bluuebunny/crossref_metadata_2025 huggingface dataset:
A pH‐sensitive, strong double‐network hydrogel: Poly(ethylene glycol) methyl ether methacrylates–poly(acrylic acid)
Authors: Naficy Sina , Razal Joselito M. , Whitten Philip G. , Wallace Gordon G. , Spinks Geoffrey M.
Published: December 2011

We here describe new double network (DN) hydrogels with excellent mechanical strength and high sensitivity to pH changes. The first polymer network has a bottle brush structure and is formed from oligo‐monomers of poly(ethylene glycol) methyl ether methacrylate (PEGMA). Poly(acrylic acid) (PAA) is used as the second network. This double network features strong intermolecular interactions between the neutral poly(ethylene glycol) (PEG) side chains of PPEGMA and the non‐ionized carboxylic acid groups of the PAA second network. When immersed in solutions with a pH below ∼4 the DN hydrogels have a low swelling ratio and are opaque as a result of solvent‐polymer phase separation driven by the formation of dense hydrogen‐bonded clusters. The compression strength (∼8 MPa) is at least 14 times higher than the analogous single networks. When immersed in solutions with a pH >4, the hydrogels are transparent and exhibit a high swelling ratio with a compression strength of ∼1 MPa. The PEG side chain length can be readily controlled without greatly altering the overall DN topology by choosing PEGMA monomers having different PEG side chain lengths. Longer PEG side branches give higher compression and tensile strengths at pH <4 when hydrogen bonded clusters form. The robust nature of these DN gels over a wide pH range may be useful for applications such as artificial muscles and controlled release devices. © 2011 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys, 2012
Auxiliaries: To's company
Authors: LEVINE ROBERT D.
Published: December 2011

In a 1982 paper in the journal Glossa, Pullum outlined a set of arguments for treating English infinitival to as a defective auxiliary verb. Twenty years later, in his entry on infinitival constructions in The Cambridge Grammar of the English Language (CGEL, 2002), Huddleston argues that several distributional facts about auxiliaries fit poorly with this hypothesis. He proposes, on the basis of significant structural parallels, that to is a subordinator (complementizer). I show that Huddleston's arguments constitute a flawed analysis in CGEL's otherwise superb coverage of English descriptive grammar, and that the facts run strongly counter to his claims, often falling out independently from generalizations about auxiliaries that Huddleston overlooks. Several of these points were anticipated in Pullum's paper, but recent research on an idiosyncratic auxiliary-specific pattern of English nonrestrictive relative clause formation provides a powerful new argument in support of the auxiliary claim. In this respect, as in all others, the assignment of to to the class of auxiliaries provides the simplest and broadest account of its syntactic behavior.
Environmental effects on coral growth and recruitment in the Caribbean
Authors: Crabbe M. James C.
Published: December 2011

Knowledge about factors that are important in coral reef growth help us to understand how reef ecosystems react following major anthropogenic and environmental disturbances. In addition, they may help the industry understand how aquarists can improve the health of their corals. I have studied environmental and climate effects on corals on fringing reefs in Jamaica. Radial growth rates (mm/yr) of non-branching corals calculated on an annual basis from 2000–2008 showed few significant differences either spatially or temporally along the north coast, although growth rates tended to be higher on reefs of higher rugosity and lower macroalgal cover. I have also reconstructed recruitment patterns, using growth modelling, for non-branching corals at sites on the north coast of Jamaica near Discovery Bay, and near Kingston Harbour, on the south coast. For all the sites, recruitment of non-branching corals was lowered due to hurricanes or severe storms. For 1560 non-branching corals at sites along the north coast of Jamaica, from Rio Bueno to Pear Tree, there was a significant difference in estimated coral recruitment in years when there were no storms or hurricanes by comparison to years when storms and hurricanes impacted the area. For 347 non-branching corals at sites in the Port Royal Cays on the south coast, there was a significant difference in estimated coral recruitment in years when there were no storms or hurricanes by comparison to years when storms and hurricanes impacted the area. Interestingly, recruitment of Siderastrea siderea on to the side of the ship channel at Rackham's Cay (~100 m from the path taken by large ships) outside Kingston Harbour had been consistent since its construction. These findings have important implications for better understanding the impacts of tropical storms on coral reefs and for aquarists to better maintain coral reef species in artificial environments.
Patterns of and hypotheses for infection-related cancers in a Chinese population with rapid economic development
Authors: CHUNG R. Y. , LEUNG G. M. , COWLING B. J. , SCHOOLING C. M.
Published: December 2011

With economic development, non-communicable diseases replace infectious diseases as the leading cause of death; how such transition occurs for infectious diseases with long latency has rarely been considered. We took advantage of a Chinese population with rapid economic development in the mid-20th century to study changing patterns of infection-related cancers. We used sex-specific Poisson regression to estimate age, period and cohort effects on adult deaths 1976–2005 from eight infection-related cancers in Hong Kong. Cervical, head and neck, and oesophageal cancers, associated with sexually transmitted infections, decreased for the first birth cohorts with sexual debut in a more developed environment. Leukaemia and non-Hodgkin's lymphoma, associated with vertically transmitted infections, decreased for the first cohorts born into a more developed environment. Birth cohort patterns were unclear for nasopharyngeal, stomach and liver cancers. Mortality rates for cancers related to early infections may depend on population history, with delayed reductions for some infection-related cancers.
On the so-called Logic of Practical Inference
Authors: Price A. W.
Published: December 2011

Different questions generate different forms of practical reasoning. A contextually unrestricted ‘What shall I do?’ is too open to focus reflection. More determinately, an agent may ask, ‘Shall I do X, or Y?’ To answer that, he may need to weigh things up—as fits the derivation of ‘deliberation’ fromlibra(Latin for ‘scales’). Ubiquitous and indispensable though this is, I mention it only to salute it in passing. Or he may ask how to achieve a proposed end: if his end is to do X, he may ask ‘How shall I do X?’ Or he may ask how to apply a universal rule or particular maxim. Aristotle supplies examples inDe Motu Animalium(7.701a7 ff.), whose wording I freely adapt to my own purposes:

A1 reasons to a necessary means to achieving an end:

I will make a cloak.

