Rural Development Administration Research Articles

154 THE EFFECT OF ZINC ON PORCINE IN VITRO MATURATION AND SUBSEQUENT EMBRYONIC DEVELOPMENT AFTER IN VITRO FERTILIZATION

Zinc (Zn) is one of the abundant transition metals in biology and is an essential component of most cells. However, there are few reports about the effect of Zn in porcine oocytes. The objective was to investigate the effects of supplementary Zn during in vitro maturation (IVM) of porcine oocytes. We investigated nuclear maturation, intracellular glutathione (GSH) levels, reactive oxygen species (ROS) levels, and subsequent embryonic development after IVF. Before the experiment, Zn concentrations in IVM medium and body fluids were measured using inductively coupled plasma spectrophotometer (sensitivity: 1 μM) and treatment concentrations were determined. Zinc concentration was 12.6 μM in porcine plasma and 12.9 μM in porcine follicular fluid. We confirmed that Zn was not detected in IVM medium. A total of 541 cumulus–oocyte complexes (COC) were used for the evaluation of nuclear maturation. The COC were matured in TCM-199 medium supplemented with various concentrations of Zn (0, 6, 12, 18, and 24 μM). After 44 h of IVM, no significant difference was observed in all groups (metaphase II rate: 85.7, 88.7, 90.4, 90.3, and 87.2%, respectively). A total of 100 matured oocytes were examined for the effects of different Zn concentrations (0, 6, 12, 18, and 24 μM) on porcine oocyte intracellular GSH and ROS levels, which were measured through fluorescent staining and image analysis program. The groups of 12, 18, and 24 μM showed a significant (P < 0.05) increase in intracellular GSH levels (1.45, 1.67, and 1.78, respectively) compared with the control and 6 μM group (1.00 and 1.08, respectively). The intracellular ROS level of oocytes matured with 12, 18, and 24 μM (0.82, 0.68, and 0.55) were significantly (P < 0.05) decreased compared with the control and 6 μM groups (1.00 and 1.03, respectively). Finally, the developmental competence of oocytes matured with different concentrations of Zn (0, 6, 12, 18, and 24 μM) was evaluated after IVF. There were no significantly different in cleavage rates. However, cleavage patterns and blastocyst (BL) formation were different. Fragmented embryo ratio of the 12 μM group (14.9%) was significantly lower than that of the other groups (control, 6, 18, and 24 μM: 26.4, 17.8, 18.4, and 18.0%, respectively). Oocytes treated with 12 μM Zn during IVM had a significantly higher BL formation rate (28.2%) after IVF compared with the control (19.8%). In conclusion, these results indicate that Zn treatment as body fluid concentration during IVM improved the developmental potential of IVF in porcine embryos by increasing the intracellular GSH concentration and decreasing the ROS level. This work was supported, in part, by a grant from the Next-Generation Bio Green 21 Program (No. PJ00956901), Rural Development Administration, and the National Research Foundation of Korea Grant funded by the Korean Government (NRF-2012R1A1A4A01004885, NRF-2013R1A2A2A04008751), Republic of Korea.

Development of a fatty acids database using the Korea National Health and Nutrition Examination Survey data

Purpose: The objective of this study was to develop a fatty acid database (DB) for estimation of intake levels of fatty acids in the Korean population, using data from the Korea National Health and Nutrition Survey (KNHANES). Methods: Analytical values of fatty acids in foods were collected from food composition tables of national institutions (National Fisheries Research & Development Institute, Rural Development Administration), Japan Ministry of Education, Culture, Sports, Science and Technology, US Department of Agriculture, and journal articles that previously reported analytical fatty acid content of some Korean foods. The coverage of fatty acids was C14:0, C16:0, C18:0, C18:1, C18:2 n-6, C18:3 n-3, C20:5 n-3 (EPA), C22:6 n-3 (DHA), SFA, MUFA, and PUFA (n-3, n-6, n-9). The fatty acids DB covered a total of 5,144 food items used in the KNHANES nutrition survey. The food items were preferentially filled with analytical values of the collected data source. An analytical value for each food item was selected based on the priority criteria and the quality evaluation of data sources. Missing values were replaced with calculated or imputed values using the analytical values of similar food items from the data source. Results: A total of 1,545 analytical values, 2,589 calculated values, and 1,010 imputed values were included in the fatty acid DB. The developed fatty acid DB was applied to 2,112 food items available for 2011 KNHANES data. Mean intake levels of total fatty acids and saturated fatty acids were 40.3 g/day and 13.2 g/day, respectively. The estimation of total fatty acid intake was 84.3% (men 83.2%, women 86.0%) of daily total fat intake. Conclusion: This newly developed fatty acid DB would be helpful in determining the association of fatty acids intake and related health concerns in the Korean population.

