내건성이 우수하다고 판명되어진 비수리(Lespedeza cuneata), 새(Arundinella hirta), 소리쟁이 (Rumex crispus), 장구채(Silene firma), 매듭풀(Kummerowia striata) 5수종의 기내대량증식 조건을 구명하였다. 5종의 선발종 중 비수리는 WPM배지에서 길이생장이 가장 좋았으며, 새는 MS배지, 소리쟁 이는 LP배지에서 장구채는 LP배지, 매듭풀은 SH배지에서 좋은 길이생장을 보였다. 뿌리발생도 수종에 따라 배양배지에 따라 다르게 나타났다. 비수리의 경우 기내발근은 모든 배지에서 뿌리가 발생하지 않 았으며, 새는 MS배지, 소리쟁이는 WPM배지, 장구채는 MS배지, 매듭풀은 1/2MS배지에서 가장 좋은 뿌리생장을 보였다. 잎 갈변은 비수리는 B5배지, 새는 WPM배지, 소리쟁이는 B5배지, 매듭풀은 LP배 지에서 가장 심한 갈변을 보였다. 생존율은 비수리, 소리쟁이, 장구채는 모든배지에서 100% 생존율을 보였으며, 새는 WPM배지에서 25% 생존율을, 매듭풀은 LP배지에서 25%의 낮은 생존율을 나타냈다. 선 발종의 다경줄기를 유도하기 위해 cytokinin을 처리한 결과 비수리의 경우 TDZ 0.5mg/L 처리구, 새는 BA 0.5mg/L, 소리쟁이는 TDZ 0.1mg/L, 장구채는 BA 0.5mg/L, 매듭풀은 BA 0.5mg/L 처리구에서 가장 많은 다경줄기를 형성하였다. cytokinin 처리는 줄기생장, 기내발근 및 다경줄기 유도에 영향을 미쳤다. 기내에서 배양된 식물체는 4종의 인공상토에서 성공적으로 순화되었다. 본 연구 결과들은 내건 성을 가진 5종의 유용식물의 품종개발 및 대량증식에 크게 기여할 수 있을 것으로 판단된다.

반하(Pinellia ternata(Thunb.) Breit)는 천남성과에 속하는 다년생 초본식물로서 조직배양을 이용하 여 대량번식 방법 연구가 활발히 진행되어 왔다. 하지만 대량생산된 괴경들의 토양 순화 및 적응을 위 한 환경 조건의 확립의 연구는 미비하다. 따라서 본 연구에서는 기내에서 증식된 반하의 괴경을 이용하 여 우수한 품질의 약재와 건전묘의 대량생산을 위해 생육에 적합한 토양조건을 탐색하였다. 본 연구에 서는 액체배지에서 현탁배양 한 괴경(Type 1)과 고체배지에 치상하여 배양된 괴경(Type 2)두 종류를 비 교하였다. 토양은 3개의 조성으로 조합하여 생육을 비교하였으며, 코코피트, 피트모스, 버미큘라이트, 펄라이트 및 제오라이트를 배합하여 사용하였다. 기내 배양 조건이 다른 처리구들의 토양 조성별 순화 율 측정하였으며, 생육의 차이를 확인하기 위해 8주동안 생육 후 초장, 잎 수, 마른잎 수, 괴경 수, 괴 경 크기, 생체중 및 건물중을 측정하였다. 또한 주아의 생성율을 확인하기 위하여 4주와 8주에 측정을 진행하였다. 그 결과 Type 2가 펄라이트를 20%증량한 상토 B에서 가장 우수한 생육을 보였다. 괴경의 비대에 영향을 주고 묘로서 이용을 위하여 필요한 잎의 수는 상토 B에서 가장 많은 1.7개로 나타났고 가장 잎의 출현이 없었던 Type 1의 상토 C가 0.9개로 나타나 1.8배의 차이를 나타냈다. 또한 같은 처 리구에서 건물중이 통계적으로 유의한 차이를 보이며 우수한 것으로 나타났다. 반하의 주아 생성율의 차이는 상토 B에서 1.1개로 우수하였으며, 가장 저조한 처리구의 수치보다 1.2배 높은 생성율을 확인할 수 있었다. 이러한 결과로 기내에서 배양된 반하의 토양 순화 및 적응기에 괴경의 비대를 유도하여 우 수한 한약재 생산이 가능 할 것으로 생각되며 차후 토양에서 재배 시 반하의 대량번식에도 도움이 될 것이라 사료된다.

