The objective of this study was to investigate the effects of NEAA and leptin supplemented to in vitro culture medium on the developmental competence of porcine embryos after intracytoplasmic sperm injection (ICSI), and to modify the culture condition to improve the quality and the development of ICSI-derived porcine embryos in vitro. After ICSI, the putative zygotes were then cultured in PZM-3 medium with/without NEAA or leptin. The proportion of embryos that developed to the blastocyst stage significantly increased when 1% NEAA (24.62%) was added to the medium compared with 2% NEAA and no NEAA group (17.24% and 20.24%, respectively, p<0.05). The effect of different concentration of leptin (0, 10, 100, 500 ng/ml) was evaluated on the development of porcine ICSI embryos cultured in vitro. In case of blastocyst formation, 100 ng/ml group (27.05%) showed significantly higher rate than 10, 500 ng/ml, and control group (23.45%, 17.99%, and 19.68%, respectively, p<0.05). We also evaluated the effects of different NEAA and leptin treatment time on the development of porcine embryos after ICSI. Among groups of embryos cultured in the presence of NEAA or leptin for whole 7 days (D 1-7), first 4 days (D 1-4), the subsequent 3 days (D 5-7), both NEAA (27.13%, 21.17 %, and 17.56%, respectively, p<0.05) and leptin (25.60%, 20.61%, and 16.53%, respectively, p<0.05) showed that supplementation for whole 7 days significantly increased the blastocyst formation rate compared with the other groups of D1-4 and D5-7. We further evaluated the combination effect of 1% NEAA and 100 ng/ml leptin compared with the effect of each supplementation with 1% NEAA or 100 ng/ml leptin or no supplementation on development of embryos. For blastocyst formation, combination group of NEAA and leptin (24.78%) showed significantly higher rate than other three groups (18.37%, 20.44 %, and 13.27%, respectively, p<0.05). We further evaluated the expression of proapoptosis genes such as BAX and BAK and anti-apoptosis genes, BCL-XL and BCL-2 in blastocysts cultured in the presence of 100 ng/ml leptin. RT-PCR analysis revealed that leptin supplementation significantly decreased the expression of pro-apoptosis genes as well as increased the expression of anti-apoptosis genes. These results of present study demonstrate that NEAA and leptin could improve the in vitro development of ICSI- derived porcine embryos with optimal concentration of each reagent. Furthermore, the optimal culture condition could increase the quality of ICSI-derived embryos in vitro.

Chicken Insulin-like Growth Factor-1 (cIGF-1), one of the most important hormone for regulating physiological function includes body growth, muscle volume, bone density, chicken cell development and metabolism. In order to find in vitro Knokdown expression of cIGF-1, this study introduced tetracycline inducible RNA interference expression system (TetRNAi system). Tet system can inductively control high expression of extrinsic genes and expression of intrinsic genes. So it has advantages such as minimized physiological side-effects any cell and low cytotoxicity. RNAi system is proving to be a powerful experimental tool for inhibition of gene expression and post-transcriptional mechanism of gene silencing. RNAi is mediated by small interfering RNA (siRNA) consisting of 19- to 23- nucleotide double-stranded RNA duplexes that promote specific endonucleolytic cleavage of mRNA targets through an RNA-induced silencing. Then, this study RNAi-based gene knockdown can be achieved by retroviral-based expression systems. Stable integration of our inducible siRNA vector allowed the production of siRNA on doxycycline induction, followed by specific down regulation of chicken IGF-1 gene. Analyses of Real-time PCR to determine expression of the cIGF-1 gene showed successful from chicken embronic fibroblast (CEF) cells with the reduced rate of an approximately 92%. Our results demonstrate the successful regulation of cIGF-1 knockdown expression in CEF cells and support the application of an tetracycline inducible RNAi expression system in transgenic Mini chicken production. This research was supported by Bio-industry Technology Development Program, Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea.

