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.
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...
Hamza E, Steinbach F, Marti E.Insect bite hypersensitivity (IBH) is an IgE-mediated dermatitis caused by bites of midges from the genus Culicoides. We have shown previously that peripheral blood mononuclear cells (PBMC) from IBH-affected horses produce higher levels of IL-4 and lower levels of IL-10 and TGF-β1 than those from healthy horses, suggesting that IBH is associated with a reduced regulatory immune response. FoxP3 is a crucial marker of regulatory T cells (Tregs). Here we have determined the proportion of CD4(+)CD25(+)FoxP3(+) T cells by flow cytometry in PBMC directly after isolation or after stimulation with Cu...
Corradetti B, Lange-Consiglio A, Barucca M, Cremonesi F, Bizzaro D.Umbilical cord matrix (UCM) has been recently proposed as an alternative source of mesenchymal stem cells (MSCs). The aim of this study was to isolate and characterize presumptive stem cells from intervascular and perivascular equine UCM and to obtain homogeneous subpopulations from both sites. Methods: Umbilical cords were processed for retrieval of MSCs. Unsieved cells from intervascular and perivascular portions were evaluated for cell cycle analysis and for immunophenotyping by flow cytometry. Cells from each site were separated into larger and smaller sieved populations using multi-dishes...
Dougherty SS, Santangelo KS, Bertone AL.To evaluate 2 commercially available transfection reagents for transfection efficiency and distribution of small interfering RNA (siRNA) molecules to chondrocytes in monolayer cultures and full-thickness cartilage explants from guinea pigs and horses. Methods: Cartilage explants from 5 one-month-old and 3 adult guinea pigs and 5 adult clinically normal horses. Methods: Monolayer chondrocytes and uniform cartilage explants were exposed to 1 of 2 siRNA transfection complexes according to manufacturers' protocols (1μM [1×]). Additionally, monolayer chondrocytes were exposed to 2× the suggested...
Moran G, Buechner-Maxwell VA, Folch H, Henriquez C, Galecio JS, Perez B, Carrasco C, Barria M.Recurrent airway obstruction (RAO, also known as equine heaves) is an inflammatory condition similar to human asthma caused by exposure of susceptible horses to poorly ventilated stable environments. The disease is characterized by neutrophilic airway inflammation, mucus hypersecretion and reversible bronchoconstriction. This inflammatory process is mediated by several factors, including antibodies, cytokines, resident cells of the airway and inflammatory cellular components that arrive in the respiratory tract. An increasing body of evidence has lent support to the concept that a dysregulatio...
Kwon S, Gewirtz AT, Hurley DJ, Robertson TP, Moore JN, Vandenplas ML.As sentinel cells of the innate immune system, neutrophils and mononuclear phagocytes use specific TLRs to recognize the conserved molecular patterns that characterize microbes. This study was performed to compare the responses of equine neutrophils and mononuclear phagocytes to LPS and flagellin, components of bacteria that are recognized by TLR4 and TLR5, respectively. Neutrophils and mononuclear phagocytes isolated from healthy horses were incubated in vitro with LPS, flagellin, or pronase-inactivated flagellin in the presence or absence of polymyxin B. Production of reactive oxygen species...
Oldenhof H, Blässe AK, Wolkers WF, Bollwein H, Sieme H.The aim of this study was to determine the osmotic tolerance limits of stallion sperm as well as the osmotic behavior of different sperm subpopulations, including viable and non-viable cells as well as viable cells of different average sizes. A flow cytometric approach was used for simultaneous assessment of cell volume and permeability of the plasma membrane for the fluorescent dye propidium iodide while exposing the cells to media with different solute concentrations. Equine spermatozoa have limited osmotic tolerance limits: exposure to hypotonic conditions below approximately 240 mOsm kg(-1...
