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.
Ambruosi B, Uranio MF, Sardanelli AM, Pocar P, Martino NA, Paternoster MS, Amati F, Dell'Aquila ME.Phthalates are ubiquitous environmental contaminants because of their use in plastics and other common consumer products. Di-(2-ethylhexyl) phthalate (DEHP) is the most abundant phthalate and it impairs fertility by acting as an endocrine disruptor. The aim of the present study was to analyze the effects of in vitro acute exposure to DEHP on oocyte maturation, energy and oxidative status in the horse, a large animal model. Cumulus cell (CC) apoptosis and oxidative status were also investigated. Cumulus-oocyte complexes from the ovaries of slaughtered mares were cultured in vitro in presence of...
Owens SD, Burges J, Johns JL, Carrade DD, Galuppo LD, Librach F, Borjesson DL.The therapeutic use of bone marrow-derived mononuclear cells (MNCs) and mesenchymal stem cells for the treatment of soft tissue and orthopedic injuries in equine patients is expanding. After collection, bone marrow must be reduced in volume and depleted of RBCs for immediate therapeutic use or to prepare cells for culture or cryopreservation and storage. The MarrowXpress (MXP) System is an automated, closed, sterile system designed to process human bone marrow samples. Objective: The purpose of this study was to evaluate the capacity of the MXP System to process equine bone marrow to reduce vo...
Everett E, Barrett JG, Morelli J, DeVita R.To compare in vitro biomechanical properties of a novel suture pattern to a current standard for primary repair of equine superficial digital flexor tendon (SDFT) laceration. Methods: In vitro randomized, paired design. Methods: Cadaveric equine forelimb SDFT (n = 24). Methods: The 3-loop pulley (3LP) and 6-strand Savage (SSS) suture patterns were applied to transected equine SDFT. Ultimate failure load, stiffness, mode of failure, and load required to form a 3-mm gap were obtained using a materials testing system and synchronized high-speed video analysis. Statistical comparisons were made us...
de Grauw JC.Diseases affecting synovial joints are a major cause of chronic disability both in humans and in companion animal species, most notably dogs and horses. As progressive deterioration of the articular cartilage is the hallmark of degenerative joint disease or osteoarthritis, research efforts traditionally tended to focus primarily on cartilage pathology. However, in recent years it has become clear that synovial joints should be considered intricate organs in their own right, with each of the constituent tissues (cartilage, bone, and synovial membrane) interacting with each other both in health ...
Mössner LD, Schmitz A, Theurillat R, Thormann W, Mevissen M.To identify and characterize cytochrome P450 enzymes (CYPs) responsible for the metabolism of racemic ketamine in 3 mammalian species in vitro by use of chemical inhibitors and antibodies. Methods: Human, canine, and equine liver microsomes and human single CYP3A4 and CYP2C9 and their canine orthologs. Methods: Chemical inhibitors selective for human CYP enzymes and anti-CYP antibodies were incubated with racemic ketamine and liver microsomes or specific CYPs. Ketamine N-demethylation to norketamine was determined via enantioselective capillary electrophoresis. Results: The general CYP inhibit...
Guschlbauer M, Feige K, Geburek F, Hoppe S, Hopster K, Pröpsting MJ, Huber K.To determine whether administration of lidocaine during ischemia and reperfusion in horses results in concentrations in smooth muscle sufficient to protect against the negative consequences of ischemia-reperfusion injury on smooth muscle motility. Methods: 12 horses. Methods: Artificial ischemia and reperfusion injury of jejunal segments was induced in vivo in conjunction with lidocaine treatment during ischemia (IRL) or without lidocaine treatment (IR). Isometric force performance was measured in vitro in IRL and IR smooth muscle preparations with and without additional in vitro application o...
Klein C, Troedsson M.During the second and third week of pregnancy, the equine conceptus is covered by an acellular glycoprotein capsule. This capsule contains glycoproteins resembling those of the mucin family with sialic acid making up a high proportion of the carbohydrate. Coinciding with conceptus fixation, a marked decline in sialic acid content of the capsule occurs, which has been proposed to contribute to cessation of conceptus mobility. Herein, we describe the expression of neuraminidase 2 (NEU2) by pre-implantation stages of equine conceptus development. NEU2 transcript abundance was examined in conceptu...
