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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.
Hypercalcemia associated with malignancy in a horse. Hypercalcemia associated with malignancy was diagnosed in a 2-year-old Thoroughbred filly admitted because of weight loss and reduced exercise tolerance of approximately 2 months' duration. Laboratory findings included hypercalcemia, hypophosphatemia, anemia, marked neutrophilia with lymphopenia and eosinopenia, and normal immunoreactive parathyroid hormone concentration. At necropsy, a 53.6-kg tumor was located in the cranioventral aspect of the abdominal cavity. Gross renal lesions were not noticed. Bone tissue appeared to be normal on gross and histologic examinations. The parathyroid gland...
Urinary enzyme concentrations in healthy horses. Urinary concentrations of gamma glutamyl transferase (GGT), alkaline phosphatase (AP), aspartate aminotransferase (AsAT), and alanine aminotransferase (AAT) were measured in 32 healthy horses (16 geldings and 16 females) at the same time on 2 consecutive days. The subjects were divided into 4 ages groups, each comprising 8 horses (4 of each sex). In 10 of the geldings, urine was collected continuously for 72 hours, with catheterized samples being obtained at 0800, 1400 and 2000 hours, and an aliquot of the pooled urine being taken every 24 hours. Urinary enzyme activity was found to be unrelat...
D-xylose absorption in the growing foal. Seven healthy foals (five ponies and two horses) were maintained on grass pasture with their dams. All foals had normal faeces at the time of testing. An oral xylose absorption test was performed on each foal at one, two and three months of age. Following an 8 h fast, 0.5 g/kg D-xylose as a 10 per cent solution was given via a nasogastric tube. Control and 30 min interval plasma samples were collected for 3 h and the plasma was analysed for xylose using the phloroglucinol microassay technique. Maximum xylose concentration levels were reached between 30 and 60 mins for each of the foals. The me...
Purification of a sialic acid-specific lectin from the Indian scorpion Heterometrus granulomanus. A sialic acid-specific lectin, scorpin, has been purified to apparent homogeneity from the Indian scorpion Heterometrus granulomanus by affinity chromatography on equine submandibular gland glycopeptides linked to Sepharose and gel filtration on Sephadex G-200. The lectin has a molecular mass of 500 000 Da and was dissociated into single polypeptide chains of 15 000 Da, as determined by SDS gel electrophoresis in the presence of 2-mercaptoethanol. Scorpin is a glycoprotein containing 2.8% sugars. Its specificity was investigated by the inhibition of hemagglutination with various derivatives of...
Triplet-singlet energy transfer in the complex of auramine O with horse liver alcohol dehydrogenase. Triplet-singlet energy transfer has been studied in the complex formed between auramine O (AO) and horse liver alcohol dehydrogenase with optically detected magnetic resonance (ODMR) spectroscopy. The results show that Trp-15 and Tyr residues transfer triplet energy mainly by a trivial process, whereas Trp-314 transfers triplet energy by a Förster process with two observed lifetimes at 77 K of 170 and 50 ms. The different Förster energy-transfer lifetimes are ascribed either to quenching of the two Trp-314 residues of the dimer by a single asymmetrically bound AO or to two distinct conformat...
Effects of urea and guanidine hydrochloride on the activity and dynamical structure of equine liver alcohol dehydrogenase. The inactivation of equine liver alcohol dehydrogenase by guanidine hydrochloride and urea has been studied by monitoring the intrinsic tryptophan fluorescence and phosphorescence emission. The use of triplet-state lifetimes to probe the flexibility of protein structure at the site of tryptophan-314 reveals a distinct behavior between the two denaturants. At predenaturational concentrations, the loss of enzyme activity in guanidine hydrochloride is associated with a loosening of intramolecular interactions resulting in a greater fluidity of the interior region of the macromolecule. In contrast...
