bluuebunny/crossref_metadata_2025 huggingface dataset:
A composite scaffold of MSC affinity peptide-modified demineralized bone matrix particles and chitosan hydrogel for cartilage regeneration
Authors: Meng Qingyang , Man Zhentao , Dai Linghui , Huang Hongjie , Zhang Xin , Hu Xiaoqing , Shao Zhenxing , Zhu Jingxian , Zhang Jiying , Fu Xin , Duan Xiaoning , Ao Yingfang
Published: December 2015

Articular cartilage injury is still a significant challenge because of the poor intrinsic healing potential of cartilage. Stem cell-based tissue engineering is a promising technique for cartilage repair. As cartilage defects are usually irregular in clinical settings, scaffolds with moldability that can fill any shape of cartilage defects and closely integrate with the host cartilage are desirable. In this study, we constructed a composite scaffold combining mesenchymal stem cells (MSCs) E7 affinity peptide-modified demineralized bone matrix (DBM) particles and chitosan (CS) hydrogel for cartilage engineering. This solid-supported composite scaffold exhibited appropriate porosity, which provided a 3D microenvironment that supports cell adhesion and proliferation. Cell proliferation and DNA content analysis indicated that the DBM-E7/CS scaffold promoted better rat bone marrow-derived MSCs (BMMSCs) survival than the CS or DBM/CS groups. Meanwhile, the DBM-E7/CS scaffold increased matrix production and improved chondrogenic differentiation ability of BMMSCs in vitro. Furthermore, after implantation in vivo for four weeks, compared to those in control groups, the regenerated issue in the DBM-E7/CS group exhibited translucent and superior cartilage-like structures, as indicated by gross observation, histological examination and assessment of matrix staining. Overall, the functional composite scaffold of DBM-E7/CS is a promising option for repairing irregularly shaped cartilage defects.
Detection and characterisation of sub-critical nuclei during reactive Pd metal nucleation by X-ray absorption spectroscopy
Authors: Chang S.-Y. , Gründer Y. , Booth S. G. , Molleta L. B. , Uehara A. , Mosselmans J. F. W. , Cibin G. , Pham V.-T. , Nataf L. , Dryfe R. A. W. , Schroeder S. L. M.
Published: December 2015

The interfacial reduction of aqueous [PdCl4]2− at the interface with an organic solution of ferrocene has been characterised by X-ray absorption fine structure (XAFS) spectroscopy.
Gadolinium(iii)-mediated multi-component confinement of imidazolium cations in p-sulfonated calix[4]arene
Authors: Ling Irene , Sobolev Alexandre N. , Raston Colin L.
Published: December 2015

Multi-component materials having supermolecules made up of mono- or bis-imidazolium cations shrouded by two p-sulfonated calix[4]arenes essentially form molecular capsules as part of the extended structure, with their formation mediated by Gd(iii), which is incorporated only in complex I.
Fragment quantum chemical approach to geometry optimization and vibrational spectrum calculation of proteins
Authors: Liu Jinfeng , Zhang John Z. H. , He Xiao
Published: December 2015

Geometry optimization and vibrational spectra (infrared and Raman spectra) calculations of proteins are carried out by a quantum chemical approach using the EE-GMFCC (electrostatically embedded generalized molecular fractionation with conjugate caps) method (J. Phys. Chem. A, 2013, 117, 7149).
Doping liquid crystals with nanoparticles. A computer simulation of the effects of nanoparticle shape
Authors: Orlandi Silvia , Benini Erika , Miglioli Isabella , Evans Dean R. , Reshetnyak Victor , Zannoni Claudio
Published: December 2015

Molecular-scale Monte Carlo simulations of liquid crystal-nanoparticle dispersions show the effect on the orientational order and on the clearing temperature of shape and concentration of the dopant nanoparticles.
Microwave catalytic effect: a new exact reason for microwave-driven heterogeneous gas-phase catalytic reactions
Authors: Xu Wentao , Zhou Jicheng , Su Zhiming , Ou Yingpiao , You Zhimin
Published: December 2015

The microwave catalytic effect (lowering of activation energy) under microwave irradiation results in NO conversion exceeding that realized through conventional heating.
Effect of CeO2 for a high-efficiency CeO2/WO3–TiO2 catalyst on N2O formation in NH3-SCR: a kinetic study
Authors: Geng Yang , Shan Wenpo , Xiong Shangchao , Liao Yong , Yang Shijian , Liu Fudong
Published: December 2015

The effect of CeO2 for a high-efficiency CeO2/WO3–TiO2 catalyst on N2O formation in NH3-SCR reaction was investigated using a kinetic method.
Structural and magnetic modulations of copper(ii) azido complexes: unexpected in situ reactions of mono-N-donor pyridine-based co-ligands
Authors: Zhao Jiong-Peng , Xie Yabo , Li Jian-Rong , Bu Xian-He
Published: December 2015

Hydrothermal reactions of azide anions, Cu(ii) ions, and fourmono-N-donor pyridine ligands lead to four new copper(ii) azides with different structures and magnetic properties, in which three co-ligands were generatedin situby decomposition, substitution, and oxidation reactions.
Synthesis and structure of the extended phosphazane ligand [(1,4-C6H4){N(μ-PNtBu)2NtBu}2]4−
Authors: Sevilla Raquel , Less Robert J. , García-Rodríguez Raúl , Bond Andrew D. , Wright Dominic S.
Published: December 2015

Reaction of (1,4-C6H4)[N(PCl2)2]2 with tBuNH2 in the presence of Et3N gives the bridged phosph(iii)azane (1,4-C6H4)[N(μ-PNHtBu)2NtBu]2 (1H4), which can be deprotonated to give the tetraanion (1,4-C6H4)[N(μ-PNtBu)2NtBu]24− (14−). The latter represents a new type of phosphazane ligand.
Efficient band structure tuning, charge separation, and visible-light response in ZrS2-based van der Waals heterostructures
Authors: Zhang Xirui , Meng Zhaoshun , Rao Dewei , Wang Yunhui , Shi Qi , Liu Yuzhen , Wu Haiping , Deng Kaiming , Liu Hongyang , Lu Ruifeng
Published: December 2015

