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.
Levitt NH, Miller HV, Pedersen CE, Eddy GA.The development of a new diagnostic procedure for the identification of Venezvelan, eastern and western equine encephalomyelitis (VEE, EEE, WEE) viruses is described. The procedure utilizes virus precipitation with reference fluorescein-conjugated gamma globulin, followed by cellulose acetate electrophoresis. Clinical specimens containing varying concentrations of virus yielded, in primary duck embryo cell culture, sufficient virus for detection within 22 to 44 hours. Identification of VEE, EEE and WEE virus in specimens was accomplished by microprecipitation within this time. In contrast to c...
Blackmore DJ, Elton D.This paper records the concentrations of aspartate amino transferase (A.A.T.), creatine kinase (C.P.K.), sorbitol dehydrogenase (S.D.H.), alpha-hydroxybuturate dehydrogenase (alpha-H.B.D.) and alkaline phosphatase (A.P.) activity observed in the sera of Thoroughbred horses in the United Kingdom, at rest and during training. The methods of analysis have been selected to achieve the optimum precision when used for horse serum. During training A.A.T., C.P.K. and alpha-H.B.D. are related and demonstrate intermittent periods of increasing activity. S.D.H. remains unchanged but demonstrates increase...
Allen PZ, Dalton EJ.Donkey IgGa was isolated in purified form from normal and immune donkey sera by column chromatography on DEAE-cellulose. Isolated donkey IgGa and mixtures of (IgGa+IgGb) were used as antigens to prepare rabbit reagents specific for equine IgGa or IgGb. Antibodies present in sera obtained from a single donkey at various times during the course of hyperimmunization with BSA were isolated by immuno-adsorption. The class or subclass of immunoglobulins present among isolated, donkey anti-BSA antibodies was determined by use of specific rabbit anti-equine immunoglobulin reagents. The homologues of h...
Wasyl Z.1. Horse liver acid phosphatase was separated into two partially purified fractions differing in molecular weight (enzyme I about 100 00, enzyme II about 25 000). 2. Enzyme I was separated into several subfractions by DEAE-cellulose chromatography and isoelectric focusing. 3. Molecular weight, sedimentation coefficient and effective molecular radii were determined for acid phosphatases I and II by gel filtration and density-gradient centrifugation.
Boorman J, Mellor PS, Penn M, Jennings M.Seven-day-old embryonated hen eggs were infected with African Horse Sickness virus by the yolk sac and intravenous routes. Virus reached a high titre in the blood of infected embryos. Culicoides variipennis midges which took a blood meal from infected eggs became infected with virus, and after 7 days at 26 degrees - 27 degrees C transmitted African Horse Sickness virus to uninfected eggs. C. variipennis may therefore be considered a biological vector of African Horse Sickness virus in the laboratory.
Salminen K.The levels of serum vitamin B were determined on 16 mature partly warm-blooded, partly Finnish rural-race horses by the radioisotopic competitive inhibition assay method. The mean value from three samplings carried out in dupli- or triplicate was 1.54 ± 0.16 ng/ml. The utilization of serum inorganic cobalt for cyanocobalamin synthesis was studied on two geldings, which received a dose of 200 µCi CoGl i.v. A Sephadex G-100 gel filtration was carried out with the serum proteins from serial blood samplings at different time intervals 15 min. to 48 hrs. after administration. The gel filtration s...
Milgrom F, Loza U, Kano K.Double diffusion tests in gel were employed for studies of reactions between infectious mononucleosis sera and extracts of bovine, sheep and equine erythrocyte stromata. The extracts were obtained by ultrasonication of stromata prepared from trypsin-digested erythrocytes. The reaction with bovine stroma extract was composed, in many instances, of two lines. A single line was observed in reactions with sheep and equine stroma extracts. This line merged into a reaction of partial or complete identy with one of the lines formed with bovine stroma extract. Evidence was obtained that some infectioo...
