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.
Baumber J, Vo A, Sabeur K, Ball BA.Contaminating leukocytes in the ejaculate are an important source of reactive oxygen species (ROS) in human semen. When present in sufficient numbers, they can have a detrimental influence on sperm function in humans. Unfortunately, there is little published information regarding the importance of leukocytes in stallion semen. The objectives of this study were to determine the production of hydrogen peroxide (H2O2) by activated equine neutrophils and to examine the effect of this ROS production on equine sperm motility in vitro. Motile equine spermatozoa (two ejaculates each from four stallion...
Bøgh IB, Bézard J, Duchamp G, Baltsen M, Gérard N, Daels P, Greve T.In the mare, rates of fertilization and development are low in oocytes matured in vitro, and a closer imitation of in vivo conditions during oocyte maturation might be beneficial. The aims of the present study were, therefore, to investigate whether (1) equine oocytes can be matured in vitro in pure equine preovulatory follicular fluid, (2) priming of the follicular fluid donor with crude equine gonadotrophins (CEG) before aspiration of preovulatory follicular fluid promotes the in vitro maturation rate, (3) the in vitro maturation rate differs between oocytes aspirated during estrus and those...
Clark P, Mogg TD, Tvedten HW, Korcal D.Delayed analysis of blood samples may be caused by restricted access to laboratories. Artifactual changes may occur in the measured analytes as a consequence of delayed analysis and may complicate interpretation of the data. Objective: The purpose of this study was to characterize artifactual changes in equine blood, due to storage, using the Advia 120 hematology analyzer. Methods: Samples of blood from 5 horses were analyzed using the Advia 120 soon after collection and again after 24 and 48 hours of storage at either 4 degrees C or ambient laboratory temperature ( approximately 24 degrees C)...
Call GB, Wolfe MW.Activation of the luteinizing hormone beta (LHbeta) promoter by gonadotropin-releasing hormone (GnRH) via the transcription factor early growth response protein-1 (Egr1) has been well characterized. To determine the mechanisms affecting Egr1 regulation of LHbeta, we analyzed five different species of LHbeta promoters (equine, mouse, rat, bovine and human). Electrophoretic mobility shift assays (EMSAs) identified multiple transcription factors binding to the Egr regions on the LHbeta promoter. Species-specific differences existed in the binding affinity for Sp1, Sp3, steroidogenic factor-1 (SF-...
Gray AW, Davies ME, Jeffcott LB.Cathepsin K and cathepsin B were immunolocalised in equine osteoclasts (OC s) present in ex vivo cartilage/subchondral bone samples. Samples were obtained post mortem from the lateral trochlear ridge (LTR) of six horses and ponies aged between 303 days gestation to 8 months. Strong expression of cathepsin K was detected in OC s, particularly those located at the osteochondral junction, apparently involved in the resorption of calcified cartilage. Cathepsin K expression was also detected in hypertrophic chondrocytes and in the endothelial cells of some blood vessels penetrating the hypertrophic...
Gray AW, Davies ME, Jeffcott LB.Equine osteoclast-like cells (OCLs) were generated from the bone marrow (BM) of two ponies and one horse in the presence of RANKL, the receptor activator of NF kappa B ligand and macrophage colony-stimulating factor (M-CSF). The phenotype of these cells was confirmed by demonstration of characteristics typical of osteoclasts (OCs) including: the expression of tartrate-resistant acid phosphatase (TRAP), the vitronectin receptor (VNR) and the calcitonin receptor (CTR), the demonstration of responsiveness to calcitonin (CT) and the ability to form resorption lacunae on ivory slices and calcium ph...
Merchant KA, Thompson DE, Xu QH, Williams RB, Loring RF, Fayer MD.Two-dimensional (2D) infrared vibrational echoes were performed on horse heart carbonmonoxymyoglobin (MbCO) in water over a range of temperatures. The A(1) and A(3) conformational substates of MbCO are found to have different dephasing rates with different temperature dependences. A frequency-frequency correlation function derived from molecular dynamics simulations on MbCO at 298 K is used to calculate the vibrational echo decay. The calculated decay shows substantial agreement with the experimentally measured decays. The 2D vibrational echo probes protein dynamics and provides an observable ...
Bacon SJ, Ellis SA, Antczak DF.In most mammals, the fetus limits its presentation of paternal antigens to the mother by suppressing the cell-surface expression of proteins of the major histocompatibility complex (MHC) on trophoblast. In the horse, however, functional, polymorphic MHC class I antigens are expressed at high levels on the invasive trophoblast cells of the chorionic girdle between Days 32 and 36 of pregnancy, although not on the adjacent noninvasive trophoblast of the chorion and allantochorion membranes. In this study, the control of MHC class I gene expression was investigated in invasive and noninvasive hors...
