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Topic:Flow Cytometry
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.
Lymphocyte immunophenotyping and concentration of MMP-9 in transudates and exudates in horses. This study is the first to provide information on the lymphocyte subpopulations in peritoneal effusions in horses. Peritoneal transudates (n = 12), peritoneal exudates (n = 6) and a pleural exudate (n = 1) were analyzed. The total nucleated cell count (TNCC), total protein (TP) and matrix metalloproteinase-9 (MMP-9) concentration determined by ELISA were measured and routine cytological evaluation was performed. CD3, CD4, CD8 and CD21 positive cells were detected by flow cytometry. A higher percentage of neutrophils (P < 0.05) and higher MMP-9 (P < 0.01) levels were found in ex...
Heterologous Seminal Plasma Reduces the Intracellular Calcium and Sperm Viability of Cryopreserved Stallion Spermatozoa. Despite the vital role of seminal plasma (SP) in maintaining sperm function and aiding gamete interaction in many species, SP is usually removed before cryopreservation of stallion sperm to improve cryosurvival of sperm. The present study assessed if the vital sperm functional parameters of genetically superior stallions producing poor quality semen can be enhanced by the supplementation of heterologous SP from the stallion producing high quality semen. Spermatozoa from poor quality semen producing stallions were divided into three aliquots: two aliquots were supplemented with SP obtained from...
Equine bronchial epithelial cells are susceptible to cell entry with a SARS-CoV-2 pseudovirus but reveal low replication efficiency. To examine the susceptibility of cultured primary equine bronchial epithelial cells (EBECs) to a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pseudovirus relative to human bronchial epithelial cells (HBECs). Methods: Primary EBEC cultures established from healthy adult horses and commercially sourced human bronchial epithelial cells (HBECs) were used as a positive control. Methods: Angiotensin-converting enzyme 2 (ACE2) expression by EBECs was demonstrated using immunofluorescence, western immunoblot, and flow cytometry. EBECs were transduced with a lentivirus pseudotyped with ...
A distinct variant of the SzM protein of Streptococcus equi subsp. zooepidemicus recruits C1q independent of IgG binding and inhibits activation of the classical complement pathway. Streptococcus equi subsp. zooepidemicus (SEZ) is a major equine pathogen that causes pneumonia, abortion, and polyarthritis. It can also cause invasive infections in humans. SEZ expresses the M-like protein SzM, which recruits host proteins such as fibrinogen to the bacterial surface. Equine SEZ strain C2, which binds only comparably low amounts of human fibrinogen in comparison to human SEZ strain C33, was previously shown to proliferate in equine and human blood. As the expression of SzM_C2 was necessary for survival in blood, this study investigated the working hypothesis that SzM_C2 inhibi...
Lactate as the sole energy substrate induces spontaneous acrosome reaction in viable stallion spermatozoa. Equine spermatozoa appear to differ from spermatozoa of other species in using oxidative phosphorylation preferentially over glycolysis. However, there is little information regarding effects of different energy sources on measured parameters in equine spermatozoa. Objective: To determine the effect of three individual energy substrates, glucose, pyruvate, and lactate, on motion characteristics, membrane integrity, and acrosomal status of stallion spermatozoa. Methods: Freshly ejaculated stallion spermatozoa were incubated with combinations of glucose (5 mm), pyruvate (10 mm), and lactate (1...
Concurrent chronic lymphocytic leukemia and primary hyperparathyroidism in a mule. A 26-year-old mule gelding was evaluated for chronic weight loss and decreased appetite. The mule had been losing weight and intermittently hypophagic for approximately 7 months. Laboratory analysis of whole blood and plasma identified severe total hypercalcemia, marked hypophosphatemia, markedly increased parathyroid hormone concentration, and marked lymphocytosis. A sestimibi scan intended to identify parathyroid gland tissue was nondiagnostic. Results of flow cytometry and PCR for antigen receptor rearrangement (PARR) were consistent with a B cell lymphoproliferative disorder, likely chro...
Effect of gentamicin on CD3+ T-lymphocyte proliferation for treatment of equine recurrent uveitis: An in vitro study. The objective of the study was to determine the effect of gentamicin on CD3+ T-lymphocyte proliferation and cell viability using an in vitro cell culture model as a means of investigating the mechanism of action of low-dose intravitreal gentamicin injection. Methods: Three adult horses with no evidence of ophthalmic or systemic disease. Methods: Peripheral blood lymphocytes were treated with gentamicin at concentrations 37.5 μg/mL, 112.5 μg/mL, 187 μg/mL, 375 μg/mL, or 750 μg/mL then stimulated to proliferate with concanavalin A (ConA). 4',6-diamidino-2-phenylindole (DAPI) and c...
