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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.
Theoretical relationship between the post-administration time and plasma or urinary concentration of a metabolite and the unchanged drug. Administration of caffeine to horses. In a doping test for racing horses, it is useful for the elucidation of the illegal use of drugs if one can estimate the time at which the detected drug was administered. In order to estimate the time which has elapsed after the administration of caffeine (CA) into horses, the ratios of concentration for the respective metabolites to the unchanged CA in the plasma or the urine were determined. These ratios have been known to be independent of the dose of CA. The relationship between [plasma or urinary concentration of a metabolite]/ [plasma or urinary concentration of the unchanged drug] and t...
Excitatory prejunctional beta 2-adrenoceptor distribution within equine airway cholinergic nerves. We examined the effect of activation of beta 2-adrenoceptor (AR) by isoproterenol (ISO) on acetylcholine (ACh) release evoked by electrical field stimulation (EFS: 20 V, 0.5 Hz, 0.5 msec) from cholinergic nerves in five regions of equine airways. We also tested if the effect of ISO was dependent on epithelium or prostanoids by examining the effect of ISO on ACh release in the presence and absence of epithelium or cyclooxygenase inhibition. Trachealis strips or bronchial rings were preincubated for 60 min with 10(-7) M atropine, 10(-6) M neostigmine, and 10(-5) M guanethidine. The ACh amount wa...
Nucleologenesis and ribonucleic acid synthesis in preimplantation equine embryos. The nucleolus is believed to be the active site of rRNA synthesis in all eukaryotic cells. In preimplantation embryos, the embryonic genome is apparently more or less silent up to a species-specific developmental stage at which a major burst of transcription occurs. Here we report on nucleologenesis and some ultrastructural aspects of the onset of RNA synthesis in equine embryos during in vivo development. The zygotes and embryos up to blastocyst stages were surgically recovered from normally cycling mares. Mares were induced to ovulate by treatment with 3000 IU hCG and inseminated 20 and 34 h...
Equine cytochrome P450 aromatase exhibits an estrogen 2-hydroxylase activity in vitro. Aromatase (estrogen synthetase) is a steroidogenic enzyme complex which catalyzes the conversion of androgens to estrogens (termed aromatization). This enzyme was purified from adult equine testis to homogeneity by five chromatographic steps. The ability of purified and reconstituted equine aromatase to exhibit an estrogen 2-hydroxylase activity was tested and compared to testosterone aromatization. Enzymatic activities were assessed by tritiated water release from labelled estradiol and testosterone. Kinetic analysis of estradiol 2-hydroxylation showed an apparent K(m) of 23 microM and a V(ma...
Accuracy of Accusport for measurement of lactate concentrations in equine blood and plasma. The aim of this study was to investigate correlations between lactate concentrations in equine whole blood and plasma measured with Accusport1 and Yellow Springs Instruments (YSI2) (2300) methods. The effect of packed cell volume (PCV) on the accuracy of Accusport was also investigated. Blood samples were collected from Thoroughbred horses at 5-10 min intervals after a treadmill exercise test. Blood was added to NaEDTA (for PCV measurement) and to 2 tubes containing lithium heparin anticoagulant (for lactate assays). At concentrations greater than 10 mmol/l, Accusport1 greatly underestimated t...
Development of a diagnostic DNA probe to detect Setaria digitata: the causative parasite of cerebrospinal nematodiasis in goats, sheep and horses. Two repetitive sequences (IpSdM and IpSdS) have been cloned and sequenced from the genome of Setaria digitata. When IpSdM (214 bp) and IpSdS (201 bp) were aligned, a high degree of homology (85%) was observed, indicating that they belong to the same family of repeats. IpSdM represents a complete repeating element while IpSdS consists of two partial repeating elements arranged in tandem. The elements are present in about 10 000 copies comprising 2.8% of the S. digitata genome. As a diagnostic probe IpSdM detects as little as 100 pg DNA of both S. digitata and S. labiato-papillosa. It can also d...
