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Topic:Laboratory Methods
Laboratory methods in equine research encompass a variety of techniques and procedures used to analyze biological samples from horses to study health, disease, genetics, and physiology. These methods include hematological analyses, biochemical assays, molecular biology techniques, and microbiological cultures. Commonly utilized laboratory techniques involve blood tests for complete blood count (CBC) and serum chemistry, polymerase chain reaction (PCR) for genetic and infectious disease studies, and enzyme-linked immunosorbent assays (ELISA) for detecting specific proteins or antibodies. These methods provide valuable data that contribute to understanding equine health and disease mechanisms. This page compiles peer-reviewed research studies and scholarly articles that explore the application, development, and outcomes of laboratory methods in the context of equine research.
Comparison of Tube, Gel, and Immunochromatographic Strip Methods for Evaluation of Blood Transfusion Compatibility in Horses. Assessment of blood compatibility, typically by tube agglutination (TUBE) and hemolysis crossmatch or, less commonly, by blood typing and alloantibody screening, often is performed before blood transfusion in horses. In contrast, gel column (GEL) and immunochromatographic strip (STRIP) techniques are preferred for compatibility testing in dogs and cats. Objective: To determine the accuracy of novel and standard crossmatch and typing methods. Methods: Thirty-eight healthy horses, previously blood typed and alloantibody screened. Methods: TUBE and GEL crossmatches were performed on 146 different...
Nitrite coordination in myoglobin. The coordination of nitrite in myoglobin (Mb) has been characterized by resonance Raman spectroscopy and the frequencies of the nitrite bound to the heme Fe as well to the 2-vinyl have been computed by density functional theory (DFT) calculations. The DFT Natural Bond Orbital (NBO) analysis and the extensive isotope-labeling in the resonance Raman experiments indicate that NO (O1NO2) is bound to the heme Fe via O1. Based on the vibrational characterization of the reversible transition between low and high spin FeONO/2-nitrovinyl species, we suggest that the key step that triggers the spin-chan...
Substrate Specificity of Equine and Human Influenza A Virus Sialidase to Molecular Species of Sialic Acid. Most equine influenza A viruses (IAVs) show strong binding to glycoconjugates containing N-glycolylneuraminic acid (Neu5Gc) as well as N-acetylneuraminic acid (Neu5Ac). Therefore, the progeny of equine IAV is thought to be released from the infected cell surface through removal of sialic acids by the viral sialidase. In the present study, equine IAV sialidases showed significantly lower substrate affinity than that of human IAV sialidases to artificial and natural Neu5Gc-conjugated substrates. The substrate specificity of equine IAV sialidases is in disagreement with their binding specificity ...
Identification of the most abundant proteins in equine amniotic fluid by a proteomic approach. Characterisation of the physiologic equine amniotic fluid (AF) proteome is a prerequisite to study its changes during diseases and discover new biomarkers. The aim of this study was to identify by a proteomic approach the most abundant proteins of equine AF. AF samples were collected at parturition from 24 healthy mares that delivered healthy foals. All samples were subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) on 4-12% gels. A pool of the 24 samples, after SDS-PAGE, was cut in 25 slices, trypsin-digested and analysed by mass spectrometry (MS) for protein i...
Intracytoplasmic Sperm Injection, Embryo Culture, and Transfer of In Vitro-Produced Blastocysts. Intracytoplasmic sperm injection is becoming a common clinical procedure in the horse, but little information is available on techniques for its performance. Each laboratory uses different procedures and different media for the steps involved with in vitro embryo production. This article outlines the procedures used in the Clinical Equine Intracytoplasmic Sperm Injection Program at Texas A&M University for in vitro blastocyst production during the past 3 years.
