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.
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...
Wagner B, Burton A, Ainsworth D.Cytokines produced by T helper (Th) cells are important in orchestrating the immune response during health and disease. Recent reports indicated that cytokine mRNA expression in foals is often quantitatively lower than that of adult horses suggesting that foal T cells are not fully mature. Here, peripheral blood mononuclear cells from foals and adult horses were stimulated with phorbol 12-myristate 13-acetate and analyzed for intracellular interferon-gamma (IFN-gamma), interleukin-4 (IL-4) and IL-10 production, representing the Th1, Th2 and regulatory TR1 cell phenotypes respectively, by flow ...
Go YY, Zhang J, Timoney PJ, Cook RF, Horohov DW, Balasuriya UB.Extensive cell culture passage of the virulent Bucyrus (VB) strain of equine arteritis virus (EAV) to produce the modified live virus (MLV) vaccine strain has altered its tropism for equine CD3(+) T lymphocytes and CD14(+) monocytes. The VB strain primarily infects CD14(+) monocytes and a small subpopulation of CD3(+) T lymphocytes (predominantly CD4(+) T lymphocytes), as determined by dual-color flow cytometry. In contrast, the MLV vaccine strain has a significantly reduced ability to infect CD14(+) monocytes and has lost its capability to infect CD3(+) T lymphocytes. Using a panel of five re...
Krakowski L, Krawczyk CH, Wrona Z, Dabrowski R, Jarosz Ł.The objective of the present study was to analyse the immune status of mares in the early stages of pregnancy and mares which experienced early embryonic death (EED). Forty-eight fertile purebred Arabian mares were used for the study. The mares were divided into two groups. Group I was the control group composed of 32 mares in the early stages of a normal pregnancy. Group II was composed of 12 mares which had experienced EED. In both groups, subpopulations of T lymphocytes containing CD2(+), CD4(+) and CD8(+) were characterized using flow cytometry. The percentages of CD3(+) lymphocytes (addin...
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...
Pickles KJ, Brooks AC, Rickards KJ, Cunningham FM.N-terminal peptides derived from the anti-inflammatory peptide, annexin-1, inhibit neutrophil function but can also induce pro-inflammatory effects. Although equine annexin-1 has been sequenced, its cellular expression and properties have not been reported. This study has examined whether annexin-1 is present in equine leucocytes and how the N-terminal peptide, Ac2-26, affects equine neutrophil superoxide production. Annexin-1 expression in equine neutrophils and mononuclear cells and the ability of Ac2-26 to activate neutrophil p42/44 MAPK were determined by immunoblotting. Equine neutrophil ...
Wagner B, Stokol T, Ainsworth DM.Transfer of maternal IgE antibodies to the neonate with the colostrum has been described in different mammalian species. Previous work in horses has shown that IgE bound to the surface of neonatal basophils is solely of maternal origin. However, the functional role of the maternal IgE transfer remained unclear. We hypothesized that maternal IgE mediates the onset of innate IL-4 production in equine neonatal basophils. Intracellular IL-4 production was measured in PBMC of newborn and older foals by flow cytometric analysis. A small population of IL-4(+) cells was observed in the peripheral bloo...
Minervini F, Lacalandra GM, Filannino A, Nicassio M, Visconti A, Dell'Aquila ME.The purpose of this study was to assess the natural exposure of male horses (Equus caballus) to the mycotoxin zearalenone (ZEA) by using the ELISA test and to evaluate the effects of in vitro exposure of sperm cells to mycotoxin-containing urine extracts on sperm chromatin structure stability. Because of their occurrence in urine samples, ZEA and its derivatives were tested by sperm chromatin structure assay (SCSA) at natural levels detected by ELISA. Thirty-eight urine extracts of Italian (n = 11) and northeastern European (n = 27) horses were tested on frozen-thawed spermatozoa to evaluate t...
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...