Foster ML, Love CC, Varner DD, Brinsko SP, Hinrichs K, Teague S, Lacaze K, Blanchard TL.Sperm membrane integrity (SMI) is thought to be an important measure of stallion sperm quality. The objective was to compare three methods for evaluating SMI: flow cytometry using SYBR-14/propidium iodide (PI) stain; an automated cell counting device using PI stain; and eosin-nigrosin stain. Raw equine semen was subjected to various treatments containing 20 to 80% seminal plasma in extender, with differing sperm concentrations, to simulate spontaneous loss of SMI. The SMI was assessed immediately, and after 1 and 2 d of cooled storage. Agreement between methods was determined according to Blan...
da Silva CM, Macías-García B, Miró-Morán A, González-Fernández L, Morillo-Rodriguez A, Ortega-Ferrusola C, Gallardo-Bolaños JM, Stilwell G....Lipid peroxidation (LPO) has been claimed as a major factor involved in stallion damage during storage or cryopreservation. Because melatonin is a well-known potent antioxidant, the aim of the present study was to investigate the effect of melatonin during in vitro incubation. Furthermore, we investigated the presence of specific melatonin receptors (MT1 and MT2) using specific polyclonal antibodies and western blotting. Stallion spermatozoa were incubated up to 3 hr at 37°C in the presence of different concentrations of melatonin (0, 50 pm, 100 pm, 200 pm, or 1 μm). At the beginning and at ...
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...
Doty A, Buhi WC, Benson S, Scoggin KE, Pozor M, Macpherson M, Mutz M, Troedsson MH.Equine spermatozoa induce a uterine inflammatory response characterized by a rapid, transient influx of polymorphonuclear neutrophils (PMNs). Seminal plasma proteins have been shown to modulate the interaction between spermatozoa and PMNs, but a specific protein responsible for this function has not been identified. The objective of this study was to isolate and identify a protein in equine seminal plasma that suppresses binding between spermatozoa and PMNs. Seminal plasma was pooled from five stallions, and proteins were precipitated in 60% (w/v) ammonium sulfate and dialyzed (3500 MW cutoff)...
Park J, Sutradhar BC, Hong G, Choi SH, Kim G.To compare the chondrotoxicity of bupivacaine, lidocaine, and mepivacaine in equine articular chondrocytes in vitro. Methods: Prospective, experimental study. Methods: Equine articular chondrocytes. Methods: Primary cultured equine chondrocytes were exposed to 0.5% bupivacaine, 2% lidocaine, or 2% mepivacaine for 30 or 60 minutes. After treatment, cell viability was evaluated by trypan blue exclusion and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay in a dose dependent manner. Apoptosis and necrosis of chondrocytes were analyzed with the double stain...
Foster ML, Varner DD, Hinrichs K, Teague S, Lacaze K, Blanchard TL, Love CC.Increasing seminal plasma concentrations in extended stallion semen were utilized to model decreasing sperm motility over time. Level of agreement was determined between flow cytometric measurement of sperm membrane integrity, using a combination of SYBR-14 and propidium iodide, and computer-assisted analysis of sperm motility. Values for total sperm motility (TMOT;%) and membrane integrity (SMI;%) were similar (∼80%) at Time 0 within all sperm treatments. However, TMOT was lower than SMI after 24 and 48 h of storage in treatments with >20% seminal plasma. At Time 0, agreement (bias and a...
Macías García B, González Fernández L, Ortega Ferrusola C, Morillo Rodríguez A, Gallardo Bolaños JM, Rodríguez Martinez H, Tapia JA....Fatty acids and plasmalogens were extracted from the phospholipids of the plasma membrane of stallion spermatozoa, to determine their relation with sperm quality after freezing and thawing. Sperm quality was rated using a quality index that combined the results of the analysis of sperm motility and velocity (CASA analysis), membrane status and mitochondrial membrane potential (flow cytometry) post thaw. Receiving operating system (ROC) curves were used to evaluate the value of specific lipid components of the sperm membrane herein studied as forecast of potential freezeability. From all parame...
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...
