It is well established that mitochondrial genome is strictly maternally inherited in mammalian, despite the fact that paternal mitochondria enter into oocyte during fertilization. To date, although some mechanisms have been extrapolated to interpret the elimination of paternal mitochondria, the exact mechanism still is unclear. Recent studies suggest that autophagy process and the ubiquitin-mediated degradation pathway may be involved in elimination of paternal mitochondria. However, the dynamic profiles of autophagy and ubiquitination associated with paternal mitochondria degradation have not been determined in mouse model. Through immunostaining with specific antibody LC3 and Ubiquitin and confocal microscopy, we investigated the dynamic profiles of LC3 and Ubiquitin signals in mouse embryos during preimplantation development. In addition, embryos were stained with MitoTracker Red for tracking the degradation process of paternal mitochondria. Our results showed that paternal mitochondria gradually degraded during postfertilization development, and sporadic paternal mitochondria were found at least in 16 cell embryos. LC3 and Ubiquitin signals appeared in the midpiece of sperm at 3 h postfertilization, and they were strictly colocalizated with paternal mitochondria from zygote to 2 cell embryo. Nevertheless, the colocalization became loose at 4 cell embryos, and gradually disappeared beyond 4 cell embryos. Our results confirmed that autophagy process and the ubiquitin-mediated degradation pathway may take part in the postfertilization remove of paternal mitochondria.

Superovulation, or ovarian stimulation is a commonly used ART for treatment of human infertility/subfertility. Recent studies suggest that superovulation unaffects methylated imprints acquisition in mouse oocytes during oogenesis, whereas disrupts DNA methylation maintenance in embryos during preimplantation development. However, the mechanisms of defects in methylation maintanence caused by superovulation remain largely unclear. We hypothesized that superovulation may disrupt the expression of DNA methyltransferases (Dnmts), the enzymes which catalyze DNA methylation acquisition and maintenance. The mice were subjected to superovulate with low (6 IU) and high (10 IU) dosage hormone. We examined the global DNA methylation levels in zygotes and DNA methylation of repeated sequences (IAP and Line 1) in blastocyst stage embryos. In addition, we investigated the expression of Dnmts (Dnmt3a, Dnmt3b, Dnmt3l and Dnmt1o) in ovulated oocytes and zygotes. Through staining with antibody 5mC and Di-H3K9 coupled with confocal microscopy, we found that global methylation profiles in zygotes derived from females after low or high dosage hormone treatment were not affected when compared to control counterpart. Moreover, methylation at IAP in blastocysts also was unaffected by superovulation, irrespective of hormone dosage. In contrast, methylation level at Line 1 decreased when the females were administered by high dosage hormone. Furthermore, expression of de novo DNA methyltransferase Dnmt3a, Dnmt3b, Dnmt3L, as well as maintenance Dnmt1o in MII oocytes and zygotes was not disrupted by superovulation. Given superovulation adversely affected methylation maintenance in blastocysts during preimplantation development but with normal expression of Dnmts in oocytes and zygotes, it is indicated that defects of embryonic methylation didn’t originate from abnormal expression of Dnmts.

Although evidences showed that histone deacetylation plays an important role in the mitotic and meiotic cell cycle, but the mechanisms are still unclear. Level of histone acetylation can be easily changed by deacetylase inhibitors (HDACi) i.e trichostatin A (TSA) and valporic acid. In this study, we determined whether the inhibition of histone deacetylation by TSA could affect porcine oocyte maturation and aging process. Our results showed that treated COCs with 100 nM TSA significantly increase the GVBD in each time group than 0, 5, 50 nM but no significantly different from that of higher concentration (200 nm or 300 nM). No significant differences on maturation, blastocyst development, MAPK pattern and expressions of apoptosis gene when treated oocytes with 100 nM TSA for the first 24h of IVM compared with control and 5, 50 nM TSA. However, in the oocytes treated with 200 nM and 300 nM TSA for first 24 h, MAPK significantly decreased and abnormal spindle were observed. But, in prolonged (64 h) of TSA treated group has no significantly different in control. Another data observed that after 24h TSA-treat to prolonged group were significantly decreased of MAPK activation and normal spindle than the other group. We concluded that TSA played a critical role in meiotic progression in porcine oocytes through the regulation of arrest GVBD, which prolonging the in vitro maturation time, but unaffected the subsequent pre-implantation embryo developmental potential and embryonic qualities. Moreover, the histone deacetylase inhibitor TSA may artificially control porcine oocyte maturation time and delay porcine oocyte aging process.

