The aim of this study is to investigate the effect of porcine epididymal fluid (pEF) on in vitro-maturation and subsequent fertilization of porcine follicular oocytes. Porcine cumulus-oocytes complexes retrieved from antral follicles were cultured in tissue culture medium (TCM)-199 supplemented with pEF of different concentrations. At 48 h after culture, development of oocytes to germinal vesicle (GV) breakdown, metaphase I, anaphase-telophase I, and metaphase II were examined. Significant (p<0.05) increase in the proportion of oocytes developed to MII stage was observed in oocytes cultured in pEF-containing TCM-199 than in oocytes cultured in pEF-free TCM-199 (46.2% vs 16.7%), which was a dose-dependent manner. Subsequently, the proportion of monospermic fertilization were significantly (p<0.05) increased in oocytes cultured in the TCM supplemented with pEF than those cultured in pEF-free TCM-199 (51.0% vs 24.1%). In the second series of experiment, the percentage of MII oocytes was significantly (p<0.05) increased after exposure of oocytes to pEF during the first 22 h period of culture than after exposure of oocytes to pEF during the next 24 h of culture, while no significant difference in the percentage of monospermy was observed. The results of this study demonstrate that pEF contains at least enhancing component(s) for nuclear maturation.

The aim of present experiment was to examine commercial synthetic extender(AndroMed) for semen cryopreservation of Korean Black Bull. Semen was collected from a Korean Black Bull using an artificial vagina and transported to the laboratory. The semen was diluted 1:1 by AndroMed. The pellect was diluted to final sperm concentration of by doubling in every 10 minutes at cold chamber. The semen was equilibrated for 1 hr at cold chamber and packed to 0.5 ml straw. The semen straws were located above 5 cm of liquid nitrogen for 5 minutes, above 5 cm for 10 minutes and above 10 cm for 10 min. And then the frozen straw was plunged to . The presented straws were examined the viability and motility after thawed at water bath. Hanwoo semen was used as KPN (Korea Proven Bull Number) in this experiment. The survival rates was significantly higher in fresh semen than frozen semen (). However, the motility rates was similar (80.7% and 66.4%). The survival and motility rates were higher in 5cm, 10 min treatment group than the other two groups in straw-located height and duration above ( and 70.7% vs, 33.18% and vs, 30.14% and 65.7%, respectively). The development rates to cleavage was higher in Black Cow than Hanwoo semen (62.2%, 64.4%), However, The development rates to blastocyst was higher in Hanwoo than Black cow semen (25.9%, 23.0%). In conclusion. The present results that acceptable fertilization and cryopreservation could be obtained by in vitro fertilization with frozen-thawed semen using a synthetic semen extender (AndroMed).

This study was carried out to investigate the effective genetic resources preservation system using the frozen boar semen. The porcine oocytes were matured for 44 hours in NCSU-23 medium with or without 10% Porcine Follicle Fluid (PFF), 0.5 porcine FSH, 0.5 equine LH, 1.0 17 -estradiol () and 10 ng/ml Epidermal Growth Factor (EGF) under mineral oil at in humidified atmosphere of 5% in air. After 44 h of culture, the oocytes were inseminated with frozen-thawed semen and fresh semen prepared with mTBM medium for 6 h. Later, set of 50 presumptive zygotes were transferred into 4-well dish (500 ) of IVC medium. for embryos freezing, slow-freezing and vitrification methods were used as a cryopreservation. Differences among treatments were analyzed using General Linear Model Procedure by SAS Package (version 6.12) differences were considered significant when p<0.05. Following IVF and IVC, the rates of cleavage and blastocysts formation were significantly higher (p<0.05) in hormone supplemented group than that of hormone-free group (25.7 vs, 12.1). The development rates to cleavage and blastocysts were significantly higher in PZM-5 group than NCSU-23 group (60.3%, 46.6% vs 27.4%, 11.1%). Further improvement was achieved when PZM-5 was supplemented with FBS. Cleavage rates was significantly higher in fresh semen source group than frozen semen (66.7% vs 43.7%). However in blastocysts rates was similar two groups. Post-thaw survival rates of embryos were 1.2% and 2.2% in slow-frezing and vitrification groups, respectively. The results of our study suggest that it is still possible to improve the culture conditions and boar semen cryopreservation for enhance reproductive technology and animal genetic resources conservation.

