bluuebunny/crossref_metadata_2025 huggingface dataset:
Improving the performance of organic thin film transistors formed on a vacuum flash-evaporated acrylate insulator
Authors: Ding Z. , Abbas G. A. , Assender H. E. , Morrison J. J. , Sanchez-Romaguera V. , Yeates S. G. , Taylor D. M.
Published: December 2013

A systematic investigation has been undertaken, in which thin polymer buffer layers with different ester content have been spin-coated onto a flash-evaporated, cross-linked diacrylate gate-insulator to form bottom-gate, top-contact organic thin-film transistors. The highest device mobilities, ∼0.65 cm2/V s and ∼1.00 cm2/V s for pentacene and dinaphtho[2,3-b:2′,3′-f]-thieno[3,2-b]thiophene (DNTT), respectively, were only observed for a combination of large-grain (∼1–2 μm) semiconductor morphology coupled with a non-polar dielectric surface. No correlation was found between semiconductor grain size and dielectric surface chemistry. The threshold voltage of pentacene devices shifted from −10 V to −25 V with decreasing surface ester content, but remained close to 0 V for DNTT.
Quantifying thickness-dependent charge mediated magnetoelectric coupling in magnetic/dielectric thin film heterostructures
Authors: Zhou Z. , Nan T. X. , Gao Y. , Yang X. , Beguhn S. , Li M. , Lu Y. , Wang J. L. , Liu M. , Mahalingam K. , Howe B. M. , Brown G. J. , Sun N. X.
Published: December 2013

Precise quantification of the magnetoelectric coupling strength in surface charge induced magnetoelectric effect was investigated in NiFe/SrTiO3thin film heterostructures with different ultra-thin NiFe thicknesses through voltage induced ferromagnetic resonance. The voltage induced ferromagnetic resonance field shifts in these NiFe/SrTiO3thin films heterostructures showed a maximum value of 65 Oe at an intermediate NiFe layer thickness of ∼1.2 nm, which was interpreted based on the thin film growth model at the low-thicknesses and on the charge screening effect at large thicknesses. The precise quantification and understanding of the magnetoelectric coupling in magnetic/dielectric thin filmsheterostructures constitute an important step toward real applications.
Room-temperature observations of the weak localization in low-mobility graphene films
Authors: Han Junhao , Wang Shanyue , Qian Di , Song Fengqi , Wang Xinran , Wang Xuefeng , Wang Baigeng , Han Min , Zhou Jianfeng
Published: December 2013

We report room-temperature observations of the quantum conductance corrections caused by the weak localization in graphene films synthesized using solid-state-source chemical vapor deposition. Both Raman spectroscopy and Hall measurements showed strong disorder in the samples with a low mobility of ∼430 cm2/V s. The emergence of weak localization at room temperature arises from the competition between the valley-dependent scattering and the thermal dephasing in such low-quality samples, although quantum effects normally appear in the samples that have an ideal structure at cryogenic temperatures. The large disorder in our low-mobility samples unexpectedly preserved the quantum mechanical weak localization.
Barrier performance optimization of atomic layer deposited diffusion barriers for organic light emitting diodes using x-ray reflectivity investigations
Authors: Singh Aarti , Klumbies Hannes , Schröder Uwe , Müller-Meskamp Lars , Geidel Marion , Knaut Martin , Hoßbach Christoph , Albert Matthias , Leo Karl , Mikolajick Thomas
Published: December 2013

The importance of O3 pulse duration for encapsulation of organic light emitting diodes (OLEDs) with ultra thin inorganic atomic layer deposited Al2O3 layers is demonstrated for deposition temperatures of 50 °C. X-ray reflectivity (XRR) measurements show that O3 pulse durations longer than 15 s produce dense and thin Al2O3 layers. Correspondingly, black spot growth is not observed in OLEDs encapsulated with such layers during 91 days of aging under ambient conditions. This implies that XRR can be used as a tool for process optimization of OLED encapsulation layers leading to devices with long lifetimes.
Electron transfer at the contact between Al electrode and gold nanoparticles of polymer: Nanoparticle resistive switching devices studied by alternating current impedance spectroscopy
Authors: Ouyang Jianyong
Published: December 2013

Electron transfer at the contact between an Al electrode and Au nanoparticles of polymer:nanoparticle devices is studied by ac impedance spectroscopy. The devices have a polystyrene layer embedded with Au nanoparticles capped with conjugated 2-naphthalenethiol sandwiched between Al and MoO3/Al electrodes, and they exhibit electrode-sensitive resistive switches. The devices in the pristine or high resistance state have high capacitance. The capacitance decreases after the devices switch to a low resistance state by a voltage scan. The change in the capacitance is attributed to the voltage-induced change on the electronic structure of the contact between the Al electrode and Au nanoparticles.
Characterization of metal contacts for two-dimensional MoS2 nanoflakes
Authors: Walia Sumeet , Balendhran Sivacarendran , Wang Yichao , Ab Kadir Rosmalini , Sabirin Zoolfakar Ahmad , Atkin Paul , Zhen Ou Jian , Sriram Sharath , Kalantar-zadeh Kourosh , Bhaskaran Madhu
Published: December 2013

While layered materials are increasingly investigated for their potential in nanoelectronics, their functionality and efficiency depend on charge injection into the materials via metallic contacts. This work explores the characteristics of different metals (aluminium, tungsten, gold, and platinum) deposited on to nanostructured thin films made of two-dimensional (2D) MoS2 flakes. Metals are chosen based on their work functions relative to the electron affinity of MoS2. It is observed, and analytically verified that lower work functions of the contact metals lead to smaller Schottky barrier heights and consequently higher charge carrier injection through the contacts.
Upward ferroelectric self-poling in (001) oriented PbZr0.2Ti0.8O3 epitaxial films with compressive strain
Authors: Luo Ying , Li Xueyan , Chang Lei , Gao Wenxiu , Yuan Guoliang , Yin Jiang , Liu Zhiguo
Published: December 2013

