bluuebunny/crossref_metadata_2025 huggingface dataset:
Surface reflectance of Mars observed by CRISM/MRO: 1. Multi‐angle Approach for Retrieval of Surface Reflectance from CRISM observations (MARS‐ReCO)
Authors: Ceamanos X. , Douté S. , Fernando J. , Schmidt F. , Pinet P. , Lyapustin A.
Published: December 2012

This article addresses the correction for aerosol effects in near‐simultaneous multi‐angle observations acquired by the Compact Reconnaissance Imaging Spectrometer for Mars (CRISM) aboard the Mars Reconnaissance Orbiter. In the targeted mode, CRISM senses the surface of Mars using 11 viewing angles, which allow it to provide unique information on the scattering properties of surface materials. In order to retrieve these data, however, appropriate strategies must be used to compensate the signal sensed by CRISM for aerosol contribution. This correction is particularly challenging as the photometric curve of these suspended particles is often correlated with the also anisotropic photometric curve of materials at the surface. This article puts forward an innovative radiative transfer‐based method named Multi‐angle Approach for Retrieval of Surface Reflectance from CRISM Observations (MARS‐ReCO). The proposed method retrieves photometric curves of surface materials in reflectance units after removing aerosol contribution. MARS‐ReCO represents a substantial improvement regarding previous techniques as it takes into consideration the anisotropy of the surface, thus providing more realistic surface products. Furthermore, MARS‐ReCO is fast and provides error bars on the retrieved surface reflectance. The validity and accuracy of MARS‐ReCO is explored in a sensitivity analysis based on realistic synthetic data. According to experiments, MARS‐ReCO provides accurate results (up to 10% reflectance error) under favorable acquisition conditions. In the companion article, photometric properties of Martian materials are retrieved using MARS‐ReCO and validated using in situ measurements acquired during the Mars Exploration Rovers mission.
Analytical solution and simplified analysis of coupled parent‐daughter steady‐state transport with multirate mass transfer
Authors: Haggerty Roy
Published: December 2012

Key Points

Ratio of daughter to parent concentrations is a simple function

Ratio information that characterizes bed exchange and respiration
Investigation of Zr(iv) and 89Zr(iv) complexation with hydroxamates: progress towards designing a better chelator than desferrioxamine B for immuno-PET imaging
Authors: Guérard François , Lee Yong-Sok , Tripier Raphaël , Szajek Lawrence P. , Deschamps Jeffrey R. , Brechbiel Martin W.
Published: December 2012

Crystallography, quantum chemistry and potentiometry of Zr(iv) hydroxamate complexes: a combination of tools to improve the development of 89Zr chelation for nuclear imaging.
Evolution of intracellular compartmentalization
Authors: Diekmann Yoan , Pereira-Leal José B.
Published: December 2012

Cells compartmentalize their biochemical functions in a variety of ways, notably by creating physical barriers that separate a compartment via membranes or proteins. Eukaryotes have a wide diversity of membrane-based compartments, many that are lineage- or tissue-specific. In recent years, it has become increasingly evident that membrane-based compartmentalization of the cytosolic space is observed in multiple prokaryotic lineages, giving rise to several types of distinct prokaryotic organelles. Endosymbionts, previously believed to be a hallmark of eukaryotes, have been described in several bacteria. Protein-based compartments, frequent in bacteria, are also found in eukaryotes. In the present review, we focus on selected intracellular compartments from each of these three categories, membrane-based, endosymbiotic and protein-based, in both prokaryotes and eukaryotes. We review their diversity and the current theories and controversies regarding the evolutionary origins. Furthermore, we discuss the evolutionary processes acting on the genetic basis of intracellular compartments and how those differ across the domains of life. We conclude that the distinction between eukaryotes and prokaryotes no longer lies in the existence of a compartmentalized cell plan, but rather in its complexity.
Bio-inspired approach of the fluorescence emission properties in the scarabaeid beetle Hoplia coerulea (Coleoptera): Modeling by transfer-matrix optical simulations
Authors: Van Hooijdonk Eloise , Berthier Serge , Vigneron Jean-Pol
Published: December 2012

Scales of the scarabaeid beetle Hoplia coerulea (Coleoptera) contain fluorescent molecules embedded in a multilayer structure. The consequence of this source confinement is a modification of the fluorescence properties, i.e., an enhancement or inhibition of the emission of certain wavelengths. In this work, we propose a bio-inspired approach to this problem. In other words, we use numerical simulations based on the one-dimensional transfer-matrix formalism to investigate the influence of a Hoplia-like system on emission characteristics and, from the results, we deduce potential technical applications. We reveal that depending on the choice of some parameters (layer thickness, dielectric constant, and position of the emitting source in the structure), it is possible to enhance or inhibit the fluorescence emission for certain wavelengths. This observation could be of great interest to design new optical devices in the field of optoelectronic, solar cells, biosensors, etc.
GaN-based platforms with Au-Ag alloyed metal layer for surface enhanced Raman scattering
Authors: Weyher J. L. , Dzięcielewski I. , Kamińska A. , Roliński T. , Nowak G. , Hołyst R.
Published: December 2012

The fabrication procedure of efficient surface enhanced Raman scattering (SERS) platforms is demonstrated based on reproducibly photo-etched GaN epitaxial layers covered by a thin Au-Ag layer and subjected to partial de-alloying. Using a gold-rich Au57Ag43 alloy (70/30 wt. %), it is possible to preserve about 19 at. % of silver in the layer, even after etching in nitric acid for up to 24 h. A large enhancement of the Raman signal from the test 4-mercaptobenzoic acid molecules attached to such porous Au-Ag metal layer is obtained due to the presence of a high percentage of Ag. For the optimal etching time, in the range of 6–24 h, an enhancement factor (EF) greater than 107 was obtained. The contribution of different features of our SERS platforms such as the size of pores, nano-roughness, and the chemical composition of the metal layer for the EF parameter are critically discussed.
Nonempirical Rung 3.5 density functionals from the Lieb-Oxford bound
Authors: Janesko Benjamin G.
Published: December 2012

