bluuebunny/crossref_metadata_2025 huggingface dataset:
Vortical structures over rectangular cavities at low speed
Authors: Ashcroft Graham , Zhang Xin
Published: December 2004

Experiments have been performed to acquire qualitative and quantitative flow-field data for an open cavity flow using the particle image velocimetry technique. The study focuses on the time-mean and instantaneous development of the turbulent flow structures in the cavity shear layer. The effects of geometry (length-to-depth ratio) and flow speed on these structures are investigated. The shear layers are found to be characterized by coherent vortical structures whose size and rate of growth vary with geometry. The smaller scales of the flow are investigated using a large-eddy decomposition method. Results show these stochastic structures to predominate primarily in the shear layer and aft wall regions.
Hybrid solar cells based on porous Si and copper phthalocyanine derivatives
Authors: Levitsky I. A. , Euler W. B. , Tokranova N. , Xu B. , Castracane J.
Published: December 2004

We demonstrate a solar cell based on n-type nanoporous Si (PSi) filled with copper phthalocyanine (CuPC) and its derivatives (including a discotic liquid crystal form). The CuPC device shows conversion efficiency up to 2% under white light illumination (20–30mW∕cm2), distinct from cells filled with CuPC derivatives with alkyl chains attached to the core. It is concluded that a critical issue for efficient photocarrier generation is the distance between the CuPC core and the PSi surface. Both organic and inorganic components contribute to photoinduced charge transfer and transport processes. The influence of the PSi structure and pore filling on the solar cell performance is discussed.
Band tail hopping conduction mechanism in highly conductive amorphous carbon nitride thin films
Authors: Lazar G. , Zellama K. , Clin M. , Godet C.
Published: December 2004

Nitrogenated amorphous carbon (a-C:N) films have been synthesized by radio frequency (rf) magnetron sputtering at various substrate temperatures and rf discharge powers. The temperature dependence of the electrical conductivity has been measured in order to identify the dominant conduction mechanism. The conductivity was found to increase over ten orders of magnitude with increasing growth temperature and discharge power. In the ohmic regime, the conductivity of the investigated a-C:N films is likely dominated by hopping conduction, in good agreement with a model for hopping in localized bandtail states, rather than by extended states band conduction.
Stimulated emission and optical gain in LaF3:Nd nanoparticle-doped polymer-based waveguides
Authors: Dekker R. , Klunder D. J. W. , Borreman A. , Diemeer M. B. J. , Wörhoff K. , Driessen A. , Stouwdam J. W. , van Veggel F. C. J. M.
Published: December 2004

We report experiments which show evidence that stimulated emission at 863nm takes place in hybrid monomode Si3N4 waveguides where LaF3:Nd nanoparticle-doped polymethylmethacrylate (PMMA) was used as a top cladding material. Furthermore, optical gain at 1319nm in LaF3:Nd nanoparticle dispersed PMMA (0.1dB∕cm) and photodefinable epoxy (Microchem SU-8) multimode waveguides has been observed at pump powers below 10mW. This class of composite materials based on polymers with dispersed nanoparticles shows promising properties for planar optical amplifiers. Simulation showed that optical gain in the order of 10dB can be achieved at 100mW pump power in a 20cm long monomode waveguide.
Coherent plasmons in InSb
Authors: Hasselbeck Michael P. , Schlie L. A. , Stalnaker D.
Published: December 2004

Far-infrared electromagnetic radiation is emitted by coherent plasmon oscillations in bulk (111) InSb. The oscillations are excited by near-infrared ultrashort laser pulses and characterized as a function of temperature. The coherent plasmon frequency is determined by the intrinsic electron concentration and donor doping density. The amplitude of the oscillations decreases with increasing temperature due to a weakening of the photo-Dember starting mechanism.
Postfabrication annealing of pentacene-based photovoltaic cells
Authors: Mayer Alex C. , Lloyd Matthew T. , Herman David J. , Kasen Todd G. , Malliaras George G.
Published: December 2004

We studied the effects of postfabrication annealing on heterojunction photovoltaic cells made from vacuum deposited pentacene and C60. The maximum power conversion efficiency under 115mW∕cm2 illumination increases from 0.45% to 1.07% after annealing the cells at 200°C. The increased performance is a result of better molecular ordering, which leads to an increased shunt resistance and built-in potential.
Focused-ion-beam-induced deposition of superconducting nanowires
Authors: Sadki E. S. , Ooi S. , Hirata K.
Published: December 2004

Superconducting nanowires, with a critical temperature of 5.2K, have been synthesized using an ion-beam-induced deposition, with a gallium focused ion beam and tungsten carboxyl, W(CO)6, as precursor. The films are amorphous, with atomic concentrations of about 40%, 40%, and 20% for W, C, and Ga, respectively, 0K values of the upper critical field and coherence length of 9.5T and 5.9nm, respectively, are deduced from the resistivity data at different applied magnetic fields. The critical current density is Jc=1.5×105A∕cm2 at 3K. This technique can be used as a template-free fabrication method for superconducting devices.
Whole-genome approaches for large-scale gene identification and expression analysis in mammalian preimplantation embryos
Authors: Adjaye James
Published: December 2004

The elucidation, unravelling and understanding of the molecular basis of transcriptional control during preimplantion development is of utmost importance if we are to intervene and eliminate or reduce abnormalities associated with growth, disease and infertility by applying assisted reproduction. Importantly, these studies should enhance our knowledge of basic reproductive biology and its application to regenerative medicine and livestock production. A major obstacle impeding progress in these areas is the ability to successfully generate molecular portraits of preimplantation embryos from their minute amounts of RNA. The present review describes the various approaches whereby classical embryology fuses with molecular biology, high-throughput genomics and systems biology to address and solve questions related to early development in mammals.
Commercial applications of nuclear transfer cloning: three examples
Authors: Forsberg Erik J.
Published: December 2004

