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.
Budnitskaya P, Gapanhuk E, Henriques OB.The kininogenase activity of trypsin, plasmin, plasma kallikrein and heated Bothrops venom was compared, using fresh, heated and heat-acid-denatured horse plasma as source of kininogen. The venom kininogenase was found to have the highest activity on fresh horse plasma, followed by plasmin and trypsin which were equally active, and plasma kallikrein which was half as active as plasmin on these substrates. Plasmin and trypsin released more kinin from heat-treated than from fresh plasma whereas kallikrein released half as much as it liberates from fresh plasma. On heat-aciddenatured plasma equal...
Helms CM, Allen PZ.Immunodiffusion analysis of papain digestion products, heavy and light chains of horse IgG-globulins with several rabbit and anti-horse IgG sera, have permitted the demonstration of five antigenic specificities (Fc1, Fc2, L, Lsp and Fabsp) associated with these equine antigens. Reactivity with anti-Fc1 is shown by both F′c and Fc fragments, while anti-Fc2 reactivity is shown only by Fc fragment.
Absorption of anti-Fab serum with L chain Fc fragment provides a reagent (anti-Fabsp) which precipitates only with Fab fragment, IgG-globulin or reduced and alkylated IgG. Upon exposure to deterge...
Correa WM.Smears and imprints were made from the liver of 27 equine fetuses, believed to have aborted as a result of Equine Virus Abortion (EVA) infection. Several different fixatives and staining techniques were employed for the demonstration of typical intra-nuclear inclusion bodies in these preparations, and the following conclusions were reached. Methanol proved to be the best fixative and Pappenheim's panoptic method was the best staining technique, giving good contrast and definition of the inclusion bodies. Cytological methods provided a simple and rapid means of diagnosis, but histological secti...
Richter GW, Moppert GA, Lee JC.1. Fingerprints of tryptic digests of apoferritins from a human liver, horse spleens and ACI rat livers were made by means of electrophoresis and chromatography on microcrystalline cellulose, and were compared.
2. All tryptic peptides also present in apoferritins from the human liver and the horse spleens were also present in apoferritin from the rat livers.
3. In the digests of horse and of rat apoferritin there was a peptide that was not present in the digests of human apoferritin. Another peptide was obtained from human and from rat apoferritin, but not from horse apoferritin.
4. T...
Sundararaman B, Vershinina AO, Hershauer S, Kapp JD, Dunn S, Shapiro B, Green RE.Hybridization capture approaches allow targeted high-throughput sequencing analysis at reduced costs compared to shotgun sequencing. Hybridization capture is particularly useful in analyses of genomic data from ancient, environmental, and forensic samples, where target content is low, DNA is fragmented and multiplex PCR or other targeted approaches often fail. Here, we describe a DNA bait synthesis approach for hybridization capture that we call Circular Nucleic acid Enrichment Reagent, or CNER (pronounced 'snare'). The CNER method uses rolling-circle amplification followed by restriction dige...
Moore RM, Venugopalan CS, Sedrish SA, Holmes EP.To determine in vitro contractile responses of equine colonic veins to various vasoconstrictor agents. Methods: Colonic veins collected from 8 adult horses. Methods: Veins were cut into 4-mm-wide rings, placed in organ baths at 37 C, and attached to a force-transducer interfaced with a polygraph; 2 g of tension was applied, and rings were allowed to equilibrate for 45 minutes. Bath solution was replaced, and tension was reapplied at 15-minute intervals. Cumulative concentration responses (10(-8) to 10(-4) M) were determined for each agent, using separate rings (n = 8). Three vein groups were e...
Accogli G, Douet C, Ambruosi B, Martino NA, Uranio MF, Deleuze S, Dell'Aquila ME, Desantis S, Goudet G.Glycoprotein oligosaccharides play major roles during reproduction, yet their function in gamete interactions is not fully elucidated. Identification and comparison of the glycan pattern in cumulus-oocyte complexes (COCs) from species with different efficiencies of in vitro spermatozoa penetration through the zona pellucida (ZP) could help clarify how oligosaccharides affect gamete interactions. We compared the expression and localization of 12 glycosidic residues in equine and porcine in vitro-matured (IVM) and preovulatory COCs by means of lectin histochemistry. The COCs glycan pattern diffe...
