Genetically modified (GM) animals are unique mutants with an enormous scientific potential. Cryopreservation of pre-implantation embryos or spermatozoa is a common approach for protecting these lines from being lost or to store them in a repository. A mutant line can be taken out of a breeding nucleus only if sufficient numbers of samples with an appropriate level of quality are cryopreserved. The quality of different donors within the same mouse line might be heterogeneous and the cryopreservation procedure might also be error-prone. However, only limited amounts of material are available for analysis. To improve the monitoring of frozen/thawed spermatozoa, commonly used in vitro fertilization (IVF) followed by embryo transfer were replaced with animal-free techniques. Major factors for assessing spermatozoa quality (i.e., density, viability, motility, and morphology) were evaluated by fluorescence microscopy. For this, a live/dead cell staining protocol requiring only small amounts of material was created. Membrane integrity was then examined as major parameter closely correlated with successful IVF. These complex analyses allow us to monitor frozen/thawed spermatozoa from GM mice using a relatively simple staining procedure. This approach leads to a reduction of animal experiments and contributes to the 3R principles (replacement, reduction and refinement of animal experiments).
Animals ; Benzimidazoles/chemistry ; Cryopreservation/veterinary ; Embryo Transfer/veterinary ; Female ; Fertilization in Vitro/veterinary ; Fluorescent Dyes/chemistry ; Male ; Mice ; Mice, Transgenic ; Microscopy, Fluorescence/*methods/veterinary ; Propidium/chemistry ; Semen Analysis/*methods/veterinary ; Semen Preservation/veterinary ; Spermatozoa/*physiology

The generation of reactive oxygen species (ROS) and subsequent mitochondrial and DNA damage in bovine somatic cell nuclear transfer (SCNT) embryos were examined. Bovine enucleated oocytes were electrofused with donor cells and then activated by a combination of Ca-ionophore and 6-dimethylaminopurine culture. The H2O2 and .OH radical levels, mitochondrial morphology and membrane potential (DeltaPsi), and DNA fragmentation of SCNT and in vitro fertilized (IVF) embryos at the zygote stage were analyzed. The H2O2 (35.6 +/- 1.1 pixels/embryo) and .OH radical levels (44.6 +/- 1.2 pixels/embryo) of SCNT embryos were significantly higher than those of IVF embryos (19.2 +/- 1.5 and 23.8 +/- 1.8 pixels/embryo, respectively, p < 0.05). The mitochondria morphology of SCNT embryos was diffused within the cytoplasm. The DeltaPsi of SCNT embryos was significantly lower (p < 0.05) than that of IVF embryos (0.95 +/- 0.04 vs. 1.21 +/- 0.06, red/green). Moreover, the comet tail length of SCNT embryos was longer than that of IVF embryos (515.5 +/- 26.4 microm vs. 425.6 +/- 25.0 microm, p < 0.05). These results indicate that mitochondrial and DNA damage increased in bovine SCNT embryos, which may have been induced by increased ROS levels.
Animals ; *Apoptosis ; Caspase 3/metabolism ; Cattle ; Colorimetry/veterinary ; Comet Assay/veterinary ; *DNA Damage ; DNA, Mitochondrial/*genetics/metabolism ; Embryo Transfer/veterinary ; Embryo, Mammalian/*cytology/embryology ; Fertilization in Vitro/veterinary ; In Situ Nick-End Labeling/veterinary ; Membrane Potential, Mitochondrial ; Microscopy, Confocal/veterinary ; Microscopy, Fluorescence/veterinary ; Mitochondria/*metabolism ; Nuclear Transfer Techniques/*veterinary ; Reactive Oxygen Species/*metabolism

This study evaluated the meiotic competence of buffalo oocytes with different layers of cumulus cells. A total of 588 oocytes were collected from 775 ovaries averaging 0.78 oocytes per ovary. Oocytes with homogenous cytoplasm (n = 441) were selected for in vitro maturation (IVM) and divided into four groups based on their cumulus morphology: a) oocytes with > or == 3 layers of cumulus cells, b) 1-2 layers of cumulus cells and oocytes with partial remnants or no cumulus cells to be cocultured c) with or d) without cumulus cells. Oocytes in all four groups were matured in 100 microL drop of TCM-199 supplemented with 10microgram/mL follicle stimulating hormone (FSH), 10microgram/mL luteinizing hormone (LH), 1.5microgram/mL estradiol, 75microgram/mL streptomycin, 100 IU/mL penicillin, 10 mM Hepes and 10% FBS at 39degrees C and 5% CO2 for 24 hours. After IVM, cumulus cells were removed from oocytes using 3 mg/mL hyaluronidase, fixed in 3% glutaraldehyde, stained with DAPI and evaluated for meiotic competence. The oocytes with > or ==3 layers of cumulus cells showed higher maturation rates (p <0.05: 64.5%) than oocytes with partial or no cumulus cells (8.6%) and oocytes co-cultured with cumulus cells (34.5%) but did not differ from oocytes having 1-2 layers of cumulus cells (51.4%). The degeneration rates were higher (p < 0.05) for oocytes with partial or no cumulus cells (51%) than rest of the groups (range: 13.8% to 17.4%). These results suggest that buffalo oocytes with intact layers of cumulus cells show better IVM rates than oocytes without cumulus cells and the co-culture of poor quality oocytes with cumulus cells improves their meiotic competence.
Animals ; Buffaloes/*physiology ; Female ; Fluorescent Dyes/chemistry ; Indoles/chemistry ; Meiosis/*physiology ; Microscopy, Fluorescence/veterinary ; Oocytes/cytology/growth&development/*physiology