Robbin MG, Wagner B, Noronha LE, Antczak DF, de Mestre AM.Several distinct T lymphocyte subpopulations with immunoregulatory activity have been described in a number of mammalian species. This study performed a phenotypic analysis of cells expressing regulatory T cell (Treg) markers in the peripheral blood of a cohort of 18 horses aged 6 months to 23 years, using antibodies to both intracellular and cell surface markers, including Forkhead box P3 (FOXP3), CD4, CD8, CD25, interferon gamma (IFNγ) and interleukin 10 (IL-10). In peripheral blood, a mean of 2.2 ± 0.2% CD4+ and 0.5 ± 0.1% CD8+ lymphocytes expressed FOXP3. The mean percentage of CD4+FOXP...
Wauters J, Martens A, Pille F, Dumoulin M, Gasthuys F, Sys S, Meyer E.Synovial fluid samples from culture-confirmed infected joints (n=13), joints with pronounced non-infectious synovitis (n=11) and healthy joints (n=14) were collected from 24 equine patients and seven slaughterhouse horses. The samples from the joints with non-infectious synovitis and healthy joints served as negative controls. After isolation, counting and identification of neutrophils, the percentage viability, and the proportion apoptotic and necrotic neutrophils were determined by flow cytometry. Viability was significantly higher in infected samples compared to the controls. A significant ...
Brunso L, Segura D, Monreal L, Escolar G, White JG, Diaz-Ricart M.Studies in animal models are useful to understand the basic mechanisms involved in hemostasis and the functional differences among species. Ultrastructural observations led us to predict differences in the activation and secretion mechanisms between equine and human platelets. The potential mechanisms involved have been comparatively explored in the present study. Equine and human platelets were activated with thrombin (0.5 U/ml) and collagen (20 µg/ml), for 90 seconds, and samples processed to evaluate: i) ultrastructural changes, by electron microscopy, ii) actin polymerization and cy...
Hackett CH, Flaminio MJ, Fortier LA.A long-term goal of mesenchymal progenitor cell (MPC) research is to identify cell-surface markers to facilitate MPC isolation. One reported MPC feature in humans and other species is lack of CD14 (lipopolysaccharide receptor) expression. The aim of this study was to evaluate CD14 as an MPC sorting marker. Our hypothesis was that cells negatively selected by CD14 expression would enrich MPC colony formation compared with unsorted and CD14-positive fractions. After validation of reagents, bone marrow aspirate was obtained from 12 horses. Fresh and cultured cells were analyzed by flow cytometry ...
Braun J, Hack A, Weis-Klemm M, Conrad S, Treml S, Kohler K, Walliser U, Skutella T, Aicher WK.To evaluate the proliferative behavior, telomere length, immunophenotype, and differentiation capacity of equine adipose tissue-derived mesenchymal stem cells (AT-MSCs). Methods: 6 adult racing horses treated for articular Injury but otherwise healthy. Methods: AT-MSCs were Isolated from horses and expanded In Dulbecco modified Eagle medium enriched with fetal bovine serum and antimicrobials. Expression of cell surface antigens and telomere length were Investigated via flow cytometry Differentiation of MSCs Into chondrocytes, osteoblasts, and adipocytes was Induced In vitro by specific stimuli...
Park SB, Seo MS, Kang JG, Chae JS, Kang KS.Amniotic fluid (AF) is a well-known source of stem cells. However, there have been no reports regarding equine AF stem cells. We have isolated equine AF-derived multipotent stem cells (MSC) (eAF-MSC) and show that these cells exhibit self-renewal ability and multilineage differentiation. Methods: AF was obtained from thoroughbred mares and mononuclear cells (MNC) were isolated by Ficoll-Paque density gradient. We measured the cumulative population doubling level (CPDL) and characterized the immunophenotype by flow cytometry. To investigate differentiation ability, a trilineage differentiation ...
Owens SD, Johns JL, Walker NJ, Librach FA, Carrade DD, Tablin F, Borjesson DL.To evaluate N-hydroxysuccinimide (NHS)-biotin labeling of equine RBCs and determine posttransfusion survival of autologous equine RBCs stored in citrate phosphate dextrose adenine-1 (CPDA-1) for 0, 1, 14, and 28 days. Methods: 13 healthy adult Thoroughbreds. Methods: Serial dilutions of biotin and streptavidin-phycoerythrin (PE) were evaluated in vitro in blood collected from 3 horses. One horse was used to determine RBC distribution and recovery. Twelve horses were allocated to 4 groups for in vivo experiments in which blood was collected into CPDA-1. Blood was labeled with biotin and reinfus...
