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.
Boone CW, Harell GS, Bond HE.Large-scale separation of mixtures of mammalian cells was obtained with the A-1X zonal centrifuge rotor and density gradients consisting of Ficoll dissolved in modified Eagle's MEM suspension-culture medium. The cells remained viable as tested by plating efficiency or by motility observed with time-lapse photography. Rabbit thymocyte and HeLa cell mixtures were separated with 99 and 89 per cent purity, respectively. Mixtures of thymocytes and suspension-cultured, human acute leukemia cells (Roswell Park strain LKID) were separated with 93 and 91% purity, respectively. HeLa cells were isolated ...
Hampil B, Melnick JL.This paper summarizes the results of the second part of co-operative studies undertaken by the WHO International Reference Centre for Enteroviruses and a number of WHO Regional Virus Reference Centres, WHO Virus Collaborating Laboratories and other laboratories in a comprehensive testing programme of enterovirus equine antisera. The studies were designed to appraise the specificity of immune serum prepared in horses against 16 representative prototype enteroviruses (polioviruses 2 and 3, coxsackieviruses A7, B1, B2, B4, B5 and B6 and echoviruses 1, 2, 3, 5, 7, 9, 12 and 17). Tests for neutrali...
Abrahams M.The mechanical behaviour of horse and human tendon, as characterised by the stress-strain curve, has been examined with respect to load-strain cycling and strain rate. It was found that the tendon stress-strain curve for successive cycles was reporducible provided that strain on the specimen did not exceed 2·0–4·0%. If this strain level was exceeded, a permanent deformation occurred. This phenomenon was verified by histological studies on strained tendon which showed that some of the collagen fibres did not return to their original orientation. Variation in the rate of strain was found to ...
Breckenridge WC, Kuksis A.The triglyceride compositions of the milk fats of man, dog, guinea pig, cow, sheep, goat, and horse were compared by gas-liquid chromatography of the intact triglycerides and of the butyl esters of the component fatty acids. The milk fats of man, dog, and guinea pig, which were largely made up of long-chain fatty acids, showed a common pattern with major contributions made by the glycerides with 48-54 acyl carbon atoms. The milk fats of cow, sheep, and goat, which were rich in short-chain acids, showed significant proportions of triglycerides with 28-54 acyl carbon atoms. Horse milk, which con...
Schneider C, Müller FP, Bertschi I.We describe the utilization of the Abbott Vision system in a horse clinic and a comparison with the Compur M 2000 CS (Bayer Diagnostics and Electronics). Discrepancies were found in respect to precision and accuracy of results. Both systems showed good practicability during routine operation but different cost-effectiveness.
Kizaki T, Mizuno Y, Takasawa T, Shiokawa H.Analysis of the quantitative precipitin reaction of acylphosphatase from porcine skeletal muscle with rabbit antiserum indicated the presence of at least two antigenic determinants on the porcine enzyme molecule. Immunological cross-reactivities of acylphosphatases from equine and rabbit skeletal muscles were examined. In double immunodiffusion with the antiserum, the precipitin lines of the porcine and equine enzymes completely fused, while the rabbit enzyme gave no precipitin line. The reaction between the 125I-labeled porcine enzyme and its antibody was inhibited to the same extent by the p...
Burton AG, Clark KC, Borjesson DL, Carrade DD, Burges J, Owens SD.Volume reduction and RBC depletion of equine bone marrow specimens are necessary processing steps for the immediate therapeutic use of bone marrow (BM)-derived mesenchymal stem cells (MSC), and for MSC expansion in culture. Objective: The purpose of the study was to evaluate the ability of the PrepaCyte-CB processing system to reduce volume, deplete RBC, and recover mononuclear cells (MNC) from equine BM specimens. Methods: One hundred and twenty mL of heparinized BM were obtained from each of 90 horses. A CBC was performed on the BM pre- and post-PrepaCyte-CB processing. Volume and RBC reduct...
