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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.
Pancreatic colipase: crystallographic and biochemical aspects. A detailed study of the crystallization of hog and horse colipases has been undertaken. Several crystallographic varieties have been obtained and a 0.3-nm resolution structure determination is actually in progress. The sequence of the A form of horse colipase (one methionine) is given. From spectrophotometric experiments and sequence comparisons, the involvement of the aromatic residue in position 52 in the micelle binding site has been demonstrated.
Chemotaxis of horse polymorphonuclear leukocytes to N-formyl-L-methionyl-L-leucyl-L-phenylalanine. Horse polymorphonuclear leukocytes (PMN) isolated from horse blood by sedimentation and isotonic lysis and having about 25% accompanying lymphocytes were as effective at chemotaxis as nearly pure PMN isolated by density gradient techniques. N-Formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP), used as a representative of the formylmethionyl peptides (produced by prokaryocytic organisms), was effective as a chemoattractant only at the high concentration of 10(-4) M. When serum was preincubated with FMLP at concentrations as low as 10(-8) M, the serum attracted horse PMN. This activity was not g...
Isolation and characterization of beta- and gamma-caseins from horse milk. Three groups of casein components were isolated from horse milk. Group I is almost insoluble at acid and neutral pH, and is rather heterogeneous on alkaline gels with or without sodium dodecyl sulphate. Group II shows strong similarity to beta-casein from other species, as concluded from its amino acid composition and its N- and C-terminal sequences. This group consists of five electrophoretically distinguishable forms, all containing ester phosphate groups but no carbohydrate. Group III is composed of C-terminal fragments of the beta-like (group II) fraction and probably arises from the actio...
Assessment of the calcium and phosphorus nutrition in horses by analysis of urine. Studied were made to determine if a practical assessment of the calcium and phosphorus nutrition of horses could be obtained from an analysis of urine samples. The concentrations of Ca and P in urine samples changed markedly when groups of 4 mares were fed diets containing from 1.0 to 3.9 g Ca/kg and from 1.5 to 6.1 g P/kg, but serum concentrations of Ca and P remained relatively constant. The concentrations in single urine samples were considered unreliable indicators of excretion of the minerals because of variations in water excretion, and two methods to overcome this problem were examined....
Comparative measurement of equine influenza virus antibodies in horse sera by single radial hemolysis, neutralization, and hemagglutination inhibition tests. Single radial hemolysis (SRH), neutralization (NT), and hemagglutination inhibition (HI) tests were carried out on sera from horses immunized against the Prague and Miami strains of equine influenza virus. The HI and NT tests demonstrated good sensitivity; the sensitivity of the SRH test was somewhat lower. The NT titers of individual sera were correlated very closely with the HI titers, although the NT titers were higher. SRH zone diameters of individual sera also showed significant correlation with the NT and NI titers. The SRH test appears to be suitable for large-scale serological surveys ...
Deuteromethylation of dimethylxanthines: a gas chromatographic mass spectrometric method for confirmatory analysis in horse urine extracts. The methylated xanthines caffeine and/or theobromine are commonly encountered in drug-positive samples from racehorses and their metabolism and excretion in the horse and their analysis in urinary extracts has been of particular interest in this laboratory. Due to their polar nature the dimethylxanthines theobromine, theophylline and paraxanthine give unsatisfactory gas chromatographic performance and require derivatization prior to analysis by gas chromatography mass spectrometry. The present paper describes a simple deuteromethylation procedure to render the compounds amenable to analysis by...
A linkage group composed of three coat color genes and three serum protein loci in horses. The equine coat color genes chestnut (e) and roan (Rn) have been tested for linkage to 15 protein and blood group loci. Data showing close or fairly close linkage to the serum albumin locus (Al) and loose linkage to the serum esterase locus (Es) for both e and Rn are presented. This means that three coat color genes (To, e and Rn) and three serum protein loci (Al, Gc, and Es) are linked in the same linkage group. The gene order can tentatively be written Al, Gc, Rn, To-e-Es. The implications of the results for studies on coat color inheritance in horses are discussed. The possibility of using ...
Isolation of an adenovirus antigenically distinct from equine adenovirus type 1 from diarrheic foal feces. Adenovirus was isolated in equine fetal kidney cell cultures from the feces of 2 foals with diarrhea that also had large numbers (greater than 10(6)/g) of rotavirus particles in their feces. Unlike equine adenovirus type 1 (EAdV1), the fecal EAdV did not hemagglutinate human O, rhesus macaque, or equine RBC. By serum neutralization, the fecal viruses were identical with each other, but showed no relationship to EAdV1. Antiserum prepared against the fecal viruses did not contain hemagglutination-inhibiting antibody to EAdV1. It is proposed that the fecal viruses be considered prototypic of EAdV...
Chemotaxis of radiolabeled equine neutrophils. A method for the isolation of equine neutrophils was developed using metrizamide cushions. A purity of greater than 95% was routinely obtained with greater than 90% viability. These cells were radiolabeled and tested for their chemotactic response in Boyden chambers to zymosan-activated equine serum, the partially purified equine complement component C5a, and formyl-L-methionyl-L-leucyl-L-phenylalanine. The time and ionic requirements for chemotaxis of radiolabeled equine neutrophils were investigated and maximal movement was observed at 2 hours' incubation and 1.0 mM Ca and 0.5 mM Mg. Dinitro...