The aim of this study was to examine the effect of acteoside (the cyclin-dependent kinase inhibitor) on the SCNT efficiency with adult fibroblasts in dog. Canine adult fibroblasts were obtained from muscle and cell cycle of fibroblasts was synchronized by culturing to confluency, serum starvation and treating with 30 μM acteoside for 48 h. Cell cycle stages, cell cytotoxicity (apoptosis) and, prduction of reactive oxygen species (ROS) were analyzed using flow cytometry. The canine cells, prepared by confluent-cell culture or treating with 30 μM acteoside for 48 h, were injected into enucleated in vivo matured oocytes, the couplets were electrical fused and activated by calcium ionomycin. SCNT embryos using acteoside-treated fibroblasts were surgically transferred into oviducts of estrus cycle synchronized recipient dogs. In cell cycle synchronization (G0/G1), there was no significant difference between serum starvations (83.9%) and acteoside treated groups (81.3%) that were higher than confluent group (78.5%). In production of apoptosis, confluent and acteoside treated groups (4.3 and 4.5%, respectively) were generated less than serum starvation group (21.8%). In case of ROS, serum starvation group was induced a significantly higher than other groups. After synchronization of the donor cell cycle, either confluent or acteoside treated, cells were placed with enucleated in vivo-matured dog oocytes, fused by electric stimulation, activated, and transferred into naturally estrus-synchronized surrogates. Fusion and cleavage rate of acteoside treated group were 64.1 and 41.5%, which were higher than those of confluent group (53.9 and 20.6%, respectively). The reconstructed embryo development rates to 4-cell and 8-cell in acteoside treated group were 29.5 and 14.8%, respectively, while confluent group showed 11.1 and 3.2%, respectively. Total 54 SCNT embryos using acteoside-treated fibroblasts were transferred into oviducts of 2 recipient dogs and one recipient finally delivered one puppy, whereas din`t detected pregnancy on transfer of cloned embryos reconstructed with confluent cells in 6 surrogate dogs. In conclusion, the results of the current study demonstrated that canine fibroblasts could be successfully arrested at the G0/G1 stage with reduced the formation of ROS and apoptosis after acteoside treatment. This results may contribute to improve the effi-ciency of canine SCNT. * This research was supported by iPET (Grants 110056-3), Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea.

Acteoside acts as an anti-oxidative activity and anti-apoptosis in the cells. But, it has been not studied on maturation and development of porcine oocytes. The aims of the present study were to examine the effects of acteoside on the morphological progress of meiosis, developmental competence, and ROS in porcine oocytes. Oocytes were matured in tissue culture medium-199, supplemented with acteoside at various concentrations: 0 (control), 10, 30 and 50 μM. The oocytes maturation rates of groups supplemented with acteoside were no significantly different (81.13, 85.96, 82.95 and 83.68%, respectively). Level of ROS was significantly decreased in acteoside treated group. Furthermore, the parthenogenetic blastocyst rate was significantly improved in 10 μM acteoside treated group compared with control group (44.83 vs. 27.75%). And we investigated effect of acteoside on the oocytes condition represented by cytoplasmic maturation by homogeneous distribution and formation of cytoplasmic organelles and regulation of apoptosis-related genes. In the results. during IVM, 10 μM acteoside treated oocytes showed that the mitochondria and lipid droplet were smaller and homogeneous distribution in cytoplasm compare with control oocytes. And reverse transcription polymerase chain reaction (RTPCR) of parthenogenetic blstocysts revealed that acteoside increased the anti-apoptotic genes (Mcl-1, Bcl-2 and Bcl-xL), whereas reduced the expression of pro-apoptotic genes (Bax and Bak). In conclusion, based on the results, the effect of acteoside on IVM was not attractive. However, in acteoside treated group, cytoplasmic maturation seemed to be improved with morphologically uniform distribution of cytoplasmic organelles. Furthermore, embryonic development in acteoside treated group was significantly highly increased than that of non-treated group. Our results represents that addition of acteoside to the IVM medium has a beneficial effect in physiology of porcine oocytes, providing a improved method for porcine oocytes in vitro. * This work was supported by a grant (Code# PJ008148) from BioGreen21 Program, Rural Development Administration, Republic of Korea.