156 EFFECTS OF MORPHOLOGY TYPE OF POLAR BODY ON PORCINE OOCYTE QUALITY AND DEVELOPMENTAL POTENTIAL AFTER IN VITRO FERTILIZATION

Evaluation of morphology of first polar body (1st PB) could be a method for the oocyte's quality and developmental competence. The developmental potential of oocyte with fragmented PB after in vitro maturation (IVM) is a controversial issue. The aim of this study is to investigate the effects of PB morphology type on oocyte quality and developmental competence after IVF. Porcine ovaries were obtained from prepubertal gilts at a local slaughterhouse and transported to the laboratory within 2 h in physiological saline supplemented with 100 IU mL–1 penicillin G and 100 mg mL–1 streptomycin sulfate. The cumulus–oocyte complexes (COC) were aspirated using an 18-gauge needle attached to a 10-mL disposable syringe from superficial follicles 3 to 6 mm in diameter followed by IVM. After IVM, oocytes were classified into 3 types as follows, oocytes with normal PB (A type), oocytes with a little of fragmented PB (B type), and oocytes with separated 2 PBs (C type), respectively. As classification of PB types, we analysed the distribution ratio of each PB type after IVM, and then performed IVF for analysis of fertilization rate and developmental potential. The ratio of oocyte with A type (73%) was significantly (P < 0.05) higher than that of B type (24.5%) or C type (2.5%) after IVM. Only mature oocytes were selected from A and B type and were subjected to IVF because of a small number of oocytes with C type. In the IVF experiment, the efficiency of monospermy and fertilization were significantly higher in oocytes of A type (46.7%) than those of type B (20.0%). The cleavage rate of oocytes with A type (63.9%) was significantly (P < 0.05) higher than the oocytes with B type (43.8%). Embryonic developmental competence to the blastocyst stage after IVF was significantly (P < 0.05) higher in the A-type oocytes (26.3%) than in the B-type oocytes (16.9%). The levels of glutathione and reactive oxygen species were not affected by the morphological classification of the PB. In summary, these results suggest that polar body morphology could be a marker of oocyte quality after IVM. We are currently studying gene expression of each oocytes and blastocysts. This work was supported, in part, by a grant from the Next-Generation BioGreen 21 Program (No. PJ00956901), Rural Development Administration, and the National Research Foundation of Korea Grant funded by the Korean Government (NRF-2012R1A1A4A01004885, NRF-2013R1A2A2A04008751), Republic of Korea.

153 ZINC INSUFFICIENCY DURING PORCINE OOCYTES IN VITRO MATURATION CAUSED MEIOTIC BLOCK AND DEVELOPMENTAL FAILURE

The objective was to investigate the effects of zinc (Zn) insufficiency during in vitro maturation (IVM) of porcine oocytes. Zinc insufficiency was induced by treatment of Zn chelator, N,N,N′,N′-tetrakis-(2-pyridylmethyl)-ethylendiamine (TPEN). In experiment 1, we investigated the effect of duration of Zn insufficiency in IVM on oocytes maturation and subsequent embryonic development after parthenogenetic activation (PA). First, 10 μM TPEN was added to the IVM medium for 0, 7, 15, or 22 h. After TPEN treatment, 10 μM Zn were supplemented on IVM medium except in the 0 h group. Reductions in the nuclear maturation rates were dependent on TPEN duration. The 0-h-treated oocytes showed 83.9 ± 3.9% metaphase II (MII) rate; the 7-h-treated oocytes had significantly lower MII rate (44.8 ± 3.0%) than 0-h-treated oocytes. The majority of 15- and 22-h-treated oocytes were arrested at metaphase I (MI rate: 98.0 ± 1.0 and 97.2 ± 1.7%, MII rate: 0 and 0%, respectively). Embryonic developmental competence was similar to maturation results. Reduction in cleavage and blastocyst (BL) rates were also dependent on duration of TPEN treatment (cleavage rate: 65.3 ± 1.4, 42.6 ± 4.8, 2.6 ± 0.1, and 3.0 ± 1.6%; BL formation rate: 29.3 ± 2.8, 9.2 ± 1.5, 0, and 0% for 0, 7, 15, and 22 h). Total cell number of BL was also significantly different. Total cell number of BL in the 0-h-treated group (51.4 ± 4.5) was significantly higher than that in the 7-h-treated group (23.2 ± 1.6). In experiment 2, to confirm that the Zn insufficiency caused oocyte immaturities and loss of developmental competence in TPEN-treated oocytes, we investigated nuclear maturation and subsequent embryonic development following 3 groups: (1) non treatment (control); (2) 10 μM TPEN treatment during 22 h of IVM; (3) 10 μM TPEN + 10 μM Zn treatment during 22 h of IVM. Only TPEN-treated oocytes and TPEN+Zn-treated oocytes showed contrasting results. Oocyte maturation rates and subsequent embryonic development competence in TPEN with Zn-treated oocytes were similar to control (MII rate: 93.0 ± 1.2 and 92.7 ± 1.8%, BL formation rate: 42.0 ± 6.7 and 40.0 ± 7.5% for TPEN+Zn-treated oocytes and control). These results were significantly different compared with only TPEN-treated oocytes’ results (MII rate: 0.61 ± 0.61%, BL formation rate: 0%). In conclusion, Zn is an essential element for successful oocyte maturation and embryo development in porcine. Zinc insufficiency caused meiotic block and had lasting effects on early embryo development. This work was supported, in part, by a grant from the Next-Generation BioGreen 21 Program (No. PJ00956901), Rural Development Administration, and the National Research Foundation of Korea Grant funded by the Korean Government (NRF-2012R1A1A4A01004885, NRF-2013R1A2A2A04008751), Republic of Korea.

