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.
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...
[Properties of alpha-1,4 leads to -glucosyltransferase from the muscles and blood serum of horses]. Alpha-1,4 leads to 1,4-glucosyltransferase preparations from horse muscles and serum were studied. The enzyme proteins from both tissues are very similar. Both proteins have a molecular weight of 240 000 and consist of four subunits of 60 000 daltons each. pH 5,0 is optimal for the activity. Only substrates with alpha-1, 4-linkages can serve as glucosyl donors for transferase reactions. Km values for both enzymes differ very slightly. At low substrate concentrations the hydrolytic activity can be found in addition to transferase reactions. At the concentration of the substrate higher than 40 m...
Isolation and some properties of equine alpha 1-antitrypsin. 1. Equine alpha 1-antitrypsin was isolated from horse plasma by a combination of ammonium sulfate and acidification precipitation followed by ion-exchange chromatography on DEAE-cellulose, molecular sieve chromatography on Sephadex G-200 and affinity chromatography on Cibacron Blue-agarose. 2. The purified protein showed a single precipitin arc on immunoelectrophoresis in agarose but gave two bands on discontinuous polyacrylamide gel electrophoresis (PAGE). 3. Both bands appeared to interact equally with trypsin and were thought to represent two isoinhibitors of equine alpha 1-AT.
Subcellular distribution of particle-associated enzymes in horse neutrophil leukocytes. The subcellular components of purified neutrophil leukocytes from horse blood were fractionated by isopyknic equilibration in sucrose and metrizamide gradients. Five classes of particles have been identified: dense azurophil granules containing the bulk of the lysosomal acid hydrolase and peroxidase activity (A); less dense particles, containing all the lysozyme activity, but not resolved from a second population of azurophils B, and particles of low density, biochemically characterized as a plasma membrane fraction (C). Isopyknic equilibration in sucrose disclosed a minor membrane fraction (D...
Interactions of different albumins and animal sera with insolubilized Cibacron Blue, Evaluation of apparent affinity constants. 1. A high concentration Cibacron Blue-Sepharose derivative has been used to study the affinity chromatography of albumin from eight animal species. 2. The apparent affinity constants for albumin varies between 3.9 x 10(4) M-1 and 0.9 x 10(4) M-1, in the order: Human greater than rabbit greater than horse greater than pig = dog greater than bovine greater than rat greater than chicken. 3. Other serum proteins were also bound to the gel, particularly lipoproteins and alpha 2-macroglobulin.
The optimum pH of renal adenosine triphosphatase in rats: influence of vanadate, noradrenaline and potassium. In the presence of vanadate, the optimum pH of renal (Na+, K+)-ATPase in rats is reduced and lies in the range of intracellular pH. This explains the difference in optimum pH observed with ATP extracted from equine muscle. Removal of vanadate from such ATP (with noradrenaline) raises the optimum to the accepted range obtained with synthetic ATP. Changes in the sensitivity of the enzyme to potassium concentration contribute to the alterations in optimum pH. The optimum pH of Mg-ATPase is unaffected by vanadate. Since vanadate may be an intracellular regulator of (Na+, K+)-ATPase changes of opti...
Comparison of receptor properties of erythrocyte membrane glycoproteins. Membrane glycoproteins from horse, sheep, goat and bovine erythrocytes were solubilized and purified. These glycoproteins could be placed in three groups based on their degrees of glycosylation: The major bovine erythrocyte glycoprotein (BGII) had 77% sugar, the minor bovine glycoprotein (BGI) had 27% sugar and the others had approximately 50% sugar. Four of the glycoproteins aggregated in a uniform way in aqueous solution--one, BGII, did not. Four had similar subunit sizes of 25-34,000 daltons, but BGII was larger--55,000 daltons. Receptor functions (for plant and invertebrate lectins, antibo...
Relaxin activity in foaling mares. Plasma relaxin concentrations were measured hourly by radio immunoassay in 4 pregnant mares from 11 days before until 4 days after natural foaling. Pre-partum levels ranged from 4 to 7 ng/ml without any surge until the second stage of labour when they increased rapidly to about 11 ng/ml. In 3 of these mares, relaxin activity declined immediately after the expulsion of the placenta and was below detectable levels within 36 h. In the other mare relaxin activity did not fall until after the mechanical removal of the placenta 7 h after foaling. Eight mares were induced to foal by the administratio...
Radioimmunoassay and in-vitro bioassay of serum LH throughout the equine oestrous cycle. Mares were bled once daily throughout a cycle, or 3 times daily from the first day of oestrus to the 2nd day after ovulation. LH was measured by heterologous radioimmunoassay and by an in-vitro bioassay based on LH-stimulated testosterone production by mouse Leydig cells. The patterns of bio- and immuno-active LH during the oestrous cycle were similar but not identical, so that in both groups of mares the ratio of biological: immunological (B:I) activity during the LH surge was significantly higher before than after ovulation (P less than 0 . 001). Considerable individual variation in cycle me...
Erythrocyte volume distribution analysis in healthy dogs, cats, horses, and dairy cows. Erythrocyte volume distribution curves (erythrograms) were determined on a total of 300 blood samples from healthy dogs, cats, horses, and cattle (dairy cows). An index of anisocytosis was determined for these animals. Erythrograms were highly reproducible, and the mean corpuscular volumes determined from erythrograms compared well with those determined from hemograms. Bovine and equine erythrocyte volumes were found to be stable after the blood was stored at 4 C for 24 hours. Under the same conditions, canine and feline erythrocytes increased slightly in volume. After incubation of blood dilu...
Vitamin A profiles of equine serum and milk. Serum and milk samples from mares and serum samples from their foals were taken at parturition and on d 1, 2, 4, 7, 14 and 21 postpartum. The samples were assayed for retinyl (r.) palmitate, r. acetate and retinol by high performance liquid chromatography. Peak vitamin A activity in milk occurred 1 d postpartum and preceded by 3 d the maximum vitamin A activity in foal serum and the lowest vitamin A activity in the mare serum. Mare serum contained approximately a 65:35 ratio of retinol:r. palmitate and less than 1% r. acetate. Retinyl palmitate was the predominant form of vitamin A in milk unt...