Background: Sperm quality and the number of sperm introduced into the uterus during artificial insemination (AI) are pivotal factors influencing pregnancy outcomes. However, there have been no reports on the relationship between sperm concentration at AI and sperm quality in Hanwoo cattle. In this study, we examined sperm quality and pregnancy rates after AI using sperm inseminated at different concentrations. Methods: We evaluated the motility, viability, and acrosomal membrane integrity of sperm at different concentrations (10, 15, 18, and 20 million sperm/straw) in 0.5-mL straws. Subsequently, we compared the pregnancy rates after AI with different sperm concentrations. Results: After freeze-thawing, sperm at the assessed concentrations showed similar viability and acrosomal membrane integrity. After AI, cattle in the 10 million group had significantly lower pregnancy rates compared to those in the 18 and 20 million groups. Conversely, there were no statistically significant variances observed between cattle in the 10 and 15 million groups. Conclusions: Sperm at concentrations of 10, 15, 18 and 20 million per straw exhibited comparable motility, viability, and acrosomal membrane integrity. However, a concentration of at least 18 million sperm per straw is required to achieve a consistent rate of pregnancy rate in Hanwoo cattle after AI.

The ovum pick up(OPU) technique can be used to produce embryos after in vitro culture of ovarian oocytes, can be used for early securement for effective herd early proliferation and excellent Hanwoo genetic resources, It is attracting attention as a very important technique for establishing technology. In addition to in vitro culture techniques, the number of oocytes retrieved depends on the environment and timing of the OPU. This study was conducted to compare and examine seasonal effect to the differences in the number of recovered oocytes, recovery rate and embryo development rate using Korean cattle kept in animal genetic resource research center by OPU technique. The grade of COCs was evaluated by microscopic examination. Grade A had 3 or more layers of cumulus cell and compact cytoplasm. Grade B had 1~3 layers of cumulus cell and compact cytoplasm. Grade C had 1 layers cumulus cell and compact cytoplasm. Grade D was denuded oocyte and poor cytoplasm. The recovery rate was 47.8±3.4% in summer (June to August) and 51.6±3.8% in autumn (September to October). The number of oocytes was 5.7±0.6 in summer and 5.2±0.7 in autumn. Oocyte grade A and B was 46.2%±6.3% in summer and 51.1±5.0% in autumn. The cleavage rate was 46.1±7.1% in summer and 65.0±11.3% in autumn. Blastocyst development rate was 19.9±9.4% in summer and 29.0±8.7% in autumn. There was no difference the recovery rate of oocytes and the number of embryos between summer and autumn. Cleavage rate and blastocyst rate of autumn was higher than summer. we will investigate to study the appropriate method for the production of Hanwoo embryos and the systematic comparison.

This study was carried out to investigate the effects of semen on reproductive ability in crossbred Korean native chicken (KNC, 58-wk old). The body weight, volume of semen and concentration of spermatozoa, were 2.96 g, 0.40 ml, 36.58×108/ml, respectively, in KNC. The fertility and hatchability were 94.8% and 78.8% respectively in crossbred KNC. KNC(Y) was high compared to other strains in fertility. The other strains were not significantly different among 6 strains. The results of this experiment indicated that hatchability of (G) was high compared to other strains. The result of this study could be available to genetic improvement of reproductive traits as a basic reference in KNC strains. To achieve the more effective improvement of reproductive traits, addition research such as genetic parameter evaluation should be performed.

