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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.
Comparison of Salmonella enterica serovar Abortusequi isolates of equine origin by pulsed-field gel electrophoresis and fluorescent amplified-fragment length polymorphism fingerprinting. Equine paratyphoid is caused by Salmonella enterica serovar Abortusequi, and manifests mainly as abortion in the mare. We compared S. Abortusequi strains isolated in Japan and other countries using pulsed-field gel electrophoresis (PFGE) and fluorescent amplified-fragment length polymorphism (FAFLP) analysis. PFGE analysis of S. Abortusequi strains gave 21-27 fragments ranging in size from 33 to 602kb. Although two PFGE profiles were observed among the 20 S. Abortusequi isolates in Japan, the restriction fragments originating from the chromosome were common between the two profiles. The simila...
Matrix-assisted laser-desorption time-of flight ionisation and high-performance liquid chromatography-electrospray ionisation mass spectral analyses of two glycosylated recombinant epoetins. Mass spectrometric analyses of the recombinant proteins in Eprex and Aranesp were undertaken with the goal of producing reference mass spectra and evaluating strategies to improve its applicability as a method for equine and canine doping control of these substances. A simple, low chemical noise deglycosylation reaction removed microheterogeneity due to post-translational carbohydrate attachment and both proteins were detectable using MALDI-TOF-MS. Deglycosylated human erythropoietin (hEPO) was also detected using HPLC-ESI-MS. This is the first time that spectra of deglycosylated Eprex and Ara...
Potentiation of the extracellular release of equine neutrophil elastase and alpha-1-proteinase inhibitor by a combination of two bacterial cell wall components: fMLP and LPS. Lipopolysaccharide (LPS) and N-formyl-methionyl-leucyl-phenylalanine (fMLP)-like peptides are Gram-negative bacterial cell wall components which, when released into the peripheral circulation in endotoxaemia, have the potential to activate leucocytes. In vitro, equine neutrophils require priming with LPS in order to generate reactive oxygen intermediates (ROI) in response to fMLP. Objective: The aim of this study was to examine whether the release of other neutrophil products is similarly dependent on prior priming with LPS. In particular, neutrophil elastase (NE), a potent proteolytic enzyme,...
Clostridium difficile infections in animals with special reference to the horse. A review. In human medicine, Clostridium (C.) difficile is since many years a well-known cause of nosocomial diarrhea induced by antibiotic treatment. In horses, C. difficile was recently suggested as a possible enteric pathogen. The bacterium is associated with acute colitis in mature horses following treatment with antibiotics. C. difficile, and/or its cytotoxin, is also associated with acute colitis in mares when their foals are being treated with erythromycin and rifampicin for Rhodococcus equi pneumonia. The colitis can have resulted from an accidental ingestion of erythromycin by the mares. In an ...
Temporal effects of freezing on plasma nitric oxide concentrations in ponies. The purpose of this study was to compare concentrations of nitric oxide (NO) in fresh plasma versus frozen plasma, and determine the temporal effects of freezing on jugular venous plasma NO concentrations in clinically healthy ponies. Twenty-eight helminth-naive ponies, aged from 4 to 6 mo, were raised and maintained under parasite-free conditions. Blood was collected from the jugular vein, centrifuged, and the plasma supernatant was analyzed fresh for NO concentrations using a chemiluminescent method. The remaining samples were aliquoted into 12 samples and stored at -70 degrees C until they ...
Pregnancies attained after collection and transfer of oocytes from ovaries of five euthanatized mares. After euthanasia, ovaries were removed from 5 horses and shipped to a laboratory where 46 oocytes were collected. The oocytes were cultured for 24 to 30 hours, and 36 oocytes were transferred to 10 recipient mares via flank laparotomies. Recipient mares were inseminated with semen from various stallions. Sixteen days after transfer, 4 of the recipients were pregnant with at least 1 embryonic vesicle. Embryonic death occurred in 3 recipients, whereas a healthy live foal was born from 1 recipient. Ovaries from valuable mares can be a source of viable oocytes after death of the mare. For shipping...
Effect of ovary storage and oocyte transport method on maturation rate of horse oocytes. Two experiments were conducted to determine the effects of storage on equine ovaries or isolated oocytes. Ovaries were collected at an abattoir and were maintained at room temperature during collection and transport (3-9h total). After arrival at the laboratory, ovaries were divided into three groups: immediate oocyte collection (control), storage at room temperature overnight (15-18 h) before oocyte collection, or storage at 4 degrees C overnight before oocyte collection. Collected oocytes were cultured in maturation medium for 24h. There was a significant increase in the proportion of oocyte...