Methemoglobinemia Caused by an Inert Ingredient after Intentional Ingestion of Pesticide

We report two cases of toxic methemoglobinemia caused by an inert ingredient in pesticide product after intentional ingestion of pesticide. First, 51-year-old male visited to the emergency department (ED) after the ingestion of pesticide in a suicide attempt. Initial methemoglobin (MetHb) level was 25.6%. We did not know the cause of methemoglobinemia at that time. Second, 56-year-old female visited to the ED after the ingestion of the same pesticide in a suicide attempt. MetHb level after 30 minutes was 16.1%. The patients were treated with methylene blue. We contacted to the Korean Rural Development Administration and estimated that magnesium nitrate was more likely to cause methemoglobinemia. This report highlights the importance of considering the possibility of methemoglobinemia caused by inert ingredient in pesticide and early antidotal therapy.

Leaf Rot and Leaf Ring Spot Caused by Rhizoctonia solani in Chinese Cabbage

Organic Agriculture Division, National Academy of Agricultural Science, Rural Development Administration, Suwon 441-707, Korea(Received on November 15, 2013; Revised on November 25, 2013; Accepted on November 26, 2013)This study was conducted to determine the occurrence of leaf rot and leaf ring spot, caused by Rhizoctoniasolani in Chinese cabbage under seedling nursery and cultivation greenhouses. Symptoms of leaf rot and leafring spot were found in three Chinese cabbage cultivars, Brassica campestris subsp. pekinensis, ‘Ryeokgwang’,‘Daetong’, and ‘CR mat’. In Hwacheon, the disease incidence was 73.8% in the seedling stage of the Chinesecabbage. In Icheon, the symptoms were observed on the upper leaves of the Chinese cabbage cultivar,‘Norangmini’ with 20.5% of disease incidence. The symptoms appeared as primary lesions consisting ofsmall, circular necrotic ring spots with gray color, 1.4 −3.0 mm in diameter, accompanied by secondary rotlesions with large irregular borders of leaves. The color of mycelial mat of 20 isolates was dark brown andlight brown. The average hyphal diameter of all the isolates was within 5.01 −11.12 µm. Among the 20 strainsisolated from Chinese cabbage, 16 isolates and four isolates anastomosed with the AG-1 (IB) and AG-1 (IC),respectively. Twenty isolates tested were only virulent on foliage parts of Chinese cabbage leaves but wereavirulent on stem parts of the plants. Based on the mycological characteristics and pathogenicity test on hostplants, the fungus was identified as Rhizoctonia solani. Keywords : Anastomosis group, Bottom rot, Brassica campestris subsp. pekinensis, Rhizoctonia solani

우리나라 벼 도열병균의 대표 균주 및 벼의 저항성 유전자형 선발

Rice Breeding and Cultivation Research Division, National Institute of Crop Science, Rural Development Administration, Iksan 570-080, Korea(Received on September 3, 2013; Revised on November 22, 2013; Accepted on November 28, 2013)Rice blast is one of the most serious disease threatening stable production of rice. Breeding of resistantcultivars has been used as the most effective and useful method to controll rice blast caused by Magnaportheoryzae. To collect rice blast isolates in fields and test their pathogenicity on new cultivars are important forestablishment of new resistant cultivars breeding program of rice. Pathotypes of Korean rice blast isolateshave been categorized to Korean differential race system developed in 1985. However, it is little known aboutgenetic background of Korean differential cultivars, so that it is hard to understand for relationship betweeneach pathogen and each host plant at genetic level. In this study, we suggested necessity of a new differentialsystem by analyzing pathogenic responses between 24 monogenic rice lines and 200 Korean rice blast isolates.In addition, we determined the nine representative resistant genes based on the resistance responses of themonogenic lines to rice blast isolates, indexed resistant responses of the monogenic lines to ten representativerice blast isolates and selected 30 Korean representative rice blast isolates proper to Korean system. We thinkthe newly developed differential race system can be broadly used to select resistant cultivars to rice blast inKorea.Keywords : Differential system, Magnaporthe oryzae, Monogenic lines, Resistance, Rice blast

식품 영양 연구, 정책, 산업의 기반이 되는 국가표준식품성분표의 활용

The National Standard Food Composition Table published by the Rural Development Administration (RDA) provides the foundations in research, nutrition monitoring, policy and dietary practices in Korea. This databases consists of several sets of data including food descriptions, nutrients, portion weights, and source of data. The National Standard Food Composition Table have been published since 1970 and, recently, new version (8th edition) of Food Composition Table which has quantitative and qualitative nutrient data is released in 2011. In addition, the User-friendly Food Composition Table is divided into adult, children, and elderly categories depending on the subjects because we need different nutrients according to various ages. The Tables of Food Functional Composition is firstly edited in 2009. RDA published the minerals and fatty acids composition table, tables of amino acid, fat-soluble vitamin composition table, and the cholesterol table. The resulting database will be widely used. The users of the databases are from diverse fields, includeing federal agencies, the food industry, health professionals, restaurants, software application developers, academia and research organizations, international organizations, and foreign governments ect. Therefore, consistent improvements of the database is important, so that people can better address such health challenges by providing reliable and accurate data.

