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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.
A new surface marker on equine peripheral blood lymphocytes. I. Subpopulations of lymphocytes with receptors for Helix pomatia A hemagglutinin (HP). Untreated and neuraminidase-treated equine peripheral blood lymphocytes were analysed for binding of the A hemagglutinin of the snail Helix pomatia (HP). For optimal staining by direct immunofluorescence, the concentration of neuraminidase had to be increased as compared to that needed for other species. Moreover, higher concentrations of HP were required for optimal staining of equine lymphocytes as compared to lymphocytes from other species. Even so, the maximal number of equine lymphocytes exhibiting positive staining was only about 20%. No, or very few, HP-positive lymphocytes were seen wh...
Microquantitative determination of the distribution patterns of alcohol dehydrogenase activity in the liver of rat, guinea-pig and horse. Microquantitative measurements of ADH-activity were carried out on the livers of male and female rats, guinea-pigs and horses (two geldings and a mare). Lyophilized cryostat sections of liver parenchyma were microdissected the whole way along the sinusoidal length from the terminal afferent vessels to the terminal efferent venule. ADH activity in samples of about 50-150 ng was measured in a microbiochemical assay using the oil-well technique without enzymatic cycling, by direct luminometric determination of NADH. On the basis of the single measurements, mean values of total hepatic ADH activit...
Qualitative detection of corticosteroids in equine biological fluids and the comparison of relative dexamethasone metabolite/dexamethasone concentration in equine urine by micro-liquid chromatography-mass spectrometry. Several important corticosteroids were qualitatively determined in the plasma and urine of horses by micro-liquid chromatography-mass spectrometry (micro-LC-MS). The sensitivity and specificity of micro-LC-MS are demonstrated as is the ability of micro-LC-MS to deal with endogenous interferences. In turn, the relative amount of dexamethasone and its major unconjugated metabolite were determined in equine urine by micro-LC-MS; the conclusions drawn are reported.
Unfolding-refolding transition of a hinge bending enzyme: horse muscle phosphoglycerate kinase induced by guanidine hydrochloride. The unfolding-refolding transition of horse muscle phosphoglycerate kinase induced by guanidine hydrochloride was studied under equilibrium conditions using four different signals: fluorescence intensity at 336 nm, UV difference absorbance at 286 and 292 nm, ellipticity at 220 nm, and enzyme activity. From the following arguments, we found that the process deviates from a two-state model and intermediates are significantly populated even at equilibrium: (1) the noncoincidence of the transition curves and (2) the asymmetry of the transition curve obtained from CD measurements. From these differ...
Specificity of pseudorabies virus serotests. Pigs experimentally inoculated with bovine herpesvirus-1 or equine herpesvirus-1 developed mild clinical disease signs. Regression of clinical disease was accompanied by development of specific virus-neutralizing antibodies. These antibodies did not react positively with pseudorabies antigens in the serum-virus neutralization test, an indirect radioimmunoassay, or a microimmunodiffusion test.
The primary structure of monomeric beta-lactoglobulin I from horse colostrum (Equus caballus, Perissodactyla). beta-Lactoglobulin-like proteins were detected in horse colostrum and normal milk using immunological techniques. In contrast to the beta-lactoglobulins sequenced so far these proteins are monomeric and genetically not homogenous. In this paper we report the first primary structure of a monomeric beta-lactoglobulin from horse colostrum. By means of an automatic liquid-phase sequenator the sequence of peptides obtained by tryptic digestion and by cyanogen bromide cleavage was determined. A limited tryptic digestion and hydrolysis with chymotrypsin provided the necessary overlapping peptides. Th...
Cadmium/zinc relationships in kidney cortex and metallothionein of horse and red deer: histopathological observations on horse kidneys. Cadmium and zinc were determined in kidney cortex of 63 horses and 51 red deer (Cervus elaphus). Cadmium and zinc were also determined in protein fractions obtained by Sephadex chromatography of kidney cortex from 10 horses and 4 red deer. Histopathological parameters in kidney cortex of horses were compared to cadmium content. The metal contents (on wet weight basis) in kidney cortex of the horses were 0.31 +/- 0.22 mmole Cd/kg (range 0.03-1.21) and 0.63 +/- 0.17 mmole Zn/kg (range 0.36-1.23). The Zn content increased with the Cd content, the Zn increase being less at higher concentrations. N...
Nutritionally variant streptococci from corneal ulcers in horses. Of 24 isolates of nutritionally variant streptococci recovered from equine corneal ulcers, 22 were tested for growth requirements, physiological and biochemical reactions, and susceptibility to different antimicrobial agents. Satisfactory growth was obtained by supplementing blood agar and Todd-Hewitt broth with pyridoxal hydrochloride, and all of the media for the culture and the biochemical testing were supplemented with 0.002% of this substance. Biochemical patterns of 12 of the isolates resembled those of two viridans streptococcal species, Streptococcus intermedius and Streptococcus const...
