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.
Robinson RC, Burtnick LD.Vitamin D binding protein (DBP) was isolated from horse plasma in a four-step procedure that involved Affi-Gel Blue affinity chromatography, gel filtration, hydroxylapatite chromatography, and anion exchange high-pressure liquid chromatography. The yield of DBP from 80 mL of plasma was 6-7 mg. Horse plasma DBP closely resembles other plasma DBPs, being a tryptophan-free protein of Mr 53,000. It is able to bind to and block the polymerization of monomeric actin. The secondary structure of DBP was calculated from circular dichroism measurements to be 39% alpha-helix, 42% beta-sheet, and 19% rand...
Lear TL, Trembicki KA, Ennis RB.Giemsa-11 (G-11) staining and in situ hybridization were used to identify the equine chromosome complement of horse x mouse somatic cell hybrids. The presence of horse chromosomes in somatic cell hybrids was determined by differential G-11 staining. The slides were then destained and hybridized with biotinylated total horse (Equus caballus) genomic DNA without suppression. Fluorescence detection permitted rapid confirmation of horse chromosomal DNA in the hybrid cells.
Adeyefa CA, Durojaiye OA.Serum samples obtained from 107 Polo horses showing clinical signs of viral respiratory disease were tested for precipitating antibodies to adenovirus by agar gel precipitation test and counter-immunoelectrophoresis method. The results obtained demonstrate serological evidence of adenovirus infection in Polo horses in Nigeria. The counter-immunoelectrophoresis method was observed to be about 3 times more sensitive than the agar gel precipitation test with 19.3 vs 64.5%. It could thus be used to screen a large number of serum samples within a short period.
Chowdhary BP, Harbitz I, Davies W, Gustavsson I.In situ hybridization techniques were used to localize regionally the calcium release channel (CRC) gene on cattle and horse chromosomes, using a porcine CRC cDNA probe. In cattle, the hybridization signal peaked on the 18q23-q26 bands and in horse on the 10pter region. Previous studies have shown that the glucose phosphate isomerase (GPI) gene localizes at the same site in both species, indicating that the two loci are syntenic. As CRC and GPI are syntenic in human, pig and mouse, the present results in cattle and horse represent another example of synteny conservation in the evolution of mam...
Ellegren H, Andersson L, Johansson M, Sandberg K.A synthetic polynucleotide (TG)n was hybridized to equine DNA digested with HinfI and hypervariable hybridization patterns were obtained. Mendelian inheritance of these DNA fingerprinting patterns was confirmed by pedigree analysis. Estimates of the probabilities of identical band patterns in unrelated individuals of different breeds (Swedish Trotters, North Swedish Trotters, Thoroughbreds and Arabians) were in the range 1 x 10(-4) - 7 x 10(-6). The variability derived with the (TG)n probe in horses was higher than what we obtained with several other commonly used probes for DNA fingerprinting...
Ericson KK, Yang TJ.During our studies of cytostatic cytokines in the mixed leukocyte culture, we found that horse serum in the medium control contained a tumor cell growth-inhibitory factor. The fraction isolated by molecular sieving and ion exchange chromatography inhibited the growth and DNA synthesis of the primary culture and passaged cell line of the canine transmissible venereal sarcoma, murine T (L5178Y) and B (P3-X63-Ag8.653) lymphoid tumor cells, murine mammary tumor cells (RIII), bovine lymphoid tumor cells (BL3), and the nontransformed cell line of baby hamster kidney cells. Nontransformed cell lines ...
Kaneko JJ, Rudolph WG, Wilson DW, Carlson GP.Serum bile acids were fractionated by high-performance liquid chromatography (HPLC) in 13 control and 8 cases of liver disease in horses. The severity and type of liver injury was determined by histopathological examination of biopsy and/or necropsy specimens. The total serum bile acids (tSBA) were determined in these horses by an enzymatic method (SBA-EA) and by summation of the bile acids (SBA-LC) as fractionated by the HPLC. The SBA-LC were generally higher than the SBA-EA in both the controls and liver disease and they did not parallel each other. The primary bile acids, total cholates and...
Schuberth HJ, Anders I, Pape U, Leibold W.The cells of 60 randomly selected Hannoveranian warm-blooded horses were subjected to one-dimensional isoelectric focusing and immunoblotting with a cross-reacting monoclonal antibody (Bo 1) recognizing bovine class I antigens. The banding patterns were correlated with the serologically defined specificities of the ELA-A locus. ELA-A2 was correlated with four bands, while ELA-A5, ELA-W18, ELA-A6, ELA-A14 and ELA-A9 were correlated with a single band each. The complexity of the pattern and additional polymorphic bands which could not be correlated to any of the known ELA specificities may indic...