Upward self-poling phenomenon was observed in PbZr0.2Ti0.8O3 ferroelectric films which were grown on (001) SrTiO3 substrate with either p-type La0.7Sr0.3MnO3 or n-type SrRuO3 buffered layer, or on n-type (001) Nb-SrTiO3 substrate. Both upward self-poling and epitaxial strain are strong in the super-thin PbZr0.2Ti0.8O3 epitaxial films, while they become weak and finally disappear in thick epitaxial films or thin epitaxial films with high-density oxygen vacancies. Besides, both of them disappear in PbZr0.2Ti0.8O3 polycrystalline films on Pt/TiO2/SiO2/Si substrate. Therefore, the main origin of upward self-poling is epitaxial strain rather than p-n/Schottky junction or charged vacancies in PbZr0.2Ti0.8O3 epitaxial films here.
Detection of Trace Amounts of Water in Organic Solvent by 8-Hydroxypyrene-1,3,6-Trisulfonic Acid Trisodium Salt
Authors: Bobe Sharad R. , Raynor Aaron Michael , Bhosale Sheshanath V. , Bhosale Sidhanath V.
Published: December 2013

A colorimetric and fluorescent sensor was developed based on 8-hydroxy-1,3,6-pyrenetrisulfonic acid trisodium salt (HPTS) for the detection of trace amounts of water in polar organic solvents. Fluorescence and UV-visible absorption spectra of HPTS were measured in a variety of organic solvents (DMF, DMSO, ethanol, methanol). Fluorescence maxima comparable with maximum/minimum ratios were determined by using UV-visible absorption spectroscopy. The HPTS sensor exhibits high sensitivity for water with a detection limit as low as 0.0001 to 0.0005 wt-%. In addition, naked-eye inspection of solutions of HPTS sensor in organic solvents before and after addition of water showed dramatic changes in colour from blue to green. These findings can be applied in the use of HPTS as a molecular probe for trace amounts of water in organic solvents.
Role of stem cells in large animal genetic engineering in the TALENs–CRISPR era
Authors: Park Ki-Eun , Telugu Bhanu Prakash V. L.
Published: December 2013

The establishment of embryonic stem cells (ESCs) and gene targeting technologies in mice has revolutionised the field of genetics. The relative ease with which genes can be knocked out, and exogenous sequences introduced, has allowed the mouse to become the prime model for deciphering the genetic code. Not surprisingly, the lack of authentic ESCs has hampered the livestock genetics field and has forced animal scientists into adapting alternative technologies for genetic engineering. The recent discovery of the creation of induced pluripotent stem cells (iPSCs) by upregulation of a handful of reprogramming genes has offered renewed enthusiasm to animal geneticists. However, much like ESCs, establishing authentic iPSCs from the domestic animals is still beset with problems, including (but not limited to) the persistent expression of reprogramming genes and the lack of proven potential for differentiation into target cell types both in vitro and in vivo. Site-specific nucleases comprised of zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regulated interspaced short palindromic repeats (CRISPRs) emerged as powerful genetic tools for precisely editing the genome, usurping the need for ESC-based genetic modifications even in the mouse. In this article, in the aftermath of these powerful genome editing technologies, the role of pluripotent stem cells in livestock genetics is discussed.
109 DEVELOPMENT OF A SPERM CRYOPRESERVATION PROTOCOL FOR THE ARGENTINE BLACK AND WHITE TEGU (TUPINAMBIS MERIANAE)
Authors: Young C. , Curtis M. , Ravida N. , Mazotti F. , Durrant B.
Published: December 2013

Only 891 of the approximately 5600 lizard species have been evaluated by the International Union for Conservation of Nature (IUCN). Of those, at least one-third are threatened with extinction. However, there is no organised effort to preserve their genetic diversity through semen banking. As part of an invasive species monitoring program, Argentine black and white tegus were captured in the Florida Everglades. Following postmortem examination, sperm was collected by flushing the vas deferens and used as a model for the development of sperm cryopreservation protocols for related endangered lizards. Initial motility score (IMS; % motile × speed of progression2), plasma membrane integrity (IPL) and acrosome integrity (IAC) were recorded before freezing. Sperm was extended in TES and Tris (TEST)-yolk buffer with a final glycerol or dimethyl sulfoxide (DMSO) concentration of 8, 12, or 16%, and frozen in vials at 0.3, 1, or 6.3°C min–1. Vials were thawed at 37°C for 90 s. Cryoprotectant (CPA) was removed by centrifugation and resuspension of the sperm pellet in M199, at which time (T0) all variables were assessed and expressed as the percentage of initial (%IMS, %IPL, and %IAC). Statistical tests included multivariate ANOVA (MANOVA) and Student's t-test. Over all CPA concentrations and freeze methods, DMSO was significantly better than glycerol in maintaining %IMS (P = 0.01; 37.32 ± 3.5 and 25.44 ± 3.09, respectively) and %IAC (P < 0.01; 81.45 ± 3.45 and 22.99 ± 3.03, respectively). The 2 CPA were equally successful in protecting %IPL (P = 0.77; 56.61 ± 5.62 and 54.42 ± 4.93, respectively). The slowest freeze rate of 0.3°C min–1 was more successful than 1 and 6.3°C min–1 in preserving %IMS (P = 0.01; 37.85 ± 3.29, 26.03 ± 4.45, and 21.91 ± 4.45, respectively) and %IPL (P < 0.01; 77.43 ± 2.54, 27.99 ± 3.44, and 42.32 ± 3.44, respectively). The %IAC was not significantly affected by freeze rate (P = 0.14; 58.06 ± 6.89, 36.14 ± 9.33, and 42.99 ± 9.33, respectively). The interaction between CPA and freeze method affected %IMS (P < 0.01) and %IAC (P < 0.01), but did not affect %IPL (P = 0.28). All variables were affected (P < 0.05) by concentration of cryoprotectant as well as the interaction between freeze method and cryoprotectant concentration. To simplify these analyses and to determine the best overall freeze method for this species, a sperm quality index (SQI) was calculated, giving equal weight to each of the 3 measured indicators of cryosurvival. Table 1 depicts the product of %IMS, %IPL, and %IAC for each treatment. Because there were significant interactions between treatment parameters, each treatment was compared with all others (a–f). The SQI analysis revealed that tegu sperm frozen at 0.3°C min–1 in 8% DMSO exhibited a significantly higher post-thaw viability compared with all other treatments.

