bluuebunny/crossref_metadata_2025 huggingface dataset:
Polyethyleneimine‐Mediated Chemical Extraction of Cytoplasmic His‐Tagged Inclusion Body Proteins from Escherichia coli
Authors: Nian Rui , Tan Lihan , Choe Woo‐Seok
Published: January 2008

The selectivity of polyethyleneimine (PEI) in DNA precipitation during chemical extraction was investigated. Chemical extraction was used to recover two His‐tagged model proteins: gloshedobin, a thrombin‐like enzyme from snake venom, and IbpA, a molecular chaperone, which were expressed mainly in the form of inclusion bodies. High DNA removal efficiency (more than 90%) was achieved at various cell densities (with OD600 ranging from 30 to 150) without affecting the solubility of host cell proteins. Compared to spermine‐induced precipitation method reported elsewhere, PEI provided a higher DNA precipitation efficiency at a significantly lower cost. Moreover, PEI obviated the use of EDTA, which has been reported to be essential for the chemical extraction methods, hence exhibiting dual roles in replacing cost‐prohibitive spermine and EDTA. The residual PEI in the post‐extraction mixture was efficiently counteracted by addition of Mg2+, allowing the streamlined application of the extraction broth to immobilized metal affinity chromatography. Taken together, the PEI‐mediated chemical extraction method provides a simpler and more economically viable processing route for the production of recombinant proteins whose expression is hampered by IB formation.
Two‐dimensional observations of overshielding during a magnetic storm by the Super Dual Auroral Radar Network (SuperDARN) Hokkaido radar
Authors: Ebihara Y. , Nishitani N. , Kikuchi T. , Ogawa T. , Hosokawa K. , Fok M.‐C.
Published: January 2008

Two‐dimensional observations of ionospheric plasma flows possibly caused by overshielding are reported for the first time. The observations were made by the midlatitude Super Dual Auroral Radar Network Hokkaido radar in Japan during a major magnetic storm on 15 December 2006. The magnetosphere was exposed continuously to a southward interplanetary magnetic field (IMF) for several hours during the main phase of the storm. Immediately following the subsequent northward turning of the IMF, an antisunward plasma flow was observed for about 14 min in the predusk sector at magnetic latitudes of 50°–60°, reaching a maximum line‐of‐sight speed of 70–80 m/s. These features are consistent with a simulation of coupling between the ring current and the ionosphere associated with an overshielding condition. Within 1 h of the first observation, a similar antisunward flow was observed during a period of southward oriented IMF. However, the simulation cannot account for the antisunward flow in this case. It is suggested that the shielding/overshielding condition is not simply caused by the northward turning of IMF. This second overshielding‐like condition is attributable to a sudden contraction of the polar cap associated with the substorm or to a sudden strengthening of the inertial current converted from the abrupt injection of magnetospheric ions. However, neither fully accounts for the observations.
Observations and simulations of the ionospheric and thermospheric response to the December 2006 geomagnetic storm: Initial phase
Authors: Lei Jiuhou , Wang Wenbin , Burns Alan G. , Solomon Stanley C. , Richmond Arthur D. , Wiltberger Mike , Goncharenko Larisa P. , Coster Anthea , Reinisch Bodo W.
Published: January 2008

We have investigated the thermospheric and ionospheric response to the 14–15 December 2006 geomagnetic storm using a Coupled Magnetosphere Ionosphere Thermosphere (CMIT) 2.0 model simulation. In this paper we focus on observations and simulations during the initial phase of the storm (about 8 h), when the shock was driving changes in geospace. The global ionospheric maps of total electron content (TEC), ionosonde data at four stations and Millstone Hill incoherent scatter radar (ISR) observations are compared with the corresponding simulation results from the CMIT model. The observations showed significant positive storm effects occurred in the Atlantic sector after the onset of this storm. The CMIT model is able to capture the temporal and spatial variations of the ionospheric storm effects seen in the GPS TEC observations, although the model slightly underestimates the daytime positive ionospheric storm in the South American sector. The simulations are also in agreement with the ionosonde and ISR ionospheric measurements. Term analysis of the ion continuity equation demonstrates that changes in the electric fields play a dominant role in generating the observed ionospheric positive storm effect in the American sector during the initial phase, although neutral winds and composition changes also contribute. The difference in the strength of the enhancements over North and South America can be explained by the slope of the topside electron density profiles in the two hemispheres. In the southern hemisphere electron densities decrease slowly with altitude, whereas the decrease is much more rapid in the northern (winter) hemisphere. The electric fields, therefore, cannot cause large increases in electron density by uplifting the plasma, so positive storm effects are small in the southern hemisphere compared with the northern hemisphere, even though the increase in hmF2 is greater in the southern hemisphere. Nighttime changes in electron density in other longitude sectors are small, because the topside electron densities also decrease slowly with altitude at night.
Onset of water stress, hysteresis in plant conductance, and hydraulic lift: Scaling soil water dynamics from millimeters to meters
Authors: Siqueira Mario , Katul Gabriel , Porporato Amilcare
Published: January 2008

