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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 hematologic and biochemical results on blood obtained by jugular venipuncture as compared with intravenous catheter in adult horses. During hospitalization, horses typically undergo frequent blood sampling for diagnostic testing and monitoring. The need for numerous samples in hospitalized horses makes acquisition from an intravenous catheter (IVC) both convenient and less stressful to the patient. Objective: We hypothesized that there would be no significant difference in the plasma chemistry and CBC variables from blood samples obtained from a jugular catheter as compared with direct jugular venipuncture. Methods: Fifty adult hospitalized horses; 25 receiving constant rate crystalloid therapy, and 25 receiving low volume ...
Direct observation of myoglobin structural dynamics from 100 picoseconds to 1 microsecond with picosecond X-ray solution scattering. Here we report structural dynamics of equine myoglobin (Mb) in response to the CO photodissociation visualized by picosecond time-resolved X-ray solution scattering. The data clearly reveal new structural dynamics that occur in the timescale of ∼360 picoseconds (ps) and ∼9 nanoseconds (ns), which have not been clearly detected in previous studies.
Comparative evaluation of four competitive/blocking ELISAs for the detection of influenza A antibodies in horses. New Zealand is free from equine influenza and has never experienced an incursion in its horse population. As part of New Zealand's preparedness to an incursion of an exotic animal disease, it was considered necessary to select the most accurate test for equine influenza (EI) from the array of those available. Four readily available blocking/competitive enzyme-linked immunosorbent assays (ELISA), originally developed and marketed for the detection of antibodies against the avian influenza virus, were evaluated using serum samples from New Zealand non-infected, non-vaccinated horses (n=365), and...
Electrochemical study of gelatin as a matrix for the immobilization of horse heart cytochrome c. The aim of this paper is to emphasize the strength of gelatin as a stable matrix for redox enzymes. Cyclic voltammetry has been applied for a detailed electrochemical study of horse heart cytochrome c (HHC) entrapped in a gelatin matrix immobilized on a gold electrode. The influence of the HHC concentration, the mass percentage of the gelatin and the nature of the gelatin on the electrochemical behaviour of HHC have been described in detail. In addition, attenuated total reflection infrared (ATR-IR) spectroscopy was used to prove the immobilization on a qualitative and conformational level. Th...
Definite coordination arrangement of organometallic palladium complexes accumulated on the designed interior surface of apo-ferritin. Apo-ferritin (apo-Fr) mutants are used as scaffolds to accommodate palladium (allyl) complexes. Various coordination arrangements of the Pd complexes are achieved by adjusting the positions of cysteine and histidine residues on the interior surface of the apo-Fr cage.
Use of blood culture medium enrichment for synovial fluid culture in horses: a comparison of different culture methods. Standard methods for culturing equine synovial fluid (SF) are often unrewarding. Evidence-based information on the relative efficiency of different systems used for optimisation of isolation of microorganisms from equine SF is lacking. Objective: To compare the results of different culture systems performed in parallel on SF samples from horses clinically diagnosed with synovial sepsis. Methods: Synovial fluid specimens were collected between February 2007 and October 2008 from all horses admitted to a referral hospital that were clinically diagnosed with synovial sepsis and from control horse...
Colic in geriatric compared to mature nongeriatric horses. Part 1: Retrospective review of clinical and laboratory data. It is the impression of some surgeons that geriatric horses have a lower survival rate compared to mature nongeriatric horses following colic surgery. One possible reason for this is that geriatric horses may be more critically ill at admission and have more severe disease than mature nongeriatric horses. Objective: To compare admission historical, physical examination and laboratory data for geriatric and mature nongeriatric horses referred for signs of colic. Methods: Medical records of horses admitted with a presenting complaint of colic between 2000 and 2006 were reviewed. Geriatric horses...
Comparison of methods for depletion of albumin and IgG from equine serum. Disease-specific biomarkers hold diagnostic promise in both human and veterinary medicine, but serum biomarkers in low concentrations may be masked by the presence of abundant proteins, mostly albumin and IgG. Methods to deplete albumin and IgG exist, but efficacy of these methods for depleting equine serum of these proteins has not been established. Objective: The aim of this study was to determine if albumin and IgG could be depleted from equine serum using several commercially available kits and procedures. Methods: One-dimensional gel electrophoresis followed by densitometry was used to de...