Maintenance of equine articular cartilage explants in serum-free and serum-supplemented media, compared with that in a commercial supplemented medium. To evaluate the effects of a commercially defined, serum-free medium additive on equine articular cartilage explants, compared with effects of serum-free and serum-supplemented media. Methods: Articular cartilage from a 3-year-old, mixed breed horse euthanatized for reasons other than musculoskeletal disease or sepsis. Methods: Media were changed every 48 hours, and the glycosaminoglycan (GAG) content was determined in media collected at each time point. Glycosaminoglycan synthesis by explant chondrocytes, and residual GAG content of articular cartilage (as a measure of explant GAG loss) were ...
A 16S rRNA-based PCR assay for detection and identification of granulocytic Ehrlichia species in dogs, horses, and cattle. A PCR-based assay was developed for detecting DNA of granulocytic ehrlichiae in blood samples from dogs, horses, and cattle, Primers were designed from 16S rRNA sequence information to specifically amplify DNA from a newly identified Swedish Ehrlichia species. The 16S rRNA nucleotide sequence of this Swedish species differs in only two and three positions from the sequences of Ehrlichia phagocytophila and Ehrlichia equi, respectively, which were also amplified by this PCR system. For evaluation, PCR results were compared with microscopic examination of stained blood smears for the detection of...
Improved isolation of Clostridium perfringens from foal faeces. There have been several case reports of foal diarrhoea associated with Clostridium perfringens. However, there has been no epidemiological assessment of the strength of the association of these bacteria with foal diarrhoea or of their relative importance. To prepare methods for such a study, the success of different cultural techniques for the isolation of C perfringens was examined with respect to the various physiological states of the bacteria. The germination and growth of C perfringens NCTC 8239 endospores of differing maturity were favoured by different pre-treatments which failed to rec...
Molecular diffusion into horse spleen ferritin: a nitroxide radical spin probe study. Electron paramagnetic resonance spectroscopy and gel permeation chromatography were employed to study the molecular diffusion of a number of small nitroxide spin probes (approximately 7-9 A diameter) into the central cavity of the iron-storage protein ferritin. Charge and polarity of these radicals play a critical role in the diffusion process. The negatively charged radical 4-carboxy-2,2,6,6-tetramethylpiperidine-N-oxyl (4-carboxy-TEMPO) does not penetrate the cavity whereas the positively charged 4-amino-TEMPO and 3-(aminomethyl)-proxyl radical and polar 4-hydroxy-TEMPO radical do. Unlike th...
Quantification of normal head morphometry of stallion spermatozoa. The heads of stallion spermatozoa were analysed by computer automated sperm head morphometry and the morphometric values of the major subpopulations of sperm heads were assessed. The criteria for normal dimensions of stallion sperm heads are proposed based on the analysis of these measurements. Semen samples were collected from 10 fertile and 10 subfertile stallions, processed by a standard method, smeared onto microscope slides and stained using haematoxylin. At least 200 properly digitized sperm heads were analysed from each stallion. The measurements for length, width, area, perimeter and w...
Calcium buffering is required to maintain bone stiffness in saline solution. This work determined whether mineral dissolution due to prolonged testing or storage of bone specimens in normal saline would alter their elastic modulus. In one experiment, small pieces of equine third metacarpal bone were soaked in normal saline supplemented with varying amounts of CaCl2. Changing Ca ion concentrations in the bath were monitored and the equilibrium concentration was determined. In a second experiment, the elastic moduli of twenty 4 x 10 x 100 mm equine third metacarpal beams were determined non-destructively in four-point bending. Half the beams were then soaked for 10 days ...
Measurement of bone specific alkaline phosphatase in the horse: a comparison of two techniques. For many years total alkaline phosphatase (AP) activity in serum has been used to monitor bone metabolism in different species. However, total AP lacks bone specificity because the total activity in serum is made up of several isoenzymes, of which the liver and bone isoforms predominate. The aim of the present study was to evaluate an immunoradiometric assay for measuring bone specific alkaline phosphatase (BAP) in horses. BAP, a specific marker of bone formation, was measured in sera from thoroughbred horses by using a previously characterised wheat germ lectin (WGL) precipitation assay and a...
