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.
Montgomery PC, Dorrington KJ, Rockey JH.Three independent methods have been used
to determine the molecular weights of the heavyand light-polypeptide chain subunits of equine yGab-,
yGc-, and yT-immunoglobulins. Extensively reduced
and alkylated proteins were filtered through standard
columns of Sephadex G-100 or G-200 in 8 M urea405
M propionic acid. Subunit molecular weights were obtained from the linear elution volume, V,, us. logarithm
molecular weight relationship defined for each column
with rabbit yG-globulin heavy and light chains and
horse heart cytochrome c. Molecular weights also were
determined by equilibrium se...
Wormstrand A.An immunological gel-diffusion test for the diagnosis of pregnancy in the mare is described. 56 blood samples from 50 different mares were tested. Control tests were made both by the Ashheim-Zondek method and by clinical examination. The accuracy of the immunological method was 96.4 %. No false positive reactions were observed. It is recommended to draw the blood sample at approximately 45 days or more after the last service. The immunological method is simple, cheap and accurate and is recommended as a routine test for the diagnosis of pregnancy in mares.
Stirk SA.A 5 year old Thoroughbred stallion with diarrhoea of unknown aetiology was referred to Davis. Treatment was aimed at terminating diarrhoea and restoring normal fluid status. Laboratory aids were utilised to establish where inbalance and deficits were present. Antibiotics and corticosteroids were used as an adjunct to fluid therapy. The case history and rationale of treatment of fluid disorders resulting from diarrhoea are discussed.
Yamada S, Suruga K, Ogawa M, Hama T, Satoh T, Kawachi R, Nishio T, Oku T.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...
Daniel GB, Tucker RL, Buckman T, Daniel SL.Isolated equine granulocytes (WBC), radiolabeled with 99mTc-hexamethylpropyleneamine oxime (99mTc-HMPAO) or 111In-oxine, were evaluated in vitro for their labeling characteristics, viability, and phagocytic function over a 6-hour postlabeling period. Mean +/- SD labeling efficiency for 111In-oxine-WBC was 62.2 +/- 15.3%, which was significantly (P less than 0.001) higher than that for 99mTc-HMPAO-WBC (32.0 +/- 17.0%). In vitro elution of radiolabel from cells was significantly (P less than 0.02) greater for 99mTc-HMPAO-WBC at 0.5, 2, and 4 hours, but was not significantly different from elutio...
Yee S, Peyton DH.The redistribution of the initially-formed myoglobin heme-insertion isomers from the initially formed 50/50 mixture to the equilibrium ratio of 90/10 has long been assumed to occur by one of two mechanisms, both of which require the rupture of the heme iron-protein bond (La Mar, G.N., Toi, H. and Krishnamoorthi, K. (1984) J. Am. Chem. Soc. 106, 6395-6401). In this study we compared the use of nuclear magnetic resonance and optical spectroscopic techniques as methods for studying the reorientation of heme within myoglobin. We found that kinetics determinations of the heme insertion isomer redis...
Lensing HH, Behr-Gross ME, Daas A, Spieser JM.The European Directorate for the Quality of Medicines (EDQM) has organised an international collaborative study, divided into two phases, aimed at producing and establishing two suitable reference sera for serological potency testing of tetanus vaccines for veterinary use for batch consistency demonstration. In phase I pools of sera were produced by immunising guinea pigs and rabbits with tetanus toxoid using the immunisation schedule prescribed by the European Pharmacopoeia (Ph. Eur.) for potency testing of tenanus vaccines for veterinary use. Following aliquoting and freeze-drying, character...
Ramírez A CE, Hurtado-Macías A, Talamantes R, Flores E, Ladrón de Guevara HP, Delgado JI, Estrella RA, Riestra JM, Montes JM, Esmonde-White K....Guided bone regeneration surgeries are based on grafting a scaffold in the site to be repaired. The main focus of the scaffold is to provide mechanical support to newly formed blood vessels and cells that will colonize the grafted site, achiving bone regenertation. In this regards, the aim of this study was to characterize the anatomy, structular, surface morphologycal, chemical composition, and nanomechanical properties of ostrich and equine trabecular bone. Ostrich and equine specimens were obtained from a local abattoir and bone was obtained by blunt dissection, n = 5. Tissue bone anatomy...
Makhaeva GF, Shataeva GA, Iankovskaia VL, Fetisov VI, Loshadkin NA.The interaction of human erythrocyte acetylcholinesterase, horse serum butyrylcholinesterase and rat liver carboxylesterase with insecticides (RO)2P(O)SCH(COOEt)SP(O)(OR)2 (I) and (RO)2P(O)SCH(COOEt)OP(S)(OR)2 (II) was studied. The type I and II compounds were not hydrolyzed by carboxylesterase and inhibited the esterases irreversibly. A complex pattern of inhibition of acetylcholinesterase and butyrylcholinesterase by these compounds was caused by kinetically-manifested formation of an enzyme-inhibitor complex. The compounds I and II were more selective towards butyrylcholinesterase than towa...