Cholesteryl sulfate: the major polar lipid of horse hoof. The lipids of horse hoof have been analyzed by quantitative thin-layer chromatography. The major components include cholesterol (37-40%), six groups of ceramides (10-15%), and cholesteryl sulfate (15-20%). Free fatty acids are abundant (15.8%) in the outer fully keratinized hoof, but are present at only low levels (3.1%) in the softer hyponychium. The material identified as cholesteryl sulfate was isolated by preparative thin-layer chromatography and characterized by a combination of chemical, chromatographic, and spectroscopic methods. The infrared spectrum of the isolated material had absorp...
Substrate-dependent kinetic behavior of horse plasma cholinesterase: evidence for kinetically distinct populations of active sites. The inhibition of horse plasma cholinesterase by propranolol showed characteristics which depended upon the identity of the substrate used. With butyrylthiocholine as substrate, the inhibition showed a first-order dependence on inhibitor concentration, and was characterized by a Ki of 8 microM (pH 7.4, 20 degrees C). With p-nitrophenylbutyrate as substrate, a biphasic v-1 versus [I] relationship was obtained. The biphasic curve could be resolved into two components, with apparent Ki's of 9 microM and 1.3 mM. Use of butyrylthiocholine as alternative substrate resulted in partial inhibition of p...
DNA sequences from the quagga, an extinct member of the horse family. To determine whether DNA survives and can be recovered from the remains of extinct creatures, we have examined dried muscle from a museum specimen of the quagga, a zebra-like species (Equus quagga) that became extinct in 1883 (ref. 1). We report that DNA was extracted from this tissue in amounts approaching 1% of that expected from fresh muscle, and that the DNA was of relatively low molecular weight. Among the many clones obtained from the quagga DNA, two containing pieces of mitochondrial DNA (mtDNA) were sequenced. These sequences, comprising 229 nucleotide pairs, differ by 12 base substitu...
The amino acid sequence of equine alpha-lactalbumin. The amino acid sequence of equine alpha-lactalbumin has been determined with the aid of an automatic sequencer. The protein chain consists of 123 amino acids and has a Mr of 14218. Elucidation of the structure involved sequence determination of native protein (residues 1-32), cyanogen bromide fragments, and tryptic, chymotryptic and S. aureus V8 proteolytic peptides. Approximately 67% of the residues are identical with corresponding residues of bovine alpha-lactalbumin B, and there is close homology with alpha-lactalbumin of other species.
Rapid screening and confirmation for drugs and metabolites in racing animals by tandem mass spectrometry. A screening and confirmation procedure for drugs and metabolites in the blood serum and urine of racing animals was developed. Equine blood serum was spiked with low concentrations of several drugs of interest. Canine blood serum and urine were collected following oral doses of diethylcarbamazine, procaine, and phenylbutazone. Serum, urine, and extracts of each were analyzed, using a triple quadrupole mass spectrometer. Simultaneous screening of up to 50 drugs was possible in a single sample, in less than 2 minutes. Detection limits for most compounds were in the ng/ml to microgram/ml range, u...
Isolation and structural characterization of the equine erythrocyte receptor for enterotoxigenic Escherichia coli K99 fimbrial adhesin. The erythrocyte receptor for Escherichia coli K99 fimbrial adhesin was isolated from equine erythrocytes and characterized as Neu5Gc-alpha(2----3)-Galp-beta(1----4)-GLcp-beta(1----1)-Ceramide. This glycolipid acted as the receptor for K99 by four different experimental approaches: inhibition of equine erythrocyte hemagglutination by preincubation of K99-positive bacteria or purified K99 fimbriae with the isolated glycolipid; inhibition of attachment of K99-positive bacteria to porcine intestinal epithelial cells in the presence of the isolated glycolipid; induction of binding of K99-positive b...
Mare lactotransferrin: purification, analysis and N-terminal sequence determination. Mare lactotransferrin has been purified and analyzed. Its molecular mass is 81 kDa. A 28 amino acid long N-terminal sequence was established and a first series of comparisons with other transferrins was performed.
Pitfalls in immunofluorescence testing in dermatology. III. Pemphigus-like antibodies in the horse and direct immunofluorescence testing in equine dermatophilosis. Indirect immunofluorescence testing for pemphigus-like antibodies was performed on 79 horses: 28 horses with various nonpemphigus dermatologic diseases, 21 horses with various nondermatologic diseases, and 30 normal horses. Pemphigus-like antibodies were detected in 6 horses: 3 normal horses with titers of 1:40, 2 horses with dermatophilosis at titers of 1:10 and 1:80, and 1 horse with lymphosarcoma at a titer of 1:320. It was concluded that equine pemphigus-like antibodies are a potential source of misinterpretation and misdiagnosis in indirect immunofluorescence testing. Direct immunofluores...