Oeding P, Hájek V, Marsálek E.Out of 70 S. aurew strains isolated from the anterior nares of horses, 48 (69 per cent)
belonged to the E biotype. Approximately one third of these isolates were typed with factor
sera, the 6 (35 per cent) that were typable showing 5 different patterns. All strains but one
were non-typable with the basic sets of phages for typing human and bovine staphylococci
even at RTD x 100. Without any exception the equine staphylococci of the E biotype
contained polysaccharide Aa. Sixteen biochemically different strains belonged to the biotype A, B or C. A number of different serological patterns an...
Main AR, Soucie WG, Buxton IL, Arinc E.A relatively simple method is described by which cholinesterase was purified about 19000-fold starting from horse serum. Typically 20 litres of serum were processed to yield 15-18mg of electrophoretically pure cholinesterase in the form of an active salt-free dry powder. The method included two stages: fractionation with (NH(4))(2)SO(4) and ion-exchange chromatography. The (NH(4))(2)SO(4) stage included, in principle, the acid (pH3) step of the Strelitz (1944) procedure. The step took advantage of the stabilizing effect that 33%-satd. (NH(4))(2)SO(4) has on cholinesterase activity at pH3 and i...
Tangyuenyong S, Nambo Y, Nagaoka K, Tanaka T, Watanabe G.Most thyroid hormone determinations in animals are based on immunoassays adapted from those used to test human samples, which may not reflect the actual values of thyroid hormone in horses because of the presence of binding proteins. The aims of the present study were i) to establish a novel radioimmunoassay (RIA) using a more simple and convenient method to separate binding proteins for the measurement of total thyroxine (T4) in horses and ii) to validate the assay by comparing total T4 concentrations in yearling horses raised in different climates. Blood samples were collected from trained y...
Paik WK, Farooqui J, Gupta A, Smith HT, Millett F.The present observations are the continuation of our earlier study on the physicochemical mechanism of protein-lysine methylation. In this paper the electrophoretic behaviour (pI values) of two chemically modified horse heart cytochromes c at lysine-72 with trifluoromethylphenylcarbamoyl (neutral group) or carboxydinitrophenyl (acidic group) is compared with the enzymatically methylated cytochrome c. The results indicate that although both chemically modified cytochromes c have lower pI values than the unmodified cytochrome c, the enzymatic methylation appears to be much more efficient in lowe...
Whitcomb RW, Schneyer AL, Roser JF, Hughes JP.Using a LH radioligand receptor assay (RRA) previously validated for use in serum and an equine monoclonal RIA, we have distinguished a subset of subfertile stallions with an elevated RRA/RIA ratio. After purification of the active moiety by anion exchange chromatography and immunoprecipitation with the equine LH (eLH) monoclonal antibody, RRA activity remained in the supernatant. This activity was also recognized by a polyclonal LH antibody (GDN 15) with wide cross-species recognition. This active fraction was further purified by gel filtration chromatography and shown to displace labeled eLH...
Wilke K, Weimann M, Jung M, Geldermann H.10 different oligonucleotide probes were evaluated for DNA fingerprinting in horses. Five probes were able to detect polymorphic bands. The probes (GT)(8) , (GTG)(5) and (GGAT)(4) are most informative for individual identification and were used to analyze a population of Hannoveranian horses. The probability that two individuals have the same DNA fingerprint pattern is 1.2 × 10(-8) , 5.2 × 10(-10) and 1.5 × 10(-7) respectively. Using a combination of the three probes, paternity tests were performed with exclusion probabilities between 0.08% and 4%. ZUSAMMENFASSUNG: Oligonukleotide-Sonden fÃ...
