Post-ejaculation of sperms into the female reproductive tract, acquisition of sperm capacitation is an essential step in the fertilization process. Accordingly, during in-vitro fertilization, the successful fertilization requires necessarily induction of capacitation in the retrieved sperms. To date, many candidate substances have been considered as capacitation inducers. However, there were no reports about the comparison of efficiency inducing sperm capacitation among diverse capacitation inducers. Therefore, we tried to determine an inducer showing the best capacitation performance in mouse sperms by comparing the preimplantation development of in-vitro-fertilized embryos using sperms experiencing capacitation by a variety of capacitation inducers. For these, calcium, progesterone, bovine serum albumin (BSA), heparin, lysophosphaticylcholine (Lyso-PC) were used as candidate capacitation inducers. Optimized concentration of each inducer were determined by accessing ratio of sperms experiencing acrosome reaction using coomassie G-250 blue staining. Subsequently, in vitro fertilization was performed using sperms incubated in each optimized concentration inducer. The ratio of fertilized oocytes was observed. As the results, Calcium at 2.7 mM and 0.3% (w/v) BSA showed the highest fertilization rates compared to 15 μM progesterone, 50 mM heparin, and 100 μM Lyso-PC. From these results, we found that 2.7 mM calcium and 0.3% (w/v) BSA were the most effective sperm capacitation inducers of mouse sperm for in vitro fertilization. From these results, we could identify that, among diverse sperm capacitation inducers, 2.7mM calcium and 0.3% (w/v) BSA were the most effective inducers for in vitro fertilization.

The objective of this study was to establish the effect of post-activation treatment with cytoskeletal regulators of CB, CB+CHX, CB+DC, CB+6’DMAP on embryonic development of pig oocytes after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT). PA and SCNT embryos were produced by using in vitro matured pig oocytes and treated for 4 h after electric activation with cytochalasins B (7.5 μg/ml), CB+cycloheximide (10 μg/ml), CB+demecolcine (0.4 μg/ml), and CB+2mM 6-Dimethylaminopurine. Post-activation treatment of PA oocytes with CB, CB+CHX, CB+DC and CB+6’DMAP no significant differences were found in embryo cleavage (83.2～91.5%), mean cell number of blastocysts (40.6～ 42.3% cells/blastocyst) but significantly (P<0.05) differences blastocyst formation (28.6～36.4%). When PA oocytes were treated with CB, CB+CHX, CB+DC, CB+6’DMAP, blastocyst formation was significantly (P<0.05) improved by CB (36.6%) compared to CB+CHX (30.9%), CB+DC (28.6%) and CB+6’DMAP (35.2%). In SCNT, was not significantly (P<0.05) increased by post-activation treatment with CB+CHX (81.3%), CB+DC (83.9%) and CB+6’DMAP (90.0%) compared to CB (84.5%) on embryo cleavage, blastocyst formation (19.1%~23.6%) and blastocyst cell number (39.6～41.4% cells/blastocyst) also were not influenced. But increased tendency in CB+6’DMAP. In addition, we investigated survivin expression in porcine SCNT embryos during the early developmental stages. The levels of survivin mRNA in 2-4 cell stage SCNT embryos were significantly higher 6’DMAP treated group than other treatment groups of SCNT embryos. These observations suggested that 2-4 cell cleaving embryos at have high developmental competence, and which may be influenced by survivin expression in porcine SCNT embryos. Our results demonstrate that post-activation treatment with CB, CB+CHX, CB+DC, CB+6’DMAP improves pre-implantation development of SCNT embryos and the stimulating effect of cytoskeletal modifiers on embryonic development is differentially shown depending on the origin (PA or SCNT) of embryos in pigs.

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.

