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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.
Expression of equine morbillivirus (EMV) matrix and fusion proteins and their evaluation as diagnostic reagents. Full-length cDNA clones coding for the matrix (M) and fusion (F) proteins of equine morbillivirus (EMV) were isolated by RT-PCR, and expressed in Escherichia coli using two different expression systems. Western blot analysis indicated that the M and F proteins, expressed either by itself or as fusion proteins with glutathione S-transferase (GST), were insoluble and degraded after expression. Analysis of the degradation pattern of recombinant M protein suggested that the N-terminus of the matrix protein might be more stable and antigenic than the C-terminal region. Therefore a third system was ...
Separation of equine IgG subclasses (IgGa, IgGb and IgG(T)) using their differential binding characteristics for staphylococcal protein A and streptococcal protein G. Equine IgG possesses four well-defined subisotypes, designated IgGa, IgGb, IgGc and IgG(T) on the basis of their increasing anodal mobility in electrophoresis. However, the preparation of IgGa and IgGb reference proteins has not previously been reported. Certain bacterial cell wall proteins, termed protein A and protein G, have been used for purification of IgG subisotypes from the serum of domestic animals which, combined with other techniques utilising differences in the physico-chemical properties of the proteins, has allowed the purification of Ig isotypes. This paper describes purificatio...
Direct detection of equine herpesvirus DNA in tissues of aborted equine fetuses. Restriction endonuclease analysis of equine herpesviruses 1 (EHV-1) and 4 has been investigated using cultured cells infected with these viruses. The DNA cleavage patterns of these viruses were observed in the intracellular DNA after digestion with Eco RI and electrophoresis. This procedure was applied to the diagnosis of equine herpesvirus infection in aborted equine fetuses. The characteristic Eco RI restriction pattern of EHV-1 DNA was directly detectable in the emulsion of lungs collected from aborted equine fetuses.
The structure of the keratan sulphate chains attached to fibromodulin isolated from articular cartilage. Fibromodulin has been isolated from bovine and equine articular cartilage and the attached keratan sulphate chains subjected to digestion by keratanase II. The oligosaccharides generated have been reduced and subsequently isolated by strong anion-exchange chromatography. Their structures have been determined by high-field 1H-NMR spectroscopy and high-pH anion-exchange chromatography. Both alpha(2-6)- and alpha(2-3)-linked N-acetylneuraminic acid have been found in the capping oligosaccharides, and, fucose which is alpha(1-3)-linked to N-acetylglucosamine has been found as a branch in both repe...
Application of polymerase chain reaction (PCR) for diagnosis of equine herpes virus-1 (EHV-1). Fifty aborted foetus samples were diagnosed for the presence of equine herpes virus-1 (EHV-1) by polymerase chain reaction (PCR) technique. Specific primer pair for amplification of a particular segment of EHV-1 DNA in gc region having 3 Hae III restriction endonuclease sites was used. A 409 base pair segment obtained as PCR amplification product in 9 samples was digested with Hae III to confirm the presence of EHV-1 as the infectious agent in aborted tissues. It was observed that PCR technique was more sensitive, specific and rapid than the conventional virological diagnostic methods.
Studies into aromatase activity associated with fetal allantochorionic and maternal endometrial tissues of equine placenta. Identification of metabolites by gas chromatography mass spectrometry. Maternal endometrial and fetal allantochorionic tissues were separated manually from the placentae of seven healthy thoroughbred and three pony mares, ranging in gestational age from 100 to 318 days. The homogeneity of subcellular fractions prepared from these tissues was assessed initially using the marker enzymes, succinate dehydrogenase, NADPH cytochrome C reductase and lactate dehydrogenase for the mitochondrial, microsomal and cytosolic fractions, respectively. Light microscopy and histochemical analysis demonstrated that the separated fetal allantochorionic membrane, which is made up of ...
Role of carbohydrates in the attachment of equine spermatozoa to uterine tubal (oviductal) epithelial cells in vitro. To test the hypotheses that the attachment of equine spermatozoa to uterine tubal (oviductal) epithelial cells (OEC) in vitro is mediated by glycoproteins, and that proteins with carbohydrate-binding properties are present in the periacrosomal plasma membrane of equine spermatozoa. Methods: 4 reproductively sound stallions, and 1 mare in estrus. Methods: In experiment 1a, fluorescent-labeled spermatozoa were cocultured with monolayers of OEC in the presence of 50 mM glucose, fructose, galactose, mannose, N-acetyl glucosamine, N-acetyl galactosamine, or N-acetyl neuraminic acid, or 10 mg of fet...