Rashmir-Raven AM, Coyne CP, Fenwick BW, Gaughan EM, Andrews GA, DeBowes RM.The ability of polysulfated glycosaminoglycans (PSGAG) to inhibit the complement cascade was evaluated. The role of complement in inflammation and infection has been well documented. Inhibition of the complement cascade by PSGAG could explain why intra-articularly administered PSGAG diminish diarthrodial joint inflammation and potentiate septic arthritis in horses. Hemolytic complement testing was performed to evaluate the effect of PSGAG on the equine classical and alternate pathways of complement, using rabbit erythrocytes as the target cells. Concentration of PSGAG between 0.2 mg/ml and 0.6...
Karlström K, Essén-Gustavsson B, Hoppeler H, Straub R, Weishaupt M.In order to investigate possible differences in variables defining capillary supply of skeletal muscle derived from two methods, the electron-microscopical and the amylase-PAS histochemical methods were applied in a study of horse and steer muscles. Samples from several locomoter muscles were taken at slaughter from one horse and one steer, divided into two and prepared separately for the two techniques. It was found that there was no difference between the two methods in the values for the capillary-to-fibre ratio. Values for mean fibre area, on the other hand, were significantly higher in th...
Gentry PA, Feldman BF, O'Neill SL, Madigan JE, Zinkl JG.Various haemostatic analytes were systematically evaluated for four months pre-partum and five months post partum in 14 healthy mares. The plasma fibrinogen concentration and both Factor VIII:C and von Willebrand factor activity showed gradual increases from mid-gestation and reached maximal, or near maximal activity at parturition. These increases were paralleled by an increase in plasma fibronectin concentration, the appearance of fibrinogen degradation products, and a modest rise in antithrombin III concentration. In contrast, the activity of Factor VII and Factor IX, and the one-stage prot...
Borovanský J, Vedralová E, Hach P.Concentration of melanosomes in various tissues has been unknown because of the impracticability of their direct quantification. Using an indirect approach comprising the estimation of melanin both in freeze-dried tissue samples and in isolated melanosomes, we obtained data on the amount of melanosomes in various pigment tissues. The concentrations of melanosomes found in the tissues were relatively high, not only reflecting the dark color of pigment tissues but also explaining their capacity to perform various functions ascribed to the presence of melanin.
Okumura M, Fujinaga T, Yamashita K, Tsunoda N, Mizuno S.Ceruloplasmin (Cp) was isolated from fresh equine plasma by precipitation, cellulose chromatography, and improved ion-exchange chromatography. Purified equine Cp is a glycoprotein having a molecular weight of approximately 115,000. In electrophoresis, equine Cp migrated to the alpha 1-globulin region, its isoelectric point was about 4.15 and consisted of about 890 amino acid residues. Serum Cp concentration was measured by use of the single radial immunodiffusion method. In clinically normal horses, the mean (+/- SD) serum Cp concentration of newborn foals was 2.87 +/- 0.40 mg/ml and that of 3...
Tulamo RM.Two recently developed direct methods, radioassay-125I-labeled hyaluronic acid binding protein (125I-HABP)- and high-performance liquid chromatography (HPLC), were used to assess and compare the concentration of hyaluronate (HA) in synovial fluid of horses. Also determined were changes in the HA concentration in an experimental treatment model involving physiologic saline solution (PSS)-irrigated or methylprednisolone acetate-injected tarsocrural joints of clinically normal horses. Serum HA concentration was determined simultaneously, using the 125I-HABP assay. Synovial fluid HA concentration ...
Willis P, Caudle AB, Fayrer-Hosken RA.Objectives of the present research were to determine the influences of types of media, sera, time and hormones on equine oocyte in vitro maturation (IVM). The following types of media and sera were evaluated: Menezo's B2 medium (B2), modified Tissue Culture Medium 199 (TCM), Defined Medium (DM), fetal calf serum (FCS), mare serum collected on the first day of estrus (MS), and mare serum collected on the day of ovulation (MSO). Resultant oocyte maturation was compared with the control: DM with bovine serum albumin (BSA). Effect of culture time (0, 15, and 32 hr) and the following hormones on oo...
