bluuebunny/crossref_metadata_2025 huggingface dataset:
Genetic variation in the mitochondrial cytochrome c oxidase subunit 1 within three species of Progamotaenia (Cestoda: Anoplocephalidae) from macropodid marsupials
Authors: HU M. , GASSER R. B. , CHILTON N. B. , BEVERIDGE I.
Published: December 2004

Sequence variation within 3 morphologically defined species of the anoplocephalid cestode genus Progamotaenia (P. ewersi, P. macropodis and P. zschokkei) was investigated using the cytochrome c oxidase subunit 1 gene. The magnitude of genetic variation detected within each morphospecies suggests that, in each instance, several cryptic species are present. Within P. ewersi, 5 genetically distict groups of cestodes were detected, 1 shared by Macropus robustus and M. parryi in Queensland, 1 in M. agilis from Queensland, 1 in Petrogale assimilis from Queensland, 1 in Macropus fuliginosus from South Australia and 1 in Wallabia bicolor from Victoria. In P. macropodis, cestodes from M. robustus from Queensland, Western Australia and the Northern Territory, M. parryi from Queensland and M. eugenii from South Australia were genetically distinct from those in Wallabia bicolor from Queensland and Victoria and from M. fuliginosus from South Australia. P. zschokkei consisted of a number of genetically distinct groups of cestodes, 1 in Lagorchestes conspicillatus and L. hirsutus from Queensland and the Northern Territory respectively, 1 in Petrogale herberti, P. assimilis and M. dorsalis from Queensland, 1 in Onychogalea fraenata from Queensland, 1 in M. agilis from Queensland and 1 in Thylogale stigmatica and T. thetis from Queensland. In general, genetic groups within each morphospecies were host specific and occurred predominantly in a particular macropodid host clade. Comparison of genetic relationships of cestodes with the phylogeny of their hosts revealed examples of colonization (P. zschokkei in M. agilis) and of host switching (P. zschokkei in M. dorsalis).
Tectonic implications of a dense continuous GPS velocity field at Yucca Mountain, Nevada
Authors: Wernicke Brian , Davis James L. , Bennett Richard A. , Normandeau James E. , Friedrich Anke M. , Niemi Nathan A.
Published: December 2004

A dense, continuous GPS network was established in the Yucca Mountain area in 1999 to provide the most reliable measurements possible of geodetic strain patterns across the nation's only proposed permanent repository for high‐level radioactive waste. The network lies astride a boundary between the geodetically stable central Great Basin and the active western Great Basin, which at the latitude of Yucca Mountain is undergoing distributed right‐lateral shear at a rate of ∼60 nstrain/yr. Monitoring from 1999 to 2003 (3.75 years) yields a velocity field characterized by nearly homogenous N20°W right‐lateral shear of 20 ± 2 nstrain/yr (net velocity contrast of ∼1.2 mm/yr across a 60 km aperture) in the vicinity of the proposed repository site. Comparison of time series of continuous results with earlier campaign surveys indicating ∼50 nstrain/yr of west‐northwest extension from 1991 to 1997 suggests that the more rapid rates were in part transient motions associated with the 1992 Ms 5.4 Little Skull Mountain earthquake. Postseismic motions do not appear to affect the 1999–2003 velocity field in either campaign or continuous data. The magnitude of the velocity contrast across the area, the overall linearity of the gradient, and the large area of undeforming crust to the east of Yucca Mountain are difficult to explain by elastic bending of the crust associated with the Death Valley fault zone, a major right‐lateral strike‐slip fault about 50 km west of the repository site. These observations, along with apparent local variations in the velocity gradient, suggest that significant right‐lateral strain accumulation, with displacement rate in the 1 mm/yr range, may be associated with structures in the Yucca Mountain area. The absence of structures in the area with equivalent late Quaternary displacement rates underscores the problem of reconciling discrepancies between geologic and geodetic estimates of deformation rates.
Tsunamis in the Black Sea: Comparison of the historical, instrumental, and numerical data
Authors: Yalçiner Ahmet , Pelinovsky Efim , Talipova Tatiana , Kurkin Andrey , Kozelkov Andrey , Zaitsev Andrey
Published: December 2004

The tsunami hazard in the Black Sea is discussed by comparing historical, instrumental data and numerical results. There are 22 tsunami events in the Black Sea documented since the first century, and nine of them have occurred in twentieth century. The numerical simulations of tsunami propagation for the 1966 and 1939 events are performed by using the framework of the shallow‐water theory. The instrumental data from tide gauge records are used to compare and validate the simulation results and estimate the wave motion at other locations where the instrumental data are unavailable. The distribution of maximum positive tsunami amplitudes along the north and south coasts are obtained for each event. The arrival time, tsunami amplitudes, and directivity of tsunamis in the Black Sea are studied. The probable source and target areas of tsunamis are discussed.
Solar activity variations of equivalent winds derived from global ionosonde data
Authors: Liu Libo , Luan Xiaoli , Wan Weixing , Lei Jiuhou , Ning Baiqi
Published: December 2004