Harkins JD, Karpiesiuk W, Lehner A, Woods WE, Dirikolu L, Carter WG, Boyles J, Tobin T.This report evaluates the pharmacological responses, urinary detection and mass spectral confirmation of ropivacaine in horses. Ropivacaine, a potent local anesthetic (LA) recently introduced in human medicine, has an estimated highest no-effect dose (HNED) of about 0.4 mg/site as determined in our abaxial sesamoid block model. Apparent ropivacaine equivalents were detectable by ELISA screening using a mepivacaine ELISA test after administration of clinically effective doses. Mass spectral examination of postadministration urine samples showed no detectable parent ropivacaine, but a compound i...
Jensen K.Examination of nasopharyngeal secretion and organ material from clinical cases of respiratory diseases in horses, using inoculation of embryonated hen eggs and rabbit and horse kidney cell cultures, resulted in the isolation of influenza virus and herpes virus. In 2 cases, both viruses were present in the same specimen. On the basis of the physio-chemical, cytological and serological criteria, the viruses were found to be identical with influenza virus type A equi 2 and herpes virus equi type 1. The methods for serological diagnosis and characterization of the influenza and herpes viruses are ...
Hänni K, Hesford F, Lazary S, Gerber H.Genomic DNA isolated from 20 horses was digested with up to six restriction endonucleases and subjected to southern blot hybridization analysis using various human class II alpha- and beta-chain cDNA probes. A high degree of restriction fragment length polymorphism (RFLP) was found for the DQ alpha, DP beta, DQ beta and DR beta probes, about 20 polymorphic bands being detected for each. DR alpha showed 2-4 polymorphic bands, whereas no evidence for DP alpha-like genes was found. A number of correlations of RFLPs with individual alloantisera were apparent.
Raeside JI, Christie HL.C(18) neutral steroid formation by cytochrome P450 aromatase has been recorded for several equine and porcine tissues. High activity of P450 aromatase is reflected in the quantities of estrogens in yolk-sac (y-s) fluid of early equine conceptuses. In a previous study of y-s fluid we detected large amounts of androgens by radioimmunoassay (RIA), using an antiserum for androstenedione (A(4)). Here, we report that RIA, following chromatography, gave tentative identification of the major peak as norandrostenedione (19-norA) not as A(4). Furthermore, even greater quantities of 19-norA seemed to be ...
Koupai-Abyazani MR, Esaw B, Laviolette B.A high-performance liquid chromatographic method was used for the detection of etodolac in equine serum and urine. The method consisted of a one-step liquid-liquid extraction, separation on a reversed-phase (RP-18) column and detection using an ultraviolet detector. Additional confirmation methods included a HPLC coupled with an atmospheric pressure chemical ionization mass spectrometer (APCI-MS). Free (unbound) etodolac and its conjugates were present in the samples. Concentrations of the drug in the serum and urine samples collected from four standardbred mares after a single oral administra...
Van Heerden J, Dauth J, Dreyer MJ, Nichas E, Marshall C, De Waal DT.Selected haematological, blood chemical and serological variables were investigated in healthy Thoroughbreds (n = 45) in training. Haemoglobin concentration, haematocrit, red, white and differential cell counts as well as serum concentrations of total and ionized calcium, sodium, potassium, chloride, urea, creatinine, total protein, albumin, inorganic phosphorus, total bilirubin, iron, glucose, magnesium, alkaline phosphatase, gamma-glutamyltransferase, lactate dehydrogenase, aspartate transaminase, alanine transaminase and creatine kinase were found to be within ranges previously reported for...
Vaughn DM, Smyth GB.The serotonin metabolite, 5-hydroxyindoleacetic acid (5-HIAA) and the dopamine metabolite, homovanillic acid (HVA) in the cerebrospinal fluid (CSF) of seven clinically normal horses were evaluated with reverse phase high pressure liquid chromatography and electrochemical detection. Comparisons of the neurotransmitter metabolite concentrations were made on CSF collected simultaneously from the atlanto-occipital and lumbosacral regions. There were significantly higher amounts of 5-HIAA and HVA in atlanto-occipital CSF than in lumbosacral CSF. Mean 5-HIAA concentrations in atlanto-occipital and l...