The objective of the current study was to describe in vitro embryo production in Hanwoo, analyzing oocytes yield and embryo production. The effects of oocytes production and the number of OPU procedures per animal on embryo production were also evaluated. OPU was done every 3～4 days during experimental period and collected oocytes were fertilized in vitro in both OPU and needle puncture groups. First, we compared the recovery rate of oocytes based on OPU session (Experiment 1). The average of collected oocytes was calculated from every 10 session. The average number of total oocytes recovered per animalonsessionwas 5.16 (mean). Second, we compared the recovery rate base on collection period of OPU (Experiment 2). The following results show the difference of the number of recovered oocytes in every month during the procedure between the months of session. Every animal shows the constant number of recovered oocytes for the first 5 months. However, the recovery rate of oocytes was decreased from month 6 to 8. Third, we compared the developmental rate to blastocyst in two groups (Experiment 3). Oocytes by needle puncture were fertilized with frozen-thawing semen; the cleavage rate 24～48 h after in vitro fertilization (IVF) was 75.8% and blastocyst development rate was 18.8% in needle puncture group. Even though there is lower cleavage rate after IVF in OPU group (61.1%), blastocyst development rate was higher compared with needle puncture group (28.4%). In conclusion, Blastocyst developmental rate could be increased by OPU than classical method of needle puncture. Improvement of bio- technique in collecting oocytes could be applied to understand the reproductive physiology in cattle, expecially Hanwoo. Therefore, further investigation should be done to clarify the efficiency and advantage of OPU involved in reproduction in animals and human being.

비모란 선인장 ‘이홍’ 품종은 진적색의 계통 ‘DR’을 모본으로 진적색의 ‘설홍’ 품종을 부본으로 2005년 교 배하여 개발되었다. 파종된 종자는 기내에서 접목되어 6개월 동안 100 mL 시험관에서 6개월 동안 자랐다. 2006년 재접목되어 온실에 정식되어 2009년까지 다양한 특징이 조사되었다. 편원형의 모양과 진적색의 구색을 가지고 있으며, 또한, 8~10개의 결각과 갈색의 직립형 가시를 가지고 있다. 온실에 정식 후 10개월째에 ‘이 홍’ 품종의 구직경은 44.0 mm였으며 ‘설홍’ 품종에 비해 큰 편이었다. 자구수는 10개월째에 15.8개로 ‘설 홍’ 품종에 비해 많았으나 유의성은 없었다.

The objective of this study was to evaluate in vitro production of bovine embryos in Hanwoo. Oocytes were collected by ovum pick up (OPU) from ovaries of genetically high-value Hanwoo or by needle puncture from ovaries of slaughtered cattle. OPU was done every 3 4 days duing experimental period and collected oocytes were fertilized in vitro in both OPU and needle puncture groups. First, We compared the in vitro maturation rate in two groups (Experiment 1). 545 oocytes were recoverd from 4 females by 32 trials of OPU and then 433 oocytes were shown MⅡ stage after in vitro maturation (79.4%). In case of needle puncture group, 1905 oocytes were collected and then 1420 oocytes were matured to MⅡ stage during in vitro culture(74.5%). Second, we compared the developmental rate to blastocyst in two groups (Experiment 2). 1420 oocyte by needle puncture were fertilized with frozen-thawing semen; the cleavage rate 24 48 h after in vitro fertilization (IVF) was 88.6% and blastocyst development rate was 20.5% in needle puncture group. Even though there is lower cleavage rate after IVF in OPU group (84.8%), blastocyst development rate was higher compared with needle puncture group (26.4%). In conclusion, Blastocyst developmental rate could be increased by OPU than classical method of needle puncture. Improvement of bio-technique in collecting oocytes could be applied to understand the reproductive physiology in cattle, expecially Hanwoo. Therefore, further investigation should be done to clarify the efficiency and advantage of OPU involved in reproduction in animals and human being. This research was suppoted by Imsil-gun agricultural technology service center.