Rakotoarisoa L, Mironneau C, Sayet I, Mironneau J.Specific binding of the Ca++ antagonist desmethoxyverapamil, (-)-[3H]D888, to cell membranes of equine portal vein smooth muscle was inhibited in a concentration-dependent manner by guanosine 5'-O-(gamma-thio)triphosphate and ATP but was little affected by guanosine 5'-O-(beta-thio)diphosphate, noradrenaline or phorbol 12-myristate 13-acetate ester. Inhibition constants for GTP and ATP were in the range of 0.1 to 0.3 mM. From Scatchard plots and dissociation kinetic experiments, it is proposed that D888 high affinity binding sites are transferred into low affinity sites. In intact strips of ra...
Hubbard CD, Shoupe TS.A transient phase for the hydrolysis of indophenyl acetate by the commercial preparation of horse serum cholinesterase was observed on a stopped-flow spectrophotometer. It was found that the transient process is a reaction of the ester with a major component of the preparation and is not caused by the serum cholinesterase enzyme. This noncholinesterase component was isolated and the dependence of its concentration and that of the ester upon the transient liberation of the indophenolate ion were determined. Studies with the isolated component and subsequent analyses have led to the tentative id...
Niyom S, Mama KR, King M, Contino E, Ferris D, Valdes-Martinez A, Frisbie DD, McIlwraith W, Zumbrunnen J.This study investigated the influence of changing recumbency and mode of ventilation over repeated anesthesias on the alveolar to arterial oxygen tension gradient (PO) and laboratory analytes in eight horses during a year-long imaging study. Anesthesia was induced with xylazine, diazepam or guaifenesin, and ketamine and maintained with isoflurane. Horses were positioned in right or left lateral recumbency for computed tomography. Ventilation was controlled during 47% of the anesthetics. Blood was sampled from an arterial catheter prior to (30 ± 5 min from connection to anesthetic circuit), wi...
Collins ND, LeRoy BE, Vap L.Extremely high bicarbonate (HCO3-) and anion gap values were measured in two horses and a calf using the Hitachi 911 automated serum biochemistry analyzer. All three animals had severe muscle disease as evidenced by markedly increased aspartate aminotransferase and creatine kinase activities. Laboratory error was suspected as the source of the increased HCO3- because values calculated from blood gas analysis were normal. It was hypothesized that increased serum lactate dehydrogenase (LDH) activity and pyruvate concentration overwhelmed the oxamate LDH inhibitor in the enzymatic HCO3- assay, re...
Elkhawagah AR, Nervo T, Poletto M, Martino NA, Gallo D, Bertero A, Vincenti L.The aim of the study was to ascertain effects of different concentrations of relaxin added to extender medium during the pre-freezing incubation periods on quality variables of stallion frozen-thawed spermatozoa. Semen samples collected from three stallions were filtered, diluted with skim milk, and centrifuged at 600g for 10 min. Sperm pellets were suspended in BotuCrio freezing medium to a final concentration of 50 × 10 sperm/mL. The diluted semen was divided into five experimental groups supplemented with 0 (control), 12.5, 25, 50, or 100 ng/mL of relaxin. The semen samples were transferre...
Battut I, Bézard J, Palmer E.A culture for equine oviduct epithelial cells is described. Primary cultures reached confluence in 5-8 days, forming a monolayer of polygonal cells and remaining morphologically intact for about 20 days. Subcultures were obtained by collecting cells detached spontaneously from the monolayers, and confluence was reached again after 5-7 days. Cells frozen before primary culture were confluent 10-15 days after thawing. Dishes containing confluent cells also were frozen, and some cohesive monolayers formed after thawing. Equine embryos, collected 2 days after ovulation, were cultured alone or with...
Arnhold S, Elashry MI, Klymiuk MC, Geburek F.Adipose tissue-derived mesenchymal stem cells (ASCs) offer a promising cell source for therapeutic applications in musculoskeletal disorders. The appropriate selection of ASCs from various fat depots for cell-based therapy is challenging. The present study aims to compare stemness and multipotency of ASCs derived from retroperitoneal (RP), subcutaneous (SC), and lipoma (LP) fat to assess their usefulness for clinical application. Equine ASCs from the three fat tissue sources were isolated and characterized. The cell viability, proliferation, and self-renewal were evaluated using MTT, sulforhod...
