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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 acetate and glucose incorporation into rat and horse skin lipids. The relative efficiency of acetate and glucose as substrates for the biosynthesis of lipids in the skin of the rat and horse was examined using in vivo pulse labelling of skin with [1-14C]acetate and [U-14C]glucose by intradermal injections. The resulting radiolabelled lipids were recovered in the rat by punch biopsy as well as by daily, long-term skin surface lipid collections and in the horse by punch biopsy of the injection sites. The lipids were examined by liquid scintillation and by a combination of thin-layer chromatography and autoradiography. In both species the recovery of radiolabel...
Erythrogram and red cell distribution width of Equidae with experimentally induced anemia. The erythrogram (erythrocyte histogram) and red cell distribution width (RDW) were evaluated in 5 purebred horses and 1 pony of mixed breeding with experimentally induced anemia. Four horses were studied for 6 weeks after 20% of their estimated blood volume was removed on each of 2 consecutive days (40% total blood loss; acute blood-loss group). Two horses were given acetylphenyl hydrazine IV daily, until acute Heinz body hemolytic anemia was induced; the 2 horses were then evaluated for 6 weeks. One horse and the pony had 20% of their estimated blood volume removed via phlebotomy once each we...
Methods of assigning accurate values to reference serum. Part 2. The use of definitive methods, reference laboratories, transferred values and consensus values. Eight analytes (Ca, Cl, cholesterol, glucose, Mg, K, Na and urate) have been determined in one horse serum masterlot by up to six different procedures: (i) by so-called definitive methods; (ii) by a group of reference laboratories using a variety of analytical methods; (iii) using the results of two independent external quality assessment schemes; (iv) by transferring values from a human serum standard reference material analysed by definitive methods; (v) by similar transfer of values from several batches of horse reference serum previously analysed by definitive methods; and (vi) as in (v) b...
Sequences of papillomavirus DNA in equine sarcoids. DNA was extracted from 14 equine sarcoids, electrophoresed and hybridised with a radioactively labelled probe of bovine papillomavirus type I (BPV 1) DNA under conditions of low stringency. Twelve sarcoids contained sequences of DNA that hybridised with the probe and that comigrated with BPV 2 DNA. The viral DNAs in four of these sarcoids differed from BPV 1 and BPV 2 DNA on restriction endonuclease analysis. One of four cell lines derived from sarcoids also contained BPV 1 related DNA. The results confirm the frequent presence in equine sarcoids of unintegrated papillomaviral DNA and suggest ...
Gas chromatography-mass spectrometry of androgens in equine ovarian follicles at ultrastructurally defined stages of development. Identification of 19-nortestosterone in follicular fluid. Follicular fluid was obtained from equine follicles at different stages of development as determined by ultrastructural study. Gas chromatography-mass spectrometry associated with stable isotope dilution permitted the demonstration of high levels of 4-estrene-3,7-dione and 17 beta-hydroxy-4-estren-3-one, 17 beta-hydroxy-4-estren-3-one levels often being about 10 times higher than those of testosterone. These findings suggest that in the mare ovary, an aromatizing pathway may proceed using these 19-norsteroids as intermediates. As a consequence of this high level of 19-norsteroids, testosterone...
Fibronectin enhances transfection of Staphylococcus aureus. The factor in normal sera primarily responsible for the enhancement of transfection (and transformation) of Staphylococcus aureus was identified as fibronectin. Serum samples which were depleted of fibronectin by affinity chromatography showed a marked decrease in enhancing activity. Fibronectin isolated from sera of several animal species demonstrated enhancing activity.
Stimulation of equine eosinophil migration by hydroxyacid metabolites of arachidonic acid. Lipoxygenase products of arachidonic acid are important mediators of inflammation, affecting several aspects of cell function. Monohydroxyeicosatetraenoic acid (mono-HETE) and 5,12-dihydroxyeicosatetraenoic acid (LTB4) enhance migration of both neutrophils and eosinophils in several species. The relative ability of positional isomers of HETE and of LTB4 to affect migration of equine eosinophils was studied. The 5, 8, 9, 11, 12, and 15 isomers of HETE were prepared by autooxidation of arachidonic acid, separated by sequential normal phase and reverse phase high performance liquid chromatography...
