Flow cytometry is a technology used to analyze the physical and chemical characteristics of cells or particles in horses. This technique involves suspending cells in a fluid stream and passing them through a laser beam, where they are individually measured for various parameters such as size, complexity, and fluorescence intensity. In equine research, flow cytometry is applied to study immune cell populations, assess cellular responses to pathogens, and evaluate hematological parameters. It is a valuable tool for veterinary diagnostics, allowing for detailed analysis of blood and tissue samples. This page compiles peer-reviewed research studies and scholarly articles that explore the applications, methodologies, and findings related to flow cytometry in equine health and disease.
Hirsch G, Lavoie-Lamoureux A, Beauchamp G, Lavoie JP.Neutrophils are generally considered less responsive to glucocorticoids compared to other inflammatory cells. The reported increase in human neutrophil survival mediated by these drugs partly supports this assertion. However, it was recently shown that dexamethasone exerts potent anti-inflammatory effects in equine peripheral blood neutrophils. Few comparative studies of glucocorticoid effects in neutrophils and other leukocytes have been reported and a relative insensitivity of neutrophils to these drugs could not be ruled out. Objective: We assessed glucocorticoid-responsiveness in equine an...
Ranera B, Remacha AR, Álvarez-Arguedas S, Romero A, Vázquez FJ, Zaragoza P, Martín-Burriel I, Rodellar C.Mesenchymal stem cells (MSCs) derived from bone marrow (BM-MSCs) and adipose tissue (AT-MSCs) are being applied to equine cell therapy. The physiological environment in which MSCs reside is hypoxic and does not resemble the oxygen level typically used in in vitro culture (20% O2). This work compares the growth kinetics, viability, cell cycle, phenotype and expression of pluripotency markers in both equine BM-MSCs and AT-MSCs at 5% and 20% O2. Results: At the conclusion of culture, fewer BM-MSCs were obtained in hypoxia than in normoxia as a result of significantly reduced cell division. Hypoxi...
Echigoya Y, Morita S, Itou T, Sakai T.To evaluate effects of extracellular lactate on viability, shape change, lactate metabolism, and reactive oxygen species (ROS) production in equine polymorphonuclear leukocytes (PMNs). Methods: PMNs isolated from equine venous blood samples. Methods: PMNs were incubated with 0 to 300mM lactate for 30 minutes before each experiment. Viability was assessed via trypan blue exclusion. Shape change was assessed via flow cytometry and light microscopy. Relative quantification of monocarboxylic acid transporter and lactate dehydrogenase lactate dehydrogenase (LDH) isotype mRNAs was performed with a r...
El Garch H, Crafford JE, Amouyal P, Durand PY, Edlund Toulemonde C, Lemaitre L, Cozette V, Guthrie A, Minke JM.A recombinant canarypox virus vectored vaccine co-expressing synthetic genes encoding outer capsid proteins, VP2 and VP5, of African horse sickness virus (AHSV) serotype 4 (ALVAC(®)-AHSV4) has been demonstrated to fully protect horses against homologous challenge with virulent field virus. Guthrie et al. (2009) detected weak and variable titres of neutralizing antibody (ranging from <10 to 40) 8 weeks after vaccination leading us to hypothesize that there could be a participation of cell mediated immunity (CMI) in protection against AHSV4. The present study aimed at characterizing the CMI ind...
Brainard BM, Epstein KL, LoBato DN, Kwon S, Darien BJ, Hurley DJ, Moore JN.Inflammation-induced P-selectin (CD62P) expression on platelets and endothelial cells facilitates interactions among platelets and polymorphonuclear leukocytes (PMN), and can also promote coagulation. The effects of clopidogrel and aspirin (ASA) on equine platelet CD62P expression were investigated. Six horses were treated in a cross-over design with clopidogrel (2mg/kg PO q 24) or ASA (5mg/kg PO q 24h) for 5 days. Platelets collected at 24, 72, 96, 120, and 168 h after the initiation of therapy were stimulated using 0.1 μg/mL thrombin, followed by flow cytometric analysis using anti-CD41/61 ...
Noronha LE, Harman RM, Wagner B, Antczak DF.The immunoreceptor NKp46 is considered to be the most consistent marker of NK cells across mammalian species. Here, we use a recombinant NKp46 protein to generate a panel of monoclonal antibodies that recognize equine NKp46. The extracellular region of equine NKp46 was expressed with equine IL-4 as a recombinant fusion protein (rIL-4/NKp46) and used as an immunogen to generate mouse monoclonal antibodies (mAbs). MAbs were first screened by ELISA for an ability to recognize NKp46, but not IL-4, or the structurally related immunoreceptor CD16. Nine mAbs were selected and were shown to recognize ...
da Silveira JC, Veeramachaneni DN, Winger QA, Carnevale EM, Bouma GJ.Proper cell communication within the ovarian follicle is critical for the growth and maturation of a healthy oocyte that can be fertilized and develop into an embryo. Cell communication within the follicle involves many signaling molecules and is affected by maternal age. Recent studies indicate that cell communication can be mediated through secretion and uptake of small membrane-enclosed vesicles. The goals of this study were to 1) identify cell-secreted vesicles (microvesicles and exosomes) containing miRNAs and proteins within ovarian follicular fluid and 2) determine if miRNA level differ...