The equivalent winds at the F layer peak are derived from global ionosonde data to investigate their solar activity variations. With increasing solar activity, the derived equivalent winds are found of nonlinearly decreased diurnal amplitudes in all seasons at most stations. This implies that the increase in ion drag more than compensates for pressure gradients and thus restrains the diurnal amplitude at high solar activity. The diurnal phase of the derived equivalent winds generally shifts later at higher solar activity. It is the first time to explicitly report this striking feature that emerged at so many stations. Another pronounced feature is that the diurnal phase has a summer‐winter difference. The diurnal phases at most stations in the Northern Hemisphere are later in winter than in summer at higher solar activity. Furthermore, a decrease in the semidiurnal amplitudes of equivalent winds with increasing solar activity is evident in winter over most stations considered and in other seasons at stations with a lower dip, but the decrease trend becomes weak in other seasons at stations with a larger dip. However, complicated dependences on solar activity can be found in the diurnal mean and the semidiurnal phases of equivalent winds at stations considered.
Spatial Dispersion of Action Potential Duration Restitution Kinetics Is Associated with Induction of Ventricular Tachycardia/Fibrillation in Humans
Authors: PAK HUI‐NAM , HONG SOON JUN , HWANG GYO SEUNG , LEE HYUN SOO , PARK SANG‐WEON , AHN JEONG CHEON , MOO RO YOUNG , KIM YOUNG‐HOON
Published: December 2004

Introduction: Action potential duration restitution (APDR) plays a role in initiation and maintenance of ventricular tachycardia (VT)/ventricular fibrillation (VF). We hypothesized that the steeply sloped APDR and its spatial heterogeneity contribute to VT/VF inducibility in patients with ventricular arrhythmia.

Method and Results: After programmed ventricular stimulation (PVS) for evaluation of clinically documented VT, patients (n = 20, 15 male, age 52.5 ± 9.5 years) were divided into two groups: inducible sustained VT/VF (IVT, n = 10) and noninducible VT/VF (NVT, n = 10). Data were compared with the corresponding results obtained from normal controls (C, n = 10). Right ventricular (RV) monophasic action potential duration at 90% repolarization (APD90) and ventricular effective refractory period (VERP) in the right ventricular apex (RVA) and right ventricular outflow tract (RVOT) were determined. APDR was acquired by scanning diastole with premature ventricular beats during a pacing cycle length of 600 msec (S1‐S2) in all patients and by rapid pacing at the cycle lengths that induced APD alternans in three patients. Maximal slopes (Smax) of the APDR curves and ΔAPD90 (APD90 at S2 400 ms – APD90 at the shortest S2) were measured. VERP and APD90 at each RV site did not differ among the three groups. Smax obtained by S1‐S2 (1.6 ± 0.6) did not differ from Smax obtained by rapid pacing (1.2 ± 0.7), with a significant correlation noted between these values (r = 0.92, P < 0.01). The IVT group had a higher spatial dispersion of Smax (Smax at RVOT – Smax at RVA) compared to the C group (P < 0.05), with no difference between the NVT group and the IVT or C groups. The IVT group had a higher spatial dispersion of ΔAPD90 compared to the NVT and C groups (P < 0.01, respectively). Smax at the RVOT (2.7 ± 1.9) was steeper than that at the RVA (1.9 ± 1.2, P < 0.05). Inducibility of sustained VT/VF was greater at the RVOT (83.3%) than at the RVA (50.0%, P < 0.05).

Conclusion: In patients with ventricular arrhythmia, VT/VF is highly inducible under conditions of greater spatial dispersion of ventricular refractoriness and APDR.
Value of Entrainment Mapping in Determining the Isthmus‐Dependent Nature of Atrial Flutter in the Presence of Amiodarone
Authors: FATEMI MARJANEH , MANSOURATI JACQUES , ROSU RADU , BLANC JEAN‐JACQUES
Published: December 2004

Introduction: Entrainment mapping is a useful procedure for localizing macroreentrant tachycardia circuits. In patients with isthmus‐dependent atrial flutter, entrainment mapping from the isthmus during tachycardia results in postpacing intervals (PPI) close to the tachycardia cycle length (TCL). However, the influence of antiarrhythmic drugs on the method's value is not clearly established. The aim of our study was to assess the value of entrainment mapping in the presence of amiodarone in patients undergoing radiofrequency ablation (RFA) of isthmus‐dependent atrial flutter.

Methods and Results: The study consisted of 83 patients with isthmus‐dependent atrial flutter: 52 were taking amiodarone at the time of RFA (group 1) and 31 were in a drug‐free state (group 2). Entrainment mapping was performed from the cavotricuspid isthmus, and PPI minus TCL was determined. The two groups had similar baseline clinical characteristics. In all patients, RFA of the isthmus resulted in termination of tachycardia, confirming the isthmus‐dependent nature of the flutter. TCL was significantly longer in group 1 than in group 2 (263 ± 31 msec vs 238 ± 27 msec, P < 0.0002). PPI minus TCL at the isthmus was significantly longer in group 1 than in group 2 (17 ± 17 msec vs 8 ± 4 msec, P < 0.01). More patients in group 1 had PPI‐TCL>20 msec compared to group 2 (37% vs 10%, P = 0.01).

Conclusion: Amiodarone significantly alters the entrainment mapping response from the isthmus. In this setting, long return cycles exceeding the TCL by >20 msec do not exclude isthmus‐dependent atrial flutter.
Catheter Ablation of Atrial Flutter after Orthotopic Heart Transplantation
Authors: HEIST E. KEVIN , DOSHI SHEPHAL K. , SINGH JAGMEET P. , SALVO THOMAS DI , SEMIGRAN MARC J. , REDDY VIVEK Y. , KEANE DAVID , RUSKIN JEREMY N. , MANSOUR MOUSSA
Published: December 2004

Introduction: Atrial arrhythmias, including atrial flutter, are common in orthotopic heart transplant recipients. However, only a small number of individual case reports describe the electrical circuit and catheter ablation of atrial flutter after heart transplantation.

Methods and Results: Detailed electrophysiologic evaluation and radiofrequency ablation of atrial flutter were performed in three patients after orthotopic heart transplantation. All cases involved a counterclockwise flutter circuit around the tricuspid annulus. All were successfully ablated at the isthmus between the tricuspid valve and the atrial anastomosis adjacent to the inferior vena cava.

