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.
Tharasanit T, Colenbrander B, Stout TA.Oocyte cryopreservation is a potentially valuable technique for salvaging the germ-line when a valuable mare dies, but facilities for in vitro embryo production or oocyte transfer are not immediately available. This study examined the influence of maturation stage and freezing technique on the cryopreservability of equine oocytes. Cumulus oocyte complexes were frozen at the immature stage (GV) or after maturation in vitro for 30 hr (MII), using either conventional slow freezing (CF) or open pulled straw vitrification (OPS); cryoprotectant-exposed and untreated nonfrozen oocytes served as contr...
Murphy BA, Vick MM, Sessions DR, Cook RF, Fitzgerald BP.The master mammalian pacemaker in the brain controls numerous diverse physiological and behavioral processes throughout the organism. Timing information is continually transmitted from the master clock to peripheral organs to synchronize rhythmic daily oscillations of clock gene transcripts and control local physiology. To investigate the presence of peripheral clocks in the horse, quantitative real-time RT-PCR assays were designed to detect levels of equine clock genes. Expression profiles for Per2, Bmal1 and Cry1 were first determined in a synchronized equine cell line. Subsequently, express...
Werners AH, Bull S, Vendrig JC, Smyth T, Bosch RR, Fink-Gremmels J, Bryant CE.The inter- and intra-species differences in the response to lipopolysaccharides (LPS) are well recognised in mammalian species. It has been hypothesized that these differences can be attributed to genetic polymorphisms in the components involved in LPS signal transduction. These components include the cluster of differentiation factor 14 (CD-14), a membrane bound protein on the surface of mononuclear cells that recognises LPS and a receptor complex consisting of Toll-like receptor-4 (TLR-4) and myeloid differentiation factor-2 (MD-2). Sequencing of these three proteins in humans and mice revea...
Coombs DK, Patton T, Kohler AK, Soboll G, Breathnach C, Townsend HG, Lunn DP.Protecting equids against equine herpesvirus-1 (EHV-1) infection remains an elusive goal. Repeated infection with EHV-1 leads to protective immunity against clinical respiratory disease, and a study was conducted to measure the regulatory cytokine response (IFN-gamma and IL-4) in repeatedly infected immune ponies compared to non-immune ponies. Two groups of four ponies were established. Group 1 ponies had previously been infected on two occasions, and most recently 7 months before this study. Group 2 ponies had no history no vaccination or challenge infection prior to this study. Both groups w...
Goodman LB, Wagner B, Flaminio MJ, Sussman KH, Metzger SM, Holland R, Osterrieder N.Equine herpesvirus type 1 (EHV-1) is a ubiquitous alphaherpesvirus of horses which causes rhinopneumonitis, abortion and myeloencephalopathy. To test the efficacy of commercial vaccines in protection against neurological EHV-1 challenge, groups of five horses were immunized with modified-live virus or an inactivated vaccine, or received placebo. Horses were challenged by aerosol with a recent virus isolate obtained from a case of paralytic EHV-1. The duration of fever decreased significantly in the modified-live virus vaccine group. Three animals in each of the inactivate and control groups sh...
Wagner B, Hillegas JM, Antczak DF.Interleukin-4 (IL-4) is secreted by T helper type 2 cells, mast cells, basophils and eosinophils. Detection of IL-4 can contribute the evaluation of cellular immune responses during infectious diseases, immunological disorders or vaccination. We used recombinant equine IL-4 to generate a monoclonal antibody (mAb) to equine IL-4. The mAb detected recombinant IL-4 in mammalian cells transfected with different plasmids containing IL-4 cDNA. After mitogen stimulation of equine peripheral blood mononuclear cells, an intracellular protein was recognized by the new mAb in 1-2% of lymphocytes using fl...
Thomas E, Thomas V, Wilhelm C.Cefquinome is known for its use as an antibacterial drug in cattle and pigs. The objective of this study was to evaluate the antibacterial activity of cefquinome against equine pathogenic bacteria. The minimum inhibitory concentration (MIC) of cefquinome was determined for a total of 205 strains, which had recently been isolated in Europe from diseased horses (respiratory infection, foal septicaemia). The bactericidal activity was tested against 19 strains using the time killing method. The post-antibiotic effect (PAE) and post-antibiotic sub-MIC effect (PA SME) were determined against 12 stra...
Lin YL, Moolenaar H, van Weeren PR, van de Lest CH.To determine effects of microcurrent electrical tissue stimulation (METS) on equine tenocytes cultured from the superficial digital flexor tendon (SDFT). Methods: SDFTs were collected from 20 horses at slaughter. Methods: Tenocytes were isolated following outgrowth from explants and grown in 48-well plates. Four methods of delivering current to the tenocytes with a METS device were tested. Once the optimal method was selected, current consisting of 0 (negative control), 0.05, 0.1, 0.5, 1.0, or 1.5 mA was applied to cells (8 wells/current intensity) once daily for 8 minutes. Cells were treated ...
