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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.
Use of an antineoepitope antibody for identification of type-II collagen degradation in equine articular cartilage. To develop an antibody that specifically recognizes collagenase-cleaved type-II collagen in equine articular cartilage. Methods: Cartilage specimens from horses euthanatized for problems unrelated to the musculoskeletal system. Methods: A peptide was synthesized representing the carboxy- (C-) terminus (neoepitope) of the equine type-II collagen fragment created by mammalian collagenases. This peptide was used to produce a polyclonal antibody, characterized by western analysis for reactivity to native and collagenase-cleaved equine collagens. The antibody was evaluated as an antineoepitope anti...
Effects of 8-epi-PGF2alpha on isolated bronchial smooth muscle of healthy and heaves-affected horses. 8-Epi-PGF2alpha, a prostaglandin-like compound generated by oxidative stress, has been shown to be an in vitro bronchoconstrictor in airways from healthy laboratory animals and healthy humans, but it has never been studied in diseased airways. Here, the bronchoconstrictive capacity of 8-epi-PGF2alpha on isolated bronchial rings (BR) of healthy and heaves-affected horses was evaluated by comparing the maximal effect and the potency of 8-epi-PGF2alpha to those of (1) acetylcholine (ACh), (2) its stereoisomer PGF2alpha and (3) its synthetic receptor agonist, U46619. Furthermore, the potential cap...
A biomechanical comparison of headless tapered variable pitch and AO cortical bone screws for fixation of a simulated lateral condylar fracture in equine third metacarpal bones. To compare drilling, tapping, and screw-insertion torque, force, and time for the 4.5-mm AO and 6.5-mm Acutrak Plus (AP) bone screws, and to compare the mechanical shear strength and stiffness of a simulated complete lateral condylar fracture of the equine third metacarpal bone (MC3) stabilized with either an AO or AP screw. Methods: In vitro biomechanical assessment of screw-insertion variables, and shear failure tests of a bone-screw-stabilized simulated lateral condylar fracture. Methods: Eight pairs of cadaveric equine MC3s Methods: Metacarpi were placed in a fixture and centered on a biax...
Inflammatory mediators induce endothelium-dependent adherence of equine eosinophils to cultured endothelial cells. Accumulation of equine eosinophils at sites of parasite infestation or allergic inflammation depends upon their adherence to vascular endothelial cells and subsequent migration through the endothelium and extracellular matrix. This study has examined whether cytokines, which cause endothelial cell-dependent eosinophil adherence in other species, and histamine and substance P, which increase adherence of equine eosinophils to protein coated plastic, induce equine eosinophil adherence to cultured equine digital vein endothelial cell (EDVEC) monolayers. The EDVEC monolayers were stimulated with r...
The effects of pyrantel tartrate on Sarcocystis neurona merozoite viability. Sarcocystis neurona is the etiologic agent of equine protozoal myeloencephalitis, a neurologic disease of horses. The present study was designed to test the hypothesis that pyrantel tartrate can kill S. neurona merozoites growing in equine dermal cell culture. Sarcocystis neurona merozoites were exposed to a range of concentrations of pyrantel tartrate or sodium tartrate ranging from 0.001 to 0.01 M. Merozoites were then placed onto equine dermal cell cultures and incubated for 2 weeks to check for viability. At 1 and 2 weeks after inoculation, plaque counts were compared between treatments an...
Activation of cumulus-free equine oocytes: effect of maturation medium, calcium ionophore concentration and duration of cycloheximide exposure. Two different culture media (TCM-199 and follicular fluid), two activation treatments (10 and 50 micromol calcium ionophore l(-1)) and three culture periods with cycloheximide were evaluated to find effective culture conditions for activation of cumulus-free equine oocytes. Oocytes were collected by scraping the follicle walls of ovaries obtained from an abattoir. Oocytes with expanded cumuli were matured at 38.2 degrees C in a humidified atmosphere of 5% CO(2) in air, in either TCM-199 with 10% fetal bovine serum (FBS) and 5 microU FSH ml(-1), or in 100% follicular fluid derived from a preovu...