Matthews JB, McArthur C, Robinson A, Jackson F.Cyathostomins are the primary parasitic pathogens of equids. For over 40 years, these nematodes have been controlled using broad spectrum anthelmintics. Three classes of anthelmintic are currently available for this use but, unfortunately, resistance to each of these has now been recorded in cyathostomin populations. As part of an optimal strategy to control cyathostomin infections in the field, it will be important to identify drug-resistant worms at as early a stage as possible. This objective needs to be supported by methodologies that will allow the accurate comparison of anthelmintic resi...
Papa FO, Felício GB, Melo-Oña CM, Alvarenga MA, De Vita B, Trinque C, Puoli-Filho JN, Dell'Aqua JA.The objective of this study was to determine whether replacing the egg yolk with soybean lecithin in the Botu-Crio® cryodiluent would maintain the fertility of cryopreserved stallion sperm. Two experiments were performed to evaluate cell freezability. In experiment 1, sperm from 15 stallions were frozen in Botu-Crio® (BC) or Botu-Crio® which contained 45g/L soybean lecithin (BCLS45) in place of the egg yolk. In experiment 2, we compared different concentrations of soybean lecithin: 0, 10.0, 12.5, 15.0, 17.5 and 20.0g/L (BC, BCLS10, BCLS12.5, BCLS17.5 and BCLS20, respectively). In experiment...
Lara PC, Fabrino HJ, Germano A, Silva JB.A method for the determination of As, Cd and Pb in bovine, equine and poultry liver by ICP-MS was developed and validated. Samples were digested in a microwave oven using a 10% HNO(3) solution. A set of experiments was made according to a central composite design (CCD) for optimisation of the plasma argon flow, nebuliser argon flow and radiofrequency power applied to the plasma. During the validation, Rh and Ru were evaluated as internal standards and, after validation, the best was Rh for Pb and Cd analysis, but for As better results were obtained without an internal standard. The method allo...
Griffeth GC, Callori N, Rankin SC, Boston RC, Morris DO.Meticillin-resistant Staphylococcus aureus (MRSA) causes serious skin and soft-tissue infections of humans and animals. Multiple strains of MRSA have been characterized, and one in particular, designated as strain USA 500, causes infections predominantly of horses and the people who work with them. The purpose of this study was to optimize an assay which could subsequently be used to compare the relative avidity of different S. aureus strains for equine corneocytes. Corneocytes were collected from the perineal skin of 10 healthy horses onto adhesive discs. The discs were then incubated at 37°...
Violini S, Gorni C, Pisani LF, Ramelli P, Caniatti M, Mariani P.Stem cells represent an important tool in veterinary therapeutic field such as tissue engineering. In the present study, equine amnion-derived mesenchymal stromal cells were investigated for applications in veterinary science as an alternative source to bone marrow mesenchymal stem cells and adipose stem cells. Amnion stromal cells isolation and characterization protocol is described; the in vitro cell growth rate was calculated by measuring viable cell number over 20 days. The expression of stem cell markers such as Oct-4, Nanog, Sox-2 and CD105 was assessed by retrotranscription quantitativ...
Go YY, Bailey E, Cook DG, Coleman SJ, Macleod JN, Chen KC, Timoney PJ, Balasuriya UB.Previously, we have shown that horses could be divided into susceptible and resistant groups based on an in vitro assay using dual-color flow cytometric analysis of CD3+ T cells infected with equine arteritis virus (EAV). Here, we demonstrate that the differences in in vitro susceptibility of equine CD3+ T lymphocytes to EAV infection have a genetic basis. To investigate the possible hereditary basis for this trait, we conducted a genome-wide association study (GWAS) to compare susceptible and resistant phenotypes. Testing of 267 DNA samples from four horse breeds that had a susceptible or a r...