Conclusion: Atrial flutter involving a counterclockwise circuit around the tricuspid annulus is common in the heart transplant population. Based on the patients described in this study and other cases reported in the literature, this arrhythmia often is treated successfully by ablation of the isthmus between the tricuspid valve and the atrial anastomosis near the inferior vena cava.
On the surface magnetization of a conductor in an alternating electric field
Authors: Gokhfel’d V. M.
Published: December 2004

The nonequilibrium contribution to the electron paramagnetic susceptibility of a conductor in an alternating electric field applied to the surface of the conductor is calculated.
The electron g factor for one-band and two-band extended models of the electron energy spectrum
Authors: Mikitik G. P. , Sharlai Yu. V.
Published: December 2004

At present, explicit expressions for the electron g factor in crystals are known only for the following two cases: when the Fermi energy εF of the electrons lies at the edge of the electron energy band, ε(kex), or when the electron energy spectrum of a crystal can be approximated by the two-band model. Here we obtain explicit formulas for the g factor in situations when the Fermi level εF is close to but does not coincide with the band edge and when the two-band model of the spectrum includes small corrections from other electron energy bands. In particular, we derive expressions that describe the dependences of the g factor on εF−ε(kex) and on the magnetic field direction for doped semiconductors. The results are applied to III–V semiconductors and to bismuth.
Influence of an admixture of H2 molecules on the structure and parameters of a Ne lattice
Authors: Gal’tsov N. N. , Prokhvatilov A. I. , Strzhemechnyı̆ M. A.
Published: December 2004

X-Ray investigations of solid solutions formed by condensation of mixtures of normal hydrogen and neon gases are performed for concentrations ranging from 2 to 60 mol.% nH2 and temperatures ranging from 5 K to the melting temperature of the sample. The structure of the vacuum condensates Ne-nH2 immediately after the samples are obtained is investigated. The boundary of single-phase solutions of hydrogen in neon is established to be 2 mol.%. At high H2 concentrations a hexagonal hcp2 phase forms in addition to a cubic fcc phase. The lattice volumes of these phases are somewhat larger but close to the volume of a pure-neon cell. The hexagonal hcp2 phase vanishes when the condensates are heated to a temperature of the order of the melting temperature of neon. This metastable hexagonal phase in the neon-rich mixtures studied is probably identical in nature to the previously observed hcp2 phase in hydrogen-rich solid mixtures. Both phases have one symmetry and the same cell volume. Information on the phase composition of the condensates is obtained from data on the concentration and temperature variations of the x-ray reflection intensities. It is shown that as the concentration of hydrogen molecules in the initial gas mixtures increases, the amount of the fcc phase in the condensates decreases almost linearly and the amount of the hcp2 phase increases. A combined analysis of the data obtained in the present work and previous measurements established the phase boundaries in the entire concentration range of the condensates. Evidently, because the molecular parameters of the components are close the Ne-nH2 mixtures do not form gel-like states, which are characteristic for quench-condensed Kr–H2 condensates.
Direct numerical simulation of homogeneous turbulence with hyperviscosity
Authors: Lamorgese A. G. , Caughey D. A. , Pope S. B.
Published: December 2004

We perform direct numerical simulations (DNS) of the hyperviscous Navier–Stokes equations in a periodic box. We consider values of the hyperviscosity index h=1, 2, 8, and vary the hyperviscosity to obtain the largest range of lengthscale ratios possible for well-resolved pseudo-spectral DNS. It is found that the spectral bump, or bottleneck, in the energy spectrum observed at the start of the dissipation range becomes more pronounced as the hyperviscosity index is increased. The calculated energy spectra are used to develop an empirical model for the dissipation range which accurately represents the bottleneck. This model is used to predict the approach of the turbulent kinetic energy k to its asymptotic value, k∞, as the hyperviscosity tends to zero.
Interaction between Sulfur(IV) Autoxidation Transients and Bioorganic Compounds
Authors: Pasiuk-Bronikowska Wanda , Bronikowski Tadeusz , Ulejczyk Marek
Published: December 2004

Environmental Context. Sulfur dioxide has long been known as a source of acidity in precipitation and of cloud condensation nuclei, but more recently it has been shown a source of radicals highly reactive with respect to various organic compounds. These radicals are formed as intermediates wherever sulfur dioxide, oxygen, and water come into contact, such as in a sulfur dioxide polluted environment (tropospheric clouds, surface waters, soil) and in living organisms (lungs, digestive tract). This work focusses on the destructive action of such intermediates upon organic compounds essential for life (such as enzymes and vitamins).
Abstract. The oxidation of sulfur dioxide in aqueous media by molecular oxygen is here exploited as a source of sulfoxy radical anions. Experimental evidence is given for the chemical interaction between these radical anions and bioorganic compounds: lysozyme (Lys), haemoglobin (Hb), and cyanocobalamin (vitamin B12). Two opposite effects are described, one leading to the elimination of sulfoxy radical anions and the other resulting in multiplication of the radicals. The former effect is due to lysozyme and haemoglobin, which behave as sulfoxy radical scavengers, whereas the latter is caused by cyanocobalamin, which actively contributes to the formation of these radicals. Possible consequences of the effects for life are briefly discussed with a view to the noxious role of tropospheric sulfur dioxide.
100 DIET AND FATTY ACID COMPOSITION OF BOVINE PLASMA, GRANULOSA CELLS, AND CUMULUS - OOCYTE COMPLEXES
Authors: Adamiak S. , Ewen M. , Rooke J. , Webb R. , Sinclair K.
Published: December 2004

