Artificial insemination (AI) has been performed widely in swine industry using fresh liquid sperm instead of frozen type of sperm. However fresh sperm are not able to preserve more than three days with optimal motility and other sperm parameters for the successful fertilization, since in vitro stored sperm has an oxidative stress that resulted increase of abnormality and acrosome reation. To overcome these major problems, novel preservative formulation is needed to neutralize the oxidative stress and to provide suitable physiological environment for sperm in in vitro. In this study, naturally derived substances such as Poncirus trifoliate (Trifoliate orange), Garcinia mangostana (Mangosteen), pig placenta and testis extracts were tested as sperm preservative agents. Placenta extracts (PE), trifoliate orange extracts (TOE), testes extracts (TE) and mangosteen extracts (ME) were applied to analyze specific parameters for sperm motion characteristics individually and combinatorial. Each individual extract treatment can accelerate the sperm motility but noticeably TOE, TE and ME treatments exhibited the considerable and significant preservation of sperm motility. PE, TE and ME showed a significant (p<0.05) increase in ALH after one week. Further we evaluated the five different combinations of these extracts on sperm motility and its motion characteristics. Surprisingly even after one week ME, TOE and TE combination significantly preserved the sperm motility about 75%. It is noteworthy that unlike individual extract treatment, combination of ME, TOE and TE simultaneously protect the sperm motility and its motion characteristics. Taken together these data conclude that addition of ME, TOE and TE can be effective for preservation of pig sperm.

Inter-granular Bright Points (igBPs) are small-scale objects in the Solar photosphere which can be seen within dark inter-granular lanes. We present a new algorithm to automatically detect and extract igBPs. Laplacian and Morphological Dilation (LMD) technique is employed by the algorithm. It involves three basic processing steps: (1) obtaining candidate "seed" regions by Laplacian; (2) determining the boundary and size of igBPs by morphological dilation; (3) discarding brighter granules by a probability criterion. For validating our algorithm, we used the observed samples of the Dutch Open Telescope (DOT), collected on April 12, 2007. They contain 180 high-resolution images, and each has a 85×68arcsec2 85×68arcsec2 field of view (FOV). Two important results are obtained: first, the identified rate of igBPs reaches 95% and is higher than previous results; second, the diameter distribution is 220±25km 220±25km , which is fully consistent with previously published data. We conclude that the presented algorithm can detect and extract igBPs automatically and effectively.

The button mushroom (Agaricus bisporus) is one of the most widely cultivated important edible mushroom species. In the breeding of new button mushroom, ‘Seolgang’ was developed by crossing two monokaryons ‘CM020913-27’ and ‘SSU423-31’. Because of the secondarily homothallism, only a small percentage of the basidia produce 3 or 4 spores, which are mostly haploid (n) and do not fruit. Single spore cultures derived from these types of spores produce a vegetative mycelium that also contain a variable number of genetically identical nuclei per cell called monokaryon. The lack of clamp connections between monokaryon and dikaryon required a series of mycelial culture and fruiting test. After crossing, hybrids were cultivated on a small scale and on a commercial scale at a farm. For this, the spawn was made by a commercial spawn producer and the spawned compost by a commercial compost producer. Mycelial growth of ‘Seolgang’ on CDA was better at 20℃ and 25℃ when it was compared with that of ‘505 Ho’. The mature cap shape of new strain ‘Seolgang’ is oblate spheroid and the immature cap shape is round to oblate spheroid. The cap diameter was 41.2 mm on average. In comparison with white strain ‘505 Ho’, the strain had a yield that was 9% higher. It produced fruiting bodies which had a higher weight on average per fruiting body and were 19% firmer with a good shelf life. Days of fruiting body were 3-4 days later than those of ‘505 Ho’. The physical characteristics such as elasticity, chewiness, adhesiveness were better than that of ‘505 Ho’. Genetic analysis of the new strain ‘Seolgang’ showed different profiles compared to ‘505 Ho’, CM02913-27, SSU413-31, when RAPD primers A02 and O04 were used.

Varieties of Bacillus thuringiensis (Bt) crystal proteins, Cry proteins, have so far been found as one of the most successful biological control agents which are safe to natural environments for a long time. Recently, cry genes encoding these Cry proteins have been widely applied for construction of transgenic crops resistant to pest insects. In this study, through the 3D structure prediction and accompanying mutagenesis study for the Mod-Cry1Ac, 7 and 16 amino acid residues from domain I and II, respectively, responsible for its insecticidal activity against larvae of Spodoptera exigua and Ostrinia furnacalis were identified. To construct novel cry genes with improved insecticidal activity, we randomly mutated these 23 amino acid sequences by in vitro muti site-directed mutagenesis, resulting in totally 24 mutant cry genes. For further characterization, these mutant cry genes were expressed as a fusion protein with polyhedrin using baculovirus expression system. SDS-PAGE analysis of the recombinant polyhedra revealed that expressed Cry proteins was occluded into polyhedra and activated stably to 65 kDa by trypsin. In the further study, we plan to investigate their insecticidal activity against Plutella xylostella, S. exigua and O. furnacalis larvae.

