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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.
An in vitro biomechanical comparison of a locking compression plate fixation and kerf cut cylinder fixation for ventral arthrodesis of the fourth and the fifth equine cervical vertebrae. To (1) define mechanical properties in flexion, extension, and left lateral bending of cadaveric equine 4th and 5th cervical (C4-C5) articulations, (2) compare biomechanical properties of C4-C5 when stabilized with a kerf cut cylinder (KCC) compared with a ventrally placed 4.5 mm locking compression plate (LCP). Methods: In vitro biomechanical investigation. Methods: Cadaveric adult equine cervical vertebral columns (n=54). Methods: Cervical vertebrae aged by horse dentition and size measured from radiographs were divided into 3 age groups then randomly allocated to 3 groups. The C4-C5 articul...
Cyclooxygenase-2 expression in animal cancers. Cyclooxygenase (COX; also known as prostaglandin endoperoxide synthase) is a key enzyme in the biochemical pathway leading to the synthesis of prostaglandins. A large amount of epidemiological and experimental evidence supports a role for COX-2, the inducible form of the enzyme, in human tumorigenesis, notably in colorectal cancer. COX-2 mediates this role through the production of PGE(2) that acts to inhibit apoptosis, promote cell proliferation, stimulate angiogenesis, and decrease immunity. Similarly, COX-2 is believed to be involved in the oncogenesis of some cancers in domestic animals. H...
Meniscal translocation and deformation throughout the range of motion of the equine stifle joint: an in vitro cadaveric study. By study of the translocation and deformation of equine menisci throughout the range of motion, it may be possible to identify potential mechanical factors in the pathogenesis of injury to the cranial horn of the medial meniscus. Objective: To quantitatively document meniscal translocation and deformation using radiographic and MR imaging, and to evaluate for potential variation between the medial and lateral menisci. Methods: Radiographic markers were embedded in the periphery of the menisci in 6 cadaver stifles. Proximal-distal radiographs were taken at 15° intervals ranging from full flexi...
Virally and physically transgenized equine adipose-derived stromal cells as a cargo for paracrine secreted factors. Adipose-Derived Stromal Cells have been shown to have multiple lineage differentiation properties and to be suitable for tissues regeneration in many degenerative processes. Their use has been proposed for the therapy of joint diseases and tendon injuries in the horse. In the present report the genetic manipulation of Equine Adipose-Derived Stromal Cells has been investigated. Results: Equine Adipose-Derived Stromal Cells were successfully virally transduced as well as transiently and stably transfected with appropriate parameters, without detrimental effect on their differentiation properties...
Isolation and characterization of equine amniotic fluid-derived multipotent stem cells. Amniotic fluid (AF) is a well-known source of stem cells. However, there have been no reports regarding equine AF stem cells. We have isolated equine AF-derived multipotent stem cells (MSC) (eAF-MSC) and show that these cells exhibit self-renewal ability and multilineage differentiation. Methods: AF was obtained from thoroughbred mares and mononuclear cells (MNC) were isolated by Ficoll-Paque density gradient. We measured the cumulative population doubling level (CPDL) and characterized the immunophenotype by flow cytometry. To investigate differentiation ability, a trilineage differentiation ...
Gene delivery in the equine cornea: a novel therapeutic strategy. To determine if hybrid adeno-associated virus serotype 2/5 (AAV5) vector can effectively deliver foreign genes into the equine cornea without causing adverse side effects. The aims of this study were to: (i) evaluate efficacy of AAV5 to deliver therapeutic genes into equine corneal fibroblasts (ECFs) using enhanced green fluorescent protein (EGFP) marker gene, and (ii) establish the safety of AAV5 vector for equine corneal gene therapy. Methods: Primary ECF cultures were harvested from healthy donor equine corneas. Cultures were maintained at 37°C in humidified atmosphere with 5% CO(2). Metho...
Investigation of the effects of prostaglandin E₂ on equine superficial digital flexor tendon fibroblasts in vitro. To evaluate the effects of prostaglandin E₂ (PGE₂) treatment on the metabolism of equine tendon fibroblasts in vitro to aid in investigating the response of tendon fibroblasts to injury and novel therapeutics. Methods: Superficial digital flexor tendon fibroblasts isolated via collagenase digestion from six young adult horses were grown in monolayer in four concentrations of PGE₂ (0, 10, 50, 100 ng/ml) for 48 hours. Cells and medium were harvested for gene expression (collagen types I and III, cartilage oligomeric matrix protein [COMP], decorin, and matrix metalloproteinase-1, -3, and -1...