Enzyme-linked immunosorbent assay for diagnosis of Corynebacterium (Rhodococcus) equi infection in foals. An enzyme-linked immunosorbent assay (ELISA) was used to diagnose Corynebacterium (Rhodococcus) equi infection in foals. In tests done with different antigen-extraction procedures (sodium dodecyl sulfate, sodium deoxycholate, polyoxy-ethylene [9] p-tert-octylphenol, polyoxy-ethylene [9-10] p-tert-octylphenol, sonification, homogenization, and heat treatment at 121 C), Tween 20 was a satisfactory reactive antigen. Using hyperimmune rabbit sera or infected foal sera, we investigated the specificity and the sensitivity of the ELISA with the Tween 20 antigen of the different serotypes or of the is...
Solubilization and characterization of [3H] 5HT high affinity binding sites (5HT1 and 5HT3). The solubilization of the serotonergic 5HT1 and 5HT3 sites was performed with digitonin and sodium cholate at 1% (final concentration). Two binding sites for [3H]5HT were observed on rat or horse brain synaptosomal membranes solubilized with these detergents. The corresponding dissociation constants (KD) were 1-3 nM and 13-30 nM respectively. These values were closely similar to those corresponding to 5HT1 and 5HT3 sites located in intact membranes. The solubilized sites specifically bound 5HT. The effect of GTP decreasing the binding to 5HT1 sites was lost on solubilized 5HT1 sites; it was re...
Horse kidney neutral alpha-D-glucosidase: purification of the detergent-solubilized enzyme; comparison with the proteinase-solubilized forms. Neutral alpha-D-glucosidase (alpha-D-glucoside glucohydrolase, EC 3.2.1.20) from horse kidney brush-border membranes was solubilized using Emulphogene BC 720 and purified by an affinity chromatography technique. The enzyme preparation (390-fold purified), which was free of other known microvillus hydrolases, exhibited one precipitate line in crossed immunoelectrophoresis and migrated as a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Several criteria (charge-shift crossed immunoelectrophoresis and hydrophobic chromatography) revealed the purified detergent form of t...
Complexities in the denaturation of horse metmyoglobin by guanidine hydrochloride. The denaturation of horse metmyoglobin by guanidine hydrochloride was studied at pH 6.4 and 25 degrees C. Measurements of both the peptide circular dichroism and the absorbance in the Soret region suggest that the extent of renaturation strongly depends on the time interval during which the protein is exposed to concentrated solutions of the denaturant. From the equilibrium measurements of the absorption in the Soret region, it is concluded that the unfolding of metmyoglobin is complex. This is further supported by kinetic studies of denaturation which suggest the occurrence of the least four ...
Equine whole saliva: variability of some major constituents. Whole saliva was collected from six horses over a period of five weeks in sufficient volume for the analysis of 10 constituents. There was considerable variation in the concentration of the analytes both between horses and between different days in the same horse. The most variable constituent was sodium, and the least variable was glucose, but this was derived from the sweet used to stimulate salivation. The use of whole saliva as a fluid for investigation would depend on achieving the minimum variability possible.
High resolution R-bands produced in equine chromosomes after incorporation of bromodeoxyuridine. Cell synchronization was used to obtain an adequate percentage of very long chromosomes in equine mitotic spreads. Reported here is our variation, adapted to horse chromosomes, of a method using excess thymidine followed by bromodeoxyuridine incorporation. This technique routinely yields excellent quality cells, predominantly in prometaphase and prophase. Among other differences with the standard technique, this method does not use Colcemid, which, in addition to inhibiting spindle fiber formation, also increases chromosome contraction resulting in thicker and thus fewer bands. Consequently, h...
The isoelectric focusing of keratins in hair followed by silver staining. An isoelectric focusing method followed by silver staining has been developed for the study of keratins which is as effective as two-dimensional electrophoresis and fluorography for hair species identification. Hair from dogs, rabbits, horses, cows, guinea-pigs, donkeys, sheep and cats were successfully identified. Narrow pH ranges were used to observe heterogeneity in human hair. Although this heterogeneity may be affected by environmental conditions, it may be of use in criminalistics.
