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.
Fransson LA, Havsmark B.The structure of dermatan sulfate-chondroitin sulfate
copolymers, isolated from horse aorta, has been examined.
It was found that a large proportion of the galactosaminoglycans of this tissue was obtained as a discrete polysaccharide fraction with an L-iduronic acid to D-glucuronic
acid ratio of approximately 1: 2. This finding together with
infrared data indicated that the polymer contained approximately equimolar proportions of the three repeating disaccharide units glucuronosyl-N-acetylgalactosamine 4-sulfate
(A), iduronosyl-N-acetylgalactosamine 4-sulfate (B), and
glucuronosyl-N-acet...
Moore RW, Redmond HE, Katada M, Wallace M.A continuous-passage horse leukocyte culture [V.B. 40, abst. 4672] was susceptible to the virus of equine infectious anaemia, as determined by cyto-pathic effect and viral titre. Ultrafiltration studies indicated that the virus was less than 32 mμ in diameter, which agrees with previous reports. Susceptible horses developed clinical signs and lesions of the disease when they were inoculated with both unfiltered and ultrafiltered culture virus.
Yamaguchi M, Miyazawa Y, Sekine T.Myosin was prepared from smooth muscle of horse esophagus in good yield
(about 15 ° mg/Ioo g tissue) and was designated myosin S. Its properties were compared
with those of myosin A from skeletal muscle.
The ratio of the absorption of myosin S at 280 nm to that at 26o nm was about
1.8, and the amount of contaminating phosphorus was only o.91 g/io 5 g of myosin S,
indicating that the latter is free of nucleic acid. The purity of this protein was examined
by ultracentrifugation, gel filtration in the presence of 0.5 M KC1 and 6 M urea and
chromatography on DEAE-cellulose columns. These e...
Hoffsis GF, Murdick PW, Tharp VL, Ault K.The plasma concentration of cortisol and corticosterone in the normal horse was determined by competitive protein-binding analysis (radiostereoassay). The mean plasma concentration of the 2 steroids in resting horses was 5.12 μg./ml., with standard deviation of 1.67 μg. There were no significant differences in the mean values according to age, sex, and pregnancy. Seemingly, diurnal variations in concentrations of the steroids existed in horses; values were high in the morning and low in the evening. Plasma values were increased in horses given corticotropin (acth) and were decreased in those...
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...
Auer DE, Ng JC, Seawright AA.Polymorphonuclear leukocytes (PMNLs) were isolated from an inflammatory exudate induced in the intercarpal joints of horses by an administration of carrageenin. Their superoxide production at rest and following stimulation with either serum-treated zymosan (STZ) or phorbol myristate acetate (PMA) was measured by cytochrome-c reduction. Stimulation of the cells increased the cytochrome-c reduction 10-15 times that of resting cells. The maxima were 20 nmol of reduced cytochrome-c per 10(6) cells per ml at 120 min (STZ) and 35 nmol of reduced cytochrome-c per 10(6) cells per ml at 60 min (PMA). T...
Hisey EA, Ross PJ, Meyers S.As standard in vitro fertilization is not a viable technique in horses yet, many different techniques have been used to create equine embryos for research purposes. One such method is parthenogenesis in which an oocyte is induced to mature into an embryo-like state without the introduction of a spermatozoon, and thus they are not considered true embryos. Another method is somatic cell nuclear transfer (SCNT), in which a somatic cell nucleus from an extant horse is inserted into an enucleated oocyte, creating a genetic clone of the donor horse. Due to limited availability of equine oocytes in t...
van den Hoven R, Meijer AE, Wensing T, Breukink HJ.Gluteal muscle specimens were taken from 4 horses. From 1 of the 4 gluteal muscles, serial sections were prepared. Individual muscle fibers were identified and studied, using photomicrographs of sections stained by different enzyme histochemical methods. In specimens in which cytoplasmic soluble enzymes were studied, use was made of the semi-permeable membrane technique to hamper enzyme diffusion into reaction fluids. Enzymes involved in glycogenolysis, glycolysis, the tricarboxylic acid cycle, synthesis of reduced nicotinamide adenine dinucleotide phosphate, the pentose phosphate cycle, the a...