Studies of quinapyramine-resistance of Trypanosoma brucei evansi in China. In the present article, we summarize our studies of antrycide-resistance of Trypanosoma brucei evansi in four aspects in the last recent several years, the analysis of quinapyramine-sensitive situation of T. b. evansi in China, biological characteristics of T. b. evansi population in quinapyramine-resistance and biological materials of quinapyramine-resistance in T. b. evansi population. Firstly, the correlative assays of effective dosage of quinapyramine on T. b. evansi disease between in vivo and in vitro methods showed that their relationship was parabolic with positive correlation. On the ...
Detection of reaginic antibodies against Faenia rectivirgula from the serum of horses affected with Recurrent Airway Obstruction by an in vitro bioassay. Reaginic antibodies, mainly of the IgE and some IgG subclasses, play an important role in the induction of type I immediate hypersensitivity reactions. These antibodies bind through their Fc fragment to high affinity receptors (FcεRI) present in the membrane of mast cells and basophils. Previously, several studies have investigated the role of reaginic antibodies in the pathogenesis of RAO. However, whereas immunological aspects of RAO have been extensively studied, the precise sequence of events is still not well understood and role of IgE in this disease still remains controversial. Therefo...
Lipoprotein complex of equine lysozyme with oleic acid (ELOA) interactions with the plasma membrane of live cells. Recent evidence supports the idea that early aggregates, protein, and lipoprotein oligomers but not large aggregates like fibrils that are formed at late stages of the aggregation process are responsible for cytotoxicity. Oligomers can interact with the cellular plasma membrane affecting its structure and/or dynamics or may be taken up by the cells. In either case, disparate cascades of molecular interactions are activated in the attempt to counteract the disturbance induced by the oligomers. If unsuccessful, cell death follows. Here, we study the molecular and cellular mechanisms underlying P...
Electrochemical study of gelatin as a matrix for the immobilization of horse heart cytochrome c. The aim of this paper is to emphasize the strength of gelatin as a stable matrix for redox enzymes. Cyclic voltammetry has been applied for a detailed electrochemical study of horse heart cytochrome c (HHC) entrapped in a gelatin matrix immobilized on a gold electrode. The influence of the HHC concentration, the mass percentage of the gelatin and the nature of the gelatin on the electrochemical behaviour of HHC have been described in detail. In addition, attenuated total reflection infrared (ATR-IR) spectroscopy was used to prove the immobilization on a qualitative and conformational level. Th...
In vitro selection and differentiation of ivermectin resistant cyathostomin larvae. Cyathostomins are considered to be the primary helminth pathogen of horses and macrocyclic lactones (ML) are the most frequently used anthelmintics. Therefore, ML resistance is a serious threat for the control of these parasites. In the present study ivermectin resistant cyathostomin L3 were in vitro selected, using a reiterative larval migration inhibition assay (rLMIA) and differentiated by reverse line blot (RLB). Larvae were obtained from two populations, one from a never treated, free-roaming horse population in the nature reserve Oostvaardersplassen (OVP) and the other from regularly ive...
Definite coordination arrangement of organometallic palladium complexes accumulated on the designed interior surface of apo-ferritin. Apo-ferritin (apo-Fr) mutants are used as scaffolds to accommodate palladium (allyl) complexes. Various coordination arrangements of the Pd complexes are achieved by adjusting the positions of cysteine and histidine residues on the interior surface of the apo-Fr cage.
Cytogenetic and genetic studies in a hypospadic horse (Equus caballus, 2n = 64). A 4-year-old male horse of Friesian breed with normal body conformation, development and libido, and showing an evident ventral penis deviation with hypospadias, underwent both cytogenetic and genetic investigation. Although the karyotype showed normal male arrangement (2n = 64,XY), one telomere of horse (ECA) chromosome 1 was shorter than both the other one and those of a normal horse (control), as revealed by CBA- and RBA-banding, and by Ag-NOR and FISH-mapping techniques using telomere PNA probes. Genetic investigation of the SRY and MAMLD1 coding sequences revealed a normal SRY sequence an...
Isolation of equine bone marrow-derived mesenchymal stem cells: a comparison between three protocols. There is a need to assess and standardise equine bone marrow (BM) mesenchymal stem cell (MSC) isolation protocols in order to permit valid comparisons between therapeutic trials at different sites. Objective: To compare 3 protocols of equine BM MSC isolation: adherence to a plastic culture dish (Classic) and 2 gradient density separation protocols (Percoll and Ficoll). Methods: BM aspirates were harvested from the sternum of 6 mares and MSCs isolated by all 3 protocols. The cell viability after isolation, MSC yield, number of MSCs attained after 14 days of culture and the functional characteri...
