The Japanese oak silkmoth, Antheraea yamamai Guérin-Méneville 1861 (Lepidoptera: Saturniidae), is one of the important natural resources possessing industrial value for silk fiber production. In this study, ten microsatellite markers and two mitochondrial DNA (mtDNA) gene sequences (COI and ND4) were used to investigate the genetic variation and geographic structure of A. yamamai populations in South Korea. Two mtDNA gene sequences revealed very low total genetic variation and resultant low geographic variation, validating to use further variable molecular markers. Population-based FIS, FST, RST, and global Mantel test consistently support that A. yamamai populations are overall well interconnected with a relatively high gene flow. Nevertheless, STRUCTURE analysis using microsatellite data and mtDNA sequences coincidently indicate the presence of two genetic pools in many populations.

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.

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.

To obtain information on the sanitary of indoor environment in greenhouses used for shiitake cultivation, bacteria associated with larvae and imagoes of Sciaridae flies, pest to shiitake mushroom, were isolated and identified. A total of 1,048 bacterial colonies were isolated from the flies’ larvae and 984 bacterial colonies were isolated from the flies’ imagoes. Based on molecular analysis of the 16S rDNA sequence, Achromobacter xylosoxidans and Tetrathiobacter kashmirensis of β-proteobacteria, Enterobacter asburiae and Raoultella ornithinolytica of γ -proteobacteria, Curtobacterium sp. and Microbacterium thalassium of Actinobacteria, and Penibacillus taichungensis of Firmicutes were identified from the colonies of the flies’ larvae. While, Bacillus megaterium, B. thuringiensis, Lysinbacillus sphaericus and L. fusifomis of Firmicutes, Microbacterium thalassium and Citricoccus parietis of Actinobacteria, and Enterobacter asburiae of γ-proteobacteria were identified from the flies’ imagoes. Some of the isolated bacterial species were known be human pathogens. Overall, the results of this study suggested that mushroom fly carrying human pathogenic bacteria is one of sources impact on the sanitary of indoor environment of greenhouses used for shiitake cultivation.

Distillers dried grain (DDG) and makgeolli spent grain (MSG) are agricultural by-product to produce alcoholic beverage. However, they are known to contain enough nutrients. Mealworm is a promising insect resource for an animal feed ingredient as well as alternative human food. With low cost, DDG and MSG were investigated as a feed ingredient for rearing high quality mealworms. DDG and MSG were mixed with wheat bran and compared to control feed (only wheat bran) for its effects on larval survivorship, larval weight, duration for larval development, pupation rate, and pupal weight. Adding DDG on wheat bran showed positive results for larval weight, duration for larval developmental period, and pupation rate. However, adding MSG made longer duration for larval development, but it also improved larval weight, pupal weight with more than 90% pupation rate. We confirmed that adding 30～50% of DDG or MSG to conventional wheat bran have a strong potential to replace the conventional wheat bran insect feed for quality insect production.

Insect is an important player in the ecosystem as a prey for animals. Moreover, they are a valuable candidate food source for rearing animal. Tenebrio molitor (Coeloptera: Tenebrionidae) larvae are known as a good food source with high protein, unsaturated fatty acid, minerals. Therefore, it has strong potential to substitute the conventional meat consumption. To utilize T. molitor as a feed, the standard mass-rearing protocol is required. To make standard mass-rearing protocol, we tested different temperature(17.5, 20, 22.5, 25, 27.5 and 30°C) conditions for egg, larvae, pupae and adult T. molitor to identify the optimal rearing condition. Hatching was occurred within 15~32.5°C range. However, 17.5~27.5°C was required to get more than 70 % hatching rete. When the eggs were treated in 22.5~27.5°C, all eggs were hatched within 10 days. As larval development, shorter developmental period, higher pupation and eclosion rates were observed within 25~27.5°C temperature range. In addition, we compared the number of egg, oviposition duration and time required to start egg-laying. The minimum egg-laying(258.40±10.86) was observed at 17.5°C, but the maximum(749.10±7.45) was at 27.5°C. The maximum oviposition duration (137.00±12.73 day (mean±S.D.)) was achieved at 27.5°C, but the minimum (87.50±3.54 day (mean±S.D.)) was at 30°C. The time required to start egg-laying was less than 10 days at 17.5, 27.5, and 30°C. To consider all the factors, we concluded that the optimal temperature is 27.5 °C.