Ski protein is implicated in proliferation/differentiation in a variety of cells. We had previously reported that Ski protein is present in granulosa cells of atretic follicles, but not in preovulatory follicles, suggesting that Ski has a role in apoptosis of granulosa cells. The alternative fate of granulosa cells other than apoptosis is to differentiate to luteal cells, however, it is unknown whether Ski is expressed and has a role in granulosa cells undergoing luteinization. Thus, the aim of the present study was to locate Ski protein in the rat ovary during luteinization to predict the possible role of Ski. In order to examine the expression pattern of Ski protein along with the progress of luteinization, follicular growth was induced by administration of equine chorionic gonadtropin to immature female rat, and luteinization was induced by human chorionic gonadtropin treatment to mimic luteinizing hormone (LH) surge. While no Ski-positive granulosa cells were present in preovulatory follicle, Ski protein expression was induced in response to LH surge, and was maintained after the formation of corpus luteum (CL). Though Ski protein is absent in granulosa cells of preovulatory follicle, its mRNA (c-ski) was expressed and the level was unchanged even after LH surge. Taken together, these results demonstrated that Ski protein expression is induced in granulosa cells upon luteinization, and suggested that its expression is regulated post-transcriptionally.

Sloan-Kettering virus gene product of a cellular protooncogene c-Ski is an unique nuclear pro-oncoprotein and belongs to the Ski/Sno proto-oncogene family. Ski plays multiple roles in a variety of cell types, it can induce both oncogenic transformation and terminal muscle differentiation when expressed at high levels. The aim of the present study was to locate Ski protein in rat ovaries in order to predict the possible involvement of Ski in follicular development and atresia. First, expression of c-Ski mRNA in the ovaries of adult female rats was confirmed by RT-PCR. Then, ovaries obtained on the day of estrus were subjected to immunohistochemical analysis for Ski and proliferating cell nuclear antigen (PCNA) in combination with terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL). Ski was expressed in granulosa cells that were positive for TUNEL, but negative for PCNA, regardless of the shape and size of follicles. Expression of Ski in TUNEL-positive granulosa cells, but not in PCNA-positive granulosa cells, was also verified in immature hypophysectomized rats having a single generation of developing and atretic follicles by treatment with equine chorionic gonadotropin (eCG). These results indicate that Ski is profoundly expressed in the granulosa cells of atretic follicles, but not in growing follicles, and suggest that Ski plays a role in apoptosis of granulosa cells during follicular atresia.

In pesticide registration, the most of applicants are manufacturing companies which have richly experience in applying registration. With their experience, registration applications of pesticide shows excellent ratio of pass, but still some of application still fail to pass, and are judged as disqualifications or to be returned for supplement. In 2009, There were 6 items failed in registrational reviews. Two of them were for their active ingredient contents lower than specifications, Other two were for trials conducted wrong, so sent back to applicants to add supplement. But the rest of them, two items showed low efficiency in one trial, and were judged as disqualifications, which mean that ingredient cannot be registered for that target crop forever. Disqualification from low efficiency has highly strict criterion and usually don't have any flexibility to lower efficiency than criteria, unless there was no pesticide available for that pest. The way to remedy this criterion is very delicate and controversial issue, and needs more reconciliation of various views.

An endoparasitoid wasp, Cotesia plutellae, parasitizes larvae of the diamondback moth, Plutella xylostella, with its symbiotic polydnavirus, C. plutellae bracovirus (CpBV). This study analyzed the role of Inhibitor-kB (IkB)-like genes encoded in CpBV in suppressing host antiviral and antimicrobial responses. Identified eight CpBV-IkBs are scattered on different viral genome segments and showed high homologies with other bracoviral IkBs in their amino acid sequences. Compared to an insect ortholog (e.g., Cactus of Drosophila melanogaster), they possessed a shorter ankyrin repeat domain without any regulatory domains. The eight CpBV-IkBs are, however, different in their promoter components and expression patterns in the parasitized host. To test their inhibitory activity on host antiviral response, a midgut response of P. xylostella against baculovirus infection was used as a model reaction. When the larvae were orally fed the virus, they exhibited melanotic responses of midgut epithelium, which increased with baculovirus dose and incubation time. Parasitized larvae exhibited a significant reduction in the midgut melanotic response, compared to nonparasitized larvae. Micro-injection of each of the four CpBV genome segments containing CpBV-IkBs into the hemocoel of nonparasitized larvae showed the gene expressions of the encoded IkBs and suppressed the midgut melanotic response in response to the baculovirus treatment. When nonparasitized larvae were orally administered with a recombinant baculovirus containing CpBV-IkB, they showed a significant reduction in midgut melanotic response and an enhanced susceptibility to the baculovirus infectivity. The transiently expressed CpBV-IkB3 inhibited expression of hemolin, but did not those of lysozyme and cecropin in P. xylostella, while both lysozyme and cecropin were inhibited in the treated Spodoptera exigua. When the recombinant AcNPV was mixed with Bacillus thuringiensis subsp. kurstaki (Bt), the bacterial pathogenicity was significantly enhanced in a dose-dependent manner, compared to a Bt mixture with an AcMNPV recombined with an enhanced green fluorescence protein gene.