Potential applications of cloning go well beyond the popularly envisioned replication of valuable animals. This is because targeted genetic modifications can be made in donor cells before nuclear transfer. Applications that are currently being pursued include therapeutic protein production in the milk and blood of transgenic cloned animals, the use of cells, tissues and organs from gene-modified animals for transplantation into humans and genetically modified livestock that produce healthier and safer products in an environmentally friendly manner. Commercial and social acceptance of one or more of these early cloning applications will lead to yet unimagined applications of nuclear transfer technology. The present paper summarises progress on three additional applications of nuclear transfer, namely the development of male livestock that produce single-sex sperm, the transfer of immune responses from animals to their clones to permit the production of unlimited supplies of unique polyclonal antibodies, and the generation of genetically modified animals that accurately mimic human diseases for the purpose of developing new therapies. However, the myriad applications of cloning will require appropriate safeguards to ensure safe, humane and responsible outcomes of the technology.
Derivation characteristics and perspectives for mammalian pluripotential stem cells
Authors: Trounson Alan
Published: December 2004

Pluripotential stem cells have been derived in mice and primates from preimplantation embryos, postimplantation embryos and bone marrow stroma. Embryonic stem cells established from the inner cell mass of the mouse and human blastocyst can be maintained in an undifferentiated state for a long time by continuous passage on embryonic fibroblasts or in the presence of specific inhibitors of differentiation. Pluripotential stem cells can be induced to differentiate into all the tissues of the body and are able to colonise tissues of interest after transplantation. In mouse models of disease, there are numerous examples of improved tissue function and correction of pathological phenotype. Embryonic stem cells can be derived by nuclear transfer to establish genome-specific cell lines and, in mice, it has been shown that embryonic stem cells are more successfully reprogrammed for development by nuclear transfer than somatic cells. Pluripotential stem cells are a very valuable research resource for the analysis of differentiation pathways, functional genomics, tissue engineering and drug screening. Clinical applications may include both cell therapy and gene therapy for a wide range of tissue injury and degeneration. There is considerable interest in the development of pluripotential stem cell lines in many mammalian species for similar research interests and applications.
12 CASA PARAMETERS OF FRESH BULL SEMEN COLLECTED BY ARTIFICIAL VAGINA OR ELECTROEJACULATION IN ARGENTINA
Authors: Brogliatti G. , Garcia Migliaro F. , Cavia R. , Larraburu G. , Albrecht A.
Published: December 2004

The latest entry in the field of semen evaluation is computer assisted semen analysis (CASA). Its greatest advantages are elimination of the subjective nature of routine semen evaluation and the addition of detailed motion analysis unquantifiable by visual examination. The objective of this study was to evaluate CASA motility parameters of fresh bull semen collected by artificial vagina (AV) or electroejaculation (EE) from a total of 56 beef different bulls. Semen samples from a total of 45 beef bulls were collected by AV from winter to the end of spring (740 collections), and from 11 beef bulls by EE (120 collections) in the same period. First and second AV collections were analyzed as individual data. EE collection was performed only one. Means and standard deviations for each characteristic were calculated, compared, and statistically analyzed. A sample of the collection was diluted 1:20 in a semi-defined semen extender (Andromed, Minitüb, Tiefenbach, Germany) and held in a glass tube at 36°C for 5 min before analysis. The sample was loaded into 20-μm chambers, and six microscope fields from each chamber were analyzed. The following sperm motility parameters were determined with the Ceros 12.1 sperm analyzer (Hamilton Thorne Biosciences, Inc., Beverly, MA, USA) on at least 1000 spermatozoa:concentration (CONC), velocity average path (VAP), velocity straight line (VSL), curvilinear velocity (VCL), amplitude lateral head (ALH), beat cross frequency (BCF), straightness (STR), linearity (LIN), and percentage of rapid or statics cells. There were no significant differences (P > 0.05) in VAP, VSL, VCL, ALH, STR, LIN, and the percentage of rapid and static cells of semen collected by AV or EE. The concentration (sperm/mL) of the AV-collected sperm was significantly higher than for the sperm collected by EE. Results from the analysis indicate that semen collected by artificial vagina have motility characteristics similar to those collected by electroejaculation. More research needs to be done to evaluate motility parameters of frozen/thawed semen collected by electroejaculation and by artificial vagina.

This research was supported by Centro Genético Bovino de EOLIA sa Argentina.
121 HEAT SHOCK TO PIG OOCYTES DOES NOT INDUCE APOPTOSIS BUT REDUCES EMBRYO DEVELOPMENT
Authors: Tseng J.-K. , Tang P.-C. , Ju J.-C.
Published: December 2004