Table 1.Sperm quality index (SQI) of thawed tegu sperm
130 REGULATION OF CALCIUM TRANSPORT CHANNELS IN THE DUODENUM, KIDNEY, AND PLACENTA OF A CATECHOL-O-METHYLTRANFERASE-INHIBITED MOUSE MODEL
Authors: Yang H. , Shin E. K. , Jeung E. B.
Published: December 2013

Preeclampsia is a pregnancy-specific disease characterised by concurrent development of hypertension, proteinuria, and oxidative stress in the placenta. Preeclampsia-like genetic models were also developed by modification of preeclampsia-related genes, such as catechol-O-methyltranferase (COMT). To investigate the pathophysiological conditions associated with preeclampsia, we divided pregnant mice into 4 groups (n = 20): vehicle, COMT inhibitor, vehicle plus 2% supplemental calcium diet, and COMT inhibitor plus 2% supplemental calcium diet. The pregnancy mice were treated with the specific COMT inhibitor (ro41–0960, 2.5 mg kg–1) from gestation Day 15 to 19. In parallel with treatment, the pregnant mice were fed with the 2% supplemental calcium diet or the normal diet for 5 days. All mice were sacrificed at 24 h after last injection. Total RNA was extracted using Trizol regent according to the manufacturer's instructions. Using a quantitative reverse transcription-PCR (RT-qPCR), we measured the mRNA levels of several calcium transporters (TRPV5, TRPV6, PMCA1, and CaBP-9k) in the placenta, duodenum, and kidney. Placental TRPV5, TRPV6, and PMCA1 mRNA was significantly reduced by the COMT inhibitor, and the reduced PMCA1 mRNA was restored by calcium supplementation. Duodenal TRPV5, TRPV6, and PMCA1 mRNA was decreased in the COMT-inhibited mice, and slightly recovered after calcium supplementation. Although renal TRPV5, TRPV6, and PMCA1 mRNA was also decreased by COMT inhibition, their expression were regained by calcium supplementation compared with those of the control group. Taken together, these results indicate that physiological changes of the response to COMT inhibitor were similar to symptoms of preeclampsia, which may be related to disturbance of calcium metabolism during pregnancy. All statistical analyses were performed using Graphpad software (GraphPad, San Diego, CA, USA).
152 MITOCHONDRIAL DYNAMICS IN PRE- AND POSTPUBERTAL PIG OOCYTES BEFORE AND AFTER IN VITRO MATURATION
Authors: Pedersen H. S. , Løvendahl P. , Nikolaisen N. K. , Holm P. , Hyttel P. , Nyengaard J. R. , Chen F. , Callesen H.
Published: December 2013

Oocytes from prepubertal (PRE) or postpubertal (POST) pigs are used in, for example, somatic cell nuclear transfer and in vitro fertilization. Here we describe mitochondrial dynamics in pig oocytes of different sizes before and after in vitro maturation (IVM), isolated from PRE or POST animals. In PRE oocytes, inside-zona pellucida diameter was measured before and after IVM (μm; small: ≤110, medium: >110, large: ≥120) and used for evaluation of (1) mitochondrial numbers before maturation and (2) mitochondrial morphology and location before and after maturation in comparison with POST oocytes. Oocytes were processed for transmission electron microscopy (Acta Anat. 129:12). For assessment of mitochondrial numbers, paired dissector sections were collected at uniform intervals throughout the oocyte, and in each set of dissector sections a known area fraction was sampled for mitochondrial counting in physical dissectors (J. Microsc. 134:127). Total number of mitochondria was calculated, and oocyte volume was estimated by Cavalieri estimator (J. Microsc. 147:229). Data were analysed by ANOVA. Mitochondrial morphology was classified as elongated, round, shell-like, or compartmentalized; mitochondrial cristae as transverse or peripheral; and mitochondrial location as cortical, subcortical, or central. Before IVM, small PRE presented elongated and round mitochondria with transverse cristae; medium and large PRE presented round mitochondria with peripheral and transverse cristae; POST presented round mitochondria with peripheral cristae in all cases. After IVM, small and medium PRE had round mitochondria with peripheral cristae; medium PRE and POST had shell-like mitochondria with peripheral cristae; large PRE had compartmentalized mitochondria with peripheral cristae. Before IVM, small PRE displayed cortical mitochondrial location, whereas the location in other groups was cortical and central. After IVM, mitochondria were located centrally in some large PRE and in all POST. Mitochondrial number increased during oocyte growth proportional to the increase in oocyte volume (Table 1). Shell-like and compartmentalized mitochondria indicate (1) dividing mitochondria (increasing mitochondrial numbers during maturation), or (2) apoptosis-related mitochondrial fission (compromised oocytes after maturation). After IVM, mitochondria seemed to reach the final central position most consistently in POST. These differences may partly explain the higher developmental competence in larger PRE and POST oocytes.