INSL3 ligand-receptor system in the equine testis. We employed molecular and immunological techniques to investigate the expression of INSL3, a member of the insulin-like superfamily, in prepubertal testis, postpubertal testes exhibiting normal and disturbed spermatogenesis, and cryptorchid testes of male horses. In addition, the partial cDNA coding sequences of the equine homologue of the human relaxin/INSL3-receptor Lgr8 were determined. Nonradioactive in-situ hybridization with a cRNA probe for equine Insl3 and immunohistochemistry with a specific rabbit INSL3 antiserum localized Insl3 transcripts and immunoreactive INSL3 ligand to Leydig c...
A simple and highly sensitive spectrophotometric method for the determination of cyanide in equine blood. An epidemiological association among black cherry trees (Prunus serotina), eastern tent caterpillars (Malacosoma americana), and the spring 2001 episode of mare reproductive loss syndrome in central Kentucky focused attention on the potential role of environmental cyanogens in the causes of this syndrome. To evaluate the role of cyanide (CN (-)) in this syndrome, a simple, rapid, and highly sensitive method for determination of low parts per billion concentrations of CN (-) in equine blood and other biological fluids was developed. The analytical method is an adaptation of methods commonly in ...
Muscarinic receptors in equine airways. The distribution of muscarinic receptors in equine airways was investigated using autoradiography. Frozen sections of tissue from six different levels in the bronchial tree, from the trachea to the distal bronchioles, were incubated in vitro with 1.5 nmol/L of the muscarinic receptor antagonist 1-[N-methyl-3H]scopolamine methyl chloride (3H-NMS). In addition, the subtype pattern of muscarinic receptors was investigated in equine tracheal smooth muscle using radioligand binding with methoctramine, tripinamidc, 4-DAMP-methiodide and pirenzipine as competitors against the binding of 1.3 nmol/L 3H...
Surfactant proteins in bronchoalveolar lavage fluid of horses: assay technique and changes following road transport. An enzyme-linked immunosorbent assay (ELISA) was developed for equine surfactant proteins SP-A and SP-D in bronchoalveolar lavage fluid (BALF). Anti-equine SP-A or SP-D monoclonal antibodies (mAb) were produced by hybridoma technology, purified by the antibody purification reagent, and analysed by Western blotting analysis. The immunoreaction (two-site sandwich ELISA) with a mAb, peroxidase-labelled mAb and BALF sample was carried out simultaneously and analytical recovery and precision were assayed. Six mAb for SP-A and four mAb for SP-D were successfully cloned in limiting dilution to monocl...
The development of a competitive PCR-ELISA for the detection of equine herpesvirus-1. Equine herpesvirus-1 (EHV-1) infection is of significant animal welfare and economic importance. Yet, no standardised molecular techniques are available for diagnosis or confirmation of viral infection. The purpose of this study was to develop a standardised and quantitative assay system for the reliable detection of EHV-1 infection which was capable of eliminating the likelihood of false negative results. A region within the EHV-1 glycoprotein B gene was amplified by polymerase chain reaction (PCR), cloned and subjected to site-directed mutagenesis to generate a control plasmid, amplifiable b...
The use of laser microdissection for the preparation of chromosome-specific painting probes in farm animals. Laser microbeam microdissection and laser pressure catapulting procedure were used for the construction of chromosome-specific painting probes, arm-specific probes and probes for chromosomal subfragments. We report on a method for generation of fluorescence in-situ hybridization probes from laser dissected chromosomes of farm animals. So far, using the described method, a set of chromosome-specific painting probes has been obtained for all porcine chromosomes, 17 chromosomes of cattle and selected equine chromosomes. It is concluded that the laser technology appears to be a useful and powerful...
Plasma and synovial fluid endothelin-1 and nitric oxide concentrations in horses with and without joint disease. To compare plasma and synovial fluid endothelin-1 (ET-1) and nitric oxide (NO) concentrations in clinically normal horses and horses with joint disease. Methods: 36 horses with joint disease, and 15 horses without joint disease. Methods: Horses with joint disease were assigned to 1 of the 3 groups (ie, synovitis, degenerative joint disease [DJD], or joint sepsis groups) on the basis of findings on clinical and radiographic examination and synovial fluid analysis. Endothelin-1 and NO concentrations were measured in plasma from blood samples, collected from the jugular vein and ipsilateral cepha...
