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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.
Nucleation rate determination by a concentration pulse technique: application on ferritin crystals to show the effect of surface treatment of a substrate. The nucleation of horse spleen ferritin (HSF) crystals on substrates was investigated using a new modification of the double pulse technique. The influence of three different structureless substrates (glass, glass covered by methyl groups and poly-L-lysin template) on the nucleation was studied. The boundaries in the phase-diagram, which separate zones of crystal nucleation and growth were obtained by keeping pH = 5.0, and using CdSO(4) as crystallizing agent. The steady-state nucleation rates were determined. The energy required for critical nuclei formation was evaluated (10(-13) erg) and th...
Correlation between the osmotic second virial coefficient and solubility for equine serum albumin and ovalbumin. The Haas - Drenth - Wilson (HDW) (Haas et al., 1999) theoretical model was used to correlate osmotic second virial coefficient (B) values with solubility (S) values for equine serum albumin (ESA) and ovalbumin for corresponding solution conditions. The best fit from the theoretical model was compared to experimental S versus B data. B values were experimentally measured using static light scattering. Solubilities of ESA were estimated using a sitting drop method. When the experimental data for S versus B were plotted, an excellent fit for ESA was obtained according to the HDW model. The result...
Presence of in vitro electrical activity in the ileum of horses with enteric nervous system pathology: equine dysautonomia (grass sickness). Equine dysautonomia (grass sickness) is a frequently fatal disease of horses characterised by intestinal stasis. Interstitial cells of Cajal (ICC) are the pacemakers and mediators of neurotransmission in the gastrointestinal tract. Impaired ICC-mediated control of motility has been implicated in intestinal disorders in laboratory mammals, humans and in equine grass sickness. The aim of this study was to compare the in vitro electrical properties of ileum from grass sickness cases with horses free from gastrointestinal disease. Intracellular microelectrode recordings were made from smooth muscl...
Selection of recently isolated colicinogenic Escherichia coli strains inhibitory to Escherichia coli O157:H7. Escherichia coli strains were screened for their ability to inhibit E. coli O157:H7. An initial evaluation of 18 strains carrying previously characterized colicins determined that only colicin E7 inhibited all of the E. coli O157:H7 strains tested. A total of 540 strains that had recently been isolated from humans and nine different animal species (cats, cattle, chickens, deer, dogs, ducks, horses, pigs, and sheep) were tested by a flip-plating technique. Approximately 38% of these strains were found to inhibit noncolicinogenic E. coli K12 strains. The percentage of potentially colicinogenic E...
Animal models for skin blistering conditions: absence of laminin 5 causes hereditary junctional mechanobullous disease in the Belgian horse. Recent achievements in the genetic correction of keratinocytes isolated from patients with junctional epidermolysis bullosa have paved the way to a gene therapy approach for the disease. Because gene therapy protocols require preclinical validation in animals, we have characterized spontaneous animal models of junctional epidermolysis bullosa. In this study we have elucidated the genetic basis of the hereditary junctional mechanobullous disease in the Belgian horse, a condition characterized by blistering of the skin and mouth epithelia, and exungulation (loss of the hoof). Immunofluorescence ...
Capacitation-like changes in equine spermatozoa throughout the cryopreservation process. Chlortetracycline (CTC) fluorescence staining analysis was used to investigate cryopreservation-induced capacitation-like changes in equine spermatozoa. Freshly ejaculated spermatozoa were found to display a high proportion of F pattern cells (uncapacitated; 93.6%) and a lower proportion of B pattern (capacitated; 5.4%) and AR pattern (acrosome-reacted; 1%) cells. Following cryopreservation in modified Kenney's medium, capacitation-like changes were observed. There was a significant increase in the proportion of spermatozoa displaying the B pattern (64.8%; P<0.001) and AR pattern (32.8%; P&...
