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.
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...
Augustinsson KB, Bartfai T, Mannervik B.The steady-state kinetics of the butyrylcholinesterase-catalysed hydrolysis of butyrylthiocholine and thiophenyl acetate were shown to deviate from Michaelis-Menten kinetics. The ;best' empirical rate law was selected by fitting different rate equations to the experimental data by non-linear regression methods. The results were analysed in view of two alternative interpretations: (1) the reaction is catalysed by a mixture of enzymes, or (2) the activity is due to a single enzyme displaying deviations from Michaelis-Menten kinetics. It was concluded that the second alternative applies, and this...
Margalit R, Schejter A.The specific binding of cations to horse heart ferrocytochrome c has been studied, using the gel
filtration method. The cations investigated were: Mg2+, Co2+, cinchonine and proflavine. The stability
constants are in the range of 5-8 mM-1, and the number of binding sites per protein molecule are
1 to 2. The temperature dependence of the stability constant for the Mg2+-ferrocytochrome system
was measured. The thermodynamic parameters were found to be: dH&s = 4-12 kcal/mol, dG;,,
(25 "C) = -5.6 kcal/mol and AS&, = +57 calxmol-lx K-I.
Wilgus H, Stellwagen E.Changes in the visible absorbance spectra of complexes of horse heart cytochrome c hemopeptide 1-65, peptide 66-104, and their guanidinated counterparts are compared with those characteristic of native and fully guanidinated ferricytochrome c over the pH range 7 to 11. Upon raising the pH, the methionine ligand in the guanidinated hemopeptide 1-65.peptide 66-104 complex is replaced by a strong field ligand. By contrast, the methionine ligand in the hemopeptide 1-65.guanidinated peptide 66-104 is replaced by a weak field ligand. These results demonstrate that lysine 13 does not ligate with the ...
Woolcock JB.A cell wall component of Streptococcus equi analogous to the M protein of group A streptococci has been identified and purified. A highly purified product has been obtained from cells by hot acid extraction, followed by acid precipitation, ammonium sulfate fractionation, and column chromatography. This product reacts with S. equi antiserum. The existence of this fraction in S. equi has been confirmed by the failure of trypsin-treated cells and their extracts to remove the long-chaining capacity of S. equi antiserum. The antigenicity of this M-like protein when incorporated in adjuvant has been...
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...
Kaneps AJ, Craig AM, Walker KC, True JE.To determine whether iontophoretic administration of dexamethasone to horses results in detectable concentrations in synovial fluid, plasma, and urine. Methods: 6 adult mares. Methods: Iontophoresis was used to administer dexamethasone. Treatments (4 mA for 20 minutes) were administered to a tarsocrural joint of each mare. The drug electrode contained 3 ml of dexamethasone sodium phosphate at a concentration of 4 or 10 mg/ml. Samples of synovial fluid, blood, and urine were obtained before and 0.5, 4, 8, and 24 hours after each treatment. All samples were tested for dexamethasone using an ELIS...
Legrottaglie R, Agrimi P.Electrophoretic analysis in polyacrylamide gel (PAGE) of the equine rotavirus 106/88/LI/EQ, isolated from the diarrhea of an 18 day old foal was compared to the bovine strain NCDV. There was a notable difference in the migration of some segments of the viral RNA. Bands 2 and 3 of the equine rotavirus comigrated while there was a clear separation of segments 7, 8 and 9. Moreover, the migration of segments 1, 4 and 5 revealed a lower molecular weight than the corresponding segments of NCDV.
Thompson DL, McNeill DR, Wiest JJ, St George RL, Jones LS, Garza F.Sequential samples of blood were drawn via jugular catheters every 15 min for 24 h from four mares in each of five reproductive states: intact anestrous mares in winter, intact diestrous mares in summer, intact estrous mares in summer, ovariectomized mares in winter and ovariectomized mares in summer. Estrous mares were sampled on d 4 or 5 of estrus and diestrous mares on d 10 or 11 of diestrus. Each sample of plasma was assessed for concentrations of luteinizing hormone (LH) and follicle stimulating hormone (FSH) in two independent radioimmunoassays. A computer program was developed that dete...