Fluorimetric determination of unsubstituted and 9(8)-O-acetylated sialic acids in erythrocyte membranes. A method is described for all quantitative determination of free or glycosidically bound sialic acids with special reference to erythrocyte membranes. Sialic acids, unsubstituted in their side chains, quantitatively yield formaldehyde after mild periodate oxidation (1 mM NaIO4, 15 min, 4 degrees C, in the dark). The formaldehyde is determined by the reaction with acetylacetone and ammonium acetate which leads to a sensitive fluorogen (F 410/510 nm). Sialic acids O-acetylated at C-9 or C-8 are not oxidized under these conditions. Therefore, they can be determined quantitatively by measuring the...
Recovery of microorganisms from synovial and pleural fluids of animals using hyperosmolar media. L-phase (CWD) broth and plate media were used in parallel with conventional microbiological media during a 3-year period for culturing synovial and pleural fluids of animals. Two kinds of recoveries were obtained where parallel conventional methods were negative: (1) parent or normal bacteria, in very low numbers; and (2) Type B CWD variants in equally low numbers. Organisms in group 1 were: Streptococcus zooepidemicus from horses (2x); beta-hemolytic streptococci, Lancefield Gp. G (2x); Staphylococcus aureus; Actinobacillus, and Actinomyces viscosus. Group 2 consisted of Bacteroides sp., Prop...
Purification of horse eosinophil peroxidase. Eosinophil peroxidase (donor: hydrogen-peroxidase oxidoreductase, EC 1.11.1.7) was isolated in a highly purified form (415/280 nm ratio, 1.05) from horse peripheral blood eosinophil. Eosinophil peroxidase was extracted from intact eosinophils (98-100% purity) or isolated eosinophil granules with 0.05 M acetate buffer (pH 4.7)/0.18 M NaCl and purified by chromatography on Sephadex G-200 and carboxymethylcellulose. Final elution was with 0.05 M acetate buffer (pH 4.7)/ 1 M NaCl. Horse eosinophil peroxidase is a strongly basic protein with bacterial properties when combined with H2O2 and iodide, ...
Prostaglandins in stallion semen. The purpose of the experiment was to obtain preparatory information about the presence of prostaglandins in semen collected from various types of horses after different periods of sexual rest. Semen was collected with an artificial vagina. Prostaglandin-like activity was estimated by the bioassay procedure described by Vane (1). Results are expressed in ng/ml PGE(2) of seminal plasma. The total concentration of prostaglandins in the full ejaculate averaged 43.73 +/- 4.93 ng/ml of plasma while the total amount of prostaglandins in the ejaculate was 1076 ng. Taking into consideration the period ...
Identification of immunoglobulin heavy-chain isotypes of specific antibodies of horse 46 group B meningococcal antiserum. Hyperimmune horse serum from a single animal (horse 46) immunized with group B (strain B-11) meningococcal vaccine provides a standardized, readily available diagnostic reagent used in primary isolation medium and for serogrouping of meningococci. Identification of the heavy-chain isotypes of specific anticapsular polysaccharide and anti-lipopolysaccharide isolated from horse 46 serum revealed a differential distribution in the occurrence of immunoglobulin classes. Meningococcal anticapsular antibodies of horse 46 serum were restricted predominately to the immunoglobulin M (IgM) class, with on...
Leukotriene generation by eosinophils. Horse eosinophils purified to greater than 98% generated slow reacting substance (SRS) when incubated with the calcium ionophore A23187. On a per cell basis, eosinophils generated four to five times the SRS produced by similarly treated horse neutrophils. Eosinophil SRS production was inhibited by 5,8,11,14-eicosatetraynoic acid and augmented by indomethacin and arachidonic acid, suggesting that it was a product(s) of the lipoxygenase pathway of arachidonic acid metabolism. Compounds with SRS activity were purified by high-pressure liquid chromatography (HPLC) and identified by ultraviolet spe...
The cryo-jaw, a clamp designed for in vitro rheology studies of horse digital flexor tendons. A clamp designed for holding tendons in force/elongation studies is described. No slippage occurred when tensile forces up to 13,800 N were applied to horses digital flexor tendons fixed in this clamp.
Purification of horse muscle acylphosphatase antibodies by affinity chromatography. Horse muscle acylphosphatase antibodies were obtained by immunizing rabbits with the highly purified antigen cross-linked with glutaraldehyde. Specific antibodies were purified from the immunoglobulin fraction by affinity chromatography using a matrix coupled with the pure antigen as immunoadsorbent. The purified antibodies were partially characterized by immunodiffusion and immunoprecipitin techniques. These antibodies could be used to study aspects of the muscle acylphosphatase structure, localization and other biological properties.
[Purification of alpha-1,4 leads to 1,4-glucosyltransferase from horse blood serum]. The purification of alpha-1,4-1,4-glucosyltransferase from the equine serum is presented. Ion-exchange chromatography on DE-11, DE-32 and CM-32 celluloses was applied in the successive steps of isolation. Gel-filtration on Bio-Gel P-200 was the last step of purification; it gave the protein which was homogeneous on disc polyacrylamide gel electrophoresis. The purification degree was of the order 2100 at about 40% yield.
Isolation and identification of steroids from gonadal vein blood of the fetal horse. Direct connection of the artery of a fetal ovary to the carotid artery of the mare allowed collection of a large volume of blood over a 30-min period. Extraction of steroids and their fractionation was followed by separation of the steroids by alumina adsorption chromatography, and Sephadex LH-20 and Celite partition chromatography. Further resolution of the material by HPLC led to the identification of dehydroepiandrosterone (DHA) by nuclear magnetic resonance and mass spectrometry. Other compounds were isolated, which remain to be identified fully, but in the 8th month of pregnancy the princ...
