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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.
Identification of 3 beta-hydroxy-5,7-androstadien-17-one as a secretory product of the fetal horse gonad in vivo and in vitro. Isolation of 3 beta-hydroxy-5,7-androstadien-17-one, as a major component of steroids extracted from vein blood of the fetal gonads of the horse, supports the proposed role for the compound as a precursor for equilin formation in the placenta of the mare. The 5,7-diene was extracted from blood collected from gonadal veins of fetal ovaries and testes in situ, and from a fetal testis connected to an artery in the neck region of the mare. Perfusion of fetal gonads in the laboratory was carried out to allow longer periods of collection. In addition, isolated cell preparations from a fetal testis w...
Nitrogen utilization in bacterial isolates from the equine cecum. A total of 114 bacterial isolates were obtained from the cecal contents of two mature cecally fistulated horses on a habitat-simulating medium containing 40% energy-depleted cecal fluid. Of these isolates, 108 were maintained in pure cultures and were tentatively grouped on the basis of cell morphology and physiological characteristics. Gram-negative rods (50.9%), gram-positive rods (22.8%), and gram-positive cocci (21.9%) represented the largest groups isolated from these animals. Fifty isolates were tested for their ability to grow in media containing urea, ammonia, peptones, or amino acids ...
Methods of assigning accurate values to reference serum. Part 2. The use of definitive methods, reference laboratories, transferred values and consensus values. Eight analytes (Ca, Cl, cholesterol, glucose, Mg, K, Na and urate) have been determined in one horse serum masterlot by up to six different procedures: (i) by so-called definitive methods; (ii) by a group of reference laboratories using a variety of analytical methods; (iii) using the results of two independent external quality assessment schemes; (iv) by transferring values from a human serum standard reference material analysed by definitive methods; (v) by similar transfer of values from several batches of horse reference serum previously analysed by definitive methods; and (vi) as in (v) b...
In vitro mechanical properties of equine tendons in relation to cross-sectional area and collagen content. The mechanical properties of the deep digital flexor tendon (DDFT), the superficial digital flexor tendon (SDFT) and the suspensory ligament (SL) of the hindlimb of the horse were studied in vitro. The tendons were observed at several morphologically distinct sites. The loaded tendon is homogeneously strained, in spite of large variations in cross-sectional area. Consequently the modulus of elasticity was inversely proportional to the corresponding cross-sectional area and ranged from 738 MPa (megaPascal, N mm-2) to 1398 MPa within the DDFT, from 1000 MPa to 1282 MPa within the SDFT and from 5...
Fibronectin enhances transfection of Staphylococcus aureus. The factor in normal sera primarily responsible for the enhancement of transfection (and transformation) of Staphylococcus aureus was identified as fibronectin. Serum samples which were depleted of fibronectin by affinity chromatography showed a marked decrease in enhancing activity. Fibronectin isolated from sera of several animal species demonstrated enhancing activity.
Stimulation of equine eosinophil migration by hydroxyacid metabolites of arachidonic acid. Lipoxygenase products of arachidonic acid are important mediators of inflammation, affecting several aspects of cell function. Monohydroxyeicosatetraenoic acid (mono-HETE) and 5,12-dihydroxyeicosatetraenoic acid (LTB4) enhance migration of both neutrophils and eosinophils in several species. The relative ability of positional isomers of HETE and of LTB4 to affect migration of equine eosinophils was studied. The 5, 8, 9, 11, 12, and 15 isomers of HETE were prepared by autooxidation of arachidonic acid, separated by sequential normal phase and reverse phase high performance liquid chromatography...
In vitro phagocytosis and killing of Corynebacterium equi by alveolar macrophages of foals. Bronchoalveolar lavage was performed 5 times, sequentially, on 3 healthy foals while each foal was 6 to 63 days of age. Phagocytosis and bactericidal assays were performed on recovered alveolar macrophages. Corynebacterium equi and alveolar macrophages at a ratio of 10:1 were incubated for 1 hour in medium containing 1% heat-inactivated rabbit anti-C equi serum. After incubation, greater than 90% of the alveolar macrophages contained at least 1 ingested bacterium and each alveolar macrophage contained 9.4 +/- 1.0 bacteria (mean +/- SE). After alveolar macrophages and C equi were incubated for ...
