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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.
In vivo activation of equine eosinophils and neutrophils by experimental Strongylus vulgaris infections. Eosinophils and neutrophils from ponies with Strongylus vulgaris-induced eosinophilia (eosinophilic ponies; activated eosinophils and neutrophils) were assayed in vitro for chemotactic and chemokinetic responses to zymosan-activated serum (ZAS) using the filter system in Boyden chambers, for Fc and complement (C) receptors using the EA and EAC-rosette assays, respectively, and for phagocytic and bactericidal activities using opsonized Escherichia coli and the acridine orange method. The responses of activated eosinophils and neutrophils in the above assays were compared with those of eosinophi...
Effect of phenytoin on the clinical signs and in vitro muscle twitch characteristics in horses with chronic intermittent rhabdomyolysis and myotonia. In vitro twitch characteristics of the semimembranosus muscle were evaluated in 9 clinically normal horses, in 15 horses with chronic intermittent rhabdomyolysis (CIR) and in 2 horses with myotonia. Effects of phenytoin on in vitro muscle twitch and clinical signs of CIR and myotonia were evaluated in these same horses. Times to 90% relaxation were prolonged in the horses with CIR (mean +/- SEM, 186 +/- 5.9 ms) and in 2 horses with myotonia (197 and 177 ms) compared with those in clinically normal horses (mean +/- SEM, 146 +/- 2.1 ms). Horses with CIR also had significantly (P less than 0.05) ...
Prokinetic effects of cisapride, naloxone and parasympathetic stimulation at the equine ileo-caeco-colonic junction. The electromyogram of the terminal ileum, the caecum and the proximal right ventral colon was recorded in fasted conscious ponies receiving intravenously equiactive doses of pilocarpine (0.05 mg/kg) and carbachol (0.01 mg/kg) as acetylcholine analogues; cisapride (0.1 mg/kg) and metoclopramide (2 mg/kg) facilitating acetylcholine release from myenteric neurones and naloxone (0.05 mg/kg) as an antagonist of the endogenous inhibitory opioid system. Both cisapride and naloxone induced typical migrating spike bursts in the colon associated with contractions of caecal body and caecal base. Both pil...
Pharmacokinetics, bioavailability, and in vitro antibacterial activity of rifampin in the horse. The pharmacokinetics and bioavailability of rifampin were determined after IV (10 mg/kg of body weight) and intragastric (20 mg/kg of body weight) administration to 6 healthy, adult horses. After IV administration, the disposition kinetics of rifampin were best described by a 2-compartment open model. A rapid distribution phase was followed by a slower elimination phase, with a half-life (t1/2[beta]) of 7.27 +/- 1.11 hours. The mean body clearance was 1.49 +/- 0.41 ml/min.kg, and the mean volume of distribution was 932 +/- 292 ml/kg, indicating that rifampin was widely distributed in the body....
Monoclonal antibody recognizes a conformational epitope in a random coil protein. The antigenic determinants for two monoclonal antibodies directed against horse apo-cytochrome c, a protein of disordered structure, as judged by spectroscopic and hydrodynamic criteria, have been studied by a combination of methods: antigen competition in solution by radio immunoassay and enzyme-linked immunoassay, and differential acetylation of free and antibody-bound antigen. In the latter method the accessibility of lysine residues of the antigen in the antigen-antibody complex is compared to the accessibility in the free antigen. The two antibodies against the heme-free protein do not re...
Opsonins in uterine washings influencing in vitro activity of equine neutrophils. Uterine washings were found to promote neutrophil mediated killing of Streptococcus zooepidemicus. Depletion of complement and/or specific antibody from the washings significantly reduced bactericidal activity. Phagocytosis of yeast by uterine washings was complement dependent. Inhibition of the classical pathway significantly reduced opsonic activity indicating that, in addition to direct activation via the alternate pathway, antibody may also be involved in yeast phagocytosis.
Use of a monoclonal antibody to evaluate integrity of the plasma membrane of stallion sperm. Transmission electron microscopy was used to confirm that a monoclonal antibody (F79.3E2; class IgG1 kappa) was specifically localized to an antigen in the acrosomal ground substance of stallion sperm. This antibody was used to develop and validate an indirect immunofluorescent procedure to evaluate integrity of the plasma-acrosomal membranes of stallion sperm. The concept was that primary monoclonal antibody would be "shielded" from its acrosomal antigen by an intact plasma membrane. Conversely, sperm with damaged plasma-acrosomal membranes would exhibit green acrosomal fluorescence when view...
