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.
Martino NA, Marzano G, Mastrorocco A, Lacalandra GM, Vincenti L, Hinrichs K, Dell Aquila ME.Time-lapse imaging was used to establish the morphokinetics of equine embryo development to the blastocyst stage after invitro oocyte maturation (IVM), intracytoplasmic sperm injection (ICSI) and embryo culture, in oocytes held overnight at room temperature (22-27°C; standard conditions) before IVM. Embryos that developed to the blastocyst stage underwent precleavage cytoplasmic extrusion and cleavage to the 2-, 3- and 4-cell stages significantly earlier than did embryos that arrested in development. We then determined the rate of blastocyst formation after ICSI in oocytes held for 2 days at ...
Lancheros-Buitrago J, Rodriguez-Villamil P, Gregory J, Bastos H, Camacho CA, Caballeros JE, Cazales N, Barros E, José de Jesus Silva M, Pimentel A....Characterization of the uterine proteome before the entry of the conceptus to the uterus is essential to know the factors involved in the physiological events of gestation. The objective of the study was to compare proteomic profile of uterine fluid collected on day 5 post ovulation of cyclic and inseminated mares. Samples of endometrial secretion were recovered over 2 cycles during the fifth day post ovulation. The first cycle constituted the Cyclic group and in the following cycle, the same mares were inseminated and considered as the Inseminated group. All the samples were subjected to two-...
Michio Clark J, Connor TA, Post A, Blaine Hoshizaki T, Ní Annaidh A, Gilchrist MD.The performance of equestrian helmets to protect against brain injuries caused by fall impacts against compliant surfaces such as turf has not been studied widely. We characterize the kinematic response of simulated fall impacts to turf through field tests on horse racetracks and laboratory experiments. The kinematic response characteristics and ground stiffness at different going ratings (GRs) (standard measurement of racetrack condition) were obtained from 1 m and 2 m drop tests of an instrumented hemispherical impactor onto a turf racetrack. The "Hard" rating resulted in higher peak lin...
Kumar R, Yadav SC, Kumar S, Dilbaghi N.A rapid flow-through immunoassay using protein A labeled gold nanoparticles (GNPs) for the qualitative detection of trypanosomosis in equine serum samples was developed. The flow-through device consisted of antigen-coated nitrocellulose membrane fixed on absorbent pads. The GNPs based detection probe was used as the colored marker. The assay can be employed for the detection of specific antibodies at 14 days post-infection in the infected pooled serum samples of equines. The assay can be conducted in < 5 min. The results of the flow-through assay were comparable with the methods like E...
Mei M, Chen R, Gao X, Cao Y, Weng W, Duan Y, Tan X, Liu Z.Meat fraud and adulteration incidents occur frequently in almost all regions of the globe, especially with the increase in the world's population. To ensure the authenticity of meat products, we developed a 10-plex xMAP assay to simultaneously detect ten animal materials: bovine, caprine, poultry, swine, donkey, deer, horse, dog, fox and mink. Results: This method was investigated by analyzing DNA extracts from raw muscle, muscle mixtures, meat products and animal feeds. Our results indicated that the species of interest can be identified, differentiated and detected down to 1 g kg in bina...
Miglio A, Morelli C, Maresca C, Felici A, Di Gianbattista A, Antognoni MT.Serum protein electrophoresis (SPE) reference intervals (RIs) have been evaluated in different horses, but no specific values are shown for equine breeds as previously described in other species (dogs, cats), and no studies have been performed on SPE in draft horses. Objective: This study aimed to determine RIs for SPE in heavy draft horses (Italian Heavy Draft Horse-IHDH) living in central Italy. A comparison between different physiologic states (pregnancy and no pregnancy) and ages (foals and adults) was executed. Methods: Blood samples were collected from 215 apparently healthy horses (mare...
Ghosh S, Serafini R, Love CC, Teague SR, Hernández-Avilés C, LaCaze KA, Varner DD.Effects of different media and promoters on lipid peroxidation (LPO) in viable stallion sperm have not been reported. Aims of this study were to determine effects of three media (INRA-96™, Equipro CoolGuard™, and Biggers, Whitten and Whittingham [BWW]), and promoters (iron sulfate-Fe; ultraviolet light-UV; or control-no exposure to promoters) on viable sperm LPO using four different flow cytometric assays (i.e., BODIPY, Liperfluo, 4-hydroxylnonenal [4HNE], malonaldehyde [MDA]). Significant media x promoter interactions were detected using the Liperfluo, 4HNE, and MDA assays (P < 0.05)...