Lysophosphatidic acid (LPA) is an important signaling molecule which mediates many different cellular responses. The purpose of this study was to investigate the effect of in vitro culture (IVC) medium supplemented with LPA on the preimplantation embryonic development of porcine embryos derived from in vitro fertilization (IVF). Embryos derived from IVF were cultured in PZM-3 medium supplemented with 30 μM LPA on Day 1 to Day 7, Day 1 to Day 3 (early stage), or Day 4 to Day 7 (late stage), or without LPA. Moreover, the messenger RNA (mRNA) expression of obtaining blastocysts from each group were analyzed. Data were analyzed by ANOVA followed by Duncan using SPSS (Statistical Package for Social Science) mean ± SEM. There was a significantly higher cleavage rate in Day 1 to Day 7 than control (71.25% and 57.46%, respectively) and significantly higher total cell number of blastocysts in Day 1 to Day 3 and Day 4 to Day 7 than control (56.07,56.53 and 45.19, respectively). The results also showed that the mRNA expression level of PCNA, Bcl-2 and Bax in Day 1 to Day 7 group blastocysts were significantly higher than control and the expression level of Bax in Day 1 to Day 3 was also significantly higher than control. Moreover, it also showed that Bcl-2/Bax mRNA ratio in D1-3 group was significantly lower than control but D4-7 and D1-7 groups were comparable to control group. In conclusion, our results suggest that treatment with 30 μM LPA during IVC improves the porcine early embryo cleavage and the blastocyst total cell number after IVF and regulating the mRNA expression of blastocysts during blastocyst formation.

The nature of molecular mechanisms governing embryo development is largely unknown, but recent reports have demonstrated that proper execution of programmed cell death is crucial for this process. The main objective of this study is to examine the mode of programmed cell death during nuclear transfer embryos development in porcine. In particular, the relative employment of two major pathways in programmed cell death; e.g. apoptosis (type I) and autophagy (type II) was compared. Oocytes use in the study was matured in vitro in the presence of 10% FBS maturation medium. After nuclear transfer embryos were cultured for each programmed cell death control factor [Cysteamine(Cyst : 0.4mM), 3-methyladenine(3MA : 2.5mM) and Rapamycin(RP : 100nM)] in TCM-199 medium supplemented with 0.1% BSA. In this study results of among the blastocysts development in 3MA; PCNA, MAP1LC3A and ATG5 RNA gene expression level increased in the order of IVF<Cyst < 3MA < RP. However Casp-3 and TNF-r RNA gene expression level decreased in the order of IVF < 3MA and RP< Cyst. The expression of mTOR showed a pattern opposite to that of MAP1LC3A. That is, its expression was the lowest in Cyst group. And next experiments analysis of MMP expression patterns. Analysed this MMPs enzyme activation to evaluate the effectiveness of high quality brastocyst culture in porcine. In this results of the enzymatic activity of MMP-2 and MMP-9 was assessed in culture, the level of active MMP-9 was higher expression in the medium of each 3MA and RP treatment group, with the level of another treatment group being relatively higher. These results suggest that the autophagy activation culture medium is more effective for stable and innovative nuclear transfer embryos development.

