• Current Research on Agriculture and Life Sciences
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• v.8
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• pp.45-50
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• 1990

This study was carried out ot investigate the effects of fetal calf serum (FCS) and gonadotropins supplemented to the medium on maturation and fertilization in vitro of porcine follcular oocytes. Ovaries were obtained from gilts at local slaughter-house. Oocyte-cumulus complexes were recovered by puncturing the ovarian follicles(3~5 mm in diameter). The complexes from individual ovaries were pooled in a $0.4m{\ell}$ droplet of medium covered with paraffin oil, then washed twice in fresh droplet and cultured for 36hrs in culture media according to experimental conditions. Boar epididymal spermatozoa were capacitated by preincubation for 4hrs in m-KRB medium and the preincubated spermatozoa were insemenated in the fertilization medium containing the cultured oocytes. The results obtained in this study are summarized as follows: 1. The maturation rates of oocytes cultured in m-KRB and m-KRB supplemented to 10% FCS were 82 and 37%, respectively. When PMSG, hCG. and PMSGt hcG($10Iu/m{\ell}$) were added to the media supplemented to 10% FCS, the maturation rates were 66, 58 and 68%, respectively. 2. Expansion of cumulus cells was not occured in m-KRB and m-KRB supplemented to 10% FCS. However, when PMSG, hCG and PMSG+hCG($10Iu/m{\ell}$) were added to m-KRB supplemented to 10% FCS, the expansion rates of cumulus cell layers were 92, 13 and 91%, respectively. 3. When oocytes were mltured in m-KRB, the rates of penetration and formation of male pronucle: were 93 and 7%, respectively. By adding FCS and gonadotropin to m-KRB, the penetration and formation of male pronuclei were 100 80%, respectively.

• Korean Journal of Animal Reproduction
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• v.23 no.2
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• pp.141-148
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• 1999

This study was to test whether the viability of bovine hatched blastocysts (HBs) can be maintained after vitrification and thawing. The HBs were produced in vitro at Day 9 and Day 10 after IVF, and they were classified to small (S-HBs; ø$\leq$300 ${\mu}{\textrm}{m}$) and large(L-HBs; ø＞300 ${\mu}{\textrm}{m}$) on the basis of embryo diameter using eyepiece micrometer. As freezing solution, we used EFS35 which consisted of 35% ethylene glycol (EG), 18% ficoll, 0.3 M sucrose and 10% FBS added in mDPBS. Vitrification was taken by two-step method, the HBs were equilibrated in 10% EG for 5 minutes and then shortly exposed in EFS35 and plunged into L$N_2$for 30~45 sec. After thawing, the survival rates were assessed by the re-expansion of the blastocoel during 2 h and 16 h of culture. The results obtained in these experiments were summarized as follows; 1) When the blastocysts(40.8%) recovered at Day 8 after IVF were further cultured for 24 h(Day 9 after IVF) and 48 h(Day 10 after IVF), the rates of HBs were 20.5% and 6.7%, respectively. Also, the total cell number of HBs on Day 9 was significantly higher than that of HBs on Day 10 (p＜0.01). 2) When the effects of freezing solution to the survival of Day 9 L-HBs were examined, the rate of vitrified group (75.7%) was significantly lower than 100% of control and exposed group(p＜0.05). 3) When the survival rates of vitrified HBs according to size and developmental age were examined, the data of L-HBs (75.5%) and S-HBs(63.6%) on Day 9 were slightly higher than those of L-HBs(64.3%) and S-HBs(60.7%) on Day 10. 4) Also, when the in vitro survival of Day 9 HBs was evaluated under different culture condition after thawing, the result in culture medium only (79.3%) was significantly higher than 43.2% in co-culture group (p＜0.05). These results demonstrated that bovine HBs can be successfully cryopreserved by two-step vitrification method using EFS35.