Equine alveolar macrophages and monocyte-derived macrophages respond differently to an inflammatory stimulus. Alveolar macrophages (AMs) are the predominant innate immune cell in the distal respiratory tract. During inflammatory responses, AMs may be supplemented by blood monocytes, which differentiate into monocyte-derived macrophages (MDMs). Macrophages play important roles in a variety of common equine lower airway diseases, including severe equine asthma (SEA). In an experimental model, an inhaled mixture of Aspergillus fumigatus spores, lipopolysaccharide, and silica microspheres (FLS), induced SEA exacerbation in susceptible horses. However, whether equine AMs and MDMs have differing immunopheno...
Isolation and cultivation as well as in situ identification of MSCs from equine dental pulp and periodontal ligament. The lifelong eruption places a great demand on the dental pulp and periodontal ligament (PDL) of horse teeth. Cells within the pulp and PDL seem to play a key role during this remodeling. Unassigned: In this study, we isolated and cultivated MSCs (medicinal signaling cells) from dental pulp, PDL and retrobulbar fat of four horses. Subsequently, we analyzed them by flow cytometry and immunohistochemistry to determine and compare their characteristics. In addition, we localized these cells within the tissue structure via immunohistochemistry of histological sections. For these analyses, several ...
Seasonality does not influence cortisol or testosterone production, or seminal quality of stallions located at low latitudes. The effects of seasonality on the reproduction of stallions vary based on the latitude. Although previous studies have shown the influence of seasonality in raw semen quality in south-eastern Brazil, data regarding the influence of seasonality in cooled and frozen stored semen in Brazil is limited. Therefore, in this study, we have analysed if seasonality influences the hormone production (i.e., cortisol and testosterone), spermatogenesis, and quality of fresh, cooled, and frozen semen of stallions in central Brazil, and established the season most suitable for semen cryopreservation in a lati...
Deciphering sperm chromatin properties to predict stallion sperm fertility. 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...
T Helper 17-Like Regulatory T Cells in Equine Synovial Fluid Are Associated With Disease Severity of Naturally Occurring Posttraumatic Osteoarthritis. Infiltration of cluster of differentiation (CD) 3 (CD3) T cells into the synovium and synovial fluid occurs in most patients with posttraumatic osteoarthritis. During disease progression, proinflammatory T helper 17 cells and anti-inflammatory regulatory T cells infiltrate the joint in response to inflammation. This study aimed to characterize the dynamics of regulatory T and T helper 17 cell populations in synovial fluid from equine clinical patients with posttraumatic osteoarthritis to determine whether phenotype and function are associated with potential immunotherapeutic targets. An imbala...
IgE+ plasmablasts predict the onset of clinical allergy. IgE+ plasmablasts develop following allergen exposure and B cell activation. They secrete IgE and therefore are directly linked to maintain the mechanisms of IgE-mediated allergies. Here, we show that the presence of IgE+ plasmablasts in peripheral blood not only coincides with clinical allergy, but also predicts the upcoming development of clinical disease. Using an equine model of naturally occurring allergy, we compared the timing of allergen exposure, arrival of IgE+ plasmablasts in peripheral blood, and onset of clinical disease. We found that IgE+ plasmablasts predict the development of ...
Buprenorphine has a concentration-dependent cytotoxic effect on equine chondrocytes in vitro. To investigate the cytotoxic effects of 2 different concentrations of buprenorphine and compare them with bupivacaine and morphine on healthy equine chondrocytes in vitro. Methods: Primary cultured equine articular chondrocytes from 3 healthy adult horses. Methods: Chondrocytes were exposed for 0 and 2 hours to the following treatments: media (CON; negative control); bupivacaine at 2.2 mg/mL (BUPI; positive control); morphine at 2.85 mg/mL (MOR); buprenorphine at 0.12 mg/mL (HBUPRE); or buprenorphine at 0.05 mg/mL (LBUPRE). Chondrocyte viability was assessed using live/dead staining, water-sol...