Csellner H, Walker C, Love DN, Whalley JM.An equine herpesvirus 1 (EHV-1) mutant was constructed by inserting a lacZ expression cassette into the intergenic region upstream of gene 62 (glycoprotein L; gL) and downstream of gene 63 (a homologue of the herpes simplex virus transcriptional activator ICP0). The recombinant lacZ62/63-EHV-1 had similar growth kinetics in cell culture to those of the parental wild type (wt) virus, with indistinguishable cytopathic effects and plaque morphology. Reverse transcriptase PCR confirmed that the lacZ insertion did not interfere with transcription of gL and immunoblot analysis indicated there was no...
Kolmakova MV, Kuskova ZR, Ratner GM, Laptakova LM.Horse serum albumin has been shown to meet the requirements to protein preparations for microanalysis and thus to be suitable for use in kits of reagents for the radioimmunological determination of insulin and myoglobin, for the determination of tick-borne encephalitis virus antigen by the method of the enzyme immunoassay and for the stabilization of proteins in the hemagglutination test and the hemagglutination inhibition test.
Word TA, Larsen RW.Here the molar volume and enthalpy changes associated with the early events in the folding of ferrocytochrome c (Cc) at high pH have been examined using time resolved photoacoustic calorimetry (PAC). The data reveal an overall volume change of 1.3 ± 0.3 mL mol and an enthalpy change of 13 ± 7 kcal mol occurring subsequent to photodissociation of the unfolded CO bound Cc species in <∼20 ns. Two additional kinetic phases are observed that are associated with non-native His binding (ΔH and ΔV of 2 ± 4 kcal mol and -0.5 mL mol, τ ∼ 2.5 μs ) and Met binding (ΔH an...
Marsan C, Goff AK, Sirois J, Betteridge KJ.Horse conceptuses were recovered non-surgically at Day 12-Day 15 and were dissociated with collagenase. Separation of the cells on a 31.8% Percoll gradient gave two bands of cells and indirect evidence suggests that the low density cells (LDC) are endoderm and the higher density cells (HDC) are trophectoderm. Each band was incubated for 24 h in Minimum Essential Medium and concentrations of oestradiol and progesterone in the medium were measured by radioimmunoassay (RIA). The LDC secreted predominately progesterone (log oestradiol/progesterone = -0.994 +/- 0.141; N = 15) whereas the HDC secret...
Worthington RW, Mülders MS.Antibodies against epsilon toxin were isolated from hyperimmune horse serum by affinity chromatography. Purified epsilon prototoxin covalently bound to Affigel 202 was used as immunosorbent, and antibodies were eluted with 6.0 M guanidine chloride. In a single run 80 mg of antibody could be recovered from a 20 microliter column of immunosorbent. The antibody was shown to belong to the IgG(T) class of immunoglobulins.
Duvivier DH, Chiap P, Crommen J, Lekeux P.The aim of this study was to determine the effect of breathing pattern, air humidity and position of the device on the delivery of an aerosol generated by a dry powder inhalation (DPI) device (Inhalator M). The in vitro inhalation study was performed using the cascade impaction method (Andersen Sampler) adapted to imitate nasal breathing. The amount of ipratropium found in the device, the artificial upper airways and the six stages of the Andersen Sampler was measured using high precision liquid chromatography. Stage 1 of the Andersen Sampler was considered to be the respirable fraction and st...
Fidani M, Casagni E, Montana M, Pasello E, Pecoraro C, Gambaro V.Bacteria frequently found in equine urine samples may cause degradation of 17beta-OH steroids. A simple liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed to evaluate the microbiological contamination of equine urine as a marker of poor storage conditions. Norethandrolone was used as the internal standard, and the linearity, sensitivity, precision and accuracy of the method were evaluated. 17beta-OH oxidation was demonstrated for testosterone, nandrolone, trenbolone and boldenone, but did not occur in alpha-epimers such as alpha-boldenone and epitestosterone, d...
