Somatic cell nuclear transfer (SCNT) has been considered for preserving genetically valuable or endangered animals. Sapsaree is a Natianal Monument in Korea to maintian a pure pedigree. The aim of this study was to produce azoospermia Sapsaree using SCNT and identify normal reproductive ability of cloned azoospermia Sapsaree. Ear skin biopsy was performed on a thirteen-year-old azoospermia Sapsaree and ear skin fibroblasts were isolated for SCNT as donor cells. The fibroblasts were injected into enucleated in vivo matured oocytes, the couplets were electrical fused by two pulse of direct current (55 V for 15 μs) using titanium and platinum fusion needle and activated by calcium ionophore. Cloned embryos were surgically transferred into oviducts of natuarally estrus cycle synchronized recipient dogs. The fusion rate of platinum needle was 70%, which was higher than those of titanium needle (64.1%). Developmental rate to the 8 cells and 10 cell stages was higher in platinum needle group (24% and 16%, respectively) than those of platinum needle group (14.8% and 3.1%, respectively). Total 35 SCNT embryos were transferred into oviducts of 3 recipient dogs and one recipient finally delivered a puppy by caesarean section. As results, this study demonstrated that platinum fusion needle could be successfully make the reconstructed embryos and improve the efficiency of canine SCNT. Cloning azoospermia Sapsaree may contribute to conserve genetically valuable and unique pedigree. And further study should be confirm whether cloned live dog is azoospermia.

Prolonged communication between oocytes and the surrounding somatic cells is one of the unique reproductive physiology in canine. Paracrine Kit ligand (KITL) signaling is a well-known communication between granulosa cells and the oocyte. KITL is a cytokine growth factor secreted by granulosa cells that signals via the c-kit receptor expressed by oocytes. Paracrine factors, including growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), exert their effects by binding with the kinase receptors expressed on the granulosa cells. However, the regulations of GDF9 and BMP15 in the canine KITL expression are currently poorly understood. Therefore, we investigated the effects of GDF9 and BMP15 on the expression of KITL in canine ovarian granulosa cells in vitro. In Annexin V assay recombinant GDF9 and BMP15 did not induce apoptosis in the cultured ovarian granulosa cells. When treated, FSH significantly increased KITL expression, and hCG suppressed its expression. When both FSH and hCG were treated, the expression of KITL was affected by GDF9 and BMP15 in dose and time dependent manner in the luteal granulosa cells. GDF9 (10 ng/mL) significantly decreased KITL expression after12 h. BMP15 (10 ng/mL) significantly also decreased KITL expression after 24 h. Western blot and immunochemistry results indicate that GDF9 activated Smad2/3. After blocking ALK 4/5/7 receptors by SB, GDF9 failed to activate Smad2/3, also BMP15 did not activate Smad1/5/8 after blocking ALK 2/3/6 receptors by DM. So GDF9 exerts its effects via using ALK 4/5/7 receptors to activate SMAD2/3 signaling, and BMP15 binds ALK 2/3/6 receptors to activate SMAD1/5/8 signaling. The expression of KITL was not changed by SB or DM treatment. However, the effect of GDF9 and BMP15, which decreased the expression of KITL, was suppressed by SB or DM treatment. In conclusion, this study provides the first evidence that recombinant GDF9 and BMP15 decrease KITL expression in canine ovarian granulosa cells.

The cancer and Parkinson's disease associated protein DJ-1 is multifunctional protein that involves in diverse cellular process. DJ-1 protein has a cellular protective role and promoted cell survival under an oxidative stress. However, the cellular protective mechanism of DJ-1 is not fully understand, and we needs to be further study their functions in novel organisms. In the present study, we investigated the protective role of DJ-1 against induced oxidative stress in canine cell line. On the basis of these experiments, canine DJ-1 overexpressing and null cell lines were established. The stable overexpression and down regulation of DJ-1 efficiency confirmed by the western blot analysis. Subsequently, the DJ-1 gene transfected cell lines and control cells were subjected to induced the oxidative stress, and then cell viability, cell proliferation assay, cellular apoptosis detection analysis (Annexin V and TUNEL assay), intracellular ROS and mitochondrial activity were measured appropriately. The results showed that DJ-1 overexpressed cells were up-regulated cell viability under oxidative stress conditions induced by the rotenone and hydrogen peroxide (H2O2), whereas loss of DJ-1 cells were down-regulated the cell survival activity. Additionally, overexpression of DJ-1 cells increased cell resistance to oxidative stress and inhibited the elevation of cell death and cellular ROS induced apoptosis. Moreover, DJ-1 overexpressed cells was increased mitochondrial functions by using confocal microscopy with MitoTracker staining. On the contrary to this, DJ-1 null cells show defective cellular protection and mitochondria activity against oxidative stress conditions. Our data indicate that canine DJ-1 protein attenuates cellular apoptosis and ROS generation, enhances the cellular survival activity and promote mitochondrial function under the oxidative stress, likewise other mammalian cells. Importantly, DJ-1 overexpression may be an important part of a protective strategy as a sensor for oxidative stress.

