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.
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 ...
Bhide M, Yilmaz Z, Golcu E, Torun S, Mikula I.The aim of the study was to determine the seroprevalence of anti-Borrelia burgdorferi antibodies in a population of Turkish dogs and horses, as well as to compare the sensitivity of novel flow-cytometry-based borreliacidal antibody test (BAT) with ELISA assay. Serum samples collected from 400 dogs and 300 horses were tested with enzyme-linked protein A/G assay (ELPAGA), using Borrelia whole cell antigens. ELPAGA test showed 93 dogs (23.2%) and 18 horses (6%) serologically positive for anti-Borrelia antibodies. In parallel testing of sera with BAT, we found 27.75% positive dogs and 6.33% positi...
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...
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...
Meng Q, Li S, Liu L, Xu J, Liu Y, Zhang Y, Zhang X, Shao Y.Equine Infectious Anemia Virus (EIAV), like other lentiviruses, has a transmembrane glycoprotein with an unusually long cytoplasmic tail (CT). Viral envelope (Env) proteins having CT truncations just downstream the putative membrane-spanning domain (PMSD) are assumed to exist among all wild-type budded virions, and also in some cell-adapted strains. To determine whether CT-truncated Env proteins can cause particularly deleterious effects on the Env expressing cells and/or their neighboring cells, plasmids encoding codon-optimized env gene including full-length (pE863) or CT-truncated (pE686* a...
Morrell JM, Johannisson A, Dalin AM, Hammar L, Sandebert T, Rodriguez-Martinez H.Artificial insemination is not as widely used in horses as in other domestic species, such as dairy cattle and pigs, partly because of the wide variation in sperm quality between stallion ejaculates and partly due to decreased fertility following the use of cooled transported spermatozoa. Furthermore, predictive tests for sperm fertilising ability are lacking. The objective of the present study was to assess sperm morphology and chromatin integrity in ejaculates obtained from 11 warmblood breeding stallions in Sweden, and to evaluate the relationship of these parameters to pregnancy rates to i...
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...
Rendle DI, Durham AE, Thompson JC, Archer J, Mitchell M, Saunders K, Millere J, Paillot R, Smith KC, Kydd JH.Lymphoid leukaemia (LL) is rare in equids. In man, immunophenotypic classification identifies distinct leukaemic types with different treatment strategies. Improved understanding and classification of equine LL may allow similar advances. Objective: To document the clinical, immunophenotypic and functional characteristics in 6 cases of equine LL of T-cell origin. Methods: The clinical records and pathological findings from 6 cases of equine LL were analysed. Immunohistochemistry to identify T or B lymphocytes was performed on paraffin embedded tissues in 4 cases. Peripheral blood mononuclear c...
Dunkel B, Rickards KJ, Page CP, Cunningham FM.Platelet activation occurs in human obstructive airway diseases and in laboratory animal models. However, there is limited evidence that platelets may be involved in equine recurrent airway obstruction (RAO) and other inflammatory diseases. This study investigated whether platelet activation also occurred in RAO. Objective: Platelet function is altered in ponies with active RAO. This alteration can be detected ex vivo by measuring platelet adhesion. Methods: An in vitro platelet adhesion assay measuring acid phosphatase (AcP) activity colorimetrically was adapted for use with equine platelets ...
Agrícola R, Carvalho H, Barbosa M, Pereira M, Medeiros JA, Ferreira-Dias G.The aim of this study was to evaluate peripheral blood lymphocyte subpopulations, neutrophil phagocytic capacity and proteinogram characteristics in mares, during the last trimester of pregnancy and in postpartum. Measurement of phagocytosis and quantification of T-lymphocyte subsets were done by flow cytometry. Quantification of T-lymphocyte subsets was performed with monoclonal antibodies specific for CD2, CD3, CD4 and CD8 cell markers. Natural killer and B-cell counts were estimated mathematically. Serum proteinogram was obtained by electrophoresis. No significant differences were observed ...