Kabithe E, Hillegas J, Stokol T, Moore J, Wagner B.CD14 is a receptor for the complex of lipopolysaccaride (LPS) and LPS-binding protein. Binding of this complex to CD14 in association with Toll-like receptor 4 provides a major pathway for the initiation of innate immune responses to bacterial pathogens. We used a mammalian expressed extracellular region of equine CD14 (rCD14) derived from an IgG fusion protein to produce monoclonal antibodies (mAbs) to CD14. Eight mAbs were tested by flow cytometric analysis of equine leukocytes and by immunoblotting using rCD14 indicating that the mAbs recognized at least three different epitopes on equine C...
Flores J, Corvera Poiré E, del Rio JA, López de Haro M.We consider a simple model to give a plausible mechanical explanation of what are the actual resting heart rates of mammals optimized for. We study what is the optimal frequency for a viscoelastic fluid circulating in a pulsatile way through a network of tubes and conclude that the heart rate is not optimized to transport blood through the whole net. Rather, actual resting heart rates of mammals happen at frequencies that optimize flow in vessels of radii that correspond to large arteries, which bring oxygenated blood rapidly far away from the heart, towards head and limbs. Our results for the...
Scheinberg P, Marte M, Nunez O, Young NS.Clones of glycosylphosphatidylinositol-anchor protein-deficient cells are characteristic in paroxysmal nocturnal hemoglobinuria and are present in about 40-50% of patients with severe aplastic anemia. Flow cytometry has allowed for sensitive and precise measurement of glycosylphosphatidylinositol-anchor protein-deficient red blood cells and neutrophils in severe aplastic anemia. Methods: We conducted a retrospective analysis of paroxysmal nocturnal hemoglobinuria clones measured by flow cytometry in 207 consecutive severe aplastic anemia patients who received immunosuppressive therapy with a h...
Minervini F, Lacalandra GM, Filannino A, Garbetta A, Nicassio M, Dell'aquila ME, Visconti A.Fumonisin B1 (FB1) is a mycotoxin produced by Fusarium species that exerts its toxic effect through interference with the sphingolipid pathway by inhibiting ceramide synthase. A FB1-dependent sperm toxicity was reported in boars. No information on FB1-related reproductive toxicity in stallions, the most sensitive animal species, has been reported. The aim of the present study was to assess the in vitro toxicity of FB1 on fresh and frozen-thawed equine spermatozoa by analyzing sperm viability, chromatin stability (SCSA) and reactive oxygen species (ROS) production by flow cytometry and sperm mo...
Dunkel B, Rickards KJ, Werling D, Page CP, Cunningham FM.To determine whether expression of equine platelet activation-dependent surface markers is influenced by phospodiesterase (PDE) isoenzyme activity and whether antigen challenge alters platelet PDE activity in horses with recurrent airway obstruction (RAO). Methods: 16 horses. Methods: 7 healthy horses were used for in vitro experiments, 6 horses with RAO were used for antigen challenge, and 6 healthy horses were used as control animals. Three of the healthy horses had also been used in the in vitro experiments. Effects of PDE inhibition and activation of adenylyl cyclase on CD41/61 and CD62P e...
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...
Dunkel B, Rickards KJ, Werling D, Page CP, Cunningham FM.Recurrent airway obstruction (RAO) in mature horses is characterized by reversible airway obstruction and neutrophilic inflammation; there is also functional activation of circulating platelets and neutrophils. This study was undertaken to determine if changes in activation marker expression and heterotypic aggregate formation can be used as an indicator of this increased functional responsiveness. In vitro conditions for flow cytometric measurement of CD13, CD41/61 and CD62P expression on activated cells and heterotypic aggregate formation were established. Values were then compared before an...