Comparison of three methods of DNA extraction from peripheral blood mononuclear cells and lung fragments of equines. We compared three different protocols for DNA extraction from horse peripheral blood mononuclear cells (PBMC) and lung fragments, determining average final DNA concentration, purity, percentage of PCR amplification using beta-actin, and cost. Thirty-four samples from PBMC, and 33 samples from lung fragments were submitted to DNA extraction by three different protocols. Protocol A consisted of a phenol-chloroform and isoamylic alcohol extraction, Protocol B used alkaline extraction with NaOH, and Protocol C used the DNAzol((R)) reagent kit. Protocol A was the best option for DNA extraction from...
Development of a new primer-probe energy transfer method for the differentiation of neuropathogenic and non-neuropathogenic strains of equine herpesvirus-1. Equine herpesvirus-1 (EHV-1) is a major pathogen of horses with worldwide distribution that can cause various clinical signs ranged from mild respiratory disease to neurological symptoms. Comparison of neuropathogenic and non-neuropathogenic EHV-1 strains revealed that a single non-synonymous nucleotide substitution (A/G2254) in the ORF30 region is associated with the altered functions of the viral DNA polymerase and therefore the neuropathogenicity of EHV-1 virus strains. The aim of the present study was the development of a new differentiation method of this particular single nucleotide poly...
Development of a DNA microarray for detection of expressed equine classical MHC class I sequences in a defined population. Development of an accurate and efficient molecular-based equine MHC class I typing method would facilitate the study of T lymphocyte immune responses in horses. Here, a DNA microarray was designed to detect expressed classical MHC class I genes comprising serologically defined equine leukocyte antigen (ELA)-A haplotypes represented in a closed Arabian horse breeding herd. Initially, cloning and sequencing of RT-PCR products were used to identify sequences associated with the ELA-A1, A4, and W11 haplotypes, and one undefined haplotype, in six horses. Subsequently, sequence-specific, conserved (...
Effects of antifungal drugs and delivery vehicles on morphology and proliferation of equine corneal keratocytes in vitro. To evaluate the effects of topical antifungal drugs and delivery vehicles on the morphology and proliferation rate of cultured equine keratocytes. Methods: 16 corneas obtained from 8 apparently ophthalmologically normal horses < 0.5 hours after euthanasia for reasons unrelated to the study. Methods: Primary cultures of equine keratocytes were obtained from corneal stroma and were exposed to several concentrations of 3 commonly used, topically applied antifungals: natamycin, itraconazole, and miconazole. In addition, effects of drug delivery vehicles DMSO, benzalkonium chloride, and carboxym...
Microbead electrochemiluminescence immunoassay for detection and identification of Venezuelan equine encephalitis virus. An electrochemiluminescence (ECL) immunoassay, incorporating chemically biotinylated and ruthenylated antibodies down-selected from a panel of monoclonal and polyclonal reagents, was developed to detect and identify Venezuelan equine encephalitis virus (VEEV). The limit of detection (LOD) of the optimized ECL assay was 10(3)pfu/ml VEEV TC-83 virus and 1 ng/ml recombinant (r) VEEV E2 protein. The LOD of the ECL assay was approximately one log unit lower than that of a sandwich enzyme-linked immunosorbent assay (ELISA) incorporating the same immunoreagents. Repetition of ECL assays over time and...
Release kinetics of VEGF165 from a collagen matrix and structural matrix changes in a circulation model. Current approaches in bone regeneration combine osteoconductive scaffolds with bioactive cytokines like BMP or VEGF. The idea of our in-vitro trial was to apply VEGF165 in gradient concentrations to an equine collagen carrier and to study pharmacological and morphological characteristics of the complex in a circulation model. Methods: Release kinetics of VEGF165 complexed in different quantities in a collagen matrix were determined in a circulation model by quantifying protein concentration with ELISA over a period of 5 days. The structural changes of the collagen matrix were assessed with lig...
The use of a simple backflush technology to improve sample throughput and system robustness in routine gas chromatography tandem mass spectrometry analysis of doping control samples. A simple, low cost system for the backflushing of capillary gas chromatography (GC) columns has been investigated and integrated into a method for the detection of anabolic steroids in equine urine. The modification to the method was simple to make and quick to setup and optimize. The use of backflushing technology was found to offer significant benefits in terms of sample throughput and improved system robustness.
Molecular and immunohistochemical distinction of equine sarcoid from schwannoma. Ten equine skin tumors that had been classified as schwannomas on routine histological examination were analyzed by polymerase chain reaction for bovine papillomavirus DNA. All 10 were positive for bovine papillomavirus 1 or 2, and all 10 were immunohistochemically negative for S-100 protein and strongly positive for vimentin. Nine tumors were moderately positive for laminin and 8, for smooth muscle actin. Five tumors were variably and weakly positive for type IV collagen. The lack of S-100 protein expression made Schwann cells an unlikely cell of origin, as opposed to peripheral nerve sheath ...
