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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.
Effects of tendon grip technique (frozen versus unfrozen) on in vitro surface strain measurements of the equine deep digital flexor tendon. To determine effects of tendon grip technique on in vitro surface strain measurements of equine deep digital flexor tendon (DDFT) when loaded in tension. Methods: 12 hind limb DDFT from 8 adult horses (mean age, 9.8 years [range, 4.5 to 17 years]; mean body weight, 472 kg [range, 450 to 509 kg]), with no clinical evidence of hind limb lameness. Methods: After calibration, liquid mercury strain gauges were sutured to plantar surfaces of the tendons at distal (position 1), middle (position 2), and proximal (position 3) metatarsal regions. Each tendon was affixed to a materials testing machine (d...
Cytofluorescent assay to quantify adhesion of equine spermatozoa to oviduct epithelial cells in vitro. To facilitate the study of interactions between equine spermatozoa and homologous oviduct epithelial cells, we developed an assay to count labelled spermatozoa bound to oviduct epithelial cell (OEC) monolayers and used the assay to compare the binding ability of spermatozoa from different stallions. Washed spermatozoa from three stallions were incubated with the fluorochrome Hoechst 33342 (5 micrograms/ml) for 1 min. Spermatozoa were then layered over confluent monolayers of oviduct epithelial cells in 2 cm2 culture wells. Coculture treatments comprised five concentrations of spermatozoa (10(5...
The vasomotor effects of 5-hydroxytryptamine on equine basilar arteries in vitro. The vasomotor effects of 5-hydroxytryptamine (5-HT) on isolated equine basilar arteries were studied. 5-HT induced contractions of equine basilar arteries in a concentration-dependent manner, with a pEC50 value (with 95% confidence limits) of 7.35 (7.08-7.62). Similar results were obtained with endothelium-denuded basilar arteries. Contractions were not competitively inhibited by the 5-HT2 receptor antagonist ketanserin at low concentrations of 5-HT. Conversely, at high concentrations of 5-HT, contractions were inhibited by ketanserin in a concentration-dependent manner, with a pA2 value of 8....
An in vitro biomechanical study of a multiplanar circular external fixator applied to equine third metacarpal bones. The biomechanical characteristics of a 4-ring circular multiplanar fixator applied to equine third metacarpal bones with a 5 mm mid-diaphyseal osteotomy gap were studied. Smooth Steinmann pins, either 1/8 inch, 3/16 inch, or 1/4 inch, were driven through pilot holes in the bone in a crossed configuration and full pin fashion and fastened to the fixator rings using cannulated fixation bolts. The third metacarpal bone fixator constructs were tested in three different modes (cranial-caudal four-point bending, axial compression, and torsion). Loads of 2,000 N were applied in bending and axial comp...
The inhibition of adenylate cyclase in equine platelets by collagen and by platelet-activating factor. Equine platelet aggregation was stimulated by collagen fibres or platelet-activating factor. The action of both ligands was blocked by forskolin or prostaglandin E(1) agents which are known to activate adenylate cyclase. Equine platelet membranes were found to contain adenylate cyclase activity which was inhibited in dose-dependent fashion by both collagen and platelet-activating factor. Platelet-activating factor-induced inhibition was antagonised by WEB2086.
Effect of growth factors on the characteristics of cells associated with equine wound healing and sarcoid formation. Wound healing in equidae is delayed and more complicated than in other species. These complications arise from a condition known as exuberant granulation tissue formation. The lower limb of the horse is frequently slower to heal than other parts of the body and has a particular tendency to produce excess (exuberant) granulation tissue. Sarcoids are tumor-like lesions of the skin which often appear at the site of wounds. This study compared the growth characteristics of the sarcoid and granulation tissue-derived cells with normal dermal fibroblasts grown from primary cell cultures. All three ce...
[Reproductive medicine in transition: new developments in embryo transfer]. Successful application of embryo transfer (ET) has become common practice in cattle, horses, sheep, goats and a variety of other species held in captivity. Yet in cattle only has the technique been established commercially. In 1994 more than 100,000 bovine embryos have been transferred in European countries. Important progress in transvaginal ovum pick up (OPU), in vitro production (IVP) and cryopreservation have further improved the applicability of ET. Direct transfer simplifies the procedure considerably allowing individual transfers and eliminating the need of synchronizing recipients. In ...
Biosynthesis and distribution of leucocyte elastase inhibitor. Production of recombinant inhibitor. The horse leucocyte elastase inhibitor (HLEI), present in neutrophils, monocytes and bone marrow cells, is apparently a cytoplasmic protein which is not released from cells even in response to stimulation with lipopolysaccharide, phorbol ester, tumour necrosis factor alpha, interleukin-1 or elastin degradation products. Although no expression of the inhibitor was detected in neutrophils, both monocytes and bone marrow cells were efficient in its synthesis. Using a new expression vector pREST5d, recombinant inhibitor was produced in a large quantity in a soluble form, with a yield of 88 mg per ...
