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.
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.
Walter I, Klein M, Handler J, Aurich JE, Reifinger M, Aurich C.To evaluate changes of glycoconjugate in uterine glands of endometrial tissues obtained from mares. Methods: adult mares. Methods: Uterine biopsy samples were collected during the breeding season and analyzed histologically for signs of chronic endometrial degeneration. Stage of the estrous cycle was established, using clinical examination and determination of hormonal status. Uterine tissue samples were analyzed, using lectin histochemical and immunohistochemical techniques (estrogen and progesterone receptors). Connective tissues were stained to determine alterations of ground substance in p...
Haruta N, Aki M, Ozaki S, Watanabe Y, Kitagawa T.Conformational change of myoglobin (Mb) accompanied by binding of a ligand was investigated with 244 nm excited ultraviolet resonance Raman Spectroscopy (UVRR). The UVRR spectra of native sperm whale (sw) and horse (h) Mbs and W7F and W14F swMb mutants for the deoxy and CO-bound states enabled us to reveal the UVRR spectra of Trp7, Trp14, and Tyr151 residues, separately. The difference spectra between the deoxy and CO-bound states reflected the environmental or structural changes of Trp and Tyr residues upon CO binding. The W3 band of Trp7 near the N-terminus exhibited a change upon CO binding...
Hines R, Maury W.In vivo, tissue macrophages have been implicated as an important cell for the replication of equine infectious anemia virus (EIAV). Laboratory investigations of EIAV/macrophage interactions, however, have been hampered by the laborious blood monocyte isolation procedures. In addition, adherent equine macrophage cultures generally have poor long-term viability and are resistant to transfection. This report describes an adherent canine macrophage-like cell line, DH82, that supports the replication of EIAV. This cell line was easily transfectable and supported EIAV Tat transactivation of the LTR....
Lieto LD, Cothran EG.A cDNA library was built using RNA extracted from the skin tissue of an adult horse. The library was primed with oligo (dT) and sequences were directionally inserted in order to produce an expression library. The library has 5.8X 10(5) plaque forming units with 99.6% recombinant phage. The average insert size is 1.3 Kbp. Three hundred and thirteen expressed sequence tags (ESTs) were generated from sequencing of the 5 prime end of randomly selected skin cDNA clones. The ESTs were sequenced on an ABI 377 using Big-Dye chemistry. A similarity search was performed on each EST using the NCBI non-re...
Suire S, Stewart F, Beauchamp J, Kennedy MW.The equine conceptus is surrounded by a fibrous capsule that persists until about day 20 of pregnancy, whereupon the capsule is lost, the conceptus attaches to the endometrium and placentation proceeds. Before attachment, the endometrium secretes in abundance a protein of the lipocalin family, uterocalin. The cessation of secretion coincides with the end of the period during which the conceptus is enclosed in its capsule, suggesting that uterocalin is essential for the support of the embryo before direct contact between maternal and foetal tissues is established. Using recombinant protein and ...
Kirschvink N, Fiévez L, Dogné S, Bureau F, Art T, Lekeux P.Solute analysis in bronchoalveolar lavage fluid involves the use of dilutional markers to correct for variable recovery of pulmonary epithelial lining fluid (PELF). Urea is the best characterised endogenous marker, whereas inulin appears to meet the requirements of an exogenous marker. In horses, the use of inulin has never been investigated and the impact of lower airway diseases such as heaves, on PELF recovery is unknown. In this study, five healthy and five heaves-affected horses underwent airway endoscopy and bronchoalveolar lavage. PELF recovery from bronchoalveolar lavage was calculated...
Goubran Botros H, Poncet P, Rabillon J, Fontaine T, Laval JM, David B.A new allergen from horse dander, Equ c 5 has been purified. Its biochemical and biophysical properties have been characterized and compared with those of Equ c 1, Equ c 2 and Equ c 4. Their molecular masses, determined by mass spectrometry, were 22 kDa for Equ c 1, 16 kDa for Equ c 2, 18.7 kDa for Equ c 4 and 16.7 kDa for Equ c 5. Their pI values were between 3.8 and 5.25. Equ c 2 and Equ c 5 are not glycosylated, while Equ c 4 contains a tri-antennary tri-sialylated N-linked glycan. Linkages of terminal N-acetylneuraminic acid to galactose were: alpha-(2-->6) in Equ c 4, and both alpha-(2...
