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.
Validation of the indirect fluorescent antibody and the complement fixation tests for the diagnosis of Theileria equi. The indirect fluorescent antibody (IFA) test for Theileria equi was evaluated to assess test's suitability for the serological diagnosis of equine piroplasmosis, to provide performance parameters for the purpose of test validation, and to compare it with the complement fixation (CF) test. Using a protocol that included Evan's blue, the specificity of the IFA test was estimated at 99.0% for T. equi by the classical method of analysis, and 96.6% by the Bayesian method. The use of Evan's blue in the test protocol increased test specificity and contributed to an excellent test agreement between tw...
Analysis of exogenous nandrolone metabolite in horse urine by gas chromatography/combustion/carbon isotope ratio mass spectrometry. Nandrolone (17beta-hydroxy-4-estren-3-one, NAD) is an endogenous steroid hormone; thus, the detection of its metabolites is not conclusive of NAD doping in racehorses. NAD doping control in male horses is based on the threshold, namely, the concentration ratio of 5alpha-estran-3beta,17alpha-diol (ETA) to 5(10)-estren-3beta,17alpha-diol (ETE). The ETA/ETE ratio of 1/1 was determined based on statistical data of authentic horses in International Federation of Horseracing Authorities. To individuals with complex metabolic disorders, however, such a threshold might not be applicable. The aim of th...
Pharmacokinetic studies on tobramycin in horses. The objective of the study was to evaluate the pharmacokinetics of tobramycin in plasma and urine in the horse (n = 7) after intravenous administration of a dose of 4 mg/kg b.w. Plasma tobramycin concentrations were assayed microbiologically and by means of HPLC analyses. Pharmacokinetic parameters, calculated on the basis of concentrations determined with the microbiological assay were not statistically different from those obtained when data from HPLC analysis were used, but the microbiological assay was more sensitive in the detection of low plasma and urine values. The values of the total ...
Comparative evaluation of Rose Bengal plate agglutination test, mallein test, and some conventional serological tests for diagnosis of equine glanders. The Rose Bengal plate agglutination test (RBT) was evaluated for the diagnosis of equine glanders, and its diagnostic efficiency was compared with that of mallein and other serological tests, including indirect hemagglutination test (IHAT), complement fixation test (CFT), and modified counter immunoelectrophoresis test (mCIET). Sera from 70 naturally infected culture-positive, 96 potentially exposed cohorts, and 110 healthy equines were tested. All tests but mCIET showed 100% specificity when testing the sera from glanders-negative equines. The calculated sensitivities of RBT, IHAT, CFT, mCIET...
Ejaculate and type of freezing extender affect rates of fertilization of horse oocytes in vitro. In vitro fertilization (IVF) was performed on in vitro-matured equine oocytes in three experiments. Frozen-thawed sperm were prepared using swim-up separation and heparin treatment. In Experiment 1, fertilization was achieved with sperm from only one frozen ejaculate of four obtained from the same stallion. Within this ejaculate, fertilization rates were higher with fresh media, as compared to media held for 6-8 days before use (39.6% versus 7.3%, respectively; P<0.001). The type of bovine serum albumin used affected fertilization rates (4% versus 39.6%; P<0.001). To determine if IVF rates wer...
Screening of anti-human leukocyte monoclonal antibodies for reactivity with equine leukocytes. Three hundred and seventy-nine monoclonal antibodies (mAbs) against various human CD molecules supplied to the HLDA8 animal homologues section (including four isotype controls) were analysed for cross-reactivity with equine leukocytes. First, flow cytometric identification of positively reacting mAbs was performed in one laboratory. Thereafter, a second round of flow cytometric evaluation was performed, involving three laboratories participating in the study. The first test-round indicated 17 mAbs as potentially positive. After the second round of flow cytometric analysis, 14 mAbs remained (di...
Non-HLDA8 animal homologue section anti-leukocyte mAbs tested for reactivity with equine leukocytes. In addition to the 379 monoclonal antibodies (mAbs) tested in the animal homologues section of HLDA8, another 155 mAbs were screened at the Institute for Zoo and Wildlife Research, Berlin for cross-reactivity with equine leukocytes. For this purpose, one colour flow-cytometric analysis was performed as screening test. This additional screening indicated further 16 mAbs as positive with staining homologous to human pattern, 1 mAb with weak (positive) reactivity, 11 mAbs with positive, but likely not valuable staining, 12 mAbs with alternate expression pattern from that expected from human immun...