Badenhorst M, de Heus P, Auer A, Rümenapf T, Tegtmeyer B, Kolodziejek J, Nowotny N, Steinmann E, Cavalleri JV.Prevalence studies have demonstrated a global distribution of equine hepacivirus (EqHV), a member of the family Flaviviridae. However, apart from a single case of vertical transmission, natural routes of EqHV transmission remain elusive. Many known flaviviruses are horizontally transmitted between hematophagous arthropods and vertebrate hosts. This study represents the first investigation of potential EqHV transmission by mosquitoes. More than 5000 mosquitoes were collected across Austria and analyzed for EqHV ribonucleic acid (RNA) by reverse transcription quantitative polymerase chain reacti...
Centromeres exert an inhibitory effect on meiotic recombination, but the possible contribution of satellite DNA to this "centromere effect" is under debate. In the horse, satellite DNA is present at all centromeres with the exception of the one from chromosome 11. This organization of centromeres allowed us to investigate the role of satellite DNA on recombination suppression in horse spermatocytes at the stage of pachytene. To this aim we analysed the distribution of the MLH1 protein, marker of recombination foci, relative to CENP-A, marker of centromeric function. We demonstrated that the sa...
Camargo Garbin L, McIlwraith CW, Frisbie DD.Platelet-rich plasma (PRP) as well as other platelet-derived products have been used as a potential disease-modifying treatment for musculoskeletal diseases, such as osteoarthritis (OA). The restorative properties of such products rely mainly on the high concentrations of growth factors, demonstrating encouraging results experimentally and clinically. Yet, the autologous blood-derived nature of the PRP product lead to limitations that precludes it's widespread use. The main limitations for PRP use are; product variability, the need for minimum laboratory settings in most cases, and the need fo...
Krueger K, Marr I, Dobler A, Palme R.Non-invasive methods enable stress evaluation through measuring fecal glucocorticoid metabolites (FGMs), and immunoglobulin A (IgA) in the feces avoiding stressful blood drawing or stressful restraining of animals in the field. However, FGMs and IgA are mostly analysed in freshly frozen samples, which is difficult when fresh samples cannot be frozen immediately or frozen samples cannot be stored or transported. Good results were also derived from air-dried fecal samples, which are hampered by unstable air humidity in the field. These difficulties may be overcome, when drying of samples could b...
Haffner JC, Neal DL, Hoffman RM, Grubbs ST.We investigated the stability of adrenocorticotropic hormone (ACTH) in plasma after freezing for different lengths of time. The plasma ACTH concentrations of 12 horses were measured on day 0 (baseline) and over time, after stimulation with thyrotropin-releasing hormone. Samples were stored at -80°C for 3, 7, 30, 60, and 90 d, or at -20°C for 3, 7, 30, and 60 d, or between ice packs at -20°C for 3 and 7 d prior to determination of ACTH concentration. ACTH concentrations were compared to baseline (non-frozen day 0 plasma) for each storage method using a mixed model with repeated measure...
Ortiz I, Dorado J, Morrell JM, Diaz-Jimenez MA, Pereira B, Consuegra C, Hidalgo M.The aim of this study was to compare the post-thaw distribution of motile sperm subpopulations, following simple or colloid centrifugation. A new analysis was used to evaluate the available number of sperm from each subpopulation after each centrifugation protocol. Frozen/thawed semen samples were divided into the following after-thawing treatments: uncentrifuged control (UDC), sperm washing (SW) and two colloid centrifugation procedures (Equipure, SLC-E, and Androcoll, SLC-A). Percentage of total and progressive motility (TM and PM), as well as sperm motility kinematics, distribution of motil...
Meyers S, Burruel V, Kato M, de la Fuente A, Orellana D, Renaudin C, Dujovne G.In this study we examined the timeline of mitotic events of invitro-produced equine embryos that progressed to blastocyst stage using non-invasive time-lapse microscopy (TLM). Intracytoplasmic sperm injection (ICSI) embryos were cultured using a self-contained imaging incubator system (Miri®TL; Esco Technologies) that captured brightfield images at 5-min intervals that were then generated into video for retrospective analysis. For all embryos that progressed to the blastocyst stage, the initial event of extrusion of acellular debris preceded all first cleavages and occurred at mean (±s.e.m.)...
Arnhold S, Elashry MI, Klymiuk MC, Geburek F.Adipose tissue-derived mesenchymal stem cells (ASCs) offer a promising cell source for therapeutic applications in musculoskeletal disorders. The appropriate selection of ASCs from various fat depots for cell-based therapy is challenging. The present study aims to compare stemness and multipotency of ASCs derived from retroperitoneal (RP), subcutaneous (SC), and lipoma (LP) fat to assess their usefulness for clinical application. Equine ASCs from the three fat tissue sources were isolated and characterized. The cell viability, proliferation, and self-renewal were evaluated using MTT, sulforhod...
