Vibrio spp. and Streptococcus spp. have caused a considerable disease of farmed fish and economic loss in fish farming and seafood industry. In this study, the efficacy of an aquatic disinfectant tablet composed to calcium hypochlorite was evaluated against V. anguillarum and S. iniae. A bactericidal efficacy test by broth dilution method was used to determine the lowest effective dilution of the disinfectant following exposure to test bacteria for 30 min at 4oC. An aquatic disinfectant tablet and test bacteria were diluted with distilled water (DW), hard water (HW) or organic matter suspension (OM) according to treatment condition. V. anguillarum on the DW, HW and OM condition was completely inactivated with 16,000 15,000 and 13,000 fold dilutions of the disinfectant, respectively. On the DW, HW and OM condition, S. iniae was absolutely inactivated with 17,000 16,000 and 14,000 fold dilutions of the disinfectant, respectively. As an aquatic disinfectant tablet possesses bactericidal efficacy against fish pathogenic bacteria such as V. anguillarum and S. iniae this disinfectant solution can be used to control the spread of fish infective bacterial diseases.

Bovine brucellosis causes abortion and infertility. The authors conducted this study in order to determine pathological lesions of Korean native cows and fetuses who received experimental vaccination with Brucella abortus RB51 and were challenged with Brucella abortus 2308. Gross and histopathological lesions in endometrium and placenta were observed in cows of the vaccinated group. Twenty-five percent of pregnant cattle in the vaccinated group showed endometritis and placentitis, which was three times lower, compared with the non-vaccinated group. The pathological lesions in the uterus and placenta in both groups were consistent with previous reports. Therefore, vaccination in heifers using Brucella abortus RB51 may not provide adequate protection against infection with Brucella abortus virulent strain.

The purpose of this experiment was to compare the pregnancy rate (PR) according to the state of the ovaries and uterus, according to the number of embryos transferred from cows and heifers and to investigate the method of artificial twin induction with Hanwoo in vitro fertilized (IVF) embryos by embryo transfer (ET). Looking at the PR according to the condition of the ovaries and uterus, the result was not influenced by the condition of the ovaries, but was significantly influenced by the state of the uterus. The PR according to the number of embryos transferred from cows was 36.8%, 53.0%, 50.5% for 1, 2, and 3 embryos, respectively and although there was a higher frequency of twin calves with 3 embryos than 2, the calving rate was the highest with 2 embryos. In case of heifers, the transfer of 1 embryo showed the best pregnancy and calving rate, and although the PR was similar with 2 embryos (67.7 versus 66.4), in case of 2 embryos transferred there was high frequency of embryonic loss (6.1%) occurred when a cow was diagnosed at 28 and 53 d after ET, total loss (21.3%); sum of fetal death, abortion and stillbirth after pregnant diagnosis at 60 day.

Since embryonic stem cells (ESCs) were first established from explant cultures of in vivo day 3.5 mouse embryos, the establishment of ESCs from species such as primates and rat has been developed. However, this success relies on the development of culture medium suitable for human and rat cells, which has different requirements from the murine ESC. In general, the establishment of ESC from pig and cow is of great interest both the agricultural perspective and for biomedical application. Large animal models, particularly pig, are likely to provide models of human genetic diseases and transplantation research where rodent models are inappropriate. However, establishment of ESCs establishment from pigs has remained an elusive goal. In the present study, we focused on signaling transduction regulation in pig epiblast stem cells (pEpiSCs). Pig epiblasts were isolated from early tubular stage embryos collected in vivo day 10.5～12 after insemination. Epiblasts were separated from trophoblast and underlying primitive endoderm using 21G needles and fine forceps. Epiblasts were cultured on mitomycin C (10 μl/ml) treated mouse embryonic feeder cells in Dulbecco’s modified Eagle’s medium (DMEM) containing 1% minimal essential medium (MEM) nonessential amino acids, 1% penicillin/ streptomycin, 1% glutamine, 0.007% β-mercaptoethanol, 5 ng/ml bFGF and 1 ng/ml LIF. After plating rapid differentiation of isolated epiblasts to extraembryonic cell types was visualized in most cultures but stem cells were enclosed by these differentiated cells. We have established seven pig epiblast stem cells lines (pEpiSC1-7) from Days 10.5–12 pig embryos. pEpiSC expressed the pluripotent markers including OCT4, NANOG, SOX2 and NODAL at 3-5 passage. In addition, the modification of culture condition by the inclusion of particular protein kinase inhibitor such as Akt inhibitor, PD0325091(PD), delyed rapid differentiation of pEpiSCs. These results showed that stemness of pEpiSCs can be maintained by regulation of signaling pathway. * This work was partly supported by a grant from the NPR (2011-0013703) and the Next-Generation BioGreen 21 Program (No. PJ008209), Rural Development Administration, Republic of Korea.