Jensen TK, Boye M, Bille-Hansen V.Fluorescent in situ hybridization, immunohistochemistry, and Grocott's methenamine-silver nitrate staining were compared as diagnostic methods for Pneumocystis carinii pneumonia in formalin-fixed lung tissue from foals and pigs. An oligonucleotide probe targeting 18S ribosomal RNA of P. carinii was designed for in situ hybridization, and a commercially available monoclonal antibody was used for immunohistochemistry. Samples from six foals and 10 pigs with P. carinii pneumonia, as verified by Grocott's methenamine-silver nitrate staining, were examined concurrently with samples from seven anima...
Martens A, De Moor A, Ducatelle R.The purpose of this study was to examine if bovine papilloma virus (BPV) DNA can be detected in superficial swabs or scrapings from equine sarcoids. Samples were obtained from 92 sarcoids and 20 non-sarcoidal control lesions. The polymerase chain reaction technique was used with a first primer set to check whether DNA extraction was successful, and with a second primer set specific for BPV-DNA. DNA isolation was successful in 88% of the swabs and 93% of the scrapings. All control lesions were negative for BPV-DNA.
Tetens J, Barker SA, Waguespack M, Hosgood G.The objectives were to use high performance liquid chromatography (HPLC) to validate an established method for adenine nucleotide separation in equine colonic mucosal tissue, to determine the inherent variability in the tissue and extraction method, and to determine the stability of ATP, ADP, and AMP in the tissue with time. Equine colonic mucosal tissue obtained from a single horse was immediately submersed in liquid nitrogen, and stored at -70 degrees C. Samples were lyophilized, extracted, and separated by HPLC. The limit of quantitation was 0.05 microg/mL. The coefficient of variation for ...
Theoret CL, Barber SM, Moyana TN, Gordon JR.To map the expression of transforming growth factor (TGF)-beta(1), TGF-beta(3), and basic fibroblast growth factor (bFGF) in full-thickness skin wounds of the horse. To determine whether their expression differs between limbs and thorax, to understand the pathogenesis of exuberant granulation tissue. Methods: Six wounds were created on one lateral metacarpal area and one midthoracic area of each horse. Sequential wound biopsies allowed comparison of the temporal expression of growth factors between limb and thoracic wounds. Methods: Four 2- to 4-year-old horses. Methods: Wounds were assessed g...
Nagarajan MM, Simard C.A nested polymerase chain reaction (PCR) amplifying a region of the gag gene of equine infectious anemia virus (EIAV) was developed for the rapid and direct detection of proviral DNA from the peripheral blood of naturally infected horses and was compared with the Coggins test. DNA prepared from white blood cells of 122 field horses from 15 stables with reported cases of EIAV and one seronegative stable were analysed. Amplifications of expected size fragments were obtained by nested PCR for 88 horses using two different sets of primers targeting the gag region. The specificity of the amplified ...
Lorch G, Hillier A, Kwochka KW, Saville WJ, Kohn CW, LeRoy BE.To compare a radioallergosorbent test and 2 ELISA with intradermal testing for the determination of environmental allergen hypersensitivity in horses with and without atopic diseases. Methods: Prospective clinical study. Methods: 10 horses with recurrent urticaria, 7 with atopic dermatitis, 16 with chronic obstructive pulmonary disease, and 22 without atopy. Methods: History, physical examination, hemogram, serum biochemical analyses, bronchoalveolar lavage, and an intradermal test (used as the criterion standard) with a regional panel of 73 allergens were performed in all horses. Serum was an...
Zhou EM, Riva J, Clavijo A.An immunoglobulin M (IgM) capture enzyme-linked immunosorbent assay (MC-ELISA) was developed for the detection of primary infection of vesicular stomatitis virus (VSV) in equine and swine sera. The test was based on the use of biotinylated sheep antibodies against equine or swine IgM molecules bound to a streptavidin-coated ELISA plate. The captured IgM antibodies were detected by application of antigens prepared from the New Jersey and the Indiana VSV serotypes (VSV-NJ and VSV-IN, respectively) and mouse polyclonal antibodies against VSV-NJ and VSV-IN. The MC-ELISA was compared to a competiti...
McDaniel TK, Dewalt KC, Salama NR, Falkow S.Because of limited genetic tools for use in Helicobacter pylori, tests routinely applied in other bacteria for demonstrating a gene's role in viability and other phenotypes have not been applied to this organism. In a mutational study of putative response regulator genes, we aimed to develop such tools for H. pylori. Methods: We attempted to mutate five response regulator genes by allelic exchange insertional mutagenesis. For genes that yielded no viable mutants, a second copy of the gene was inserted into the chromosome via a suicide vector, and it was seen if providing the second copy would ...