De Schauwer C, Piepers S, Van de Walle GR, Demeyere K, Hoogewijs MK, Govaere JL, Braeckmans K, Van Soom A, Meyer E.During recent years, cell-based therapies using mesenchymal stem cells (MSC) are reported in equine veterinary medicine with increasing frequency. In most cases, the isolation and in vitro differentiation of equine MSC are described, but their proper immunophenotypic characterization is rarely performed. The lack of a single marker specific for MSC and the limited availability of monoclonal antibodies (mAbs) for equine MSC in particular, strongly hamper this research. In this study, 30 commercial mAbs were screened with flow cytometry for recognizing equine epitopes using the appropriate posit...
Morillo Rodriguez A, Balao da Silva C, Macías-García B, Gallardo Bolaños JM, Tapia JA, Aparicio IM, Ortega-Ferrusola C, Peña FJ.A total of 42 ejaculates were used in the experiment; six ejaculates per stallion, obtained from seven Pure Spanish stallions (PRE), were split and frozen in freezing media with different concentrations and combinations of cryoprotectant (CPA): (i) Cáceres (skim milk based extender) containing 2.5% glycerol (2.5GL), (ii) Cáceres containing 1.5% glycerol and 1.5% dimethylformamide (1.5%GL-1.5%DMFA), (iii) Cáceres extender supplemented with 1.5% glycerol and 2.5% dimethylformamide (1.5%GL-2.5%DMFA) and (iv) Cáceres extender supplemented with 4% dimethylformamide (4%DMFA). After at least 4 we...
Noronha LE, Harman RM, Wagner B, Antczak DF.The low-affinity Fc receptor CD16 plays a central role in the inflammatory and innate immune responses of many species, but has not yet been investigated in the horse. Using the predicted extracellular region of equine CD16 expressed as a recombinant fusion protein with equine IL-4 (rIL-4/CD16), we generated a panel of mouse monoclonal antibodies (mAbs) that recognize equine CD16. Nine mAbs were chosen for characterization based upon recognition of CD16, but not IL-4, in ELISA. All nine mAbs recognized full-length, cell-surface CD16 expressed as a GFP fusion protein by CHO cells, but not the c...
Wagner B, Hillegas JM, Babasyan S.CD23, also called FcεRII, is the low-affinity receptor for IgE and has first been described as a major receptor regulating IgE responses. In addition, CD23 also binds to CD21, integrins and MHC class II molecules and thus has a much wider functional role in immune regulation ranging from involvement in antigen-presentation to multiple cytokine-like functions of soluble CD23. The role of CD23 during immune responses of the horse is less well understood. Here, we expressed equine CD23 in mammalian cells using a novel IL-4 expression system. Expression resulted in high yield of recombinant IL-4/...
Pretorius A, Van Kleef M, Van Wyngaardt W, Heath J.African horsesickness (AHS) is an infectious but noncontagious viral disease affecting all species of Equidae. The recall immune response of AHSV naïve horses immunised with an attenuated African horsesickness virus serotype 4 (AHSV4) was characterised using immune assays including ELISPOT, real-time PCR (qPCR) and flow cytometry. The recall immune response detected in PBMC isolated from three inoculated horses showed an upregulation of circulating B lymphocytes that correlated with elevated IL-4 mRNA expression indicative of humoral immunity, but reduced frequency of CD4⁺ cells. In additio...
Lavoie-Lamoureux A, Beauchamp G, Quessy S, Martin JG, Lavoie JP.To compare innate immune responses of peripheral blood leukocytes from healthy and asymptomatic heaves-affected horses. Methods: Heaves-affected horses (n=5-6) and healthy controls (n=4-5) kept under low dust environments (pasture or shavings and pellets). Methods: Blood neutrophil and neutrophil-depleted cell populations were isolated using MACS system. Cells were incubated with or without bacterial products (lipopolysaccharide (LPS), 100 ng/mL and fMLP, 5 ng/mL, 5 h). Cytokine (IL-1β, IL-8, TNF, IL-4, INFγ and IL-10) and receptor (TLR4) mRNA expression was assessed by qPCR. TNF concentrati...
Gallardo Bolaños JM, Miró Morán Á, Balao da Silva CM, Morillo Rodríguez A, Plaza Dávila M, Aparicio IM, Tapia JA, Ortega Ferrusola C, Peña FJ.Apoptosis has been recognized as a cause of sperm death during cryopreservation and a cause of infertility in humans, however there is no data on its role in sperm death during conservation in refrigeration; autophagy has not been described to date in mature sperm. We investigated the role of apoptosis and autophagy during cooled storage of stallion spermatozoa. Samples from seven stallions were split; half of the ejaculate was processed by single layer centrifugation, while the other half was extended unprocessed, and stored at 5°C for five days. During the time of storage, sperm motility (C...
