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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.
Expression of Toll-like receptors 2, 3, 4, 6, 9, and MD-2 in the normal equine cornea, limbus, and conjunctiva. Human corneal cells have detectable levels of TLRs 1-10. TLRs 2 and 4 are the major corneal receptors, recognizing the PAMPs associated with fungal invasion in humans. The conjunctiva and limbus contain TLRs 2, 4, and 9. Our purpose was to determine the expression of TLRs 2, 3, 4, 6, 9, and MD-2 in the normal equine cornea, conjunctiva, and limbus. Methods: Corneal, limbal, and conjunctival tissues were collected from seven euthanized horses having no evidence of ocular disease. RNA extraction with DNase-1 digestion was performed followed by RT-PCR to determine expression of TLRs 2, 3, 4, 6, 9...
Evaluation of the in vitro effects of aqueous black walnut extract on equine mononuclear cells. To evaluate effects of black walnut extract (BWE) on equine mononuclear cells and determine whether BWE has direct proinflammatory effects. Methods: Mononuclear cells separated from blood samples from 8 horses. Methods: Aqueous BWE was prepared and processed to eliminate contamination with particulates and microbes. A Limulus amoebocyte lysate assay was used to detect lipopolysaccharide (LPS) contamination in the BWE. Mononuclear cells were incubated in minimal essential medium with or without the addition of 0.6% to 10% (vol/vol) BWE. These mononuclear cells were assessed for viability, activ...
An in vitro biomechanical comparison of equine proximal interphalangeal joint arthrodesis techniques: two parallel transarticular headless tapered variable pitch screws versus two parallel transarticular AO cortical bone screws inserted in lag fashion. To compare the mean number of cycles to failure under axial compression of equine proximal interphalangeal (PIP) joint arthrodesis constructs created by 2 parallel transarticular Acutrak Plus screws (AP-TS) or 2 parallel transarticular 5.5 mm cortical screws inserted in lag fashion (AO-TLS). Methods: Paired in vitro biomechanical testing of 2 methods of stabilizing cadaveric adult equine forelimb PIP joints. Methods: Cadaveric adult equine forelimbs (n=5 pairs). Methods: For each forelimb pair, 1 PIP joint was stabilized with AP-TS and 1 with AP-TLS. The 5 construct pairs were tested for cycli...
Induced pluripotent stem cell lines derived from equine fibroblasts. The domesticated horse represents substantial value for the related sports and recreational fields, and holds enormous potential as a model for a range of medical conditions commonly found in humans. Most notable of these are injuries to muscles, tendons, ligaments and joints. Induced pluripotent stem (iPS) cells have sparked tremendous hopes for future regenerative therapies of conditions that today are not possible to cure. Equine iPS (EiPS) cells, in addition to bringing promises to the veterinary field, open up the opportunity to utilize horses for the validation of stem cell based therapi...
Ethyl pyruvate decreases proinflammatory gene expression in lipopolysaccharide-stimulated equine monocytes. Monocytes are among the initial cells that interact with circulating LPS. Binding of LPS to monocyte surface receptors triggers an intracellular signaling cascade and results in the production of proinflammatory cytokines. Ethyl pyruvate, a stable derivative of pyruvate, has been effective in mitigating LPS induced alterations in isolated human monocytes. We hypothesized that ethyl pyruvate would suppress proinflammatory gene expression in LPS-stimulated equine monocytes without affecting cell viability. Equine monocytes were isolated from whole blood using a sediment-gradient centrifugation p...
High throughput proteomic analysis of the secretome in an explant model of articular cartilage inflammation. This study employed a targeted high-throughput proteomic approach to identify the major proteins present in the secretome of articular cartilage. Explants from equine metacarpophalangeal joints were incubated alone or with interleukin-1beta (IL-1β, 10ng/ml), with or without carprofen, a non-steroidal anti-inflammatory drug, for six days. After tryptic digestion of culture medium supernatants, resulting peptides were separated by HPLC and detected in a Bruker amaZon ion trap instrument. The five most abundant peptides in each MS scan were fragmented and the fragmentation patterns compared to m...