Incorporation of L-75Se-cystine in tissue fragments from the matrix of the hoof and the claw–a tool for studying the pathogenesis of laminitis? An in vitro method has been designed and used to study the incorporation of 75Se-cystine into matrix fragments from hooves and claws of healthy horses and cattle. Tissue fragments from the zone of keratinisation were incubated with L-75Se-cystine in a tissue culture medium for 4 to 6 h, during which time there was continuous incorporation of the labelled selenocystine. The incorporation was greatly decreased by adding L-cystine to the incubation mixture. It is concluded that the incorporation of 75Se-cystine depends on the presence of a specific receptor for cystine in the tissue fragments stu...
The influence of dietary selenium levels on blood levels of selenium and glutathione peroxidase activity in the horse. Twenty mature geldings, averaging 535 kg, were used to determine the influence of dietary selenium (Se) on the blood levels of Se and Se-dependent glutathione peroxidase (SeGSH-Px) activity in the horse. Horses were randomly assigned within breed to four treatments consisting of five horses each and fed a basal diet containing .06 ppm of naturally occurring Se. Diets were supplemented with .05, .10 and .20 ppm Se, as sodium selenite. Blood was drawn for 2 wk before, and for 12 wk following, the inclusion of supplement Se in the diets. Whole blood and plasma Se concentrations and plasma SeGSH-P...
Cell synchronization and dynamic G-banding of equine chromosomes by bromodeoxyuridine. Both dynamic G-banding and cell synchronization produced by bromodeoxyuridine (BrdU), were applied to equine chromosomes. BrdU incorporated during the first half of the S-phase is taken up into the R-bands that are early replicating. These bands, which have incorporated BrdU, cannot contract as usual and remain elongated; only the other regions of the chromosome, i.e., the G-bands, contract normally and are sharply defined. BrdU also can be used for cell synchronization. The addition of BrdU in a high concentration, 15 hours before harvest, and its removal 11 hours later, has two effects: init...
Demonstration of a COOH-terminal extension on equine lutropin by means of a common acid-labile bond in equine lutropin and equine chorionic gonadotropin. The beta subunits of equine lutropin and equine chorionic gonadotropin were incubated in 0.013 N HCl for 30 min at 110 degrees C and separated into two fragments by reverse-phase high performance liquid chromatography. The amino acid and carbohydrate compositions of both fragments from each subunit were analyzed. The results demonstrated that equine lutropin-beta has a glycosylated COOH-terminal extension that differs only in carbohydrate composition from the COOH-terminal portion of equine chorionic gonadotropin-beta. This is the first demonstration of a glycosylated COOH-terminal extension i...
Kinetic studies of the unfolding-refolding of horse muscle phosphoglycerate kinase induced by guanidine hydrochloride. The kinetics of the unfolding and refolding of horse muscle phosphoglycerate kinase were studied with three different signals: fluorescence emission intensity at 336 nm (excitation at 292 nm), ellipticity at 220 nm, and enzyme activity. The results corroborate the conclusion on the existence of intermediates in the folding pathway obtained from equilibrium studies. Kinetic studies showed at least two phases of refolding, as revealed by fluorescence as well as by circular dichroism measurements. During the fast phase, an intermediate was formed with a fluorescence intensity higher than that of ...
The use of the glutaraldehyde coagulation test for detection of hypogammaglobulinaemia in neonatal foals. The effectiveness of the glutaraldehyde coagulation test (GCT) in detecting failure to acquire colostral immunoglobulin in neonatal foals was investigated. This was achieved by comparing and correlating results from the GCT with those obtained by single radial immunodiffusion (SRID) of equine IgG. The GCT was found to be a practical, inexpensive, semiquantitative test with a high specificity and sensitivity at critical IgG levels.