The effect of frog pressure and downward vertical load on hoof wall weight-bearing and third phalanx displacement in the horse–an in vitro study. A shoe was designed to combine the advantages of a reverse shoe and an adjustable heart bar shoe in the treatment of chronic laminitis. This reverse even frog pressure (REFP) shoe applies pressure uniformly over a large area of the frog solar surface. Pressure is applied vertically upward parallel to the solar surface of the frog and can be increased or decreased as required. Five clinically healthy horses were humanely euthanased and their dismembered forelimbs used in an in vitro study. Frog pressure was measured by strain gauges applied to the ground surface of the carrying tab portion of t...
Effectiveness of an antagonist to gonadotrophin releasing hormone on the FSH and LH response to GnRH in perifused equine pituitary cells, and in seasonally acyclic mares. We wish to use a gonadotrophin-releasing hormone (GnRH) antagonist in the mare as a tool for investigating the control of the oestrous cycle. The aim of this study was to test the effectiveness of the antagonist cetrorelix by testing both in vitro, using perifused equine anterior pituitary cells, and in vivo in seasonally acyclic mares. Pituitary cells were prepared and after 3-4 days incubation, loaded onto columns and given four pulses of GnRH (at 0, 30, 60 and 90 min; dose-response study). After the second GnRH pulse, infusion of cetrorelix began (0, 100, 1000 and 2000 pmol/l) and continued...
Pregnancies from vitrified equine oocytes collected from super-stimulated and non-stimulated mares. The objectives were to compare embryo development rates after transfer into inseminated recipients, vitrified thawed oocytes collected from super-stimulated versus non-stimulated mares. In vivo matured oocytes were collected by transvaginal, ultrasound guided follicular aspiration from super-stimulated and non-stimulated mares 24-26 h after administration of hCG. Oocytes were cultured for 2-4 h prior to vitrification. Cryoprotectants were loaded in three steps before oocytes were placed onto a 0.5-0.7 mm diameter nylon cryoloop and plunged directly into liquid nitrogen. Oocytes were thawed and...
Effects of anti-arthritic drugs on proteoglycan synthesis by equine cartilage. The concentration-effect relationships of phenylbutazone, indomethacin, betamethasone, pentosan polysulphate (PPS) and polysulphated glycosaminoglycan (PSGAG), on proteoglycan synthesis by equine cultured chondrocytes grown in monolayers, and articular cartilage explants were measured. The effect of PSGAG on interleukin-1beta induced suppression of proteogycan synthesis was also investigated. Proteoglycan synthesis was measured by scintillation assay of radiolabelled sulphate (35SO4) incorporation. Polysulphated glycosaminoglycan and PPS stimulated proteoglycan synthesis in chondrocyte monolay...
A comparison of ultra-high-molecular weight polyethylene cable and stainless steel wire using two fixation techniques for repair of equine midbody sesamoid fractures: an in vitro biomechanical study. To compare the monotonic tensile and fatigue strength of 16-gauge stainless steel wire (SSW) to ultra-high-molecular weight polyethylene (UHMWPE) cable using a transfixed cerclage technique in an in vitro midbody sesamoid osteotomy model. Endoscopic modifications to Martins transfixed cerclage technique were developed. A new suture technique of fixation was compared with the transfixed cerclage technique by measuring gap formation after cyclic testing. Methods: An in vitro biomechanical paired equine cadaver limb study. Methods: Twenty-one paired cadaveric adult equine forelimbs. Methods: Unia...
Effects of a P2Y(12) receptor antagonist on the response of equine platelets to ADP. Comparison with human platelets. Horses show susceptibility to platelet-related disorders. Equine platelets differ from human platelets in some of their responses, so information available about human platelets must be validated in the horse. Aggregation of platelets by ADP involves both P2Y(1) and P2Y(12) receptors on the platelet surface. We have compared the effect of the P2Y(12) antagonist, AR-C67085, on equine and human platelets in vitro using turbidimetric aggregometry to measure the rate and final extent of aggregation. Aggregation profiles, concentration-response curves and pA(2) values show that the rate of aggregat...