Satué K, Hernández A, Lorente C, O'Connor JE.Assessment of lymphocyte subsets is an effective method for characterizing disorders such as leukemia, lymphomas, autoimmune and infectious diseases. In order to clinically interpret these parameters, normal reference values should be set, estimating age- and gender-related variations. This research aimed to: (1) characterize lymphocyte subpopulations in Andalusian horse, and (2) evaluate age and gender-related variations of lymphocyte subsets. Jugular blood samples were obtained from 159 animals, 77 males and 82 females, belonging to four age groups-1: 1-2 years (N=39; 21 males and 18 females...
Radcliffe CH, Flaminio MJ, Fortier LA.Mesenchymal progenitor cells (MPCs) are often characterized using surface markers after expansion and treatment in culture. There are no studies directly comparing gene and protein markers in undifferentiated samples during the very early phases of culture. The goal of this study was to evaluate temporal gene and protein expression changes during establishment of equine MPC cultures. Bone marrow aspirate was obtained from 35 horses and processed by density gradient centrifugation. In freshly isolated bone marrow, mononuclear cells had variable expression of CD44, CD11a/CD18, CD90, and CD45RB c...
Ortega Ferrusola C, González Fernández L, Macías García B, Salazar-Sandoval C, Morillo Rodríguez A, Rodríguez Martinez H, Tapia JA, Peña FJ.The ability of stallion spermatozoa to produce nitric oxide (NO) before (fresh) and after freezing and thawing (FT) was evaluated by means of flow cytometry after loading the sperm suspension with the probe, 4,5-diaminofluorescenin diacetate. The presence of NO synthase (NOS) was investigated by Western blotting using anti-NOS1, anti-NOS3, or anti-universal NOS antibodies (Abs). While NO was detected both in fresh and FT sperm suspensions, its production increased after cryopreservation only when egg yolk was removed from the extender. Anti-NOS1 Ab intensively labeled a single band with an app...
de Mattos Carvalho A, Alves AL, Golim MA, Moroz A, Hussni CA, de Oliveira PG, Deffune E.The purpose of this work was to isolate and cultivate mesenchymal stem cells (MSC) derived from equine adipose tissue and conduct cellular characterization with the following markers: CD90, CD44 and CD13. Adipose tissue collection was performed at the base of the horses' tails, followed by immediate isolation and cultivation of the MSC and posterior characterization by flow cytometry for the interspecies reaction test using mouse anti-rat CD90 monoclonal antibody (mAb), fluorescein isothiocyanate (FITC), and tests with specific mAb mouse anti-horse CD13 and mouse anti-horse CD44. The technique...
Lima-Neto JF, Fernandes CB, Alvarenga MA, Golim MA, Landim-Alvarenga FC.This experiment aimed to study equine fibroblasts in culture analyzing and the cell cycle and viability of cells pre- and post-freezing. Skin fragments were obtained from 6 horses and cultured in DMEM high glucose + 10% FCS in 5% CO(2) until the beginning of confluence. Two passages were performed before freezing. Cells subjected to serum starvation (0.5% FCS) were analyzed for viability and cell cycle at 24, 48, 72, 96, 120, 144 and 168 h of culture. For the confluent groups, cells were analyzed at the moment they achieved confluence. Cellular viability was assisted with Hoescht 33342 and pro...
Lilliehöök I, Tvedten H.The Sysmex XT-2000iV is a laser-based, flow cytometric hematology system that stains nucleic acids in leukocytes with a fluorescent dye. A 4-part differential is obtained using side fluorescence light and laser side scatter. Objective: The purpose of this study was to validate the Sysmex XT-2000iV for determining differential leukocyte counts in blood from ill dogs, cats, and horses. Methods: Blood samples from diseased animals (133 dogs, 65 cats, and 73 horses) were analyzed with the Sysmex XT-2000iV (Auto-diff) and the CELL-DYN 3500. Manual differentials were obtained by counting 100 leukocy...
Lilliehöök I, Tvedten H.The Sysmex XT-2000iV is a laser-based, flow cytometric hematology system that has been introduced for use in large and referral veterinary laboratories. Objective: The purpose of this study was to validate the Sysmex XT-2000iV for counting erythrocytes, reticulocytes, platelets, and total leukocytes in blood from ill dogs, cats, and horses. Methods: Blood samples from diseased animals (133 dogs, 65 cats, and 73 horses) were analyzed with the Sysmex XT-2000iV and the CELL-DYN 3500. Manual reticulocyte counts were done on an additional 98 canine and 14 feline samples and manual platelet counts w...