Endotoxin-induced procoagulant activity in equine peripheral blood monocytes. Increasing evidence has demonstrated the importance of monocyte procoagulant activity (PCA) in the pathogenesis of coagulopathies in a variety of diseases. Because endotoxin precipitated coagulopathies are common sequelae to intestinal ischemia/endotoxemia in the equine species, we investigated the ability of equine peripheral blood monocytes to express PCA. Monocytes isolated from five healthy adult horses were incubated in vitro with Escherichia coli endotoxin (10 micrograms), and the PCA was measured by the ability of cellular lysates to accelerate the clotting times of equine plasma in a m...
In-vitro and in-vivo responsiveness of the corpus luteum of the mare to gonadotrophin stimulation. Dispersed horse luteal cells were used to evaluate the ability of horse LH, hCG and PMSG to stimulate progesterone secretion in vitro. Morphological characterization of these cells before gonadotrophin stimulation indicated the presence of two populations of cells based on cell diameters. In luteal cells incubated as suspended cells, horse LH and hCG stimulated (P less than or equal to 0.05) progesterone production at all levels of treatment. Stimulation of progesterone secretion by hCG was greater (P less than or equal to 0.05) than by horse LH over the range of concentrations utilized. When ...
Purification of specific heterologous F(ab)2 fragments with DEAE-Zeta-Prep cartridges for ion-exchange chromatography. A simple two-step procedure for purifying F(ab)2 fragments of horse immunoglobulins is described. In the first step, the horse plasma is diluted, made up to 12% (w/v) with ammonium sulphate and digested with pepsin. In the second step, the previously dialyzed solution is chromatographed. Instead of a normal ion-exchange resin, a DEAE-cellulose, covalently linked to a synthetic vinyl polymer, was used (DEAE-Zeta-Prep). With this assembly it is possible to perform chromatography at a high flow-rate without the problems related to the use of large columns. The yield and purity of the final produc...
The role of intestinal microflora in the metabolism of trichothecene mycotoxins. The role of faecal and intestinal microflora on the metabolism of trichothecene mycotoxins was examined in this study. Suspensions of microflora obtained from the faeces of horses, cattle, dogs, rats, swine and chickens were incubated anaerobically with the trichothecene mycotoxin, diacetoxyscirpenol (DAS). Micro-organisms from rats, cattle and swine completely biotransformed DAS, primarily to the deacylated deepoxidation products, deepoxy monoacetoxyscirpenol (DE MAS) and deepoxy scirpentriol (DE SCP). By contrast, faecal microflora from chickens, horses and dogs failed to reduce the epoxide ...
Chemotactic and chemokinetic activity of Streptococcus faecalis culture supernatant for equine neutrophils. Although equine neutrophils did not respond towards formylated methionyl peptides, Streptococcus faecalis culture supernatant caused an in vitro stimulation of equine neutrophil motility when measured by an under-agarose assay. The migration of neutrophils towards the culture supernatant increased sigmoidally with the logarithmic concentration of the culture supernatant in the chemoattractant wells. The streptococcal culture supernatant was chemokinetic because it stimulated the random motility of the phagocytes. Because granulocytes migrated further towards the supernatant than could be expla...
Epidermal growth factor-mediated effects on equine vascular smooth muscle cells. Epidermal growth factor (EGF) receptor binding kinetics and EGF-mediated stimulation of DNA synthesis and cellular proliferation were studied in cultured vascular smooth muscle cells (VSMC) from the equine thoracic aorta. Binding studies, using murine 125I-labeled EGF, indicate the presence of a single class of high-affinity binding sites (apparent KD = 2.8 X 10(-11) M), with an estimated maximal binding capacity of 5,800 sites/cell. EGF stimulated [3H]thymidine uptake in confluent quiescent monolayers in a dose-dependent fashion, half-maximal stimulation occurring at 7.5 X 10(-11) M. Likewise...
Analysis of the in vitro translation products of the equine herpesvirus type 1 immediate early mRNA. Equine herpesvirus type 1 (EHV-1) gene expression is coordinately regulated in an alpha, beta, gamma fashion. Viral alpha gene products include a 6.0-kb immediate early (IE) mRNA species (W. L. Gray et al., 1987, Virology 158, 79-87) and at least four closely related IE polypeptides (IEPs) (G.B. Caughman et al., 1985, Virology 145, 49-61). In this report, we describe results obtained from a series of in vitro translation experiments which were performed in an effort to characterize the IEPs and identify the mechanism by which individual IE protein species are generated. Our data indicate that ...