To make a cloak I must do A.

So, I will do A.
Climate change portal established
Authors: Showstack Randy
Published: December 2011

The World Bank has developed a Climate Change Knowledge Portal as a kind of “onestop shop” for climate‐related information, data, and tools. The portal provides access to global, regional, and national data and reports with an aim to providing a resource for learning about climate information and increasing knowledge on climate change—related actions. For more information, see http://sdwebx.worldbank.org/climateportal/.
Arctic Ocean tides measured by satellite
Authors: Balcerak Ernie
Published: December 2011

The Gravity Recovery and Climate Experiment (GRACE) measures Earth's gravity field by gauging the acceleration of two satellites that travel about 220 kilometers apart—slight differences in acceleration between the two satellites provide information about the distribution of mass below the satellites. To get an accurate picture of Earth's gravity field, it is necessary to know how tides are contributing to GRACE measurements. However, tidal corrections have usually been made using data from satellite altimeters that do not cover the Arctic. To get around this problem, Killett et al. developed a new method of analyzing GRACE data that enabled them to extract Arctic Ocean tides. They suggest that applying their method could help improve GRACE data processing. (Journal of Geophysical Research‐Oceans, doi:10.1029/2011JC007111, 2011)
Identifying sources of methane in the Arctic
Authors: Balcerak Ernie
Published: December 2011

Methane, a potent greenhouse gas that contributes to climate change, enters the atmosphere from a variety of sources—it can leak from industrial gas fields or pipelines, escape from submarine hydrates that decompose with warming temperatures, or be released from decaying organic matter. Methanes from different sources have different isotopic compositions, allowing researchers to identify the source of methane in the air, as Fisher et al. have done in a new study. The researchers analyzed the isotopic composition of methane in the air off Spitsbergen, Norway, in 2008 and 2009. They found that in the summer, wetlands were the dominant methane source. Methane is being released to the water column from gas hydrates on the seabed, but so far, the study indicates, this methane has not reached the atmosphere. Wetlands are likely to release more methane as temperatures warm, feeding further climate change. (Geophysical Research Letters, doi:10.1029/2011GL049319, 2011)
Time domain parallelization for computational geodynamics
Authors: Samuel Henri
Published: December 2011

I present a time domain parallelization approach for geodynamic modeling. This algorithm, named parareal, is based on the use of coarse sequential and fine parallel propagators to predict and to iteratively correct the solution of the governing equations over a given time interal. Although the method has been successfully used to solve differential equations, in various scientific areas, it has not been applied to model solid‐state convective motions relevant to the Earth and other planetary mantles. In that case, the time‐dependence of the velocity is only implicit, which requires modifications to the original algorithm. The performances of this adapted version of the parareal algorithm were investigated using theoretical model predictions in good agreement with numerical experiments. I show that under optimum conditions, the parallel speedup increases linearly with the number of processors, and speedups close to 10 were measured, using only few tens of CPUs. This parareal approach can be used alone or combined with any spatial parallel algorithm, allowing significant additional increase in speedup with increasing number of processors.
Observations of the vertical structure of turbulent oscillatory boundary layers above fixed roughness beds using a prototype wideband coherent Doppler profiler: 1. The oscillatory component of the flow
Authors: Hay Alex E. , Zedel Len , Cheel Richard , Dillon Jeremy
Published: December 2011

Results are presented from an experimental investigation of rough turbulent oscillatory boundary layers using a prototype wideband bistatic coherent Doppler profiler. The profiler operates in the 1.2 MHz to 2.3 MHz frequency band and uses software‐defined radio technologies for digital control of the frequency content and shape of the transmit pulse and for digital complex demodulation of the received signals. Velocity profiles are obtained at sub‐millimeter range resolution and 100 Hz profiling rates (each profile being an ensemble average of 10 pulse pairs). The measurements were carried out above beds of fixed sand or gravel particles, with median grain diameters of 0.37 mm and 3.9 mm, respectively, oscillating sinusoidally at a 10 s period through excursions of 0.75 m to 1.5 m. The resulting vertical profiles of horizontal velocity magnitude and phase, with the vertical axis scaled by ℓ = κu∗m/ω, are comparable to similarly scaled profiles obtained using laser Doppler anemometry by Sleath (1987) and Jensen (1988). A key objective of the comparisons between the previous experiments and those reported here was to establish how close to the bed reliable velocity measurements can be made with the sonar. This minimum distance above the bed is estimated to be 5 ± 1 mm, a value approaching the 3 to 4 mm limit set by the path of least time.
Culture Media and Methods for the Isolation of Listeria monocytogenes
Authors: Beumer R. R. , Curtis G. D. W.
Published: December 2011