Semen can be divided into two parts. One is cellular part which contains sperms the other is liquid part which is called by seminal plasma. The seminal plasma is a nutritive and protective medium for the sperms. Fructose, which is major energy source, is supplied to sperms swim to female oocyte. Alkalic property protects sperms from hostile environment of female reproductive organ. Also, seminal plasma induces tolerance to preexisted immune cells, and changes intra‐uterine environment to better conditions for fertilized embryos to implant. However, the effects of seminal plasma in in vitro culture of fertilized embryos are unclear. Second fraction of fresh semen was obtained from a normal farm pig. The semen was centrifuged to remove sperms, and then supernatant was filtrated. The filtered seminal plasma was stored in — 30℃. In this study, electrically activated and chemically activated porcine embryos were employed to investigate the developmental rate after 2 hours treatment of none, 0.1%, 0.5%, and 1% seminal plasma in culture media by two days of activation. Both electrically and chemically activated embryos, cleavage rate and cell numbers of blastocysts were not significant difference within four groups. Blastocyst formation rate of electrically activated embryos also did not show significant difference within any groups. However 0.1% seminal plasma treatment group showed significantly increase of blastocyst formation rate in chemically activated group (None; 24.8%, 0.1%; 31.7%, 0.5%; 19.4, and 1%; 16.5%, respectively. p<0.05).

Poly(ADP-ribosyl)ation is post-translational modification of cellular proteins related to cell survival, cell death, cellular proliferation and epigenetic events. It has recently been shown to be important for pre-implantation development of mouse embryos. However, its function during early embryonic development of pig is not clear. This study investigated the importance of poly(ADP-ribosyl)ation during in vitro development of pig embryos produced by in vitro fertilization(IVF) or parthenogenetic activation (PA). Results showed that, chemical inhibition of PARP by 3-aminobenzamide (3-AB) did not influence the in vitro development of pig embryos up to morula stage (20±3.1 vs. 28.1±1.2%; p>0.05) but significanlty reduced the rate of blastocyst formation (5.2±2.1 vs. 20±3.1%; p<0.05) when compared to non-treated controls. Furthermore, culture of morula stage embryos in the pressence of 3-AB for 24h significantly reduced the rate of blastocyst formation (19.6± 4.6 vs. 41.4±5.3%; p<0.05) and expansion (4.7±3.0 vs. 28.1±6.1; p<0.05). The proportion of large-sized blastocyst (>200 μm) having higher blastocoel volume (15.3×106 μm3) was significantly reduced (p<0.05) in treatment group (32.2±7.8%) compared to non-treated control group (65.7±9.0%). TUNEL assay revealed that poly(ADP-ribosyl)ation-inhibited blastocyst had significantly increased indices of apoptosis than those of non-treated controls (10.88±0.02 vs. 2.71±0.01; p<0.05). These data suggest that Poly(ADP-ribosyl)ation may be important for blastocyst formation in pig embryo.