Foster NM, Barber TL, Walton TE.Venezuelan equine encephalomyelitis (VEE) TC-84 vaccinal virus, from 10-1. quantities of infected duck embryo fibroblast cell culture fluids, was isolated by combined continuous-flow centrifugation with isopycnic banding in sucrose. Most of the recovered infectivity and hemagglutinating activity were in a single band at a buoyant density (rho) of 1.2. About 90% of the total input protein (450-520 mg) was removed with the effluent, whereas most of the remaining 10% also banded at a rho of 1.2. Infectivity was inactivated with formalin at a final concentration of 0.05% at 37 degrees C for 24 hr....
Wang X, Bou G, Zhang X, Tao L, Shen Y, Na R, Liu G, Ren H, Ren X, Song L, Su S, Bai D, Zhao Y, Li B, Dugarjaviin M.Having considered that the current methods are costly and time-consuming, we designed an only 3 pairs primer-based PCR test to accurately identify the species and gender in horses, donkeys, mules and hinnies. Through a thorough sequence comparison between horse and donkey's highly similar genomes, and a vast amount of preliminary confirmation, we found that three fragments, CNGB3 gene on an autosome, displacement loop region on mitochondrial DNA and SRY genes on chromosome Y, within these equine DNA, are enough to enable us achieving our goal. The PCR test described here would be an economical...
Brinkmann J, Koudelka T, Keppler JK, Tholey A, Schwarz K, Thaller G, Tetens J.The production and consumption of mare's milk in Europe has gained importance, mainly based on positive health effects and a lower allergenic potential as compared to cows' milk. The allergenicity of milk is to a certain extent affected by different genetic variants. In classical dairy species, much research has been conducted into the genetic variability of milk proteins, but the knowledge in horses is scarce. Here, we characterize two major forms of equine αS2-casein arising from genomic 1.3 kb in-frame deletion involving two coding exons, one of which represents an equid specific duplicati...
Jeronimo C, Archambault D.Equine arteritis virus (EAV), an enveloped positive-stranded RNA virus, is the prototype of the arterivirus group. In a previous paper (A. Kheyar, S. Martin, G. St.-Laurent, P. J. Timoney, W. H. McCollum, and D. Archambault, Clin. Diagn. Lab. Immunol. 4:648-652, 1997), we have shown that the unglycosylated membrane (M) protein, which is composed of 162 amino acids (aa), is a major target of equine antibody to EAV. In order to determine the antigenic regions of the M protein, the cDNA encoding the M protein of EAV was inserted into the procaryotic expression vector pGEX-4T-1 to produce recombin...
Samper JC, Morris CA.Various factors affect the success of AI with frozen-thawed semen in horses. Stallion variability is thought to be one of the major factors, but semen processing and evaluation techniques, thawing protocols, packaging systems and timing of insemination are far from standardized among laboratories. Our objective was to survey current methods for stallion semen cryopreservation used commercially around the world. From the answers to the questions in the survey, we attempted to provide an overview of procedures that are standard as well as those that are used by only few laboratories and to revie...
Rumpler MJ, Sams RA, Colahan P.We describe a validated, rapid, sensitive, and specific UHPLC-MS/MS method to detect and quantify glycopyrrolate in 0.5 mL of horse urine. Further, we investigated the elimination of glycopyrrolate in urine after both intravenous and oral administration of clinically relevant doses to Thoroughbred horses. Quantification was performed by weighted, linear regression analysis using a deuterated analogue of glycopyrrolate as internal standard (IS). The method was characterized by a linear range of 5-2500 pg/mL, a lower limit of quantification of 5 pg/mL and a limit of detection of 1 pg/mL. The int...