Solubilization and characterization of [3H] 5HT high affinity binding sites (5HT1 and 5HT3). The solubilization of the serotonergic 5HT1 and 5HT3 sites was performed with digitonin and sodium cholate at 1% (final concentration). Two binding sites for [3H]5HT were observed on rat or horse brain synaptosomal membranes solubilized with these detergents. The corresponding dissociation constants (KD) were 1-3 nM and 13-30 nM respectively. These values were closely similar to those corresponding to 5HT1 and 5HT3 sites located in intact membranes. The solubilized sites specifically bound 5HT. The effect of GTP decreasing the binding to 5HT1 sites was lost on solubilized 5HT1 sites; it was re...
Horse kidney neutral alpha-D-glucosidase: purification of the detergent-solubilized enzyme; comparison with the proteinase-solubilized forms. Neutral alpha-D-glucosidase (alpha-D-glucoside glucohydrolase, EC 3.2.1.20) from horse kidney brush-border membranes was solubilized using Emulphogene BC 720 and purified by an affinity chromatography technique. The enzyme preparation (390-fold purified), which was free of other known microvillus hydrolases, exhibited one precipitate line in crossed immunoelectrophoresis and migrated as a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Several criteria (charge-shift crossed immunoelectrophoresis and hydrophobic chromatography) revealed the purified detergent form of t...
Complexities in the denaturation of horse metmyoglobin by guanidine hydrochloride. The denaturation of horse metmyoglobin by guanidine hydrochloride was studied at pH 6.4 and 25 degrees C. Measurements of both the peptide circular dichroism and the absorbance in the Soret region suggest that the extent of renaturation strongly depends on the time interval during which the protein is exposed to concentrated solutions of the denaturant. From the equilibrium measurements of the absorption in the Soret region, it is concluded that the unfolding of metmyoglobin is complex. This is further supported by kinetic studies of denaturation which suggest the occurrence of the least four ...
High resolution R-bands produced in equine chromosomes after incorporation of bromodeoxyuridine. Cell synchronization was used to obtain an adequate percentage of very long chromosomes in equine mitotic spreads. Reported here is our variation, adapted to horse chromosomes, of a method using excess thymidine followed by bromodeoxyuridine incorporation. This technique routinely yields excellent quality cells, predominantly in prometaphase and prophase. Among other differences with the standard technique, this method does not use Colcemid, which, in addition to inhibiting spindle fiber formation, also increases chromosome contraction resulting in thicker and thus fewer bands. Consequently, h...
Incorporation of L-75Se-cystine in tissue fragments from the matrix of the hoof and the claw–a tool for studying the pathogenesis of laminitis? An in vitro method has been designed and used to study the incorporation of 75Se-cystine into matrix fragments from hooves and claws of healthy horses and cattle. Tissue fragments from the zone of keratinisation were incubated with L-75Se-cystine in a tissue culture medium for 4 to 6 h, during which time there was continuous incorporation of the labelled selenocystine. The incorporation was greatly decreased by adding L-cystine to the incubation mixture. It is concluded that the incorporation of 75Se-cystine depends on the presence of a specific receptor for cystine in the tissue fragments stu...
Causative ehrlichial organisms in Potomac horse fever. An ehrlichia was consistently isolated from the peripheral blood leukocyte fraction of ponies that had been experimentally infected with Potomac horse fever by whole blood transfusion from naturally infected horses. The organism was propagated in a human histiocyte cell line for 3 to 5 weeks and then inoculated intravenously or intradermally into healthy adult ponies. Clinical signs of Potomac horse fever, which varied in the degree of severity, occurred 9 to 14 days post-inoculation in all of the ponies. One pony died 20 days post-inoculation. The ehrlichial organism was reisolated in the hum...
Cell synchronization and dynamic G-banding of equine chromosomes by bromodeoxyuridine. Both dynamic G-banding and cell synchronization produced by bromodeoxyuridine (BrdU), were applied to equine chromosomes. BrdU incorporated during the first half of the S-phase is taken up into the R-bands that are early replicating. These bands, which have incorporated BrdU, cannot contract as usual and remain elongated; only the other regions of the chromosome, i.e., the G-bands, contract normally and are sharply defined. BrdU also can be used for cell synchronization. The addition of BrdU in a high concentration, 15 hours before harvest, and its removal 11 hours later, has two effects: init...
Cefadroxil in the horse: pharmacokinetics and in vitro antibacterial activity. Sodium cefadroxil was administered as a single intravenous dose (25 mg/kg) to six healthy adult mares. Plasma samples were collected over a 24-h period and cefadroxil concentrations were measured by microbiological assay. The pharmacokinetic behavior of the drug was appropriately described in terms of a one-compartment open model. Values for the major pharmacokinetic terms were: extrapolated initial plasma concentration = 59.2 +/- 15.0 micrograms/ml; half-life = 46 +/- 20 min; apparent volume of distribution = 462 +/- 191 ml/kg; and body clearance = 7.0 +/- 0.6 ml/min.kg. In a subsequent study...