돈분액비를 시용한 녹비보리 및 헤어리베치의 혼입시기가 벼 수량에 미치는 영향

Effect of Incorporation Times of Green Barley and Hairy Vetch on Rice Yield in Paddy Soil with Liquid Pig Manure Se-Won Kang, Dong-Cheol Seo, Sang-Gyu Lee, Young-Jin Seo, Ju-Wang Park, Jin-Hee Ryu, Min-Tae Kim, Hang-Won Kang, Jong-Soo Heo and Ju-Sik Cho (Department of Bio-Environmental Sciences, Sunchon National University, National Institute of Crop Science, Rural Development Administration, Division of Applied Life Science(BK21 program) & Institute of Agriculture and Life Science, Gyeongsang National University)

복령(Poria cocos) 재배기술의 변천과 실용화

The cultivation of Poria cocos on tree-stumps and logs, was first described at the beginning of the 13th century. Poria cocos has been used for medicinal purposes to treat physical and mental recuperation, promote diuresis, forgetfulness and physical weakening. The artificial cultivation techniques of P. cocos was reported in China in 1957. are reported. The basic study on morphological characteristics and artificial cultivation method of Poria cocos with pine tree log buried under ground were initiated by Rural Development Administration. To widen the utilization of harvested sclerotia of Poria cocos as new food stuffs, powder of the sclerotia were used for producing new soft drinks and supplemented to wheat flour to make functional bread. Last 30 years, the supply of P. cocos in Korea through cultivation could not meet the domestic demand, Korea collected P. cocos in the mountainous areas and also import the sclerotia from China. Since the domestic demand of the sclerotia in China increased, the price of imported sclerotia of P. cocos also increased. Recently, high-quality and high-yielding cultivation methods were developed in Gyeongbuk Agricultural Research and Extension Services (GARES). The new cultivation methods of P. cocos developed by GARES was to change traditional cultivation method from under ground to above ground cultivation house, and shortened cultivation period by half, and improved the quality of the sclerotia. Therefore, it is suggested that advanced new technology for production of sclerotia of P. cocos from under ground and above ground must be developed for fullfil the domestic demand of the sclerotia.

'신고'배 재배지 내 수체 및 토양의 탄소 및 질소 저장량

IPCC 2006 guideline에서 제시한 Tier 3 수준에 맞게 배나무 과수원의 온실가스 저장량을 산정하기 위하여, 전라남도 나주에 위치한 농촌진흥청 배시험장 재배포장(<TEX>$35^{\circ}01^{\prime}27.70N$</TEX>, <TEX>$126^{\circ}44^{\prime}53.50^{\prime\prime}E$</TEX>, 표고 6m)에 <TEX>$5.0{\times}3.0m$</TEX> 간격으로 재식된 Y자 수형의 15년생 배 '신고(Pyrus pyrifolia Nakai cv. Niitaka)' 재배지를 대상으로 과수와 재배지 토양의 총 탄소 및 질소 저장량을 측정하였다. 이를 위해 2012년 8월, 3반복으로 과수를 굴취하여 주간, 주지, 측지, 잎, 과일, 뿌리로 분류하였고, 과수 주간으로부터 약 0.5m 떨어진 지점에서 0.6m 깊이까지 0.1m 간격으로 토양을 채취하였다. 나무 한그루당 건물중은 주간이 4.7kg, 주지가 13.3kg, 측지가 13.9kg, 잎이 3.7kg, 과일이 6.7kg, 뿌리가 14.1kg이었다. 단위 질량당 총 탄소 및 질소의 함량은 주간에서 각 2.3kg과 0.02kg, 주지에서 6.4kg과 0.07kg, 측지에서 6.4kg과 0.09kg, 뿌리에서 6.5kg과 0.07kg, 잎에서 1.7kg과 0.07kg, 과일에서 3.2kg과 0.03kg 이었다. 재식밀도(667trees/ha)를 기준으로, 탄소와 질소는 재배지 토양에 각각 138.29 및 <TEX>$13.31Mg{\cdot}ha^{-1}$</TEX>가 저장되어 있었고, 과수에 각각 17.66 및 <TEX>$0.23Mg{\cdot}ha^{-1}$</TEX>씩 보유되어 있었다. 이를 종합한 결과 과수 재배지에서 탄소는 <TEX>$155.95Mg{\cdot}ha^{-1}$</TEX>, 질소는 <TEX>$13.54Mg{\cdot}ha^{-1}$</TEX>가 저장되어 있었다. To report country-specific carbon and nitrogen stocks data in a pear orchard by Tier 3 approach of 2006 IPCC guidelines for national greenhouse gas inventories, an experimental pear orchard field of the Pear Research Station, National Institute of Horticultural & Herbal Science, Rural Development Administration, Naju, Korea (<TEX>$35^{\circ}01^{\prime}27.70N$</TEX>, <TEX>$126^{\circ}44^{\prime}53.50^{\prime\prime}E$</TEX>, 6 m altitude), where 15-year-old 'Niitaka' pear (Pyrus pyrifolia Nakai cv. Niitaka) trees were planted at a <TEX>$5.0m{\times}3.0m$</TEX> spacing on a Tatura trellis system, was chosen to assess the total amount of carbon and nitrogen stocks stored in the trees and orchard soil profiles. At the sampling time (August 2012), three trees were uprooted, and separated into six fractions: trunk, main branches, lateral branches (including shoots), leaves, fruits, and roots. Soil samples were collected from 0 to 0.6 m depth at 0.1 m intervals at 0.5 m from the trunk. Dry mass per tree was 4.7 kg for trunk, 13.3 kg for main branches, 13.9 kg for lateral branches, 3.7 kg for leaves, 6.7 kg for fruits, and 14.1 kg for roots. Amounts of C and N per tree were respectively 2.3 and 0.02 kg for trunk, 6.4 and 0.07 kg for main branches, 6.4 and 0.09 kg for lateral branches, 6.5 and 0.07 kg for roots, 1.7 and 0.07 kg for leaves, and 3.2 and 0.03 kg for fruits. Carbon and nitrogen stocks stored between the soil surface and a depth of 60 cm were 138.29 and <TEX>$13.31Mg{\cdot}ha^{-1}$</TEX>, respectively, while those contained in pear trees were 17.66 and <TEX>$0.23Mg{\cdot}ha^{-1}$</TEX> based on a tree density of 667 <TEX>$trees{\cdot}ha^{-1}$</TEX>. Overall, carbon and nitrogen stocks per hectare stored in a pear orchard were 155.95 and 13.54 Mg, respectively.