Rae C, Board PG, Kuchel PW.In mammalian red blood cells the metabolism of methylglyoxal, and some alpha-ketoaldehydes, takes place via two, generally, highly active enzymes, glyoxalase 1 and 2. The 1H NMR spin-echo spectra of horse erythrocytes, and the various reactants in the glyoxalase system, were characterized as a prelude to obtaining series of spectra in time courses of methylglyoxal metabolism. We characterized the kinetics of the enzyme system in red cells from a normal horse and also from one which had very low activity of glyoxylase 2. The kinetics of the reaction scheme, with methylglyoxal as the starting su...
Derse D, Carvalho M, Carroll R, Peterlin BM.Transcriptional regulatory mechanisms found in lentiviruses employ RNA enhancer elements called trans-activation responsive (TAR) elements. These nascent RNA stem-loops are cis-acting targets of virally encoded Tat effectors. Interactions between Tat and TAR increase the processivity of transcription complexes and lead to efficient copying of viral genomes. To study essential elements of this trans activation, peptide motifs from Tats of two distantly related lentiviruses, equine infectious anemia virus (EIAV) and human immunodeficiency virus type 1 (HIV-1), were fused to the coat protein of b...
Tkachuk-Saad O, Prescott J.Fifty-four strains of Rhodococcus equi from different clinical sources (mainly horses and pigs) were examined for their plasmid content by two screening methods. Plasmids were detected in 49 of 54 strains. A plasmid of approximately 80 kb was isolated from 21 of 22 isolates from horses and 20 of 28 isolates from pigs, and a 105-kb plasmid was isolated from 7 of 28 isolates from pigs. The 80-kb plasmid was significantly associated with strains of equine rather than porcine origin, and the 105-kb plasmid was significantly associated with strains of porcine origin. The type strain, ATCC 6939, con...
Su XZ, Morris DD, McGraw RA.We report the molecular cloning and nucleotide sequence of the equine gene encoding tumor necrosis factor alpha. The 2610-bp genomic sequence was derived from three overlapping polymerase chain reaction products.
Livesey JH, Carne A, Irvine CH, Ellis J, Evans MJ, Smith R, Donald RA.A 41 amino acid peptide, probably identical in structure to human corticotropin releasing factor, was isolated from 70 equine hypothalami by methanol extraction, immunoaffinity chromatography and single step of reverse phase HPLC. The amino acid sequence was determined by gas phase sequence analysis. Probable carboxyl terminal amidation was demonstrated by similar retention times for equine and human corticotropin releasing factor on reverse phase HPLC at pH 8. The likely structure of equine corticotropin releasing factor is: Ser-Glu-Glu-Pro-Pro- Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu...
Rautschka R, Stanek C, Knezevic PF.In seven sound horses histamine plasma levels were evaluated by means of a radioenzymatic method using the enzyme histamine-N-methyltransferase (mean = 4.46 nmol/l, SD = 1.55 nmol/l). Histamine levels of laminitic horses were significantly higher (n = 11; mean = 12.46 nmol/l, SD = 3.24 nmol/l). In three sound horses plasma histamine levels were determined over a two week period following a double administration of 10 mg dexamethasone-21-isonicotinate/100 kg body weight. Histamine levels remained within the range of sound control horses.
Szenci O, Brydl E, Bajcsy CA.The stability of blood ionized calcium (Ca2+) and acid-base variables in equine, bovine, ovine, and canine venous blood samples (n = 15, in each group) stored at 4 C for 3, 6, 9, 24, or 48 hours was studied. Variables included blood Ca2+ and standard ionized calcium (Ca2+ corrected to pH 7.4) concentrations, pH, blood carbon dioxide and oxygen tensions, base excess, bicarbonate concentration, and total carbon dioxide content. Results indicate that storage of blood samples at 4 C for up to 48 hours, despite appreciable acid-base changes, is associated with less than 1.5% change in equine, bovin...
Morozova L, Haezebrouck P, Van Cauwelaert F.The thermal denaturation of Ca(2+)- and apo-forms of equine lysozyme was followed by using far and near UV circular dichroism and intrinsic fluorescence methods. The difference found between the temperature dependence of the ellipticity at 222 nm and 287 nm, which show two stages in the thermal transition, and those at 228 nm and 294 nm, which indicate only one stage over a wide range of temperatures reflects that different subdivisions of the protein molecule are characterized by a different stability, cooperativity and pathway of denaturation. The first transition, reflected in the increase ...