Kingston JK, Bayly WM, Sellon DC, Meyers KM, Wardrop KJ.To investigate the effects of sodium citrate, low molecular weight heparin (LMWH), and prostaglandin E1 (PGE1) on aggregation, fibrinogen binding, and enumeration of equine platelets. Methods: Blood samples obtained from 4 Thoroughbreds. Methods: Blood was collected into syringes in the ratio of 9 parts blood:1 part anticoagulant. Anticoagulants used were sodium citrate, LMWH, sodium citrate and LMWH, or 300 nM PGE1/ml of anticoagulant. Platelet aggregation in response to ADP, collagen, and PGE1 was assessed, using optical aggregometry. Platelet activation was evaluated, using flow cytometry, ...
Tobiasch E, Kehm R, Bahr U, Tidona CA, Jakob NJ, Handermann M, Darai G, Giese M.Equine arteritis virus (EAV) is a member of the Arteriviridae family, that includes lactate dehydrogenase-elevating virus (LDV), porcine reproductive and respiratory syndrome virus (PRRSV), and simian haemorrhagic fever virus (SHFV). Equine arteritis is a contagious disease of horses and is spread via respiratory or reproductive tract. The objective of the present study is to evaluate the possibility for developing a model system for prevention horses against an EAV infection by DNAvaccination. A cDNA bank from the RNA of EAV was established. This gene library contains the translation unit of ...
The Journal of parasitologyApril 25, 2001
Volume 87, Issue 2 345-353 doi: 10.1645/0022-3395(2001)087[0345:COTOIO]2.0.CO;2
Dubey JP, Liddell S, Mattson D, Speert CA, Howe DK, Jenkins MC.Neospora hughesi was isolated in cell cultures inoculated with homogenate of spinal cord from a horse in Oregon. Tachyzoites of this Oregon isolate of N. hughesi were maintained continuously by cell culture passage and tachyzoites were infective to immunosuppressed mice. Gamma interferon gene knockout (KO) mice injected with tachyzoites developed fatal myocarditis and numerous tachyzoites were seen in lesions. Gerbils (Meriones unguiculatus) inoculated with tachyzoites developed antibodies (> or = 1:500) as indicated by the Neospora caninum agglutination test but did not develop clinical si...
Rho JY, Currey JD, Zioupos P, Pharr GM.The equine radius is a useful subject for examining the adaptation of bone histology to loading because in life the anterior cortex is loaded almost entirely in tension, the posterior cortex in compression. The histology of the two cortices is correspondingly different, the osteones and the interstitial lamellae in the posterior cortex having a more transversely oriented fibre arrangement than those in the anterior cortex. Presumably as a result of this histological difference, the posterior cortex is stronger in compression than the anterior cortex; the anterior cortex is stronger in tension ...
Walter I.Uterine tubes from cow, sheep, horse, pig, dog, cat, rabbit, and guinea pig were examined immunohistochemically for presence and distribution of contractile cells in the mucosal lamina propria. We tested for the presence of alpha-smooth muscle actin, tropomyosin, desmin, smooth muscle myosin (heavy chain) and cytoskeletal proteins vimentin and cytokeratin. Immunostaining for alpha-smooth muscle actin showed two distribution patterns: either positive cells were freely dispersed in the mucosal lamina propria or a distinct subepithelial layer of positive cells was seen. The first distribution pat...
Nunez R, Gomes-Keller MA, Schwarzwald C, Feige K.RATIONALE: Thrombocytopenia is a platelet associated process that occurs in human and animals as result of i) decreased production; ii) increased utilization; iii) increased destruction coupled to the presence of antibodies, within a process know as immune-mediated thrombocytopenia (IMT); or iv) platelet sequestration. Thus, the differentiation of the origin of IMT and the development of reliable diagnostic approaches and methodologies are important in the clarification of IMT pathogenesis. Therefore, there is a growing need in the field for easy to perform assays for assessing platelet morpho...
Mariat D, Oustry-Vaiman A, Cribiu EP, Raudsepp T, Chowdhary BP, Guérin G.In order to increase the number of markers on the horse cytogenetic map and expand the integration with the linkage map, an equine BAC library was screened for genes and for microsatellites. Eighty-nine intra-exon primers were designed from consensus gene sequences in documented species. After PCR screening, 38 clones containing identified genes were isolated and FISH mapped. These data allowed us to refine the available Zoo-FISH results, to define ten new conserved cytogenetic segments and expand two others, thus leading to the identification of a total of 26 conserved segments between horse ...