Umair M, Claes A, Buijtendorp M, Cuervo-Arango J, Stout TAE, Henning H.Artificial insemination with chilled stallion semen is hampered by a limited period of maximum fertility maintenance (24-48 h). This study used multiparametric flow cytometry to simultaneously measure reactive oxygen species (ROS) production, mitochondrial function or [Ca ] and plasma membrane fluidity in viable, acrosome-intact spermatozoa, with the aim of providing insight into changes in sperm function during storage at 5°C. High proportions of viable and acrosome-intact spermatozoa (71 ± 8%) remained after 96 h of storage demonstrating that the basic integrity of the cells was we...
Nouri H, Shojaeian K, Jalilvand G, Kohram H.The objective was to assess the influence of pomegranate seed oil supplementation on the quality of fresh, cooled and frozen-thawed Arabian breed stallion semen. Eight stallions (n = 4 per group) received their normal diet (control group) or normal diet top dressed with 200 ml of pomegranate seed oil (PSO group). Semen was collected every fifteen days for 90 days. Stallions were reversed across the treatments after a sixty-day interval. In cooled and stored condition (2, 12 and 24 hr), spermatozoa motion characteristics, membrane integrity, viability, morphology and lipid peroxidation wer...
Kavak A, Johannisson A, Lundeheim N, Rodriguez-Martinez H, Aidnik M, Einarsson S.Methods to evaluate the quality of frozen-thawed stallion semen are still needed, particularly those considering the sperm function. The present study evaluated sperm motility, membrane and acrosome integrity and the capacitation status of frozen-thawed spermatozoa from seven Tori and six Estonian breed stallions by way of computer assisted sperm analysis (CASA), a triple fluorophore stain combination and Merocyanine 540, respectively, the latter ones using flow cytometry. Two ejaculates from each stallion were cryopreserved using the Hannover method in 0.5 ml plastic straws. Two straws per ej...
Oliveira CH, Vasconcelos AB, Souza FA, Martins-Filho OA, Silva MX, Varago FC, Lagares MA.Addition of cholesterol to sperm membranes improved equine sperm stability during semen cryopreservation; however, it also reduced in vivo fertility. The objective of the present study was to determine the effects of adding cholesterol to stallion sperm prior to freezing, and subsequently removing it from frozen-thawed sperm. Semen from 12 stallions was subjected to four treatments: (T1) control, semen was diluted with Kenney extender, centrifuged, and resuspended to 100 x 10(6)spermatozoa/mL in INRA 82 freezing extender, packaged into 0.5-mL straws, cooled to 5 degrees C, and cryopreserved in...
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...
Elzinga SE, Rohleder B, Schanbacher B, McQuerry K, Barker VD, Adams AA.Extracts derived from the leaves of the stevia plant (stevioside) are commonly used as sweeteners for humans and horses. Stevioside appears to be safe for human consumption, including for individuals with insulin dysregulation. In the horse, the safety or metabolic effects of stevioside on normal animals or on those with metabolic dysfunction are unknown. Furthermore, the inflammatory response to a glycemic challenge or to stevioside in horses is not well defined. Therefore, the objective of this study was to measure the effects of stevioside and a glycemic challenge on insulin, glucose, and i...
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...
Gutmann S, Zawatzky R, Müller M.Interferon-gamma (IFN-gamma) is a key cytokine in cell-mediated immunity. To measure IFN-gamma production of equine lymphocytes (eqIFN-gamma), we developed a quantitative ELISA. Monoclonal antibodies (mAb) were produced against bacterially derived eqIFN-gamma. The mAbs recognised recombinant and lymphocyte-derived eqIFN-gamma in ELISA, Western blotting, as well as flow cytometric and microscopic analysis. In contrast to bacterially derived material, mammalian and insect cell-derived eqIFN-gamma was biologically active but could be neutralised by one of the monoclonal antibodies. Unexpectedly, ...
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...
Serpa PBS, Brooks MB, Divers T, Ness S, Birschmann I, Papich MG, Stokol T.Horses with inflammatory and infectious disorders are often treated with injectable heparin anticoagulants to prevent thrombotic complications. In humans, a new class of direct oral acting anticoagulants (DOAC) appear as effective as heparin, while eliminating the need for daily injections. Our study in horses evaluated apixaban, a newly approved DOAC for human thromboprophylaxis targeting activated factor X (Xa). Our goals were to: (1) Determine pharmacokinetics and pharmacodynamics of apixaban after oral (PO) and intravenous (IV) administration in horses; (2) Detect any inhibitory effects of...
