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.
Cohen ND, Bourquin JR, Bordin AI, Kuskie KR, Brake CN, Weaver KB, Liu M, Felippe MJ, Kogut MH.Neutrophils play an important role in protecting against infection. Foals have age-dependent deficiencies in neutrophil function that may contribute to their predisposition to infection. Thus, we investigated the ability of a CpG-ODN formulated with Emulsigen to modulate functional responses of neutrophils in neonatal foals. Eighteen foals were randomly assigned to receive either a CpG-ODN with Emulsigen (N = 9) or saline intramuscularly at ages 1 and 7 days. At ages 1, 3, 9, 14, and 28, blood was collected and neutrophils were isolated from each foal. Neutrophils were assessed for basal and R...
O'Shea CM, Werre SR, Dahlgren LA.(1) To compare the performance of 4 platelet counting technologies in equine platelet concentrates and (2) to evaluate the ability of the Magellan platelet rich plasma (PRP) system to concentrate equine platelets. Methods: Experimental study to assess method agreement. Methods: Adult mixed breed horses (n = 32). Methods: Acid citrate dextrose-A anti-coagulated whole blood was collected and PRP produced using the Magellan system according to the manufacturer's instructions. Platelets were quantified using 4 counting methods: optical scatter (Advia 2120), impedance (CellDyn 3700), hand counting,...
Balao da Silva CM, Ortega Ferrusola C, Gallardo Bolaños JM, Plaza Dávila M, Martín-Muñoz P, Morrell JM, Rodriguez Martínez H, Peña FJ.Flow cytometry is considered the only reliable method for the separation of X and Y chromosome bearing spermatozoa in equines. The MoFlo SX DP sorter is highly efficient, allowing the production of foals of the desired sex. However, to achieve acceptable pregnancy rates the currently used protocol requires working with fresh semen obtained close to, or at, the sorting facility. An alternative protocol was tested during two consecutive breeding seasons. Fresh stallion semen was cooled for 20 h, during which staining with Hoechst 33342 took place. On the following day, this sample was flow sort...
Petersen GF, Hilbert B, Trope G, Kalle W, Strappe P.Equine adipose-derived mesenchymal stem cells (EADMSC) provide a unique cell-based approach for treatment of a variety of equine musculoskeletal injuries, via regeneration of diseased or damaged tissue, or the secretion of immunomodulatory molecules. These capabilities can be further enhanced by genetic modification using lentiviral vectors, which provide a safe and efficient method of gene delivery. We investigated the suitability of lentiviral vector technology for gene delivery into EADMSC, using GFP expressing lentiviral vectors pseudotyped with the G glycoprotein from the vesicular stomat...
Springer NL, Smith E, Brooks MB, Stokol T.To develop a flow cytometric assay to quantify platelet-derived microparticles (PMPs) in equine whole blood and plasma. Methods: Citrate-anticoagulated whole blood from 30 healthy adult horses. Methods: Platelet-poor plasma (PPP) was prepared from fresh whole blood by sequential low-speed centrifugation (twice at 2,500 × g). Samples of fresh whole blood and PPP were removed and stored at 4° and 24°C for 24 hours. Platelet-derived microparticles were characterized in fresh and stored samples on the basis of the forward scatter threshold (log forward scatter < 10(1)) and labeling with anne...
Braune S, Walter M, Schulze F, Lendlein A, Jung F.For in vitro studies assessing the interaction of platelets with implant materials, common and standardized protocols for the preparation of platelet rich plasma (PRP) are lacking, which may lead to non-matching results due to the diversity of applied protocols. Particularly, the aging of platelets during prolonged preparation and storage times is discussed to lead to an underestimation of the material thrombogenicity. Here, we study the influence of whole blood- and PRP-storage times on changes in platelet morphology and function. Blood from apparently healthy subjects was collected according...
Murata D, Miyakoshi D, Hatazoe T, Miura N, Tokunaga S, Fujiki M, Nakayama K, Misumi K.Cartilage regeneration with cell therapy following arthroscopic surgery could be used in racehorses with intra-articular fractures (IAF) and osteochondritis dissecans (OCD). The aims of this study were to investigate the origin and multipotency of stromal cells in the synovial fluid (SF) of horses with intra-articular injury and synovitis, and to provide a new strategy for regeneration of lost articular cartilage. Mesenchymal stromal cells were isolated from SF of horses with IAF and OCD. Multipotency was analysed by RT-PCR for specific mRNAs and staining for production of specific extracellul...