Henderson K, Stewart J.A dipstick, competitive immunoassay for rapidly measuring serum oestrone sulfate (OS) concentrations in horses was developed to distinguish mares 100 or more days pregnant from non-pregnant animals. 6-Ketoestrone 6-carboxymethyloxime conjugated to bovine serum albumin (oestrone CMO-BSA) was 'dotted' 25 mm from the bottom edge of 45 x 5 mm strips of polyester-film-supported cellulose nitrate membrane, pore size 3 microm. The strips were blocked, dried and a 15 x 5-mm cellulose absorbent sink attached 10 mm from the top of each strip. The manufactured dipsticks were stored with desiccant at room...
Nie GJ, Wenzel JG.Hypoosmotic swelling (HOS) is used for assessing plasma membrane function and fertilizing capacity of human spermatozoa. However, HOS solutions and methodologies have not been evaluated specifically for assessing stallion spermatozoa. The objective of this study was to identify a HOS solution and assay conditions specifically for stallions that would maximize spermatozoal plasma membrane swelling. The HOS solutions and assay conditions, including incubation time (15 to 180 min), temperature (25 degrees vs 37 degrees C), and total number of cells examined (100, 200 or 500) were evaluated. Assay...
Iametti BS, Tedeschi G, Oungre E, Bonomi F.In this work the purification and the complete primary structure of kappa-casein from equine milk are reported for the first time. Mares' milk casein was separated by RP-HPLC into four fractions. Complete primary sequence was obtained by sequence analysis of the protein in the fastest eluting peak isolated by chromatography. This sequence was 95% identical to that reported for the C-terminal portion of the zebras' kappa-casein and showed high similarity with kappa-caseins from sources other than Equidae, confirming that this protein was indeed kappa-casein in equine milk. The presence of post-...
Thougaard AV, Jaliashvili I, Christiansen M.The glycoprotein tetranectin (TN) found in human serum is a 90-kDa homotrimeric C-type lectin binding Ca2+, heparin and plasminogen kringle 4. TN is suggested as being implicated in tissue remodelling. The antigenic reactivity of putative TN was examined in serum from 14 different animal species using three sandwich enzyme immunoassays for human TN. Crab-eating macaque serum showed the strongest reaction, followed by horse and cat. Serum from cow, goat, pig, mouse and chicken reacted weakly, while dog, trout, and the amphibian and the reptile species did not react. The TN-like protein from mac...
Lindgren G, Breen M, Godard S, Bowling A, Murray J, Scavone M, Skow L, Sandberg K, Guérin G, Binns M, Ellegren H.We report fluorescence in-situ hybridization (FISH) and somatic cell hybrid mapping data for 13 different horse genes (ANP, CD2, CLU, CRISP3, CYP17, FGG, IL1RN, IL10, MMP13, PRM1, PTGS2, TNFA and TP53). Primers for PCR amplification of intronic or untranslated regions were designed from horse-specific DNA or mRNA sequences in GenBank. Two different horse bacterial artificial chromosome (BAC) libraries were screened with PCR for clones containing these 13 Type I loci, nine of which were found in the libraries. BAC clones were used as probes in dual colour FISH to confirm their precise chromosom...
Peterman BF, Morton RA.The research explores how different binding ions affect the oxidation speed of horse heart ferrocytochrome c, a protein, by potassium ferricyanide at a constant ionic strength. Studying the Ion Effect […]
Davis JL, Marshall JF, Papich MG, Blikslager AT, Campbell NB.The purpose of this study was to determine the pharmacokinetics of deracoxib following oral administration to horses. In addition, in vitro equine whole blood cyclooxygenase (COX) selectivity assays were performed. Six healthy adult horses were administered deracoxib (2 mg/kg) orally. Plasma samples were collected prior to drug administration (time 0), and 10, 20, 40 min and 1, 1.5, 2, 4, 6, 8, 12, 24, and 48 h after administration for analysis with high pressure liquid chromatography using ultraviolet detection. Following PO administration, deracoxib had a long elimination half-life (t(...
