Omneity® Pellets
All-In-One Vitamin & Mineral Pellet
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.
Cloning and pharmacological characterization of the equine adenosine A3 receptor. The aim of this study was to establish a heterologous expression system for the equine adenosine A(3) receptor (eA(3)-R) in an effort to ascertain its pharmacologic profile. Initially, radioligand binding assays identified clones expressing the eA(3)-R in human embryonic kidney cells (HEK) based on the specific binding of [(125)I]AB-MECA. Subsequently, adenylate cyclase assays were utilized to demonstrate functional coupling of the eA(3)-R to the G-protein/adenylate cyclase system. Equilibrium competition binding assays were then performed using selective and non-selective A(3) agonists and an...
In vitro inhibition of matrix metalloproteinase activity in tracheal epithelial lining fluid from horses with recurrent airway obstruction. To evaluate inhibitory effects of synthetic matrix metalloproteinase (MMP) inhibitors in vitro on gelatinolytic and collagenolytic activities in tracheal epithelial lining fluid (TELF) of horses with recurrent airway obstruction (RAO). Methods: 10 horses with RAO and 5 healthy control horses. Methods: Substrate-based functional assays, collagen I and gelatin degradation, were used to measure endogenous collagenolytic and gelatinolytic activities in TELF. In vitro inhibition of MMP activity in TELF with 2 chemically modified tetracyclines (CMTs; CMT-3 and CMT-8) and 2 bisphosphonates (BPs; zole...
Evaluation of permissiveness and cytotoxic effects in equine chondrocytes, synovial cells, and stem cells in response to infection with adenovirus 5 vectors for gene delivery. To evaluate host cell permissiveness and cytotoxic effects of recombinant and modified adenoviral vectors in equine chondrocytes, synovial cells, and bone marrow-derived mesenchymal stem cells (BMD-MSCs). Methods: Articular cartilage, synovium, and bone marrow from 15 adult horses. Methods: Equine chondrocytes, synovial cells, and BMD-MSCs and human carcinoma (HeLa) cells were cultured and infected with an E-1-deficient adenovirus vector encoding the beta-galactosidase gene or the green fluorescent protein gene (Ad-GFP) and with a modified E-1-deficient vector with the arg-gly-asp capsid pepti...
Use of an in vitro culture system to detect Theileria equi strains from infected equids and/or reservoirs. A horse erythrocyte culture technique, partly modifying that originally developed by Holman, was used to detect the presence of Theileria equi strains in 12 horse and 2 mule blood samples. The animals were placed into four groups on the basis of their case history and laboratory test results: the mules and two horses were considered as infected and included in the 'recent infection' group, four horses with a history of past infection were included in the 'past infection' group and four animals subjected to anti-theileria treatment formed the 'treated animals' group. The final group consisted o...
von Willebrand factor A1 domain can adequately substitute for A3 domain in recruitment of flowing platelets to collagen. Binding of von Willebrand factor (VWF) to platelet GPIbalpha and to collagen is attributed to VWF A1 and A3 domains, respectively. Objective: Using VWF, VWF lacking A1 (DeltaA1-VWF) or A3 (DeltaA3-VWF) and VWF with defective A3 (H1786A-VWF), in combination with recombinant A1 (residues 1262-1492) or A3 (residues 1671-1878), fused to glutathione-S-transferase (GST-A1 and GST-A3), we have re-investigated the role of A1 in platelet recruitment to surfaces of collagen. Results: In flow, measurable binding of DeltaA3-VWF occurred to horse tendon, but also to human type III collagen. GST-A1 and GST-...
Miscibility of binary monolayers at the air-water interface and interaction of protein with immobilized monolayers by surface plasmon resonance technique. The miscibility and stability of the binary monolayers of zwitterionic dipalmitoylphosphatidylcholine (DPPC) and cationic dioctadecyldimethylammonium bromide (DOMA) at the air-water interface and the interaction of ferritin with the immobilized monolayers have been studied in detail using surface pressure-area isotherms and surface plasmon resonance technique, respectively. The surface pressure-area isotherms indicated that the binary monolayers of DPPC and DOMA at the air-water interface were miscible and more stable than the monolayers of the two individual components. The surface plasmon re...