Enantioselective analysis of ketamine and its metabolites in equine plasma and urine by CE with multiple isomer sulfated beta-CD. CE with multiple isomer sulfated beta-CD as the chiral selector was assessed for the simultaneous analysis of the enantiomers of ketamine and metabolites in extracts of equine plasma and urine. Different lots of the commercial chiral selector provided significant changes in enantiomeric ketamine separability, a fact that can be related to the manufacturing variability. A mixture of two lots was found to provide high-resolution separations and interference-free detection of the enantiomers of ketamine, norketamine, dehydronorketamine, and an incompletely identified hydroxylated metabolite of no...
Hsp90 mediates insulin-like growth factor 1 and interleukin-1beta signaling in an age-dependent manner in equine articular chondrocytes. Many metabolic processes in chondrocytes thought to contribute to age-related changes in the extracellular matrix are influenced by known roles of Hsp90. Age-related decreases in the level of Hsp90 have been documented in numerous cell types and could contribute to cartilage degeneration. The aim of this study was to investigate the roles of age and Hsp90 in insulin-like growth factor 1 (IGF-1) and interleukin-1beta (IL-1beta) signaling in chondrocytes. Methods: Levels of Hsp90 messenger RNA (mRNA) and protein, with respect to age, were determined by quantitative real-time polymerase chain rea...
The efficacy of a single chain recombinant equine luteinizing hormone (reLH) in mares: induction of ovulation, hormone profiles, and inter-ovulatory intervals. The objectives of this study were to determine the efficacy of recombinant equine luteinizing hormone (reLH) in shortening the time to ovulation in cycling mares and to determine the effects of treatment on endogenous hormones and inter-ovulatory intervals. In study 1, mares of light horse breeds (3-20 years) were treated with either a vehicle, various doses of reLH, or human chorionic gonadotropin (hCG). Cycling mares were examined by palpation and ultrasound per rectum daily or every 12h from the time of treatment to ovulation. In studies 2 and 3, jugular blood samples were collected daily o...
A bottom-up approach in estimating the measurement uncertainty and other important considerations for quantitative analyses in drug testing for horses. Quantitative determination, particularly for threshold substances in biological samples, is much more demanding than qualitative identification. A proper assessment of any quantitative determination is the measurement uncertainty (MU) associated with the determined value. The International Standard ISO/IEC 17025, "General requirements for the competence of testing and calibration laboratories", has more prescriptive requirements on the MU than its superseded document, ISO/IEC Guide 25. Under the 2005 or 1999 versions of the new standard, an estimation of the MU is mandatory for all quantitativ...
In vitro-produced equine embryos: production of foals after transfer, assessment by differential staining and effect of medium calcium concentrations during culture. Viability of equine embryos produced by oocyte maturation, intracytoplasmic sperm injection and embryo culture to the blastocyst stage in vitro was evaluated after transfer of embryos to recipient mares. No pregnancies were produced after transfer of five blastocysts that had been cultured in G media. Transfer of 10 blastocysts cultured in modified DMEM/F-12 medium produced five pregnancies and three live foals; the two lost pregnancies developed only trophoblast (based on transrectal ultrasonography). To evaluate the status of the inner cell mass, equine blastocysts produced in vivo and in vi...
Relevance of using a human microarray to study gene expression in heaves-affected horses. Environmental causes of heaves are well described, but the molecular mechanisms of the disease remain unclear. Previous studies have highlighted the implications of variations in gene expression, most using reverse transcription polymerase chain reaction (RT-PCR). This well-known technique limits the number of genes that can be studied in a single assay. Microarray appears to be a valuable tool to by-pass this limitation, but so far there has been no equine-specific microarray available on the market. The present study was performed to determine whether a human microarray could be used to stud...
Morphometric classification of Spanish thoroughbred stallion sperm heads. This work used semen samples collected from 12 stallions and assessed for sperm morphometry by the Sperm Class Analyzer (SCA) computer-assisted system. A discriminant analysis was performed on the morphometric data from that sperm to obtain a classification matrix for sperm head shape. Thereafter, we defined six types of sperm head shape. Classification of sperm head by this method obtained a globally correct assignment of 90.1%. Moreover, significant differences (p<0.05) were found between animals for all the sperm head morphometric parameters assessed.
