• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.123-123
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• 2003

This study was conducted to determine the ability of nuclear development of canine oocytes depend on the kind of maturation media and addition of serum sources. Ovaries were collected from a bitches at various stages of estrus cycle by an ovariohysterectomy. Oocytes were collected of cumulus oocytes complexes after slicing of ovaries with blade. The maturation medium was containing 0.6 mM/ml cysteine, 0.2 mM pyruvic acid, 20 ng/ml $E_2$ and 1 $\mu g/ml$ rbST Exp. 1, the oocytes were matured in four different maturation medium as follows: 1) TCM-199, 2) DMEM, 3) NCSU37 and 4) modified-NCSU37 with 10% FBS. Exp. 2: the oocytes were matured in mNCSU37 supplemented with different protein sources (10% FBS, 10% EDS, 0.3% BSA and 0.1% PVA) to select the optimal one. Oocytes were matured in a humidified atmosphere containing 5% $CO_2$ at $39{\circ}C$ for 72 hrs. The maturation rate were analyzed by Duncan's multiple range test using General Linear Models procedure in SAS. The rates of meiotic resumption to MI-MII depend on different culture media were achieved with TCM-199 (5.2%), DMEM (5.0%), NCSU37 (7.2%) and m-NCSU37 (5.9%), respectively. The rates of meiotic resumption to MI-MII according to addition of protein source were 10% FBS (13.3%), 10% EDS (25.0%), 0.3% BSA (25.0%) and 0.1% PVA (15.4%), respectively. In conclusion, the results obtained showed that in vitro maturation media and protein supplement to m-NCSU37 culture medium tested did not promote the final steps of IVM in canine oocytes.

• Journal of Embryo Transfer
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• v.22 no.2
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• pp.137-141
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• 2007

Antioxidants were well known to be essential supplements in the complex media and serve as a reservoir of oxygen. In this study, Hanwoo COCs (cumulus oocytes complexes) were matured and developed in L-cysteine-TCM199 and analyzed metaphase chromosome. Maturation rate of Hanwoo COCs were 73.4%, 94.6% in 0.1% PVA, 0.1 mM L-cysteine, respectively and showed significantly different between the treatments (p<0.05). Blastocyst formation were revealed 20.3%, 10.0% in 5% FBS+TCM199, 0.1 mM L-cysteine+1% BSA, respectively. There were no significant difference among treatment groups. Metaphase chromosome were showed 18.3%, 12.0% in 5% FBS-TCM199, 0.1 mM L-cysteine, respectively and analyzable chromosome were 6.1%, 4.0% and had no differences between the treated groups. In the case of in vitro developmental stages, metaphase chromosome were showed 18.3%, 12.0% in $4{\sim}16$ cells stage, 43.1%, 13.0% in morulae stage and 94.8%, 100.0% in blastocyst stage. These results suggested L-cysteine has beneficial role for in virto maturation and development in Hanwoo COCs.

• Asian-Australasian Journal of Animal Sciences
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• v.14 no.6
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• pp.759-764
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• 2001

This study was carried out to investigate the effect of activation timing, cell cycle and passage on the development of embryos produced by cumulus cell nuclear transfer in Hanwoo (Korean cattle). Nuclear donor cumulus cells were cultured in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum at $38.5^{\circ}C$ in a humidified atmosphere of 5% $CO_2$ in air. The 1~6 passages of serum deprived or actively dividing cumulus cells were isolated and used as donor cells. The in vitro matured oocytes were enucleated and then the isolated donor cells were introduced. One pulse of 180 volts for $15{\mu}s$ was applied to induce the fusion between karyoplast and cytoplast. The activation was done before or after the fusion. To activate, oocytes were treated with $10{\mu}M$ calcium ionophore for 5 min immediately followed by 2 mM 6-dimethylaminopurine for 3 h. The nuclear transfer embryos were cultured in $500{\mu}l$ of modified CRlaa supplemented with 3 mg/ml BSA in four well dish covered with mineral oil. After 3 days culture, culture medium was changed into modified CRlaa medium containing 1.5 mg/ml BSA and 5% FBS for 4 days. The incubation environment was 5% $CO_2$, 5% $O_2$, 90% $N_2$ at $38.5^{\circ}C$. There was no blastocyst formation when the nuclear transfer embryos were activated before the fusion, whereas, 29.9% of blastocyst formation was shown when the nuclear transfer embryos were activated after the fusion. When serum deprived and actively dividing cumulus cells were used as nuclear donor cells, the developmental rates to blastocyst were 38.5% and 40.6%, respectively. There was no significant difference between serum deprived and actively dividing cells in the developmental rates. The developmental rates to blastocyst according to 1~6 passages were 37.5~44.4%. However, there were no significant differences among passages. These results indicate that 1~6 passage cumulus cell irrespective of cell cycle could support development of nuclear transfer embryos activated after the fusion.

