In vitro research involving horses refers to the study of equine cells, tissues, or biological molecules outside their normal biological context, typically in controlled laboratory environments. This research approach allows scientists to investigate cellular processes, molecular interactions, and the effects of various treatments without the ethical and logistical complexities of in vivo studies. In vitro studies contribute to understanding equine physiology, pathology, and pharmacology by providing insights into cellular responses to pathogens, drugs, and other stimuli. This page compiles peer-reviewed research studies and scholarly articles that explore various in vitro methodologies and their applications in equine science, including cell culture techniques, molecular assays, and drug efficacy testing.
Ojo KK, Dangoudoubiyam S, Verma SK, Scheele S, DeRocher AE, Yeargan M, Choi R, Smith TR, Rivas KL, Hulverson MA, Barrett LK, Fan E, Maly DJ....Sarcocystis neurona is the most frequent cause of equine protozoal myeloencephalitis, a debilitating neurological disease of horses that can be difficult to treat. We identified SnCDPK1, the S. neurona homologue of calcium-dependent protein kinase 1 (CDPK1), a validated drug target in Toxoplasma gondii. SnCDPK1 shares the glycine "gatekeeper" residue of the well-characterized T. gondii enzyme, which allows the latter to be targeted by bumped kinase inhibitors. This study presents detailed molecular and phenotypic evidence that SnCDPK1 can be targeted for rational drug development. Recombinant ...
Rader K, Choi YH, Hinrichs K.Intracytoplasmic sperm injection is becoming a common clinical procedure in the horse, but little information is available on techniques for its performance. Each laboratory uses different procedures and different media for the steps involved with in vitro embryo production. This article outlines the procedures used in the Clinical Equine Intracytoplasmic Sperm Injection Program at Texas A&M University for in vitro blastocyst production during the past 3 years.
Carnevale EM.Assisted reproductive techniques that are based on oocyte manipulations have gained acceptance in the equine industry. Methods to collect and handle immature or maturing oocytes have been developed, and systems to ship oocytes now allow for collection in one location and intracytoplasmic sperm injection (ICSI) in another. Subsequently, ICSI-produced embryos can be transferred onsite, shipped to another location, or cryopreserved. Methods for the collection, identification, culture, maturation, and shipment of equine oocytes are reviewed, with an emphasis on procedures from laboratories providi...
Varner DD.Subfertility can be a confusing term because some semen of good quality can have reduced fertility following cooled transport if the semen is processed in an improper manner. General procedures aimed at processing stallion semen for cooled transport are well described. An array of factors could exist in reduced fertility of cool-transported semen. This article focuses on centrifugation techniques that can be used to maximize sperm quality of stallions whose semen is intended for cooled transport. Clinical cases are also provided for practical application of techniques.
Pouran B, Arbabi V, Zadpoor AA, Weinans H.The metabolic function of cartilage primarily depends on transport of solutes through diffusion mechanism. In the current study, we use contrast enhanced micro-computed tomography to determine equilibrium concentration of solutes through different cartilage zones and solute flux in the cartilage, using osteochondral plugs from equine femoral condyles. Diffusion experiments were performed with two solutes of different charge and approximately equal molecular weight, namely iodixanol (neutral) and ioxaglate (charge=-1) in order to isolate the effects of solute's charge on diffusion. Furthermore,...
Word TA, Larsen RW.Treatment of horse heart Cytochrome-c (Cc) with N-chloro-4-toluosulfonamide (Chloramine-t, CT) results in the oxidation of methionine (Met) residues to the corresponding sulfoxide including the distal heme ligand, Met80. The resulting Fe-sulfoxide coordination is sufficiently labile in the ferrous form to be displaced by gaseous ligands, including CO. Photolysis of the CO-CT-Cc complex provides an opportunity to examine ligand binding dynamics that are associated with a relatively rigid distal heme pocket. In this work, photoacoustic calorimetry (PAC) was utilized to obtain the kinetics as wel...
Szcześniak KA, Ciecierska A, Ostaszewski P, Sadkowski T.β-Hydroxy-β-methylbutyrate (HMB) is a popular ergogenic aid used by human athletes and as a supplement to sport horses, because of its ability to aid muscle recovery, improve performance and body composition. Recent findings suggest that HMB may stimulate satellite cells and affect expressions of genes regulating skeletal muscle cell growth. Despite the scientific data showing benefits of HMB supplementation in horses, no previous study has explained the mechanism of action of HMB in this species. The aim of this study was to reveal the molecular background of HMB action on equine skeletal m...