Schejter A, Ryan MD, Blizzard ER, Zhang C, Margoliash E, Feinberg BA.Contrary to most heme proteins, ferrous cytochrome c does not bind ligands such as cyanide and CO. In order to quantify this observation, the redox potential of the ferric/ferrous cytochrome c-cyanide redox couple was determined for the first time by cyclic voltammetry. Its E0' was -240 mV versus SHE, equivalent to -23.2 kJ/mol. The entropy of reaction for the reduction of the cyanide complex was also determined. From a thermodynamic cycle that included this new value for the cyt c cyanide complex E0', the binding constant of cyanide to the reduced protein was estimated to be 4.7 x 10(-3) L M(...
Deeg CA, Amann B, Raith AJ, Kaspers B.To test the hypothesis that inter- and intramolecular spreading to S-antigen (S-Ag) and interphotoreceptor retinoid binding protein (IRBP)-derived epitopes occurs in a spontaneous model of recurrent uveitis in the horse. Methods: The immune response of eight horses with equine recurrent uveitis (ERU) was compared with that of five control horses with healthy eyes. Lymphocytes derived from peripheral blood (PBLs) were tested every 8 weeks for their reactivity against S-Ag and various S-Ag and IRBP-derived peptides for 12 to 39 months (median, 22 months). During uveitic episodes, additional bloo...
Weston MC, Collins ME, Cunningham FM.To assess the biological effects of purified recombinant equine CCL11 on equine eosinophil function. Methods: Following stimulation of eosinophils from normal horses, the polymerised form of actin was measured by flow cytometry using fluorescently labelled phalloidin. Migration was determined in a 96 well plate chemotaxis assay using 8 microm pore membranes, and adherence of eosinophils to serum-coated plastic was assessed using a colorimetric assay for eosinophil peroxidase. Superoxide generation was measured by the reduction of cytochrome C in a colorimetric assay. Results: Equine CCL11 indu...
Kareskoski AM, Reilas T, Andersson M, Katila T.With the aim of investigating properties of stallion seminal plasma to eventually improve semen-handling techniques, sperm motility and plasma membrane integrity were analysed in different fractions of the ejaculates after storage. Semen was collected using a computer-controlled automated phantom that separates the ejaculates into five successive cups. Samples containing seminal plasma and skim milk extender were compared with samples stored in skim milk extender after the removal of seminal plasma by centrifugation. Fractionated ejaculates were stored cooled for 24 h after dilution with exten...
Zerpa H, Bailey SR, Berhane Y, Elliott J.Alpha-adrenoceptors mediate contractile responses in equine digital veins (EDVs) and arteries. Vascular smooth muscle alpha(1)-adrenoceptor subtypes have been implicated in a number of conditions, such as acute equine laminitis, and are therapeutic targets for the treatment of this condition. Digital veins, rather than arteries, were investigated in the present study because they have been specifically implicated in the pathophysiology of acute laminitis. The order of potency of a series of alpha(1)-adrenoceptor-selective agonists and antagonists was determined in isolated rings of EDVs under ...
Mills PC, Cross SE.Little is known about the transdermal penetration of hydrocortisone in the horse and, although commercial formulations containing hydrocortisone are registered for topical use in the horse, there have been no studies investigating the movement of this glucocorticoid through different regions of equine skin. Skin was harvested from the thorax, groin and leg (dorsal metacarpal) regions of five Thoroughbred geldings and frozen (-20 degrees C) until required. Defrosted skin was placed in Franz-type diffusion cells and the amount of radiolabelled ((3)H) hydrocortisone, in a saturated solution of un...
Dias GM, Retamal CA, Tobella L, Arnholdt AC, López ML.'The highly packed chromatin of mature spermatozoa results from replacement of somatic-like histones by highly basic arginine- and cysteine-rich protamines during spermatogenesis, with additional conformational changes in chromatin structure during epididymal transit. The objective of the present study was to compare the nuclear characteristics of immature and mature epididymal stallion spermatozoa, using a variety of experimental approaches. Resistance to in vitro decondensation of chromatin, following exposure to SDS-DTT and alkaline thioglycolate, increased significantly in mature spermatoz...