Influence of magnetic field on aqueous two-phase extraction of horse ferritin in the polyethylene glycol/hydroxyethyl starch system. The presented experiments show the model of expectation of equine spleen ferritin extraction in a new aqueous two-phase system which was formed by mixing polyethylene glycol (PEG) and hydroxyethyl starch (HES). The tendency of the protein to migrate in the analyzed systems was dependent on the concentrations of HES and PEG as well as PEG molecular weight. The highest concentration of ferritin in the top phase (rich in PEG) was recorded in the system composed of 6% PEG 3000 and 3% HES. The obtained concentration was 0.88 mg mL(-1). The lowest concentration was 0.42 mg mL(-1) in the system compo...
The use of in vitro technologies and high-resolution/accurate-mass LC-MS to screen for metabolites of ‘designer’ steroids in the equine. Detection of androgenic-anabolic steroid abuse in equine sports requires knowledge of the drug's metabolism in order to target appropriate metabolites, especially where urine is the matrix of choice. Studying 'designer' steroid metabolism is problematic since it is difficult to obtain ethical approval for in vivo metabolism studies due to a lack of toxicological data. In this study, the equine in vitro metabolism of eight steroids available for purchase on the Internet is reported; including androsta-1,4,6-triene-3,17-dione, 4-chloro,17α-methyl-androsta-1,4-diene-3,17β-diol, estra-4,9-diene-...
Assessing permethrin resistance in the stable fly (Diptera: Muscidae) in Florida by using laboratory selections and field evaluations. Insecticide resistance in the stable fly, Stomoxys calcitrans (L.) (Diptera: Muscidae),has been demonstrated previously, but mostly with insecticides that are no longer used, such as the organochlorines. Resistance to commonly used pyrethroids has been evaluated twice, but only in the midwestern United States. Stable fly susceptibility to a commonly used pyrethroid, permethrin, was determined in Florida to assess the possibility of resistance development. Diagnostic concentration evaluations of three stable fly field strains demonstrated a maximum of 57 and 21% survival to permethrin residues ...
In vitro effect of ventriculocordectomy before laryngoplasty on abduction of the equine arytenoid cartilage. To determine whether ventriculocordectomy (VCE) performed before prosthetic laryngoplasty (PL) results in increased rima glottidis size compared with PL alone. Methods: Experimental study. Methods: Equine cadaver larynges (n=13). Methods: Right arytenoid cartilages were maximally abducted using a standard PL technique. Standard PLs were then performed on the left side and the force required to maximally abduct the left arytenoid cartilage recorded (F(max) ). Photographs were taken of the rima glottidis at zero force and at five equal levels of force up to F(max) . The force applied was release...
Modulating effects of acepromazine on the reactive oxygen species production by stimulated equine neutrophils. To investigate the effect of acepromazine (ACP) on reactive oxygen species (ROS) production by stimulated equine neutrophils. Methods: Ex vivo biochemical experiments. Methods: Isolated neutrophils from healthy untreated horses. Methods: Neutrophils were incubated with ACP at concentrations of 10(-4), 10(-5) or 10(-6) M and then stimulated with phorbol-myristate-acetate (PMA) before measurement of lucigenin-enhanced chemiluminescence (CL). In a second experiment neutrophils were incubated in the presence of α-keto-γ methylthiobutyric acid (KMB) and treated with ACP at concentrations of 10(-4...
Comparison of the cytotoxic effects of bupivacaine, lidocaine, and mepivacaine in equine articular chondrocytes. To compare the chondrotoxicity of bupivacaine, lidocaine, and mepivacaine in equine articular chondrocytes in vitro. Methods: Prospective, experimental study. Methods: Equine articular chondrocytes. Methods: Primary cultured equine chondrocytes were exposed to 0.5% bupivacaine, 2% lidocaine, or 2% mepivacaine for 30 or 60 minutes. After treatment, cell viability was evaluated by trypan blue exclusion and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay in a dose dependent manner. Apoptosis and necrosis of chondrocytes were analyzed with the double stain...