This study investigated the effect of Charcoal:Dextran Stripped fetal bovine serum (CDS FBS) and heat-inactivated FBS (HI FBS) in embryo culture medium on their ability to support in vitro development of bovine embryos. The developmental ability and quality of bovine embryos were determined by assessing their cell number, lipid content, mitochondrial activity, gene expression, and cryo-tolerance. The percentages of embryos that underwent cleavage and formed a blastocyst were significantly (P<0.05) higher in medium containing CDS FBS than in medium containing HI FBS (42.84 ± 0.78% vs. 36.85 ± 0.89%, respectively). Furthermore, the beneficial effects of CDS FBS on embryos were associated with a significantly reduced intracellular lipid content, as identified by Nile red staining, which increased their cryo-tolerance. The post-thaw survival rate of blastocysts was significantly (P<0.05) higher in the CDS FBS than in the HI FBS group (85.33 ± 4.84% vs. 68.67 ± 1.20%). Quantitative real-time PCR showed that the mRNA levels of acyl-CoA synthetase long-chain family member 3, acyl-coenzyme A dehydrogenase long-chain, hydroxymethylglutaryl-CoA reductase, and insulin-like growth factor 2 receptor were significantly increased upon culture with CDS FBS. Moreover, the mRNA levels of sirtuin 1, superoxide dismutase 2, and anti-apoptotic associated gene B-cell lymphoma 2 in frozen-thawed blastocysts were significantly (P<0.05) higher in the CDS FBS group than in the HI FBS group, however, the mRNA level of the pro-apoptotic gene BCL2-associated X protein was significantly reduced. Taken together, these data suggest that supplementation of medium with CDS FBS improves in vitro bovine embryo developmental competence and cryo-tolerance.

Growth differentiation factor8 (GDF8) is a member of the transforming growth factor-β that has been identified as a strong physiological regulator. Overall of the current studies, the GDF8 is detected in oviduct fluid and uterus which led us to suggest that the GDF8 may effect on preimplantation embryonic development and act paracrine role to correlate with successful late-blastocyst implantation in in vivo. The purpose of this study is the effects of GDF8 on porcine parthenogenesis (PA) embryo development during in vitro culture (IVC). We were investigated the effect of GDF8 supplement during PA embryo IVC by cleavage and blastocyst formation rate and patterning analysis. Data were analyzed by on way ANOVA, followed by Tukey’s range test. Respectively 0.2, 2 and 20 ng/mL of GDF8 were added during IVC followed experiment design as control, 0.2, 2, and 20 GDF8 supplement groups. After 48h of embryo culture time, no significant difference was observed on cleavage rate from the different concentration (0, 0.2, 2, and 20 ng/ml) of GDF8 supplement groups (65.7%, 66.0%, 66.3%, and 65.8%, respectively). After 120h of embryo culture time, the 0.2 and 2 group showed significantly (p<0.05) higher blastocyst formation rate than control (40.4% and 36.4% VS 40.4%, respectively). In embryo developmental pattern analysis, the 0.2 ng/ml GDF8 supplement groups showed significantly higher (p<0.05) 2-3 cell cleavage- and early blastocyst pattern compared with control (12.0% and 10.4% VS 6.6% and 6.2%, respectively). However there are no significantly different pattern was observed in other groups. In conclusion, the 0.2 ng/ml of GDF8 supplementation during porcine PA embryo IVC significantly changed embryonic developmental patterns. However there are further studies are required such as analysis of blastocyst total number, specific gene transcription pattern, and ICM/TE rate to make clarify and support the conclusion.