Doping control analysis of anabolic steroids in equine urine by gas chromatography-tandem mass spectrometry. Anabolic steroids are banned substances in equine sports. Gas chromatography-mass spectrometry (GC-MS) has been the traditional technique for doping control analysis of anabolic steroids in biological samples. Although liquid chromatography-mass spectrometry (LC/MS) has become an important technique in doping control, the detection of saturated hydroxysteroids by LC-MS remains a problem due to their low ionization efficiency under electrospray. The recent development in fast-scanning gas-chromatography-triple-quadrupole mass spectrometry (GC-MS/MS) has provided a better alternative with a sign...
Confirmatory analysis of etanercept in equine plasma by LC-MS for doping control. Etanercept is a protein-based medication for the treatment of human patients with rheumatoid arthritis and other autoimmune-based diseases; its pharmacological action is to inhibit and antagonize tumour necrosis factor alpha. Etanercept was rumoured to be used in horse racing in North America. To detect such use, the aim of this study was to develop a liquid chromatography-mass spectrometry (LC-MS) method for confirmation of etanercept in equine plasma. Etanercept was extracted from plasma by anti-human IgG antibody linked to magnetic beads. The analyte was reduced and alkylated, and then dige...
Photoacoustic calorimetry studies of CO photo-dissociation from chloramine-T modified horse heart cytochrome-c. Treatment of horse heart Cytochrome-c (Cc) with N-chloro-4-toluosulfonamide (Chloramine-t, CT) results in the oxidation of methionine (Met) residues to the corresponding sulfoxide including the distal heme ligand, Met80. The resulting Fe-sulfoxide coordination is sufficiently labile in the ferrous form to be displaced by gaseous ligands, including CO. Photolysis of the CO-CT-Cc complex provides an opportunity to examine ligand binding dynamics that are associated with a relatively rigid distal heme pocket. In this work, photoacoustic calorimetry (PAC) was utilized to obtain the kinetics as wel...
Characterisation of equine satellite cell transcriptomic profile response to β-hydroxy-β-methylbutyrate (HMB). β-Hydroxy-β-methylbutyrate (HMB) is a popular ergogenic aid used by human athletes and as a supplement to sport horses, because of its ability to aid muscle recovery, improve performance and body composition. Recent findings suggest that HMB may stimulate satellite cells and affect expressions of genes regulating skeletal muscle cell growth. Despite the scientific data showing benefits of HMB supplementation in horses, no previous study has explained the mechanism of action of HMB in this species. The aim of this study was to reveal the molecular background of HMB action on equine skeletal m...
Equine hepatocytes: isolation, cryopreservation, and applications to in vitro drug metabolism studies. Despite reports of the successful isolation of primary equine hepatocytes, there are no published data regarding the successful cryopreservation of these isolated cells. In this study, a detailed description of the procedures for isolation, cryopreservation, and recovery of equine hepatocytes are presented. Furthermore, the intrinsic clearance (Cl) and production of metabolites for three drugs were compared between freshly isolated and recovered cryopreserved hepatocytes. Primary equine hepatocytes were isolated using a two-step collagenase perfusion method, with an average cell yield of 2.47Â...
Characterization and angiogenic potential of xenogeneic bone grafting materials: Role of periodontal ligament cells. Adequate revascularization is a prerequisite for successful healing of periodontal bone defects. This study characterized three different xenogeneic bone grafting materials: Gen-Os of equine and porcine origins, and anorganic Bio-Oss. We also investigated their angiogenic potential. All materials were composed of poorly crystalline calcium oxide phosphate, with Bio-Oss exhibiting a carbonated phase and larger particle size and both Gen-Os showing the presence of collagen. Both Gen-Os materials significantly enhanced vascular endothelial growth factor (VEGF) secretion by PDL cells. A significan...
Cytokine and chemokine profiles of aqueous humor and serum in horses with uveitis measured using multiplex bead immunoassay analysis. To determine whether horses with clinically diagnosed Equine Recurrent Uveitis (ERU) and those with Leptospirosis infection have a specific cytokine profile in their aqueous humor (AH) and serum that differs from horses with uveitis secondary to other ocular inflammatory processes and from horses with normal eyes. Methods: Twenty-five client-owned horses with uveitis that were presented to the North Carolina State University Ophthalmology Service, and four University-owned horses without history or clinical signs of ocular disease. Methods: Samples of AH and serum were obtained from horses wit...
