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.
LeBlanc PH, Eberhart SW, Robinson NE.In horses with noninduced, reversible airway obstruction (heaves), pulmonary function is improved after sedation with the alpha 2-adrenergic agonist xylazine. The mechanism of this effect is undetermined. Because the predominant excitatory innervation of equine airways is cholinergic, the influence of alpha 2-adrenergic receptor stimulation on the response of isolated distal airways to cholinergic stimulation was determined. Distal bronchial segments from 22 healthy horses were suspended in isolated organ baths where their mechanical responses to various stimuli could be studied. Each tissue w...
Ellington JE, Ball BA, Yang X.The objective of this study was to determine whether coculture of stallion spermatozoa and mare oviductal (uterine tubal) epithelial cells induced sperm cell capacitation in vitro. Capacitation as determined by zona binding and chlortetracycline staining of the sperm cells was compared for stallion spermatozoa: (1) incubated with medium alone (negative control), (2) treated with calcium ionophore A23187 (positive control) or (3) cultured with mare oviductal epithelial cells (OEC) for 4 h. Chlortetracycline staining patterns of sperm cells bound to the zonae were used to group spermatozoa as un...
Scepek S, Lindau M.We have investigated the granule fusion events during exocytosis in horse eosinophils by time-resolved patch-clamp capacitance measurements. Stimulation with intracellular GTP gamma S leads to a stepwise capacitance increase by 4.0 +/- 0.9 pF. At GTP gamma S concentrations < 20 microM the step size distribution is in agreement with the granule size distribution in resting cells. Above 80 microM the number of steps is reduced and very large steps occur. The total capacitance increase, however, is unaffected. These results show that at high GTP gamma S concentrations granule--granule fusion o...
Threadgill DS, Steagall WK, Flaherty MT, Fuller FJ, Perry ST, Rushlow KE, Le Grice SF, Payne SL.The putative dUTPase domain was deleted from the polymerase (pol) gene of equine infectious anemia virus (EIAV) to produce a recombinant delta DUpol Escherichia coli expression cassette and a delta DU proviral clone. Expression of the recombinant delta DUpol polyprotein yielded a properly processed and enzymatically active reverse transcriptase, as determined by immunoblot analysis and DNA polymerase activity gels. Transfection of delta DU provirus into feline (FEA) cells resulted in production of virus that replicated to wild-type levels in both FEA cells and fetal equine kidney cells. In con...
de Haas GH, Dijkman R, Lugtigheid RB, Dekker N, Van den Berg L, Egmond MR, Verheij HM.The inhibitory power (Z) of a number of (R)-1-alkyl-2-acylamino phospholipid analogues was determined for three mammalian phospholipases A2 from pig, ox and horse pancreas. All three enzymes display a clear preference for anionic (phosphoglycol) inhibitors over the zwitterionic (phosphocholine) derivatives; this effect is most pronounced for the bovine enzyme. Upon variation of the 1-alkyl chain length, the bovine and equine phospholipases, like the porcine enzyme in previous studies, show an optimum in Z for a six-carbon alkyl group. The introduction of a double bond in the 2-acylamino group ...
Tözsér J, Friedman D, Weber IT, Bláha I, Oroszlan S.The proteinase of the equine infectious anemia virus (EIAV), a lentivirus closely related to human immunodeficiency virus (HIV), was purified from concentrated virus. The specificity of the enzyme was characterized using oligopeptides representing naturally occurring cleavage sites in the Gag and Gag-Pol polyproteins. The length of the substrate binding pocket was found to be 1-2 residues longer than that of HIV proteinases. Although the EIAV and HIV proteinases cleaved most of the peptides at the same bond, some were hydrolyzed by only the EIAV enzyme. Oligopeptides representing cleavage site...
Inaba M, Goto I, Sato K, Maede Y.This report describes a unique response of the anion transporter in equine erythrocytes to the fluorescent substrate N-(2-aminoethyl sulfonate)-7-nitrobenz-2-oxa-3-diazole (NBD-taurine). Equine erythrocytes showed fluxes of NBD-taurine both inward and outward at rates considerably slower than those in human cells. These fluxes were completely abolished by a typical anion transport inhibitor, 4,4'-diisothiocyanostilbene-2,2'-disulfonate. Furthermore, NBD-taurine competitively inhibited the uptake of phosphate in equine red cells with an inhibition constant of phosphate that was slightly higher ...