The first isolation methods for the detection of Listeria spp. were based on the direct culture of samples on simple agar media, but isolation of the pathogenic Listeria monocytogenes was difficult. In time, new media were developed based on a variety of selective and elective agents in enrichment and isolation enumeration media. The recovery of low numbers of L. monocytogenes from foods and environmental samples requires the use of enrichment cultures followed by selective plating. In this chapter the development of selective media, including so-called differential media on which L. monocytogenes can be distinguished from other, non-pathogenic, species, are discussed. Little attention is paid to pre-enrichment media, recommended for the recovery of injured organisms, because in our opinion the presence of listeriae in products that receive a listericidal treatment is mainly due to post-process contamination. The introduction of chromogenic media, such as agar Listeria according to Ottoviani and Agosti (ALOA) allows the recovery of Listeria and L. monocytogenes from a variety of foods with relative ease.
Culture Media for the Detection and Enumeration of Bifidobacteria in Food Production
Authors: Barrett Eoin , Mattarelli Paola , Simpson Paul J. , W. O’Toole Paul , Fitzgerald Gerald F. , Ross R. Paul , Stanton Catherine
Published: December 2011

Bifidobacterium spp. are becoming increasingly popular in food products, in particular dairy products, due to their history of safe use and the accumulating clinical evidence for their human health-promoting properties as probiotics. If probiotic cultures are incorporated into fermented milks with a specific health claim, a minimum of 106 cfu g−1 are required and therefore reliable methods are essential to monitor their survival and viability following production and during storage. Since most commercially available probiotic products contain a number of bacterial species, it is important to use a medium which promotes the growth of the bacterial group of interest while also inhibiting the growth of starter and other probiotic bacteria present. Indeed, for any culture-dependent enumeration method, the basic prerequisite is its ability to elect and select the target microorganism from the background microflora. This review will detail the elective and selective media used to enumerate bifidobacteria in foods with emphasis on selective media, given the likelihood of water and food products having a bifidobacteria component that is not numerically dominant. Numerous selective agents used for the isolation of bifidobacteria from various environments and food samples are also discussed, as is their suitability for enumerating bifidobacteria from foods and water. It is apparent that no single medium is suitable for the enumeration of all species of bifidobacteria from water and food; however, it appears that the following media, supplemented with mupirocin, are suitable for the selective enumeration of bifidobacteria: de Man Rogosa and Sharpe agar (de Man et al., 1960) supplemented with cysteine hydrochloride, trans-galactosylated oligosaccharide propionate agar, trypticase phytone yeast or reinforced clostridial agar.
Photosynthetic Light-Harvesting Complexes
Authors: Collins Aaron M. , Wen Jianzhong , Blankenship Robert E.
Published: December 2011

The light-harvesting antenna systems found in photosynthetic organisms function to collect light and transfer energy in the photon to a reaction center, where electron transfer gives rise to long-term energy storage. The antenna systems found in different types of photosynthetic organisms adapt the organisms to very different photic environments, and almost certainly have been invented multiple times during evolution. The diverse collection of photosynthetic antenna systems is described in terms of their pigment and protein components and their organization in the photosystem. The Förster theory is described as the physical basis of energy transfer in photosynthetic antennas, although in many systems it is not adequate to describe energy transfer in complexes with closely interacting pigments. Regulatory aspects of antennas are described, including the process of non-photochemical quenching.
Shape and orientation effects on the ballistic phonon thermal properties of ultra-scaled Si nanowires
Authors: Paul Abhijeet , Luisier Mathieu , Klimeck Gerhard
Published: December 2011

The effect of geometrical confinement, atomic position, and orientation of silicon nanowires (SiNWs) on their thermal properties are investigated using the phonon dispersion obtained using a Modified Valence Force Field (MVFF) model. The specific heat (Cν) and the ballistic thermal conductance (κlbal) shows anisotropic variation with changing cross-section shape and size of the SiNWs. The Cν increases with decreasing cross-section size for all the wires. The triangular wires show the largest Cν due to their highest surface-to-volume ratio. The square wires with [110] orientation show the maximum κlbal because they have the highest number of conducting phonon modes. At the nano-scale a universal scaling law for both Cν and κlbal are obtained with respect to the number of atoms in the unit cell. This scaling is independent of the shape, size, and orientation of the SiNWs, revealing a direct correlation of the lattice thermal properties to the atomistic properties of the nanowires. Thus, engineering the SiNW cross-section shape, size, and orientation open up new ways of tuning the thermal properties in the nanometer regime.
Effect of strain and deadlayer on the polarization switching of ferroelectric thin film
Authors: Wang Jie , Xia Yifan , Chen Long-Qing , Shi San-Qiang
Published: December 2011

The effect of misfit strain and deadlayer on the polarization switching of epitaxial ferroelectric thin film is investigated using a phase field model. Simulation results show that a compressive misfit strain increases the coercive field and remanent polarization of ferroelectric thin film with deadlayers, whereas a tensile misfit strain decreases these factors. The presence of a deadlayer between the ferroelectric thin film and the electrode prevents charge compensation on the ferroelectric surface and reduces the coercive field and remanent polarization of the film. A periodic a/c/a/c multiple domain structure is found in ferroelectric thin film with tensile misfit strain when there is no deadlayer. However, when a deadlayer is present in the film, the c domains vanish and the out-of-plane component of polarization degrades. The degradation of the out-of-plane polarization makes ferroelectric thin films with tensile misfit strain and deadlayers lose their ferroelectric property in the thickness direction, which can be attributed to the combined effects of the deadlayer and tensile strain. The coercive field and remanent polarization of ferroelectric thin film decrease with the thickness of the deadlayer.
Effect of shell thickness on two-photon absorption and refraction of colloidal CdSe/CdS core/shell nanocrystals
Authors: Zhu Bao-Hua , Zhang Hui-Chao , Zhang Zong-Yan , Cui Yi-Ping , Zhang Jia-Yu
Published: December 2011