Wilson SM, Pediani JD, Ko WH, Bovell DL, Kitson S, Montgomery I, Brown UM, Smith GL, Elder HY, Jenkinson DM.When sweat glands isolated from samples of horse skin were explanted and cultured under favourable conditions, they could exhibit cellular outgrowth. This growth could be maintained for 2-4 weeks and these primary cultures were then disaggregated and the resultant cell suspensions used to initiate epithelial cell lines. Secretion from intact equine sweat glands is regulated by beta 2-adrenoceptors and appears to be mediated by cyclic AMP, but there is evidence that calcium may also play a role. Adrenaline could increase the cyclic AMP content of the cultured cells and this response was mediate...
Weston MC, Collins ME, Cunningham FM.To assess the biological effects of purified recombinant equine CCL11 on equine eosinophil function. Methods: Following stimulation of eosinophils from normal horses, the polymerised form of actin was measured by flow cytometry using fluorescently labelled phalloidin. Migration was determined in a 96 well plate chemotaxis assay using 8 microm pore membranes, and adherence of eosinophils to serum-coated plastic was assessed using a colorimetric assay for eosinophil peroxidase. Superoxide generation was measured by the reduction of cytochrome C in a colorimetric assay. Results: Equine CCL11 indu...
Houghton E, Ginn A, Teale P, Dumasia MC, Copsey J.The stereochemistry of an isomer of 5-estrane-3,17 alpha-diol, the major metabolite of 19-nortestosterone in horse urine has been established by the use of methylene unit (MU) values. The empirical MU values of the bis-trimethylsilyl (TMS) derivatives of the eight available isomers of 5-androstane-3,17-diol and four isomers of 5-estrane-3,17 beta-diol were determined by capillary gas chromatography using three different columns. From this data the theoretical MU values for the bis-TMS derivatives of the four 5-estrane-3,17 alpha-diol isomers were predicted. Comparison of the experimentally det...
BMC research notesDecember 16, 2009
Volume 2 255 doi: 10.1186/1756-0500-2-255
Rendo F, Iriondo M, Manzano C, Estonba A.The Cantabrian Coast horse breeds of the Iberian Peninsula have mainly black or bay colored coats, but alleles responsible for a chestnut coat color run in these breeds and occasionally, chestnut horses are born. Chestnut coat color is caused by two recessive alleles, e and e(a), of the melanocortin-1 receptor gene, whereas the presence of the dominant, wild-type E allele produces black or bay coat horses. Because black or bay colored coats are considered as the purebred phenotype for most of the breeds from this region, it is important to have a fast and reliable method to detect alleles caus...
Knych HK, Stanley SD, McKemie DS, Steinmetz SJ.Romifidine is an alpha-2 adrenergic agonist used for sedation and analgesia in horses. As it is a prohibited substance, its purported use at low doses in performance horses necessitates further study. The primary goal of the study reported here was to describe the serum concentrations and pharmacokinetics of romifidine following low-dose administration immediately prior to exercise, utilizing a highly sensitive liquid chromatography-tandem mass spectrometry assay that is currently employed in many drug testing laboratories. An additional objective was to describe changes in heart rate and rhyt...
Foster NM, Barber TL, Walton TE.Venezuelan equine encephalomyelitis (VEE) TC-84 vaccinal virus, from 10-1. quantities of infected duck embryo fibroblast cell culture fluids, was isolated by combined continuous-flow centrifugation with isopycnic banding in sucrose. Most of the recovered infectivity and hemagglutinating activity were in a single band at a buoyant density (rho) of 1.2. About 90% of the total input protein (450-520 mg) was removed with the effluent, whereas most of the remaining 10% also banded at a rho of 1.2. Infectivity was inactivated with formalin at a final concentration of 0.05% at 37 degrees C for 24 hr....