Mischke R, Junker J, Deegen E.The sensitivity of commercial prothrombin time (PT) tests was assessed based on a dilution series of equine pooled plasma (EPP) (experiment 1) and on 40 equine plasma samples with reduced activity of coagulation factors II, V, VII and X (experiment 2). Two different PT reagents (reagent 1, human placental thromboplastin; reagent 2, recombinant human tissue factor) were used according to the manufacturers' instructions (standard test, PT([ST])) and compared to a modified test procedure (modified test, PT([MT])) using sample dilution and fibrinogen addition. In all samples, sensitivity was lower...
Yamamoto K.Monospecific (MSp-) antisera against E1 and E2 glycoproteins of western equine encephalitis (WEE) virus were prepared and examined for binding activities to whole virions, hemagglutination-inhibition (HI), neutralization (NT) and protection. Both anti-E1 and anti-E2 MSp-Abs protected mice against WEE virus challenge. A competition experiment with monoclonal antibodies showed that these MSp-antisera appear to lack the antibody population for some epitopes involved in viral neutralization.
Bai Y, Tong TG, Zhang WJ, Xu SL, Wang Q, Liu GL, Wu DL.To develop a quantitative ELISA by measuring interferon (IFN-gamma) of equine lymphocytes. Methods: Sandwich ELISA for equine IFN-gamma was developed using mAb A5 as a capture antibody and biotinylated mAb SB10 as a detection antibody. Results: The detection limit of the sandwich ELISA for equine IFN-gamma was 1 microg/L and did not show cross-reactivity with recombinant equine IL-18. Equine IFN-gamma was detected by ELISA in culture medium of the peripheral blood mononuclear cells (PBMCs) stimulated with ConA or PMA/Ionomycin. Conclusions: This method can be used to help understand the role o...
Ho EN, Kwok WH, Wong AS, Wan TS.Quaternary ammonium drugs (QADs) are anticholinergic agents some of which are known to have been abused or misused in equine sports. A recent review of literature shows that the screening methods reported thus far for QADs mainly cover singly-charged QADs. Doubly-charged QADs are extremely polar substances which are difficult to be extracted and poorly retained on reversed-phase columns. It would be ideal if a comprehensive method can be developed which can detect both singly- and doubly-charged QADs. This paper describes an efficient liquid chromatography/tandem mass spectrometry (LC/MS/MS) m...
Matteri RL, Baldwin DM, Lasley BL, Papkoff H.Previous studies from this laboratory have described the properties of purified luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from horse and donkey anterior pituitary glands. The present study afforded the opportunity to further characterize these previously purified hormone preparations and to compare them with enriched gonadotropin fractions from zebra pituitary glands. Although a single LH and FSH fraction was usually obtained for each pool of pituitaries, two separate zebra LH and two donkey FSH preparations were generated. Purified hormone preparations from the horse wer...
Hagedorn HW, Schulz R, Jaeschke G.An enzyme linked immunosorbent assay (ELISA) was developed to detect the anabolic steroid boldenone in equine blood and urine. The polyclonal antiserum was raised in rabbits, employing boldenone-17-hemisuccinate-bovine serum albumin as antigen. Boldenone-17-hemisuccinate-horseradish peroxidase served as enzyme conjugate. Sensitivity of the assay was 26.0 +/- 3.0 pg/well. Among the endogenous steroids tested only progesterone and testosterone exhibited moderate cross-reactivities, 3.4 and 2.5%, respectively. These cross-reactivities are of no importance for the boldenone assay. For the reductio...
Henion JD, Maylin GA.A sensitive, quantitative method has been developed for the determination of hydrochlorothiazide in equine plasma and urine. Thin-layer chromatography is used to screen for the presence of the drug in unknown samples. The TLC screening methods described provide minimum detection limits of 50 ng/mL in plasma and 25 ng/mL in urine. A silica micro chromatography column is used to clean up ethyl acetate extracts for HPLC analysis and mass spectral confirmation. An internal standard, trichloromethiazide, is used to derive quantitative data at concentrations as low as 25 ng/mL for plasma disappearan...
Monkos K.This paper presents the results of viscosity determinations on aqueous solutions of equine, porcine and rabbit serum albumin over a wide range of concentrations and at temperatures ranging from 5 degrees C to (42-45) degrees C. The results are compared with human and bovine serum albumin previously studied. Viscosity-temperature dependence is discussed on the basis of the modified Arrhenius formula. The effective specific volume, the activation energy and entropy of viscous flow for all investigated albumins are compared. Viscosity-concentration dependence, in turn, is discussed on the basis o...