Chopineau M, Maurel MC, Combarnous Y, Durand P.In order to localize the epitopes of equine chorionic gonadotropin (eCG) involved in interaction with luteinizing hormone (LH) and follicle-stimulating hormone (FSH) receptors, we used 14 monoclonal anti-eCG antibodies (mAbs). Different effects of these mAbs on the bioactivities of eCG were observed in in vitro bioassays, but the effects of each mAb on the two bioactivities were similar for all but four mAbs. All mAbs were found to inhibit the binding of eCG to LH receptors except 3A3 mAb, in radioreceptor assay. Six mAbs, which were strong inhibitors of eCG binding to LH receptors and of both...
Carvalho M, Derse D.Equine infectious anemia virus (EIAV) is a lentivirus that causes a chronic disease of horses characterized by cyclic episodes of fever, anemia, and viremia. Although the genome and promoter of EIAV are much less complex than those of its relatives the primate immunodeficiency viruses, the cellular proteins that activate and regulate transcription of EIAV have not yet been identified. In this report, we show by electrophoretic mobility shift assays and DNase I footprinting that the EIAV promoter contains multiple binding sites for ubiquitous, cell type-specific, and inducible cellular proteins...
Holman PJ, Frerichs WM, Chieves L, Wagner GG.Culture of horse blood for Babesia caballi identified four carrier horses among nine previously infected horses. Three of the carriers had no detectable parasitemias on stained blood smears, and sera from two carrier horses were complement fixation test negative. Three cultures were continuously cultivated. Cryopreserved fourth-passage B. caballi was successfully reestablished in vitro. Blood from a 10th horse previously subinoculated with blood from a suspected carrier was cultured, with negative results.
Casey PJ, Robertson KR, Liu IK, Espinoza SB, Drobnis EZ.Subfertility in stallions is common, and methodologies are needed to increase the fertility in these animals. In other species, removal of the dead sperm from semen increases the quality and fertility of semen. With horse semen we evaluated 48 combinations of column separation techniques using micro-spin chromatography columns. The greatest improvement in motility was observed with glass wool, whereas glass beads exhibited the greatest recovery of motile sperm. Although centrifugation time did not influence recovery rate or percent motility, a column length of 2 cm was superior for recovery of...
Poole AW, Heath MF, Evans RJ.Pre-incubation of equine platelets in platelet-rich plasma with adenosine 5'-diphosphate (ADP) induced a reduction in aggregation responsiveness to subsequent additions of ADP. The desensitisation was shown to be homologous since the responsiveness to platelet-activating factor, thrombin, collagen, 5-hydroxytryptamine or ionomycin remained unchanged. Adenosine 5'-(beta-thio)-diphosphate (ADP beta S), a non-hydrolysable analogue of ADP, was shown to act as an agonist inducing aggregation by interaction with the ADP receptor. ADP beta S was then used in the desensitisation studies in which resid...
Wang Z, Robinson NE, Yu M.This study was conducted to determine the effects of stimulation parameters and muscle preload on acetylcholine (ACh) release induced by electrical field stimulation (EFS) of horse airway cholinergic nerves. Trachealis strip bundles were prepared and suspended in 2-ml tissue baths. The tissues were stimulated three to five times for 30 min each. Increasing frequency (0.5-16 Hz) and voltage (5-20 V) increased ACh release; increasing pulse duration (0.5-3 ms) had only a minor effect. Alterations in muscle preload (2-20 g) had no effect on ACh release. ACh release was fairly constant for up to fi...
Hoffman DW, Colvin RA, Garcia-Blanco MA, White SW.A 25-nucleotide RNA with the sequence of the trans-activation response (TAR) element of equine infectious anemia virus (EIAV) was analyzed by biochemical methods and by one- and two-dimensional NMR spectroscopy. NMR, nuclease probing, and polyacrylamide gel migration rates show that the RNA consists of an A-helical stem capped by two non-Watson-Crick U-G base pairs and a compact four-nucleotide loop. The loop is stabilized by base stacking, with loop nucleotides C12 and C15 stacked upon U11 and G16, respectively. Near the 5' end of the molecule, the stem contains a bulge at nucleotide C2, most...
Auer DE, Ng JC, Seawright AA.Free radical oxidation products, namely conjugated dienes, ultraviolet fluorescence (excitation 325 nm, emission 395 nm) and visible fluorescence (excitation 360 nm, emission 460 nm) were measured in equine synovial fluid exposed to free radicals in vitro and in the plasma and synovial fluids of horses with synovial effusions. The synovial effusions were induced by intra-articularly administered carrageenin (0.3 ml, 1%), which rarely resulted in clinical lameness. The free radicals were generated in vitro by mixtures of iron and ethylene diamine tetra acetate (Fe/EDTA) or mixtures of hypoxanth...