Dhawedkar RG, Jain NC, Mount ME, Bowling AT, Vegad JL.An enzyme-linked immunosorbent assay (ELISA) was standardised and applied for the detection of antiplatelet and antineutrophil antibodies using a heterologous system consisting of equine platelets or neutrophils and antisera raised in rabbits. The standardised technique consisted of using Immulon type 3 plate, 1 per cent gelatine as a blocking solution, poly-L-lysine buffer as a coating solution, unfixed antigen, 90 microliters test serum, horseradish peroxidase conjugated antibody and o-phenylenediamine dihydrochloride as a substrate. The number of unfixed platelets or neutrophils required fo...
Stanley S, Jeganathan A, Wood T, Henry P, Turner S, Woods WE, Green M, Tai HH, Watt D, Blake J.We have raised antibodies to morphine and etorphine and developed one-step enzyme-linked immunosorbent assays (ELISA) for these drugs as part of a panel of post race tests for drugs in racing horses. These tests are simple, can be completed in 2 h, and can be read by visual inspection. The morphine ELISA has an I50 for morphine of about 1.5 ng/mL, while the etorphine ELISA has an I50 for etorphine of 250 pg/mL. Cross-reactivity studies show that the antimorphine antibody cross-reacts well with levorphanol, hydromorphone, and oxycodone, while the anti-etorphine antibody showed no cross-reactivi...
Freeman DA, Butler JE, Weber JA, Geary RT, Woods GL.Oviductal and uterine embryos were collected from mares at 5 to 7 days following ovulation 1) to evaluate the effects of oviductal tissue explants on in vitro growth and development of equine embryos and 2) to study the morphologic development of equine embryos in culture. Embryos were incubated for 5 days in a medium (control group) or in medium supplemented with oviductal tissue explants (co-culture group). Embryos were evaluated and the media changed daily. Following 5 days in culture, 10 10 (100%) control embryos and 27 29 (93%) co-cultured embryos had doubled in diameter. All embryos that...
Houpt KA.Some of the techniques that may be used to study social, reproductive, and ingestive behavior in horses are described in this paper. One of the aspects of equine social behavior is the dominance hierarchy or patterns of agonistic behavior. Paired or group feeding from a single food source may be used to determine dominance hierarchies quickly. Focal animal studies of undisturbed groups of horses may also be used; this method takes longer, but may reveal affiliative as well as agonistic relationships among the horses. Reproductive behavior includes flehmen, the functional significance of which ...
Morris DD, Moore JN, Crowe N.Over a 24-month period, serum tumor necrosis factor (TNF) activity was determined in 289 horses with colic attributable to gastrointestinal tract disease. Serum TNF activity was quantitated by use of a modified in vitro cytotoxicity bioassay, using WEHI 164 clone-13 murine fibrosarcoma cells. Causes for colic, determined by clinical and laboratory evaluation, exploratory celiotomy, or necropsy included: gastrointestinal tract rupture (GTR); ileal impaction; small intestinal strangulating obstruction (SIO); proximal enteritis (PE); transient small intestinal distention; large-colon displacement...
Weber JA, Freeman DA, Vanderwall DK, Woods GL.This study was conducted to identify embryonic products whose secretion was temporally associated with the oviductal transport period of the mare. Chemicals secreted by oviductal-transport-stage equine embryos were identified by incubating Day 6 or Day 7 early uterine embryos with 35S-methionine/cysteine, 3H-progesterone, or 3H-arachidonic acid for 24 h, and subsequently identifying radioactively labeled proteins (SDS-PAGE; n = 3 embryos), steroids (HPLC; n = 3 embryos), or prostaglandins (HPLC; n = 3 embryos) in the culture medium. Early uterine embryos secreted 116.1 +/- 45.5 pg of prostagla...
Biswas B, Mukherjee D, Mattingly-Napier BL, Dutta SK.Genomic amplification by the polymerase chain reaction (PCR) was used to identify a unique genomic sequence of Ehrlichia risticii directly in DNA isolated from peripheral-blood buffy coat cells of E. risticii-infected horses (Potomac horse fever) and from infected cell cultures. A specific primer pair, selected from a cloned, species-specific, 1-kb DNA fragment of the E. risticii genome as a template, was used for the amplification of the target DNA of 247 bp. The optimal number of 40 PCR cycles, determined by analyzing an amplification profile obtained with a constant Taq polymerase concentra...