We developed and characterized five polymorphic microsatellites of Nilaparvata lugens from hybridization method using biotin enrichment strategy and two polymorphic microsatellites from Next Generation Sequencing. Also 11 microsatellites that developed by Sun et al. (2011) are employed to carry out genetic analysis of N. lugens in Southeast Asia. The number of alleles per locus ranged from 2 to 12 with an average of 4.63 alleles per locus. The mean observed heterozygosity of the eleven populations ranged from 0.031 to 0.938 and the expected hetetrozygosity ranged from 0.031 to 0.881. Signifiant genetic differentiation was detected among the three N. lugens populations as the FST ranged from 0.028 (Cheong Do and Ha Long) to 0.161 (CH and BN). The results of microsatellite marker suggested that found N. lugens migrated to Korea at least two times in different period or once. Genetic distance of N. lugens between Korea and Hi Pong were mostly closed and genetic distance of Ha Long and HCM were relatively closed. In this study, development of microsatellites should facilitate the study of future population genetics of N. lugens, and eventually elucidate the route of N. lugens migration to Korea. Thus, combining satisfactory microsatellite markers and intensive surveillance methods in paddy field could be easy to understand to the N. lugens migration mechanism.

Interferon induced transmembrane protein-1 (Ifitm-1) has been reported to have an important role in primordial germ cell formation, and it has expressed in female reproductive organ. In the present study, Ifitm-1 gene expression was identified in testes and all part of epididymis using western immunoblot and immunohistochemistry. Interestingly, Ifitm-1 expression was observed on the head of spermatozoa. To investigate the role of Ifitm-1 gene expression in behavior of spermatozoa after acrosome reaction, fresh sperm was incubated with calcium ionophore to induce acrosome reaction, whereas the expression of Ifitm-1 was not altered after the acrosome reaction. Then to identify the effect of Ifitm-1 in sperm motility and other seminal parameters, different concentration of Ifitm-1 antibody was incubated with spermatozoa, and seminal parameters were assessed using computer-assisted semen analysis (CASA). Interestingly, motility, progressive, and VAP were increased in the sperm with Ifitm-1 antibody treated compared to rabbit serum, however other parameters such as straightness were not changed. In order to identify the functional significance of Ifitm-1 in fertilization, capacitated spermatozoa were pre-incubated with anti- Ifitm-1 antibody and subsequently examined the ability to adhere to mouse oocytes. However, any defection or alteration in sperm-egg fusion was not found, Ifitm-1 antibody treated or non-treated spermatozoa showed a normal penetration. Although the precise role of Ifitm-1 in sperm motility and following fertilization need to be elucidated, this study suggests that the activation of Ifitm-1 on the sperm may enhance the motility of spermatozoa in mice.

Pleurotus ostreatus ‘Miso’ is a mutant strain showing white color in pileus from the known parent strain ‘Wonhyeong 1’. Shape and several other characters also vary with culture conditions. Mating experiments were performed to understand interstrain mating relationship using monokaryons of the parent and the mutant strains. All monokaryons were grown from single spores isolated from freshly collected fruit bodies. Pairings were performed in 90 mm petri dishes on PDA. They were allowed to grow at 25 until two fronts of the advancing mycelia met and developed a conspicuous contact zone. The contact zone and the outer edges of paired colonies on each plate were examined for clamp connections. The parent and the mutant resulted in tetrapolar incompatibility in intrastrain crosses. In interstrain crosses, each monokaryotic tester strain of the parent strain was out-crossed to monokaryotic tester strains of the mutant. As a result of these crosses it was found that both strains share the same A and B incompatibility factors yielding 25% compatibility.