Sequence variability of Borna disease virus open reading frame II found in human peripheral blood mononuclear cells. A cDNA fragment of the Borna disease virus (BDV) open reading frame II (ORF-II), which encodes a 24-kDa phosphoprotein (p24 [P protein]), was amplified from total RNA of peripheral blood mononuclear cells (PBMC) from three psychiatric inpatients. The amplified cDNA fragments were cloned, sequenced, and analyzed. A total of 15 clones, 5 from each patient, were studied. Intrapatient divergencies of the BDV ORF-II nucleotide sequence were 4.2 to 7.3%, 4.8 to 7.3%, and 2.8 to 7.1% for the three patients, leading to differences of 7.7 to 14.5%, 10.3 to 17.1%, and 6.0 to 16.2%, respectively, in the ...
The synthesis of 19-norandrostenedione from dehydroepiandrosterone in equine placenta is inhibited by aromatase inhibitors 4-hydroxyandrostenedione and fadrozole. 19-Norandrostenedione was synthesized in vitro from dehydroepiandrosterone by explants of equine full-term placenta. The synthesis of 19-norandrostenedione was inhibited by two specific aromatase inhibitors, 4-hydroxyandrostenedione and fadrozole.
The dose-related effects of phenylbutazone and a methylprednisolone acetate formulation (Depo-Medrol) on cultured explants of equine carpal articular cartilage. The dose-related effects of phenylbutazone and Depo-Medrol on chondrocyte viability and chondrocyte-mediated synthesis and depletion of proteoglycans were investigated using cultured explants of equine middle carpal joint articular cartilage. Explants from 12 horses (941 x 3 mm diameter) were cultured for a total of 5 days, which included 3 days' exposure to either phenylbutazone (0, 2, 20, 200 or 2000 micrograms/mL) or Depo-Medrol (0, 20, 200 or 2000 micrograms/mL). For each explant, amino sugar content was used as a measure of proteoglycan content, 35S incorporation as a measure of the rate ...
Effect of coculture with stallion spermatozoa on de novo protein synthesis and secretion by equine oviduct epithelial cells. Adhesion of equine spermatozoa to homologous oviduct epithelial cells (OEC) in vitro results in specific changes in spermatozoa and OEC function. To test the hypothesis that adhesion of spermatozoa affects protein synthesis and secretion by OEC, the following treatment groups were established in culture: OEC with culture medium only; control spermatozoa in culture medium only; OEC in coculture with spermatozoa; and OEC and spermatozoa in coculture, but physically separated by a microporous membrane. The experiment was replicated within each of 4 ejaculates from 3 stallions. De novo protein sec...
Evidence for endothelium-derived relaxing factor/nitric oxide in equine digital arteries. To test the hypothesis that endothelium-derived nitric oxide modulates vasomotor reactivity in equine digital arteries. Methods: Digital arteries were isolated from adult horses, and their vasodilator properties were examined in an in vitro controlled environment. Methods: Five adult horses (1 gelding, 4 mares) without evidence of hoof or vascular disease were studied. Methods: Arterial rings with or without endothelium were exposed to endothelium-dependent vasodilator drugs in the presence or absence of a pharmacologic inhibitor of the enzyme nitric oxide synthase. Results: Vasodilator effect...
Abnormalities in oxygenation, coagulation, and fibrinolysis in colonic blood of horses with experimentally induced strangulation obstruction. To measure arterial and venous blood gas, coagulation, and fibrinolysis variables in blood from isolated segments of control and ischemic large colons for the purpose of identifying variables for rapid, indirect assessment of colonic mucosal injury. Methods: Variables were determined at specific intervals during the 4-hour study (3 hours of ischemia and 1 hour of reperfusion). Methods: Seven clinically normal horses between 2 and 15 years old. Methods: Horses underwent laparotomy and occlusion of the lumen and vasculature of the mid-portion of the pelvic flexure of the large colon. During isch...
Effect of various extenders and taurine on survival of stallion sperm cooled to 5 degrees C. Stallion semen was diluted in five different extenders (dimitro-poulus onze (Dimitro's), Kenney's modified tryode (Kenney's), modified INRA82 (INRA82), egg yolk-citrate-taurine (Citrate) and EZ-Mixin) and evaluated for motility after cooling and storage at 5 degrees C for 0, 24, 48, 72 and 96 h. EZ-Mixin extender was used as control while 70 and 100 mM of taurine were added to Dimitro's, Kenney's and INRA82 to study its effect under conditions of storage at 5 degrees C and varying processing modifications. Motility in INRA82 was 57.0, 58.4, 61.1, and 56.1% after 24, 48, 72 and 96 h, respective...