Stratford CH, Pemberton A, Cameron L, McGorum BC.Although a presumptive diagnosis of acute grass sickness (AGS) can be made on the basis of clinical signs, a definitive ante mortem diagnosis currently requires histological examination of enteric ganglia. Development of an accurate noninvasive ante mortem diagnostic test is therefore warranted. The objective of this study was to determine whether quantification of the plasma concentrations of the heavily phosphorylated form of major neurofilament subunit NF-H (pNF-H), which mirror the degree of axonal degeneration in some human and animal neurodegenerative disorders, could distinguish AGS-aff...
Yi R, Sandhu J, Zhao S, Lam G, Loganathan D, Morrissey B.Efaproxiral (RSR 13) is an experimental synthetic allosteric modifier of haemoglobin (Hb) that acts by increasing the release of oxygen from Hb to the surrounding tissues. It has been shown to increase maximum oxygen uptake (VO(2max)) in a canine skeletal muscle model. The ability to increase maximal muscle oxygen uptake makes efaproxiral a potential performance-enhancing agent and is therefore prohibited by the World Anti-Doping Agency. In this study, a method for the detection and elimination of efaproxiral in equine plasma and urine after a 2.5 g intravenous administration of efaproxiral ...
McGuire TC.Isolated equine immunoglobulin (Ig)G(T) antibodies to equine infectious anemia virus P26 antigen did not precipitate with antigen when the ratio of antibody to antigen was high. However, at lower ratios of antibody to antigen precipitation occurred. In addition, complement-fixation by IgG and P26 antigen was inhibited by high concentrations of IgG(T). The unusual reaction pattern noted with IgG(T) antibodies was still detectable by the immunodiffusion test for equine infectious anemia virus. In situations of nonprecipitability by IgG(T), the adjacent positive control line was inhibited, and th...
Brunso L, Segura D, Monreal L, Escolar G, White JG, Diaz-Ricart M.Studies in animal models are useful to understand the basic mechanisms involved in hemostasis and the functional differences among species. Ultrastructural observations led us to predict differences in the activation and secretion mechanisms between equine and human platelets. The potential mechanisms involved have been comparatively explored in the present study. Equine and human platelets were activated with thrombin (0.5 U/ml) and collagen (20 µg/ml), for 90 seconds, and samples processed to evaluate: i) ultrastructural changes, by electron microscopy, ii) actin polymerization and cy...
Estepa JC, Garfia B, Gao PR, Cantor T, Rodriguez M, Aguilera-Tejero E.Parathyroid hormone (PTH) plays a critical role in the regulation of mineral metabolism in mammals. Until recently, the standard method for PTH measurement has been the 2nd generation intact-PTH (I-PTH) assay. Current evidence indicates that the I-PTH assay binds to the PTH molecule and to an inactive N-terminally truncated PTH fragment that tends to accumulate in the blood of uraemic patients. Therefore, a new 3rd generation PTH assay that detects only the whole PTH molecule (W-PTH; cyclase-activating PTH [CAP]) has been developed. Objective: To validate this more specific W-PTH assay for mea...
Hernández-Avilés C, Ramírez-Agámez L, Pearson M, Beckham AMN, Varner DD, Love CC.Analysis of sperm morphology is an important part of the stallion breeding soundness evaluation since it provides an objective measure of a stallion's sperm quality and is one of many factors that estimate a stallion's fertility potential. To describe the effect of sperm quality level on the technique (Differential Interference Contrast - DIC; Phase-contrast - PH; Dip-Quick staining - DQ; and eosin-nigrosin staining - EN; semen samples fixed in buffered-formal saline) and evaluator (three evaluators; using only DIC), stallions were categorized based on sperm quality into three categories: High...
Dennis VA, Klei TR, Chapman MR, Jeffers GW.Eosinophils and neutrophils from ponies with Strongylus vulgaris-induced eosinophilia (eosinophilic ponies; activated eosinophils and neutrophils) were assayed in vitro for chemotactic and chemokinetic responses to zymosan-activated serum (ZAS) using the filter system in Boyden chambers, for Fc and complement (C) receptors using the EA and EAC-rosette assays, respectively, and for phagocytic and bactericidal activities using opsonized Escherichia coli and the acridine orange method. The responses of activated eosinophils and neutrophils in the above assays were compared with those of eosinophi...
Brown JE, Corstvet RE, Stratton LG.Two experiments incorporating 13 mares were conducted for the purpose of producing and monitoring intrauterine infection with Klebsiella pneumoniae. In the pilot study, the infection was produced with strains of K pneumoniae type 68 and type 10 isolated from the genital tract of stallions with a history of breeding problems. In the principal study, K pneumoniae type 68 was used to produce the infection. Tampons and guarded culture swabs were used to obtain uterine samples in the pilot study. In comparing the efficacies of isolation of K pneumoniae with the tampons and isolation with standard g...