Comparison of opioid receptor binding in horse, guinea pig, and rat cerebral cortex and cerebellum. To compare the density and binding characteristics of opioid receptor subtypes in horse, rat, and guinea pig cerebral cortex and cerebellum. Methods: Prospective receptor binding study. Methods: Whole brains were obtained from four neurologically normal adult horses during necropsy. Rat and guinea pig brains were obtained commercially. Methods: The cerebellum and cerebral cortex were dissected from each brain, and tissue homogenates prepared. A radioligand binding technique with the highly selective ligands [(3)H]-DAMGO, [(3)H]-U69593, and [(3)H]-DPDPE was used to identify the mu- (mu), kappa-...
Mechanism of translesion synthesis past an equine estrogen-DNA adduct by Y-family DNA polymerases. 4-Hydroxyequilenin (4-OHEN)-dC is a major, potentially mutagenic DNA adduct induced by equine estrogens used for hormone replacement therapy. To study the miscoding property of 4-OHEN-dC and the involvement of Y-family human DNA polymerases (pols) eta, kappa and iota in that process, we incorporated 4-OHEN-dC into oligodeoxynucleotides and used them as templates in primer extension reactions catalyzed by pol eta, kappa and iota. Pol eta inserted dAMP opposite 4-OHEN-dC, accompanied by lesser amounts of dCMP and dTMP incorporation and base deletion. Pol kappa promoted base deletions as well as ...
Effects of syringe type and storage temperature on results of blood gas analysis in arterial blood of horses. Results of arterial blood gas analysis can be biased by pre-analytical factors, such as time to analysis, syringe type, and temperature during storage. However, the acceptable delay between time of collection and analysis for equine arterial blood gas remains unknown. Objective: Dedicated plastic syringes provide better stability of arterial blood gases than multipurpose plastic syringes. Methods: Eight mares, 1 stallion, and 1 gelding, ages 3 to 10 years old. Methods: Arterial blood samples were collected in a glass syringe, a plastic syringe designated for blood gas collection, and a multipu...
Fluorescent in situ hybridization mapping of the epidermal growth factor receptor gene in donkey. The physical localization of the epidermal growth factor receptor (EGFR) gene was performed on donkey chromosomes. Bacterial artificial chromosome DNA containing the equine EGFR gene was used to map this gene by fluorescent in situ hybridization on donkey metaphase chromosomes. The gene was mapped on donkey 1q21.1 region.
The mechanisms determining the nucleolar-organizing regions inactivation of domestic horse chromosomes. Cytogenetic investigations of the nucleolar-organizing regions (NORs) show that there is variation in the transcriptional activity of rDNA in many organisms. As a consequence, genetic polymorphism of these regions has been detected. The aim of the present study was to evaluate the hypothetic genetic mechanisms determining the NORs polymorphism of the domestic horse chromosomes. Molecular cytogenetic analyses were carried out on Hucul horses and the following techniques were used: fluorescence in situ hybridization (FISH), telomere primed in situ synthesis (PRINS), in situ nick-translation with...
Molecular characterization of the equine herpesvirus 1 strains RacL11 and Kentucky D. The pathogenicities of RacL11 and Kentucky D strains of equine herpesvirus 1 in the hamster infection model are different from those of Ab4p and the Japanese isolates. Virus genome restriction fragment length polymorphism analysis and sequence comparison of an intergenic region, glycoproteins and tegument genes showed higher conservation but with some strain-specific differences. These results indicate that point nucleotide differences in RacL11 and Kentucky D might be responsible for their pathogenicity in rodent models.
Evaluation of activation of protein kinase C during agonist-induced constriction of veins isolated from the laminar dermis of horses. To determine the effects of the protein kinase C (PKC) inhibitor, Ro-31-8220, on agonist-induced constriction of laminar arteries and veins obtained from horses. Methods: Laminar arteries and veins obtained from 8 adult mixed-breed horses. Methods: Laminar arteries and veins were isolated and mounted on small vessel myographs for the measurement of isometric tension. Concentration-response curves were then obtained for the vasoconstrictor agonists phenylephrine, 5-hydroxytryptamine, prostaglandin F(2), and endothelin-1. All responses were measured with or without the addition of Ro-31-8220 (3 ...
Metabolic studies of mesterolone in horses. Mesterolone (1alpha-methyl-5alpha-androstan-17beta-ol-3-one) is a synthetic anabolic androgenic steroid (AAS) with reported abuses in human sports. As for other AAS, mesterolone is also a potential doping agent in equine sports. Metabolic studies on mesterolone have been reported for humans, whereas little is known about its metabolic fate in horses. This paper describes the studies of both the in vitro and in vivo metabolism of mesterolone in racehorses with an objective to identify the most appropriate target metabolites for detecting mesterolone administration. In vitro biotransformation st...