van der Meulen K, Caij B, Pensaert M, Nauwynck H.In vitro studies demonstrated that most equine herpesvirus 1 (EHV-1)-infected peripheral blood mononuclear cells (PBMC) do not expose viral envelope proteins on their surface. This protects them against antibody-dependent lysis. We examined whether viral envelope proteins are also undetectable on infected PBMC during cell-associated viremia. Further, surface expression of major histocompatibility complex (MHC)-I was examined, since MHC-I assists in making infected cells recognizable for cytotoxic T-lymphocytes (CTL). Four ponies, previously exposed to EHV, and two ponies that had no contact wi...
von Samson-Himmelstjerna G.Conventional and real time polymerase chain reaction-based tests have been developed for the diagnosis of anthelmintic resistance (AR) in populations of several small and large ruminant as well as horse gastro-intestinal nematode species. To date, molecular markers that correlate well with AR are available only for the detection of benzimidazole resistance. Recently, however, a single nucleotide polymorphism was found in vitro to be of functional relevance for reduced drug efficacy to macrocylic lactones. The focus of the present review, therefore, is the molecular mechanism of action of these...
Baselgia S, Doherr MG, Mellor P, Torsteinsdottir S, Jermann T, Zurbriggen A, Jungi T, Marti E.Insect bite hypersensitivity (IBH) is an IgE-mediated allergic dermatitis caused by bites of Culicoides and Simulium species, and improved means of diagnosis are required. Objective: The cellular antigen simulation test (CAST) with C. nubeculosus and S. vittatum extracts was assessed in a population of IBH-affected and healthy horses. Variations in test results over a one year period and possible cross-reactivity between different insect extracts was studied. Methods: A total of 314 mature horses were studied using the CAST. Influence of severity of clinical signs, gender and age were evaluate...
Rossignol F, Perrin R, Desbrosse F, Elie C.To compare in vitro the load necessary for a partial and complete rupture of the muscular process arytenoid cartilage when a suture prosthesis is positioned by a bone trocar versus a trocar point needle and to compare failure mode. Methods: Experimental using cadaver specimens. Methods: Larynges from 18 Thoroughbred race horses, aged 2-20 years. Methods: Arytenoid cartilages were separated randomly into 2 groups: group 1-suture prosthesis inserted directly through the muscular process using a curved trocar point needle and group 2-suture passed through a hole predrilled with a 3 mm bone trocar...
Christopherson PW, Insalaco TA, van Santen VL, Livesey L, Bourne C, Boudreaux MK.Glanzmann thrombasthenia (GT) is an inherited, intrinsic platelet defect characterized by a quantitative or qualitative change in the platelet glycoprotein complex IIb-IIIa (integrin alpha(IIb)beta3). The subunits are encoded by separate genes and both subunits must be expressed for a stable complex to form on the platelet surface; therefore, a defect in either gene can result in GT.
Borland C, Dunningham H, Bottrill F, Vuylsteke A.To model lung nitric oxide (NO) and carbon monoxide (CO) uptake, a membrane oxygenator circuit was primed with horse blood flowing at 2.5 l/min. Its gas channel was ventilated with 5 parts/million NO, 0.02% CO, and 22% O2 at 5 l/min. NO diffusing capacity (Dno) and CO diffusing capacity (Dco) were calculated from inlet and outlet gas concentrations and flow rates: Dno = 13.45 ml.min(-1).Torr(-1) (SD 5.84) and Dco = 1.22 ml.min(-1).Torr(-1) (SD 0.3). Dno and Dco increased (P = 0.002) with blood volume/surface area. 1/Dno (P < 0.001) and 1/Dco (P < 0.001) increased with 1/Hb. Dno (P = 0.01...
Apprich V, Spergser J, Rosengarten R, Stanek C.Keratinolytic properties of two dermatophytes (Microsporum gypseum, Trichophyton mentagrophytes) and three moulds (Scopulariopsis brevicaulis, Alternaria alternata, Geotrichum candidum) isolated from diseased equine hooves were examined to improve the understanding of pathogenic mechanisms leading to equine onychomycosis. Equine hoof horn material and skin, as well as hoof keratin and dermal keratin extracted from corresponding tissues, were used as sole carbon and nitrogen sources in five test tubes for each fungus. Within 18 days, supernatants of all tubes were repeatedly examined for kerati...
Scotty NC, Evans TJ, Giuliano E, Johnson PJ, Rottinghaus GE, Fothergill AW, Cutler TJ.Lufenuron is a benzoylphenyl urea-derived insecticide that has been recently introduced as a novel treatment for fungal infections in horses. The purposes of this study were to determine (1) the in vitro efficacy of lufenuron against Aspergillus and Fusarium spp. and (2) the ability of lufenuron to reach efficacious blood concentrations after PO administration in horses. Fungal colonies isolated from diseased equine corneas were tested against lufenuron solutions up to 700 microg/mL. Twenty-one adult horses received 1 of 3 PO lufenuron treatment regimens: 5 mg/kg body weight (BW) q24h for 3 da...