Nelis SA, Sievers C, Jarrett M, Nissen LM, Kirkpatrick CM, Shaw PN.In this paper, a method for the sensitive and reproducible analysis of lignocaine and its four principal metabolites, monoethylxylidide (MEGX), glycylxylidide (GX), 3-hydroxylignocaine (3-HO-LIG), 4-hydroxylignocaine (4-HO-LIG) in equine urine and plasma samples is presented. The method uses liquid chromatography coupled to tandem mass spectrometry operating in electrospray ionisation positive ion mode (+ESI) via multiple reaction monitoring (MRM). Sample preparation involved solid-phase extraction using a mixed-mode phase. The internal standard adopted was lignocaine-d(10). Lignocaine and its...
Benoit E, Jaussaud P, Besse S, Videmann B, Courtot D, Delatour P, Bonnaire Y.A benzhydrolic metabolite of ketoprofen, formed by reduction of the keto group of the drug, has been identified by gas chromatography-mass spectrometry in equine plasma and urine. After partial synthesis, its structure has been confirmed by UV, IR and 1H NMR spectroscopy. The kinetics of ketoprofen and this metabolite have been monitored in plasma by high-performance liquid chromatography. The two products were quantified in plasma up to 4 and 3 h, respectively, and were detected in urine up to 72 and 24 h, respectively, after a single intravenous administration to horses at the dose of 2.2 mg...
Kravtsov AL.Compare the content of bactericidal granules (BG) in blood phagocytes of animals, that differ by species sensitivity to plague infection, under the conditions of measuring, that ensure automatic differentiating by this parameter of monocytes and granulocytes of human blood. Methods: Human whole blood leukocytes were studied, as well as from 7 animal species: mice, guinea pigs, golden hamsters, white rats, rabbits, dogs and horses. Acridine orange (AO) was used for supra-vital staining in primary (bactericidal) granule cells. Relative BG content was measured in separate cells in conventional un...
Petry S, Breuil MF, Duquesne F.Contagious equine metritis (CEM) detection by PCR is recognized by the European Union according to Commission Implementing Regulation (EU) No 846/2014, and real-time PCR is now recommended by the World Organisation for Animal Health Terrestrial Manual at the same level as the culture method. The present study highlights the creation of an efficient network of approved laboratories in France in 2017 for CEM detection by real-time PCR. The network currently consists of 20 laboratories. A first proficiency test (PT) was organized by the national reference laboratory for CEM in 2017 to evaluate th...
George JW, Duncan JR, Mahaffey EA.Pyrimidine 5'nucleotidase (P5N) activities of erythrocytes for Mongolian gerbils, cats, dogs, sheep, horses, and calves were measured, using a radiometric technique with [14C]cytidine monophosphate as the substrate. Erythrocytes of gerbils had the highest activity [1,177.1 +/- 133.6 mU/g of hemoglobin (Hb)]. Feline erythrocytes had 327.4 +/- 204.4 mU/g of Hb. Canine erythrocytes had 148.0 +/- 19.8 mU/g of Hb. Ovine erythrocytes (44.3 +/- 20.9 mU/g of Hb), equine erythrocytes (30.0 +/- 15.9 mU/g of Hb), and bovine erythrocytes (14.1 +/- 6.9) had relatively low P5N activity. The P5N activity was...
Shen Y, Wang WJ, Fu M, Xu GQ, Zhou X, Liu B.To identify the original components of Asini Colla Corii and its raw material hides provides a guarantee for authenticity of Asini Colla Corii. It is urgent for Asini Colla Corii production enterprises and market supervision departments to develop effective identification methods of Asini Colla Corii and hides derived from horses, donkeys, mules and hinnies. This study screened species-specific DNA sequences of nuclear and mitochondrial genomes as detection targets, designed horse and donkey specific primers and established multiple PCR identification methods for identifying the animal hides (...
