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Topic:In Vitro Research
In vitro research involving horses refers to the study of equine cells, tissues, or biological molecules outside their normal biological context, typically in controlled laboratory environments. This research approach allows scientists to investigate cellular processes, molecular interactions, and the effects of various treatments without the ethical and logistical complexities of in vivo studies. In vitro studies contribute to understanding equine physiology, pathology, and pharmacology by providing insights into cellular responses to pathogens, drugs, and other stimuli. This page compiles peer-reviewed research studies and scholarly articles that explore various in vitro methodologies and their applications in equine science, including cell culture techniques, molecular assays, and drug efficacy testing.
Lactobacillus equigenerosi strain Le1 invades equine epithelial cells. Lactobacillus equigenerosi strain Le1, a natural inhabitant of the equine gastrointestinal tract, survived pH 3.0 and incubation in the presence of 1.5% (wt/vol) bile salts for at least 2 h. Strain Le1 showed 8% cell surface hydrophobicity, 60% auto-aggregation, and 47% coaggregation with Clostridium difficile C6. Only 1% of the cells adhered to viable buccal epithelial cells and invaded the cells within 20 min after contact. Preincubation of strain Le1 in a buffer containing pronase prevented adhesion to viable epithelial cells. Preincubation in a pepsin buffer delayed invasion from 20 min to...
Freezing or adding trypsin inhibitor to equine intestinal contents extends the lifespan of Clostridium perfringens beta toxin for diagnostic purposes. Clostridium perfringens type C causes necrotizing enteritis mostly in neonatal animals of several species, including horses. The virulence of C. perfringens type C is mostly mediated by beta toxin (CPB). This toxin is highly sensitive to the action of trypsin and other proteases, which explains the increased susceptibility of neonatal animals to type C infections. Final confirmation of type C disease diagnosis should be based on detection of CPB in the intestinal content of affected animals. However, because CPB is so sensitive to the action of proteases, it is believed that this toxin persist...
Restoration of seminal plasma to stallion spermatozoa selected by colloid centrifugation increases sperm progressive motility but is detrimental to chromatin integrity. There is controversy about whether the presence of some seminal plasma (SP) in an equine insemination dose is necessary for promoting fertility. A new technique for improving stallion sperm quality, single layer centrifugation (SLC) using a species-specific colloid, Androcoll-E, selects a sperm subpopulation that is highly motile with normal morphology, intact membranes and good chromatin integrity from the rest of the ejaculate and removes SP. The present study was designed to investigate the effect of restoring homologous SP (5% and 10%) on the progressive motility, velocity, and chromatin i...
Comparison of the efficacy of alpha-lactalbumin from equine, bovine, and human milk in the growth of intestinal IEC-6 cells. Native alpha-lactalbumins (α-LA) from equine, bovine, and human milk were not cytotoxic. However, after treatment with trifluoroethanol (TFE), all three α-LAs exhibited cytotoxicity. Toxic potencies were distinctly different among them. Equine α-LA was the most robust, bovine α-LA was moderate, and human α-LA was weak. There were no significant structural changes as between the native and the TFE-treated α-LAs.
Androcoll-E large selects a subset of live stallion spermatozoa capable of producing ROS. The aim of this study was to elucidate if SLC after 24 h storage selects the subpopulation of spermatozoa that better withstands osmotic shock. To test this hypothesis, viability, mitochondrial membrane potential (MMP) and superoxide anion (O(2)(·-)) production of uncentrifuged (UC) and single layer centrifugation (SLC) - selected spermatozoa were analyzed following SLC after storage of the semen. An aliquot of the extended ejaculate (100×10(6) spermatozoa/mL) was centrifuged through a single layer of a silane-coated silica based colloid formulation optimized for equine spermatozoa (Androcol...