The effects of altering dietary carbohydrates and lipids to oocyte donors during superovulation and ovum pickup (OPU) on in vitro embryo production was reported previously (Adamiak et al. 2004 Reprod. Dev. Fert. 16, 193–194). Here we report the effects of these dietary treatments on the fatty acid (FA) composition of plasma, granulosa cells (GCs), and cumulus–oocyte complexes (COCs) from the 32 heifers used in that study. Blood samples were collected by jugular venipuncture. COCs and GCs were harvested from each heifer by OPU as described previously but were pooled between pairs of heifers within treatment to provide adequate material for FA analysis. Both GCs and COCs were washed twice in PBS supplemented with 0.3% (w/v) BSA (FA-free) before being transferred into 2 : 1 (v/v) chloroform : methanol solution for FA extraction. FA composition was determined using gas chromatography as described previously (Reis et al. 2002, Theriogenology 57, 507). Data were analyzed by ANOVA. Total plasma FA content averaged 1.12 μg/mL and was unaffected by body condition score (BCS). Low BCS heifers had more saturated (54.0 ± 1.76 vs. 49.2 ± 1.74%) and monounsaturated (22.4 ± 1.08 vs. 18.2 ± 0.69%) FA, but less polyunsaturated FA (PUFA) (23.7 ± 1.75 vs. 32.8 ± 2.21%) in plasma than moderate BCS heifers (P < 0.01). Animals fed high relative to low fiber diets had greater plasma FA (1.3 ± 0.15 vs. 1.0 ± 0.12 μg/mL) and PUFA (31.2 ± 2.56 vs. 25.3 ± 2.19%), but less monounsaturated FA (18.8 ± 1.04 vs. 21.5 ± 1.09%) (P < 0.01). Dietary protected lipid (Ca soaps of FA) increased plasma FA (1.6 ± 0.07 vs. 0.6 ± 0.04 μg/mL) and PUFA (30.0 ± 1.92 vs. 26.4 ± 3.03%) (P < 0.05), but reduced plasma saturated FA (48.9 ± 1.12 vs. 54.2 ± 2.14%; P < 0.01) in the animals fed high compared to low fiber diets, respectively. In contrast to their effects in plasma, BCS and diet had little effect on FA composition of GCs and COCs. Although low BCS and dietary lipids both increased FA in COCs (78.3 ± 2.01 vs. 69.2 ± 2.80, P < 0.01; and 77.1 ± 2.96 vs. 70.5 ± 2.26 ng/COC; P < 0.05, respectively), neither factor significantly altered the FA composition of COCs. Across treatments, the FA composition of GCs and COCs differed markedly from that of plasma (Table 1), confirming the presence of a selective uptake mechanism in the follicle that can moderate dietary induced fluctuations in FA supply from peripheral circulation.

Table 1.
Concentrations of fatty acids in plasma, GCs, and COCs expressed as mean percentages (w/w) of total fatty acids

This work was supported by Defra and The Perry Foundation.
117 CYTOPLASMIC FACTORS INFLUENCE DEVELOPMENTAL POTENTIAL OF SAMP1/Yit MOUSE EMBRYOS
Authors: Otsuka J. , Funabashi H. , Kono T.
Published: December 2004

Nuclear transplantation is an efficient means to investigate nucleo-cytoplasmic interactions of mammalian embryos during early development. A recent study has shown that the developmental potential of embryos is affected by the type of cytoplasm. The SAMP1/Yit mouse, an inbred strain that develops spontaneous chronic ileitis resembling Crohn's disease (Matsumoto S 1999 Bioscience Microflora 18, 1–9), has poor reproductive performance, and the developmental ability of embryos is low (unpublished data). Therefore we need to enhance productivity of the SAMP1/Yit mouse. Recently it was reported that cytoplasm of F1 mouse egg supported the development of embryos which have low developmental ability (Muggleton-Harris A et al. 1982 Nature 299, 460–462). In the present study, we examined the influences of the nucleus and cytoplasm on the development of reconstructed embryos in vitro and in vivo, using reciprocal nuclear transplantation between SAMP1/Yit and B6P1F1 (C57BL/6J × SAMP1/Yit) mouse embryos. We evaluated the developmental ability of reconstructed embryos by the development rate into blastocysts in vitro and by the rate of offspring after transfer of blastocysts to recipient mice. Pronuclear transplantation was carried out as reported previously (McGrath J and Solter D 1983 Science 220, 1300–1302). Briefly, karyoplasts from one-cell SAMP1/Yit embryos were introduced into enucleated B6P1F1 zygotes (SAMP1/B6P1F1) and fused by addition of inactivated HVJ (2700 U L−1). The other group of reconstructed embryos (B6P1F1/SAMP1) was manipulated similarly. After fusion, reconstructed embryos were cultured in drops of KSOM medium for 120 h at 37°C in 5% CO2 in humidified air. Some reconstructed and control (unmanipulated) embryos that developed to the blastocyst stage were transferred to the uteri of recipient mice. Data were compared using chi-square test; differences were considered significant at P < 0.01. The development rate of [SAMP1/B6P1F1] embryos to the blastocyst stage was significantly (P < 0.01) higher (75.0%) than that of SAMP1/Yit controls (39.1%). The rate of offspring in [SAMP1/B6P1F1] was also significantly (P < 0.01) higher (47.5%) than that of SAMP1/Yit controls (22.1%). On the other hand, [B6P1F1/SAMP1] embryos showed low developmental potential compared to B6P1F1 control embryos. These results indicate that the source of the cytoplasm strongly influences the development of reconstructed embryos containing SAMP1/Yit karyoplasts.