Estimation of water uptake by plants and subsequent water stress are complicated by the need to resolve the soil‐plant hydrodynamics at scales ranging from millimeters to meters. Using a simplified homogenization technique, the three‐dimensional (3‐D) soil water movement dynamics can be reduced to solving two 1‐D coupled Richards' equations, one for the radial water movement toward rootlets (mesoscale, important for diurnal cycle) and a second for vertical water motion (macroscale, relevant to interstorm timescales). This approach allows explicit simulation of known features of root uptake such as diurnal hysteresis in canopy conductance, hydraulic lift, and compensatory root water uptake during extended drying cycles. A simple scaling analysis suggests that the effectiveness of the hydraulic lift is mainly controlled by the root vertical distribution, while the soil moisture levels at which hydraulic lift is most effective is dictated by soil hydraulic properties and surrogates for atmospheric water vapor demand.
Insights learned from pBTAi1, a 229-kb accessory plasmid from Bradyrhizobium sp. strain BTAi1 and prevalence of accessory plasmids in other Bradyrhizobium sp. strains
Authors: Cytryn Eddie J , Jitacksorn Siriluck , Giraud Eric , Sadowsky Michael J
Published: January 2008

In silico, physiological and in planta analyses were used to characterize pBTAi1, a 229-kb accessory plasmid from Bradyrhizobium sp. strain BTAi1, and assess its potential ecological function under free-living and symbiotic growth conditions. Sequence analysis revealed the presence of an uptake hydrogenase system, a repABC family plasmid replication module and open reading frames encoding type IV secretion system, TraI and TraR autoinducer proteins and several copper resistance-related proteins. Bradyrhizobium sp. BTAi1 was capable of growing in 200 mg l−1 CuCl2. In contrast, the closely related, plasmid-free Bradyrhizobium sp. strain ORS278 could not grow at copper concentrations exceeding 100 mg l−1. The plasmid-localized hydrogenase genes were phylogenetically distinct from those typically found in other rhizobial species, and were most related to hup genes from Thiobacillus denitrificans. The induction of the plasmid-borne hydrogenase genes during symbiosis was significantly lower than the two chromosomal-borne hydrogenase clusters. CHEF-pulsed-field gel electrophoresis was used for a comprehensive analysis of the diversity, abundance and genetic composition of accessory plasmids in other Bradyrhizobium strains. Plasmids were detected in 11 of 46 (23.9%) geographically diverse Bradyrhizobium japonicum and Bradyrhizobium elkanii strains, isolated from the United States, China and Thailand. Plasmid size was heterogeneous, ranging from 75 to 330 kb, with only two strains (DASA01244 and DASA01265) harboring plasmids with identical (240 kb) size. None of the plasmids harbored nodulation or hydrogenase genes. Taken together, our results indicate that while plasmids having ecologically significant functions may be detected in Bradyrhizobium sp. strains, they lack genes necessary for symbioses with legumes.
Arsenite-dependent photoautotrophy by an Ectothiorhodospira-dominated consortium
Authors: Budinoff Charles R , Hollibaugh James T
Published: January 2008

Over the past decade numerous lineages of bacteria have been shown to obtain energy for growth through redox transformations of arsenic. However, phototrophic growth using reduced arsenic as an electron donor has not been described. Here we report the light-dependent oxidation of arsenite to arsenate, coupled with autotrophic growth, by an Ectothiorhodospira-dominated consortium of bacteria from alkaline, hypersaline Mono Lake, California. Pure cultures of the Mono Lake Ectothiorhodospira were not capable of phototrophic arsenite oxidation under the culture conditions tested. Electron micrographs of the culture showed a close association between consortia members, although the specific contribution of the individual bacteria is currently unknown. This report extends the list of compounds known to support anoxygenic photosynthesis and documents a previously unknown pathway in arsenic geochemistry.
3D FISH for the quantification of methane- and sulphur-oxidizing endosymbionts in bacteriocytes of the hydrothermal vent musselBathymodiolus azoricus
Authors: Halary Sébastien , Riou Virginie , Gaill Françoise , Boudier Thomas , Duperron Sébastien
Published: January 2008

Dual endosymbioses involving methane- and sulphur-oxidizing bacteria occur in the gills of several species of mussels from deep-sea hydrothermal vents and cold seeps. Variations of total and relative abundances of symbionts depending on local environmental parameters are not yet understood, due to a lack of reliable quantification of bacteria in the host tissue. Here, we report the first attempt to quantify volumes occupied by each type of symbiont in bacteriocyte sections from a vent mussel, Bathymodiolus azoricus, using fluorescence in situ hybridization (FISH) coupled to three dimentional microscopy and image analysis carried out by a dedicated software, which we developped. Bacteriocytes from mussels recovered at different vent sites displayed significantly different abundances of bacteria. Specimens kept in aquaria at atmospheric pressure and exposed to an artificial pulse of sulphur displayed an increase in absolute and relative abundance of sulphur oxidizers within their bacteriocytes. Distributions of all measured parameters fitted normal distributions, indicating that bacteriocytes from a specimen tend to display similar behaviours. This study shows that symbiont volume quantification is tractable using 3D FISH, and confirms the impact of local environmental parameters on symbiont abundances.
The electron photodetachment spectrum of c-C4F8−: A test case for the computation of Franck-Condon factors of highly flexible molecules
Authors: Borrelli Raffaele , Peluso Andrea
Published: January 2008

The long (∼20000cm−1) vibrational progression observed in the electron photodetachment spectrum of c-C4F8− anion is analyzed in terms of the Franck-Condon factors between the ground electronic states of the anionic and neutral species. The observed spectrum is correctly reproduced and its vibronic structure is assigned in detail. The very simple structure of the spectrum, consisting of a sequence of evenly spaced peaks, at 355cm−1 each other, is assigned to a series of overlapping progressions associated with vibrational excitations of two totally symmetric modes rather than to a single mode progression, as originally proposed. The underlying continuum observed in the experimental spectrum is traced back to the excitations of a low frequency ring-puckering vibration, which, in the neutral species, exhibits a double minimum potential energy profile.
Delay-induced inward and outward spiral waves in oscillatory medium
Authors: Hu Hai Xiang , Ji Lin , Li Qian Shu
Published: January 2008