Bartosik J, Łojek J, Długosz E, Górski P, Zygner W.Tapeworm infections in Konik Polski horses from Biebrza National Park were investigated in this study. Faecal samples were collected 10 times: in 2012 - 1 time, in 2013 - 4 times, in 2014 - 4 times and in 2015 - 1 time. In total, 162 faecal samples were collected and tested. Faecal egg counts (FECs) method was used in the study. Positive samples with cestode eggs were noted only twice - in October 2012 and December 2013 in two adult mares (9 and 11 years old). The determined prevalence was surprisingly low comparing to other studies, 4.3% in October 2013 and 28.5% in December 2013. Parasite ge...
Yu NH, Ho EN, Wan TS, Wong AS.Recombinant human erythropoietin (rhEPO), darbepoetin alfa (DPO) and methoxy polyethylene glycol-epoetin beta (PEG-EPO) are synthetic analogues of the endogenous hormone erythropoietin (EPO). These erythropoiesis-stimulating agents have the ability to stimulate the production of red blood cells and are commercially available for the treatment of anaemia in humans. These drugs are understood to have performance-enhancing effects on human athletes due to their stimulation of red blood cell production, thereby improving delivery of oxygen to the muscle tissues. Although their effect on horses has...
Dubin A, Potempa J, Turyna B.Horse blood leucocyte cytosol exhibits a broad inhibitory activity against serine proteinases. The purified inhibitor was exposed to investigated enzymes (trypsin, chymotrypsin, elastases and serine proteinase from S. aureus) for variable time and the products were analyzed by gradient polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The molar ratio I:E, association rate constants k on and inhibition constants Ki for the enzymes and inhibitor were determined. The examined elastases form stable, stoichiometric complexes with the inhibitor (Ki less than 10(-10) M), ...
Rubessa M, Feugang JM, Kandel ME, Schreiber S, Hessee J, Salerno F, Meyers S, Chu I, Popescu G, Wheeler MB.The capacity for microscopic evaluation of sperm is useful for assisted reproductive technologies (ART), because this can allow for specific selection of sperm cells for in vitro fertilization (IVF). The objective of this study was to analyze the same sperm samples using two high-resolution methods: spatial light interference microscopy (SLIM) and atomic force microscopy (AFM) to determine if with one method there was more timely and different information obtained than the other. To address this objective, there was evaluation of sperm populations from boars and stallions. To the best of our k...
Rifkind JM.The oxidation of horse hemoglobin by Cu(II) has been followed by the changes in the electron spin resonance spectra of copper. By stopped-flow and freeze-quenching techniques, it is shown that the second-order rate constant for the binding of Cu(II) to hemoglobin is greater than 5 X 10(5) mol-1 s-1 and the apparent first-order rate for the reduction of Cu(II) to Cu(I) is 0.051 s-1. It is also shown that the binding of Cu(II) to hemoglobin is followed by an alteration of the Cu(II) spectrum, decreasing the g values. This process has an apparent rate constant of 17 s-1 and presumably involves a ...
Cota LO, Júnior OS, Oliveira APL, Paz CFR, Maróstica TP, Duarte PC, Leme FOP, Faleiros RR.Systemic inflammatory response syndrome (SIRS) is a common condition in horses with gastrointestinal disorders. If not prevented or controlled, SIRS promotes multiple organ dysfunctions that may culminate in serious disabilities or even death. The objective of this study was to evaluate the effects of Lithothamnion supplementation on systemic inflammatory response and organ function variables in horses undergoing oligofructose overload (OFO) intake. Twelve healthy horses were randomly divided into control and treated groups. The treated group received Lithothamnion (100 mg/kg bw PO BID) for 7 ...
Baxter GM, Moore JN, Tackett RL.The in vitro responses of isolated vascular preparations of digital arteries and veins obtained from healthy anaesthetised horses were determined for dopamine and fenoldopam. The digital vessels were harvested, cut into 4 mm vascular segments, suspended in tissue baths and attached to force-displacement transducers. Dose-response studies between 10(-8) and 10(-4)M concentrations were performed for all drugs. The change in tension of each vascular ring was measured in grams of force. The reactivity between palmar and plantar digital vessels and baseline vascular responses were determined for do...
