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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.
[3H]5-HT binding sites and 5-HT-sensitive adenylate cyclase in glial cell membrane fraction. Glial cell membrane fractions were prepared using glial cells preparations isolated from horse brain striatum. [3H]5-HT binding was measured by the filtration technique and the adenylate cyclase activity determined by measuring the cAMP production using a radioimmunoassay. Serotonin binds to glial membrane fractions with an affinity corresponding to a dissociation constant Kd = nM. The corresponding site is serotoninergic specific: [3H]5-HT binding is inhibited by 5-HT agonists (5 OH NM-DMT, 5-MeOHT, 5-MeOH-DMT, NN-DMT) or antagonists (cinanserine, cyproheptadine, methysergide, LSD) and not (o...
Isolation of Yersinia enterocolitica from selected animal species. Yersinia enterocolitica was isolated from 5 of 1,002 fecal samples taken from laboratory rats and mice, hamsters, dogs, cats, pigs, cattle, horses, and deer. Two isolates were from dogs (2 of 202; 1%) and 1 from a pig (1 of 107; 0.9%). The 3 isolates were biotype 1. Atypical environmental Y enterocolitica was isolated from a cow (1 of 141; 0.7%) and a horse (1 of 101; 1%). Isolates were not recovered from the other animal species.
Hypogammaglobulinaemia in foals: prevalence on Victorian studs and simple methods for detection and correction in the field. The prevalence of hypogammaglobulinaemia in 82 young foals was determined. Twelve foals were considered clinically abnormal at birth and ten died within two weeks. All of these foals were hypogammaglobulinaemic. Seven (10%) of the other 70 apparently normal foals were hypogammaglobulinaemic despite having suckled normally. Three of these foals developed significant disease and one died at one month of age. Rapid detection of foals with low serum immunoglobulin levels was achieved by adapting the zinc sulphate turbidity test to partially evacuated blood collection tubes. This permitted test to ...
Characterization of gangliosides from equine kidney and spleen. Gangliosides were isolated from equine kidney and spleen, and their carbohydrate and lipid moieties were characterized. Among the long-chain bases, considerable proportions of trihydroxy bases (42.3 to 61.2% of the total bases), in which phytosphingosine was predominant were found in all the ganglioside classes. The other major base was sphingosine. Among the constituent fatty acids, long-chain acids (with a carbon number of more than 20), comprised approximately half the total acids, with some alpha-hydroxy and mono-unsaturated acids. By means of sequential hydrolysis with glycosidases couple...
Moldy sweetclover poisoning in a horse. A six year old Percheron mare was presented with a history of spontaneous unilateral epistaxis of 24 hours duration. The blood one stage prothrombin and partial thromboplastin times were markedly prolonged. A diagnosis of moldy sweetclover poisoning was made on the basis of the history and clinical and laboratory findings. A single whole blood transfusion and four daily intravenous injections of vitamin K(3) proved to be a successful treatment.
Horse erythrocyte gangliosides: preparation of the major hematoside NeuNG1-Lac-Cer. A simple method for the isolation of hematoside NeuNG1-Lac-Cer from horse erythrocytes is described. An aliquot of the crude ganglioside fraction was labeled by tritiated sodium borohydride after mild periodate oxidation. The compounds obtained were used as radioactive tracers in column chromatography. Gangliosides were applied onto a silicic acid column and eluted stepwise by solvents of steadily increasing polarity. The major ganglioside, NeuNG1-Lac-Cer, was eluted in a high yield by the solvent mixture chloroform/methanol/water (60:35:8, v/v/v).
Contagious equine metritis: isolation and characterization of the etiologic agent. Uterine, cervical, and clitoral specimens on swabs from pony mares infected with contagious equine equine metritis (CEM) bacteria were streaked on agar plates. Colonies of CEM bacteria were observed under CO2 incubation in 2 days on Eugon chocolate agar and Eugon blood agar plates. The diameter of the colonies varied from 0.2 mm to 1 mm in 2 days which increased to 0.3 mm to 2.0 mm on day 4. The colonies on Eugon chocolate agar plates on days 2 to 4 were shiny, brown, round, and convex, and easily glided when pushed with a loop. The diameter of the colonies on chocolate and blood agar plates m...
Maturation of equine epididymal spermatozoa. Spermatozoa from four regions of the epididymis and from ejaculated semen were evaluated for their resistance to cold shock, progressive motility, and structural stability. Spermatozoa were incubated at 38 C and their percentage of eosinophilia was compared with that of spermatozoa cooled to 0 C in 2 minutes, 10 C in 12 minutes, or 4 C in 22 minutes. Spermatozoa motility was estimated visually under phase-contrast microscopy and was recorded by cinematography. Structural stability of spermatozoa incubated in 0.05 M sodium borate buffer, 0.035 M sodium dodecyl sulfate (SDS), 0.002 M dithiothrei...