Diaz-Espiñeira M, Escolar E, Bellanato J, De La Fuente MA.Atomic spectrometry has been used in 20 samples of equine urinary sabulous deposits in order to detect minor elements accompanying the predominant element, calcium, which is present in the form of calcium carbonate (calcite and/or vaterite). The elements measured have been (besides calcium) magnesium, sodium, potassium, iron, copper and manganese. Phosphates, sulphates and silica are frequently present as minor constituents of equine urinary sabulous deposits and uroliths, but their detection can be difficult by infrared (IR) spectroscopy in the original samples due to overlapping with the ban...
Petrov KK, Dicks LM.The aim of this study was to determine which of the two species, Fusobacterium necrophorum or Dichelobacter nodosus, are associated with hoof thrush in horses. Fourteen hoof samples, collected from eight horses with thrush and 14 samples collected from eight horses with healthy hooves, were examined for the presence of F. necrophorum, Fusobacterium equinum and D. nodosus. Only isolates with phenotypic characteristics representing Fusobacterium could be cultured. Total DNA extracted from the 28 hoof samples was amplified by using DNA primers designed from gene lktA, present in F. necrophorum su...
Dunkel B, Rickards KJ, Page CP, Cunningham FM.Platelet activation occurs in human obstructive airway diseases and in laboratory animal models. However, there is limited evidence that platelets may be involved in equine recurrent airway obstruction (RAO) and other inflammatory diseases. This study investigated whether platelet activation also occurred in RAO. Objective: Platelet function is altered in ponies with active RAO. This alteration can be detected ex vivo by measuring platelet adhesion. Methods: An in vitro platelet adhesion assay measuring acid phosphatase (AcP) activity colorimetrically was adapted for use with equine platelets ...
Bailly-Chouriberry L, Cormant F, Garcia P, Kind A, Popot MA, Bonnaire Y.Cobra venom (Naja kaouthia) contains a toxin called α-cobratoxin (α-Cbtx). This toxin is a natural protein containing 71 amino acids (MW 7821 Da) with a reported analgesic potency greater than morphine. In 2007, in USA, this substance was found in the barns of a thoroughbred trainer and since then till date, the lack of a detection of this molecule has remained a recurring problem for the horseracing industry worldwide. To solve this problem, the first method for the detection of α-cobratoxin in equine plasma has now been developed. Plasma sample (3 mL) was treated with ammonium sulfate at ...
Kwok WH, Leung DK, Leung GN, Wan TS, Wong CH, Wong JK.A rapid liquid chromatography-tandem mass spectrometry (LC-MS-MS) method was developed for the simultaneous screening of 19 drugs of different classes in equine plasma using automated on-line solid-phase extraction (SPE) coupled with a triple quadrupole mass spectrometer. Plasma samples were first protein precipitated using acetonitrile. After centrifugation, the supernatant was directly injected into the on-line SPE system and analysed by a triple quadrupole LC-MS-MS in positive electrospray ionisation (+ESI) mode with selected reaction monitoring (SRM) scan function. On-line extraction and c...
Scarth JP, Spencer HA, Hudson SC, Teale P, Gray BP, Hillyer LL.In this study, the use of equine liver/lung microsomes and S9 tissue fractions were used to study the metabolism of the androgenic/anabolic steroid stanozolol as an example of the potential of in vitro technologies in sports drug surveillance. In vitro incubates were analysed qualitatively alongside urine samples originating from in vivo stanozolol administrations using LC-MS on a high-resolution accurate mass Thermo Orbitrap Discovery instrument, by LC-MS/MS on an Applied Biosystems Sciex 5500 Q Trap and by GC-MS/MS on an Agilent 7000A. Using high-resolution accurate mass full scan analysis o...
Berg LC, Lenz J, Kjelgaard-Hansen M, Thomsen PD, Jacobsen S.More sensitive and specific diagnostic methods for early detection of changes in the joint cartilage are needed. Cartilage-derived retinoic acid-sensitive protein (CD-RAP) is a potential marker of cartilage synthesis and regeneration. This is the first study on equine CD-RAP. Objective: To evaluate the ability of a commercially available human sandwich ELISA assay to detect equine CD-RAP in synovial fluid from healthy and diseased joints. Methods: Synovial fluid was collected from 28 horses with no signs of joint disease and from 5 with induced inflammatory arthritis. CD-RAP concentrations wer...