In the present study, we investigated the effect of porcine follicular fluid (PFF) concentration (10% vs. 1%) and protein-free media (PFF 0%) on maturation of porcine oocytes in vitro and analysed difference in gene expression in resulting blastocysts following parthenogenetic activation. Three groups were tested; 1) 10% PFF: Tissue culture medium (TCM) 199+10% PFF; 2) 1% PFF: TCM 199+1% PFF; and 3) 0.1% PVA: TCM 199+0.1 PVA. Cumulus-oocyte-complexes were cultured in the respective media containing gonadotrophin (1 ug/ml), epidermal growth factor (10 ng/ml), cystein (0.57 mM), sodium pyruvate (0.91 mM), insulin (5 ug/ml), 9-cis retinoic acid (5 nM) for 20～22 h and then without hormonal supplements for an additional 20-22 h. Data was analyzed using statistical analysis system(SAS) program. There was no significant difference in oocyte maturation rate. However, significantly higher (p<0.05) proportions of embryos developed to the blastocyst stage when oocytes were matured in 10% PFF group (45%) than in the 1% PFF group (31.1%). The total cell numbers were not significantly different among groups (52 ± 1.3 vs. 54.6±3.1 vs. 54.4±2.5, respectively). The relative abundance (ratio to beta-actin mRNA) of gene transcripts related to apoptosis in blastocysts was measured by real- time PCR. The expression of anti-apoptotic gene (BclxL) was up-regulated and the expression of pro-apoptotic gene (Bax) was down-regulated in 10% PFF group than in the other groups. Therefore, it can be concluded that supplementation of 10% PFF during in vitro maturation improves embryo development by reduction of apoptosis. * This study was supported by IPET (#311011-05-1-SB010), RNL Bio (#550-20120006), MKE (#10033839-2011-13), Institute for Veterinary Science, the BK21 program and TS Corporation.

Embryo transfer (ET) is the final procedure for getting pregnancy through assisted reproductive technology such as IVF (in vitro fertilization), SCNT (somatic cell nuclear transfer). In our laboratory, the porcine cloned embryos loaded in ET medium are carried for 3 hours by portable incubator because of the great distance from the laboratory to the experimental farm. Thus, before transferring into recipient, porcine cloned embryos are exposed in vitro condition for long time. Medium which is used in this process is the TALP (Tyrode’s medium supplemented with 10 mM HEPES), but it includes little nutrients for embryo. Thus, the aim of this study is to determine whether ET media containing nutrients affect the in vitro development of embryos compared to TALP. For the experiment, porcine zygote medium (PZM)-5 which has amino acids for developing embryo was chosen as ET medium containing nutrients, added 10 mM Hepes as PZM-5 does not contain buffering system. For experiment, we carried out parthenogenesis through a chemical method using Thi/DTT. Parthenogenetic embryos were cultured in PZM-5 for 2 days, and then they were randomly divided into two group; loaded in a straw with TALP or PZM-5-Hepes, respectively. They were stored in a portable incubator for 3 hours to simulate the time consumed in ET, thereafter embryos in both TALP and PZM-5-Hepes groups were respectively cultured in PZM-5 for additional 5 days. All experiments were repeated 5 times. In result, blastocyst formation rate were 22.46%±1.47 and 23.17%± 2.13, respectively and total cell number were 32.9±2.22 and 37.09±2.18, respectively. There is no significant difference between TALP and PZM-5-Hepes groups. * Further study will investigate effect of PZM-5-Hepes on in vivo development of porcine cloned embryo. This study was supported by IPET (#311011-05-1-SB010), RNL Bio (#550-20120006), Institute for Veterinary Science, the BK21 program and TS Corporation.

Adipose tissue-derived mesenchymal stem cells (ASCs) are very interesting in several laboratory animals and humans because they are easy to harvest and expand to generate millions of cells from a small quantity of fat. ASCs are known as useful materials for clinical applications in human cell therapy and as a donor cell in somatic cell nuclear transfer (SCNT). Here, we investigated if 1) minipig ASCs can be isolated, self-renewed and differentiated into multiple tissue lineages, 2) ASCs can be a suitable donor cell type for generation of cloned pig. In order to isolate ASC, adipose tissues were collected from inguinal region of a 6-year-old female minipig. The ASCs were attached to the culture dish with a fibroblast-like morphology. They expressed cell-surface marker characteristics of stem cell, underwent osteogenic, adipogenic, myogenic, neurogenic and chondrogenic differentiation when exposed to specific differentiation-inducing conditions. To investigate its potential as donor cell for cloning, we respectively carried out SCNT using ASC, adult skin fibroblast (ASF) and fetal fibroblast (FF) derived from same minipig. The ratio of blastocysts to 2-cell embryos and total cell number of blastocysts were monitored as experimental parameters. In results, cleavage and developmental competence to blastocysts rate showed no significant difference among the three groups. On the other hand, total cell numbers of blastocysts derived from ASC and FF were significantly higher than in ASF (89±7.9 and 105±5.5 vs. 57.5±5.2, respectively). Our results demonstrated that ASC have potential compared to ASF and FF in terms of the in vitro development and blastocyst formation ability. In further study, we will investigate the in vivo developmental ability of ASC as donor cell for pig cloning. * This study was supported by IPET (#311011-05-1-SB010), RNL Bio (#550-20120006), Institute for Veterinary Science, the BK21 program, TS Corporation and Optifarm Solution.