Growth differentiation factor 8 (GDF8) is a member of the transforming growth factor-β that has been identified as a strong physiological regulator. The purpose of this study is to investigate the effects of GDF8 on porcine oocytes during in vitro maturation (IVM). We investigated a specific gene transcription levels in oocytes and cumulus cells (CC) after IVM by realtime PCR arry, and specific protein expression and activation levels in matured CCs by western blotting. Each concentration (0, 1, 10, and 100 ng/ml) of GDF8 was added in maturation medium (TCM199) during process of IVM. Data were analyzed by ANOVA followed by Duncan using SPSS (Statistical Package for Social Science). Data are presented as the mean and Differences were considered significant at P < 0.05. After 44 h of IVM, oocytes are mechanically denuded from CCs with 0.1% of hyaluronidase, and then the separated oocytes and CCs were sampled following each group. To assess the effect of GDF8 on specific gene transcription level changes as a dose response during IVM, the realtime PCR array was performed. In CCs the 1- and 10 ng/ml of GDF8 supplement group showed the transcription co-factors CBP and SP1, cell metabolic regulator MAPK1, and cumulus expansion related genes Has2, Cox-2, Ptx3 and Areg transcription levels were significantly distinguished with control when hierarchically clustered by Euclidean distance with average linkage method after IVM. In matured oocytes the 10- and 100 ng/ml of GDF8 supplement group showed the maternal factors JMJD3 and Zar1, transcriptional regulator FOXO1, Sirt1 and Sirt2, mitochondrial activity factor Sirt3, ACSL3 and ACADL, anti-apoptosis gene BCL-2, and oocyte secrete factor BMP15 mRNA transcription levels were significantly distinguished compared with control. To determine effect of GDF8 supplement during IVM, the GDF8 down steam canonical regulator SMAD2/3 protein phosphorylation levels analyzed in CCs by western blotting. The 10- and 100 ng/ml supplement groups showed significantly increase phosphorylated (P)-SMAD3 (1.56 and 1.34 times higher than control) protein levels (P < 0.05). In conclusion, supplement of GDF8 during IVM activates FOXO homolog transcription and induced cumulus cells expansion via activation of SMAD3 signaling in CCs. While process of IVM, the transcriptional landscape changes in CCs may consequently result maternal factors accumulation and mitochondrial activation in oocytes.

Several species show low sensitivity to IVM, and the development of optimized medium possible oocyte quality and stable growth. Furthermore, adding additive to the medium can effectively reducing development cost and leads to easy handling of oocytes. Isoliquritigenin and formononetin are extracts found in licorice. Previous studies reported that isoliquritigenin and formononetin affected the activity of sperm, but the oocytes are unknown. This study adds isoliquritigenin or formononetin to αMEM to mature oocytes under simple IVC conditions. Recovered oocytes are cultured in αMEM, isoliquritigenincontaining medium and formononetin-containing medium. In study we proved that in addition to the medium, above the quality of oocytes cultured when specific additives were added, more stable growth is possible. collection and IVM of oocyte. SD rats at 6 to 8 wks of age are injected is intraperitoneal with 30 IU/mL of PMSG and 48 hrs later, HCG 50 IU/mL is intraperitoneal injected. Oocytes are collected ovary after 17 hrs. Collected oocytes are cultured for 16 hrs with 200 μL αMEM and 200 μL αMEM containing isoliquritigenin or formononetin at 0, 0.01, 0.02, 0.04, 0.1 mg/mL. Also, isoliquritigenin and formononetin were mixed with 200 μL αMEM at a ratio of 0.25: 0.75, 0.50: 0.50, and 0.75: 0.25 mg/mL respectively. Oocytes supplemented with isoliquritigenin and Formononetin had high quality than oocytes cultured with αMEM and showed an increase in the IVF fertility rate. Our experimental results indicate that using isoliquritigenin, formononetin when cell culture, rather than used only in medium, more effective oocyte quality and stable growth.