Sanada Y, Noda H, Nagahata H.A fluorometric assay was applied to evaluate blastogenesis of equine lymphocytes. Optimal culture conditions were as follows; concentrations of phytohaemagglutinin-P (PHA), concanavalin A (Con A) and pokeweed mitogen (PWM) were 1 microgram/ml, 40 micrograms/ml and 10 micrograms/ml, respectively, when 5 X 10(5) lymphocytes were incubated with culture medium containing 20% pooled horse serum (PHS) for 120 hours. The relative mean stimulation index of healthy non-pregnant mares were 5.107 +/- 0.323 (M +/- SE) with PHA, 4.019 +/- 0.183 with Con A and 3.610 +/- 0.131 with PWM. Sequentially the blas...
Sutton DG, Preston T, Love S.Validation of a reliable, noninvasive clinical test for quantification of equine orocaecal transit time (OCTT) is required. This would facilitate an evidence-based approach to investigation and treatment of equine small intestinal disorders. Objective: 1) Comparison of the lactose (13) C-ureide breath test (LUBT) with the hydrogen breath test (H(2) BT) for OCTT measurement. 2) Identification of the characteristics of gastrointestinal microbial glycosylureide hydrolase activity in vitro. 3) Production of an optimised protocol for the LUBT for in vivo measurement of equine OCTT. Objective: Signi...
Gahne B, Juneja RK.Cattle and horse plasma samples of known post-albumin types were radiolabelled with 14C-vitamin D3. These samples were then analysed by polyacrylamide gel electrophoresis, followed by autoradiography. The patterns observed were identical to those of post-albumin variants. The polymorphic post-albumin protein of cattle and horse was thus identified as the vitamin D binding protein and homologous to the Gc protein of human plasma.
Kimball FA, Wyngarden LJ.Preliminary studies indicate the presence of PGF2alpha specific binding sites in membrane fractions prepared from equine corpora lutea. The equilibrium binding data indicate an apparent dissociation constant of 3.2 X 10(-9)M and the concentration of binding sites of -0.1 pmoles/mg membrane protein. Competition of several natural prostaglandins for equine luteal PGF2alpha specific binding sites indicates specificity for the 9alpha-hydroxyl moiety and the 5,6-cis doublebond. Significant increases in relative binding affinities were demonstrated for PGF2alpha analogs with a phenyl ring introduced...
Wassall DA, Gregory RJ, Phipps LP.The detection of antibodies against Trypanosoma equiperdum in 689 equid sera was compared by enzyme-linked immunosorbent assay (ELISA), the complement fixation test (CFT) and an indirect immunofluorescent test (IIF). CFT was the least sensitive technique, susceptible to anti-complementary factors and the most technically demanding. IIF was more sensitive, but was only suitable for testing limited numbers of samples. In this study, ELISA was the most sensitive test, the least labour intensive and lends itself to a considerable degree of automation. It is suggested that ELISA would be relatively...
Boorman S, Hanson RR, Velloso Alvarez A, Zhong K, Hofmeister E, Boone LH.To determine the effect of concurrent versus delayed treatment with corticosteroid on equine articular tissues also treated with local anesthetic in vitro in the presence of inflammatory mediators. Methods: Controlled laboratory study. Methods: Five geldings, one mare (aged 3-18 years). Methods: From each horse, 24 synovial and 12 osteochondral explants were cultured in a 12-well plate (2 wells/group, 2 synovial and 1 osteochondral explant/well, total 216 explants in the study). Explants were stimulated in culture medium with 10 μg/ml recombinant equine interleukin-1β and 10 μg/ml tumor...
Hewson J, Arroyo LG.Evaluation of the upper and lower respiratory tract of horses requires strategic selection of possible diagnostic tests based on location of suspected pathologic lesions and purpose of testing and must also include consideration of patient status. This article discusses the various diagnostic modalities that may be applied to the respiratory system of horses under field conditions, indications for use, and aspects of sample collection, handling, and laboratory processing that can impact test results and ultimately a successful diagnosis in cases of respiratory disease.
Miserez R, Frey J, Krawinkler M, Nicolet J.A polymerase chain reaction (PCR) for identification of Taylorella equigenitalis was developed. The oligonucleotide primers are based on the DNA sequence of the rrs gene of T. equigenitalis, encoding for the 16S ribosomal RNA. Analysis of 21 strains of T. equigenitalis from England, USA and Switzerland showed an amplification product of 410 bp with identical Sau3A restriction profile. The sensitivity of the PCR-Assay was estimated to detect 50 to 500 bacteria of T. equigenitalis in a mixture with frequently found contaminants. Further analysis of culture from 60 genital swabs, taken in the cou...