Watts AE, Ness SL, Divers TJ, Fubini SL, Frye AH, Stokol T, Cummings KJ, Brooks MB.To evaluate the effects of clopidogrel on clinical and clinicopathologic variables in healthy horses with experimentally induced endotoxemia. Methods: 12 adult mares. Procedures-Horses were assigned with a randomization procedure to receive clopidogrel (4 mg/kg, once, then 2 mg/kg, q 24 h; n = 6) or a placebo (6) through a nasogastric tube. After 72 hours of treatment, horses received lipopolysaccharide (LPS; 30 ng/kg, IV). Heart rate, respiratory rate, rectal temperature, CBC variables, plasma fibrinogen concentration, serum tumor necrosis factor-α concentration, plasma von Willebrand factor...
Gallardo Bolaños JM, Balao da Silva C, Martín Muñoz P, Plaza Dávila M, Ezquerra J, Aparicio IM, Tapia JA, Ortega Ferrusola C, Peña FJ.To investigate the mechanisms inducing sperm death after ejaculation, stallion ejaculates were incubated in BWW media during 6 h at 37°C. At the beginning of the incubation period and after 1, 2, 4 and 6 h sperm motility and kinematics (CASA), mitochondrial membrane potential and membrane permeability and integrity were evaluated (flow cytometry). Also, at the same time intervals, active caspase 3, hydrogen peroxide, superoxide anion (flow cytometry) and Akt phosphorylation (flow cytometry) were evaluated. Major decreases in sperm function occurred after 6 h of incubation, although after 1 h ...
Paebst F, Piehler D, Brehm W, Heller S, Schroeck C, Tárnok A, Burk J.Horses are an approved large animal model for therapies of the musculoskeletal system. Especially for tendon disease where cell-based therapy is commonly used in equine patients, the translation of achieved results to human medicine would be a great accomplishment. Immunophenotyping of equine mesenchymal stromal cells (MSCs) remains the last obstacle to meet the criteria of the International Society for Cellular Therapy (ISCT) definition of human MSCs. Therefore, the surface antigen expression of CD 29, CD 44, CD 73, CD 90, CD 105, CD 14, CD 34, CD 45, CD 79α, and MHC II in equine MSCs from a...
Gulati BR, Kumar R, Mohanty N, Kumar P, Somasundaram RK, Yadav PS.Tendon injuries are common in race horses, and mesenchymal stem cells (MSCs) isolated from adult and foetal tissue have been used for tendon regeneration. In the present study, we evaluated equine amniotic fluid (AF) as a source of MSCs and standardised methodology and markers for their in vitro tenogenic differentiation. Plastic-adherent colonies were isolated from 12 of 20 AF samples by day 6 after seeding and 70-80% cell confluency was reached by day 17. These cells expressed mesenchymal surface markers [cluster of differentiation (CD)73, CD90 and CD105] by reverse transcription (RT)-polyme...
Barberini DJ, Freitas NP, Magnoni MS, Maia L, Listoni AJ, Heckler MC, Sudano MJ, Golim MA, da Cruz Landim-Alvarenga F, Amorim RM.Studies with mesenchymal stem cells (MSCs) are increasing due to their immunomodulatory, anti-inflammatory and tissue regenerative properties. However, there is still no agreement about the best source of equine MSCs for a bank for allogeneic therapy. The aim of this study was to evaluate the cell culture and immunophenotypic characteristics and differentiation potential of equine MSCs from bone marrow (BM-MSCs), adipose tissue (AT-MSCs) and umbilical cord (UC-MSCs) under identical in vitro conditions, to compare these sources for research or an allogeneic therapy cell bank. Methods: The BM-MS...