Inward and outward spiral waves as well as inward target waves are induced by local delay feedback in a reaction-diffusion system exhibiting a Turing hexagon pattern spontaneously. The system gives rise to large-amplitude sinusoidal oscillations when the inward spiral waves are observed. Compared with the inward spirals, the outward spiral waves usually possess longer wavelength and exhibit larger amplitude relaxation oscillations. Varying the feedback parameters continuously, the inward spiral waves are transformed into outward spirals through a turbulent state. The dispersion analysis about the delayed system reveals that there is an unstable band on the dispersion curve, where the turbulent state arises. The dispersion curve is divided into two parts by the unstable band. The inward spiral waves exist in the left part with negative group velocity, i.e., dω∕dk<0, while the outward spiral waves are stable in the right part with dω∕dk>0 (ω is frequency and k wavenumber).
On the mapping of time-dependent densities onto potentials in quantum mechanics
Authors: Baer Roi
Published: January 2008

The mapping of time-dependent densities on potentials in systems of identical quantum mechanical particles is examined. This mapping is of significance ever since Runge and Gross [Phys. Rev. Lett. 52, 997 (1984)] established its uniqueness, forming the theoretical basis for time-dependent density functional theory. Beyond uniqueness there are two important issues: existence, often called v-representability, and stability. We show that v-representability for localized densities in turn-on situations is not assured and we give a simple example of nonexistence. As for stability, we discuss an inversion procedure and by computing its Lyapunov exponents we demonstrate that the mapping is unstable with respect to fluctuations in the initial state. We argue that such instabilities will plague any inversion procedure.
Synthesis and magnetic characterizations of manganite-based composite nanoparticles for biomedical applications
Authors: Pradhan A. K. , Bah R. , Konda R. B. , Mundle R. , Mustafa H. , Bamiduro O. , Rakhimov R. R. , Wei Xiaohui , Sellmyer D. J.
Published: January 2008

We report chemically synthesized highly crystalline lanthanum strontium manganite (LaSrMnO3) and Eu-doped Y2O3 and their composites. The synthesis yields nanoparticles of size 30–40nm. Magnetic measurements performed on nanoparticles and composites show magnetic transition at about 370K with a superparamagnetic behavior at room temperature. The ferromagnetic resonance studies of the nanoparticles show large linewidth due to surface strains. The composite nanoparticles also display luminescent behavior when irradiated with ultraviolet light. The manganites as well their composite with the luminescent nanoparticles may be very useful for biomedical applications.
Cavity effects on light extraction in organic light emitting devices
Authors: Lee Jaewon , Chopra Neetu , So Franky
Published: January 2008

We have demonstrated that the light extraction efficiency of an organic light emitting device is a strong function of device geometry. Specifically, we have found that the ratio of the extracted mode to the substrate-guided mode varies from 22% to 55% depending on the location of the recombination zone. Our simulation results also indicate that more light is trapped in the substrate as the optical length of device increases. We further show that the light intensity profile varies from a Lambertian shape to a non-Lambertian shape depending on the device geometry due to the cavity effect.
Magnetostrictive strains in polycrystalline FePdRh alloy
Authors: Jen S. U. , Chen Yuan-Tsung , Tsai T. L. , Lin Y. C.
Published: January 2008

The conventional magnetostrictive property was studied in the polycrystalline Fe–30Pd–4Rh alloy. The alloy was solution treated at 950°C for 1.5h and then water quenched. At room temperature, the alloy has a mixed adaptive (or intermediate) martensite structure with ao=3.784Å and co=3.514Å. At a lower temperature (e.g., T=150K), the full martensitic transformation is finally completed, and an expansion of the c axis with Δc∕co≃6.8% was observed. The thermal expansion coefficient, (1∕T)(Δa∕ao), is +3.84×10−6K−1 between 300 and 150K. From the electrical resistance (R) measurement between 300 and 4K, we confirm that the second, or final, martensitic transformation temperature (TIM) is approximately 170K. In addition, at room temperature, the saturation magnetization Ms(300)=655G, while at T=5K, Ms(5)=808G. Because an external field (H) may rotate Ms in the sample plane of Fe–Pd–Rh alloy, we find that, at T=300K, its saturation magnetostriction λs=62ppm, and at T=77K, λs=115ppm. There are at least two advantages on using the Fe–Pd–Rh alloy as the material for a magnetic-type microactuator or spring: (1) the alloy is ductile, and (2) at room temperature, the magnetostrictive susceptibility (Δλ)∥s∕(ΔH) is high, about 0.8ppm∕Oe at low field (H=50Oe).
Correlations between morphology, crystal structure, and magnetization of epitaxial cobalt-platinum films grown with pulsed laser ablation
Authors: Rakshit R. K. , Bose S. K. , Sharma R. , Budhani R. C. , Vijaykumar T. , Neena S. J. , Kulkarni G. U.
Published: January 2008