Smith RF, Jackson LS, Moore A.An 125I radioimmunoassay to determine the pattern of urinary excretion of etorphine (a semisynthetic opiate agonist) after its administration to horses is described. Three thoroughbred horses were each given 5, 15, 30 and 100 micrograms of etorphine intramuscularly. Urine was collected for up to 72 after administration. The maximum etorphine concentration after administration of a dose of 5 micrograms was 711 pg ml-1 (concentrations were greater than 100 pg ml-1 after 23 h in all three horses); a 15 micrograms gave 2661 pg ml-1 (levels remained above 100 pg ml-1 for more than 44 h in each hors...
da Silva TRO, Gonçalves PNC, Marcus VB, Mucellini CI, Dos Santos IR, Kommers G, Driemeier D, Flores EF, Cargnelutti JF, Flores MM.For approximately one decade, a novel papillomavirus termed Equus caballus papillomavirus-2 (EcPV-2) has been associated with equine penile/preputial papillomas and squamous cell carcinomas (SCCs). It is currently believed that the virus has a carcinogenic activity, being able to induce such neoplastic lesions. After being first described, EcPV-2 has been detected in many countries from North America, Europe, and Asia; however, to date, it has not been reported in Brazil. The aim of this research was to investigate the presence of EcPV-2 in penile/preputial papillomas and SCCs of Brazilian hor...
Ishiyama T, Nishimori T, Kato M, Yamada H, Sato K, Sentsui H.Restriction endonuclease analysis of equine herpesviruses 1 (EHV-1) and 4 has been investigated using cultured cells infected with these viruses. The DNA cleavage patterns of these viruses were observed in the intracellular DNA after digestion with Eco RI and electrophoresis. This procedure was applied to the diagnosis of equine herpesvirus infection in aborted equine fetuses. The characteristic Eco RI restriction pattern of EHV-1 DNA was directly detectable in the emulsion of lungs collected from aborted equine fetuses.
Hernandez SM, Begum L, Keller LE, Fu Q, Zhang S, Fortier LA.To identify chondroprotective factors as potential disease-modifying osteoarthritis treatments using an unbiased, bottom-up proteomics approach. Methods: Paired equine cartilage explants and synovial membrane were collected postmortem from 4 horses with no history of lameness and grossly normal joints at necropsy. Methods: Six groups were established: cartilage, synoviocytes, and cartilage + synoviocytes (coculture), all with or without interleukin (IL)-1β. The catabolic effect of IL-1β was verified by glycosaminoglycan (GAG) released from cartilage into media by 1,9-dimethyl-methylene blue ...
Read NG, Trist DG.The uptake of mepacrine by isolated horse polymorphonuclear leucocytes (PMN) was measured using spectrophotofluorimetry. Two phases of uptake were observed, the first, rapid fraction, essentially complete by 10 min, and a second, slow fraction, which was still proceeding after 60 min. The appearance of mepacrine within the PMN was also visualized by fluorescence microscopy. Discrete yellow points of fluorescence were observed in the cytoplasm of PMN within 30 s. These discrete points corresponded both in size and number to the PMN granules. After 5 min, the nuclei showed faint fluorescence whi...
Tomić S, Sesartić L, Tomasić J.1. 14C-labelled methyl 2,6-di-O-pivaloyl-alpha-D-glucopyranoside (1) was used as a substrate for esterases from rabbit, guinea pig, mouse, donkey, pig, horse, sheep and human sera. 2. Stepwise de-esterification of the diester substrate 1 occurred with rabbit, guinea pig and mouse serum. Data on time-course experiments and kinetic data are reported. 3. The use of donkey, pig, horse, sheep and human serum led to the migration of the 2-O-pivaloyl group in substrate 1 to the position 4- in the sugar molecule, followed by stepwise de-esterifications of both 1 and the newly formed methyl 4,6-di-O-pi...