The spotted-wing drosophila (SWD), Drosophila suzukii (Diptera: Drosophilidae), is an economically damaging pest that feeds on most thin-skinned fruits. In this study, we sequenced portions of the mitochondrial (mt) COI and ND4 genes from a total of 195 individuals collected mainly from Korea. A total of 139 haplotypes were obtained from the concatenated COI and ND4 sequences. A dataset combining GenBank sequences with our own data identified a total of 94 worldwide COI haplotypes with a maximum sequence divergence of 5.433% (32 bp). A rough estimate of genetic diversity in each country showed higher diversity in ancestral distributional ranges, but the invasion over Asian countries seems to have been substantial because haplotype diversity was only 2.35-3.97-fold lower in the USA, Canada, and Italy than that in the populations ancestral ranges.

Grapholita molesta occur four times a year and Carposina sasakii occur twice a year, and both pests do damage on stone fruits such as peach, apple, plum, apricot, etc. Grapholita molesta is worldwide distributed in temperate and subtropical areas including South Korea. But, Carposina sasakii distributed in South Korea, Japan, China and Asia, and has been managed as an important import quarantine pest by the authorities of United States, Canada and Taiwan. Forecasting of both pests in Korea is currently done through the investigation of 1,000 fruits per 10 trees (100 fruits / tree) in designated peach orchard. However, this method is very difficult to observe the pest by bagging of peach and require too much time and labor. Therefore, we tried to carry out a new forecasting method by using of sex pheromone traps for newly standardized method as an alternative. Using sex pheromone trap, attractiveness of G. molesta was proved to be 2.5 > 1.5 > 0.5 m by the height and the border => outside > center by the position. Attractiveness of C. sasakii made no difference in height, but, more trapped at the center and border than outside in position at peach orchard.

Recently an outbreaked pest belongs to Hemiptera: Recaniidae, Ricanula sp. is greatly concerned about the outspreading throughout the South of Korea by wide range of host, including Cornelian cherry, Jujube, and peach trees. In Chungbuk province, this pest was first occurred at Jincheon and Okcheon in 2012, Cheongju in 2013 and now found out at Eumseong and Goesan in 2015. Ricanula sp. was oviposited directly into one-year twig, did damages on fruit-bearing formation and finally withered the host. This study was performed to understand the ovipositional characteristics and to develop the standardized forecasting method. Oviposition by Ricanula sp. was abundant in tree than in bush, adult laid eggs on new inner twigs and then covered with wax compound. Total no. of oviposited egg-mass was 10 to 318, and that of on new twig was 5 to 185 per tree, with different to host trees. Thickness of oviposited twigs were done within 2 ~ 5.5 mm and the height was mostly founded with range of 1 to 2 m, founded with highest height over 3 m. Oviposited no. of egg-mass within 30 cm twig was appeared differently from 2 to 7 every host. From based on this investigated result, we provide this for standardized forecasting method. This pest will need to control when egg-mass will occur over 2 at new twig, within 30 cm from the tip, set as total 25 point/ 5 plants (5 point per plant).

To compensate for the critical shortage of human organs for allotransplantation, xenotransplantation studies using genetically modified pigs are being performed in Korea. Two types of pigs that are used are α1,3-galactosyltransferase gene knockout (GalT KO) pigs and GalT KO+hCD46 (human complement regulatory protein) pigs. The present study measured the gestation time, birth weight, daily growth rate, and heart weight of both kinds of transgenic minipigs. The gestation period for both types of pigs was 117∼119 days. There was no difference in the body weight of GalT KO (—/+) and GalT KO (—/—) piglets, but GalT KO+hCD46 (—hCD46+/+) pigs were significantly heavier at birth than were GalT KO+hCD46 (—hCD46+/—hCD46+) pigs. During the first 10 weeks of life, the daily weight gain of GalT KO+hCD46 (—hCD46+/—CD46+) piglets, which are considered the optimal type for xenotransplantation, was 0.19 kg. The weight of hearts from GalT KO piglets up to two months of age was affected more by body weight than by age. Transgenic pigs showed no differences in gestation period or reproductive ability compared with normal pigs. These results comprise basic data that may be used in xenotransplantation studies and transgenic animal production in Korea.