• Korean Journal of Plant Tissue Culture
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• v.21 no.4
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• pp.239-246
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• 1994

The genotypic (2n, 3n, 4n) response of watermelon in vitro shoot tip culture was evaluated. Different genotypes had similar response in terms of shoot formation and growth. Shoot formation was better at lower concentration of 0.3 mg/L BA and higher concentration of 5-10.0 mg/L 2iP and kinetin, but growth of newly formed shoot was inhibited. With further subculture, kinetin did not promote shoot formation Better shoot formation was observed at 0.3-0.5 mg/L BA. Combination of 0.3 mg/L BA and 0.3-0.5 mg/L BA was effective in shoot multiplication, growth and induction of more internodes. Varrying levels of light intensity and agar concentration did not affect the performance of tetraploid plants. Higher light intensity and agar concentrations decreased the number of shoot formed in triploid plane. Growth in both genotype, however was inhibited. Higher light intensity was found to promote leaf senescence in all genotypes. All growth inhibitors decreased the number of shoots formed and slowed plant growth there by prolonging duration of cultures. Growth inhibitors were to observed to decrease incidence of hyperhydricity in culture. No difference in shoot formation was observed in each of the concentrations used in Ancymidol, TIBA, CCC and PP333. Shoot formation and growth was more inhibited in ABA treatments. Leaf expansion and growth was poor in all treatments.

• Journal of Korean Society of Forest Science
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• v.97 no.6
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• pp.650-655
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• 2008

In an attempt to establish the mass proliferation system, Eucalyptus pellita, a 5-year-old plus tree, was cultured with three different culture types in 1L vessels: solid culture without ventilation (conventional culture), liquid culture without ventilation and open-type liquid culture with forced ventilation. Then the culture scale was subsequently increased from 1L to 10L in vessel volume. After 4 weeks of 1L-scale culture, the best growth was obtained by culturing plantlets on open-type liquid culture, suggesting that the in vitro plantlets growth can be enhanced by liquid medium and ventilation. In open-type large scale culture in 10L vessel, plantlets growth resulted in a 370% increase in the number of nodes, 3.6 times increase in leaf expansion, and 3.3 times increase in shoot length, while the conventional culture suppressed shoot growth due to the callusing on the leaves and lack of $CO_2$. The results indicated that the open-type large scale culture system was effective for enhancing productivity by improving growth of the plantlets in clonally proliferated plus tree, Eucalyptus pellita.

• Clinical and Experimental Reproductive Medicine
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• v.27 no.4
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• pp.345-351
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• 2000

Objective: Hatching of the blastocyst from the zona pellucida (ZP) is a key event in mammalian implantation. In vivo, two factors have been identified as possible mediators of hatching: lysis of the ZP by substances elaborated either from the embryo or female reproductive tract and pressure exerted on the zona by expansion of the blastocyst. Two methods of zona manipulation were already in use to enhance the ability of embryos to hatch: mechanical PZD and chemical ZD by acidic Tyrode's solution. But several controversies of each method have been reported. The purpose of this study was to investigate the effect of pronase for mouse embryo hatching. Methods: Mouse embryos were obtained following ovulation induction of $F_1$ animals. Fresh and cryo-thawed morula embryos were exposed to 0.5, 1.0, 2.0, 5.0 ${\mu}g/ml$ pronase in Ham's F10 for 72 hrs. Main outcome measures were the rates of partial hatching and completely hatched blastocysts, and cell number of it. Results: In fresh and cryo-thawed group, the rates of completely hatched blastocyst were significantly higher in 5 ${\mu}g/ml$ pronase treatment group than control group. There was no difference in completely hatched blastocyst total cell number between pronase treatment group and control group. This suggest that pronase treatment did not harmful in mouse embryo development. In pronase treatment group, zona pellucida were thinner than control group. Conclusion: The addition of pronase to culture media may accelerate the hatching of embryo. So, enzymatic treatment of the zona may provide a valuable and effective assisted hatching technique for human in-vitro fertilization-embryo transfer.