Gallicano KD, Park HC, Young LM.A sensitive method was required to analyze low levels of camphor in equine urine and plasma. Camphorated oil (20% w/w camphor) was administered topically (6 g) and intratracheally (1 g) to standardbred mares. The drug was extracted from urine and plasma by diethyl ether and analyzed as its 2,4-dinitrophenylhydrazone derivative by reverse phase HPLC with UV detection. The UV detector was set at 368.5 nm and the samples were eluted from the C18 column by 82% acetonitrile in water. The detection limit achieved was about 10 ng/mL urine and about 20 ng/mL plasma. After topical administration, only ...
Scarth JP, Clarke AD, Teale P, Pearce CM.Effective detection of the abuse of androgenic-anabolic steroids in human and animal sports often requires knowledge of the drug's metabolism in order to target appropriate urinary metabolites. 'Designer' steroids are problematic since it is difficult to obtain ethical approval for in vivo metabolism studies due to a lack of a toxicological profile. In this study, the in vitro metabolism of estra-4,9-diene-3,17-dione is reported for the first time. This is also the first study comparing the metabolism of a designer steroid in the three major species subject to sport's doping control; namely th...
Laroucau K, Colaneri C, Jaÿ M, Corde Y, Drapeau A, Durand B, Zientara S, Beck C.To evaluate the routine complement fixation test (CFT) used to detect Burkholderia mallei antibodies in equine sera, an interlaboratory proficiency test was held with 24 European laboratories, including 22 National Reference Laboratories for glanders. The panels sent to participants were composed of sera with or without B mallei antibodies. This study confirmed the reliability of CFT and highlighted its intralaboratory reproducibility. However, the sensitivity of glanders serodiagnosis and laboratory proficiency may be improved by standardising critical reagents, including antigens, and by dev...
Hussain KA, Issel CJ, Schnorr KL, Rwambo PM, West M, Montelaro RC.Monoclonal antibodies (MCAbs) were used to dissect the antigenic sites of the surface glycoproteins of the prototype cell-adapted Wyoming strain of equine infectious anemia virus (EIAV). Serologic reactivities of these MCAbs were determined by ELISA, additive ELISA, competitive ELISA, and Western blot assays. The results indicated that antigenic reactivity of gp90 was localized on at least four distinct epitopes, two of which were important in neutralization. Our studies also revealed that these epitopes were localized on overlapping antigenic sites on gp90. On the other hand, only two distinc...
Thway TM, Wolfe MW.Equids and primates are the only species known to express the placental hormone chorionic gonadotropin (CG). CG is a member of the heterodimeric glycoprotein family and is composed of an alpha subunit linked to a hormone-specific beta subunit. Previously, we have reported that epidermal growth factor (EGF) regulates the equine glycoprotein hormone alpha subunit promoter through a protein kinase C (PKC)/mitogen-activated protein kinase (MAPK) signal transduction pathway in trophoblasts. The current study investigates the regulatory element/factors involved in the induction of equine glycoprotei...
Morrell JM, Dalin AM, Rodriguez-Martinez H.A new, simpler, technique of colloidal centrifugation has recently been developed, designated single layer centrifugation (SLC). This technique requires evaluation by comparison with a density gradient for its ability to select the best quality spermatozoa and its practicality of use on studfarms. Objective: To compare the effect of 2 methods of colloidal centrifugation, density gradient centrifugation and single layer centrifugation, on stallion sperm motility, yield and survival, using freshly collected extended stallion semen. Methods: Aliquots of extended stallion semen from 10 stallions (...
Hans A, Gaudaire D, Manuguerra JC, Leon A, Gessain A, Laugier C, Berthet N, Zientara S.This study shows that an unbiased amplification method applied to equine arteritis virus RNA significantly improves the sensitivity of the real-time reverse transcription-quantitative PCR (RT-qPCR) recommended by the World Organization for Animal Health. Twelve viral RNAs amplified using this method were hybridized on a high-density resequencing microarray for effective viral characterization.