Colloidal CdSe nanocrystals (NCs) are successively overcoated with CdS monolayers (MLs), and the effective two-photon absorption (TPA) coefficient and refractive index of this series of CdS/CdSe core/shell NCs are measured by a Z-scan technique at 800 nm wavelength. The TPA and nonlinear refraction are enhanced dramatically with the CdS shell growth towards 3 MLs, but decreased with the further shell growth. The effects of surface states, local field, and intrinsic piezoelectric polarization are discussed to explain the optical nonlinearity of the colloidal core/shell NCs.
X-ray nanodiffraction of tilted domains in a poled epitaxial BiFeO3 thin film
Authors: Hruszkewycz S. O. , Folkman C. M. , Highland M. J. , Holt M. V. , Baek S. H. , Streiffer S. K. , Baldo P. , Eom C. B. , Fuoss P. H.
Published: December 2011

We present measurements of crystallographic domain tilts in a (001) BiFeO3 thin film using focused beam x-ray nanodiffraction. Films were ferroelectrically pre-poled with an electric field orthogonal and parallel to as-grown tilt domain stripes. The tilt domains, associated with higher energy (010) vertical twin walls, displayed different nanostructural responses based on the poling orientation. Specifically, an electric field applied perpendicular to the as-grown domain stripe allowed the domain tilts and associated vertical twin walls to persist. The result demonstrates that thin film ferroelectric devices can be designed to maintain unexpected domain morphologies in working poled environments.
Effects of interface roughness scattering on radio frequency performance of silicon nanowire transistors
Authors: Kim Sung Geun , Luisier Mathieu , Boykin Timothy B. , Klimeck Gerhard
Published: December 2011

The effects of an atomistic interface roughness in n-type silicon nanowire transistors (SiNWT) on the radio frequency performance are analyzed. Interface roughness scattering (IRS) is statistically investigated through a three dimensional full–band quantum transport simulation based on the sp3d5s* tight–binding model. As the diameter of the SiNWT is scaled down below 3 nm, IRS causes a significant reduction of the cut-off frequency. The fluctuations of the conduction band edge due to the rough surface lead to a reflection of electrons through mode-mismatch. This effect reduces the velocity of electrons and hence the transconductance considerably causing a cut-off frequency reduction.
Atom probe study of Cu-poor to Cu-rich transition during Cu(In,Ga)Se2 growth
Authors: Couzinie-Devy F. , Cadel E. , Barreau N. , Arzel L. , Pareige P.
Published: December 2011

Atomic scale chemistry of polycrystalline Cu(In,Ga)Se2 (CIGSe) thin film has been characterized at key points of the 3-stage process using atom probe tomography. 3D atom distributions have been reconstructed when the layer is Cu-poor ([Cu]/([Ga] + [In]) < 1), Cu-rich ([Cu]/([Ga] + [In]) > 1), and at the end of the process. Particular attention has been devoted to grain boundary composition and Na atomic distribution within the CIGSe layer. Significant variation of composition is highlighted during the growing process, providing fundamental information helping the understanding of high efficiency CIGSe formation.
Highly unidirectional Y-junction-coupled S-section quantum-dot ring lasers
Authors: Withers Nathan J. , Cao Hongjun , Smolyakov Gennady A. , Osiński Marek
Published: December 2011

Fabrication and characterization of Y-junction-coupled S-section InAs/InGaAs/GaAs quantum-dot ring lasers with high unidirectionality is reported. Stable unidirectional operation of the ring lasers is ensured by a new design that suppresses the unwanted counterpropagating modes more effectively than it was possible in the previous S-section-racetrack design.
Time- and vector-resolved Kerr microscopy of hard disk writers
Authors: Gangmei P. , Keatley P. S. , Yu W. , Hicken R. J. , Gubbins M. A. , Czoschke P. J. , Lopusnik R.
Published: December 2011

Time-resolved scanning Kerr microscopy has been used to make wafer level measurements of magnetization dynamics within the yoke and pole piece of partially built hard disk writer structures. Three Cartesian components of the vector magnetization were recorded simultaneously using a quadrant photodiode polarization bridge detector. The rise time, relaxation time, and amplitude of each component has been related to the magnetic ground state, the initial torque, and flux propagation through the yoke and pole piece. Dynamic images reveal “flux-beaming” in which the magnetization component parallel to the symmetry axis of the yoke is largest along that axis.
Leaf development, radiation interception and radiation-use efficiency of kale crops supplied with different rates of banded or broadcast phosphorus fertiliser
Authors: Chakwizira E. , Moot D. J. , Scott W. R. , Fletcher A. L. , Maley S.
Published: December 2011