The objective of this study was to assess the effect on post-thawed sperm motility, viability and acrosome integrity of boar semen frozen in the freezing extender with chicken or duck egg yolks. The Sperm rich fraction of ejaculates from three Duroc boars were collected by a glove-hand technique. Samples with more than 80% motile sperm were used for this experiment. Semen was diluted with freezing extender (LEY) containing 11% (v/v) lactose, 20% (v/v) hen egg yolk with 3.5% (v/v) glycerol, and 0.5% (v/v) Orvus Es Paste(OEP, Nova Chemical Sales Inc., Scituate, MA. USA) to yield a final sperm concentration of 5×108 cells/ml. Following complete dilution, semen samples were loaded in 0.5 ml French medium straws (IMV technologies, France) and transferred to programmable semen freezer (SY-LAB Gerate GmbH, Austria). For freezing the semen samples, each straw was cooled from 5℃ to — 5℃ at 6℃/min, auto-seeding at — 5℃ and held for 60sec, samples were then cooled from — 5 to — 80℃ at 40℃/min, and thereafter from — 80℃ to — 150℃ at 60℃/min. The yolks used were sourced from fresh chicken and duck eggs. To evaluate the post-thaw sperm quality, semen was thawed at 38℃ for 20 sec and sperm motility, viability and acrosome integrity were assessed. Motility was assessed for %motile cell characteristics using computer-assisted semen analysis (CASA; SAIS SI-100, Medical supply, Korea). The percentage of sperm viability was assessed using LIVE/DEAD® sperm viability kit (Molecular probes, Eugene, OR, USA). The acrosome integrity was assessed by FITC-PNA staining. Sperm quality in terms of motility, viability and acrosome integrity showed higher after freezing in medium containing duck yolk than chicken yolk. However, there was no significant difference in sperm quality for the different types of yolk(p>0.05). * The result of this study showed that there was no significant difference between the egg yolk types when considering the sperm motility, viability and acrosome integrity of boar semen frozen in the freezing extender with chicken or duck egg yolks.

There are diverse methods of cryopreservation of mammalian embryos with variable degrees of success. Although cryopreservation technique of mammalian embryos has been advanced, freezing stress affect to cellular event such as apoptosis and autophage in embryos. The objective of the study is to investigate the affection of to survival, development, live offspring, apoptosis and autophagy on embryo. Mouse embryos were vitrified and thawed using normal straw and modified cut standard straw (M-CSS), then in vitro cultured until blastocyst stage and transferred to recipient. Recovery rates (100 vs 99.2%), survival rates (99.2 vs 78.6%), developmental rates (18.4 vs 10.7%), total cell numbers (45 vs 37), preganacy rates (34.5 vs 25%) and offspring numbers (10.1 vs 4.9 %) of M-CSS group are significantly higher than those of normal straw vitrified group. Also, rate of apoptosis in blastocysts developed using M-CSS (1.9%) was significantly lower than using normal straw vitrification (2.7%). Apoptosis-related gene, caspase 3, was expressed at the highest level in blastocysts derived from normal straw group. However, no differences of autophagy related gene, Atg6 and expression of LC3 between normal straw and M-CSS groups were observed. In conclusion, the standard vitrification procedure induces mitochondrial apoptosis in zygotes in an autophagy-independent manner, whereas the novel M-CSS procedure may improve embryo vitrification.

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.