Bundgaard L, Jacobsen S, Dyrlund TF, Sørensen MA, Harman VM, Beynon RJ, Brownridge PJ, Petersen LJ, Bendixen E.The aim of this study was the development of a quantitative assay that could support future studies of a panel of acute phase proteins (APPs) in the horse. The assay was based on a quantification concatamer (QconCAT) coupled to selected reaction monitoring methodology. Thirty-two peptides, corresponding to 13 putative or confirmed APPs for the Equus caballus (equine) species were selected for the design of a QconCAT construct. The gene encoding the QconCAT was synthesized and expressed as an isotope-labeled chimaeric protein in Escherichia coli. The QconCAT tryptic peptides were analyzed on a ...
Mantovani R, Rora A, Falomo ME, Bailoni L, Vincenti L.The aims of this study were to compare glycerol (G) at customary concentrations and ethylene glycol (EG) as cryoprotectants for stallion semen in a skimmed milk (SM) extender, to test different EG concentrations and to compare the results of manual and computerized analysis with the hypoosmotic swelling (HOS) test. Ejaculates from two stallions were collected over 3 weeks (6 ejaculates per stallion), diluted in a SM based extender, divided into 4 fractions, centrifuged and diluted again to a concentration of 100 x 10(6) mL(-1) progressive motile spermatozoa (PMS) in addition with the cryoprote...
Stockert JC, Trigoso CI, Tato A, Ferrer JM.The structure of specific granules from horse eosinophil leukocytes is still largely unknown. In this work, electron microscopical studies of horse eosinophils reveal that the large cytoplasmic granules contain an external membrane, a matrix of less density, and a dense (non crystalline) core. Round vacuolar inclusions of matrix materials were often observed within the cores. Horse eosinophil granules showed a considerable heterogeneity, and three morphological types could be identified according to structural features of the core and matrix.
Popot MA, Ho ENM, Stojiljkovic N, Bagilet F, Remy P, Maciejewski P, Loup B, Chan GHM, Hargrave S, Arthur RM, Russo C, White J, Hincks P, Pearce C....Cobalt is an essential mineral micronutrient and is regularly present in equine nutritional and feed supplements. Therefore, cobalt is naturally present at low concentrations in biological samples. The administration of cobalt chloride is considered to be blood doping and is thus prohibited. To control the misuse of cobalt, it was mandatory to establish an international threshold for cobalt in plasma and/or in urine. To achieve this goal, an international collaboration, consisting of an interlaboratory comparison between 5 laboratories for the urine study and 8 laboratories for the plasma stud...
Anestad G, Maagaard O.During an epizootic of equine influenza in Norway caused by influenza A/equine (H3N8) virus the efficacy of rapid virus diagnosis by the indirect immunofluorescence technique was evaluated. The antiserum used in the test was a polyclonal influenza A virus antiserum with reactivity directed mainly against the common nucleoprotein and matrix protein. This antiserum possessed sufficient reactivity for the detection of virus-infected exfoliated nasopharyngeal cells. Nasopharyngeal smear samples from 92 horses were examined and a positive diagnosis was obtained for 57 (62 per cent). Paired serum sa...
Yu NH, Ho EN, Tang FP, Wan TS, Wong AS.A multi-target high-throughput liquid chromatography-tandem mass spectrometry (LC-MS-MS) method for the detection of low ppt to low ppb levels of anabolic steroids, corticosteroids, anti-diabetics, and non-steroidal anti-inflammatory drugs (NSAIDs) in equine plasma was developed for the purpose of doping control. Plasma samples were first deproteinated by addition of trichloroacetic acid. Drugs were then extracted by solid-phase extraction (SPE) using Bond Elut Certify cartridges, and the extracts were analysed by a triple-quadrupole/linear ion trap LC-MS-MS instrument in positive electrospray...
Scott TW, Olson JG, All BP, Gibbs EP.Sensitivity and specificity of an antigen-capture ELISA vs virus isolation in cell culture were evaluated for the detection of eastern equine encephalomyelitis (EEE) virus in the brain tissue of naturally infected equids. Brain specimens from 16 equids with neurologic disease were examined by ELISA and by inoculation onto baby hamster kidney cell cultures. Of 10 brain samples from which virus was isolated in the cell culture bioassay, all were correctly identified as containing EEE virus antigen by ELISA. None of the remaining 6 specimens, without detectable infectious EEE virus, contained det...
Machtinger ET, Geden CJ.House flies (Musca domestica L.) and stable flies (Stomoxys calcitrans (L.)) (Diptera: Muscidae) are common pests in equine and cattle facilities. Pupal parasitoids, primarily in the genera Spalangia and Muscidifurax (Hymenoptera: Pteromalidae), can be purchased for biological control of these flies. However, little is known about the host-habitat preferences associated with host-seeking by these parasitoids. The preferences of two Spalangia and two Muscidifurax species to odors associated with house fly hosts in equine and bovine manure were investigated in the laboratory using a Y-tube olfac...