Myoplasmic calcium regulation in myotubes from horses with recurrent exertional rhabdomyolysis. To determine whether alterations in myoplasmic calcium regulation can be identified in muscle cell cultures (myotubes) and intact muscle fiber bundles derived from Thoroughbreds affected with recurrent exertional rhabdomyolysis (RER). Methods: 6 related Thoroughbreds with RER and 8 clinically normal (control) Thoroughbred or crossbred horses. Methods: Myotube cell cultures were grown from satellite cells obtained from muscle biopsy specimens of RER-affected and control horses. Fura-2 fluorescence was used to measure resting myoplasmic calcium concentration as well as caffeine- and 4-chloro-m-c...
Excessive degradation of type II collagen in articular cartilage in equine osteochondrosis. Articular osteochondrosis (OCD) occurs in both man and animals. The etiology remains to be determined. Studies of OCD lesions in animals may provide clues as to its pathogenesis. The aim of our study was to determine whether there was evidence for increased degradation namely proteoglycan (PG) release and type II collagen cleavage in articular cartilage harvested from OCD lesions. We examined ex vivo explants at post-mortem from equine OCD lesions and macroscopically normal site and age matched cartilage. These were cultured over a 10 day period in serum-free medium. Type II collagen cleavage ...
Mapping of equine potassium chloride co-transporter (SLC12A4) and amino acid transporter (SLC7A10) and preliminary studies on associations between SNPs from SLC12A4, SLC7A10 and SLC7A9 and osmotic fragility of erythrocytes. Consensus DNA sequences from human, mouse and/or rat were used to design oligonucleotide primers for equine homologues of exons 16, 17 and 20-23 of potassium chloride co-transporter (SLC12A4) and exons 10, 11 and 3, 4, respectively, for two amino acid transporters (SLC7A10 and SLC7A9). DNA sequences of the PCR products showed high sequence identity to these regions. Equine BAC clones were obtained for SLC12A4 and SLC7A10 and mapped to equine chromosomes ECA3p13 and ECA10p15, respectively, by fluorescence in situ hybridization (FISH). Several single nucleotide polymorphisms (SNP) were found. Su...
Molecular characterization of the equine testis-specific protein 1 (TPX1) and acidic epididymal glycoprotein 2 (AEG2) genes encoding members of the cysteine-rich secretory protein (CRISP) family. The cysteine-rich secretory protein (CRISP) family consists of three members called acidic epididymal glycoprotein 1 (AEG1), AEG2, and testis-specific protein 1 (TPX1), which share 16 conserved cysteine residues at their C-termini. The CRISP proteins are primarily expressed in different sections of the male genital tract and are thought to mediate cell-cell interactions of male germ cells with other cells during sperm maturation or during fertilization. Therefore, their genes are of interest as candidate genes for inherited male fertility dysfunctions and as putative quantitative trait loci fo...
Effect of (auto)phosphorylation on the kinetic behavior of the Ca2+/calmodulin-dependent protein kinase II from horse brain. After extraction and purification, the kinetic behavior of the Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II) from horse brain was investigated as a function of ATP and a synthetic substrate, syntide-2. Both phospho- and dephospho- forms of the enzyme obey a bi-bi random mechanism. The K(M)s for ATP (K(M,ATP)) and syntide-2 (K(M,syntide-2)) were determined as equal to 80 and 30 microM, respectively. However, the maximum reaction yield is decreased by 50% when the enzyme is (auto)phosphorylated. In addition, this phosphorylated form of the enzyme leads to the formation of a total...
Development and validation of a specific radioimmunoassay for equine osteocalcin. This study describes for the first time the development and validation of a sensitive and specific radioimmunoassay (RIA) for equine osteocalcin (OC) quantification using purified equine OC as standard, tracer, and immunogen for antibody formation in rabbits. The assay allowed to measure equine serum OC levels with a sensitivity of 0.2 ng/mL. Immunoreactive serum OC values of clinically normal, different-aged horses ranged from 3.68 to 127.31 ng/mL. Intra- and inter-assay coefficients of variation (CV) were 6.2 and 8.2%, respectively. Serial equine serum sample dilutions were linear. The recov...
Appearance of nitrite reducing activity of cytochrome c upon heat denaturation. The appearance of NO2- reducing activity of cytochrome c (Cyt c) upon heat denaturation was investigated with equine heart Cyt c. Denatured equine heart Cyt c (dCyt c), which was treated at 100 degrees C for 30 min, had NO2- reducing activity in the presence of dithionite and methylviologen in an aqueous solution under anaerobic conditions. In contrast, hemoglobin and myoglobin had no such activity under the same conditions. Using spectroscopic methods, we found that the appearance of this activity in the Cyt c was due to the following intramolecular changes: unfolding of the peptide chain, ex...
