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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.
CE provides evidence of the stereoselective hydroxylation of norketamine in equines. CE with multiple isomer sulfated-CD as selector was used for the simultaneous analysis of the stereoisomers of ketamine, norketamine, 5,6-dehydronorketamine and hydroxylated metabolites of norketamine in liquid/liquid extracts of (i) in vitro incubations with ketamine or norketamine and equine liver microsomes and (ii) plasma and urine of ponies receiving a target-controlled infusion of ketamine under isoflurane anesthesia. Hydroxynorketamine metabolites with the hydroxy group at the cyclohexanone ring could be shown to be formed stereoselectively both in vitro and in vivo. Due to the lack of ...
Characterization of bradykinin-induced endothelium-independent contraction in equine basilar artery. We investigated the effect of bradykinin (BK) on isolated equine basilar arterial rings with and without endothelium. BK induced concentration-dependent contraction of resting arterial rings and no relaxation when the rings were precontracted by prostaglandin F(2alpha). The maximal response and pD(2) value were 161.2 +/- 28.1% (to 60 mm KCl-induced contraction) and 8.24 +/- 0.25 respectively. The cumulative concentration-response curve for BK was not shifted to the right by des-Arg(9)-[Leu(8)]-BK (a B(1)-receptor antagonist), HOE140 (a B(2)-receptor antagonist) or NPC567 (another B(2)-receptor...
Electrophysiological characterization of electrolyte and nutrient transport across the small intestine in horses. The aim of this study was to characterize the transport mechanisms of electrolytes and nutrients across the jejunum of nine healthy horses electrophysiologically. The stripped mucosa was mounted in Ussing chambers and tissue conductances (G(t)) and short circuit currents (I(sc)) were continuously monitored. After blocking the sodium and potassium channels with amiloride, tetraethylammonium chloride (TEA) and barium, chloride secretion was stimulated by carbachol and forskolin. Subsequently, chloride channels were inhibited by 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid, 5-nitro-2-(3-ph...
Expression and purification of active recombinant equine lysozyme in Escherichia coli. Equine lysozyme (EL) is a calcium (Ca)-binding lysozyme and is an intermediary link between non-Ca-binding C-type lysozyme and alpha-lactalbumin. The feature of lysozymes to assemble into the fibrils has recently gained considerable attention for the investigation of the functional properties of these proteins. To study the structural and functional properties of EL, a synthetic gene was cloned and EL was overexpressed in Escherichia coli as a fused protein. The His-tagged recombinant EL was accumulated as inclusion bodies. Up to 50 mg/l of the recombinant EL could be achieved after purificati...
Ex vivo serotype-specific transduction of equine joint tissue by self-complementary adeno-associated viral vectors. Cell transplantation for the treatment of joint disease is an important clinical tool. Genetic modification of cells before transplantation has shown enhanced healing. Ex vivo genetic modification of joint tissue cells with various adeno-associated virus (AAV) serotypes has not been investigated. The transduction efficiencies of self-complementary AAV serotypes (1-6 and 8) were determined in joint tissue containing chondrocytes and synoviocytes isolated from equine models. When comparing scAAV serotypes for efficient transduction ex vivo, in chondrocytes versus synoviocytes, serotypes 6 and 2,...
Lactobacillus equicursoris sp. nov., isolated from the faeces of a thoroughbred racehorse. We previously isolated five strains of putative lactobacilli from the faeces of a thoroughbred horse (a 4-year-old male). Of the five strains, four were identified as members of existing Lactobacillus species; however, sequence analysis of the 16S rRNA gene revealed that the fifth isolate, DI70(T), showed approximately 97 % identity (1325/1366 bp) with the type strain of Lactobacillus delbrueckii. Therefore, we considered the possibility that DI70(T) represents a novel species of the genus Lactobacillus. Cells of strain DI70(T) were Gram-stain-positive, catalase-negative, non-spore-forming, no...