Nitta K, Tsuge H, Iwamoto H.Unfolding profiles of two calcium-binding lysozymes, equine milk lysozyme and pigeon egg-white lysozyme, were obtained by circular dichroism and proton NMR measurements. Equine lysozyme unfolds through a stable molten globule intermediate. The molten globule of equine lysozyme was characterized as more ordered than that of bovine alpha-lactalbumin. On the other hand, pigeon lysozyme unfolds by a two-state mechanism and the intermediate could not be observed in guanidine or thermal unfolding, the same as with conventional non-calcium-binding lysozymes. Thus, from the point of view of the unfold...
Hinrichs K, Schmidt AL, Friedman PP, Selgrath JP, Martin MG.The chromatin configuration of resting horse oocytes and the time course of in vitro oocyte maturation was characterized using a fluorescent, DNA-specific label. Oocytes were classified as having either compact (CP) or expanded (EX) cumuli at the time of collection. Centrifugation of oocytes was effective in allowing visualization of the germinal vesicle. Two main chromatin configurations were found in oocytes known to have a germinal vesicle: condensed chromatin (CC), in which the chromatin formed a dense mass surrounding the nucleolus; and fluorescing nucleus (FN), in which the entire nucleu...
Pérez MD, Puyol P, Ena JM, Calvo M.The interaction of sheep, horse, pig, human and guinea-pig whey proteins with fatty acids has been studied. Using gel filtration and autoradiography, it was found that sheep beta-lactoglobulin and serum albumin from all species had the ability to bind fatty acids in vitro. Sheep beta-lactoglobulin, isolated from milk, had approximately 0.5 mol fatty acids bound per mol monomer protein, and albumin from sheep, horse and pig contained approximately 4.5, 2.9 and 4.7 mol fatty acids/mol protein respectively. However, beta-lactoglobulin from horse and pig milk had neither fatty acids physiologicall...
Simmons TR, Cook JA, Moore JN, Halushka PV.Thromboxane (TX) A2 has been implicated as an important pathophysiologic mediator of a variety of cardiovascular diseases. Monocytes synthesize TXA2 and it modulates their function. This study sought to characterize monocyte TXA2 receptors. Radioligand binding studies were performed on membranes prepared from equine peripheral blood monocytes using [125I]BOP, a TXA2 receptor agonist. [125I]BOP bound to a single class of binding sites (Kd = 1.0 +/- 0.3 nM and Bmax = 389 +/- 191 fmol/mg protein; n = 5). Several TXA2 receptor agonists and antagonists competed for binding with [125I]BOP. I-BOP pro...
Gospodarek E.Direct and intermediate hemolytic activity of 526 strains of Acinetobacter was investigated. Their ability to produce lipase and lecithinase was also studied. Measurements were performed parallely on human, horse, sheep and bovine erythrocytes. Direct hemolytic activity was exhibited by 16% of tested strains (17 out of 24 strains of A. haemolyticus). Human, sheep and bovine erythrocytes were useful for testing the hemolytic activity of Acinetobacter. The hemolysis was occurring faster and was visible more frequently during incubation at 37 degrees C. Indirect hemolytic activity was observed in...
Poole AW, Heath MF, Sage SO, Evans RJ.Equine platelets in platelet-rich plasma were incubated with the fluorescent indicator dye, Fura-2-AM (Fura-2-acetoxymethyl ester) and the degree of loading of the cells with the dye and the extent of hydrolysis of the ester was assessed by quantitative fluorimetry and by thin-layer chromatography respectively. Under these conditions the cells loaded poorly with Fura-2 to a concentration of 4 microM. The technique was validated by demonstrating adequate loading of human platelets with Fura-2, to a concentration of 250-300 microM, using the same method. The removal of plasma from the extracellu...
García-Alfonso C, Martínez-Galisteo E, Llobell A, Bárcena JA, López-Barea J.1. Purification of horse-liver glutathione reductase was obtained by affinity chromatography on N6-(6-aminohexyl)-adenosine-1'5'-bisphosphate Sepharose (N6-2'5'-ADP-Sepharose) and Reactive Red-120-Agarose, and chromatography on DEAE-Sephadex and Sephacryl S-300. 2. The final preparation had 248 U/mg specific activity after 11,174-fold purification with 47% final recovery, and was homogeneous by SDS-electrophoresis. It showed charge heterogeneity in non-denaturing electrophoresis and chromatofocusing, with several peaks of pI between 5.7 and 6.7. 3. The enzyme was homodimeric (107,000 native MW...
Salloum RM, Duckworth D, Madison JB, Souba WW.The sodium-dependent transporter system responsible for L-glutamine uptake by brush border membrane vesicles prepared from equine jejunum was characterized. Vesicle purity was ascertained by a 14- to 17-fold increase in activity of the brush border enzyme markers. Glutamine uptake was found to occur into an osmotically active space with negligible membrane binding. The sodium-dependent velocity represented approximately 80% of total uptake and demonstrated overshoots. Kinetic studies of sodium-dependent glutamine transport at concentrations between 5 microM and 5 mM revealed a single saturable...