Fedorka CE, Scoggin KE, Woodward EM, Squires EL, Ball BA, Troedsson M.In the horse, breeding induces a transient endometrial inflammation. A subset of mares are unable to resolve this inflammation, and they are considered susceptible to persistent mating-induced endometritis PMIE Select seminal plasma proteins cysteine-rich secretory protein-3 (CRISP-3) and lactoferrin have been shown to affect the innate immune response to sperm in vitro. The objective of this study was to determine whether the addition of CRISP-3 and lactoferrin at the time of insemination had an effect on the mRNA expression of endometrial cytokines in susceptible mares after breeding. Six ma...
Shibany KA, Tötemeyer S, Pratt SL, Paine SW.Despite reports of the successful isolation of primary equine hepatocytes, there are no published data regarding the successful cryopreservation of these isolated cells. In this study, a detailed description of the procedures for isolation, cryopreservation, and recovery of equine hepatocytes are presented. Furthermore, the intrinsic clearance (Cl) and production of metabolites for three drugs were compared between freshly isolated and recovered cryopreserved hepatocytes. Primary equine hepatocytes were isolated using a two-step collagenase perfusion method, with an average cell yield of 2.47Â...
Rombouts C, Jeanneau C, Camilleri J, Laurent P, About I.Adequate revascularization is a prerequisite for successful healing of periodontal bone defects. This study characterized three different xenogeneic bone grafting materials: Gen-Os of equine and porcine origins, and anorganic Bio-Oss. We also investigated their angiogenic potential. All materials were composed of poorly crystalline calcium oxide phosphate, with Bio-Oss exhibiting a carbonated phase and larger particle size and both Gen-Os showing the presence of collagen. Both Gen-Os materials significantly enhanced vascular endothelial growth factor (VEGF) secretion by PDL cells. A significan...
Swegen A, Lambourne SR, Aitken RJ, Gibb Z.Media used for equine sperm storage often contain relatively high concentrations of glucose, even though stallion spermatozoa preferentially utilize oxidative phosphorylation (OXPHOS) over glycolysis to generate ATP and support motility. Rosiglitazone is an antidiabetic compound that enhances metabolic flexibility and glucose utilization in various cell types, but its effects on sperm metabolism are unknown. This study investigated the effects of rosiglitazone on stallion sperm function in vitro, along with the possible role of AMP-activated protein kinase (AMPK) in mediating these effects. Sp...
Maumus M, Roussignol G, Toupet K, Penarier G, Bentz I, Teixeira S, Oustric D, Jung M, Lepage O, Steinberg R, Jorgensen C, Noel D.Mesenchymal stem cells isolated from adipose tissue (ASC) have been shown to influence the course of osteoarthritis (OA) in different animal models and are promising in veterinary medicine for horses involved in competitive sport. The aim of this study was to characterize equine ASCs (eASCs) and investigate the role of interferon-gamma (IFNγ)-priming on their therapeutic effect in a murine model of OA, which could be relevant to equine OA. Methods: ASC were isolated from subcutaneous fat. Expression of specific markers was tested by cytometry and RT-qPCR. Differentiation potential was evaluat...
Biedrzycki AH, Grant BG, Nemke B, Morello SL, Markel MD.OBJECTIVE To evaluate the biomechanical properties of 4 methods for fusion of the centrodistal and tarsometatarsal joints in horses and compare them among each other and with control tarsi. SAMPLE 24 sets of paired tarsi without substantial signs of osteoarthritis harvested from equine cadavers. PROCEDURES Test constructs (n = 6/type) were prepared from 1 tarsus from each pair to represent surgical drilling; 2 medially to laterally placed kerf-cut cylinders (MLKCs); a single large, dorsally applied kerf-cut cylinder (DKC); and a dorsomedially applied locking compression plate (DMLCP). Construc...
Sharpe AN, Seeto WJ, Winter RL, Zhong Q, Lipke EA, Wooldridge AA.OBJECTIVE To evaluate optimal isolation of endothelial colony-forming cells (ECFCs) from peripheral blood of horses. SAMPLE Jugular and cephalic venous blood samples from 17 adult horses. PROCEDURES Each blood sample was divided; isolation was performed with whole blood adherence (WBA) and density gradient centrifugation (DGC). Isolated cells were characterized by uptake of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate-labeled acetylated low-density lipoprotein (DiI-Ac-LDL), vascular tubule formation, and expression of endothelial (CD34, CD105, vascular endothelial growth ...