Animal biotechnology is the use of science and engineering to modify living organisms. Examples of animal biotechnology include creating transgenic animals (animals with one or more genes introduced by human intervention), using gene knock out technology to make animals with a specific inactivated gene and producing nearly identical animals by somatic cell nuclear transfer (or cloning). Animal biotechnology in use today is based on the science of genetic engineering. Under the umbrella of genetic engineering exist other technologies, such as transgenics and cloning, that also are used in animal biotechnology. Th main purpose of production of transgenic animals are to produce pharmaceutical proteins for human use. Scientists use reproductive cloning techniques to produce multiple copies of mammals that are nearly identical copies of other animals, including transgenic animals, genetically superior animals and animals that produce high quantities of milk or have some other desirable trait. To date, cattle, sheep, pigs, goats, horses, mules, cats, rats and mice have been cloned, beginning with the first cloned animal, a sheep named Dolly, in 1996. In addition to the use of transgenics and cloning, scientists can use gene knock out technology to inactivate, or “knock out,” a specific gene. It is this technology that creates a possible source of replacement organs called xenotransplantastion. In this talk I deal with current and future of animal biotechnolgy. In addition I would like to introduce Center for the Animal Bioreactors and Xenotranplantaion supported by Rural Development Administration, Korea.

Methyl bromide (MB) is a widely used fumigant in most of the countries for quarantine purpose. However, MB has been phasing out and under control in many countries because it is listed as an ozone-depleting substance under the Montreal Protocol. In this study, we have investigated the effectiveness of phosphine fumigation on wood pests for developing an MB alternatives. We evaluated two bioassay methods; wooden cube (10×10×10 cm) and normal fumigation procedures in comparison with effectiveness of phosphine (PH3) penetrations into the timber block. Fumigation to adults of Reticulitermes speratus was carried in a desiccator system for 24hr at 5 and 15 ℃. As a result, LC99 of PH3 to R. speratus in wooden cubes and insect breeding dish at 5℃ was 0.183 and 0.177 mg L-1, respectively. LC99 of PH3 in wooden cubes and insect breeding dish at 15℃ was 0.077 and 0.078 mg L-1, respectively.

The cotton aphid, Aphis gossypii Glover is an important sap-sucking pest of many pant, including cucumber and pepper. The objective of the present study was to determine the effects of sublethal concentrations of two insecticides (imidacloprid and flonicamid) and the action mechanisms on the feeding behavior of A. gossypii. The median lethal concentrations (LC50) of imidacloprid and flonicamid for adult A. gossypii were 2.01 and 1.92 ppm, respectively. The sublethal concentrations of imidacloprid were 0.22 ppm (LC10) and 0.82 ppm (LC30), and those of flonicamid were 0.094 ppm (LC10) and 0.56 ppm (LC30). The developmental period of A. gossypii nymphs at LC30 was 3.6 days for both insecticide which shorter than controls (4.2 days). Adult longevities at LC10 and LC30 of imidacloprid were 15.2 and 13.6 days, respectively. Adult longevity at LC10 and LC30 of flonicamid was 11.1 and 9.9 days, respectively. Control adult longevity was 15.5 days. Total fecundity was decreased at both sublethal concentration of two insecticides. Feeding behavior analysis using an electrical penetration graph showed that sublethal doses of imidacloprid and flonicamid had significant effects on the duration of phloem ingestion. However, higher doses of flonicamid induced starvation by inhibition of phloem ingestion and higher doses of imidacloprid induced contact toxicity rather than inhibition of feeding behavior.

The influence of electron beam irradiation on each developmental stage and reproduction of Spodoptera litura were examined. Eggs, larvae, pupae, and adults were irradiated at target doses of 30, 50, 100, 150, 200, and 250 Gy. When eggs were irradiated with 100 Gy, egg hatching was perfectly inhibited. When irradiated to the larvae, pupation was inhibited at 100 Gy and larval period was delayed. When irradiated to the pupae, emergence was inhibited at 100 Gy and above. When irradiated to the adults, longevity and fecundity did not show any differences. However, egg hatching was strongly decreased at 100 Gy and above. Also, electron beam irradiation was not induced the instantaneous death of S. litura. Reciprocal crosses between irradiated and unirradiated moths demonstrated that males were more radiotolerant than females. Adult longevity was not affected in all stages. The levels of DNA damage in S. litura adults were evaluated using the alkaline comet assay. Our results indicate that electron beam irradiation increased levels of DNA damage. These results suggest that electron beam irradiation induced abnormal development and reproduction by DNA damage in S. litura.