Table 1.Mitochondrial number and oocyte volume in pre- and postpubertal pigs
157 LEUKEMIA INHIBITORY FACTOR IMPROVES OOCYTE MATURATION AND DEVELOPMENTAL COMPETENCE IN PIGS
Authors: Haraguchi S. , Dang-Nguyen T. Q. , Kikuchi K. , Tanihara F. , Bodo S. , Somfai T. , Akagi S. , Hirao Y. , Watanabe S. , Nagai T.
Published: December 2013

A variety of growth factors and cytokines that are present in follicular fluid provide oocytes with a suitable environment for their maturation. One such cytokine is leukemia inhibitory factor (LIF). Although LIF-supplemented medium enhances embryo development in human, mouse, and bovine, studies investigating the effects of LIF on in vitro maturation (IVM) and subsequent embryo development are inconclusive. Additionally, the underlying mechanisms of LIF in oocyte maturation and embryo development after IVF have not been studied yet. In the present study, we examined the effect of recombinant porcine LIF (pLIF), produced in our laboratory, on porcine oocyte maturation and the mechanism of how LIF involves in oocyte maturation process at molecular level. The biological activity of pLIF was evaluated by sustenance of mouse embryonic stem (ES) cells with an undifferentiating state in ES medium supplemented with pLIF, and the final concentration (1 : 200, equivalent to 1000 U mL–1 of mouse LIF) was determined by serial dilution. Porcine cumulus–oocyte complexes (COC) were cultured in modified NCSU-37 medium supplemented with pLIF during the first 22 h [pLIF (+, –)], the latter 22 h [pLIF (–, +)], or whole 44 h [pLIF (+, +)] of IVM and the proportion of metaphase II (M-II) stage oocytes was observed. Oocyte maturation was enhanced in each group by supplementation with pLIF [pLIF (+, –): 76.1%, n = 138; pLIF (–, +): 82.1%, n = 140; pLIF (+, +): 86.6%, n = 127], when compared with control [pLIF (–, –): 69.6%, n = 112], in which a significant increase of M-II rate (P < 0.05 by ANOVA) and cumulus expansion were observed in the pLIF (+, +) group. The effect of pLIF was only seen for COC but not for denuded oocytes. When oocytes were subjected to IVF (Kikuchi et al. 2002), those matured in pLIF (+, +)-supplemented medium demonstrated higher blastocyst developmental rates (21.1% v. 16.2%; P = 0.07) with increased cell numbers (50.2 cells v. 45.0 cells; P = 0.12) compared with pLIF (–, –) on Day 6 of embryo culture (IVF = 0). Examination of transcripts and proteins of the LIF signalling pathway revealed that mRNA and protein levels of LIF, LIF receptors, and signal transducer and activator of transcription 3 (STAT3) were similar in both pLIF (–, –) and pLIF (+, +) samples. However, notable phosphorylation of STAT3 was observed in the pLIF (+, +) sample. These results suggest that the LIF/STAT3-pathway is functional during oocyte maturation in pigs. Therefore, supplementation of maturation medium with pLIF could improve the developmental competence of oocytes by activation of this pathway.
This project was supported by JSPS and HAS under the Japan-Hungary Research Cooperative Program.
158 ASTAXANTHIN EFFECTS ON MATURATION, FERTILIZATION, AND DEVELOPMENT OF PORCINE OOCYTES CULTURED UNDER HEAT STRESS
Authors: Do L. T. K. , Luu V. V. , Sato Y. , Taniguchi M. , Otoi T.
Published: December 2013

Heat stress can engender various disorders in reproductive functions such as impairment of oocyte maturation, fertilization, and embryonic development. Astaxanthin, an extremely common carotenoid, is a typical fat-soluble antioxidant that scavenges ROS and blocks lipid peroxidation. Moreover, astaxanthin has been shown to improve the development of embryos exposed to heat stress by a reduction in stress-inducible genes. This study was conducted to investigate the effects of astaxanthin supplementation on the meiotic competence, fertilization, and development of porcine oocytes exposed to high temperature (41°C) during maturation culture. Cumulus–oocyte complexes (COC) collected from ovaries were transferred into maturation medium supplemented with astaxanthin (0, 0.25, 0.5, or 1.0 ppm) and were then cultured for 46 h at 41°C or 38.5°C. After maturation culture, the COC were subjected to IVF and embryo culture to evaluate the fertility and development of oocytes. The total cell number and DNA fragmentation in the blastocysts were assessed using terminal deoxynucleotidyl transferase dUTP nick end labelling and Hoechst 33342 staining. The total numbers of oocytes matured at 41°C and 38.5°C in each treatment group were 432 to 470 and 426 to 444, respectively. Data were analysed using ANOVA, followed by Fisher's protected least significant difference test. Exposure to elevated temperatures during maturation culture significantly reduced the proportions of oocytes that reached metaphase II. When the COC were cultured in the maturation medium supplemented with 0.5 and 1.0 ppm of astaxanthin under heat stress conditions (41°C), the supplementation of astaxanthin significantly improved the proportions of maturation, fertilization, and blastocyst formation compared with the control group (0 ppm) (50–52%, 45–55%, and 11–12% v. 17, 25, and 6%, respectively). The supplementation of the maturation medium with 0.25 ppm of astaxanthin improved only blastocyst formation (9.6%). Similarly, the supplementation of astaxanthin at 1.0 ppm improved the proportions of maturation, fertilization, and blastocyst formation of oocytes matured at 38.5°C s compared with the control group (67, 57, and 18% v. 48, 33, and 12%, respectively). However, no beneficial effect of astaxanthin supplementation was found in the total cell number or DNA fragmentation in the blastocysts, irrespective of culture temperature. Our findings show that the supplementation of astaxanthin to maturation medium improves maturation, fertilization, and embryo development of porcine oocytes exposed to heat stress during maturation culture.
170 INFLUENCE OF OOCYTE QUALITY AND DIFFERENT MEDIA SUPPLEMENT ON IN VITRO MATURATION, CLEAVAGE, AND EMBRYO DEVELOPMENT OF BUFFALO (BUBALUS BUBALIS) OOCYTES
Authors: Waheed M. M. , El-Shahat K. H. , Hammam A. M.
Published: December 2013