This study was carried to develop new white cultivars that are suitable export using back cross method of Hypsizygus marmoreus. At first, we did select mother strains(Hm3-8 as brown and Hm0-7 as white) that have excellent cultivational caracteristics and morphology. The two strains are collected spore and selected 150 monokaryon mycelia by dilution method, and then picked out, 10 monokaryon strains, that havn‘t clamp connection and different mycelial morphology on plate. The selected monokaryon mycelia of GPHm3-8 and GPHm0-7 was crossed in PDA plate and the, acquired 100 dikaryon strains as mating rate is 100%. The 100 dikaryon strains is cultivated by sawdust media. And, we are selected 5 strains (BW16, BW41, BW56, BW76, BW96). To practice back cross, we selected 10 monokaryon mycelia that didn't clamp connection from the 5 stains(BW16, BW41, BW56, BW76, BW96). And then, we were carried out back cross with Hm0-7. The mating rate were investigated 31%, 33%, 33%, 34%, and 47% within BW16, BW41, BW56, BW76, and BW96, each other. We obtained 15 hybrid strains as brown line, and 23 hybrid stains as white line among 178 hybrid strains, and picked up 23 white strains because 15 brown strains were not suitable our object to develop new white cultivars among 38 strains. To select the best white strain , cultivated by standard growing condition, and BW76W-13 strain has been selected because it had good quality and morphology, finally. We were carried RAPD to verify difference between mother strains. We confirmed that band pattern of BW76W-13 had different pattern compared with mother strains.

Mushroom is cultivated as one of the major economical crops in many areas in Korea. The total production has steadily increased from approximately 186,400 M/T in 2007 to 198,563 M/T in 2009. Several bacteria are known as the causal agents of diseases of the cultivated button mushroom (Agaricus bisporus) and oyster mushroom (Pleurotus ostreatus). Pseudomonas tolaasii is the causal agent of brown blotch disease of commercial mushrooms. Colonization of mushroom caps by the bacterium results in development of brown or cream lesions on pileus and stipe. These lesions are slightly concave spots and can be round or spreading. Antagonists against P. tolaasii, HC1 were selected and their control efficacy of brown blotch disease was investigated in this study. The HC1 isolate was selected as an inhibitor of tolaasin activity by bioassay on potato and it was identified as Pseudomonas sp. by the cultural, physiological and biochemical properties and analysis of the 16S rRNA. Control efficacy of brown blotch disease by HC1 treatment was 69% on Agaricus bisporus, 68% on Flammulina velutipes and 55% on pleurotus ostreatus respectively.

The total production has steadily increased from approximately 186,400 M/T in 2007 to 198,563 M/T in 2009. Winter mushroom, Flammulina velutipes, with 61,057913 M/T in 2009, showed the highest production. Several bacteria are known as the causal agents of diseases of the cultivated button mushroom (Agaricus bisporus) and oyster mushroom (Pleurotus ostreatus). Pseudomonas tolaasii is the causal agent of brown blotch disease of commercial mushrooms. Antagonists against P. tolaasii, HC5 were selected and their control efficacy of brown blotch disease was investigated in this study. After proceeding antagonistic test, HC5 was selected as a strong antagonist against P. tolaasii and the HC5 strain was identified as P. azotoformans with the cultural, physiological and biochemical properties and analysis of the 16S rRNA. Control efficacy of brown blotch disease by HC5 treatment was 73% on Agaricus bisporus, 78% on Flammulina velutipes and 71% on pleurotus ostreatus respectively.