Spermatogonial stem cells(SSCs) only are responsible for the generation of progeny and for the transmission of genetic information to the next generation in male. Other in vitro studies have cultured SSCs for proliferation, differentiation, and genetic modification in mouse and rat. Currently, information regarding in vitro culture of porcine Germline Stem Cell(GSC) such as gonocyte or SSC is limited and is in need of further studies. Therefore, in this study, we report development of a successful culture system for gonocytes of neonatal porcine testes. Testis cells were extracted from 10～14-day-old pigs. These cells were harvested using enzymatic digestion, and the harvested cells were purified with combination of percoll, laminin, and gelatin selection techniques. The most effective culture system of porcine gonocytes was established through trial experiments which made a comparison between different feeder cells, medium, serum concentrations, temperatures, and O2 tensions. Taken together, the optimal condition was established using C166 or Mouse Embryonic Fibroblast(MEF) feeder cell, Rat Serum Free Medium(RSFM), 0% serum concentration, 37℃ temperature, and O2 20% tension. Although we discovered the optimal culture condition for proliferation of porcine gonocytes, the gonocyte colonies ceased to expand after one month. These results suggest inadequate acquirement of ingredients essential for long term culture of porcine GSCs. Consequently, further study should be conducted to establish a successful long-term culture system for porcine GSCs by introducing various growth factors or nutrients.

This study was conducted to examine the effects of human follicular fluid and gonadotropin (FSH+HCG+rhEGF) on in vitro maturation, fertilization and development of human immature oocytes. Cumulus-oocyte complexes (COCs) were collected following for in vitro fertilization and embryo transfer (IVF-ET) cycles of the patients. At the time of oocytes collection, oocytes were classified into MII, MI and GV in accordance with their appearance (MII: Fully mature oocyte at metaphase II of meiosis; MI: Nearly mature oocytes at metaphase I of meiosis; GV: Immature oocytes at prophase I of meiosis). After controlled ovarian stimulation using gonadotropin(FSH) and human chorionic gonadotropin (HCG) in 70 ICSI cycles, 158 MI to MII matured oocytes were intracytoplasmic sperm injection (ICSI) h after in vitro culture and 553 MII oocytes were ICSI after denudation. The aspirated MI and GV oocytes were cultured in culture medium containing 10% (v/v) serum protein substitute (SPS), 10% (v/v) human follicular fluid (hFF) and 10% (v/v) serum protein substitute (SPS)+1 IU/ml FSH+10 IU/ml HCG+10 ng/ml recombinant human epidermal growth factor (rhEGF). The maturation rate of immature oocytes was similar among the three group. When maturation medium was supplemented with 10% SPS, 10% hFF or gonadotropins, the fertilization rate of in vitro matured oocytes was higher in 10% SPS (80.0%), but there was no statistical significance (78.2%; hFF, 76.9%; gonadotropin, p>0.05). The development rate of human embryos developed to cells were not significant difference in the medium containing SPS, hFF and gonadotropins (65.6%, 65.9% and 66.7%). The results of these study suggest that human follicular fluid and gonadotropins supplemented in the culture medium was not effected on the in vitro maturation, fertilization and development of human immature oocytes.

We conducted a series of in vitro experiments to evaluate the anticancer effect of photodynamic therapy using hypericin and 532㎚ DPSS (diode pumped solid state laser). The cultured KB cells were treated with serial concentrations of hypericin ranging from 0.01㎍/㎖ to 5㎍/㎖ (two-fold dilution) with variable laser dosage (10J, 20J, 30J). The cell viability was evaluated by MTT assay. The type of cell death was detected by fluorescent microscope using Hoechst 33342 / PI (propidium iodide) stain methods. In this study, IC50 value with hypericin-mediated PDT with 10J DPSS laser was 35 ng/ml. The maximum cytotoxicity with Photofrin II-based PDT was observed at high drug concentrations(> 90 ng/ml) independent with laser dose. And the in vitro PDT effects depended on the laser dose and drug concentrations were displayed by the difference in the type of cell death, namely apoptosis or necrosis. According to this result, the hypericin based photodynamic therapy with DPSS laser was effective photodynamic therapy.

마치현을 압출성형 및 효소분해 처리할 경우 원료에 비하여 수용성 고분자 다당류 및 아라비노갈락탄 함량이 증가하였다. 마치현 수용성 다당류 중 아라비노스와 갈락토오스의 함량이 원료 마치현보다 1.5배 증가하였으며, 람노스 함량도 2.6배 증가한 유의적인 결과를 보였다. 압출성형 처리효과로 고분자 분획(I)은 Ext I, Ext II 및 Ext III 시료에서 각각 37, 29 및 26% 정도 저분자 분획(II)으로 분자 재배열이 발생함과 동시에 66,000-74,000 Da범위의 분자량을 갖는 다당체로 구조변형 되었다. 특히, 저분자 분획의 분자량과 조성비에 있어서 압출성형 처리한 마치현은 처리하지 않은 원료에 비하여 9-13% 정도 증가하여 유의성이 있었다. 이같은 다당류의 붕괴 및 변형 정도는 압출성형 처리시 투입된 기계적 소모 에너지와 비례적인 상관성을 보였다. 압출성형 처리를 한 수용성 다당류의 경우 압출성형 처리온도 120oC 및 140oC인 경우 자유 라디칼 소거활성능이 압출성형 처리하지 않은 원료에 비하여 높게 증가하였다. 상기와 같은 마치현 유래 아라비노갈락탄의 항산화 활성 기능의 결과에 비추어볼 때 보다 폭 넓은 범위의 분자량을 갖는 분획물 제조 및 생리활성 평가실험을 지속적으로 추진한다면 새로운 기능성 식품소재로 활용할 가치가 있다고 기대된다.