Murphy C, English AM, Holden SA, Fair S.An unacceptable proportion of stallion sperm do not survive the freeze-thaw process. The hypothesis of this study was that adding cholesterol to a stallion semen extender would stabilise the sperm membrane, resulting in an improved post-thaw semen quality in terms of increased sperm viability, membrane integrity and fluidity, and reduced oxidative stress. Semen was collected from three stallions and diluted in four extenders: TALP; TALP+0.75mg methyl-β-cyclodextrin-cholesterol (MβCD)/mL (MβCD0.75); TALP+1.5mg MβCD-cholesterol/mL (MβCD1.5); and Equipro. Following 15min incubation, samples ...
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 ...
Schnabel LV, Pezzanite LM, Antczak DF, Felippe MJ, Fortier LA.The horse is a valuable species to assess the effect of allogeneic mesenchymal stromal cells (MSCs) in regenerative treatments. No studies to date have examined recipient response to major histocompatibility complex (MHC)-mismatched equine MSCs. The purposes of this study were to immunophenotype MSCs from horses of known MHC haplotype and to compare the immunogenicity of MSCs with differing MHC class II expression. Methods: MSCs and peripheral blood leukocytes (PBLs) were obtained from Thoroughbred horses (n=10) of known MHC haplotype (ELA-A2, -A3, and -A9 homozygotes). MSCs were cultured thro...
Mohanty N, Gulati BR, Kumar R, Gera S, Kumar P, Somasundaram RK, Kumar S.Mesenchymal stem cells (MSCs) isolated from umbilical cord blood (UCB) in equines have not been well characterized with respect to the expression of pluripotency and mesenchymal markers and for tenogenic differentiation potential in vitro. The plastic adherent fibroblast-like cells isolated from 13 out of 20 UCB samples could proliferate till passage 20. The cells expressed pluripotency markers (OCT4, NANOG, and SOX2) and MSC surface markers (CD90, CD73, and CD105) by RT-PCR, but did not express CD34, CD45, and CD14. On immunocytochemistry, the isolated cells showed expression of CD90 and CD73...
Henríquez C, Perez B, Morales N, Sarmiento J, Carrasco C, Morán G, Folch H.Recurrent airway obstruction (RAO) is an equine immune-mediated disease with a high incidence worldwide. The aim of this work was to contribute to the understanding of RAO pathogenesis by studying T cells bearing regulatory markers in peripheral blood (PB) and in bronchoalveolar lavage fluid (BALF) recovered from the same group of susceptible horses before and after exposure to moldy hay, which has been shown to induce RAO signology in our horse herd. With this purpose, mononuclear cells were obtained from the BALF and PB from horses before and after antigenic challenge and were stained with f...
Carvalho AM, Yamada AL, Golim MA, Álvarez LE, Hussni CA, Alves AL.Mesenchymal stem cells (MSCs) have been used to treat equine tendonitis with promising results; however, little is known about the potential migration of these cells. Objective: To assess the possible migration of MSCs from an implantation site in the superficial digital flexor tendon (SDFT) to a lesion in the SDFT of the contralateral limb. Methods: In vivo experimental study. Methods: Adipose-derived MSCs were isolated from 4 healthy horses. Lesions were induced in the SDFTs of both forelimbs, followed by intralesional implantation of autologous adipose-derived MSCs labelled with nanocrystal...
Baghi HB, Laval K, Favoreel H, Nauwynck HJ.The nasal mucosa surface is continuously confronted with a broad variety of environmental antigens, ranging from harmless agents to potentially harmful pathogens. This area is under rigorous control of professional antigen presenting cells (APCs), such as dendritic cells (DCs) and macrophages. Mucosal APCs play a crucial role in inducing primary immune responses and the establishment of an immunological memory. In the present study, a detailed characterization of CD172a(+) cells, containing the APCs residing in the equine nasal mucosa was performed for the first time. CD172a(+) cells were isol...
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...
Schnabel CL, Wagner S, Wagner B, Durán MC, Babasyan S, Nolte I, Pfarrer C, Feige K, Murua Escobar H, Cavalleri JM.Research on equine cytokines is often performed by analyses of mRNA. For many equine cytokines an analysis on the actual protein level is limited by the availability of antibodies against the targeted cytokines. Generation of new antibodies is ongoing but time consuming. Thus, testing the reactivity of commercially available antibodies for cross-reactivity with equine cytokines is of particular interest. Fifteen monoclonal antibodies against IL-1β, IL-6, IL-8, IL-12, IL-18 and Granulocyte Macrophage Colony stimulating factor (GM-CSF) of different species were evaluated for reactivity with the...