Reproducibility of a non-invasive ultrasonic technique of tendon force measurement, determined in vitro in equine superficial digital flexor tendons. A non-invasive ultrasonic (US) technique of tendon force measurement has been recently developed. It is based on the relationship demonstrated between the speed of sound (SOS) in a tendon and the traction force applied to it. The objectives of the present study were to evaluate the variability of this non-linear relationship among 7 equine superficial digital flexor (SDF) tendons, and the reproducibility of SOS measurements in these tendons over successive loading cycles and tests. Seven SDF tendons were equipped with an US probe (1MHz), secured in contact with the skin overlying the tendon me...
Molecular characterization and expression pattern of the equine lactate dehydrogenase A and B genes. The species-specific properties of LDH isozymes are essentially determined by M (muscle) and H (heart) subunit proteins encoded by the LDHA and LDHB genes, respectively. In the present study, we molecularly characterized the full-length equine lactate dehydrogenase A (eLDHA) and B (eLDHB) cDNAs. The eLDHA cDNA consisted of a 999-bp open reading frame (ORF), while the eLDHB and newly acquired bat LDHB consisted of a 1002-bp ORF, which is 3 bp shorter than the LDHB ORF of other registered mammals. The alignment of amino acid sequences showed that eLDHA acquired positively charged His 88 and 226,...
Osmotic tolerance limits and membrane permeability characteristics of stallion spermatozoa treated with cholesterol. Stallion spermatozoa exhibit osmotic damage during the cryopreservation process. Recent studies have shown that the addition of cholesterol to spermatozoal membranes increases the cryosurvival of bull, ram and stallion spermatozoa, but the exact mechanism by which added cholesterol improves cryosurvival is not understood. The objectives of this study were to determine if adding cholesterol to stallion sperm membranes alters the osmotic tolerance limits and membrane permeability characteristics of the spermatozoa. In experiment one, stallion spermatozoa were treated with cholesterol-loaded cycl...
Comparative study of the characteristics and properties of tendinocytes derived from three tendons in the equine forelimb. The aim of this study was to determine the characteristic differences in tendinocytes derived from tendons in the equine forelimb, superficial digital flexor tendon (SDFT), deep digital flexor tendon (DDFT) and common digital extensor tendon (CDET), in morphology, proliferation, collagen production ability and ability for synthesis of matrix metalloproteinases (MMPs). Significant differences were observed in cell number in vivo. The cellular number was largest in the SDFT and smallest in the CDET. The values of in vitro proliferation ratios and ability for synthesis of collagen and MMPs were l...
Temporal analysis of equine bone marrow aspirate during establishment of putative mesenchymal progenitor cell populations. Mesenchymal progenitor cells (MPCs) are often characterized using surface markers after expansion and treatment in culture. There are no studies directly comparing gene and protein markers in undifferentiated samples during the very early phases of culture. The goal of this study was to evaluate temporal gene and protein expression changes during establishment of equine MPC cultures. Bone marrow aspirate was obtained from 35 horses and processed by density gradient centrifugation. In freshly isolated bone marrow, mononuclear cells had variable expression of CD44, CD11a/CD18, CD90, and CD45RB c...
Further Development of an Equine Cell Line that can be Propagated over 100 Times. Cell lines originating from horses are necessary for isolation and propagation of equine herpesviruses (EHV). Although we established an equine-derived cell line, FHK-Tcl3, propagation ceased after fewer than 40 passages. In this study, FHK-Tcl3 cell propagation continued beyond 40 passages, achieving over 100 passages. FHK-Tcl3 cells were then cloned by limiting dilution at the 100th passage. Cloned cells were termed FHK-Tcl3.1. FHK-Tcl3.1 cells grew well and were propagated every 3 to 4 days by splitting 1:5. In addition, EHV-1, -2 and -4 showed a clear cytopathic effect (CPE) in FHK-Tcl3.1 ...
