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.
Van den Oord AH, Wesdorp JJ, Van Dam AF, Verheij JA.In commercial samples of equine myoglobin and samples of equine and bovine myoglobin prepared in the laboratory, a small amount of the protein was present as an aggregate. The presence of the myoglobin aggregate could be demonstrated by gel filtration on Sephadex G-100 Superfine, which also provided a means of isolating it. Gel filtration on Sephadex G-100 showed the molecular weights of the equine and bovine moyglobin aggregates to be about 35000 and 34000 respectively, thus supporting the hypothesis that they are dimers. This was confirmed for the equine myoglobin by ultracentrifugation meas...
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...
Barban PS, Gol'din RB, Misenzhnikov AV, Prusakova ZM, Pantiukhina AN.The authors present the results of immunization of horses-producers with a commercial antigen and the yolk culture of the living R. sibericus (strain K1) for the purpose of obtaining specific immune sera for many purposes. It was shown that the original combined scheme of immunization and reimmunization of horses, successfully approved in the preparation of immune sera to Rickettsia prowazeki also proved to be highly effective for obtaining the antisera to R. sibericus. Sera obtained after the primary immunization of horses could be successfully used as diagnostic sera, but they were of no use...
Pérez-Marín CC, Requena FD, Arando A, Requena L, Requena F, Agüera EI.The aim of this study was to determine the effects of various abiotic factors, such as light, physical stress (pipetting) and thermal shock, on the quality of fresh and cooled equine sperm. In experiment I, four sperm aliquots were subjected to different light exposures: (i) protected control samples (CTRL), (ii) exposed to UV light at 10 cm (UV10), (iii) exposed to UV light at 20 cm (UV20) and (iv) exposed to laboratory lighting (LAB). In experiment II, four semen aliquots were subjected to repeated pipetting for 0, 10, 20 and 30 times (CTRL, P10, P20 and P30, respectively). In experiment I...
Kaminski M.The detection of the recessive null allele of horse serum esterase (Es) is possible in heterozygotes Es+/EsO which by starch gel electrophoresis appear like homozygotes Es+/Es+. Two methods are proposed, the titration of enzymatic activity of esterase and the immunochemical titration of esterase as antigen. These methods can be applied to solve the cases of suspect parentage or in population studies.
McCann ME, Watson TD, Boudinot FD, Moore JN.We evaluated the pharmacokinetics of IV administered sodium heparin and the pharmacodynamic effect of heparin on lipoprotein lipase (LPL) activity. Horses were allotted to 3 groups. Plasma samples were obtained from each horse before and at various times for 6 hours after heparin administration for determination of heparin concentration, LPL activity, and activated partial thromboplastin time (APTT). The disposition of heparin was dose dependent. The area under the plasma heparin concentration vs time curve (AUC) increased more than proportionally with dose, indicating that heparin elimination...
Dryburgh EL, Marsh AE, Dubey JP, Howe DK, Reed SM, Bolten KE, Pei W, Saville WJ.Sarcocystis neurona is considered the major etiologic agent of equine protozoal myeloencephalitis (EPM), a neurological disease in horses. Raccoon ( Procyon lotor ) is considered the most important intermediate host in the life cycle of S. neurona in the United States; S. neurona sarcocysts do mature in raccoon muscles, and raccoons also develop clinical signs simulating EPM. The focus of this study was to determine if sarcocysts would develop in raccoons experimentally inoculated with different host-derived strains of in vitro-cultivated S. neurona merozoites. Four raccoons were inoculated wi...
Romagnano A, Richer CL, Messier PE, Jean P.Silver staining shows the presence in the domestic horse of six NORs located on chromosomes 1, 26 and 31 as identified after R-banding. Following electron microscopy, the argyrophilic material was observed outside the terminal secondary constrictions (satellite stalks) on the terminal portion of the short arm of chromosome 1, outside the secondary constrictions on the proximal region of the long arms of chromosome 31, and beside the proximal region of the long arms of chromosome 26. Satellite staining applied to these chromosomes appears to reveal only the active NORs.
Schenk I, Broussou D, Roques B, Lagershausen H, Machnik M, Röttgen H, Toutain PL, Thevis M.The combination of sulfadoxine (SDO) with trimethoprim (TMP) is widely used in veterinarian medicine. The aim of the present study was to compare excretion profiles and detection time windows of SDO and TMP in plasma and urine by means of a validated quantitative method. Eight horses received a single intravenous (i.v.) dose of 2.7 mg TMP and 13.4 mg SDO per kg bodyweight. Plasma and urine samples were collected up to 15 and 70 days post-administration, respectively. While urine samples underwent an enzymatic hydrolysis, plasma samples were proteolysed before further analysis. After solid-...
Ahmed JS, Schmid G, Hörchner F.Pony peripheral blood lymphocytes (PBL) were stimulated with a soluble fraction of Trypanosoma (T.) evansi (SF). As determined by 3H-thymidine incorporation, the cells underwent a proliferative response and were able to: a) produce a factor having the biological activities of interleukin 2 (IL-2) since their supernatants could support the in vitro growth of pony PBL stimulated with concanavalin A (Con A-blasts); b) undergo a further proliferative response when incubated in short term cultures with SF, human recombinant IL-2 (hrIL-2), or both c) bind specifically radiolabelled hrIL-2 (125I-hrIL...
Carstanjen B.Effective, non-invasive bone assessment methods for screening, diagnosis and follow-up of the skeleton are more and more requested in veterinary medicine. In contrast to clinical parameters, invasive methods and imaging techniques, indices of bone turnover is a tool for bone metabolism evaluation of the whole skeleton. Biochemical bone markers therefore provide a more real-time assessment of the bone status with simple blood- or urine-analysis. This article surveys currently available biochemical marker of bone metabolism used in veterinary medicine. Additionally, information is provided about...
Basova NE, Kormilitsin BN, Perchenok AIu, Rozengart EV, Saakov VS, Suvorov AA.There was studied action of aliphatic alcohols (ethanol, propanol, isopropanol, n-butanol, isobutanol, secbutanol, tretbetanol) and pH on various kinds of reactional capability the serum cholinesterase. At the alcohols-affected inhibition of the cholinesterase hydrolytic activity, the determining role was played not the total number carbon atoms in the alcohol molecule, but by the "effective length" of the carbohydrate chain. The fact that the presence of alcohols did not affect parameters of the reverse cholinesterase inhibition with onium ions tetramethylammonium and choline allows suggestin...
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...
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...