Odashima and Capelle (OC) recently presented a nonempirical, nonlocal, approximate exchange-correlation density functional constructed from exact exchange and the Lieb-Oxford bound [M. M. Odashima and K. Capelle, Phys. Rev. A 79, 062515 (2009)]10.1103/PhysRevA.79.062515. Our “Rung 3.5” exchange energy, built from the product of the Kohn-Sham one-particle density matrix and a semilocal model density matrix, provides an upper bound to exact exchange via the Cauchy-Schwarz inequality. The present work combines this bound with the OC construction to yield nonempirical Rung 3.5 functionals. Numerical tests indicate that these functionals provide reasonable treatments of many molecular properties, and suggest areas for improvement. The results illustrate the promise of the OC construction and the utility of Rung 3.5 exchange in constraint satisfaction.
L2,3 edge photoabsorption spectra of bulk V2O5: A two components relativistic time dependent density functional theory description with finite cluster model
Authors: Fronzoni Giovanna , De Francesco Renato , Stener Mauro
Published: December 2012

The two-component relativistic time dependent density functional theory method to treat the core electron excitations has been applied to the bulk V2O5 for the description of X-ray absorption at the L edges. The theoretical method has proven accurate to reproduce the experimental NEXAFS spectrum, thanks to the inclusion of the most relevant physical effects: the crystal field, the configuration mixing, and the spin-orbit coupling. The method has been applied by taking into account suitable cluster models, accurately chosen in order to simulate at best the electronic structure of the condensed phase.
A proposed 2D framework for estimation of pore size distribution by double pulsed field gradient NMR
Authors: Benjamini Dan , Katz Yaniv , Nevo Uri
Published: December 2012

Reconstructing a pore size distribution of porous materials is valuable for applications in materials sciences, oil well logging, biology, and medicine. The major drawback of NMR based methods is an intrinsic limitation in the reconstruction which arises from the ill-conditioned nature of the pore size distribution problem. Consequently, while estimation of the average pore size was already demonstrated experimentally, reliable evaluation of pore size distribution remains a challenging task. In this paper we address this problem by analyzing the mathematical characteristics that create the difficulty and by proposing an NMR methodology and a numerical analysis. We demonstrate analytically that an accurate reconstruction of pore size distribution is problematic with the current known strategies for conducting a single or a double pulsed field gradient (s-PFG, d-PFG) experiment. We then present a method for choosing the experimental parameters that would significantly improve the estimation of the size distribution. We show that experimental variation of both q (the amplitude of the diffusion gradient) and φ (the relative angle between the gradient pairs) is significantly favorable over single and double-PFG applied with variation of only one parameter. Finally, we suggest a unified methodology (termed Concentric d-PFG) that defines a multidimensional approach where each data point in the experiment is characterized by φ and q. The addition of the angle parameter makes the experiment sensitive to small compartment sizes without the need to use strong gradients, thus making it feasible for in-vivo biological applications.
On the periodic solutions of the static, spherically symmetric Einstein-Yang-Mills equations
Authors: Buzzi Claudio A. , Llibre Jaume
Published: December 2012

We prove that the static, spherically symmetric Einstein-Yang-Mills equations do not have periodic solutions when r > 0.
Ablation driven by hot electrons generated during the ignitor laser pulse in shock ignition
Authors: Piriz A. R. , Rodriguez Prieto G. , Tahir N. A. , Zhang Y. , Liu S. D. , Zhao Y. T.
Published: December 2012

An analytical model for the ablation driven by hot electrons is presented. The hot electrons are assumed to be generated during the high intensity laser spike used to produce the ignitor shock wave in the shock ignition driven inertial fusion concept, and to carry on the absorbed laser energy in its totality. Efficient energy coupling requires to keep the critical surface sufficiently close to the ablation front and this goal can be achieved for high laser intensities provided that the laser wavelength is short enough. Scaling laws for the ablation pressure and the other relevant magnitudes of the ablation cloud are found in terms of the laser and target parameters. The effect of the preformed plasma assembled by the compression pulse, previous to the ignitor, is also discussed. It is found that a minimum ratio between the compression and the ignitor pulses would be necessary for the adequate matching of the corresponding scale lengths.
Suppression of antiferroelectric state in NaNbO3 at high pressure from in situ neutron diffraction
Authors: Mishra S. K. , Gupta M. K. , Mittal R. , Chaplot S. L. , Hansen Thomas
Published: December 2012

We report direct experimental evidence of antiferroelectric to paraelectric phase transition under pressure in NaNbO3 using neutron diffraction at room temperature. The paraelectric phase is found to stabilize above 8 GPa and its crystal structure has been determined in orthorhombic symmetry with space group Pbnm. The variation of the structural parameters of the both orthorhombic phases as a function of pressure was determined. We have not found evidence for structural phase transition around 2 GPa as previously suggested in the literature based on Raman scattering experiments; however, significant change in Nb-O-Nb bond angles are found around this pressure. The response of the lattice parameters to pressure is strongly anisotropic with a largest contraction along 〈100〉. The structural phase transition around ∼8 GPa is followed by an anomalous increase in the orthorhombic strain and tilt angle associated with the R point (q = ½ ½ ½). Ab-initio calculation of the enthalpy in the various phases of NaNbO3 is able to predict the phase transition pressure well.
Cylindrical Fresnel lenses based on carbon nanotube forests
Authors: Butt Haider , Rajesekharan Ranjith , Dai Qing , Sarfraz Sohab , Vasant Kumar R. , Amaratunga Gehan A. J. , Wilkinson Timothy D.
Published: December 2012