Diffusion of contrast medium after four different techniques for analgesia of the proximal metacarpal region: an in vivo and in vitro study. There is limited information on potential diffusion of local anaesthetic solution after various diagnostic analgesic techniques of the proximal metacarpal region. Objective: To document potential distribution of local anaesthetic solution following 4 techniques used for diagnostic analgesia of the proximal metacarpal region. Methods: Radiodense contrast medium was injected around the lateral palmar or medial and lateral palmar metacarpal nerves in 8 mature horses, using 4 different techniques. Radiographs were obtained 0, 10 and 20 min after injection and were analysed subjectively. A mixture ...
Antioxidant effect of different vitamins on methemoglobin production: An in vitro study. Nitrite intoxication occurs frequently in ruminants and equines. The most common treatment of this disorder is administration of 1% methylene blue, although the use of some antioxidant agents e.g. vitamins and complementary treatment may also be useful. The aim of this study was to evaluate the in vitro antioxidative effects of some vitamins on methemoglobinemia induced by sodium nitrite. For this purpose the blood sample of a healthy dairy cattle was pre-incubated with three different concentrations (5, 10, 20 mmol L(-1)) of each vitamin (E, C, B1, A and a combination of vitamin E and vitamin...
Post-thaw addition of seminal plasma reduces tyrosine phosphorylation on the surface of cryopreserved equine sperm, but does not reduce lipid peroxidation. The objective was to verify the relationship between equine semen cryopreservation and changes related to increased lipid peroxidation. Also, addition of autologous or homologous seminal plasma from a stallion with a good freezing response to post-thawed sperm was tested to determine whether it would confer protection. Frozen-thawed sperm were evaluated and allocated into three groups: without plasma addition, and supplemented with either homologous or autologous seminal plasma. All groups were evaluated at 0, 60 and 120 min after incubation at 37 °C. Cryopreservation did not increase plasma ...
Towards an inhalative in vivo application of immunomodulating gelatin nanoparticles in horse-related preformulation studies. Delivering active ingredients using biocompatible and biodegradable carriers such as gelatin nanoparticles (GNPs) to the lung constitutes a promising non-invasive route of administration. However, the pulmonary delivery of nanoparticle-based immunotherapy is still a field that requires more clarification. In this study, GNPs loaded with cytosine-phosphate-guanine oligodeoxynucleotides (CpG-ODN)-loaded and plain GNPs were aerosolised either by a conventional pressured metered dose inhaler (pMDI) or by active or passive vibrating-mesh (VM) nebulisers. GNP sizes after nebulisation by active and p...
Pyrethroid tolerance in the chewing louse Bovicola (Werneckiella) ocellatus. Equine pediculosis is a significant health and welfare issue, particularly in elderly and chronically debilitated animals. Currently infestation is controlled predominantly using topically applied pyrethroid insecticides, allowing limited scope for the rotation of drugs and increasing the risk of selection for resistance. Here the insecticidal efficacies of two pyrethroid-based products against the louse Bovicola (Werneckiella) ocellatus collected from donkeys were examined in vitro. The products were cypermethrin (Deosect™, Pfizer Ltd., 5% (w/v) cypermethrin, cutaneous spray) and permethrin...
Elimination half-life of intravenously administered equine cardiac troponin I in healthy ponies. To date, no information is available on the true biological elimination half-life (T(1/2) ) of cardiac troponin I (cTnI) in the equine species. Such data are required to better evaluate the optimal time to acquire the cTnI sample following acute myocardial injury. Objective: To determine the T(1/2) of equine cTnI. Methods: Four healthy ponies received i.v. injections of recombinant equine cTnI. Plasma cTnI concentrations were measured with a point-of-care cTnI analyser at multiple time points after injection. Standard pharmacokinetic analysis was performed to establish the T(1/2) of cTnI. Resu...