SDS-PAGE characterization of the proteins in equine seminal plasma. The aims of this project were to document the protein profile of equine seminal plasma and determine the variability between stallions in the relative composition of proteins in the ejaculate. A single ejaculate was obtained from 14 stallions of varying breed and age. The gel fraction was removed by an in-line filter. The semen was centrifuged and the supernatant seminal plasma aspirated without disturbing the sperm pellet. The seminal plasma was recentrifuged and stored in cryovials at -70 degrees C. Samples were thawed, recentrifuged, assayed for protein concentration (BCA protein assay), di...
Positive selection of EqCD8+ precursors increases equine lymphokine-activated killing. Lymphokine activated killing (LAK) is an example of natural cytotoxicity, and as such is a critical means of defense against diseases such as viral infection and neoplasia. Despite this important role, the specific molecular interactions involved in LAK or other forms of natural cytotoxicity are only partially understood. In some species, cells capable of mediating natural cytotoxicity express the CD8 molecule, although no specific role has been demonstrated for CD8 in non-MHC restricted cytotoxicity. In this study the role of the EqCD8 equine homolog of CD8 in LAK cell activity was examined. ...
Further characterization of IgE-binding antigens in horse dander, with particular emphasis on glycoprotein allergens. IgE-binding components in an extract of horse dander were analyzed, especially with regard to the glycoprotein allergens. After SDS-PAGE under reducing conditions and blotting, several of the glycoprotein IgE-binding components, including two distinct bands of 27 and 31 kDa, were detected. Together with several other bands, they were shown to bind to the lectins Sambucus nigra agglutinin (SNA) and Datura stramonium agglutinin (DSA), indicating terminal sialic acid linked alpha 2 --> 6 to galactose, and galactose linked beta 1 --> 4 to N-acetylglucosamine, respectively. Carbohydrate analy...
Echinococcus granulosus in Spain: strain differentiation by SDS-PAGE of somatic and excretory/secretory proteins. A comparison was made, by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), of excretory/secretory (ES)-crude and immunopurified (with the corresponding anti-host serum) hydatid fluids-and somatic (S)-protoscoleces-proteins, from several ovine, equine, swine, bovine and human Echinococcus granulosus Spanish isolates. Likewise, the host influence on parasitic ES protein expression was studied, comparing purified hydatid fluids from ovine and equine cysts obtained from natural hosts and in RNMI mice. Purified hydatid fluids patterns, under reducing conditions, yielded the mos...
Fibronectin mRNA splice variant in articular cartilage lacks bases encoding the V, III-15, and I-10 protein segments. Fibronectin is an extracellular matrix glycoprotein encoded by a single gene. Alternative RNA splicing has been reported at three sites, ED (extra type III domain)-A, ED-B, and the variable or V region. Articular cartilage fibronectin monomers are rarely (ED-A)+, but approximately 25% are (ED-B)+. RNA gel electrophoresis and Northern blot analysis identified two (ED-B)+ and two (ED-B)- fibronectin transcripts in cartilage, each pair differing by approximately 750 bases. This difference results from a previously unreported RNA splicing pattern that eliminates not only the V region but also nucl...
[Antibody titers against Borrelia in horses in serum and in eyes and occurrence of equine recurrent uveitis]. In Germany very little is known about antibody titers against Borrelia burgdorferi in the horse. In the USA there exist some studies on the titer levels and symptoms due to borrelia infections. Beside lameness, fever, polyarthritis, pneumonia and dullness there is a study showing a connection between panuveitis and Borrelia infection in the horse. In human medicine the infection with Borrelia burgdorferi becomes more and more important. Uveitis and other eye diseases due to Borrelia burgdorferi are proved and documented. The goal of this study was to find a connection between antibodies to Bor...