Kirschvink N, Art T, Lekeux P, Roberts C, Gustin P.8-Epi-PGF2alpha, a prostaglandin-like compound generated by oxidative stress, has been shown to be an in vitro bronchoconstrictor in airways from healthy laboratory animals and healthy humans, but it has never been studied in diseased airways. Here, the bronchoconstrictive capacity of 8-epi-PGF2alpha on isolated bronchial rings (BR) of healthy and heaves-affected horses was evaluated by comparing the maximal effect and the potency of 8-epi-PGF2alpha to those of (1) acetylcholine (ACh), (2) its stereoisomer PGF2alpha and (3) its synthetic receptor agonist, U46619. Furthermore, the potential cap...
Zierz J, Wintzer HJ.To judge acute processes of pain objectively the results are told of a determination of adrenaline and noradrenaline in the plasma of 30 horses suffering from pain. Besides a scheme basing on an awarding of points is developed to ascertain changes of physiological and ethological parameters caused by pain. These results in changes of behaviour are compared to results determined by laboratory experiments. Concerning pain of medium and high level a relation to the concentration of catecholamines is noticed. Therefore the total of certain clinical observations is suitable for graduating acute pai...
Pusterla N, Mapes S, Leutenegger CM.A questionnaire was developed to document the knowledge base of large-animal diplomates of the American College of Veterinary Internal Medicine (ACVIM) regarding polymerase chain reaction (PCR) technology and to identify the common use of this technology in equine practice. Ninety-three of the 278 mailed questionnaires were returned, for an overall response rate of 33.4%. Ninety respondents (99%) reported being familiar with the general principles of nucleic acid probe technology; however, only 52 (57%) knew the difference between conventional (traditional) and real-time (second-generation) PC...
Bridges CG, Edington N.Equine sera were used to immunoprecipitate radiolabelled virus-infected cell proteins; subsequent resolution with polyacrylamide gel electrophoresis identified the EHV-1 polypeptides VP 2, 10a, 11, 13, 14, 15, 16, 20, 21 and 23a. The humoral support of ADCC by these sera was examined in vitro. Cytotoxicity could be demonstrated against both subtypes irrespective of the immunising isolate. The implications of these results are discussed.
Gerdemann R, Deegen E, Kietzmann M, Venner M.In the present study the significance of eicosanoids in the development of shock in horses on the basis of ileus has been investigated using the prostanoids thromboxane B2 (TXB2) and prostaglandine E2 (PGE2) as indicators. The prostanoid synthesis inhibitor flunixin meglumine was to be examined regarding its efficacy in the effective blockade of the synthesis of these mediators within the peri-operative timeframe as well as its effects on clinical signs and laboratory parameters. 21 horses suffering from ileus and ready for surgical intervention received an intravenous flunixin dosis of 1.1 mg...
Constable PD, Tinkler SH, Couëtil LL.To determine the degree of agreement between 2 analyzers for measurement of total CO₂ concentration (ctCO₂) in equine plasma. Methods: 6 healthy untrained horses, 6 trained Standardbreds undergoing a simulated race protocol, and 135 trained Standardbreds at a racetrack. Methods: Jugular venous blood samples were obtained from all horses. Two analyzers (commonly used analyzer A and less expensive analyzer B) were used to measure plasma ctCO₂ in each sample. Validation of both analyzers was conducted in accordance with guidelines established by the Clinical and Laboratory Standards Institu...
Serafini R, Ghosh S, Love CC, Medrano JMR, Teague SR, LaCaze KA, Varner DD.Commercially available vaginal lubricants, typically labeled as non-spermicidal, are used to lubricate equine artificial vaginas prior to semen collection. Improper type or amount of lubricant might affect stallion sperm quality, either after short-time exposure or following cooled storage of extended semen previously exposed to lubricant. The aim of this study was to evaluate stallion sperm quality following exposure to lubricant-containing extender for 1 h (T1h) or 24 h (T24h). Three ejaculates were collected from each of four stallions using a small volume of petrolatum to lubricate a...