The green peach aphid, Myzus persicae (Sulzer), is one of the most serious pest in cabbage cultivation. Field survey was carried out to know the insecticide resistance levels in five main cabbage cultivation regions (Pyeong-chang, Hong-cheon, Bong-wha, Mu-ju and Je-ju) in 2009~2011. The green peach aphid can resist a wide range of insecticides in five surveyed local populations. Among the nine tested insecticides, four chemicals (methomyl, bifenthrin, pymetrozine and flonicarmid) showed less than 60% mortality in the recommended concentration in most years of local populations. Multi resistant (MR) strain was selected from these populations and modified AChE (MACE: StoF mutation), MtoL mutation in para-type sodium channel, esterase over-expression were observed in almost of all populations including MR strain. Especially, StoF and MtoL mutations were highly correlated with resistance ratio and population based quantitative sequencing results. Therefore, these results suggested that molecular-biology based resistance monitoring can applied resistance management in M. persicae.

We investigated the spatial distribution of ground beetle species from the edge of secondary forests in Hwaseong-si, Gyeonggi-do. Nine secondary forests were selected, and 81 pitfall traps for collecting ground beetles were placed along forest interior―forest edge―forest exterior gradients during 14 June to 9 November, 2011. A total of 32 species belonging to 13 genera of 8 subfamilies were identified from 3710 collected ground beetles. Ten dominant species were selected for analysis. Five species such as Chlaenius micans (ANOVA, with Tukey's test, F2, 78=3.11, P=0.0502), Chlaenius ocreatus (F2, 78=2.76, P=0.0692), Dolichus halensis halensis (F2, 78=9.80, P=0.0002), Harpalus eous (F2, 78=2.73, P=0.0712), and Harpalus tridens (F2, 78=6.74, P=0.0020) were abundant at forest exterior. Three species such as Synuchus cycloderus (F2, 78=7.91, P=0.0007), Synuchus nitidus (F2, 78=9.72, P=0.0002), and Synuchus sp.1 (F2, 78=4.50, P=0.0142) were abundant at forest edge and forest interior. Coptolabrus smaragdinus branickii (F2, 78=3.24, P=0.0444) was abundant at forest edge and exterior. Finally, Chlaenius naeviger (F2, 78=1.39, P=0.2542) showed a wide distribution in this study. This study showed a significant edge effect on dominant ground beetles. Many environmental variables and prey items may be important factors for the spatial pattern of ground beetles. Because forest edges are generally important for many predaceous arthropods including ground beetles, the conservation and management of forest edges are important for maintaining biodiversity and ecosystem functions.

The cotton aphid, Aphis gossypii (Glover), is one of the most serious pest in seed potato and various vegetable cultivation. The imidacloprid-resistant strain (IR) was over 300-fold more resistant to imidacloprid compared to a susceptible strain (S) as judged by LC50 values. A highly imidacloprid-resistant local field population (L) was collected from cucumber at Gangwha island in 4th August 2011. Even though neonicotinoid insecticides especially imidacloprid were sprayed six times during June and July, aphid density was too high to be counted. To identify differentially expressed genes in IR or L, comparative transcriptome analyses based on GS-FLX were conducted using total RNAs extracted from IR, L and S strains. Futhermore, to search the resistance associated proteins in IR or L, comparative proteome analyses based on 2DE were conducted using total proteins extracted from IR, L and S strains. Few common candidate genes detected among IR and L such as ABC genes. Comparison of the nucleotide sequence of six nicotinic acetylcholine receptor (nAChR) subunit (alpha 1-5, beta 1) genes from IR, L and S strain revealed a point mutation in the loop D region of the nAChR beta 1 subunit of the IR, causing an arginine to threonine substitution (R81T). These mechanisms also reported in Myzus persicae and this amino acid change confers a vertebrate-like character to the insect nAChR and results in reduced sensitivity to neonicotinoids.

The Diadegma fenestrale was known as parasitoid on potato tuber moth, Phthorimaea operculella and diamondback moth, Plutella xylostella. This species, genus Diadegma are first reported from Korea. DfIV showed typical ichnovirus shape which two membranes surround virus capsids. The genome contents of DfIV consist about sixteen double-stranded DNA segments ranging 2 to 6 kb. To identify DfIV genes, whole genome sequencing based on GS-FLX was conducted using purified total viral DNA extracted from D. fenestrale calyx. About sixty ORFs were analyzed and several typical polydna virus gene family detected such as cys-motif, rep, vinnexin and vankyrin. This is the first report of DfIV and these lepidopteran host immune suppression genes will be deeply identified.