Sievert M, Krohn J, Wehrend A.Due to the special structure of the equine placenta, foals depend on an adequate intake of high-quality colostrum post natum in order to ensure the development of passive immunity. The quality of the colostrum is determined, among other things, by the IgG content. This may be evaluated in the colostrum by direct and indirect methods (density and refractive index). The density of the colostrum is measured by a colostrometer and should amount to at least 1060 g/l. Refractometry is suitable for assessing the relative density or refractive index. Good equine colostrum has a Brix value of at leas...
Kellon EM, Gustafson KM.Hyperinsulinemia associated with equine metabolic syndrome and pituitary pars intermedia dysfunction is a risk factor for laminitis. Research in other species has shown elevated body iron levels as both a predictor and consequence of insulin resistance. In humans, this is known as dysmetabolic hyperferritinemia. To explore the relationship between equine hyperinsulinemia and body iron levels. We reviewed case histories and laboratory results from an open access database maintained by the Equine Cushing's and Insulin Resistance Group Inc. (ECIR). We identified 33 horses with confirmed hyperinsu...
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%)...
Pereira B, Dorado J, Diaz-Jimenez M, Consuegra C, Ortiz I, Gosalvez J, Hidalgo M.The acquisition of equine oocyte developmental capacity is ensured by the follicular environment, such as granulosa cells, which could reflect the meiotic development potential of immature oocytes. This study evaluated the relationship between DNA fragmentation of granulosa cells, using the chromatin dispersion test, and equine oocyte meiotic development after in vitro maturation. Granulosa cells and cumulus-oocytes complexes (n = 50) were recovered from slaughterhouse-derived ovaries. Oocytes were in vitro matured, stained and evaluated under fluorescence microscopy. Maturation rates were c...
Leschke DH, Muir GS, Hodgson JK, Coyle M, Horn R, Bertin FR.Diseases associated with insulin dysregulation (ID), such as equine metabolic syndrome and pituitary pars intermedia dysfunction, are of interest to practitioners because of their association with laminitis. Accurate insulin concentration assessment is critical in diagnosing and managing these diseases. Objective: To determine the effect of time, temperature, and collection tube type on insulin concentrations in horses at risk of ID. Methods: Eight adult horses with body condition score >6/9. Methods: In this prospective study, subjects underwent an infeed oral glucose test 2 hours before...
Assunção P, Barbosa T, Yonezawa L, Barbosa L, Watanabe M, Kohayagawa A, Schmidt E.High-intensity exercise can be associated with the occurrence of muscle injury, as well as the induction of an acute-phase response (APR). The present study aims to investigate the synthesis and profile of serum proteins in horses before and after participating in 2 different exercise protocols and to relate this profile to the presence or absence of muscular injury caused by exercise. Ten purebred Arabian (n = 5) and Criollo (n = 5) horses were subjected to 2 different tests on a treadmill, one consisting of short-duration and rapid-acceleration training (TRA) that was mostly anerobic and the...
Stack JD, Cousty M, Steele E, Handel I, Lechartier A, Vinardell T, David F.Synovial fluid analysis is utilized to diagnose septic synovitis. However, not all cases are clearly and rapidly discernible with the diagnostic tools available in the laboratory. Serum amyloid A (SAA), an acute phase protein, has been shown to be elevated in synovial fluid from inflamed synovial structures. The goal of this study is to describe the correlation between two diagnostic tests measuring equine SAA levels in septic and non-septic synovial structures and to understand the correlation between an elevated SAA result and synovial sepsis. Prospective estimation of sensitivity (Se) and s...
Cappelli K, Gialletti R, Tesei B, Bassotti G, Fettucciari K, Capomaccio S, Bonfili L, Cuccioloni M, Eleuteri AM, Spaterna A, Laus F.Guanylate cyclase-C (GC-C) is a multifunctional receptor encoded by the gene, representing an attractive target for therapy in several gastrointestinal diseases in humans. Little is known about this system in horses. We investigated for the first time the gene expression of guanylin, uroguanylin and GC-C receptors in different horse's gastrointestinal tracts. Tissue samples from stomach, duodenum, jejunum, ileum, head and body of cecum, left and right dorsal colon, left and right ventral colon, pelvic flexure, transverse colon, descending colon and rectum were collected from adult horses with...