Table 1.
125 CHANGES OF PLASMA MACROPHAGE COLONY-STIMULATING FACTOR LEVELS AND ITS GENE EXPRESSION IN PERIPHERAL WHITE BLOOD CELLS DURING PREGNANCY IN JAPANESE BLACK CATTLE
Authors: Oshima K. , Yoshihara K. , Watanabe H. , Kojima T. , Komatsu M. , Yamamoto N.
Published: December 2004

Macrophage colony-stimulating factor (M-CSF) is a hemopoietic cytokine that plays a primary role in placental physiology. Gene expression of M-CSF in bovine intercaruncular endometrium shows an upward trend in mid-pregnancy. The objective of this study was to determine the plasma M-CSF levels and the M-CSF gene expression levels in maternal peripheral white blood cells (PWBCs) during pregnancy using ELISA and quantitative RT-PCR. In Experiment 1, the plasma M-CSF levels in 112 Japanese Black heifers or cows were determined. Animals were divided into four groups according to pregnancy stage: first- (n = 29), second- (n = 33), third- (n = 26) trimester, and non-pregnant (n = 24). ELISA for bovine M-CSF established by Yoshihara et al. (2003 Vet. Immunol. Immunopathol. 95, 103–111) was used according to their instructions. The absorbance was measured at 405 nm in the Biomek Plate Reader (Beckman Coulter, Fullerton, CA, USA). In Experiment 2, the plasma M-CSF levels and M-CSF gene expression levels in PWBCs during pregnancy were determined. The plasma samples for ELISA were obtained from 8 heifers and 3 cows every 1 and/or 2 weeks. The PWBCs samples for quantitative RT-PCR were obtained from 4 heifers every 1 and/or 4 weeks. All quantitative RT-PCR protocols were carried out according to the previous report (Oshima et al. 2003 Theriogenology 60, 1217–1226). The quantitative PCR assay used an ABI Prism 7700 Sequence Detector (Applied Biosystems, Foster City, CA, USA). Signals were detected according to the manufacturer's instructions. The relative level of M-CSF expression was calculated on the basis of glyceraldehyde-phosphate-dehydrogenase (GAPDH) quantity (in the method of calculation, the relative level = M-CSF quantity/GAPDH quantity). Data were analyzed by Kruskal-Wallis test. In Experiment 1, the plasma M-CSF level in second-trimester cows was significantly higher than those in other stages (P < 0.05). In Experiment 2, the plasma M-CSF levels were significantly higher in gestational age from −4 to 1 weeks compared with the last stage of pregnancy (P < 0.05). The levels decreased until 6 weeks, appeared to temporarily increase, and were relatively constant until 35 weeks. Macrophage colony-stimulating factor genes were expressed in all samples examined; the levels were relatively constant in early pregnancy, and then were widely varied until parturition. These results suggest that plasma M-CSF levels may be related to the maternal condition of pregnancy and to a slight extent to M-CSF gene expression in PWBCs.
This work was supported in part by a Grant-in-aid from the Recombinant Cytokine Project (RCP2002-2110), provided by the Ministry of Agriculture, Forestry, and Fisheries, Japan.
143 IN VITRO DEVELOPMENT OF OVINE OOCYTES FROM EWES OF CONTRASTING VITAMIN B12 STATUS
Authors: Mitchell L.M. , McCallum G. , Mackie K. , Ewen M. , Ainslie A. , McEvoy T.G.
Published: December 2004

Suboptimal circulating concentrations of vitamin B12 are commonly found in cattle and sheep grazing cobalt-deficient pastures. Vitamin B12 is a co-factor for enzymes involved in energy metabolism (methylmalonyl CoA mutase) and DNA synthesis/methylation (methionine synthase), and vitamin B12 status may therefore impact on cell division and gene expression in early embryos. The aim of this study was to determine the effect of vitamin B12 status on the in vitro development of ovine oocytes to the blastocyst stage. Mature Scottish Blackface ewes from cobalt-deficient farms were housed for ∼ four months and fed a cobalt-deficient diet (0.06 mg cobalt kg DM−1). At housing, 55 of the ewes were given an intra-ruminal slow-release cobalt bolus to compensate for the dietary deficit, and 52 remained untreated. The ovaries of all ewes were recovered at slaughter within the natural breeding season. Oocytes were aspirated and those with evenly granulated cytoplasm and >3 layers of cumulus cells were pooled according to ewe cobalt treatment, matured, fertilized, and cultured in vitro (∼20 oocytes per 50-μL drop under mineral oil). Oocytes were matured for 24 h in M199 + 10% fetal calf serum at 38.5°C in a humidified atmosphere of 5% CO2 in air prior to co-incubation for 18 h with frozen-thawed semen from a single ejaculate (1 × 106 live sperm mL−1). Presumptive zygotes were cultured for 7 Days in synthetic oviduct fluid + 0.4% fatty acid-free BSA (5% CO2, 5% O2, 90% N). Blastocysts formed at the end of the culture period were fixed and stained (Hoechst 33258) to count cell numbers. Data were analyzed by ANOVA and chi-square. For cobalt-supplemented and non-supplemented ewes, circulating concentrations of vitamin B12 at the time of slaughter were 1244 ± 52.5 and 372 ± 27.9 pmol L−1 (P < 0.001), respectively. Numbers of small (<5 mm) follicles per ewe were 17.6 ± 1.22 and 17.1 ± 1.31, and large (>5 mm) follicles per ewe were 1.8 ± 0.16 and 1.6 ± 0.18, respectively (NS). Cobalt-supplemented ewes yielded a lower proportion of matured oocytes that cleaved but an increased proportion of cleaved oocytes that formed blastocysts by Day 6 of the culture period (Table 1). The proportion of grade 1 and 2 blastocysts was also increased but cobalt treatment did not affect blastocyst cell numbers. In conclusion, results suggest that cleaved eggs derived from ewes of adequate, compared to suboptimal vitamin B12 status have improved developmental competence in vitro.