Morris CM, Field HJ.Tissue specimens obtained from equine herpesvirus-1 (EHV-1), subtype 1-infected aborted foetuses were analysed for the presence of virus DNA by means of Southern blot and dot blot hybridisations. The specificity of the methods was confirmed although the sensitivity was inferior to classical techniques such as virus isolation. However, the possibility of detecting the state of the virus DNA and the ability to distinguish between subtypes were important features, and the dot blot method was shown to have potential for a rapid diagnostic test. This report demonstrates some potential practical app...
Wong JKY, Leung DKK, Curl P, Schiff PJ, Lam KKH, Wan TSM.The detection of boldenone, nandrolone, 5(10)-estrene-3β,17α-diol, and 4-estrene-3,17-dione in a urine sample collected from a gelding having been treated with testosterone (500 mg 'Testosterone Suspension 100', single dose, injected intramuscularly) in 2009 led the authors' laboratory to suspect that these 'testicular' steroids could be minor metabolites of testosterone in geldings. Administration trials on six castrated horses with Testosterone Suspension 100 confirmed that low levels of boldenone, nandrolone, 5(10)-estrene-3β,17α-diol, and 4-estrene-3,17-dione could indeed be detected ...
Ainsworth DM, Dodds WJ, Brown CM.A 16-year-old gelding was examined because of weight loss, inappetence, and intermittent fever of 2 months' duration. Preliminary laboratory findings revealed anemia, hypoproteinemia, thrombocytopenia, and prolongation of the activated partial thromboplastin time. A deficiency or inhibition of coagulation factor XI, factor XII, or high molecular weight kininogen was diagnosed. This defect was not associated with a bleeding diathesis, but should be considered as a cause of prolongation of the activated partial thromboplastin time.
Morris DD, Crowe N, Moore JN.The purpose of this study was to determine if a structurally novel dual inhibitor of arachidonic acid metabolism, SK & F 86002, would inhibit the endotoxin-induced production of tumor necrosis factor (TNF) activity by equine peritoneal macrophages. Equine peritoneal macrophages were variously pretreated for 0, 0.5 and 2 h with SK & F 86002 at 10(-9) to 10(-4) molar final concentrations or were left untreated. Then, the macrophages were cultured in vitro in the presence of endotoxin (5 ng/mL). Supernatant media were collected after 4 h and stored at -70 degrees C until assayed for TNF a...
Delbeke FT, Baert K, De Backer P.Urinary and plasma concentrations of the nonsteroidal anti-inflammatory drug tiaprofenic acid were determined following oral and intramuscular administration of a dose of 1 g to five fasted horses. Quantitation was performed by high-performance liquid chromatography (HPLC). The limit of quantitation (LOQ) was 0.1 microg/ml and 0.5 microg/ml in 2 ml plasma and 1 ml urine, respectively. Assay precision and extraction recovery were between acceptable values. Tiaprofenic acid pharmacokinetics were described by non-compartment analysis of the data. Absorption was faster after oral administration as...
Whan G, Phillips J, Bullock S, Runciman RJ, Pearce S, Hurtig M.A novel, multi-use, low-stiffness and low-cost transducer for measuring in vitro strains has been developed and tested. Currently available strain measurement methods are either too expensive, too complicated or too inflexible for multi-use strain measurement. The stainless-steel modular strain measurement clip introduced here was instrumented with four 350 Omega axial strain gauges in a full Wheatstone bridge configuration to take advantage of commonly available strain gauge amplifier equipment. Adjustable extension arms were designed to allow greater application versatility. The clip was cal...
Carstanjen B, Amory H, Youssao I, Remy B.The purpose of this study was to validate an equine-specific osteocalcin (OC) radioimmunoassay (RIA) for use in donkeys and to establish age-related changes in serum OC concentrations in healthy donkeys. Serial dilutions of donkey serum showed parallelism with standard curves obtained with the equine-specific OC RIA. There was a tight linear regression between donkey serum OC values obtained with the equine specific OC RIA and a commercially available bovine-specific OC RIA. Serum OC levels of 27 healthy donkeys, analysed with the equine-specific OC RIA, showed a tight negative logarithmic reg...