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...
Treonze KM, Alves K, Fischer P, Hagmann WK, Hora D, Kulick A, Vakerich K, Smith ND, Lingham RB, Maniar S, Reger TS, Zunic J, Munoz B, Prasit P....The purpose of this study was to characterize the alpha(4)beta(1) receptor (CD49d/CD29, very late antigen-4, VLA-4) on circulating equine leukocytes and to evaluate the intrinsic potency of an alpha(4)beta(1) receptor antagonist (Compound B) in the horse. Ultimately, these studies would allow us to determine the suitability of treating recurrent airway obstruction (RAO; heaves) affected horses by blocking the cellular recruitment of lymphocytes and neutrophils into the lung. The data demonstrates the alpha(4)beta(1) integrin is present on horse lymphocytes and neutrophils (fluorescence-assiste...
Mari G, Rizzato G, Merlo B, Iacono E, Bucci D, Seren E, Tamanini C, Galeati G, Spinaci M.Little information is available on the quality of stallion spermatozoa after sex sorting. The objectives of the present study were to assess the quality of sex-sorted stallion spermatozoa and determine its fertilizing ability after hysteroscopic low dose insemination. Ejaculates from four stallions were collected and sorted by a MoFlo SX flow cytometer/sperm sorter. Before and after sorting, spermatozoa were evaluated for motility by Computer Assisted Sperm Analysis, viability (SYBR 14-propidium iodide), mitochondrial function (JC-1) and acrosomal status (fluorescein isothiocyanate Pisum sativ...
Lin YZ, Deng XL, Shen N, Lü XL, Zhao LP, Kong XG, Shao YM, Zhou JH.To develop a flow cytometry using (5-carboxyfluorescein diacetate succinmidyl ester, CFSE) to detect the proliferation of specific T lymphocytes from equine infectious anemia virus (EIAV). Methods: Peripheral blood mononuclear cells (PBMC) were isolated, stained with CFSE and incubated with EIAV for 5 days. After interacted with either CD4(+) or CD8(+) antibody, the cells were detected for proliferated population, which contained serially 2-fold reduced CFSE in CD4(+) and CD8(+) T lymphocytes. Results: The concentration of CFSE, and the type, concentration and reaction time of EIAV-specific an...
Ortega-Ferrusola C, García BM, Gallardo-Bolaños JM, González-Fernández L, Rodríguez-Martinez H, Tapia JA, Peña FJ.In an attempt to identify valuable markers for potential freezeability of the equine spermatozoa, three ejaculates were collected from five Andalusian stallions and frozen using a standard protocol. Before freezing, three apoptotic cell markers were studied by flow cytometry (early changes in sperm membranes, mitochondrial membrane potential and caspase activity). Post-thaw, spermatozoa were again evaluated for these parameters. Sperm kinematics using CASA were also studied before and after freezing and thawing. Receiving operating system curves were used to evaluate the relative value of the ...
Lecoq L, Vincent P, Lavoie-Lamoureux A, Lavoie JP.Glucocorticoids have potent anti-inflammatory properties and are frequently used for the treatment of domestic animal species, including horses. They induce a down-regulation of multiple inflammatory pathways through both genomic and non-genomic effects. Currently, little is known on the effects of glucocorticoids on equine peripheral blood neutrophils. Objective: Dexamethasone (DEX), a potent synthetic glucocorticoid, inhibits the functions of equine peripheral blood neutrophils through both genomic and non-genomic effects. Methods: Six healthy adult mixed breed female horses. Methods: To ass...