Rumpler MJ, Sams RA, Colahan P.We describe a validated, rapid, sensitive, and specific UHPLC-MS/MS method to detect and quantify glycopyrrolate in 0.5 mL of horse urine. Further, we investigated the elimination of glycopyrrolate in urine after both intravenous and oral administration of clinically relevant doses to Thoroughbred horses. Quantification was performed by weighted, linear regression analysis using a deuterated analogue of glycopyrrolate as internal standard (IS). The method was characterized by a linear range of 5-2500 pg/mL, a lower limit of quantification of 5 pg/mL and a limit of detection of 1 pg/mL. The int...
Dubin A, Potempa J, Turyna B.Horse blood leucocyte cytosol exhibits a broad inhibitory activity against serine proteinases. The purified inhibitor was exposed to investigated enzymes (trypsin, chymotrypsin, elastases and serine proteinase from S. aureus) for variable time and the products were analyzed by gradient polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The molar ratio I:E, association rate constants k on and inhibition constants Ki for the enzymes and inhibitor were determined. The examined elastases form stable, stoichiometric complexes with the inhibitor (Ki less than 10(-10) M), ...
Rifkind JM.The oxidation of horse hemoglobin by Cu(II) has been followed by the changes in the electron spin resonance spectra of copper. By stopped-flow and freeze-quenching techniques, it is shown that the second-order rate constant for the binding of Cu(II) to hemoglobin is greater than 5 X 10(5) mol-1 s-1 and the apparent first-order rate for the reduction of Cu(II) to Cu(I) is 0.051 s-1. It is also shown that the binding of Cu(II) to hemoglobin is followed by an alteration of the Cu(II) spectrum, decreasing the g values. This process has an apparent rate constant of 17 s-1 and presumably involves a ...
Cota LO, Júnior OS, Oliveira APL, Paz CFR, Maróstica TP, Duarte PC, Leme FOP, Faleiros RR.Systemic inflammatory response syndrome (SIRS) is a common condition in horses with gastrointestinal disorders. If not prevented or controlled, SIRS promotes multiple organ dysfunctions that may culminate in serious disabilities or even death. The objective of this study was to evaluate the effects of Lithothamnion supplementation on systemic inflammatory response and organ function variables in horses undergoing oligofructose overload (OFO) intake. Twelve healthy horses were randomly divided into control and treated groups. The treated group received Lithothamnion (100 mg/kg bw PO BID) for 7 ...
Baxter GM, Moore JN, Tackett RL.The in vitro responses of isolated vascular preparations of digital arteries and veins obtained from healthy anaesthetised horses were determined for dopamine and fenoldopam. The digital vessels were harvested, cut into 4 mm vascular segments, suspended in tissue baths and attached to force-displacement transducers. Dose-response studies between 10(-8) and 10(-4)M concentrations were performed for all drugs. The change in tension of each vascular ring was measured in grams of force. The reactivity between palmar and plantar digital vessels and baseline vascular responses were determined for do...
Gallicano KD, Park HC, Young LM.A sensitive method was required to analyze low levels of camphor in equine urine and plasma. Camphorated oil (20% w/w camphor) was administered topically (6 g) and intratracheally (1 g) to standardbred mares. The drug was extracted from urine and plasma by diethyl ether and analyzed as its 2,4-dinitrophenylhydrazone derivative by reverse phase HPLC with UV detection. The UV detector was set at 368.5 nm and the samples were eluted from the C18 column by 82% acetonitrile in water. The detection limit achieved was about 10 ng/mL urine and about 20 ng/mL plasma. After topical administration, only ...
Scarth JP, Clarke AD, Teale P, Pearce CM.Effective detection of the abuse of androgenic-anabolic steroids in human and animal sports often requires knowledge of the drug's metabolism in order to target appropriate urinary metabolites. 'Designer' steroids are problematic since it is difficult to obtain ethical approval for in vivo metabolism studies due to a lack of a toxicological profile. In this study, the in vitro metabolism of estra-4,9-diene-3,17-dione is reported for the first time. This is also the first study comparing the metabolism of a designer steroid in the three major species subject to sport's doping control; namely th...