Millington WR, Dybdal NO, Mueller GP, Chronwall BM.beta-Endorphin is post-translationally processed to both N-acetylated and C-terminally shortened derivatives in the anterior lobe of the horse pituitary, a processing pattern qualitatively different from that of the rat and virtually every other mammalian species. Thus, separation of the molecular forms of beta-endorphin using gel filtration and ion exchange chromatography showed that the horse anterior lobe primarily contains beta-endorphin-1-31 and N-acetyl-beta-endorphin-1-27 along with smaller amounts of beta-lipotropin, beta-endorphin-1-27, and N-acetyl-beta-endorphin-1-31 and -1-26, in c...
Carrier SP, Boulanger P, Bannister GL.The comparative values of the direct, the indirect complement-fixation and the agar-gel immunodiffusion tests were assessed for the diagnosis of equine infectious anemia. Antibodies were detected on the agar-gel immunodiffusion test as early as 18 days post-inoculation in the serums of experimentally infected horses and were readily detectable in all the subsequent bleedings. Complement-fixing antibodies, demonstrable by the direct method, were detected commencing about the same time. However, these were not long-lasting and were replaced by the non-complement-fixing antibodies demonstrable by...
Ahmed SM, Broad SC, Edington N.Sera from two sibling groups of ponies experimentally infected with Equid herpesvirus 1 or 4 (EHV-1 or 4) were used to investigate which viral polypeptides (VPs) of EHV-1 and EHV-4 were recognised. Recognition was detected as early as 8 d.p.i. and thereafter. The polypeptides of EHV-1 (labelled with 35S-methionine) immunoprecipitated (IIP) by sera from both groups had Mr of 148, 138, 123, 117, 110, 77-79, 70, 55, 49-50, 47, 40 and 35-37 kDa respectively. Of these VP148K (VP9 nucleocapsid) gave the maximum precipitation, followed by 117 and 77-79 kDa. The latter were confirmed by monoclonal ant...
Coyne CP, Smith JE, DeBowes RM.Several pharmaceutical compounds were evaluated for their ability to selectively inhibit activated coagulation factor-XIII-like enzyme activity (eg, XIIIa*) in pooled equine plasma. Presence of coagulation factor-XIIIa*-like enzyme activity in plasma was established by assay procedures involving incorporation of the fluorescent amine compound, monodansylcadaverine, into purified casein, which served as a protein substrate. Pharmaceuticals inhibitory to coagulation factor-XIIIa*-like enzyme activity were recognized by plasma gel formation of high spectrophotometric transmittance (transparency),...
Batt RM, Burgess D, Edwards GB, van de Waal S, Sørensen SH, Hart CA.To examine the postnatal development of equine small intestine, biopsy specimens of jejunal mucosa from 8 ponies, between 6 and 28 weeks old, were subjected to analytical subcellular fractionation and assay of organelle marker enzymes. Fractionation revealed a reduction in the particulate brush border component of beta-galactosidase (lactase) activity between 6 and 28 weeks, and a corresponding increase in soluble activity, although the reduction in mean specific activity was not significant. There also was a decrease in the proportion of brush border to soluble aminopeptidase N activity, a re...
Alexander S, Irvine CH.Mares were bled once daily throughout a cycle, or 3 times daily from the first day of oestrus to the 2nd day after ovulation. LH was measured by heterologous radioimmunoassay and by an in-vitro bioassay based on LH-stimulated testosterone production by mouse Leydig cells. The patterns of bio- and immuno-active LH during the oestrous cycle were similar but not identical, so that in both groups of mares the ratio of biological: immunological (B:I) activity during the LH surge was significantly higher before than after ovulation (P less than 0 . 001). Considerable individual variation in cycle me...
Brom-de-Luna JG, Salgado RM, Felix MR, Canesin HS, Stefanovski D, Diaw M, Hinrichs K.In vitro production of horse embryos via intracytoplasmic sperm injection (ICSI) is a useful clinical and research technique. Current rates of blastocyst production are typically sub-optimal, and few methods to increase the rate of equine blastocyst development have been reported. Factors that might improve blastocyst production in a horse embryo culture system were explored. Myo-inositol is found in the horse oviduct and improves blastocyst development in other species, thus Experiment 1 was conducted to assess the effect of 10 mM myo-inositol added to Day 0-5 embryo culture medium, using hor...