Davidson TR, Chamberlain CS, Bridges TS, Spicer LJ.Little is known regarding the hormonal regulation of granulosa cell steroidogenesis and the ovarian insulin-like growth factor (IGF) system in the mare. The objectives of this study were to determine, first, if estradiol, insulin, and/or FSH affect steroid production by equine granulosa cells (experiment 1) and, second, if the components of the IGF system are produced by equine granulosa cells in culture as well as whether estradiol, insulin, and/or FSH affects IGF and/or IGF-binding protein (IGFBP) production by equine granulosa cells (experiment 2). Granulosa cells from small (6-15 mm), medi...
Müller-Schöttle F, Bogusz A, Grötzinger J, Herrler A, Krusche CA, Beier-Hellwig K, Beier HM.After its original description as a steroid-dependent protein in the rabbit uterus, uteroglobin became one of the best characterized proteins. However, detailed knowledge of its physiological role remains an enigma. In this study we investigate how its structure is phylogenetically conserved in the horse compared to other mammalian species. Northern blot analysis showed that in horses, the main expression of uteroglobin appears in lung, uterus, and prostate tissues. Western blot analysis demonstrated that the dimeric form of uteroglobin is found predominantly in biological compartments. Using ...
Bentley AP, Barton MH, Lee MD, Norton NA, Moore JN.To determine which antimicrobials that are used to treat neonatal foals with septicemia attributable to Escherichia coli will minimize endotoxin release from bacteria and subsequent activity of inflammatory mediators while maintaining bactericidal efficacy. Methods: Blood samples from 10 healthy foals. Methods: Escherichia coli isolates A and B were isolated from 2 septicemic foals, and minimal inhibitory concentrations (MIC) were determined for 9 antimicrobials. Five of these antimicrobials were tested in vitro at 2 and 20 times their respective MIC. Whole blood or mononuclear cells grown in ...
Gokbulut C, Nolan AM, McKellar QA.Oxibendazole (OBZ) was administered to eight horses at an oral dose of 10 mg kg(-1) bodyweight each. Parent OBZ could only be detected in plasma at the 0.5 and 1.0 hours post administration sampling times and the mean maximum plasma concentration was 0.008 microg ml(-1). Parent OBZ was detected in faeces between 12 and 72 hours after administration and the highest dry faecal concentration was detected at 24 hours. An unidentified metabolite was detected in plasma between 0.5 and 72 hours. The unidentified metabolite in the plasma of treated horses corresponded to the second eluted metabolite i...
Armstrong S, Lees P.Chondrocytes and synoviocytes harvested from the joints of healthy horses were maintained in tissue culture. Production of the cytokines interleukin-1 (IL-1), interleukin-6 (IL-6) and tumour necrosis factor-alpha (TNF-alpha) in response to lipopolysaccharide (LPS), and the effects of addition of carprofen (racemate and R and S enantiomers) were determined. Lipopolysaccharide failed to stimulate TNF-alpha activity in both cell types but concentrations of IL-1 and IL-6 were both increased in a concentration and time-related manner. Both carprofen enantiomers and the racemic mixture attenuated th...
Knoll U, Strauhs P, Schusser G, Ungemach FR.Olsalazine sodium (Dipentum*) has been used therapeutically against inflammatory bowel disease in human medicine as an alternative to sulphasalazine over the past 20 years. Bacteria in the colon split this prodrug into two molecules of the locally effective 5-aminosalicylic acid (5-ASA). Considering the potential therapeutic use in equine colitis, the pharmacokinetics of olsalazine (OLZ) after single oral administration to six horses at a dosage of 30 mg/kg was investigated. Plasma concentrations of OLZ, 5-ASA, and its main metabolite N-acetyl-5-aminosalicylic acid (Ac-5-ASA) were analysed by ...
Theoret CL, Barber SM, Moyana TN, Gordon JR.To determine whether transforming growth factor (TGF)-beta1 and -beta3 expression differs between equine limb wounds healing normally and those healing with experimentally induced exuberant granulation tissue (EGT). Methods: Six wounds were created on the lateral aspect of both metacarpi of each horse; one forelimb was untreated, and the other was bandaged to stimulate the development of EGT. Sequential wound biopsies allowed comparison of growth factor expression between the two types of wound. Methods: Four horses (2 to 4 years of age; 350 to 420 kg). Methods: Wounds were assessed grossly, h...