Kinetic studies of the unfolding-refolding of horse muscle phosphoglycerate kinase induced by guanidine hydrochloride. The kinetics of the unfolding and refolding of horse muscle phosphoglycerate kinase were studied with three different signals: fluorescence emission intensity at 336 nm (excitation at 292 nm), ellipticity at 220 nm, and enzyme activity. The results corroborate the conclusion on the existence of intermediates in the folding pathway obtained from equilibrium studies. Kinetic studies showed at least two phases of refolding, as revealed by fluorescence as well as by circular dichroism measurements. During the fast phase, an intermediate was formed with a fluorescence intensity higher than that of ...
Proteinase inhibitors of horse seminal plasma. A high molecular mass, acid-soluble proteinase inhibitor. Horse seminal plasma does not possess a proteinase inhibitor corresponding to human HUSI-I (human seminal plasma inhibitor). Instead a protein complex of high relative molecular mass (Mr) containing proteinase inhibitory activity was detected, which was called horse seminal plasma protein complex or HSPC. The compound had a broad enzyme-inhibiting spectrum. Its Mr was estimated to be 800 000 and it was composed of 7 different polypeptides with Mr values ranging from 11 000 to 30 000. Its carbohydrate content was between 3.5% and 5%. Despite the high molecular mass, the complex was soluble in d...
In vitro development of Strongylus edentatus to the fourth larval stage with notes on Strongylus vulgaris and Strongylus equinus. Strongylus edentatus was successfully cultured in vitro to the fourth larval stage (L4). Some growth continued for periods of 40-50 days at which time reductions in viability were observed in some of the culture systems tested. Various combinations of media, sera, buffers and organ explant cultures were tested. All cultures were incubated at 37 C in an atmosphere of 95% air and 5% CO2. Larvae underwent growth and differentiation to the L4 in all medium-serum combinations with and without organ explant cultures. Development and growth did occur but viability was reduced to insignificant levels ...
Pre-alpha 2-elastase inhibitor of the horse: a hybrid molecule between alpha 1-proteinase inhibitor and alpha 2-beta 1-glycoprotein. Pre-alpha 2-elastase inhibitor of horse plasma has recently been isolated in our laboratory. In this article we demonstrate that the inhibitor is a composite structure built of alpha 1-proteinase inhibitor and alpha 1-beta 1-glycoprotein. The compound inhibitor is biologically active, although it has previously been shown that its enzyme specificity is different from that of free alpha 1-proteinase inhibitor. Our observations are based on immunochemical cross-reactions between pre-alpha 1-elastase inhibitor and antibodies to alpha 2-beta 1-glycoprotein as well as antibodies to alpha 1-proteina...
Functional and biochemical characterization of immunologically derived equine platelet-activating factor. Antigen-specific challenge of equine leukocytes induced the non-lytic release of a platelet-activating factor in vitro. The equine platelet-activating factor stimulated the release of serotonin from equine platelets in a dose-responsive manner, independent of the presence of cyclo-oxygenase pathway inhibitors such as indomethacin. Rabbit platelets were also responsive to equine platelet-activating factor. The release of equine platelet-activating factor was a rapid reaction with near maximal secretion taking place in 30 seconds. Addition of equine platelet-activating factor to washed equine pl...
Enhanced prostacyclin biosynthesis and decreased thromboxane formation by 3-dimethylamino 5-(2′,6′-dichlorobenzylidene) 6-methyl (4H)-pyridazine (PC 89). The effects of 3-dimethylamino 5-(2',6'-dichlorobenzylidene) 6-methyl (4H)-pyridazine (PC 89) on the biosynthesis of PG I2 and TX A2 using horse aorta and horse platelet microsomes as sources of enzymes and arachidonic acid as substrate, were investigated. PC 89 (1.10(-6) M- 1.10(-3) M) dose-dependently - enhanced the biosynthesis of PG I2: the AD50 was 6.8 X 10(-6) M +/- 1.2 X 10(-9) M, the Vmax did not vary significantly with concentrations: PC 89 increased the affinity of enzyme for substrate - but inhibited TX A2 biosynthesis (ID50 = 3.31 X 10(-3) M +/- 4.8 X 10(-7) M): this inhibiting act...
A direct technique for the preparation of chromosomes from early equine embryos. A technique is described for the preparation of banded chromosomes from early equine embryos cultured for less than 10 h in a medium containing bromodeoxyuridine. In addition to standard Giemsa staining and C-banding, chromosomes thus prepared can also be R-banded by either the RBA or the RB-FPG methods. This technique is rapid, repeatable, and limits cell loss, making it ideal for the preparation of early embryos.