The forests of carbon nanotubes have been termed as the darkest man-made materials. Such materials exhibit near-perfect optical absorption (reflectance ∼ 0.045%) due to low reflectance and nanoscale surface roughness. We have demonstrated the utilization of these perfectly absorbing forests to produce binary amplitude cylindrical Fresnel lenses. The opaque Fresnel zones are defined by the dark nanotube forests and these lenses display efficient focusing performance at optical wavelengths. Lensing performance was analyzed both computationally and experimentally with good agreement. Such nanostructure based lenses have many potential applications in devices like photovoltaic solar cells.
Increasing recoverable energy storage in electroceramic capacitors using “dead-layer” engineering
Authors: McMillen M. , Douglas A. M. , Correia T. M. , Weaver P. M. , Cain M. G. , Gregg J. M.
Published: December 2012

The manner in which ultrathin films of alumina, deposited at the dielectric-electrode interface, affect the recoverable energy density associated with (BiFeO3)0.6–(SrTiO3)0.4 (BFST) thin film capacitors has been characterised. Approximately 6 nm of alumina on 400 nm of BFST increases the maximum recoverable energy of the system by around 30% from ∼13 Jcc−1 to ∼17 Jcc−1. Essentially, the alumina acts in the same way as a naturally present parasitic “dead-layer,” distorting the polarisation-field response such that the ultimate polarisation associated with the BFST is pushed to higher values of electric field. The work acts as a proof-of-principle to illustrate how the design of artificial interfacial dielectric “dead-layers” can increase energy densities in simple dielectric capacitors, allowing them to compete more generally with other energy storage technologies.
Role of barrier structure in current collapse of AlGaN/GaN high electron mobility transistors
Authors: DasGupta Sandeepan , Biedermann Laura B. , Sun Min , Kaplar Robert , Marinella Matthew , Zavadil Kevin R. , Atcitty Stan , Palacios Tomas
Published: December 2012

Simultaneous measurements of surface potential by Kelvin force microscopy and drain current detrapping transients in AlGaN/GaN high electron mobility transistors are performed on devices with two different epitaxial structures to understand if the trapped charges are predominantly in the AlGaN bulk, at the AlGaN surface, or in the GaN buffer. The results show that the predominant location of charge trapping is affected more strongly by the design of the device and the operating voltages than by surface passivation or buffer defects. The experiments also show that the dominant component of current collapse in devices with thick AlGaN barrier layers comes from AlGaN traps.
Universal access to ambulance does not increase overall demand for ambulance services in Queensland, Australia
Authors: Tippett Vivienne C. , Toloo Ghasem (Sam) , Eeles David , Ting Joseph Y. S. , Aitken Peter J. , FitzGerald Gerard J.
Published: December 2012

Objective.
To determine the impact of the introduction of universal access to ambulance services via the implementation of the Community Ambulance Cover (CAC) program in Queensland in 2003–04.

Method.
The study involved a 10-year (2000–01 to 2009–10) retrospective analysis of routinely collected data reported by the Queensland Ambulance Service (QAS) and by the Council of Ambulance Authorities. The data were analysed for the impact of policy changes that resulted in universal access to ambulance services in Queensland.

Results.
QAS is a statewide, publically funded ambulance service. In Queensland, ambulance utilisation rate (AUR) per 1000 persons grew by 41% over the decade or 3.9% per annum (10-year mean = 149.8, 95% CI: 137.3–162.3). The AUR mean after CAC was significantly higher for urgent incidents than for non-urgent ones. However projection modelling demonstrates that URs after the introduction of CAC were significantly lower than the projected utilisation for the same period.

Conclusions.
The introduction of universal access under the Community Ambulance Cover program in Queensland has not had any significant independent long-term impact on demand overall. There has been a reduction in the long-term growth rate, which may have been contributed to by an ‘appropriate use’ public awareness program.

What is known about the topic?
It is generally well accepted that the demand for emergency health services is increasing however the drivers for demand are poorly understood. In Queensland in particular, growth in demand for services exceeds that seen in other states and territories. Some commentators have pointed at service funding policy and costs to end users as potential reasons for excess demand for services.

What does this paper add?
The assumption that forced subsidisation creates a perception of entitlement amongst consumers is challenged in this paper. We are able to demonstrate that demand for emergency health services did not increase beyond what would have been expected under a mandatory subscription system known as Community Ambulance Cover (CAC). This paper contributes to the developing body of knowledge about drivers for emergency health service demand.

What are the implications for practitioners?
There is a need to continue analysis of the system to determine drivers for demand and develop an evidence base on which to formulate emergency health policy, including funding models, for the future. Purely economic drivers for service demand seem unlikely to hold up and policy makers need to understand the complex relationships between service systems; end user perceptions and health literacy; and the costs of services in order to effect policy reform.
Reproductive and productive performances of the stress-negative Piétrain pigs in the tropics: the case of Vietnam
Authors: Luc D. D. , Bo H. X. , Thomson P. C. , Binh D. V. , Leroy P. , Farnir F.
Published: December 2012