The effects of growth rate (Gr), deposition temperature (Td), film thickness (tF), and substrate-induced strain (ϵ) on morphological, crystallographic, and magnetic characteristics of equiatomic CoPt epitaxial films synthesized with pulsed laser deposition are investigated. The (001)-oriented single-crystal substrates of MgO, SrTiO3, and LaAlO3 provide different degrees of epitaxial strain for growth of the disordered face-centered cubic (fcc) and ordered face-centered tetragonal (L10) phases of CoPt. The films deposited at Td≈600 °C on all three substrates are fcc with in-plane magnetization and a narrow hysteresis loop of width ≈200 Oe. The L10 phase, stabilized only at Td≥700 °C, becomes predominantly c-axis oriented as Td is increased to 800 °C. While the crystallographic structure of the films depends solely on the Td, their microstructure and magnetization characteristics are decided by the growth rate. At the higher Gr(≈1 Å/s) the L10 films have a maze-like structure which converts to a continuous film as the tF is increased from 20 to 50 nm. The magnetic coercivity of these films increases as the L10 phase fraction grows with Td and its orientation becomes out of the film plane. The evolution of microstructure with Td is remarkably different at a lower growth rate (≈0.4 Å/s). Here, the structure changes from a self-similar fractal pattern to a disordered assembly of nanodots as the Td is raised from 700 to 800 °C, and is understood in terms of the imbalance between strain and interfacial energies. Magnetic force microscopy of such films reveals no distinct domain walls within the nanoislands, while a clear contrast is seen between the islands of reversed magnetization. Magnetic relaxation measurements on these assemblies of single-domain islands show a negligible decay of magnetization unless a reverse field close to the coercive field (Hc≈30 kOe) is applied. The simple picture of coherent rotation of moment appears incompatible with the time dependence of the remanent magnetization in these films.
Thermoelectric power in the double exchange model
Authors: Kim Beom Hyun , Yu Unjong , Kim Kyoo , Min B. I.
Published: January 2008

Employing the Monte Carlo method and the exact diagonalization, we have investigated the temperature dependence of the thermoelectric power (TEP) for the double exchange model in the dilute carrier concentration limit. We have found that the TEP follows the Heikes formula in the high temperature regime, whereas, in the intermediate temperature regime, the TEP is suppressed by the exchange coupling between itinerant electrons and local spins. In the low temperature regime, the TEP exhibits an anomalous peak and dip feature near the magnetic transition temperature TC which can be understood based on the magnetic polaron state. We have also found that the TEP, in the presence of the magnetic field, shows the positive magnetothermoelectric power near TC.
Enhanced optical nonlinearity of surfactant-capped CdS quantum dots embedded in an optically transparent polystyrene thin film
Authors: Gao Y. , Tonizzo A. , Walser A. , Potasek M. , Dorsinville R.
Published: January 2008

Nanocomposites of surfactant-capped CdS quantum dots embedded in polystyrene thin film have been prepared and characterized by z-scan measurements. Large values of the two- and three-photon absorption coefficients were observed at 532nm (252cm∕GW) and 1064nm (160cm3∕GW2). The cross sections of absorption were ∼1×10−44cm4s/photon (532nm) and 8×10−73cm6s2∕photon2 (1064nm). These results are among the highest values of nonlinear absorption reported and orders of magnitude larger than CdS quantum dots capped by inorganic materials or in other matrix material. The high multiphoton absorption suggests that this class of materials is extremely promising for optical applications.
Investigation of the vortex order-disorder phase transition line in Bi2Sr2CaCu2O8+x using ac techniques
Authors: Sochnikov I. , Shaulov A. , Yeshurun Y.
Published: January 2008

ac magnetic measurements were employed in the study of the vortex phase diagram in Bi2Sr2CaCu2O8+x. The Bragg-glass to vortex-glass (solid-solid) and the Bragg-glass to liquid (melting) transitions are manifested in these measurements by an increase or a decrease in the screening signal, respectively. The ac measured solid-solid transition line extends to high temperatures where a melting transition is observed in dc measurements. Increasing the frequency of the ac field can further enlarge this region of the solid-solid transition. These results confirm that the solid-solid and the solid-liquid transition lines are two segments of the same order-disorder phase transition line.
Synthesis and interfacial phenomena in ultra-thin yttria-doped zirconia films grown by alloy oxidation under photon irradiation
Authors: Tsuchiya Masaru , Ramanathan Shriram
Published: January 2008

We report on synthesis, structural evolution, and high temperature properties of ultrathin yttria-doped-zirconia (YDZ) films grown by alloy oxidation under ultraviolet (UV) irradiation. From high-resolution studies on interfacial layer growth kinetics, we found that oxygen diffusivity maximum in UV-YDZ film is located at higher yttria concentration than that in thermally oxidized films. It is likely that the oxygen-rich nature of UV-grown films enabled by photon irradiation is responsible for the observed differences. The results demonstrate routes for synthesis of oxide films with controlled structure and stoichiometry.
Acoustic-assisted magnetic-field-induced strain and stress output of Ni–Mn–Ga single crystal
Authors: Techapiesancharoenkij Ratchatee , Kostamo Jari , Simon Jesse , Bono David , Allen Samuel M. , O’Handley Robert C.
Published: January 2008

The application of acoustic energy input from a piezoelectric actuator is observed to increase the magnetic-field-induced strain (MFIS) and decrease the threshold field in Ni–Mn–Ga ferromagnetic shape memory alloys (FSMAs). To appreciate better the benefit of the acoustic assistance, the stress and strain outputs of the quasistatic FSMA actuation under acoustic assistance are studied systematically. With acoustic assistance, the maximum reversible strain increases from 3% to 4.5% and appears in a broader range of stress output between 0.4 and 1.2MPa. Elastic stress wave analysis explains quantitatively the effect of acoustic assistance on MFIS.
Simultaneous color and luminance control of organic light-emitting diodes for mood-lighting applications
Authors: Köhnen Anne , Meerholz Klaus , Hagemann Malte , Brinkmann Matthias , Sinzinger Stefan
Published: January 2008

