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.
Kemeny L, Pearson JE.An agent which possessed the properties of herpesviruses was isolated from the leukocytes of 71 out of 80 (88.7%) apparently normal Iowa horses. It was ether- and heat-sensitive, DNA type, and produced type-A intranuclear inclusion bodies in cell cultures. Electron micrographs revealed a virion of typical herpesvirus structure. Leukocyte isolate virus could be differentiated from equine rhinopneumonitis virus (ERV) by serum neutralization, by growth differences in rabbit kidney cells, and by fluorescent antibody staining. Specific neutralizing antibody against this agent was found in a pooled ...
Srivastava SK, Beutler E.1. Erythrocytes from normal and glucose 6-phosphate dehydrogenase-deficient humans were subjected to hydrogen peroxide diffusion to oxidize the GSH. Studies were carried out in the presence and absence of chromate to inhibit glutathione reductase and with or without the addition of glucose. 2. The GSH content of erythrocytes from other species was oxidized by subjecting them to hydrogen peroxide diffusion in the presence of chromate and glucose. 3. Chromate (1.3mm) inhibited glutathione reductase by about 80%, whereas glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, hexokin...
Williams MA, Harrison PM.Horse ferritin was fractionated both by starch-gel electrophoresis and by gel filtration on Sephadex G-200. Monomer fractions contained up to 98% of monomer and oligomer fractions up to 76% of oligomers as determined by quantitative electron microscopy. Percentages obtained from electron micrographs correlated well with analytical starch-gel electrophoretograms and ultracentrifuge patterns. Amino acid analyses of monomer- and oligomer-enriched fractions showed no significant differences. Ferritin oligomers did not apparently dissociate on dilution for electron microscopy or on storage. Apoferr...
Turner GA, Taylor DM.The interactions between tetravalent plutonium and horse serum proteins were studied in vitro by electrophoresis on cellulose acetate and by gel filtration. The results show that in horse serum, as in other mammalian sera, the plutonium is associated principally with the transferrin component of the beta1-globulins. The formation of the plutonium-transferrin complex requires the presence of HCO3-, and plutonium is displaced from the complex by excess iron, thus indicating that similar binding sites may be involved in the complexing of iron and plutonium. The plutonium complex is considered to ...
O'Callaghan DJ, Hyde JM, Gentry GA, Randall CC.Infection of exponential-phase suspension cultures of mouse fibroblast cells (L-M) with equine abortion virus (EAV) resulted in inhibition of cell growth and marked alterations in host metabolic processes. The synthesis of deoxyribonucleic acid (DNA) and ribonucleic acid was inhibited within 4 hr after infection and was suppressed by more than 90% by the time of maximal virus replication (14 to 18 hr). The overall rate of protein synthesis, however, was similar in uninfected and virus-producing cells as determined by measurements of net protein and isotope incorporation. The time course of vir...
Delesalle CJ, Callens C, Van Colen I, Lefebvre RA.Selective 5-HT4 receptor agonists such as prucalopride are used as human prokinetics, since activation of 5-HT4 receptors on intestinal cholinergic neurons facilitates acetylcholine release. 5-HT4 receptors, linked to adenylyl cyclase, act via generation of cAMP. None of the 4 in vitro studies on 5-HT in horses provided evidence for neuronal 5-HT4 receptors, but none used the protocol as described in human studies [1-4]. Objective: To investigate whether functional 5-HT4 receptors are present in the equine small intestine. Methods: In vitro organ bath set up, applying electrical field stimulat...
Theodorakis JL, Armes LG, Margoliash E.beta-Thiopropionyl derivatives of horse cytochrome c singly modified at each of 18 different lysine epsilon-amino groups have been prepared using sulfosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate and purified to homogeneity by high-pressure liquid chromatography. These derivatives were characterized by determination of: (i) the location of the modification; (ii) reduction potentials; (iii) visible and NMR spectra: and by (iv) measurement of electron transfer activity with cytochrome-c oxidase. No significant changes in structure were indicated, except for the ferric forms of the deri...
Kasza A, Korpula-Mastalerz R, Rose-John S, Dubin A.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 ...