The study was conducted to evaluate the effects of microbial culture supplements on ruminal fermentation and fermentative quality of Italian ryegrass silage (IRGS) both in vitro and in situ. Three species of microbes (Lactobacillus casei (LC), Bacillus subtilis (BS), and Saccharomyces cerevisiae (SC)) were used in this study. They were applied to IRGS at 30 days after silage manufacture. Various items were measured using in vitro and in situ incubation technique after each microbial supplement was inoculated into IRGS at 0.5×104 CFU/g. In the first experiment, in vitro ruminal fermentation characteristics of IRGS were evaluated at 0, 12, 24, 48, and 72 hours after microbes were inoculated into IRGS. In the second experiment, in situ fermentation characteristics were investigated at 0, 1, 3, and 5 days after the inoculation of each microbial supplement. In vitro ruminal NH3-N content was significantly (p<0.05) increased in LC-, BS-, and SC-IRGS at 12 hrs post incubation compared to that in control IRGS. In vitro ruminal total VFA concentration and dry matter digestibility (DMD) of IRGS were not significantly difference among LC-, BS-, and SC-IRGS, although they were numerically increased in LC-IRGS than those of the other IRGS. In addition, this study evaluated the fermentation characteristics and in situ DMD of IRGS with the lapse of incubation time up to 5 days. Throughout the incubation times from 1 day to 5 days, the pH value was significantly (p<0.05) lower in BS-, LC-, and SC-IRGS than that in control IRGS. Lactate was significantly (p<0.05) higher, and significantly (p<0.05) butyrate was lower in LC-IRGS than that in other treatments at 0 day. It was higher (p<0.05) in control IRGS than that of BS-, LC-, and SC-IRGS at 1-5 days. In situ DMD tended to increase in BS-, LC-, and SC-IRGS compared to that in control IRGS. Especially, DMD was higher in SC-IRGS than that in other treatments at 0 day. It tended to be higher in LC-IRGS at all incubation time. Taken together, these results suggest that it might be useful to select a microorganism by considering the feeding time of IRGS to ruminants because organic acids and DMD of IRGS were affected by the incubation time of each microorganism with IRG silage, especially for L. casei decreased the content of acetate and butyrate in IRGS.

This study was conducted to establish a propagation system by shoot tip culture of Hosta plantaginea ‘Joseon’, of which production has increased as landscape plants and cut foliage. We evaluated the effects of culture methods (drum shaker in liquid, filter paper bridge in liquid and solid media), media (1/2MS , 1MS, 2MS), cytokinin (BA, kinetin, TDZ), and light quality (fluorescent lamp, red LED, blue LED, red LED + blue LED) on shoot multiplication and rooting. In the treatment by culture methods, the highest number of shoots (2.4 shoots per explant) was obtained on liquid media with drum shaker (BA 4.0 mg·L-1+ NAA 0.2 mg·L-1) and solid medium (BA 4.0 mg·L-1+ NAA 0.2 mg·L-1), but the shoots showed vitrification symptoms in liquid media. In media treatment, underground growth was good in 1/2MS and 1MS: overground growth was good in 1MS and 2MS but shoot length of 2MS tended to be shortened. In the treatment by cytokinin, kinetin 2.0 mg·L-1+ NAA 0.2 mg·L-1 and k inetin 4.0 mg·L-1+ NAA 0.2 mg·L-1 showed the highest number of shoots (3.4 and 3.3 shoots per explant, respectively). In the treatment by light qualities, red LED showed the highest number of shoots, shoot length, leaf number, fresh weight, dry weight, and leaf area. Red LED enhanced shoot length elongation, but chlorophyll content was lower. The content of chlorophyll was highest in blue LED, but the number of the shoots was lower. Shoots were rooted easily on basa MS medium without NAA, showing 100% of rooting. As a result, it might be considered that roughly 6,561 plants can be produced through subculture for one year in vitro from only one shoot tip of H. plantaginea ‘Joseon’ in MS semi-solid medium with BA 4.0 mg·L-1+ NAA 0.2 mg·L-1 or with kinetin 4.0 mg·L-1+ NAA 0.2 mg·L-1.