Method for preparing thin sections of untreated equine hoof horn for electron microscopic examination. The preparation of hard tissues such as the equine hoof horn for electron microscopic examination is very difficult. In particular the penetration of fixatives and chemicals used during fixation and embedding is a problem. The objective of this study was to find and implement an alternative method enabling the preparation of high-quality thin sections of hoof horn and other hard tissue, which maintains the hard tissue ultrastructure and can be used for immuno-labeling. Compared to commonly used fixation and embedding techniques, the preparation of thin sections from untreated material method s...
Characterisation of the biological activity of recombinant equine eotaxin in vitro. The chemokine eotaxin (CCL11) is a key player in the trafficking of eosinophils to normal tissues and in the tissue eosinophilia associated with human allergic disease. We have recently cloned equine eotaxin and here we report the production of rEq eotaxin, with and without a C-terminal fusion peptide, in a novel expression system utilising stably transfected insect cells. rEq eotaxin induced equine eosinophil migration and superoxide production in vitro. A shape change in human eosinophils that could be blocked by 7B11, a monoclonal antibody against human CCR3, was also observed. Biological a...
Spectroscopic and electrochemical studies of horse myoglobin in dimethyl sulfoxide. This paper reports the first report of rapid, reversible direct electron transfer between a redox protein, specifically, horse myoglobin, and a solid electrode substrate in nonaqueous media and the spectroscopic (UV-vis, fluorescence, and resonance Raman) characterization of the relevant redox forms of myoglobin (Mb) in dimethyl sulfoxide (DMSO). In DMSO, the heme active site of metmyoglobin (metMb) appears to remain six-coordinate high-spin, binding water weakly. Changes in the UV-fluorescence spectra for metMb in DMSO indicate that the protein secondary structure has been perturbed and sugge...
An activator protein-1 complex mediates epidermal growth factor regulation of equine glycoprotein alpha subunit expression in trophoblast cells. Equids and primates are the only species known to express the placental hormone chorionic gonadotropin (CG). CG is a member of the heterodimeric glycoprotein family and is composed of an alpha subunit linked to a hormone-specific beta subunit. Previously, we have reported that epidermal growth factor (EGF) regulates the equine glycoprotein hormone alpha subunit promoter through a protein kinase C (PKC)/mitogen-activated protein kinase (MAPK) signal transduction pathway in trophoblasts. The current study investigates the regulatory element/factors involved in the induction of equine glycoprotei...
Effect of prolonged water immersion on equine hoof epidermis in vitro. To evaluate the effect of prolonged water exposure on tissue mass and solutes of outer and inner layers of the stratum medium, sole, frog, and the stratum medium (SMZA) zona alba layer of horses' hooves. SPECIMEN POPULATION: 10 hooves from 10 horses without foot abnormalities. Methods: Hoof wall tissue specimens were obtained and immersed for 10 days in distilled deionized water. Serial changes in mass were recorded during the immersion period. Subsequently, osmolarity and Na+, K, Cl-, and protein concentrations of the immersion solution were quantified. Results: Fully cornified outer hoof wal...
Effects of anti-arthritis preparations on gene expression and enzyme activity of cyclooxygenase-2 in cultured equine chondrocytes. To determine the effects of recombinant equine interleukin -1beta (reIL-1beta) and 4 anti-inflammatory compounds on the expression and activity of cyclooxygenase (COX)-2 in cultured equine chondrocytes. Methods: Articular cartilage from 9 young adult horses. Methods: Reverse transcriptase-polymerase chain reaction methods were used to amplify a portion of equine COX-2 to prepare a cDNA probe. Northern blot analysis was used to quantify the expression of COX-2 in first-passage cultures of equine articular chondrocytes propagated in media containing dexamethasone (DEX), phenylbutazone (PBZ), pol...