Modulation of peripheral markers of the serotoninergic system in healthy horses. To evaluate the changes in plasma and platelet serotonin (5-HT) as markers of the serotoninergic system in equines, 5-HT content was measured by high performance liquid chromatography (HPLC) in deproteinized plasma obtained from peripheral blood samples of 12 clinically healthy horses, before and after feeding. 5-HT was measured in platelet rich plasma (PRP) and in platelet poor plasma (PPP). 5-HT in platelets (p5-HT) was obtained by subtracting free 5-HT in PPP (f5-HT) from 5-HT in PRP. After food ingestion, significant increases in p5-HT and f5-HT (p < 0.001), and no changes in the f5-HT/p5-...
Novel keratins identified by quantitative proteomic analysis as the major cytoskeletal proteins of equine (Equus caballus) hoof lamellar tissue. The dermo-epidermal interface that connects the equine distal phalanx to the cornified hoof wall withstands great biomechanical demands, but is also a region where structural failure often ensues as a result of laminitis. The cytoskeleton in this region maintains cell structure and facilitates intercellular adhesion, making it likely to be involved in laminitis pathogenesis, although it is poorly characterized in the equine hoof lamellae. The objective of the present study was to identify and quantify the cytoskeletal proteins present in the epidermal and dermal lamellae of the equine hoof by ...
Total RNA isolation from stallion sperm and testis biopsies. Sperm mRNA transcriptional profiles can be used to evaluate male fertility, yet differences between species in sperm attributes and packaging require adjustments in sperm RNA isolation protocols. The objective was to optimize RNA isolation methodology for fresh, frozen, and extended ejaculates, and epididymal sperm of stallions. Additionally, a protocol for RNA isolation from testis biopsies was established. Separation of sperm from somatic cells was critical for assuring the isolation of sperm-specific RNA. The highest purity was obtained by centrifuging ejaculates and epididymal sperm at 200...
Equus caballus major histocompatibility complex class I is an entry receptor for equine herpesvirus type 1. In this study, Equus caballus major histocompatibility complex class I (MHC-I) was identified as a cellular entry receptor for the alphaherpesvirus equine herpesvirus type 1 (EHV-1). This novel EHV-1 receptor was discovered using a cDNA library from equine macrophages. cDNAs from this EHV-1-susceptible cell type were inserted into EHV-1-resistant B78H1 murine melanoma cells, these cells were infected with an EHV-1 lacZ reporter virus, and cells that supported virus infection were identified by X-Gal (5-bromo-4-chloro-3-indolyl-beta-d-galactopyranoside) staining. Positive cells were subjected t...
Optimization of a procedure to accurately detect equine TNFα in serum samples. The systemic component of chronic inflammatory diseases may lead to clinical complications. High levels of TNFα, a pro-inflammatory cytokine, are found in human patients with COPD and asthma. Horses are also susceptible to an array of chronic inflammatory disorders possibly associated with systemic inflammation, including respiratory diseases. Currently, there is no commercially available ELISA validated to assess TNFα in equine serum samples. Moreover, the reported normal mean concentration of serum TNFα in horses vary greatly. Hence, we sought to optimize and validate a procedure to quant...
Analysis of Ca2+/Mg2+ selectivity in alpha-lactalbumin and Ca(2+)-binding lysozyme reveals a distinct Mg(2+)-specific site in lysozyme. The triggering of Ca(2+) signaling pathways relies on Ca(2+)/Mg(2+) specificity of proteins mediating these pathways. Two homologous milk Ca(2+)-binding proteins, bovine alpha-lactalbumin (bLA) and equine lysozyme (EQL), were analyzed using the simplest "four-state" scheme of metal- and temperature-induced structural changes in a protein. The association of Ca(2+)/Mg(2+) by native proteins is entropy-driven. Both proteins exhibit strong temperature dependences of apparent affinities to Ca(2+) and Mg(2+), due to low thermal stabilities of their apo-forms and relatively high unfavorable enthalpi...