Evaluation of Compass as a comparative mapping tool for ESTs using horse radiation hybrid maps. Loci for 9322 equine expressed sequence tags (ESTs) were predicted using the Comparative Mapping by Annotation and Sequence Similarity (Compass) strategy in order to evaluate the programme's ability to make accurate locus predictions in species with comparative gene maps. Using human genome sequence information from Build 35 (May 2004) and published marker information from the radiation hybrid (RH) maps for equine chromosomes (ECA) 17 and X, 162 ESTs were predicted to locations on ECA17 and 328 ESTs to locations on ECAX by selection of the 'top blast hit'. The locations of 30 ESTs were assesse...
The quantitation of procaine in equine plasma by liquid chromatography-linear ion trap mass spectrometry. A method for the extraction and quantitation of procaine in equine plasma was developed for use with liquid chromatography-mass spectrometry (LC-MS). Procaine was isolated from equine plasma by liquid-liquid extraction at pH 11 with dichloromethane using procaine-d10 as an internal standard. Quantitation was achieved by LC-MS using a 3-microm C-18 column coupled to an electrospray ionization source on a linear ion-trap mass spectrometer. The limit of detection and limit of quantitation was determined to be 50 and 200 pg/mL, respectively. The lowest limit of detection determined by previous met...
Glanzmann thrombasthenia in an Oldenbourg filly. An 18-month-old Oldenbourg filly was presented with a bleeding diathesis. Laboratory testing included platelet count, gingival bleeding time, prothrombin time (PT), activated partial thromboplastin time (aPTT), von Willebrand factor (vWf) antigen, clottable fibrinogen, clot retraction time, PFA-100 closure time, platelet aggregometry (on platelet-rich plasma), and thrombelastography (TEG). TEG was performed by using kaolin and tissue factor as coagulation activators. Expression of the platelet receptor for fibrinogen was assessed by flow cytometry by using anti CD41 (alpha(IIb) or glycoprotein...
Clinical evaluation of the CA530-VET hematology analyzer for use in veterinary practice. The CA530-VET is a completely automated impedance cell hematology analyzer, which yields a 16-parameter blood count including a 3-part leukocyte differential. Objective: The aim of this study was to examine the operational potential of the CA530-VET and its value for use in veterinary practice. Methods: The analyzer was tested for blood carry-over, precision, and accuracy. Comparison methods included the CELL-DYN 3500, microhematocrit centrifugation, manual platelet (PLT) counting for feline and equine species, and a 100-cell manual WBC differential. Blood samples for comparison of the methods...
Genes and respiratory disease: a first step on a long journey. This review highlights the critical importance of phenotype definition in the understanding of the pathogenesis of respiratory disease in horses. The general approach to genetic studies is discussed and comparative studies of recurrent airway obstruction (RAO) conditions, such as asthma, described in the context of learning more about equivalent equine conditions. The availability of methods to study genetic tests have previously relied on DNA sequence knowledge from man, laboratory and domesticated animals, but recent data from the horse genome sequence are now available. This should facilita...
Equine lysozyme: the molecular basis of folding, self-assembly and innate amyloid toxicity. Calcium-binding equine lysozyme (EL) combines the structural and folding properties of c-type lysozymes and alpha-lactalbumins, connecting these two most studied subfamilies. The structural insight into its native and partially folded states is particularly illuminating in revealing the general principles of protein folding, amyloid formation and its inhibition. Among lysozymes EL forms one of the most stable molten globules and shows the most uncooperative refolding kinetics. Its partially-folded states serve as precursors for calcium-dependent self-assembly into ring-shaped and linear amyloi...
Quantitative HPLC-UV method for the determination of firocoxib from horse and dog plasma. A sensitive reversed-phase HPLC-UV method was developed for the determination of firocoxib, a novel and highly selective COX-2 inhibitor, in plasma. A 1.0 mL dog or horse plasma sample is mixed with water and passed through a hydrophobic-lipophilic copolymer solid-phase extraction column to isolate firocoxib. Quantitation is based on an external standard curve. The method has a validated limit of quantitation of 25 ng/mL and a limit of detection of 10 ng/mL. The validated upper limit of quantitation was 2500 ng/mL for horses and 10,000 ng/mL for dogs. The average recoveries ranged from 88-93% ...