The hock as a potential site for non-invasive bone measurement. An in vitro study on the calcaneus of adult horses (n = 5) and foals (n = 10) was carried out using radiographic photodensitometry, single photon absorptiometry, transmission ultrasound velocity and chemical analysis. Data for trabecular bone content, ash, calcium and phosphorus levels were obtained. As techniques for assessing bone quality, ultrasound velocity was not sufficiently sensitive nor accurate and radiographic photodensitometry was found to be limited value. Photon absorptiometry was both accurate and reproducible, although some variation in bone mineral content and bone mineral den...
Ultrasonic transmission velocity and single photon absorptiometric measurement of metacarpal bone strength: an in vitro study in the horse. Ten pairs of third metacarpal bones from Thoroughbred horses aged two to 12 years were used to estimate bone strength. Measurements of transverse cortical ultrasound velocity, cortical cross sectional area and bone mineral content were made using ultrasonic transmission velocity and single photon absorptiometry. These data were used to determine bone mineral density, compact bone density and modulus of elasticity. The results were compared with those measured by direct means or chemical analysis and satisfactory correlations were obtained between estimated and measured values. Single photon ab...
Effect of ovarian steroids on migration of uterine lumenal neutrophils and on chemokinetic factors in uterine secretions from mares. Incubation of blood neutrophils with uterine flushings collected from ovariectomised mares treated with oestradiol, stimulated migration under agarose, whereas flushings from mares treated with progesterone or oily vehicle, inhibited migration. After intra-uterine infusion of bacteria, however, flushings from oestradiol-treated and vehicle-treated mares inhibited migration, whereas progesterone treatment stimulated migration. Migration of uterine-derived neutrophils under agarose was less than that of blood neutrophils and was not influenced by treatment with ovarian steroids. Uterine suscepti...
The effect of drugs used in the treatment of osteoarthrosis on stromelysin (proteoglycanase) of equine synovial cell origin. There is increasing evidence that the proteoglycan-degrading neutral metalloproteinase, stromelysin, is a key enzyme in the pathogenesis of osteoarthrosis. Equine synovial lining cells were stimulated in vitro to produce stromelysin, and phenylbutazone, flunixin, betamethasone, sodium hyaluronate and polysulphated glycosaminoglycan (PSGAG) were tested for their ability to inhibit the action of this enzyme on 14C-labelled casein substrate. Only PSGAG possessed inhibitory activity at concentrations likely to be achieved therapeutically in the equine fetlock joint.
Immunoassay detection of drugs in racing horses. VII. Detection of acepromazine in equine urine and blood by ELISA and PCFIA. We have developed and evaluated a one step enzyme-linked immunosorbent assay (ELISA) test and a particle concentration fluorescence immunoassay (PCFIA) test for acepromazine as part of a panel of pre- and post-race tests for illegal medications in racing horses. These tests are rapid, sensitive and economical and development of the tests occurred in less than seven months. The ELISA test detects acepromazine with an I-50 of about 150 pg/ml. In vivo, it readily detects the presence of acepromazine or its metabolites in equine blood and urine from 8 to 72 hours or longer, respectively, after adm...
Characterisation of IgE-mediated histamine release from equine basophils in vitro. In vitro IgE-mediated histamine release by equine blood basophils was characterised as the basis for a screening test for immediate hypersensitivity responses in horses. The responses are initiated by inducing agents that are capable of crosslinking or bridging the membrane-bound IgE molecules. The release process is complete within 40 mins. In vitro histamine release is dose-dependent, with a submaximal response at less or greater than the optimal dose of inducing agent. Exogenous calcium is required but not magnesium; the optimal release calcium concentration is 1.0 to 1.5 mM. If an IgE-medi...
In vitro evaluation of a sustained-release veterinary peroral pellet preparation. In a preceding in vivo study in horses, wide interindividual variation was found in the extent of bioavailability and time to reach peak concentration after peroral administration of one specific theophylline sustained-release dosage form. The purpose of the present study was to investigate the factors of potency, the pH of dissolution medium, the enzymes in the dissolution medium, and the crushing of the pellets on in vitro performance. The results show a wide variation in potency for the individual units, an increase in release rate with increasing pH, and an increase in release rate if the ...