Scharf A, Holmes SP, Thoresen M, Mumaw J, Stumpf A, Peroni J.Ultrasound-guided intralesional injection of mesenchymal stem cells (MSCs) is held as the benchmark for cell delivery in tendonitis. The primary objective of this study was to investigate the immediate cell distribution following intralesional injection of MSCs. Unilateral superficial digital flexor tendon (SDFT) lesions were created in the forelimb of six horses and injected with 10 × 10 MSCs labeled with superparamagnetic iron oxide nanoparticles (SPIOs) under ultrasound guidance. Assays were performed to confirm that there were no significant changes in cell viability, proliferation, migra...
de la Torre J, Sánchez-MartÃn P, Gosálvez J, Crespo F.Sperm quality was assessed in normozoospermic human (n = 10) and Spanish breed stallion (n = 10) after sperm fractionation during ejaculation. The first ejaculated fraction was separated from the second. A third sample was reconstituted using equivalent proportion of both fractions (RAW). Fraction 1, Fraction 2 and RAW semen were incubated for 30 min at 37°C to homogenise the impact of iatrogenic damage between both species. Sperm concentration, motility and sperm DNA damage were assessed in each fraction and RAW semen. The results showed two important facts: (i) spermatozoa confined at ...
Merlo B, Iacono E, Bucci D, Spinaci M, Galeati G, Mari G.In vitro embryo production in the horse is still not as efficient as in other species. Oxidative stress negatively affects oocyte and embryo culture. To attenuate/minimize the oxidative stress, antioxidants such as low-molecular thiol compounds can be added to culture media. Beta-mercaptoethanol (BME) has been shown to improve maturation and embryo development in different species. The aim of this study was to investigate whether the addition to maturation medium of BME at common (0.1Â mM) and high (0.7Â mM) concentration could improve oocyte maturation also in the horse. Equine oocytes recove...
Zucca E, Corsini E, Galbiati V, Lange-Consiglio A, Ferrucci F.Data obtained in both animal models and clinical trials suggest that cell-based therapies represent a potential therapeutic strategy for lung repair and remodeling. Recently, new therapeutic approaches based on the use of stem cell derivatives (e.g., conditioned medium (CM) and microvesicles (MVs)) to regenerate tissues and improve their functions were proposed. The aim of this study was to investigate the immunomodulatory effects of equine amniotic mesenchymal cell derivatives on lipopolysaccharide (LPS)-induced cytokine production in equine alveolar macrophages, which may be beneficial in lu...
Leemans B, Gadella BM, Stout TA, De Schauwer C, Nelis H, Hoogewijs M, Van Soom A.In contrast to man and many other mammalian species, conventional in vitro fertilization (IVF) with horse gametes is not reliably successful. The apparent inability of stallion spermatozoa to penetrate the zona pellucida in vitro is most likely due to incomplete activation of spermatozoa (capacitation) because of inadequate capacitating or fertilizing media. In vivo, the oviduct and its secretions provide a microenvironment that does reliably support and regulate interaction between the gametes. This review focuses on equine sperm-oviduct interaction. Equine sperm-oviduct binding appears to be...
Cruz Villagrán C, Schumacher J, Donnell R, Dhar MS.Transplantation of mesenchymal stromal cells (MSCs) to sites of experimentally created nerve injury in laboratory animals has shown promising results in restoring nerve function. This approach for nerve regeneration has not been reported in horses. In this study, we first evaluated the ability of equine bone marrow-derived MSCs (EBM-MSCs) to trans-differentiate into Schwann-like cells and subsequently tested the MSCs for their potential to regenerate a transected nerve after implantation. The EBM-MSCs from three equine donors were differentiated into SCLs for 7 days, , in the presence of s...
Chen Q, Tong Q, Ge H, Li W, Liu J, Wang Y, Guo Z, Quan F, Zhang Y.SP110 has previously shown to be a genetic determinant of host resistance to the intracellular pathogen infection in mouse and human. However, its relevant biological information in large non-primate animals still remains unknown. Here we report the novel discovery and characterization of three transcript variants of horse SP110. The transcript variant 1 (Tv1) of horse SP110 with the longest open reading frame has four domains (Sp100, SAND, PHD and Bromo domain). Tv2 and Tv3 share the same N-terminal sequence as Tv1, which contains Sp100 and SAND. We show that Tv2 is generated from alternative...
Warnken T, Huber K, Feige K.Exact analysis of equine insulin in blood samples is the key element for assessing insulin resistance or insulin dysregulation in horses. However, previous studies indicated marked differences in insulin concentrations obtained from sample analyses with different immunoassays. Most assays used in veterinary medicine are originally designed for use in human diagnostics and are based on antibodies directed against human insulin, although amino acid sequences between equine and human insulin differ. Species-specific assays are being used more frequently and seem to provide advantages compared to ...