• Reproductive and Developmental Biology
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• v.28 no.2
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• pp.113-117
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• 2004

The present study was carried out to examine the effects of $O_2$ concentrations and culture media (North Carolina State University (NCSU)-23, porcine zygote medium(PZM)-3 or PZM-4) on in vitro development of porcine IVM/IVF embryos. Porcine oocyte-cumulus complexes were cultured in BSA-free NCSU-23 medium containing porcine follicular fluid (10%), cysteine (0.9 mM), $\beta$-mercaptoethanol (25 $\mu\textrm{g}$/$m\ell$), epidermal growth factor (10 ng/$m\ell$) and hormonal supplements (PMSG and hCG: 10 IU/$m\ell$) for 20∼22 h. They were then cultured in the same medium but without hormonal supplements for an additional 20∼22 h. After culture, cumulus-free oocyte were coincubated with liquid boar spermatozoa for 5∼6h. Putative zygotes were transferred to NCSU-23, PZM-3 and PZM-4 medium under the condition of 5% $O_2$ or 20% $O_2$ concentrations. At 48 h, no mean differences were found in cleavage rates. However, the rates of blastocyst formation at day 7 after in vitro fertilization were significantly higher in PZM-3 medium under the condition of 5% $O_2$ concentration than other treatments (19.9$\pm$2.4 vs. 11.1$\pm$2.0 to 16.0$\pm$2.5%, P<0.05). The total cell numbers of blastocysts were significantly higher in 5% $O_2$ than in 20% O2 (P<0.05). However, no differences was found among the culture media within each $O_2$ concentrations. In conclusion, the use of PZM-3 medium in 5% $O_2$ concentration was effective on in vitro development of porcine IVM/IVF embryos.

• Microbiology and Biotechnology Letters
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• v.18 no.5
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• pp.460-465
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• 1990

Regeneration of protoplast was effective by preincubating spore suspension containing 30$\mu g$/ml of 2-DG for 4 hours, and CBE medium containing casamino acid, bovine serum albumin, ergosterol and myoinositol was found to be more efficient than any other regeneration medium tested in this experiment. The regeneration frequency was about 30%. Optimal conditions for conidial protoplast fusion were obtained by treatment of protoplasts with 10 mM $CaCl_2$ and 30% polyethylene glycol 4000 (pH 7.5) as fusogenic agent at $37^{\circ}C$ for 10 minutes. The fusion frequency was $8.2\times 10^{-4}$. The higher productivity of enzyme of fusant FWN-56 was achived: 2.3-fold for CMCase, 1.5-fold for avicelase, 1.8-fold for $\beta$-glucosidase and 2.5-fold for xylanase compared to that obtained in two parental strains. The genetic stability of fusant after maintenance on minimal medium for more than 4 weeks was high because segregant rate was below 1%. The conidial DNA content of fusant was 1.4-1.6 times higher than that of the parental strains, The nucleus size of fusants were also higher than that of each parental strains.