Effect of cryopreservation on nitric oxide production by stallion spermatozoa. The ability of stallion spermatozoa to produce nitric oxide (NO) before (fresh) and after freezing and thawing (FT) was evaluated by means of flow cytometry after loading the sperm suspension with the probe, 4,5-diaminofluorescenin diacetate. The presence of NO synthase (NOS) was investigated by Western blotting using anti-NOS1, anti-NOS3, or anti-universal NOS antibodies (Abs). While NO was detected both in fresh and FT sperm suspensions, its production increased after cryopreservation only when egg yolk was removed from the extender. Anti-NOS1 Ab intensively labeled a single band with an app...
Small interfering RNA targeting bovine papillomavirus type 1 E2 induces apoptosis in equine sarcoid transformed fibroblasts. Equine sarcoids are skin tumours of horses caused by infection with BPV-1 or 2. Maintenance and replication of the viral genome depend upon the viral proteins E1 and E2. We examined the effects of an E2 specific siRNA on E2 and E1 viral gene expression, viral load and cell growth in BPV-1 transformed sarcoid-derived cells. Transfection with E2-siRNA caused a reduction in E2 and E1 mRNA expression as well as viral load, growth inhibition and decreased anchorage-independent growth. siRNA treated cells showed significantly higher apoptosis rates than control cells. Thus sequence specific targetin...
Single-layer centrifugation with Androcoll-E can be scaled up to allow large volumes of stallion ejaculate to be processed easily. The objective of the current study was to optimize the volumes of Androcoll-E and sperm sample used in various sizes of centrifuge tube to scale up single-layer centrifugation (SLC) for routine use in the field. Although sperm suspensions of equivalent quality were produced using Androcoll-E in small and large tubes, the sperm yield was much lower in the latter (P<0.001). In contrast, in 200-mL tubes (XL), the yields were approximately 25% higher than those for the small tubes. An increased volume (4.5 mL) of extended ejaculate in small tubes (SLC-Inc) or 15 to 18 mL extended ejaculate on 1...
New insights into the mechanisms of fertilization: comparison of the fertilization steps, composition, and structure of the zona pellucida between horses and pigs. The mechanism of fertilization remains largely enigmatic in mammals. Most studies exploring the molecular mechanism underlying fertilization have been restricted to a single species, generally the mouse, without a comparative approach. However, the identification of divergences between species could allow us to highlight key components in the mechanism of fertilization. In the pig, in vitro fertilization (IVF) and polyspermy rates are high, and spermatozoa penetrate easily through the zona pellucida (ZP). In contrast, IVF rates are low in the horse, and polyspermy is scarce. Our objective was ...
Protein oligomerization induced by oleic acid at the solid-liquid interface–equine lysozyme cytotoxic complexes. Protein oligomeric complexes have emerged as a major target of current research because of their key role in aggregation processes in living systems and in vitro. Hydrophobic and charged surfaces may favour the self-assembly process by recruiting proteins and modifying their interactions. We found that equine lysozyme assembles into multimeric complexes with oleic acid (ELOA) at the solid-liquid interface within an ion-exchange chromatography column preconditioned with oleic acid. The properties of ELOA were characterized using NMR, spectroscopic methods and atomic force microscopy, and showed...
Freezing equine semen: the effect of combinations of semen extenders and glycerol on post-thaw motility. We evaluated combinations of two commercial semen extenders and three concentrations of glycerol to determine the combination that yielded the highest post-thaw sperm motility. Methods: A randomised 2 x 3 block design was used. Methods: Semen was collected from four stallions (6 collections per stallion). The sample was diluted with either a dried skim-milk glucose extender (EZ Mixin Original Formula) or a chemically defined, milk-free diluent (INRA 96), and each was used in combination with 2%, 3% or 4% glycerol in standard commercial freezing medium. Sperm motility was assessed by microscopy...