Electrophoretically mediated microanalysis for the evaluation of interspecies variation in cholinesterase metabolism. This study describes an electrophoretically mediated microanalysis method, suitable for the preclinical evaluation of the hydrolysis of ester drugs by the serum of different animals and for further characterization of human-animal correlation. Dog, cat, cow, horse, sheep, rat and human serum were diluted (25%) in the appropriate buffer and replaced the enzyme solution usually used in electrophoretically mediated microanalysis methods for the study of enzyme kinetics. They were then compared in terms of the ability to hydrolyze acetylthiocholine and butyrylthiocholine (0.25 mM) by in-capillary ...
Biomarkers of in vivo fertility in sperm and seminal plasma of fertile stallions. The global proteome of sperm and seminal plasma of fertile stallions was investigated to determine whether associations with relative in vivo fertility exist. Seven stallions at stud in a commercial breeding station were collected throughout the breeding season and bred to a total of 164 mares to determine conception rates. On three occasions during the breeding season, raw semen was obtained from a regular collection for proteomic analysis using two-dimensional electrophoresis and also assessed for routine semen quality end points. First cycle conception rate was negatively related to ejacula...
A reverse genetics system of African horse sickness virus reveals existence of primary replication. African horse sickness virus (AHSV), a member of the orbivirus genus of the family Reoviridae, is an insect-vectored pathogen of horses of concern to the equine industry. Studies on AHSV replication and pathogenesis have been hampered by the lack of reverse genetics allowing targeted mutation of viral genomes. We demonstrate that AHSV single-stranded RNA synthesized in vitro (core transcripts) is infectious and that there are distinct primary and secondary stages of the replication cycle. Transfection with a mixture of core transcripts from two different serotypes or a mixture of core transcri...
Delta opioid receptor on equine sperm cells: subcellular localization and involvement in sperm motility analyzed by computer assisted sperm analyzer (CASA). Opioid receptors and endogenous opioid peptides act not only in the control of nociceptive pathways, indeed several reports demonstrate the effects of opiates on sperm cell motility and morphology suggesting the importance of these receptors in the modulation of reproduction in mammals. In this study we investigated the expression of delta opioid receptors on equine spermatozoa by western blot/indirect immunofluorescence and its relationship with sperm cell physiology. Methods: We analyzed viability, motility, capacitation, acrosome reaction and mitochondrial activity in the presence of naltri...
Single layer centrifugation of stallion spermatozoa through Androcoll™-E does not adversely affect their capacitation-like status, as measured by CTC staining. This study was designed to evaluate the effect of single layer centrifugation (SLC) and subsequent cold storage on stallion sperm capacitation-like status and acrosome reaction. Three stallions were included in the study, with three ejaculates per stallion. The samples were examined 4, 24 and 72 h after collection, extension and SLC, with storage at 6°C. Sperm capacitation-like status was investigated using the fluorescent dye chlortetracycline (CTC). There was no difference in capacitation-like status between colloid-selected and non-selected spermatozoa. Sperm motility decreased significant...
Protein composition of seminal plasma in fractionated stallion ejaculates. Seminal plasma (SP) contains several types of compounds derived from the epididymides and accessory glands. The aim of this study was to examine the protein composition of different ejaculate fractions. Trial I: fractionated ejaculates were collected from two normal and two subfertile stallions. Samples containing pre-sperm fluid and the first sperm-rich jets (HIGH-1), the main sperm-rich portion (HIGH-2), the jets with low sperm concentrations (LOW), and a combined whole-ejaculate (WE) sample was centrifuged, and the SP was filtered and frozen. A part of each SP sample was stored (5°C, 24 h)...
Determining the source of equine bloodstains by dinucleotide repeats. A novel multiplex of independent dinucleotide tandem repeat (DTR) loci was previously described that is capable of not only discriminating human and equine DNA, but of identifying a single equine source. We report a case in which a bloodstained syringe and two needles were found during inspection of a barn by inspectors of the Pennsylvania Racing Commissions. Using the multiplex and single-locus detection, all 21 equine DTR markers were detected in a suspect horse and two evidence samples, indicating the evidence samples came from the suspect animal. Only six markers were detected in the third...
A morphological and quantitative immunohistochemical study of the interstitial cells of Cajal in the normal equine intestinal tracts. The interstitial cells of Cajal (ICC) play a key role in the control of intestinal motility and have been implicated in several human gastrointestinal dysmotility syndromes, in equine grass sickness and in other intestinal disorders where a significant reduction in ICC density was observed. Objective: To investigate the density of ICC in clinically normal horses, ICC c-Kit expression was evaluated by image analysis in order to obtain numerical data. Methods: Intestinal samples from the jejunum to small colon from 5 clinically normal horses were studied. Immunohistochemical labelling of ICC was...