Murgiano L, Waluk DP, Towers R, Wiedemar N, Dietrich J, Jagannathan V, Drögemüller M, Balmer P, Druet T, Galichet A, Penedo MC, Müller EJ....We investigated a family of horses exhibiting irregular vertical stripes in their hair coat texture along the neck, back, hindquarters, and upper legs. This phenotype is termed "brindle" by horse breeders. We propose the term "brindle 1 (BR1)" for this specific form of brindle. In some BR1 horses, the stripes were also differentially pigmented. Pedigree analyses were suggestive of a monogenic X-chromosomal semidominant mode of inheritance. Haplotype analyses identified a 5 Mb candidate region on chromosome X. Whole genome sequencing of four BR1 and 60 nonbrindle horses identified 61 private v...
Nazmutdinov RR, Bronshtein MD, Zinkicheva TT, Hansen NS, Zhang J, Ulstrup J.Outer-sphere electron transfer (ET) between optically active transition-metal complexes and either other transition-metal complexes or metalloproteins is a prototype reaction for kinetic chirality. Chirality as the ratio between bimolecular rate constants of two enantiomers mostly amounts to 1.05-1.2 with either the Λ or Δ form the more reactive, but the origin of chirality in ET parameters such as work terms, electronic transmission coefficient, and nuclear reorganization free energy has not been addressed. We report a study of ET between the Λ-/Δ-[Co(Ox)3](3-) pair (Ox = oxalate) and hor...
Mirzai S, Safi S, Mossavari N, Afshar D, Bolourchian M.The present study was conducted to establish a Loop-mediated isothermal amplification (LAMP) technique for the rapid detection of B. mallei the etiologic agent of glanders, a highly contagious disease of equines. A set of six specific primers targeting integrase gene cluster were designed for the LAMP test. The reaction was optimized using different temperatures and time intervals. The specificity of the assay was evaluated using DNA from B.pseudomallei and Pseudomonas aeruginosa. The LAMP products were analyzed both visually and under UV light after electrophoresis. The optimized conditions w...
Ma L, Sang M, Zhang J, Zhang S.Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily and plays multifunctional roles in the immune system. In the present study, a homolog of TRAIL from the Mongolian horse (named ecTRAIL) was identified and characterized. The 870-bp open reading frame encodes a polypeptide of 289 amino acid residues with a predicted molecular weight of 33.47Â kDa and pI of 8.47. The genomic structure of ecTRAIL shares a five-exon/six-intron arrangement similar to its orthologs. Multiple alignments show that ecTRAIL is a type II transmembrane protein with a t...
Suganuma K, Narantsatsral S, Battur B, Yamasaki S, Otgonsuren D, Musinguzi SP, Davaasuren B, Battsetseg B, Inoue N.Trypanosoma equiperdum causes dourine via sexual transmission in Equidae. T. equiperdum is classified under the subgenus Trypanozoon along with the T. brucei sspp. and T. evansi; however, the species classification of Trypanozoon remains a controversial topic due to the limited number of T. equiperdum reference strains. In addition, it is possible that some were misclassified T. evansi strains. Thus, there is a strong need for a new T. equiperdum strain directly isolated from the genital mucosa of a horse with a clinically- and parasitologically-confirmed dourine infection. Trypanosomes isolat...
Han X, Zhang P, Yu W, Xiang W, Li X.The Chinese EIAV vaccine is an attenuated live virus vaccine obtained by serial passage of a virulent horse isolate (EIAV) in donkeys (EIAV) and, subsequently, in donkey cells in vitro. In this study, we compare the env gene of the original horse virulent virus (EIAV) with attenuated strains serially passaged in donkey MDM (EIAV) and donkey dermal cells (EIAV). Genetic comparisons among parental and attenuated strains found that vaccine strains contained amino acid substitutions/deletions in gp90 that resulted in a loss of three potential N-linked glycosylation sites, designated g5, g9, and g1...
Wu H, Bi X, Cao F, Zhu C, Liu H, Song J, Ma L, Ma L, Zhang Y, Zhao D, Liu H, Xu X, Zhang S.A proliferation inducing ligand (APRIL) is a member of the TNF superfamily. It shares two receptors with B-cell activating factor (BAFF), B-cell maturation antigen (BCMA), and transmembrane activator and CAML interactor (TACI). Herein, the equine APRIL was identified from equine adipose-derived stem cell (ASC), and the protein expression of APRIL and its related molecules were detected during the adipogenic differentiation of equine ASC in vitro. The equine APRIL gene was located on chromosome 11, spans 1852 base pairs (bp). Its open reading frame covers 753Â bp, encoding a 250-amino acid prot...