Hamza E, Gerber V, Steinbach F, Marti E.Horses are particularly prone to allergic and autoimmune diseases, but little information about equine regulatory T cells (Treg) is currently available. The aim of this study therefore was to investigate the existence of CD4(+) Treg cells in horses, determine their suppressive function as well as their mechanism of action. Freshly isolated peripheral blood mononuclear cells (PBMC) from healthy horses were examined for CD4, CD25 and forkhead box P3 (FoxP3) expression. We show that equine FoxP3 is expressed constitutively by a population of CD4(+) CD25(+) T cells, mainly in the CD4(+) CD25(high)...
Filannino A, Stout TA, Gadella BM, Sostaric E, Pizzi F, Colenbrander B, Dell'Aquila ME, Minervini F.The aim of this study was to investigate the in vitro effects of the Fusarium fungus-derived mycotoxin, zearalenone and its derivatives alpha-zearalenol and beta-zearalenol on motility parameters and the acrosome reaction of stallion sperm. Since the toxic effects of zearalenone and its derivatives are thought to result from their structural similarity to 17beta-estradiol, 17beta-estradiol was used as a positive control for 'estrogen-like' effects. Methods: Stallion spermatozoa were exposed in vitro to zearalenone, alpha-zearalenol, beta-zearalenol or 17beta-estradiol at concentrations ranging...
McCarthy HE, Bara JJ, Brakspear K, Singhrao SK, Archer CW.A chondrocyte progenitor population isolated from the surface zone of articular cartilage presents a promising cell source for cell-based cartilage repair. In this study, equine articular cartilage progenitor cells (ACPCs) and equine bone marrow-derived stromal cells (BMSCs) were compared as potential cell sources for repair. Clonally derived BMSCs and ACPCs demonstrated expression of the cell fate selector gene, Notch-1, and the putative stem cell markers STRO-1, CD90 and CD166. Chondrogenic induction revealed positive labelling for collagen type II and aggrecan. Collagen type X was not detec...
Hagen J, Hartnett C, Houchins JP, Giguère S, Kalyuzhny AE.Human and mouse immune system cells are the most frequently used specimens in ELISPOT assays. In an effect to expand the application of ELISPOT assay to other species, we developed matched antibody pairs for ready-to-use kits designed for studying the frequency of equine IFNγ- and IL-4-secreting peripheral blood mononuclear cells (PBMCs). Equine PBMCs were stimulated with either concanavalin A (Con A) or calcium ionomycin mixed with phorbol 12-myristate 13-acetate (CaI + PMA). We found that Con A, in general, had a more profound stimulating effect than CaI + PMA on IL-4 secretion, whereas bot...
Wauters J, Franck T, Pille F, Martens A, Demeyere K, Sys S, Serteyn D, Gasthuys F, Meyer E.Myeloperoxidase (MPO) is a protein of interest due to its involvement in equine pathologies. Until now, results in equine diagnostic research were achieved through extracellular MPO detection. However, studying the cellular MPO content in neutrophils has revealed important insights in human diseases. This study aimed to develop a technique for the specific detection of MPO on the single cell level defining a flow cytometric protocol for the detection of both equine surface-bound and cellular MPO. Both indirect and direct labeling techniques are described which include the comparison of two sec...
Azab W, El-Sheikh A, Abdel-Gawad A.Equine herpesvirus 4 (EHV-4) is an important pathogen that causes respiratory tract disease in horse populations worldwide. Glycoprotein G (gG) homologs have been identified in several alphaherpesviruses as minor non-essential membrane-anchored glycoproteins. In this study, EHV-4 gG deletion mutant has been generated by using bacterial artificial chromosome technology to investigate the role of gG in viral pathogenesis. Our findings reported here revealed no significant difference between parental EHV-4 and gG-negative strain in their replication cycle in cell culture. Furthermore, virus titer...
Deschene K, Céleste C, Boerboom D, Theoret CL.Exuberant granulation tissue (EGT), a fibrotic healing disorder resembling the human keloid, occurs almost exclusively in limb wounds of horses and may be caused in part by a relative state of hypoxia within the wound. Objective: The objectives of this study were therefore to (1) assess the effects of hypoxia on equine dermal fibroblast (EDF) proliferation and apoptosis, (2) study the effects of hypoxia on the expression of key extracellular matrix (ECM) associated proteins and determine if such effects are dependent on hypoxia-inducible factor (HIF), and (3) determine if EDFs from the body or...