Pemberton DH, Thomas KW, Terry MJ.The prevalence of hypogammaglobulinaemia in 82 young foals was determined. Twelve foals were considered clinically abnormal at birth and ten died within two weeks. All of these foals were hypogammaglobulinaemic. Seven (10%) of the other 70 apparently normal foals were hypogammaglobulinaemic despite having suckled normally. Three of these foals developed significant disease and one died at one month of age. Rapid detection of foals with low serum immunoglobulin levels was achieved by adapting the zinc sulphate turbidity test to partially evacuated blood collection tubes. This permitted test to ...
Mogielnicka-Brzozowska M, Woźniak MJ, Cichowska AW, Fraser L, Kraziński B, Strzezek R, Zielińska D.Seminal plasma (SP) proteins are responsible for sperm functional quality. Developing a reliable method to determine the degree of oxidative damage of these proteins is important for establishing semen fertilizing ability. The main aim of the study was to verify the applicability of protein carbonyl derivatives measurement in the SP of canine and stallion, using a method with 2,4-dinitrophenylhydrazine (DNPH). The research material consisted of ejaculates obtained from eight English Springer Spaniels, and from seven half-blood stallions during the breeding and non-breeding season. The content ...
Inoue OJ, Freeman DE, Wallig M.To study effects of hypochlorous acid (HOCl) on equine colonic mucosa in vitro, and determine whether addition of ascorbic acid protects against the effects. Methods: 6 healthy horses and ponies. Methods: Short-circuit current was measured in mucosa mounted in Ussing chambers. Incubation conditions were: control (no additions); 5 mM HOCl; 1 mM HOCl; same and 5 mM ascorbic acid; 3 mM HOCl; 3 mM HOCl and 5 mM ascorbic acid; 7 mM HOCl; and 7 mM HOCl plus 5 mM ascorbic acid. Permeability was measured with [3H]mannitol and, at the conclusion of each experiment, tissues were examined microscopically...
Pu F, McKinney AR, Stenhouse AM, Suann CJ, McLeod MD.A study of the equine phase II metabolism of the anabolic agent boldenone is reported. Boldenone sulfate, boldenone glucuronide and their C17-epimers were synthesised as reference standards in our lab and a method was developed for their detection in a horse urine matrix. Solid phase extraction was used to purify the analytes, which were then detected by ion trap LC/MS. Negative and positive ionisation mode MS(2) were used for the detection of sulfate and glucuronide conjugates, respectively. Boldenone sulfate and 17-epiboldenone glucuronide were detected as the major and minor phase II metabo...
Pihl TH, Jacobsen S, Olsen DT, Højrup P, Grosche A, Freeman DE, Andersen PH, Houen G.OBJECTIVE To purify and characterize equine vitamin D-binding protein (VDBP) from equine serum and to evaluate plasma concentrations of VDBP in healthy horses and horses with gastrointestinal injury or disease. ANIMALS 13 healthy laboratory animals (8 mice and 5 rabbits), 61 healthy horses, 12 horses with experimentally induced intestinal ischemia and reperfusion (IR), and 59 horses with acute gastrointestinal diseases. PROCEDURES VDBP was purified from serum of 2 healthy horses, and recombinant equine VDBP was obtained through a commercial service. Equine VDBP was characterized by mass spectr...
Gu X, Meleka-Boules M, Chen CL.A capillary electrophoresis technique was developed for the separation of synthetic glucocorticoids and the determination of dexamethasone and flumethasone in horse urine. Pretreatment of the sample using a dexamethasone affinity column resulted in low background that enabled the authors to detect levels as low as 1.1 ng/mL and 2.7 ng/mL for dexamethasone and flumethasone in horse urine, respectively. The developed method was used to detect dexamethasone in horse urine samples after the injection of a therapeutic dose of dexamethasone for up to 12 hr postinjection. The optimum conditions for c...