Beech J, Lindborg S, Fletcher JE, Lizzo F, Tripolitis L, Braund K.Muscle from horses with intermittent exercise associated rhabdomyolysis was examined to determine if calcium regulation was abnormal. In vitro studies on semimembranosus muscle fibre bundles showed the time to 50 per cent relaxation of caffeine-induced contractures was shorter and the electrically elicited twitch longer in horses with exercise associated rhabdomyolysis. Substitution of strontium for calcium eliminated the difference in caffeine contracture between the normal and rhabdomyolysis horses. The threshold of calcium-induced calcium release was lower than normal in terminal cisternae-...
Hay WP, Tulleners EP, Ducharme NG.An extralaryngeal approach to partial arytenoidectomy in the horse was developed by in vitro experiments on isolated larynges and then on intact equine cadavers. The goals of the approach were to preserve the laryngeal mucosa, eliminate the need for a laryngotomy or tracheotomy, and minimize postoperative complications. The new approach was evaluated in seven horses with normal upper respiratory tracts. Left laryngeal hemiplegia was surgically created, and, after a 30-day convalescence, left partial arytenoidectomy was performed using an extralaryngeal approach. The left-to-right hemilaryngeal...
Pelegrino JL, Vázquez S, Morier L, Castillo A, Guzmán MG, Kourí G.We present the results attained in the identification of Eastern equine encephalomyelitis virus isolations in Vero and XL-2 cell systems, using a double-antibody ELISA technique and the indirect immunofluorescence method. The results attained through these two techniques coincided by 100% with identification through neutralization. With the former, the virus was detected within 6-8 hours after inoculation. Better results were attained with XL-2 cells.
White IG.When sperm of the ram, bull, boar and stallion are cold-shocked by rapid cooling to near freezing point, motility and metabolic activity are irreversibly depressed and the acrosome and plasma membrane disrupted. Ram sperm become susceptible to cold shock in the proximal corpus region of the epididymis when the cytoplasmic droplet has moved backwards to the distal portion of the sperm midpiece. The membrane constituents phospholipids and cholesterol are important in cold shock which causes loss of lipid from sperm. The susceptibility of sperm to cold shock is linked with a high ratio of unsatur...
Rojas G, Vargas M, Robles A, Gutiérrez JM.Twenty batches of polyvalent antivenom produced at the Instituto Clodomiro Picado were analyzed for turbidity, both before and after freezing-thawing and lyophilization. Eight batches became turbid upon freezing-thawing, and this change correlated with high levels of cholesterol, triglycerides and lipoproteins, especially beta-lipoprotein. Since normal horse serum does not become turbid after freezing-thawing, despite the fact that it has high lipoprotein levels, the possibility was raised that phenol, used as a preservative during serum fractionation, might affect lipoproteins, inducing the a...
Fletcher JE, Tripolitis L, Beech J.The modulation of Ca2+ release by a cardiotoxin (CTX) from Naja naja kaouthia snake venom was examined in terminal cisternae-containing fractions from equine and human skeletal muscle. Pretreatment with CTX (10 microM) decreased by 27% (human muscle), or had no effect on (equine muscle), the threshold of Ca(2+)-induced Ca2+ release. If terminal cisternae fractions were first preloaded with Ca2+ to greater than 65% of the threshold of Ca(2+)-induced Ca2+ release and then CTX added, an immediate and sustained release of Ca2+ occurred in preparations from both species. Addition of CTX after a Ca2...
Reisen WK, Meyer RP, Presser SB, Hardy JL.The extrinsic incubation rate (inverse of the time in days from infection to median transmission) of western equine encephalomyelitis (WEE) and St. Louis encephalitis (SLE) viruses by laboratory strains of Culex tarsalis Coquillett increased as a linear function of incubation temperatures from 10 to 30 degrees C. The estimated temperatures for zero transmission thresholds (intercept of the X axis) were 10.9 and 14.9 degrees C, and the number of degree days above these thresholds required for median transmission (inverse of the slope) was 67.6 and 115.2, respectively. Although the bodies of mos...