Epidermal growth factor regulation of equine glycoprotein hormone alpha subunit expression in trophoblast cells. Primates and equids are the only species known to express the placental glycoprotein hormone, chorionic gonadotropin (CG), a heterodimeric glycoprotein composed of an alpha subunit linked to a hormone-specific beta subunit. The regulatory mechanisms involved in the induction of equine glycoprotein alpha subunit gene expression have not been identified. Epidermal growth factor (EGF) receptor is known to transduce signals that alter a number of different cellular functions (cell proliferation, differentiation, hormone secretion, and gene regulation). In the present study, we investigated the reg...
The effect of sodium heparin on equine articular cartilage. This study compared the effect of sodium heparin and gentamicin sulphate on equine articular cartilage (AC) explants in order to investigate the possible use of sodium heparin in the treatment of infectious arthritis. Six concentrations of sodium heparin and gentamicin sulphate were tested. The supernatant and explant digest were assayed for glycosaminoglycan (GAG) content with the dimethyl-methylene blue assay and the per cent loss of GAG was calculated. A significant (P< 0.001) increase in percentage GAG loss was noted for the sodium heparin groups when compared to the control, whilst no ...
Differential regulation of gelatinases by transforming growth factor beta-1 in normal equine chondrocytes. Cartilage destruction in osteoarthritis (OA) is associated with increased levels of several matrix metalloproteinases (MMPs), including the gelatinases MMP-2 and MMP-9. While increases in some MMPs may be destructive, up-regulation of others may result from increases in normal tissue turnover. The production of MMP-2 and MMP-9 by the anabolic transforming growth factor beta-1 (TGF-beta1) in normal equine chondrocytes was investigated. Methods: Equine chondrocytes from clinically normal femoropatellar joints were maintained in alginate beads. After serum deprivation, cells were exposed to TGF-b...
Evaluation of tear film proteinases in horses with ulcerative keratitis. Ulcerative keratitis is a common and potentially blinding ocular disease of horses, capable of progressing to corneal perforation in as little as 24 h. This rapid stromal degeneration is mediated in part by exogenous and endogenous proteinases. We measured and compared the concentrations of two matrix metalloproteinases (MMP-2 and MMP-9) and a serine proteinase (neutrophil elastase) present in the precorneal tear film of normal horses and horses with rapidly progressing ulcerative keratitis. Precorneal tear film samples were collected from 23 ulcerated and 21 unaffected eyes of 23 horses with ...
DH82 cells: a macrophage cell line for the replication and study of equine infectious anemia virus. In vivo, tissue macrophages have been implicated as an important cell for the replication of equine infectious anemia virus (EIAV). Laboratory investigations of EIAV/macrophage interactions, however, have been hampered by the laborious blood monocyte isolation procedures. In addition, adherent equine macrophage cultures generally have poor long-term viability and are resistant to transfection. This report describes an adherent canine macrophage-like cell line, DH82, that supports the replication of EIAV. This cell line was easily transfectable and supported EIAV Tat transactivation of the LTR....
Influence of co-culture during maturation on the developmental potential of equine oocytes fertilized by intracytoplasmic sperm injection (ICSI). The influence of co-culture with either oviduct epithelial cells or fetal fibroblast cells on in vitro maturation of equine oocytes and their potential for development to blastocysts and fetuses after intracytoplasmic sperm injection (ICSI) was investigated. The oocytes were obtained from ovaries from abattoirs and were matured in vitro for 28-30 h in TCM-199 only, or in TCM-199 co-culture with oviduct epithelial cells or fetal fibroblast cells. Metaphase II oocytes were subjected to ICSI with an ionomycin-treated spermatozoon. The injected oocytes were cultured for 7-9 days in Dulbecco's modi...