The amino-acid sequence of beta-lactoglobulin II from horse colostrum (Equus caballus, Perissodactyla): beta-lactoglobulins are retinol-binding proteins. beta-Lactoglobulin isolated from horse colostrum is heterogeneous and contains two components: beta-lactoglobulin I and beta-lactoglobulin II. These two proteins are monomeric and show differences in their electrophoretic mobilities, chain lengths and primary structures. The complete amino-acid sequence of beta-lactoglobulin II was determined by automated Edman degradation of the intact protein and of the peptides derived from these by digestion with trypsin or chymotrypsin and by chemical cleavage with cyanogen bromide. Unlike other beta-lactoglobulins which contain 162 amino acids, horse bet...
Photomicrographic evaluation of stallion spermatozoal motility characteristics. A photomicrographic method for evaluation of stallion spermatozoal motility was developed, and spermatozoal image and velocity characteristics were defined. The photomicrographic method was compared with visual estimation of motility in the same semen sample over time. Using photomicrography, velocities and percentages of individual spermatozoal image characteristics were determined. Although there was a high correlation between results of the 2 methods, results of the photomicrographic method were more repeatable than were those of the visual method.
An investigation, in vitro, of the actions of three Western Australian snakes on the blood coagulation of the dog, cat, horse and wallaby. Venoms of the tiger snake and brown snake were procoagulant, in vitro, when tested with cat, dog, horse and wallaby plasma. In the absence of calcium and phospholipid the coagulant activity of tiger snake venom was minimal. In contrast, brown snake venom alone had marked procoagulant activity. This activity, however, was enhanced by the presence of calcium and phospholipid. Death adder venom exerted an anticoagulant effect. Apparent species' differences in susceptibility to the coagulant venoms were noted. However, the probable explanation of these differences was attributed to variation in th...
Equine alternative pathway activation by unsensitized rabbit red blood cells. The equine alternative complement pathway has been partially characterized and compared to the equine classical activation pathway. A dose-dependent lysis of RbRBC was observed with peak lytic values noted within 10 minutes at 37 degrees C when rabbit red blood cells (RbRBC) were used as an alternative pathway activator. Sheep red blood cells (SRBC) sensitized with rabbit hemolysin or partially purified equine IgM antibodies were equally sensitive to lysis. Dilution of the commercial hemolysin by 1/5 reduced lysis from 90% to 38% in the presence of constant cell numbers. Hemolysis of SRBC peak...
Failure of superoxide dismutase to alter equine arachidonic acid-induced platelet aggregation, in vitro or ex vivo. Superoxide dismutase (SOD), a free radical scavenger with anti-inflammatory activity, was administered IM to horses. Ex vivo platelet aggregation in response to arachidonic acid was monitored to determine whether exogenous SOD altered equine platelet prostaglandin metabolism. Preparations of platelet-rich plasma obtained before SOD administration were incubated with different concentrations of SOD and were aggregated with arachidonic acid. Superoxide dismutase did not exert a demonstrable effect, either ex vivo or in vitro. Aspirin abolished arachidonic acid-induced platelet aggregation in vit...
Comparison of peripheral blood and uterine-derived polymorphonuclear leukocytes from mares resistant and susceptible to chronic endometritis: chemotactic and cell elastimetry analysis. The functional competence of peripheral blood and uterine-derived polymorphonuclear leukocytes (PMN) from 12 mares were analyzed for chemotactic responsiveness using a chemotactic chamber (filter) assay and for deformability by cell elastimetry analysis. Peripheral blood PMN obtained from control mares and from 8 mares experimentally inoculated via the uterus with 1 x 10(9) Streptococcus zooepidemicus had similar normal chemotactic responsiveness and were highly deformable before and at 12 hours after inoculation. Uterine PMN obtained 12 hours after uterine inoculation with S zooepidemicus fro...
Hybrids from equine LH: alpha enhances, beta diminishes activity. LH hybrids were prepared by combining eLH alpha and eLH beta with the corresponding subunits of oLH, pLH and hCG. Recombinants were isolated by gel filtration and assessed by SDS-polyacrylamide gel electrophoresis under both dissociating and non-dissociating conditions. All combinations of subunits produced hybrid LH molecules. Hybrids prepared by combining eLH beta with oLH alpha, pLH alpha or hCG alpha were very inactive in rat radioligand and Leydig cell in vitro bioassays. Hybrids prepared with eLH alpha were very active in both assays. The greatest potentiating activity was observed when ...