• Polymer(Korea)
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• v.34 no.3
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• pp.253-260
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• 2010

In this study, we prepared and evaluated a series of biocompatible and biodegradable block copolymer hydrogels with a delayed swelling property for tissue expander application. The hydrogels were synthesized via a radical crosslinking reaction of poly(ethylene glycol) (PEG) diacrylate and poly(D,L-lactide-co-glycolide)-poly(ethylene glycol)-poly(D,L-lactide-co-glycolide)(PLGA-PEG-PLGA) triblock copolymer diacrylate as a swelling/degradation controller (SDC). For the synthesis of various SDCs that can lead to different degradation and swelling properties, various PLGA-PEG-PLGA triblock copolymers with different LA/GA ratios and different PLGA block lengths were synthesized and modified to have terminal acrylate groups. The resultant hydrogels were flexible and elastic even in the dry state. The in vitro degradation tests showed that the delayed swelling properties of the hydrogels could be modulated by varying the chemical composition of the biodegradable crosslinker (SDC) and the block ratio of SDC/PEG. The histopathologic observation after implantation of hydrogels in mice was performed and evaluated by macrography and microscopy. Any significant inflammation or necrosis was not observed in the implanted tissues. Due to their biocompatibility, elasticity, sufficient swelling pressure, delayed swelling and controllable degradability, the hydrogels could be useful for tissue expansion and other biomedical applications.

• Korean Journal of Poultry Science
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• v.36 no.2
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• pp.177-186
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• 2009

It is well-known that unregulated over-expression of foreign gene may have unwanted physiological or toxic effects in transgenic animals. To circumvent these problems, we constructed retrovirus vector designed to express the foreign gene under the control of the tetracycline-inducible promoter. However, gene expressions in the tetracycline-inducible expression system (Tet system) are not completely regulated but a little leaky due to the inherent defects in conventional Tet-based systems. A more tightly controllable regulatory system can be achieved when the advanced versions ($rtTA2^SM2$) of rtTA and a minimal promoter in responsive components (pTRE-tight) are used in combination therein. In this study, we tried to produce human thrombopoietin (hTPO) from various target cells and transgenic chickens using the retrovirus vector combined with Tet system. hTPO is the primary regulator of platelet production and has an important role in the survival and expansion of hematopoietic stem cells. In a preliminary experiment in vitro, higher hTPO expression and tighter expression control were observed in chicken embryonic fibroblast (CEF) cells. We also measured the biological activity of the hTPO using Mo7e cells whose proliferation is dependant on hTPO. The biological activity of the recombinant hTPO from CEF was higher than both its commercial counterpart and hTPO from other target cells. The recombinant retrovirus was injected beneath the blastoderm of non-incubated chicken embryos (stage X). Out of 138 injected eggs, 15 chicks hatched after 21 days of incubation. Among them, 8 hatched chicks were hTPO positive. When the Go transgenic chicken was fed doxycycline (0.5 mg per 1 gram of feed), a tetracycline derivative, hTPO concentration of the transgenic chicken blood was 200 ng/mL. Germline transmission of the transgene was confirmed in sperm of the Go transgenic roosters. These results are informative to establish transgenic chickens as bioreactors for the mass production of commercially valuable and biological active human cytokine proteins.

• Development and Reproduction
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• v.6 no.2
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• pp.97-104
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• 2002

In most mammals, mature oocyte-cumulus complexer(OCCs) ovulate into the oviduct where fertilization by sperm takes place. However, the complex that fail to fertilize eventually undergoes degeneration while they reside in the oviduct. Yet there is no blown mechanism how both oocyte and cumulus cells degenerate. Using human follicular fluid (hFF), bovine oviductal tissue extract (BOX) and mouse OCC, the present study aimed to find how the oviduct influence the viability of the oocyte and cumulus cells in vitro. There was no difference of oocyte maturation rate between the control and BOX-treated groups. However, there was a significant difference in the survival of cumulus cells between two groups. Cumulus cells cultured in the presence of hFF alone underwent initially expansion and then they formed monolayer in the culture dish. Even after 72 hr, they proliferated well and showed fibroblast-like morphology. Cumulus cells cultured in the presence of both hFF and BOX also expanded after 24 hr, however, after 72 hr culture, they eventually detached and degenerated. Cumulus cells cultured in the BOX alone gave a similar drastic result. When the cumulus cells cultured in the presence of BOX were stained with DAPI, their nuclei showed partial condensation and fragmentation. After detailed analysis of these cells by TUNEL assay, many nuclei of them exhibited well stained spots indicating the signs of apoptosis. Based upon these observations, it is suggested that BOX might possess a factor that leads mouse cumulus cells to undergo apoptosis in vitro.