Wise LM, Bodaan CJ, Mercer AA, Riley CB, Theoret CL.Wounds in horses often exhibit sustained inflammation and inefficient vascularization, leading to excessive fibrosis and clinical complications such as "proud flesh". Orf virus-derived proteins, vascular endothelial growth factor (VEGF)-E and interleukin (ovIL)-10, enhance angiogenesis and control inflammation and fibrosis in skin wounds of laboratory animals. Objective: The study aimed to determine if equine dermal cells respond to VEGF-E and ovIL-10. Equine dermal cells are expected to express VEGF and IL-10 receptors, so viral protein treatment is likely to alter cellular gene expression an...
Fortuna PRJ, Bielefeldt-Ohmann H, Ovchinnikov DA, Wolvetang EJ, Whitworth DJ.Horses are susceptible to a number of neurotropic viruses, including West Nile virus (WNV), which is a pathogen of global significance in both horses and humans. However, there are no in vitro models with which to study infectious neuropathic diseases in the horse. In an effort to redress this, we have generated neurons from equine induced pluripotent stem cells (equiPSCs) that express a range of cortical neuron-specific markers, in addition to the membrane-bound ligand ephrin B3, which plays an important role in axon guidance as well as functioning as the receptor through which henipaviruses,...
Avarzed A, Igarashi I, De Waal DT, Kawai S, Oomori Y, Inoue N, Maki Y, Omata Y, Saito A, Nagasawa H, Toyoda Y, Suzuki N.Monoclonal antibody (MAb) BEG3 was produced against Babesia equi parasites to define a species-specific antigen for diagnostic use. The MAb reacted with single, paired, and Maltese cross forms of B. equi, and no reaction was observed with this MAb on acetone-fixed Babesia caballi, Babesia ovata, or Babesia microti parasites in the indirect immunofluorescent antibody test. Confocal laser and immunoelectron microscopic studies showed that the antigen which was recognized by this MAb was located on the surface of B. equi parasites. This MAb recognized a 19-kDa protein of B. equi antigen and did n...
van der Harst MR, Bull S, Laffont CM, Klein WR.The aminoglycoside gentamicin is often used in equine practice. Despite its clinical use, concerns remain regarding the potential toxic side-effects, such as nephrotoxicity, in equine patients, particularly after repeated dosing. The aim of the study was to investigate first in vitro the mechanisms contributing to the renal toxicity of gentamicin and to identify sensitive biomarkers indicating proximal tubule damage. To this end, the kidney-derived cell lines LLC-PKI and MDCK were treated with gentamicin at different concentrations. Toxicity was assessed by measuring the release of gamma-gluta...
Brooks DE, Andrew SE, Dillavou CL, Ellis G, Kubilis PS.To evaluate in vitro susceptibility to topical antifungal medications, as measured by minimum inhibitory concentration (MIC) and 50% inhibitory concentration (IC50%), of fungal isolates from horses with ulcerative keratomycosis in Florida; to compare results with those of other studies to identify differences in susceptibility patterns among fungi isolated from horses in different geographic regions; and to note indications of fungal resistance to drugs tested in other studies. Methods: Corneal fungal cultures from client-owned horses from Florida with ulcerative keratomycosis (n = 22). Method...
Tablin F, Walker NJ, Hogle SE, Pratt SM, Norris JW.To determine whether platelet growth factors are preserved in supernatants obtained from rehydrated trehalose-stabilized, freeze-dried (lyophilized) equine platelets and whether those growth factors stimulate fibroblast proliferation and migration and enhance fibroblast-associated contraction in a collagen gel assay. Methods: 6 clinically normal adult horses. Methods: Blood samples were obtained from 6 horses, and washed platelets were prepared via differential centrifugation. Washed platelets were freeze-dried in a physiologic buffer with a mixture of trehalose and polyethylene glycol 4000. R...
Leemans B, Stout TAE, Soom AV, Gadella BM.Procaine directly triggers pH-dependent cytokinesis in equine oocytes and induces hypermotility in stallion spermatozoa, an important event during capacitation. However, procaine-induced hyperactivated motility is abolished when sperm is washed to remove the procaine prior to sperm-oocyte co-incubation. To understand how procaine exerts its effects, the external Ca2+ and Na+ and weak base activity dependency of procaine-induced hyperactivation in stallion spermatozoa was assessed using computer-assisted sperm analysis. Percoll-washed stallion spermatozoa exposed to Ca2+-depleted (+2 mM EGTA) p...