Richards CM, Aucken HA, Tucker EM, Hannant D, Mumford JA, Powell JR.Studies were carried out to determine the optimum conditions for the production of equine monoclonal antibodies (MAbs). Lymphocytes from ponies immunised with influenza A equine 2 virus, isolate A/Equine/Newmarket/79 (H3N8) were fused with mouse myeloma (NSO) cells and with horse-mouse heterohybridomas made aminopterin-sensitive by selective growth in 8-azaguanine. Although all fusions initially resulted in heterohybridoma colonies that secreted equine immunoglobulin, many of these were unable to maintain secretion for longer than a few weeks. Increasing the time between immunisation and the b...
Kiku Y, Kusano K, Miyake H, Fukuda S, Takahashi J, Inotsume M, Hirano S, Yoshihara T, Toribio RE, Okada H, Yoshino TO.Cellular activation and functional cell surface markers were evaluated during experimentally-induced endotoxemia in healthy horses. Eight healthy adult horses were infused a low dose of endotoxin (lipopolysaccharide from Escherichia coli O26: B6, 30 ng/kg of body weight, IV) and five control horses were given an equivalent volume of sterile saline solution. Venous blood samples were collected for flow cytometric analysis of peripheral blood mononuclear cells (PBMCs) and to measure plasma endotoxin concentrations. Clinical signs of endotoxemia were recorded at 10, 20, 30, 40, 50 min, 1, 2, 3, 4...
Terio KA, Stalis IH, Allen JL, Stott JL, Worley MB.Coccidioidomycosis is a rare, often subclinical infection in domestic animals caused by the fungus Coccidioides immitis. Because of an apparent high incidence of coccidioidomycosis in Przewalski's horses (Equus przewalskii) housed at a single facility, necropsy records and biomaterials from animals that died between 1984 and 2000 were reviewed (n = 30, 15 males, 15 females). Coccidioidomycosis was the leading cause of death (33%) in this population with lesions in the lungs and tracheobronchial lymph nodes of all animals and variable involvement of the skeletal muscle, heart, kidney, liver, sk...
Hartmann C, Gerner W, Walter I, Saalmüller A, Aurich C.In the mare, early pregnancy loss is common, but involvement of the maternal immune system in the pathogenesis of this condition has not been investigated in detail so far. In the present study, we assessed effects of exposure of the endometrium to semen or seminal plasma in oestrous mares on the response of regulatory T lymphocytes (Tregs) in the peripheral circulation as well as in the endometrium. Raw semen, seminal plasma or PBS (control) were introduced into the uterus of oestrous mares (n = 12). Blood was collected immediately before insemination or PBS infusion (time 0), and 12, 24 ...
Lai SW, Lin JH, Lai SS, Wu YL.The characteristic ingredients of Ganoderma lucidum, such as polysaccharides, triterpenoids, nucleic acids and small proteins, have been found and proved to have many special pharmacological properties. Mice and rats have been extensively used to investigate the effects of G. lucidum. Experiments with horses as an animal model for investigating the effects of G. lucidum have never been reported. The purpose of this investigation was to understand the influence of G. lucidum feeding on blood biochemistry and immunocompetence in horses. Complete blood count (CBC) and blood biochemistry were surv...
Prieto JMB, Tallmadge RL, Felippe MJB.Identification and classification of B cell subpopulations has been shown to be challenging and inconsistent among different species. Our study tested aspects of ontogeny, phenotype, tissue distribution, and function of equine CD5 B cells, which represented a greater proportion of B cells early in development and in the peritoneal cavity. CD5 and CD5 B cells differentially expressed B cell markers (CD2, CD21, IgM) measured using flow cytometry, but similar mRNA expression of signature genes (DGKA, FGL2, PAX5, IGHM, IL10) measured using quantitative RT-PCR. Sequencing lambda light chain segment...