Balao da Silva C, Macías-García B, Morillo Rodriguez A, Gallardo Bolaños JM, Tapia JA, Aparicio IM, Morrell JM, Rodriguez-Martínez H....The only known means of effectively separating populations of X and Y bearing sperms is the Beltsville sexing technology. The technology implies that each individual sperm is interrogated for DNA content, measuring the intensity of the fluorescence after staining the spermatozoa with Hoechst 33342. Because there are no data regarding the effect of the staining on stallion sperm, ejaculates were incubated up to 90 min in presence of 0, 4.5, 9, 22.5, 31.5, 45, 54, 67.5, 76.5 and 90 μM of Hoechst 33342, in two media, KMT or INRA-Tyrodes. After 40 and 90 min of incubation, motility (CASA) and mem...
Mudge MC, Walker NJ, Borjesson DL, Librach F, Johns JL, Owens SD.Post-transfusion survival of allogeneic RBCs has been reported to be much shorter in horses than in other species. We hypothesized that post-transfusion survival of biotinylated allogeneic equine RBCs would be greater than the survival previously reported from studies using radioactive RBC-labeling techniques. Objective: The study objective was to determine post-transfusion survival of N-hydroxysuccinimide (NHS)-biotin-labeled allogeneic equine RBCs transfused into adult horses. Methods: Horses were adults and included 5 donors and 5 recipients. All horses were blood-typed, and donors were pai...
Brooks AC, Rickards KJ, Cunningham FM.Neutrophil activation, whilst a key component of host defence, must be tightly regulated in order to avoid an inappropriate cellular response. Annexin-1, which is present in large amounts in neutrophils, and its N-terminal peptides, reduce neutrophil accumulation but annexin peptides have also been shown to exhibit neutrophil activating properties. We have recently shown annexin-1 to be present in equine neutrophils and demonstrated that the annexin-1-derived peptide, Ac2-26, can both reduce superoxide production by these cells in response to other stimuli and directly induce free radical prod...
Morillo-Rodríguez A, Macías-García B, Tapia JA, Ortega-Ferrusola C, Peña FJ.Ejaculates from six pure Spanish stallions were split, and one subsample frozen in a commercial extender supplemented with the lipid soluble antioxidant butylated hydroxytoluene (BHT), while the other subsample served as control. After at least 4 weeks of storage, samples were thawed and post-thaw sperm quality analysed: sperm motility and kinematics using a CASA system, membrane and acrosome integrity and mitochondrial membrane potential using flow cytometry. The outcome of cryopreservation varied significantly among stallions. However, the supplementation with 1 mm BHT had no significant eff...
Dawson DR, Nydam DV, Price CT, Graham JE, Cynamon MH, Divers TJ, Felippe MJ.To investigate the effect of opsonization of Rhodococcus equi with R. equi-specific antibodies in plasma on bacterial viability and phagocyte activation in a cell culture model of infection. Methods: Neutrophils and monocyte-derived macrophages from 6 healthy 1-week-old foals and 1 adult horse. Methods: Foal and adult horse phagocytes were incubated with either opsonized or nonopsonized bacteria. Opsonization was achieved by use of plasma containing high or low concentrations of R. equi-specific antibodies. Phagocyte oxidative burst activity was measured by use of flow cytometry, and macrophag...
Wong DM, Alcott CJ, Clark SK, Jones DE, Fisher PG, Sponseller BA.A 3-day-old Thoroughbred colt was originally presented for treatment of neonatal isoerythrolysis, which was treated with a blood transfusion. However, persistent neutropenia was observed despite the absence of detectable infection. Subsequently, a granulocyte agglutination test was performed by incubating the colt's neutrophils with the mare's serum; results were positive, leading to a clinical diagnosis of alloimmune neonatal neutropenia. The diagnosis was further supported via flow cytometric analysis. The colt was hospitalized and treated prophylactically with antimicrobials and 4 separate ...
Filannino A, Stout TA, Gadella BM, Sostaric E, Pizzi F, Colenbrander B, Dell'Aquila ME, Minervini F.The aim of this study was to investigate the in vitro effects of the Fusarium fungus-derived mycotoxin, zearalenone and its derivatives alpha-zearalenol and beta-zearalenol on motility parameters and the acrosome reaction of stallion sperm. Since the toxic effects of zearalenone and its derivatives are thought to result from their structural similarity to 17beta-estradiol, 17beta-estradiol was used as a positive control for 'estrogen-like' effects. Methods: Stallion spermatozoa were exposed in vitro to zearalenone, alpha-zearalenol, beta-zearalenol or 17beta-estradiol at concentrations ranging...