Bilayer ZrS2-based vdW heterostructures are proposed as efficient photocatalysts for water splitting under visible light.
A transition metal oxofluoride offering advantages in electrocatalysis and potential use in applications
Authors: Svengren H. , Torapava N. , Athanassiadis I. , Ali S. I. , Johnsson M.
Published: December 2015

The recently described solid solution (Co,Ni,Mn)3Sb4O6F6has proved stable and efficient as a catalyst for electrocatalytic water oxidation. The end component Co3Sb4O6F6was found to be most efficient, maintaining a current density ofj= 10 mA cm−2at an overpotential of 443 mV with good capability. At this current density, O2and H2were produced in the ratio 1 : 2 without loss of faradaic current against a Pt-cathode. A morphological change in the crystallite surface was observed after 0.5 h, however, even after 64.5 h, the overall shape and size of the small crystallites were unaffected and the electrolyte contained only 0.02 at% Co. It was also possible to conclude fromin situEXAFS measurements that the coordination around Co did not change. The oxofluorides express both hydrophilic and hydrophobic surface sites, incorporate a flexible metalloid element and offer the possibility of a mechanism that differs from other inorganic catalytic pathways previously described.
Combinational concentration gradient confinement through stagnation flow
Authors: Alicia Toh G. G. , Yang Chun , Wang Zhiping , Nguyen Nam-Trung
Published: December 2015

A concentration gradient device generates stagnation flows which confine combinational concentration gradients within velocity wells, thereby suppressing shear stress effects.
Osmotic pressure-triggered cavitation in microcapsules
Authors: Shang Luoran , Cheng Yao , Wang Jie , Yu Yunru , Zhao Yuanjin , Chen Yongping , Gu Zhongze
Published: December 2015

Cavitation was found in solid microcapsules with a membrane shell and a liquid core by treating these microcapsules with hypertonic solutions.
Unexpected photoluminescence properties from one-dimensional molecular chains
Authors: Yuan Ye , Yao Mingguang , Chen Shuanglong , Liu Shijie , Yang Xigui , Zhang Weiwei , Yao Zhen , Liu Ran , Liu Bo , Liu Bingbing
Published: December 2015

Unlike bulk iodine, iodine molecular chains formed inside one dimensional (1D) nanochannels of AlPO4-5 (AFI) single crystals show unexpected PL behavior.
Defect-free thin InAs nanowires grown using molecular beam epitaxy
Authors: Zhang Zhi , Chen Ping-Ping , Lu Wei , Zou Jin
Published: December 2015

Controlled growth of defect-free thin InAs nanowires using MBE.
Sugarcane bagasse delignification with potassium hydroxide for enhanced enzymatic hydrolysis
Authors: Paixão S. M. , Ladeira S. A. , Silva T. P. , Arez B. F. , Roseiro J. C. , Martins M. L. L. , Alves L.
Published: December 2015

Alkali pretreatment of sugarcane bagasse biomass was shown to be effective for producing sugar-rich hydrolysates for biotechnological applications.
Valence-tautomeric infinite coordination polymer nanoparticles for encapsulation of rhodamine B and its potential application for colorimetric and fluorescence dual mode sensing of hypochlorite
Authors: Zhang Xiaolei , Deng Jingjing , Shi Guoyue , Zhou Tianshu
Published: December 2015

The colorimetric and fluorescence ClO− sensing based on the stimulus response of valence-tautomeric RhB@{Co(3,5-dbsq)(3,5-dbcat)(bix)} ICP nanoparticles.
Catalytic properties of γ-Al2O3 supported Pt–FeOx catalysts for complete oxidation of formaldehyde at ambient temperature
Authors: Cui Weiyi , Yuan Xiaoling , Wu Ping , Zheng Bin , Zhang Wenxiang , Jia Mingjun
Published: December 2015

A Pt–FeOx/Al2O3 catalyst with suitable Fe/Pt ratio showed excellent catalytic activity and stability for complete oxidation of formaldehyde at ambient temperature.
Dispersive suspended-solidified floating organic droplet microextraction of nonsteroidal anti-inflammatory drugs: comparison of suspended droplet-based and dispersive-based liquid-phase microextraction methods
Authors: Barfi Behruz , Asghari Alireza , Rajabi Maryam , Mirkhani Nasim
Published: December 2015

A dispersive suspended-solidified floating organic droplet microextraction method was developed for determination of some nonsteroidal anti-inflammatory drugs in human plasma and urine samples.
Nearly warm white-light emission of silicon-rich amorphous silicon carbide
Authors: Tai Hung-Yu , Cheng Chih-Hsien , Wang Po-Sheng , Wu Chih-I , Lin Gong-Ru
Published: December 2015

An amorphous Si-rich SiC film with nearly warm white-light photoluminescence is synthesized to serve as a solid-state phosphorous material for white-lighting applications.
Optimization of ultrasonic-assisted alkaline extraction of polysaccharides from Phellodendron amurense Rupr. pollen using response surface methodology and its structure features
Authors: Wang Xuefei , Zhang Hua , Wang Zhenyu , Bai Haina
Published: December 2015

To investigate extraction technology and determine the optimal extraction conditions of polysaccharides from Phellodendron amurense Rupr. pollen (PARPP), an alkaline extraction method was carried out to extract PARPP with ultrasonic treatment.
Comparative study of the inverse versus normal bicontinuous cubic phases of the β-d-glucopyranoside water-driven self-assemblies using fluorescent probes
Authors: Zahid N. Idayu , Abou-Zied Osama K. , Nabila Saari N. A. , Hashim Rauzah
Published: December 2015