Caetano AR, Lyons LA, Laughlin TF, O'Brien SJ, Murray JD, Bowling AT.Comparative anchor tagged sequences (CATS) from human Chromosome 5 (HSA5) were used as PCR primers to produce molecular markers for synteny mapping in the horse. Primer sets for 21 genes yielded eight horse-specific markers, which were mapped with the UC Davis horse-mouse somatic cell hybrid panel into two synteny groups: UCD14 and UCD21. These data, in conjunction with earlier human chromosome painting studies of the horse karyotype and synteny mapping of horse microsatellite markers physically mapped by FISH, confirm the assignment of UCD21 to ECA21 and suggest that UCD14 is located on ECA14...
Ceusters JD, Serteyn DA, Minguet G, de la Rebière de Pouyade G, Romainville J, Deby-Dupont GP, Mouithys-Mickalad AA, Franck TJ.Horses are particularly sensitive and exposed to excessive inflammatory responses evolving toward an important stimulation of polymorphonuclear neutrophils (PMNs). The aim of this work was to stimulate equine neutrophils in whole blood and to evaluate their response by measuring the release of total and active myeloperoxidase (MPO) and total elastase, considered as markers of neutrophil stimulation and degranulation. Because of the critical importance of the concomitant presence of LPS and TNF-α in equine pathological situations, we combined these two natural mediators to stimulate PMN and co...
Ortiz I, Felix M, Resende H, Ramírez-Agámez L, Love CC, Hinrichs K.To define the effect of sperm agglutination, associated with incubation under capacitating conditions, on accuracy of membrane assessment via flow cytometry and to develop methods to mitigate that effect. Methods: Sperm motility was measured by CASA. Sperm were stained with PI-PSA or a novel method, LD-PSA, using fixable live/dead stain and cell dissociation treatment, before flow-cytometric analysis. Using LD-PSA, acrosome reaction and plasma membrane status were determined in equine sperm treated with 10 μm A23187 for 10 min, followed by 0, 1, or 2 h incubation in capacitating conditions. R...
Goudet G, Belin F, Młodawska W, Bézard J.The effect of epidermal growth factor (EGF) on the in vitro maturation rate of equine oocytes was examined. Oocytes were collected from an abattoir (Expt 1) or using ultrasound-guided follicular puncture in vivo (Expt 2). All oocytes with a compact or expanded cumulus at recovery were cultured for 30 h in: medium 1 (TCM199 + fetal calf serum (FCS) + crude equine gonadotrophin (CEG) + oestradiol + antibiotics); medium 2 (TCM199 + EGF); medium 3 (medium 1 without FCS + EGF); or medium 4 (medium 1 without CEG + EGF). In Expt 1, 84% (37/44) and 87% (40/46) cumulus expansion (P > 0.05), and 39% (22...
Zhang K, Zhou R, Huang H, Ma W, Qi Y, Li B, Zhang D, Li K, Chu H.The aim of this study was to identify the aggregation sites and transmission characteristics of Gasterophilus pecorum, the dominant pathogen of endangered equines in desert steppe. Therefore, we tested with a four-arm olfactometer the olfactory response of the G. pecorum adults to the odors that have a great impact on their life cycle, and also investigated the occurrence sites of the adults in the area where the Przewalski's horse (Equus przewalskii) roam frequently during the peak period of G. pecorum infection. The results of four-directional olfactory test showed that the fresh horse feces...
Aguilera-Tejéro E, Pascoe JR, Amis TC, Kurpershoek CJ, Woliner MJ.A rebreathing method for measurement of pulmonary diffusing capacity for carbon monoxide (DLCO) and functional residual capacity (FRC) was evaluated in conscious horses. Horses were manually ventilated through an endotracheal tube, using a custom-made syringe filled with a gas mixture containing 18-carbon monoxide (18CO) and helium (He). The 18CO and He concentrations were continuously monitored by use of a mass spectrometer connected to the rebreathing circuit. Values for DLCO and FRC were calculated from changes in the concentration of these 2 gases. In 11 Thoroughbreds, mean (+/- SD) DLCO w...