Wauters J, Franck T, Pille F, Martens A, Demeyere K, Sys S, Serteyn D, Gasthuys F, Meyer E.Myeloperoxidase (MPO) is a protein of interest due to its involvement in equine pathologies. Until now, results in equine diagnostic research were achieved through extracellular MPO detection. However, studying the cellular MPO content in neutrophils has revealed important insights in human diseases. This study aimed to develop a technique for the specific detection of MPO on the single cell level defining a flow cytometric protocol for the detection of both equine surface-bound and cellular MPO. Both indirect and direct labeling techniques are described which include the comparison of two sec...
Casella S, Giudice E, Giannetto C, Marafioti S, Piccione G.The purpose of this study was to evaluate in vitro the effects of hydrocortisone and aminophylline on adenosine diphosphate (ADP)-induced platelet aggregation in horses. Blood samples from 30 healthy Thoroughbred horses were collected by via jugular venipuncture to assess platelet aggregation. Platelet-rich and platelet-poor plasma were prepared from all samples by centrifugation and divided into three different aliquots. In the first aliquot, platelet aggregation was measured after platelet activation with 1 µM and 0.5 µM ADP (Group A). In the other two aliquots, the effect of a 10 min prei...
De Schauwer C, Meyer E, Cornillie P, De Vliegher S, van de Walle GR, Hoogewijs M, Declercq H, Govaere J, Demeyere K, Cornelissen M, Van Soom A.Mesenchymal stromal cells (MSC) represent a promising population for supporting new clinical concepts in cellular therapy. A wide diversity of isolation procedures for MSC from umbilical cord blood (UCB) has been described for humans. In contrast, a few data are available in horses. In the current study, a sedimentation method using hydroxyethyl starch and a method based on the lysis of red blood cells using ammonium chloride (NH(4)Cl) were compared with two density gradient separation methods (Ficoll-Paque and Percoll). Adherent cell colonies could be established using all four isolation meth...
Ibrahim S, Steinbach F.Earlier studies investigating the cross-reactivity of antibodies submitted to the HLDA8 had used flow cytometry as a method of choice to screen mAbs for reactivity with equine leukocytes, including two-color flow-cytometry to characterize the lymphocyte population they detect. In addition, immuno-histochemistry (IHC) was used to detect distribution of positive cells in lymphoid tissue sections. In this study we performed immunoprecipitation (IP) to complement the previous results and add valuable information regarding the molecules detected by the cross-reacting antibodies. Surface molecules f...
Pascucci L, Curina G, Mercati F, Marini C, Dall'Aglio C, Paternesi B, Ceccarelli P.In the last decades, multipotent mesenchymal progenitor cells have been isolated from many adult tissues of different species. The International Society for Cellular Therapy (ISCT) has recently established that multipotent mesenchymal stromal cells (MSCs) is the currently recommended designation. In this study, we used flow cytometry to evaluate the expression of several molecules related to stemness (CD90, CD44, CD73 and STRO-1) in undifferentiated, early-passaged MSCs isolated from adipose tissue of four donor horses (AdMSCs). The four populations unanimously expressed high levels of CD90 an...
Rodríguez AM, Ferrusola CO, García BM, Morrell JM, Martínez HR, Tapia JA, Peña FJ.Ejaculates from 7 stallions were split and simultaneously frozen in three different extenders, INRA 96 egg yolk glycerol, Ghent and the newly developed extender Caceres. After thawing, samples were evaluated for motility (CASA system) sperm membrane integrity and early membrane changes (YoPro-1/Eth staining), acrosome integrity (FICT-PNA), and mitochondrial membrane potential (JC-1) (flow cytometry). Samples frozen in Caceres extender consistently showed the best results in post-thaw motility (increases ranging from 11 to 17%, p<0.05) and velocity (p<0.05), membrane integrity (increases ...
Ranera B, Lyahyai J, Romero A, Vázquez FJ, Remacha AR, Bernal ML, Zaragoza P, Rodellar C, Martín-Burriel I.Bone marrow and adipose tissue are the two main sources of mesenchymal stem cell (MSC). The aim of this work was to analyse the immunophenotype of 7 surface markers and the expression of a panel of 13 genes coding for cell surface markers in equine bone marrow and adipose tissue-derived MSCs obtained from 9 horses at third passage. The tri-lineage differentiation was confirmed by specific staining. Equine MSCs from both sources were positive for the MSC markers CD29 and CD90, while were negative for CD44, CD73, CD105, CD45 and CD34. The gene expression of these molecules was also evaluated by ...
Mérant C, Sheoran A, Timoney JF.Streptococcus equi (Se), the cause of equine strangles, is highly resistant to phagocytosis by neutrophils and is usually classified as an extracellular pathogen. Large numbers of the organism in tonsillar tissues during the acute phase of the disease are completely eliminated during convalescence by mechanisms not yet understood. In this study we demonstrate in an opsono-bactericidal assay and by cytometry and confocal microscopy that Se is interiorized and killed by equine blood monocytes. This finding supports the hypotheses that adaptive immune clearance is mediated by tonsillar macrophage...