Desantis S, Ventriglia G, Zizza S, De Santis T, Di Summa A, De Metrio G, Dell'aquila ME.Equine cumulus-oocyte complexes (COCs) were analyzed by means of 13 lectins to evaluate their glycoconjugate patterns and to verify differences between COCs recovered with compact (Cp) and expanded (Exp) cumulus. Cumulus cells showed a similar staining pattern in both Cp and Exp COCs with all lectins used, except for a higher reactivity with SNA and GSA II in Cp COCs and SBA in Exp COCs. The zona pellucida (ZP) showed (1) uniform staining with MAL II, RCA(120), and SBA in both Cp and Exp COCs, (2) trilaminar binding pattern with WGA as well as higher Con A reactivity in the outer region of bot...
Morgan SJ, Storts RW, Stromberg PC, Sowa BA, Lay JC.Factors involved in the proliferation of equine vascular smooth muscle cells were studied in vitro. The most prominent proliferative responses in cultured vascular smooth muscle cells were induced by Strongylus vulgaris larval antigen extract (LAE) and platelet-derived factors. Less significant proliferative responses were obtained with conditioned media from S. vulgaris LAE stimulated and from unstimulated equine mononuclear leukocytes. Additionally, vascular smooth muscle cells exposed to S. vulgaris LAE developed numerous perinuclear vacuoles and were more spindle-shaped than control or smo...
Dippert KD, Jasko DJ, Seidel GE, Squires EL.Embryo recovery per ovulation has been shown to be lower in superovulated mares than in untreated controls. The objectives of this study were to 1) determine whether follicles stimulated with superovulatory treatment ovulate or luteinize without ovulation, 2) determine fertilization rates of oocytes in oviducts of superovulated and control mares, and 3) evaluate viability of early stage embryos from superovulated and control mares when cultured in equine oviductal cell-conditioned medium. Cyclic mares were randomly assigned to 1 of 2 groups (n=14 per group) on the day of ovulation (Day 0): Gro...
Rydevik A, Bondesson U, Hedeland M.Bupivacaine is a local anaesthetic prohibited in equine sports. It is highly metabolized in the horse but a thorough description of its metabolite profile is lacking. An administration study should find appropriate analytical targets for doping control. Furthermore, knowledge of an in vitro system for production of metabolites would be beneficial. Methods: Marcain® (bupivacaine hydrochloride) was administered subcutaneously to a horse and urine samples were collected. In vitro metabolic systems consisting of the fungi Cunninghamella elegans and Cunninghamella blakesleeana were incubated with ...
van Dixhoorn ID, Meershoek LS, Huiskes R, Schamhardt HC.Motion of the navicular bone might play a role in the development of navicular disease in horses but is difficult to asses. In the present study, 3-dimensional motion of this bone was determined using roentgen stereophotogrammetric analysis. Tantalum pellets were inserted, in vitro, in the bones of 6 forelimbs of mature Shetland ponies and kinematics were measured during vertical loading up to 2 kN. The motions of the navicular bone and coffin joint were limited to flexion/extension, there were no substantial out-of-plane motions. There was only little flexion between the navicular bone and th...
Tessier GJ, Lackner PA, O'Grady SM, Kannan MS.The role of epithelium in the modulation of contractile responses to electrical field stimulation (EFS), acetylcholine (ACh), and KCl were studied in vitro in strips of equine tracheal smooth muscle (TSM). EFS with 0.5 ms pulses of voltage (70 V) resulted in frequency dependent contractions of equine TSM that were sensitive to tetrodotoxin (TTX) and atropine. In TSM without epithelium, preincubation with indomethacin significantly potentiated contractile responses to EFS. The potentiating effect of indomethacin on EFS contractions was abolished by the addition of 3 nM prostaglandin E2 (PGE2). ...
Pessoa GA, Martini AP, Trentin JM, Minela T, Fiorenza MF, Rubin MIB.The aim of this study was to compare the sperm separation technique using filtration through glass wool compared with just diluted cooled semen. Eighteen ejaculates were collected from 6 pony stallions of the Brazilian pony breed. Evaluations were done on pH, osmolarity, total motility, membrane functionality (HOST), membrane integrity (CFDA/PI), morphology and mitochondrial viability (MTT) in fresh, 24 and 48 h of cooled semen at 5°C. After dilution, the half of the extended semen was cooled (control group). The other half was cooled after filtration trough glass wool (filtered group). Reta...
Waller CC, Cawley AT, Suann CJ, Ma P, McLeod MD.Furazadrol ([1',2']isoxazolo[4',5':2,3]-5α-androstan-17β-ol) is a designer anabolic androgenic steroid that is readily available via the internet. It contains an isoxazole fused to the steroid A-ring which offers metabolic stability and noteworthy anabolic activity raising concerns over the potential for abuse of this compound in equine sports. The metabolism of furazadrol was studied by in vivo and in vitro methods for the first time. Urinary furazadrol 17-sulfate and furazadrol 17-glucuronide metabolites were detected in vivo after a controlled administration and compared with syntheticall...