• Journal of Plant Biotechnology
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• v.34 no.4
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• pp.331-338
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• 2007

One-node stem pieces ca. 1 cm in length containing a axillary bud and a fully expanded leaf were obtained from it in vitro plants of potato (Solanum tuberosum L.). Leaves were removed and the nodes were cultured on the MS medium to investigate the effects of temperature, day length, sucrose, and CCC in microtuber formation and development. The fresh weight of microtubers after 80 days increased significantly at 8% sucrose and $20^{\circ}C$ compared with $28^{\circ}C$. The tuberization and development were reduced at $28^{\circ}C$ except short-day treatment of 8 hours at 8% sucrose. The fresh weight and diameter were increased on the culture medium added CCC 500 mg/L. The potato tuberization was promoted under short daylength, and it showed great effect by treatment with the CCC. Though the tuberization was promoted at low temperature of $20^{\circ}C$ in a histologic change of an axillary bud part cell of a potato, the cells were able to observe the swelling growth. Swelling growth of tissue was stimulated in the darkness and was more remarkable by addition of CCC. In particular, in the visual ratio of cell division for each position in the tissue, the cortex part showed larger ratio of cell expansion than that of the pith part. The effect of CCC was identified at 8% sucrose in the darkness. The effect of CCC was not showed in sucrose 3% under long daylength of 16 hours. As a result, the fact of a substance with AGPase important for starch composition was certified by the result with the inclose of AGPase activity on high concentration of sucrose, CCC, and dark treatment by which tuber formation and development are promoted.

• Korean Journal of Animal Reproduction
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• v.20 no.1
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• pp.27-34
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• 1996

The objective of this study was to examine the cell number of Total, ICM and TE cells of bovine blastocysts according to development progression cultured in CR1 medium, which was reported as successfully supporting medium for preimplantaion bovine embryo development to the blastocyst stage, by differential labelling of the nuclei with immunosurgery and polynucleot-ide-specific fluorochromes. Blastocysts were obtained at day 8 after in vitro fertilization and classified to early, middle, expanded stage according to the developmental morphology; blastocoel expansion and zona thickness. Also, bias tocysts in the same category were divided into two parts to check the Total cell number by using bisbenzimide only and ICM, TE and Total cell number by using immunosurgery and two polynucleotide-specific fluorochromes. 1) The development rate of blastocysts at day 8 after in vitro fertilization was 29.3% and classified bIas tocysts to early, middle, expanded and hatching stage were 8.7, 9.9, 7.6 and 3.1%, respectively. 2) The numbers of total blastomere using bisbenzimide in the classified blastocysts to early, middie and expanded were 46.9${\pm}$8.6, 66.2${\pm}$12.5 and 122.8 ${\pm}$ 14.4, respectively. This indicated that CR1 is a appropriate culture medium for bovine embryo development. 3) The count of ICM and TE cell number by using differential labelling with immunosurgery and polynucleotide-specific fluorochromes in the classified blastocysts to early, middle and expanded; ICM cell numbers of were 12.8${\pm}$5.9, 26.3${\pm}$8.4 and 35.5${\pm}$15.0, respectively and TE cell numbers were 30.5${\pm}$5.0, 4 41.3${\pm}$8.2 and 81.1${\pm}$13.4, respectively. These results presented that the increase of ICM and TE cell numbers averaged two and three doublings between early and expanded blastocyst stage and also total cell number counted from ICM nuclei and TE nuclei by using differential label-ling showed the increase pattern with development advance level and the results were similar to total cell number obtained from bisbenzimide treatment only. Therefore, the differential labelling of ICM and TE nuclei in situ is a very useful technique to evaluate embryo qualities and can be used as an indicator on study of preim-plantation embryo development.