Oocytes are susceptible to heat shock (HS) during the maturation process. It has been demonstrated that HS induces apoptosis and/or the expression of HS protein 70 (hsp 70) in in vitro-produced oocytes and embryos. The objectives of this study were to analyze the effects of HS on the development and apoptosis of pig oocytes and embryos. Porcine ovaries were collected from a local slaughterhouse and the cumulus-oocyte complexes (COCs) were aspirated from follicles 3–6 mm in diameter and subjected to standard in vitro maturation procedures at 39°C for 42 h. The in vitro matured oocytes were then randomly allocated to different HS treatments at 41.5°C for 0 (control, C0h), 1 (HS1h), 2 (HS2h), or 4 h (HS4h). An additional control group of oocytes was cultured for 4 h without HS (C4h). Data were analyzed by chi-square test. In Experiment 1, anti-hsp 70 (SPA-810AP, Stressgen, San Diego, CA, USA) and Western blotting were used to examine the expression of hsp 70. Results indicated that no significant difference of hsp 70 expression in metaphase II porcine oocytes occurred between controls and HS groups (P > 0.05, 7 replicates). In Experiment 2, apoptosis of metaphase II oocytes after HS was identified by annexin V-FITC (Sigma, St. Louis, MO, USA) staining and TUNEL (Roche, Indianapolis, IN, USA). No significant apoptotic signal was detected in the HS groups compared to the controls. The intensity of annexin V staining was not affected by HS, but it increased with the time of culture (P < 0.05, n = 24–37). In Experiment 3, the apoptotic rate and developmental competence of the HS-oocytes were evaluated by TUNEL assay (n = 123–137, 4 replicates). Parthenogenetic activation (n = 123–137) was performed by an electric pulse (2.2 kV cm−1) combined with 6-dimethyaminopurine treatment (6-DMAP, 2.5 μM, 4 h, Sigma). The cleavage rates in HS2h (43 ± 29%) and HS4h (35 ± 28%) decreased (P < 0.05) compared to those in C0h (62 ± 12%) and C4h (66 ± 8%). In addition, the blastocyst formation rates and total cell numbers reduced (P < 0.05) after 2 h (11 ± 10%, 20 ± 16) and 4 h (11 ± 8%, 19 ± 8) of HS treatments compared to those in C0h (23 ± 14%, 32 ± 22) and C4h (21 ± 11%, 27 ± 17), all respectively. The numbers of blastocysts with TUNEL-positive signals were not significantly different between the HS and control groups, but the signals increased (P < 0.05) before the 8-cell stage in HS groups (22–24%) compared to the C0h and C4h controls (16 and 11%), respectively. These results indicate that reduction in developmental competence of in vitro-matured pig oocytes after heat shock is not closely correlated to the expression of hsp 70 in the oocytes and to the apoptotic cell numbers in the blastocyst. Whether detection of apoptosis by TUNEL or annexin V-FITC in oocytes is a good indicator requires further investigation.
127 VILLOUS ARCHITECTURE AND FETO-MATERNAL INTERDIGITATION IN THE AFRICAN BUFFALO (SYNCERUS CAFFER) DURING DIFFERENT GESTATION STAGES
Authors: Schmidt S. , Soley J.T. , Aire T.A. , Gerber D.
Published: December 2004

Anchorage of the ruminant placenta is achieved by complementary indentation of chorionic villi into endometrial crypts. Geometrical patterns of interdigitation vary greatly among ruminants. Fetal villosity has been studied in other bovid species (Bos taurus, Bubalus bubalis) closely related to the African buffalo. The present study provides first information on villous architecture and feto-maternal anchorage in the African buffalo. Tissue samples from six pregnant cows (crown/rump length in cm: 2, 3, 14, 17, full-term) were collected during disease eradication programs in the Hluhluwe Game Reserve and from a buffalo breeding centre at Phalaborwa (afterbirths, n = 3). Tissue was fixed with 2.4% glutaraldehyde in Millonig's phosphate buffer by immersion or, if possible, via vascular perfusion through uterine and umbilical arteries prior to separation of feto-maternal tissue for scanning electron microscopy (SEM) preparation. Samples were further prepared for light microscopy and SEM using standard techniques. SEM samples were examined in a Philips XL 20 Microscope operated at 7 kV. Fetal villi consisted of a vascularized mesenchymal core covered by trophoblast epithelium. At a crown/rump length of 2 and 3 cm, the cotyledons comprised numerous short finger-like villi (length = 0.15–0.46 mm) originating from the chorionic plate. Some of these primary (stem) villi divided longitudinally a short distance from their origin. These relative simple villi were accommodated within corresponding caruncular crypts. At later gestation (crown/rump length: 14 and 17 cm), fetal villi had lengthened (length = 2–7.5 mm) and lateral (secondary) branching had started. The latter appeared in the form of bulbous or elongated structures projecting at approximately right angles from the stem villus. During the last month of gestation and in post-partum samples, primary (8–12 mm) and secondary villi had developed considerably in length. Secondary villi displayed a complex array of tertiary (terminal) villi of variable shape and size. A rich capillary network was observed within the connective tissue core of the tertiary villi as well as in the corresponding endometrial septae forming the walls of the caruncular crypts. Fully developed, each primary villus with its attendant secondary and tertiary villi formed a slender conical-shaped unit, also referred to as a cotyledonary villous tree. This paper provides the first description of villous architecture and placentome development in the African buffalo. The specific branching pattern of cotyledonary villi and complementary caruncular crypts in the African buffalo placenta, as described in this study, appears less complex when compared to that in the placenta of related Bos and Bubalus species.
157 HOLSTEIN-CHINESE YELLOW HYBRID RECIPIENT OOCYTES RECOVERED BY OVUM PICKUP CAN IMPROVE THE DEVELOPMENT OF CLONED BOVINE EMBRYOS
Authors: Yang X.Y. , Zhao J.G. , Li H.W. , Li H. , Liu H.F. , Zeng Y.T. , Huang S.Z.
Published: December 2004