Sonographic-anatomic correlation and imaging protocol for the kidneys of horses. Sonographic and anatomic observations were made of the kidneys of 23 Thoroughbreds or Standardbreds. In an in vitro study of 16 horses, precise correlations were established between the gross anatomic features of the kidneys and their sonographic appearance in images obtained in dorsal, sagittal, transverse, and transverse oblique anatomic planes. The renal cortex had a uniformly mottled echogenicity, and the renal medulla was relatively hypoechogenic, compared with the cortex. Acoustic anisotropy was observed in the cortex and medulla of the cranial and caudal extremities of each kidney. The ...
Effects of hydrogen peroxide on isolated trachealis muscle of horses. During acute bouts of recurrent airway obstruction (heaves) in horses, neutrophils that are capable of increased production of reactive oxygen species accumulate in the airways. In the study reported here, the effect of hydrogen peroxide (H2O2; 1 microM to 0.1M), one of these reactive oxygen species products, on the responses of isolated trachealis muscle of horses was determined. Before and after incubation with H2O2, contractile responses to acetylcholine, electrical field stimulation (EFS), 127 mM KCl, and relaxation responses to isoproterenol and activation of the nonadrenergic noncholiner...
Initiation of transcription and nucleologenesis in equine embryos. The time of activation of the embryonic genome (maternal-embryonic transition) in equine embryos was investigated by assessing incorporation of 3H-uridine and nucleolar development. In Experiment 1, embryos were recovered from the oviduct (n = 15) and the uterus (n = 3). Recovered embryos were assessed for morphologic development and quality score. Recovered embryos with less than 8 cells (two cells, n = 4; four cells, n = 5; five cells, n = 2) were incubated with 3H-uridine (560 microCi/ml) for 10 hr, while eight-cell embryos (n = 2), morulae (n = 2), and blastocysts (n = 3) were incubated wi...
Protein tyrosine phosphorylation in equine platelets: the effect of stimulation by thrombin and platelet-activating factor (PAF). Protein tyrosine phosphorylation (PTP) in thrombin- and platelet-activating factor (PAF)-stimulated equine platelet activation was investigated in the absence and presence of 2 protein tyrosine kinase inhibitors (PTKIs), methyl 2,5-dihydroxycinnamate (MDHC) and genistein. Washed equine platelets aggregated irreversibly in response to thrombin or PAF in an agonist concentration dependent fashion. MDHC produced an MDHC concentration and time dependent inhibitory effect on rate and extent of thrombin- and PAF-induced aggregations, whereas the effect of genistein on the same parameters was only ge...
Demonstration of tissue-specific promoters in nonprimate species that express aromatase P450 in placentae. Conversion of androgens to estrogens is catalyzed by aromatase P450 (P450arom; the product of the CYP19 gene). Regulation of tissue-specific expression of P450arom in humans is due, in part, to alternative transcriptional start sites that arise as a consequence of the use of granulosa cells and placental tissue from cows, horses, and pigs (ungulates) in order to determine whether these species, like the human, utilize tissue-specific promoters to drive P450arom expression. The majority of transcripts in the placenta have 5'-termini that differ from those in the ovary upstream of a common site ...
Cryopreservation reduces the ability of equine spermatozoa to attach to oviductal epithelial cells and zonae pellucidae in vitro. Two bioassays were used to evaluate the interaction of fresh and cryopreserved equine semen with oviductal epithelial cells (OEC) and with the zona pellucida (ZP). Split ejaculates were either stored at room temperature or frozen and thawed. In experiment 1, progressive motility and membrane integrity were evaluated for each treatment. Fluorescent labeled spermatozoa were cocultured with monolayers of OEC for 30 minutes, and the number of sperm attached to OEC was counted by fluorescence microscopy and analysis of digitized images. Motility of spermatozoa attached to OEC was observed at 0.5, 3...
Molecular cloning of equine interleukin-1 alpha and -beta cDNAs. Equine interleukin-1 alpha (IL-1 alpha) and IL-1 beta were molecularly cloned to establish a basis for research on inflammatory and immune responses in the horse. Equine peripheral blood mononuclear cells (PBMC) were stimulated with lipopolysaccharide (LPS), and cDNA clones of equine IL-1 alpha and IL-1 beta covering the whole coding sequences were isolated from them. These equine IL-1 alpha and IL-1 beta clones contained open reading frames encoding 271 and 269 amino acids, respectively. The deduced amino acid sequence of equine IL-1 alpha showed 71.6% and 60.2% similarity with that of human ...