The objective of the present study is to investigate the reproduction, production and semen traits of the stress-negative Piétrain line of pigs in the north of Vietnam, as well as to identify various factors (halothane genotype, generation-origin, parity, sex and month of the year) acting on these traits. The experiment was carried out on a purebred nucleus on Dong Hiep farm, Haiphong province in Vietnam. The reproduction performances and the sperm quality measures were based on 12 sows and 5 boars imported from Belgium as well as on 29 sows and 14 boars born in Vietnam, while the production performances were recorded only from 251 animals born in Vietnam. No significant differences were observed between halothane genotypes of sows for reproduction traits (P > 0.0578). At the age of 7.5 months, bodyweight, backfat thickness and longissimus depth (7.5) of homozygous-dominant pigs were higher (P < 0.05) than in heterozygotes. Homozygous-dominant boars had higher total number of spermatozoa than heterozygote boars (P < 0.05); similarly, Vietnam boars also presented higher total number of spermatozoa values than Belgium boars (P < 0.001). Vietnam sows had longer duration of pregnancy, more piglets born alive and heavier litter weight of weaning than Belgium sows (P < 0.01). Bodyweight at birth, at weaning, at 2, 5.5 and 7.5 months of age increased from the first to third parity (P < 0.05). Gilts had more lean meat than intact boars (P < 0.01). Compared with observations made in temperate countries, the reproduction and production performances, sperm concentration and total number of spermatozoa of stress-negative Piétrain pigs were reduced, whereas lean meat percentage was not affected. This might be due to the different climate, although improving values for animals born in Vietnam might indicate adaptation of the animals and/or of the technicians with that breed. The results support the interest of the use of this new Piétrain line in tropical countries with ecological and climatic conditions similar to Vietnam.
Genetic structure and diversity of introduced eastern mosquitofish (Gambusia holbrooki) in south-eastern Australia
Authors: Ayres Renae M. , Pettigrove Vincent J. , Hoffmann Ary A.
Published: December 2012

The closely related eastern mosquitofish (Gambusia holbrooki) and western mosquitofish (Gambusia affinis) have been introduced into many countries and collectively represent the most widely distributed freshwater fish in the world. We investigated genetic patterns associated with the spread of G. holbrooki in its introduced range in south-eastern Australia, by sampling 60 G. holbrooki populations (n = 1771) from major regions where G. holbrooki was initially introduced into Australia, and characterising the genetic diversity and population structure of G. holbrooki, using five polymorphic nuclear microsatellite loci and sequences from two mitochondrial genetic markers. Results were compared with published data on American and European Gambusia samples and historical records. Low microsatellite diversity and strong population genetic structuring were found within G. holbrooki in south-eastern Australia. Observed heterozygosity and allelic richness declined regionally in the order Sydney, Brisbane, Canberra, Melbourne and Adelaide. Microsatellite variation in Australia was reduced compared with native populations. Two mitochondrial DNA haplotypes of G. holbrooki were found; one was common, whereas the other was detected in one Sydney population and one Melbourne population. Cytochrome b sequence diversity was reduced compared with native and European ranges, and sequences were identical to two haplotypes previously identified. Microsatellite diversity of G. holbrooki in south-eastern Australia validates historical records of its spread, beginning north and moving south. Mitochondrial sequencing confirms that G. holbrooki is present in Australia, but the origins of Australian G. holbrooki populations remain unclear.
122 OXYTOCIN-INDUCED PROLONGED DIOESTRUS DOES NOT DOWNREGULATE THE EXPRESSION OF PROSTAGLANDIN-ENDOPEROXIDASE SYNTHASE 2 OR OXYTOCIN RECEPTOR IN EQUINE ENDOMETRIUM
Authors: Klein C. , Troedsson M. H. T.
Published: December 2012

Lifespan of the corpus luteum is regulated through the release of PGF2α from the endometrium. The endocrine factors regulating the timed release of prostaglandin during luteolysis and the abrogation thereof during pregnancy is only partially understood in the mare. Vanderwall et al. (2007 J. Am. Vet. Med. Assoc. 231, 1864–1867) reported that twice-daily administration of oxytocin during early dioestrus results in prolongation of luteal function. We hypothesised that the aforementioned repeated administration of oxytocin during early dioestrus results in downregulation of oxytocin receptor transcript while prostaglandin-endoperoxide synthase 2 transcript abundance is unaffected. The current study, therefore, compared the expression oxytocin receptor (OXTR) and prostaglandin-endoperoxide synthase 2 (PTGS2) in endometrial samples obtained 16 days after ovulation from nonpregnant mares (n = 5), pregnant mares (n = 5), and mares displaying prolonged dioestrus after the repeated administration of oxytocin (n = 6). Prolonged dioestrus was observed in all mares treated with oxytocin, evident by plasma progesterone levels above 6 ng mL–1 at the time of sample collection. Using quantitative real-time PCR analysis, PTGS2 showed a 4.37-fold lower abundance in pregnant v. nonpregnant mares (P = 0.009), while OXTR abundance was not affected by pregnancy status. Repeated administration of oxytocin did not lower expression levels of PTGS2 or OXTR when compared to levels observed in nonpregnant mares. As hypothesised, PTGS2 abundance was not affected by the treatment with oxytocin. Furthermore, we failed to prove the hypothesis that repeated administration of oxytocin leads to lowered expression of OXTR 16 days after ovulation in mares displaying prolonged dioestrus following repeated administration of oxytocin. A regulation of the oxytocin receptor at the post-transcriptional level, such as uncoupling of the receptor from its G-protein or possibly phosphorylation of the receptor, cannot be excluded and might account for the observed prolongation of luteal lifespan.
126 TIME OF FIRST CLEAVAGE AND DEVELOPMENT TO BLASTOCYST OF BOVINE IVF EMBRYOS MONITORED BY TIME-LAPSE IMAGING AFTER IN VITRO OR IN VIVO MATURATION
Authors: Beck A. , Reichenbach M. , Reichenbach H. D. , Habermann F. , Arnold G. J. , Wolf E.
Published: December 2012