Characterization of expressed sequence tags generated from skin cDNA clones of Equus caballus by single pass sequencing. A cDNA library was built using RNA extracted from the skin tissue of an adult horse. The library was primed with oligo (dT) and sequences were directionally inserted in order to produce an expression library. The library has 5.8X 10(5) plaque forming units with 99.6% recombinant phage. The average insert size is 1.3 Kbp. Three hundred and thirteen expressed sequence tags (ESTs) were generated from sequencing of the 5 prime end of randomly selected skin cDNA clones. The ESTs were sequenced on an ABI 377 using Big-Dye chemistry. A similarity search was performed on each EST using the NCBI non-re...
Uterocalin, a lipocalin provisioning the preattachment equine conceptus: fatty acid and retinol binding properties, and structural characterization. The equine conceptus is surrounded by a fibrous capsule that persists until about day 20 of pregnancy, whereupon the capsule is lost, the conceptus attaches to the endometrium and placentation proceeds. Before attachment, the endometrium secretes in abundance a protein of the lipocalin family, uterocalin. The cessation of secretion coincides with the end of the period during which the conceptus is enclosed in its capsule, suggesting that uterocalin is essential for the support of the embryo before direct contact between maternal and foetal tissues is established. Using recombinant protein and ...
Biochemical characterization and surfactant properties of horse allergens. A new allergen from horse dander, Equ c 5 has been purified. Its biochemical and biophysical properties have been characterized and compared with those of Equ c 1, Equ c 2 and Equ c 4. Their molecular masses, determined by mass spectrometry, were 22 kDa for Equ c 1, 16 kDa for Equ c 2, 18.7 kDa for Equ c 4 and 16.7 kDa for Equ c 5. Their pI values were between 3.8 and 5.25. Equ c 2 and Equ c 5 are not glycosylated, while Equ c 4 contains a tri-antennary tri-sialylated N-linked glycan. Linkages of terminal N-acetylneuraminic acid to galactose were: alpha-(2-->6) in Equ c 4, and both alpha-(2...
Confocal microscopy of germinal vesicle-stage equine oocytes. The objectives were to compare cumulus type with nucleus form in equine cumulus oocyte complexes (COCs), to define the percentage of germinal vesicle (GV)-stage oocytes within a population of mares, and to further define GV nucleus shapes of equine oocytes. Cumulus types were as follows: 1) compact (56/208, 26.9%), 2) slightly expanded (37/208, 17.8%), 3) moderately expanded (27/208, 13.0%), 4) greatly expanded (15/208, 7.2%), or 5) denuded (73/208, 35.1%). One hundred thirty of 208 COCs (62.5%) were GV-stage, 21/208 (10.1%) were condensed chromatin-stage, 8/208 (3.8%) were polar body-stage, 4...
Increasing culture time from 48 to 96 or 144 hours increases the proportions of equine cumulus oocyte complexes with negative or fragmented nucleus morphology. The objective was to test the hypothesis that increasing equine oocyte culture time from 48 to 96 or 144 h increases nucleus maturation of equine oocytes. The hypothesis was not supported because condensed chromatin-stage oocytes decreased (P<0.01) from 33/126 (26.2%) at 48 h or 34/95 (35.8%) at 96 h to 11/117 (9.4%) at 144 h, and polar body-stage oocytes decreased (P<0.01) from 65/126 (51.6%) at 48 h to 25/95 (26.3%) at 96 h and (P<0.01) to 1/117 (0.9%) at 144 h. Negative (non-staining) oocytes increased (P<0.01) from 16/126 (12.7%) at 48 h or 15/95 (15.8%) at 96 h to 39/117 (33.3...
Effects of enrofloxacin and ciprofloxacin hydrochloride on canine and equine chondrocytes in culture. To study chondrotoxic effects of enrofloxacin (ENR) and ciprofloxacin hydrochloride (CFX) on canine and equine articular chondrocytes in culture and to compare the effects with that of cultivation in Mg2+-free medium. Methods: Chondrocytes from articular cartilage of 4- and 6 -month old dogs and 2- to 4- year-old horses. Methods: Chondrocytes were cultivated with 10, 40, 80, and 160 microg of CFX/ml, 10, 50, 100, and 150 microg of ENR/ml, or in Mg2+-free medium. A live-to-dead test was performed to test cytotoxic effects. Morphologic changes were evaluated by electron microscopy. An attachment...