Comparison of methods for depletion of albumin and IgG from equine serum. Disease-specific biomarkers hold diagnostic promise in both human and veterinary medicine, but serum biomarkers in low concentrations may be masked by the presence of abundant proteins, mostly albumin and IgG. Methods to deplete albumin and IgG exist, but efficacy of these methods for depleting equine serum of these proteins has not been established. Objective: The aim of this study was to determine if albumin and IgG could be depleted from equine serum using several commercially available kits and procedures. Methods: One-dimensional gel electrophoresis followed by densitometry was used to de...
Is FAS/Fas ligand system involved in equine corpus luteum functional regression? Proapoptotic factor Fas ligand (FASL) and its cell surface receptor FAS are tumor necrosis factor superfamily members that trigger apoptosis in different cell types. However, their influence on luteal steroidogenesis is not clearly understood. The aim of the present work was to determine (i) the presence of the cytokine FASL and its receptor FAS in the mare's corpus luteum (CL) throughout the luteal phase, as well as (ii) the influence of FASL alone, or together with the cytokines tumor necrosis factor alpha (TNF) and interferon gamma (IFNG), on equine luteal cell production of luteotrophic an...
Comparison of three methods of DNA extraction from peripheral blood mononuclear cells and lung fragments of equines. We compared three different protocols for DNA extraction from horse peripheral blood mononuclear cells (PBMC) and lung fragments, determining average final DNA concentration, purity, percentage of PCR amplification using beta-actin, and cost. Thirty-four samples from PBMC, and 33 samples from lung fragments were submitted to DNA extraction by three different protocols. Protocol A consisted of a phenol-chloroform and isoamylic alcohol extraction, Protocol B used alkaline extraction with NaOH, and Protocol C used the DNAzol((R)) reagent kit. Protocol A was the best option for DNA extraction from...
Comparison of in vitro digestibility estimates using the DaisyII incubator with in vivo digestibility estimates in horses. The objective of this study was to determine if in vitro methodologies developed for the Ankom Daisy(II) incubator could produce accurate estimates of in vivo equine DM digestibility (DMD) and NDF digestibility (NDFD) when equine feces were used as the inoculum source. Four mature geldings were utilized in a 4 × 4 Latin square design experiment with a 2 × 2 factorial arrangement of dietary treatments (timothy hay, alfalfa hay, timothy hay plus oats, and alfalfa hay plus oats), in which the geldings were individually housed and fed. During each 5-d total fecal collection period, feces were co...
Efficacy of tuohy needle in oocytes collection from excised mare ovaries. TWO METHODS HAVE BEEN DESCRIBED TO RECOVER OOCYTES FROM EQUINE FOLLICLES IN EXCISED OVARIES: aspiration and scraping. Aim of this work was to develop an effective method for collecting equine oocytes using Tuohy needle and comparing this technique to aspiration and scraping, with or without tunica albuginea removal. This hollow hypodermic needle, usually employed for inserting epidural catheters, is designed with a slightly curved tip, shaped similar to a small curette. In unpeeled ovaries, the recovery rates of Tuohy needle group was higher (P < .05) than in the 16 g needle aspiration and ...
Development of a DNA microarray for detection of expressed equine classical MHC class I sequences in a defined population. Development of an accurate and efficient molecular-based equine MHC class I typing method would facilitate the study of T lymphocyte immune responses in horses. Here, a DNA microarray was designed to detect expressed classical MHC class I genes comprising serologically defined equine leukocyte antigen (ELA)-A haplotypes represented in a closed Arabian horse breeding herd. Initially, cloning and sequencing of RT-PCR products were used to identify sequences associated with the ELA-A1, A4, and W11 haplotypes, and one undefined haplotype, in six horses. Subsequently, sequence-specific, conserved (...
In vitro comparison of two centrally threaded, positive-profile transfixation pin designs for use in third metacarpal bones in horses. To compare heat generation during insertion, pullout strength, and associated microdamage between a self-tapping positive profile transfixation pin (STTP) and nontapping positive profile transfixation pin (NTTP). Methods: 30 pairs of third metacarpal bones (MC3s) from adult equine cadavers. Methods: One MC3 of each pair was assigned to the STTP group; the other was assigned to the NTTP group. The assigned pin was inserted into the diaphysis in a lateral to medial direction. Bone temperature increase during pilot-hole drilling and pin insertion was recorded at 1 mm from the final thread positio...