The black-veined white, Aporia crataegi (Lepidoptera: Papilionoidea), is nearly extinct in South Korea, although substantial numbers of dried specimens are available. One of the common practices for such species is to launch re-introduction program after proper amount of genetic information are analyzed from donor and donee populations. In this study, we sequenced complete mitochondrial genome (mitogenome) of A. crataegi to design species-specific primers for subsequent population works and to further understand the mitogenome evolution in lepodiopteran Papilionoidea. The 15,140-bp long A. crataegi mitogenome that has typical sets of 37 genes is smallest among true butterfly species with overall slightly smaller size in genes and regions throughout the genome. Arrangement of the genome is identical to those of other lepidopteran mitogenomes, in which tRNA cluster located between the A+T-rich region and ND2 gene is translocated into tRNAMet, tRNAIle, and tRNAGln from ancestral arrangement, tRNAIle, and tRNAGln, tRNAMet. The A/T content of the genome at 81.3% is the highest in Pieridae, but lower than that of lycaenid species (81.7% ~ 82.7%) The high A/T content in the genome is also reflected in codon usage, accounting for 41.69% of A/T-composed codons (TTA, ATT, TTT, and ATA). Unlikely the diversified or modified usage of anticodon for tRNASer(AGN) the species of Pieridae including A. crataegi all unanimously have GCT that has been hypothesized as ancestral for Lepidoptera. A total of 111 bp of non-coding sequences are dispersed in 13 regions, ranging in size from 1–49 bp. Among them relatively longer ones (≥ 16 bp) all have relatively higher sequence identity to other regions of the genome, suggesting partial duplication of the sequences during A. crataegi evolution. As has been reported in some species of Lepidoptera, the A. crataegi A+T-region also has typically found conserved sequences (e.g., poly-T stretch, ATAGA motif, ATTTA element, microsatellite-like A/T sequence, and poly-A stretch) and one tRNA-like sequence, and this feature was commonly found in true butterfly species.

The phylogenetic relationships among the Nymphalidae (Lepidoptera: Papilionoidea) have been controversial in several perspective. The present study sequenced a total of ~ 3,500 bp from cytochrome oxidase subunit I (COI), 16S ribosomal RNA (16S rRNA), and elongation factor-1 alpha (EF-1α) in 80 nymphalid species belonging to seven subfamilies (Linmenitidinae, Heliconiinae, Nymphalinae, Apaturinae, Libytheinae, Satyrinae, and Danainae), along with those of six lycaenid species as outgroups. Phylogenetic analyses via Bayesian Inference (BI) and Maximum Likelihood (ML) algorithms concordantly supported the subfamilial relationships of (((((Linmenitidinae + Heliconiinae) + (Nymphalinae + Apaturinae)) + Libytheinae) + Satyrinae) + Danainae), with high nodal support for monophyletic subfamilies and tribes. This result is largely consistent with a previous study performed with a substantially large sequence information and morphological characters, except for the position of Libytheinae that has previously been placed as the sister to all reminder of Nymphalidae.

The Samia cynthia ricini (Lepidoptera: Saturniidae) is a commercial silk-producing insect belonging to an insect family Saturniidae in Bombycoidea. The species that has presumably been originated in India, is distributed in India, China, and Japan. Unlikely domestic silkworm the prime host plant for the species is a castor-oil plant (Ricinus communis in Euphorbiaceae). Recently, the eri-silkworm also is reared in Korea and is expected to be utilized for a diverse purpose. In this report, we present the complete mitochondrial genome of the species with the emphasis of a few major characteristics. The 15,384-bp long S. cynthia ricini (Lepidoptera: Saturniidae) mitochondrial genome was amplified into three long overlapping fragments (from COI ~ ND4, ND5 ~ lrRNA, and lrRNA ~ COI) and subsequent several short fragments using the long fragments as temperate. The primers for both long and short fragments were designed solely for lepidopteran genomes, without any species-specific primers. As a usual the genome is composed of 37 genes: 13 protein-coding genes (PCGs), two rRNA genes, and 22 tRNA genes, and one large non-coding region termed the A+T-rich region. Arrangement of the genome is identical to those of other lepidopteran mitochondrial genome, but this differs from the common arrangement found in a diverse insect order, by the movement of tRNAMet to a position 5’- up stream of tRNAIle. Unlikely previous report on the start codon for COI gene in Lepidoptera S. cynthia ricini COI gene starts with typical ATT codon located between tRNATyr and the beginning region of COI gene. The 22 tRNAs that are interspersed throughout the mitogenome ranged in length from 62 to 71 bp. All tRNAs but tRNASer(AGN) were shown to be folded into the expected cloverleaf secondary structures. More detailed structural and phylogenetic analyses among Bombycidae and Saturniidae in connection with other families in the Bombycoidea will be performed soon

