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.

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.

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.

Attacin is a well-studied glycine-rich antibacterial protein in insect immune response, which has limitary antibacterial effect to some Gram-negative bacteria. A cDNA encoding the attacin gene was screened and isolated from the immunized larvae of the swallowtail butterfly, Papilio xuthus. The complete P. xuthus attacin cDNA is 949 nucleotides encoding a 250 amino acid precursor that contains a putative 18-residue signal peptide, a common 42-residue propeptide sequence and a presumed 190-residue mature protein with a theoretical mass of 19904.01 and a pI of 9.13. The putative mature protein of P. xuthus attacin showed 48%~52% and 24%~30% identity in amino acid sequences with that of lepidopteran and dipteran insects, respectively. The attacin transcript was induced at significant level after injection with bacterial lipopolysaccharide (LPS). Recombinant attacin was highly expressed in E. coli BL21 (DE3) cells by fusing with an N-terminal S-tag/thrombin cleavage site configuration protein to avoid the cell death during induction. The expressed fusion protein was purified by Ni-NTA immobilized metal affinity chromatography (IMAC). After desalting and cleavage with thrombin, the recombinant attacin was released and showed considerably antibacterial activity against Gram-negative bacteria, E. coli ML 35. Our results proved that this protein family with a potent antibacterial activity may play a role in the immune response of butterflies.

The use of natural paint for the application to walls and furnishings is now increasing to improve indoor air quality, thereby the natural paint could be a significant source of biogenic volatile organic compounds (BVOCs) in indoor environments. Recent studies have shown that gas-phase reactions between terpenes and ozone can generate sub-micron size particles and toxic volatile organic compounds such as aldehydes and ketones. In this research, we have studied the formation of particles and secondary organic compounds during the reaction of ozone with terpenes emitted from commercial natural paint. The paint applied onto stainless steel was dried and oxidized in a teflon chamber. Two monoterpenes (α- and β-pinenes) were identified by FTIR and GC/MS. Several tests were performed to evaluate the effects of ozone concentration on particle formation. Increased ozone levels significantly affect the increase of particle number concentration (monitored with SMPS), which results in the increase of particle counts ranging from 8,000 to 70,000 particles/㎤. Gas-phase products such as formaldehyde, acetaldehyde, acetone + acrolein, and propionaldehyde were identified during the terpene/ozone reactions by HPLC. These compounds are potential hazardous chemical compounds having harmful health effects to animals and plants. The results obtained from this study provide an insight on the adverse effect of eco-friendly natural product on indoor air quality (IAQ).

치과 콘빔 전산화단층 검사 시 갑상선 부위의 방사선 피폭선량을 최소화하기 위하여 보루스 (Bolus)를 이용한 차폐체를 제작하고, 방사선 차폐 효과와 영상의 적정성을 평가하였다. 치과용 콘빔 전산화단층검사 장비를 사용하여 치과방사선 두부 팬텀을 대상으로 갑상선의 좌, 우측 부위에 유리선량계를 부착시켜 방사선량을 측정하였다. 차폐체의 두께를 각각 10 mm, 20 mm, 30 mm로 다르게 하여 각 차폐체별로 흡수선량을 측정하여 차폐체를 사용하지 않았을 경우와 비교하였다. 8명의 평가자가 진료영상의 적정성을 여부를 평가하였다. 왼쪽 갑상선 부위에 30 mm Bolus 차폐체를 사용한 경우에는 결과값이 평균 342.67 μ㏉로 Bolu s 차폐체를 사용하지 않고 측정한 결과값의 평균 431.22 μ㏉보다 20.7% 감소하였고, 오른쪽 갑상선 부위에 30 mm Bolus 차폐체를 사용한 경우에는 평균 424.56 μ㏉로 21.9%의 선량감소효과를 보였다. 진료 영상의 적정성은 평가자 모두 사용 가능하다 판단하였다. 결론적으로, 치과 콘빔 전산화단층검사 시 갑상선 부위의 방사선 피폭선량을 최소화하기 위해 제작된 Bolus 차폐체는 장해음영 없이 적정한 진단 영상 처리가 가능하고, 방사선차폐 효과가 있어 유용한 차폐체로 적용 가능할 것으로 사료된다.

The morphometric truss characteristics and classical dimensions of the marine medaka, Oryzias dancena, that might distinguish diploid and triploid fish were examined. Significant differences in all the classical and truss dimensions of the diploid and triploid fish were observed in both sexes (p<0.01). All the dimensions of the triploid fish were greater than those of the diploid fish. The triploid marine medaka shows sexual dimorphism in these characters, and the sexual dimorphism of the triploid marine medaka is similar to that of the diploid marine medaka. Thus, when their classical dimension and truss dimension was measured, the growth of triploid marine medaka is faster than that of the diploid fish, and it displays clear sexual dimorphism, with male fish having longer dorsal and anal fins than female fish.