As part of a study on structural, molecular, and functional deficiencies of both in vitro and in vivo matured oocytes, the outcome of in vitro fertilization and embryo culture was evaluated by noninvasive time-lapse monitoring. Oocytes for IVM were recovered from abattoir-derived ovaries (G1). In vivo matured oocytes were obtained by ovum pick-up from six FSH (Pluset®, Laboratorios Calier, Spain) superstimulated Simmental heifers (Reichenbach et al. 2010 Reprod Domest Anim. 45, 41) shortly before ovulation (G2). Gonadotropin-releasing hormone (GnRH; Receptal®, MSD, Germany) was applied 18 h before ovum pickup (OPU). After IVM (G1) for 23 h or directly after OPU (G2), all oocytes were fertilized and cultured in vitro for 7 days. To continuously monitor multiple individual embryos in parallel, a well-of-wells culture dish system and a transmitted-light microscope equipped with a digital camera placed in the incubator (Primo Vision, Cryo Innovation Inc., Budapest, Hungary) was used. Each embryo was photographed every 5 min over the whole culture period to precisely determine the time and morphological pattern of the first cleavage and to observe the development up to the hatching blastocyst stage. In a first set of experiments, 128 (G1) and 64 (G2) embryos were cultured and monitored under identical conditions. In a statistical analysis (ANOVA, SAS, SAS Institute Inc., Cary, NC, USA) only embryos with a morphologically regular first and second cleavage (G1: n = 86; G2; n = 42; Total n = 128) were included. Fixed effects in the model were time of first cleavage (class 1: ≤26 hours post-insemination (hpi), class 2: >26 to 28 hpi, class 3: >28 to 30 hpi, class 4: >30 to 32 hpi, class 5: >32 hpi) and in vitro versus ex vivo maturation on blastocyst outcome. The total blastocyst rate was lower after in vitro maturation (40/86) than after in vivo maturation (29/42), while the time of first cleavage was not different. The time until the onset of the first cytokinesis ranged in both oocyte groups from 25–37 hpi. Notably, zygotes that cleaved between 26 and 28 hpi (class 2) showed the highest blastocyst rate (35/47), while the rate decreased in a statistically significant way (P < 0.01), when the first cleavage was observed between 30 and 32 hpi (class 4) (4/15). Now we turn to the investigation of the causes of a delay and the aberrations of the first cleavage cycle. An important step is the use of noninvasive microscopic monitoring to select embryos according to their developmental stage and history for 3D fluorescence microscopy, proteome analyses, and functional studies.
This work is supported by the Deutsche Forschungsgemeinschaft (DFG FOR1041).
127 THYMIDINE SYNCHRONIZATION OF IN VITRO DEVELOPMENT OF BOVINE EMBRYOS
Authors: Rubessa M. , Iannuzzi A. , Peretti V. , Pauciullo A. , Cosenza G. , Ramunno L. , Iannuzzi L. , Rubes J. , Di Berardino D.
Published: December 2012

Excess thymidine is capable of synchronizing lymphocyte cultures in vitro by acting with a feed-back mechanism during the S phase of the cell cycle (Harper 2005 Methods Mol. Biol. 296, 157–166). The possibility to synchronize the embryonic growth can be a good strategy for future epigenetic studies. The present study was undertaken to test whether excess thymidine could also synchronize in vitro development of bovine embryos. Abattoir-derived cumulus–oocyte complexes (COC) of the Agerolese breed of cattle were matured in vitro using standard procedures. After maturation, COCs were transferred in drops of 300 µL of IVF-TALP (25/drop) and covered with mineral oil. Frozen sperm from a bull were selected by centrifugation on a Percoll discontinuous gradient (45 to 80%). The pellet was diluted in IVF medium and added to the COC at the concentration of 1 × 106 sperm mL–1. After 18–20 h of gametes co-incubation, presumptive zygotes were denuded and cultured in SOF medium containing different concentrations of thymidine (0, 300, 600, 1200, 2400 µg mL–1, final concentrations). The day after (Day 2) presumptive zygotes were washed four times in fresh SOF, classified morphologically under a stereomicroscope as not divided (n.d.), 2 cells, 3–8 cells, and 9–32 cells and cultured in standard SOF at 39°C in a humidified mixture of 5% CO2,7% O2, and 88% N2. On Day 3, the embryos were again examined for the growth stage in relation to the synchronization effects. On Day 7, the embryos were evaluated for the final growth efficiency (cleavage stage and blastocyst formation). The experiment was replicated 4 times (except the 2400 µg mL–1 condition, which was replicated 2 times because of its clear toxic effect). Data were analyzed by ANOVA test. At Day 2, there were no differences between groups whatever the concentration (n.d.–2 cells: 59.7 ± 11.2, 52.9 ± 26.3, 56.1 ± 14.6, 66.0 ± 2.8, 38.0 ± 11.3; 3–8 cells : 40.3 ± 11.2, 47.1 ± 26.3, 43.9 ± 14.6, 34.0 ± 2.8, 59.9 ± 9.5; respectively for 300, 600, 1200, 2400, and control), while differences at Day 3 and 7 are shown in Table 1. The dosage of 300 µg mL–1 slowed embryo development without altering the developmental rate, whereas the other dosages were somewhat toxic to the zygotes, affecting the final percentage of blastocysts.