Table 1.
Effect of cobalt/vitamin B12 status on the in vitro development of ovine oocytes

This work was funded by the Scottish Executive Environment and Rural Affairs Department.
146 THE EFFECT OF CULTURE TEMPERATURE ON THE CLEAVAGE, DEVELOPMENT, AND GENE TRANSCRIPTION PATTERNS OF BOVINE EMBRYOS
Authors: Palasz A.T. , Beltrán Breña P. , Ramírez M.A. , Pérez-Garnelo S. , Gutiérrez-Adán A. , De la Fuente J.
Published: December 2004

Bovine oocytes matured and fertilized at 39°C had significantly higher rates of fertilization than at 37°C (Lenz et al. 1983 Biol. Reprod. 29, 173–179). However, embryo culture temperature that may affect molecular composition and metabolism of embryo membranes was not investigated. The objective of this study was to determine the effect of two culture temperatures, 37 and 39°C on the cleavage, in vitro development, and gene transcription patterns in bovine embryos. A total of 794 oocytes (5 replicates) were matured in TCM-199 medium containing 10% FCS and 10 ng/mL epidermal growth factor and inseminated with 1 × 106/mL spermatozoa in groups of 50 in 250 μL in Fert-Talp supplemented with 25 mM bicarbonate, 22 mM sodium lactate, 1 mM sodium pyruvate, 6 mg/mL BSA-FAF, and 10 μg/mL heparin at 39°C. After 24 h of oocyte-sperm co-incubation, presumptive zygotes were divided into two groups and cultured at 37 or 39°C under paraffin oil and 5% CO2 in humidified air. Embryos were cultured in SOFaa medium supplemented with 4% BSA in 50-μL drops (25 zygotes per drop). Cleavage rates were recorded on Day 2 and zygote development on Days 4, 7, 8, and 9. At least 5 blastocysts from each replicate from each treatment were frozen for evaluation of gene expression patterns. Poly(A) mRNA was prepared from 6 groups of pools of 5–7 embryos. A total of 11 developmentally important mRNA transcripts were examined. The quantification of all gene transcripts was performed by real time quantitative RT-PCR in three replicates. Embryo development was analyzed by chi-square analysis and data on mRNA expression by one-way repeated-measures ANOVA. There was no effect of culture temperature on embryo cleavage (65.2 and 72.0% for 37 or 39°C, respectively) but at Day 4 significantly (P < 0.05) more embryos developed to <8 cells at 39°C (32.5%) than at 37°C (25.9%). There was no difference in the total number of blastocysts produced in either temperature (22.4% at 37°C and 22.1% at 39°C), and significantly (P < 0.05) more zygotes that were at <8-cell stage at Day 4 progressed to the blastocyst stage at 37°C (86.4%) than at 39°C (66.6%). Transcript levels for genes related to response to stress (SOX, IFNτ) and glucose metabolism (Glut-1, G6pd) were higher (P < 0.05) in the blastocysts cultured at 39°C, and levels of Glut-5 (glucose metabolism) and Oct-4 (factor related to pluripotency) were higher (P < 0.05) in the blastocysts cultured at 37°C. No difference was found in mRNA transcription of genes related to apoptosis (BAX), response to heat (Hsp70), compaction (Ecad, Na/K, DcII), and cell connection (Cx43) at either temperature tested. It can be concluded that culture temperature may affect embryo molecular composition and the kinetics of embryo development.
174 ISOLATION, CULTURE AND POTENTIAL USE OF THE PORCINE NEURAL AND EPIDERMAL STEM CELLS
Authors: Motlik J. , Vodicka P. , Klima J. , Smetana K. , Liu F. , Gabius H.
Published: December 2004

The mammalian brain and epidermis contain stem cells, so-called neural stem cells (NSC) and epidermal stem cells (EpSC). To achieve the full therapeutic potential of stem cells, appropriate animal models have to be used to establish the sequence of pathological changes and to test potential therapies to block these changes. In the following studies miniature pigs were used as a biomedical model. We isolated multipotent cells from brains of porcine fetuses for future use in allotransplantation experiments in the inbred miniature pig strain. Brain tissue from 40- and 80-day-old porcine fetuses was mechanically dissociated, and cells were cultured in serum-free F12/DMEM medium with B27 and N2 supplements, EGF and bFGF. In 3–5 days some cells divided and formed floating spheres that were dissociated to single cell suspension and formed secondary spheres in culture. At all time points tested, the spheres represented mixtures of undifferentiated cells stained with nestin and Ki-67 antibodies and already differentiated neurons (Tu-20, MAP2) and glia (GFAP). After being plated on laminin/fibronectin coated coverslips and cultured in medium containing 2% FBS or 1 μM retinoic acid, the spheres adhered to the surface, and flattened, and cells started to migrate out. After immunofluorescence staining with antibodies to neuronal markers Tu-20 and MAP2, glial marker GFAP and oligodendrocyte marker CNPase showed that all the three cell types were present among differentiated cells. The EpSC are characterized by a slow and unlimited proliferation rate and, therefore, they retain labelled precursors of DNA more extensively than other keratinocytes. The main pool of EpSC is located in the bulge region of the hair follicle root sheath. A new procedure to isolate porcine hair follicles including their root sheaths was developed. The keratinocytes that migrated from hair follicles in the presence of feeder cells were poorly differentiated and specifically expressed galectin-1 or galectin-1-binding sites in their nuclei in co-localization with ΔNp63α. The exclusion of feeder cells from experimental system induced formation of spheroid bodies from these keratinocytes. Approximately one-third of these spheroids were able to adhere to a surface precolonized with feeder cells and to start forming normally growing colonies. Porcine hair follicles represent an excellent model for study of the functional phenotype of hair follicle-originated keratinocytes, and the endogenous lectin Gal-1 seems to be a potential marker of the porcine stem cell compartment of the hair follicle under in vitro conditions.
176 IMPROVED GROWTH OF HUMAN EMBRYONIC STEM CELLS IN A REDUCED OXYGEN ATMOSPHERE
Authors: Peura T. , Bosman A. , Stojanov T.
Published: December 2004