Insect pollinators of the endanger orchid Cypripedium japonicum were surveyed and identified during two years, as a part of a conservation project of the orchid at Jukyeup-san and Hwaak-san (Mt.), South Korea. In total 40 individuals of 16 species in 4 families were identified. The dominant family was Halictidae, and Lasioglossum exiliceps Vachal visited the most frequently C. japonicum during the surveys. The average visiting frequency was 2.5 individuals per hour and the highest 4.3, from 12:00 – 13:00 in a day. After 15:00 insects did not visit the flowers at all. However, all of the visiting insects were found to not carry a pollinium or pollens of the orchard on their bodies; pollen carryover by any of the visiting insects did not occur at all. The orchid seems to require certain pollinators in particular body thickness due to its unique pollination mechanism. The orchid has two exit route openings, around 1 cm in diametre, where the entrapped insects can exit and an anther is situated just in front of each opening. It was inferred that a pollen carrier should be around 1 cm in body thickness. Therefore, the candidate species as the proper pollen carriers can be Tetralonia nipponensis Perez, Xylocopa appendiculata circumvolans Smith and Bombus consobrinus Dahlbom among the surveyed visitors.

Smoking is a risk factor for oral leukoplakia and oral cancer, as well as lung cancer, cardiovascular diseases and many other systemic diseases. Smoking is considered increasing factor of some oral diseases involved indigenous bacteria. In addition, a relationship between smoking and infection of Human papillomavirus (HPV), which is associated with oropharyngeal cancer, remains unclear. The aim of this study is to assess whether smoking has an impact on increase of bacteria inducing oral disease such as dental caries and periodontitis, and HPV infection. DNA of saliva gathered from smokers and non-smokers, consisted of men and women, was analyzed using PCR. Oral disease-causing bacteria were more detected in men smokers than men non-smokers and HPV was most found in women non-smokers. Taken together, this study suggests smoking is related with variation of oral microorganism existence in some way.

The fact that flip-flops, one of many different types of unstable shoes, are light and relatively easy to put on, accounts for their popularity among people. But because flip-flops rely heavily on the support of a single thong between your first and second toes, they impose a huge amount of pressure onto lower leg. Thus in the following experiment we tried to examine the different effects of flip-flops and running shoes in terms of their effect on muscle activity and fatigue of tibialis anterior and gastrocnemius during walking. In order to measure an electromyogram we used Free EMG system. 10 men and 10 women in running shoes ran on treadmills for 15 minutes at 4.8km/h, 2 days later the same experiment was carried out, but this time, in flip-flops. p value turned out to be greater than .05 and thus there was no considerable difference between the effects of flip-flops and running shoes on muscle activity and fatigue during walking. Therefore we conclude that despite the fact that flip-flops are considered unstable, their effects on muscle activity and fatigue of tibialis anterior and gastrocnemius are negligible.

Mealworms, Tenebrio molitor (L.) is used as an important animal feed additive for growth promotion and health management, but potentially exposes to fungal infection. In this work, virulence of two species of entomopathogenic fungi against the insect, and the relationship between abiotic features and virulence were investigated. Secondly our consideration was also given to the effect of chemical fungicides on conidial germination for risk control. Between Beauveria bassiana (Bb) and Metarhizium roberstii (Mr) (previously M. anisopliae), Bb isolates had much higher virulence (~100% mortality in 3~4 days after the treatment), rather than Mr isolates in laboratory assays. Next, fungus-treated mealworms were kept at wheat bran at 20, 25, 30 and 35℃ with 3, 6, 9 times of water spray to the feeds for set-up of different humidity conditions. Inoculation of fungi to mealworms was conducted by fungal spray and feeding methods, which resulted in higher virulence in feeding method. In the feeding method, all temperature treatments except 35℃ showed high virulence against mealworms, but any significant relationship between virulence and humidity was not observed. In the chemical fungicide screening, fluazinam (CAS No. 79622-59-6) and mancozeb (8018-01-7) significantly inhibited the germination of Bb and Mr conidia. This work suggest that contamination of wheat bran with fungal pathogens, particularly B. bassiana may induce mycosis of mealworms, but introduction of effective fungicides possibly reduce fungal infection.

True hermaphrodites are animals of equivocal sex in which both male and female gonads develop simultaneously. The frequency of true hermaphroditism is higher in pigs than in other domestic animals. Two Korean pigs were diagnosed with true hermaphroditism showing ovotestes, epididymes, penes, and uteri. Histomorphologically, the testicular tissues consisted of Sertoli cells that were devoid of spermatogenic germ cells and showed proliferation of interstitial cells. However, the uteri were of normal architecture and had well-developed uterine endometrial glands. The samples were 38, XX female karyotype without the sex-determining region Y (SRY) gene. The findings of this study could contribute to the understanding of true hermaphroditism in the Korean pig industry. * This work was supported by a grant (Code# PJ008148) from BioGreen21 Program, Rural Development Administration, 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.