We used the subspecies hybrid F1 oocytes (Holstein/Chinese Yellow cattle) recovered by ovum pickup (OPU) as recipient cytoplasts to improve the development of bovine cloned embryos. Ten Holstein cattle, four Chinese Yellow cattle, and four hybrid F1 bovines were subjected to OPU once a week. In total, 44, 110, and 42 OPU sessions were respectively performed for hybrid cattle, Holstein cattle and Chinese Yellow cattle. The mean numbers of punctured follicles for hybrid and Yellow cattle were higher than for Holstein cattle (11.4 ± 0.5 and 11.7 ± 0.5 vs. 10.1 ± 0.7, mean ± SE), but the recovery rate for Holstein cattle was higher than that for hybrid and Yellow cattle (76.2% vs. 70.3% and 66.6%); therefore, recovered oocytes per session were similar in hybrid, Holstein, and Yellow cattle (8.0 ± 0.5, 7.7 ± 0.4 and 7.8 ± 0.5, respectively). No difference was shown in the quality of the recovered oocytes among the three breeds. The three kinds of recipient oocytes had a maturation rate of 72–73% (256/353, 614/847, and 238/327, respectively). Matured oocytes were used as recipients without selection, and same batch cumulus cells collected from Holstein cow were used as donor cells. The nuclear transfer procedure was essentially as described by Park et al. (2004 Mol. Reprod. Dev. 69, 365–374). Cleavage rate of reconstructed embryos was similar in the hybrid, Holstein, and Yellow groups (66%, 66%, and 75%, respectively). However, the blastocyst rate from the cleavage embryos (51% vs. 37% and 27%), cell number of each blastocyst on Day 8 (135 ± 4.1 vs. 116 ± 3.6, and 101 ± 4.2), and the percentage of Grade A blastocysts (54% vs. 42% and 29%) in the hybrid group were higher than in the Holstein and Yellow groups. The proportion of blastocyst production on Day 7 was greater in the hybrid group than in the Holstein and Yellow groups (89% vs. 71% and 63%). The blastocyst rate from morula in the hybrid group (84%, 37/44) was significantly higher than in the Holstein group (63%, 52/83) and the Yellow group (59%, 24/43). Taken together, these results strongly show that hybrid recipient oocytes can improve the development of cloned bovine embryos. It is suggested that the genetic heterogeneity of the hybrid recipient cytoplasm would lead to more possibilities of reprogramming and embryo development.

Table 1.
Effect of different recipient oocytes on development of cloned embryos

This work was supported by the Chinese “863” High-Tech Plan Program (Grant No. 2002AA206201).
168 ESTABLISHMENT AND MOLECULAR CHARACTERIZATION OF PIG PARTHENOGENETIC EMBRYONIC STEM CELLS
Authors: Brevini T.A.L. , Cillo F. , Gandolfi F.
Published: December 2004

Parthenogenetic embryonic stem cells have been obtained in mouse and in primates. However, it would be desirable to have an alternative experimental model that could be used to investigate the therapeutic potential of these cells. For this purpose, we generated parthenogenetic pig blastocysts from in vitro-matured oocytes activated by sequential exposure to 10 μM ionomycin for 5 min and 2 mM 6-DMAP for 3 h. Inner cell masses were isolated by immunosurgery and plated on mitotically inactivated STO fibroblast feeder layers in 4-well dishes. Cells were incubated in 5% CO2 at 37°C in low glucose DMEM/F10 medium supplemented with 1000 IU/mL of mouse recombinant LIF, 10% Knockout serum replacer (Gibco, Italy), and 5% FBS. Within 3 days, circular colonies with distinct margins of small round cells were observed on both substrates. When a colony enlarged enough to cover half or more of the well surface, cells were trypsinized in clumps never reaching single-cell suspension and passaged to a newly prepared well. The expression of a gene panel was examined by RT-PCR on a portion of the cells at each passage. Oct-4 and nanog were used as markers of pluripotency. Interferon-τ, α-Amilase, Bone Morphogenetic Protein-4, and Neurofilament were used as markers of trophectoderm, endoderm, mesoderm, and ectoderm differentiation respectively. After 4 passages, three colonies expressed Oct-4 and nanog and were negative for all four differentiation markers. Two colonies at the 5th and 7th passages maintained nanog but not Oct-4 expression, while remaining negative to all of the other genes. To induce the formation of embryoid bodies (EBs), cells were cultured in 50-μL droplets of medium without LIF. Initiation of differentiation of EBs was confirmed through both morphological examination and molecular analysis; mesodermal, ectodermal, and endodermal markers were all expressed by Day 9 of culture and Oct-4 and nanog expression was completely down-regulated. Interestingly, when EBs were returned to adherent culture conditions patches of differentiated cells tended to form, spontaneously differentiating into mesodermal, endodermal, or neuroectodermal cell monolayers. The present data suggest that it is possible to establish putative embryonic stem cells from pig parthenotes. Further studies are in progress to determine their ability to stably maintain the undifferentiated state.
This work was supported by MIUR COFIN 20022074357 and Fondazione CARIPLO.
173 EFFECT OF CULTURE SYSTEM FOR IVM-IVF PIG EMBRYOS ON THE ICMS ABILITY TO PRODUCE OUTGROWTHS FOR EMBRYONIC STEM CELL DERIVATION
Authors: Lazzari G. , Lagutina I. , Crotti G. , Turini P. , Colleoni S. , Duchi R. , Galli C.
Published: December 2004