Rakhit S, Murdoch R, Wilson SM.Adrenaline, forskolin and ATP all evoked accumulation of cyclic AMP in equine sweat gland epithelial cells, although the response to adrenaline was more transient than that to forskolin and ATP. Cells preincubated in adrenaline (10 micromol l-1, 32 min) showed essentially complete, homologous desensitisation, and this phenomenon reversed slowly (half-time 6.3+/-0.9 h). After 10 min of recovery from preincubation in adrenaline, isobutylmethylxanthine (IBMX, 5 mmol l-1) had no effect upon the desensitisation and the cells showed no loss of sensitivity to ATP and forskolin. After 10 h, however, t...
Sod GA, Riggs LM, Mitchell CF, Martin GS, Gill MS.To compare the monotonic biomechanical properties and fatigue life of a 5.5-mm-broad locking compression plate (5.5 LCP) fixation with a 4.5-mm-broad locking compression plate (4.5 LCP) fixation to repair osteotomized equine 3rd metacarpal (MC3) bones. Methods: In vitro biomechanical testing of paired cadaveric equine MC3 with a middiaphyseal osteotomy, stabilized by 1 of 2 methods for fracture fixation. Methods: Fifteen pairs of adult equine cadaveric MC3 bones. Methods: Fifteen pairs of equine MC3 were divided into 3 test groups (5 pairs each) for (1) 4-point bending single cycle to failure ...
Haussler KK, McGilvray KC, Ayturk UM, Puttlitz CM, Hills AE, McIlwraith CW.Sacroiliac joint injuries can cause poor performance; however, the interaction between pelvic mechanics and the sacroiliac joint is poorly understood. Objective: To measure pelvic displacement during 3D sacroiliac joint loading. Methods: Nine reflective triads were attached rigidly to bony prominences in sacropelvic specimens harvested from 14 horses for stereophotogrammetric analysis of triad displacements and joint kinematics. The sacrum was coupled to a load cell and mounted vertically within a material testing system (MTS). A pneumatic actuator was used to apply 90 Nm moments to the ischia...
Reubel GH, Studdert MJ.Based on sequence homology with human adenovirus 2 (HAdV2), the hexon gene of equine adenovirus 1 (EAdV1) was identified. HindIII restriction fragments containing the hexon and other viral genes were cloned into the plasmids pUC19 and pBlueScript SK(-) and sequenced. The nucleotide sequence of the hexon gene was completely determined and partial sequence data were obtained for seven other EAdV1 genes. Amino acid (aa) sequence comparison with published adenovirus (AdV) proteins identified the genes for the IIIa, penton, pVII, PVI, 23K proteinase, DNA binding and 100K proteins. The eight EAdV1 g...
Chodkowski M, Słońska A, Gregorczyk-Zboroch K, Nowak-Zyczynska Z, Golke A, Krzyżowska M, Bańbura MW, Cymerys J.Mitochondria are key cellular organelles responsible for many essential functions, including ATP production, ion homeostasis and apoptosis induction. Recent studies indicate their significant role during viral infection. In the present study, we examined the effects of equine herpesvirus type 1 (EHV-1) infection on the morphology and mitochondrial function in primary murine neurons in vitro. We used three EHV-1 strains: two non-neuropathogenic (Jan-E and Rac-H) and one neuropathogenic (EHV-1 26). The organization of the mitochondrial network during EHV-1 infection was assessed by immunofluores...
Tetens J, Venugopal CS, Holmes EP, Koch CE, Hosgood G, Moore RM.To evaluate the in vitro effects of adenosine tryphosphate (ATP) on vasomotor tone of equine colonic vasculature. Methods: Arteries and veins from the left ventral colon of 14 mixed-breed horses euthanatized for reasons unrelated to cardiovascular or gastrointestinal tract disease. Methods: Endothelium-intact and -denuded arterial and venous rings were precontracted with 10(-7) and 1.8 x 10(-8) M endothelin-1, respectively. In 1 trial, endothelium-intact rings were also incubated with 10(-4) M N omega-nitro-L-arginine methyl ester (L-NAME) to inhibit nitric oxide (NO) production. Adenosine tri...
Bertero A, Ritrovato F, Evangelista F, Stabile V, Fortina R, Ricci A, Revelli A, Vincenti L, Nervo T.The purpose of this study was to observe -matured equine oocytes with an objective computerized technique that involves the use of a polarized light microscope (PLM) in addition to the subjective morphological evaluation obtained using a classic light microscope (LM). Equine cumulus-oocyte complexes (COCs, = 922) were subjected to different maturation times (24, 36 or 45 h), however, only 36-h matured oocytes were analyzed using CLM. The 36-h matured oocytes that reached maturity were parthenogenetically activated to evaluate the quality and meiotic competence. Average maturation perce...