Galuppo LD, Stover SM, Aldridge A, Hewes C, Taylor KT.To compare monotonic mechanical properties of gap-ostectomized third metacarpal bones (MC3) stabilized with an MP35N interlocking nail system with contralateral intact bones. Methods: Twenty-four pairs of cadaveric equine MC3s. Methods: Third metacarpal bones were divided into 4 mechanical testing groups (6 pairs per group): compression, palmarodorsal (PD) and mediolateral (ML) 4-point bending, and torsion. One MC3 from each pair was randomly selected as an intact specimen, and the contralateral gap ostectomized bone was stabilized with a 4-hole, 14-mm-diameter, 250-mm-long, MP35N intramedulla...
Galuppo LD, Stover SM, Jensen DG.To compare bone fragment compression and the mechanical pushout strength and stiffness of 6.5-mm Acutrak Plus (AP) and 4.5-mm AO cortical (AO) bone screws after stabilization of a simulated equine third metacarpal (MC3) bone complete lateral condylar fracture. Methods: In vitro biomechanical paired study of screw insertion variables, bone fragment compression, and screw pushout tests using a bone screw stabilized simulated lateral condylar fracture model. Methods: Six pairs of cadaveric equine MC3s. Methods: Metacarpi were placed in a fixture and centered on a biaxial load cell in a materials ...
Andrea CR, Stover SM, Galuppo LD, Taylor KT, Rakestraw PC.To compare screw insertion characteristics and pullout mechanical properties between self-tapping (ST) and non-self-tapping (NST) AO 4.5-mm cortical bone screws in adult equine third metacarpal bone (MC3). Methods: In vitro biomechanical experiment. Methods: Seven pairs of adult equine MC3. Methods: Bicortical holes were drilled transversely in proximal metaphyseal, diaphyseal, and distal metaphyseal locations of paired MC3. NST screws were inserted in pre-tapped holes in 3 sites of one bone pair, and ST screws were inserted in non-tapped holes of contralateral MC3. Tapping and screw insertion...
Martínez AC, Prieto D, Hernández M, García-Sacristán A, Benedito S.The present investigation was designed to evaluate the effect of histamine on isolated rings of horse deep dorsal penile vein. Under precontracted or basal conditions, histamine evoked an endothelium-independent contraction. Preincubation of the vein rings with the selective H1 receptor antagonist, mepyramine, shifted the concentration-response curve for histamine and to the H1 receptor agonist 2-pyridylethylamine to the right in a competitive manner. Pretreatment with cimetidine, a specific H2 receptor antagonist, did not modify the pEC50 and maximal contraction of the histamine response. Cim...
The testicular and epididymal fluids of ram, boar, and stallion were analyzed by means of one-dimensional and two-dimensional gelatin gel zymography. Five main gelatinolytic bands were revealed in the ram and at least seven were observed in the boar and stallion. These proteolytic bands showed regionalized distribution throughout the organs. The two main proteolytic activities at around 54-66 kDa retrieved in all three species were inhibited by EDTA and phenanthroline, indicating that they were metallo-dependent enzymes. The activity of some of the low-molecular-weight gelatinases was also dec...
Li X, Morris LH, Allen WR.This study investigated the basic conditions required for the production of horse embryos by the transfer of the nuclei of fetal and adult fibroblast cells to enucleated oocytes. Cumulus-oocyte complexes were recovered from abattoir ovaries and matured in vitro in groups of 20-30 for 28-30 h in tissue culture medium 199 containing 20% v:v fetal bovine serum in coculture with equine oviduct epithelial cells. Fetal fibroblast cells (FFC) were derived from a 32-day-old Thoroughbred x Pony fetus, and adult skin fibroblast cells (SFC) were obtained from subdermal biopsies recovered from a 4-yr-old ...
Hinrichs K, Love CC, Choi YH, Varner DD, Wiggins CN, Reinoehl C.Germinal vesicle (GV)-stage horse oocytes with diffuse chromatin are meiotically incompetent and degenerate in culture, whereas horse oocytes having condensed chromatin within the GV are meiotically competent. Degeneration of incompetent oocytes in culture may be related to premature GV breakdown, which could possibly be prevented by inhibition of m-phase protein activity. We examined the effects of 6-dimethylaminopurine (6-DMAP), butyrolactone and roscovitine on GV-stage horse oocytes. Culture in the presence of 2 mM 6-DMAP for 24 h suppressed meiosis (2% MI or MII compared with 38% for untre...