Sod GA, Hubert JD, Martin GS, Gill MS.To compare the monotonic biomechanical properties of a prototype equine third metacarpal dynamic compression plate (EM-DCP) fixation with a double broad dynamic compression plate (DCP) fixation to repair osteotomized equine third metacarpal (MC3) bones. Methods: In vitro biomechanical testing of paired cadaveric equine MC3 with a mid-diaphyseal osteotomy, stabilized by 1 of 2 methods for fracture fixation. Methods: Twelve pairs of adult equine cadaveric MC3 bones. Methods: Twelve pairs of equine MC3 were divided into 3 test groups (4 pairs each) for (1) 4-point bending single cycle to failure ...
Sod GA, Hubert JD, Martin GS, Gill MS.To compare the monotonic biomechanical properties and fatigue life of a broad, limited contact, dynamic compression plate (LC-DCP) fixation with a broad, dynamic compression plate (DCP) fixation to repair osteotomized equine 3rd metacarpal (MC3) bones. Methods: In vitro biomechanical testing of paired cadaveric equine MC3 with a mid-diaphyseal osteotomy, stabilized by 1 of 2 methods for fracture fixation. Methods: Twelve pairs of adult equine cadaveric MC3 bones. Methods: Twelve pairs of equine MC3 were divided into 3 test groups (4 pairs each) for (1) 4-point bending single cycle to failure t...
Hansen N, Buchner HH, Haller J, Windischbauer G.To evaluate using strain gauges, a hoof cast with heel wedge, and a therapeutic shoe with unsupported toe for their effectiveness in redistribution of load from the dorsal hoof wall. Methods: In vitro biomechanical study. Methods: Twenty forelimb specimens. Methods: Rosette strain gauges were placed on the dorsal and lateral hoof wall of 20 normal shaped hooves. Limbs were loaded vertically using a tensile testing machine with a 1 Hz sinusoidally cycling load up to 3000 N during 15 seconds. Mean values of principal strain and direction at 2500 N load were calculated for 3 experimental conditio...
Marti M, Mevissen M, Althaus H, Steiner A.The goal of this study was to investigate the effect of bethanechol (BeCh) on contractility patterns of smooth muscle preparations of equine duodenum descendens, jejunum, caecum and pelvic flexure in vitro. Concentration-response relationships were developed for BeCh using in vitro assays with and without preincubation of muscarinic (M) receptor antagonists for M2 and M3 receptors. BeCh induced a significant, concentration-dependent increase in contractile response in equine intestine in specimens with circular orientation. The maximal effect was largest for jejunal specimens with no differenc...
Sod GA, Hubert JD, Martin GS, Gill MS.To evaluate the effects of plate luting on the biomechanical properties of a broad limited contact-dynamic compression plate (LC-DCP) fixation to repair osteotomized equine 3rd metacarpal (MC3) bones. Methods: In vitro biomechanical testing of paired cadaveric equine MC3 with a mid-diaphyseal osteotomy, stabilized by LC-DCP fixation, with 1 of the pair luted with polymethylmethacrylate (PMMA). Methods: Ten pairs of adult equine cadaveric MC3 bones. Methods: Ten pairs of equine MC3 were divided into 2 test groups (5 pairs each) for (1) palmarodorsal 4-point bending single cycle to failure testi...
Robertson TP, Peroni JF, Lewis SJ, Moore JN.To determine the effects of induction of capacitative Ca2+ entry on tone in equine laminar arteries and veins. Methods: Laminar arteries and veins from 6 adult mixed-breed horses. Methods: Arteries and veins were isolated and mounted on small vessel myographs for the measurement of isometric tension. Capacitative Ca2+ entry was induced by incubating the vessels with the specific Ca2+-ATPase inhibitor thapsigargin (100nM) in a Ca2+-free physiologic salt solution. Capacitative Ca2+ entry-associated contractile responses were determined by the subsequent addition of 2mM Ca2+ to the solution bathi...
Neil KM, Orth MW, Coussens PM, Chan PS, Caron JP.To determine whether glucosamine and chondroitin sulfate (CS) at concentrations approximating those achieved in plasma by oral administration would influence gene expression of selected mediators of osteoarthritis in cytokine-stimulated equine articular chondrocytes. Methods: Samples of grossly normal articular cartilage obtained from the metacarpophalangeal joint of 13 horses. Methods: Equine chondrocytes in pellet culture were stimulated with a subsaturating dose of recombinant equine interleukin (reIL)-1beta. Effects of prior incubation with glucosamine (2.5 to 10.0 microg/mL) and CS (5.0 t...