Althogh Spermatogonial stem cells (SSCs) are widely studied in mice, study of pig SSCs is not sufficient for the isolation, long-term culture, and characterization. To identify the effect of growth factor in cultured pig SSC, newly generated pSSCs like cell from neonatal 5days porcine testis were cultured and investigated for the pSSCs like cell formation. Glial derived neurotrophic gactor (GDNF), fibroblast growth factor (FGF), leukemia inhibitory factor (LIF), and epidermal growth factor (EGF) were applied to culture media to identify the pSSC like cell growth and stem cell formation. The criteria for the determining of stem cell characters, morphology, number of colonies, putative stem cell marker were analysed by microspic, polymerase chain reaction (PCR) and immunocytochemistry (ICC) methods. Most of the pSSCs like cells were formed approximately 100 μm size with sphere shape. Most of the feeder cells were highly dependent on FGF that feeder cells were not stably attached on plate without FGF and colony formation of pSSC was not observed consequently. Immunocyto chemistry data revealed that this cells expressed the ubiquitin-C-terminal hydrolase 1 (UCHL-1, PGP9.5) and Dolichos Biflorus Agglutinin (DBA) in addition of 20 ng/ml EGF, 10 ng/ml FGF, 10 ng/ml GDNF, 10 U3/ml LIF. In addition, Alkaline Phosphatase ()was positive in all period of culture. This study suggest that various growth factorsinp SSC culture system is important to regulate and maintain the pSSC. In conculsion, although the precise role of growth factor in pSSC proliferation need to be clarified, combination of growth factor might be critical in order to derivation and proliferation of neonatal pSSCs and spermatogenesis.

Spermatogonial stem cells (SSC) undergo self-renewal division and generate spermatogenesis to produce mature spermatozoa. Very recently in some species, include rodent and farm animals, SSC has been isolated and cultured in vitro. In this study, we analysed SSC marker of both 5 days old and pubertal pig testis by histological method. In 5day pig testis, prior to set of spermatogenic differentiation, the seminiferous tubules contain a relatively large number of SSCs than in pubertal pig testis. Then putative pig SSCs were successfully isolated from 5 day pig testis, and cultured long term using CD34 positive cell culture media contained GDNF, bFGF, LIF and EGF. The SSC colonies were appeared at 3 days after cells were seed. The SSC colonies were alkaline phosphatase positive and strongly expressed PGP 9.5, PLZF and DBA, but not expressed GATA4 and LH receptors. The SSC colonies were stably proliferated in GDNF, bFGF, LIF and EGF contained CD34 positive cell culture media up to 60 days. This study will be facilitated to study of in vitro and ex vivo spermatogenesis and of production of transgenic pigs using sperm vector.

INTRODUCTION In rodent, molecular markers of spermatogonia, spermatocyte, spermatid and sperm have been identified. It has been reported that DBA, PGP 9.5 and NanpG can be the markers for spermatogonia in pig. For further understanding the spermatogenesis of the pig on morphological and molecular level, we report identification of testicular cells in neonatal and pubertal pig testis, and a putative marker for spermatogonia and spermatid in pig testis. MATERIALS AND METHODS Neonatal (3 day) and pubertal testis (150 day) was cut and fixed in Bouin’s solution for immunohistochemistry (IHC). Gonocytes were isolated from neonatal testis for immunocytochemistry (ICC). Based on references (Phillips et al., 2010), thirteen antibodies (VASA, Oct4, NanoG, PGP 9.5, DAZL, SCF, GFR-alpha 1, PLZF, c-kit, integrin-beta1, Thy1, Sohlh1 and CD9) were used for IHC and ICC. Paraffin section was performed for IHC. Gonocytes were attached to the APS-coated slides for ICC. HRP-conjugated and florescent-labeled secondary antibody was used for IHC and ICC, respectively. RESULTS In histological analysis, spermatogonia and Sertoli cells, which are enclosed by seminiferous tubule and connective tissue, were observed in neonatal testis. However, complete spermiogenesis, including spermatocyte, spermatid and spermatozoa, was not observed in neonatal testis. Spermatocyte, spermatid and elongated spermatid were observed in pubertal testis but matured spermatozoa were not observed. As a result, two antibodies (PGP 9.5 and GFR-alpha1) of thirteen antibodies were available for IHC and ICC. As reported in other studies, PGP 9.5 protein was detected in spermatogonia of ne-onatal in IHC. In addition, it was observed in spermatogonia of pubertal testis. GFR- alpha1 protein was detected in spermatogonia of neonatal testis and spermatid of pubertal testis. In ICC, PGP 9.5 protein was detected in gonocytes as reported in other studies. GFR-alpha1 was also observed in gonocyte isolated from pig testis. In this study, we found that 1) only spermatogonia exist in neonatal pig testis (3 day), 2) GFR-alpha1 is a new marker for spermatogenesis in pig testis.