An in vitro biomechanical comparison of two interlocking-nail systems for fixation of ostectomized equine third metacarpal bones. To compare the mechanical properties of 2 interlocking-nail systems for fixation of ostectomized equine third metacarpi (MC3): (1) a standard interlocking nail with 2 parallel screws proximal and distal to a 1-cm ostectomy; and (2) a modified interlocking nail with 2 screws proximal and distal to a 1-cm ostectomy with the screws offset by 30 degrees. Methods: Twelve pairs of adult equine forelimbs intact from the midradius distally. Methods: Twelve pairs of equine MC3 were divided into 2 test groups (6 pairs each): torsion and caudocranial 4-point bending. Standard interlocking nails (6-hole, ...
Generation of reactive oxygen species by equine spermatozoa. To characterize generation of reactive oxygen species (ROS) by equine spermatozoa. Methods: Multiple semen samples collected from 9 stallions. Methods: Equine spermatozoa were separated from seminal plasma on a discontinuous polyvinylpyrrolidone (PVP)-coated silica gradient and resuspended in a modified Tyrode albumin-lactate-pyruvate medium. Amount of hydrogen peroxide (H2O2) generated was assayed by use of a 1-step fluorometric assay, using 10-acetyl-3,7-dihydroxyphenoxazine as a probe for detection of H2O2 in a microplate assay format. Concentration of H2O2 was determined by use of a fluore...
New approaches for validation of lethal phenotypes and genetic reversion in Helicobacter pylori. Because of limited genetic tools for use in Helicobacter pylori, tests routinely applied in other bacteria for demonstrating a gene's role in viability and other phenotypes have not been applied to this organism. In a mutational study of putative response regulator genes, we aimed to develop such tools for H. pylori. Methods: We attempted to mutate five response regulator genes by allelic exchange insertional mutagenesis. For genes that yielded no viable mutants, a second copy of the gene was inserted into the chromosome via a suicide vector, and it was seen if providing the second copy would ...
Effects of sodium citrate, low molecular weight heparin, and prostaglandin E1 on aggregation, fibrinogen binding, and enumeration of equine platelets. To investigate the effects of sodium citrate, low molecular weight heparin (LMWH), and prostaglandin E1 (PGE1) on aggregation, fibrinogen binding, and enumeration of equine platelets. Methods: Blood samples obtained from 4 Thoroughbreds. Methods: Blood was collected into syringes in the ratio of 9 parts blood:1 part anticoagulant. Anticoagulants used were sodium citrate, LMWH, sodium citrate and LMWH, or 300 nM PGE1/ml of anticoagulant. Platelet aggregation in response to ADP, collagen, and PGE1 was assessed, using optical aggregometry. Platelet activation was evaluated, using flow cytometry, ...
Embryo production by ovum pick up from live donors. Embryo production by in vitro techniques has increased steadily over the years. For cattle where this technology is more advanced and is applied more, the number of in vitro produced embryos transferred to final recipients was over 30,000 in 1998. An increasing proportion of in vitro produced embryos are coming from oocytes collected from live donors by ultrasound-guided follicular aspiration (ovum pick up, OPU). This procedure allows the repeated production of embryos from live donors of particular value and is a serious alternative to superovulation. Ovum pick up is a very flexible technique...
Characterization of the Oregon isolate of Neospora hughesi from a horse. Neospora hughesi was isolated in cell cultures inoculated with homogenate of spinal cord from a horse in Oregon. Tachyzoites of this Oregon isolate of N. hughesi were maintained continuously by cell culture passage and tachyzoites were infective to immunosuppressed mice. Gamma interferon gene knockout (KO) mice injected with tachyzoites developed fatal myocarditis and numerous tachyzoites were seen in lesions. Gerbils (Meriones unguiculatus) inoculated with tachyzoites developed antibodies (> or = 1:500) as indicated by the Neospora caninum agglutination test but did not develop clinical si...