In vitro maturation (IVM) systems have become indispensable for the production of large numbers of competent oocytes in domestic species. The quality of in vitro matured oocyte is one of the important factors determining the success of assisted reproductive technologies (ARTs) including intracytoplasmic sperm injection (ICSI), in vitro fertilization (IVF), and somatic cell nuclear transfer (SCNT) in human and livestock. Incomplete cytoplasmic maturation of oocytes can lead not only to a failure of fertilization but also to a developmental arrest after ARTs. Thus, establishment of a stable IVM system to produce a large number of high quality oocytes, especially in domestic animals, is essential for improvement of ARTs efficiency by producing high quality embryos. The morphological characteristics are commonly used to predict the developmental potential of oocytes and embryos. Usually, normal oocytes shrink when exposed to a hypertonic medium, and recover their morphology when returned to an isotonic medium. During this process, oocytes show various morphologic changes, such as shrinkage in spherical (SSP) or irregular shapes (SIR). In the first study, we investigated whether the shrinkage pattern of oocytes that was observed after hyperosmotic treatment could be used as a morphologic characteristic to predict the quality of IVM oocytes in pigs. We found that SSP oocytes showed improved developmental competence after PA and SCNT. This improved embryonic development was most likely because of the more advanced nuclear and cytoplasmic maturation in SSP oocytes compared with SIR oocytes. Pig oocytes shows a wide variation in the size of perivitelline space (PVS) after IVM. Based on this finding, we examined in the second study whether or not there was any correlation between the PVS size of IVM oocytes and their developmental competence after PA and SCNT. Our results demonstrated that in vitro developmental competence to the blastocyst stage positively correlated with the size of the PVS of oocytes after IVM. In addition, we observed that mature oocytes with a larger PVS showed higher levels of intracellular GSH content and transcription factor expression. Furthermore, enlargement of the PVS by culturing in reduced NaCl medium improves the embryonic development after PA and SCNT. In the third study, we investigated the effects of a hypotonic medium with reduced NaCl (61.6 mM) compared with an isotonic medium (108.0 mM NaCl) on oocyte maturation and embryonic development after PA and SCNT. In addition, we attempted to optimize our IVM system using a hypotonic maturation medium by examining the effects of hypotonic medium during various stages of IVM on oocyte maturation and subsequent embryonic development. Our results demonstrated that maturation of pig oocytes in hypotonic medium with reduced NaCl during the last 11 hr of IVM increased the developmental competence of oocytes after PA and SCNT. These beneficial effects was also shown in a commercial medium (a minimum essential medium; aMEM) in which the NaCl concentration was reduced to 61.6 mM. In addition, IVM of oocytes in medium with reduced NaCl increases the proportion of SSP oocytes in pigs. In summary, our results demonstrate that IVM of pig oocytes in a hypotonic medium with low-NaCl is better able to support embryonic development after PA and SCNT, most likely by improving the cytoplasmic maturation via increased intraoocyte GSH content and widened PVS. Based on these results, the newly developed IVM system using a hypotonic medium with reduced NaCl can produce high quality oocytes and be considered a new strategy for improving ARTs efficiency in pigs.

The gastro-intestinal behaviors of foods influence their physiological functions in the human body. In-vitro methods simulating digestion processes have been extensively used to study the gastro-intestinal behaviors of foods due to more rapid and less expensive advantages. However, there is a lack of systematic studies to monitor the rheological changes of the food digesta in real time. In this study, rice-based products (specifically, extruded noodles) were prepared with three varieties of rice flours with different contents of amylose and their physicochemical properties and in-vitro digestibility were then characterized from a rheological point of view. The rice flours with higher amylose contents exhibited greater stability to dual mixing and higher degrees of starch gelatinization and retrogradation in thermo-mechanical measurements. In addition, greater elastic properties were clearly observed in the high amylose rice samples. The noodles which were produced with high amylose rice flour had a harder texture and reduced cooking loss. When the rheological changes of the extruded rice noodles were monitored in real time during the in-vitro starch digestion, the rice noodle digesta with higher amylose content exhibited greater viscosities throughout the simulated oral-gastric-intestinal digestion steps. The flow behavior of the rice noodle digesta consisted of the Power-law region and infinite shear plateau that were satisfactorily characterized by the Sisko model (R2>0.99). Hence, this study was conducted to investigate the physicochemical and in-vitro digestibility of extruded rice noodles with different amylose contents. These results can provide a promising opportunity for the food industry to research in-vitro digestion and physicochemical characteristics of rice-based products.

The aim of this study was to use an in vitro method and estimate glycaemic index (GI) from porridges to determine the digestibility of porridges. Glycaemic index’s concept is to classify foods on the basis of their postprandial blood glucose response. The GI of a foodstuff is generally measured by determining the increment in blood glucose concentration after the consumption of a test meal over a set period of time and comparing it with an isoglucosidic control meal (normally white bread or glucose) and expressed as a percentage. In this study, the 5 porridges were studied for their starch digestibility. The available starch contents of the samples varied from 65~85 g/100 g dry solids. From in vitro digestion, the porridge samples were Medium glycaemic index foods with calculated GIs ranging from 56 to 67.