금(Au)나노물질은 광학 및 바이오 분야에서 다양하게 응용될 수 있을 뿐만 아니라 식품 농작물 재배에 무기비료제 로 사용 시 기능성 및 품질 향상에 긍정적인 영향을 줄 수 있는 것으로 알려지면서 금 나노입자를 이용한 건강기능식 품 또는 기능성 농작물 재배에 대한 연구개발이 진행되고 있다. 따라서 식품 농작물 잔류와 체내 흡수 가능성에 따른 식품 및 생체 내에서의 정확하고 정밀한 분석법 확립과 안전성검증이 요구된다. 본 연구에서는 유도결합플라즈마 질 량분석기(ICP-MS)를 이용한 금 나노입자의 정량 분석을 확립하고자 전처리 방법을 최적화하고, 회수율, 정확성 및 정 밀성을 분석하여Au의 분석법을 확립하였다. 확립된 Au 정량 분석법을 이용하여 생체시료 내에서 금 나노입자의 정량 분석을 수행하여 생체 매트릭스의 영향을 확인하였다. 이러한 분석법을 바탕으로 장내 micro fold (M) 세포를 모사한 follicle associated epithelium (FAE) 모델과 일반 장관 상피세포 조건을 모사한 Caco-2 monolayer 모델을 이용하여 금 나노입자의 유입량을 비교 연구하였다. 그 결과, 생체 매트릭스 존재에 따른 금 나노입자 정량분석에 미치는 영향은 없는 것으로 나타났으며, 금 나노입자는 장관 상피세포 내 M 세포에 의해 에너지 의존적 endocytosis 기작에 의해 체 내 유입될 수 있는 것으로 확인되었다. 이러한 식품 농작물 무기비료로서 금 나노입자의 식품 및 체내 분석법 확립연 구 및 체내 흡수기작 규명은 추후 안전성 평가에 기초자료로 활용될 수 있을 것이다.

Carbon source, an essential nutrient for plant growth, mainly includes exogenous sugar and CO2 of the environment in vitro. Therefore, the exogenous sugar and CO2 of the environment make the important roles in tissue culture. The aim of this study is to investigate the effects of different sugar concentrations (0, 10, 15 and 30 g·L-1) on the growth of colored Zantedeschia in vitro under certain CO2 concentration and explore the optimal sugar concentration. The plantlets in vitro of colored Zantedeschia had the largest root number, root weight, and root vigor under 0 g·L-1 (sugar-free culture) treatment. And they had the largest plant height, leaf length and leaf chlorophyll content, but p oor r oot v igor under 3 0 g·L-1 sugar. This study indicated that the optimal condition for proliferation and seedling culture of colored Zantedeschia plantlets in vitro was MS medium with 30 g·L-1 sugar, and the suitable medium for rooting culture and transplanting of colored Zantedeschia was MS medium with sugar-free culture under CO2 enrichment condition.

The objectives of the present study were to select an effective basic medium including its hormone and protein supplementation for IVM of oocytes of indigenous zebu cows. The ovaries of cows were collected from slaughter house and the follicular fluid was aspirated from 2 to 8 mm diameter follicles. The COCs with more than 3 cumulus cell layers and homogenous cytoplasm were selected for maturation. The oocytes were matured in media for 24 hrs at 39℃ with 5% CO2 in humidified air. The maturation of oocytes was evaluated by examining the presence of first polar body under microscope. An efficient basic medium was determined after culturing COCs in either TCM 199 or SOF medium in Experiment 1. An efficient hormone supplementation was determined after culturing COCs in either FSH or gonadotrophin supplemented TCM 199 in Experiment 2. An efficient protein supplementation was determined after culturing COCs in either FBS or Oestrous cow serum (OCS) supplemented TCM 199 in Experiment 3. The oocyte recovery rate per ovary was 3.35. The overall rate of IVM was 74.6%. The maturation rate was 75.5±3.9 and 62.2±20.2% in TCM and SOF medium, respectively (P>0.05). The maturation rate of oocytes was significantly higher (76.6±13.2%) in FSH supplemented medium than gonadotrphin supplemented counterpart (69.7±10.8%) (P<0.05). The maturation rates of oocytes were 81.7±12.9 and 85.7±12.7% in medium supplemented with FBS and OCS, respectively (P>0.05). In conclusions, both TCM 199 and SOF supplemented with either FBS or OCS, and FSH may be used as medium for IVM of indigenous zebu oocytes in Bangladesh.