Equid herpesvirus 1 infection of endothelial cells requires activation of putative adhesion molecules: an in vitro model. Antisera to activated equine endothelial cells, which detected surface molecules of 116 kD, 97 kD, 42 kD and 38 kD, were made to investigate the role of endothelial adhesion molecules in equid herpes virus 1 infection. These putative adhesion molecules could be induced by 17-beta oestradiol, chorionic gonadotrophin, or IL-2, as well as by LPS and PWM. In an in vitro flow system, using equine veins or arteries, equid herpesvirus 1 in leucocytes was only transferred to infect endothelial cells if both leucocytes and endothelial cells expressed these surface molecules. Blocking of the membrane mo...
Evaluation of microbial culture techniques for the isolation of Pythium insidiosum from equine tissues. The purpose of this study was to evaluate the effects of sample handling, storage, and culture techniques on the isolation of Pythium insidiosum from infected equine tissues. Tissue and kunker samples obtained immediately posteuthanasia from a horse with subcutaneous pythiosis were used to assess the effects of sample type (kunkers vs. tissues), media type (selective vs. nonselective), storage technique, and storage time on P. insidiosum isolation rate. Overall, isolation rates were higher from fresh kunkers (94.6%) and stored kunkers (76.4%) than from fresh tissues (8.3%) or stored tissues (4...
Flow cytometry: clinical applications in equine medicine. The use of flow cytometry in veterinary diagnostics is becoming a valuable clinical tool with a broad range of applications. Physical characteristics of cells can be determined by the flow cytometer laser and electronics through the measurement of changes in light scatter properties. Other components and functions of cells can be defined through the application of fluorochrome dyes that have an affinity for cellular components. Traditionally, common clinical applications are immunophenotyping of cells of the hematopoietic system with fluorescent-labeled antibodies raised against specific cell ...
A PCR-RFLP for KIT associated with tobiano spotting pattern in horses. An MspI polymorphism was identified in intron 13 of the equine homologue of proto-oncogene c-kit (KIT) by comparing DNA sequences from horses with solid coat colour and horses homozygous for the tobiano spotting (To) gene. The allele associated with solid coat colour was designated KM0, while the allele associated with the tobiano pattern created an additional MspI restriction site and was designated KM1. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) studies using DNA from hair follicles demonstrated that all 129 of 129 tobiano patterned horses possessed the KM1...
Expression of interleukin-1 (IL-1) system genes in equine cumulus-oocyte complexes and influence of IL-1beta during in vitro maturation. A growing body of evidence suggests that the ovary is a site of inflammatory reactions, and thus, ovarian cells could represent sources and targets of the interleukin-1 (IL-1) system. The aim of the present work was to investigate the expression of IL-1alpha, IL-1beta, IL-1ra, IL-1R1, and IL-1R2 genes in equine cumulus cells and oocytes. Moreover, the influence of IL-1beta on in vitro maturation of cumulus-oocytes complexes (COCs) was examined. COCs were collected using ultrasound-guided follicular puncture in vivo. Oocytes and cumulus cells were isolated from preovulatory and subordinate foll...
Production of nuclear transfer horse embryos by Piezo-driven injection of somatic cell nuclei and activation with stallion sperm cytosolic extract. We investigated the use of direct nuclear injection using the Piezo drill and activation by injection of stallion sperm cytosolic extract for production of cloned equine embryos. When metaphase II horse oocytes were injected with either of two dosages of sperm extract and cultured 20 h, similar activation rates (88% vs. 90%) and cleavage rates (49% vs. 46%) were obtained. The successful reconstruction rate of horse oocytes with horse somatic cell donor nuclei after direct injection using the Piezo drill was 82%. Four dosages of sperm extract (containing 59, 176, 293, or 1375 microg/ml protein)...