This work investigates the head group region of the inverse and normal bicontinuous cubic phases (Ia3d space group) of the glucopyranoside/water system using 2-(2′-hydroxyphenyl)benzoxazole and its derivatives as fluorescent probes.
Ruthenium/base-catalyzed ortho-selective C–H arylation of acylarenes with halogenated arylboronates
Authors: Yamamoto Tetsuya , Yamakawa Tetsu
Published: December 2015

Ruthenium-catalyzed ortho-selective C–H arylation of acylarenes with halogenated arylboronates was promoted by the catalytic amount of bases and provided desired halogenated biaryls in excellent yields under 0.2–1 mol% ruthenium catalyst loading.
Catalytic wet oxidation of phenol with Fe–ZSM-5 catalysts
Authors: Yan Ying , Jiang Songshan , Zhang Huiping
Published: December 2015

Systematic study of catalytic wet oxidation of phenol over Fe–ZSM-5 and Fe2O3/ZSM-5 catalysts.
A modification of a conventional technique for the synthesis of hydrazones of racemic carbonyls: prevention of spontaneous chiral inversion
Authors: Singh Manisha , Bhushan Ravi
Published: December 2015

Conventionally hydrazones and other derivatives of carbonyls are synthesized under acidic conditions when spontaneous chiral inversion is a common problem if the carbonyl compound is chiral.
Probing the structural and electronic properties of doped gallium oxide and sulfide, M(GaX2)2 where M = alkali or coinage metal; X = O, S
Authors: Seeburrun N. , Alswaidan I. A. , Fun H.-K. , Archibong E. F. , Ramasami P.
Published: December 2015

A theoretical study on the equilibrium geometries, vibrational frequencies and electronic features of a series of doped gallium oxide and sulfide clusters, M(GaX2)2 (M = alkali or coinage metal; X = O or S), was conducted.
A comprehensive comparison of bacterial and fungal aerobic granules: formation, properties, surface modification, and biosorption of metals
Authors: Wang Li , Wang Ya-yi , Liu Xiang , Chen Xiao-feng , Lee Duu-Jong , Tay Joo-Hwa , Zhang Yi , Wan Chun-li
Published: December 2015

Aerobic granules, a relatively new form of microbial aggregate, can be formed with bacteria or fungi as the dominant population, depending on operational conditions. They have respective advantages and disadvantages in characteristic and functions.
A new avenue to the Dakin reaction in H2O2–WERSA
Authors: Saikia Bishwajit , Borah Parinita
Published: December 2015

We have developed a novel protocol to realize the Dakin reaction in H2O2–WERSA at room temperature and the system does not require activation, toxic ligand, additive/promoter, transition metal catalyst, base, organic solvent and so on.
The assembly of polyethyleneimine-entrapped gold nanoparticles onto filter paper for catalytic applications
Authors: Liu Lei , Zhao Yili , Chen Qian , Shi Xiangyang , Shen Mingwu
Published: December 2015

Polyethyleneimine-entrapped gold nanoparticles can be assembled onto filter paper via electrostatic interaction for high-performance catalytic applications.
Multicolour electroluminescence from light emitting diode based on ZnO:Cu/p-GaN heterojunction at positive and reverse bias voltage
Authors: Wang Yumei , Liu Nishuang , Chen Ying , Yang Congxing , Liu Weijie , Su Jun , Li Luying , Gao Yihua
Published: December 2015

The ZnO:Cu/p-GaN LED made by simply CVD method emitted different multicolour EL light at positive and reverse bias voltage.
New Securinega alkaloids with anti-HIV activity from Flueggea virosa
Authors: Zhang Hua , Zhang Chuan-Rui , Han Ying-Shan , Wainberg Mark A. , Yue Jian-Min
Published: December 2015

A series of Securinega alkaloid hybrids and oligomers with anti-HIV activity were isolated and identified from Flueggea virosa.
Radiosynthesis and evaluation of N-(3,4-dimethylisoxazol-5-yl)piperazine-4-[4-(4-fluorophenyl)thiazol-2-yl]-1-[11C]carboxamide for in vivo positron emission tomography imaging of fatty acid amide hydrolase in brain
Authors: Shimoda Y. , Yui J. , Zhang Y. , Hatori A. , Ogawa M. , Fujinaga M. , Yamasaki T. , Xie L. , Kumata K. , Zhang M.-R.
Published: December 2015

[11C] DPFC is a promising PET radiotracer forin vivoimaging of fatty acid amide hydrolase in brain.
Bio-conversion of apple pomace into fumaric acid in a rotating drum type solid-state bench scale fermenter and study of the different underlying mechanisms
Authors: Das Ratul Kumar , Lonappan Linson , Brar Satinder Kaur , Verma Mausam
Published: December 2015

Apple pomace was bio-converted into fumaric acid in a rotating drum type solid-state bench scale fermenter and bio-conversion mechanisms were uncovered.
Copper promoted synthesis of substituted quinolines from benzylic azides and alkynes
Authors: Luo Ching-Zong , Gandeepan Parthasarathy , Wu Yun-Ching , Chen Wei-Chen , Cheng Chien-Hong
Published: December 2015

A novel method for the synthesis of substituted quinolines from benzylic azides and internal alkynes using Cu(OTf)2is described. The reaction features a broad substrate scope, high product yields and excellent regioselectivity.
DMSO-based PbI2precursor with PbCl2additive for highly efficient perovskite solar cells fabricated at low temperature
Authors: Zhang Zhirong , Yue Xiaopeng , Wei Dong , Li Meicheng , Fu Pengfei , Xie Bixia , Song Dandan , Li Yingfeng
Published: December 2015