기온, 강수량, 이산화탄소농도 변화에 따른 CERES-Barley 국내품종의 종실수량 반응

Our understanding of the sensitivities of crop responses to changes in carbon dioxide, temperature, and water is limited, which makes it difficult to fully utilize crop models in assessing the impact of climate change on future agricultural production. Genetic coefficients of CERES-Barley model for major domestic cultivars in South Korea (Olbori at Suwon, Albori at Milyang, Saessalbori at Iksan, and Samdobori at Jinju) were estimated from the observed data for daily weather and field trials for more than 10 years by using GenCalc in DSSAT. Data from 1997-2002 annual crop status report (Rural Development Administration, RDA) were used to validate the crop coefficients. The sitecalibrated CERES-Barley model was used to perform crop growth simulation with the 99 treatments of step change combinations in temperature, precipitation and carbon dioxide concentration with respect to the baseline climate (1981-2010) at four sites. The upper boundary corresponds to the 2071-2100 climate outlook from the RCP 8.5 scenario. The response surface of grain yield showed a distinct pattern of model behavior under the combined change in environmental variables. The simulated grain yield was most sensitive to CO2 concentration, least sensitive to precipitation, and showing a variable response to temperature depending on cultivar. The emulated impacts of response surfaces are expected to facilitate assessment of projected climate impacts on a given cultivar in South Korea.

Comparison of Seed Viability Among 42 Species Stored in a Genebank

This study was conducted to compare seed viability among 42 species after ten years of storage in the midterm storage complex (<TEX>$4^{\circ}C$</TEX>, 30-40% RH) at the National Agrobiodiversity Center (NAC) Korean genebank maintained by the Rural Development Administration (RDA), Republic of Korea and to suggest the relative seed longevity and suitable monitoring intervals. The germination data from initial tests and after ten years of storage were compared to measure changes in viability during storage. The decline in seed viability varied greatly among seeds from -11.5% for Triticum sp. to 80% for melon. Coriander, crowndaisy, safflower, cosmos, Chinesebellflower, waxgourd, melon, castorbean, Welch-onion, hollyhock, wild barley, and tallfescue showed significant decreases in viability of 34.2%, 73.4%, 36.5%, 30.0%, 40.2%, 71.3%, 80.0%, 65.9%, 45.5%, 51.4%, 53.0%, and 33.5%, respectively. Gardenpea, soybean, perilla, onion, wild rice, Italian-ryegrass, and pepper showed a 15-30% decline in viability, while the viability of morningglory, adzukibean, maize, and Capsicum sp. decreased by 15% to 5%. Chicory, radish, Chinese-cabbage, bottlegourd, watermelon, cucumber, pumpkin, Cucurbita sp., groundnut, kidneybean, clubwheat, sesame, wheat, Triticum sp., rice, barley, orchardgrass, buckwheat, and wild tomato showed changes in viability of <5%. The changes in storage viability also varied within families. The wild types of rice and barley showed rapid viability loss and presented different aspects from cultivars. Since seed viability of species, classified as index 1 or 2, showed germination losses >15% after ten years of storage, a viability test should be conducted with five year intervals, while species with germination loss of <15% (in index 3 or 4) can be retested at ten year intervals.

Botrytis cinerea에 의한 소엽풍란 잿빛곰팡이병

Horticultural & Herbal Crop Environment Division, National Institute of Horticultural & Herbal Science, Rural Development Admin-istration, Suwon 441-440, KoreaABSTRACT : In February 2010, the grayish fungus was found on Neofinetia falcata at Namsa-myeon, Yongin city, Gyeonggi-do,Korea. The symptoms start mainly on the leaves and stems, and the infected stems were rotten. Many conidia appeared on thelesions under humid condition. Colonies were grayish brown color and sclerotial formation was observed on potato dextrose agar(PDA). Conidia were mostly ellipsoidal to ovoid in shape, hyaline, one-celled, and 5.3-16.5×3.8-11.0 µm in size. Based onmorphological characteristics and pathogenicity, the causal fungus was identified as Botrytis cinerea Pers. This is the first report ofgray mold on N. falcata caused by B. cinerea in Korea.KEYWORDS :

4 PRODUCTION OF 11β-HYDROXYSTEROID DEHYDROGENASE TYPE 1 (11β-HSD1) OVER-EXPRESSED PIGS FOR THE STUDY OF METABOLIC SYNDROME DISEASE