Schulze-Tanzil G, Müller RD, Kohl B, Schneider N, Ertel W, Ipaktchi K, Hünigen H, Gemeinhardt O, Stark R, John T.For lack of sufficient human cartilage donors, chondrocytes isolated from various animal species are used for cartilage tissue engineering. The present study was undertaken to compare key features of cultured large animal and human articular chondrocytes of the knee joint. Primary chondrocytes were isolated from human, porcine, ovine and equine full thickness knee joint cartilage and investigated flow cytometrically for their proliferation rate. Synthesis of extracellular matrix proteins collagen type II, cartilage proteoglycans, collagen type I, fibronectin and cytoskeletal organization were ...
Adams AA, Breathnach CC, Katepalli MP, Kohler K, Horohov DW.A number of model systems have been employed to investigate age-associated changes in immune function. The purpose of the current study was to characterize senescent T cells and to investigate the inflamm-aging phenomenon both in vitro and in vivo using the old horse as a model. We examined whether decreased T cell proliferation induced by Con A is caused by increased apoptosis. We also utilized intracellular CFSE to analyze changes within each round of cell proliferation, in particular cytokine production. Intracellular staining with flow cytometry, RT-PCR, and ELISA were used to measure pro-...
Lin Y, Deng X, Shen N, Zhao L, Meng Q, Max J, Wang J, Shao Y, Zhou J.The attenuated vaccine of equine infectious anemia virus (EIAV) is the first lentiviral vaccine that provides solid protection against the infection of EIAV virulent strains. Study of the immune response induced by EIAV vaccine is an important approach to understand the immunity to other lentiviruses. IFN-gamma expressed by specifically stimulated lymphocytes is an important indicator for the evaluation of T cell-mediated immunity. A flow cytometry based assay was established in this study to accurately and effectively detect IFN-gamma expression in different subtypes of T lymphocytes in EIAV-...
Clulow JR, Evans G, Morris LH, Maxwell WM.Intrinsic differences between stallions exist for semen traits such as motility, morphology fertility and the ability of spermatozoa to survive cryopreservation processes. Ejaculates from 11 stallions were used to test the differences between stallions when selecting X- and Y-chromosome bearing spermatozoa using a modified flow cytometer. Data on orientation and viability of spermatozoa were collected during sex-sorting, and motility characteristics of sex-sorted and non-sorted (control) spermatozoa were assessed before and after cryopreservation. An index was created to rank each stallion in ...
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...
Cassart D, Fett T, Sarlet M, Baise E, Coignoul F, Desmecht D.The morphopathological picture of a subset of equine myopathies is compatible with a primary mitochondrial disease, but functional confirmation in vivo is still pending. The cationic dye JC-1 exhibits potential-dependent accumulation in mitochondria that is detectable by a fluorescence shift from green to orange. As a consequence, mitochondrial membrane potential can be optically measured by the orange/green fluorescence intensity ratio. A flow cytometric standardized analytic procedure of the mitochondrial function of equine peripheral blood mononuclear cells is proposed along with a critical...
Gaitskell-Phillips G, Martín-Cano FE, Ortiz-Rodríguez JM, Silva-Rodríguez A, Gil MC, Ortega-Ferrusola C, Peña FJ.The identification of stallions and or ejaculates that will provide commercially acceptable quality post-thaw before cryopreservation is of great interest, avoiding wasting time and resources freezing ejaculates that will not achieve sufficient quality to be marketed. Our hypothesis was that after bioinformatic analysis, the study of the stallion sperm proteome can provide discriminant variables able to predict the post-thaw quality of the ejaculate. At least three ejaculates from 10 different stallions were frozen following a split sample design. Half of the ejaculate was analyzed as a fresh ...
Yang Q, Pinto VMR, Duan W, Paxton EE, Dessauer JH, Ryan W, Lopez MJ.Damage to an ectodermal-mesodermal interface like that in the equine hoof and human finger nail bed can permanently alter tissue structure and associated function. The purpose of this study was to establish and validate culture of primary progenitor cell isolates from the ectodermal-mesodermal tissue junction in equine hooves, the stratum internum, with and without chronic inflammation known to contribute to lifelong tissue defects. The following were evaluated in hoof stratum internum cell isolates up to 5 cell passages (P): expansion capacity by cell doublings and doubling time; plasticity ...