Watts AE, Nixon AJ, Yeager AE, Mohammed HO.A consistent and clinically relevant model for the induction of core lesions confined to the mid-metacarpal superficial digital flexor tendon (SDFT) has not been previously reported. Injection of bacterial collagenase is commonly used but often results in large, irregular and inconsistent lesions that disrupt the superficial tendon layers and epitenon. Objective: To develop and evaluate a new injection technique for collagenase induction of SDFT injury. Methods: Collagenase gel was injected into a physical columnar defect created by longitudinally placing a curved 16 gauge 8.89 cm needle in ...
Smits K, Govaere J, Hoogewijs M, Piepers S, Van Soom A.Intracytoplasmic sperm injection (ICSI) is the method of choice for the in vitro production (IVP) of equine embryos. However, conventional ICSI has been associated with mechanical damage to the oocyte caused by the deformation of the zona pellucida (ZP) and exposure of the oolemma to negative pressure during injection. Introduction of the less traumatic and more efficient piezo drill-assisted ICSI (PDAI) yielded higher cleavage rates and more consistent results. Nevertheless, PDAI is also associated with disadvantages such as the use of mercury and possible DNA damage. This led us to explore a...
Fowlie J, Arnoczky S, Lavagnino M, Maerz T, Stick J.To evaluate the magnitude and distribution of joint contact pressure on the medial tibial condyle after grade III cranial horn tears of the medial meniscus. Methods: Experimental study. Methods: Cadaveric equine stifles (n = 6). Methods: Cadaveric stifles were mounted in a materials testing system and electronic pressure sensors were placed between the medial tibial condyle and medial meniscus. Specimens were loaded parallel to the longitudinal axis of the tibia to 1800 N at 130°, 140°, 150°, and 160° stifle angle. Peak pressure and contact area were recorded from the contact maps. Testing...
Böer U, Lohrenz A, Klingenberg M, Pich A, Haverich A, Wilhelmi M.Decellularized equine carotid arteries (dEAC) may represent a reasonable alternative to alloplastic materials in vascular replacement therapy. Acellularity of the matrix is standardly evaluated by DNA quantification what however may not record sufficiently the degree of matrix immunogenicity. Thus, our aim was to analyze dEAC with a low DNA content for residual cellular proteins. A detergent-based decellularization protocol including endonuclease treatment resulted in dEAC with 0.6 ± 0.15 ng DNA/mg dry weight representing 0.33 ± 0.14% of native tissue DNA content. In contrast, when matrices ...
Lange-Consiglio A, Corradetti B, Bizzaro D, Magatti M, Ressel L, Tassan S, Parolini O, Cremonesi F.The aim of this work was to isolate, for the first time, progenitor-like cells from the epithelial (AECs) and mesenchymal (AMCs) portions of the horse amniotic membrane, and to define the biological properties of these cells. AECs displayed polygonal epithelial morphology, while AMCs were fibroblast-like. Usually, six to eight passages were reached before proliferation decreased, with 13.08 and 26.5 cell population doublings attained after 31 days for AECs and AMCs, respectively. Immunocytochemical studies performed at passage 3 (P3) showed that both cell populations were positive for the expr...
Leon PM, Campos VF, Kaefer C, Begnini KR, McBride AJ, Dellagostin OA, Seixas FK, Deschamps JC, Collares T.The gene expression of Bax, Bcl-2, survivin and p53, following in vitro maturation of equine oocytes, was compared in morphologically distinct oocytes and cumulus cells. Cumulus-oocyte complexes (COC) were harvested and divided into two groups: G1 - morphologically healthy cells; and G2 - less viable cells or cells with some degree of atresia. Total RNA was isolated from both immature and in vitro matured COC and real-time reverse transcription polymerase chain reaction (qRT-PCR) was used to quantify gene expression. Our results showed there was significantly higher expression of survivin (P &...