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...
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...
Hamza E, Akdis CA, Wagner B, Steinbach F, Marti E.Insect bite hypersensitivity (IBH) is a recurrent allergic dermatitis of horses with similarities to human atopic eczema, caused by bites of insects of the genus Culicoides. Previous studies suggested a dysregulated T cell tolerance to Culicoides allergen in IBH-affected horses. Objective: We have investigated whether the suppressive function of CD4(+) CD25(high) cells is impaired in IBH-affected horses and possible ways to restore it. Methods: CD4(+) CD25(-) cells sorted from peripheral blood mononuclear cells (PBMC) were stimulated with irradiated autologous PBMC pulsed with Culicoides or te...
Balao da Silva CM, Spinaci M, Bucci D, Giaretta E, Peña FJ, Mari G, Galeati G.The interest on sex sorting by flow cytometry on the equine industry has been increasing over the years. In this work, three different tests were performed in order to evaluate the membrane status of sorted stallion spermatozoa: assessment of binding ability to porcine oocytes, evaluation of acrosome integrity after stimulation with A23187, and detection of tyrosine phosphorylation. These evaluations were made after incubation for 0h, 1.5h and 3h in a capacitating medium. Sorted stallion spermatozoa attached similarly to the porcine oocytes, when compared with control samples. Sorted spermatoz...
Sarmiento J, Perez B, Morales N, Henriquez C, Vidal L, Folch H, Galecio JS, Morán G.A reduction in inflammatory cell apoptosis is an important concept in the maintenance of inflammation and a potential target for the resolution of inflammation in many inflammatory diseases. Dysregulation of apoptosis has been implicated in a range of diseases, including tumors, neurodegenerative disorders and autoimmunity, and may also be implicated in allergic asthma. In horses, recurrent airway obstruction (RAO) is an asthma-like condition that is characterized increased survival neutrophil bronchial. Tamoxifen is a synthetic, non-steroidal, anti-estrogen agent that is widely used for treat...
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...
Morrell JM, Winblad C, Georgakas A, Stuhtmann G, Humblot P, Johannisson A.There are anecdotal reports that equine fertility may decline towards the end of the breeding season. Previous studies have examined differences in sperm quality between the breeding season and non-breeding season but few studies have investigated the proportions of superoxide or peroxide containing spermatozoa at different times during the breeding season. The purpose of this study was to measure the content of these reactive oxygen species (ROS) at the beginning and end of the Swedish breeding season, using flow cytometric analysis of the fluorescence produced after staining with hydroethidi...
Radtke CL, Nino-Fong R, Esparza Gonzalez BP, Stryhn H, McD￮ LA.To characterize equine muscle tissue- and periosteal tissue-derived cells as mesenchymal stem cells (MSCs) and assess their proliferation capacity and osteogenic potential in comparison with bone marrow- and adipose tissue-derived MSCs. Methods: Tissues from 10 equine cadavers. Methods: Cells were isolated from left semitendinosus muscle tissue, periosteal tissue from the distomedial aspect of the right tibia, bone marrow aspirates from the fourth and fifth sternebrae, and adipose tissue from the left subcutaneous region. Mesenchymal stem cells were characterized on the basis of morphology, ad...
Maia L, Landim-Alvarenga FC, Da Mota LS, De Assis Golim M, Laufer-Amorim R, De Vita B, Barberini DJ, Listoni AJ, De Moraes CN, Heckler MC, Amorim RM.The aim of this study was to isolate, culture, and characterize mesenchymal stem cells (MSCs) from horse bone marrow (BM) using the techniques of flow cytometry, immunocytochemistry, cytogenetics, and electron microscopy. Immunophenotypic analysis revealed the presence of MSCs with high expression of the CD90 marker, lower expression of the CD44 marker, and absent expression of the CD34 marker. In assays of differentiation, the positive response to osteogenic (OST), chondrogenic (CDG), and adipogenic (ADP) differentiation signals was observed and characterized by deposition of calcium-rich ext...