Rozovsky I, Hoving S, Anderson CP, O'Callaghan J, Finch CE.Premarin, which contains several equine estrogens, as well as estradiol (E2) as a minor component, is widely used for replacement therapy of estrogen deficits, but little is known of its direct actions on brain cells. In mixed glial cultures, apolipoprotein E (apoE) and glial fibrillary acidic protein (GFAP) are induced by estrogens. GFAP induction showed an inverted-U shape E2 dose response, with a maximum induction at 1 pM, whereas apoE mRNA induction was greatest at 100 pM. GFAP and ApoE mRNAs were induced by equine estrogens in the following order: E2=equilin>estrone>17 alpha-dihydro...
Liu X, Yao J, Pisha E, Yang Y, Hua Y, van Breemen RB, Bolton JL.Excessive exposure to synthetic and endogenous estrogens has been associated with the development of cancer in several tissues. 4-Hydroxyequilenin (4-OHEN), a major metabolite of equine estrogens present in estrogen replacement formulations, has been shown to induce cytotoxic/carcinogenic effects. In the present study, we have found that 4-OHEN caused DNA damage in breast cancer cells, and cells that contain estrogen receptor alpha (S30) are more sensitive to 4-OHEN-mediated DNA damage as compared to estrogen receptor negative cells (MDA-MB-231). For example, concentration-dependent increases ...
Iqbal J, Purewal AS, Edington N.The aim of this study was to investigate the role of immediate early gene (gene63) in the pathogenesis of equine herpesvirus 1 (EHV-1) acute and latent infections in equine and murine models. EHV-1 gene63 mutant virus (g63mut) along with EHV-1 (Ab4) was used for intracerebral and intranasal infection of 3 and 17-day-old mice. Both viruses were recovered at the same frequency from tissues after infection. Two Welsh ponies were infected via the intranasal route with each of the viruses. Acute infection was monitored by virus isolation from nasal swabs and peripheral blood leukocytes. Six weeks p...
Kingston JK, Bayly WM, Sellon DC, Meyers KM, Wardrop KJ.To investigate the potential use of fluorescent-labeled annexin V, anti-human fibrinogen antibody, and anti-human thrombospondin antibody for detection of the activation of equine platelets by use of flow cytometry. Methods: Platelets obtained from 6 Thoroughbreds. Methods: Flow cytometry was used to assess platelet activation as indicated by detection of binding of fluorescent-labeled annexin V, anti-human fibrinogen antibody, and anti-thrombospondin antibody to unactivated and ADP-, collagen-, platelet activating factor (PAF)-, and A23187-activated equine platelets. Human platelets were used...
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...
Takafuji VA, McIlwraith CW, Howard RD.To evaluate the effects of equine recombinant interleukin-1alpha (rEqIL-1alpha) and recombinant interleukin-1beta (rEqIL-1beta) on proteoglycan metabolism and prostaglandin E2 (PGE2) synthesis by equine articular chondrocytes in explant culture. Methods: Near full-thickness articular cartilage explants (approx 50 mg) harvested from stifle joints of a 3-year-old and a 5-year-old horse. Methods: Expression constructs containing cDNA sequences encoding EqIL-1alpha and EqIL-1beta were generated, prokaryotically expressed, and the recombinant protein purified. Near full-thickness articular cartilag...
Flaminio MJ, Rush BR, Davis EG, Hennessy K, Shuman W, Wilkerson MJ.This paper describes a method for simultaneously measuring phagocytosis and oxidative burst activity in equine peripheral blood leukocytes by flow cytometry. Opsonized propidium iodide-labelled Staphylococcus aureus (PI-Sa) was used to measure the uptake of bacteria by equine phacocytes and the oxidative burst activity by oxidation of dihydrorhodamine 123. The requirements to achieve optimal activity of phagocytosis and oxidative burst are described. The advantage of the simultaneous technique is that it provides both independent and comparative values for phagocytosis and the oxidative burst,...
Feughelman M, Lyman DJ, Willis BK.Recent Fourier transform infrared spectroscopy (FTIR) with attenuated total reflection technique (ATR) has been applied to alpha-keratin fibers (horse-hair) extended in water both at 21 and 95 degrees C. Infrared absorption bands in the Amide 1 region indicated that at extensions to 40-50% strain in water at 21 degrees C alpha-helices had completely disappeared and parallel beta-sheets were formed [Appl. Spectrosc. 55 (2001) 552]. However, when the hair fibers were extended to the same strain at 95 degrees C in water the result was the formation of anti-parallel beta-sheets. These results sugg...