Diagnosis and treatment of cardiac arrhythmias. Cardiac arrhythmias are probably more common in horses than in any other domestic animal species. The most frequent clinical complaint associated with cardiac arrhythmias is exercise intolerance. Physical examination is characterized by auscultation abnormalities such as fast or slow heart rate, irregular rhythm, extra sounds, long pauses, or abnormal heart sounds. The electrocardiogram is used to make a definitive diagnosis of the dysrhythmia. Other laboratory and cardiac function tests are employed to determine the etiology and to assess the significance of the arrhythmia. Antiarrhythmic the...
Enzyme histochemical features of equine gluteus muscle fibers. Gluteal muscle specimens were taken from 4 horses. From 1 of the 4 gluteal muscles, serial sections were prepared. Individual muscle fibers were identified and studied, using photomicrographs of sections stained by different enzyme histochemical methods. In specimens in which cytoplasmic soluble enzymes were studied, use was made of the semi-permeable membrane technique to hamper enzyme diffusion into reaction fluids. Enzymes involved in glycogenolysis, glycolysis, the tricarboxylic acid cycle, synthesis of reduced nicotinamide adenine dinucleotide phosphate, the pentose phosphate cycle, the a...
Pre-alpha 2-elastase inhibitor of the horse: a hybrid molecule between alpha 1-proteinase inhibitor and alpha 2-beta 1-glycoprotein. Pre-alpha 2-elastase inhibitor of horse plasma has recently been isolated in our laboratory. In this article we demonstrate that the inhibitor is a composite structure built of alpha 1-proteinase inhibitor and alpha 1-beta 1-glycoprotein. The compound inhibitor is biologically active, although it has previously been shown that its enzyme specificity is different from that of free alpha 1-proteinase inhibitor. Our observations are based on immunochemical cross-reactions between pre-alpha 1-elastase inhibitor and antibodies to alpha 2-beta 1-glycoprotein as well as antibodies to alpha 1-proteina...
Equine leucocyte antigen system: progress and potential. Leucocyte antigens are cell-surface glycoproteins, the structure of which is under the genetic control of a chromosome region called the major histocompatibility complex. Progress in the study of the equine leucocyte antigen (ELA) system has been achieved in two ways; first by the fact that the ELA system is intensively investigated in different laboratories all over the world and parallels can be drawn to the information gained from research in more extensively studied species, and secondly by the collaborative efforts of the participants in three international workshops. The potential applic...
Deficiency of the contact phase of intrinsic coagulation in a horse. A 16-year-old gelding was examined because of weight loss, inappetence, and intermittent fever of 2 months' duration. Preliminary laboratory findings revealed anemia, hypoproteinemia, thrombocytopenia, and prolongation of the activated partial thromboplastin time. A deficiency or inhibition of coagulation factor XI, factor XII, or high molecular weight kininogen was diagnosed. This defect was not associated with a bleeding diathesis, but should be considered as a cause of prolongation of the activated partial thromboplastin time.
Use of enzyme-linked immunosorbent assay for the diagnosis of equine Histoplasmosis farciminosi (epizootic lymphangitis). An enzyme-linked immunosorbent assay was evaluated for the detection of antibody in sera of equine naturally infected with Histoplasma farciminosum 'epizootic lymphangitis'. Ten sera from naturally infected horses were tested. A hydrogen peroxide ABTS mixture constituted the substrate. The reactions were read as the absorbance values measured at 405 nm using a spectrophotometer. The standard deviation and the average percentage of the absorbance values of the different serum samples were considered in the interpretation of the results. All sera were proved positive with variations in the diffe...
Functional and biochemical characterization of immunologically derived equine platelet-activating factor. Antigen-specific challenge of equine leukocytes induced the non-lytic release of a platelet-activating factor in vitro. The equine platelet-activating factor stimulated the release of serotonin from equine platelets in a dose-responsive manner, independent of the presence of cyclo-oxygenase pathway inhibitors such as indomethacin. Rabbit platelets were also responsive to equine platelet-activating factor. The release of equine platelet-activating factor was a rapid reaction with near maximal secretion taking place in 30 seconds. Addition of equine platelet-activating factor to washed equine pl...