Invasion, and short- and long-term survival of Babesia divergens (Phylum Apicomplexa) cultures in non-bovine sera and erythrocytes. In order to explore the feasibility of producing a Babesia divergens live vaccine free of bovine material contaminants the parasite's ability to grow in human, sheep and horse erythrocytes and serum and serum-free medium was investigated. B. divergens was successfully maintained in bovine erythrocytes overlaid with serum-free HL-1 medium. Supplementation of the culture medium with bovine or sheep serum improved parasite growth (monitored by measuring parasitaemia and uptake of tritiated hypoxanthine) whereas horse and human sera reduced parasite growth. As assessed by Giemsa's stained and FITC...
Evaluation of the influence of prostaglandin E2 on recombinant equine interleukin-1beta-stimulated matrix metalloproteinases 1, 3, and 13 and tissue inhibitor of matrix metalloproteinase 1 expression in equine chondrocyte cultures. To determine the effects of prostaglandin E2 (PGE2) on recombinant equine interleukin (IL)-1beta-stimulated expression of matrix metalloproteinases (MMP 1, MMP 3, MMP 13) and tissue inhibitor of matrix metalloproteinase 1 (TIMP 1) in vitro. Methods: Cultured equine chondrocytes. Methods: Stationary monolayers of first-passage chondrocytes were exposed to graduated concentrations of PGE2 with or without a subsaturating dose (50 pg/ml) of recombinant equine IL-1beta (reIL-1beta) to induce expression of MMP 1, MMP 3, MMP 13, and TIMP 1, followed by RNA isolation and northern blotting. In subseque...
Molecular characterization of the equine AEG1 locus. Acidic epididymal glycoprotein 1 (AEG1), also called cysteine-rich secretory protein 1 (CRISP1), is a member of the CRISP protein family which is characterized by 16 conserved cysteine residues at the C-terminus. The CRISP proteins are expressed in the male genital tract and are thought to be involved in sperm-egg fusion. Therefore, their genes are of interest as candidate genes for inherited male fertility dysfunctions and as putative quantitative trait loci for male fertility traits. In this report, the cloning and DNA sequence of 90 kb of horse genomic DNA from equine chromosome 20q22 conta...
Expression of a chemokine by ciliary body epithelium in horses with naturally occurring recurrent uveitis and in cultured ciliary body epithelial cells. To determine whether a chemokine (RANTES)-like protein expressed by ciliary epithelium plays a role in uveitis. Methods: 3 clinically normal horses intradermal, 5 eyes from 5 horses with recurrent uveitis, and 10 normal eyes from 5 age- and sex-matched horses. Methods: Cross-reactivity and sensitivity of recombinant human (rh)-regulated upon activation, normal T-cell expressed and secreted (RANTES) protein were evaluated in horses by use of intradermal hypersensitivity reactions and a chemotaxis assay. Aqueous humor and ciliary body of eyes from clinically normal horses and horses with uveitis...
Contribution of heme-propionate side chains to structure and function of myoglobin: chemical approach by artificially created prosthetic groups. Horse heart myoglobin was reconstituted with mesohemin derivatives methylated at the 6- or 7-position to evaluate the role of the heme-6-propionate or heme-7-propionate side chain in the protein. The association and dissociation of the O(2) binding for the deoxymyoglobin with 6-methyl-7-propionate mesoheme are clearly accelerated. Furthermore, the myoglobin with 6-methyl-7-propionate mesoheme shows fast autoxidation from oxymyoglobin to metmyoglobin compared to the myoglobin with 6-propionate-7-methyl heme and the reference protein. These results indicate the 6-propionate plays an important ph...
Two bi-allelic single nucleotide polymorphisms within the promoter region of the horse tumour necrosis factor alpha gene. Primers based on GenBank sequences within the 5' untranslated region (UTR) of the human and horse tumour necrosis factor alpha (TNF-alpha) genes were designed and used to amplify a 522-bp product. Sequencing of five clones derived from five independent PCRs obtained from three different animals of three different breeds (Old Kladruber, Akhal-Teke and Shetland Pony) revealed a high level of sequence identity to the TNF-alpha promoter regions of other species. The existing GenBank horse sequences were confirmed and extended upstream by 230 nucleotides. Based on the sequence obtained, a new horse...