Sex diagnosis of equine preimplantation embryos using the polymerase chain reaction. A rapid and reliable method for sex determination of preimplantation-stage equine embryos has not been available. The aim of the present study was to find an enzyme which would distinguish sexes in the horse by finding a polymorphic restriction site between the ZFY and ZFX homologues amplified by the polymerase chain reaction (PCR). Altogether, 38 different restriction enzymes were tested using female and male DNA extracted from blood. The primers used for amplification were selected from conserved sequences between human ZFY and ZFX genes and mouse Zfy-1 and Zfy-2 genes. Nine enzymes cut the ...
Relation between stallion sperm binding to homologous hemizonae and fertility. The hemizona assay (HZA) has been developed as a diagnostic test to predict the fertilisation potential of human spermatozoa. The aim of this study was to develop an HZA for stallion spermatozoa and to investigate a possible relationship between fertility and the outcome of the HZA in this species. Equine oocytes were obtained from ovaries collected at a slaughterhouse and by transvaginal, ultrasound-guided follicle aspiration. They were then denuded from cumulus cells and stored in salt solution at 4 degrees C until use. On the day of the experiments the oocytes were bisected, thus providing ...
Molecular cloning of DNA for inhibin alpha-subunit from equine ovary. cDNA encoding equine inhibin alpha-subunit precursor protein was isolated from an equine ovarian cDNA library. For screening, the DNA probe was amplified by the RT-PCR using primers designed based on the rat inhibin alpha-subunit cDNA sequence. Out of 1.2 x 10(5) plaques screened, 19 positive clones were isolated, and one of these clones (Eq-alpha-11) contained a complete open reading frame encoding 367 amino acids. The similarity of the deduced amino acid sequences of both equine inhibin alpha-subunit precursor protein and the mature protein were greater than 80% to those of other six mammali...
Metalloproteinase activity has a role in equine chorionic girdle cell invasion. Chorionic girdle cells are a highly invasive subpopulation of trophoblast cells of the equine conceptus. By Day 35 (Day 0 = day of ovulation), cells of the chorionic girdle adhere to the uterine epithelium and begin to invade the endometrial wall. Invasive cells must attach to extracellular matrix proteins, secrete proteinases capable of degrading matrix, and migrate through the degraded matrix; invasion is largely dependent on the proteinase activity of the cells. The objective, therefore, was to develop an in vitro system to examine the mechanisms of equine chorionic girdle cell invasion thr...
Ultrasonography as a method to determine tendon cross-sectional area. Ultrasonographic cross sectional area (CSA) measurements of equine superficial digital flexor (SDF) tendon were obtained to determine the feasibility of ultrasonography for CSA measurement of tendon in vivo and in vitro. Ultrasonographic measurements were compared with a more traditional CSA measurement method, ink-blot analysis. In addition, values for ultrasonographic SDF tendon mean echogenicity were obtained in vivo and in vitro. The left forelimb SDF tendons of 23 horses were evaluated ultrasonographically. Cross sectional images were acquired at 4-cm intervals distal to the base of the a...
Antibacterial susceptibility patterns for microbial isolates associated with infectious keratitis in horses: 63 cases (1986-1994). Seventy-three aerobic bacterial isolates were cultured from 64 eyes of 63 horses with infectious keratitis. Forty-two (58%) of the organisms isolated initially were gram-positive (g+, 10 genera) and 31 (42%) were gram-negative (g-, 5 genera). After local antimicrobial treatment, repeat cultures from samples obtained from 15 eyes of hospitalized horses yielded 21 secondary bacterial isolates. Staphylococci spp and Streptococci spp were the most common g(+) isolates and accounted for 79% of g(+) organisms isolated initially. Antibiograms revealed ticarcillin to be the most efficacious antibiotic...
Effect of different protein supplements on motility and plasma membrane integrity of frozen-thawed stallion spermatozoa. Three experiments were conducted to evaluate the effect of different macromolecule components (egg yolk, skim milk, and BSA) in a widely employed extender for cryopreservation of horse semen. Spermatozoal motility (MOT) and the percentage of spermatozoa with an intact plasma membrane (IPM) were evaluated in frozen-thawed samples. In the first experiment (four Draft Horse stallions, four ejaculates each) a standard freezing extender containing 20% whole egg yolk was modified by replacing extender components (glucose-EDTA solution, 11% lactose solution) with an increasing volume of a skim milk d...
The unfolding thermodynamics of c-type lysozymes: a calorimetric study of the heat denaturation of equine lysozyme. The energetics of the temperature-induced unfolding of equine lysozyme was studied calorimetrically and compared with that of two structurally homologous proteins: hen egg white lysozyme and alpha-lactalbumin. The structure of each of these proteins is characterized by the presence of a deep cleft that divides the molecule into two regions called the alpha and beta domains. In equine lysozyme and alpha-lactalbumin the latter domain specifically binds Ca2+. It is shown that, in contrast to hen egg white lysozyme in which the alpha and beta domains unfold as a single cooperative unit, in equine ...