Survival of equine herpesvirus-4, feline herpesvirus-1, and feline calicivirus in multidose ophthalmic solutions. To determine survival over time of infectious equine herpesvirus-4, feline herpesvirus-1, and feline calicivirus in three commercially available and commonly used ophthalmic solutions (eyewash, fluorescein, and proparacaine HCl). Methods: Viruses used in this study were originally isolated from eyes of animals referred to the University of Illinois. Equine herpesvirus-4 was propagated in MDBK cells and feline herpesvirus-1 and feline calicivirus in CRFK cells. Methods: After separately inoculating a designated solution with a specific titer of an individual virus, solutions were incubated per ...
Expression of biologically active recombinant equine interferon-gamma by two different baculovirus gene expression systems using insect cells and silkworm larvae. The full-length equine interferon-gamma (eIFN-gamma) cDNA, including the secretion signal peptide coding region, was recloned into baculovirus transfer vector pAcYM1. This vector was co-transfected with Autographa californica nuclear polyhedrosis virus DNA or hybrid nuclear polyhedrosis virus DNA into Spodoptera frugiperda cells. The recombinant viruses, named AcEIFN-gamma and HyEIFN-gamma, were then recovered. Recombinant eIFN-gamma (reIFN-gamma) was accumulated in the culture fluid of the AcEIFN-gamma or HyEIFN-gamma infected Tricoplusia ni -derived cell line, BTI TN 5B1-4, and hemolymph of ...
Serum fluoride concentrations, biochemical and histopathological changes associated with prolonged sevoflurane anaesthesia in horses. The volatile anaesthetic sevoflurane is degraded to fluoride (F-) and a vinyl ether (Compound A), which have the potential to harm kidney and liver. Whether renal and hepatic injuries can occur in horses is unknown. Cardiopulmonary, biochemical and histopathological changes were studied in six healthy thoroughbred horses undergoing 18 h of low-flow sevoflurane anaesthesia. Serum F- concentrations were measured and clinical laboratory tests performed to assess hepatic and renal function before and during anaesthesia. Necropsy specimens of kidney and liver were harvested for microscopic examinat...
Comparative study of tyrosine radicals in hemoglobin and myoglobins treated with hydrogen peroxide. The reactions of hydrogen peroxide with human methemoglobin, sperm whale metmyoglobin, and horse heart metmyoglobin were studied by electron paramagnetic resonance (EPR) spectroscopy at 10 K and room temperature. The singlet EPR signal, one of the three signals seen in these systems at 10 K, is characterized by a poorly resolved, but still detectable, hyperfine structure that can be used to assign it to a tyrosyl radical. The singlet is detectable as a quintet at room temperature in methemoglobin with identical spectral features to those of the well characterized tyrosyl radical in photosystem...
Quantification and distribution of equine oocyte cortical granules during meiotic maturation and after activation. In vitro fertilization (IVF) is being routinely used in humans and several domestic species, however, limited success has been achieved in the horse. Although immature equine oocytes are capable of completing meiosis in vitro, subsequent fertilization, and embryonic development of those oocytes are questionable. The lack of development of these oocytes could be attributed to an impaired cytoplasmic maturation. In the horse, the study of oocyte cytoplasmic maturation and post-fertilization development has been hindered by the lack of progress in IVF. In mammalian oocytes, migration of cortical ...
Myosin heavy chain-based fibre types in red cell hyper- and normovolaemic Standardbred trotters. An assumed link between red cell hypervolaemia, an excessive amount of training and impaired performance of hypervolaemic horses has led to a theory that the muscle fibres could be affected. Myosin heavy chain (MHC)-based fibre type composition in gluteus medius muscle of red blood cell normo- (NV) and hypervolaemic (HV) Standardbred trotters was evaluated using immunohistochemistry. Muscle biopsies were obtained from 13 NV and 16 HV horses. Serial transverse sections were cut and reacted with antibodies against different isoforms of the myosin heavy chains MHCI, MHCIIA and MHCIIX. Sections we...
Comparison between glycerol and ethylene glycol for the cryopreservation of equine spermatozoa: semen quality assessment with standard analyses and with the hypoosmotic swelling test. The aims of this study were to compare glycerol (G) at customary concentrations and ethylene glycol (EG) as cryoprotectants for stallion semen in a skimmed milk (SM) extender, to test different EG concentrations and to compare the results of manual and computerized analysis with the hypoosmotic swelling (HOS) test. Ejaculates from two stallions were collected over 3 weeks (6 ejaculates per stallion), diluted in a SM based extender, divided into 4 fractions, centrifuged and diluted again to a concentration of 100 x 10(6) mL(-1) progressive motile spermatozoa (PMS) in addition with the cryoprote...