A comparison of three-dimensional ultrasound, two-dimensional ultrasound and dissections for determination of lesion volume in tendons. The purpose of this work was to evaluate the accuracy and precision of a freehand three-dimensional (3-D) ultrasonography system in the determination of lesion volume in tendons. The accuracy and precision of a 3-D ultrasonography system was assessed by performing repeated measurements on a phantom of known volume. Volume measurements of tendon lesions performed with 3-D ultrasonography were compared with measurements based on a series of two-dimensional (2-D) ultrasound (US) scans and to direct measurements from dissections. A novel method for the creation of tendon lesions in vitro was devel...
EIAV vector-mediated delivery of endostatin or angiostatin inhibits angiogenesis and vascular hyperpermeability in experimental CNV. We evaluated the efficacy of equine infectious anaemia virus (EIAV)-based lentiviral vectors encoding endostatin (EIAV.endostatin) or angiostatin (EIAV.angiostatin) in inhibiting angiogenesis and vascular hyperpermeability in the laser-induced model of choroidal neovascularisation (CNV). Equine infectious anaemia virus.endostatin, EIAV.angiostatin or control (EIAV.null) vectors were administered into the subretinal space of C57Bl/6J mice. Two weeks after laser injury CNV areas and the degree of vascular hyperpermeability were measured by image analysis of in vivo fluorescein angiograms. Compar...
Effect of fetal bovine serum and heat-inactivated fetal bovine serum on microbial cell wall-induced expression of procoagulant activity by equine and canine mononuclear cells in vitro. To determine the effect of fetal bovine serum (FBS) and heat-inactivated FBS (HI-FBS) on lipopolysaccharide (LPS)- and zymosan-induced procoagulant activity of equine and canine mononuclear cells. Methods: Mononuclear cells from 18 horses and 3 dogs. Methods: Cells were incubated with various concentrations of FBS, HI-FBS, LPS, zymosan, polymyxin B, and anti-LPS-binding protein monoclonal antibody or combinations of these constituents. A 1 stage recalcification assay was used to determine procoagulant activity. Results: Addition of FBS to media significantly increased procoagulant activity; eq...
Assessment of the catabolic effects of interleukin-1beta on proteoglycan metabolism in equine cartilage cocultured with synoviocytes. To evaluate the effects of interleukin (IL)-1beta on proteoglycan metabolism in equine cartilage explants when cultured in the presence of synoviocytes. Methods: Samples of cartilage and synovium collected from the femoropatellar joints of three 2- to 3-year-old horses. Methods: 3 experimental groups were established: cartilage explants only, synoviocytes only, and cartilage explants-synoviocytes in coculture. In each group, samples were cultured with or without IL-1beta (10 ng/mL) for 96 hours. Glycosaminoglycan (GAG) content of cartilage and medium samples was measured by use of a spectropho...
Pharmacokinetics of voriconazole after oral and intravenous administration to horses. To characterize pharmacokinetics of voriconazole in horses after oral and IV administration and determine the in vitro physicochemical characteristics of the drug that may affect oral absorption and tissue distribution. Methods: 6 adult horses. Methods: Horses were administered voriconazole (1 mg/kg, IV, or 4 mg/kg, PO), and plasma concentrations were measured by use of high-performance liquid chromatography. In vitro plasma protein binding and the octanol:water partition coefficient were also assessed. Results: Voriconazole was adequately absorbed after oral administration in horses, with a s...
