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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.
Sequence analysis and polymerase chain reaction amplification of small subunit ribosomal DNA from Sarcocystis neurona. To identify Sarcocystis neurona-specific DNA sequences in the nuclear small subunit ribosomal RNA (nss-rRNA) gene that could be used to distinguish S neurona from other closely related protozoal parasites, and to evaluate a polymerase chain reaction (PCR) test, using broad based primers and a unique species-specific probe on CSF for detection of S neurona in equids. Methods: Sequencing of the nuclear small subunit ribosomal RNA gene from a new S neurona isolate (UCD 1) was performed. The sequence was compared with that of other closely related Sarcocystidae parasites. From this sequence, conse...
Experimental model for the study by chemiluminescence of the activation of isolated equine leucocytes. The activation of human polymorphonuclear leucocytes (the respiratory burst) can be studied by measuring their chemiluminescent response. This technique was adapted to equine leucocytes to investigate the effects of cell number, activator concentration, enhancers of chemiluminescence, pH, temperature and inhibitors. Leucocytes were isolated from citrated blood from healthy horses and chemiluminescence was measured with a Bio-Orbit luminometer sensitive to 900 nm light. The optimal cell density for the maximal chemiluminescent response ranged from 10(6) to 10(7) leucocytes 600 microliters-1. Ch...
A cb-type cytochrome-c oxidase terminates the respiratory chain in Helicobacter pylori. A Helicobacter pylori membrane fraction oxidized yeast and equine cytochrome c, and N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD). When ascorbate was used as reductant, the Vmax and apparent Km values were 612 nmol electron min-1 (mg protein)-1 and 14 microM for yeast, and 419 nmol electron min-1 (mg protein)-1 and 19 microM for equine cytochrome c, respectively. For TMPD oxidation, the Vmax and Km values were 640 nmol electron min-1 (mg protein)-1 and 182 microM, respectively. These oxidase activities showed a high affinity for oxygen. Inhibition of both cytochrome-c and TMPD oxidase activi...
Ionic mechanisms of Ca(2+)-dependent electrolyte transport across equine sweat gland epithelium. 1. The ionic mechanism involved in Ca(2+)-stimulated electrolyte transport in cultured equine sweat gland epithelial cells was studied using the short-circuit current (ISC) technique. 2. Microscopy revealed that the cultured cells grown on Millipore filters formed polarized monolayers with tight junctions. Monolayers exhibited a mean transepithelial resistance of 333.9 +/- 40.4 omega cm2. 3. Ca(2+)-mobilizing agents, A23187 (1 microM) or thapsigargin (0.01-1 microM), stimulated ISC while forskolin exerted little effect on the ISC. 4. Replacement of external Cl- by gluconate significantly reduc...
Influence of glycerol on the structure and stability of ferric horse heart myoglobin: a SAXS and circular dichroism study. The influence of glycerol on the structural properties of Fe(III)-horse heart myoglobin has been investigated by absorbance, CD and SR-SAXS spectroscopy. The results obtained indicate that both the tertiary and the secondary (alpha-helix) conformations of the protein are influenced by glycerol; in particular, an increase of approx. 8% in helical content was observed. Further, analysis of both the acid- and guanidine-induced denaturation transitions points to a glycerol-induced decreased stability of the tertiary structure; conversely, the alpha-helix conformation is found to be stabilized by t...
Looking for residues involved in the muscle acylphosphatase catalytic mechanism and structural stabilization: role of Asn41, Thr42, and Thr46. Asn41, Thr42, and Thr46 are invariant residues in both muscle and erythrocyte acylphosphatases isolated so far. Horse muscle acylphosphatase solution structure suggests their close spatial relationship to Arg23, the main substrate binding site. The catalytic and structural role of such residues, as well as their influence on muscle acylphosphatase stability, was investigated by preparing several gene mutants (Thr42Ala, Thr46Ala, Asn41Ala, Asn41Ser, and Asn41Gln) by oligonucleotide-directed mutagenesis. The mutated genes were cloned and expressed in Escherichia coli, and the mutant enzymes were...
Use of adverse conditions to stimulate a cellular stress response by equine articular chondrocytes. To determine the response of equine articular cartilage cells to heat and calcium stresses. Methods: Analysis of newly synthesized, [35S]methionine-labeled proteins after treatment of isolated primary equine chondrocytes. Methods: Primary cultures of equine articular chondrocytes were incubated at temperatures ranging from 37 to 42 C for heat stress experiments or incubated in the presence or absence of the intracellular calcium pump inhibitor, thapsigargin, for calcium stress experiments. Patterns of new protein synthesis were determined by incubating with [35S]methionine followed by separati...