Yager JA, Foster SF, Zink MC, Prescott JF, Lumsden JH.Polymorphonuclear leukocytes from adult horses were separated from whole blood, using a 2-step Percoll gradient, and were tested for bactericidal function against Corynebacterium equi. Staphylococcus aureus, an organism against which equine neutrophils have proved efficacy, was a positive control. The percentage of uptake after a 15-minute preincubation of the neutrophils and bacteria in the presence of normal horse serum was also calculated. The results indicated that equine neutrophils effectively phagocytosed and killed C equi and S aureus. The percentage of uptake for S aureus (95% +/- 3%)...
Berg LC, Mata X, Thomsen PD.Cartilage-derived retinoic acid sensitive protein (CD-RAP) also known as melanoma inhibitory activity (MIA) has already been established as a marker for chondrocyte differentiation and a number of cancerous conditions in humans. Studies have also shown that CD-RAP/MIA is a potential marker of joint disease. The objective of this study was to characterize the equine CD-RAP/MIA gene and thus make it available as a marker in cartilage research and clinical studies. Gene analysis revealed that the equine gene (GenBank accession no. EF679787) consists of four exons and three introns, and the homolo...
Mancheño JM, Jayne S, Kerfelec B, Chapus C, Crenon I, Hermoso JA.Horse pancreatic lipase-related proteins PLRP1 and PLRP2 are produced by the pancreas together with pancreatic lipase (PL). Sequence-comparison analyses reveal that the three proteins possess the same two-domain organization: an N-terminal catalytic domain and a C-terminal domain, which in PL is involved in colipase binding. Nevertheless, despite the high level of sequence identity found, they exhibit distinct enzymatic properties. The intrinsic sensitivity of the peptide bond between Ser245 and Thr246 within the flap region of PLRP2 to proteolytic cleavage probably complicates PLRP2 crystalli...
Sandbaumhüter FA, Theurillat R, Bettschart-Wolfensberger R, Thormann W.The combination of ketamine and an α -receptor agonist is often used in veterinary medicine. Four different α -receptor agonists, medetomidine, detomidine, xylazine, and romifidine, which differ in their chemical structure and thus in selectivity for the α -receptor and in the sedative and analgesic potency, are typically employed during surgery of equines. Recovery following anesthesia with ketamine and an α -receptor agonist is dependent on the α -receptor agonist. This prompted us to investigate (i) the inhibition characteristics for the N-demethylation of ketamine to norketamine and (...
Hu WG, Alvi AZ, Fulton RE, Suresh MR, Nagata LP.A recombinant gene encoding a single-chain variable fragment (scFv) antibody against Venezuelan equine encephalitis virus (VEE) was cloned into a prokaryotic T7 RNA polymerase-regulated expression vector. A streptavidin-binding peptide gene fused to a 6His tag was attached downstream to the scFv gene. The recombinant fusion protein was expressed in bacteria as inclusion bodies that were subsequently solubilized with 8 M urea and renatured by an arginine system. Purification of the fusion protein was achieved by immobilized metal affinity chromatography. Enzyme-linked immunosorbent assay (ELISA...
Contreras MJ, Arias ME, Silva M, Cabrera P, Felmer R.Cryopreservation of stallion semen is less efficient than other species such as bovine. This is mainly because of the greater susceptibility of stallion sperm to the freezing damage that generates oxidative stress and plasma membrane injury, resulting in DNA fragmentation and cell death. These data suggest the need to develop new strategies of sperm cryopreservation that can improve the efficiency of this technique in stallions by reducing or preventing membrane damage and cell death. The present study aimed to evaluate the effect of adding membrane stabilizers to the freezing medium and asses...
de Rebière de Pouyade G, Salciccia A, Ceusters J, Deby-Dupont G, Serteyn D, Mouithys-Mickalad A.The endothelium plays an active role in ischemia/reperfusion injuries. Herein, we report the effect of a single or successive cycles of anoxia/reoxygenation (A/R) on the mitochondrial respiratory function of equine endothelial cells (cultured from carotids) monitored by high resolution oxymetry, and on their production of reactive oxygen species (ROS). ROS were measured by electron paramagnetic resonance (ESR) using POBN and DMPO spin traps, and by gas chromatography (GC) of ethylene released by ROS-induced α-keto-γ-(methylthio)butyric acid (KMB) oxidation. The oxygen consumption significant...