Attempts to derive true embryonic stem cells in large farm animals rely on the supply of good quality embryos. In these species, including the pig, pre-implantation-stage embryos can be produced by in vitro techniques from slaughterhouse ovaries. The objective of this study was to evaluate the ability of the inner cell masses (ICMs) of pig embryos, produced in vitro by different methods, to provide viable initial outgrowths of ICM cells that could be subsequently subcultured and expanded. Porcine oocytes were recovered from slaughtered donors and matured in vitro for 40–44 h in DMEM-F12 supplemented with 10% FCS, 0.05 IU LH and FSH (Menogon, Ferring, Milan, Italy), 0.3 mM cystine, 0.5 mM cysteamine, 50 ng/mL long-EGF, 100 ng/mL long-IGF1, 5 ng/mL bFGF (Sigma-Aldrich, Milan, Italy) in 5% CO2 at 38.5°C. Boar frozen-thawed semen was separated on a percoll gradient and diluted in TALP medium with PHE (penicillamine, hypotaurine, epinefrine) to a concentration ranging from 0.05 to 0.1 million sperm per mL. Oocytes were partially decumulated, co-incubated with sperm for 24 h, and finally denuded and cultured in microdrops of mSOFaa or NCSU. After cleavage, approximately half of the cleaved embryos were surgically transferred into the sheep oviduct for 4 days of in vivo culture and the remaining embryos were left in vitro in the two media. On Day +6 in vivo-cultured embryos were recovered from the sheep oviduct. Blastocyst formation and quality were comparatively evaluated in the three culture groups. Quality specifically referred to the morphology/size of the ICM according to the following criteria: ICM A (large/prominent), ICM B (flat), and ICM C (non-visible). All embryos with a visible inner cell mass were subjected to microdissection with needles to recover the ICMs that were then plated on feeder-layers of mitomycin-treated STO fibroblasts. Attachment and outgrowth was evaluated 48–72 h post-plating. Results are presented in Table 1. Our data indicate that in vivo culture of pig embryos in the sheep oviduct greatly enhance both blastocyst development and ICM quality. As a consequence the efficiency of outgrowth formation, following plating for ES cell derivation, was significantly higher with ICMs derived from IVM-IVF pig embryos cultured in vivo as compared to their in vitro-cultured counterparts. Within the two culture media tested for in vitro culture, SOF and NCSU, the rate of blastocyst formation was similar but the quality of SOF-cultured embryos is higher. In conclusion, embryo/ICM quality represents a fundamental requirement for the derivation of ES cell lines, and in vivo culture in the sheep oviduct provides the most efficient source of high quality IVM-IVF pig embryos.

Table 1.
Blastocyst development and ICM quality of in vitro-produced pig embryos

This work was supported by the Istituto Superiore di Sanità, Programma Nazionale Cellule Staminali, Rome, Italy, grant No. CS 11.
182 DISINFECTION OF DRY (VAPOR) SHIPPERS ('DEWARS') FROM MICROBIAL CONTAMINATION ASSOCIATED WITH CRYOPRESERVED GERMPLASM
Authors: Bielanski A.
Published: December 2004

Cryopreservation, storage, and transport of cryopreserved germplasm without the risk of disease transmission is of great concern to animal and human health authorities. Here we report on the efficacy of microbial decontamination of liquid nitrogen (LN) dry (vapor) shippers used for short-term storage and transportation of germplasm and other biological specimens. Dry shippers containing either a hydrophobic or a nonhydrophobic LN absorbent were experimentally contaminated with high titers of cultures of Pseudomonas aeruginosa, Escherichia coli, Staphylococus aureus, bovine viral diarrhea virus (BVDV), and bovine herpesvirus-1 (BHV-1). Biocidals with broad-spectrum antimicrobial activity and gas vapors of formalin and ethylene oxide were used for disinfection of the shippers. All biocide solutions were diluted with milli-Q water to the concentrations recommended by the manufacturer and poured directly into the chamber of the dry shippers. The dry shipper was filled with the disinfectant for 30 min, drained, washed three times with sterile water, and drained before testing the residue for microbial contaminant. Among the biocidals used, treatment with sodium hypochlorite solution (30% of household bleach), a quaternary ammonium-based disinfectant (100% Expel), and peracetic acid (30%) were the most effective and useful for dry shippers with a hydrophobic LN absorbent. None of the bacterial or viral microorganisms were detected in samples of semen and embryos stored in dry shippers following their disinfection with these biocides. Other disinfectants (Virkon, Roccal, 1-Stroke, Expel Odor) were not effective due to their foaming properties. Some other disinfectants (Viralex and 70% ethanol) caused irreversible damage to the permeability of the LN-absorbent hydrophobic membrane. Gas sterilization by ethylene oxide was effective for both types of dry shipper.
The author thanks Mr. A. Hanniman for his technical support and Ms. B. Phipps-Todd for processing samples for the scanning electron microscopy.
187 ANALYZING DISEASE TRANSMISSION RISKS FROM ABATTOIR-DERIVED IN VITRO-PRODUCED BOVINE EMBRYOS
Authors: Perry G.
Published: December 2004

While thousands of in vitro-produced (IVP) bovine embryos have been transferred commercially with no reports of disease transmission, such risks must be considered. Due to differences in their zonae pellucidae, the disease risks with IVP embryos are known to be higher than with in vivo-derived embryos. Possible sources of infection include the oocytes, spermatozoa, serum, and co-culture cells. The Terrestrial Animal Health Code of the Office International des Epizooties (OIE, 2003) stipulates that disease risk management should meet standards set by the World Trade Organization. These standards include subjecting the IVP procedures to quantitative risk assessment to evaluate disease transmission risk. The purpose of the present work was to measure the risks of transmitting disease with IVP embryos obtained from abattoir-derived tissues. A simulation model was developed using Microsoft Excel spreadsheets with the Palisade @RISK (London, UK) software program. The model incorporates probability distributions, the shapes of which reflect the random nature of some of the data (e.g. fluid volumes in cultures and washes) and the conjectural nature of some of the scientific information (e.g. on disease agents). The model is adaptable so that, when accurate data or information become available, variability estimates and degrees of uncertainty can be replaced with fixed values. The model assumes: (1) the IVP method is as described in the IETS Manual (1998); (2) there are five possible sources of infection; donor cow, donor bull, fetal calf serum, bovine serum albumin, and co-culture cells; (3) the disease agents can survive and/or proliferate during in vitro maturation, fertilization and culture; (4) fluid volumes in cultures and washes follow “known” normal distributions; (5) uncertainties in current knowledge of IVP embryos and disease agents can be taken into account by use of appropriate probability distributions; (6) different methods of in vitro fertilization do not affect the level of risk; and (7) different methods of in vitro culture can affect the level of risk. The model as constructed fits comfortably into a single workbook with one worksheet allocated for the model itself and another serving to store data on diseases of interest. Data on oocytes, blastocyst numbers, etc., and on media and wash fluid volumes are held within the model while information relating to particular diseases can be selected from a drop-down list at the top of the first worksheet. The relevant data stored in the database are then retrieved and used for modelling, using Monte Carlo simulation. The model estimates the final titer of the disease agent in IVP embryos and the probability of at least one infective transmission to a recipient, expressed as distributions.
196 IN VITRO DEVELOPMENT OF RECONSTRUCTED WATER BUFFALO (BUBALUS BUBALIS) OOCYTES AFTER FETAL SKIN FIBROBLAST CELL NUCLEAR TRANSFER
Authors: Meena C.R. , Das S.K.
Published: December 2004