Eight individual and blended chemical disinfectants were screened for preliminary evaluation of efficacy against duct organisms. Viable counts of surviving bacteria were determined after 30 min contact with each disinfectant. The single and blended disinfectants with high microcidal activity included glutaraldehyde/formaldehyde-based, glutaraldehyde/formaldehyde-based, cresol-based, organic acid-based and potassium peroxymonosulphate-based products. Iodophors of disinfectants showed an inconsistent and low anti-microbial effect.

This study was to research the relationships between rice straw degradation and changes of fibrolytic bacteria population during the in vitro rumen fermentation. Dry matter(DM) digestion of rice straw and population of fibrolytic bacteria were measured at the 0. 4, 8, 12 and 48 hours during the incubation. The populations of F. succinogenes. R. albus and R. flavefaciens were defined as log copy number of 16S rDNA by technical method of Quantitative real-time PCR. Total population of F. succinogenes, R. flavefaciens and R. albus was sum of bactera attached on rice straw and suspended in medium. It's population was increased with incubation, reached top level of 29.0 Log copy No at the 24 hour and then decreased. In the meantime, DM digestion of rice straw showed the higher increasement from the 8 hour to the 24 hour than from the 0 hour to the 8 hour, and then a slowdown in increasing trend of digestibility. Attachments of F. succinogenes, R. flavefaciens and R. albus were detected immediately after start of in vitro rumen incubation. At the same time, the colonized bacterial share were respectively 34.5%, 84.4% and 67.9% in total population. All of them was reached the highest colonized bacterial share above 94.7% at the 4 hour incubation. However population of attached bacteria was shown the highest level at the 12 hour or the 24 hour incubation. Kinetics of colonization were formed area of top speed from the 12 hour to the 24 hour and respectively reached 10.33, 9.28 및 8.30 Log copy No/h/g DM at the 24 hour by F. succinogenes, R. flavefaciens and R. albus. The kinetics of rice straw degradation was formed top level of 0.95% DM/h at the 24 hour. The present results gave clear evidence that degradation of rice straw was increased with the development of total fibrolytic bacteria in process of rumen fermentation. Also, their attachment was largely occurred immediately after insertion of rice straw, the colonized bacteria was actively proliferated, and then degradation of rice straw was maximized.

Ganglioside GD1a is specifically formed by the addition of sialic acid to ganglioside GM1a by ST3 β- galactoside α -2,3-sialyltransferase 2 (ST3GAL2). Above all, GD1a are known to be related with the functional regulation of several growth factor receptors, including activation and dimerization of epidermal growth factor receptor (EGFR) in tumor cells. The activity of EGF and EGFR is known to be a very important factor for meiotic and cytoplasmic maturation during in vitro maturation (IVM) of mammalian oocytes. However, the role of gangliosides GD1a for EGFR-related signaling pathways in porcine oocyte is not yet clearly understood. Here, we investigated that the effect of ST3GAL2 as synthesizing enzyme GD1a for EGFR activation and phosphorylation during meiotic maturation. To investigate the expression of ST3GAL2 according to the EGF treatment (0, 10 and 50 ng/ml), we observed the patterns of ST3GAL2 genes expression by immunofluorescence staining in denuded oocyte (DO) and cumulus cell-oocyte-complex (COC) during IVM process (22 and 44 h), respectively. Expression levels of ST3GAL2 significantly decreased (p<0.01) in an EGF concentration (10 and 50 ng/ml) dependent manner. And fluorescence expression of ST3GAL2 increased (p<0.01) in the matured COCs for 44 h. Under high EGF concentration (50 ng/ml), ST3GAL2 protein levels was decreased (p<0.01), and their shown opposite expression pattern of phosphorylation-EGFR in COCs of 44 h. Phosphorylation of EGFR significantly increased (p<0.01) in matured COCs treated with GD1a for 44 h. In addition, ST3GAL2 protein levels significantly decreased (p<0.01) in GD1a (10 μM) treated COCs without reference to EGF pre-treatment. These results suggest that treatment of exogenous ganglioside GD1a may play an important role such as EGF in EGFR-related activation and phosphorylation in porcine oocyte maturation of in vitro.