Growth kinetics of equine respiratory tract viruses in cell and organ cultures. Growth kinetics of equine influenza virus-A1, equine herpesvirus-1, and equine rhinovirus-1 were determined in susceptible cell monolayers and in organ cultures of equine fetal tracheal and nasal turbinate epithelium. Equine influenza virus-A1 was replicated in cell and organ cultures and was released more readily and for longer periods from nasal turbinate epithelium than from tracheal epithelium. Equine herpesvirus-1 was also replicated in cell and organ cultures. During the first 24 hours after inoculation, equine herpesvirus-1 was released more readily from tracheal epithelium than from na...
Effect of tyrosine modification on the biological and immunological properties of equine chorionic gonadotropin. The tyrosine residues of equine chorionic gonadotropin have been nitrated with tetranitromethane and the resulting effects on the biological and immunological activities of the hormone studied. All of the tyrosine residues in equine chorionic gonadotropin were found to react with tetranitromethane when a 100-fold molar excess of reagent was used or with an 8.6 molar excess in the presence of 5 M guanidine hydrochloride. Complete nitration abolished the biological activities and decreased the immunological activity of the hormone. The nitration of one tyrosine residue resulted in the loss of 70...
Isolation and partial characterization of bovine and equine factor D. Bovine and equine factor D were purified to apparent homogeneity as evidenced by a single protein staining band on 7.5-17.5% SDS-PAGE slab gels under both reducing and non-reducing conditions. An apparent mol. wt of 15,000 for bovine D and 22,500 for equine D were noted after SDS-PAGE gel analysis of both reduced and non-reduced preparations. A single polypeptide chain for both proteins was evidenced by the lack of any change in the electrophoretic mobility under each of these conditions. The bovine and equine D were enriched 3347- and 9447-fold, with a 20 and 29% yield of hemolytic activity, ...
[Interaction of bis-phosphorylated methanes with mammalian esterases]. The interaction of human erythrocyte acetylcholinesterase, horse serum butyrylcholinesterase and rat liver carboxylesterase with insecticides (RO)2P(O)SCH(COOEt)SP(O)(OR)2 (I) and (RO)2P(O)SCH(COOEt)OP(S)(OR)2 (II) was studied. The type I and II compounds were not hydrolyzed by carboxylesterase and inhibited the esterases irreversibly. A complex pattern of inhibition of acetylcholinesterase and butyrylcholinesterase by these compounds was caused by kinetically-manifested formation of an enzyme-inhibitor complex. The compounds I and II were more selective towards butyrylcholinesterase than towa...
Extenders for preservation of canine and equine spermatozoa at 5 degrees C. Two experiments were conducted to evaluate the effects of six extenders and three glycerol levels on the motility of sperm stored at 5 degrees C. Using a split-ejaculated design, semen from 10 dogs and 12 stallions was extended with egg-yolk-tris (EYT), egg-yolk-bicarbonate (EGB), Beltsville F-3 (BF-3), Cornell University (CUE), caprogen (CAP) and heated skim milk (SM) extenders. After cooling to 5 degrees C, additional extender containing 0% to 12% glycerol was added to provide a final concentration of 0%, 3% or 6% glycerol. Regardless of glycerol level, a higher (P<0.05) percentage of can...
Biotin-labeled antigen sandwich enzyme-linked immunosorbent assay (BLA-S-ELISA) for the detection of Japanese encephalitis antibody in human and a variety of animal sera. A biotin-labeled antigen (BLA) was adapted to a sandwich enzyme-linked immunosorbent assay (S-ELISA) for detection of Japanese encephalitis (JE) antibody in a variety of animal sera. JE antigen was fixed on the wells of a microplate and became bound to the specific antibody which could react with a peroxidase-labeled avidin conjugate and azino-di-(3-ethylbenzthiazolin sulfonic acid) (ABTS) as a substrate. The BLA-S-ELISA could simultaneously detect JE antibody in all hemagglutination inhibition (HI) positive sera from man, swine, monkey, horse, cattle, rabbit, rat, mouse and pigeon by using th...
Counterimmunoelectrophoresis for identification of equine urine. Counterimmunoelectrophoresis was evaluated as a method to distinguish urine of human origin from that of equine origin. The procedure used anti-equine serum and anti-human serum antibodies that had been solid-phase absorbed to eliminate species cross-reactivity. Counterimmunoelectrophoresis reliably detected contamination of equine urine by human urine to a level of 10% with a minimum sensitivity to about 2% contamination. Compared with double diffusion, counterimmunoelectrophoresis was approximately 10 to 15 times more sensitive in the detection of urine proteins.