Rational design of synthetic peptides to generate antibodies that recognize in situ CD11c(+) putative dendritic cells in horse lymph nodes. A three-dimensional model of the alphaX I-domain of the horse integrin CD11c from dendritic cells provided information for selecting two segments of the primary structure for peptide synthesis. Peptide 1 contains 20 amino acids and peptide 2 has 17 amino acid residues. The first spans from position Thr229 to Arg248 of an alpha-helix segment of the structure, whereas peptide 2 goes from Asp158 to Phe174 and corresponds to an exposed segment of the loop considered to be the metal ion-dependent adhesion site. Murine polyclonal antisera against both peptides were generated and assayed in periphera...
Diffusion of contrast medium after perineural injection of the palmar nerves: an in vivo and in vitro study. Proximal diffusion of local anaesthetic solution after perineural anaesthesia may lead to the desensitisation of structures other than those intended. However, there is no evidence-based study demonstrating the potential distribution and diffusion of local anaesthetic solution after perineural analgesia in the distal limb. Objective: To document the potential diffusion of local anaesthetic solution using a radiopaque contrast model and to evaluate the influence of walking compared with confinement in a stable after injection. Methods: Radiopaque contrast medium was injected subcutaneously over...
In vivo and in vitro evidence of the involvement of CXCL1, a keratinocyte-derived chemokine, in equine laminitis. C-X-C motif ligand 1 (CXCL1) is an important chemokine of epithelial origin in rodents and humans. Objective: To assess in vivo and in vitro the regulation of CXCL1 in equine laminitis. Methods: Twenty adult horses. Methods: Real-time quantitative polymerase chain reaction (PCR) was used to assess expression of CXCL1 in samples of laminae, liver, skin, and lung from the black walnut extract (BWE) model of laminitis, and in cultured equine epithelial cells (EpCs). Tissue was obtained from control animals (CON, n = 5), and at 1.5 hours (early time point [ETP] group, n = 5), at the onset of leuko...
Isolation and immunophenotypic characterization of mesenchymal stem cells derived from equine species adipose tissue. The purpose of this work was to isolate and cultivate mesenchymal stem cells (MSC) derived from equine adipose tissue and conduct cellular characterization with the following markers: CD90, CD44 and CD13. Adipose tissue collection was performed at the base of the horses' tails, followed by immediate isolation and cultivation of the MSC and posterior characterization by flow cytometry for the interspecies reaction test using mouse anti-rat CD90 monoclonal antibody (mAb), fluorescein isothiocyanate (FITC), and tests with specific mAb mouse anti-horse CD13 and mouse anti-horse CD44. The technique...
Effect of age and mitogen on the frequency of interleukin-4 and interferon gamma secreting cells in foals and adult horses as assessed by an equine-specific ELISPOT assay. Peripheral blood mononuclear cells (PBMCs) were obtained from 6 foals <1 week of age, 6 foals between 3 and 4 months of age, and 10 adult horses. PBMCs were stimulated with concanavalin A (ConA) or calcium ionomycin-phorbol myristate acetate (CaI-PMA) and the frequency of interferon IFN-gamma and IL-4 secreting cells was measured using an equine-specific ELISPOT assay. The number of IFN-gamma secreting cells was significantly lower in both groups of foals than in adult horses regardless of the mitogen used for stimulation. The number of IFN-gamma secreting cells was significantly higher in ...
Expression of toll-like receptor 9 in horse lungs. Toll-like receptor 9 (TLR9) has been found to be the main receptor to respond to bacterial DNA in a wide variety of species. Recent work has shown that TLR9 is expressed in a diverse set of cells within the lung. However, much of this data has been centered on human and mouse cell culture lines or primary cultures and very little is known of TLR9 expression in intact lung, especially that of the horse. Here we show that TLR9 is expressed in the lungs of horses in a wide variety of cells. In particular, we note expression in pulmonary intravascular macrophages (PIMs), alveolar macrophages, bron...