Ranera B, Ordovás L, Lyahyai J, Bernal ML, Fernandes F, Remacha AR, Romero A, Vázquez FJ, Osta R, Cons C, Varona L, Zaragoza P, Martín-Burriel I....Mesenchymal stromal cells (MSCs) represent an attractive source for regenerative medicine. However, prior to their application, fundamental questions regarding molecular characterisation, growth and differentiation of MSCs must be resolved. Objective: To compare and better understand the behaviour of equine MSCs obtained from bone marrow (BM) and adipose tissue (AT) in culture. Methods: Five horses were included in this study. Proliferation rate was measured using MTT assay and cell viability; apoptosis, necrosis and late apoptosis and necrosis were evaluated by flow cytometry. The mRNA expres...
Podico G, Canisso IF.This study aimed to assess the parameters of epididymal sperm harvested by retrograde flushing (RF) followed by slicing float-up (SF). Epididymides from donkeys (n = 18) and horses (n = 28) were subjected to RF with a freezing extender and then SF technique. The retrieved sperm after RF and SF was evaluated for volume, concentration, and total sperm and then cryopreserved separately. Post-thaw total motility (TM) and progressive motility (PM) were evaluated with CASA. Sperm membrane integrity (SMI) and mitochondrial membrane potential (MMP) were assessed with flow cytometry. Sperm concentratio...
Schnabel CL, Babasyan S, Freer H, Wagner B.C-X-C motif ligand 10 (CXCL10) is a pro-inflammatory chemokine and has been extensively evaluated in people and mice. In horses, CXCL10 and its involvement in host immunity has rarely been analyzed due to the lack of specific antibodies. We generated a mAb specific for the equine chemokine CXCL10 using hybridoma technology. Antibody specificity was confirmed by intracellular staining and flow cytometric analysis of Chinese Hamster Ovary (CHO) cells expressing equine rCXCL10, while CHO cells expressing equine rCXCL9 were not detected. Native CXCL10 expression in PBMC from horses of different ag...
Ball BA, Sabeur K, Allen WR.Sperm-mediated gene transfer has been reported as a method for production of transgenic animals in a variety of species, and this technique represents a possible method for production of transgenic equids. Objective: To evaluate the uptake of exogenous DNA (enhanced green fluorescent protein; pEGFP) by equine spermatozoa and to assess the ability of transfected spermatozoa to introduce this transgene into early equine embryos. Methods: To evaluate incorporation of pEGFP into equine spermatozoa, washed spermatozoa were incubated with 32P-pEGFP, with or without lipofection. Spermatozoa were also...
Wilhelm KM, Graham JK, Squires EL.Computer-assisted motion analyses (CASA) and flow cytometry were used to evaluate stallion spermatozoa prior to and after cryopreservation. Spermatozoa were pretreated with: (1) Hepes-buffered medium (SHB); (2) phosphatidylserine (PS) liposomes; or (3) liposomes composed of both PS and cholesterol (PSCH) prior to dilution in either SHB or skim milk-egg yolk extender (SMEY). After cooling to 5 degrees C in SHB, PS and PSCH pretreatment (23%). Spermatozoal motion parameters were higher for spermatozoa diluted in SMEY than dilution in SHB. In Experiment 2, motion parameters were compared for sper...
Kwirant LADA, De La Corte FD, Cantarelli C, Cargnelutti JF, Martins M, Cabral MW, Maciel N, Rubin MIB.Equine platelet-rich plasma (PRP) has been used in horses to repair bone, articular and tendinous lesions, laminitis, and even endometritis. However, platelets have a very limited lifespan, which makes it difficult to prepare and use PRP, except in loco. With the aim to produce PRP with higher platelet viability for clinical purposes, the effects of the cryoprotectants dimethyl sulfoxide (DMSO) and trehalose were evaluated on cooled (4°C) and cryopreserved (-196°C) equine PRP. The protocols of cooling and cryopreservation were performed independently, comparing the following treatments: fres...
Rossi M, Gonzalez-Castro R, Falomo ME.Different additives have been tested in cooled stallion sperm, in order to maintain sperm quality and to ameliorate the decrease in sperm fertility potential. In several species, caffeine and pentoxifylline promote sperm motility by increasing energy production. We evaluate the effects of caffeine and pentoxifylline when added to stallion sperm before or after cooling. Three ejaculates from five stallions each were processed and resuspended in skim milk extender. Caffeine (5 mM), pentoxifylline (3.5 mM), or both additives combined were included to sperm before or after cooling (4°C for 24 ...
