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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.
Lymphocyte transformation test in veterinary clinical immunology. Lymphocyte transformation test is a powerful tool in laboratory testing of immunologic competence of animals. The impaired function of the lymphocytes or presence of mitogenesis suppressing factors in the patient serum were detected by comparing lymphocyte transformation (expressed as thymidine incorporation) obtained in media containing either autologous, homologous, or fetal calf serum additions. Most valuable results were obtained by using at least two, preferably three, different phytomitogens: concanavalin A (Con A), pokeweed mitogen (PWM), and phytohemagglutinin (PHA) at optimal concentr...
In vitro host range of equine infectious anemia virus. Equine infectious anemia virus (EIAV) was successfully inoculated onto cell cultures of canine and feline origin, resulting in chronic infections in these cultures. Infection of equine cell cultures, which were the previous sole in vitro source demonstrated for virus production, was also performed for comparative purposes. Determination of the nature of the virus produced in the heterologous as well as the equine cells was accomplished in several ways. SDS-PAGE of purified virus from the different cell lines indicated very similar protein composition. Immunological identity was observed in gel...
Determination of plasma fibrinogen concentration in the horse. The microhematocrit heat-precipitation methods of Millar et al (1971) and Schalm et al (1975) were compared with the reference clottable protein method of Ratnoff and Menzie (1951) in the measurement of plasma fibrinogen concentration in horses. The millar et al method was more precise and accurate and showed better positive correlation with the reference method than did the Schalm et al method. There was no significant difference in the plasma fibrinogen concentration between healthy Thoroughbreds and healthy horses of other breeds. Horses with bacterial pneumonia and abscesses had significan...
[Some clinical biochemical features of ‘tying up’ in horses (author’s transl)]. The changes in the activities of lactate dehydrogenase (LDH) and creatine phosphokinase (CPK) in de blood of thirty-three horses with 'tying up' were compared. The extent to which the serum enzymes LDH, CPK and glutamic oxaloacetic transaminase (GOT) and the changes in the activities of these enzymes after suitable labour can be used in the diagnosis of 'tying up' and in following the recovery of patients was studied.
Fractionation and partial characterization of alpha-1-protease isoinhibitors of horse. The principal alpha-1-protease inhibitor of horse was fractionated by classical methods and analysed with a modified fibrinogen-agarose gel electrophoretic method of high sensitivity and resolving power. Starting with an electrophoretically homogeneous inhibitor in unfractionated serum, two isoinhibitor bands became apparent after fractionation with (NH4)2SO4 and DEAE-cellulose DE-52 ion-exchange chromatography. The isoinhibitors differed in electrophoretic migration and in the elution pattern from Sephadex G-100 gel filtration, but possessed identical antigenic determinants and enzyme specifi...
[Effect of 2 methods of demineralization on the on the preservation of glycoproteins and proteoglycans in the intertubular and peritubular dentin in the horse]. The effect of 2 methods of demineralization on the preservation of proteoglycans and glycoproteins was studied in the intertubular and peritubular dentine of the horse. The specimens embedded in Epon were demineralized with a 2% acid formic solution (Bonucci and Gheradi, 1975). Other fragments were treated with an organic solution of EDTA alkylammonium salt (Scott and Kyffin, 1979). These methods preserved in a satisfactory way these labile organic components. In the intertubular dentine, glycoproteins and proteoglycans were also identified, either associated with collagen fibres as a glue and...
Helix packing and subunit conformation in horse spleen apoferritin. An electron density map of horse spleen apoferritin at 0.28-nm (2.8 A) resolution and its preliminary interpretation have been described previously. Rigorous examination of this and newer maps at the same nominal resolution but calculated from more extensive data sets, including model building in a Richards' comparator, now allows us to report on structural features in more detail. We list inter-helical angles within and between neighbouring subunits, and describe a new short region of inter-subunit anti-parallel pleated sheet. A short section of electron density not properly accounted for in ...
[The immunological relation between human and equine Gc proteins (author’s transl)]. The immunological comparison of human and equine Gc proteins showed partial identical reactions between both species. Immunizations of goats and rabbits with horse serum produced antisera able to recognize human Gc proteins.
Comparison of milk and blood plasma progesterone concentrations in cycling and pregnant mares. Progesterone concentrations were measured in milk and blood plasma for 15 mares throughout a normal estrous cycle and early pregnancy to determine the feasibility of utilizing progesterone in milk as an indicator of pregnancy. Samples were obtained daily from foaling until diagnosis of pregnancy by rectal examination at 30 to 35 days of gestation. Progesterone in milk and blood plasma was quantified by radioimmunoassay. Mean progesterone concentrations (+/- SE) in plasma from foaling to foal heat and during estrus, luteal phase and pregnancy were .51 +/- .09 ng/ml, .53 +/- .08 ng/ml, 3.88 +/- ...
Conformation of Immunoglobulin M. III. Structural requirements of antigen for complement fixation by equine IgM. Complexes of IgM equine anti-dansyl antibodies and different dansyl substituted carriers were tested for their ability to fix complement (C). Only dansyl92-Ficoll and dansyl12-poly-L-lysine were found to be effective. Dansyl13-bovine serum albumin, dansyl127-keyhole limpet hemocyanin, and reduced and alkylated dansyl10-ribonuclease were all ineffective. Lack of C fixation by the dansyl-ribonuclease was not due to lack of antibody-antigen complex formation, since binding at the concentrations employed for C fixation was established. However, in contrast, polymerized dansyl-ribonuclease (polydis...
Selected ion monitoring assay for bromhexine in biological fluids. A method has been developed for quantification of bromhexine in plasma using gas chromatography mass spectrometry with selected ion monitoring. A deuterium labelled analogue was synthesized and used as the internal standard. To evaluate the gas chromatographic electron capture detection method described earlier, 23 plasma samples have been analysed by both techniques. Although a good correlation was shown, selected ion monitoring was superior to the electron capture detection method for levels below 3 ng ml-1. The mass spectrometric method has also been used to set up a pharmacokinetic study o...
Sequence of the low activity equine erythrocyte carbonic anhydrase and delineation of the amino acid substitutions in various polymorphic forms. the sequence of the low activity form of equine erythrocyte carbonic anhydrase has been determined. The most common electrophoretic form, designated D, has been found to have five substitutions. Amino acid exchanges in the electrophoretic variants known as A1, A2, B, and T have been found at six other positions. The data do not permit calculation of the number of polymorphic forms of this enzyme. The equine D isozyme and the analogous human enzyme are quite homologous, 211 of their 260 residues, or 81%, being identical.
[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...