목 적 : 경피적 방사선 위루술(percutaneous radiologic gastrotomy, PRG)에서 위고정(gastropexy)을 위해 사용되는 T-fastener에 대해 자기장에 의한 수평인력, 회전력을 표준방법에 의해 평가하고, 돼지고기 모델을 이용하여 환자에게 사용되었을 때의 안전성에 대해 평가하였다. 대상 및 방법 : T-fastener는 Cope(Cook, Bloomington, USA)와 Saf-T-Pexy(Kimberly-Clark, Roswell, USA)모델을 사용하였으며, 길이는 각각 21 mm, 10 mm이다. MRI장치는 1.5 T(Avanto MRI, Siemens, Germany)와 3.0 T(Ingenia, Philips Medical System, Netherland)을 사용하였다. T-fastener에 대한 자기유도 수평인력은 주자장이 가장 큰 지점인 96cm지점에 측정장치를 위치하여 최대 변위각을 3회씩 측정하였고, 회전력은 원형용기 내부의 45°간격의 실선 위에 놓았 을 때 나타난 회전형태를 정성적 평가기준으로 측정하였다. 돼지고기 삽겹살에 T-fastener를 설치하여 본원의 복부 MRI sequence로 1.5 T와 3.0 T에서 scan 후 움직임 또는 회전상태를 관찰하였다. 결 과 : 자기유도 수평인력의 측정결과 변위각의 평균값은 Cope 제품의 경우 1.5 T와 3.0 T에서 각각 139.3°, 145.8°로 나타났고, Saf-T-pexy제품은 각각 131.3°, 141.7°로 나타났다. 자기유도 회전력의 평가결과 회전력은 두 제품 모두 1.5 T와 3.0 T에서 +4 (very strong torque), 즉 주자장에 대해 매우 빠르게 정렬되었다. 돼지고기 모델에서 1.5 T와 3.0 T에서 Scan 후 움직임 또는 회전상태는 scan 전과 후의 변화가 없었다. 결 론 : 이번 연구에서는 T-fastener에 대한 1.5 T와 3.0 T에서의 MR 안전성을 수평인력과 회전력을 통해 평가했으며, 두 제품 모두 변위각 130° 이상, 회전력 +4 이상 나타났다. 하지만 돼지고기 모델을 이용한 시험에서 충분한 유지력이 평가되어 MR환경에 사용되어도 환자안전에 영향이 없을 것으로 사료된다.

The oocyte undergoes various events during In vitro maturation (IVM) and subsequence development. One of the events is production of reactive oxygen species (ROS) that is a normal process of cell metabolism. But imbalances between ROS production and antioxidant systems induce oxidative stress that negatively affect to mammalian reproductive process. In vitro environments, In vitro matured oocytes have many problems, such as excessive production of ROS and imperfect cytoplasmic maturation. Therefore, In vitro matured oocytes still have lower maturation rates and developmental competence than in vivo matured oocytes. In order to improve the IVM and In vitro culture (IVC) system, antioxidants, vitamins were added to the IVM, IVC medium. Antioxidant supplementation was effective in controlling the production of ROS and it continues to be explored as a potential strategy to overcome mammalian reproductive disorders. Based on these studies, we expect that the use of antioxidants in porcine oocytes could improved maturation and development rates.

We report the use of carrot, a new and inexpensive biomaterial source, for preparing high quality carbon dots (CDs) instead of semi-conductive quantum dots for bioimaging application. The as-derived CDs possessing down and up-conversion photoluminescence features were obtained from carrot juice by commonly used hydrothermal treatment. The corresponding physiochemical and optical properties were investigated by electron microscopy, fluorescent spectrometry, and other spectroscopic methods. The surfaces of obtained CDs were highly covered with hydroxyl groups and nitrogen groups without further modification. The quantum yield of as-obtained CDs was as high as 5.16%. The cell viability of HaCaT cells against a purified CD aqueous solution was higher than 85% even at higher concentration (700 μg mL−1) after 24 h incubation. Finally, CD cultured cells exhibited distinguished blue, green, and red colors, respectively, during in vitro imaging when excited by three wavelength lasers under a confocal microscope. Offering excellent optical properties, biocompatibility, low cytotoxicity, and good cellular imaging capability, the carrot juice derived CDs are a promising candidate for biomedical applications.