Olson LE, Perkowski SZ, Mason DE, Muir WW.Strips of trachealis muscle were dissected from the midcervical portion of the trachea of horses that were free of respiratory tract disease, and the overlying epithelium and mucosa were removed. Muscle strips were suspended in tissue baths that were filled with Krebs-bicarbonate solution, aerated with 5% CO2 in oxygen and maintained at 37 C. Isometric tension was continuously recorded. The increase in active isometric tension was concentration dependent when acetylcholine (10(-9) to 10(-4) M) or histamine (10(-9) to 10(-4) M) was added to the tissue baths in 0.5-logarithmic increments. When t...
Zielińska-Dawidziak M, Błaszak R, Piasecka-Kwiatkowska D.The presented experiments show the model of expectation of equine spleen ferritin extraction in a new aqueous two-phase system which was formed by mixing polyethylene glycol (PEG) and hydroxyethyl starch (HES). The tendency of the protein to migrate in the analyzed systems was dependent on the concentrations of HES and PEG as well as PEG molecular weight. The highest concentration of ferritin in the top phase (rich in PEG) was recorded in the system composed of 6% PEG 3000 and 3% HES. The obtained concentration was 0.88 mg mL(-1). The lowest concentration was 0.42 mg mL(-1) in the system compo...
Shivers CA, Liu IK.Sera and follicular fluid from mares previously determined as having antibodies to the zona pellucida were exposed to porcine ovarian oocytes before insemination with boar spermatozoa in vitro. There was a dramatic decline in the number of spermatozoa bound to zonae compared to treatments with sera and follicular fluid from mares negative for zona antibodies. These data suggest that antibodies reactive with the zona pellucida may be responsible for reduced or even complete infertility in some mares. In a group of 50 randomly selected mares tested for antizona antibodies, 2 pregnant mares were ...
Le Goff D, Hannan J, Maboundou JC, Ayrault-Jarrier M.Equine plasma lipoproteins were fractionated into VLDL, LDL-1, LDL-2 and HDL by density gradient ultracentrifugation. From each lipoprotein fraction, five apo C like peptides of approx. M(r) 1400, 10000, 9500, 9000 and 8000 were detected by SDS-polyacrylamide gel electrophoresis. After partial purification by Sephadex G-75, one fraction, showing a strong activation of lipoprotein lipase, was further purified by Mono Q anion exchange column. Two of the apo C like peptides (M(r) 10000 and 8000) activated the bovine milk lipoprotein lipase in vitro; only one (M(r) 9500) inhibited the lipolytic ac...
Word TA, Larsen RW.Treatment of horse heart Cytochrome-c (Cc) with N-chloro-4-toluosulfonamide (Chloramine-t, CT) results in the oxidation of methionine (Met) residues to the corresponding sulfoxide including the distal heme ligand, Met80. The resulting Fe-sulfoxide coordination is sufficiently labile in the ferrous form to be displaced by gaseous ligands, including CO. Photolysis of the CO-CT-Cc complex provides an opportunity to examine ligand binding dynamics that are associated with a relatively rigid distal heme pocket. In this work, photoacoustic calorimetry (PAC) was utilized to obtain the kinetics as wel...
Leblová S, Galociová J, Cerovská N.The studied herbicides (terbutylazine, simazine) inhibit the activity of plant, animal, and yeast alcohol dehydrogenases. The inhibition constant Ki for alcohol dehydrogenase (ADH) isolated from peas and bakers' yeast equals approximately 10(-4) M, and that for ADH isolated from horse liver is of the order of 10(-5) M. The character of inhibition for all the herbicides studied for the reaction catalyzed by pea, liver, and yeast ADH is always noncompetitive toward ethanol and competitive with respect to NAD. The inhibition constants for the enzyme isolated from peas are pH independent. The inte...
Sahmi F, Nicola E, Price CA.The objective was to establish a cell line-based bioassay for FSH in horse serum for screening samples with high eCG bioactivity. A cell line (HEK293) was transiently cotransfected with an FSH reporter expression plasmid and a cAMP-responsive β-galactosidase reporter plasmid. Cells were bulk frozen, and thawed for assay purposes. This assay was specific for FSH, with no cross-reaction with LH or insulin-like growth factor-1. Standard curves (eCG) and serum samples from pregnant mares passed parallel line bioassay validity tests (linearity and parallelism). Estimates of bioactivity with this b...