Beef marbling is known as one of the most important beef-quality traits in Korea. It is likely that marbling derived from fatty acids, mainly propionate, is mediated by rumen. Recently micro-agents were studied to enhance marbling, although many parts of that were digested in rumen. Therefore, this study was conducted to screen candidate materials to effect on beef quality with in vitro ruminal incubation. The materials such as saponin, chitosan, Zn compounds (4), vitamin C sources (2), Korean herb cocktail and garlic sources (2) were added to rumen fluid to 1.25% of substrate volume at 0 and 24 h incubation time. Total gas production in intact vitamin C source increased but that in all Zn compounds decreased (P＜0.05). Total gas production in Zn sulfate compound less decreased than in other Zn sources. Propionate in Zn sulfate increased than the other candidate compounds at all incubation time (P＜0.05). Experiment two was conducted to clarified effect of additives such as vitamin C sources (2), garlic lyophilized, Korea herb cocktail and Zn sulfate were supplemented with 2.5% volume at the 0, 3, 6, 12 and 24 h incubation time. Total gas in Zn sulfate was lower than any other treatments. Propionate in garlic, herb and Zn sulfate appeared to be lower than control and vitamin C sources at all incubation time, although significant difference was not observed in total VFA among control and all treatment. This study suggested that micro-agent might be used to improve beef quality with minute level.

In the present study, effects of concentration of cryoprotectant solutions on the nuclear maturation of vitrifiedthawed porcine oocytes were examined. Oocytes were cultured in TCM-199 medium supplemented with 5% FBS at C in 5% and air. The percentage of monospermy in the toxicity group and vitrification group (22.0 3.0% and 31.5 3.5%) was decreased compared with that of the control group (44.0 4.0%). The percentage of in vitro development to blastocyst in the toxicity group and vitrification group (12.0 2.5% and 14.8 2.8%) was decreased compared with that of the control group (28.0 3.0%, p<0.05). The survival and in vitro developmental rate of oocytes vitrification-thawed with EDS and EDT + TCM-199 medium supplemented with 0.1% PVA were 46.3 3.0%, 54.5 3.8% and 14.8 2.5%, 16.4 2.7%, respectively. This results were lower than the control group (28.0 3.5%). The in vitro developmental rate of embryos vitrified with EDS and EDT supplemented PVA did not have a significant difference. The survival and in vitro developmental rate of vitrified-thawed morula and blastocyst embryos were 44.2 3.5%, 17.3 3.0% and 48.1 4.2%, 18.5 3.5%, respectively. Vitrified morulae and blastcyst embryos had a lower survival and developmental rates than their control counterparts.

This study was designed to investigate the effect of kinetin on in vitro development of parthenogenetic porcine oocytes exposed to demecolcine prior to activation. In vitro matured metaphase II stage oocytes were incubated in 0 or 2 g/ml demecolcine supplemented defined culture medium for 3 h and the oocytes were activated electrically. The parthenogenetic porcine embryos were then cultured in 0 or 200 M kinetin supplemented defined culture medium for 7 days. Regardless of demecolcine treatment, kinetin supplementation increased blastocyst rates significantly (7.0% versus 12.1% and 4.9% versus 8.5%; Control versus Kinetin and Demecolcine versus Kinetin + Demecolcine, respectively, p<0.05). Demecolcine treatment before activation tended to decrease blastocyst rates regardless of kinetin supplementation although it is not statistically significant. Total cell numbers in the blastocysts also tended to be elevated in embryos when supplemented with kinetin, however only the result between Kinetin and Demecolcine groups is statistically significant (37.6 7.2 versus 28.1 9.5, respectively, p<0.05). In conclusion, the present report shows that kinetin enhances developmental competence of parthenogenetic porcine embryo regardless of demecolcine pre-treatment before parthenogenetic activation when they were developed in defined culture condition.