Matthews GL, Keegan KG, Graham HL.To determine effects of tendon grip technique on in vitro surface strain measurements of equine deep digital flexor tendon (DDFT) when loaded in tension. Methods: 12 hind limb DDFT from 8 adult horses (mean age, 9.8 years [range, 4.5 to 17 years]; mean body weight, 472 kg [range, 450 to 509 kg]), with no clinical evidence of hind limb lameness. Methods: After calibration, liquid mercury strain gauges were sutured to plantar surfaces of the tendons at distal (position 1), middle (position 2), and proximal (position 3) metatarsal regions. Each tendon was affixed to a materials testing machine (d...
Fachal MV, Furlan M, Clark R, Card CE, Chedrese PJ.The objective of this investigation was to clone and express the elk and horse common alpha-subunit and FSH beta-subunit cDNAs, and to produce recombinant FSH from both species in vitro. The RNAs extracted from elk and horse pituitary glands were reverse-transcribed and amplified by polymerase chain reaction. The cDNAs corresponding to both subunits of elk and horse were cloned into the expression vector pBudCE4.1 and transfected into CRL-9096 cells. Expression of both genes was determined in the transfected cells by Northern and Western blot analysis. Recombinant elk and horse FSH secreted in...
Morillo-Rodríguez A, Macías-García B, Tapia JA, Ortega-Ferrusola C, Peña FJ.Ejaculates from six pure Spanish stallions were split, and one subsample frozen in a commercial extender supplemented with the lipid soluble antioxidant butylated hydroxytoluene (BHT), while the other subsample served as control. After at least 4 weeks of storage, samples were thawed and post-thaw sperm quality analysed: sperm motility and kinematics using a CASA system, membrane and acrosome integrity and mitochondrial membrane potential using flow cytometry. The outcome of cryopreservation varied significantly among stallions. However, the supplementation with 1 mm BHT had no significant eff...
Clegg PD, Coughlan AR, Carter SD.Matrix metalloproteinases (MMPs) are the main enzymes involved in connective tissue turnover. Regulation of MMPs is achieved by controlling production, activation of the pro-enzymes together with the presence of inhibitors, such as, tissue inhibitors of metalloproteinases (TIMPS). The presence of TIMPs in equine synovial fluid was assessed by the ability of the fluid to inhibit equine MMP-9 activity using a gelatin degradation ELISA. The cellular source of the TIMPs was determined using culture supernatants of resident articular cells (chondrocytes and synovial fibroblasts) and invading inflam...
Vittoria A, Cocca T, La Mura E, Cecio A.The aim of this study is to describe the presence of neuroendocrine (NE) cells (paraneurons), producing biogenic amines and/or peptidergic hormones, in the female urethra of cattle, sheep, pigs, and horses, by means of histochemical and double labeling immunofluorescent techniques. 5-Hydroxy-tryptamine-, chromogranin A-, cholecystokinin- and somatostatin-containing NE cells are present in the urethral epithelium of all the species studied, with the unique exception of the lack of somatostatin cells in the horse. Paraneurons containing 5-hydroxytryptamine colocalized with chromogranin A or chol...
Minguet G, Franck T, Joris J, Ceusters J, Mouithys-Mickalad A, Serteyn D, Sandersen C.Volatile anaesthestics have shown to modulate the oxidative response of polymorphonuclear neutrophils (PMNs). We investigated the effects of isoflurane and sevoflurane on the degranulation of total and active myeloperoxidase (MPO) from horse PMNs and their direct interaction with MPO activity. Whole blood from horse was incubated in 1 and 2 minimal alveolar concentrations (MAC) of isoflurane or sevoflurane for 1h and PMNs were stimulated with cytochalasin B (CB) plus N-formyl-méthionyl-leucyl-phenylalanine (fMLP). After stimulation, the plasma was collected to measure total and active MPO by ...
Fanelli D, Tesi M, Monaco D, Diaz-Jimenez M, Camillo F, Rota A, Panzani D.In the literature, the very low pregnancy rates after artificial insemination (AI) with frozen semen in donkeys were improved in one study after re-extension of the frozen-thawed semen in autologous seminal plasma. The aims of our study were (1) to describe in vitro post-thaw parameters of donkey jackass semen after re-extension in seminal plasma (SP) or in INRA96 and (2) to compare pregnancy rates in jennies bred with frozen-thawed semen using two different AI protocols. Semen collected from two Amiata donkey stallions, known to be fertile, was frozen in INRA96 supplemented with 2% egg yolk, ...