Duran MC, Willenbrock S, Müller JM, Nolte I, Feige K, Murua Escobar H.Interleukin-12 (IL-12) and interferon gamma (IFN-γ) are key cytokines in immunemediated equine melanoma therapy. Currently, a method for accurate simultaneous quantification of these equine cytokines is lacking. Therefore, we sought to establish an assay that allows for accurate and simultaneous quantification of equine IL-12 (eIL-12) and IFN-γ (eIFN-γ). Methods: Several antibodies were evaluated for cross-reactivity to eIL-12 and eIFN-γ and were used to establish a bead-based Luminex assay, which was subsequently applied to quantify cytokine concentrations in biological samples. Results: ...
Wilks CR, Coggins L.Twenty-two ponies were examined for serum-neutralizing (SN) antibody to equine herpesvirus type 1 and for in vitro lymphocyte transformation in the presence of viral antigen. Six ponies had undetectable levels of neutralizing antibody (titer less than 1:2) and had lymphocytes which did not respond in culture with viral antigen (stimulation index less than 2.0). Four ponies which had SN antibody to equine herpesvirus type 1 did not manifest lymphocyte transformation in vitro. The 12 remaining seropositive ponies had lymphocyte transformation with viral antigen in vitro (stimulation indexes from...
Martinez RE, Leatherwood JL, Bradbery AN, Silvers BL, Fridley J, Arnold CE, Posey EA, He W, Bazer FW, Wu G.In livestock species, the enterocytes of the small intestine are responsible for the synthesis of citrulline and arginine from glutamine and proline. At present, little is known about de novo synthesis of citrulline and arginine in horses. To test the hypothesis that horses of different age groups can utilize glutamine and proline for the de novo synthesis of citrulline and arginine, jejunal enterocytes from 19 horses of three different age groups: neonates (n = 4; 7.54 ± 2.36 d of age), adults (n = 9; 6.4 ± 0.35 yr), and aged (n = 6; 22.9 ± 1.0 yr) with healthy gastrointestinal tracts were...
Albornoz A, Morales N, Uberti B, Henriquez C, Burgos RA, Alarcon P, Moran G.Neutrophils participate in innate immunity as the first line of host defence against microorganisms. However, persistent neutrophil activity and delayed apoptosis can be harmful to surrounding tissues; this problem occurs in diverse inflammatory diseases, including asthma-affected horses. Previous studies in horses with acute lung inflammation indicated that treatment with tamoxifen (TX), a selective oestrogen receptor modulator, produces a significant decrease in bronchoalveolar lavage fluid (BALF) neutrophil content. The aim of this study was to investigate the effect of tamoxifen and its me...
Schumacher J, Wilson AM, Pardoe C, Easter JL.A prosthesis, composed of a steel cable and stress-reducing washers, was developed to prevent failure of laryngoplasty, a common treatment for horses affected by recurrent laryngeal neuropathy. Laryngoplasties were performed on 15 cadaveric larynges using a polyester suture on one side and the cable prosthesis on the other. Each prosthesis was distracted at a displacement rate of 20 mm/s using a servohydraulic materials testing machine until laryngoplasty failed. Distraction force and actuator displacement were recorded and analysed. All 15 laryngoplasties performed with a suture failed at the...
Wunn D, Wardrop KJ, Meyers K, Kramer J, Ragle C.To develop methods to isolate, culture, and characterize equine hoof endothelial cells (EC) and keratinocytes. Methods: Cells harvested from the forelimbs of 8 horses. Methods: EC were obtained via catheters placed in the palmar digital arteries of the disarticulated lower portion of the forelimbs from 4 horses that had been heparinized prior to euthanasia. Phosphate-buffered saline solution was used to remove and discard RBC from blood vessels, and collagenase was used to loosen and flush EC from the vasculature. Hoof keratinocytes were obtained from 4 recently euthanatized horses by use of d...
Lange-Consiglio A, Arrighi S, Fiandanese N, Pocar P, Aralla M, Bosi G, Borromeo V, Berrini A, Meucci A, Dell'Aquila ME, Cremonesi F.There is no published information about follicular-fluid leptin concentrations or the presence of leptin and leptin receptor in the equine ovary or oocyte. Three groups of mares - adult draft mares, draft fillies and adult Standardbred mares - were included in the study. Leptin and leptin receptor were detected in all immature oocytes by immunofluorescence with higher intensity in oocytes from draft mares compared with draft fillies and Standardbred mares. After in vitro maturation a higher proportion of oocytes reached metaphase II in draft mares than in draft fillies and Standardbred mares, ...