Clegg PD, Jones MD, Carter SD.Loss of articular cartilage, which is the most important pathological lesion occurring in osteoarthritis, has been shown to be enzymatically mediated. The matrix metalloproteinases (MMPs) are a group of enzymes which have been implicated in this degradation of articular cartilage matrix. The use of pharmacological agents to inhibit this catabolic process in the joint is a potential route for therapeutic intervention. The gelatinase MMPs, MMPs-2 and 9, were purified by affinity chromatography from equine cell cultures. The ability of phenylbutazone, flunixin, betamethasone, dexamethasone, methy...
Bewley TA, Li CH.The conformations of human, equine, and porcine pituitary prolactins, as evidenced by various optical properties, have been compared. The alpha-helix contents of all three proteins are essentially identical to each other (60 +/- 5%), as well as to prolactins isolated from other mammalian species. Direct absorption (zero and second-order), difference absorption, fluorescence emission, and circular dichroism spectra suggest that the majority of tyrosine and tryptophan side chains in these three proteins exist in very similar microenvironments within the folded forms of the hormones. Thus, the ge...
Weidolf LO, Chichila TM, Henion JD.Methods for screening by thin-layer chromatography, quantification by high-performance liquid chromatography with ultraviolet detection and confirmation by gas chromatography-mass spectrometry of boldenone sulfate in equine urine after administration of boldenone undecylenate (Equipoise) are presented. Sample work-up was done with C18 liquid-solid extraction followed by solvolytic cleavage of the sulfate ester. Confirmatory evidence of boldenone sulfate in equine urine was obtained from 2 h to 42 days following a therapeutic intramuscular dose of Equipoise. The use of 19-nortestosterone sulfat...
Martin G, Plowright R, Chen C, Kault D, Selleck P, Skerratt LF.Hendra virus (HeV) is lethal to humans and horses, and little is known about its epidemiology. Biosecurity restrictions impede advances, particularly on understanding pathways of transmission. Quantifying the environmental survival of HeV can be used for making decisions and to infer transmission pathways. We estimated HeV survival with a Weibull distribution and calculated parameters from data generated in laboratory experiments. HeV survival rates based on air temperatures 24 h after excretion ranged from 2 to 10 % in summer and from 12 to 33 % in winter. Simulated survival across the distri...
Adolf GR, Traxler E, Maurer-Fogy I.Equine interferon-beta 1 (EqIFN-beta 1) was purified from extracts of recombinant Escherichia coli by sequential chromatography on hydroxylapatite, anion-, and cation-exchangers. The resulting protein was greater than 98% pure as determined by sodium dodecylsulfate gel electrophoresis, gel permeation HPLC, and reverse-phase HPLC. Amino-terminal amino acid sequencing revealed that essentially all molecules contained an additional amino-terminal methionine. The specific antiviral activity of EqIFN-beta 1 determined on equine dermal fibroblasts challenged with vesicular stomatitis virus (VSV) was...
Schnabel LV, Mohammed HO, Miller BJ, McDermott WG, Jacobson MS, Santangelo KS, Fortier LA.Platelet rich plasma (PRP) has recently been investigated for use in tissue regeneration studies that seek to utilize the numerous growth factors released from platelet alpha-granules. This study examined gene expression patterns, DNA, and collagen content of equine flexor digitorum superficialis tendon (SDFT) explants cultured in media consisting of PRP and other blood products. Blood and bone marrow aspirate (BMA) were collected from horses and processed to obtain plasma, PRP, and platelet poor plasma (PPP). IGF-I, TGF-beta1, and PDGF-BB were quantified in all blood products using ELISA. Ten...
Pellegrini A, Hägeli G, Fretz D, von Fellenberg R.An electrophoretic procedure for the qualitative and quantitative assay of protein protease inhibitors is reported. This assay is particularly suited for investigations of crude biological materials when specific antisera are not available. The supporting medium consists of agarose into which denatured fibrinogen is incorporated as the substrate for proteases. The processing then is divided into two steps: (1) electrophoretic resolution of the inhibitor containing material and (2) detection of the inhibitor bands through their protease inhibiting activity. The inhibitor position is thus made v...