The Riptortus-Burkholderia symbiosis is a newly emerging insect-bacterium symbiotic system. This symbiosis system has a good merit as an experimental model system to produce the non-symbiotic (apo) and symbiotic (sym) host insect. In recent reported papers, the symbionts play important biological roles for the host insects. Meanwhile, juvenile hormone (JH) is one of major hormone synthesized corpora allata(CA) to control many physiology of insect. However, the study for cross-talk mechanism between symbionts and host hormones to control important physiological phenomenon of insects is almost none. In this study, we found that Riptortus speed up adult emerging and increase egg laying on presence of symbiont Burkholderia. Also we found that hexamerin proteins, which were controlled the expression by JH, were accumulated in sym-Riptortus hemolymph compare with apo-Riptortus. According as combined results, we hypothesized that the gut symbiont Burkholderia can control JH titer to conclude out beneficial effects such as development and reproduction of R. pedestris. To verify this hypothesis, we examined measurement of JH titer, expression of hexamerins as JH response genes and RNAi for hexamerin protein during whole Riptortus life on presence or absence of symbiont Burkholderia. All results demonstrated that gut symbiont controlled JH titer of Riptortus. Controlled JH amount by symbiont Burkholderia in host midgut regulated hexamerin protein expression for speeding up adult emerging and increasing egg production.

Urbanization is one of the leading causes of habitat loss, habitat degradation, and fragmentation. Urban development negatively affects biodiversity. This study aimed to clarify the change of butterfly communities on effect of urbanization in urban green areas. Butterfly survey was conducted using the line transect methods from April to October in 2012. A total of 59 species and 1,465 individuals of butterflies were observed in four urban green areas: Namsan Park (NS), Ewha Womans University (EW), Bukseoul Dream Forest (BD), and Hongneung Forest (HF), and natural forest: Gwangneung Forest (GF). The category of land use around study site was determined based on GIS data. Species richness and abundance of niche breadth and habitat type in urban green areas differed significantly from those in GF. Estimated species richness and species diversity (H’) in four urban green areas were significantly lower than those in GF. Species richness and abundance of forest interior species and specialist were positively correlated with paddy, field, and forest, whereas those of forest interior species and specialist were negatively correlated with urban area and road. Butterfly communities in four urban green area differed from that in GF. The result suggests that the decrease of paddy, field, and forest associated with increase of urban area and road negatively influences species composition and changes butterfly communities.

Ampelopsis brevipedunculata, a porcelain berry that is a kind of Korean domestic wild berry (Viticeae), has been known to be resistant to diseases and insects. A total of 2,622 unigenes containing 912 contigs and 1,710 singletons were obtained by sequencing 5,839 expressed sequence tag (EST) clones derived from the cDNA library of wild grape, A. brevipedunculata. In the gene ontology analysis, 1,175 genes related to biological process, 1,516 genes related to molecular function, and 1,413 ones related to cell components were annotated. Among the genes showing molecular function, 17 clones were classified into defense-related genes, and 102 were known to be related with responses to stress in plants. Domains, such as leucine-rich repeat, Serine/threonine protein kinase-related, WD40 repeat, EF-hand calcium-binding, pentatricopeptide repeat, and pathogenesis-related transcriptional factor were highly expressed. Genes encoding defense related proteins, such as chitinase, catalase, protein-serine/threonine kinases, were also clustered into an abundant group in cDNAs from A. brevipedunculata. Approximately, 80 simple sequence repeats with 2~5 nucleotides were detected in the cDNAs of A. brevipedunculata. These data could provide useful information for the genetic analysis of wild grapevines and in programs for breeding grape cultivars.

For more than two decades, the intracytoplasmic sperm injection (ICSI) technique has been used as a valuable tool to provide opportunities for studying fertilization, treating human infertility, and producing transgenic animals. Not only in facilitating fertilization but also in propagating mammalian species, ICSI has enhanced the potential of assisted reproductive technologies in human. Polyspermic fertilization has been one of major problems in pig reproduction, but the ICSI helped to solve the problem, and used widely to generate transgenic piglets. Although the ICSI technique is considered to be a very useful tool in assisted reproductive technologies, including generation of transgenic animals, there are some disadvantages using the technique. In this review, we describe the ICSI technique and its application in animal production and human infertility, and discuss advantage and disadvantage of the technique in mammals.