Orsztynowicz M, Pawlak P, Podstawski Z, Nizanski W, Partyka A, Gotowiecka M, Kosiniak-Kamysz K, Lechniak D.Predicting male fertility on non-invasive sperm traits is of big importance to human and animal reproduction strategies. Combining the wide range of parameters monitored by computer-assisted sperm analysis (CASA) with some molecular traits (e.g. mtDNA content) may help to identify markers of the male fertility. The aim of this study was to characterize variation in the mtDNA copy number in equine sperm and to investigate whether mtDNA content is correlated with quality traits of stallion spermatozoa and the age of the male. Ejaculates collected from 53 fertile stallions were divided into four ...
Breuer J, Müller U, Locher L, Spallek A, Recknagel S, Uhlig A, Schusser GF.Proinflammatoric cytokines are released extracellularly during necrosis. These lead to inflammation and destruction of surrounding tissues. The aim of this study was to compare the number of viable, apoptotic and necrotic cells in the bronchoalveolar lavage fluid (BALF) of normal horses and horses with recurrent airway obstruction (RAO) and to determine if fluorescence microscopy is a reliable method for this examination. A group of six normal horses and a group of ten horses with RAO were examined. Samples were assessed using annexin-V and propidium iodide immunofluorescence assay and examine...
Brooks AC, Rickards KJ, Cunningham FM.Neutrophil activation, whilst a key component of host defence, must be tightly regulated in order to avoid an inappropriate cellular response. Annexin-1, which is present in large amounts in neutrophils, and its N-terminal peptides, reduce neutrophil accumulation but annexin peptides have also been shown to exhibit neutrophil activating properties. We have recently shown annexin-1 to be present in equine neutrophils and demonstrated that the annexin-1-derived peptide, Ac2-26, can both reduce superoxide production by these cells in response to other stimuli and directly induce free radical prod...
Zhang YW, Davis EG, Blecha F, Wilkerson MJ.Innate immunity relies on a series of germline-encoded pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs), to detect conserved microbial components. TLR9 is typically expressed intracellularly in immune cells such as dendritic cells and recognizes unmethylated bacterial or viral cytosine-phosphate-guanine DNA (CpG-DNA). To investigate innate immune responses through TLR9 signaling pathway in horses, we cloned and characterized equine TLR9. Protein sequence analysis shows that equine TLR9 has a typically conserved cytosolic Toll/interleukin-1 receptor (TIR) domain, three l...
McKelvie J, Foster AP, Cunningham FM, Hamblin AS.Circulating lymphocyte numbers are elevated in horses with the allergic skin disease sweet itch and skin lesions are typified by an infiltrate of eosinophils and mononuclear cells, the latter of which have not been fully characterised. The aim of the present study was to characterise the lymphocyte subpopulations in the circulation and skin of ponies with sweet itch by flow cytometry and a newly developed modified alkaline phosphatase immunohistochemical technique. Sweet itch ponies were found to have significantly greater numbers of circulating CD5+ and CD4+ T-lymphocytes than normal animals....
Salter MM, Seeto WJ, DeWitt BB, Hashimi SA, Schwartz DD, Lipke EA, Wooldridge AA.To isolate and characterize endothelial colony-forming cells (ECFCs; a subtype of endothelial progenitor cells) from peripheral blood samples of horses. Methods: Jugular venous blood samples from 24 adult horses. Methods: Blood samples were cultured in endothelial cell growth medium. Isolated ECFCs were characterized by use of functional assays of fluorescence-labeled acetylated low-density lipoprotein (DiI-Ac-LDL) uptake and vascular tubule formation in vitro. Expression of endothelial (CD34, CD105, vascular endothelial growth factor receptor 2, and von Willebrand factor) and hematopoietic (C...
Flaminio MJ, Yen A, Antczak DF.Observations in early equine pregnancy clearly reveal maternal immune recognition of and response to the presence of the conceptus. Nevertheless, both maternal cellular and humoral responses appear ineffective in destroying the developing placenta and fetus in early pregnancy. Our previous studies had shown that the pre-conditioned medium generated from the culture of equine invasive trophoblast inhibited mitogen-induced lymphocyte proliferation and the expression of cytokine messenger RNA in vitro. Those findings also suggested that lymphocytes might have been halted in the G0/G1 phase of the...
Kirk ES, Squires EL, Graham JK.Assessing the fertilizing potential of a semen sample is important for effective stallion management and for rapid progress in evaluating new cryopreservation technologies. Unfortunately, sperm motility does not estimate fertility well. These experiments established assays to measure cell viability, acrosomal integrity and mitochondrial function for cryopreserved stallion spermatozoa, using flow cytometry, and determined the variability associated with these assays. Correlations between results for these laboratory assays and stallion fertility were also determined. The inter-assay variability...