Davis EG, Wilkerson MJ, Rush BR.The use of flow cytometry in veterinary diagnostics is becoming a valuable clinical tool with a broad range of applications. Physical characteristics of cells can be determined by the flow cytometer laser and electronics through the measurement of changes in light scatter properties. Other components and functions of cells can be defined through the application of fluorochrome dyes that have an affinity for cellular components. Traditionally, common clinical applications are immunophenotyping of cells of the hematopoietic system with fluorescent-labeled antibodies raised against specific cell ...
Ness SL, Frye AH, Divers TJ, Rishniw M, Erb HN, Brooks MB.OBJECTIVE To determine effects of oral administration of Yunnan Baiyao on platelet activation, coagulation, and fibrinolysis in healthy horses. ANIMALS 12 healthy adult horses. PROCEDURES In a randomized blinded crossover study that included a 4-week washout period between treatments, horses were orally administered a paste containing Yunnan Baiyao (15 mg/kg) or placebo at 12-hour intervals for 3 days. Blood samples were collected before start of treatment (time 0) and at 24 and 72 hours for a CBC, measurement of fibrinogen concentration, coagulation screening tests, and a panel of assays to a...
Metz GE, Abeyá MM, Serena MS, Panei CJ, Echeverría MG.Equine arteritis virus (EAV) induces apoptosis in infected cells. Cell death caused by EAV has been studied mainly using three cell lines, BHK-21, RK-13 and Vero cells. The mechanism of apoptosis varies among cell lines and results cannot be correlated owing to differences in EAV strains used. We evaluated different markers for apoptosis in BHK-21, RK-13 and Vero cell lines using the Bucyrus EAV reference strain. Acridine orange/ethidium bromide staining revealed morphological changes in infected cells, while flow cytometry indicated the extent of apoptosis. We also observed DNA fragmentation,...
Bulkeley E, Santistevan AC, Varner D, Meyers S.Low levels of intracellular reactive oxygen species (ROS) are essential for normal sperm function and are produced by sperm mitochondria as a byproduct of metabolism, but in excess, ROS can cause catastrophic cellular damage and has been correlated with infertility, poor sperm motility and abnormal morphology in humans. Stallion sperm motility is fueled predominantly by oxidative phosphorylation-produced ATP, requiring high basal rates of mitochondrial function. Consequently, whether elevated ROS production by stallion sperm is an indicator of dysfunctional or highly motile cells has been deba...
Crawford PC, Hanel RM, Levy JK.To evaluate equine IgG as a treatment for kittens with failure of passive transfer of immunity (FPT). Methods: 13 specific pathogen-free queens and their 77 kittens. Methods: Kittens were randomized at birth into 9 treatment groups. One group contained colostrum-fed (nursing) kittens; the other groups contained colostrum-deprived kittens that were administered supplemental feline or equine IgG PO or SC during the first 12 hours after birth. Blood samples were collected at serial time points from birth to 56 days of age for determination of serum IgG concentrations. The capacity of equine IgG t...
Sugiura T, Nakamura H.The zinc content in platelets from rabbits, humans and horses was determined, and the levels of zinc were found to be significantly higher (3 micrograms/10(10) cells) than those in other peripheral blood cells. About 70% of the zinc in the supernatants of platelet lysates could be detected. From the results of gel filtration analysis, the zinc in platelet lysates was found to be bound with a low-molecular-weight protein (MW 6,000-8,000) detected as metallothionein (MT) on the basis of antigenic properties determined by enzyme-linked immunoassay and immunoblotting analysis using monoclonal anti...
Santos VHD, Pfeifer JPH, Souza JB, Stievani FC, Hussni CA, Golim MA, Deffune E, Alves ALG.Osteoarthritis is an incapacitating disease characterized by pain and a progressive decrease in joint mobility. The implantation of mesenchymal stem cells (MSCs) has shown promising results for its treatment. The challenge remains to keep the cells longer at the site of action, increasing their therapeutic potential. The aim of this study was to evaluate the effectiveness of the Qtracker® 655 nanocrystal marking on allogeneic synovial membrane (SM) MSCs, encapsulated in alginate hydrogel, evaluating the migration of these cells. The 10 radiocarpal joints were submitted to arthroscopic surgery...