The advantages of both doping chloride and using dimethylsulfoxide (DMSO) as solvent are combined, by which the performance of the as-prepared perovskites solar cells is significantly improved.
The carbonization of polyethyleneimine: facile fabrication of N-doped graphene oxide and graphene quantum dots
Authors: Zhou Xi , Pan Yumei , Xu Jian , Wang Anqi , Wu Shishan , Shen Jian
Published: December 2015

In this study, N-doped graphene oxide (N-GO) and graphene quantum dots (N-GQDs) were synthesized by the carbonization of branched polyethyleneimine (PEI) in the presence of H2O2.
H2S gasotransmitter-responsive polymer vesicles
Authors: Yan Qiang , Sang Wei
Published: December 2015

A type of new polymeric vesicle self-assembly byo-azidomethylbenzoate-containing diblock copolymer can respond to the cell signaling molecule hydrogen sulfide (H2S). The intracellular H2S gasotransmitter can trigger biomimetic polymersome disruption for targeted drug delivery.
A Research-Based Clinical Tutorial in Adolescent Stuttering: Response to Coleman, Miller, and Weidner (2015)
Authors: Bothe Marcotte Anne , Santus Nina M.
Published: December 2015

Coleman, Miller, and Weidner's (2015) recent clinical vignette and tutorial described one very common, broad-based, and theory-driven approach to treating stuttering in a 14-year-old boy. This response suggests that methods based on prolonged speech are better supported in our research literature; are effective, efficient, and well-received by clients; and allow clinicians using the principles of evidence-based practice to address and solve the concerns that clients have raised.
Causes and consequences of oxidative stress in spermatozoa
Authors: Aitken Robert John , Gibb Zamira , Baker Mark A. , Drevet Joel , Gharagozloo Parviz
Published: December 2015

Spermatozoa are highly vulnerable to oxidative attack because they lack significant antioxidant protection due to the limited volume and restricted distribution of cytoplasmic space in which to house an appropriate armoury of defensive enzymes. In particular, sperm membrane lipids are susceptible to oxidative stress because they abound in significant amounts of polyunsaturated fatty acids. Susceptibility to oxidative attack is further exacerbated by the fact that these cells actively generate reactive oxygen species (ROS) in order to drive the increase in tyrosine phosphorylation associated with sperm capacitation. However, this positive role for ROS is reversed when spermatozoa are stressed. Under these conditions, they default to an intrinsic apoptotic pathway characterised by mitochondrial ROS generation, loss of mitochondrial membrane potential, caspase activation, phosphatidylserine exposure and oxidative DNA damage. In responding to oxidative stress, spermatozoa only possess the first enzyme in the base excision repair pathway, 8-oxoguanine DNA glycosylase. This enzyme catalyses the formation of abasic sites, thereby destabilising the DNA backbone and generating strand breaks. Because oxidative damage to sperm DNA is associated with both miscarriage and developmental abnormalities in the offspring, strategies for the amelioration of such stress, including the development of effective antioxidant formulations, are becoming increasingly urgent.
Role of progesterone in embryo development in cattle
Authors: Lonergan Pat , Forde Niamh , Spencer Thomas
Published: December 2015

Progesterone (P4) from the corpus luteum is critical for the establishment and maintenance of pregnancy and plays a major role in regulating endometrial secretions essential for stimulating and mediating changes in conceptus growth and differentiation throughout early pregnancy in ruminants. Numerous studies have demonstrated an association between elevated systemic P4 and acceleration in conceptus elongation. A combination of in vivo and in vitro experiments found that the effects of P4 on conceptus elongation are indirect and mediated through P4-induced effects in the endometrium. Despite effects on elongation, data on the effects of post-insemination supplementation with P4 on pregnancy rates are conflicting. This review highlights the effects of P4 on conceptus development and examines strategies that have been undertaken to manipulate P4 concentrations to increase fertility.
Measuring embryo metabolism to predict embryo quality
Authors: Thompson Jeremy G. , Brown Hannah M. , Sutton-McDowall Melanie L.
Published: December 2015

Measuring the metabolism of early embryos has the potential to be used as a prospective marker for post-transfer development, either alone or in conjunction with other embryo quality assessment tools. This is necessary to maximise the opportunity of couples to have a healthy child from assisted reproduction technology (ART) and for livestock breeders to efficiently improve the genetics of their animals. Nevertheless, although many promising candidate substrates (e.g. glucose uptake) and methods (e.g. metabolomics using different spectroscopic techniques) have been promoted as viability markers, none has yet been widely used clinically or in livestock production. Herein we review the major techniques that have been reported; these are divided into indirect techniques, where measurements are made from the embryo’s immediate microenvironment, or direct techniques that measure intracellular metabolic activity. Both have strengths and weaknesses, the latter ruling out some from contention for use in human ART, but not necessarily for use in livestock embryo assessment. We also introduce a new method, namely multi- (or hyper-) spectral analysis, which measures naturally occurring autofluorescence. Several metabolically important molecules have fluorescent properties, which we are pursuing in conjunction with improved image analysis as a viable embryo quality assessment methodology.
Breeding animals for quality products: not only genetics
Authors: Chavatte-Palmer Pascale , Tarrade Anne , Kiefer Hélène , Duranthon Véronique , Jammes Hélène
Published: December 2015

The effect of the Developmental Origins of Health and Disease on the spread of non-communicable diseases is recognised by world agencies such as the United Nations and the World Health Organization. Early environmental effects on offspring phenotype also apply to domestic animals and their production traits. Herein, we show that maternal nutrition not only throughout pregnancy, but also in the periconception period can affect offspring phenotype through modifications of gametes, embryos and placental function. Because epigenetic mechanisms are key processes in mediating these effects, we propose that the study of epigenetic marks in gametes may provide additional information for domestic animal selection.
1 GENERATION OF A STABLE TRANSGENIC SWINE MODEL FOR CELL TRACKING AND CHROMOSOME DYNAMIC STUDIES
Authors: Sper R. , Simpson S. , Zhang X. , Collins B. , Piedrahita J.
Published: December 2015