Brooks MB, Divers TJ, Watts AE, Ness SL, Frye AH, Stokol T, Fubini SL.To evaluate the platelet activation response before and after treatment with clopidogrel in horses. Methods: 12 healthy adult mares. Methods: In a masked study, horses (6/group) were randomly allocated to alternately receive placebo or clopidogrel via nasogastric tube at a loading dose of 4 mg/kg followed by 2 mg/kg every 24 hours. Blood samples were collected before and 72 hours after initiation of treatment for ADP- and collagen-induced light transmission aggregometry; determination of closure time in collagen-ADP cartridges; modified thrombelastography for comparison of maximal amplitudes g...
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...
Mundy LN, Ishihara A, Wellman ML, Bertone AL.To assess efficiency of gravity filtration to enhance recovery of equine bone marrow elements including stem and progenitor cells. Methods: 12 healthy adult horses. Methods: Bone marrow aspirates were collected from the fifth sternebral body and filtered by gravitational flow to obtain bone marrow elements. Raw and harvested bone marrow and marrow effluent were evaluated for WBC and platelet counts, automated and cytomorphologic cell differential counts, mesenchymal stem cell CFUs, cell viability, and differentiation capacity. Isolated cells were analyzed for CD90 and major histocompatibility ...
Siedek EM, Honnah-Symns N, Fincham SC, Mayall S, Hamblin AS.Monoclonal antibodies (mAbs) recognizing equine macrophages are scarce. The present study compared the immunocytochemical staining of various equine tissues (lymphoid tissue, lung, liver, small intestine, skin and blood leucocytes) by an antibody, Ki-M6, which detects CD68 in human macrophages and dendritic cells, and by a new anti-equine mAb, JB10, with staining produced by two previously described anti-equine macrophage mAbs, CZ2.2 and CZ3.3. Ki-M6 was shown to identify equine macrophages, which had a distribution different from those identified by CZ2.2 and CZ3.3. JB10 identified equine mac...
Hanel RM, Crawford PC, Hernandez J, Benson NA, Levy JK.To determine whether passive transfer of IgG in neonatal kittens affects plasma opsonic capacity and neutrophil phagocytic and oxidative burst responses to bacteria in vitro. Methods: 22 kittens from 6 specific pathogen-free queens. Methods: Kittens were randomized at birth into the following treatment groups: colostrum-fed, colostrum-deprived, or colostrum-deprived supplemented with feline or equine IgG. Blood samples were collected at intervals from birth to 56 days of age. Plasma IgG concentrations were determined by radial immunodiffusion assay. Neutrophil function was assessed by a flow c...
Gil MC, Ferrusola CO, Anel-López L, Ortiz-Rodriguez JM, Alvarez M, de Paz P, Anel L, Peña FJ.Spermatozoa undergo apoptotic changes during the cryopreservation process. These changes, recently termed spermptosis, resemble the cryopreservation induced delayed onset of cell death observed after thawing of somatic cells. Due to its importance in cryobiology, methods to easily identify spermptotic cells are warranted. In this study, a well-validated method for identification of spermatozoa with caspase 3 activity was compared with use of the combination of Hoechst 33342 (H-42) and ethidium homodimer (Eth-1). Live, dead and apoptotic spermatozoa assessed with each method were compared using...
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...
Foerster RJ, Wolf G.Equine blood polymorphonuclear leukocytes (PMN) were isolated by buffy coat and hypotonic lysis of residual erythrocytes. A highly reproducible method is described for measuring the uptake of opsonized latex microspheres by equine PMN using flowcytometry. The use of cytochalasin D allowed for differentiation of ingested from attached particles. The kinetics of phagocytosis in vitro is shown for different experimental conditions. We developed an assay for evaluation of phagocytic capacity of PMN which allows the assessment of drugs for their influence on phagocytosis in vivo as well as in vitro...
Carter EI, Valli VE, McSherry BJ.The peripheral blood cells from Standard bred horses were subjected to procedures which will separate equine peripheral blood cells with good precision and efficiency into red cell, leukocyte, and platelet fractions. The separated cells have normal morphology and the differential count of the separated granulocytes and lymphocytes is unchanged from that of the original sample.