The regenerative medicine laboratory: facilitating stem cell therapy for equine disease. This article focuses on the emerging field of equine regenerative medicine with an emphasis on the use of mesenchymal stem cells (MSCs) for orthopedic diseases. We detail laboratory procedures and protocols for tissue handling and MSC isolation, characterization, expansion, and cryopreservation from bone marrow, fat, and placental tissues. We provide an overview of current clinical uses for equine MSCs and how MSCs function to heal tissues. Current laboratory practices in equine regenerative medicine mirror those in the human field. However, the translational use of autologous and allogeneic M...
Agreement between measures of total motility and membrane integrity in stallion sperm. Increasing seminal plasma concentrations in extended stallion semen were utilized to model decreasing sperm motility over time. Level of agreement was determined between flow cytometric measurement of sperm membrane integrity, using a combination of SYBR-14 and propidium iodide, and computer-assisted analysis of sperm motility. Values for total sperm motility (TMOT;%) and membrane integrity (SMI;%) were similar (∼80%) at Time 0 within all sperm treatments. However, TMOT was lower than SMI after 24 and 48 h of storage in treatments with >20% seminal plasma. At Time 0, agreement (bias and a...
Analysis of bioactive eicosanoids in equine plasma by stable isotope dilution reversed-phase liquid chromatography/multiple reaction monitoring mass spectrometry. Oxidative metabolites of arachidonic acid (AA) are implicated in inflammation. Thus, we evaluated cycloxygenases (COXs) and lipoxygenases (LOs) mediated metabolism of AA to eicosanoids in equine plasma. Eicosanoids were extracted from plasma by two liquid-liquid extraction (LLE) steps; first was by chloroform/isopropanol and second by methyl-tert-butyl ether. For identification and quantification of 25 eicosanoids, a highly specific, selective and sensitive stable isotope dilution liquid chromatography (LC) multiple reaction monitoring (MRM) mass spectrometric (MS) method was developed. To avo...
Effect of sod (superoxide dismutase) protein supplementation in semen extenders on motility, viability, acrosome status and ERK (extracellular signal-regulated kinase) protein phosphorylation of chilled stallion spermatozoa. New studies are underway to find new methods for supporting longer storage of cooled stallion semen. It is known that high concentrations of reactive oxygen species (ROS) cause sperm pathology. The metalloprotein superoxide dismutase (SOD) is responsible for H(2)O(2) and O(2) production, by dismutation of superoxide radicals. The aim of this study is to assess the quality of chilled stallion semen processed with extenders containing SOD at different concentrations as antioxidant additives. A total of 80 ejaculates collected from 5 standardbred stallions was divided into 5 aliquots treated as: ...
Evaluation of the cyclooxygenase selectivity of robenacoxib and its effect on recovery of ischemia-injured jejunal mucosa in horses. To determine the cyclooxygenase (COX) selectivity of robenacoxib and its effect on recovery of jejunal mucosa following ischemic injury in horses. Methods: 12 healthy horses. Methods: Half the maximal inhibition (EC₅₀) of robenacoxib for COX-1 and COX-2 activity was established in bloods samples from 6 horses via measurement of thromboxane B₂ (TXB₂) and prostaglandin E₂ concentrations, respectively; COX selectivity was subsequently calculated. Six other horses were anesthetized, and ischemia was induced in the jejunum for 2 hours. Control and ischemia-injured mucosa were collected an...
Effects of preparation method, shear force, and exposure to collagen on release of growth factors from equine platelet-rich plasma. To test the hypotheses that preparation method, exposure to shear force, and exposure to collagen affect the release of growth factors from equine platelet-rich plasma (PRP). Methods: PRP obtained from 6 horses. Methods: PRP was prepared via 2 preparation methods (tube and automated) and subjected to 6 treatment conditions (resting, detergent, exposure to shear via 21- and 25-gauge needles, and exposure to collagen [10 and 20 μg/mL]). Concentrations of platelet-derived growth factor, isoform BB (PDGF-BB); transforming growth factor β, isoform 1 (TGFβ₁); and insulin-like growth factor, iso...