A series of 4 factorial-arranged experiments were conducted to study the effect of oocyte quality and different in vitro maturation (IVM) media supplements on IVM, cleavage, and embryo development of buffalo (Bubalus bubalis) oocytes. Buffalo ovaries were collected at a local abattoir in a warm (32–35°C) saline (0.9% NaCl), and oocytes were aspirated using an 18-gauge needle. In experiment 1, oocytes (n = 320) were classified according to the number of cumulus cell layers and morphology of ooplasm as excellent, good, or fair. Oocytes were cultured for IVM, fertilization, and embryo culture (IVMFC) in TCM-199 + 10% FCS. In experiment 2, excellent quality oocytes (n = 237) were subjected to IVM in TCM-199 enriched with either 10% FCS or oestrous buffalo serum (EBS; 20–40 pg mL–1) and then fertilized using frozen–thawed buffalo semen capacitated in Bracket and Oliphant's (BO) medium containing heparin (0.02 mg mL–1) and sodium caffeine benzoate (3.89 mg mL–1). In experiment 3, oocytes (n = 290) were classified into 2 groups; Group 1, without gonadotropins, served as a control; Group 2, in which IVM medium was supplemented with 20 IU mL–1 equine chorionic gonadotropins (eCG). Experiment 4 was carried out to examine the suitable capacitating agent added to BO medium, either heparin or caffeine or both (n = 210 fertilized oocytes). In all experiments (multiple replicates), oocytes (2–6 mm in diameter) were kept at 39°C under 5% CO2 for IVMFC and examined several times for cleavage and embryo development (morula and blastocyst). Statistical analysis was carried out using Chi-squared test. Excellent and good quality oocytes produced a higher (P < 0.05) maturation, cleavage, and morula development rates than poor quality oocytes (70% and 65% v. 33.3%), (50% and 46.2% v. 25%), and (42.9% and 33.3% v. 10%), respectively. Blastocyst production rate was also higher (P < 0.05) for excellent compared with good quality oocytes (28.6% v. 16.7%, respectively). In experiment 2, the IVM and cleavage rates were significantly higher (P < 0.05) in IVM medium plus 10% EBS than those cultured in 10% FCS (73% v. 45% and 50% v. 33.3%, respectively). In experiment 3, the addition of eCG to maturation medium increased (P < 0.05) developmental competence of buffalo oocytes (IVMFC) compared with control medium (16% v. 4%). In experiment 4, the addition of heparin together with caffeine to BO medium produced significantly higher (P < 0.05) cleavage and embryo developmental rates compared with heparin or caffeine alone (56.3% v. 33.3% and 35.7%, respectively; 22.2% v. 10% and 8%, respectively). In conclusion, excellent quality oocytes cultured in IVM medium supplemented with either EBS or eCG and fertilized with capacitated buffalo spermatozoa in BO medium enriched with heparin and caffeine progressively enhanced developmental competence of buffalo oocytes.
178 PORTABLE AUTOMATED MICROFLUIDIC DEVICE FOR RAPID DETERMINATION OF SPERM COUNTS
Authors: Kjelland M. E. , Stroud T. , Ayliffe H. E. , Dittami G.
Published: December 2013

The primary objective of this research was to determine whether an automated electronic cell counter using microfluidic technology (Moxi Z) could be effective for determining sperm concentrations. Validation consisted of comparing frozen-thawed conventional bovine sperm counts using the Moxi Z to Coulter Z2 counter results. Specific goals were to determine (1) if the Moxi Z could count intact motile sperm, (2) if differences in media containing egg yolk lipids would affect counting, and (3) evaluate sample concentration requirements for Moxi Z accuracy. Cryopreserved straws (n = 20) of conventional bovine semen (Brangus), were obtained from Stroud Veterinary Embryo Services Inc. Straws were thawed (~30 s, 38°C) in a water bath, dried, and cut open. Semen was expelled into 15-mL conical tubes (BD Falcon) using a straw plunger. Thereafter, samples were centrifuged (400 × g, 10 min). The supernatant was removed and replaced with PBS (media 1) or Tris A semen extender (20% vol/vol egg yolk; media 2). Sperm samples exhibited ~70% motility for the experiments, based on subjective microscopy by placing 10-μL sample volumes on 25 × 75 × 1-mm microscope slides (#2951–001, Thermo Scientific, Waltham, MA, USA) with 24 × 50-mm #1.5 coverslips (#M6047–9, Baxter, McGraw Park, IL, USA) and viewing with a Axio Scope.A1 microscope (Zeiss, Thornwood, NY, USA) using 10× (M Plan Apo NIR, Mitutoyo Am, Aurora, IL, USA) and 40× (LD-Plan NeoFluor, Zeiss) lenses (i.e. 100–400×). The protocol for each sample was to establish an initial concentration of ~2.5 × 106 cells mL–1 and perform a serial dilution assay, using modified Hank's Balanced Salt Solution (Sigma, St. Louis, MO, USA). Counts (n = 3) for each dilution level (e.g. 1.0, 0.8, 0.6, 0.4, 0.2, 0.1, 0.05, 0.025) were made simultaneously on each instrument, Moxi Z (v3.6 OS, Orflo) and Coulter Z2 (Beckman Coulter, Fullerton, CA, USA). Data points were compared to linear fitted curves. The Moxi Z gated range was set at 2.88 to 6.54 μ to approximate the count range of the Z2 system (2.91–6.46 μ). The sperm samples provided similar results on both the Moxi Z and the Z2 instruments. Specifically, data for the 2 media using serial dilutions of the sperm samples were analysed, and the Moxi Z counts correlated well with the Z2 counts for media 1 (R2 > 0.9469) and media 2 (R2 > 0.9771). Overall the Moxi Z can provide rapid bovine sperm counts (<15 s) using small sample volumes. The degree of precision and accuracy depends strongly on proper gate setting and on the ability to reduce sample debris. Increased size resolution will be realised by future improvements to the Moxi Z signal processing (v 4.0), as well as by slowing the system flow rate; thereby improving cell peak discrimination. The portability of the Moxi Z may be an asset for field work and will be tested in future research. The data and dilution ranges herein demonstrate the utility of the Moxi Z for diluted conventional semen and lower dose concentrations, such as that of sexed semen.
180 SPERM SELECTION OF STALLION PONIES THROUGH GLASS WOOL
Authors: Pessoa G. A. , Trentin J. M. , Martini A. P. , Dotto D. R. , Centeno L. A. M. , Jardim M. L. , Aires K. V. , Rubin M. I. B.
Published: December 2013