Transgenic pigs are an attractive research model in the field of translational research, regenerative medicine, and stem cell therapy due to their anatomic, genetic, and physiological similarities with humans. The development of a transgenic murine model with a fusion of green fluorescent protein (GFP) to histone 2B protein (H2B, protein of nucleosome core) resulted in an easier and more convenient method for tracking cell migration and engraftment levels after transplantation as well as a way to better understand the complexity of molecular regulation within cell cycle/division, cancer biology, and chromosome dynamics. Up to now the development of a stable transgenic large animal model expressing H2B-GFP has not been described. Our objective was to develop the first transgenic porcine H2B-GFP model via CRISPR-CAS9 mediated recombination and somatic cell nuclear transfer (SCNT). Porcine fetal fibroblasts were cotransfected with CRISPR-CAS9 designed to target the 3′ untranslated region of ACTB locus and a targeting vector containing 1Kb homology arms to ACTB flanking an IRES-H2B-GFP transgene. Four days after transfection GFP cells were fluorescence activated cell sorted. Single cell colonies were generated and analysed by PCR, and heterozygous colonies were used as donor cells for SCNT. The custom designed CRISPR-CAS9 knockin system demonstrated a 2.4% knockin efficiency. From positive cells, 119 SCNT embryos were generated and transferred to a recipient gilt resulting in three positive founder boars (P1 generation). Boars show normal fertility (pregnancies obtained via AI of wild type sows). Generated P1 clones were viable and fertile with a transgene transmission rate of 55.8% (in concordance with Mendel’s law upon chi-square test with P = 0.05). Intranuclear H2B-GFP expression was confirmed via fluorescence microscopy on 8-day in vitro cultured SCNT blastocysts and a variety of tissues (heart, kidney, brain, bladder, skeletal muscle, stomach, skin, and so on) and primary cultured cells (chondrocytes, bone marrow derived, adipocyte derived, neural stem cells, and so on) from P1 cloned boars and F1 42-day fetuses and viable piglets. In addition, chromosome segregation could be easily identified during cell cycle division in in vitro cultured stem cells. Custom designed CRISPR-CAS 9 are able to drive homologous recombination in the ACTB locus in porcine fetal fibroblasts, allowing the generation of the first described viable H2B-GFP porcine model via SCNT. Generated clones and F1 generation expressed H2B-GFP ubiquitously, and transgene transmission rates were with concordance of Mendel’s law. This novel large animal model represents an improved platform for regenerative medicine and chromosome dynamic and cancer biology studies.
102 EFFECTS OF LINOLEIC ACID/LINOLENIC ACID RATIO ON OOCYTE MATURATION AND FERTILIZATION IN PREPUBERTAL SHEEP
Authors: Ghaffarilaleh V. , Ghaffarilaleh R. , Ghaffarilaleh G.
Published: December 2015

The aim of this study was to clarify the effects of linoleic acid (LA)/alpha-linolenic acid (ALA) ratio on oocyte maturation and fertilization. Prepubertal sheep ovaries were sliced, then cumulus-oocyte complexes were matured in a media based on TCM-199 supplemented with FSH, LH, epidermal growth factor, BSA fraction V, cysteamine, glutamine, and sodium pyruvate for 24 h in different ratios of LA/ALA (0, 1 : 1, 2 : 1, 4 : 1, and 10 : 1). After maturation, 169 oocytes stained for maturation rate. In the second experiment, 176 presumptive zygotes from 227 fertilized oocytes were stained for fertilization rate. In the third experiment, 570 oocytes were used for embryo development. The generalized linear model was used for analysis. The results showed that LA/ALA at the ratios of 1 : 1, 2 : 1, and 4 : 1 significantly (P < 0.05) improved the MII percentages and the 10 : 1 ratio was detrimental. Fertilization rate was not significant between treatments, but the two pro-nuclei percentage was significantly increased for the ratios of 1 : 1, 2 : 1, and 4 : 1 (P < 0.05). Cleavage rate for the ratio of 2 : 1 and 4 : 1 was significantly higher (P < 0.05). The ratio of 10 : 1 was detrimental for embryo development, and none of the cleaved embryos reached the blastocyst rate. In conclusion, the ratio of 10 : 1 LA/ALA was detrimental and resulted in degenerated oocytes and subsequently for prepubertal sheep embryos, whereas lower ratios of LA/ALA were beneficial for oocyte competition and fertilisation.
106 COMPARISON OF IN VITRO EMBRYO PRODUCTION IN PANAMA USING HOLSTEIN OOCYTES WITH BOS INDICUS SEXED SEMEN FROM DONORS IN DIFFERENT LOCATIONS: FIELD DATA
Authors: Rodriguez S. J. R. , Ramirez Y. E. , Gomes E. , Nasser L. F. , Pontes J. H. F. , Rubessa M. , Wheeler M. B.
Published: December 2015