Whitesell K, Stefanovski D, McDonnell S, Turner R.The stallion Breeding Soundness Examination (BSE), as proposed by the Society for Theriogenology, recommends that a stallion produce a minimum of one billion progressively motile, morphologically normal sperm (PMMNS) in the second of two ejaculates collected 1 h apart to be classified as a Satisfactory Prospective Breeder. With this in mind, the first objective of this study was to determine if the classification outcome of the traditional BSE differs depending on the methods used to evaluate sperm motility, morphology and concentration. We hypothesized that application of Computer Assisted ...
Yaneselli K, Barrachina L, Remacha AR, Algorta A, Vitoria A, Cequier A, Romero A, Vázquez FJ, Maisonnave J, Rodellar C.Propagation ex vivo of mesenchymal stem cells (MSCs) requires culture medium supplementation. Fetal bovine serum (FBS) has long been the gold standard supplement, but its use is being questioned mainly due to ethical and safety issues. The use of platelet lysate (PL) as substitute of FBS has been proposed but little is known about its effects on equine MSCs characteristics including their immune profile. The aim of this work was to investigate for the first time the effect of allogenic PL on the immunogenic and immunomodulatory gene expression profile of equine bone marrow derived MSCs (eBM-MS...
Norris JK, Slusarewicz P, Nielsen MK.The efficacy of anthelmintic treatments against populations of endoparasites infecting livestock throughout the world is decreasing. To mitigate this, the use of fecal egg counts is recommended to determine both the necessity, and to ensure the appropriate choice, of anthelmintic treatment. Traditionally, and in order to facilitate easier identification and/or enumeration, samples are analysed after separating eggs from other fecal particulates by exposing them to a solution with a density higher than that of the eggs, but lower than the remaining fecal contents. While many parasite egg flotat...
Pechacek D, Hwangbo M, Moreland R, Liu M, Ramsey J. 4s is a Gram-negative bacterium found in the equine intestinal ecosystem alongside diverse other coliform bacteria and bacteriophages. This announcement describes the complete genome of the T7-like 4s podophage Penshu1. From its 39,263-bp genome, 54 protein-encoding genes and a 179-bp terminal repeat were predicted.
Willingham-Lane JM, Berghaus LJ, Berghaus RD, Hart KA, Giguère S.The soil-dwelling, saprophytic actinomycete is a facultative intracellular pathogen of macrophages and causes severe bronchopneumonia when inhaled by susceptible foals. Standard treatment for disease is dual-antimicrobial therapy with a macrolide and rifampin. Thoracic ultrasonography and early treatment with antimicrobials prior to the development of clinical signs are used as means of controlling endemic infection on many farms. Concurrently with the increased use of macrolides and rifampin for chemoprophylaxis and the treatment of subclinically affected foals, a significant increase in t...
Scholtzek AD, Hanke D, Walther B, Eichhorn I, Stöckle SD, Klein KS, Gehlen H, Lübke-Becker A, Schwarz S, Feßler AT.The detection of borderline oxacillin-resistant (BORSA) represents a challenge to both, veterinary and human laboratories. Between 2015 and 2017, 19 equine with elevated minimal inhibitory concentrations for oxacillin were detected in routine diagnostics. The aim of this study was to characterize these isolates to identify factors possibly associated with the BORSA phenotype. All were subjected to antimicrobial susceptibility testing and whole genome sequencing (WGS). A quantifiable β-lactamase activity assay was performed for a representative subset of 13 isolates. The WGS data analysis o...
Poirier M, Smith OE, Therrien J, Rigoglio NN, Miglino MA, Silva LA, Meirelles FV, Smith LC.Cell reprogramming by somatic cell nuclear transfer and in induced pluripotent stem cells is associated with epigenetic modifications that are often incompatible with embryonic development and differentiation. For instance, aberrant DNA methylation patterns of the differentially methylated region and biallelic expression of H19-/IGF2-imprinted gene locus have been associated with abnormal growth of fetuses and placenta in several mammalian species. However, cloned horses are born with normal sizes and with no apparent placental anomalies, suggesting that H19/IGF2 imprinting may be epigenetical...
Dobrinski I, Ignotz GG, Thomas PG, Ball BA.To test the hypotheses that the attachment of equine spermatozoa to uterine tubal (oviductal) epithelial cells (OEC) in vitro is mediated by glycoproteins, and that proteins with carbohydrate-binding properties are present in the periacrosomal plasma membrane of equine spermatozoa. Methods: 4 reproductively sound stallions, and 1 mare in estrus. Methods: In experiment 1a, fluorescent-labeled spermatozoa were cocultured with monolayers of OEC in the presence of 50 mM glucose, fructose, galactose, mannose, N-acetyl glucosamine, N-acetyl galactosamine, or N-acetyl neuraminic acid, or 10 mg of fet...