Organic light-emitting diodes (OLEDs) using polymer blends as a single emissive layer often suffer from large color shifts with changing operational voltage. Until now, such devices cannot stand the critical demands of lighting industry. In this contribution, we introduce a pulse-width-modulation-based driver concept, enabling the user to simultaneously and independently adjust color and luminance of a single device with two contacts. This concept makes color-shifting OLEDs highly interesting for “mood-light” applications.
High-performance visible-blind GaN-based p-i-n photodetectors
Authors: Butun Bayram , Tut Turgut , Ulker Erkin , Yelboga Tolga , Ozbay Ekmel
Published: January 2008

We report high performance visible-blind GaN-based p-i-n photodetectors grown by metal-organic chemical vapor deposition on c-plane sapphire substrates. The dark current of the 200μm diameter devices was measured to be lower than 20pA for bias voltages up to 5V. The breakdown voltages were higher than 120V. The responsivity of the photodetectors was ∼0.23A∕W at 356nm under 5V bias. The ultraviolet-visible rejection ratio was 6.7×103 for wavelengths longer than 400nm.
Slow cooling prevents cold-induced damage to sperm motility and acrosomal integrity in the black-footed ferret (Mustela nigripes)
Authors: Santymire R. M. , Marinari P. E. , Kreeger J. S. , Wildt D. E. , Howard J. G.
Published: January 2008

The endangered black-footed ferret (Mustela nigripes) has benefited from artificial insemination; however, improved sperm cryopreservation protocols are still needed. The present study focused on identifying factors influencing gamete survival during processing before cryopreservation, including: (1) the presence or absence of seminal plasma; (2) temperature (25°C v. 37°C); (3) type of medium (Ham’s F10 medium v. TEST yolk buffer [TYB]); (4) cooling rate (slow, rapid and ultra-rapid); and (5) the presence or absence of glycerol. Seminal plasma did not compromise (P > 0.05) sperm motility or acrosomal integrity. Sperm motility traits were maintained longer (P < 0.05) at 25°C than at 37°C in Ham’s or TYB, but temperature did not affect (P > 0.05) acrosomal integrity. Overall, TYB maintained optimal (P < 0.05) sperm motility compared with Ham’s medium, but Ham’s medium maintained more (P < 0.05) intact acrosomes than TYB. Slow cooling (0.2°C min–1) was optimal (P < 0.05) compared to rapid cooling (1°C min–1), and ultra-rapid cooling (9°C min–1) was found to be highly detrimental (P < 0.05). Results obtained in TYB with 0% or 4% glycerol were comparable (P > 0.05), indicating that 4% glycerol was non-toxic to ferret sperm; however, glycerol failed to ameliorate the detrimental effects of either rapid or ultra-rapid cooling. The results of the present study demonstrate that the damage observed to black-footed ferret spermatozoa is derived largely from the rate of cooling.
Dissection of culture media for embryos: the most important and less important components and characteristics
Authors: Gardner David K.
Published: January 2008

Improvements in culture media formulations have led to an increase in the ability to maintain the mammalian embryo in culture throughout the preimplantation and pre-attachment period. Amino acids and specific macromolecules have been identified as being key medium components, whereas temporal dynamics have been recognised as important media characteristics. Furthermore, other laboratory factors that directly impact embryo development and viability have been identified. Such factors include the use of a reduced oxygen tension, an appropriate incubation system and an adequate prescreening of all contact supplies. With rigourous quality systems in place, it is possible to obtain in vivo rates of embryo development in vitro using new media formulations while maintaining high levels of embryo viability. The future of embryo culture will likely be based on novel culture chips capable of providing temporal dynamics while facilitating real-time analysis of embryo physiology.
102 IDENTIFICATION OF CONTRASTED PHENOTYPES IN THE BOVINE FROM REPEATED IN VIVO AND IN VITRO EMBRYO PRODUCTION FOLLOWING SUPEROVULATION
Authors: Guyader-Joly C. , Ponchon S. , Gonzalez C. , Marquant-Le Guienne B. , Clément L. , Dalbiès-Tran R. , Mermillod P. , Humblot P.
Published: January 2008

This study was initiated to evaluate maternal influence on in vivo and in vitro bovine embryo production and identify animals with contrasted phenotypes for reproductive parameters. Nine Montbéliard cows raised on the same farm and with various genetic origins were included in the study. In vivo-derived embryos were collected nonsurgically from superovulated cows on day 7 after AI (34 collections). Immature oocytes were collected by ovum pickup from the same (superovulated) cows (36 sessions) then matured, fertilized (day 0) with the same bull, and cultured in vitro until day 7 on Vero cell monolayers in B2 medium. Grade 1 to 3 in vivo and grade 1 and 2 in vitro produced embryos deemed viable according to IETS criteria. The mean numbers of blastocysts and viable blastocysts per session per cow were, respectively, 8.3 ± 5.5 and 4.8 ± 3.6 in the in vivo system and 2.5 ± 2.6 and 1.8 ± 2.2 in the in vitro system. Individual cow data of in vivo and in vitro embryo production were analyzed by ANOVA (GLM program in SAS; SAS Institute Inc., Cary, NC, USA). Results are presented in Table 1: mean ± SD. Quantity and quality of produced embryos varied significantly among females, and production in vivo and in vitro was not systematically related. Contrasted phenotypes were identified according to their viable blastocyst rates in both systems (in vivo: no viable/recovered; in vitro: no viable/inseminated). Two females presented a relatively high percentage of viable blastocysts in both systems (over 30% in vitro and over 70% in vivo, Table 1). On the contrary, 2 females showed low percentages of blastocysts in the 2 systems (<10% in vitro and <50% in vivo). For most other females, the percentage of in vivo-produced blastocysts was relatively high (>50%), but in vitro development rates were low. Only one female (C3) presented the inverse situation. Oocytes collected from animals with contrasted phenotypes will be analysed for gene expression to identify marker genes associated with oocyte developmental competence.