The present study was undertaken to explore the feasibility of using buffalo fetal skin fibroblasts as donor nuclei and to determine the developmental competence of embryos following transfer of these nuclei to in vitro-matured enucleated buffalo oocytes. Skin cells were isolated from 1–2-month-old fetuses, obtained from an abattoir, by enzymatic digestion (0.5% w/v trypsin + 0.05% w/v collagenase in Dulbecco's PBS) for 15–20 min. The cells were washed four times with Dulbecco's PBS and then once with RPMI-1640 medium + 10% FBS by centrifugation at 600g. The cells were then cultured in the same medium in a CO2 incubator (5% CO2 in air) at 38.5°C for 2–3 days. COCs collected from slaughterhouse buffalo ovaries were subjected to IVM in the IVM medium (TCM-199 + 1 μg mL−1 E-β + 5 μg mL−1 FSH-P + 10 μg mL−1 LH + 10% FBS) for 22–24 h in a CO2 incubator (5% CO2 in air) at 38.5°C. Oocytes were denuded with 0.1% trypsin followed by repeated pipetting and then enucleated by aspirating the first polar body and 10–15% of nearby cytoplasm with a micromanipulator. Two different types of donor cells (growing cells and those arrested with cytochalasin-B) were used for reconstruction of oocytes. The reconstructs were electrofused and incubated in the activation medium (TCM-199 + 8 μg mL−1 cytochalasin-B + 10% FBS) for 4 h. These were then cultured in IVC medium (TCM-199 + 10% FBS) in a CO2 incubator (5% CO2 in air) at 38.5°C for 48 h. Next, the cleaved embryos were co-cultured with buffalo oviductal cells in embryo development medium. Out of 119 denuded matured oocytes which were enucleated and reconstructed with growing cells, 78 (65.5%) were electrofused, activated and cultured, out of which 4 (5.1%) reconstructs cleaved and developed to the 2-cell stage, 3 (3.8%) reached the 4-cell stage, and 1 (1.3%) reached the 8-cell stage. In the synchronized group, out of 100 denuded matured oocytes which were reconstructed with cytochalasin-B blocked cells, 40 (40%) were electrofused, activated, and cultured, out of which 4 (10%) developed to the 2-cell stage, 3 (7.5%) to the 4-cell stage, 2 (5.0%) to early morula stage, and 1 (2.5%) to blastocyst stage. These results suggest that buffalo fetal skin fibroblasts could be used as donor nuclei for the production of buffalo embryos after nuclear transfer to enucleated in vitro-matured buffalo oocytes.
228 EXPRESSION OF zag1 IN MOUSE PRE-IMPLANTATION EMBRYOS
Authors: Matsumoto K. , Uenoyama A. , Matsuoka T. , Saeki K. , Hosoi Y. , Iritani A.
Published: December 2004

Embryonic gene activation (EGA) first occurs during the second half of the mouse 1-cell embryo (Latham KE 1999 Int. Rev. Cytol. 193, 71–124). Moreover, precise regulation of EGA is considered to be essential for normal embryo development. To understand the molecular basis for the regulation of EGA, we have focused on the identification and functional characterization of genes activated at the late 1-cell stage of the mouse embryo. Recently, we have identified and isolated a novel gene, termed zag1 (zygotic activating gene 1), transcribed specifically at the EGA, using a fluoro-differential display method with oocytes and embryos at 15 h post-insemination. Messenger RNA of zag1 expressed at lower level in the oocyte than that in the embryo at 15 h post-imsemination. In this study, we investigated the potential function of zag1 by analysis of mRNA expression and protein distribution in mouse tissues and pre-implantation embryos. Nucleotide sequence analysis of zag1 cDNA revealed that the open reading frame of 1726 bps encodes a protein of 575 amino acids with a predicted molecular mass of 66 kDa. The deduced amino acid sequence indicated that zag1 protein might be a soluble protein with a bipartite nuclear targeting sequence, a NACHT NTP domain, and an APT/GTP binding site motif as a predicted functional domain. Two μg of Poly(A)+ RNA from various tissues of adult mice were subjected to Northern blot analysis using the mouse zag1 cDNA probe. We detected this gene abundantly expressed in mouse testis and ovary by approximately 2- to 3-fold compared with one in other mouse tissues (heart, liver, kidney, lung, brain, skeletal muscle, and spleen). zag1 transcript and protein, as assessed by RT-PCR and immunoblotting, respectively, were slightly present in ovulated oocytes, gradually decreased in the early 1-cell embryos, but re-expressed in the late 1-cell and early 2-cell stage embryos which coincided with the mouse EGA. Subsequent to microinjection of an expression vector encoding zag1-enhanced green fluorescent protein (EGFP), fused protein into male pronucleus of 1-cell embryos was detected in the nuclei of 2-cell embryos. These findings suggest that zag1 may be functionally associated with early embryonic development.
This study was supported by a Grant-in-Aid for the 21st Century COE Program of the Japan MEXT, and by a grant from the Wakayama Prefecture Collaboration of Regional Entities for the Advancement of Technological Excellence of the JST.
249 EFFECT OF REFREEZING BULL SEMEN ON IVF SUCCESS RATE
Authors: McCue P. , Kelly J. , Ashworth S. , Kleemann D. , Walker S.
Published: December 2004