Pereira GR, Lorenzo PL, Carneiro GF, Bilodeau-Goeseels S, Kastelic J, Liu IK.The objectives of this study were firstly to determine whether the stimulatory function of equine growth hormone (eGH) on equine oocyte maturation in vitro is mediated via cyclic adenosine monophosphate (cAMP); and secondly if the addition of eGH in vitro influences oocyte nuclear maturation and if this effect is removed when GH inhibitors are added to the culture. Cumulus-oocyte complexes (COCs) were recovered from follicles <25 mm in diameter and randomly allocated as follows: (i) control (no additives); and (ii) 400 ng/ml of eGH. A specific inhibitor against cyclic AMP-dependent protein ...
Watts EJ, Rose MT.Horses are more prone to complications in the wound healing process than other species, and problems such as chronic inflammation, delayed epithelialization, poor wound contraction, and exuberant granulation tissue are commonly seen, particularly in wounds on the distal limbs. In comparison, wounds of the oral mucosa heal rapidly in a scarless fashion with a high degree of wound contraction. The effect of platelet-derived growth factor BB (PDGF), insulin-like growth factor (IGF)-1, and transforming growth factor beta1 (TGFbeta1) on the contraction of a fibroblast-populated collagen matrix (FPC...
Kamel M, Pavulraj S, Osterrieder K, Azab W.Primary infection and pathogenesis of equine herpesvirus type 1 (EHV-1) require an intricate interaction of virus with the mucosal epithelium, mononuclear cells and the vascular endothelium. Studies on EHV-1 have been facilitated by the development of different models that recapitulate the tissue complexity. The available assays can be categorized into (i) models mimicking the epithelium-peripheral blood mononuclear cell (PBMC) interaction, which include mucosal (nasal and vaginal) explants and equine respiratory epithelial cells (EREC) cultures; and (ii) PBMC-endothelium mimicking models,...
Morillo Rodriguez A, Balao da Silva C, Macías-García B, Gallardo Bolaños JM, Tapia JA, Aparicio IM, Ortega-Ferrusola C, Peña FJ.A total of 42 ejaculates were used in the experiment; six ejaculates per stallion, obtained from seven Pure Spanish stallions (PRE), were split and frozen in freezing media with different concentrations and combinations of cryoprotectant (CPA): (i) Cáceres (skim milk based extender) containing 2.5% glycerol (2.5GL), (ii) Cáceres containing 1.5% glycerol and 1.5% dimethylformamide (1.5%GL-1.5%DMFA), (iii) Cáceres extender supplemented with 1.5% glycerol and 2.5% dimethylformamide (1.5%GL-2.5%DMFA) and (iv) Cáceres extender supplemented with 4% dimethylformamide (4%DMFA). After at least 4 we...
Wagner B, Siebenkotten G, Radbruch A, Leibold W.IgE is the dominant immunoglobulin isotype involved in type I hypersensitivities in mammals. The heavy chain constant region domains of equine IgE are encoded by a single gene, the Cvarepsilon gene. By restriction analysis of cDNA from 15 unrelated horses, we have now identified two Cvarepsilon alleles, characterised by a Sma I restriction fragment length polymorphism, which we designated Cvarepsilon(a) and Cvarepsilon(b). Sequence analysis of both, Cvarepsilon(a) and Cvarepsilon(b) cDNA, showed in addition two single base exchanges resulting in two amino acid substitutions. Both sequences hav...
Kingston JK, Bayly WM, Sellon DC, Meyers KM, Wardrop KJ.To investigate the effects of formaldehyde fixation on equine platelets using flow cytometric methods to evaluate markers of platelet activation. Methods: Blood samples from 6 Thoroughbreds. Methods: The degree of fluorescence associated with binding of fluorescein isothiocyanate (FITC)-conjugated anti-human fibrinogen antibody and FITC-annexin V in unactivated and adenosine diphosphate (ADP)-, platelet activating factor (PAF)-, and A23187-activated platelet samples in unfixed and 0.5, 1.0, and 2.0% formaldehyde-fixed samples was assessed by use of flow cytometry. Results: In samples incubated...
Magistrini M, Guitton E, Levern Y, Nicolle JC, Vidament M, Kerboeuf D, Palmer E.New staining methods and automated instruments are now available to evaluate the sperm cell in vitro. Individual compartments of the sperm cell, such as the nucleus and the plasma and acrosomal membranes, may be investigated, as well as the cell function as shown by mitochondria activity and capacitation. Various probes are used and they can be analyzed by direct light or fluorescent microscopy or by flow cytometry. The automated instruments allow objective and accurate analysis and quantification as well as the ability to evaluate large population of cells in a shorter time, thus providing ac...