Traditional cell culture conditions entail the use of gas atmosphere consisting of 5–6% CO2 in air. These same conditions have also been used universally for the culture of human embryonic stem cells (hES), despite the natural milieu of the embryos, from which these cells are derived, being slightly hypoxic. The aim of this work was to examine if human embryonic stem cells would benefit from the reduced oxygen culture environment, as used for human pre-implantation embryos. In Experiment 1, the relative growth in high and low oxygen atmosphere was compared by cutting undifferentiared hES-colonies into equal-size pieces and transferring them to two dishes, one into the conventional incubator in 6% CO2 in air (HIGH), the other into K-MINC-1000 (Eight Mile Plains, Queensland) mini-incubator in 5% O2, 6% CO2 and 89% N2 (LOW). After 8 days the colony sizes and differentiation status were measured. In Experiment 2, the absolute growth in high and low oxygen atmosphere was compared by cutting exactly same size fragments from undifferentiated hES-colonies and distributing them to two treatments as described. All colonies were measured and passaged at Day 7 and final measurements taken at Day 14. In Experiment 1, improved growth was observed in reduced oxygen, mean percentage of undifferentiated growth from original colony (assigned as 100%) being 217% for HIGH and 482% for LOW over three replicates. In Experiment 2, the total of 16.9 mm2 of undifferentiated colonies in both treatment groups (24 colonies per group over 3 replicates) had by Day 7 grown to total of 51.1 and 79.5 mm2 (P < 0.001), and by Day 14 to 216.8 and 373.3 mm2 (P < 0.0001) in HIGH and LOW, respectively (see Table 1). In neither experiment were there differences in the differentiation status of the colonies between the treatments (mean 6% and 5% at Day 7, and 9% and 9% at Day 14 in HIGH and LOW, respectively). We conclude that culture in reduced oxygen improves growth of human embryonic stem cells.

Table 1.
Growth of hES colonies on high and low oxygen atmosphere (data from 3 replicates)

We like to acknowledge Bresagen Ltd. for the supply of hesBGN-01 cell line.
19 LOW-DOSE DEEP INTRAUTERINE INSEMINATION IN SOWS UNDER CONDITIONS: INCIDENCE OF UNILATERAL FERTILIZATIONS
Authors: Martinez E.A. , Vazquez J.M. , Parrilla I. , Cuello C. , Gil M.A. , Tornel J.A. , Roca J. , Vazquez J.L.
Published: December 2004

A new procedure for nonsurgical deep intrauterine insemination (DUI) in non-sedated sows has recently been reported (Martinez et al. 2002 Reproduction 123, 163–170). In comparison to traditional artificial insemination (AI), using this procedure, a 20-fold reduction in the number of spermatozoa inseminated can be used without a decrease in fertility when hormonally treated post-weaning estrous sows are used. The aim of the present study was to evaluate the effectiveness of DUI under field conditions. In Experiment 1, crossbred sows (2–6 parity) were weaned at 20.75 ± 0.06 days. Estrous detection was performed once per day, beginning 3 days after weaning. Sows with a weaning to estrus interval of 4–5 days were selected to be inseminated. A total of 190 sows were inseminated at 12, 24, and 36 h after onset of estrus using one of the following two regimes: (1) DUI with 150 × 106 fresh spermatozoa in 5 mL of BTS (n = 95) and (2) Traditional AI with 3 × 109 fresh spermatozoa in 100 mL of BTS (n = 95) prepared from the same semen samples used for the DUI group. Farrowing rates (FR) and litter sizes (LTS; mean ± SEM) from both groups were compared using chi-squared test and ANOVA, respectively. There was no significant difference in the FR between groups (83.2 and 86.3% for DUI and AI groups, respectively). However, a decrease (P < 0.001) in the LTS was observed in sows inseminated by the DUI procedure (9.8 ± 0.29 and 10.9 ± 0.17, respectively). In Experiment 2, seventy one natural post-weaning estrus sows were used. Fifty-five sows were DUI inseminated three times with 150 (n = 17), 300 (n = 19), or 600 (n = 19) × 106 spermatozoa in 5, 10, or 20 mL of BTS, respectively. The remaining sows (n = 16) were traditionally inseminated. On Day 6 after estrus, sows were subjected to laparotomy and the tips of both uterine horns were flushed in order to evaluate pregnancy rate (PR: percentage of sows with at least 4 viable embryos) and fertilization rate (ratio of viable embryos to the total number of embryos and oocytes). PR was similar in all the groups, ranging from 84.2% (DUI 300 × 106 spermatozoa group) to 94.7% (DUI 600 × 106 spermatozoa group). Fertilization rate and the percentage of bilateral fertilization after DUI with 600 × 106 spermatozoa did not differ from those of the AI group (97.8 and 100% vs. 98.4 and 100%, respectively), but a significant decrease in both parameters (P < 0.05; chi-square test) was observed in sows inseminated with 300 (94.3 and 87.5%) or 150 (84.4 and 66.7%) × 106 spermatozoa. In conclusion, DUI with 150 × 106 spermatozoa offers similar FR but a lower LTS in sows with natural estrus in comparison with those parameters obtained when traditional AI is used. The lower litter size could be related to the low percentage of bilateral fertilization observed in that group.
This work was supported by CDTI 020003.
195 CHANGES OF BLESBOK AND BLUE WILDEBEEST EPIDIDYMAL SPERM AFTER INCUBATION AT 37°C
Authors: Martinez-Pastor F. , Olivier F. , Spies T. , Anel L. , Bartels P.
Published: December 2004