Sharpe AN, Seeto WJ, Winter RL, Zhong Q, Lipke EA, Wooldridge AA.OBJECTIVE To evaluate optimal isolation of endothelial colony-forming cells (ECFCs) from peripheral blood of horses. SAMPLE Jugular and cephalic venous blood samples from 17 adult horses. PROCEDURES Each blood sample was divided; isolation was performed with whole blood adherence (WBA) and density gradient centrifugation (DGC). Isolated cells were characterized by uptake of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate-labeled acetylated low-density lipoprotein (DiI-Ac-LDL), vascular tubule formation, and expression of endothelial (CD34, CD105, vascular endothelial growth ...
Tangyuenyong S, Viriyakhasem N, Peansukmanee S, Kongtawelert P, Ongchai S.Cartilage erosion in degenerative joint diseases leads to lameness in affected horses. It has been reported that andrographolide from Andrographis paniculata inhibited cartilage matrix-degrading enzymes. This study aimed to explore whether this compound protects equine cartilage degradation in the explant culture model and to determine its effect on matrix metalloproteinase-2 (MMP-2) expression, a matrix-degrading enzyme, in equine chondrocyte culture. Equine articular cartilage explant culture was induced by 25 ng/mL interleukin-1β, a key inducer of cartilage degeneration, in cultures with...
Blancquaert AM, Colgan SP, Bruyninckx WJ.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...
Dobrinski I, Ignotz GG, Fagnan MS, Yudin SI, Ball BA.The periacrosomal plasma membrane of spermatozoa is involved in sperm binding to oviductal epithelial cells and to the zona pellucida. A protein of 68-70 kD molecular mass was purified biochemically from the isolated periacrosomal plasma membrane of equine spermatozoa as a possible receptor for adhesion of spermatozoa to oviductal epithelial cells. A polyclonal antibody raised in rabbits against the purified equine sperm membrane protein recognized the 70 kD and an antigenically related to 32 kD protein in preparations of isolated periacrosomal sperm plasma membrane and in detergent extracted ...
Swanstrom MD, Stover SM, Hubbard M, Hawkins DA.To determine the relative contributions of the muscles, tendons, and accessory ligaments to the passive force-length properties of the superficial (SDF) and deep digital flexor (DDF) myotendinous complexes. Methods: 8 cadaveric forelimbs from 6 adult Thoroughbreds. Methods: In vitro, limb configurations during slack position and myotendinous lengths during subsequent axial loading of forelimbs were recorded before and after transection of accessory ligaments. Expressions were derived to describe the force-length behavior of each muscle, tendon, and accessory ligament-tendon unit; linear stiffn...
Horspool LJ, McKellar QA.A high performance liquid chromatography (HPLC) method was developed for the determination of seven short-chain fatty acids in equine caecal liquor. Samples were cleaned up on a Sep-pak (C18) cartridge, and the analyte was eluted from the extraction cartridge and filtered through a 0.45 micron cellulose nitrate filter. The analyte was chromatographed by ion exchange HPLC. Detection was by UV at 210 nm. Recovery from phosphate buffer (0.05 M, pH 7.0) and equine caecal liquor was 76.95% (lactic), 76.76% (valeric). The limit of (propionic), 89.35% (isobutyric), 88.73% (butyric), 80.33% (isovaleri...
Derochette S, Serteyn D, Mouithys-Mickalad A, Ceusters J, Deby-Dupont G, Neven P, Franck T.Excessive neutrophil stimulation and reactive oxygen species (ROS) production are involved in numerous human or horse pathologies. The modulation of the neutrophil NADPH oxidase (NOX) has a great therapeutic potential since this enzyme produces superoxide anion whose most of the other ROS derive. The measurement of NOX activity by cell-free systems is often used to test potential inhibitors of the enzyme. A major drawback of this technique is the possible interferences between inhibitors and the probe, ferricytochrome c, used to measure the activity. We designed the "EquiNox2", a new pharmacol...
Gutiérrez-Cepeda L, Crespo F, Blazquez JC, Serres C.The Purebred Spanish Horse, according to our clinical experience, is characterized by having a high number of stallions that do not meet the international commercial recommendations for equine-sperm cryopreservation. This means that artificial insemination with frozen semen from these stallions is less widespread than in other breeds. In this study, we investigated if the incorporation of single-layer colloidal centrifugation prior to cryopreservation in clinical conditions could increase the number of ejaculates of Purebred Spanish stallions suitable for this processing, observing the influen...