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...
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, ...
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...
Krista KM, White NA, Barrett JG, Furr MO, Buechner-Maxwell VA.To quantify peripheral blood neutrophil apoptosis in equine patients with acute abdominal disease (ie, colic) caused by strangulating or nonstrangulating intestinal lesions and compare these values with values for horses undergoing elective arthroscopic surgery. Methods: 20 client-owned adult horses. Methods: Peripheral blood was collected from horses immediately prior to and 24 hours after surgery for treatment of colic (n = 10) or elective arthroscopic surgery (10), and neutrophils were counted. Following isolation by means of a bilayer colloidal silica particle gradient and culture for 24 h...
J Lacy K, Parlane NA, Riley CB, Gee EK, Roberts JM, McIlwraith CW.CellTrace Violet™ is a commonly used fluorescent dye used with flow cytometry to identify cell proliferation. Activated equine lymphocytes were examined using flow cytometry, microscopy and tritiated thymidine proliferation assays. CellTrace Violet™ was incorporated into the equine lymphocytes effectively. Equine lymphocytes proliferated when activated with pokeweed mitogen, but did not proliferate when previously stained with CellTrace Violet™. Serial dilutions of CellTrace Violet™ did not eliminate the inhibition of activated lymphocytes. Equine lymphocyte viability was greater than ...
Segura D, Monreal L, Pérez-Pujol S, Alonso A, Díaz-Ricart M, Brugués R, Ordinas A, Escolar G.The aim of this study was to investigate whether etamsylate produces equine platelet activation. In vitro and in vivo studies were designed in which seven and eight adult healthy horses were included, respectively. In the in vitro study, citrated blood was incubated with different concentrations of etamsylate, and P-selectin expression and annexin V binding were determined by flow cytometry. In the in vivo study, blood was collected before and 1 and 2h after IV administration of etamsylate, and P-selectin expression was evaluated. In the in vitro study, a significant increase in P-selectin exp...
Filho JS, Corcini CD, Santos FCC, Anciuti AN, Gatti NLS, Anastacio E, Mielke R, Nogueira CEW, Curcio BR, Varela AS. BACKGROUND: Supplementation of sperm diluents to reduce the damage caused by the freeze-thaw cycle is broadly used in equine semen cryopreservation. Objective: The present study aimed at determining the most appropriate quercetin supplementation in equine freezing extender. Methods: Quercetin at four different concentrations (0.25, 0.5, 0.75 or 1 mM) was added in the sperm freezing diluent before the freeze-thaw cycle. The spermatozoa population was analyzed by flow cytometry and a statistical analysis was conducted to detect significant differences between control and treated samples. R...
Bell SC, Savidge C, Taylor P, Knottenbelt DC, Carter SD.A putative immunodeficiency, causing mortality in UK Fell pony foals (Fell pony syndrome), was studied in affected foals and compared with healthy, age-matched foals. Differential cell counts of peripheral blood indicated that the syndrome foals were lymphopenic (P<0.05). Flow cytometric analysis of circulating leucocytes showed a reduced MHC II expression (P<0.01) on lymphocytes but not on polymorphonuclear cells in affected foals. There were no changes in the percentages of CD4+ or CD8+ T cells. There was an increased (P<0.05) expression of CD11a/18 by the lymphocytes of the syndrom...
Ortiz I, Felix M, Resende H, Ramírez-Agámez L, Love CC, Hinrichs K.To define the effect of sperm agglutination, associated with incubation under capacitating conditions, on accuracy of membrane assessment via flow cytometry and to develop methods to mitigate that effect. Methods: Sperm motility was measured by CASA. Sperm were stained with PI-PSA or a novel method, LD-PSA, using fixable live/dead stain and cell dissociation treatment, before flow-cytometric analysis. Using LD-PSA, acrosome reaction and plasma membrane status were determined in equine sperm treated with 10 μm A23187 for 10 min, followed by 0, 1, or 2 h incubation in capacitating conditions. R...