Taylor SE, Clegg PD.Mesenchymal stromal cells (MSC) are derived from adult mesenchymal tissues and have the ability to undergo differentiation into bone, cartilage, and fat, and have therefore attracted great interest in regenerative medicine. Many isolation and culture methods have been described, making comparison between laboratories and quality-control protocols difficult. A uniform protocol to characterize equine MSC has recently been proposed, aiming to introduce consistency across the equine stem cell research field. This article reviews the published techniques for collection and propagation of equine MSC...
Kisiday JD, Goodrich LR, McIlwraith CW, Frisbie DD.To compare the mesenchymal stem cell (MSC) yield and chondrogenic and osteogenic differentiation from 5- and 50-mL bone marrow aspirates from horses. Methods: Six 2- to 5-year-old mixed-breed horses. Procedures-2 sequential 5-mL aspirates were drawn from 1 ilium or sternebra. A single 50-mL aspirate was drawn from the contralateral ilium, and 2 sequential 50-mL aspirates were drawn from a second sternebra. The MSC yield was determined through the culture expansion process. Chondrogenesis and osteogenesis were evaluated by means of conventional laboratory methods. Results: The second of the 2 s...
Ball BA, Gravance CG, Medina V, Baumber J, Liu IK.To characterize the activity of catalase in equine semen. Methods: 15 stallions of known and unknown reproductive history. Methods: Seminal plasma was collected from raw equine semen by centrifugation, and samples of seminal plasma were frozen prior to assay for catalase activity. Tissue samples (n = 3 stallions) from the bulbourethral gland, prostate gland, vesicular gland, and testis were homogenized, and cauda epididymal fluid was collected for determination of catalase activity. Catalase activity was determined as an enzyme kinetic assay by the disappearance of H2O2 as measured by ultravio...
Knych HK, Baden RW, Gretler SR, McKemie DS.Despite their widespread popularity as sport and companion animals and published and anecdotal reports of vast difference in drug disposition and pharmacokinetics between individuals, studies describing equine drug metabolism are limited. It has been theorized that similar to humans, members of the CYP2D family in horses may be polymorphic in nature leading to differences in metabolism of substrates. This study aims to build on the limited current knowledge regarding P450 mediated metabolism in horses by describing the metabolism of the polymorphic CYP2D6 probe drug codeine in vitro. Codeine, ...
Kayis SA, Atli MO, Kurar E, Bozkaya F, Semacan A, Aslan S, Guzeloglu A.The aim was an evaluation of a set of housekeeping genes (HKGs) to be used in the normalization of gene expression in the equine endometrium. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hypoxanthine ribosyl transferase 1 (HPRT1), ubiquitin B (UBB), tubulin alpha 1 (TUBA1), ribosomal protein L32 (RPL32), beta-2-microglobulin (B2M), 18S rRNA (18S), and 28S rRNA (28S) HKGs were evaluated using real-time PCR and were compared in different physiological stages of the endometrium. Endometrial biopsies were obtained from mares on day of ovulation (d0, n=4), at late diestrus (LD, n=4), after lut...
Bousfield GR, Ward DN.A method has been developed for the purification of equine lutropin (eLH) and equine follitropin (eFSH) from horse pituitary glands which attains high yields of both hormones in contrast to previous methods that were devoted to one or the other with inferior recovery of the hormones. Two-pass chromatography over CM-Sephadex was used to separate eLH from eFSH. Subsequent steps employing QAE (quaternary amino-ethyl)-Sephadex chromatography and gel filtration on Sephacryl S-200 produced highly purified hormone preparations. Yields of purified eLH and eFSH were 110 and 60 mg/kg of frozen pituitari...
Espina M, Jülke H, Brehm W, Ribitsch I, Winter K, Delling U.Background. Mesenchymal stromal cells (MSCs) are increasingly used for clinical applications in equine patients. For MSC isolation and expansion, a laboratory step is mandatory, after which the cells are sent back to the attending veterinarian. Preserving the biological properties of MSCs during this transport is paramount. The goal of the study was to compare transport-related parameters (transport container, media, temperature, time, cell concentration) that potentially influence characteristics of culture expanded equine MSCs. Methods. The study was arranged in three parts comparing (I) fiv...