Cryopreserved semen from valuable sires may be available in limited quantities in some situations. A large percentage of the spermatozoa in a thawed straw is potentially wasted since a relatively small number of spermatozoa are required for most assisted reproduction techniques. The goal of this study was to determine the effect of dilution and refreezing of bull semen on fertilization and blastocyst development rates following in vitro fertilization. The hypothesis was that frozen bull semen that was thawed, diluted, and refrozen could be used successfully for IVF. Oocytes were harvested from cow ovaries collected from a local abattoir and matured in vitro for 24 hours. Ova were subsequently assigned to one of four in vitro fertilization treatment groups. Group 1 ova (n = 158) were fertilized with bull semen frozen at a concentration of 20 × 106 spermatozoa per 0.25 mL straw. Group 2 ova (n = 157) were fertilized with semen frozen at an initial concentration of 2 × 106 spermatozoa. Group 3 ova (n = 157) were fertilized with semen that had been thawed and refrozen at a concentration of 20 × 106 spermatozoa. Group 4 ova (n = 150) were fertilized with semen that had initially been frozen at a concentration of 20 × 106 spermatozoa and then thawed, diluted to a concentration of 2 × 106 spermatozoa, and refrozen. IVF was performed in a medium volume of 100 μL using 1 × 106 spermatozoa/mL. Cleavage and blastocyst rates were determined 2 days and 7 days, respectively after IVF. Cleavage rates following IVF was highest with semen frozen at 20 × 106 spermatozoa (89.9%), intermediate with semen frozen at 2 × 106 spermatozoa or refrozen at 20 × 106 spermatozoa (71.3% and 73.9%, respectively), and lowest with semen refrozen at 2 × 106 spermatozoa (38.7%) (P < 0.05). Blastocyst development rate was not significantly different (P > 0.05) among treatment groups. This study confirmed the hypothesis that refrozen bovine semen can be used successfully for in vitro fertilization. Although the overall IVF efficiency was lower using diluted refrozen semen, multiple IVF procedures could theoretically be performed over time from one initial straw. Consequently, if a limited amount of frozen semen is available, thawing of a single straw followed by dilution, re-allocation into multiple straws, and refreezing should be considered to facilitate the more efficient use of semen in future assisted reproduction endeavors.
This study was supported by the PEG Program, Colorado State University.
257 EFFECTS OF GLUTAMINE AND HYPOTAURINE ON OXIDATIVE STRESS OF PORCINE EMBRYOS CULTURED IN VITRO
Authors: Suzuki C. , Yoshioka K.
Published: December 2004

We previously developed an in vitro production system for porcine embryos and reported that the addition of glutamine and hypotaurine during in vitro culture improved blastocyst yield and the total number of cells in the blastocysts. Glutamine and hypotaurine might reduce oxidative stress, allowing the development of embryos cultured in vitro, because glutamine reportedly protects embryos against oxidative stress by helping to maintain intracellular levels of cysteine, a precursor of glutathione (GSH), and hypotaurine is a potent antioxidant. In the present study we evaluated the effects of the presence of glutamine and hypotaurine from Day 2 (Day 0 = the day of in vitro fertilization) to Day 3 on oxidative stress during in vitro development of porcine embryos. Porcine cumulus-oocytes complexes from prepubertal gilts were matured and fertilized in vitro using frozen-thawed ejaculated boar semen (Yoshioka et al. 2003 Biol. Reprod. 69, 2092–2099). Presumptive zygotes were cultured in porcine zygote medium (PZM)-5 (Suzuki et al., 2002) containing 2 mM of glutamine and 5 mM of hypotaurine as a basal culture medium until Day 2. The cleaved embryos were then transferred into one of four media prepared as follows: (1) containing no glutamine or hypotaurine (G−H−), (2) containing glutamine (G+H−), (3) containing hypotaurine (G−H+), (4) containing glutamine and hypotaurine (G+H+) (= PZM-5), and cultured for 24 h. After culture, the total number of cells, intracellular GSH content, and level of hydrogen peroxide (H2O2), which is a reactive oxygen species, in the cleaved embryos were examined. Some cleaved embryos were cultured in PZM-5 from Day 3 until Day 5 and the percentage of embryos that developed into blastocysts and the total number of cells in the blastocysts were investigated. Intracellular GSH content and H2O2 level on Day 3 were determined by a dithionitrobenzoic acid-glutathione disulfide (DTNB-GSSG) reductase recycling assay and dichlorohydrofluorescein diacetate (DCHFDA)-based assay, respectively. Data were statistically analyzed by ANOVA and Fisher's PLSD test. The total number of cells (4.3 to 4.4 cells) and intracellular GSH content (2.3 to 2.9 pmol/embryo) in the cleaved embryos on Day 3 did not differ among treatments. On Day 3, the intracellular H2O2 level of the cleaved embryos cultured in G+H+ decreased by 49% compared with those cultured in G−H− (100%) (P < 0.05). On Day 5, the percentage of embryos that developed into blastocysts in G+H+ (52%, 47/90) and G+H− (41%, 36/88) was significantly higher than in G−H− (11%, 11/90) and G−H+ (21%, 19/89) (P < 0.05). The total number of cells in the Day 5 blastocysts from G+H+ (34.5 cells) was significantly (P < 0.05) greater than in those from G−H− (25.8 cells). These results suggest that the presence of glutamine and hypotaurine in PZM-5 from Day 2 to Day 3 improves the subsequent development of porcine embryos into blastocysts by reducing intracellular H2O2 levels.
This work was supported by MAFF, Japan.
259 INVOLVEMENT OF Ca2+/CALMODULIN-DEPENDENT PROTEIN KINASE II IN THE SPONTANEOUS ACTIVATION IN RAT OOCYTES
Authors: Yoo J.G. , Smith L.C.
Published: December 2004