Yagisawa S.One mole of horse hemoglobin tetramer reacts with 2 moles of 2-chloromercuri-4-nitrophenol (MNP) at beta 93 cysteine. The difference spectra between NMP-bound hemoglobin and hemoglobin, measured with the aid of ascorbic acid and ascorate oxidase [EC 1.10.3.3] as deoxygenation reagents, indicate that the pK of the phenolic hydroxyl group of MNP increases by 0.6 to 0.8 pH unit on deoxygenation of the hemoglobin. The Hill constant of the modified hemoglobin changes with pH. It decreases from about 2.4 at pH 6.8 to about 1.0 at pH 9.0 This effect of the reagent is interpreted as inherent to the re...
Yoshimura T, Sogabe T, Aki K.Electron transfer between horse heart and Candida krusei cytochromes c in the free and phosvitin-bound states was examined by difference spectrum and stopped-flow methods. The difference spectra in the wavelength range of 540-560 nm demonstrated that electrons are exchangeable between the cytochromes c of the two species. The equilibrium constants of the electron transfer reaction for the free and phosvitin-bound forms, estimated from these difference spectra, were close to unity at 20 degrees C in 20 mM Tris-HCl buffer (pH 7.4). The electron transfer rate for free cytochrome c was (2-3).10(4)...
Landim-Alvarenga FC, Alvarenga MA, Seidel GE, Squires EL, Graham JK.Experiments evaluated the ability of follicular fluid (FF), dilauroylphosphatidylcholine (PC12) and the calcium ionophore A23187 (A23187) to induce capacitation in stallion and bull spermatozoa, determined by the ability of the spermatozoa to penetrate zona-free hamster, bovine and equine oocytes. Spermatozoa suspensions were incubated at 37 degrees C in one of the following treatments: 1) a modified Tyrode's medium (BGM3) alone; 2) BGM3 + FF; 3) BGM3 + PC12; 4) BGM3 + FF + PC12; 5) BGM3 + A23187; and 6) BGM3 + FF + A23187. Treated spermatozoa were incubated with zona-free hamster, bovine and ...
Theiner E, Weber C, Müller E, Venner M, Vervuert I.This study aimed to establish reference intervals for Mn in whole blood, plasma and serum of healthy, adult warmblood horses with known dietary Mn intake and to compare 2 methods of analysis. Methods: Between May 2018 and November 2019 a single blood sample was taken from a total of 270 clinically healthy horses (age: 3-25 years) in 3 stud farms. In lithium-heparin (LH) whole blood, LH plasma and serum Mn concentrations were analyzed by means of atomic absorption spectrometry (AAS) and inductively coupled plasma mass spectrometry (ICP-MS). The reference intervals were calculated according to t...
Cartoni GP, Montanaro M.The results and the improvement of the analytical procedures adopted for the control of doping in horses will be reported. This control has been systematically carried out in Italy for about 10 years in the laboratories of Italian Federation of Sport and Medicine in which the biological samples for the control of doping in various sport activities (football, cycling, athletics etc.) are also examined. In this way it is possible to use the same instruments for all these similar problems and compare the results. The analytical procedure is based on the following steps: 1) Extraction of the sampl...
Rodríguez H, Bustos-Obregón E.Epididymal sperm maturation in the stallion was analysed using eight epididymides and deferent ducts from healthy animals. Samples were obtained in June-July and October-November (resting and breeding periods, respectively). Epididymides were divided into head, body and tail. Sperm samples were submitted to a routine seminogram, chromatin decondensation test (Lung, 1972) and sperm velocity determination (Makler, 1980). Results demonstrate that stallion spermatozoa achieve maturation in the transition between the head and body of the epididymis as revealed by chromatin decondensation. Objective...
Bhutia WD, Gupta S, Rani R, Batra K, Sethi K, Kumar S, Kumar R.Trypanosoma evansi is a causative agent of chronic wasting and fatal disease of livestock and wild animals known as surra. In this study, repurposing approach based on drug target was used to investigate the efficacy of kinase inhibitors (Barasertib-HQPA, BAR and Palbociclib isethionate, PAL) and protease inhibitors (Z-pro-prolinal, Z-PRO and Leupeptin hemisulphate, LEU) against T. evansi in HMI-9 medium. BAR, PAL and Z-PRO exhibited IC values of 13.52 µM, 0.6375 µM and 63.20 µM against T. evansi in terms of growth inhibition, in the contrary, LEU failed to exhibit a significant growth i...