Hoffmann C, Bazer FW, Klug J, Aupperle H, Ellenberger C, Schoon HA.Although alterations in patterns of protein secretion revealed in uterine flushings from mares suffering from endometrosis have been described, little is known about alterations at the cellular level. Hence, the aim of this study was to characterize deviations in patterns of uterine gland secretion patterns using endometrial biopsies, histochemical and newly established immunohistochemical methods. Forty-eight endometrial biopsies were obtained from mares suffering from various types of endometrosis (active and inactive, destructive and non-destructive) and degree (mild to severe) were analyze...
Ortved KF, Begum L, Stefanovski D, Nixon AJ.Following joint trauma, a posttraumatic inflammatory cascade drives degeneration of the joint. We aimed to assess whether transduction of chondrocytes with AAV5 overexpressing the immunomodulatory cytokine IL-10 would have protective effects in pellet cultures stimulated with IL-1β. Chondrocytes were isolated from 3 healthy horses and were transduced with AAV5-IL-10 at a dose of 1 x 105vg/cell. Chondrocyte pellets were formed by centrifugation and were stimulated with IL-1β starting 48 hours following transduction. After 2, 6 and 14 days in culture, supernatants were collected for cytokine a...
Zamboulis DE, Marr N, Lenzi L, Birch HL, Screen HRC, Clegg PD, Thorpe CT.Energy storing tendons such as the human Achilles and equine superficial digital flexor tendon (SDFT) are prone to injury, with incidence increasing with aging, peaking in the 5 decade of life in the human Achilles tendon. The interfascicular matrix (IFM), which binds tendon fascicles, plays a key role in energy storing tendon mechanics, and aging alterations to the IFM negatively impact tendon function. While the mechanical role of the IFM in tendon function is well-established, the biological role of IFM-resident cell populations remains to be elucidated. Therefore, the aim of this study was...
Zhang B, Sun C, Jin S, Cascio M, Montelaro RC.The equine lentivirus receptor 1 (ELR1), a member of the tumor necrosis factor receptor (TNFR) protein family, has been identified as a functional receptor for equine infectious anemia virus (EIAV). Toward defining the functional interactions between the EIAV SU protein (gp90) and its ELR1 receptor, we mapped the gp90 binding domain of ELR1 by a combination of binding and functional assays using the EIAV SU gp90 protein and various chimeric receptor proteins derived from exchanges between the functional ELR1 and the nonbinding homolog, mouse herpesvirus entry mediator (murine HveA). Complement...
Birch-Machin I, Rowan A, Pick J, Mumford J, Binns M.The nucleotide sequence of the nonstructural protein NS1 of the influenza virus A/equine 2/Suffolk/89 was determined and found to be 97% identical to that of A/equine 2/Miami/63. A similar level of identity was shown for the deduced NS1 amino acid sequence. The NS1 gene was expressed, in its entirety and in part, as fusion proteins with glutathione S-transferase using the pGEX-3X expression vector. Antibodies to NS1 protein were detected in serum samples from ponies experimentally infected with influenza virus, but not in animals vaccinated with whole inactivated virus or in unprimed control a...
Wong CH, Leung DK, Tang FP, Wong JK, Yu NH, Wan TS.Liquid chromatography/mass spectrometry (LC/MS) has been successfully applied to the detection of anabolic steroids in biological samples. However, the sensitive detection of saturated hydroxysteroids, such as androstanediols, by electrospray ionisation (ESI) is difficult because of their poor ability to ionise. In view of this, chemical derivatisation has been used to enhance the detection sensitivity of hydroxysteroids by LC/MS. This paper describes the development of a sensitive ultra-high-performance liquid chromatography/tandem mass spectrometry (UHPLC/MS/MS) method for the screening of a...
Johnson A, Smith R, Saxne T, Hickery M, Heinegård D.Previous experiments have shown that addition of fragmented fibronectin can induce cartilage chondrolysis. In this study we investigated the fate of the collagen- and cell-binding molecules Cartilage oligomeric matrix protein (COMP) and chondroadherin. Methods: Equine articular cartilage explants were stimulated with the C-terminal and the N-terminal heparin-binding fragments of fibronectin respectively, and the conditioned media were analysed by both quantitative (ELISA) and qualitative (mass spectrometry, Western blots) methods. Results: Both COMP and chondroadherin were released in a dose-d...
Capomaccio S, Verini-Supplizi A, Galla G, Vitulo N, Barcaccia G, Felicetti M, Silvestrelli M, Cappelli K.A large proportion of mammalian genomes is represented by transposable elements (TE), most of them being long interspersed nuclear elements 1 (LINE-1 or L1). An increased expression of LINE-1 elements may play an important role in cellular stress-related conditions exerting drastic effects on the mammalian transcriptome. To understand the impact of TE on the known horse transcriptome, we masked the horse EST database, pointing out that the amount is consistent with other major vertebrates. A previously developed transcript-derived fragments (TDFs) dataset, deriving from exercise-stimulated hor...