Table 1.State of development of zygotes on Day 3 and Day 7 (cleavage and embryo rate)
129 POST-HATCHING DEVELOPMENT AND GENE EXPRESSION OF IN VITRO-PRODUCED BOVINE EMBRYOS IN RECIPIENT UTERUS AFTER MULTIPLE TRANSFER
Authors: Machado G. , Ferreira A. , Pivato I. , Fidelis A. , Srpicigo J. F. , Franco M. , Dode M.
Published: December 2012

This study aimed to compare post-hatching development of Day 7 in vitro and in vivo embryos cultured in recipient uterus until Day 14. For producing in vitro embryos (IVP), oocytes were matured, fertilized (Day 0) and cultured in vitro for 6 days (Day 7) in synthetic oviduct fluid medium supplemented with 5% of fetal bovine serum and incubated at 39°C in 5% CO2 in air. At Day 7, part of IVP blastocysts was transferred to recipient uterus and part was stored for gene expression analysis. As a control group, in vivo embryos were produced after ovarian stimulation, insemination and uterine flushing on Day 7 post insemination. Similarly to the IVP embryos, part of embryos was transferred to recipient uterus and part was stored for gene expression analysis. Day 7 in vivo (n = 53) and IVP (n = 64) expanded blastocysts were transferred to synchronized recipients (10/horn) and were collected by uterine flushing 7 days after transfer (Day 14). Recovered embryos were measured using Motic Image Plus software and evaluated for presence and size of embryonic disc (ED). A trophoblast biopsy was removed and stored for gene expression analysis. For the molecular profile evaluation of Day 7 and Day 14 in vivo and in vitro embryos, 8 genes related with placentation, implantation, oxidative stress, and glucose metabolism (PLAC8, CD9, GLUT-1, GLUT-3, KRT8, MnSOD, HSP70, and INFT, respectively) were quantified by RT-qPCR using ΔΔCT method and CYC-A gene as endogenous control. The recovery rate of Day 14 embryos, analyzed by chi-square test, was higher (P < 0.05) for in vitro than for in vivo embryos, being 50.0% (64/128) and 38.6% (53/137), respectively. No differences (P > 0.05; t-test) were observed in embryo length when comparing Day 14 in vitro (19.1 ± 2.4 mm) and in vivo embryos (24.2 ± 3.7 mm). ED was detected in 25% (16/64) of in vitro and in 26% (14/53) of in vivo embryos. No differences were found (P > 0.05; t-test) in diameter between the two types of embryos (0.3 ± 0.0 mm/in vitro and 0.3 ± 0.0 mm/in vivo). Regarding gene expression, Day 7 IVP embryos showed higher (P < 0.05, Mann–Whitney test) expression of HSP70 and SCL2A1 than in vivo embryos. However, at Day 14 no differences between embryos were observed in transcript levels for any of the studied genes. Therefore, the present study showed that although differences in Day 7 in vitro embryos were observed at the molecular level compared to in vivo counterpart, after transfer to the uterine environment, they showed similar morphology and gene expression profile. These results highlight the importance of evaluating embryos produced by assisted reproductive techniques in later stages of development to have a more precise evaluation of their quality.
Financial support: Embrapa, CNPq, CAPES.
130 EFFECT OF HYALURONIC ACID SUPPLEMENTATION ON OVINE EMBRYO DEVELOPMENT IN VITRO AND ON SURVIVAL AFTER VITRIFICATION
Authors: Kelly J. M. , Kleemann D. O. , Walker S. K.
Published: December 2012

Studies have shown that supplementation with hyaluronic acid (HA), a glycosaminoglycan found in mammalian follicular, oviduct, and uterine fluids, improves in vitro development and post-thaw survival of bovine embryos. In this study, we examined the effect of HA supplementation on ovine embryo development and on survival after vitrification using the minimum volume cooling (MVC) cryotop method. Abattoir sourced ovine oocytes were in vitro matured and fertilized as per routine procedures (Walker et al. 1996 Biol. Reprod. 55, 703–708). In Experiment 1 (5 replicates), presumptive zygotes were randomly allocated to IVC medium supplemented with 0.8 mg mL–1 BSA, amino acids, and 0, 0.5, 1.0, 1.5 or 2.0 mg mL–1 HA. Cleavage rates were recorded and blastocyst development evaluated on Day 7 (Day 0 = day of IVF). In Experiment 2 (3 replicates), presumptive zygotes were placed in in vitro culture (IVC) medium with or without 1.0 mg mL–1 HA. Embryos were vitrified using the MVC cryotop method (Kelly et al. 2004 Reprod. Fert. Dev. 16, 172) on either Day 5 (morula–blastocyst stages), Day 6 (compact morula–hatching blastocyst stages), or Day 7 (blastocyst–hatching blastocyst stages). Vitrified embryos were thawed 7 days later and placed into IVC medium. Embryo survival (assessed by blastocoele re-expansion) and hatching rates were recorded on Day 8. Variables were assessed using procedure CATMOD in SAS (SAS Institute Inc., Cary, NC, USA). In Experiment 1, the addition of HA did not affect cleavage or blastocyst formation rates but hatching rates were significantly (P < 0.05) improved at concentrations of 0.5 to 1.5 mg mL–1 (Table 1). In Experiment 2, HA supplementation (1.0 mg mL–1) compared with control medium did not affect cleavage (96.8 and 97.1%, respectively) or blastocyst formation rates (68.6 and 70.2%, respectively). HA significantly (P < 0.05) improved survival after thawing of embryos vitrified on Day 5 (100 v. 85.6%, n = 85 and 90). However no effect was observed when embryos were vitrified on either Day 6 (97.8 v. 97.8%, n = 91 and 92) or Day 7 (96.7 v. 97.9%, n = 92 and 94). Within day, HA supplementation did not affect hatching rate compared with control medium (Day 5, 54.1 v. 53.2%; Day 6, 62.9 v. 65.6%; Day 7, 71.9 v. 62.0%, respectively). These results demonstrate that HA supplementation of IVC medium significantly improves hatching rate of ovine embryos and we speculate that this improvement may correlate with comparable improvements in pregnancy rates after transfer. Hyaluronic acid binds to CD44, a glycoprotein expressed on the surface of preimplantation ovine embryos and shown to play a role on embryo development (Luz et al. 2012 Genet. Mol. Res. 11, 799–809). Hyaluronic acid plays a role in cell migration and we suggest that, in the early blastocyst, it affords an advantage to the trophectoderm cells.