The objective of this study was to determine the mitotic intervals (τ0) of two consecutive cell divisions and synchronous embryonic cleavage in grass puffer, Takifugu niphobles at different water temperatures (18, 20, 22, and 24℃). The color of the fertilized egg was light yellowish. The egg type was demersal and unadhesive. Egg weight was 0.09±0.002 mg. The sizes of unfertilized eggs were smaller than fertilized eggs in major axis and minor axis at 20℃ (p<0.05). The size of the fertilized egg of 18℃ water temperature group at the blastodisc stage was the smallest (p<0.05), but no significant differences were observed in the other water temperatures group except 18℃ water temperature group (p>0.05). The first cleavage stages at 18, 20, 22, and 24℃ were at 75, 90, 105, and 120 mins, respectively. As water temperature was increased, embryonic development and formation time of the first cleavage furrow were accelerated. There were negative correlation between τ0 and water temperature for grass puffer (Y=–1.225X+70.05, R2=0.988, n=10, where Y was τ0 and X was temperature). This study confirmed that successful hatching of grass puffer was related to water temperature. Chromosome manipulation will be helpful for this species using cleavage frequency and τ0.

Background : The purpose of this study was to evaluate methods to high quality food ramie rice cake, thereby increasing farm income. This study investigated the effects of different Split Application and Adapted Fertilizer Culture for Stable Production with Edible Leaf in Ramie (Boehmeria nivea L.). Methods and Results : Method of fertilizer application were conducted under three condition compose to standard application (N-P-K-Compost applied at 27-9-27-1,500 ㎏․ 10 a−1), 25% increased application (N-P-K-Compost applied at 34-11-34-1,500 ㎏․10 a−1), 50% increased application (N-P-K-Compost applied at 41-14-41-1,500 ㎏․ 10 a−1) cultivation. Compost and fused phosphate applied at 100% of basal fertilizer in March 25. The split application level of standard application (N-K) applied at 50% of basal fertilizer in March 25, 50% of top dressing were five times application (N-K) in May 4, July 5, September 5, October 5, 20% respectively. Transplanting with Yeongkwang varieties in April 12 by growing pot seedling for 35 days in 2011. Planting density were spaced 60 ㎝ apart in rows 25 ㎝ apart with rice straw cutting covered open cultivation. Plot design randomized block 3 repetition. Conclusion : Organic matter, available phosphate, K2O, CaO, and MgO of soil increased in all plots with the addition of fertilizers culture, specifically in 25% and 50% increased application. Growth characteristics by 50% of top dressing were five times application (N-K) in May 4, July 5, September 5, October 5, compared to standard application (N-K) applied at 100% basal fertilizer, aerial part as a result were so many such amount of growth as number of branch and leaf per plant, etc., Length, width of leaf and leaf colorimetry of aboveground part growth increment rising highest. Green leaf yields compared to standard fertilizer 100% application (1,344 kg․ 10 a−1). 25% and 50% increased application (N-K) applied at 50% basal fertilizer, 50% of top dressing were five times application increased by 9%, 17%. respectively.

Optimum concentrations of anesthetic clove oil and anesthetic lidocaine-HCl were determined for a species of adult marine medaka, Oryzias dancena, over a range of salinity conditions, and investigated in a transport simulation experiment by analyzing various water and physiological parameters. Research indicated that the higher the concentration of anesthetic at each salinity, the shorter the anesthesia time at each salinity. At each concentration, fish were anesthetized slower at water salinities over 10 ppt (P<0.05). Anesthesia time at 10 ppt was faster than any other salinity. In 10 ppt salinity, the dissolved oxygen (DO) concentrations and respiratory frequencies of the clove-oil-administered groups decreased until 48 hours (P<0.05), whereas the NH4 + and CO2 concentrations increased until 48 hours (P<0.05). In same period, the DO, NH4 +, and CO2 concentrations and respiratory frequencies all decreased as the clove oil concentration increased (P<0.05). The trends in the DO, NH4 +, and CO2 concentrations and respiratory frequencies in the lidocaine-HCl-administered groups were similar to those in the clove-oil-administered groups. In conclusion, clove oil and lidocaine-HCl are effective anesthetics, improving the transportation of the marine medaka. The results from this study will contribute to safe laboratory handling of the marine medaka, which are commonly required by many research studies and experiments.

The marine medaka, Oryzias dancena is a suitable sample as a laboratory animal because it has a small size and clearly distinguishes between female and male. Data on the growth and maturity of the diploid and triploid sea cucurbit species suitable for laboratory animals are very useful for studying other species. Triploidy was induced in the marine medaka by cold shock treatment (0°C) of fertilized eggs for 45 min, applied two minutes after fertilization. The diploid and triploid male fish were larger than their female counterparts (P<0.05), and the concentrations of thyroid stimulating hormone (TSH) and thyroxine (T4) were higher in the induced triploids over 1 year (P<0.05). In both the diploid and tri-ploid groups the concentrations of TSH and T4 were higher in the male fish than in the females (P<0.05), while the testo-sterone and estradiol-17ß concentrations in the induced triploids were lower than in the diploids (P<0.05). The gonadosomatic index (GSI) of the triploid fish was lower than that for the diploids, and the GSI for females in each ploidy group were higher than that for the males. For both groups the GSI was highest at 4 months of age, and decreased thereafter to 12 months. Analysis of the gonads of one-year-old triploid fish suggested that the induction of triploidy probably causes sterility in this species; this effect was more apparent in females than in males.