Alexander AJ, Bailey E, Woodward JG.Fourteen Standardbred horses homozygous for one of six equine lymphocyte antigen (ELA) specificities (A1, A3, A4, A5, A6, or A10) were analyzed by Southern blot hybridization using DNA probes derived from the mouse major histocompatibility complex (MHC). Total genomic DNA from peripheral lymphocytes was digested with the restriction enzymes Hind III, Pvu II, or Eco RI. Twenty-three to thirty-three bands were generated for individual horses with the class I cDNA probe. The resulting band patterns revealed 12-14 nonpolymorphic fragments, which is consistent with the highly conserved Qa/Tla genes...
Alanazi AD, Said AE, Morin-Adeline V, Alyousif MS, Slapeta J.Equine piroplasmosis is the most important tick-borne disease of horses. Regulations on movement of horses into disease-free countries are in place to preserve international trade. Introduction of infectious disease, such as equine piroplasmosis, into non-endemic countries remains a substantial risk owing to the wide-spread distribution of vectors. Identification and restriction of movement of Theileria equi persistently infected horses is an integral part of control strategies, because persistently infected horses with low parasitaemia are an important reservoir. We used real-time PCR for dia...
Christensen H, Bisgaard M, Olsen JE.Members of Bisgaard taxon 11 have been isolated from horses. These bacteria are of importance in the veterinary clinic and also to the medical profession, since they may be isolated from infected wounds of humans bitten by horses. Six strains from different continents were identified as taxon 11, with 16S rRNA similarities between 98.0 and 99.7%. A single isolate that represented the so-called (+)L-arabinose-positive Actinobacillus equuli isolated from a diseased foal showed 99.9% 16S rRNA similarity to the type strain of A. equuli. DNA-DNA hybridizations showed that (+)L-arabinose-positive st...
Hayden SS, Blanchard TL, Brinsko SP, Varner DD, Hinrichs K, Love CC.Dilution of semen to less than 20 × 10(6) sperm/mL has been reported to decrease sperm quality in multiple species, a phenomenon known as the semen "dilution effect." Critical evaluation of stallion semen diluted to these concentrations, however, has not been reported. This study evaluated sperm motion characteristics (percent total motility [TMOT], percent progressive motility [PMOT], curvilinear velocity [μm/s], and percent straightness) and plasma membrane integrity (percent plasma membrane intact [PMI]) in semen samples diluted to 2.5 × 10(6) sperm/mL with the addition of 0%, 7.5%, ...
Marr N, Hopkinson M, Hibbert AP, Pitsillides AA, Thorpe CT.Three-dimensional imaging modalities for optically dense connective tissues such as tendons are limited and typically have a single imaging methodological endpoint. Here, we have developed a bimodal procedure utilising fluorescence-based confocal microscopy and x-ray micro-computed tomography for the imaging of adult tendons to visualise and analyse extracellular sub-structure and cellular composition in small and large animal species. Results: Using fluorescent immunolabelling and optical clearing, we visualised the expression of the novel cross-species marker of tendon basement membrane, lam...
Todd FG, Stermitz FR, Schultheis P, Knight AP, Traub-Dargatz J.Horses in a few, localized northern Colorado pastures exhibited weight loss and colic. At post mortem, intestinal fibrosis and vascular sclerosis of the small intestine was identified. The pastures where the affected horses grazed were overrun by field bindweed (Convolvulus arvensis). Bindweed from the pasture was found to contain the tropane alkaloids tropine, pseudotropine, and tropinone and the pyrrolidine alkaloids cuscohygrine and hygrine. Laboratory mice readily ate C. arvensis and exhibited a variety of abnormal clinical signs depending on the amount eaten. Similar alkaloids have been f...
Mohammadi-Sangcheshmeh A, Held E, Ghanem N, Rings F, Salilew-Wondim D, Tesfaye D, Sieme H, Schellander K, Hoelker M.Efficiencies for in vitro production of equine embryos are still low due to highly variable developmental competences of equine immature oocytes. In contrast to the equine, in vitro developmental competence of immature oocytes has been predicted successfully by the activity of glucose-6-phosphate dehydrogenase (G6PDH) indicated by brilliant cresyl blue (BCB) dye in a range of different species. Therefore, the aim of the present study was to test the association between G6PDH activity in equine oocytes with: (1) cumulus morphology and oocyte properties in terms of diameter and volume; (2) matur...