Den Hartog SM, Back W, Brommer H, van Weeren PR.Insight into the loading pattern of the articular cartilage surface during the complete stride is important as biomechanical factors play a pivotal role in the pathogenesis of joint trauma and osteoarthritis (OA). Objective: To determine the loading pattern in the equine MCP articulation in vitro during simulated walk. Methods: Eight cadaveric limbs from mature Dutch Warmblood horses were loaded in a pneumatic loading device in 6 different positions (A1-A6). The pressure distribution on the articular surface of the proximal phalanx (P1) was measured at 7 sites (S1-7) using intra-articularly pl...
Hetherington VJ, Kawalec JS, Dockery DS, Targoni OS, Lehmann PV, Nadler D.One means of treating osteoarthritis is with autologous or allogeneic osteochondral grafts. The purpose of this study was to evaluate the innate immunological response in humans toward xeno-derived osteochondral grafts that have been partially or entirely treated by the photooxidation process. Methods: The antigens tested included bovine, porcine, ovine and equine osteochondral samples that have been treated in successive steps of photooxidation. ELISPOT assays were used to evaluate the production of IL-1, IL-4, IL-6, IL-10, IL-12 and TNF-alpha by human monocytes in response to the antigens. R...
Van Hoogmoed LM, Nieto JE, Snyder JR, Harmon FA.To evaluate the efficacy of intraluminal administration of a customized solution during low-flow ischemia and reperfusion in the jejunum of horses. Methods: Segments of jejunum obtained from 13 healthy adult horses. Methods: In isolated segments of jejunum maintained in an extracorporeal circuit, arterial flow was reduced to 20% of baseline for 40 minutes (ischemia) followed by 60 minutes of reperfusion. In 2 groups, a customized solution (concentrations, 12.5 and 25%, respectively) was placed in the lumen prior to low-flow ischemia and maintained during reperfusion. The control group received...
Tappenbeck K, Hoppe S, Geburek F, Feige K, Huber K.By blocking the enteric nervous system (ENS) using tetrodotoxin (TTX), previous studies have documented the contractility-enhancing (CE) effects of lidocaine in equine intestinal smooth muscle (SM) at the level of SM cells and/or interstitial cells of Cajal (ICC). The present study examined the impact of ENS deactivation on CE lidocaine effects, and investigated the effects of lidocaine on ENS activity. TTX application did not affect the CE effects of lidocaine, indicating that these were not mediated by TTX-sensitive sodium channels. Application of TTX or ≥100 mg/L lidocaine reduced ENS a...
Ortiz I, Dorado J, Morrell JM, Diaz-Jimenez MA, Pereira B, Consuegra C, Hidalgo M.The aim of this study was to compare the post-thaw distribution of motile sperm subpopulations, following simple or colloid centrifugation. A new analysis was used to evaluate the available number of sperm from each subpopulation after each centrifugation protocol. Frozen/thawed semen samples were divided into the following after-thawing treatments: uncentrifuged control (UDC), sperm washing (SW) and two colloid centrifugation procedures (Equipure, SLC-E, and Androcoll, SLC-A). Percentage of total and progressive motility (TM and PM), as well as sperm motility kinematics, distribution of motil...
Choi YH, Chung YG, Walker SC, Westhusin ME, Hinrichs K.This study was conducted to evaluate the effects of insulin-like growth factor I (IGF-I) and other media factors during oocyte maturation, and the presence of different compositions of amino acids in embryo culture medium, on the development of equine embryos. Oocytes recovered from slaughterhouse-derived ovaries were matured in vitro for 24 h and those with a polar body were subjected to intracytoplasmic sperm injection (ICSI) or nuclear transfer with adult fibroblasts (NT). For ICSI embryos, there were no significant differences in rates of morphological cleavage, cleavage with normal nuclei...
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...
van Hoogmoed LM, Harmon FA, Stanley S, White J, Snyder J.The objective of this study was to determine if a correlation exists between the presence of nitric oxide and prostaglandin release in the equine ventral colon smooth muscle, since this relationship may accentuate the inflammatory process during intestinal injury. Tissue was collected from the ventral colon, cut into muscle strips oriented along the circular, longitudinal and taenial layers, and mounted in a tissue bath system. Samples of the bath fluid were collected before, following electrical field stimulation (EFS), and following EFS in the presence of L-NAME, a nitric oxide synthase inhi...
Kumar CS, Sivaramakrishna D, Ravi SK, Swamy MJ.Seminal fibronectin type-II (Fn-II) proteins interact with choline phospholipids present on the sperm plasma membrane and play a crucial role in sperm capacitation. Crystal structure of phosphorylcholine (PrC) complex of PDC-109, the major bovine Fn-II protein, together with fluorescence spectroscopic studies has shown that tryptophan residues are crucial for its specific interaction with choline phospholipids. In the present study, the heterogeneity and microenvironment of tryptophan residues in HSP-1/2, a major protein of horse seminal plasma (which is homologous to PDC-109) were investigate...