Gene transfer by adenovirus in smooth muscle cells. We report adenovirus-mediated gene transfer into airway smooth muscle cells in cultured cells and organ-cultured tracheal segments. Incubation of cultured rat tracheal myocytes with virus (5 x 10(8) pfu/ml) for 6 h resulted in beta-galactosidase expression in 94.8 +/- 2.5% of cells (n = 4). Following incubation of thin (less than 200 microns diameter) equine trachealis muscle segments with virus in organ culture (5 x 10(8)-5 x 10(10) pfu/ml) the average expression of the Lac Z gene was approximately 19 +/- 10% (n = 9). Expression was markedly improved, however, in segments from neonatal rats (...
Surface plasmon resonance studies of complex formation between cytochrome c and bovine cytochrome c oxidase incorporated into a supported planar lipid bilayer. II. Binding of cytochrome c to oxidase-containing cardiolipin/phosphatidylcholine membranes. Complex formation between horse heart cytochrome c (cyt c) and bovine cytochrome c oxidase (cco) incorporated into a supported planar egg phosphatidylcholine membrane containing varying amounts of cardiolipin (CL) (0-20 mol%) has been studied under low (10 mM) and medium (160 mM) ionic strength conditions by surface plasmon resonance (SPR) spectroscopy. Both specific and nonspecific modes of cyt c binding are observed. The dissociation constant of the specific interaction between cyt c and cco increases from approximately 6.5 microM at low ionic strength to 18 microM at medium ionic strength, ...
Identification of Rhodococcus equi using the polymerase chain reaction. Two regions in the gene coding for 16S rRNA in Rhodococcus equi were selected as species-specific primer sequences for the polymerase chain reaction (PCR). PCR using these primers was tested against 10 strains of R. equi (including the type strain) and gave positive results for all but was negative for all other tested species of Rhodococcus; representatives of the most closely related genera and a number of other bacterial species. This method could therefore be used to identify this species which can infect the lungs or other organs of horses, pigs, humans and other animals.
Comparison of media and sera used for in vitro culture of equine superficial digital flexor tendon explants. To compare the effects of different commercial nutrient media and sera on protein synthesis and maintenance of cellular density in cultures of the equine superficial digital flexor tendon (SDFT). Methods: 8 healthy 2- to 4-year-old horses. Methods: First Dulbecco's modified Eagle's medium, Ham's F12 nutrient mixture, RPMI 1640 medium, minimum essential medium with Earle's salts, minimum essential medium with Hanks' salts, and a Dulbecco's modified Eagle's medium/Ham's F12 nutrient mixture with 10% fetal bovine serum (FBS) were compared. Then FBS, fetal equine serum, and donor horse serum, each...
Structure of equine infectious anemia virus proteinase complexed with an inhibitor. Equine infectious anemia virus (EIAV), the causative agent of infectious anemia in horses, is a member of the lentiviral family. The virus-encoded proteinase (PR) processes viral polyproteins into functional molecules during replication and it also cleaves viral nucleocapsid protein during infection. The X-ray structure of a complex of the 154G mutant of EIAV PR with the inhibitor HBY-793 was solved at 1.8 A resolution and refined to a crystallographic R-factor of 0.136. The molecule is a dimer in which the monomers are related by a crystallographic twofold axis. Although both the enzyme and t...
Immunohistochemical analysis of an equine model of synovitis-induced arthritis. To use lipopolysaccharide (LPS) to create synovitis in the midcarpal joint of ponies, and to assess the morphologic, histochemical, and immunohistochemical effects of synovitis on articular cartilage of the third carpal bone. Methods: 2- to 3-year-old ponies, 6 control (group 1) and 6 treated (group 2). Methods: Synovitis was induced in 1 midcarpal joint of group-2 ponies by intra-articular injections of LPS (0.02 micrograms/kg of body weight), morphine (0.1 mg/kg), and saline solution (group 2a) and a morphine and saline solution alone in the contralateral midcarpal joint (group 2b). Articula...