Gadella BM, Rathi R, Brouwers JF, Stout TA, Colenbrander B.During sexual reproduction, the sperm and oocyte must fuse before the production of a diploid zygote can proceed. In mammals such as equids, fusion depends critically on complex changes in the plasma membrane of the sperm and, not surprisingly, this membrane differs markedly from that of somatic cells. After leaving the testes, sperm cease to synthesize plasma membrane lipids or proteins, and vesicle-mediated transport stops. When the sperm reaches the female reproductive tract, it is activated by so-called capacitation factors that initiate a delicate reorientation and modification of molecul...
Kamm JL, Riley CB, Parlane N, Gee EK, McIlwraith CW.Despite significant immunosuppressive activity, allogeneic mesenchymal stromal cells (MSCs) carry an inherent risk of immune rejection when transferred into a recipient. In naïve recipients, this immune response is initially driven by the innate immune system, an immediate reaction to the foreign cells, and later, the adaptive immune system, a delayed response that causes cell death due to recognition of specific alloantigens by host cells and antibodies. This review describes the actions of MSCs to both suppress and activate the different arms of the immune system. We then review the surviva...
Fowlie JG, Arnoczky SP, Stick JA, Pease AP.By study of the translocation and deformation of equine menisci throughout the range of motion, it may be possible to identify potential mechanical factors in the pathogenesis of injury to the cranial horn of the medial meniscus. Objective: To quantitatively document meniscal translocation and deformation using radiographic and MR imaging, and to evaluate for potential variation between the medial and lateral menisci. Methods: Radiographic markers were embedded in the periphery of the menisci in 6 cadaver stifles. Proximal-distal radiographs were taken at 15° intervals ranging from full flexi...
Quist EE, Hokin LE.A potent inhibitor of (Na+ + K+)-ATPase activity was purified from Sigma equine muscle ATP by cation- and anion-exchange chromatography. The isolated inhibitor was identified by atomic absorption spectroscopy and proton resonance spectroscopy to be an inorganic vanadate. The isolated vanadate and a solution of V2O5 inhibit sarcolemma (Na+ + K+)-ATPase with an I50 of 1 micrometer in the presence of 1 mM ethyleneglycol-bis-(beta-aminoethylether)-N,N'-tetraacetic acid (EGTA), 145 mM NaCl, 6mM MgCl2, 15 mM KCl and 2 mM synthetic ATP. The potency of the isolated vanadate is increased by free Mg2+. ...
Ahern BJ, Schaer TP, Terkhorn SP, Jackson KV, Mason NJ, Hankenson KD.To evaluate effects of apheresis on mesenchymal stem cells (MSCs) and compare those MSCs with MSCs obtained from adipose tissue or bone marrow (BM). Methods: Samples obtained from 6 adult horses. Methods: Samples of blood from a peripheral vein, adipose tissue, and BM aspirate were obtained from each horse. Samples were processed via apheresis of blood and techniques reported elsewhere for adipose tissue and BM. Cultures were maintained until adherence and subsequently were subjected to differentiation protocols to evaluate adipogenic, osteoblastogenic, and chondrogenic potential. Results: Aph...
Uhl PB, Szober CM, Amann B, Alge-Priglinger C, Ueffing M, Hauck SM, Deeg CA.Retinal pigment epithelium (RPE) builds the outer blood-retinal barrier of the eye and plays an important role in pathogenesis of the sight threatening disease equine recurrent uveitis (ERU). ERU is a spontaneous autoimmune mediated inflammatory disease characterised by the breakdown of the outer blood-retinal barrier and an influx of autoaggressive T-cells into the inner eye. Therefore, identification of molecular mechanisms contributing to changed function of blood-retinal barrier in ERU is important for the understanding of pathophysiology. Cell surface proteins of RPE collected from health...
Buss DG, Sharma A, Giuliano EA, Mohan RR.Mitomycin C (MMC) is used clinically to treat corneal scarring in human patients. We investigated the safety and efficacy of MMC to treat corneal scarring in horses by examining its effects at the early and late stages of disease using an in vitro model. Methods: An in vitro model of equine corneal fibroblast (ECF) developed was used. The ECF or myofibroblast cultures were produced by growing primary ECF in the presence or absence of transforming growth factor beta-1 (TGFbeta1) under serum-free conditions. The MMC dose for the equine cornea was defined with dose-dependent trypan blue exclusion...