Stege PW, Lapierre AV, Martinez LD, Messina GA, Sombra LL.In this study we developed an interesting alternative to HPLC-mass spectrometry for the quantification of seven important drugs of abuse in racehorses. The procedure proposed in this work is a combination of single-drop microextraction (SDME) and an open tubular capillary electrochromatography (OT-CEC) using multi-wall carbon nanotubes (MWCTs) immobilized into a fused-silica capillary as a stationary phase. The SDME showed to be a powerful tool for extraction/preconcentration of the seven drugs analyzed in the study, showing an enrichment factor between 38- and 102-fold depending on the drug. ...
Watt B, Tenza D, Lemmon MA, Kerje S, Raposo G, Andersson L, Marks MS.PMEL is a pigment cell-specific protein that forms physiological amyloid fibrils upon which melanins ultimately deposit in the lumen of the pigment organelle, the melanosome. Whereas hypomorphic PMEL mutations in several species result in a mild pigment dilution that is inherited in a recessive manner, PMEL alleles found in the Dominant white (DW) chicken and Silver horse (HoSi)--which bear mutations that alter the PMEL transmembrane domain (TMD) and that are thus outside the amyloid core--are associated with a striking loss of pigmentation that is inherited in a dominant fashion. Here we show...
Pillet E, Labbe C, Batellier F, Duchamp G, Beaumal V, Anton M, Desherces S, Schmitt E, Magistrini M.Egg yolk is normally used as a protective agent to freeze semen of equine and other species. However, addition of egg yolk in extenders is not without disadvantages and the demand to find cryoprotective alternatives is strong. The objective of this study was to test the cryoprotective capacities of liposomes composed of egg yolk phospholipids. Two experiments were conducted: 1) the first to determine the optimal composition and concentration of liposomes to preserve post-thaw motility and membrane integrity of spermatozoa; 2) the second to assess in vivo the cryoprotective capacities of these ...
Bahrami A, Divar MR, Azari M, Kafi M.In the present study, we aimed to evaluate the possible protective effects of the nicotinic acid (NA) at three concentrations (10, 20, and 40 mM) on the equine cooled and frozen-thawed spermatozoa quality markers including viability, plasma membrane or acrosome integrity, DNA fragmentation, lipid peroxidation, and total oxidant levels. We also evaluated the effects of NA on preservation of the post-thaw sperm quality after 6 hours of cold storage before freezing. Five stallions were used for semen collections. The current experiment was repeated six times using pooled semen samples from two ...
Baker CB, Adams MH, McDowell KJ.Horse conceptuses were collected on Days 13, 15, 20 and 25 after ovulation. Whole conceptuses (Days 13 and 15) or extra-embryonic membranes (Days 20 and 25) were homogenized and poly-adenylated RNA (poly A RNA) was isolated by binding to oligo (dT)-cellulose. Poly A RNA (1 microgram/well) was separated by size on a denaturing 1% agarose gel and blotted onto nitrocellulose filters (northern blotting). DNA probes were prepared from plasmids containing equine alpha 1, omega 1 and omega 2 interferons and human beta actin. The presence of messenger RNA (mRNA) was detected by specific hybridization ...
Gargouri Y, Pieroni G, Rivière C, Sugihara A, Sarda L, Verger R.We report further investigations on protein inhibition of pancreatic and microbial lipases carried out with the monolayer technique. When beta-lactoglobulin A, melittin, serum albumin, myoglobin, and a protein inhibiting lipase from soybean were preincubated with a dicaprin film at a surface pressure of 35 dynes/cm, no activity was detected with horse pancreatic or Rhizopus delemar lipases. By contrast, Rhizopus arrhizus and Geotrichum candidum lipase activities were not impaired under the same conditions. Experiments using mixed lipid-protein film transfer clearly show that the inhibition of ...
Sabeur K, Ball BA.To identify the generation of the superoxide anion by equine spermatozoa. Methods: Multiple ejaculates collected from 3 Thoroughbred stallions. Methods: Induced superoxide production by reduced nicotinamide adenine dinucleotides (NAD[P]H; ie, reduced nicotinamide adenine dinucleotide [NADH] and reduced nicotinamide adenine dinucleotide phosphate [NADPH]) was measured by use of a nitroblue tetrazolium (NBT) reduction assay on whole spermatozoa and a cytochrome c reduction assay on isolated membrane fractions of spermatozoa. Localization of superoxide generation was determined by use of NBT cyto...