The effects of oxygenation upon the Cl-dependent K flux pathway in equine red cells. The effects of oxygen tension (PO2) upon the K influx pathways of equine red cells have been studied using 86Rb+ as congener for K. Equilibration of cells in 100% nitrogen led to a low and Cl-independent K flux. Change to an atmosphere of 100% air led to a rapid sixfold increase in K flux. The oxygen-activated flux was entirely Cl dependent and was maintained for up to 3 h. Oxygenation-evoked activation was dependent upon PO2 over the physiological range with little effect up to 70% saturation of haemoglobin with oxygen but significant effects between 70 and 100%. K flux at low PO2 was unaffec...
Comparison of polymerase chain reaction and microbiological culture for detection of salmonellae in equine feces and environmental samples. To compare the sensitivity of polymerase chain reaction (PCR) with microbiological culture for detecting salmonellae in equine fecal samples and equine environmental swab specimens. Methods: Samples and specimens were tested by PCR and microbiological culture. Methods: A fecal sample from each of 152 horses admitted consecutively to the clinic for evaluation by the outpatient service, 282 fecal samples from 110 hospitalized horses that had been submitted to the clinical microbiology laboratory, and 313 environmental swab specimens were examined. Methods: Each sample and specimen in the study w...
Rapid analysis of four bilirubins in domestic animal sera using high-performance liquid chromatography. A rapid method was developed to analyze delta-bilirubin (B delta), diconjugated bilirubin (DCB), monoconjugated bilirubin (MCB), and unconjugated bilirubin (Bu) by direct injection of sera using high-performance liquid chromatography (HPLC) with an internal-surface reversed-phase silica support (ISRP) column. Sharp bilirubin peaks were obtained using a simple mobile phase of acetonitrile: 0.5 M Tris-HCl buffer (20:80, v/v, pH 7.2). A variable-wavelength detector set at 450 nm, 0.01 absorbance unit full scale (AUFS), and a recorder set at 4 mm/min were used for detection. Peaks for B delta, DCB...
Nested polymerase chain reaction for detection of Ehrlichia equi genomic DNA in horses and ticks (Ixodes pacificus). A nested polymerase chain reaction for detecting Ehrlichia equi in horses and ticks (Ixodes pacificus) was developed. A major second-round PCR product of 928 bp could be readily visualized in ethidium bromide-stained agarose minigels. An internal probe was used to verify the identity of the amplified product by non-radioactive (digoxigenin-based) Southern blotting; additional confirmation was provided by DNA sequence analysis. A dilution study testing the sensitivity of the PCR indicated that DNA derived from 3 infected neutrophils was sufficient to generate a PCR signal. The specificity of t...
Detection of equine infectious anemia viral RNA in plasma samples from recently infected and long-term inapparent carrier animals by PCR. Control of equine infectious anemia (EIA) is currently based on detection of anti-EIA virus (EIAV) antibodies. However, serologic diagnostic methods may give false-negative results in infected horses that fail to respond adequately or are in the early stages of infection. We developed a reverse transcriptase nested PCR (RT-nPCR) assay for the detection of viral gag gene sequences in plasma from EIAV-infected horses. The ability of RT-nPCR to detect field strains of EIAV was investigated by assaying plasma samples from 71 horses stabled on EIA quarantine ranches. Positive PCR signals were detec...
Detection of quinine and its metabolites in horse urine by gas chromatography-mass spectrometry. After oral administration of quinine sulfate to a thoroughbred mare, seven urine samples were obtained over a 45.5 h period. Using gas chromatography -electron impact ionization and positive-ion chemical ionization mass spectrometry, quinine and five putative metabolites were detected and tentatively identified in enzyme-hydrolysed post-administration urine; all metabolites involved some form of oxidation. The parent drug could be detected for about 16 h and some phase I biotransformation products for up to 40 h post-administration.
Detection of bacteria in equine synovial fluid by use of the polymerase chain reaction. Equine synovial fluid aliquots were inoculated with Salmonella enteritidis, Escherichia coli, Actinobacillus equuli, Staphylococcus aureus, and Streptococcus zooepidemicus to obtain approximate concentrations of 1000, 100, 10, and 1 colony forming U/mL. Synovial fluid aliquots were also inoculated with an unquantitated inoculum of Bacteroides fragilis and Clostridium perfringens. Inoculated synovial fluid was incubated in trypticase-soy broth or Columbia broth for approximately 12 hours. Then aliquots were removed for DNA extraction and polymerase chain reaction (PCR) analysis for detection of...