The effects of cannabidiol on immune function and health parameters in senior horses. Cannabidiol (CBD) has potential to reduce pain and inflammation in humans leading to the interest of use in equine. The purpose of this study was to determine the effects of CBD on immune function by measuring inflammatory cytokines and antibody responses to vaccination, as well as other health parameters in senior horses. Horses were orally-dosed with CBD (2 mg/kg: 13 horses) or control (soy oil: 14 horses) daily for 90 days, from July 2021 to November 2021. Peripheral blood samples were collected on days 0, 30, 60, and 90 before administering treatments. On day 90 all horses were kept on tr...
Immunogenicity analysis of BPV-1 positive equine sarcoid-derived cultured fibroblasts. Sarcoids are the most common equine skin tumours Although they do not metastasize, they can be locally aggressive and cause significant clinical symptoms in affected horses. Despite being common, very little is known about the host immune response and the biological mechanisms underlying persistence and recurrence of equine sarcoids. The latter reflects the need for further research in this field. This in-vitro study used sarcoid explants from horses with naturally occurring sarcoids (n = 12) to evaluate the induction of a humoral immune response directed against equine sarcoid-derived bovin...
The Autophagy Marker LC3 Is Processed during the Sperm Capacitation and the Acrosome Reaction and Translocates to the Acrosome Where It Colocalizes with the Acrosomal Membranes in Horse Spermatozoa. Despite its importance in somatic cells and during spermatogenesis, little is known about the role that autophagy may play in ejaculated spermatozoa. Our aim was to investigate whether the molecular components of autophagy, such as microtubule-associated protein 1 light chain 3 (LC3), are activated in stallion spermatozoa during the capacitation and acrosome reaction and if this activation could modulate these biological processes. To analyze the autophagy turnover, LC3I and LC3II proteins were assessed by western blotting, and the ratio between both proteins (LC3II/LC3I) was calculated. In so...
Binding of Equine Seminal Lactoferrin/Superoxide Dismutase (SOD-3) Complex Is Biased towards Dead Spermatozoa. Sperm-neutrophil binding is an important facet of breeding and significantly impacts fertility. While a specific seminal plasma protein has been found to reduce this binding and improve fertility (CRISP-3), additional molecule(s) appear to promote binding between defective sperm and neutrophils. Recent work has suggested one of these proteins is lactoferrin (LF), an 80 kDa iron-binding protein found throughout the body, but the purity of the protein was not confirmed. It is unknown if LF binds to sperm selectively based on viability, and if receptors for LF are located on equine sperm. To eval...
In vitro aging of stallion spermatozoa during prolonged storage at 5°C. Artificial insemination with chilled stallion semen is hampered by a limited period of maximum fertility maintenance (24-48 h). This study used multiparametric flow cytometry to simultaneously measure reactive oxygen species (ROS) production, mitochondrial function or [Ca ] and plasma membrane fluidity in viable, acrosome-intact spermatozoa, with the aim of providing insight into changes in sperm function during storage at 5°C. High proportions of viable and acrosome-intact spermatozoa (71 ± 8%) remained after 96 h of storage demonstrating that the basic integrity of the cells was we...
Populations of NK Cells and Regulatory T Cells in the Endometrium of Cycling Mares-A Preliminary Study. Endometrial immune cells are essential to support uterine functions across the estrous cycle and in preparation for pregnancy. It has been acknowledged that changes in phenotype and/or numbers of lymphocytes, such as regulatory T cells (Tregs) and NK cells, might result in lower fertility in women and mice. Little is known about equine endometrial immune cells across the estrous cycle. Here, we compared the populations of endometrial Tregs and NK cells in estrus and diestrus in mares. Endometrial biopsy and blood samples were taken in estrus and diestrus from 11 mares ages 4-12 years. Flow cyt...
The stallion sperm acrosome: Considerations from a research and clinical perspective. 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...
Flow cytometry analysis of CD11c-positive peripheral blood mononuclear cells in horses. Horses have played a prominent role in shaping our modern world, with important effects on health. Unfortunately, better characterization of the horse immune system is still needed. In this report, using flow cytometry techniques, four monoclonal antibodies against horse CD11c integrin were characterized and described for their ability to provide a positive recognition signal in peripheral blood mononuclear cells. Further immune cell phenotype experiments were performed using MHC-II, CD14, TLR4 and the specific anti-horse CD11c monoclonal antibody (1C4). With this staining panel, it was possib...