Adaptation of the hypoosmotic swelling test to assess functional integrity of stallion spermatozoal plasma membranes. Hypoosmotic swelling (HOS) is used for assessing plasma membrane function and fertilizing capacity of human spermatozoa. However, HOS solutions and methodologies have not been evaluated specifically for assessing stallion spermatozoa. The objective of this study was to identify a HOS solution and assay conditions specifically for stallions that would maximize spermatozoal plasma membrane swelling. The HOS solutions and assay conditions, including incubation time (15 to 180 min), temperature (25 degrees vs 37 degrees C), and total number of cells examined (100, 200 or 500) were evaluated. Assay...
Lipid fatty acid and protein pattern of equine prostasome-like vesicles. The semen of several mammals contains vesicles of different composition and origin. We have recently reported on the presence of lipoprotein vesicles in stallion semen. To a certain extent, these resemble human prostasomes, but differ from them in amount and composition. These horse-semen prostasome-like vesicles may be important, not only in horse reproductive physiology, but also in view of stallion semen cryopreservation. In this paper, we have studied horse-semen prostasome-like vesicles and found that they possess less saturated fatty acid than human prostasomes. Moreover, their protein p...
West Nile virus recombinant DNA vaccine protects mouse and horse from virus challenge and expresses in vitro a noninfectious recombinant antigen that can be used in enzyme-linked immunosorbent assays. Introduction of West Nile (WN) virus into the United States in 1999 created major human and animal health concerns. Currently, no human or veterinary vaccine is available to prevent WN viral infection, and mosquito control is the only practical strategy to combat the spread of disease. Starting with a previously designed eukaryotic expression vector, we constructed a recombinant plasmid (pCBWN) that expressed the WN virus prM and E proteins. A single intramuscular injection of pCBWN DNA induced protective immunity, preventing WN virus infection in mice and horses. Recombinant plasmid-transform...
In vitro evidence for effects of magnesium supplementation on quinolone-treated horse and dog chondrocytes. Quinolones and magnesium deficiency cause similar lesions in joint cartilage of young animals. Chondrocytes cultivated in the presence of quinolones and in Mg-free medium show severe alterations in cytoskeleton and decreased ability to adhere to the culture dish. We investigated whether Mg2+ supplementation can prevent quinolone-mediated effects on chondrocytes in vitro. Chondrocytes cultivated in Dulbecco's modified Eagle's medium/HAM's F-12 medium were treated with ciprofloxacin (80 and 160 microg/ml) and enrofloxacin (100 and 150 microg/ml). Mg2+ was added at a concentration of 0.0612 mg/ml...
Cytokeratins of the matrices of the chestnut (torus carpeus) and periople in horses with acute laminitis. To determine whether there is a change in the expression of cytokeratins in the epidermal cells of the non-weight-bearing parts of the limb in horses with acute laminitis and thus determine whether the morphologic changes that develop in the periople and chestnut (torus carpeus) of horses early in acute laminitis are caused by inhibition of keratinocyte differentiation. Methods: 8 horses with acute laminitis. Methods: Tissue specimens were obtained from the chestnuts of all 8 horses and from the stratum externum of the hoof wall of 3 horses. Tissue specimens were obtained within 48 hours of th...
Variations in cartilage catabolism in different equine joints in response to interleukin-1 in vitro. An explant system was used to investigate the hypothesis that cartilage from different equine joints might respond differently to challenge with interleukin-1alpha (IL-1alpha). Pairs of normal cartilage samples were taken from the metacarpophalangeal, proximal interphalangeal and distal interphalangeal joints of six horses. One of each pair was stimulated with 10 ng/ml human recombinant IL-1alpha for three days, and the supernatants and remaining cartilage explants were analysed for their total content of glycosaminoglycans. A significantly higher percentage of glycosaminoglycans was released ...