Johannisson A, Al-Essawe EM, Al-Saffar AK, Karkehabadi S, Lima-Verde I, Wulf M, Aurich C, Morrell JM.The mechanism by which the content of the major groups of seminal plasma proteins in stallion semen changes between the breeding and non-breeding seasons remains unknown. Here, we investigated the proportions of non-heparin-binding, phosphorylcholine-binding, and heparin-binding proteins in seminal plasma with the aim of relating them to sperm quality and testosterone levels in good and bad freezer stallions. Only minor variations in the major protein groups were found between the breeding and non-breeding seasons. In the non-breeding season, a higher content of a subset of non-heparin binding...
Dominguez-Medina CC, Rash NL, Robillard S, Robinson C, Efstratiou A, Broughton K, Parkhill J, Holden MTG, Lopez-Alvarez MR, Paillot R, Waller AS.Bacterial superantigens (sAgs) are powerful activators of the immune response that trigger unspecific T cell responses accompanied by the release of proinflammatory cytokines. () and () produce sAgs that play an important role in their ability to cause disease. Strangles, caused by , is one of the most common infectious diseases of horses worldwide. Here, we report the identification of a new sAg of , SpeS, and show that mutation of the putative T cell receptor (TCR)-binding motif (YAY to IAY) abrogated TCR-binding, whilst maintaining interaction with major histocompatibility complex (MHC) c...
Caselles AB, Miro-Moran A, Morillo Rodriguez A, Gallardo Bolaños JM, Ortega-Ferrusola C, Salido GM, Peña FJ, Tapia JA, Aparicio IM.Apoptosis in the testis is required to ensure an efficient spermatogenesis. However, sometimes, defective germ cells that are marked for elimination during this process escape elimination in the testes, giving rise to ejaculates with increased percentages of abnormal and apoptotic spermatozoa and a high percentage of apoptotic bodies. Apoptosis markers in the ejaculate have been associated with low fertility, either in animals or humans. Therefore, the goal of this study was to investigate whether fresh equine semen contains apoptotic bodies [initially named Merocyanine 540 (M540) bodies] and ...
Segabinazzi LGTM, Scheeren VFDC, Freitas-Dell'Aqua CP, Papa FO, Alvarenga MA, Dell'Aqua JA, Canisso IF.The present study aimed to compare semen parameters and fertility of cooled donkey semen extended in a commercially available skim milk (SKM) based extender and the same extender with cholesterol-loaded cyclodextrin (SKM-CLC). In Experiment 1, thirty-five ejaculates from seven jacks were split in SKM and SKM-CLC, extended at 50 million sperm/mL and stored at 5°C for 48 hours. Total motility (TM), progressive motility (PM), percentage of sperm with rapid motility (RAP) were assessed with CASA. Plasma membrane stability (PMS), and high mitochondrial membrane potential (HMP) were assessed with t...
Loureiro KC, Lima-Verde IB, Johannisson A, Ntallaris T, Jager A, Štěpánek P, da Costa Mendonça M, Severino P, Morrell JM.Cryopreservation of stallion spermatozoa tends to cause plasma membrane damage due to the low ratio of cholesterol to phospholipids. Gums have been suggested as an alternative cryoprotectant to glycerol for stallion spermatozoa. Therefore, the present experiment was designed to verify whether the effect of addition of cashew gum (CG), or nanoparticles (NP) containing CG, to the extender before cooling on sperm quality in stallion semen. Ejaculates from 6 stallions were extended and split between six treatment groups (control, a-tocopherol [TOC], CG1, CG0.5, NP1 and NP0.5), stored in cryotubes ...
Clulow JR, Evans G, Morris LH, Maxwell WM.Intrinsic differences between stallions exist for semen traits such as motility, morphology fertility and the ability of spermatozoa to survive cryopreservation processes. Ejaculates from 11 stallions were used to test the differences between stallions when selecting X- and Y-chromosome bearing spermatozoa using a modified flow cytometer. Data on orientation and viability of spermatozoa were collected during sex-sorting, and motility characteristics of sex-sorted and non-sorted (control) spermatozoa were assessed before and after cryopreservation. An index was created to rank each stallion in ...