Nazmutdinov RR, Bronshtein MD, Zinkicheva TT, Hansen NS, Zhang J, Ulstrup J.Outer-sphere electron transfer (ET) between optically active transition-metal complexes and either other transition-metal complexes or metalloproteins is a prototype reaction for kinetic chirality. Chirality as the ratio between bimolecular rate constants of two enantiomers mostly amounts to 1.05-1.2 with either the Λ or Δ form the more reactive, but the origin of chirality in ET parameters such as work terms, electronic transmission coefficient, and nuclear reorganization free energy has not been addressed. We report a study of ET between the Λ-/Δ-[Co(Ox)3](3-) pair (Ox = oxalate) and hor...
Cremonesi F, Anderson K, Lange-Consiglio A.TWO METHODS HAVE BEEN DESCRIBED TO RECOVER OOCYTES FROM EQUINE FOLLICLES IN EXCISED OVARIES: aspiration and scraping. Aim of this work was to develop an effective method for collecting equine oocytes using Tuohy needle and comparing this technique to aspiration and scraping, with or without tunica albuginea removal. This hollow hypodermic needle, usually employed for inserting epidural catheters, is designed with a slightly curved tip, shaped similar to a small curette. In unpeeled ovaries, the recovery rates of Tuohy needle group was higher (P < .05) than in the 16 g needle aspiration and ...
Schellenberg LM, Bonicelli J, Hochheim I, Regenthal R, Abraham G.The β-adrenergic receptor (β-AR) plays an important role in regulating a variety of cell and organ functions in different animal species and is an important target in asthma pathogenesis and therapy. The β-AR expression and function in equine bronchial epithelial cells (EBEC) were not known but innervation and significant decrease in receptor level were reported in the equine bronchial tissues from asthmatic horses. I-iodocyanopindolol (ICYP) binding studies were undertaken in primary freshly isolated and cultured EBEC to identify the presence of the β-ARs. The receptor distribution was as...
Wang Y, Du X.The miscibility and stability of the binary monolayers of zwitterionic dipalmitoylphosphatidylcholine (DPPC) and cationic dioctadecyldimethylammonium bromide (DOMA) at the air-water interface and the interaction of ferritin with the immobilized monolayers have been studied in detail using surface pressure-area isotherms and surface plasmon resonance technique, respectively. The surface pressure-area isotherms indicated that the binary monolayers of DPPC and DOMA at the air-water interface were miscible and more stable than the monolayers of the two individual components. The surface plasmon re...
Pérez-Castrillo S, González-Fernández ML, Gutiérrez-Velasco L, Villar-Suárez V.To characterize the ultrastructure of mesenchymal stem cells (MSCs) that were harvested from the adipose tissue (AT-MSCs) and bone marrow (BM-MSCs) of horses and transfected with green fluorescent protein. Methods: MSCs from adipose tissue and bone marrow of 6 adult female Hispano-Bretón horses. Methods: Harvested equine MSCs were cultivated and transfected with green fluorescent protein, and the immunophenotypes of the MSCs were characterized by use of anti-CD90 and anti-CD105 monoclonal antibodies. When stable transfection of MSCs was achieved, the morphological and ultrastructural characte...
Thomas KL, Carmalt JL, Burnett WD, Arjmand H, Johnston JD.OBJECTIVE To compare strain at the bone-pin and cast-pin interfaces among 3 transfixation pin-cast constructs applied to equine forelimbs. ANIMALS 15 forelimbs from 15 adult horses. PROCEDURES Limbs were randomly assigned to 1 of 3 constructs. Centrally threaded positive-profile pins were used for all constructs, and the most distal pin was placed just proximal to the epicondyles of the third metacarpal bone. Construct 1 consisted of two 6.3-mm-diameter pins spaced 4 cm apart at 30° to each other. Construct 2 was the same as construct 1 except the pins were placed 5 cm apart. Construct 3 cons...