Insecticidal susceptibilities of 16 registered insecticides on each developmental stages of Phthorimaea operculella were investigated and further examined the contact, oral and residual toxicities after chosen from insecticides showing good effect. Mortality, longevity and effect on reproduction of 16 insecticides to P. operculella adults were also investigated. To the eggs and pupae, only spinosad showed 71.1% inhibition rate of egg hatch and 66.7% inhibition rate of emergence. To the 3rd nymphs, fenitrothion (LC50 336.6 ppm), esfenvalerate (LC50 8.6 ppm), ethofenprox (LC50 35.7 ppm), and emamectin benzoate (LC50 0.05 ppm) showed oral toxicity over 90% and esfenvalerate (LC50 0.87 ppm), ethofenprox (LC50 16.5 ppm), emamectin benzoate (LC50 0.53 ppm), and spinosad (LC50 2.48 ppm) showed the contact toxicity over 90%. To the adults, mortalities of insecticides were showed as below: deltamethrin and spinosad showed perfect mortality 48 h after treatment; esfenvalerate, ethofenprox, and thiamethoxam showed 40 - 60% mortalities; but the others are not showed any effect. Fecundities of female adults were inhibited by esfenvalerate, emamectin benzoate, and dinotefuran, compare to that of the control, but there were no statistical differences to that of ethofenprox, benfuracarb, thiamethoxam, clothianidin, and diflubenzuron. Adult longevity was showed no difference compare to that of the control. Residual effect of emamectin benzoate showed perfect insecticidal activity at 14 days after treatment and the next ethofenprox showed over 90% at 7 days after treatment.

The blueberry gall midge, Dasineura oxycoccana (Johnson)(Diptera: Cecidomyiidae), has known as a key pest of blueberries in the southeastern United States, Europe and Canada. It can cause considerable damage to developing blueberry flower buds and also injure vegetative growth by distorting and blackening shoot tips. Similar symptoms of damage, which mighty be caused by D. oxycoccana, have been investigated on blueberry plants(damage rate: 20~80% of total shoot tips), Vaccinium spp., in Hwaseong-si of Gyounggi-do in 2010. And, followed by an investigation on the occurrences of D. oxycoccana and its damages in 2010~2012. We emphasize caution concerning the possibilities that D. oxycoccana could infest flower buds and shoot on blueberries and developed monitoring methods and environment-friendly management strategies for D. oxycoccana on buleberry.

Ooencyrtus nezarae Ishii and Gryon japonicum (Ashmead), egg parasitoids of Riptortus pedestris (Fabricius), coexist despite direct competition for host eggs. As asymmetrical pattern of seasonal occurrence, i.e., more G. japonicum during spring-summer and more O. nezarae during summer-fall, has been reported, host resource partitioning may occur in temporal scales. To test this hypothesis, we demonstrated the interspecific competition between the two species by measuring parasitism in nine combinations of host densities and exposure times. To reflect gregarious-solitary dichotomy, three O. nezarae and one G. japonicum mated females were used in each experiment. O. nezarae was better competitor when exposure time was longer than 1 day irrespective of host densities. Parasitism rate and progeny emergence of O. nezarae was 1.6-2.8 and 4.7-7.3 times higher than G. japonicum. O. nezarae has higher potential rate of increase than G. japonicum due to gregariousness, and be more successful in larval competition inside multiparasitized host egg as it acts as a facultative hyperparasitoid. Although G. japonicum was more effective in host finding (as they showed relatively higher per capita parasitism and progeny emergence), their progeny suffered high mortality from the larval competition with O. nezarae. These results may explain the asymmetrical occurrence pattern in the field.

Teratocytes are originated from embryonic serosal membrane of some endoparasitoid wasps. Cotesia plutellae eggs release teratocytes in parasitoid host hemocoel at hatch in about 150 cells per egg. Teratocytes of C. plutellae were cultured in an insect culture medium for at least 14 days. Teratocytes cultured in vitro showed no increase in cell numbers but increased in cell size. Similarly,teratocytes in parasitized larvae did not increase cell numbers, but increased their cell size. Microinjection of invitro cultured teratocytes in to third instar larvae of nonparasitized Plutella xylostella showed a dose-dependently inhibitory effect on development and larval-pupal metamorphosis. In addition, teratocytes prolonged the immature developmental period and reduced the pupation rate, in which young aged host larvae were more sensitive to teratocytes treatment than old larvae. These results suggest that teratocytes play a crucial role in successful parasitization of C.plutellae by altering host developmental program.