Evidence for multiple foci of eastern equine encephalitis virus (Togaviridae:Alphavirus) in central New York State. A regional surveillance system for eastern equine encephalitis (EEE) virus was established in central New York in 1984 after the 2nd human EEE fatality occurred in 1983. Extensive mosquito surveillance activities were coordinated with the rapid laboratory processing of mosquito specimens for EEE virus. Active surveillance for EEE infections in humans and equines also was initiated. Results of long-term surveillance detected the presence of multiple Culiseta breeding swamps. A 6-yr interepizootic period (1984-1989) was followed by 2 yr of equine EEE. In 1990, there were 7 equine cases and a rec...
Evaluation of a one-step test for rapid, in practice detection of rotavirus in farm animals. An immunochromatographic test for the detection of group A rotavirus was evaluated against a reference group A rotavirus ELISA, by using a panel of 161 bovine, porcine and equine faecal samples submitted for routine examination. The sensitivity of the test was 89 per cent and the specificity 99 per cent compared with the ELISA. Its reproducibility was 100 per cent. The simplicity and rapidity of the test procedure make it suitable for use in practice.
In vitro induction of acrosome reactions in stallion spermatozoa by heparin and A23187. The ability of the glycosaminoglycan, heparin, and the calcium ionophore, A23187, to induce acrosome reaction in equine spermatozoa was assessed using semen from 3 warmblood stallions of known high fertility. After collection of semen, the spermatozoa were washed and incubated in vitro with heparin or A23187. Incubation periods were 0, 4, 6 or 8 h with 0, 1, 10 or 100 microg/ml heparin or 0, 10, 30 or 60 min with 0, 0.01, 0.1, 1 or 10 microM A23187, respectively. Acrosome reactions were determined by staining the spermatozoa with naphthol yellow S plus erythrosin B, and sperm viability was ass...
Voltage-dependent calcium currents and cytosolic calcium in equine airway myocytes. 1. The relationship between voltage-dependent calcium channel current (I(Ca)) and cytosolic free calcium concentration ([Ca2+]i) was studied in fura-2 AM-loaded equine tracheal myocytes at 35 degrees C and 1.8 mM Ca2+ using the nystatin patch clamp method. The average cytosolic calcium buffering constant was 77 +/- 3 (n = 14), and the endogenous calcium buffering constant component is likely to be between 15 and 50. 2. I(Ca) did not evoke significant calcium-induced calcium release (CICR) since (i)[Ca2+]i scaled with the integrated I(Ca) over the full voltage range of evoked calcium currents, ...
Determination of phenylbutazone and oxyphenbutazone in plasma and urine samples of horses by high-performance liquid chromatography and gas chromatography-mass spectrometry. A method is described for the qualitative and quantitative determination of phenylbutazone and oxyphenbutazone in horse urine and plasma samples viewing antidoping control. A horse was administered intravenously with 3 g of phenylbutazone. For the qualitative determination, a screening by HPLC was performed after acidic extraction of the urine samples and the confirmation process was realized by GC-MS. Using the proposed method it was possible to detect phenylbutazone and oxyphenbutazone in urine for up to 48 and 120 h, respectively. For the quantitation of these drugs the plasma was deprotein...
Cryopreservation of equine embryos. Principles and procedures for cryopreservation of equine embryos are described. Embryos less than 250 microM in diameter can be cryopreserved successfully if glycerol is used as the cryoprotectant. Cooling is takes place in such a way that most of the water leaves the cells before intracellular ice forms, and glycerol is removed after thawing without undue osmotic swelling of cells. Vitrification procedures also show promise for small embryos. Satisfactory procedures for cryopreserving embryos of more than 250 microM in diameter are not yet available.
Methods for induction of capacitation and the acrosome reaction of stallion spermatozoa. Methodologies to capacitate bovine spermatozoa, induce the acrosome reaction, and fertilize bovine oocytes in vitro have been established. The capability to do the same with stallion spermatozoa, however, is not available. Several different methods have been used to capacitate stallion spermatozoa with variable results. More basic research needs to be done to establish in vitro conditions necessary to capacitate and induce an acrosome reaction in stallion spermatozoa. Although much progress can be expected in this area, it is unlikely that the general practitioner will use these technologies i...
Intracellular calcium concentration in equine spermatozoa attached to oviductal epithelial cells in vitro. Interaction of spermatozoa with oviductal epithelial cells (OEC) in the oviductal isthmus prolongs the life span of spermatozoa. The hypothesis that the interaction of equine spermatozoa with OEC affects their intracellular calcium concentration ([Ca2+]i) was tested in a sperm-OEC coculture model. Changes in [Ca2+]i in spermatozoa loaded with the fluorescent calcium indicator indo-1 acetoxymethylester (AM) were determined for spermatozoa attached to OEC or to Matrigel, as well as for free-swimming spermatozoa incubated without oviductal epithelium. [Ca2+]i was determined before incubation and ...