The UL49.5 gene of most herpesviruses is conserved and encodes glycoprotein N. However, the UL49.5 protein of duck enteritis virus (DEV) (pUL49.5) has not been reported. In the current study, the DEV pUL49.5 gene was first subjected to molecular characterization. To verify the predicted intracellular localization of gene expression, the recombinant plasmid pEGFP-C1/pUL49.5 was constructed and used to transfect duck embryo fibroblasts. Next, the recombinant plasmid pDsRed1-N1/glycoprotein M (gM) was produced and used for co-transfection with the pEGFP-C1/pUL49.5 plasmid to determine whether DEV pUL49.5 and gM (a conserved protein in herpesviruses) colocalize. DEV pUL49.5 was thought to be an envelope glycoprotein with a signal peptide and two transmembrane domains. This protein was also predicted to localize in the cytoplasm and endoplasmic reticulum with a probability of 66.7%. Images taken by a fluorescence microscope at different time points revealed that the DEV pUL49.5 and gM proteins were both expressed in the cytoplasm. Overlap of the two different fluorescence signals appeared 12 h after transfection and continued to persist until the end of the experiment. These data indicate a possible interaction between DEV pUL49.5 and gM.
Animals ; Ducks/virology ; Genes, Viral/genetics ; Mardivirus/*genetics ; Membrane Glycoproteins/*genetics ; Microscopy, Fluorescence ; Phylogeny ; Polymerase Chain Reaction/veterinary ; Viral Envelope Proteins/*genetics

Studies on Marek's disease virus (MDV)-unique genes are important for understanding the biological nature of the virus. Based on complete DNA sequence analyses of the MDV genomes, the MDV genomes contain presumably at least five MDV-unique genes, which are commonly conserved among the three MDV serotypes. A recombinant baculovirus that contains the MDV serotype 2 (MDV2)-unique gene, ORF873, under the polyhedrin promoter was constructed and designated rAcORF873. Polyclonal and monoclonal antibodies, which recognize the recombinant MDV2 ORF873 protein in Spodoptera frugiperda clone 9 (Sf9) cells infected with rAcORF873, were prepared by immunizing mice with a recombinant fusion protein expressed in Escherichia coli. Immunoblot analyses with the antibodies revealed a major protein band with a molecular mass of 108-kDa in both MDV2-infected chick embryo fibroblasts (CEF) and rAcORF873-infected Sf9 cells. By indirect immunofluorescence analyses using monoclonal antibody, the authentic ORF873 protein was localized in the cytoplasm of MDV2-infected CEF cells. The monoclonal and polyclonal sera, which were generated in the present study and reacted effectively to MDV2 ORF873 protein, are considered to be useful reagents for further studying the role(s) of the ORF873 protein in MDV2 infection.
Animals ; Cell Line ; *Chickens ; DNA, Viral/chemistry/genetics ; Herpesvirus 3, Gallid/*genetics/*metabolism/pathogenicity ; Immunoblotting/veterinary ; Marek Disease/*virology ; Mice ; Mice, Inbred BALB C ; Microscopy, Fluorescence/veterinary ; Open Reading Frames/*genetics ; Polymerase Chain Reaction/veterinary ; Recombinant Proteins/genetics ; Specific Pathogen-Free Organisms ; Transfection/veterinary ; Viral Proteins/genetics/*metabolism

Chicken anemia virus (CAV) is an important viral pathogen that causes anemia and severe immunodeficiency syndrome in chickens worldwide. In this study, a potential diagnostic monoclonal antibody against the CAV VP1 protein was developed which can precisely recognize the CAV antigen for diagnostic and virus recovery purposes. The VP1 gene of CAV encoding the N-terminus-deleted VP1 protein, VP1Nd129, was cloned into an Escherichia (E.) coli expression vector. After isopropyl-beta-D-thiogalactopyronoside induction, VP1Nd129 protein was shown to be successfully expressed in the E. coli. By performing an enzyme-linked immunoabsorbent assay using two coating antigens, purified VP1Nd129 and CAV-infected liver tissue lysate, E3 monoclonal antibody (mAb) was found to have higher reactivity against VP1 protein than the other positive clones according to the result of limiting dilution method from 64 clones. Using immunohistochemistry, the presence of the VP1-specific mAb, E3, was confirmed using CAV-infected liver and thymus tissues as positive-infected samples. Additionally, CAV particle purification was also performed using an immunoaffinity column containing E3 mAb. The monoclonal E3 mAb developed in this study will not only be very useful for detecting CAV infection and performing histopathology studies of infected chickens, but may also be used to purify CAV particles in the future.
Animals ; Antibodies, Monoclonal/biosynthesis/genetics/*immunology ; Antigens, Viral/analysis ; Capsid Proteins/genetics/*immunology ; Chicken anemia virus/genetics/*immunology ; *Chickens ; Circoviridae Infections/blood/immunology/*veterinary/virology ; Escherichia coli/genetics ; Immunohistochemistry/veterinary ; Liver/virology ; Mice ; Mice, Inbred BALB C ; Microscopy, Fluorescence/veterinary ; Poultry Diseases/blood/immunology/*virology ; Specific Pathogen-Free Organisms ; Thymus Gland/virology