Immunohistochemical examination of light-chain expression (lambda/kappa ratio) in canine, feline, equine, bovine and porcine plasma cells. Five species of domestic animals were examined immunohistochemically and the light-chain ratios of the immunoglobulins produced by plasma cells analysed. Forty dogs, 11 cats, 10 horses, 11 cattle and 14 pigs were tested using the sequential indirect immunoperoxidase and immunophosphatase methods. Tissues from the tonsils, spleen and cervical lymph nodes were analysed. It could be seen that the lambda/kappa ratio in dogs, cats, horses and cattle is largely dominated by the lambda chains (lambda/kappa ratio in dogs: 91/9%, in cats; 92/8%; in horses: 96/4%; in cattle: 91/9%). A more or less balan...
Modulation of matrix metalloprotease 13 (collagenase 3) gene expression in equine chondrocytes by interleukin 1 and corticosteroids. To determine whether matrix metalloprotease 13 (MMP-13; collagenase 3) is produced by equine chondrocytes and to investigate modulation of its expression by recombinant human interleukin 1 beta (rhIL-1 beta) and corticosteroids. Methods: Equine chondrocytes in monolayer culture were stimulated with rhIL-1 beta. Total RNA was extracted, purified, and reverse transcribed into DNA. Using appropriate primers, a putative MMP-13 fragment was amplified by polymerase chain reaction, and cloned into a bacterial vector. The resultant fragment was purified and sequenced, then was used to prepare a digoxi...
Purification, crystallization and preliminary crystallographic analysis of mare lactoferrin. Lactoferrin is an iron-binding glycoprotein with a molecular weight of 80 kDa. The protein has two iron binding sites. It has two structural lobes, each housing one Fe(3+) and the synergistic CO(3)(2-) ion. The protein was isolated from the colostrum/milk of mares maintained at National Research Centre on Equines, Hisar, India. The purified samples of the protein were crystallized using a microdialysis method. The protein was dialysed against low ionic strength buffer solution. Several crystal forms were obtained, out of which three were characterized which have cell dimensions as follows. For...
Use of peanut agglutinin to assess the acrosomal status and the zona pellucida-induced acrosome reaction in stallion spermatozoa. Peanut agglutinin (PNA) was used to assess the sperm acrosomal status and the acrosome reaction during gamete interaction in the equine species. PNA exclusively binds to the outer acrosomal membrane of stallion spermatozoa, as was established by transmission electron microscopy. Fluorescein isothiocyanate-PNA (FITC-PNA) labeling was used to monitor sperm acrosomal changes during a prolonged incubation period of 24 hours and during a 2-hours incubation in the presence of 5 microM calcium ionophore A23187. In addition, after a 4-hours preincubation in SP-TALP medium, sperm samples were incubated...
Study of the haemolytic process and receptors of thermostable direct haemolysin from Vibrio parahaemolyticus. The haemolytic action of 125I-labelled thermostable direct haemolysin from Vibrio parahaemolyticus was studied on human and equine erythrocytes. In the first step, the haemolysin bound to the membranes of both erythrocyte species. This binding seemed temperature-independent. Then, for human erythrocytes, haemolysin produced cell disruption, and haemoglobin was released. Following this step, haemolysin was also released in a temperature-dependent manner. In contrast, equine erythrocytes were not disrupted, and no release of haemolysin occurred. The receptors of labelled haemolysin were analysed...
Identification of an alternatively spliced transcript of equine interleukin-1 beta. Using lipopolysaccharide (LPS)-stimulated equine peripheral blood mononuclear cell (PBMC) cDNA as a template, we performed polymerase chain reaction (PCR) amplification with equine interleukin-1 beta (IL-1 beta) specific primers. Electrophoresis of the PCR product on agarose gel revealed an additional smaller fragment that hybridized with an equine IL-1 beta cDNA probe. Sequencing of this fragment demonstrated that it was shorter than normal equine IL-1 beta cDNA by 162 nucleotides, which corresponded to exon 5 of the human and murine IL-1 beta genes. The deletion of 162 nucleotides did not re...
Use of two in vitro methods for the detection of benzimidazole resistance in equine small strongyles (Cyathostoma spp.). Ten stables were included in a study to evaluate two in vitro methods for the detection of anthelmintic resistance in cyathostomes by comparing a faecal egg count reduction test (FECRT) to a larval development assay (LDA) and an egg hatch assay (EHA). The LDA was used in seven stables and EHA in the last three. On the basis of FECR values, resistance to benzimidazoles was detected in eight of the ten small strongyle populations. Resistance to pyrantel pamoate and ivermectin was not detected. The mean concentrations that inhibited hatching in 50% of the eggs (EC50), using thiabendazole (TBZ) in...
Solid-phase extraction and derivatisation methods for beta-blockers in human post mortem whole blood, urine and equine urine. This paper details various rapid and sensitive methods for the extraction and derivatisation of propranolol, metoprolol, sotalol, atenolol, pindolol, timolol, oxprenolol, alprenolol and penbutolol in equine urine and in human post mortem whole blood and urine. Three solid-phase extraction methods are described involving the use of either XtrackT XRDAH515, Bond Elut Certify or Sep-Pak C18 cartridges. Two derivatisation methods are also described involving the formation of cyclised silyl or pentafluoropropionate derivatives with either chloromethyldimethylchlorosilane or pentafluoropropionic anh...