de Vasconcelos Franco JS, Faheem M, Chaveiro A, Moreira da Silva F.The effects of supplementation of α-tocopherol and different freezing rates (FRs) on the ability of stallion sperm to fertilize bovine oocytes with intact zona pellucida were investigated, in an attempt to develop a model to assess cryopreserved sperm function. Semen was obtained from four purebred Lusitano stallions (n = 4). Each ejaculate was subjected to cryopreservation with a commercial extender (Ghent, Minitub Iberia, Spain), without any supplementation (control) or supplemented with 2-mM α-tocopherol. The semen was exposed to two different FRs between 5 °C and -15 °C: slow (5 °C...
equine sarcoids are the most frequent skin tumors in equidae worldwide. It is well known that delta bovine papillomaviruses are their causative agents. We have recently shown the presence in equine sarcoids of abnormal vessel structures, which could cause a hypoxic condition. The aim of this study was to analyze the expression of hypoxia-inducible factor-1 alpha (HIF-1α) in a subset of BPV positive equine sarcoids and explore the relationship with vascular endothelial growth factor (VEGF) expression. Results: 80% of equine sarcoids showed strong cytoplasmic staining in >60% of neoplastic f...
Ababneh MM, Troedsson MH, Michelson JR, Seguin BE.Equine conceptuses are thought to produce antiluteolytic factors that inhibit endometrial PGF2alpha and, thus, prevent luteolysis in pregnant mares. The aim of the present study was to characterize partially the chemical nature of the prostaglandin inhibitory factor (PIF) produced by equine conceptuses in vitro. Embryos were collected from pregnant mares 13 +/- 0.5 days after ovulation and were cultured for 24 h. Harvested equine conceptus conditioned media (CCM) were assayed for antiluteolytic activity by determining the inhibition of endometrial PGF2alpha synthesis in vitro. Significant anti...
Haouz A, Glandieres JM, Zentz C, Pin S, Ramstein J, Tauc P, Brochon JC, Alpert B.The effects of the solvent conditions (buffer pH 9, 8, or 7 or buffer pH 6.5 alone or mixed with 3.2% ethanol or 6.2% formamide) on the protein dynamics of horse apomyoglobin were investigated through tryptophan fluorescence quenching, spectra, and decay properties. Raising the pH (which induces discontinuous protein conformation changes) increases the structural fluctuations inside the hydrophobic A, G, and H helix core. Mixed solutions containing either 3.2% ethanol or 6.2% formamide (which redistribute water molecules on the protein surface) produce protein dynamics changes in the vicinity ...
Iyer LK, Qasba PK.Alpha-lactalbumins (LAs) and c-type lysozymes (LYZs) are two classes of proteins which have a 35-40% sequence homology and share a common three dimensional fold but perform different functions. Lysozymes bind and cleave the glycosidic bond linkage in sugars, where as, alpha-lactalbumin does not bind sugar but participates in the synthesis of lactose. Alpha-lactalbumin is a metallo-protein and binds calcium, where as, only a few of the LYZs bind calcium. These proteins consist of two domains, an alpha-helical and a beta-strand domain, separated by a cleft. Calcium is bound at a loop situated at...
Szober CM, Hauck SM, Euler KN, Fröhlich KJ, Alge-Priglinger C, Ueffing M, Deeg CA.The purpose of this study was to characterize the cell surface proteome of native compared to cultured equine retinal pigment epithelium (RPE) cells. The RPE plays an essential role in visual function and represents the outer blood-retinal barrier. We are investigating immunopathomechanisms of equine recurrent uveitis, an autoimmune inflammatory disease in horses leading to breakdown of the outer blood-retinal barrier and influx of autoreactive T-cells into affected horses' vitrei. Cell surface proteins of native and cultured RPE cells from eye-healthy horses were captured by biotinylation, an...
Mills PC.The effects of three vehicles, phosphate-buffered saline (PBS), ethanol (50% in PBS w/w) and propylene glycol (50% in PBS w/w) on in vitro transdermal penetration of testosterone was investigated in the horse. Skin was harvested from the thorax of five Thoroughbred horses after euthanasia and stored at -20 degrees C until required. The skin was then defrosted and placed into Franz-type diffusion cells, which were maintained at approximately 32 degrees C by a water bath. Saturated solutions of testosterone, containing trace amounts of radiolabelled [14C]testosterone, in each vehicle were applie...
McMonagle EL, Taylor S, van Zuilekom H, Sanders L, Scholtes N, Keanie LJ, Hopkins CA, Logan NA, Bain D, Argyle DJ, Onions DE, Schijns VE, Nicolson L.Interleukin-12 (IL-12) is a key cytokine in the development of cell-mediated immune responses. Bioactive IL-12 is a heterodimeric cytokine composed of disulphide linked p35 and p40 subunits. The aim of this study was to verify biologically activity of the products expressed from equine interleukin-12 (IL-12) p35 and p40 cDNAs and to establish whether equine IL-12 could be expressed as a p35/p40 fusion polypeptide, as has been reported for IL-12a of several mammalian species. We report production of equine IL-12 through expression of p35 and p40 subunits in mammalian and insect cells and of a p...