Characterization of Berne virus genomic and messenger RNAs. From 380S particles of Berne virus (proposed family Toroviridae) one species of polyadenylated RNA was isolated. Using agarose gel electrophoresis its length was estimated as 20 kb or greater. When assayed under hypertonic transfection conditions genomic RNA was found to be infectious; RNase treatment destroyed the infectivity. The positive polarity of the molecule was confirmed by filter spot hybridization using cDNA prepared against poly(A)-selected RNA from infected cells. In embryonic mule skin cells infected with Berne virus the presence of five virus-specific, polyadenylated RNA species ...
Pre-chemotactic and chemotactic properties of uterine fluid from mares with experimentally induced endometritis. Streptococcal endometritis was induced experimentally in pony mares during oestrus. Uterine fluid was collected 30, 60, 120 or 240 minutes later and tested for its effect on the in vitro morphology and chemotaxis of equine neutrophils by two independent methods. The maximal response occurred between 30 and 60 minutes after infection and persisted until 240 minutes. The chemo-attractant contained both heat labile and heat stable components and the latter appeared to be active at low concentrations.
Molecular mechanics calculation of geometries of NAD+ derivatives, modified in the nicotinamide group, in a ternary complex with horse liver alcohol dehydrogenase. The geometry of seven NAD+ analogues bound to horse liver alcohol dehydrogenase (LADH) modified only in their nicotinamide group, have been studied using AMBER molecular mechanics energy-minimization procedures. Starting geometries were taken from X-ray crystallographic data for NAD+/Me2SO/LADH reported by Eklund and co-workers. In this study the NAD+ analogues were encaged by the constituent amino acids of the enzyme within a range of 0.6 nm from the initial NAD+/Me2SO/Zn2+ complex. The calculational method used is able to rationalize individual substituent effects and to evaluate the essenti...
Platelet function testing in the pony. Platelet isolation techniques and platelet function were evaluated in 35 adult ponies. Platelet recovery from whole blood was consistent and the preparation of platelet rich plasma was facilitated by an enhanced erythrocyte sedimentation rate. All platelet samples aggregated in response to 10 microM ADP. However, concentrations of ADP as high as 100 microM did not elicit significant 14C-serotonin release. Collagen induced irreversible platelet aggregation and 14C-serotonin release in all samples. The threshold dose for collagen in most ponies was 1.5 micrograms. Arachidonic acid (500 microM) f...
Characterization of an equine herpesvirus type 1 gene encoding a glycoprotein (gp13) with homology to herpes simplex virus glycoprotein C. The molecular structure of the equine herpesvirus type 1 (EHV-1) gene encoding glycoprotein 13 (gp13) was analyzed. The gene is contained within a 1.8-kilobase AccI-EcoRI restriction fragment mapping at map coordinates 0.136 to 0.148 in the UL region of the EHV-1 genome and is transcribed from right to left. Determination of the nucleotide sequence of the DNA fragment revealed a complete transcriptional unit composed of typical regulatory promoter elements upstream to a long open reading frame (1,404 base pairs) that encoded a 468-amino-acid primary translation product of 51 kilodaltons. The p...
Mechanism of binding of horse liver alcohol dehydrogenase and nicotinamide adenine dinucleotide. The binding of NAD+ to liver alcohol dehydrogenase was studied by stopped-flow techniques in the pH range from 6.1 to 10.9 at 25 degrees C. Varying the concentrations of NAD+ and a substrate analogue used to trap the enzyme-NAD+ complex gave saturation kinetics. The same maximum rate constants were obtained with or without the trapping agent and by following the reaction with protein fluorescence or absorbance of a ternary complex. The data fit a mechanism with diffusion-controlled association of enzyme and NAD+, followed by an isomerization with a forward rate constant of 500 s-1 at pH 8: E E...
Factors affecting the composition of mare uterine fluid. The influx of protein and polymorphonuclear leucocytes (PMN) into the uterine lumen was examined at different intervals after intrauterine infusion of fluids. The intrauterine infusion of both phosphate buffered saline (PBS) and a solution of lipopolysaccharide (LPS) derived from Escherichia coli resulted in a biphasic influx of protein in the uterine flushings peaking three and six hours after infusion. LPS infusion caused an additional influx of protein at 24 hours. The initial influx of protein preceded a biphasic influx of PMN which peaked six and 24 hours after both infusions. Uterine flu...