Ortega-Ferrusola C, Sotillo-Galán Y, Varela-Fernández E, Gallardo-Bolaños JM, Muriel A, González-Fernández L, Tapia JA, Peña FJ.The kinematics of the appearance of apoptotic markers was studied by flow cytometry and immunoblot assays in equine spermatozoa subjected to freezing and thawing. Caspase activity, low mitochondrial membrane potential, and increases in sperm membrane permeability were observed in all of the phases of the cryopreservation procedure. Freezing and thawing caused an increase in membrane permeability and changes in the pattern of caspase activity; decreases in mitochondrial membrane potential were observed after centrifugation and cooling to 4 degrees C and after freezing and thawing. It is propose...
Wagner B, Hillegas JM, Brinker DR, Horohov DW, Antczak DF.Interleukin-10 (IL-10) terminates inflammatory immune responses and inhibits activation and effector functions of T-cells, monocytes, macrophages and dendritic cells. IL-10 has also been found to be a key cytokine expressed by subpopulations of regulatory T-cells. In this report, we describe the generation and characterization of three monoclonal antibodies (mAbs) to equine IL-10. The antibodies were found to be specific for equine IL-10 using different recombinant equine cytokine/IgG fusion proteins. Two of the anti-equine IL-10 mAbs were selected for ELISA to detect secreted IL-10 in superna...
Segura D, Monreal L, Armengou L, Tarancón I, Brugués R, Escolar G.Mean platelet component (MPC) is a new platelet variable, measured by modern commercial complete blood count analyzers, that is reduced during platelet activation in humans and small animals. Objective: MPC decreases in horses with clinical conditions that cause platelet activation and disseminated intravascular coagulation (DIC). Methods: We obtained 418 CBCs from 100 sick and 20 healthy neonates and 178 sick and 45 sound adult horses. Sick neonates were classified into septic and nonseptic, and DIC and non-DIC groups. Adults were grouped by diagnoses (systemic inflammatory disorders, gastroi...
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...
Xu J, Cai J, Peek SF, Suresh M, Darien BJ.P-selectin glycoprotein ligand (PSGL-1) is a widely distributed adhesion molecule that plays a critical role in regulating lymphocyte homing and leukocyte trafficking during inflammation. The lack of specific reagents for equine PSGL-1 (ePSGL-1) has prevented mechanistic studies regarding its function and regulation in the horse. We synthesized a ePSGL-1 peptide to generate a monoclonal antibody (mAb), ePL1. Using flow cytometry and Western blot, we showed that ePL1 binds specifically to ePSGL-1 in transfected mammalian cells. We also demonstrated that ePL1 binds to equine leukocytes and recog...
Gardner RB, Nydam DV, Luna JA, Bicalho ML, Matychak MB, Flaminio MJ.Phagocytic activity of neonatal foals has been reported to be similar to that of adult horses, but serum opsonization capacity develops with age and may be further altered when opsonins are consumed during infection. Objective: Phagocytosis, oxidative burst activity, and serum opsonization capacity in neonatal foals admitted to an intensive care unit are reduced in comparison with control foals. Methods: Blood samples were collected from hospitalized neonatal foals and from control foals. Hospitalized foals were characterized as sick or septic on the basis of a sepsis score and received intrav...
Norris JW, Pratt SM, Hunter JF, Gardner IA, Tablin F.To measure the frequency and magnitude of reduced fibrinogen binding in a population of horses from a Thoroughbred breeding farm. Methods: 444 Thoroughbred horses, 1 to 27 years old, including 316 females, 72 geldings, and 56 sexually intact males. Methods: Blood was collected from horses into tubes containingacid citrate dextrose adenine, and washed platelets were examined by use of flow cytometry for their ability to bind fibrinogen. Results: Data regarding fibrinogen binding to activated platelets were normally distributed, with nearly identical amounts of variation regardless of sex. In 3 ...