Inadequate phosphorus (P) supply at crop establishment can reduce dry matter (DM) accumulation. A field experiment quantified the effects of banded or broadcast P fertiliser (0, 20, 40 or 60 kg P/ha) applied at establishment to moderately fertile soils on growth and development of ‘Regal’ kale (Brassica oleracea var. acephala L.) crops. DM yield increased from 8710 to ~11 400 kg/ha by the addition of P fertiliser but was unaffected by the method of P application. The control crops accumulated 630 kg DM/100 degree-day (degree-day-accumulated heat available for crop growth) compared with ~800 kg/100 degree-day for the P-fertilised crops. The yield response to P was caused by an increased rate of development of leaf area index (LAI) and consequently earlier canopy closure that led to higher accumulated radiation interception (RIcum). The maximum LAI for the control crops was 3.80 or 24% lower than for fertilised crops. At the final harvest total RIcum for P-fertilised crops was 22% higher than the 592 MJ/m2 for the control, and this accounted for 80% of their yield differences. Leaf appearance rates were unaffected by P supply, with a common phyllochron of 109 degree-day. There was a consistent relationship between light interception and LAI, with a critical LAI of 3.40, extinction coefficient of 0.90 and radiation-use efficiency of 1.56 g/MJ photosynthetically active radiation. Overall, these results support a starter P application of at least 20 kg P/ha at establishment to maximise yields for kale crops when initial soil Olsen P levels ranged from 9 to 17 mg/kg soil.
Intrafollicular conditions as a major link between maternal metabolism and oocyte quality: a focus on dairy cow fertility
Authors: Leroy J. L. M. R. , Rizos D. , Sturmey R. , Bossaert P. , Gutierrez-Adan A. , Van Hoeck V. , Valckx S. , Bols P. E. J.
Published: December 2011

Reduced oocyte and embryo quality are recognised as major factors in the problem of disappointing fertility in high producing dairy cows. This review aims to shed more light on the importance of the intrafollicular environment in the subfertility problem in dairy cows. Metabolic disturbances associated with negative energy balance (NEB) early postpartum are associated with ovarian dysfunction. Changes in the growth pattern of the ovarian follicle during a period of NEB can indirectly affect oocyte quality. Furthermore, a maternal metabolic disorder (linked with NEB or nutritionally induced) may alter the endocrine and biochemical composition of the follicular fluid, the micro-environment of the growing and maturing female gamete. The maturing oocyte is very sensitive to any perturbation in its direct environment and in vitro maturation models revealed that some of these metabolic changes reduce the oocyte’s developmental competence. Also, embryo quality is significantly reduced due to maturation in adverse conditions. Well balanced and timed oocyte metabolism and gene expression are crucial to safeguard an optimal oocyte development. In that perspective, metabolome and transcriptome parameters of the oocyte may serve to predict reproductive success rates. Finally, there is growing evidence that adverse conditions for oocyte growth and maturation may also jeopardise the health and performance of the offspring.
219 ISOLATION AND CHARACTERIZATION OF DOMESTIC CAT SPERMATOGONIAL CELLS
Authors: Powell R. H. , Biancardi M. N. , Pope C. E. , Leibo S. P. , Wang G. , Gómez M. C.
Published: December 2011

Spermatogonial stem cells (SSC) have the capacity for self-renewal and the potential of producing progenitor spermatogonia that will differentiate into spermatozoa. SSC transplantation may be a valuable alternative for the propagation of genetically important males and preservation of endangered wild felids, as recently demonstrated by the production of ocelot spermatozoa after the xeno-transplantation of a mixed germ cell population into the testis of a domestic cat (Silva et al. 2011 J. Androl.). SSC are in low numbers in the testis and have been isolated in different mammalian species by using specific cell surface markers; however, the expression of SSC-surface markers in feline species has not been characterised. In the present study, testes of domestic cats were obtained from veterinary clinics. The selected testes (n = 4) ranged in size from 1.3 to 2.0 cm in length. To obtain a suspension of a mixed population of spermatogonial cells, seminiferous tubules were enzymatically dissociated using two digestion steps followed by dual filtration through 100-μm and 40-μm nylon mesh filters and a final separation over a 5-layer Percoll™ density gradient (35, 30, 27.5, 25 and 20%). Spermatogonial cells were morphologically identified by their characteristic large round nucleus with a homogenous appearance in the bands formed at the 30%, 27.5% and 25% layers. The mean number of cells/testis collected was ∼13 × 106 ± 12.2, with an overall percentage of live cells of 97%. After isolation, cells were fixed with 4% PFA, blocked overnight and stained with primary antibodies specific for SSC-surface markers (CD49f, CD9, C-Kit, GFRα1, GPR125 and Thy-1) and pluripotent stage-specific embryonic antigen markers (SSEA-1 and SSEA-4). Fluorescence microscopy showed a positive expression of GFRα1, GPR125 and C-Kit, but not for CD49f, CD9, or Thy-1. It also revealed that a low number of cells were positive for SSEA-1 and SSEA-4. For further characterisation, molecular detection of the pluripotent gene Oct-4 and the germ cell-specific genes BOLL, DAZL and VASA was performed in germ cells isolated from one testis of four individuals. For RT-qPCR, the Cells-to-cDNA™ II kit (Ambion) was used to produce cDNA from an aliquot of ∼30 000 cells directly after isolation. RT-qPCR showed that none had detectable levels of Oct-4 within the range of the standard. Three of the four testes expressed all three germ cell-specific genes, BOLL, DAZL and VASA, while only VASA was detected in the remaining testis. These results suggest that cat SSCs and spermatogonial cells express some of the SSC markers tested. However, the positive expression of SSEA-1 and SSEA-4 in a low number of cells further supports the stem cell-like state of cat SSCs and that these markers can be used in dual staining for purifying cat SSCs from a mixed germ cell population by fluorescence-activated cell sorting.
220 NONHUMAN PRIMATE EMBRYONIC STEM CELLS SIMILAR TO THE BIOLOGICAL PROPERTIES OF MOUSE EMBRYONIC STEM CELLS
Authors: Kusanagi A. , Yamasaki J. , Iwatani C. , Tsuchiya H. , Torii R.
Published: December 2011