Merkies K, Buhr MM.Spermatozoal function is affected by the ability to regulate intracellular calcium concentrations ([Ca2+]i), and may be influenced by epididymal maturation as well as environmental components. Regulation of [Ca2+]i in ejaculated and epididymal stallion spermatozoa was monitored over time in various media. Spermatozoa from each of 5 pony stallions (3 ejaculate samples and 1 caput and cauda sample) were labeled with the fluorescent calcium indicator probe Indo-1 in a calcium-free modified Tyrode's buffer. Fluorescent emissions were monitored by a dual wavelength spectrofluorometer over 5 h. Calc...
Liberatori J, Morisio Guidetti L, Conti A.By double diffusion in agarose gel, in well defined experimental conditions, cross reactions were observed between porcine beta-lactoglobulins and anti-bovine beta-lactoglobulin antisera. The immunological reactivity between these beta-lactoglobulins from a monogastric and the ruminant anti beta-lactoglobulin antiserum thus implies a certain degree of similarity between the monomeric beta-lactoglobulins examined and the dimeric of the ruminants. With the same antisera it also proved possible to demonstrate the presence of beta-lactoglobulins in the mammary secretions of another monogastric, na...
Bresnahan DR, Lupole RE, Stilz CR, Carnevale EM.Zona pellucida (ZP) proteins are important for fertilization and sperm binding and are closely associated with cumulus cells. Communication between cumulus and oocytes is facilitated by intracellular membrane channels composed of connexins. The extent aging impacts potential differences in fertilization and reductions in fertility is not well understood. This study characterized age-related differences in transcript abundance of ZP proteins and connexins in cells from ovarian follicles. Additionally, differences in sperm binding to oocytes from old and young mares was evaluated. For experiment...
Fleitman J, Neu D, Benjamin E.A reverse phase liquid chromatographic procedure is described for the simultaneous determination of oxfendazole [2-(methoxycarbonylamino)-5-phenylsulfinylbenzimidazole] and trichlorfon [(2,2,2-trichloro-1-hydroxyethyl)phosphonic acid dimethyl ester] in equine paste. The sample is extracted by sonication in methanol. Insoluble excipients are removed by centrifugation and an aliquot plus internal standard are diluted with dilution solvent (water-acetonitrile-phosphoric acid, 80 + 20 + 1). The samples are filtered and injected onto a Partisil-5 ODS-3 column with acetonitrile-0.01 M phosphate buff...
Nic An Ultaigh S, Ryan MF.Proteinases released during the in vitro maintenance of asynchronous cultures of the free-living nematode Caenorhabditis elegans were characterized on the basis of subunit composition, fluorogenic substrate specificity, inhibitor sensitivity and pH optima. Cysteine proteinases are present in the excretory-secretory products (ESP) as indicated by the hydrolysis of cathepsin fluorogenic substrates and confirmed by immunoblotting. Serine proteinases were predominant as indicated by substrate gel analysis and inhibitor studies. The presence of metallo-proteinases was also indicated by inhibitor st...
Seidel GE.Principles for selecting future research projects include interests of investigators, fundability, potential applications, ethical considerations, being able to formulate testable hypotheses and choosing the best models, including selection of the most appropriate species. The following 10 areas of assisted reproduction seem especially appropriate for further research: efficacious capacitation of bovine spermatozoa in vitro; improved in vitro bovine oocyte maturation; decreasing variability and increasing efficacy of bovine superovulation; improved fertility of sexed semen; improving equine IV...
Magnuson NS, Perryman LE.The effect of adenosine on the mitogenic response of peripheral blood lymphocytes (PBL) and on the nucleotide pools of erythrocytes from normal horses, horses heterozygous for the combined immunodeficiency (CID) trait (carriers), and foals with CID was studied. When PBL from normal, carrier, and CID horses were stimulated by phytohemagglutinin (PHA), concanavalin A, or pokeweed mitogen, [3H]thymidine uptake was inhibited by adenosine (0.1 microM) to 1.0 mM) in a dose-dependent manner. Adenosine (100 microM) mediated inhibition of [3H]thymidine uptake was prevented in both normal and carrier ho...