Induction of pluripotency in adult equine fibroblasts without c-MYC. Despite tremendous efforts on isolation of pluripotent equine embryonic stem (ES) cells, to date there are few reports about successful isolation of ESCs and no report of in vivo differentiation of this important companion species. We report the induction of pluripotency in adult equine fibroblasts via retroviral transduction with three transcription factors using OCT4, SOX2, and KLF4 in the absence of c-MYC. The cell lines were maintained beyond 27 passages (more than 11 months) and characterized. The equine iPS (EiPS) cells stained positive for alkaline phosphatase by histochemical staining ...
Cell-secreted vesicles in equine ovarian follicular fluid contain miRNAs and proteins: a possible new form of cell communication within the ovarian follicle. Proper cell communication within the ovarian follicle is critical for the growth and maturation of a healthy oocyte that can be fertilized and develop into an embryo. Cell communication within the follicle involves many signaling molecules and is affected by maternal age. Recent studies indicate that cell communication can be mediated through secretion and uptake of small membrane-enclosed vesicles. The goals of this study were to 1) identify cell-secreted vesicles (microvesicles and exosomes) containing miRNAs and proteins within ovarian follicular fluid and 2) determine if miRNA level differ...
Expression of late viral proteins is restricted in nasal mucosal leucocytes but not in epithelial cells during early-stage equine herpes virus-1 infection. Equine herpes virus (EHV)-1 replicates in the epithelial cells of the upper respiratory tract and reaches the lamina propria and bloodstream in infected mononuclear cells. This study evaluated expression of the late viral proteins gB, gC, gD and gM in respiratory epithelial and mononuclear cells using: (1) epithelial-like rabbit kidney cells and peripheral blood mononuclear cells infected with EHV-1 in vitro; (2) an equine ex vivo nasal explant system; and (3) nasal mucosa tissue of ponies infected in vivo. The viral proteins were expressed in all late-infected epithelial cells, whereas expres...
Proliferation of equine bone marrow-derived mesenchymal stem cells in gelatin/β-tricalcium phosphate sponges. A three dimensional scaffold is essential in mesenchymal stem cells (MSCs) delivery in cell-based therapy for facilitating cell adherence, migration, proliferation, and differentiation. The objectives of this study were to evaluate the possibility of β-tricalcium phosphate incorporated gelatin sponges (Gelatin/β-TCP sponge) as scaffolds for equine MSCs and to examine the effects of seeding density and seeding method on the proliferation of equine MSCs in the Gelatin/β-TCP sponges. Mononuclear cells and MSCs isolated from bone marrow were seeded into Gelatin/β-TCP sponges at different densi...
Effects of components of semen extenders on the binding of stallion spermatozoa to bovine or equine zonae pellucidae. The effects of semen extender components on the ability of stallion sperm to bind to the zona pellucida (ZP) and the suitability of using bovine ZP for a ZP-binding assay for stallion sperm were investigated in a series of experiments. In Experiment I, binding of stallion sperm to both bovine and equine ZP was significantly increased when a skim milk-based extender (EZM) was used. In Experiment II, a threefold increase in sperm binding to ZP was observed when sperm were diluted in EZM compared with diluents, which contained no milk (TALP, LAC, and EmCare). In Experiment III, centrifuging the s...
In search for cross-reactivity to immunophenotype equine mesenchymal stromal cells by multicolor flow cytometry. During recent years, cell-based therapies using mesenchymal stem cells (MSC) are reported in equine veterinary medicine with increasing frequency. In most cases, the isolation and in vitro differentiation of equine MSC are described, but their proper immunophenotypic characterization is rarely performed. The lack of a single marker specific for MSC and the limited availability of monoclonal antibodies (mAbs) for equine MSC in particular, strongly hamper this research. In this study, 30 commercial mAbs were screened with flow cytometry for recognizing equine epitopes using the appropriate posit...
Dimethylformamide improves the in vitro characteristics of thawed stallion spermatozoa reducing sublethal damage. 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...