Platt R, Sponseller BA, Chiang YW, Roth JA.The cell-mediated immune (CMI) response of foals to virulent equine herpesvirus-1 (EHV-1) infection was evaluated by multi-parameter flow cytometry (FCM). Ten 7-8-month-old EHV-1 seronegative foals were infected intranasally with virulent EHV-1 and 10 foals served as uninfected controls. Blood samples were collected 6 and 7 weeks after infection to test for specific CMI responses to live heterologous EHV-1 recall antigen. The activation markers included major histocompatibility complex class II (MHC II), intracellular interferon gamma (IFN-gamma) and interleukin 4 (IL-4). The results from both...
Stokol T, Serpa PBS, Zahid MN, Brooks MB.Equid herpes virus type-1 (EHV-1) is a major pathogen of horses, causing abortion storms and outbreaks of herpes virus myeloencephalopathy. These clinical syndromes are partly attributed to ischemic injury from thrombosis in placental and spinal vessels. The mechanism of thrombosis in affected horses is unknown. We have previously shown that EHV-1 activates platelets through virus-associated tissue factor-initiated thrombin generation. Activated platelets participate in thrombus formation by providing a surface to localize coagulation factor complexes that amplify and propagate thrombin genera...
Lindsey AC, Schenk JL, Graham JK, Bruemmer JE, Squires EL.The objective of this experiment was to determine the effects of flow cytometric sorting and freezing on stallion sperm fertility. A 2 x 2 factorial design was used to delineate effects of flow sorting and freezing spermatozoa. Oestrus was synchronised (July-August) in 41 mares by administering 10 ml altrenogest (2.2 mg/ml) per os for 10 consecutive days, followed by 250 microg cloprostenol i.m. on Day 11. Ovulation was induced by administering 3,000 iu hCG i.v. either 6 h (fresh spermatozoa) or 30 h (frozen/thawed spermatozoa) prior to insemination. Mares were assigned randomly to one of 4 sp...
Segura D, Monreal L, Espada Y, Pastor J, Mayós I, Homedes J.The objective of this study was to assess whether a new human platelet function analyser (the PFA-100) could be used to evaluate platelet function in horses and detect acetylsalicylic acid (ASA)-induced platelet dysfunctions. Citrated blood samples from 40 healthy horses were processed to obtain reference values for closure time (CT) using cartridges with collagen-ADP (CT-ADP) and collagen-epinephrine (CT-EPI) as platelet agonists. In addition, CT-ADP and CT-EPI were also measured before and 24 h after oral ASA administration in another 12 healthy horses. The sensitivity and specificity of the...
Cian F, Tyner G, Martini V, Comazzi S, Archer J.A 16-year-old, Irish Draft mare was admitted to the referring veterinarian for an annual health check. A mild generalized lymphadenomegaly was noted. Rectal palpation and transrectal ultrasonographic examination revealed prominent mesenteric lymph nodes. A transcutaneous abdominal ultrasonographic evaluation was unremarkable. A CBC revealed a marked leukocytosis (63.06 × 10(3)/μL) and lymphocytosis (58.2 × 10(3)/μL) due to increased numbers of small lymphocytes. No evidence of anemia or thrombocytopenia was found and neutrophil counts were low-normal. Cytologic examination of fine-needle a...
Panarace M, Pellegrini RO, Basualdo MO, Belé M, Ursino DA, Cisterna R, Desimone G, Rodríguez E, Medina MJ.Flow cytometry sex-sorting technology was developed in 1989. However, it is only the bovine species in which offspring of the desired sex are obtained at a commercial level. The aim of the present work was to evaluate efficiency parameters when using fresh sexed semen in a large-scale equine commercial embryo transfer program. During the 2009, 2010 and 2011 breeding seasons, 938 synchronized cycles were artificially inseminated. One hundred (10.6%) mares failed to ovulate, and for the remaining 838 useable cycles, 887 doses of sexed semen were used, representing 1.06 doses per cycle. In genera...
Crump AL, Davis W, Antczak DF.A cell surface molecule of equine T lymphocytes was identified and characterized using a mouse monoclonal antibody, HT23A. The molecule was detected on all T cells but not on other cells in peripheral blood, with the possible exception of a small subpopulation (about 5%) of B cells, as assessed by indirect immunofluorescence and flow cytometry. HT23A labelled T cell areas of horse lymph nodes and spleen when used in an indirect immunoperoxidase assay on frozen sections. Macrophages and neutrophils were not labelled by the antibody nor were frozen sections of horse liver, kidney, or brain. HT23...
Bauer N, Moritz A.Besides flow cytometric detection of cellular hemoglobin (HGB) concentration, the ADVIA 2120 uses a novel cyanide-free colorimetric method to determine extracellular total HGB concentration. In human samples, the results are equivalent to those of the cyanmethemoglobin method on the ADVIA 120. Cyanide-free HGB measurement has not been evaluated in animal samples. Objective: The aim of this prospective study was to compare the 3 methods of HGB analysis on the ADVIA 2120 and ADVIA 120 in blood samples from dogs, cats, and horses. Methods: Consecutive fresh K(3)EDTA blood samples from 119 dogs, 1...