Two techniques of sperm concentration (centrifugation or filtering) and sperm separation technique with glass wool were applied to the sperm samples collected from 3 pony stallions (6 ejaculates; 2 from each stallion). Ejaculates were extended to a final concentration of 50 × 106 spermatozoa mL–1 using a nonfat dry milk-based extender and evaluations occurred at 24, 48, and 72 h after immediate ejaculate dilution and cooling. Each stallion was considered as a block, and semen from each stallion was assigned to 4 treatments: Group A (control): extended semen alone; Group B: extended-centrifuged semen; Group C: extended-sperm filtered semen; Group D: extended-glass wool-separated semen. All groups were tested for pH, osmolarity, motility, morphology, membrane functionality (hyposmotic), and cell viability (MTT assay). The experimental design was performed using a split-plot model. Data analysis at the level of 5% was performed using ANOVA and Bonferroni as post-hoc test. Data are presented as mean ± standard error. Group D had the highest rate of viable cells (P < 0.05) after the separation procedure (Table 1). Group B had a higher percentage of cells with tail defects after processing compared with the controls and Groups A, C, and D (P < 0.05). More than 60% of the cells retained on the filter showed defects (P < 0.001). Progressive motility was greater in group D at 0, 24, and 48 h (P < 0.05). Seventy-two hours after cooling, motility in groups A and B was lower than in Group D (P < 0.01). Group D showed a higher number of cells with mitochondrial activity during the cooling period. In conclusion, the technique of sperm selection by gravity using a glass wool filter resulted in an increased number of viable sperms after cooling pony semen for 24, 48, and 72 h.

Table 1.Effect of sperm concentration and separation techniques on mean ± standard error percent of intact sperm from 3 stallions ponies (2 ejaculates/pony) stained with eosin-nigrosin
19 LIFETIME MORTALITY OF CLONED PIGLETS
Authors: Schmidt M. , Winther K. D. , Callesen H.
Published: December 2013

Perinatal mortality of cloned animals is a well-recognised problem, but it is not clear if and how the problems continue throughout the life of cloned animals. Here we report occurrence and causes of lifetime mortality of cloned piglets from their perinatal period and onwards. From 2006 until 2013, 178 Large White (LW) sows received Day 5–6 handmade cloned morulae or blastocysts where donor cells were from LW, Yucatan, or Göttingen, and were either transgenic or not. Pregnancy rate on Day 30 was 73%, but 19% terminated before term, resulting in 104/178 deliveries. For each litter, each piglet was characterised as either alive or stillborn (i.e. dead without signs of autolysis or dying during the first minutes after birth). Data were analysed by Fisher's Exact test with a significance level of P < 0.05. In the 104 litters, stillborn piglets were found in 54 (from 1 to 7 stillborn piglets in a litter), and the highest number of stillborns was found in Göttingen piglets (P < 0.05; see Table 1). Alive piglets were followed and their mortality registered, and they either died spontaneously (arthritis, pericarditis, enteritis, septicemia, lethal malformations) or were killed if they had other malformations, no weight gain, anorexia, bristly hair coat or signs of compromised well-being. Piglet mortality was highest in the perinatal period but remained high until weaning at Day 28 (see Table 1), so almost half of the alive piglets died during the first month; this preweaning mortality was highest in Yucatan piglets (P < 0.05), which also had the highest cumulative mortality. Further analyses are needed to describe differences observed between donor cell breeds. After weaning, where the pigs were kept under standard conditions, the mortality was at the normal level for the farm. Few died of infections with no breed differences, so most were killed over the subsequent months and years for various experimental purposes. The oldest pig lived until 6 years of age. During their life, no differences compared with noncloned pigs were observed in behaviour, growth, occurrence of diseases, or viability. This study confirms the well-known high perinatal mortality of cloned piglets and shows also high mortality until weaning. However, after weaning, mortality was not different from that of noncloned piglets.

Table 1.Lifetime mortality rates in cloned piglets of different breeds and at different time periods after birth
205 ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY ON NEURAL DIFFERENTIATION IN HUMAN EMBRYONIC STEM CELLS
Authors: Kang H. S. , Jung E. M. , Jeung E. B.
Published: December 2013

Human embryonic stem cells (hESCs) have a potential for differentiation into neuronal cells. Neural differentiation of hESCs is currently used in many fields of neurological study. Therefore, evaluation of developmental neurotoxicity in hESCs is possible during the embryonic period. In the current study, we investigated the neuronal toxicity of hydroxyurea, cytosine arabinoside, and penicillin G at multiple doses (low, medium, high) for 28 days for neural differentiation. For assessment of neural toxicity, we examined the expression of marker genes that represent neural cell development. The mRNA levels of the marker genes were evaluated by real-time PCR in hESCs. Morphological changes of hESCs during neuronal differentiation were also estimated. The results for low and medium doses of hydroxylurea showed a significant increase of dopaminergic neuron marker gene (NR4A2) and GABA neuron marker gene (GAD2). However, glutamartergic neuron marker gene (SLC1A2) and oligodendrocyte marker gene (CNP) showed a significant decrease. Results for another drug, cytosine arabinoside, showed a significant decrease of glutamartergic neuron marker gene (SLC1A2) and oligodendrocyte marker gene (CNP) at a high dose. In addition, cytosine arabinoside caused a significant decrease of dopaminergic neuron marker gene (NR4A2) without significant change in GAD2. For the control, penicillin G, no significant difference in expression of neural specific-genes was observed at all tested doses. These findings suggest that neural-specific genes are perturbed by hydroxyurea and cytosine arabinoside, which may be involved in abnormal neural development during the embryonic neurogenesis period in hESCs.
222 PRODUCTION OF KNOCKOUT RATS BY USING ZINC FINGER NUCLEASES AND TAL EFFECTOR NUCLEASES
Authors: Kaneko T. , Mashimo T.
Published: December 2013