The purpose of this experiment is to compare the pregnancy rate (PR) according to the state of the ovaries and uterus, according to the number of embryos transferred from cows and heifers and to investigate the method of artificial twin induction with Hanwoo in vitro fertilized (IVF) embryos by embryo transfer (ET). Looking at the PR according to the condition of the ovaries and uterus, the result was not influenced by the condition of the ovaries, but was significantly influenced by the state of the uterus. The PR according to the number of embryos transferred from cows was 36.8%, 53.0%, 50.5% for 1, 2, and 3 embryos respectively, and although there was a higher frequency of twin calves with 3 embryos than with 2, the calving rate was the highest with 2 embryos. In case of heifers, the transfer of 1 embryo showed the best pregnancy and calving rate, and although the PR was similar with 2 embryos (67.7 versus 66.4), in case of 2 embryos transferred there was high frequency of embryonic loss( 6.1%) occurred when a cow was diagnosed at 28 and 53 d after ET, total loss (21.3%; sum of fetal death, abortion and stillbirth after pregnant diagnosis at 60 day).

Somatic cell nuclear transfer (SCNT) and induced pluripotent stem cell (iPS) experiments have generally demonstrated that a differentiated cell directly converts into a undifferentiated or pluripotent state. In SCNT experiment, nuclear reprogramming is induced by exposure of introduced donor cell nuclei to the recipient cytoplasm of matured oocytes. Although nuclear reprogramming of cells by the ex-ovo methods is not always consistent or efficient, it has been suggested that a combination of nuclear reprogramming technique may improve the efficiency or frequency of normal development of SCNT embryos. Here we hypothesized that treatment of somatic cells with extracts prepared from GV stage sturgeon's oocytes prior to their use as nuclear donors for SCNT will improve subsequent development. We reported a reversible permeabilization protocol with digitonin to deliver sturgeon oocyte exteact (SOE) to porcine fetal fibroblast cell nuclei ex ovo. Porcine fibroblasts were permeabilized by 4 μg/ml of digitonin for 2 min at 4℃ and then incubated in SOE for 7h at 15 18℃ followed by resealing of cell membrane. As results, no difference was observed in the number of fused couplets or the number of fused couplets that cleaved between the extract treated or control group. However, there was a significantly decrease in the percentage of fused couplets that developed to the blastocyst stage in the SOE treated group (p<0.05). Histone acetylation status was determined using an antibody to acetylation at lysine 9 on histone 3 (H3K9Ac). The intensity of H3K9Ac staining in 1-cell stage NT embryos was significantly increased when treated with the SOE (p<0.05), similar to that in 1-cell stage IVF embryos. In addition, porcine NT embryos reconstructed by using donor cell exposed to SOE prior to cell fusion significantly decreased developmental competence to the blastocyst stage but increased pluripotent gene expressions (Sox2, Nanog and Oct3/4) when compared with those in normal NT embryos (p<0.05).

Several studies have been conducted with the aim of establishing embryonic stem cell lines from porcine embryos. However, most researchers to date have found it difficult to maintain an ES-like state in derived cell lines, with the cells showing a strong tendency to differentiate into an epithelial or EpiSC-like state. We have also been able to derive cell lines of an EpiSC-like state and a differentiated non-ES-like state from porcine embryos of various origins, including invitro fertilized(IVF), in vivo derived, IVF aggregated and parthenogenetic embryos. In addition, we have generated induced pluripotent stem cells(piPSCs) via plasmid transfection of reprogramming factors (Oct4, Sox2, Klf4 and c-Myc) into porcine fibroblast cells. X chromosome inactivation (XCI) have recently been addressed as a hallmark to determine whether pluripotent cell is naïve or primed state. In this study, we could confirm the X chromosome inactivation status in female cell lines as well as marker expression, pluripotency and of our Epi- SC-like pESC lines along with our piPSC line. All of our cell lines showed AP activity and expressions of the genes Oct4, Sox2, Nanog, Rex, TDGF1, bFGF, FGFR1, FGFR2, Nodal and Activin-A involved in pluripotency and signaling pathways, XCI in female cell lines, in vitro differentiation potential and a normal karyotype, thus displaying similarities to epiblast stem cells or hES cells. Therefore, it may be inferred that, as a non-permissive species, the porcine species undergoes reprogramming into a primed state during the establishment of pluripotent stem cell lines.