James S, Daffy J, Cook J, Samiric T.Fluoroquinolone antibiotics are associated with increased risk of tendinopathy and tendon rupture, which can occur well after cessation of treatment. We have previously reported that the fluoroquinolone ciprofloxacin (CPX) reduced proteoglycan synthesis in equine tendon explants. This study aimed to determine the effects of CPX on proteoglycan catabolism and whether any observed effects are reversible. Equine superficial digital flexor tendon explant cultures were treated for 4 days with 1, 10, 100 or 300 µg/mL CPX followed by 8 days without CPX. The loss of [S]-labelled proteoglycans and che...
Bhuyan AK, Rao DK, Prabhu NP.Proteins meet with the stipulations of Levinthal. Two test tube variants of ferrocytochrome c (ferrocyt c) whose thermodynamic stabilities are vastly different refold to the same global minimum under a given final native condition, and they do so quickly at rates that do not reflect a strong dependence on the thermodynamic driving force. The transition-state ensemble is more unfolded-like, and the folding barrier offered is energetically sizable. The experiments involve neutral- (pH 7) and alkaline ferrocyt c pH (12.7), whose aqueous stabilities are 18 (+/-0.3) and 3 (+/-0.5) kcal mol(-)(1), r...
Amaral A, Fernandes C, Szóstek-Mioduchowska A, Lukasik K, Rebordão MR, Pinto-Bravo P, Skarzynski DJ, Ferreira-Dias G.Cathepsin G (CAT) is a protease released by neutrophils when forming neutrophil extracellular traps that was already associated with inducing type I collagen (COL1) in equine endometrium in vitro. Endometrosis is a fibrotic condition mainly characterized by COL1 deposition in the equine endometrium. The objective was to evaluate if noscapine (an alkaloid for cough treatment with anti-neoplastic and anti-fibrotic properties) would reduce transcription (evaluated by qPCR) and COL1 protein relative abundance (evaluated by western blot) induced by CAT in equine endometrial explants from follicula...
Boger BL, Manfredi JM, Yob C, Weber PSD, Jacobs CC.Diagnosis of equine septic arthritis is not straightforward, and increasing time between onset, diagnosis, and treatment can have serious consequences for quality of life. Defensins are used in diagnosis of human joint infection. The presence of beta defensins (BDs) in equine synovial fluid and their utility as a biomarker of sepsis has not been investigated; therefore, our objectives were to (1) compare in vitro gene expression of lipopolysaccharide (LPS)-stimulated equine neutrophils to unstimulated neutrophils and (2) compare BD protein expression from normal, aseptically inflamed, and sept...
Willingham-Lane JM, Berghaus LJ, Berghaus RD, Hart KA, Giguère S.The soil-dwelling, saprophytic actinomycete is a facultative intracellular pathogen of macrophages and causes severe bronchopneumonia when inhaled by susceptible foals. Standard treatment for disease is dual-antimicrobial therapy with a macrolide and rifampin. Thoracic ultrasonography and early treatment with antimicrobials prior to the development of clinical signs are used as means of controlling endemic infection on many farms. Concurrently with the increased use of macrolides and rifampin for chemoprophylaxis and the treatment of subclinically affected foals, a significant increase in t...
Alvarenga MA, Landim-Alvarenga FC, Moreira RM, Cesarino MM.The present experiments aimed to examine the substitution of glycerol (G) by ethylene glycol (E) as a cryoprotective agent for stallion spermatozoa. Two different ethylene glycol concentrations (5% and 10%) and also the association of glycerol (2%) and ethylene glycol (3%) (E/G) were studied (Experiment 1). In Experiment 2, two packing systems (0.5 x 4.0 ml) were evaluated using both cryoprotectors. In both experiments, the sperm membrane integrity after freezing was evaluated using transmission electron microscopy. The mean post-thaw motility was 34.25, 36.5, 29.25 and 34.75% for G5%, E5%, E1...
Singer E, Garcia T, Stover S.The biomechanical circumstances that promote sagittal fracture of the equine proximal phalanx (P1) are poorly understood. In order to improve our understanding of equine metacarpophalangeal joint (MCPJ) biomechanics and potential aetiologies of sagittal P1 fractures, the study objectives were to quantify P1 bone strains, collateromotion and axial rotation during MCPJ extension under controlled loading circumstances. Unilateral limbs from six cadavers were instrumented with bone reference markers for measurement of P1 movement relative to third metacarpal bone positions during axial limb loadin...