• Asian-Australasian Journal of Animal Sciences
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• v.14 no.6
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• pp.754-758
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• 2001

Bovine oocytes with compact and complete cumulus cells were cultured for up to 24h in TCM199 buffered with 25mmol/l hepes and supplemented with 10% FBS (fetal bovine serum), 1mg/ml $17{\beta}$-estradiol, 20IU/ml hCG(human chorionic gonadotropin). All of the oocytes were divided into at 6 groups depending upon incubation times (control, 0 hour, 6 hours, 12 hours, 16 hours, 18 hours). To all experimental media, $200{\mu}g/ml$ puromycin was added at different incubation times mentioned above. Following these culture times, in vitro insemination was conducted with frozen-thawed bovine spermatozoa in medium BO (Brackett and Oliphant medium for in vitro insemination) with $10{\mu}g/ml$ BSA(bovine serum albumin) and 10 mg/ml heparin added. After 22h culture, the oocytes were fixed with acetic alcohol solution and stained with orcein acetic solution to evaluate sperm nuclear progression. Addition of puromycin after 0, 6 and 12 h of culture resulted in near of oocyte maturation at the M1 stage. Contrariwise, puromycin addition after 12 h of culture led to restoration of nuclear progression to M2 stage. On the one hand, puromycin affected the synthesis of Cyclin B protein that may be involved in the oocyte maturation and sperm capacitation for in vitro fertilization. The present study suggests the participation of protein synthesis, cyclin B, in the oocyte development from M1 to M2 stages in vitro.

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.85-85
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• 2001

본 연구는 돼지 난자의 체외 수정시 sucrose 첨가가 돼지 난자의 다정자 침입율에 미치는 영향을 조사하기 위하여 실시하였다. 돼지 난포란은 0.1% PVA, 3.05 mM D-glucose, 0.91 mM sodium pyruvate, 0.57 mM cysteine, 0.5 $\mu\textrm{g}$/$m\ell$ FSH, 0.5 $\mu\textrm{g}$/$m\ell$ LH, 및 10 ng/$m\ell$ EGF가 첨가된 TCM199 배양액에서 42-44 시간 동안 배양하여 체외 성숙을 유도하였으며, 2 mM caffeine과 0.1% BSA가 첨가된 체외수정용 mTBM 배양액에 3% sucorse를 첨가하여 6시간동안 수정을 유도하였다. 수정된 난자의 다정자 침입율을 조사하기 위해 체외수정 후 12, 13 및 14 시간대에 각각 1% orcein으로 염색하여 전핵 형성율을 조사하였다. 돼지 난자의 체외수정시 3% sucorse를 첨가하였을 때, 전핵 형성율을 조사한 결과 대조구에서 미수정, 1, 2, 및 3 PN 이상의 전핵 형성율은 각각 57.6 7.6, 9.7, 및 25%로, 처리구 74.2, 7.6, 13.6, 및 4.5% 에 비교하여 다정자 침입율이 유의하게 높았다(p<0.05). 그리고 3% sucorse를 첨가한 체외수정용 배양액에서 수정을 유도한 후 12, 13, 및 14 시간대별로 나누어 전핵 형성율을 조사한 결과, 수정 후 시간이 경과되면서 다정자 침입율이 증가되는 것을 볼 수 있었다 그러나 대조구 60.7%에 비해서는 각각 32.5, 36.0, 및 33.3%로 현저히 낮은 것을 볼 수 있었다.(Table Omitted). 또한, 3% sucorse가 첨가된 배양액에서 수정된 수정란의 발달율을 조사한 결과, 배반포 생산율은 처리구 21.8%로 대조구 6.9%에 비해 유의하게 높은 것을 확인 하였다. 따라서, 돼지 난자의 체외수정시 3% sucorse첨가는 체외수정란 생산에서 문제가 되고 있는 다정자 침입율을 저하시키고 배발달율을 향상시킴으로서 수정란의 체외 대량생산 효율성은 물론 수정란 이식시 수태율을 증가시킬 수 있을 것으로 사료된다.