Garvican ER, Vaughan-Thomas A, Redmond C, Clegg PD.Our objective was to characterize the variation in gene expression for key genes associated with chondrogenic phenotype of osteochondrosis (OC)-affected and normal chondrocytes, and to identify whether OC chondrocytes can redifferentiate and regain a phenotype similar to normal chondrocytes if appropriate chondrogenic signals are given. Equine articular cartilage removed at surgery to treat clinically significant OC lesions was collected (n = 10), and the gene expression evaluated and compared to aged-matched normal samples (n = 10). Cartilage was harvested from normal (n = 4) and OC (n = 3) j...
Fingerhut L, Yücel L, Strutzberg-Minder K, von Köckritz-Blickwede M, Ohnesorge B, de Buhr N.Equine recurrent uveitis (ERU) is a common ocular disease of horses and described as a model for human autoimmune uveitis. This immune-mediated, inflammatory condition progressively destroys the eye, ultimately leading to blindness. Genetic and autoimmune factors, next to infections with Leptospira, are discussed as key factors in the pathogenesis. Furthermore, a release of neutrophil extracellular traps (NETs) by activated neutrophils is involved. NETs are composed of decondensed chromatin and proteins that can immobilize invading pathogens. However, if NETs accumulate, they can contribute to...
Sp N, Kang DY, Kim DH, Lee HG, Park YM, Kim IH, Lee HK, Cho BW, Jang KJ, Yang YM.Cortisol is a hormone involved in stress during exercise. The application of natural compounds is a new potential approach for controlling cortisol-induced stress. Tumour suppressor protein p53 is activated during cellular stress. Succinate dehydrogenase complex subunit A () and hypoxanthine phosphoribosyl transferase 1 () are considered to be two of the most stable reference genes when measuring stress during exercise in horses. In the present study cells were considered to be in a 'stressed state' if the levels of these stable genes and the highly stress responsive gene p53 were altered. It ...
Rasmussen T, Brittain T, Berks BC, Watmough NJ, Thomson AJ.Nitrous oxide reductase (N2OR) catalyses the final step of bacterial denitrification, the two-electron reduction of nitrous oxide (N2O) to dinitrogen (N2). N2OR contains two metal centers; a binuclear copper center, CuA, that serves to receive electrons from soluble donors, and a tetranuclear copper-sulfide center, CuZ, at the active site. Stopped flow experiments at low ionic strengths reveal rapid electron transfer (kobs=150 s-1) between reduced horse heart (HH) cytochrome c and the CuA center in fully oxidized N2OR. When fully reduced N2OR was mixed with oxidized cytochrome c, a similar rat...
Alm H, Torner H, Blottner S, Nürnberg G, Kanitz W.Little information is available on methods of sperm capacitation for IVF in the horse. In this study, we summarized results of several independent trials that compared acrosome reaction, hyperactivation and chromatin integrity of fresh or cryopreserved stallion spermatozoa after treatment with heparin or with calcium ionophore. We also examined the influence of spermatozoa storage (fresh vs. cryopreserved), capacitation treatment, oocyte maturation time and cumulus morphology on the penetration rate and fertilization rate. We recovered cumulus-oocyte-complexes (COCs) from ovaries by ultrasound...
Romito M, Du Plessis DH, Viljoen GJ.The ability of a DNA vaccine to elicit an immune response in a horse was evaluated. The outer capsid protein VP2 of African horsesickness virus is known to elicit protective immunity in horses. Reverse transcribed DNA of the gene encoding VP2 was placed under the transcriptional control of the cytomegalovirus immediate-early enhancer/promoter and was injected on several occasions intramuscularly into a horse. Low antibody levels could be detected by ELISA. Antibodies directed against VP2 alone were shown by Western blot while low levels of neutralizing antibodies were detected by a 50% plaque ...
Bourzac CA, Koenig JB, Link KA, Nykamp SG, Koch TG.To evaluate the efficacy and effects of labeling equine umbilical cord blood (UCB)- and bone marrow (BM)-derived multipotent mesenchymal stromal cells (MSCs) with an ultrasmall superparamagnetic iron oxide (SPIO) contrast agent and the detection of labeled MSCs by use of MRI. Methods: UCB MSCs from placental tissues of 5 foals and BM MSCs from 5 horses. Methods: UCB and BM MSC cultures were seeded in duplicate (5,000 cells/cm(2)). One duplicate was incubated with SPIO (50 μg/mL); the other was processed identically, but without SPIO. Mesenchymal stromal cells were expanded in triplicates for ...