Brazil TJ, Rossi AG, Haslett C, McGorum B, Dixon PM, Chilvers ER.The synthetic formylpeptide fMLP is widely used as a model chemoattractant and secretagogue for mammalian neutrophils. Despite possessing fMLP receptors, equine neutrophils do not produce superoxide anions in response to fMLP and there is no inflammatory reaction in the horse when fMLP is injected intradermally. The functional capability of these receptors was investigated after pretreatment with recognized priming agents. Purified neutrophils were pretreated with lipopolysaccharide (LPS), platelet-activating factor (PAF), or tumor necrosis factor alpha (TNF-alpha) and superoxide anion generat...
Pretorius A, Van Kleef M, Van Wyngaardt W, Heath J.African horsesickness (AHS) is an infectious but noncontagious viral disease affecting all species of Equidae. The recall immune response of AHSV naïve horses immunised with an attenuated African horsesickness virus serotype 4 (AHSV4) was characterised using immune assays including ELISPOT, real-time PCR (qPCR) and flow cytometry. The recall immune response detected in PBMC isolated from three inoculated horses showed an upregulation of circulating B lymphocytes that correlated with elevated IL-4 mRNA expression indicative of humoral immunity, but reduced frequency of CD4⁺ cells. In additio...
Ferreira-Dias G, Claudino F, Carvalho H, Agrícola R, Alpoim-Moreira J, Robalo Silva J.The primary objective of this study was to evaluate the possible role of leptin, body weight and immune status on reproductive activity throughout the transition period from cyclicity to seasonal anestrus, during anestrus and resumption of ovarian activity in Lusitano mares. Mares in good body condition were monthly monitored throughout 2 years (10 mares in each year) for evaluation of their reproductive status by sequential ultrasonography and plasma progesterone determinations. On the second year, all mares were weighed. Progesterone and leptin were assayed by radioimmunoassay (RIA). Paramet...
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...
Al-Kass Z, Ntallaris T, Morrell JM, Johannisson A.Although previous studies have examined the relationship between the sperm DNA fragmentation index and fertility in stallions, other aspects of chromatin structure or packaging and fertility have not been explored. In the present study, relationships between fertility and DNA fragmentation index, protamine deficiency, total thiols, free thiols and disulfide bonds in stallion spermatozoa were investigated. Ejaculates (n = 36) were collected from 12 stallions and extended to prepare semen doses for insemination. One dose from each ejaculate was sent to the Swedish University of Agricultural Sc...
Qin Z, Durand LG, Allard L, Cloutier G.In most studies that were aimed at evaluating the kinetics of red blood cell (RBC) aggregation, human blood was initially circulated at a high shear rate to disrupt the aggregates, and measurements were performed following a complete flow stoppage, during the process of rouleau formation. However, it is known that a very low shear rate can enhance the formation of aggregates, as demonstrated by the modal relationship of the shear-rate dependence of RBC aggregation. The objective of the present study was, thus, to evaluate the influence of sudden flow reductions compared to a complete flow stop...
Merkies K, Chenier T, Plante C, Buhr MM.Viability of spermatozoa can be assessed by numerous methods, but many are slow and poorly repeatable, and subjectively assess only 100 to 200 spermatozoa per ejaculate. We collected two ejaculates from each of 4 stallions, and extended them to 50x10(6) sperm/mL in a nonfat dried milk solids glucose extender (EZ Mixin). Half the ejaculate was freeze-killed by immersing in liquid nitrogen for 10 min. Aliquots using appropriate volumes of live and freeze-killed spermatozoa provided the following ratios of live:dead spermatozoa: 100:0, 75:25, 50:50, 25:75, 0:100. We determined the viability of ea...
Gröndahl G, Sternberg S, Jensen-Waern M, Johannisson A.Two of the most commonly isolated foal pathogens are Escherichia coli and Actinobacillus equuli. The hypothesis tested in this study was that young foals carry a lower opsonic capacity for these bacteria compared to adult horses. A flow-cytometric method for the phagocytosis of these by equine neutrophils was established. The opsonic capacity of serum from healthy foals from birth to age 6 weeks was evaluated and related to the concentrations of IgGa and IgGb. Phagocytosis of yeast was used as a control. Serum was required for phagocytosis, with higher concentrations for E. coli than for A. eq...