Environmental health impacts of equine estrogens derived from hormone replacement therapy. Many factors have been considered in evaluations of the risk-benefit balance of hormone replacement therapy (HRT), used for treating menopausal symptoms in women, but not its potential risks for the environment We investigated the possible environmental health implications of conjugated equine estrogens (CEEs), the most common components of HRT, including their discharge into the environment, their uptake, potency, and ability to induce biological effects in wildlife. Influents and effluents from four U.K. sewage treatment works (STWs), and bile of effluent-exposed fish, were screened for six ...
Pressure distribution between the deep digital flexor tendon and the navicular bone, and the effect of raising the heels in vitro. The objectives of this study were to topographically map pressure distribution across the palmar surface of the navicular bone in response to forces applied by the deep digital flexor tendon (DDFT), and evaluate the effect of raising the heels in vitro . A rig was constructed to hold an equine cadaver limb and apply controlled pressure to the navicular bone from the DDFT. Pressure-sensitive paper was used to quantify and map the resultant pressure. Changes in response to heel wedges (5 degrees and 10 degrees ) were recorded. The areas subject to maximum pressure from the DDFT were: the central...
In vitro and in vivo evaluation of ferric-hyaluronate implants for delivery of amikacin sulfate to the tarsocrural joint of horses. To assess the antimicrobial elution characteristics, toxicity, and antimicrobial activity of amikacin-impregnated ferric-hyaluronate implants (AI-FeHAI) for amikacin delivery to the tarsocrural joint of horses. Methods: Experimental study. Methods: AI-FeHAI implants, equine cartilage, and synovium, and horses (n=6). Methods: In vitro study: Five AI-FeHAI were placed in saline solution with daily replacement until implant degradation. Eluent was tested for amikacin concentration and bioactivity. Synovial and cartilage explants were incubated in the presence or absence of AI-FeHAI for 72 hours a...
Standardization and validation of Vero cell assay for potency estimation of diphtheria antitoxin serum. Diphtheria toxin has the capacity to block protein synthesis in cultured mammalian cells, and thus causing cell death. This capacity of diphtheria toxin was utilized for in-vitro neutralization test to determine antibody titer, using Vero cells, which have been found to be susceptible to diphtheria toxin. In the present study, a Vero cell assay was standardized and validated for potency estimation of diphtheria antitoxin serum (DATS). The results obtained by Vero cell assay were compared with in-vivo biological assay. High degree of correlation (+0.98) was found between in-vivo biological assa...
NMR and computational studies of stereoisomeric equine estrogen-derived DNA cytidine adducts in oligonucleotide duplexes: opposite orientations of diastereomeric forms. The equine estrogens equilin (EQ) and equilenin (EN) are the active components in the widely prescribed hormone replacement therapy formulation Premarin. Metabolic activation of EQ and EN generates the catechol 4-hydroxyequilenin (4-OHEN) that autoxidizes to the reactive o-quinone form in aerated aqueous solutions. The o-quinones react predominantly with C, and to a lesser extent with A and G, to form premutagenic cyclic covalent DNA adducts in vitro and in vivo. To obtain insights into the structural properties of these biologically important DNA lesions, we have synthesized site-specifically...
The uterine environment modulates trophectodermal POU5F1 levels in equine blastocysts. The reported patterns of trophectodermal expression of POU5F1 protein in blastocysts vary among species, and are possibly related to the differences in placental growth and function. This study investigated the pattern of embryonic POU5F1 expression in the horse, a species with delayed placental formation. Immature equine oocytes expressed POU5F1 protein in the cytoplasm and nucleus. Staining for POU5F1 protein in in vitro-produced (IVP) embryos decreased to day 5 of culture, then the nuclear staining increased to day 7. IVP day-7 to -11 blastocysts showed POU5F1 staining in nuclei throughout ...