The complete amino acid sequence of horse muscle acylphosphatase. The amino acid sequence of horse muscle acylphosphatase is given in the present paper. The carboxymethylated enzyme consists of a single polypeptide chain of 98 amino acid residues with an acetyl group blocking the NH2 terminus and a tyrosine at the COOH terminus. The calculated molecular weight of the native protein, a mixed disulfide with glutathione, is 11,365. The carboxymethylated protein was cleaved by cyanogen bromide. The three expected fragments were purified; moreover, an additional fragment, derived from a partial failure of cleavage at methionine-24, was purified and characterized....
Assembly of intra- and interspecies hybrid apoferritins. An intraspecies hybrid apoferritin was assembled by mixing subunits of horse heart ferritin, which consists mainly of H-type subunits, and horse spleen ferritin, in which L-type subunits predominate. Interspecies hybrid apoferritins were reconstituted from subunits of human liver-horse spleen ferritins and from rat liver-horse spleen ferritins. All the hybrid ferritins migrated as single zones with electrophoretic mobilities intermediate between those of the parent ferritins. Isoelectric focusing data and immunological patterns were consistent with the view that the reassembled apoferritins we...
Murine infection model for contagious equine metritis: a new venereal disease of horses. An infection model in laboratory mice for studying the bacterium (proposed name Haemophilus equigenitalis) causing contagious equine metritis is described. Small porous chambers were implanted subcutaneously into mice and after 1 to 3 weeks were inoculated with H equigenitalis. Infections that persisted for > 30 days were established by direct transfer of infective chamber fluid or by injection of laboratory-grown cultures. Immunization of mice with formaldehyde-treated cells induced significant, strain-related immunity to infection and did not appear to require complement as a protection medi...
Qualitative and quantitative analysis of hydrochlorothiazide in equine plasma and urine by high-performance liquid chromatography. A sensitive, quantitative method has been developed for the determination of hydrochlorothiazide in equine plasma and urine. Thin-layer chromatography is used to screen for the presence of the drug in unknown samples. The TLC screening methods described provide minimum detection limits of 50 ng/mL in plasma and 25 ng/mL in urine. A silica micro chromatography column is used to clean up ethyl acetate extracts for HPLC analysis and mass spectral confirmation. An internal standard, trichloromethiazide, is used to derive quantitative data at concentrations as low as 25 ng/mL for plasma disappearan...
Identification by gas-liquid chromatography-mass spectrometry of 4-O-acetyl-9-O-lactyl-N-acetyl-neuraminic acid, a new sialic acid from horse submandibular gland. The novel sialic acid 4-O-acetyl-9-O-lactyl-N-acetylneuraminic acid has been identified as a constituent of horse submandibular gland glycoproteins in addition to the already known equine sialic acids, N-acetylneuraminic acid, 4-O-acetyl-N-acetylneuraminic acid, 9-O-acetyl-N-acetylneuraminic acid, 4,9-di-O-acetyl-N-acetylneuraminic acid, N-glycolylneuraminic acid, 4-O-acetyl-N-glycolylneuraminic acid and 9-O-acetyl-N-glycolylneuraminic acid. The structure has been established by combined gas-liquid chromatography-mass spectrometry.
Alterations in horse blood cell count and biochemical values after halothane anesthesia. Quantitative changes in hematologic and serum biochemical values associated with prolonged general anesthesia produced by known alveolar doses of halothane in oxygen were determined in six young, healthy horses under laboratory conditions. In addition, 25 young equine patients anesthetized for shorter periods under clinical conditions were similarly (except hematologic values) prospectively evaluated. In normal horses, muscle- and hepatic-derived serum biochemical values were mildly increased immediately after anesthesia. Values after anesthesia remained at greater than base-line values for up...
Enzyme-linked immunosorbent assay, using staphylococcal protein A for detecting virus antibodies. A modification of the indirect enzyme-linked immunosorbent assay (ELISA) was developed which used staphylococcal protein A linked to horseradish peroxidase. Virus antibodies in equine, bovine, porcine, feline, canine, lagomorphic (rabbit), and human sera were detected, using the indirect ELISA in which the antiglobulin enzyme conjugate was replaced by protein A linked to horseradish peroxidase. Results of the ELISA were compared with the results of the serum-virus neutralization test. The application of the test in laboratories performing serologic assays with sera from diverse animal species ...
Purification and characterization of donkey chorionic gonadotrophin. Serum of the pregnant donkey, like that of the mare, contains a gonadotrophin of chorionic origin. The chorionic gonaditrophin of the donkey (dCG) has been isolated in purified form from the serum of pregnant donkeys using methodology previously employed for the purification of pregnant mare chorionic gonadotrophin (eCG). Unlike eCG, dCG is predominatly an LH in biological tests. In the in-vitro rat Leydig cell assay, dCG was as active as eCG, but in the in-vitro rat seminiferous tubule assay for FSH and in the augmentation assay, dCG was considerably less potent than eCG (1-10%). Specific rat...