Table 1.

This study was conducted with financial support of ‘Genanimal’ – French Ministry of Research (#03P409) and Apis-Gene.
107 CHANGES OF HIGHER ORDER CHROMATIN ARRANGEMENTS CORRELATE WITH GENOME ACTIVATION DURING EARLY BOVINE DEVELOPMENT
Authors: Koehler D. , Brero A. , Cremer T. , Wolf E. , Zakhartchenko V.
Published: January 2008

In interphase nuclei of higher eukaryotes, the genome is organized within discrete chromosome territories (CT). These CT are arranged in a nonrandom fashion depending on cell type, species, and cell cycle stage. Gene-rich CT locate more internally in the nucleus, whereas gene-poor ones are found more peripherally. So far, CT have been investigated mainly in cultured cells and to some extent in tissues, yet little is known about the origin and fate of CT during early development. To gain insights into the very early organization of CT in embryos, we developed a fluorescence in situ hybridization protocol that yields optimal results with 3 dimensionally preserved bovine embryos. Using this protocol, we investigated CT of bovine chromosome 19 and 20. These chromosomes represent the gene-richest and gene-poorest chromosomes, according to the number of annotated genes at the Ensembl database (www.ensembl.org). We could confirm a gene density-related radial nuclear distribution in bovine fibroblasts and lymphocytes with CT 19 being more interior than CT 20. Because cell cycle-dependent effects were not expected in view of published data in other species, we did not consider the cell cycle stage of the investigated specimen. In early bovine embryos generated by in vitro fertilization, we demonstrated the existence of nonoverlapping CT already in zygotic pronuclei. In day 3 embryos, but interestingly not in day 2 embryos, a gene density-related radial distribution of CT 19 and 20 was observed. Because these stages represent a transition stage during which the embryonic genome is activated, our results indicate a correlation between genome activation and changes of higher order nuclear organization. We are currently investigating the question whether transcription is a prerequisite for the establishment of a gene density-related chromosome positioning, as well as how reprogramming after nuclear transfer affects CT distribution of somatic donor nuclei.
The present work is supported by the Deutsche Forschungsgemeinschaft (ZA 425/1).
119 ENDOMETRIAL CO-CULTURE MODELS FOR THE IN VITRO INVESTIGATION OF EARLY EMBRYO - MATERNAL CROSSTALK IN CATTLE
Authors: Zitta K. , Ulbrich S. , Wolf E. , Boelhauve M.
Published: January 2008

Cellular and molecular changes in the endometrium during the preimplantation period are orchestrated by a complex network of molecules. The endometrium consists mainly of epithelial (EC) and stromal cells (SC) which produce a variety of active factors, such as prostaglandins (PG), in response to hormones and embryonic signals. However, it is unknown whether and how EC and SC influence each other in their reaction to these endo- and paracrine stimuli. To address this question, we measured the PG secretion of separately cultured EC and SC, and of EC+SC co-cultures in an insert system (0.2 µm membrane pore size) allowing exchange of secreted factors, but no direct contact between EC and SC. Bovine endometrial cells were isolated from cow uterus by a combination of mechanical and enzymatic procedures on Day 8 of the estrous cycle (ovarian morphology). Isolated EC and SC (90% purity) were cultured in DMEM/F12 containing 10% fetal bovine serum at 37�C in an atmosphere of 5% CO2 in air. PGE2 and PGF2α concentrations in media from separately cultured EC and SC or co-cultured EC+SC were measured by specific ELISAs after 24 h of stimulation with (i) 100 nm oxytocin (OXT); (ii) 100 ng mL–1 interferon tau (IFNT); or (iii) the respective solvent as non-stimulated control. EC responded to OXT stimulation with a decreased PGE2/PGF2α ratio, although the difference was not significant (PGE2/PGF2α ratio non-stimulated: 2.4 � 0.9; PGE2/PGF2α ratio stimulated: 1.5 � 0.8; P > 0.05 v. non-stimulated control, evaluated by t-test). No differences in the ratios of PGE2/PGF2α release into the medium were observed when comparing OXT-treated SC with non-treated SC. Incubating co-cultures of EC and SC with OXT resulted in a statistically significant, 2.5-fold decrease in the PGE2/PGF2α ratio as compared to non-stimulated cells (0.8 � 0.2 v. 2.0 � 0.7; P < 0.05; t-test). In contrast, IFNT stimulation of EC and SC co-cultures shifted PG secretion toward the pregnancy protective PGE2 (PGE2/PGF2α ratio non-stimulated: 0.99 � 0.44; PGE2/PGF2α ratio stimulated: 2.36 � 0.96; P < 0.05 v. non-stimulated control, by t-test). Conclusions: Our findings indicate that interactions between EC and SC regulate the responsiveness of the co-culture system to OXT and the pregnancy recognition molecule IFNT. We propose that co-culture of both cell types should be used for studying mechanisms of early embryo–maternal communication.
12 COMPUTER-ASSISTED SPERM ANALYSIS OF FRESH EPIDIDYMAL CAT SPERMATOZOA AND THE IMPACT OF COOLED STORAGE (4°C) ON SPERM QUALITY
Authors: Filliers M. , Rijsselaere T. , Bossaert P. , De Causmaecker V. , Dewulf J. , Pope C. E. , Van Soom A.
Published: January 2008