Assessment of reactive oxygen species production in cultured equine skeletal myoblasts in response to conditions of anoxia followed by reoxygenation with or without exposure to peroxidases. To culture equine myoblasts from muscle microbiopsy specimens, examine myoblast production of reactive oxygen species (ROS) in conditions of anoxia followed by reoxygenation, and assess the effects of horseradish peroxidase (HRP) and myeloperoxidase (MPO) on ROS production. Methods: 5 healthy horses (5 to 15 years old). Methods: Equine skeletal myoblast cultures were derived from 1 or 2 microbiopsy specimens obtained from a triceps brachii muscle of each horse. Cultured myoblasts were exposed to conditions of anoxia followed by reoxygenation or to conditions of normoxia (control cells). Cell p...
Evaluation of the automated hematology analyzer Sysmex XT-2000iV ™ compared to the ADVIA ® 2120 for its use in dogs, cats, and horses. Part II: Accuracy of leukocyte differential and reticulocyte count, impact of anticoagulant and sample aging. The automated laser-based hematology analyzer Sysmex XT-2000iV™ provides a 5-part differential count and specific cytograms that are of great interest for large veterinary laboratories. The aim of the study was to validate the Sysmex XT-2000iV compared to the laser-based hematology analyzer ADVIA® 2120 and manual differential in dogs, cats, and horses as well as the impact of anticoagulant (heparin, ethylenediamine tetra-acetic acid [EDTA], and citrate) and storage at 22°C and 4°C. Consecutive fresh K(3)-EDTA blood samples from 216 cats, 314 dogs, and 174 horses were included. The impact ...
Effect of environmental conditions on degree of hoof wall hydration in horses. To determine the effect of various environmental conditions on the degree of hydration in hoof wall horn tissue from feral horses and investigate the effect of short-term foot soaking on moisture content in hoof wall and sole tissue in domestic horses. Methods: 40 feral horses from 3 environments (wet and boggy [n = 10], partially flooded [20], and constantly dry desert [10]) and 6 nonferal Quarter Horses. Methods: The percentage of moisture content of hoof wall samples from feral horses was measured in vitro. In a separate evaluation, the percentage of moisture content of hoof wall and sole t...
Contamination of joints with tissue debris and hair after arthrocentesis: the effect of needle insertion angle, spinal needle gauge, and insertion of spinal needles with and without a stylet. To assess fetlock joint contamination with tissue debris and hair after arthrocentesis. Methods: Experimental. Methods: Fetlock joint tissues (n = 10 horses). Methods: Soft tissue flaps including the joint capsule were dissected from the dorsal fetlock joints of 7 anesthetized horses leaving an intact proximal base. Needles inserted through the tissue flaps were flushed into tissue cell culture plates and examined for debris. Studies were repeated on excised fetlock tissue preparations after being stored for 5 days. Variables included gauge and type of needle, insertion of spinal needles with ...
Generation and characterization of monoclonal antibodies to equine CD16. The low-affinity Fc receptor CD16 plays a central role in the inflammatory and innate immune responses of many species, but has not yet been investigated in the horse. Using the predicted extracellular region of equine CD16 expressed as a recombinant fusion protein with equine IL-4 (rIL-4/CD16), we generated a panel of mouse monoclonal antibodies (mAbs) that recognize equine CD16. Nine mAbs were chosen for characterization based upon recognition of CD16, but not IL-4, in ELISA. All nine mAbs recognized full-length, cell-surface CD16 expressed as a GFP fusion protein by CHO cells, but not the c...
Macrophage sub-populations and the lipoxin A4 receptor implicate active inflammation during equine tendon repair. Macrophages (Mφ) orchestrate inflammatory and reparatory processes in injured connective tissues but their role during different phases of tendon healing is not known. We investigated the contribution of different Mφ subsets in an equine model of naturally occurring tendon injury. Post mortem tissues were harvested from normal (uninjured), sub-acute (3-6 weeks post injury) and chronically injured (>3 months post injury) superficial digital flexor tendons. To determine if inflammation was present in injured tendons, Mφ sub-populations were quantified based on surface antigen expression of...