Isolation of endothelial colony-forming cells from blood samples collected from the jugular and cephalic veins of healthy adult horses. OBJECTIVE To evaluate optimal isolation of endothelial colony-forming cells (ECFCs) from peripheral blood of horses. SAMPLE Jugular and cephalic venous blood samples from 17 adult horses. PROCEDURES Each blood sample was divided; isolation was performed with whole blood adherence (WBA) and density gradient centrifugation (DGC). Isolated cells were characterized by uptake of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate-labeled acetylated low-density lipoprotein (DiI-Ac-LDL), vascular tubule formation, and expression of endothelial (CD34, CD105, vascular endothelial growth ...
Beta-mercaptoethanol supplementation of in vitro maturation medium does not influence nuclear and cytoplasmic maturation of equine oocytes. In vitro embryo production in the horse is still not as efficient as in other species. Oxidative stress negatively affects oocyte and embryo culture. To attenuate/minimize the oxidative stress, antioxidants such as low-molecular thiol compounds can be added to culture media. Beta-mercaptoethanol (BME) has been shown to improve maturation and embryo development in different species. The aim of this study was to investigate whether the addition to maturation medium of BME at common (0.1Â mM) and high (0.7Â mM) concentration could improve oocyte maturation also in the horse. Equine oocytes recove...
N-glycosylation proteomic characterization and cross-species comparison of milk fat globule membrane proteins from mammals. Glycosylation of proteins has been implicated in various biological functions and has received much attention; however, glycoprotein components and inter-species complexity have not yet been elucidated fully in milk proteins. N-linked glycosylation sites and glycoproteins in milk fat globule membrane (MFGM) fractions were investigated by combining N-glycosylated peptides enrichment and high-accuracy Q Exactive identification, to map the N-glycoproteome profiles in Holstein and Jersey cows, buffaloes, yaks, goats, camels, horses, and humans. A total of 399 N-glycoproteins with 677 glycosylation...
A Serological Protein Microarray for Detection of Multiple Cross-Reactive Flavivirus Infections in Horses for Veterinary and Public Health Surveillance. The genus Flavivirus in the family Flaviviridae includes some of the most important examples of emerging zoonotic arboviruses that are rapidly spreading across the globe. Japanese encephalitis virus (JEV), West Nile virus (WNV), St. Louis encephalitis virus (SLEV) and Usutu virus (USUV) are mosquito-borne members of the JEV serological group. Although most infections in humans are asymptomatic or present with mild flu-like symptoms, clinical manifestations of JEV, WNV, SLEV, USUV and tick-borne encephalitis virus (TBEV) can include severe neurological disease and death. In horses, infection wi...
Detection of single nucleotide polymorphisms (SNP) in equine coat color genes using SNaPshotTM multiplex kit or pluronic F-108 tri-block copolymer and capillary electrophoresis. Molecular methods for the detection of mammalian coat color phenotypes have expanded greatly within the past decade. Many phenotypes are associated with a single nucleotide polymorphism mutation in the genetic sequence. Traditionally, these mutations are detected through sequencing, hybridization assays or mini-sequencing. However, these techniques can be expensive and tedious. Previously, CE-SSCP using the F-108 polymer was able to distinguish SNPs for the melanocortin-1 receptor (mc1r) coat color gene in horses (Equus caballus) that differed by one nucleotide substitution. The objective of t...
Species determination using the red blood cells morphometry in domestic animals. This investigation is placed in the context of continuity of a preliminary study already published; it was conducted in cattle, sheep, goats, horses, and dogs; the main aim is to reveal and develop criteria for the animal species determination based on the morphometric parameters of red blood cells. Methods: Blood samples were taken from the jugular vein; and the smears were confectioned on slides immediately after the blood collection and stained according to the May-Gründwald Giemsa method. For the morphometric study, three parameters were considered which are: The diameter, the circumferen...