Morrell JM, Georgakas A, Lundeheim N, Nash D, Davies Morel MC, Johannisson A.Removing most of the seminal plasma (SP) from stallion semen has been shown to improve survival during cooled storage, yet adding small quantities of SP may improve pregnancy rates or cryosurvival. Furthermore, there is considerable controversy about whether the stallion's own SP or heterologous SP produces the best effect, possibly because of the variation between stallions in SP proteins or because some homologous SP remained in the sperm preparation. The SP is removed completely from stallion spermatozoa prepared by colloid centrifugation. Thus, the aim of the present study was (1) to inves...
Azab W, Tsujimura K, Kato K, Arii J, Morimoto T, Kawaguchi Y, Tohya Y, Matsumura T, Akashi H.Equine herpesvirus 4 (EHV-4) is an important equine pathogen that causes respiratory tract disease among horses worldwide. A thymidine kinase (TK)-deletion mutant has been generated by using bacterial artificial chromosome (BAC) technology to investigate the role of TK in pathogenesis. Deletion of TK had virtually no effect on the growth characteristics of WA79DeltaTK in cell culture when compared to the parent virus. Also, virus titers and plaque formation were unaffected in the absence of the TK gene. The sensitivity of EHV-4 to inhibition by acyclovir (ACV) and ganciclovir (GCV) was studied...
Rosas CT, Tischer BK, Perkins GA, Wagner B, Goodman LB, Osterrieder N.The immunogenicity in horses of a recombinant equine herpesvirus type 1 (EHV-1) vaccine expressing West Nile virus (WNV) prM and E proteins was studied. To construct the recombinant EHV-1, two-step en passant mutagenesis was employed for manipulation of a bacterial artificial chromosome (BAC) of vaccine strain RacH. Recombinant EHV-1 stably expressed the WNV prM and E proteins as demonstrated by indirect immunofluorescence and Western blotting. In addition, growth properties in vitro of the EHV-1/WNV recombinant were found to not be significantly different from those of the parental virus. To ...
Ujvari S, Schwarzwald CC, Fouché N, Howard J, Schoster A.Point-of-care (POC) diagnostic tests with good sensitivity and specificity are needed for diagnosing failure of transfer of passive immunity (FTPI) in foals. Turbidimetric immunoassays (TIA) have these characteristics and provide quantitative results. A commercially available TIA-based POC test (POC-TIA) has not been validated in horses. Objective: To validate a POC-TIA and compare results of POC-TIA, a POC-ELISA, and radial immunodiffusion (RID). Methods: Heparinized blood samples (n = 127) from 48 hospitalized foals (<12 hour to 48 days). Methods: Prospective validation study. IgG concentrat...
Emenius G, Larsson PH, Wickman M, Härfast B.The objective was to establish an ELISA to detect horse allergen in ambient air and settled dust. Methods: Monoclonal antibodies (mAbs) were produced against extracts of horse antigen. Two mAbs were selected and used in a sandwich ELISA. By the aid of portable pumps, air samples were collected in one stable and in the ambient air surrounding this stable. Furthermore, settled dust was collected by wiping spots with pieces of fabric, at sites within 500 m of the stable. Results: Extracts of horsehair could be extensively diluted and still be positive. Extracts of cat and dog allergen failed to b...
Kupczyk P, Rykala M, Serek P, Pawlak A, Slowikowski B, Holysz M, Chodaczek G, Madej JP, Ziolkowski P, Niedzwiedz A.The endocannabinoid system (ECS) is composed of cannabinoid receptors type 1 (CBR1) and type 2 (CBR2), cannabinoid-based ligands (endogenous chemically synthesized phytocannabinoids), and endogenous enzymes controlling their concentrations. Cannabinoid receptors (CBRs) have been identified in invertebrates and in almost all vertebrate species in the central and peripheral nervous system as well as in immune cells, where they control neuroimmune homeostasis. In humans, rodents, dogs, and cats, CBRs expression has been confirmed in the skin, and their expression and tissue distribution become di...
Ji S, Na L, Ren H, Wang Y, Wang X.Human myxovirus resistance protein 2 (huMxB) has been shown to be a determinant type I interferon (IFN)-induced host factor involved in the inhibition of human immunodeficiency virus type 1 (HIV-1) as well as many other primate lentiviruses. This blocking occurs after the reverse transcription of viral RNA and ahead of integration into the host DNA, which is closely connected to the ability of the protein to bind the viral capsid. To date, Mx2s derived from nonprimate animals have shown no capacity for HIV-1 suppression. In this study, we examined the restrictive effect of equine Mx2 (eqMx2) o...