Metabolic syndrome shows a complication at risk for cardiovascular disease and diabetes. With the high prevalence of obesity globally, the incidence of metabolic syndrome is increased. However, the basic mechanisms of metabolic syndrome are not completely known yet. Therefore, we attempted to develop large-animal model for the study of metabolic syndrome disease. Some studies have shown that constitutive overexpression of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) in mice leads to metabolic syndrome. Therefore, we tried to produce the 11β-HSD1 gene overexpressed pig by somatic cell nuclear transfer (SCNT). First, we established 11β-HSD1 overexpressed cells for the preparation of the transgenic (TG) donor cells. Porcine fetal fibroblasts were transfected with cytomegalovirus vector that included the porcine 11β-HSD1 gene. The 11β-HSD1-TG cell was injected into the enucleated ooplasm to produce 11β-HSD1-TG cloned embryos. In total, 833 TG porcine SCNT embryos (TG-SCNT embryos) were made. Of these, 155 TG-SCNT embryos were cultured in procine zygote medium-3 to evaluate the in vitro developmental potential of TG-SCNT embryos. Among these porcine SCNT-TG embryos, 109 embryos (70.3%) were cleaved at 48 h. On Day 7, 31 transgenic porcine SCNT embryos (20.0%) developed to the blastocyst stage. The remaining 678 TG-SCNT embryos were transferred to 6 surrogates (average 113 embryos per surrogate). On 25 days after embryo transfer, 2 surrogates were diagnosed as pregnant (pregnancy rate, 33.3%). On Day 114, we obtained 9 live piglets and 3 stillborn piglets from 2 surrogates. By PCR analysis, we confirmed that 1 live piglet and 2 stillborn piglets were integrated with 11β-HSD1 gene. We succeeded to obtain TG piglets at sixth trials, and for the re-cloning by SCNT, a stable cell line transfected with the 11β-HSD1 gene was established from a TG cloned piglet. This study presents new possibilities for large-animal model development for the study of metabolic syndrome disease. This work was supported, in part, by a grant from the Next-Generation Bio Green 21 Program (No. PJ00956901), Rural Development Administration, and the National Research Foundation of Korea Grant funded by the Korean Government (NRF-2012R1A1A4A01004885, NRF-2013R1A2A2A04008751), Republic of Korea.

188 PLURIPOTENCY OF PORCINE EMBRYONIC AND INDUCED PLURIPOTENT STEM CELLS RELATED WITH THE EXPRESSIONS OF EPITHELIAL–MESENCHYMAL TRANSITION AND APOPTOTIC MARKERS

In transgenic pig production for generating animal models of human diseases, apoptosis of an early implantation embryo disturbs the transgenic pig production. Porcine embryonic stem cells (pESC) and porcine induced pluripotent stem cells (piPSC) have an advantage for the generation of transgenic pigs; however, porcine stem cells have not yet been established. In addition, epithelial–mesenchymal transition (EMT) may play a critical role in embryo development and apoptosis. Thus, in this study we generated pESC and pIPSC and further examined the changes in EMT and apoptotic markers. We cultured pESC and piPSC in pESC media containing basic fibroblast growth factor (bFGF), doxicyclin, and leukemia inhibitory factor (LIF), and performed RT-PCR and alkaline phosphatase (AP) test to measure pluripotency markers. The RT-PCR results show that OCT-4, NANOG, and SOX2 were expressed in these stem cells, characteristic of stem cells. AP-positive cells were observed in pESCs and piPSC. In addition, we performed immunocytochemistry (ICC) to examine the expression of surface markers, such as SSEA-1 and SSEA-4. We found that pESC and piPSC expressed these markers, indicating that they have a stem cell property similar to rodent and human stem cells. Second, we treated pESC and piPSC with transforming growth factor beta (TGF-β) to examine the relationship between EMT and apoptotic markers, and confirmed a significant variation of EMT and apoptotic markers, i.e. bcl-2, bax, E-cadherin, and vimentin, by Western blot analysis. In a future study, we will examine the effect(s) of various endocrine hormones secreted by the ovary, such as E2 or P4, on the expressions of EMT and apoptotic markers in pESC and piPSC. Consequently, this study will contribute to elucidate underlying mechanism(s) of EMT and apoptosis by endocrine factors to prevent early apoptosis of pig embryos in these porcine stem cells. This work was supported by a grant from the Next-Generation BioGreen 21 Program (No. PJ009599), Rural Development Administration, Republic of Korea.