Novelli EL, Rodrigues NL, Chiacchio SB.Biochemical values are widely related with environmental agents, sex and age, and are used in disease diagnosis. Numerous reports have been published on the biochemical parameters of different breeds of horses. However, there is a paucity of information concerning Cu-Zn superoxide dismutase (SOD), ceruloplasmin, copper and zinc determinations in the serum. Blood samples from a total of 60 horses of the Mangalarga-Paulista breed, representing three age groups (0 to 4 months old, 6 to 18 months old and adult) were examined. Male horses have a higher mean value of SOD, ceruloplasmin and copper th...
Brems DN, Liu YC, Stellwagen E.The heme iron of horse heart cytochrome c was selectively removed using anhydrous HF. The product, porphyrin c, exhibits the viscosity, far ultraviolet circular dichroic, and fluorescence properties characteristic for native cytochrome c. However, porphyrin c is more susceptible to denaturation by guanidine hydrochloride and by heat than is the parent cytochrome. All of the conformational parameters of porphyrin c exhibit a common reversible transition centered at 0.95 m guanidine hydrochloride at 23 degrees C and pH 7.0. Guanidine denatured porphyrin c refolds in two kinetic phases having tim...
Stratil A, Tomásek V, Bobák P, Glasnák V.Homozygous horse transferrin (Tf O) is highly heterogeneous. In starch gel electrophoresis it gives at least 9 zones. Two main components (2a and 4b) were purified by rivanol and ammonium sulphate precipitation, DEAE-Sephadex chromatography and SP-Sephadex chromatography. Molecular weights of 75 200 and 80 500 for components 2a and 4b, respectively, were determined by sedimentation equilibrium ultracentrifugation. Amino acid compositions of the two components were similar, and there were no differences in the N-terminus (glutamic acid followed by glutamine) and the C-terminus (valine). Differe...
Williams RC.The evolutionary distance between two sets of proteins was estimated using the techniques of Miyata and Yasunaga (1980) and Kimura (1980). Human beta 2-microglobulin was compared with the homologous murine molecule, while human and equine alpha-globin were similarly treated. It was found that a large amount of molecular evolution has occurred in beta 2-microglobulin since its divergence from the common ancestor of mice and humans. Kimura's estimate of evolutionary distance, K, is 0.353, while those of Miyata and Yasunaga are KS = 0.708 and KA = 0.171. The respective values for human and equine...
Garcia P, Paris AC, Leufroy A, Popot MA, Bonnaire Y.A quantitative method, using LC/ESI-MS(n) with a quadrupole linear ion trap mass analyzer, has been developed for the analysis of ipratropium cation in horse plasma and urine. The method applies solid-phase extraction with WCX cartridges for plasma and MM2 cartridges for urine, prior to analysis by LC/ESI-MS(n). The efficiency of extraction combined with the sensitivity and the selectivity of MS(n) allows for the quantification of ipratropium cation at picogram per milliliter levels. The analytical capabilities of the method have been successfully checked by the quantitative analysis of ipratr...
Amann JF, Smith RM, Ganjam VK, Paull WK, McClure RC, Green EM, Garner HE.The distribution of cells that stain positive for beta-endorphin and ACTH immunoreactivity was studied in the pars intermedia (PI) of the hypophysis in 3 healthy horses and 2 healthy ponies. Serial sections treated with commercial antibodies generated against beta-endorphin or ACTH were processed for immunocytochemical studies, using the avidin biotin immunoperoxidase-complex method. Distribution patterns of cells reacting with antibodies were similar in cells from all equids. Cells immunostained for ACTH were numerous and widely distributed in the PI. Cells immunopositive for ACTH probably co...
Posthuma CC, Pedersen KW, Lu Z, Joosten RG, Roos N, Zevenhoven-Dobbe JC, Snijder EJ.The replication/transcription complex of the arterivirus equine arteritis virus (EAV) is associated with paired membranes and/or double-membrane vesicles (DMVs) that are thought to originate from the endoplasmic reticulum. Previously, coexpression of two putative transmembrane nonstructural proteins (nsp2 and nsp3) was found to suffice to induce these remarkable membrane structures, which are typical of arterivirus infection. Here, site-directed mutagenesis was used to investigate the role of nsp3 in more detail. Liberation of the hydrophobic N terminus of nsp3, which is normally achieved by c...
Bentinck-Smith J, Tasker JB.The following topics are discussed in this presentation: A. Recent advances in the use and interpretation and methodology of antibiotic susceptibility testing. B. Improvements in sample submittal to obtain accurate results from your laboratory. C. Staining blood, bone marrow, and cytology specimens in the office laboratory. D. Pathogenesis and differential diagnosis of lipemia. E. Differential diagnosis of abdominal effusions.