Chronic laminitis is associated with potential bacterial pathogens in the laminae. A common sequella of chronic laminitis in horses is repeated abscesses with variable lameness and drainage. It is unclear whether the exudate represents the debridement phase of a non-septic inflammatory process involving clearance of laminar tissue damaged during the acute episode of laminitis, or a response to a microbial infection developed by ascent of microbes from the environment to the tissue via the white line. The objective of this study was to evaluate the possibility that an undiagnosed microbial infection in laminar tissue is present in laminar tissue collected from chronically lam...
Effects of cell storage and passage on basal and oxytocin-regulated prostaglandin secretion by equine endometrial epithelial and stromal cells. Cell cultures are useful for determining the responses of specific cell types to various factors under controlled conditions and for obtaining a better understanding of in vivo physiologic processes. The aims of the present study were (i) to establish methodologies for isolation, culture and cryopreservation of equine endometrial epithelial and stromal cells; and (ii) to determine the effect of passage and cryopreservation on endometrial cell physiology, based on their basal and oxytocin (OT)-stimulated prostaglandin (PG) release. Epithelial and stromal cells were obtained by enzymatic digesti...
Monoclonal antibodies to equine CD23 identify the low-affinity receptor for IgE on subpopulations of IgM+ and IgG1+ B-cells in horses. CD23, also called FcεRII, is the low-affinity receptor for IgE and has first been described as a major receptor regulating IgE responses. In addition, CD23 also binds to CD21, integrins and MHC class II molecules and thus has a much wider functional role in immune regulation ranging from involvement in antigen-presentation to multiple cytokine-like functions of soluble CD23. The role of CD23 during immune responses of the horse is less well understood. Here, we expressed equine CD23 in mammalian cells using a novel IL-4 expression system. Expression resulted in high yield of recombinant IL-4/...
Cytokine production and proliferation upon in vitro oligodeoxyribonucleotide stimulation of equine peripheral blood mononuclear cells. Synthetic oligodeoxyribonucleotides (ODN) may prove useful immune modulators in equine medicine. It is however important to assess the effects of each specific ODN in the species it is intended to be used in. The present study therefore aimed to evaluate some ODN for induction of cytokine production; i.e. type I interferons (IFN), IFN-γ, tumor necrosis factor-α (TNF-α) and transforming growth factor-β (TGF-β), and proliferation of equine peripheral blood mononuclear cells (PBMC). A panel of four ODN containing unmethylated cytosine-guanosine sequences (CpG) was used: ODN 1 and ODN 8 repre...
Equine peripheral blood-derived mesenchymal stem cells: isolation, identification, trilineage differentiation and effect of hyperbaric oxygen treatment. Two studies report variability in proliferation and limited adipocyte differentiation of equine peripheral blood-derived adult mesenchymal stem cells, thus casting doubt on their adipogenic potential. Peripheral blood can be a valuable source of adult mesenchymal stem cells if cell culture conditions permissive for their adherence, proliferation and differentiation are defined. Hyperbaric oxygen treatment has been reported to mobilise haematopoietic progenitor stem cells into the peripheral blood in humans and mice, but similar experiments have not been done in horses. Objective: To optimise c...
Toxicity of glycerol for the stallion spermatozoa: effects on membrane integrity and cytoskeleton, lipid peroxidation and mitochondrial membrane potential. Glycerol is, to date, the most widely used cryoprotectant to freeze stallion spermatozoa at concentrations between 2% and 5%. Cryoprotectant toxicity has been claimed to be the single most limiting factor for the success of cryopreservation. In order to evaluate the toxic effects of the concentrations of glycerol used in practice, stallion spermatozoa were incubated in Biggers Whitten and Whittingham (BWW) media supplemented with 0%, 0.5%, 1.5%, 2.5%, 3.5%, and 5% glycerol. In two additional experiments, a hyposmotic (75 mOsm/kg) and a hyperosmotic (900 mOsm/kg) control media were included. Sp...