Type II renal tubular acidosis in a mare. Persistent, severe metabolic acidosis complicated the operative and postoperative period in a 4-year-old mare with colic. On the basis of clinical and laboratory findings, a renal tubular disorder was diagnosed. Renal tubular acidosis is rare in horses. In the only report found on the subject, type I renal tubular acidosis was described in 2 horses. Bicarbonate titration studies in our case helped document type II renal tubular acidosis in this mare.
Isolation and characterization of latherin, a surface-active protein from horse sweat. A protein, latherin, with unusual surface activity was isolated from horse sweat by gel filtration and ion-exchange chromatography. The protein has a Stokes radius, determined by gel filtration, of 2.47 nm, and in the ultracentrifuge sediments as a single species with S20,W 2.05 S, indicating an Mr of 24,400. On SDS/polyacrylamide-gel electrophoresis the molecule behaves as a single peptide chain of apparent Mr 20,000. Latherin contains a high proportion of hydrophobic amino acids (37.2%), and the leucine content (24.5%) is exceptionally high. The unusual composition of the protein may account...
Radioimmunoassay screening for etorphine in racing horses. A commercially available radioimmunoassay kit was used to screen for the presence of etorphine in post-race urines from horses racing in Kentucky. Most horse urines contained small amounts of materials which reacted positively in this immunoassay. These materials are apparently endogenous to the horse and were called apparent etorphine equivalents. The levels of these apparent etorphine equivalents in post-race urines from 70 horses were estimated. Their modal level averaged 0.1 ng/ml, the population distribution was log normal, and individual horses showed levels of up to 0.8 ng/ml.
Immunodiffusion test for diagnosing and monitoring pythiosis in horses. A practical, sensitive, and specific immunodiffusion test was developed for diagnosing and monitoring pythiosis in horses. Culture filtrates, a soluble cell mass, and trypsinized Pythium sp. antigens were evaluated against prepared rabbit anti-Pythium sp. serum and pythiosis horse case sera. The culture filtrate antigens demonstrated the greatest capacity for detecting precipitins and the greatest stability during storage. In contrast, the trypsinized antigens had the weakest capability for detecting multiple precipitins and the poorest stability. The 13 sera from horses with proven active pyt...
Generation of packaging-defective DNA molecules of equine adenovirus. Equine adenovirus (EAd) DNA prepared from infected bovine kidney (MDBK) cells contained additional sequences of about 100 to 700 bp at the left-hand end of the genome. These aberrant viral genomes were produced even after the first passage of the wild type EAd in MDBK cells and their relative amounts did not change significantly during serial passage. The left terminal fragments of two defective viral DNAs were cloned into the plasmid vector pBR322 and the nucleotide sequences of their terminal regions were analyzed. The data indicate that one viral DNA contained a duplication of the inverted ...
Determination of nefopam in equine plasma by gas chromatography-mass spectrometry with chemical ionization. This study demonstrates the development of a method using gas chromatography-mass spectrometry for determining nefopam, a non-narcotic pain reliever that is sometimes abused in horse doping, in equine plasma. Background […]
Equine endothelial cells in vitro. Certain in vitro culture conditions were determined for equine endothelial cells obtained from the aorta and pulmonary arteries. Cells were enzymatically isolated from the vessel lumen, using clostridial collagenase (2.5 mg/ml of Hanks's balanced salt solution) incubated at 37 C for 30 minutes. Cells were cultured in alpha minimum essential medium supplemented with plasma-derived and nonplasma-derived bovine fetal sera, endothelial cell-growth supplement, heparin, and antibiotics. Smooth muscle cell growth was not inhibited with nonplasma-derived animal sera, plasma-derived equine serum, or he...