The rat has been used as an important animal for understanding human diseases. Genetically engineered rat strains are used as a human disease model in various research fields. Genetically engineered rat strains are now being routinely produced, not only as transgenic animals but also using gene knockout techniques. Recently, zinc finger nucleases (ZFN) and TAL effector nucleases (TALEN) have enabled editing targeted genes without using embryonic stem cells. These techniques have been applied for production of the knockout and knockin animals. We here studied that the effects of gene targeting by ZFN and TALEN introduced into rat embryos for efficient production of knockout rats. We custom-designed ZFN and TALEN plasmids targeted rat interleukin 2 receptor gamma (Il2rg) gene. Each mRNA was transcribed in vitro from these plasmids. Final concentration of mRNA was adjusted at 10 ng μL–1 in sterilized water for microinjection. Messenger RNA was injected into rat pronuclear stage embryos. The embryos were then cultured in vitro to the 2-cell stage, and were transferred into oviducts of pseudopregnant females. The rate of development of offspring of embryos and effects of editing targeted genes were examined. Of 41 two-cell embryos introduced ZFN after embryo transfer, 9 embryos (22%) developed to offspring. Three offspring (33%) had an edited targeted gene locus. In the embryos introduced TALEN, 30% (6 offspring) of embryos developed to offspring after embryo transfer and all offspring had an edited targeted gene locus. This study demonstrated that the ZFN and TALEN mRNA was active after introduction into rat embryos. Knockout rats could be produced by introduction of ZFN and TALEN into rat embryos. ZFN and TALEN will provide a powerful new approach for targeted gene editing not only in rats but also in other animal species.
40 KNOCKOUT OF GOAT NUCLEOPORIN 155 (NUP155) GENE USING CRISPR/Cas9 SYSTEMS
Authors: Hu S. , Wang Z. , Polejaeva I.
Published: December 2013

Nucleoporin 155 (NUP155) is a major component of the nuclear pore complex involved in the cellular nucleo-cytoplasmic transport. NUP155 knockout in mice leads to atrial fibrillation and early sudden cardiac death. Due to the small size of the heart, functional electrophysiology is problematic in transgenic mice. Large transgenic animal models have the potential to offer insights into the role of nuclear pore complex in cardiovascular disease. The goal of this study was to generate NUP155 knockout goats by using the CRISPR/Cas9 systems. Two CRISPR target sites were chosen in the first exon of the goat NUP155 gene and CRISPR knockout vectors were constructed. Surveyor assay showed that the 2 CRISPR vectors efficiently induced a double-strand break with cleavage efficiencies of 30 to 50% in transfected goat fibroblast cells. Single cell-derived clones were obtained by limiting dilution of the transfected cells in 48-well plates. In total, 23 single cell clones were isolated and subjected to Surveyor assay followed by sequencing the PCR products spanning the target site. Five of 23 single cell colonies had heterozygous mutations and one carried biallelic modifications of NUP155. The heterozygous cells will be used to produce NUP155± goats by somatic cell nuclear transfer. In conclusion, we demonstrated that CRISPR/Cas9 systems are highly efficient for gene knockout applications in goats.
This work was supported by the Utah Science Technology and Research Initiative, Utah Multidisciplinary Arrhythmia Consortium and Utah Agricultural Experiment Station project #1100.
83 VITAMIN K2 SUPPLEMENTATION IMPROVES BLASTOCYST RATE BY RECOVERY OF MITOCHONDRIA IN IN VITRO-CULTURED BOVINE EMBRYOS
Authors: Baldoceda L. , Vigneault C. , Blondin P. , Robert C.
Published: December 2013

Mitochondria play an important role during early mammalian embryo development through their diverse cellular functions, in particular creating balance between production of ATP by electron transport chain and oxidative stress. Embryonic mitochondria are inherited maternally and independently of the nuclear genome. They show limited activity during the early developmental stages before embryonic genome activation. It has been shown that in vitro culture (IVC) has an adverse effect on mitochondrial function in embryos. So far several attempts have been performed to improve and rescue the impaired mitochondria. It has been shown that vitamin K2 (a membrane-bound electron carrier, similar to ubiquinone) was used to rescue mitochondrial dysfunction and resulted in more efficient ATP production in eukaryotic cells (Vos et al. 2012 Science 336, 1306–1310). Therefore, the aim of the present study was to investigate the effects of supplementation of vitamin K2 on mitochondrial activity and blastocyst rate. Cumulus–oocytes complexes (n = 687) recovered from slaughtered animals, were matured and fertilized in vitro according to our standard procedures. After fertilization, zygotes were cultured in SOF media supplemented with 10 mg mL–1 BSA. At 96 h post-fertilization, vitamin K2 was added to the culture media (n = 448 oocytes). On Day 7, treatment embryos were compared with untreated controls (n = 239 oocytes). In vitro culture was carried out at 38.5°C under 5% CO2, 7% O2, and 88% N2. Differences among groups in blastocyst yield were analysed by ANOVA. Mitochondrial activity data was analysed by unpaired 2-tailed t-tests. Results show that the vitamin K2-treated group had a significantly (P < 0.05) higher blastocyst rate (+8.6%), expanded blastocyst rate (+7.8%), as well as better morphological quality compared with the control group. Furthermore, to evaluate mitochondria activity, pools of embryos of each treatment were labelled with a specific dye for active mitochondria (Mitotracker Red). A significantly higher intensity of Mitotracker Red (P < 0.05) was observed in the vitamin K2 treatment versus control group, as measured by fluorescent microscopy. In conclusion, for the first time, our data prove that supplementation of vitamin K2 during IVC of bovine embryos increases blastocyst rates and embryo quality. Future studies will focus on gene expression to identify targets implicated in impaired mitochondrial activity in in vitro bovine embryo production.
Avalanches mediate crystallization in a hard-sphere glass
Authors: Sanz Eduardo , Valeriani Chantal , Zaccarelli Emanuela , Poon Wilson C. K. , Cates Michael E. , Pusey Peter N.
Published: December 2013