Calcium-ions are involved in erythrocyte invasion by equine Babesia parasites. Ethylene glycol bis (beta-aminoethylether)-N,N,N,N-tetraacetic acid (EGTA) is a chelating agent capable of binding to positively-charged metal ions, including a calcium-ion (Ca2+). Here, we demonstrated the inhibitory effect of the chemical on the in vitro asexual growth of the equine protozoan parasites, Babesia caballi and Babesia equi. The growth of both B. caballi and B. equi was significantly inhibited in the presence of EGTA (IC50=1.27 and 2.25 mM, respectively). Under microscopical observation, increased percentages of extracellular merozoites in the total parasites were detected in bot...
Production of horse foals via direct injection of roscovitine-treated donor cells and activation by injection of sperm extract. We evaluated the effects of different donor cell treatments and activation methods on production of blastocysts after equine nuclear transfer. Nuclear transfer was performed by direct injection of donor cells, using a piezo drill, and standard activation was by injection of sperm factor followed by culture with 6-dimethylaminopurine. There was no difference in blastocyst development between embryos produced with roscovitine-treated or confluent donor cells (3.6% for either treatment). Addition of injection of roscovitine or culture with cycloheximide at the time of activation did not affect bl...
Intermediate amyloid oligomers of lysozyme: Is their cytotoxicity a particular case or general rule for amyloid? In the current study we investigated the molecular mechanisms of cytotoxicity of amyloid oligomers of horse milk lysozyme. We have shown that lysozyme forms soluble amyloid oligomers and protofibrils during incubation at pH 2.0 and 4.5 and 57 degrees C. These structures bind the amyloid-specific dyes thioflavin T and Congo Red, and their morphology and size were analyzed by atomic force microscopy. Monomeric lysozyme and its fibrils did not affect the viability of three cell types used in our experiments including primary murine neurons and fibroblasts, as well as neuroblastoma cell line IMR-3...
New staining methods for sperm evaluation estimated by microscopy and flow cytometry. New staining methods and automated instruments are now available to evaluate the sperm cell in vitro. Individual compartments of the sperm cell, such as the nucleus and the plasma and acrosomal membranes, may be investigated, as well as the cell function as shown by mitochondria activity and capacitation. Various probes are used and they can be analyzed by direct light or fluorescent microscopy or by flow cytometry. The automated instruments allow objective and accurate analysis and quantification as well as the ability to evaluate large population of cells in a shorter time, thus providing ac...
Determination of acrosin amidase activity in equine spermatozoa. Acrosin amidase activity of spermatozoa has been been associated with in vitro fertilization success in humans and has been proposed as an additional method for assessing sperm function in vitro. In this study, acrosin amidase activity was determined in equine spermatozoa by the hydrolysis of an arginine amide substrate. This assay includes a detergent to release acrosomal enzymes into a medium of basic pH to activate proacrosin to acrosin, which subsequently hydrolyses N-alpha-benzoyl-DL-arginine para-nitroanilide-HCl (BAPNA) to a chromogenic product. Spermatozoa (n = 3 ejaculates from each o...
A novel bioerodible deep scleral lamellar cyclosporine implant for uveitis. To determine the feasibility, safety, and effectiveness of an episcleral or deep scleral lamellar sustained release cyclosporine (CsA) device in a naturally occurring animal model of uveitis. Methods: A two-compartment perfusion chamber was used to assess in vitro human and equine scleral permeability of fluorescein, dexamethasone-fluorescein, or CsA. A biodegradable, matrix-reservoir CsA implant was designed, and release rates of CsA were determined in vitro. Tissue CsA levels were measured in eyes with the implant. Horses with equine recurrent uveitis (ERU) received episcleral or deep sclera...
Effective removal of equine arteritis virus from stallion semen. A method of removing equine arteritis virus (EAV) from equine semen used for artificial insemination is urgently needed. Recent medical studies suggest that a double semen processing technique of density gradient centrifugation followed by a 'swim-up' can provide virus-free sperm preparations for assisted reproduction. Objective: To investigate the use of the double semen processing technique to obtain virus-free sperm preparations from stallion semen containing EAV. Methods: Aliquots of an ejaculate from an uninfected stallion were spiked with virus and processed by the double processing tech...