Thway TM, Wolfe MW.Equids and primates are the only species known to express the placental hormone chorionic gonadotropin (CG). CG is a member of the heterodimeric glycoprotein family and is composed of an alpha subunit linked to a hormone-specific beta subunit. Previously, we have reported that epidermal growth factor (EGF) regulates the equine glycoprotein hormone alpha subunit promoter through a protein kinase C (PKC)/mitogen-activated protein kinase (MAPK) signal transduction pathway in trophoblasts. The current study investigates the regulatory element/factors involved in the induction of equine glycoprotei...
Bewley TA, Li CH.The conformations of human, equine, and porcine pituitary prolactins, as evidenced by various optical properties, have been compared. The alpha-helix contents of all three proteins are essentially identical to each other (60 +/- 5%), as well as to prolactins isolated from other mammalian species. Direct absorption (zero and second-order), difference absorption, fluorescence emission, and circular dichroism spectra suggest that the majority of tyrosine and tryptophan side chains in these three proteins exist in very similar microenvironments within the folded forms of the hormones. Thus, the ge...
Weidolf LO, Chichila TM, Henion JD.Methods for screening by thin-layer chromatography, quantification by high-performance liquid chromatography with ultraviolet detection and confirmation by gas chromatography-mass spectrometry of boldenone sulfate in equine urine after administration of boldenone undecylenate (Equipoise) are presented. Sample work-up was done with C18 liquid-solid extraction followed by solvolytic cleavage of the sulfate ester. Confirmatory evidence of boldenone sulfate in equine urine was obtained from 2 h to 42 days following a therapeutic intramuscular dose of Equipoise. The use of 19-nortestosterone sulfat...
Pellegrini A, Hägeli G, Fretz D, von Fellenberg R.An electrophoretic procedure for the qualitative and quantitative assay of protein protease inhibitors is reported. This assay is particularly suited for investigations of crude biological materials when specific antisera are not available. The supporting medium consists of agarose into which denatured fibrinogen is incorporated as the substrate for proteases. The processing then is divided into two steps: (1) electrophoretic resolution of the inhibitor containing material and (2) detection of the inhibitor bands through their protease inhibiting activity. The inhibitor position is thus made v...
Croft MG, Fraser GC, Gaul WN.A Laboratory Information Management System (LIMS) was used to manage the laboratory data and support planning and field activities as part of the response to the equine influenza outbreak in Australia in 2007. The database structure of the LIMS and the system configurations that were made to best handle the laboratory implications of the disease response are discussed. The operational aspects of the LIMS and the related procedures used at the laboratory to process the increased sample throughput are reviewed, as is the interaction of the LIMS with other corporate systems used in the management...
Carpenter MA, Broad TE.Transferrin, the iron transport protein of the blood, is highly polymorphic in many species, including the horse. A number of sequence polymorphisms that distinguish several of the variants of horse transferrin are reported here. Previous studies indicated that exons 12 and 15 were likely to be polymorphic. Sequencing regions of exons 12 and 15 from D and R variants revealed 10 nucleotide substitutions that encoded six amino acid replacements. The F1, F2, H2, and * variants were identical to D, and the O variant was almost identical to R, in the regions studied. The data indicated that the hor...
Caffrey CR, Ryan MF.An excretory-secretory (ES) preparation derived from adult Strongylus vulgaris in vitro was assessed for proteolytic activity using azocasein and synthetic, fluorogenic, peptide substrates. Fractionation was by molecular sieve fast protein liquid chromatography (molecular sieve FPLC) and resolution by gelatin-substrate sodium dodecyl sulphate-polyacrylamide gel electrophoresis (gelatin-substrate SDS-PAGE). The cysteine proteinase activator, dithiothreitol (DTT), enhanced azocaseinolysis and hydrolysis of carbobenzoxy-phenylalanyl-arginine-7-amido-4-methylcoumarin (Z-Phe-Arg-NMec) by the ES pre...