Phosphoinositides direct equine infectious anemia virus gag trafficking and release. Phosphatidylinositol 4,5-biphosphate [PI(4,5)P(2) ], the predominant phosphoinositide (PI) on the plasma membrane, binds the matrix (MA) protein of human immunodeficiency virus type 1 (HIV-1) and equine infectious anemia virus (EIAV) with similar affinities in vitro. Interaction with PI(4,5)P(2) is critical for HIV-1 assembly on the plasma membrane. EIAV has been shown to localize in internal compartments; hence, the significance of its interaction with PI(4,5)P(2) is unclear. We therefore investigated the binding in vitro of other PIs to EIAV MA and whether intracellular association with comp...
Preliminary safety and biological efficacy studies of ethyl pyruvate in normal mature horses. Endotoxaemia causes substantial morbidity and mortality in horses with colic and sepsis. Ethyl pyruvate is a novel anti-inflammatory medication that improved survival in preclinical models of severe sepsis endotoxaemia and intestinal ischaemia and reperfusion in rodents, swine, sheep and dogs and may be a useful medication in horses. Objective: Ethyl pyruvate has no adverse effects in normal horses and is biologically active based on suppression of proinflammatory gene expression in endotoxin stimulated whole blood, in vitro. Methods: Physical and neurological examinations, behaviour scores, e...
The pharmacokinetics and in vitro cyclooxygenase selectivity of deracoxib in horses. The purpose of this study was to determine the pharmacokinetics of deracoxib following oral administration to horses. In addition, in vitro equine whole blood cyclooxygenase (COX) selectivity assays were performed. Six healthy adult horses were administered deracoxib (2 mg/kg) orally. Plasma samples were collected prior to drug administration (time 0), and 10, 20, 40 min and 1, 1.5, 2, 4, 6, 8, 12, 24, and 48 h after administration for analysis with high pressure liquid chromatography using ultraviolet detection. Following PO administration, deracoxib had a long elimination half-life (t(...
Isolation of an equine coronavirus from adult horses with pyrogenic and enteric disease and its antigenic and genomic characterization in comparison with the NC99 strain. A new equine coronavirus was isolated from the feces of adult horses with pyrogenic and enteric disease. The disease outbreak was mainly observed among 2- to 4-year-old horses living in stables of a draft-horse racetrack in Japan. On comparing the isolated virus (isolate Tokachi09) with the equine coronavirus NC99 strain, no significant differences were observed in several biological properties such as hemagglutinating activity, antigenicity (in indirect immunofluorescence and neutralization tests), and one-step growth (in cell culture). The sequences of the nucleocapsid and spike genes of iso...
Immunological characterization of the equine airway epithelium and of a primary equine airway epithelial cell culture model. Our understanding of innate immunity within the equine respiratory tract is limited despite growing evidence for its key role in both the immediate defense and the shaping of downstream adaptive immune responses to respiratory disease. As the first interface to undergo pathogen invasion, the respiratory epithelium is a key player in these early events and our goal was to examine the innate immune characteristics of equine respiratory epithelia and compare them to an in vitro equine respiratory epithelial cell model cultured at the air-fluid interface (AFI). Respiratory epithelial tissues, isol...
Evaluation of gastric pressures as an indirect method for measurement of intraabdominal pressures in the horse. To develop an indirect method for measurement of intraabdominal pressures in the standing horse using measurement of gastric pressures as a less invasive technique, and to compare this method with direct intraabdominal pressures obtained from the peritoneal cavity. Methods: Prospective, experimental study. Methods: University-based equine research facility. Methods: Ten healthy adult horses, 7 geldings and 3 mares. Methods: Gastric pressures were measured using a nasogastric tube with a U-tube manometry technique, while intraperitoneal pressures were measured with a peritoneal cannula. Measure...