The objective of this work was to compare in vitro embryo production of Bos taurus × Bos indicus cross embryos using oocytes from Holstein donors under different production and environment systems. This study also examined the possibility for in vitro production using oocytes imported and transported fresh between the USA and Panama. All animals were mature Holstein cows going through a normal lactation. The first group of donors was from the University of Illinois dairy herd and went through 3 ovum pickup sessions. The second group of donors were Holstein cows already adapted to Panama and went through 10 ovum pickup sessions. The Panamanian herd of Holstein donors were born and raised in Panama in an area of mountains, on average 1300 m above sea level. This environment does not have the typical hot and humid tropical weather seen in other regions of Panama. Both groups of donors were aspirated without stimulation during the years 2013 and 2014. Oocytes recovered from donors in Illinois were imported fresh under a special sanitary research protocol between Panama and the University of Illinois. The transport of fresh oocytes from the USA to Panama was done using a portable incubator set at 39°C (Minutube of America). Oocytes were matured during transport in 5-mL tubes (~30–35 oocytes per tube) containing 400 µL of maturation media (TCM-199) that had been equilibrated with 5% CO2. Oocytes recovered from donors in Panama were matured using the same media. For both groups, oocytes were inseminated 24 h after ovum pickup using sexed semen from the same bull. All embryo production procedures followed the protocols of the In vitro Brasil™ commercial system. At 72 h postinsemination, cleavage was evaluated. On Day 7 after insemination, embryo development to the blastocyst stage (early to expanded) was recorded. Data were analysed using Chi-squared. As shown in Table 1, there was no effect of oocyte collection location on embryo development. These results indicate that it is possible to produce a viable in vitro-produced embryo using fresh oocytes collected and transported from different countries. This work opens the possibility to access superior genetics and improve herds in countries seeking to increase their production systems and potentially improve their quality of life.

Table 1.Effect of oocyte collection location on embryo development

This project was supported by Programa de Competitividad ProCom Senacyt, Panama.
115 CHARACTERIZING NEUTROPHIL PROFILES IN HORSES FOR RHINOCEROS CAPTURE
Authors: Fayrer-Hosken R. A. , Kruger M. , Vandenplas M. , Giguere S. , Buss P.
Published: December 2015

Conservation of several African species is becoming essential, and efforts to move threatened animals are causing physiological and reproductive problems. To save these species, a more comprehensive knowledge of their biology and response to stressors is required. Capture stress of rhinoceroses has been quantified (Kruger et al. 2011 Reprod. Fertil. Dev. 23, 181–182) by evaluating leucocyte coping capacity (LCC). LCC is the measurement of the fluorescence of circulating active neutrophils, then expressed as optical density (OD)/1000 neutrophils. The LCC then provides a standardized value between species as we used identical conditions and reagents. To quantify the role of LCC in rhinoceros conservation, it is essential to characterize normal LCC profiles of healthy unstressed rhinoceroses. Horse neutrophils are very similar to rhinoceros neutrophils in their biological activity. The objective of the study was to characterize normal LCC profiles in stallions, geldings, nonpregnant mares, pregnant mares, as well as fillies and colts of various ages as a benchmark for adult and juvenile rhinoceroses. The LCC profiles are shown in Figures 1 to 3. For the colts (days, weeks, and months old) there was little difference in their LCC profile over time. For the fillies (days, weeks, and months old) the LCC response for fillies only days old was significantly (P < 0.05) greater when compared to fillies that were weeks and months old. For the adult horses, the stallions had the lowest overall LCC and were very similar to nonpregnant mares. The LCC of pregnant mares was of significantly (P < 0.5) greater magnitude than that of stallions and nonpregnant mares. The LCC response of gelding was significantly (P < 0.5) greater than that of stallions and nonpregnant mares, but significantly (P < 0.5) lower than that of pregnant mares. The stallion and pregnant mare responses mirrored the quantitative responses of breeding rhinoceros bulls and pregnant rhinoceros cows. From the data (Figure 4) we suggest that breeding males (stallions, elephants, and rhinoceros bulls) have the lowest LCC activity curves, as their breeding activities result in higher daily stresses. We hypothesized that the stressed bulls have their neutrophil activity down regulated by circulating corticosteroids. The converse is true for pregnant females (mares and cows), as they may have more vigorous neutrophils and therefore the highest LCC curves. These basic studies support and validate the role of rapid LCC in stress evaluation of wild caught rhinoceroses and horses could be applicable for captured elephant.
117 DEVELOPMENT OF A NONINVASIVE, FECAL PROTEIN PREGNANCY TEST FOR POLAR BEARS
Authors: DeLorenzo C. , Lynch B. , Roth T. , Petren K. , Curry E.
Published: December 2015

Differentiating between pregnancy and nonpregnancy noninvasively is difficult in species that experience pseudopregnancy, including polar bears (Ursus maritimus). These bears usually breed in the spring, undergo delayed implantation until late summer or early fall, and give birth in late fall. In other species, the placental protein transthyretin (TTR) has been shown to be essential for early fetal growth, responsible for transporting thyroid hormone from the mother to the fetus during early pregnancy. Preliminary data obtained via 2D-DIGE indicated that fecal TTR is elevated during polar bear pregnancy, but further research is needed to validate its use as an accurate biomarker of pregnancy. The aim of this study was to develop a bench-side assay to characterize TTR in longitudinal fecal samples from pregnant and nonpregnant polar bears. Specific objectives were to 1) develop an effective method for total fecal protein extraction; 2) identify antibodies that cross-react with polar bear fecal TTR; 3) validate an EIA for measuring fecal TTR; and, for proof of concept, 4) compare fecal TTR concentrations among pregnant, pseudopregnant, and nonpregnant polar bears. Fecal samples (n = 205) were collected from females that produced cubs (pregnant; n = 2), did not breed but exhibited a prolonged increase in progesterone (P4; pseudopregnant; n = 2), or did not breed and did not exhibit an increase in P4 (nonpregnant; n = 2). Total protein of each sample was extracted using a modified ammonium sulfate method. Protein concentrations were quantified using the Bradford assay. Western blot was used to determine commercial antibody compatibility with polar bear fecal TTR. A sandwich EIA was optimized using a standard curve ranging from 12.5 to 400 pg of human TTR/well. A parallelism was performed using two-fold serial dilutions of pooled fecal protein. Fecal TTR concentrations were measured in duplicate and are reported as femtograms of TTR per micrograms of total protein. Mean TTR concentrations in samples collected from July–December were compared among groups. Values are reported as mean ± standard error of the mean. Total fecal protein yield was 459.38 ± 284.38 µg g–1 of feces. Successful binding of 2 polyclonal antibodies to the 15-kDa subunit of the TTR protein was verified via western blot and indicated that fecal proteins can retain antibody-binding capacity. The parallelism exhibited strong correlation with the standard curve (R2 = 0.989). Data suggest that fecal TTR is higher during late summer or early fall in pregnant bears (588.40 ± 116.52) when compared to the pseudopregnant state (111.23 ± 15.98) and the nonpregnant state (250.83 ± 34.34). This is the first EIA developed to measure fecal TTR in any species. Although preliminary, these data suggest that the fecal TTR EIA may be useful in diagnosing pregnancy in polar bears.
119 HORMONE-ASSISTED REPRODUCTION IN A CAPTIVE MOUNTAIN YELLOW-LEGGED FROG POPULATION
Authors: Calatayud N. E. , Shier D. M. , Swaisgood R. R. , Durrant B. S.
Published: December 2015