The leaf beetle, Chrysolina aurichalcea (Coleoptera: Chysomelidae), is a pest damaging plants of Compositae. In order to understand the genetic diversity and geographic variation of the species we sequenced a portion of mitochondrial COI gene (658 bp) and complete nuclear internal transcribed spacer 2 (ITS2) collected from seven Korean localities. A total of 18 haplotypes (BARCA01 ~ BARCA18), with the maximum sequence divergence of 3.04% (20 bp) were obtained from COI gene sequence, whereas 17 sequence types (ITS2CA01 ~ ITS2CA17), with the maximum sequence divergence of 2.013% (9 bp) were obtained from ITS2, indicating substantially larger sequence divergence in mitochondrial gene sequence. Phylogenetically, the mitochondrial DNA has shown several haplotypes formed independent groups with substantially high node support (≥ 90%), whereas no such grouping was evidenced for ITS2, indicating different behaviors of the two molecules. Such difference may reflect a diverse dynamics of the species such as biogeographic history, mating behaviors, and also possibly different mode of inheritance of the two molecules, but requires further scrutinized examination of the dataset. In terms of population genetic perspective, overall no population subdivision was detected from both molecules, except for locality 7 (Eocheong islet) from mitochondrial DNA. As more scrutinized analysis is performed, further fruitful inference on the geographic contour of the species might be available.

본 연구는 붉은꽃인동덩굴의 적심높이(60, 120, 180 cm) 가 가지길이, 측지수 및 피복에 미치는 적심높이별 영 향을 알아 보기 위하여 수행하였다. 인동덩굴의 줄기길 이는 고절위(180 cm) 적심이 저절위(60 cm) 적심보다 길 었고, 측지수는 저절위 적심이 고절위 적심보다 많았다. 그러나 분지수는 적심처리간에 큰 차이가 없었다. 퍼골라 벽면의 피복도는 저절위 적심이 85%, 중절위 적심이 74%, 고절위 적심이 62%를 보였다. 개화는 5월 중순부터 10월 중순까지 계속되었고, 첫 개화는 60 cm 적심이 5월 중순으로 가장 빨랐다. 대체로 적심 높이가 낮고 적심시기가 빠를수록 개화가 빨랐고, 개화최성기는 8월 중순이었다. 붉은꽃인동덩굴을 이용한 퍼골라 벽면의 가로 3.0 m ×높이 2.2 m 녹화를 하기 위해서는 퍼골 라당 6주(1m당 2주)를 재식하고, 60 cm 벽면 높이에서 1회 적심하면 당년에 85%의 벽면스크린 및 녹화가 가 능하였다.

The principal objective of this study was to assess the antioxidative activities of Petasites japonicus against oxidative stress in bovine brain tissue . Petasites japonicus is found with a relatively widespread distribution, and is cultivated as a culinary vegetable in Korea. Petasites japonicus samples were dried either by freeze-drying or by hot air-convection drying (80℃), then evaluated for their antioxidative activity by measuring 1-dipheny-1，2-picrylhydrazyl (DPPH) radical scavenging, and by measuring thiobarbituric acid-reactive substances (TBARS) in brain homogenates subjected to Fe2+ -mediated lipids with or without the addition of botanical extract. Hot air convection-drying resulted in a slight increase in the extraction yie1d as compared with freeze-drying. However, total phenol and flavonoid contents in freeze-dried Petasites japonicas were significantly higher than those of hot air convection-drying. Freeze-drying increased the free radical scavenging activity of Petasites japonicas, leaves, and stems by 52.6, 28.6, and 248.0%, as compared with hot air convection-drying. Additionally, the IC50 values measured by TBARS in hot air convection-dried Petasites japonicas， leaves, and stems were increased by 36.0, 31.6, and 15.9%, as compared to those of freeze-drying. Although antioxidative activity was reduced slightly by heat processing in Petasites japonicas, freeze-drying for each portion of Petasites japonicus was the most appropriate for use as a functional food and pharmaceutical material.