zygotes exhibit limited embryonic development after in vitro culture. Even though in vitro matured oocytes achieved high fertilization and cleavage rates, these embryos appear extremly low rate of pregnancies when transferred to synchronized recipients. Development of early bovine embryos in vitro is generally arrested at the 8-to 16-cell stage. However, recent use of somatic cells such as trophoblastic vesicle, granulosa and oviduct epithelial cell for co-culture with early bovine embryos has proven effective for development of embryos, matured and fertilized in vitro, past the in vitro cell blocks. These factors clearly indicate the value of the co-culture system in promoting development of bovine oocytes matured and fertilized in vitro to morula or blastocyst stage in vitro. In addition, co-culture system may beome a tool for evaluation of viability of ova that have been manipulated by procedures such as splitting, microinjection and nuclear transfer.

• Journal of Embryo Transfer
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• v.15 no.1
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• pp.23-31
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• 2000

This study was conducted to investigate the effects of quiescent treatment of donor cells and activation treatment time of recipient cytoplasm on nuclear remodeling and in vitro development of somatic cell-cloned bovine embryos. Serum starved, confluent and nonquiescent cycling adult skin cells were teansferred into enucleated oocytes. Nuclear transfer oocytes were activated at 30 min, 1 and 2 hrs after electrofusion. Some nuclear transfer embryos(23% to 35%) extruded a polar body, which was not affected by quiescent treatment of donor cells and activiation time of recipient cytoplasm. About 68% of nuclear transfer embryos fused with a serum starved cells has a chromatin clump, but which was not different from embryos fused with confluent(51%) and nonquiescent(47%) cells. The proportion of embryos with a single chromatin clump was sightly increased when nuclear transfer embryos were activated within 30 min after fusion(69%) compared to those were activated at 1 and 2 hrs after fusion, but there was not significantly different. Development rates to the blastocyst stage were 8.6% and 15.9% when serum starved and confluent cells were transferred, which were higher than that of control group. Developmental rate to the blastocyst stage was higher in embryos were activated within 30 min after fusion (17.3%) compared to those of embryos were activated at 1 and 2 hrs after fusion (P<0.05). From the present result, it is suggested that quiescent treatment of donor cells and activation time of recipient cytoplasm can affect the in vitro development. Quiescent plasm activation within 30 min after fusion could increase the number of embryos with a normal chromation structure, which results in increased in vitro development.

• Reproductive and Developmental Biology
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• v.32 no.3
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• pp.167-173
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• 2008

Somatic cells such as oviduct epithelial cell, uterine epithelial cell, cumulus-granulosa cell and buffalo rat river cell has been used to establish an effective culture system for bovine embryos produced in in vitro. But nitric oxide (NO) metabolites secreted from somatic cells were largely arrested the development of bovine in vitro matured/ in vitro fertilized (IVM/IVF) embryos, suggesting that NO was induced the embryonic toxic substance into culture medium. The objective of this study was to investigate whether BOEC co-culture system can ameliorate the NO-mediated oxidative stress in the culture of bovine IVM/IVF embryos. Therefore, we evaluated the developmental rate of bovine IVM/IVF embryos under BOEC co-culture system in the presence or absence of sodium nitroprusside (SNP), as a NO donor, and also detected the expression of growth factor (TGF-$\beta$, EGF and IGFBP) and apoptosis (Caspase-3, Bax and Bcl-2) genes. The supplement of SNP over 5 uM was strongly inhibited blastocyst development of bovine IVM/IVF embryos than in control and 1 uM SNP group (Table 2). The developmental rates beyond morulae stages of bovine IVM/IVF embryos co-cultured with BOEC regardless of SNP supplement (40.4% in 5 uM SNP+ BOEC group and 65.1% in BOEC group) were significantly increased than those of control (35.0%) and SNP single treatment group (23.3%, p<0.05: Table 3). The transcripts of Bax and Caspase-3 genes were detected in all experiment groups (1:Isolated fresh cell (IFC), 2:Primary culture cell (PCC), 3:PCC after using the embryo culture, 4: PCC containing 5 uM SNP and 5: PCC containing 5 uM SNP after using the embryo culture), but Bcl-2 gene was not detected in IFC and PCC (Fig. 1). In the expression of growth factor genes, TGF-$\beta$ gene was found in all experimental groups, and EGF and IGFBP genes were not found in IFC and PCC (Fig. 2). These results indicate that BOEC co-culture system can increase the development beyond morula stages of bovine IVM/IVF embryos, possibly suggesting the alleviation of embryonic toxic substance like nitric oxide.

• Korean Journal of Animal Reproduction
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• v.25 no.2
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• pp.131-138
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• 2001

This experiment was designed to evaluate effects of nonspecific immunostimulator(NIS) Barodon-FX(equation omitted), anionic alkali mineral complex and far-infrared radiation solution on in vivo-produced mouse and in vitro-produced bovine embryos to blastocyst development. Proportion of mouse embryos developing into blastocyst was not greater in BSA- and Barodon-added medium than in BSA-control, but there was signifcantly different(P ＜ 0.05) in hatching and hatched blastocyst development between 0.25% Barodon-and PVP-contained medium(54.7%) than PVP-control(32.5%). BOEC and GC resulted in higher proliferation rate(24∼40% and 17∼22%, respectively) in 0.25∼0.5% Barodon-added medium than in controls, but proliferation of GC and CC greatly decreased in 1∼2% Barodon-added medium. Effect of Barodon on cell proliferation greatly varied among somatic cells. Proportion of early bovine embryos developing into morula and blastocyst was significantly greater(P ＜ 0.05) in 0.5% Barodon-added medium(50% and 63.6%) than in control(31.6% and 27.4%) under co-culture with BOEC and GC, but developmental rate was not different between other Barodon treatments and control. These data indicate that effect of Barodon on cell proliferation significantly varied between somatic cells and that addition of 0.5% Barodon in BOEC-coculture system may further improve blastocyst development in early bovine embryos.