In vivo-derived horse blastocysts show transcriptional upregulation of developmentally important genes compared with in vitro-produced horse blastocysts. In vitro-produced (IVP) equine blastocysts can give rise to successful pregnancies, but their morphology and developmental rate differ from those of in vivo-derived equine blastocysts. The aim of the present study was to evaluate this difference at the genetic level. Suppression subtractive hybridisation (SSH) was used to construct a cDNA library enriched for transcripts preferentially expressed in in vivo-derived equine blastocysts compared with IVP blastocysts. Of the 62 different genes identified in this way, six genes involved in embryonic development (BEX2, FABP3, HSP90AA1, MOBKL3, MCM7 a...
Evaluation of equine peripheral blood apheresis product, bone marrow, and adipose tissue as sources of mesenchymal stem cells and their differentation potential. To evaluate effects of apheresis on mesenchymal stem cells (MSCs) and compare those MSCs with MSCs obtained from adipose tissue or bone marrow (BM). Methods: Samples obtained from 6 adult horses. Methods: Samples of blood from a peripheral vein, adipose tissue, and BM aspirate were obtained from each horse. Samples were processed via apheresis of blood and techniques reported elsewhere for adipose tissue and BM. Cultures were maintained until adherence and subsequently were subjected to differentiation protocols to evaluate adipogenic, osteoblastogenic, and chondrogenic potential. Results: Aph...
Trophoblast stem cell marker gene expression in inner cell mass-derived cells from parthenogenetic equine embryos. Although putative horse embryonic stem (ES)-like cell lines have been obtained recently from in vivo-derived embryos, it is currently not known whether it is possible to obtain ES cell (ESC) lines from somatic cell nuclear transfer (SCNT) and parthenogenetic (PA) embryos. Our aim is to establish culture conditions for the derivation of autologous ESC lines for cell therapy studies in an equine model. Our results indicate that both the use of early-stage blastocysts with a clearly visible inner cell mass (ICM) and the use of pronase to dissect the ICM allow the derivation of a higher proportion...
In vivo confocal microscopy of equine fungal keratitis. To describe in vivo corneal confocal microscopy of horses with fungal keratitis and correlate findings with clinical, histopathological, and microbiological evaluations of clinical cases and an ex vivo experimental equine fungal keratitis model. Methods: A total of 12 horses with naturally-acquired fungal keratitis and ex vivo equine corneas experimentally infected with clinical fungal isolates. Methods: Horses with naturally-acquired fungal keratitis were examined with a modified Heidelberg Retina Tomograph II and Rostock Cornea Module. Confocal microscopy images of clinical isolates of Asper...
Transcriptional profiling of equine conceptuses reveals new aspects of embryo-maternal communication in the horse. Establishment and maintenance of pregnancy are critically dependent on embryo-maternal communication during the preimplantation period. The horse is one of the few domestic species in which the conceptus-derived pregnancy recognition signal has not been identified. To gain new insights into the factors released by the equine conceptus, transcriptional profiling analyses of conceptuses retrieved 8, 10, 12, and 14 days after ovulation were performed using a whole-genome microarray. Selected array data were confirmed using quantitative PCR, and the expression of proteins of interest was confirmed...
Core decompression of the equine navicular bone: an in vitro biomechanical study. To determine the effect of core decompression surgery and bone mineral density (BMD) on the mechanical properties of equine navicular bones. Methods: Experimental, in vitro study. Methods: Fore limb navicular bones (n=36 pairs) from sound 2-5-year-old horses with no radiographic abnormalities of the distal aspect of the forelimbs. Methods: Navicular BMD was measured using dual energy X-ray absorptiometry. One randomly assigned navicular bone from each pair served as control. The contralateral test specimen was allocated to 1 of 6 treatment groups defined by drill bit size (3.2 versus 2.5 mm di...