Postmortem recovery of epididymal spermatozoa and their preservation in Biological Resource Banks is a convenient source of germplasm, providing a possible future conservation resource for selected endangered wildlife species. It is necessary to gain knowledge of the biology of the gametes of the different species, in order to define effective protocols for cryopreservation and future assisted reproductive technology application. A pilot study on the changes in blue wildebeest (Connochaetes taurinus) and blesbok (Damaliscus dorcas phillipsi) epididymal sperm was carried out in order to provide some insight into the effects of incubation at 37°C. Chemicals were aquired from Sigma (South Africa), except JC-1 (Molecular Probes, Leiden, The Netherlands). Sperm was obtained by flushing the vas deferens and cauda epididymis of 6 adult blue wildebeests and 4 adult blesbok after the breeding season using 1 mL of Biladyl (fraction A; Minitüb, Tiefenbach, Germany). Cells were washed and resuspended in buffered medium (20 mM HEPES, 197 mM NaCl, 10 mM glucose, 2.5 mM KOH). Part of each sample was analyzed and part was incubated for 1 h at 37°C, and then analyzed. Analysis consisted of: motility (% of motile sperm, TM; and % of linear sperm, LM), vitality (fluorescent dye propidium iodide, 7 μM; % of unstained cells noted after 10 min at RT: vital, VIT), mitochondrial status (fluorescent dye JC-1, 7.5 μM; % of cells with orange midpiece noted after 30 min at 37°C: active mitochondria, MIT), and induction of acrosome reaction (15 min at 37°C in buffered medium complemented with 3 mM CaCl; % of intact acrosomes noted in control: splits no ionophore, ACR, and test: splits 1 μM calcimycin, ION). Samples were assessed using phase contrast microscopy (×400; ×200 for motility). Results are showed in Table 1. No significant differences (Wilcoxon Rank Sign test) were detected, possibly due to the low number of samples. However, LM appeared to decrease after incubation. Incubaton may increase the sensitivity of blue wildebeest sperm to ionophore (ION). Motility was least for blesbok, and the decrease of LM after incubation was more apparent. This treatment may induce different physiologycal changes between the species (different LM variation). The rest of the parameters suggest that the treatment did not induce extensive cell damage. Further research must be carried out to confirm these findings.

Table 1.
Median values for the analyzed parameters

Sponsors of this project include Vodacom, Joan St. Leger Lindburgh Charitable Trust, Tony and Lizette Lewis Foundation, Department Science and Technology (South Africa), British Airways, IMV Technologies/CBS (France), NECSA, Zeiss Microscopes, AEC-Amersham, CryoLogic (Australia), Cook Veterinary (Australia), Mazda Wildlife Fund, The Scientific Group, Genaust (Australia), and SCI – Chesapeake Chapter.
232 SPECIES-RELATED DIFFERENCES IN BLASTOCYST QUALITY ARE ASSOCIATED WITH DIFFERENCES IN RELATIVE mRNA EXPRESSION
Authors: Rizos D. , Gutierrez-Adan A. , Moreira P. , O'Meara C. , Fair T. , Evans A.C.O. , Boland M.P. , Lonergan P.
Published: December 2004

We have previously reported a species-related qualitative difference (in terms of ultrastructural morphology and cryotolerance) between bovine and ovine blastocysts produced under identical conditions of in vitro culture in synthetic oviduct fluid (SOF1, Rizos et al. 2002 Mol. Reprod. Dev. 62, 320–327). The overall objective of this study was to see if these differences were reflected at the transcript level. From each of five IVF replicates, groups of 10 bovine and 10 ovine blastocysts were used. The objective of Experiment 1 was to compare the relative transcript abundance of eight candidate genes between ovine and bovine blastocysts cultured in SOF1. Following real-time quantitative RT-PCR, transcript levels for MnSOD, survivin, and Glut-5 were significantly higher in ovine than in bovine blastocysts (ANOVA, P < 0.05), while transcripts for Cx31, IFN-tau and SOX were significantly more abundant in bovine blastocysts (P < 0.01). For the two remaining transcripts, E-cad and Na/K, there was no difference. The objective of Experiment 2 was to examine the possibility of modifying the pattern of expression in both types of blastocysts by changing the culture medium. Culture took place in SOF1 or SOF2 (Holm et al. 1999 Theriogenology 52, 683–700). Culture of bovine embryos in SOF2 resulted in a significant increase in the level of expression of MnSOD and Glut-5 (P < 0.05) compared to culture in SOF1. For all the other transcripts except survivin, there was a significant decrease in the relative abundance. Culture of ovine embryos in either SOF1 or SOF2 did not have a major influence on transcript abundance; of the eight transcripts examined, the relative abundance of only one, SOX, was significantly altered. Based on the above, the objective of Experiment 3 (3 replicates) was to determine whether the changed pattern of expression in bovine blastocysts produced in SOF2 was associated with an improvement in cryotolerance. Bovine blastocysts produced in both culture media were vitrified and warmed, and survival was assessed by re-expansion and hatching. Blastocysts produced in SOF2 had significantly higher survival rates at 24, 48, and 72 h and significantly higher hatching rates following vitrification and warming than those produced in SOF1 (P < 0.001). In conclusion, we have demonstrated that the apparent differences between ovine and bovine embryos in their adaptability to culture conditions, manifested in differences in embryo morphology and cryotolerance, are related to differences in mRNA relative abundance.
This work was supported by Science Foundation Ireland under Grant No. 02/IN1/B78 and by Ministerio de Ciencia y Tecnologia from Spain under Grant No. AGL2003-05783.