Interaction of GroEL with conformational states of horse cytochrome c. GroEL interacts with proteins in denatured states and promotes their efficient folding. To understand the conformational features required for the substrate, we studied the interactions of GroEL with various derivatives of horse cytochrome c including porphyrin-cytochrome c, apo-cytochrome c, and the three fragments containing the heme group, i.e. fragments 1-65, 1-38 and 11-21. Size-exclusion chromatography was performed, taking advantage of the heme absorption of the fluorescence label. Under low-salt conditions, significant binding to GroEL was observed for porphyrin-cytochrome c, apo-cytoc...
PCR-RFLP analysis of the cytochrome b gene in horse mitochondrial DNA. The mitochondrial DNA sequence of cytochrome b gene in a Thoroughbred horse was determined. By comparing DNA sequences between the Thoroughbred and published sequence data (two horses and one Grevyi zebra), polymerase chain reaction (PCR) primers were designed for amplification of a 590 bp DNA fragment in the cytochrome b gene, and PCR-restriction fragment length polymorphism (RFLP) analysis was studied in 140 horses of six breeds using three restriction enzymes (AciI, BamHI, RsaI). Two morphs were found using each of the three enzymes. By combining three enzymes morphs, the 140 horses examine...
Myosin heavy chain isoforms in adult equine skeletal muscle: an immunohistochemical and electrophoretic study. The aim of this study was to characterize the myosin heavy chain (MyHC) isoforms present in equine skeletal muscle. Methods: Muscle biopsies were removed from the superficial region of the gluteus medius muscle of five mature horses and analyzed by immunohistochemistry (using a battery of monoclonal antibodies specific for rat MyHC isoforms) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Results: Immunohistochemistry allowed subdivision of three different muscle fiber populations containing a single MyHC, one slow and two fast, and two hybrid populations, one containing slow an...
Activation of apical P2U purine receptors permits inhibition of adrenaline-evoked cyclic AMP accumulation in cultured equine sweat gland epithelial cells. Experiments were undertaken using cultured equine sweat gland epithelial cells that express purine receptors belonging to the P2U subclass which allow the selective agonist uridine triphosphate (UTP) to increase the concentration of intracellular free Ca2+ ([Ca2+]i). Experiments using pertussis toxin (Ptx), which inactivates certain guanine-nucleotide-binding proteins (G-proteins), showed that this response consisted of Ptx-sensitive and Ptx-resistant components, and immunochemical analyses of the G-protein alpha subunits present in the cells showed that both Ptx-sensitive (alpha i1-3) and Ptx...
Variability of cardiomyocyte DNA content, ploidy level and nuclear number in mammalian hearts. DNA content, ploidy level, cell size and nuclear number were investigated in 54 mammalian hearts from nine species. DNA content was determined biochemically and ploidy level of cells was studied by the means of Feulgen cytophotometry. Nuclear number was calculated by a new method, while cell size was determined by using ocular micrometry. In most mammals diploid cell nuclei predominate. Higher ploidy levels were found in the human and the pig hearts. The total amount of DNA correlated with the myocardial weight. Eight million heart muscle cell nuclei were found in mice (myocardial weight 160 m...
Phorbol ester stimulation of equine macrophage cultures alters expression of equine infectious anemia virus. Equine infectious anemia virus (EIAV) is a lentivirus that replicates predominantly in mature tissue macrophages. Viral expression is strongly influenced by the state of differentiation of the host cell. While blood monocytes can be infected, viral transcription is limited until the cell differentiates into a mature macrophage. Activation of mature macrophages infected with EIAV might also alter viral expression, presumably through binding of cellular transcription factors to viral nucleic acid sequences within the long terminal repeat (LTR). Using DNA amplification techniques, we compared LTR...
Species markers for equine strongyles detected in intergenic rDNA by PCR-RFLP. Five species of equine strongyle belonging to the subfamily Strongylinae (Strongylus edentatus, S. equinus, S. vulgaris, Oesophagodontus robustus and Triodontophorus serratus) and 11 species belonging to the subfamily Cyathostominae (Poteriostomum imparidentatum, P. ratzii, Cylicocyclus insignis, Cc. leptostomus, Cc. nassatus, Cylicostephanus calicatus, Cs. longibursatus, Cs. goldi, Cyathostomum catinatum, Cy. labiatum and Cy. pateratum) were characterized using a polymerase chain reaction-linked restriction fragment length polymorphism technique (PCR-RFLP). Internal transcribed spacer ribosom...
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...