Skin surface lipids of the horse. Skin surface lipids from the sides of male and female horses (Equus caballus) were collected in acetone and analyzed by thin layer chromatography and gas liquid chromatography. The sole components in both sexes were cholesterol, cholesteryl esters and the lactones of 32-, 32- and 36-carbon omega-hydroxy acids, each including a methyl group in the n-1 position. Most of the lactones were monounsaturated (either n-8 or n-10), but small amounts of saturated and dienoic species were present. A pooled sample of the skin surface lipids contained 14% cholesterol, 38% cholesteryl esters and 48% lactone...
Biochemical characterization of equine herpesvirus type 3-induced deoxythymidine kinase purified from lytically infected horse embryo dermal fibroblasts. Infection of horse KyED cells with equine herpesvirus type 3 (EHV-3) resulted in a sevenfold increase in cytosol deoxythymidine kinase (dTK) activity. The EHV-3 dTK was purified from KyED cytosol dTK by affinity chromatography on deoxythymidine-Sepharose and characterized with respect to its electrophoretic mobility, molecular weight, substrate specificity, phosphate donor specificity, and immunological specificity. The purified EHV-3 dTK migrated in polyacrylamide gels with an Rf of 0.30 and sedimented in glycerol gradients with an S value of 5.13, corresponding to a molecular weight of 83,00...
The reverse single radial immunodiffusion technique for detecting antibodies to Dermatophilus congolensis. The reverse single radial immunodiffusion technique was used to detect Dermatophilus congolensis antibody in sera collected from animals previously infected to varying levels with D congolensis. Ammonium sulphate and trichloroacetic acid extracts of five different strains of D congolensis obtained from different geographical locations were used as antigens. All the extracts showed variations in their sensitivities in detecting D congolensis antibody in the various serum samples. Multiple antibodies were detected by some extracts while some showed negative antibody reaction to all extracts. Two...
Negative contrast electron microscopic techniques for diagnosis of viruses of veterinary importance. Negative contrast electron microscopy (NCEM) was utilized as a routine tool in the diagnosis of viral infections of domestic and wild animals. Viruses identified by this technique were observed in infected culture systems or clinical specimens from several species including horses, cattle, sheep, dogs, cats, pigs, deer, Rocky Mountain bighorn sheep, antelope, and several avian species. Viruses were identified by NCEM based on their size, morphology, and symmetry and consisted of adenoviruses, herpesviruses, paramyxoviruses, myxoviruses, picornaviruses, parvoviruses, coronaviruses, reoviruses, ...
Studies on fungal flora in hair from domestic and laboratory animals suspected of dermatophytosis. I. Dematophytes. Hairsamples of domestic and laboratory animals suspected of dermatophytosis were examined for the presence of dermatophytes. A nutritionally poor base-medium developed by the author was successfully used in the isolation and identification of dermatophytes. Casein-medium supplemented with vitamins and Sabouraud-liquid medium were used in special cases. Dermatophytes were isolated in 36 of 331 samples (10.9%). The dermatophytes recovered were Microsporum canis: 13 isolates from cat. 4 from dog. 1 from horse; Trichophyton mentagrophytes var. granulare: 3 isolates from dog, 3 from horse, 2 from g...
Involvement of lysines-72 and -79 in the alkaline isomerization of horse heart ferricytochrome c. Spectrophotometric titrations of five singly modified horse heart ferricytochromes c, specifically (trifluoromethyl)phenylcarbamylated (CF3PhNHCO-) or trifluoroacetylated (CF3CO-) at lysines-13, -72, and -79, were carried out. The CF3PhNHCO-Lys-13, Lys-79, and CF3CO-Lys-79 derivatives all underwent alkaline isomerization with loss of the 695-nm band to low-spin species with an apparent pK of about 8.9, as did the unmodified cytochrome. However, modification of lysine-72 appeared to alter the reaction pathway since the CF3PhNHCO-Lys-72 derivative isomerized to a high-spin form with an apparent ...
Purification by affinity chromatography and characterization of a neutral alpha-glucosidase from horse kidney. A horse kidney neutral alpha-D-glucosidase (alpha-D-glucoside glucohydrolase, EC 3.2.1.20) was purified about 580-fold with a yield of 33% by an affinity chromatography technique using the p-aminophenyl-beta-D-maltoside, a substrate derivative, as ligand. The purified enzyme, homogeneous in polyacrylamide gel electrophoresis, was a glycoprotein with a molecular weight of 280 000 as calculated by gel filtration and its isoelectric focusing points was found to be pH 4.1. The purified enzyme was able to hydrolyze various substrates having (alpha-1,2), (alpha-1,3), (alpha-1,4), and (alpha-1,6) glu...