Severe declines of the mountain yellow-legged frog (MYLF, Rana muscosa) led to establishment of a captive population at the San Diego Zoo Institute for Conservation Research. With less than 200 adults estimated to remain in the wild, the Institute’s MYLF colony currently holds approximately 30% of the entire gene pool. Simulating natural seasonality by artificial brumation and manipulation of atmospheric temperature and light cycles is an integral part of this species’ management in captivity. However, over 5 years, the number of females that have oviposited has decreased from 80% in 2011 to 28% in 2014. It is unclear if changes in reproduction are related to husbandry or a lack of information regarding the natural reproductive cycles of these animals. Therefore, we examined the effects of hormone treatments on reproduction in the MYLF captive population. Prior to breeding, 21 females and 18 males were evenly assigned to either a control group (IP injection of PBS) or hormone-treated group [IP injection of gonadotropic releasing hormone (GnRH, D-Ala6, des-GLy10 ethylamide LHRH derivative) at 0.4 µg g–1 of body weight and metoclopramide (dopamine inhibitor) at 10 µg g–1 of body weight]. Males and females were housed in groups of 3 males and 3 females and allowed to choose their mate. Once a male had amplexed a female they were housed in pairs until oviposition occurred or until males ceased amplexus. Responses recorded during this study included amplexus, spermiation, oviposition, and embryonic cleavage. There was no significant difference in the number of eggs deposited by females treated with hormones and untreated controls (P = 0.1949) nor were there any differences between groups in the number of embryos that cleaved (P = 0.673) or survived to tadpole stage (P = 0.629). No significant differences were detected between the numbers of males that amplexed in the control or treated groups (P = 0.1120). Urine collected from 10/18 amplexed males (7 hormone-treated and 3 controls) indicated that 57% percent of hormone-treated males and 67% of control males were spermiating at the time of collection. Therefore, hormone treatments did not increase the number of eggs oviposited or the number of males spermiating or amplexing. Closer analysis of individual female reproductive histories indicated that 48% of captive female MYLF oviposited just once in 5 years, 40% in 2 consecutive years, 8% oviposited eggs in 2 consecutive years, skipped a year, and then oviposited again in 2 consecutive years, and 4% oviposited in 3 consecutive years. The number of females ovipositing in 2015 was significantly higher than 2012 (P = 0.0002), 2013 (P = 0.0001), and 2014 (P = 0.0026), but not 2011 (P = 0.0885), suggesting breeding may not occur annually in females. Understanding the breeding cycles of MYLF females will enable managers to determine if and when hormone administration is efficacious in captive amphibian breeding populations.
124 COMPARISON OF TWO EMBEDDING SYSTEMS FOR FOLLICULAR CULTURE
Authors: Polkoff K. M. , Rubessa M. , Winters R. , Lopez N. A. , Wheeler M. B.
Published: December 2015

Recent research has focused on developing secondary, preantral follicles as a new way to collect female gametes to develop in vitro or to study folliculogenesis. Secondary follicles are abundant in ovaries of almost all females, and can be isolated from the ovarian cortex of fresh or cryopreserved tissue. These follicles must be matured to obtain mature oocytes, but the mechanism for early follicle development is not well understood. Currently, culture techniques are being developed to promote the growth and quality of these follicles. The aim of this preliminary study was to compare 2 embedding systems for porcine follicles to improve preantral follicle culture. We collected secondary preantral follicles isolated from porcine ovaries recovered from the abattoir. Ovaries were transported to our laboratory in saline solution (0.9% NaCl) and cut into smaller fragments with a scalpel. Follicles were further mechanically isolated with a 23 1/2-gauge needle and placed into a dish with medium TCM199 plus Hepes (Lonza 12–117F) supplemented with 5% fetal bovine serum (FBS). Secondary follicles less than 300 µm were selected. Furthermore, only clear follicles with dark oocytes and no antrum were used. The selected follicles were randomly divided into 5 different treatment groups: 1) embedded in 0.5% alginate gel; 2) 1.0% alginate gel; 3) 1.0% agarose gel; 4) 2.0% agarose gel; and 5) control. Three follicles were placed in each well with 280 µL of α-minimal essential medium supplemented with 3.5 μg mL–1 of insulin 10 μg mL–1 of transferrin, 100 μg mL–1 of l-ascorbic acid, and 7.5% porcine serum and cultured for 4 days at 39°C at 5% CO2. Media (140 µL) from each well was changed on Day 2 and saved for metabolic and functional analysis. Initial diameters and measurements on Day 2 and 4 were taken to analyse dimensional growth. We evaluated 6 follicles per group per replicate, for a total of 3 replicates (18 per group). All recorded parameters were subjected to a 2-way ANOVA using the procedure of the Generalized Linear Model (SPSS, 18th version, SPSS Inc., Chicago, IL, USA). Independent variable was the result of the balance between the size on Day 2 and 0 and the size on Day 4 and 0 time of observation. Data were normally distributed. Least squares means tests were used to perform statistical multiple comparisons. The α level was set at 0.05. All data were expressed as quadratic means and mean standard errors. The results in the Table 1 show that at Day 2 there was no difference between groups. However, after 4 days of development, the group agar 2% and alginate 1% showed a statistical difference when compared with the control. These results suggest that there is a positive effect when the follicles are embedded during culture.