Human and mouse embryonic stem (ES) cells are derived from the inner cell mass of preimplantation blastocysts and human ES cells were long thought to be equivalent to mouse ES cells, despite clear morphological difference and different signalling pathways to maintain their pluripotency between these two ES cell types. Mouse ES cells depend on leukemia inhibitory factor (LIF) and bone morphogenic protein 4 (BMP4) signalling, whereas their human counterparts rely on basic fibroblast growth factor (bFGF) and activin A signalling. The biggest difference of two ES cells is the ability of chimera formation and mouse ES cells can contribute chimera but primate ES cells fails to do that. Monkey ES cells in primates only can be tested for chimera formation in vivo due to the ethical issue and cynomolgus monkey is the most common nonhuman primate to be used for the safety study of drug discoveries. The objective of this study was to develop novel cynomolgus monkey ES cells that have similar biological properties with mouse ES cell and our ultimate goal is to establish germline competent nonhuman primate ES cells. Ovarian stimulation and oocyte collection were carried out for the derivation of ES cells as previously described by Torii et al. Briefly, GnRH (0.9 mg/head) was administered to cynomolgus monkey and two weeks later, a micro infusion pump (iPRECIO™, Primetech Corp) contains FSH was implanted subcutaneously. Follicular aspiration was then performed 40 h after hCG injection and metaphase II oocytes were fertilized by intracytoplasmic sperm injection (ICSI). Cynomolgus monkey ES cells were then established under mouse ES cell conditions such as LIF/STAT signalling and a dome tree-dimensional (3D) morphology nonhuman primate ES cells were selected. On the other hands, ES cells that were established with the presence of basic FGF showed conventional layer-type morphology. Dome-type ES cells express pluripotent transcriptional factors such as Oct-3/4, Nonog and Sox2 as same as layer-type ES cells and both ES lines were capable of multilineage differentiations in vitro after embryoid body formation. Dome-type nonhuman ES cells can also form teratomas and differentiated into all three germ layers when grafted into immunodeficiency mice. For fluorescent gene delivery to nonhuman primate ES cells, feeder-free condition was applied and CAG-GFP vector was transfected into ES cells using Neon electroporation system (Invitrogen Inc.) for the tracing ES cells in the transplantation study. In this study, we have established dome-type ES cell lines that similar to mouse ES cells in morphology and signalling pathway. Dome-type nonhuman primate ES cells express pluripotent gene markers and prove their pluripotency both of in vitro and in vivo, in addition, these modifications would be important to create germline competent ES cells.
231 SUPEROVULATORY RESPONSE AND EMBRYO YIELD OF SANTA INES EWES SUBMITTED TO FOLLICLE STIMULATING HORMONE TREATMENTS STARTED NEAR THE TIME OF EMERGENCE OF THE FIRST AND LAST FOLLICULAR WAVE OF A PROGESTERONE-BASED PROTOCOL
Authors: Oliveira M. E. F. , D'Amato C. C. , Oliveira L. G. , Bicudo S. D. , Fonseca J. F. , Vicente W. R. R.
Published: December 2011

This study was designed to investigate if the superovulatory response and embryo yield in Santa Ines ewes are influenced by the time that FSH treatments are inititiated: near the emergence of the first or last follicular wave of a progesterone-based (P4) protocol. Days of emergence of the follicular waves was defined in a previous study that evaluated follicular dynamics in oestrus synchronization treatments (Oliveira et al. 2011 Acta Sci. Vet. 40). Twenty Santa Ines cyclic ewes were submitted to 2 superovulatory protocols according to the time that FSH treatments were initiated (G-first wave, n = 10 and G-last wave, n = 10). Thus, the FSH treatment started on Day 4 and 10 of protocol for G-first and G-last, respectively (Day 0 = onset of protocol). Ewes were treated with a P4 device (CIDR®; Pfizer-New Zealand) for 6 and 12 days for G-first and G-last, respectively. Additionally, in G-last, the CIDR was replaced by a new one on Day 7. All ewes received 2 injections of 37.5 μg of D-cloprostenol (Prolise®, Arsa-Argentina) on Day 0 and at CIDR removal. The superovulatory regimen consisted of 8 IM injections of pFSH (Folltropin®; Bioniche Animal Health, Belleville, Ontario, Canada) administrated twice daily (40, 40, 30, 30, 20, 20, 10 and 10 mg of porcine FSH). A single IM dose of 200 IU of eCG (Novormon®, Syntex, Buenos Aires, Argentina) was given concurrently with CIDR removal. Ewes were mated by a fertile ram. Embryo collections were accomplished 7 days after CIDR removal. The superovulatory response was classified in scores: (0) 4 or fewer corpora lutea (CL); (1) between 5 and 10 CL and (2) 11 or more CL. Means (± SD) were compared using Kruskal-Wallis test and percentages using Chi-square (P < 0.05). Most donors (70%, 7/10) from G-last had a superovulatory response classified as score 2 and the remainder (30%, 3/10) as score 1, whereas half of ewes from G-first were classified as score 2 and half as score 1 (P > 0.05).There was no effect between treatments (P > 0.05) for ovulation rate (G-first: 90.7 ± 10.2% vs G-last: 93.0 ± 7.2%), number of ovulations (G-first: 13.1 ± 7.3 vs G-last: 14.0 ± 5.8), or number of luteinized unovulated follicles (G-first: 1.4 ± 1.3 vs G-last: 0.9 ± 1.0). Furthermore, there was no effect between G-first and G-last (P > 0.05) in the rate of ova/embryos recovered (55.1 ± 27.1% vs 67.0 ± 19.4%), mean number of ova/embryos recovered (7.0 ± 5.5 vs 9.1 ± 3.8), number of viable embryos (3.9 ± 6.1 vs 5.7 ± 4.4), or viability rate (49.1 ± 43.7 vs 58.5 ± 34.6). In conclusion, superovulatory response and embryo yield did not differ between FSH treatments initiated during the first or last follicular wave of a progesterone-based treatment in Santa Ines ewes.
Financial support of FAPESP is acknowledged.
INDUCING PLURIPOTENCY IN RUMINANTS
Authors: Sumer Huseyin , Liu Jun , Ortega Luis Malaver , Verma Paul J.
Published: December 2011