Yamamoto M, Yamamoto I, Tanaka Y, Sugano M.1. A method for the purification of horse serum lecithin:cholesterol acyltransferase has been established. 2. The method involves the adsorption of the enzyme from diluted horse serum on DEAE-Sephadex A-50, (NH4)2SO4 fractionation, 1-butanol treatment, and chromatographic techniques of DEAE-Sepharose CL-6B, DEAE-Sephadex A-50, Affi-Gel blue and hydroxylapatite. 3. The resultant enzyme preparation essentially formed a single main band when subjected to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. 4. The final purification of the enzyme was 20,000-fold with 7% yi...
Gysens L, Martens A, Haspeslagh M.Bovine papillomavirus (BPV) types 1 and 2 are causally associated with equine sarcoid, the most common mesenchymal neoplasm of horses, but the viral load (VL) differs between lesions. Sensitive and accurate BPV detection and quantification is essential for clinicians to confirm clinical suspicion, as well as in research settings for stratifying these skin lesions. Due to the limitations of histopathology in sarcoid diagnosis, PCR screening of superficial swabs constitutes the principal sampling method for BPV detection. This study aimed to investigate the ability of superficial swabs and fine-...
Lindgren G, Swinburne JE, Breen M, Mariat D, Sandberg K, Guérin G, Ellegren H, Binns MM.A horse bacterial artificial chromosome (BAC) library was screened for 19 microsatellite markers from unassigned or non-oriented linkage groups. Clones containing 11 (AHT20, EB2E8, HMS45, LEX005, LEX014, LEX023, LEX044, TKY111, UCDEQ425, UCDEQ464 and VIASH21) of these were found, which were from eight different linkage groups. The BAC clones were used as probes in dual colour FISH to identify their precise chromosomal origin. The microsatellite markers are located on nine different horse chromosomes, four of which (ECA6, ECA25, ECA27 and ECA28) had no previously in situ assigned markers.
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...
Jacob RJ, Steiner MR.Equine herpesvirus 3 (EHV-3)-infected equine cells display a kinetics of infected cell polypeptide (ICP) synthesis at 34 degrees C that is typical of coordinate cascade gene regulation of herpesviruses. In contrast, when infected cell cultures are incubated at the restricted temperature of 39 degrees C, the shift from early (beta) gene expression to late (gamma) gene expression is perturbed, i.e., there is an accumulation of early (beta) gene products and a decrease in, or absence of, late (gamma) gene products. Some of the affected late (gamma) gene products may be glycoproteins since these I...
Jones S, Blackmore DJ.The distribution of the isoenzymes of aspartate aminotransferase (AST, E.C. 2.6.1.1.) in equine tissues has been studied to ascertain whether the organ of origin may be identified when the total AST activity of serum is raised. Most tissues contain 3 isoenzymes of cytoplasmic origin (cAST) with isoelectric points of 5.6, 5.7 and 5.9, and one isoenzyme of mitochondrial (mAST) origin with an isoelectric point of 9. Serum from horses with azoturia contained an additional cytoplasmic subform with an isoelectric point of 5.8. This form could not be generated by ageing, freezing and thawing or bindi...
Chen CL, Neilson JT, Kumar MS, Estes KS.Highly purified equine prolactin was prepared from equine pituitary glands (hypophysis) by serial extractions with water at pH 5.5, 0.1 M (NH4)2SO4 at pH 4.0, and 0.25 M (NH4)2SO4 at pH 5.5 to remove other hormones, and then finally with 70% ethanol at pH 9.3 to 10.0 to extract prolactin. Preliminary purification of the extract involved salting out other substances with 0.1% NaCl at pH 9.0. Prolactin was precipitated out by adding three times the volume of 95% ethanol at 4 C. This prolactin preparation had a biological potency of 24 IU/mg. Further purification by isoelectric focusing on a pH g...
White KL, Thomson DL, Wood TC.An indirect immunofluorescence assay was used to detect the presence of H-Y antigen on equine blastocysts. A total of 33 blastocyst stage horse embryos were collected 6 to 7 days post-ovulation by trans-cervical flush and were immediately evaluated for the presence of H-Y antigen. Additionally, 17 embryos, were collected and cultured for 72 h to the expanded blastocyst stage and similarly evaluated. Embryos were placed in medium containing monoclonal antibodies to H-Y antigen followed by incubation in medium containing 1/10 (v/v) fluorescein isothiocyanate conjugated goat anti-mouse IgM Fc spe...