Interferon induced transmembrane protein-1 (IFITM1) is one of transmembrane protein which is differentially expressed in uterus during estrus cycle and pregnancy, that IFITM1 gene is highly expressed in estrus stage by the effect of estrogen, and in parturition by the effect of PGF2 alpha. This genes are also up-regulated in cells with hyperactivation of the WNT/β-catenin signaling pathway. In this study, to identify a function of IFITM1, the binding partner of IFITM1 were determined using immunoprecipitation and LC- MASSMASS methods. 1, 3 and 5 ug of polyclonal anti-IFITM1 antisera were used for immunoprecipitation, and the 75 kDa of specific band was detected in silver stained polyacylamide gel. This band were chracterized using LC-MASS-MASS, and revealed this band is glucose regulated protein 75 (GRP75) which binds to p53 and inhibits the p53 action in nucleus. To identify the localization of GRP75 in cells, immunocytochemical approach has been applied, and GRP75 is expressed in mitochondria of L929 murine connective tissue cells. Co-localization study between IFITM1 and GRP75 in L929 cell identified that these two proteins were closely expressed in mitochondria. Although the role of the interaction of these two protein need to be clarified in various biological phenomena, this data suggest that close interaction of IFITM1 and GRP75 may regulate cellular functions in uterus on sets of estrus cycle and pregnancy.

SERPINB3 (also known Squamous cell carcinoma antigen 1, SCCA1) is involved in apoptosis, immune response, cell migration and invasiveness of cells. It has been investigated in various types of squamous cell carcinoma. Therefore we investigated the functional role of SERPINB3 gene in human epithelial ovarian cancer (EOC) using laying hens, the most relevant animal model. In 136 laying hens, EOC was found in 10 (7.4%). We compared the expression and localization of SERPINB3 using RT-PCR, quantitative RT-PCR, in situ hybridization and immunohistochemistry, and SERPINB3 activation was detected in chicken and human ovarian cancer cell lines using immunofluorescence microscopy. Thereafter, we examined the prognostic value of SERPINB3 expression in patients with EOC by multivariate linear logistic regression and Cox’ proportional hazard analyses. In present study, SERPINB3 mRNA was induced in cancerous ovaries (p< 0.01), and it was only expressed in the glandular epithelium(GE) of cancerous ovaries of laying hens. SERPINB3 protein was localized predominantly to the nucleus of glandular epithelium in cancerous ovaries of laying hens, and it was abundant in the nucleus of both chicken and human ovarian cancer cell lines. In 109 human patients with EOC, 15 (13.8%), 66 (60.6%) and 28 (25.7%) of those patients showed weak, moderate and strong expression of SERPINB3 protein, respectively. Strong expression of SERPINB3 protein was a prognostic factor for platinum resistance (adjusted OR, 5.94; 95% Confidence Limits, 1.21-29.15). Therefore SERPINB3 may play an important role in ovarian carcinogenesis and be a novel biomarker for predicting platinum resistance and a poor prognosis for survival in patients with EOC. This research was funded by the World Class University (WCU) program (R31-10056), Basic Science Research Program (2010- 0013078) through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science, and Technology and by the Next-Generation BioGreen 21 Program (No.PJ008142), Rural Development Administration, Republic of Korea.

Rabies is one of the most dreadful diseases known to human. Annually, more than 55,000 human deaths occur throughout the world. The main transmitters are dogs. In South Korea, urban rabies is eliminated after massive national vaccine programme but rabies is still present in wildlife around northern part of the country near the border. Occasionally, rabies cases are still reported and there are spill over cases from racoon dogs. No human case was reported since 2005. Therefore, risk of rabies from exporting domestic dogs and cats from South Korea is very low. Hence, foreign rabies can be introduced by importing wild carnivores and unvaccinated dogs and cats under the age of three months since the South Korean legislation does not cover them. Therefore, it is essential to update current import regulation to minimise the risk of rabies.

A new cultivar "KNR11-1" of Pleurotus eryngii was developed by the method of mono-mono crossing between monokaryotic strains derived from KNR2598 and KNR2610. The optimum temperature of mycelial growth was 25℃ and that of fruiting body development was 15 - 16℃. The period of harvesting including primordia formation of "KNR11-1" was one day faster than that of control strain KNR2312. The color of pileus and stipe surface was neutral-brown and pure white, respectively. The shape of pileus was dome and has the teeth of a comb. The yield was 90.4 g/850cc plastic bottle. The fruit body has a longer shelf life than control strain KNR2312 at 4℃. It is able to store up to 67.5 days in a cold room. Analysis of the genetic characteristics of the new commercial strain "KNR11-1" showed a different profile as that of the control strain KNR2312 when Random Amplified Polymorphic DNA primers were used. The new cultivar of Pleurotus eryngii is characterized by the improved storability after harvesting.