The generation of ES cell lines in ruminants, including cattle and sheep, has been inefficient with putative ESCs undergoing rapid spontaneous differentiation. Recently direct reprogramming of rodent and primate somatic cells to induced Pluripotent Stem Cells (iPSC) is possible simply by forcing expression of a handful of transcription factors, typically OCT3/4, SOX2, KLF4 and cMYC (OSKM). Using a similar approach we have identified that addition of NANOG to the reprogramming cocktail was essential for the generation of stable bovine induced pluripotent stem cells (biPSC). Conversely, generation of reprogrammed sheep iPS (siPSC) cells was achieved by transduction of four factors (OSKM) alone. Significantly, at passage 17 the siPS cells showed silencing of the OSKM transgenes with concomitant reactivation of endogenous pluripotent genes, including OCT4, SOX2 and NANOG. The generation of stable ruminant pluripotent cell lines paves the way for the use of these cells for both biomedical research and agricultural outcomes.
44 REACTIVE OXYGEN SPECIES IN VITRIFIED BOVINE IN VITRO-MATURED OOCYTES
Authors: De Blasi M. , Rubessa M. , Albero G. , Lavrentiadou S. , Sapanidou V. , Gasparrini B. , Tsantarliotou M.
Published: December 2011

Vitrification of in vitro-matured oocytes has important applications in fertility preservation and management of genetic resources. However, despite the increasing interest, the efficiency of oocyte vitrification needs to be improved. It was demonstrated that under stressful conditions of cryopreserving pig oocytes accumulate reactive oxygen species (ROS; Gupta et al. 2010 Fertility and Sterility 93, 2602–2607). Reactive oxygen species are known to exert harmful effects such as mitochondrial damage, ATP (ATP) depletion, altered calcium oscillation during fertilization and consequently their developmental ability may be compromised (Takahashi et al. 2003 Mol. Reprod. Dev. 66, 143–152). The aim of the present study was to evaluate whether the exposure to cryoprotectants and vitrification procedure affect ROS production in bovine in vitro-matured oocytes. Abattoir-derived bovine (n = 360, over 6 replicates) cumulus oocyte complexes (COCs), were in vitro-matured. COCs were mechanically stripped of their cumulus cells by gentle pipetting, washed and divided into 3 groups: control (C; i.e. fresh non treated oocytes), toxicity (T) and vitrification (V) groups. In group V, oocytes were exposed to 10% ethylene glycol (EG) + 10% DMSO for 3 min, then to 20% EG + 20% DMSO and 0.5 M sucrose, loaded on cryotops and plunged into liquid nitrogen within 25 s. Oocytes were warmed into a 1.25 M sucrose solution for 1 min and then to decreasing concentrations of sucrose (0.625 M, 0.42 M and 0.31 M) for 30 s each. In group T, oocytes were simply exposed to the vitrification and warming solutions. ROS determination was carried out by a spectrofluorometer at 495 nm excitation and 525 nm emission. Frozen oocytes were thawed and incubated in 500 μL of TRIS-HCl 40 mM, pH 7.0 in the presence of 5 μmol L–1 of 2′,7′-dichlorfluorescein-diacetate, for 20 min at 37°C into a shaker. After incubation, the extraction was obtained by a syringe and the samples were centrifuged at 3000 rpm for 10 min at 4°C. Data were expressed as arbitrary ROS units per oocyte per min (U) and analysed by ANOVA. The results of this study showed that in bovine oocytes ROS levels tend to increase in the T and V groups compared to group C (76.0 ± 6.4, 249.9 ± 87.3 and 147.6 ± 42.6 in C, T and V groups, respectively). However, there were no statistical differences among groups and this was mainly due to the high variability recorded in both treated groups. In conclusion, these results suggest that both exposure to cryoprotectants and vitrification of in vitro-matured oocytes may influence ROS generation. However, the high variability recorded among replicates recommends further investigations.
46 PLASMINOGEN ACTIVATOR ACTIVITY IN BUFFALO IN VITRO MATURED OOCYTES AFTER VITRIFICATION-WARMING
Authors: Tsantarliotou M. , De Blasi M. , Lavrentiadou S. , Sapanidou V. , Boccia L. , Di Francesco S. , Gasparrini B.
Published: December 2011