Table 1.Effect of hyaluronic acid (HA) supplementation in IVC medium on cleavage, blastocyst, and hatching rates
143 EFFECTS OF BOAR SEMINAL PLASMA IN IN VITRO CULTURE OF PORCINE EMBRYOS
Authors: Moon J. H. , Kim S. J. , Kang J. T. , Park S. J. , Choi J. Y. , Saadeldin I. M. , Oh H. J. , Park J. E. , Song K. Y. , Kim M. J. , Kim G. A. , Park E. J. , Choi J. , Jang G. , Lee B. C.
Published: December 2012

Seminal plasma consisting of carbohydrates, proteins, and lipids not only serves as a nutritive and protective medium for sperm cells but also play a pivotal role in inducing the tolerance to pre-existing immune cells as well as improving the intra-uterine conditions for implantation of fertilized embryos (Guerin et al. 2009 Hum. Reprod. Update 15, 517–535). However, the effects of seminal plasma in in vitro culture of fertilized embryos are unknown. In the present study, the seminal plasma was separated from the second fraction of a normal farm boar (n = 1) by centrifugation and filtered seminal plasma was stored at –30°C until use. In a preliminary experiment, the optimal activity of seminal plasma was evaluated by incubating the embryos for different time intervals. To investigate the developmental rates, electrically (EA) (triplicates, n = 490) or chemically (CA) (quintuplicates, n = 599) activated 2-day-old porcine embryos were incubated for 3 h in PZM-5 medium (Funakoshi Co., Tokyo, Japan, Catalog no. IFP0410P) containing 0% (EA: n = 122 and CA: n = 152), 0.1% (EA: n = 123 and CA: n = 148), 0.5% (EA: n = 122 and CA: n = 150), or 1% (EA: n = 123 and CA: n = 149) seminal plasma. Similarly, the developmental rate of chemically activated 2-day-old somatic cell nuclear transferred porcine embryos (quadruplicates, n = 239) was studied after incubation with 0% (n = 119) or 0.1% (n = 120) seminal plasma for 3 h. A significant difference was noticed only in the rate of blastocyst formation in the chemically activated embryos treated with 0.1% seminal plasma (31.7 v. 24.8% in the 0% group, ANOVA; P < 0.05; Prism5, GraphPad Software Inc., La Jolla, CA, USA). None of the treatments showed a significant effect on the cleavage rate and cell numbers of blastocysts. In conclusion, the seminal plasma did not show any harmful effect on early embryos development. Furthermore, the seminal plasma (0.1%) improved the rate of blastocyst formation among the chemically activated nuclear transferred embryos. The results of this preliminary study suggest that the addition of seminal plasma during embryo transfer could increase the rate of pregnancy in pig.
This study was supported by MKE (#10033839-2012-21), IPET (#311011-05-1-SB010), the Research Institute for Veterinary Science, and TS Corporation.
152 EVALUATION OF POMEGRANATE (PUNICA GRANATUM) AS REPLICATION INHIBITOR OF BoHV-1 COLORADO STRAIN DURING IN VITRO MATURATION OF INFECTED BOVINE OOCYTES
Authors: Palazzi E. G. , Gonçalves R. F. , Hansen D. , Pituco E. M. , Felicio J. D'A. , de Almeida M. L. , Alves M. F. , de Campos Nogueira A. H. , Sturaro L. G. R. , D'Angelo M.
Published: December 2012

The widespread use of biotechnologies in animal breeding in Brazil has enabled greater control over the transmission of pathogens. Nevertheless, disease transmission continues to be a significant concern and justifies the search for better control agents. The objective of this study was to evaluate whether the detrimental effects of the experimental infection of oocytes with BoHV-1 (Colorado strain, 108 TCID 50 mL–1) during in vitro maturation (24 h) could be reduced by the viral inhibitor ethanolic extract of Punica granatum (PG). Cattle ovaries were obtained from a local slaughterhouse (unknown breed), and transported to the laboratory. Cumulus–oocyte complexes (COC) were aspirated from follicles and allocated into four groups, which were exposed to: 10 µL of sterile physiological solution (G1 [control], n = 125); 10 µL of PG extract in 0.01% sterile saline solution (G2, n = 149); 10 µL of BoHV-1 virus (G3, n = 151); or 10 µL of PG extract in 0.01% and 10 µL of BoHV-1 virus (G4, n = 144). All groups were matured in 100 mL of IVM for 24 h at 37.5°C, 5% CO2 in air. After in vitro maturation, we evaluated COC expansion and presence of a polar body by optical microscope, and viral replication by titration (Reed and Muench test) after 72 h co-culture with Madin-Darby bovine kidney (MDBK) cells. Differences among groups in maturation rates were compared by chi-square test, and in titration by t-test. The G1, G2, and G4 showed steady expansion of the cumulus cells and ooplasm with uniform appearance. The G3 did not show expansion of the cumulus cells and ooplasma showed degenerative appearance. The maturation rates were as follows: G1 52% (65/125); G2 45.63% (68/149); G3 27.81% (42/151); G4 41.66% (60/144). We used the χ2 test (P ≤ 0.05) for the rate of maturation and the t-test (P ≤ 0.05) for the titration data. A reduction (P ≤ 0.05) in maturation rate was observed in G3, when compared to the G1 [control]. Oocytes exposed to BoHV-1 virus and matured in the presence of PG extract (G4) had lower (P ≤ 0.05) rates of viral replication than those matured in the absence of PG (G3). These results support the conclusion that PG extract reduces the viral rate of replication without interfering in oocyte maturation, and may be an alternative to sanitary control protocols.
154 EMBRYO RESPONSE OF SUPEROVULATED HOLSTEIN HEIFERS INSEMINATED WITH X-SORTED FROZEN SPERM
Authors: Fuentes S. , Liebana E. , de la Fuente J.
Published: December 2012