Table 1.Growth of Follicles (in μm) since Day 0 in different embedding systems
130 ANTRAL FOLLICLE COUNTS, VULVA WIDTH, AND SERUM ANTI-MULLERIAN HORMONE CONCENTRATIONS IN BOVINE FEMALES OF THE TABAPUÃ BREED
Authors: Souza J. C. , Maculan R. , Rosa R. G. , Smith L. F. , Casas P. S. , Faria B. R. M. , Alves N. , Gonçalves T. M.
Published: December 2015

Bovine genetic selection for fertility is difficult in part due to the low heritability of fertility traits and to the extension of the calving interval and the productive life length inherent to this species, which makes it harder to identify superior individuals from their records. In this regard, it is feasible to try to identify characteristics that are easy to detect and may be linked to superior reproductive efficiency as early as possible in females. The objective was to evaluate the possible relationships between vulva width, antral follicle counts (AFC), and the serum concentrations of anti-Mullerian hormone (AMH). Tabapuã (a Brazilian Zebu breed) heifers (n = 155) and cows (n = 422) from 4 herds were used in the study in which all antral follicles ≥3 mm were counted directly with an ultrasound unit. Follicle counts were classified into 2 classes, based on the methodology proposed by Burns et al. (2005) in dairy cattle, such that the low and intermediate AFC classes were merged into one class (low; n = 245) and the high and very high into another (high; n = 332). The vulva width was determined by measuring the distance between the lateral borders of the vulva with a digital caliper placed at a 90-degree angle from the half point of the rima vulvae. Single blood samples were collected from a subgroup of animals (n = 220) and the serum frozen (–20°C) after centrifugation for the AMH analysis, which was performed by specific bovine ELISA (Ansh Laboratories, Webster, TX, USA) test. All statistical analyses were performed with SAS (SAS Institute Inc., Cary, NC, USA). Data was submitted for analyses of variance using the PROCGENMOD procedure. Vulva width was larger (P = 0.02) in the class of animals of the high AFC (8.73 ± 0.11 cm) in comparison with those of the low AFC (8.39 ± 0.09 cm), considering the larger sample (n = 591). In the subsample (n = 220) the concentration of AMH was higher (P < 0.0001) in the high AFC class females (1.15 ± 0.07 ng mL–1; n = 95) in comparison with the low AFC class (0.50 ± 0.06 ng mL–1; n = 125). Vulva width, AFC, and AMH serum concentrations were interrelated and likely fertility indicative phenotypes under the present experimental conditions and should be further evaluated in larger samples in order to be considered in bovine genetic selection.
136 COMPARISON OF NCSU-23 AND ALPHA-MINIMAL ESSENTIAL MEDIA IN THE DEVELOPMENT OF ISOLATED PORCINE PREANTRAL FOLLICLES IN VITRO
Authors: Rubessa M. , Rocha R. , Lima L. , Winters R. , Figueiredo J. R. , Wheeler M. B.
Published: December 2015

To develop a preantral follicular culture system that will support follicular growth and result in fertilizable oocytes, we conducted an experiment designed to determine the best medium for culture. In our preliminary experiment, we compared 2 common base media used for porcine oocytes: α-minimal essential medium and NCSU-23. Ovaries were collected from prepubertal gilts at a local abattoir and transported to the laboratory in saline solution (0.9% NaCl) maintained at 30–35°C. The ovaries were cut into small pieces (1–3 mm), and preantral follicles were isolated mechanically. Preantral follicles from 280 to 300 μm in diameter were collected into a small dish containing medium TCM199 (Lonza 12–117F) supplemented with 5% fetal bovine serum. The follicles were transferred from the collecting medium to the culture medium that consisted of base medium (NCSU23 or α-minimal essential medium) supplemented with 3.5 μg mL–1 of insulin, 10 μg mL–1 of transferrin, 100 μg mL–1 of l-ascorbic acid, 7.5% porcine serum, and 1.5 ng mL–1 of FSH. The follicles were randomly distributed to the different experimental treatments and cultured for 6 days in 24-well cell culture plates, with 3 follicles per well in 280 μL of culture medium. The culture was carried out at 38.5°C in 5% CO2 in air. Culture medium was changed every 2 days with freshly prepared medium. The diameters of follicles were measured every 2 days, and each follicle was photographed and evaluated at 20× magnification. Forty-two follicles per group were analysed and collected in 4 replicates. Data were statistically analysed with ANOVA using the Generalized Linear Model (GLM) procedure (SPSS, version 18, SPSS Inc., Chicago, IL, USA), where the independent variable was the sample (group and day of culture). Tukey's post hoc test was used to perform multiple comparisons; the α level was set at 0.05. All data were expressed as quadratic means with standard error of the means. Only the antrum formation was evaluated by chi-square test. The results, reported in Table 1, show that there was no statistical differences between follicle size between NCSU23 or α-minimal essential media, but at Day 6 there was a positive trend (P = 0.08). Otherwise, when we compared the size inside the groups, we observed that the preantral follicles grew more in α-minimal essential media than in NCSU23. The percentages of antrum formation were 65 v. 76% (NCSU23 and α-minimal essential media, respectively). These results support the use of α-minimal essential media because it had a positive effect on the antrum formation, and that after Day 4 some follicles could undergo a regression phase. Future studies will be necessary to evaluate the molecular status and the hormone production.

Table 1.Follicle size (μm) from Day 0 to Day 6