[Determination of the standard oxyhemoglobin dissociation curve in horses. Effects of temperature, pH and diphosphoglycerate]. The equine blood oxyhemoglobin dissociation curve has been traced in its entirety in standard conditions and the effects of temperature, pH and 2,3-diphosphoglycerate on this curve have been measured. When compared to that of human blood, the curve showed a higher oxygen affinity of hemoglobin (23.8 +/- 0.8 versus 26.6 mm Hg). The effect of the pH, expressed by d log P50/dpH, was found to be identical in man and horse (-0.47). The effect of temperature, however, expressed by d log P50/dT, proved to be lower in the horse (0.016 versus 0.024). The P50 showed an increase of 1 mm Hg each time 2,3-...
Equine thyroid function assessment with the thyrotropin-releasing hormone response test. The effect of thyrotropin-releasing hormone (TRH) on equine thyroid function was determined by quantifying serum thyroxine (T4) and 3,5,3'-triiodothyronine (T3) before and after TRH administration. Thyrotropin-releasing hormone was administered IV to adult horses (n = 5) and ponies (n = 6) at a dose of 1 mg or 0.5 mg, respectively. Serum T4 and T3 concentrations were determined before and 0.25, 0.5, 1, 2, 4, 6, 8, 12, and 24 hours after TRH administration. Serum T4 increased from a basal concentration of 24.4 +/- 8.7 ng/ml (mean +/- SD) to a maximum value of 48.2 +/- 10.2 by 4 hours after TRH ...
Occurrence and distribution of 5-S-cysteinyl derivatives of dopamine, dopa and dopac in the brains of eight mammalian species. The 5-S-cysteinyl derivatives of dopamine, dopa (3,4-dihydroxyphenylalanine) and dopac (3,4-dihydroxyphenylacetic acid) were synthesized and used as reference compounds in high performance liquid chromatography analyses of extracts from various brain regions of eight mammalian species. All three metabolites were detected in the brains of all the species studied. The regional distribution of the metabolites was similar to that of dopamine; the metabolite concentrations ranged from less than 0.1 percent to more than 1 percent of the dopamine level, the highest ratios generally being found in sub...
Isolation and characterization of three forms of luteinizing hormone from the pituitary gland of the horse. Three isoforms of equine luteinizing hormone (eLH-A, eLH-B and eLH-C) have been isolated from horse pituitary glands. Separation was achieved on the basis of charge heterogeneity by ion-exchange chromatography. These charge differences were apparent after final purification, as determined by electrophoretic mobility on polyacrylamide disc gels (RF = 0.14, 0.19 and 0.26 for eLH-A, -B and -C, respectively). Apparent size differences were also noted between the isohormones by gel filtration on Sephadex G-100. Ve/Vo ratios for eLH-A, -B and -C were 1.72, 1.54 and 1.47, respectively. All 3 isoforms...
In vitro interference between equine herpesvirus types 1 and 2. Interference between equine herpesvirus types 1 (EHV-1) and 2 (EHV-2) was studied in equine dermis (ED) monolayer cell cultures and equine lymphocyte cultures. Cell cultures were infected with EHV-2, and after a short incubation period, the cultures were superinfected with EHV-1. At various intervals, different measurements of EHV-1 expression in dually infected cultures, compared with those in cultures infected with EHV-1 alone, were studied. In dually infected ED cell cultures, the EHV-1 cytopathic effect, EHV-1 titer, and EHV-1 enzyme-linked immunosorbent assay antigen titer were maximally ...
Direct demonstration of intrinsic follicle-stimulating hormone receptor-binding activity in acid-treated equine luteinizing hormone. After dissociating equine gonadotropins as a function of time at pH 3, we examined them by radioligand assay and sodium dodecyl sulfate polyacrylamide gel electrophoresis under nondissociating conditions (low, 0.1% SDS). Equine follicle-stimulating hormone (FSH) rapidly lost its receptor-binding activity, and low SDS-polyacrylamide gels demonstrated dissociation into subunits. Maximum dissociation occurred after 20-30 min of pH 3 incubation. Equine luteinizing hormone (LH), however, retained most biologic activity and was largely intact after 72 h of pH 3 incubation. Dose-response curves of ac...