Ibrahim S, Saunders K, Kydd JH, Lunn DP, Steinbach F.Three hundred and seventy-nine monoclonal antibodies (mAbs) against various human CD molecules supplied to the HLDA8 animal homologues section (including four isotype controls) were analysed for cross-reactivity with equine leukocytes. First, flow cytometric identification of positively reacting mAbs was performed in one laboratory. Thereafter, a second round of flow cytometric evaluation was performed, involving three laboratories participating in the study. The first test-round indicated 17 mAbs as potentially positive. After the second round of flow cytometric analysis, 14 mAbs remained (di...
Ibrahim S, Steinbach F.In addition to the 379 monoclonal antibodies (mAbs) tested in the animal homologues section of HLDA8, another 155 mAbs were screened at the Institute for Zoo and Wildlife Research, Berlin for cross-reactivity with equine leukocytes. For this purpose, one colour flow-cytometric analysis was performed as screening test. This additional screening indicated further 16 mAbs as positive with staining homologous to human pattern, 1 mAb with weak (positive) reactivity, 11 mAbs with positive, but likely not valuable staining, 12 mAbs with alternate expression pattern from that expected from human immun...
Hamza E, Doherr MG, Bertoni G, Jungi TW, Marti E.Equine insect bite hypersensitivity (IBH) is an immediate-type hypersensitivity reaction provoked by insect-derived allergens. Icelandic horses living in Iceland do not have IBH due to absence of relevant insects, but acquire it at high frequency after being imported to mainland Europe. In contrast, their offspring born in mainland Europe has reduced IBH incidence. T helper 1 (Th1) and Th2 cells and cytokines were determined in Icelandic horses born in Iceland and on the continent and which either have IBH or are healthy. Methods: Peripheral blood mononuclear cells (PBMC) from these horses wer...
Luce R, Shepherd M, Paillot R, Blacklawst B, Wood JL, Kydd JH.An assay has been developed that measures EHV-1 specific interferon gamma synthesis (IFNgamma), a cytokine produced following the activation of memory T lymphocytes and therefore a measure of cell mediated immunity. The method requires validation in the field. Objective: To measure the frequency of EHV-1 specific, IFNgamma synthesising peripheral blood mononuclear cells (PBMC) in a population of Thoroughbred horses, and examine its relationship with age, gender, premises and history of vaccination or field infection with EHV-1. Methods: Lymphocytes from 200 Thoroughbred horses were stimulated ...
Macieira S, Rivard GE, Champagne J, Lavoie JP, Bédard C.An 18-month-old Oldenbourg filly was presented with a bleeding diathesis. Laboratory testing included platelet count, gingival bleeding time, prothrombin time (PT), activated partial thromboplastin time (aPTT), von Willebrand factor (vWf) antigen, clottable fibrinogen, clot retraction time, PFA-100 closure time, platelet aggregometry (on platelet-rich plasma), and thrombelastography (TEG). TEG was performed by using kaolin and tissue factor as coagulation activators. Expression of the platelet receptor for fibrinogen was assessed by flow cytometry by using anti CD41 (alpha(IIb) or glycoprotein...
Hoffman AM, Oura TJ, Riedelberger KJ, Mazan MR.Horses with recurrent airway obstruction (heaves) are described as exhibiting "increased abdominal effort," but it is unknown whether this translates to an effective contribution to ventilation. Objective: We hypothesized that heaves is characterized by asynchrony between rib cage and abdominal motions, and that the abdominal component is the major contributor to ventilation. Methods: The rib cage versus abdominal motion in naturally occurring heaves (n = 15) was compared to controls at rest (n = 7) and during hyperpnea because of lobeline treatment, and the effects of histamine-induced bronch...