Krüger B, Siesenop U, Böhm KH.In 1993 and 1994 a highly increased occurrence of equine dermatophilosis was observed, and a study was initiated to determine phenotypic heterogeneity among 120 clinical isolates using biochemistry, antibiotic resistance profiles, membrane protein profiles and Western blotting. The biochemical examinations contained 1% equine serum in medium. Moreover, the API ZYM-test from bioMérieux was used. The biochemical reactions were suited to identify Dermatophilus congolensis but did not allow a differentiation among the various isolates. Antibiotic resistance in one or more isolates was observed ag...
Decloedt AI, Bailly-Chouriberry L, Vanden Bussche J, Garcia P, Popot MA, Bonnaire Y, Vanhaecke L.Traditionally, steroids other than testosterone are considered to be synthetic, anabolic steroids. Nevertheless, in stallions, it has been shown that β-Bol can originate from naturally present testosterone. Other precursors, including phytosterols from feed, have been put forward to explain the prevalence of low levels of steroids (including β-Bol and ADD) in urine of mares and geldings. However, the possible biotransformation and identification of the precursors has thus far not been investigated in horses. To study the possible endogenous digestive transformation, in vitro simulations of t...
Sandusky GE, Wightman KA.Specific cell populations in the pituitary glands of the rat, cat, pig, and human being were positive for thyroid-stimulating hormone (TSH), luteinizing hormone (LH), and follicle-stimulating hormone (FSH). When reacted with prediluted rabbit anti-human TSH, LH, and FSH, antisera were not positive for the demonstration of these hormones in the horse, cow, or dog. Immunocytochemical staining was obtained in the horse, cow, and dog by the use of a primary antiserum against a specific beta-subunit of bovine TSH. The immunocytochemical staining of TSH, LH, FSH, adrenocorticotropic hormone, growth ...
Hardee GE, Tshabalala MA, Moore JN, Gokhales RD.Separate high performance liquid chromatographic methods were developed for thiabendazole (TBZ) and 5-hydroxy thiabendazole (5-OH-TBZ) determination in horse plasma using 1-methyl-2-phenyl benzimidazole (MPBZ) as an internal standard. In both methods TBZ and 5-OH-TBZ were extracted from plasma using organic solvents, injected on to a C-18 column, and eluents monitored by a fluorescence detector. However, mobile phase composition, extraction solvent as well as detector wavelength differed in the two methods. The linear range for TBZ was 0.02 to 0.77 microgram ml-1 while that for 5-OH-TBZ was 0....
Bhavnani BR, Woolever CA.A mixture of [4,8,12-14C]farnesyl pyrophosphate and [3H]dehydroepiandrosterone was injected into a horse fetus im during laparotomy, after which maternal urine was collected for 6 days. Steroid conjugates in the urine were extracted with Amberlite XAD-2 resin, hydrolyzed, and separated into phenolic and neutral fractions. Estrone, 17 alpha-estradiol, equilin [3-hydroxy-1,3,5(10),7-estratetraen-17-one], and 17 alpha-dihydroequilin [1,3,4(10),7-estratetraene-3,17 alpha-diol] were isolated from the phenolic fraction and their radiochemical purities were established. Only estrone and 17 alpha-estr...
Mathes RL, Dietrich UM, Krunkosky TM, Hurley DJ, Reber AJ.To establish a reproducible method for the culture of primary equine corneal epithelial cells, keratocytes, and endothelial cells and to describe each cell's morphologic characteristics, immunocytochemical staining properties and conditions required for cryopreservation. Methods: Corneas from eight horses recently euthanized for reasons unrelated to this study were collected aseptically and enzymatically separated into three individual layers for cell isolation. The cells were plated, grown in culture, and continued for several passages. Each cell type was characterized by morphology and immun...