Echigoya Y, Morita S, Itou T, Sakai T.To evaluate effects of extracellular lactate on viability, shape change, lactate metabolism, and reactive oxygen species (ROS) production in equine polymorphonuclear leukocytes (PMNs). Methods: PMNs isolated from equine venous blood samples. Methods: PMNs were incubated with 0 to 300mM lactate for 30 minutes before each experiment. Viability was assessed via trypan blue exclusion. Shape change was assessed via flow cytometry and light microscopy. Relative quantification of monocarboxylic acid transporter and lactate dehydrogenase lactate dehydrogenase (LDH) isotype mRNAs was performed with a r...
Mohanty N, Gulati BR, Kumar R, Gera S, Kumar S, Kumar P, Yadav PS.Mesenchymal stem cells (MSCs) offer promise as therapeutic aid in the repair of tendon and ligament injuries in race horses. Fetal adnexa is considered as an ideal source of MSCs due to many advantages, including non-invasive nature of isolation procedures and availability of large tissue mass for harvesting the cells. However, MSCs isolated from equine fetal adnexa have not been fully characterized due to lack of species-specific markers. Therefore, this study was carried out to isolate MSCs from equine umbilical cord blood (UCB) and characterize them using cross-reactive markers. The plastic...
Mislei B, Bucci D, Malama E, Bollwein H, Mari G.Oxidative stress is regarded as an important cause of sperm damage during cryopreservation. However, seasonal changes in oxidative status in unfrozen and frozen-thawed stallion sperm have not been well established. We tested the hypothesis that sperm ROS concentrations and lipid peroxidation change between breeding and non-breeding seasons and influence quality of unfrozen and frozen-thawed sperm. Eighteen ejaculates from six Warmblood stallions (8-21 y) known to be fertile, were collected in winter and summer and processed for freezing. After 90 min at +4 °C, some straws from each ejacu...
Larson EM, Babasyan S, Wagner B.IL-8 is a potent chemokine that recruits neutrophils and basophils to promote inflammation in many species. IL-8 is produced by many cell types, including monocytes. In this study, we report a novel role for IgE-binding monocytes, a rare peripheral immune cell type, to promote allergic inflammation through IL-8 production in a horse model of natural IgE-mediated allergy. We developed a mAb with confirmed specificity for both recombinant and native equine IL-8 for flow cytometric analysis. Equine IL-8 was produced by CD14/MHC class II/CD16 monocytes, including a subpopulation of IgE-binding mon...
Casella S, Giannetto C, Giudice E, Marafioti S, Fazio F, Assenza A, Piccione G.Adenosine diphosphate (ADP)-induced platelet aggregation in fed and fasted horses after addition of tramadol hydrochloride was evaluated in vitro. On 10 horses citrated blood samples were collected 2h after feeding (fed animals) and 21 h after feeding (fasted animals). Final concentrations of ADP 1 and 0.5 μM, and tramadol hydrochloride (1, 15, 30, 45 and 60 min after the addition of tramadol) were used to determine the maximum degree and initial velocity of platelet aggregation. Repeated measures multifactor analysis of variance (MANOVA) was used to evaluate the effect of feeding/fasting con...
Ortega Ferrusola C, Anel-López L, Ortiz-Rodriguez JM, Martin Muñoz P, Alvarez M, de Paz P, Masot J, Redondo E, Balao da Silva C, Morrell JM....In order to gain insight of the modifications that freezing and thawing cause to the surviving population of spermatozoa, changes in the potential of the plasma membrane (Em) and intracellular Na content of stallion spermatozoa were investigated using flow cytometry. Moreover, caspase 3 activity was also investigated and the functionality of the Na -K ATPase pump was investigated before and after freezing and thawing. Cryopreservation caused a significant (p < 0.001) increase in the subpopulation of spermatozoa with depolarized sperm membranes, concomitantly with an increase (p < 0.0...