The purpose of this study was to compare the muscle activity of the abdominal and lumbar multifidus during unilateral prone hip extension on the floor and on a round foam roll. Fifteen healthy participants were recruited. They were instructed to perform a unilateral hip extension on the floor and on a round foam roll in the prone position. Surface electromyography (EMG) signals were recorded from bilateral lumbar multifidus (LM), external oblique (EO), and internal oblique (IO) muscles. A paired t-test was used to compare muscle activity, with the level of significance set at =.05. The results showed that bilateral LM, EO, IO EMG activity during right-hip extension on a round foam roll was greater than that on the floor, and EMG activity of bilateral LM, right EO, and left IO during left-hip extension on a round foam roll was greater than that on the floor (p<.05). These findings suggest that the unilateral hip-extension exercise on a round foam roll can be used to activate the lumbar multifidus and abdominal oblique muscles and causes a different increasing pattern between the two lifting sides.

This study aims to examine the effect of patellar taping common to patients with patellofemoral pain syndrome on the change of knee joint location. The total number of participants is 12 patients with no pain in their knee. There are three different experiments: no-taping, placebo taping, and patellar taping. After application, they squat on their hams. As a result, both the muscle activity of vastus medialis and that of vastus lateralis increased in placebo taping compared to no-taping, which wasn't statistically significant. However, the muscle activity of vastus medialis and that of vastus lateralis decreased in patellar taping compared to no-taping, which was statistically significant. This suggests that patellar taping causing the lateral attraction of knee joint is more influential to the dynamics of knee joint than skin afferent input in placebo taping. Therefore, patellar taping is effective to change the location of knee joint, affect the muscle activity of quadriceps muscle of thigh, and thus correct the misalignments of the knee joint.

This study was conducted in order to examine the safety of bee venom as an alternative for antibiotics using male ICR mice. Five-week-old male mice received a single intravenous injection of a dried honey bee venom at the concentration of 0.25 mg/kg (a clinical dose) or 0.5 mg/kg through the tail vein and various pathophysiological analyses were performed after three days. No significant differences in changes of body weight were observed between the saline-treated control group and the experimental groups. In the hematological analysis, none of the parameters were affected by bee venom. In blood biochemistry analysis, none of the markers were affected by administration of bee venom. Similarly, there were no significant effects on markers for liver, kidney, and skeletal muscle functions in all treated- groups. On macroscopic examination, no remarkable lesions were detected in these organs. Because there were no adverse effects of the bee venom in a single intravenous toxicity test for three days, it was concluded that bee venom could be a candidate for a safe natural antibiotic for use in the animal production industry.

장수명 핵분열생성물인 79Se와 99Tc는 자연수 중에서 용해도가 클 뿐더러 음이온으로 존재하여 방사성폐 기물 처분장에서 주요 관심핵종들로 고려되고 있다. 본 연구에서는 KURT 지하수의 다양한 pH와 산화-환 원 조건에서 셀레늄과 테크네튬의 Solubility Limiting Solid Phase (SLSP)로 알려진 FeSe2와 TcO2의 용해 도를 측정하였다. 또한, 지화학코드를 이용하여 실험과 유사조건에서 이들의 용해도와 주요 화학종을 계 산하였다. 실험 및 계산으로부터 pH 8∼9.5와 Eh=-0.3∼-0.4 V 조건에서 FeSe2의 용해도는 1x10-6 mol/L 이하이며, 주 용해 화학종은 HSe-로 판단된다. TcO2의 경우는 pH 6∼9.5와 Eh<-0.1 V 영역에서 용해도와 주 용해 화학종이 각각 5x10-8∼1x10-9 mol/L와 TcO(OH)2로 나타났지만, Eh=-0.35 V조건에서는 주 용해화학종이 pH가 10.5∼12와 12이상에서 각각 TcO(OH)3 -와 TcO4 -로 계산되었다.