• Korean Journal of Microbiology
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• v.24 no.1
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• pp.24-31
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• 1986

The optimal conditions for the formation and the regeneration of Pseudomonas spheroplast were measured. Pseudomonas spp. cells were transformed to spheroplasts from 99.0% to 99.9% by treatment of $100{\mu}g/ml$ lysozyme and 10mM EDTA at room temperature. The optimal pH for the spheroplast formation was pH 8.0. Magnecium chloride, calcium chloride and streptomycin were effective on the stabilization of Pseudomonas spheroplast, while $Mg^+\;and\;Na^+$ ions were effective on the formation of Pseudonomas spheroplast. Rich Regeneration Medium was used for the regeneration of Pseudonomas spheroplast. To improve regeneration frequency, Bovin Serum Albumine and cationic ions were added to the spheroplast dilution beffer and regeneration environment respectively. Treatment of 20mM calcium chloride in ehr Rich Regeneration Medium could improve the yield of regenerants as much as 28-fold. Treatment of 1% Bovin Serum Albumine in the spgeroplast formation and dilution buffer increased the yield of regenerants to 10-fold. Also, the regeneration frequency was improved to 14-fold shen Rich Regeneration Meidum containing 0.5% Gelatin was used for regeneration as well as 1% Bovin Serum Albumine.

• The Korea Journal of Herbology
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• v.32 no.3
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• pp.63-69
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• 2017

Objectives : Advanced glycation end product (AGEs) is combine formation of glucose and protein. AGEs and reactive oxygen species are potential therapeutic targets for the various disease such as diabetic complications, renal injury, skin damage. The aim of this study was investigated the AGEs inhibitory activity and antioxidant activity of water extracts from 40 Korean medicines and 5 heating-processed Korean medicines. Methods: AGEs formation inhibitory activities of Korean medicines measured using bovine serum albumin (BSA), glucose, and fructose. Then, five effective Korean medicines were selected and heated with 30% ethanol. The AGEs inhibitory activities of heated Korean medicine were measured compared with not-heated Korean medicines. The antioxidant activities were evaluated through radical scavenging assays using 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radicals. Furthermore, we examined total phenol and flavonoids contents. Results: Scutellariae Radix, Corni Fructus, Persimmon Fruit, Paeoniae Radix, Mori Folium respectively reduced AGEs production. Morever, heating-processed Scutellariae Radix has AGEs inhibitory activities better than not-processed Scutellariae Radix. Heating- processed Scutellariae Radix scavenged DPPH and ABTS effectively and $IC_{50}$ of DPPH and ABTS radical scavenging activity of Heat processed Scutellariae Radix were $15.47{\pm}0.26{\mu}g/m{\ell}$ and $12.07{\pm}1.23{\mu}g/m{\ell}$. It caused heat processing methods of Scutellariae Radix up regulated total phenol and flavonoids contents ($26.68{\pm}0.01$ to $46.15{\pm}0.10$, $20.30{\pm}0.38$ to $64.20{\pm}0.52$). Conclusion: It has AGEs inhibitory activities that 20 kind of medicinal plants of 40 medicinal plants. Especially, heat processed Scutellariae Radix has excellent AGEs inhibitory activities and antioxidant effect.

• Journal of the Korean Society of Food Culture
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• v.22 no.1
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• pp.97-103
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• 2007

In this study, physicochemical properties and the antioxidative activity of whey protein isolate(WPI) for com germ oil were measured. The pH of WPI was 6.26, and the titrable acidity was 0.18%. The WPI’s moisture content was 5.2% and each of the other element content such as lactose, crude protein, crude ash and crude fat was found to be 0.8%, 90.7%, 2.7% and 0.6%, respectively. The amounts of active SH group in WPI 9 ${\mu}$ M-g and total colony counts of bacteria was 5.9 ${\times}$ 10$^3$ CFU-g. ${\alpha}$-Lactalbumin, ${\beta}$-lactoglobulin and bovine serum albumin(BSA) were shown in WPI as major protein by electrophoresis. The antioxidative effect of WPI and other antioxidants on com germ oil used as substrate was determined by peroxide value(POV) and conjuqated dienoic acid value(CDV). By these results, the order of antioxidative effects could be defined as BHT 0.02%>ascorbic acid 0.1%>WPI 0.1%>WPI 0.02%>ascorbic acid 0.02%>control>tocopherol 0.02%>tocopherol 0.1%, respectively. Also the induction period of com germ oil added with WPI was longer by 1.6 times than that of control(none added any antioxidant). Therefore the fact suggested that WPI could be utilized as a good antioxidative agents.