Witonsky S, Gogal RM, Buechner-Maxwell V, Ahmed SA.To determine whether immune function can be accurately assessed in blood samples obtained from horses and refrigerated overnight and whether a nonradioactive lymphocyte proliferation assay can be used to evaluate samples obtained from horses. Methods: 224 blood samples from 28 clinically normal adult horses. Methods: Heparinized blood samples were collected. Each sample was divided into 2 equal aliquots. One aliquot was refrigerated overnight to simulate overnight shipping of blood samples, and the other aliquot was evaluated on the day of blood collection. Lymphocytes were isolated and enumer...
Cheng FP, Gadella BM, Voorhout WF, Fazeli A, Bevers MM, Colenbrander B.The aim of the present study was to investigate whether the induction of stallion sperm acrosome reaction (AR) by progesterone is mediated by binding of progesterone to a receptor on the sperm plasma membrane or to an intracellular progesterone receptor. Progesterone-BSA conjugate labeled with fluorescein isothiocyanate (P-BSA-FITC) in combination with a vital stain, ethidium homodimer, was applied to visualize the presence of the progesterone receptor on living spermatozoa. Alternatively, an indirect immunofluorescence technique employing a monoclonal antibody (C-262) against human intracellu...
Treulen F, Aguila L, Arias ME, Jofré I, Felmer R.During cryopreservation procedures, the spermatozoa are exposed to physical and chemical stressors that generate an increase in the intracellular concentration of reactive oxygen species (ROS). If ROS concentrations are too great, this can lead to a state of oxidative stress that are detrimental to sperm quality. The aim of this study was to ascertain the profile the ROS production and assess the effects of post-thaw supplementation of a semen extender with different antioxidant compounds on the quality and function variables of frozen-thawed stallion spermatozoa incubated in vitro. Frozen-tha...
Witkowska-Piłaszewicz O, Pingwara R, Szczepaniak J, Winnicka A.Clenbuterol, the β2-adrenoceptor agonist, is gaining growing popularity because of its effects on weight loss (i.e., chemical liposuction). It is also popular in bodybuilding and professional sports, due to its effects that are similar to anabolic steroids. However, it is prohibited by anti-doping control. On the other hand, it is suggested that clenbuterol can inhibit the inflammatory process. The cells from 14 untrained and 14 well-trained race horses were collected after acute exercise and cultured with clenbuterol. The expressions of CD4, CD8, FoxP3, CD14, MHCII, and CD5 in PBMC, and reac...
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 (...
Al-Kass Z, Spergser J, Aurich C, Kuhl J, Schmidt K, Johannisson A, Morrell JM.Contamination of semen with bacteria arises during semen collection and handling. This bacterial contamination is typically controlled by adding antibiotics to semen extenders but intensive usage of antibiotics can lead to the development of bacterial resistance and may be detrimental to sperm quality. The objective of this study was to determine the effects of antibiotics in a semen extender on sperm quality and to investigate the effects of removal of bacteria by modified Single Layer Centrifugation (MSLC) through a colloid. Semen was collected from six adult pony stallions (three ejaculates...
Hernández-Avilés C, Ramírez-Agámez L, Varner DD, Love CC.During the fertilization process, the interaction between the sperm and the oocyte is mediated by a process known as acrosomal exocytosis (AE). Although the role of the sperm acrosome on fertilization has been studied extensively over the last 70 years, little is known about the molecular mechanisms that govern acrosomal function, particularly in species other than mice or humans. Even though subfertility due to acrosomal dysfunction is less common in large animals than in humans, the evaluation of sperm acrosomal function should be considered not only as a complementary but a routine test whe...
Mudge MC, Walker NJ, Borjesson DL, Librach F, Johns JL, Owens SD.Post-transfusion survival of allogeneic RBCs has been reported to be much shorter in horses than in other species. We hypothesized that post-transfusion survival of biotinylated allogeneic equine RBCs would be greater than the survival previously reported from studies using radioactive RBC-labeling techniques. Objective: The study objective was to determine post-transfusion survival of N-hydroxysuccinimide (NHS)-biotin-labeled allogeneic equine RBCs transfused into adult horses. Methods: Horses were adults and included 5 donors and 5 recipients. All horses were blood-typed, and donors were pai...