Feline epididymal sperm is commonly used for in vitro fertilization. It also yields the opportunity to conserve genetic material from valuable males that suddenly die. Epididymal sperm quality parameters vary considerably among laboratories, implicating the need for objective evaluation methods. The aim of the present study was to describe reference values of computer-assisted sperm analysis (CASA) parameters of fresh epididymal cat sperm and to assess the effect of prolonged cooled storage (4�C) on various sample characteristics. Epididymides obtained from tomcats after routine orchiectomy (2–4 pairs/replicate) were sliced to release spermatozoa. The sperm suspension was placed on a 2-layer gradient and, after centrifugation, the sperm pellet was recovered. In Experiment 1 (20 replicates), sperm motility parameters were assessed immediately after retrieval (T0) using the Hamilton Thorne analyzer Ceros 12.1 (HTR; Hamilton Thorne Biosciences, Beverly, MA, USA). In Experiment 2, fresh (T0) sperm samples (4 replicates) were evaluated for motility parameters (HTR), acrosomal status (FITC-Pisum sativum agglutinin staining), morphology (eosin/nigrosin (E/N) staining), and membrane integrity (E/N and SYBR�-14-propidium iodide staining; Molecular Probes, Inc., Eugene, OR, USA). After addition (1:2) of a Tris-glucose-citrate diluent containing 20% egg yolk, samples were cooled and reassessed on Days 1 (T1), 3 (T3), 5 (T5), 7 (T7), and 10 (T10). Results were analyzed in a mixed linear model, with replicate as random factor and time as fixed effect (S-PLUS 7.0; Insightful Corp., Seattle, WA, USA). Results of Experiment 1 were as follows (mean � SD): motility (MOT): 80.8% � 23.5; progressive motility (PMOT): 69.9% � 23.2; velocity average pathway (VAP): 98.7 µm s–1 � 24.2; velocity straight line (VSL): 89.3 µm s–1 � 25.4; velocity curved line (VCL): 134.8 µm s–1 � 31.9; amplitude lateral head (ALH): 4.3 µm � 2.0; beat cross frequency (BCF): 34.6 Hz � 7.0; and straightness (STR): 89.6% � 6.6. In Experiment 2, MOT, PMOT, VAP, VSL, VCL, BCF, and the percentage of normal spermatozoa showed a decrease over time (P < 0.05) compared to fresh samples, starting from T1, T3, T5, T7, T5, T3, and T1, respectively. In contrast, STR, ALH, membrane integrity, and the percentage of acrosome-intact spermatozoa were not affected (P > 0.05) by cooled storage. To summarize, we have presented a set of reference values for CASA-parameters of fresh, epididymal cat spermatozoa. Cooled storage impaired most motility parameters and lowered the percentage of normal spermatozoa, but did not influence membrane integrity or acrosomal status. The effect of cooled storage on DNA fragmentation of sperm and its subsequent influence on in vitro embryo development require further investigation.
125 EXAMINING CRITERIA FOR EXTENDING BOVINE BLASTOCYST SURVIVAL IN VITRO
Authors: Cooke F. N. T. , Rodina T. M. , Hansen P. J. , Ealy A. D.
Published: January 2008

Most of the current culture procedures used for bovine in vitro embryo production terminates at the blastocyst stage. Developing procedures for extending embryo lifespan beyond this phase will provide a valuable tool for understanding events that occur during the second week of pregnancy in cattle. The overall objective of the present studies was to identify culture conditions required to support bovine blastocyst development beyond its initial formation. In the first study, individual day 8 blastocysts (day 0 = day of IVF) were cultured until day 11 in 30 µL microdrops of Potassium Simplex Optimized Medium-Bovine Embryo 2 containing 0.1 mm non-essential amino acids or Tissue Culture Medium 199 (M199). Both media were supplemented with 5% [v/v] fetal bovine serum (FBS) and incubations were in an atmosphere of either 5 or 21% (v/v) oxygen. A medium by oxygen interaction (P = 0.007) occurred when assessing cell number on day 11. Blastocysts cultured in M199 and in a 5% O2 environment had greater (P < 0.002) cell numbers (536 � 49) than blastocysts incubated in other conditions (339 � 28). Conditioned medium from blastocysts incubated in 21% O2 contained greater (P < 0.05) concentrations of bioactive interferon-tau (IFNT) than blastocysts incubated in 5% O2 regardless of medium type (70.5 � 28 v. 17.2 � 2.6 ng mL–1). In a follow-up study, blastocysts could remain morphologically viable through day 11 in M199 containing at least 2.5% FBS. To examine whether oxidative stress was responsible for the increase in IFNT production under 21% O2, blastocysts were incubated under a 5% O2 atmosphere in M199 containing 2.5% FBS and increasing concentrations of tert-butylhydroperoxide (tBH), a membrane-permeable oxidative agent. Addition of e3 nm tBH decreased cell numbers but did not increase IFNT concentrations in conditioned medium. To examine whether blastocysts could survive beyond day 11 in culture, day 11 blastocysts were transferred to 400 �L of M199 with 20% FBS under 5% oxygen and cultured from day 11 to 20–21 post-IVF. Half of the medium was replaced every 2–3 days. On day 13–14, 16.6 � 6.1% of blastocysts showed initial signs of degeneration. A portion of blastocysts (32.9 � 9.6%) began attaching to plates on days 13–15 and produced outgrowths that appeared viable on days 20–21. All of the non-attached blastocysts degenerated by day 17–18. No blastocyst elongation was detected. In conclusion, a culture system was developed that sustains blastocyst viability and IFNT production in vitro to day 11. Although this culture system allowed blastocyst survival until day 14, normal conceptus development (i.e. elongation/filamentation) was not achieved. Nonetheless, the culture system provides a useful tool for examining the initial stages of blastocyst development and IFNT production from individual bovine embryos.
128 EFFECT OF OXYGEN TENSION ON INDIVIDUAL IN VITRO BOVINE EMBRYO DEVELOPMENT IN CUMULUS CELL COCULTURE
Authors: Goovaerts I. G. F. , Leroy J. L. M. R. , Andries S. , De Clercq J. B. P. , Bols P. E. J.
Published: January 2008