Immediately after recovery from the oviduct, rat metaphase-arrested oocytes are very sensitive to spontaneous activation (SA), which is characterized by resumption of meiotic division followed by the cytoplasmic scattering of chromosomes. Although it has been shown that proteasome inhibitor is effective in stopping SA, it is likely that an upstream event is controlling SA. Constitutively active Ca2+/calmodulin-dependent protein kinase II (CaMKII) triggers cyclin B destruction and release from MII arrest. To investigate the kinetics and mechanism of rat spontaneous oocyte activation, we (1) compared SA status after different periods of aging in oviducts and examined the effects of hyalurondiase treatment, (2) compared SA between Ca2+-free and Ca2+-containing conditions, (3) examined the pattern of SA after using CaMII inhibitor and proteasome inhibitor, and (4) analyzed the activity of CaMKII at different times after oocyte collection. Oocytes were collected from three 4-wk-old PMSG-primed Sprague Dawley rats. ANOVA Tukey-Kramer HSD was used for statistical analysis using a P value of 0.05. Each experiment contained at least four replicates with at least 100 oocytes. Experiment 1: Oocytes were collected at different times post-hCG injection (14, 18, 22 h) and cumulus cells were removed before or after 2 h of in vitro culture with hyaluronidase. There was no difference in SA due to oocyte aging in oviducts or hyalurondiase treatment. Experiment 2: Oocytes were cultured in Ca2+-containing and Ca2+-free KSOM for 2 h after collection at different times (14, 18, 22 h) post hCG injection. Although there were no differences among Ca2+-containing KSOM culture groups (91%, 82%, and 90%, respectively), Ca2+-free KSOM significantly decreased SA at 14, 18, and 22 h post-hCG (19%, 43%, and 51%, respectively). Experiment 3: Oocytes were treated with different doses of inhibitor of CaMKII, myr-AIP (10, 20, 50, 100 μM), and proteosome inhibitor MG132 (10, 20, 50, 100 μM) after oocyte collection. Fifty and 100 μM myr-AIP induced significantly lower SA (36%, and 17%) than 0, 10, 20 μM (91%, 65%, and 53%, respectively). Moreover, higher concentrations of MG132 (50 and 100 μM) induced significantly lower SA rates (6% and 5%) than 0, 10, 20 μM (91%, 41%, and 22%, respectively). We also investigated the effect of short exposures to myr-AIP and MG132 during the first 2 min post-recovery. The myr-AIP group produced significantly lower SA compared to control and MG132-treated groups. Experiment4: Oocytes collected at different times (0, 10, 20, 30, 60 and 90 min) were used for CaMKII activity assay. CaMKII activity increased at 20 min and remained high for 30 min followed by decreased activity by 60 min. In conclusion, CaMKII seems to be one of the upstream signals that causes rat oocytes to spontaneously activate after recovery.
Financial support was provided by NSERC and Canada Research Chairs.
269 INACTIVATION OF MATURATION PROMOTING FACTOR AND MITOGEN-ACTIVATED PROTEIN KINASE IN PORCINE OOCYTES BY A SINGLE ELECTRICAL PULSE
Authors: Koo D.-B. , Chae J.-I. , Kim J.-S. , Wee G. , Song B.-S. , Lee K.-K. , Han Y.-M.
Published: December 2004

Activities of maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAPK) of mature oocytes should be decreased to begin subsequent development. In this study, activities of MPF and MAPK were investigated in porcine oocytes after artificial activation. To determine optimal electrical activation, porcine oocytes were exposed to 3 V AC pulse for 5 s followed by a single DC pulse of various electric field strengths (120, 150, 180, and 210 V/mm) and pulse durations (15, 30, 45, and 60 μs). For chemical activation, oocytes were exposed to 5 μM ionomycin for 5 min followed by 2 mM 6-dimethylaminopurine (6-DMAP) or 5 μg/mL cycloheximide for 4 h or 6 h. After activation, 40 to 50 oocytes were cultured in 50-μL drops of NCSU23 medium supplemented with 4 mg/mL BSA at 39°C, and 5% CO2 in air. After 6 days of culture, blastocyst formation was observed and then numbers of blastocyst nuclei were counted after staining with Hoechst 33342. in vitro development rates and numbers of blastocyst nuclei by the field strengths were not significantly different among experimental groups (P > 0.05). However, development rates to the blastocyst stage of porcine oocytes exposed to 15 and 30 μÂ­s were 27.4 and 24.4%, respectively, which were significantly higher than that (12.5%) of 60 μs (P < 0.05). Mean numbers of blastocyst nuclei in 15- and 30-μs groups (38.6 ± 13.4 and 37.9 ± 11.4, respectively) were significantly higher than that (25.8 ± 10.5) of the 60-μs group (P < 0.05). Blastocyst development after optimal electrical pulse exposure was compared with that after different chemical treatments. Electrical stimulation induced 22.9% of blastocyst formation, which was significantly higher (P < 0.01) than those induced by the chemical stimulators (3.4 and 2.7%). Based on these results, changes of constituent proteins (cdc2 and ERK) of MPF and MAPK after artificial activation were analyzed by immunoblotting using anti-PSTAIRE monoclonal antibody and anti-MAP kinase polyclonal antibody. Activities of both cdc2 and ERK in pig oocytes were reduced 4 h after electrical stimulus, but were maintained at optimal levels after treatment with ionomycin + 6-DMAP. Our results indicate that an optimal single electrical pulse is effective on the inactivation of MPF and MAPK in porcine oocytes, eventually resulting in activation of porcine oocytes produced in vitro.