Plasminogen activators (PA) are proteolytic enzymes that convert plasminogen into plasmin. Plasmin is involved in physiological processes such as ovulation (Liu 2004 Front. Biosci. 9, 3356–3373), cumulus cell layer expansion (Liu et al. 1986 Endocrinology 119, 1578–1587), oocyte maturation (Dow et al. 2002 Biol. Reprod. 66, 1413–1421) and fertilization (Huarte et al. 1993 Dev. Biol. 157, 539–546). Although the interest is increasing, buffalo oocyte cryopreservation is still inefficient, especially in terms of blastocyst development after IVF. The aim of the present study was to evaluate whether exposure to cryoprotectants and the vitrification procedure affect plasminogen activator activity (PAA) in buffalo in vitro-matured oocytes. A total number of 300 cumulus-oocyte complexes over 5 replicates were selected and in vitro-matured. Cumulus-oocyte complexes were mechanically stripped of their cumulus cells by gentle pipetting, washed and divided into 3 groups (20 oocytes/group, over 5 replicates). The control group consisted of fresh in vitro-matured oocytes. In the vitrification group, denuded oocytes were first exposed to 10% ethylene glycol (EG) + 10% dimethyl sulfoxide (DMSO) for 3 min, then to 20% EG + 20% DMSO and 0.5 M sucrose, loaded on cryotops and plunged into liquid nitrogen within 25 s. Subsequently, oocytes were warmed in a 1.25 M sucrose solution for 1 min and then in decreasing concentrations of sucrose (0.625 M, 0.42 M and 0.31 M) for 30 s each. In order to test cryoprotectant effects, oocytes were simply exposed to the vitrification and warming solutions (toxicity group). Surviving oocytes were extracted by a fine needle, centrifuged at 4000 rpm for 10 min and the supernatant was mixed with the reaction solution: TRIS-HCl 0.1 M, homologous plasminogen, the chromogenic substrate for plasmin S-2251 and incubated at 37°C for 30 min. The PAA levels were measured by a spectrophotometer (405 nm) expressed as Abs/20 oocytes. The data were analysed by the Kruskal-Wallis nonparametric test. Low levels of PAA were detected in the denuded oocytes of the control, toxicity and vitrification groups. No significant differences in mean PAA values were observed among the 3 experimental groups (0.017 ± 0.001, 0.018 ± 0.002 and 0.017 ± 0.001 units, in the control, toxicity and vitrification groups, respectively). In conclusion, cryoprotectants and the vitrification procedure do not affect the proteolytic activity linked to plasmin in in vitro-matured buffalo oocytes. The results show that the vitrification/warming procedure does not exert an effect on in vitro-matured buffalo oocytes in terms of PAA generation, a parameter that plays an important role in fertilization and in vitro embryo development. Further studies are needed to identify factors affecting the efficiency of oocyte cryopreservation.
76 DECIPHERING G-PROTEIN COUPLED RECEPTOR NETWORK DURING BOVINE CONCEPTUS DEVELOPMENT AND MATERNAL RECOGNITION OF PREGNANCY
Authors: Mamo S. , Mehta J. P. , McGettigan P. , Lonergan P.
Published: December 2011

G-protein coupled receptors (GPCR) comprise a large protein family of transmembrane receptors that sense molecules outside the cell to activate the signal transduction pathways inside and, ultimately, the cellular responses. They have a central role in various physiological systems that places them at the forefront of many drug target programs and perturbation in their activity has been cited in many diseases, including infertility (Jean-Alphonse and Hanyaloglu 2011 Mol. Cell. Endocrinol. 331, 205–214). However, little information is available on the types of GPCR and their expression in the bovine conceptus during maternal recognition of pregnancy. The aim of this study was, therefore, to take advantage of the bovine conceptus global gene expression profiles, particularly to examine the expression of various transcripts of GPCR during the window of maternal recognition of pregnancy. Global transcriptome profiling of the bovine conceptuses at 5 key stages of pre- and peri-implantation growth (Day 7, 10, 13, 16 and 19) was carried out using in vivo-derived embryos and state-of-the-art RNA sequencing techniques, following the Illumina standard procedures for library preparation and genome analyzer sequencing (Illumina, San Diego, CA, USA). Following various quality control procedures, the transcripts were mapped to the bovine genome. ANOVA was carried out to detect the differentially regulated transcripts between any 2 developmental stages followed by self-organizing map clustering to identify genes with a similar temporal expression pattern. Those transcripts differentially regulated on Day 16 (the day of maternal recognition of pregnancy in cattle) were screened and submitted to Ingenuity pathway analysis (Ingenuity Systems Inc., Redwood City, CA). Subsequently, the expression profiles of the transcripts in the GPCR network were examined across the 5 key developmental stages. Pathway analysis detected various transcript networks, including the GPCR network, comprising a large number of transcripts (∼30). These include F2RL2, GCGR, VIPR1, LPAR6, RAC2, LPHN3, PTGFR, PTH1R, CNR2, CD97, APLNR and various other GPCR family genes. Analysis of expression profiles across the 5 development stages revealed that these receptors were significantly (P < 0.05) upregulated at Day 16, compared with other time points. The significantly higher expression pattern of these GPCR during this critical stage of pregnancy may suggest an important role during this period of maternal recognition and establishment of pregnancy that contributes to embryo-maternal crosstalk during this critical period.
Supported by Science Foundation Ireland (07/SRC/B1156).