Xu J, Cai J, Anderson B, Wagner B, Albrecht R, Peek SF, Suresh M, Darien BJ.The recent molecular characterization and sequencing of equine P-selectin (ePsel), and its glycoprotein ligand, P-selectin glycoprotein ligand-1 (PSGL-1), have provided the tools for further investigation into their role in leukocyte trafficking. Here, we report the generation of a genetically engineered chimeric protein (ePsel-IgG) in which the equine P-selectin lectin and epithelial growth factor (EGF) domains were covalently linked to the equine IgG1 heavy chain constant region. The soluble ePsel-IgG was observed to bind to equine monocytes by confocal microscopy and flow cytometry. Further...
Norris JW, Pratt SM, Auh JH, Wilson SJ, Clutter D, Magdesian KG, Ferraro GL, Tablin F.Bleeding in racing horses associated with exercise appears to be multifactorial, and clinical investigation into severe cases rarely occurs. Previously, we reported a severe bleeding diathesis in a Thoroughbred mare. Herein, we describe the cellular physiology of this defect, provide a diagnostic tool for identifying it, and demonstrate that the dysfunction is heritable. Objective: The subject has a heritable defect in platelet secretion that reduces thrombin generation in the absence of additional plasma factors and delays the onset of thrombin production even in the presence of these factors...
de Bruijn CM, Veenman JN, Rutten VP, Teske E, van Nieuwstadt RA, van den Ingh TS.This study documents the clinical, histopathological, immunohistochemical and flow-cytometric findings in five horses with cutaneous non-epidermotropic malignant lymphoma (ML). The median survival time after discovery of the first subcutaneous nodules was 3.8 years (range 2-5 years: n=4). Histologically, the cutaneous ML had a pleiomorphic structure and contained a mixture of large reticulo-endothelial cells, medium-large sized lymphoid cells with a rounded nucleus and small nucleoli, many medium sized lymphoid cells with irregular nuclei, and some small lymphoid cells. Immunohistochemically (...
Ehrmann C, Engel J, Moritz A, Roscher K.In addition to maintaining hemostasis, platelets have an important role in modulating innate and adaptive immune responses. A low platelet count has been found to be a negative prognostic factor for survival in humans and horses with critical illnesses, such as sepsis or systemic inflammatory response syndrome (SIRS). Decreased platelet aggregation, caused by in vivo activation, has been found in human patients with severe sepsis. In our prospective controlled study, we assessed platelet biology in blood samples from 20 equine SIRS cases and 120 healthy control horses. Platelet variables such ...
Pintado CO, Friend M, Llanes D.This paper describes two anti-glycoprotein IIb/IIIa or CD41/61 murine monoclonal antibodies (Co.35E4 and Co.2oA1). The cellular distribution and apparent molecular weight of the antigen detected by these antibodies is consistent with their reaction with ruminant and equine glycoprotein IIb/IIIa. Biochemical analysis of the equine molecule using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) revealed bands of 24, 100 and 110 kDa under reducing conditions and 115 and 80 kDa under nonreducing conditions. Biochemical analysis of ruminant antigen revealed that the 24 kDa band...
Dascanio JJ, Zhang CH, Antczak DF, Blue JT, Simmons TR.Chronic lymphocytic leukemia (CLL) was diagnosed in two horses: an 18-year-old Quarter Horse gelding that was examined because of edema of the prepuce and ventral abdomen; and a 20-year-old mixed breed gelding that was referred because of lymphocytosis, ventral edema, and weight loss. The first horse had enlarged peripheral lymph nodes and cool nonpainful pitting edema of the ventral abdomen and prepuce. The second horse had enlarged peripheral lymph nodes, cool nonpainful pitting edema of the ventral thorax and cranial ventral abdomen, and a 3/5 holosystolic heart murmur. The diagnosis of CLL...
Meng Q, Li S, Liu L, Xu J, Liu Y, Zhang Y, Zhang X, Shao Y.Equine Infectious Anemia Virus (EIAV), like other lentiviruses, has a transmembrane glycoprotein with an unusually long cytoplasmic tail (CT). Viral envelope (Env) proteins having CT truncations just downstream the putative membrane-spanning domain (PMSD) are assumed to exist among all wild-type budded virions, and also in some cell-adapted strains. To determine whether CT-truncated Env proteins can cause particularly deleterious effects on the Env expressing cells and/or their neighboring cells, plasmids encoding codon-optimized env gene including full-length (pE863) or CT-truncated (pE686* a...