In vitro culture of murine embryos is an important step for in vitro production systems including in vitro fertilization and generations of genetically engineered mice. M16 is widely used commercialized culture media for the murine embryos. Compared to other media such as potassium simplex optimization medium, commercial M16 (Sigma) media lacks of amino acid, glutamine and antibiotics. In the present study, we optimized M16 based embryo culture system using commercialized antibiotics-glutamine or amino acids supplements. In vivo derived murine zygote were M16 media were supplemented with commercial Penicillin-Streptomycin-Glutamine solution (PSG; Gibco) or MEM Non- Essential Amino Acids solution (NEAA; Gibco) as experimental design. Addition of PSG did not improved cleavage and blastocyst rates. On the other hand, cleavage rate is not different between control and NEAA treated group, however, blastocyst formation is significantly (P<0.05) improved in NEAA treated group. Developmental competence between PSG and NEAA treated groups were also compared. Between two groups, cleavage rate was similar. However, blastocyst formation rate is significantly improved in NEAA treated group. Taken together, beneficial effect of NEAA on murine embryos development was confirmed. Effect of antibiotics and glutamine addition to M16 media is still not clear in the study.

The aim of the present study was to compare two different serum-free media, modified synthetic oviduct fluid (mSOF) and modified potassium simplex optimization medium (mKSOM) containing 20% RD (RPMI1640 + DMEM, 1:1 v/v) (RD-mKSOM), for in vitro culture (IVC) of bovine embryos. After in vitro maturation and fertilization, the presumptive zygotes were cultured in two different serum-free conditions for 7 days and 9 days to evaluate blastocyst formation and hatching, respectively. Serum supplemented conventional CR2 medium was used as control. After 7 day of culture, there was no significant difference in cleavage and blastocyst formation rates among three groups (mSOF, 59.3 and 30.1%; RD-mKSOM, 65.0 and 41.5%; control, 51.6 and 38.0%, respectively). Hatching rate was significantly higher in control (69.0%) than other experimental groups (mSOF, 22.0%; RD-mKSOM, 39.5%) (P＜0.0001 and P＜0.001, respectively). Although both serum-free conditions showed lower hatching rates than serum-added control, in serum-free groups, RD-mKSOM showed significantly higher hatching rate than mSOF (P＜0.001). In addition, one-step using RD-mKSOM may facilitate IVC procedure than two-step culture system. In conclusion, the results indicate that one-step RD-mKSOM is more suitable defined culture system for IVC of bovine embryos than two-step mSOF.

The objective of this study was to investigate the changes of oxidative stress and antioxidant enzyme during in vitro development with washing culture oil in porcine embryos. During the culture, the four types of culture oil such as paraffin oil with or without washing and mineral oil with or without washing were examined. The oil was washed with PZM-3 during 7 days and collected oil only. The embryos were stained with CellTrackerTM Red, DC-FDA and Hoechst 33342 to confirm the effects of the oil. As a results, Cleavage rates and total cell number were no difference among the four oil groups. However, ≥16 cell embryos were significantly different in fore type oil treat-ment and blastocyst rate was significantly higher washing paraffin treatment than in other group(p<0.05). Also, the expression of free radical were lower in washing paraffin oil than in other groups (p<0.05). On the other hand, the expression of glutathione were not significant different among paraffin oil with or without washing and mineral oil with or without washing, however washing paraffin oil and washing mineral groups were higher than other treat-ment groups. In conclusion, the washing oil was expected with positive effects on in vitro development in porcine embryos.

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), BaCl2, 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.

본 연구는 포화지방산과 불포화 지방산 첨가가 반추위 발효성상과 효소활력에 미치는 영향을 알아보기 위하여 Piromyces communis 곰팡이를 이용하여 in vitro 발효 시험을 실시하였다. 시험 Ⅰ과 Ⅱ의 대조구에는 지방산을 첨가하지 않았고, 처리구는 시험Ⅰ에서 palmitic acid, stearic acid, oleic acid를 첨가하였고, 실험 Ⅱ에서는 linoleic acid, linolenic acid, arachidonic acid를 첨가하였다. 두 실험 모두 39℃에서 7일간 (2, 3, 4, 6 및 7일) 실시되었다. 시험 Ⅰ의 가스발생량은 불포화 지방산인 oleic acid에서 가장 많이 생성되었고 H2가스의 비율은 가장 낮았다. 시험 Ⅱ의 linoleic acid 첨가구에서 2일에서 6일까지의 가스 발생량은 대조구에 비해 높았지만 이후에는 낮았다. 수소가스 생성비율은 linoleic acid 첨가구에서 가장 많았고 linolenic acid 첨가구에서 가장 낮았다. 또한 시험 Ⅰ의 pH는 6.63에서 5.85로, 시험 Ⅱ에서는 6.59에서 5.40으로 낮아졌다. Carboxymethylcellulase 활력은 발효시간(day) 경과에 따라 증가하는 경향이었고, 특히 arachidonic acid 첨가구의 증가폭이 컸다. 본 실험의 결과는 반추동물의 사료에 불포화지방산을 첨가하면 반추위곰팡이가 생성하는 수소를 불포화 지방산이 이용하여 메탄생성 억제에 도움이 될 것으로 기대된다.