Effects of antifungal drugs and delivery vehicles on morphology and proliferation of equine corneal keratocytes in vitro. To evaluate the effects of topical antifungal drugs and delivery vehicles on the morphology and proliferation rate of cultured equine keratocytes. Methods: 16 corneas obtained from 8 apparently ophthalmologically normal horses < 0.5 hours after euthanasia for reasons unrelated to the study. Methods: Primary cultures of equine keratocytes were obtained from corneal stroma and were exposed to several concentrations of 3 commonly used, topically applied antifungals: natamycin, itraconazole, and miconazole. In addition, effects of drug delivery vehicles DMSO, benzalkonium chloride, and carboxym...
Use of an in vitro biotinylation technique for determination of posttransfusion survival of fresh and stored autologous red blood cells in Thoroughbreds. To evaluate N-hydroxysuccinimide (NHS)-biotin labeling of equine RBCs and determine posttransfusion survival of autologous equine RBCs stored in citrate phosphate dextrose adenine-1 (CPDA-1) for 0, 1, 14, and 28 days. Methods: 13 healthy adult Thoroughbreds. Methods: Serial dilutions of biotin and streptavidin-phycoerythrin (PE) were evaluated in vitro in blood collected from 3 horses. One horse was used to determine RBC distribution and recovery. Twelve horses were allocated to 4 groups for in vivo experiments in which blood was collected into CPDA-1. Blood was labeled with biotin and reinfus...
The equine oocyte: factors affecting meiotic and developmental competence. There is currently much interest in assisted reproduction techniques in the horse, however, many aspects of oocyte maturation, fertilization, and embryo development in the horse differ from those in other species. Because of the close attachment of the equine oocyte to the follicle wall, scraping of the follicle is the most effective method for oocyte recovery. A notable feature of equine oocytes is that those with expanded cumuli (Ex oocytes), which originate from atretic follicles, have higher meiotic competence (ability to mature to metaphase II in vitro) than do oocytes with compact cumuli...
Coculture of equine mesenchymal stem cells and mature equine articular chondrocytes results in improved chondrogenic differentiation of the stem cells. Bone marrow derived mesenchymal stem cells (MSCs) can be used to repair articular cartilage defects, these cells should be properly stimulated so that they could differentiate morphologically and hold cellular synthetic features closer to maturely differentiated chondrocytes. It is well known that tissue specific environment plays an important role in cell fate determination. Once improved isolation, proliferation and differentiation protocols have been developed, the likelihood of spontaneous differentiation of MSCs into divergent lineages will be reduced, thus increasing their value for cart...
Disposition of oral telithromycin in foals and in vitro activity of the drug against macrolide-susceptible and macrolide-resistant Rhodococcus equi isolates. The objectives of this study were to determine the serum and pulmonary disposition of telithromycin in foals and to determine the minimum inhibitory concentration (MIC) of telithromycin against macrolide-susceptible and macrolide-resistant Rhodococcus equi isolates. A single dose of telithromycin (15 mg/kg of body weight) was administered to six healthy 6-10-week-old foals by the intragastric route. Activity of telithromycin was measured in serum, pulmonary epithelial lining fluid (PELF), and bronchoalveolar lavage (BAL) cells using a microbiological assay. The broth macrodilution method was u...
The effect of cooling on the contractility of equine digital small lamellar arteries: modulating role of the endothelium. The equine hoof displays thermoregulatory functions, and the blood vessels lying under the hoof wall are temperature sensitive. The aim of this study was to investigate the effect of cooling on the contractile responses to α-adrenoceptor and 5-HT receptor stimulation in equine small lamellar arteries using wire myography. The role of the endothelium in the response mediated by 5-HT was also evaluated. Moderate cooling caused a reduction of the contraction induced by depolarizing Krebs solution (DKS, containing 118 mm KCl) and the maximal contraction caused by UK-14304 (α(2)-adrenoceptor agon...
Release kinetics of VEGF165 from a collagen matrix and structural matrix changes in a circulation model. Current approaches in bone regeneration combine osteoconductive scaffolds with bioactive cytokines like BMP or VEGF. The idea of our in-vitro trial was to apply VEGF165 in gradient concentrations to an equine collagen carrier and to study pharmacological and morphological characteristics of the complex in a circulation model. Methods: Release kinetics of VEGF165 complexed in different quantities in a collagen matrix were determined in a circulation model by quantifying protein concentration with ELISA over a period of 5 days. The structural changes of the collagen matrix were assessed with lig...