Vilasi S, Dosi R, Iannuzzi C, Malmo C, Parente A, Irace G, Sirangelo I.In protein deposition disorders, a normally soluble protein is deposited as insoluble aggregates, referred to as amyloid. The intrinsic effects of specific mutations on the rates of protein aggregation and amyloid formation of unfolded polypeptide chains can be correlated with changes in hydrophobicity, propensity to convert alpha-helical to beta sheet conformation and charge. In this paper, we report the aggregation rates of buffalo, horse and bovine apomyoglobins. The experimental values were compared with the theoretical ones evaluated considering the amino acid differences among the sequen...
Mason DE, Muir WW, Olson LE.Smooth muscle strips from the midcervical portion of the trachea and bronchial smooth muscle strips from third-generation airways of horses were placed in tissue baths, and isometric contractile force was measured. Active force was measured in response to electrical stimulation and exogenous acetylcholine. Square-wave electrical stimuli were applied at various voltages (10, 12, 15, 18, 20, 25 V), frequencies (3, 5, 10, 15, 20, 25, 30 Hz), and pulse durations (0.2, 0.5, 1.0, 1.5, 2.0 ms). Isometric contractile force increased as voltage, frequency, and pulse duration increased. Maximal contract...
Hassall L, Rigsby P, Stickings P.The International Standard for Diphtheria Antitoxin Equine is essential for the standardisation of assays used to determine the potency of therapeutic diphtheria antitoxin products produced from equine serum. This paper describes the production and characterization of the 2nd International Standard for Diphtheria Antitoxin Equine and its calibration in International Units. Calibration was performed by toxin neutralization test in vivo and in vitro (Vero cell assay), and potency was expressed relative to the 1st International Standard to ensure continuity of the International Unit. The candidat...
Rathi R, Nielen M, Cheng FP, van Buiten A, Colenbrander B.Subfertility in stallions is attributed to the inability of spermatozoa to undergo the acrosome reaction in response to progesterone. In the present study, it was assessed whether there is a correlation between stallion fertility, defined on the basis of first cycle foaling rate and first cycle 'non-return rate', and the proportion of spermatozoa with exposed progesterone receptors on their plasma membranes. Semen from Dutch Warmblood (n=10) and Friesian (n=4) stallions was analysed. Progesterone 3-(o-carboxymethyl) oxime-BSA coupled with fluorescein isothiocyanate was used as a progesterone r...
Haezebrouck P, Noppe W, Van Dael H, Hanssens I.From fluorescence measurements on mixtures of bis-ANS and equine lysozyme and from Ca(2+)-dependent hydrophobic interaction chromatography of equine lysozyme, it is demonstrated that Ca2+ binding induces a conformational change upon which hydrophobic regions in the protein become less accessible. Bis-ANS fluorescence titrations in the absence of Ca2+ and in 2 mM Ca2+ are also performed with equine alpha-lactalbumin variants B and C. These variants differ by an amino-acid exchange Asp----Ile at residue 95. The fluorescence titration curves indicate that the accessibility of the probe to the Ca2...
Kyvsgaard NC, Høier R, Brück I, Nansen P.Ionizing irradiation and binary ethylenimine treatment have previously been shown to be effective for in-vitro inactivation of virus in biological material. In the present study the 2 methods were tested for possible effects on measurable concentrations of reproductive hormones in equine plasma (luteinizing hormone (LH), folliclestimulating hormone (FSH), progesterone (P4), and oestradiol-17 beta (E2)). The inactivation methods were electron beam irradiation with a dose from 11 to 44 kGy or treatment with binary ethylenimine (BEI) in concentrations of 1 and 5 mmol/L. Generally, there was a clo...
Orsolini MF, Verstraete MH, van Heule M, Orellana D, Ortega A, Meyers S, Dini P.Intracytoplasmic sperm injection (ICSI) is the only method for in vitro embryo production (IVP) in horses. Besides oocyte developmental competence, the outcome of IVP is also highly dependent on sperm quality. Therefore, it is not only essential to employ superior methods of selecting high quality sperm, but also to be able to characterize which quantifiable properties of sperm quality are most indicative of its fertility. In men, a net negative surface charge, estimated by zeta potential (ZP) is highly correlated with sperm quality and in vitro embryo developmental outcomes. However, there ...