Recent 2 decades, including in vitro maturation (IVM), assisted reproductive technologies (ARTs) achieved noteworthy development. However the efficiency of ARTs with in vitro matured oocytes is still lower than that with in vivo oocytes. To overcome those limitations, many researchers attempted to adapt co-culture system during IVM and consequently maturation efficiency has been increased. The beneficial effects of applying co-culture system is contemplated base on communication and interaction between various somatic cells and oocytes, achievement of paracrine factors, and spatial effects of extracellular matrix (ECM) from somatic cell surface. The understanding of co-culture system can provide some information to narrow the gap between in vitro and in vivo. Here we will review current studies about issues for understanding cu-culture system with various somatic cells to improve in vitro maturation microenvironment and provide bird view and strategies for further studies.

Spermatogonial stem cells (SSCs) developed into sperms through spermatogenesis have been utilized as a useful tool in the field of regenerative medicine and infertility. However, a small number of highly qualified SSCs are resided in the seminiferous tubule of testis, resulted in developing effective in-vitro culture system of SSCs for solving simultaneously quantitative and qualitative problems. Presently, SSCs can be enriched on testicular stromal cells (TSCs), but there are no systematic researches about TSC culture. Therefore, we tried to optimize culture condition of TSCs derived from mouse with different strains. For these, proliferation and viability were measured and compared by culturing ICR outbred or DBA/2 inbred mouse-derived TSCs at 35 or 37℃. In case of ICR strain, primary TSCs cultured at 37℃ showed significantly higher proliferation and viability than those at 35℃ and significant increase of proliferation and viability in sub-passaged TSCs was detected in the 35℃ culture condition. Moreover, sub-passage of primary TSCs at 35℃ induced no significant effects on proliferation and viability. In contrast, in case of DBA/2 strain, significantly improved proliferation were detected in the primary TSCs cultured at 35℃, which showed no significant difference in the viability, compared to those at 37℃. Furthermore, sub-passaged TSCs cultured at 37℃ showed no significant differences in proliferation and viability, compared to those at 35℃. However, with significant decrease of proliferation induced by sub-passage of primary TSCs at 35℃, no significant effects on proliferation and viability were resulted from sub-passage of primary TSCs at 37℃. From these results, culture temperature of primary TSCs derived from outbred and inbred strain of mouse could be separately optimized in primary culture and subculture.

In order to investigate the effects of antioxidants on the culture of mouse preantral follicles in vitro, we examined the effects of taurine, glutathione and catalase on their growth and maturation. Addition of taurine was not effective on the survival of preantral follicles. However, metaphase II rates of oocytes within preantral follicles were signifi-cantly higher in 1 mM treated group than in control and 10 mM treated group (p<0.05). Glutathione did not improved the rates of survival and metaphase II oocytes. However, metaphase II rates of oocytes progressively decreased with increasing glutathione concentration. Catalase also showed that the rates of survival and metaphase II oocytes pro-gressively decreased with increasing concentration. Especially, all of preantral follicles cultured in medium containing 100 IU/ml catalase were degenerated. These results suggest that low concentraion of taurine, as an antioxidant, have positive effect on the culture of mouse preantral follicles in vitro.