Two-dimensional 1H NMR studies of cytochrome c: assignment of the N-terminal helix. The 1H resonances of 11 sequential amino acids in the N-terminal helix of horse ferrocytochrome c were studied by two-dimensional nuclear magnetic resonance techniques. All the main-chain protons from Lys-5 through Ala-15 and many of the side-chain protons were assigned. J-Correlated spectroscopy (COSY) was used to distinguish protons on neighboring bonds and to recognize amino acid types. Nuclear Overhauser effect spectroscopy (NOESY) was used to define spatially contiguous protons and to determine amino acid sequence neighbors. The relayed coherence experiment (relay COSY) was used to resolv...
Molecular pathogenesis of equine coital exanthema: temperature-sensitive function(s) in cells infected with equine herpesviruses. Preliminary experiments have revealed that several laboratory and wild-type strains of the equine herpesvirus (EHV) triad were temperature-sensitive for growth when assayed at 39 degrees C. The efficiencies of plating (EOP) observed were 10(-2) for both EHV 1 and 2, and 1 X 10(-6) for EHV 3. The EOPs were determined by plaque assays which compared titrations at 34 degrees C and 39 degrees C on equine fetal dermal fibroblast cells. Growth yield experiments, assayed at 34 degrees C, reflected those EOP's, but did not indicate any difference in yields when infected cultures were incubated at 34 d...
A study on the role of evolutionarily invariant leucine 32 of cytochrome c. To investigate the role of evolutionarily invariant leucine 32 of horse cytochrome c, analogs of residues 28-38, (28-38), each containing a substituted amino acid at positions 32 or 35 were synthesized using Merrifield's method. Position 35 is leucine in horse cytochrome c but replaced by nonpolar amino acids in some species. The ability of the analogs to bind to the two-fragment complex of ferri- or ferro heme fragment (1-25)H and apofragment (39-104) was measured using gel filtration and equilibrium dialysis. Replacement of leucine 32 with isoleucine, for example, increased the dissociation ...
Continuous-flow centrifugation hemapheresis in the horse. In a continuous-flow centrifugation apheresis technique adapted for blood-component separation and collection in horses, hydroxyethyl starch was not required for erythrocyte sedimentation. The efficacy and separation characteristics of whole blood from 10 horses were evaluated at various gravitational forces (700 to 1,500 rpm), using a constant withdrawal rate (100 ml/min). Maximum leukocyte collection occurred at 700 rpm (P less than 0.01), and optimal neutrophil collection occurred at 700 to 750 rpm (P less than 0.01). Although neutrophil counts decreased and lymphocyte counts remained const...
Assessment of spermatozoal function using dual fluorescent staining and flow cytometric analyses. Spermatozoa from bulls, boars, dogs, horses, mice, and men were examined using a fluorogenic stain consisting of the membrane-permeant substrate carboxyfluorescin diacetate (CFDA) and the relatively membrane-impermeant nuclear stain propidium iodide (PI). Three distinct populations of spermatozoa were discernible in samples from each species upon microscopic examination. Individual spermatozoa, presumed to be viable because of their motility, retained products of the fluorescein chromophore throughout the cell. A second population of spermatozoa in which the nuclei stained red with PI retained...
Cytolytic activity of liposomes containing stearylamine. In order to develop the cytotoxic liposome, the cytolytic effect of polycationic liposome was examined. Upon incubation of the stearylamine-containing liposome (stearylamine-liposome) with rabbit erythrocyte, a significant extent of hemolysis was observed. Hemolytic activity of the liposome depends on the amount of stearylamine in the liposome membrane. The plots of the initial rate of hemolysis versus the concentration of stearylamine-liposome showed a sigmoidal curve, suggesting that stearylamine-liposomes act cooperatively on the erythrocyte membrane. Hemolytic activity of stearylamine-lipo...