O'Keefe JS, Julian A, Moriarty K, Murray A, Wilks CR.A detection system incorporating the polymerase chain reaction was compared with the use of histopathology and virus isolation to determine the presence of equid herpesvirus type 1 or equid herpesvirus type 4 in equine tissues submitted to a diagnostic laboratory. When the polymerase chain reaction was performed, these tissues had been stored for up to 3 years. Thirty-eight tissues representing 14 cases had been stored embedded in paraffin wax. Analysis of these tissues using the PCR gave predictive values of 1.0 and 0.91 for a positive and negative result respectively, and sensitivity and spe...
Dillon AM, Heath MF.The effects of the protein tyrosine kinase inhibitors tyrphostins B42 and B46 on equine platelet function and protein tyrosine phosphorylation (PTP) were assessed. Tyrphostins B42 and B46 (both at 100 microM concentration) produced significant inhibition of thrombin-stimulated equine platelet aggregation. The effect of tyrphostin B46 was also time-dependent. The same concentration of these inhibitors produced very little or no inhibition of platelet-activating factor (PAF)-induced aggregation. The effects of tyrphostins B42 and B46 on thrombin- and PAF-stimulated PTP were generally similar, al...
Wu D, Murakami K, Liu N, Konishi M, Muneta Y, Inumaru S, Kokuho T, Sentsui H.The equine interleukin-18 (IL-18) cDNA that contains the coding sequence was cloned and a recombinant baculovirus, named AcEIL-18, was constructed. The recombinant protein of the equine IL-18 was expressed by AcEIL-18 and its expression was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Insect cells infected with AcEIL-18 secreted a precursor IL-18 with 24 kilo dalton (kDa) into the culture supernatant. Western blot analysis showed that mature equine IL-18 about 18 kDa was also confirmed without co-expression of caspase-1. Culture supern...
Azuma T, Choi YH, Hochi S, Oguri N.The development of in-vitro matured and microfertilized horse oocytes was examined in vitro. Fertilized oocytes were produced by 20-h insemination of in-vitro matured and partially zona-removed oocytes with frozen spermatozoa that had been treated with caffeine/calcium ionophore A23187 (fertilization rate 34.2%, monospermy rate 76.9%). Embryonic development was assessed by the number of nuclei stained with Giemsa solution. In Experiment 1, a continuous 8-day culture of the microfertilized oocytes in TCM199 or modified synthetic oviduct fluid (m-SOF) supplemented with 10% fetal bovine serum or ...
Martín-Faivre L, Gaudaire D, Laugier C, Bouraïma-Lelong H, Zientara S, Hans A.Equine arteritis virus (EAV) is an Alphaarterivirus (family Arteriviridae, order Nidovirales) that frequently causes an influenza-like illness in adult horses, but can also cause the abortions in mares and death of newborn foals. Once primary infection has been established, EAV can persist in the reproductive tract of some stallions. However, the mechanisms enabling this persistence, which depends on testosterone, remain largely unknown. We aimed to establish an in vitro model of non-cytopathic EAV infection to study viral persistence. In this work, we infected several cell lines originating f...
Roth TL, White KL, Thompson DL, Horohov DW.It has been proposed that PGE2 is an important immunosuppressant acting at the fetal-maternal interface during pregnancy. We have previously shown that horse conceptus-conditioned medium suppresses lymphocyte proliferation. This experiment was designed to determine if horse conceptus-derived immunosuppressive activity could be attributed to PGE2 production by the trophoblast tissue. Trophoblast tissue from 21-day-old conceptuses was cut into equal sections and cultured in the presence or absence of the prostaglandin inhibitor, indomethacin. Following culture, immunosuppressive activity and the...
Nichani AK, Sharma RD, Sarup S.Efficacy of medium RPMI-1640 (supplied by Gibco USA, Centron and Hi-media) supplemented with horse, donkey, sheep and goat sera was evaluated for in vitro propagation of Theileria annulata (Hisar) infected bovine mononuclear cells. The results were compared with the growth rate in RPMI-1640 supplemented with foetal bovine serum (Gibco). RPMI-1640 (Gibco) proved to be the best medium for in vitro cultivation of the parasite infected cells. Foetal bovine serum could be easily, safely and reliably substituted with goat and sheep sera in the growth medium. Horse and donkey sera also gave comparabl...