Attia MM, Khalifa MM, Mahdy OA. larvae are of veterinary and medical importance caused specific equine intestinal myiasis. (Botfly larvae) had a wide geographical distribution. The present study explores the prevalence rate of 3 stage larvae in Egypt from January- December 2017; besides, in vitro trials to control of this larvae and evaluation of this trial using Scanning Electron Microscope (SEM) and histopathology of treated larvae. In the present study, the 3 larval stage of was found in clusters in the epithelium of the investigated stomach and infested with prevalence rate 97.2%. The highest collected numbers of lar...
Zhang Y, Long R, Warzecha CM, Coverdale JA, Latham EA, Hume ME, Callaway TR, O'Neil MR, Beier RC, Anderson RC, Nisbet DJ.The nitrotoxins 3-nitro-1-propionic acid (NPA) and 3-nitro-1-propanol (NPOH) are produced by a wide variety of leguminous plants, including over 150 different species and varieties of Astragalus potentially grazed by livestock. These toxins are known to be detoxified by at least one ruminal bacterium but detoxification by bacteria from other gut habitats is not known. In the present study, mixed populations of bovine ruminal and equine cecal microbes were enriched for NPA-metabolizing bacteria via consecutive 24-72 h culture in a basal minimal rumen fluid-based medium supplemented with 4.2 mM ...
Boyer JD, Breeden DC, Brown DL.To identify compounds in Acer rubrum that cause hemolysis or oxidation of equine erythrocytes and determine whether these toxins are found in other Acer spp. Methods: Equine erythrocytes. Methods: Washed erythrocytes were incubated with extracts and fractions of Acer spp that were separated by thin layer chromatography. Methemoglobin and hemolysis were measured spectrophotometrically. Compounds within Acer spp fractions associated with cell oxidation or hemolysis were identified by gas chromatography-mass spectrometry. Results: Erythrocytes incubated separately with either A. rubrum, A. saccha...
Mendes MA, Chies JM, de Oliveira Dias AC, Filho SA, Palma MS.Most commercial recombinant proteins used as molecular biology tools, as well as many academically made preparations, are generally maintained in the presence of high glycerol concentrations after purification to maintain their biological activity. The present study shows that larger proteins containing high concentrations of glycerol are not amenable to analysis using conventional electrospray ionization mass spectrometry (ESI-MS) interfaces. In this investigation the presence of 25% (v/v) glycerol suppressed the signals of Taq DNA polymerase molecules, while 1% (v/v) glycerol suppressed the ...
Brinsko SP, Ball BA, Miller PG, Thomas PG, Ellington JE.This study was designed to investigate the development of day 2 embryos obtained from young and aged mares, co-cultured with oviductal epithelial cells obtained from mares in each age group in a 2 x 2 crossover design. Young, fertile mares (n = 19; 2-7 years of age) and aged, subfertile, mares (n = 16; 17-24 years of age) were used as embryo and oviductal epithelial cell donors. Embryos (n = 37) were collected from the oviducts 2 days after ovulation and were paired (embryos obtained from young mares with embryos obtained from aged mares) so that eight pairs were co-cultured with young mare ov...
Kruse CJ, Stern D, Mouithys-Mickalad A, Niesten A, Art T, Lemieux H, Votion DM.Equine atypical myopathy is a seasonal intoxication of grazing equids. In Europe, this poisoning is associated with the ingestion of toxins contained in the seeds and seedlings of the sycamore maple (Acer pseudoplatanus). The toxins involved in atypical myopathy are known to inhibit ß-oxidation of fatty acids and induce a general decrease in mitochondrial respiration, as determined by high-resolution respirometry applied to muscle samples taken from cases of atypical myopathy. The severe impairment of mitochondrial bioenergetics induced by the toxins may explain the high rate of mortality obs...
Broström H, Bredberg-Rådén U, England J, Obel N, Perlmann P.Cell-mediated immunity in horses with sarcoid tumor against sarcoid antigens was studied in vitro by means of mixed lymphocyte tumor cell culture assay and lymphocyte-mediated cytotoxicity of 52Cr-labeled target cells. When Mc-1 sarcoid cells were used as stimulatory cells for peripheral blood lymphocytes in the mixed lymphocyte tumor cell assay, a clear difference in the kinetics of the generated lymphocytic proliferative response could be detected between sarcoid and control horses. With sarcoid horses, their proliferative maximum was reached 3 days earlier than that of the control horses, a...