A study was conducted within a commercial embryo transfer program to assess the appropriateness of X-sorted semen in superovulated heifers. Commercial X-sorted semen frozen at a dose of 2 million sperm/straw and unsorted semen frozen at a dose of 20 to 30 million sperm/straw was used in donors according to the owner’s genetic criteria. One-hundred 15- to 18-month-old Holstein heifers were superovulated as follows: Animals received a CIDR device (Pfizer Animal Health, New York, NY, USA) for 13 days. At the time of CIDR insertion and 5 days later, heifers received 2 mL (0.15 mg) of d-cloprostenol (Veteglan, Laboratorios Calier, Spain). Gonadotropin-releasing hormone (GnRH), 200 mcg IM (Gonasyl, SIVA, Spain) was administered twice, 36 hours after the second prostaglandin (PGF) and 24 hours after CIDR removal. Superovulatory treatments with commercial FSH (Pluset, Laboratorios Calier) were initiated 36 hours after the first GnRH, with a total dose of 11.5 mL (575 IU), given twice daily in 10 daily decreasing-dose injections. Luteolysis was induced with 0.15 mg PGF given twice, 12 h before and at the time of CIDR removal. Heifers were randomly assigned to two groups for insemination in the uterine body: Group A (n = 31) heifers were inseminated with 5 straws of X-sorted semen in three inseminations (2 straws at 12 h, 2 straws at 18 h, and 1 straw at 24 h after the last GnRH); Group B (n = 69) heifers were inseminated twice with unsorted semen (one straw at each of 12 and 24 h after the last GnRH). A total of 614 embryos (153 in Group A and 461 in Group B) were collected and frozen. There were no significant differences between groups (Mann–Whitney rank sum test, SigmaStat, Systat Software Inc., San Jose, CA, USA) in the embryo production obtained (Table 1). Eighty-four embryos frozen with ethylene glycol were thawed and transferred; 34 from Group A, where all births were females and 50 from Group B with a 0.54 sex ratio. Pregnancy rates were 55 and 54% in Groups A and B, respectively. In conclusion, with the superovulation and AI schedule utilized in this experiment, the use of X-sorted semen in heifers can achieve reproductive outcomes similar to those obtained with unsexed semen, with the benefit of an increase in females offspring.

Table 1.Mean number of recovered ova/embryos, transferable embryos, and unfertilized ova obtained in superovulated Holstein heifers inseminated with X-sorted (Group A) and unsorted (Group B) semen
156 THE USE OF THE DG29™ ENZYME-LINKED IMMUNOSORBENT ASSAY KIT TO PREDICT PREGNANCY PRIOR TO EMBRYO TRANSFER IN LACTATING HOLSTEIN COWS
Authors: Nakamura Y. , Ideta A. , Shirasawa A. , Hayama K. , Sakai S. , Urakawa M. , Imakawa K. , Aoyagi Y.
Published: December 2012

Evaluation of postpartum fertility in cows is important for the efficient management of reproduction. DG29™ enzyme-linked immunosorbent assay (ELISA) kit (Conception, Animal Reproduction Technologies, Canada) measures the level of pregnancy–related glycoproteins in blood that are linked to pregnancy in the bovine species. The proteins are known to persist in the postpartum period. Here, we investigated whether the postpartum fertility in Holstein dairy cows can be evaluated through the use of the DG29 kit. We confirmed that genital organs of lactating Holstein cows (n = 119, from Days 56 to 688 postpartum) were normal by a 5.0/7.5-MHz linear array transducer (Tringa, Pie Medical Equipment B.V., Maastricht, The Netherlands), then a progesterone releasing intravaginal device (PRID; CEVA Sante Animale, Libourne, France) was inserted (Day 0) and maintained for 9 days. On Day 7, PGF2α was administered (2 mL Dalmazine, Kyoritsu Seiyaku, Tokyo, Japan). Blood samples were collected from the tail vein or artery into vacuum tubes at the time of PRID insertion. Serum was separated and stored at –30°C until the ELISA was performed. Oestrus (Day 0) was detected by visual observation. Fresh embryos recovered from Japanese Black cows were transferred to 119 recipient cows in various parities (primiparous = 70, biparous = 27, and multiparous = 22) on Days 6 to 8 of oestrous cycle. Pregnancy was diagnosed between Days 40 to 60 by transrectal ultrasonography. The statistical significance of any differences between various parities was assessed by chi-squared and Student’s t-tests. The pregnancy rate was higher for primiparous cows than for biparous and multiparous cows (64.3, 55.6, and 54.5%, respectively), while concentrations of the pregnancy-related glycoproteins in primiparous cows (135.0 ± 29.8 pg mL–1) were significantly lower than those of biparous (389.4 ± 175.9 pg mL–1) and multiparous cows (399.2 ± 203.1 pg mL–1, mean ± SEM; P < 0.05). In primiparous and multiparous cows, the concentrations of pregnancy-related glycoproteins were significantly lower in pregnant cows compared with nonpregnant cows (primiparous: 81.1 ± 29.9 v. 232.6 ± 59.8 pg mL–1; P < 0.05; multiparous: 20.8 ± 16.2 v. 853.4 ± 411.5 pg mL–1; P < 0.05). However, there was no significant difference between pregnant and nonpregnant biparous cows. In conclusion, the DG29 kit may be useful for the prediction of postpartum fertility in lactating Holstein cows. Further studies are needed to test the validity of this observation by using a greater number of various parties’ cows.