174 RECOVERY OF LONG-BEAKED COMMON DOLPHIN OOCYTES FROM KOREAN WATERS

The aims of this study were to collect oocytes from ovaries of Delphinus capensis (long-beaked common dolphin) and to examine the morphological characteristics of their reproductive organ. Female genital organs were collected from 2 incidentally caught Delphinus capensis (ID-96: estimated 5–6 years, and ID-97: estimated 7–8 years). Collected ovaries were transported to the laboratory over 3.5 h in 0.9% saline at 39°C. To perform assisted reproductive techniques in wild animals, it is important to maximize the number of oocyte recovered per ovary. In this study, Delphinus capensis oocytes were collected by 2 methods: (1) aspiration of visible follicles (2 to 3 mm in diameter) and (2) remnant dissection, in which the ovary is finely dissected. The collected cumulus–oocyte complexes (COC) were separated by ovary of origin and collection method. Oocytes were subjected to in vitro maturation (IVM). Immature oocytes were placed into IVM medium [TCM-199 supplemented with 10% (vol/vol) fetal bovine serum, 0.5 IU mL–1 LH, and 0.5 IU mL–1 porcine FSH] in the 4-well dish for 120 h at 37°C in 5% CO2 in a humidified atmosphere. The uterus type was observed to be a uterus bicornis. Morphology of ovary was similar to the bovine ovary among land animals. In total, 114 oocytes were obtained. From the ovary of ID-97, the number of oocytes recovered by aspiration and ovary dissection were 15 and 57, respectively. However, only 42 oocytes were recovered by ovary dissection from the ovary of ID-96. The number of oocytes recovered per ovary by dissection (49.5 ± 7.5) was higher than that by the aspiration (7.5 ± 7.5) method, but not significantly different (P = 0.06). For accurate statistical analysis, additional trials will be performed. Most oocytes had either no cumulus cells or had only 1 to 2 layers attached to the zona pellucida enclosing a homogeneous cytoplasm. After IVM, all oocytes were at the metaphase I stage, with even fewer attached cumulus cells than at recovery. To summarise, we describe the gross morphology of the female reproductive tract of the long-beaked common dolphin and determined that dissection of the ovary is required for maximal recovery of oocytes. This work was supported, in part, by a grant from the Next-Generation Bio Green 21 Program (No. PJ00956901), Rural Development Administration, and the National Research Foundation of Korea Grant funded by the Korean Government (NRF-2012R1A1A4A01004885, NRF-2013R1A2A2A04008751), Republic of Korea.

155 EFFECT OF CO-CULTURE WITH CUMULUS-DERIVED SOMATIC CELLS DURING IN VITRO MATURATION ON PORCINE CUMULUS–OOCYTE COMPLEXES AND SUBSEQUENT EMBRYONIC DEVELOPMENT AFTER IN VITRO FERTILIZATION

The purpose of this study was to investigate the effects of co-culture with cumulus-derived somatic cells (CSC) during porcine in vitro maturation (IVM) and subsequent embryonic development after IVF. The CSC were cultured in Dulbecco's modified Eagle medium for 48 h with various numbers of cumulus-derived somatic cells (0.0, 2.5, 5.0, and 10.0 × 104), and then cultured in TCM-199 for 4 h before the oocytes were added. Cumulus-oocytes complexes from 3- to 6-mm follicles were matured in 500 μL of TCM-199, with eCG and hCG, for 22 h, and then cultured in M199 without hormones for 22 h. Each experiment consisted of at least 4 replicates. Statistical analyses were carried out using SPSS 17.0 software (SPSS Inc., Chicago, IL). Percentage data were compared by one-way ANOVA, followed by Duncan's multiple range test. Data were presented as means ± s.e.m. Differences were considered to be significant if the P-value was 0.05. After IVM, no significant difference (P &lt; 0.05) was observed in nuclear maturation rate among the 0.0, 2.5, 5.0, and 10.0 × 104 groups (88.0 ± 2.37, 81.5 ± 2.17, 87.0 ± 1.98 and 86.0 ± 1.93%, respectively). The 2.5 × 104 group showed a significant (P &lt; 0.05) increase in intracellular glutathione (GSH) levels compared with that of the other groups. Intracellular reactive oxygen species (ROS) levels of mature oocyte in all groups showed no significant differences. The developmental competence of matured oocytes in all groups was evaluated after IVF. The 2.5 and 5.0 × 104 groups showed significantly (P &lt; 0.05) high cleavage rates (60.0 ± 4.7 and 64.52 ± 5.9%, respectively) compared with the 0 and 10.0 × 104 groups (43.15 ± 5.0 and 53.8 ± 5.0%, respectively). The 2.5 × 104 group showed a significantly (P &lt; 0.05) higher BL formation rate (35.7 ± 2.9) than control group (21.0 ± 3.8%, respectively), and higher total cell number (127.25 ± 7.7) compared with the 0 and 10 × 104 groups (89.3 ± 4.0 and 92.6 ± 3.7, respectively). In the analysis of gene expression, IVF-BL derived from the 2.5 and 5.0 × 104 groups showed higher (P &lt; 0.05) mRNA expression of PCNA, which is an essential component of the DNA replication and repair machinery and POU5F1 has been used to evaluate developmental potential in embryos. The 10.0 × 104 group showed higher (P &lt; 0.05) mRNA expression of caspase-3 and Bak as known pro-apoptotic factors, compared with the control group IVF-BL. The results of cortical granules distribution which leads digesting sperm receptor proteins ZP2 and ZP3 to block polyspermy, showed that the 2.5 × 104 group was increased significantly (P &lt; 0.05) compared with the other co-culture groups (13.7 ± 6.1, 29.2 ± 9.5, 18.3 ± 0.8 and 19.52 ± 5.3, respectively). In conclusion, co-culture with 2.5 × 104 cumulus-derived somatic cells during IVM improved the developmental potential of porcine IVF embryos by increasing the intracellular GSH level and distribution of cortical granules during oocyte maturation. This work was supported, in part, by a grant from the Next-Generation BioGreen 21 Program (No. PJ00956901), Rural Development Administration, and the National Research Foundation of Korea Grant funded by the Korean Government (NRF-2012R1A1A4A01004885, NRF-2013R1A2A2A04008751), Republic of Korea.