Joujou-Sisic K, Granérus M, Wetterling H, Wikström K, Engström W, Jeffcott L, Schofield PN, Welin A.The expression of the insulin like growth factor (IGF) II gene has been examined in the developing equine fetus. It was found that IGF II transcripts were present in abundant quantities in third trimester embryonic and extraembryonic tissues as for example the placenta. The expression of the IGF II gene was high in the fetal liver where two prominent transcripts--4.6 and 4.1--kB were produced. However, these transcripts could not be traced in the adult liver. Instead we found two different transcripts with the sizes of 4.0 and 2.9 kB in the adult liver. These findings taken together with the d...
Arroyo-Salvo C, Sanhueza F, Fuentes F, Treulén F, Arias ME, Cabrera P, Silva M, Felmer R.Conventional in vitro fertilization has not yet been implemented in the equine species. One of the main reasons has been the inability to develop a culture medium and incubation conditions supporting high levels of stallion sperm capacitation and hyperactivation in vitro. Although different culture media have been used for this purpose, human tubal fluid (HTF) medium, widely used in the manipulation of human and mice gametes, has not been reported so far in stallion sperm culture. The first part of this study aimed to compare HTF and Whitten's media on different stallion sperm quality and capa...
Lefebvre RA, Callens C, Van Colen I, Delesalle CJG.Postoperative ileus (POI) remains an important cause of death in horses. The recently developed selective 5-HT receptor agonists such as prucalopride target 5-HT receptors on myenteric cholinergic neurons to enhance acetylcholine release and GI motility. No clearcut in vitro evaluation whether highly selective 5-HT receptor agonists enhance submaximal cholinergic neurotransmission towards the muscle layer has been performed in horses. Objective: To identify functional 5-HT receptors in equine jejunum. Methods: In vitro experimental study. Methods: Circular and longitudinal smooth muscle strips...
Sayers DE, Theil EC, Rennick FJ.Cell-specific variations in apoferritin structure correlate with variations in iron metabolism that suggest functional specificity of the protein shell. Using EPR spectroscopy, we previously showed that vanadyl binds to specific sites on apoferritin, and that VO2+ binding is reduced by Fe(II) and Fe(III) (the natural substrates) and by metals known to influence iron storage (Chasteen, N. D., and Theil, E. C. (1982) J. Biol. Chem. 257, 7672-7677). Such observations suggest that the metal-binding site is important to apoferritin function and may define a location where the influence of cell-spec...
Moslemi S, Seralini GE.In order to approach the detailed structure-function relationships of aromatase, we studied the inhibitory and inactivatory potencies of several steroidal androstenedione analogues (1: 4-hydroxyandrostenedione, 2: 4-acetoxyandrostenedione and 3: 7 alpha-(4'-amino)phenylthio-4-androstene-3, 17-dione) and non-steroidal imidazole derivatives (4: ketoconazole, 5: miconazole and 6: fadrozole) on equine aromatase in placental microsomes, a well established mammalian model. Human placental microsomes and the purified enzyme from equine testis were also used to compare inhibition by 1 and 2. In equine...
Bakala A, Karlik W, Wiechetek M.In this study a detailed description of the equine hepatocyte isolation procedure is presented. Livers were obtained from horses slaughtered at the local slaughterhouse. For blood removal and liver preservation the following steps are suggested: perfusion with the oxygenated HBSS (0-2 degrees C, with continuous flow of 500-800 ml/min for 3-6 min), protection from ischemia injury by flushing with ice-cold University of Wisconsin Solution (UW, flow rate of 500-800 ml/min), and finally immersion of the liver lobe in UW solution (2 degrees C) during its transport to the laboratory. For equine isol...
Álvarez C, González N, Luño V, Martínez F, Gil L.The aim of this study was to test and compare two new components in extenders for freezing donkey semen: mare colostrum and jenny colostrum. Colostrum was obtained from four mares and four jennies right after the foal's birth. Ejaculates were collected from five fertile donkeys. Sperm samples were pooled, diluted and cryopreserved in three different experimental extender groups: lactose supplemented with egg yolk extender (20%) as the control group, lactose supplemented with jenny colostrum extender (20%), and lactose supplemented with mare colostrum extender (20%). After thawing, we evaluated...
Becker CK, Savelberg HH, Barneveld A.The material properties of the accessory ligament of the deep digital flexor tendon (AL) of 21 forelimbs from horses between ages one day and 15 years were determined. The force (634-11416 N), failure stress (45-138 N/mm2), failure strain (7-24%) and tangent modulus (33-1639 MPa) are presented in relation to age. Tangent modulus did not indicate changes in elasticity due to age. The results demonstrate that complete ligament failures (CLF) of ALs of older horses (mean 7835 N) occur at lower forces than ALs of young adult horses (mean 8894 N). Sudden decreases, 'dips', in the force-time curves ...