Characterisation of lymphocyte subsets in the equine oviduct. The equine oviduct is the site of fertilisation and location of embryonic development during the first 5 or 6 days. It therefore has an important influence on mare fertility. Although histopathological changes have been described previously, there is limited information regarding lymphocyte subtypes present in the mucosa of the normal equine oviduct. Objective: To characterise the distribution of CD3+, CD4+, CD8+ and B lymphocytes in the equine oviduct from inseminated mares during oestrus and dioestrus, and from noninseminated mares during the immediate post ovulatory period. Methods: Oviduct...
Blastocyst development in equine oocytes with low meiotic competence after suppression of meiosis with roscovitine prior to in vitro maturation. This study was conducted to evaluate the in vitro development of equine oocytes with compact cumuli that had been subjected to a period of meiotic suppression with roscovitine before in vitro maturation. In experiment 1, oocytes were recovered from slaughterhouse-derived ovaries and held in M199 + 10% fetal bovine serum containing 66 microM roscovitine with or without an overlay of mineral oil in 5% CO2 in air at 38.2 degrees C for 16-18 or 24 h. No oocytes treated with roscovitine in the absence of an oil overlay for 16-18 h were maturing, compared with 2-4% of oocytes in other treatments. In...
Inhibition of caspase-9 reduces chondrocyte apoptosis and proteoglycan loss following mechanical trauma. Chondrocyte death, a notable feature of osteoarthritis, may play a role in the initiation of cartilage degeneration. The present study was aimed at uncovering the nature and involvement of cell death in the initiation of cartilage degeneration induced by mechanical trauma. Methods: Articular cartilage discs obtained from healthy skeletally mature horses were subjected to a single-impact load (500 g from 50 mm) using a simple drop-tower device and cultured in vitro for 48 h. Chondrocyte death was examined using two independent methods: transmission electron microscopy and caspase-3 activity ass...
Flow microcalorimetric study of butyrylcholinesterase kinetics and inhibition. The enzymatic hydrolysis of butyrylcholine, catalyzed by horse serum butyrylcholinesterase (EC 3.1.1.8), was studied at 37 degrees C in Tris buffer (pH 7.5) by flow microcalorimetry. A convolution procedure, using the Gamma distribution to represent the impulse response of the calorimeter, was developed to analyze the microcalorimetric curves. After correction for buffer protonation, the hydrolysis reaction was found to be slightly endothermic, with Delta H=+9.8 kJ mol(-1). Enzyme kinetics was studied with both the differential and integrated forms of the Michaelis equation with equivalent res...
In vitro efficacies of oseltamivir carboxylate and zanamivir against equine influenza A viruses. To investigate the possibilities of two NA inhibitors [oseltamivir carboxylate (OC) and zanamivir (ZA)] as the clinical agents for equine influenza A virus (EIV) infection, we examined the efficacies of these inhibitors against twelve EIVs in vitro. OC and ZA inhibited NA activities of all EIVs with 50% inhibitory concentrations with ranging from 0.017 to 0.130 and from 0.010 to 0.074 microM, respectively. OC and ZA inhibited plaque-forming of all EIVs in MDCK cells with 50% effective concentrations with ranging from 0.015 to 0.097 and from 0.016 to 0.089 microM, respectively, except for one s...
Effect of intraluminal distension or ischemic strangulation obstruction of the equine jejunum on jejunal motilin receptors and binding of erythromycin lactobionate. To determine whether inflammation of the jejunum of horses decreases the number of motilin receptors and amounts of motilin receptor mRNA and alters erythromycin lactobionate binding affinity to the motilin receptor in jejunal tissues. Methods: Jejunal segments in 6 adult horses. Methods: Each horse was anesthetized, and a ventral median celiotomy was performed; 2 segments of jejunum underwent a sham operation, 2 segments underwent ischemic strangulation obstruction (ISO), and 2 segments underwent intraluminal distension (ILD). Treatments were maintained for 120 minutes. From each segment, ful...