Yu NH, Ho EN, Leung DK, Wan TS.Anabolic steroids have the capability of improving athletic performance and are banned substances in the Olympic games as well as in horseracing and equestrian competitions. The control of their abuse in racehorses is traditionally performed by detecting the presence of anabolic steroids and/or their metabolite(s) in urine samples using gas chromatography-mass spectrometry (GC-MS). However, this approach usually requires tedious sample processing and chemical derivatisation steps and could be very insensitive in detecting certain steroids. This paper describes a high performance liquid chromat...
Thorsteinsdóttir L, Torsteinsdóttir S, Svansson V.Due to the slow growth of equine gammaherpesviruses, isolation of these viruses requires cells that can be propagated long term and show clear cytopathy following infection. Equine cell lines with extended lifespan were established from primary cells originating from equine fetal kidney and lung by transfecting the cells with the retroviral vector LXSN116E6E7 containing the human papilloma virus oncogenes 16 E6 and E7. The transfected equine kidney cell line and equine lung cell line can be propagated for more than 40 passages, whereas the corresponding primary cells only for 10-12 passages. T...
Mifune H, Richter R, Forssmann WG.The distribution of immunoreactivity (IR) for cardiodilatin/atrial natriuretic peptide (CDD/ANP) and brain natriuretic peptide (BNP) was examined immunohistochemically and immuno-electron-microscopically in the equine atrium, using specific antibodies. In the immunohistochemical studies, IR-CDD/ANP and IR-pBNP-26 (porcine BNP-26 immunoreactivity) was detected in the cytoplasm of the auricular cardiocytes, but IR-hBNP-32 (human BNP-32 immunoreactivity) was not. The double immunogold labelling method for IR-hBNP-28 and IR-pBNP-26 revealed that gold particles of different sizes were located in th...
Auer DE, Skelton KV, Tay S, Baldock FC.Total plasma carbon dioxide (TCO2) concentrations were measured in Standardbred horses to determine criteria to discriminate between normal horses and horses with excessive TCO2 concentrations on raceday. TCO2 concentrations from stabled horses were distributed normally with a mean of 30.2 mmol/L and a standard deviation of 1.2 (n = 192) while pre-race TCO2 concentrations were not normally distributed. The results indicate that about 50 horses per million are likely to have TCO2 concentrations greater than or equal to 35 mmol/L and that it is extremely unlikely that a normal horse would have a...
Parsons Aubone AM, Bisiau CM, McCue PM, Bouma GJ.Mammals have a circadian rhythm that is synchronized by a master clock located in the hypothalamic suprachiasmatic nucleus (SCN). The SCN regulates additional clocks located in peripheral tissues, including some involved in endocrine or reproductive functions. Studies in humans and mice report that molecular clocks also exist in the placenta. However, little is known about the presence of "Clock genes," namely Circadian Locomotor Output Cycles Kaput (CLOCK), Brain and Muscle Arnt-Like 1 (BMAL1), Period 1 (PER1), Period 2 (PER2), Cryptochrome 1 (CRY1), and Cryptochrome 2 (CRY2), in equine place...
Sukmawinata E, Sato W, Uemura R, Kanda T, Kusano K, Kambayashi Y, Sato T, Ishikawa Y, Toya R, Sueyoshi M.In this study, the occurrence of antimicrobial-resistant (AMR) enterococci was evaluated in Thoroughbred (TB) racehorses in Japan. Fecal samples were collected from 212 healthy TB racehorses at the Miho and Ritto Training Centers of the Japan Racing Association from March 2017 to August 2018. Isolation and identification were performed by enterococcus selective medium and confirmed to the species using MALDI-TOF MS. Enterococcus faecium and E. faecalis isolates were subjected to antimicrobial susceptibility test against 11 antimicrobials by minimum inhibitory concentration based on recommenda...
Shepard KN, Haffner JC, Neal DL, Grubbs ST, Pearce GL.Plasma adrenocorticotropic hormone (ACTH) concentration is used in the diagnosis of pituitary pars intermedia dysfunction (PPID) in horses. We enrolled 10 horses, 5 PPID-positive and 5 PPID-negative, in our study, September 20-22, 2016. On day 0, 5 mL of whole blood was collected into each of 6 EDTA tubes and immediately placed in a refrigerator at 7°C. One tube was centrifuged within 15 min of collection, followed by centrifugation of one tube from each horse at 4, 8, 12, 24, and 36 h following collection. At each time, centrifuged plasma was pipetted into 1.5-mL polypropylene tubes and stor...