Silberzahn P, Dehennin L, Zwaïn IH, Leymarie P.Testosterone has been identified by mass spectrometry in blood and follicular fluid aspirated from mature Graafian follicles of mares. Quantitative measurements made by gas chromatography-mass spectrometry have validated the determination of plasma testosterone made by radioimmunoassay. However, because of high levels of epitestosterone (17 alpha-hydroxyandrost-4-en-3-one) in the follicular fluid, radioimmunoassay overestimates the true concentrations of testosterone. The occurrence of testosterone in mare follicular fluid at a concentration which is two orders of magnitude higher than that in...
Nielsen AH, Gotfredsen P, Nielsen PB, Hyttel P, Poulsen K.1. Species specific problems complicating the measurement of prorenin and renin concentrations were studied in bovine, hog and horse plasma. 2. In contrast to horse renin, bovine and hog renin reacted with rat angiotensinogen, allowing measurement of the plasma renin concentration in cattle and hog with rat angiotensinogen as exogenous substrate. 3. Trypsin treatment of plasma in order to activate prorenin generated an interfering angiotensin I immunoreactive material in all three species, most extensively in horse plasma. 4. This material could be removed in bovine and hog plasma by a cation-...
Wang CC, Hartmann-Fischbach P, Krueger TR, Wells TL, Simonson A, Compton JC.Due to opiorphin's analgesic and antidepressant functions, its illicit use is rumored in some racing jurisdictions. Opiorphin is very difficult to detect due to its hydrophilic nature and rapid degradation in plasma and urine samples. METHODOLOGY & RESULTS: We have developed a sensitive, reliable method for opiorphin detection and confirmation in equine samples, using EDTA to inhibit analyte degradation between the time of collection and analysis. Opiorphin was extracted by weak cation exchange followed by analysis using HILIC-MS/MS. The method was validated and the LOD was determined to b...
Ogorevc J, Poklukar K, Dovč P.Skin-derived tissue cultures are a useful model to study molecular mechanisms of skin renewal and pathogenesis of dermal diseases. Horses often suffer from skin diseases, skin trauma and problems with proper wound healing, which could be improved by in vitro grown keratinocyte grafts. Herein we describe establishment and characterization of equine skin-derived primary cell cultures, using enzymatic and explant methods. The established cell lines of primary equine keratinocytes (peK) maintained high proliferative capacity for over five passages and expressed different epithelial/keratinocyte-s...
Wong JKY, Kwok WH, Chan GHM, Choi TLS, Ho ENM, Jaubert M, Bailly-Chouriberry L, Bonnaire Y, Cawley A, Ming Williams H, Keledjian J, Brooks L....Acadesine, 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside, commonly known as AICAR, is a naturally occurring adenosine monophosphate-activated protein kinase (AMPK) activator in many mammals, including humans and horses. AICAR has attracted considerable attention recently in the field of doping control because of a study showing the enhancement of endurance performance in unexercised or untrained mice, resulting in the term 'exercise pill'. Its use has been classified as gene doping by the World Anti-Doping Agency (WADA), and since it is endogenous, it may only be possible to control del...
Sharma AK, Kathikeyan S, Kaur P, Singh TP, Yadav MP.Lactoferrin is an iron-binding glycoprotein with a molecular weight of 80 kDa. The protein has two iron binding sites. It has two structural lobes, each housing one Fe(3+) and the synergistic CO(3)(2-) ion. The protein was isolated from the colostrum/milk of mares maintained at National Research Centre on Equines, Hisar, India. The purified samples of the protein were crystallized using a microdialysis method. The protein was dialysed against low ionic strength buffer solution. Several crystal forms were obtained, out of which three were characterized which have cell dimensions as follows. For...
Skrabalak DS, Henion JD.Betamethasone and its major unconjugated metabolite, 6-beta-hydroxybetamethasone, were detected in equine urine by thin-layer chromatography and characterized by micro-liquid chromatography/mass spectrometry (micro-LC/MS). Their structures were confirmed by a combination of infrared spectroscopy and nuclear magnetic resonance spectroscopy.