Background : Haskap berries commonly refer to fruits of Lonicera caerulea L., recognized by the Japanese aborigines as the “The elixir of life.”. Due to their recent arrival on the North American market, haskap berries have not yet been positioned among other berries and compared in terms of their phytochemical content. And haskap berries have higher ascorbic acid and anthocyanin content than other berries known for their health-promoting benefits, such as blueberries. However, no study has reported on the antioxidant and anti-cancer activity of Lonicera caerulea stem. The purpose of this study is to present the current research on the chemical content, antioxidant and anti-cancer activities of Lonicera caerulea stem. Methods and Results : The stem of Lonicera caerulea L. ware dried in the shade at room temperature and extracted with 100% methanol. The extract was suspended in deionized water and partitioned sequentially with n-hexane, chloroform, ethyl-acetate and butanol (water saturated BuOH) fractions. Antioxidant activities were measured by determination of antioxidants, DPPH (2,2-diphenyl-1-picrylhydrazyl). Cell viability was determined by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay. All cell lines were purchased from the Korean Cell Line Bank (Seoul, Korea). All results were performed with three replications were processed statistically. By DPPH assay, the Lonicera caerulea L. the highest activity was obtained from the ethyl-acetate fraction (IC50=15.46 ㎍/㎖). By MTT assay, the chloroform fraction showed a significant growth inhibiting effect on MCF-7 (Human breast cancer, IC50=225.91 ㎍/㎖), COLO 205 (Human colon cancer, IC50=179.55 ㎍/㎖), but on AGS (Human stomach cancer) and other fractions it did not show effect. Conclusion : We demonstrated that Lonicera caerulea L. stem extract and fractions has antioxidant and antiproliferation activity in vitro. Further studies should identify the active constituents in Lonicera caerulea L stem to evaluate the potential in vitro antioxidant and antiproliferation activities of the extract.

Background : Angelica dahurica Bentham et Hooker, Angelica gigas Nakai, Ostericum koreanum Maximowicz and Peucedanum japonicum Thumberg　are a major medicinal plant in north Geungbuk province. Using medicinal plants are impotant it`s ingredient. Dry condition and stroage method are not standard manual. The ingredient variation of dry condition and stroage method were not researched. Methods and Results : Using plant material were cutivated on Gyongsangbukdo Bonghwa area. It were studied ingredient variation after dry and storage condition by HPLC methods. Major ingredient of Angelica gigas Nakai are decurusin, decurusinangelate. Heated air bulk dry get more decursin than natuarl dry and decurusinangelate of natural bulk dry was higher than heated air bulk dry. Major ingredient of Ostericum koreanum Maximowicz are imperatorin and isoimperatorin.. Imperatorin of Ostericum koreanum was highest peak on 50℃ heated-air dry after plastic bag sorage and isoimperatorin was highest peak on 40℃ heated-air dry after mountain cultivation. Imperatorin is a major ingredient Angelica dahurica Bentham et Hooker. Heated air bulk dry get more decursin and decursinangelate than natuarl dry and small heated-air dry. Peucedanol-7o_glucoside is a major ingredient Peucedanum japonicum Thumberg. Natural bulk dry get more peucedanol-7o_glucoside than heated-air bulk dry. Conclusion : Ingredient of Angelica dahurica Bentham et Hooker, Angelica gigas Nakai, Ostericum koreanum Maximowicz are different under various cutivation, drying method, storage. Diffent Ingedients of Angelica gigas Nakai, Ostericum koreanum Maximowicz were not accord it’s optical conditon.

Koreans have sedentary lifestyles and use under-floor heating called Ondol as home heating systems. Sick Building Syndrome (SBS) caused by VOCs released from flooring material has been of significant interest in residential apartments. In this study, we measured exposure to VOCs by having test subjects breathe under conditions of high surface temperature. The results showed that the emission level of VOCs was high at 40oC in the early stage of the experiment. However, 20 days after the experiment began, the emission level was reduced by 0.9-4 times in each product at 40oC. The exposure to VOCs released from floor coverings was measured by taking into account the average respiratory rate, weight, and activity hour of test subjects and presuming that people are exposed to VOCs all the time while staying indoors. The level of emission from FJR of low-cost PVC floor coverings was the highest among the tested coverings, but the exposure to VOCs emission from the covering was relatively low at 0.025 mg/kg/day at 40oC. The amount of toluene detected from this experiment was the highest among VOCs emitted from floor coverings. The hazard quotient (HQ) of toluene detected in this test was over 20 times smaller than the risk characterization level of 0.1. It was, therefore, estimated that the emission of toluene from the floor surface would not be critically harmful to residents.