Validation of the sperm mobility assay in boars and stallions. The sperm mobility assay used in the present study measures the rate of sperm penetration in a biologically inert cell-separation solution (Accudenz). When a sample of sperm is overlaid in a cuvette containing Accudenz, sperm penetrate the solution and absorbance of the sample can be measured with a spectrophotometer. This assay has been successfully used to select chicken and turkey semen donors. We validated this assay for semen from boars and stallions. Absorbance was measured after overlaying fresh semen from each species in prefilled cuvettes for 1, 5, 10, 15, 20, and 40 min. There were n...
Selection of a set of reliable reference genes for quantitative real-time PCR in normal equine skin and in equine sarcoids. Real-time quantitative PCR can be a very powerful and accurate technique to examine gene transcription patterns in different biological conditions. One of the critical steps in comparing transcription profiles is accurate normalisation. In most of the studies published on real-time PCR in horses, normalisation occurred against only one reference gene, usually GAPDH or ACTB, without validation of its expression stability. This might result in unreliable conclusions, because it has been demonstrated that the expression levels of so called "housekeeping genes" may vary considerably in different t...
An in vitro comparison of two suture intervals using braided absorbable loop suture in the equine linea alba. To compare bursting strength and failure modes of ventral median abdominal incisions closed with loop suture in a simple continuous pattern using two different suture-bite intervals. Methods: In vitro experiment. Methods: Equine cadavers (n=14; weighing >318 kg; postmortem interval <2 hours). Methods: A template was used to make a 25 cm incision with suture interval and bite size of either 1.0 cm x 1.5 cm or 1.5 cm x 1.5 cm. A 200 L polyurethane bladder was inserted within the abdomen and insufflated to create abdominal wall tension. Celiotomies were closed with a #2 braided lactomer 9-1...
In situ and ex vivo evaluation of an arthroscopic indentation instrument to estimate the health status of articular cartilage in the equine metacarpophalangeal joint. To evaluate an arthroscopic indentation instrument (Artscan 200) for assessment of the health status of equine articular cartilage. Methods: In vitro experiment using equine isolated proximal phalanx (P1) specimens. Methods: P1 specimens from 39 horses (aged 1.5-22 years). Methods: Reproducibility was tested by determination of the coefficient of variation (CV). Dynamic modulus and cartilage degeneration index (CDI) values were measured at 2 predefined sites (site 1, joint margin; site 2, joint center) to assess the accuracy and to evaluate the relation with surface integrity. Results: CV was ...
Endometrial nitric oxide production and nitric oxide synthases in the equine endometrium: Relationship with microvascular density during the estrous cycle. Nitric oxide (NO) plays an important role in angiogenesis and in the regulation of the blood flow. This study was carried out to investigate (i) the effects of endogenous estrogens and progestins and exogenous progesterone (P(4)) (5 ng/ml or 1 microg/ml) or estradiol 17beta (E(2)beta) (50 pg/ml or 1 microg/ml) on in vitro endometrial NO synthesis; (ii) the presence of different isoforms of NO synthase; (iii) and their relationship to microvascular density in the equine endometrium during the estrous cycle. NOS expression was also evaluated in the myometrium. Expression of endothelial and induc...
Foals are interferon gamma-deficient at birth. The increased vulnerability of foals to specific pathogens such as Rhodococcus equi is believed to reflect an innate immunodeficiency, the nature of which remains poorly understood. Previous studies have demonstrated that neonates of many species fail to mount potent Th1 responses. The current research investigates the ability of circulating and pulmonary lymphocytes of developing foals to produce interferon gamma (IFNgamma). Peripheral blood mononuclear cells (PBMC) were prepared from up to 10 horse foals at regular intervals throughout the first 6 months of life. Bronchoalveolar lavage (BAL)...