Development and evaluation of a new lateral flow assay for simultaneous detection of antibodies against African Horse Sickness and Equine Infectious Anemia viruses. African horse sickness (AHS) and equine infectious anemia (EIA) are both notifiable equid specific diseases that may present similar clinical signs. Considering the increased global movement of horses and equine products over the past decades, together with the socio-economic impact of previous AHS and EIA outbreaks, there is a clear demand for an early discrimination and a strict control of their transmission between enzootic and AHS/EIA-free regions. Currently, the individual control and prevention of AHS or EIA relies on a series of measures, including the restriction of animal movements, v...
Comparison of three different methods for the quantification of equine insulin. Exact analysis of equine insulin in blood samples is the key element for assessing insulin resistance or insulin dysregulation in horses. However, previous studies indicated marked differences in insulin concentrations obtained from sample analyses with different immunoassays. Most assays used in veterinary medicine are originally designed for use in human diagnostics and are based on antibodies directed against human insulin, although amino acid sequences between equine and human insulin differ. Species-specific assays are being used more frequently and seem to provide advantages compared to ...
Chiral Selectivity in Inter-reactant Recognition and Electron Transfer of the Oxidation of Horse Heart Cytochrome c by Trioxalatocobaltate(III). Outer-sphere electron transfer (ET) between optically active transition-metal complexes and either other transition-metal complexes or metalloproteins is a prototype reaction for kinetic chirality. Chirality as the ratio between bimolecular rate constants of two enantiomers mostly amounts to 1.05-1.2 with either the Λ or Δ form the more reactive, but the origin of chirality in ET parameters such as work terms, electronic transmission coefficient, and nuclear reorganization free energy has not been addressed. We report a study of ET between the Λ-/Δ-[Co(Ox)3](3-) pair (Ox = oxalate) and hor...
Development of a loop-mediated isothermal amplification assay for rapid detection of Burkholderia mallei. The present study was conducted to establish a Loop-mediated isothermal amplification (LAMP) technique for the rapid detection of B. mallei the etiologic agent of glanders, a highly contagious disease of equines. A set of six specific primers targeting integrase gene cluster were designed for the LAMP test. The reaction was optimized using different temperatures and time intervals. The specificity of the assay was evaluated using DNA from B.pseudomallei and Pseudomonas aeruginosa. The LAMP products were analyzed both visually and under UV light after electrophoresis. The optimized conditions w...
Isolation, cultivation and molecular characterization of a new Trypanosoma equiperdum strain in Mongolia. Trypanosoma equiperdum causes dourine via sexual transmission in Equidae. T. equiperdum is classified under the subgenus Trypanozoon along with the T. brucei sspp. and T. evansi; however, the species classification of Trypanozoon remains a controversial topic due to the limited number of T. equiperdum reference strains. In addition, it is possible that some were misclassified T. evansi strains. Thus, there is a strong need for a new T. equiperdum strain directly isolated from the genital mucosa of a horse with a clinically- and parasitologically-confirmed dourine infection. Trypanosomes isolat...
Progestin withdrawal at parturition in the mare. Mammalian pregnancies need progestogenic support and birth requires progestin withdrawal. The absence of progesterone in pregnant mares, and the progestogenic bioactivity of 5α-dihydroprogesterone (DHP), led us to reexamine progestin withdrawal at foaling. Systemic pregnane concentrations (DHP, allopregnanolone, pregnenolone, 5α-pregnane-3β, 20α-diol (3β,20αDHP), 20α-hydroxy-5α-dihydroprogesterone (20αDHP)) and progesterone) were monitored in mares for 10days before foaling (n=7) by liquid chromatography-mass spectrometry. The biopotency of dominant metabolites was assessed using luci...