A 5-color flow cytometry panel to assess plasma membrane integrity, acrosomal status, membrane lipid organization and mitochondrial activity of boar and stallion spermatozoa following liquid semen storage. For a more practically applicable analysis of different sperm characteristics, this study aimed to develop a 5-color flow cytometry (FC) panel to concurrently analyze four sperm parameters in liquid boar and stallion semen, using also a DNA-marker for selecting sperm cell events. From each of thirty extended boar semen doses and twelve stallion semen doses, six aliquots were taken. For evaluating mitochondrial activity (A), degree of lipid disorder of plasma membrane (B), integrity of plasma membrane (C), acrosomal status (D) and marking DNA (E), five aliquots were individually stained with Rh...
Imaging flow cytometry to characterize the relationship between abnormal sperm morphologies and reactive oxygen species in stallion sperm. 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...
Comparison of Sources and Methods for the Isolation of Equine Adipose Tissue-Derived Stromal/Stem Cells and Preliminary Results on Their Reaction to Incubation with 5-Azacytidine. Physiological particularities of the equine heart justify the development of an in vitro model suitable for investigations of the species-specific equine cardiac electrophysiology. Adipose tissue-derived stromal/stem cells (ASCs) could be a promising starting point from which to develop such a cardiomyocyte (CM)-like cell model. Therefore, we compared abdominal, retrobulbar, and subcutaneous adipose tissue as sources for the isolation of ASCs applying two isolation methods: the collagenase digestion and direct explant culture. Abdominal adipose tissue was most suitable for the isolation of ASC...
Retrograde Flushing Followed by Slicing Float-Up as an Approach to Optimize Epididymal Sperm Recovery for the Purpose of Cryopreservation in Equids. This study aimed to assess the parameters of epididymal sperm harvested by retrograde flushing (RF) followed by slicing float-up (SF). Epididymides from donkeys (n = 18) and horses (n = 28) were subjected to RF with a freezing extender and then SF technique. The retrieved sperm after RF and SF was evaluated for volume, concentration, and total sperm and then cryopreserved separately. Post-thaw total motility (TM) and progressive motility (PM) were evaluated with CASA. Sperm membrane integrity (SMI) and mitochondrial membrane potential (MMP) were assessed with flow cytometry. Sperm concentratio...
Effect of aging on monocyte phagocytic and inflammatory functions, and on the ex vivo inflammatory responses to lipopolysaccharide, in horses. 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...
Comprehensive Flow Cytometric Characterization of Bronchoalveolar Lavage Cells Indicates Comparable Phenotypes Between Asthmatic and Healthy Horses But Functional Lymphocyte Differences. Equine asthma (EA) is a highly relevant disease, estimated to affect up to 20% of all horses, and compares to human asthma. The pathogenesis of EA is most likely immune-mediated, yet incompletely understood. To study the immune response in the affected lower airways, mixed leukocytes were acquired through bronchoalveolar lavage (BAL) and the cell populations were analyzed on a single-cell basis by flow cytometry (FC). Samples of 38 horses grouped as respiratory healthy or affected by mild to moderate (mEA) or severe EA (sEA) according to their history, clinical signs, and BAL cytology were ana...
Feeding a Saccharomyces cerevisiae Fermentation Product (Olimond BB) Does Not Alter the Fecal Microbiota of Thoroughbred Racehorses. Feed supplements such as fermentation products (SCFP) alter immune responses in horses. The purpose of this study was to analyze whether a prebiotic activity of the SCFP alters the gut microbiome in horses. Racehorses were fed either SCFP (Olimond BB, OLI, = 6) or placebo pellets (PLA, = 5) for 43 days. Fecal microbiota analysis was performed using 16S rRNA gene sequencing. The numbers and function of circulating immune cell subpopulations were analyzed by flow cytometry. SCFP supplementation resulted in non-consistent differences in fecal microbiota between the PLA and OLI during the feedi...
Extracellular vesicles from follicular fluid may improve the nuclear maturation rate of in vitro matured mare oocytes. The in vitro maturation (IVM) of equine oocytes is still not efficient and does not yield consistent results. The specific requirements of equine oocytes during this process are still largely unknown, which hinders the development of assisted reproductive techniques (ART) in this species. Because the ovarian follicle microenvironment supports oocytes in their acquisition of developmental competence, follicular fluid seems to be a substantial source of bioactive factors that could support the IVM process. Extracellular vesicles (EVs) are cell-secreted molecules in body fluids that are able to ...