Chemicals for cosmetics, including skin, the skin absorbs some of the research in the field of science or pharmacy recently, about the environment and the health of the heightened interest in skin absorption. Many other human attributes and absorption evaluation studies are underway in various areas. This study were used rats and carried out to find out the effects of commercial permanent wave products to skin which are composed with thioglycolic acid and bases. Results were as follows. Permanent wave penetrated to 3 hours later with steady state in skins and was not significant changeable after 20hr later. In case of neutralizer with thioglycolic acid lag time and permeability coefficient in healthy skin were 3.32hr and 0.101μg/cm2/hr, in old skin were 3.08hr and 0.117μg/cm2/hr, and in wounded skin were 3.02hr and 0.166μg/cm2/hr. In conclusion, lag time and permeability coefficient in old skin and wounded skin were faster than healthy skin. In vivo, We were studied to general time and method of permanent wave. We found out that fine wrinkle and rash of skin were changeable in the case of treating with permanent wave drugs than normal skin.

Successful in vitro embryo production heavily relies on the normal maturation and fertilisation of oocytes. We examined the normal and abnormal fertilisation of zebu cattle oocytes matured in vitro. Immature cumulus oocyte complexes (COCs) from zebu cattle ovaries at slaughter were matured in vitro (IVM) for 24 h. The oocytes were either fixed, stained and examined for nuclear changes or fertilised in vitro (IVF) with Percoll-separated, heparintreated spermatozoa (1.0 /mL) of zebu (n = 7) and crossbred bulls (n = 7). After 18 h of sperm-COCs co-incubation at C with 5% in humidified air, the presumptive zygotes were fixed, stained and examined for pronuclei. The number of oocytes retrieved per ovary was 5.4 0.7. The percentage of matured oocytes was 73.0. The difference in motility of spermatozoa before and after Percoll seperation was significant (p<0.001). The percentages of normal and abnormal fertilisation (polyspermia and oocytes with one pronucleus) varied significantly depending on individual bulls (p<0.05). A protocol for IVF of IVM oocytes in Bangladeshi zebu cattle is developed. A future study may elucidate the capacity of such IVM-IVF oocytes to develop to the blastocyst stage for transfer to surrogate mother.

Pteris cretica ‘Wilsonii’의 대량생산에 적합한 배양 환경을 구명하기 위하여 연구를 수행하였다. 포자는 7주 안에 모두 발아하였다. Knop과 Hyponex배지에서 전 엽체 증식이 왕성하였지만, 전엽체의 생육에는 Hyponex 배지가 더 유용하였다. MS배지에서는 전엽체가 괴사하 였으며, 질소급원과 sucrose의 농도를 조절한 경우에도 sucrose 무첨가구를 제외한 모든 첨가구에서 전엽체가 괴사하였다. Sucrose 1%와 agar 0.6%를 첨가한 Hyponex배지가 전엽체의 증식과 생육에 가장 적합한 것으로 나타났다. 접종방법을 달리한 결과, Hyponex배 지에서는 다져서 접종하는 전엽체의 증식 및 생육에 효과적이었지만, MS배지에서는 전엽체의 군집을 4등 분하여 접종한 처리구에서 전엽체의 증식 및 생육이 우수하였다. 고체배지에서 배양하는 것이 액체배지에서 배양하는 것보다 효과적이었다. 액체배양은 전엽체의 괴사를 유도하였다. 액체진탕배양한 전엽체는 생육은 우수하였으나 고체배양한 전엽체에 비하여 증식이 억 제되었다.

본 연구는 청나래고사리의 기내 포자체 유묘를 이용한 효율적인 대량생산 방법을 개발하기 위하여 시행되었다. 유묘의 엽신, 엽병, 근경을 곱게 다져 활성탄 0.1%를 첨가한 1/2MS배지에 배양한 결과, 근경의 절편에서만 포자체 유묘가 생산되었다. 곱게 다진 근경 절편은 1/2MS 배지에서 포자체 재생이 가장 왕성하였으며, sucrose 농도를 2%로 조절하고 NaH2PO4 50 mgL−1을 첨가할 경우 포자체 재생이 더욱 촉진되었다. Kinetin과 BA를 단용 또는 NAA, IBA와 각각 혼용하여 배양한 결과, kinetin 1 μM 단용 처리구에서 포자체 재생이 가장 왕성하였다. BA 첨가구에서는 분열조직의 증식이 왕성 하였으나. 분열조직이 포자체로 분화되지 못하는 특징을 보였다. 변형 1/2MS 배지(sucrose 2%, NaH2PO4 50mgL−1, kinetin 1 μM, pH 5.8, agar 0.8%)에 활성탄 0 또는 0.1%를 첨가하여 고체, 액체 정체, 액체 진탕배양한 결과, 포자체 재생은 액체 진탕배양시 가장 왕성하였으며, 활 성탄의 첨가는 포자체 재생을 촉진하였다.