The diamondback moth, Plutella xylostella, is reluctant to a baculovirus, Autographa california nucleopolyhedrosis virus (AcNPV) at its oral administration. However, parasitization by an endoparasitoid wasp, Cotesia plutellae, enhances the viral susceptibility. This study analyzed an antiviral activity of P. xylostella in response to the viral infection and determined the parasitic factor inhibiting the antiviral mechanism. For the analysis of antiviral activity of P. xylostella, a recombinant AcNPV expressing enhanced green fluorescence (AcNPV-EGFP) was orally adminstered to lavae of P. xylostella. After 24 h, EGFP expression was observed in the midgut tissue at a confocal-FITC mode. At the same time, a characteristic midgut melanotic response (MMR) was observed in some midgut regions under a phase contrast microscope. Thereafter, the EGFP signal was attenuated, while MMR spread on most midgut region. When the MMR was scored from 0 to 5 by the intensity of melanized cell density, it increased in time- and dose-dependent manners at the viral administration per os. These results suggest that the MMR is an antiviral activity of P. xylostella. This antiviral activity was significantly attenuated by C. plutellae parasitism. The parasitized P. xylostella showed significant decrease in the MMR score compared to nonparasitized larvae when they were orally administered with the same dose of AcNPV. To determine the parasitic factor(s) inhibiting the antiviral activity from the symbiotic polydnavirus of C. plutellae (C. plutellae bracovirus: CpBV), CpBV-IkB, which is a viral homolog of NFkB inhibitor and has been considered as an antiviral factor as in other polydnaviruses, was tested. A recombinant AcNPV expressing CpBV-IkB (AcNPV-IkB) was constructed and administered to P. xylostella larvae. As expected, AcNPV-IkB significantly decreased the antiviral activity measured by the MMR score compared to AcNPV-EGFP treatment. This study suggests that CpBV-IkB plays an antiviral parasitic role in the molecular interactions between P. xylostella and C. plutellae.

The possibility of producing transgenic embryos expressing the green fluorescence protein (GFP) gene have been evaluated after transfer of exogenous gene into the porcine zygote cytoplasm using the intracytoplasm sperm injection (ICSI) as gene delivery method. For DNA binding to sperm heads, 0.05% Triton X-100 or Lipofectin was used. After injection of the sperm bound to DNA by means of Lipofectin or Triton X-100 triturate, the blastocyst formation rates on day 6 were not significantly different from that of ICSI only group (18.8, 19.2 and 25.3%). In terms of GFP expression, more embryos were in GFP form in Triton X-100 group than in Lipofectin group (40.6 vs 36.4%), while percentage of non-mosaic embryos expressing the GFP gene in all blastomere was higher (P<0.05) in Lipofectin group than in Triton X-100 group (4.2 vs 0.9%). ICSI embryos derived from sperm treated with Lipofectin/DNA complex was transferred into 3 recipients and were collected by uterine flushing on days 5, 7 and 15 after embryo transfer, and then GFP expression was observed by a fluorescence microscopy. Over 26% of the collected embryos were normally expressed GFP gene. These results suggest that foreign gene transfer method with DNA bound sperm caused minimal damage to structure of oocytes that can result to full development of porcine embryos. This was confirmed in this study when the embryos that were transferred after ISCI of DNA bound sperm had a normal development and gene expression until preimplantation.