127 TREATMENTS WITH DIVERSE ENDOCRINE-DISRUPTING CHEMICALS RESULTED IN THE INHIBITION OF OVARIAN TUMOR PROGRESSION VIA INTERRUPTION OF TRANSFORMING GROWTH FACTOR-β IN IN VITRO AND XENOGRAFTED MOUSE MODELS

Activated oestrogen receptor (ER) signaling pathway by 17β-estadiol (E2) appeared to suppress transforming growth factor β (TGF-β) signaling pathway by cross-talk with TGF-β components in ER-positive cancer cells. In this study, we further examined the inhibitory effects of alkylphenols, including 4-nonylphenol (NP), 4-otylphenol (OP), bisphenol A (BPA), and benzophenon-1 (BP-1), in TGF-β signaling pathway. The transcriptional and translational levels of TGF-β-related genes were examined by reverse-transcription PCR (RT-PCR), Western blotting analysis in xenografted mouse models of ovarian cancer BG-1 cells. The NP, OP, and BPA induced the expression of snoN, a TGF-β pathway inhibitor. Treatment with NP, BPA, and BP-1 resulted in decreased phosphorylation of Smad3, a downstream target of TGF-β. With these 2 effects, NP and BPA stimulated the proliferation of BG-1 cells via inhibition of the TGF-β signaling pathway. In a xenograft mouse model, transplanted BG-1 ovarian cancer cells showed significantly decreased phosphorylation of Smad3 and increased expression of snoN in the ovarian tumour masses following treatment with E2, NP, or BPA. In parallel with an in vitro model, the expressions of TGF-β signaling pathway were similarly regulated by NP or BPA in a xenograft mouse model, revealing consistent results. Taken together, these results support that NP and BPA may cause the disruption of the TGF-β signaling pathway and increase the risk of oestrogen-dependent cancers such as ovarian cancer. This work was supported by a grant from the Next-Generation BioGreen 21 Program (No. PJ009599), Rural Development Administration, Republic of Korea.

125 BISPHENOL A-INDUCED GROWTH OF OVARIAN CANCER CELLS WAS REVERSED BY A PHYTOESTROGEN, GENISTEIN, BY INHIBITION OF A CROSSTALK BETWEEN ESTROGEN RECEPTOR AND INSULIN-LIKE GROWTH FACTOR 1 RECEPTOR IN IN VITRO AND XENOGRAFT MOUSE MODELS

It has been shown that oestrogen (E2) up-regulated the expression of components of insulin-like growth factor-1 (IGF-1) signaling pathway and induced the downstream of mitogenic signaling cascades via phosphorylation of insulin receptor substrate-1 (IRS-1). An interaction between oestrogen receptor (ER) and IGF-1 receptor (IGF-1R) signaling pathway plays an important role in proliferation of and resistance to endocrine therapy to oestrogen-dependent cancers (i.e. breast and endometrial cancers). In the present study, we evaluated xenoestrogenic effect of bisphenol A (BPA) and antiproliferative activity of genistein (GEN) in accordance with the influence on this crosstalk. The gene expressions in mRNA and protein levels were examined by semiquantitative RT-PCR and Western blot analysis, in which the primers for ERα, IGF-1R, and GAPDH and the antibodies against pIRS-1, pAkt, and GAPDH were used, respectively. Total RNA and protein samples were isolated from BG-1 cells treated with dimethyl sulfoxide (DMSO), estradiol (E2; 10–9 M), BPA (10–5 M), E2 (10–9 M) + GEN (10–4 M), and BPA (10–5 M) + GEN (10–4 M). The DMSO was used a vehicle of E2, BPA, and GEN in in vitro experiments. All in vitro experiments were done in triplicates. The effects on tumour growth and immunohistologic alterations were identified in in vivo mouse models. The mice were injected subcutaneously with corn oil (vehicle, n = 6), E2 (n = 6), BPA (n = 6), E2+GEN (n = 6), and BPA+GEN (n = 6) for 8 weeks. The BPA treatment resulted in up-regulation of ERα and IGF-1R mRNA, and induced phosphorylation of IRS-1 and Akt proteins compared with a control (DMSO) in BG-1 ovarian cancer cells as E2 did in triplicates. In the mouse model xenografted with BG-1 cells, BPA significantly increased a tumour burden of mice and expressions of ERα, pIRS-1, and cyclin D1 in tumour mass compared with the vehicle (corn oil), indicating that BPA induces ovarian cancer growth by promoting the crosstalk between ER and IGF-1R signals. On the other hand, GEN effectively reversed estrogenicity of BPA by reversing mRNA and protein expressions of ERα, IGF-1R, pIRS-1, and pAkt induced by BPA in cellular model with triplicates. The GEN also significantly decreased tumour growth and in vivo expressions of ERα, pIRS-1, and pAkt in a xenografted mouse model. Also, GEN was confirmed to have an antiproliferative effect by inducing apoptotic signaling cascades. Taken together, these results suggest that GEN effectively reversed the increased proliferation of BG-1 ovarian cancer by suppressing the crosstalk between ER and IGF-1R signaling pathways up-regulated by BPA or E2. This work was supported by a grant from the Next-Generation BioGreen 21 Program (No. PJ009599), Rural Development Administration, Republic of Korea.