Shang S, Zhang M, Zhao Y, Dang W, Hua P, Zhang S, Wang Z.Due to the thriving development of the modern horse industry and the occurrence of horse related crimes, the demand for methods of individual horse identification, parentage tests and other genetic analyses is increasing. Previous methods had disadvantages that decreased the accuracy of the results, lacked the inclusion of all commonly used short tandem repeats (STR) or increased the experimental cost and time. Objective: We aimed to develop a novel 13-plex STR typing system to resolve the above issues. Methods: Experimental study. Methods: Twelve autosomal and most commonly used di-nucleotide...
Henderson LL, Warren JC.Estradiol 17 alpha-dehydrogenase (EC 1.1.1.148) and estradiol 17 beta-dehydrogenase (EC 1.1.1.62) from horse placenta have been purified to homogeneity. Both enzymes are localized in the microsomal fraction and are solubilized in 1.5% sodium cholate. The 17 alpha- and 17 beta-dehydrogenases are separated by selective elution from hydroxylapatite with 0.5 and 1.0 M potassium phosphate, respectively. Subsequent purification is achieved by two affinity-absorption steps using reactive blue 2-agarose and estriol hemisuccinate-Sepharose. Homogeneous estradiol 17 alpha-dehydrogenase has a specific ac...
Kwok WH, Leung DK, Leung GN, Tang FP, Wan TS, Wong CH, Wong JK.In September 2005, our laboratory detected the presence of 4-androstene-3,17-dione and androsterone in a standard steroid screen of a post-race gelding urine sample received from an overseas authority. All other urine samples from the same batch tested negative. Subsequent gas chromatography/mass spectrometry (GC/MS) confirmatory analyses, however, repeatedly failed to detect any amount of 4-androstene-3,17-dione and androsterone in the suspicious sample. On the other hand, identical results were obtained when the initial GC/MS screening method was repeated on the suspicious sample as well as ...
Al-Ghamdi GM, Kapur V, Ames TR, Timoney JF, Love DN, Mellencamp MA.To determine whether repetitive sequence-based polymerase chain reaction (rep-PCR) could be used to differentiate Streptococcus equi isolates, to examine S equi isolates from throughout the world, and to determine whether a horse had > 1 subtype of S equi during an outbreak of disease. Methods: An initial group of 32 S equi isolates, 63 S equi isolates from various geographic areas, and 17 S equi isolates obtained during outbreaks of disease. Methods: An aliquot of S equi genomic DNA was amplified, using enterobacterial repetitive intergenic consensus primers. Gel electrophoresis was perfor...
Len JA, Beehan DP, Lyle SK, Eilts BE.It was hypothesized that optimal sperm recovery rate (RR) without damage to the sperm would be obtained after centrifugation without a cushion solution. Semen collected three times from six light breed stallions was extended to 25 × 10(6) sperm/mL and centrifuged at CON (noncentrifuged), 900NC (no-cushion), 900C (cushion), 1800NC, and 1800C × g for 10 minutes. Sperm concentration, motility (TM and PM), and intact plasma membranes (PLM) and acrosomes (ACR) pre- and postcentrifugation (D0) and after 24 hours (D1) of cooling were evaluated. The RR in the CON (100 ± 0.0), 900NC (93.7 ± 2.9), a...
Goodrich EL, Behling-Kelly E.The clinical evaluation of lipid metabolism in equids is often limited to the measurement of total cholesterol and triglyceride concentrations. This provides a limited picture of metabolic state and general health, given the continuous exchange of lipid species between various lipoproteins. Major lipoprotein classes in equids include high-density lipoprotein (HDL), intermediate-density lipoprotein (IDL), low-density lipoprotein (LDL), very low-density lipoprotein (VLDL), and chylomicrons (CM). Unlike large breed horses, donkeys are highly susceptible to hepatic lipidosis. Currently, serum trig...
Consuegra C, Crespo F, Dorado J, Diaz-Jimenez M, Pereira B, Hidalgo M.Lipids and proteins can be used for sperm vitrification to preserve the integrity of sperm membranes or to increase the viscosity of the medium. This study evaluated the effect of low-density lipoproteins (LDL) and milk serum proteins (Pronexcell) for stallion sperm vitrification. Hippex extender (Barex Biochemical Products, The Netherlands), plus 1% of bovine serum albumin and 100 mM of trehalose, was used as control for sperm vitrification. In experiment 1, different concentrations of LDL (L1 = 0.25, L2 = 0.5, L3 = 1%) and in experiment 2 of Pronexcell (P1 = 1, P2 = 5, P3 = 10%)...