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...
Michler JK, Hillmann A, Savkovic V, Mülling CKW.The easily accessible niche represented by skin and its appendages may serve as a promising source to complement modern regenerative medicine for horses. In humans and in animal models for human medicine, the hair follicle and its stem cell niches are well characterized. Since literature in this field of equine research is scarce, we sought to analyze cells of the dermal stem cell niche of the equine hair follicle morphologically and for a subset of markers useful for cell characterization via immunolabeling. We cultured equine forelock skin explants to obtain cultures with cells migrating fro...
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...
Ortiz-Rodriguez JM, Bucci D, Tovar-Pascual L, Granata S, Spinaci M, Nesci S.Sperm metabolism consists of a sophisticated network of biochemical reactions and varies between species, resulting in different metabolic strategies for ATP production to maintain sperm functionality. ATP can be produced through glycolysis or in the mitochondria by oxidative phosphorylation (OXPHOS). Since OXPHOS is the predominant metabolic pathway in horses spermatozoa, various assessments of mitochondrial activity are used to evaluate fertility, utilizing techniques such as fluorescent probes analysed via microscopy or flow cytometry, and polarographic electrode assays to measure current f...
Beaumont RE, Smith EJ, David C, Paterson YZ, Faull E, Guest DJ.Tendon injuries are a common problem in humans and horses. There is a high re-injury rate in both species due to the poor regeneration of adult tendon and the resulting formation of scar tissue. In contrast, fetal tendon injuries undergo scarless regeneration, but the mechanisms which underpin this are poorly defined. It is also unclear if tendon cells derived from embryonic stem cells (ESCs) would aid tendon regeneration. In this study we determined the responses of adult, fetal and ESC-derived equine tenocytes to a range of cytokines, chemokines and growth factors that are upregulated follow...
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.
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 ...
Marycz K, Pielok A, Kornicka-Garbowska K.Laminitis is a life threating, extremely painful and frequently recurrent disease of horses which affects hoof structure. It results from the disruption of blood flow to the laminae, contributing to laminitis and in severe separation of bone from the hoof capsule. Still, the pathophysiology of the disease remains unclear, mainly due to its complexity. In the light of the presented data, in the extremally difficult process of tissue structure restoration after disruption, a novel type of progenitor cells may be involved. Herein, we isolated and performed the initial characterization of stem pro...
Herbst AC, Reedy SE, Page AE, Horohov DW, Adams AA.Studies investigating age-related changes in the function of monocytes are currently limited for horses. Thus, the main goal of this study was to determine the effect of aging on monocyte phagocytic capacity and pro-inflammatory cytokine responses to bacterial lipopolysaccharide (LPS). A second goal of this work was to examine the effect of aging on the inflammatory cytokine responses to LPS in a whole blood ex vivo model. Seven healthy young adult (4-6 years of age) and seven healthy senior horses (>20 years of age) were enrolled. Phagocytosis of E. coli, and pro-inflammatory cytokine (TNF...
Agrícola R, Carvalho H, Barbosa M, Pereira M, Medeiros JA, Ferreira-Dias G.The aim of this study was to evaluate peripheral blood lymphocyte subpopulations, neutrophil phagocytic capacity and proteinogram characteristics in mares, during the last trimester of pregnancy and in postpartum. Measurement of phagocytosis and quantification of T-lymphocyte subsets were done by flow cytometry. Quantification of T-lymphocyte subsets was performed with monoclonal antibodies specific for CD2, CD3, CD4 and CD8 cell markers. Natural killer and B-cell counts were estimated mathematically. Serum proteinogram was obtained by electrophoresis. No significant differences were observed ...
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...
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...