This study was determined optimal condition as culture medium, pH, carbohydrate source and growth regulators through axillary buds cultures of Salix gracilistyla. S. gracilistyla shoots were obtained from axillary bud on 1/2MS basal medium after 2 weeks of culture. Highest shoot growth was showed in the MS medium(GI= 2.43). Shoot growth was highest in 9% sucrose compared to other concentration. BAP affects axillary shoot multiplication in of S. gracilistyla. Among nutrients, Ca2+ was not affected shoot number of S. gracilistyla. However, NH4+, NO3 -, and K+ is greatly influenced shoot number. However, Ca2+ was affected in shoot length of S. gracilistyla. Feeding of Mg2+ was not affected in shoot length. Root number is greatly influenced by NH4 +, NO3 -, and K+, however NH4 + and K+ was not affected root number of S. gracilistyla. NH4 + was affected root length, however Mg4 + was not affected root length. Treatment of 0.1mg/L IBA promoted in vitro rooting. In vitro rooted plantlets were successfully transferred to pots for 4weeks hardening process. Later, these plants were transferred to soil with above 90% survival rate in BS+SD(2:1). These results could be easily adopted for commercial large scale cultivation.

하수오(Polygonum multiflorum Thunb.)는 여뀌과에 속하는 초본식물로 동아시아와 북아메리카에 자생하고 있다. 하수오는 관상용으로도 이용되며, 한의학에서 뿌리를 약용으로 사용한다. 하지만 월동 이 어렵고 노동력 소모가 많아 최근 재배농가가 감소하고 있다. 본 연구에서는 하수오의 우량종묘를 짧 은 시간에 대량생산하기 위하여 수경재배에 적합한 양액과 조직배양묘의 크기를 조사하였다. 토양에 적 응이 완료된 하수오 종묘는 크기에 따라 Type 1, Type 2 및 Type 3으로 나누어 4종류의 양액에 치상 하였다. 치상 후 양액과 종묘에 따른 생육차이를 확인하기 위하여 생육조사를 실시하였다. 생육조사는 8주 동안 양액에서 생육시킨 후 줄기길이, 잎 수, 마른 잎 수, 줄기 수, 생체중과 건물중에 대해 측정 하였다. 그 결과 양액3에서 Type 1과 Type 2의 뿌리길이는 각각 33.8cm와 29.5cm로 각각 나타났다. 이와 유사한 경향으로 덩굴 길이도 CaCl2가 첨가 된 양액3에서 Type 1과 Type 2가 가장 길게 나타나 우수한 생육을 보였다. 이처럼 뿌리가 증대된 종묘는 밭으로 이식 될 때 활착을 도와 우수한 초기생육 을 나타낼 것이라 사료된다. 반면 Type 3는 잎끝마름과 고사하는 현상을 보여 추후 양액의 적정농도에 대한 추가 실험이 필요할 것이라 사료된다.

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.

NNK (4-(methylnitrosamino)―1-(3-pyridyl)-1-butanone) is a major form of nitrosamine abundant in cigarette smoke and is a powerful carcinogen. Mercury is a major component of the amalgam that is widely used as dental filling material. Concurrent exposure to these two agents may result in their interaction and alter their carcinogenic potential. The present study used an immortalized human epithelial cell system that allows continuous exposure to potential carcinogens, in an attempt to elaborate the carcinogenic potential of mercury and NNK in humans. Cytotoxicity of mercury chloride and NNK was measured by an MTT assay. Parameters of neoplastic cellular transformation such as cell saturation density, soft-agar colony formation, and cell aggregation were analyzed to examine the carcinogenic potential of mercury chloride and NNK. The study showed that exposure to mercury chloride with NNK resulted in increased soft agar colony formation and cell aggregation. ROS generation by mercury chloride was further enhanced by treatment with NNK. The apoptosis that was observed following mercury chloride exposure was further increased upon co-treatment with NNK. The interaction between these two agents was also observed in cytokine mRNA induction. In the present study, mercury alone did not seem to pose a significant threat as a carcinogen, but it may have potential to enhance the carcinogenic potential of a known carcinogen from cigarette smoke. The present study provides valuable data regarding the evaluation of potential carcinogenic risk of mercury chloride and NNK on concurrent exposure.