The green pale plant bug, Apolygus spinolae was one of the main insect pests that damaged leaves and fruit in grapes and its damage status was firstly reported in 2000 in grape orchards. This research was conducted to evaluate the distribution and difference in damage rate depending in management type of grapevine orchards (domestic sale farm vs export farm) in the export complex area of Korea (Hwangsung in Gyeonggii, Sangju and Yeongcheon in Gyeongbuk, Namwon in Junbuk and Yeongdong in Chungbuk) from 2010 to 2012. Damage by A. spinolae occurred in all 62 survey farms and damage rate differed depending on locality and individual farms in the same area. Damage rate was lower in export farms than in domestic sale farms, and damage rate of leaves was highly correlated with damage rate of new shoots. 15 species of hemipteran insect were attracted to sticky traps and A. spinolae was the dominant species. The attracted number of A. spinolae in the sticky traps differed depending on locality, and more occurred in domestic sale farms than expert farms. A. spinolae was continually attracted to sticky traps in the harvest period in grapevine orchards.

Predatory gamasid mite (Acari: Mesostigmata) is living in soil and litter. They feed on nematode, collembolan and insect larvae. This research compared diversity of gamasid mite in organic and conventional apple orchards. Soil samples were collected in spring season of 2011 to 2012 (5 for each orchard system). Fifteen families, 54 species and 15 families, 50 species were collected from organic and conventional orchards, respectively. In total 18 families and 77 species of gamasid mite were collected. Shannon Diversity was average 2.5 in organic orchard and 2.3 in conventional orchard. Major dominant species was Eugamasus fujisanus (Parasitidae) in organic orchard and Uropoda hokkaidoensis (Uropodidae) in conventional orchard. Parasitidae and Uropodidae were preferred in soil surface, large soil pores and decaying organic material litter. Future, more research on composition of gamasid mite, biotic and abiotic factors in orchard system are demanded.

This report is a part of research on pests occurring in grapevine orchards in export complexes (Hwangsung in Gyeonggi, Sangju and Yeongcheon in Gyeongbuk, Namwon in Junbuk and Yeongdong in Chungbuk) from 2010 to 2012. This research was conducted to evaluate the distribution and difference in damage rates depending on management types of grapevine orchards (domestic sale farm vs. export farm). Damage by Arbordia spp. occurred only in 2010 and differed depending on localities and individual farms in the same area. Numbers of orchards damaged by Arbordia spp. were one, two and four in Hwasung, Namwon and Sangju, respectively, and the damage rate was below 6.2%. There was no damage in the orchards in 2011 and 2012, however, Arbordia spp. were collected on sticky traps in the orchards. A. nigrigena and A. kakogawana were the dominant species in Yeongcheon and Yeongdong, respectively, in 2011. A. kakogawana, A. maculifrons and A. nigrigena were collected on sticky traps in 2012. Collected numbers of Arbordia spp. were different depending on localities and management types of the orchards (domestic sale vs. export). A. kakogawana was the dominant species in all the survey sites and the densities were higher in the domestic sale farms than in the export farms.

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.

Setdb1/Eset, a histone lysine methyltransferase, is recruited by various transcription factors to modify local chromatin. The observation that Setdb1-null blastocysts fail to produce epiblast-lineage cells suggests a role for Setdb1 in generating mouse embryonic stem cells (mESCs). When examined in mouse zygotes, Setdb1 proteins appeared as dots at perinucleolar rims of pronuclei, with the dot-shaped signals more prominent in male pronuclei. Setdb1 signals were observed diffusely in the nucleus from the two-cell stage onward and, by the blastocyst, took a punctate form, away from nucleolus. Such varying expression patterns suggest its involvement in diverse biological processes at preimplantation stage. Setdb1 appeared in Oct4-positive cells of inner-cell-mass origin but not in trophectoderm-lineage cells in blastocyst outgrowths. Setdb1 co- immunoprecipitated with Oct4 in mESCs, and Setdb1 expression was markedly reduced upon retinoic acid- induced differentiation. These observations suggest that Setdb1 has an important role in maintaining the self-renewal of mESCs through collaboration with Oct4.