Amino acid mutations in the env gp90 protein that modify N-linked glycosylation of the Chinese EIAV vaccine strain enhance resistance to neutralizing antibodies. The Chinese EIAV vaccine is an attenuated live virus vaccine obtained by serial passage of a virulent horse isolate (EIAV) in donkeys (EIAV) and, subsequently, in donkey cells in vitro. In this study, we compare the env gene of the original horse virulent virus (EIAV) with attenuated strains serially passaged in donkey MDM (EIAV) and donkey dermal cells (EIAV). Genetic comparisons among parental and attenuated strains found that vaccine strains contained amino acid substitutions/deletions in gp90 that resulted in a loss of three potential N-linked glycosylation sites, designated g5, g9, and g1...
Molecular Characterization of Equine APRIL and its Expression Analysis During the Adipogenic Differentiation of Equine Adipose-Derived Stem Cell In Vitro. A proliferation inducing ligand (APRIL) is a member of the TNF superfamily. It shares two receptors with B-cell activating factor (BAFF), B-cell maturation antigen (BCMA), and transmembrane activator and CAML interactor (TACI). Herein, the equine APRIL was identified from equine adipose-derived stem cell (ASC), and the protein expression of APRIL and its related molecules were detected during the adipogenic differentiation of equine ASC in vitro. The equine APRIL gene was located on chromosome 11, spans 1852 base pairs (bp). Its open reading frame covers 753Â bp, encoding a 250-amino acid prot...
Plasma, subcutaneous tissue and bone concentrations of ceftiofur sodium after regional limb perfusion in horses. Regional limb perfusion (RLP) is an effective treatment option for injuries and infections of the distal limb in horses. Using ceftiofur sodium in RLP has been studied due to its superior spectrum of Gram-positive organisms compared to aminoglycosides, but it is not known if this antimicrobial drug adequately penetrates subcutaneous tissue and bone. Objective: To determine the concentration of ceftiofur in plasma, subcutaneous tissue and bone in horses after RLP. Methods: Experimental prospective study. Methods: Six healthy horses were used in this study. Under standing sedation, an Esmarch to...
Diagnostic performance of molecular and conventional methods for identification of dermatophyte species from clinically infected Arabian horses in Egypt. Rapid and accurate identification of dermatophytes is crucial for the effective control of disease outbreaks. Current methods based on culture and microscopic characteristics may require weeks before positive identification is made. Objective: To (i) identify the most common pathogenic dermatophytes affecting Arabian horses; (ii) compare the performance of direct microscopy (DM), culture, PCR using hair samples (PCRhair ) and PCR based on culture isolates (PCRculture ) for the diagnosis of dermatophytosis. Methods: Samples of hair and crusts of skin lesions from Arabian horses were collected o...
Orf virus interleukin-10 and vascular endothelial growth factor-E modulate gene expression in cultured equine dermal fibroblasts. Wounds in horses often exhibit sustained inflammation and inefficient vascularization, leading to excessive fibrosis and clinical complications such as "proud flesh". Orf virus-derived proteins, vascular endothelial growth factor (VEGF)-E and interleukin (ovIL)-10, enhance angiogenesis and control inflammation and fibrosis in skin wounds of laboratory animals. Objective: The study aimed to determine if equine dermal cells respond to VEGF-E and ovIL-10. Equine dermal cells are expected to express VEGF and IL-10 receptors, so viral protein treatment is likely to alter cellular gene expression an...
PATHFAST, a novel chemiluminescent enzyme immunoassay for measuring estradiol in equine whole blood and serum. A novel chemiluminescent enzyme immunoassay system, PATHFAST, for the measurement of estradiol in horses was evaluated. The concentrations of estradiol in the whole blood and serum of mares were measured using PATHFAST and the estradiol concentrations measured by PATHFAST were compared with those measured by a time-resolved fluoro-immunoassay (FIA). To monitor physiological changes, serum estradiol concentrations in mares were measured using PATHFAST throughout the gestation period. The serum estradiol concentrations correlated highly with those in whole blood samples. The serum concentrations...