Machado TSL, Massoco CO, Silva LCLC, Fülber J, Moreira JJ, Baccarin RYA.To compare effects of platelet-rich plasma (PRP), interleukin-1 receptor antagonist protein (IRAP), autologous processed plasma (APP), and sodium hyaluronate treatments on synovial fluid cells in vitro and on synovial fluid obtained from osteochondrotic joints of horses. Methods: Synovial fluid cells from 8 healthy equine tibiotarsal joints (in vitro experiment) and synovial fluid samples from 40 tibiotarsal joints of 25 horses with osteochondrosis dissecans (in vivo experiment). Methods: Effects of various treatments on concentrations of prostaglandin (PG) E2, interleukin (IL)-1β, tumor necr...
Katepalli MP, Adams AA, Lear TL, Horohov DW.Telomeres, specialized structures present at the ends of linear eukaryotic chromosomes, function to maintain chromosome stability and integrity. Telomeres shorten with each cell division eventually leading to replicative senescence, a process thought to be associated with age-related decline in immune function. We hypothesized that shortened PBMC telomere length is a factor contributing to immunosenescence of the aged horse. Telomere length was assessed in 19 horses ranging in age from 1 to 25 years. Mitogen-induced 3H-thymidine incorporation, total serum IgG, and pro-inflammatory cytokine exp...
Clegg PD, Burke RM, Coughlan AR, Riggs CM, Carter SD.The cellular production by resident articular cells and infiltrating inflammatory cells of the gelatinase matrix metalloproteinases (MMP) was investigated by tissue culture methods and analysis of cell supernatants by gelatin zymography. Peripheral blood neutrophils in short term culture produced MMP-9, as did peripheral blood monocytes in culture. Isolated articular chondrocytes in monolayer culture produced both MMP-2 and MMP-9, although articular cartilage maintained as explant culture produced MMP-2 alone. Synovial fibroblasts grown in monolayer culture produced MMP-2 alone, although synov...
Silver K, Desormaux A, Freeman LC, Lillich JD.Non-steroidal anti-inflammatory drugs (NSAIDs) are among the most widely used drugs for the suppression of inflammation and pain. However, the analgesic properties of NSAIDs are also associated with significant negative side effects, most notably in the gastrointestinal (GI) tract. Increasingly, evidence indicates that the ulcerogenic properties of some NSAIDs are not exclusively the result of inhibition of cyclooxygenase isoforms in the GI tract, and other mechanisms, including inhibition of cell migration and epithelial restitution, are being explored. Recently, microarray analysis was used ...
How MJ, Gonzales D, Irwin A, Caro T.Of all hypotheses advanced for why zebras have stripes, avoidance of biting fly attack receives by far the most support, yet the mechanisms by which stripes thwart landings are not yet understood. A logical and popular hypothesis is that stripes interfere with optic flow patterns needed by flying insects to execute controlled landings. This could occur through disrupting the radial symmetry of optic flow via the aperture effect (i.e. generation of false motion cues by straight edges), or through spatio-temporal aliasing (i.e. misregistration of repeated features) of evenly spaced stripes. By r...
Burk SV, Dangoudoubiyam S, Brewster-Barnes T, Bryant UK, Howe DK, Carter CN, Vanzant ES, Harmon RJ, Kazacos KR, Rossano MG.Currently, diagnosis of Parascaris equorum infection in equids is limited to patent infections. The goals of this study were to culture P. equorum larvae in vitro and identify excretory-secretory (ES) products for prepatent diagnostic testing. Parascaris equorum L2/L3 larvae were hatched and cultured for up to 3 weeks for ES product collection. Fifth stage (L5) P. equorum were also cultured for ES product collection. Examination of ES fractions by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and silver stain revealed L2/L3 products ranging from 12-94 kDa and L5 products...
Phillips JC, Lembcke LM, Noltenius CE, Newman SJ, Blackford JT, Grosenbaugh DA, Leard AT.To determine the tissue-restricted expression pattern of tyrosinase mRNA in canine and equine melanocytic tumors and relative tyrosinase and major histocompatibility complex (MHC) I mRNA expression in variants of melanocytic tumors. Methods: 39 canine and 8 equine tumor samples and 10 canine and 6 equine normal tissue samples. Methods: RNA was isolated from formalin-fixed, paraffin-embedded tissues. Real-time PCR assays were designed to amplify canine and equine tyrosinase, S18 ribosomal RNA, and major histocompatibility complex I transcripts. Relative expression was determined by use of S18 a...