An in vitro production system, in which a single oocyte can be followed from the moment of retrieval up to the blastocyst stage, would be a valuable tool for studies linking developmental competence and embryo metabolism to immature oocyte quality and follicular environment. Earlier studies revealed that cumulus cell (CC) coculture during IVC enhances individual development, in contrast to group culture. These studies were performed in 5% O2, whereas generally an atmosphere of 20% oxygen is used for coculture with somatic cells. This study aimed to investigate the effect of oxygen tension on individual embryo development in CC coculture. As a control, the effect of oxygen tension on embryo group culture without CC was assessed simultaneously. Therefore, 692 COC from slaughterhouse ovaries (4 replicates) were routinely matured (TCM-199 + 20% serum) and fertilized in groups and then assigned to 4 culture treatments (SOF + 5% serum under oil). Treatments were T1 = 1 zygote in 20 μL + CC in 5% O2; T2 = 1 zygote in 20 μL + CC in 20% O2; T3 = 20 to 25 zygotes in 50 μL in 5% O2; and T4 = 20 to 25 zygotes in 50 μL in 20% O 2. Cleavage, blastocyst, and hatching rates were assessed 2, 8, and 10 days after fertilization, respectively. Possible effects of oxygen tension in individual and group culture were evaluated with binary logistic regression. No interactions between replicate and treatment could be found. Cleavage rates of individual culture showed a tendency (P < 0.1) to be lower in 5% O2 (62.1 v. 71.0% in 20% O2), whereas blastocyst rates were significantly (P < 0.05) higher in 5% O2 (26.6 v. 16.6% in 20% O2). Hatching rates did not differ significantly between the 2 individual treatments (Table 1). Oxygen tension did not have a significant effect on cleavage rates when embryos were cultured in groups, but blastocyst rates were significantly higher in 5% O2 (41.7 v. 27.6% in 20% O2). The group results confirm other studies (Yuan YQ et al. 2003 Theriogenology 59, 1585–1596). In conclusion, higher blastocyst rates can be obtained when an atmosphere of 5% O2 is used for culturing individual zygotes in CC coculture. Because cleavage rates showed a tendency to be higher in 20% O2, an alternating treatment, with 20% O2 until 2 days after fertilization, followed by 5% O2 until the blastocyst stage, should be investigated.

Table 1. Cleavage, blastocyst, and hatching rates in 5 and 20% oxygen
131 CHARACTERISTICS OF PREGNANCIES AND OFFSPRING FOLLOWING TRANSFER OF BOVINE IN VIVO EMBRYOS ASSESSED BY NANORESPIROMETRY
Authors: Lopes A. S. , Madsen S. E. , Greve T. , Callesen H.
Published: January 2008

It has been speculated whether the metabolism of the pre-implantation embryo may be reflected on the pregnancy and characteristics of the newborn animal. The present study investigated whether respiration rates of individual embryos were correlated with gestation length, type of parturition, birth weight, sex, and viability of the offspring. In-vivo embryos (n = 47) were recovered on day 7 from 11 superovulated and inseminated Holstein-Friesian cows and embryonic respiration rates were measured individually by nanorespirometry. The embryos were classified according to morphological quality (I, II, III), stage of development, and diameter and were subsequently transferred individually (n = 43) to synchronized recipients. Gestation length of the recipients (n = 22) was calculated and the type of parturition (no assistance, light traction, heavy traction, or caesarean section) recorded. Sex, weight, and condition of the calves at birth (weak, normal, or very active) were also assessed. Results were evaluated by chi-square analysis and using a linear mixed model. The pregnancy rate was 60% (26/43), and the respiration rates of individual embryos influenced gestation length as well as the interaction of weight at birth and type of parturition (P < 0.05). Embryos with higher respiration rates tended to be associated with a slightly more difficult type of parturition, which was not affected by sex. When embryos were divided into two even-sized categories according to their respiration rates (high v. low), low embryonic respiration rates were associated with low birth weight in 73% of the newborn calves (Table 1). Neither sex nor condition of the calves was directly affected by the embryonic respiration rates. The diameter and morphology of the embryo affected birth weight (P < 0.05), with quality I embryos associated with heavier calves when compared to quality III embryos. Furthermore, light traction was more frequently used to retrieve heavier animals (P < 0.05), and male calves were 3.2 kg heavier than females (P < 0.05). These results suggest that the metabolism of the embryo might affect the weight of the newborn animal and consequently the type of parturition. However, birth weight is highly influenced by the recipients and season, and thus a more accurate correlation is likely to be established when the weight of the postpubertal offspring is considered.

Table 1. Birth weight of newborn calves according to respiratory category of bovine in-vivo embryos