Significance

Glasses are formed from the supercooled liquid state when motion is arrested on the scale of the particle diameter. Such states are thermodynamically unstable: An apparently deeply arrested amorphous material can transform into a crystal (devitrification) without large-scale particle motion. The prediction and avoidance of devitrification represent major formulation issues in materials science, hence the importance of understanding its mechanism. Using molecular-dynamics simulations, we study the crystallization of the simplest glass-forming system, a hard-sphere glass. We find that crystallization is caused by a subset of particles cooperatively undergoing large rearrangements in an intermittent stochastic fashion (avalanches). Particles involved in an avalanche are not themselves crystallizing, but they induce crystallization in nearby regions that already show incipient local order.
Biphasic influence of Miz1 on neural crest development by regulating cell survival and apical adhesion complex formation in the developing neural tube
Authors: Kerosuo Laura , Bronner Marianne E.
Published: December 2013

Myc interacting zinc finger protein-1 (Miz1) is a transcription factor known to regulate cell cycle– and cell adhesion–related genes in cancer. Here we show that Miz1 also plays a critical role in neural crest development. In the chick, Miz1 is expressed throughout the neural plate and closing neural tube. Its morpholino-mediated knockdown affects neural crest precursor survival, leading to reduction of neural plate border and neural crest specifier genes Msx-1, Pax7, FoxD3, and Sox10. Of interest, Miz1 loss also causes marked reduction of adhesion molecules (N-cadherin, cadherin6B, and α1-catenin) with a concomitant increase of E-cadherin in the neural folds, likely leading to delayed and decreased neural crest emigration. Conversely, Miz1 overexpression results in up-regulation of cadherin6B and FoxD3 expression in the neural folds/neural tube, leading to premature neural crest emigration and increased number of migratory crest cells. Although Miz1 loss effects cell survival and proliferation throughout the neural plate, the neural progenitor marker Sox2 was unaffected, suggesting a neural crest–selective effect. The results suggest that Miz1 is important not only for survival of neural crest precursors, but also for maintenance of integrity of the neural folds and tube, via correct formation of the apical adhesion complex therein.
ATP increases within the lumen of the endoplasmic reticulum upon intracellular Ca2+release
Authors: Vishnu Neelanjan , Jadoon Khan Muhammad , Karsten Felix , Groschner Lukas N. , Waldeck-Weiermair Markus , Rost Rene , Hallström Seth , Imamura Hiromi , Graier Wolfgang F. , Malli Roland
Published: December 2013

Multiple functions of the endoplasmic reticulum (ER) essentially depend on ATP within this organelle. However, little is known about ER ATP dynamics and the regulation of ER ATP import. Here we describe real-time recordings of ER ATP fluxes in single cells using an ER-targeted, genetically encoded ATP sensor. In vitro experiments prove that the ATP sensor is both Ca2+and redox insensitive, which makes it possible to monitor Ca2+-coupled ER ATP dynamics specifically. The approach uncovers a cell type–specific regulation of ER ATP homeostasis in different cell types. Moreover, we show that intracellular Ca2+release is coupled to an increase of ATP within the ER. The Ca2+-coupled ER ATP increase is independent of the mode of Ca2+mobilization and controlled by the rate of ATP biosynthesis. Furthermore, the energy stress sensor, AMP-activated protein kinase, is essential for the ATP increase that occurs in response to Ca2+depletion of the organelle. Our data highlight a novel Ca2+-controlled process that supplies the ER with additional energy upon cell stimulation.
Exo70E2 is essential for exocyst subunit recruitment and EXPO formation in both plants and animals
Authors: Ding Yu , Wang Juan , Chun Lai John Ho , Ling Chan Vivian Hoi , Wang Xiangfeng , Cai Yi , Tan Xiaoyun , Bao Yiqun , Xia Jun , Robinson David G. , Jiang Liwen
Published: December 2013

In contrast to a single copy of Exo70 in yeast and mammals, the Arabidopsis genome contains 23 paralogues of Exo70 (AtExo70). Using AtExo70E2 and its GFP fusion as probes, we recently identified a novel double-membrane organelle termed exocyst-positive organelle (EXPO) that mediates an unconventional protein secretion in plant cells. Here we further demonstrate that AtExo70E2 is essential for exocyst subunit recruitment and for EXPO formation in both plants and animals. By performing transient expression in Arabidopsis protoplasts, we established that a number of exocyst subunits (especially the members of the Sec family) are unable to be recruited to EXPO in the absence of AtExo70E2. The paralogue AtExo70A1 is unable to substitute for AtExo70E2 in this regard. Fluorescence resonance energy transfer assay and bimolecular fluorescence complementation analyses confirm the interaction between AtExo70E2 and Sec6 and Sec10. AtExo70E2, but not its yeast counterpart, is also capable of inducing EXPO formation in an animal cell line (HEK293A cells). Electron microscopy confirms the presence of double-membraned, EXPO-like structures in HEK293A cells expressing AtExo70E2. Inversely, neither human nor yeast Exo70 homologues cause the formation of EXPO in Arabidopsis protoplasts. These results point to a specific and crucial role for AtExo70E2 in EXPO formation.