Siedek E, Little S, Mayall S, Edington N, Hamblin A.Despite their important role in initiating T-cell responses in other species, dendritic cells have not been studied in the horse. A method for isolating blood dendritic cells by adherence and metrizamide gradients was adapted to equine cells. A number of monoclonal antibodies (mAbs), including some which label dendritic cells in other species, were tested for immunochemical reactivity with the isolated blood dendritic cells, and sections of lymph node and spleen. 62 +/- 6% of the isolated blood cells were MHC Class II positive and had typical dendritic cell morphology and only 4 +/- 2% contain...
Bozorgmanesh R, Magdesian KG, Sutton-Burges JW, Owens SD, Tablin F.Development of equine platelet concentrate (PC) would aid management of cases requiring transfused platelets (PLTs), where adminstration of whole-blood or platelet-rich plasma (PRP) might be contraindicated. Objective: To test and validate a method for production of an equine PRP-PC product. Methods: Six healthy Thoroughbred geldings from a research herd. Methods: In this prospective experimental study, whole blood was collected and processed through multiple centrifugation steps to yield 120 mL of PC. The PC was stored at 22°C and gently and continuously agitated. Measurements of PLT count...
Meulenbroeks C, van der Meide NM, Willemse T, Rutten VP, Tijhaar E.Ponies may suffer from Insect bite hypersensitivity (IBH), an allergic IgE-mediated pruritic skin disorder, induced by allergens from biting midges of the Culicoides spp. Objective: To determine whether recombinant Culicoides obsoletus allergens are able to activate T cells of ponies exposed to C. obsoletus and whether these allergen-specific responses differ between IBH-affected and healthy ponies. Methods: Ten IBH-affected Shetland ponies and 10 age-matched healthy controls taken from the same stables, to ensure similar exposure to midges. Methods: Peripheral blood mononuclear cells (PBMC) w...
Weiss DJ, Evanson OA, Fagliari JJ, Valberg S.To determine whether platelets become activated and form platelet-neutrophil aggregates during near-maximal treadmill exercise in horses. Methods: 4 Thoroughbreds. Methods: Horses were subjected to 4 standardized exercise tests on a treadmill, and blood samples were collected before exercise, at treadmill speed of 12 m/s, and 5 minutes after exercise. Flow cytometric techniques were used to identify activated platelets, and flow cytometric and microscopic techniques were used to identify platelet-neutrophil aggregates. Results: Platelet-neutrophil aggregates increased from 2.8 +/- 0.4% at rest...
Anderson SL, Duke-Novakovski T, Robinson AR, Townsend HGG, Singh B.The objective of this study was to determine the effect of pulmonary intravascular macrophage depletion on systemic inflammation and ex vivo neutrophil apoptosis using an experimental model of intestinal ischemia and reperfusion injury in horses. Neutrophils were isolated before and after surgery from horses that were randomized to three treatment groups, namely, sham celiotomy (CEL, = 4), intestinal ischemia and reperfusion (IR, = 6), and intestinal ischemia and reperfusion with gadolinium chloride treatment to deplete pulmonary intravascular macrophages (PIMs, IRGC, = 6). Neutrophil apopt...
Gibb Z, Morris LH, Maxwell WM, Grupen CG.The low efficiency of flow cytometric sex-sorting of stallion sperm has been attributed to the use of an opaque skim milk-based diluent during Hoechst 33342 (H33342) staining. Three experiments were conducted to formulate an optically clear stallion semen diluent for use during H33342 staining, and to determine whether a clear diluent improved resolution during sorting. For Experiment 1, sperm were incubated at 34 °C in each of five diluents containing either no protein, skim milk, 0.25% Cohn's Fraction V BSA, 0.5% BSA, or 1% BSA, following an 18 h storage (15 °C) period, or shortly after co...
Banks KL, Greenlee A.Various cell populations of equine mononuclear leukocytes were identified and isolated. Mononuclear leukocytes were concentrated by isopyknic centrifugation, using a solution of Ficoll and Hypaque. Three additional techniques were explored to separate monocytes from lymphocytes, and 3 methods were used to separate lymphocyte types. Cytochemical techniques for the detection of nonspecific esterase readily distinguished equine monocytes from lymphocytes. Peripheral blood lymphocytes were separated into at least 2 populations. One population had surface traits identical to thymocytes [ie, they re...
Al-Kass Z, Brown A, Johannisson A, Ntallaris T, Morrell JM.Although single layer centrifugation (SLC) selects robust spermatozoa from stallion semen, the effect of individual variation has not been studied in detail. The objective of this study was to determine the variation among stallions in the effects of SLC on sperm quality during cooled storage for up to 48 hr. Semen samples from seven stallions (18 ejaculates) were split, with one portion being used for SLC and the other serving as a control (CON). Sperm quality (kinematics, reactive oxygen species (ROS) production, membrane integrity (MI) and chromatin integrity) were analysed at 0, 24 and 48...