Borena BM, Meyer E, Chiers K, Martens A, Demeyere K, Broeckx SY, Duchateau L, Spaas JH.Mammal skin plays a pivotal role in several life preserving processes and extensive damage may therefore be life threatening. Physiological skin regeneration is achieved through ongoing somatic stem cell differentiation within the epidermis and the hair follicle. However, in severe pathological cases, such as burn wounds, chronic wounds, and ulcers, the endogenous repair mechanisms might be insufficient. For this reason, exogenous purification and multiplication of epithelial-like stem/progenitor cells (EpSCs) might be useful in the treatment of these skin diseases. However, only few reports a...
Mondal SP, Cook RF, Chelvarajan RL, Henney PJ, Timoney PJ, Balasuriya UB.Strains of equine arteritis virus (EAV) differ in their virulence phenotypes, causing anywhere from subclinical infections to severe disease in horses. Here, we describe the in silico design and de novo synthesis of a full-length infectious cDNA clone of the horse-adapted virulent Bucyrus strain (VBS) of EAV encoding mCherry along with in vitro characterization of the progeny virions (EAV sVBSmCherry) in terms of host-cell tropism, replicative capacity and stability of the mCherry coding sequences following sequential passage in cell culture. The relative stability of the mCherry sequence duri...
Casella S, Giannetto C, Giudice E, Marafioti S, Fazio F, Assenza A, Piccione G.Adenosine diphosphate (ADP)-induced platelet aggregation in fed and fasted horses after addition of tramadol hydrochloride was evaluated in vitro. On 10 horses citrated blood samples were collected 2h after feeding (fed animals) and 21 h after feeding (fasted animals). Final concentrations of ADP 1 and 0.5 μM, and tramadol hydrochloride (1, 15, 30, 45 and 60 min after the addition of tramadol) were used to determine the maximum degree and initial velocity of platelet aggregation. Repeated measures multifactor analysis of variance (MANOVA) was used to evaluate the effect of feeding/fasting con...
Cuniberti B, Odore R, Barbero R, Cagnardi P, Badino P, Girardi C, Re G.Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit cyclooxygenases (COX), and the inhibition of COX-2 rather than COX-1 can limit the onset of NSAID-related adverse effects. The pharmacodynamic properties of eltenac, naproxen, tepoxalin, SC-560 and NS 398 in healthy horses were investigated using an in vitro whole blood assay. To predict COX selectivity in clinical use, eltenac and naproxen were also studied ex vivo after intravenous administration. SC-560 acted as a selective COX-1 inhibitor, tepoxalin as a dual inhibitor with potent activity against COX-1, and NS 398 as a preferential CO...
Malisauskas M, Darinskas A, Zamotin VV, Gharibyan A, Kostanyan IA, Morozova-Roche LA.In the current study we investigated the molecular mechanisms of cytotoxicity of amyloid oligomers of horse milk lysozyme. We have shown that lysozyme forms soluble amyloid oligomers and protofibrils during incubation at pH 2.0 and 4.5 and 57 degrees C. These structures bind the amyloid-specific dyes thioflavin T and Congo Red, and their morphology and size were analyzed by atomic force microscopy. Monomeric lysozyme and its fibrils did not affect the viability of three cell types used in our experiments including primary murine neurons and fibroblasts, as well as neuroblastoma cell line IMR-3...
Ishitani K, Listowsky I.Horse spleen ferritin was fractionated into its constituent isoferritins by isoelectric focusing. Separated isoferritins were stable and showed no tendency to redistribute when re-examined by analytical gel focusing. All of the isoferritins were immunologically indistinguishable when tested with antibodies raised against unfractionated horse spleen ferritin. The separated isoferritins also had similar conformations as determined by circular dichroism. Iron distribution studies, however, revealed a wide disparity among the isoferritins. The most acidic components had the lowest iron content but...
Bonilla-Gutiérrez AF, Castillo-Franz C, López C, Álvarez ME, Giraldo CE, Carmona JU.The aim of this study was to evaluate the release of pro- and anti-inflammatory as well as anabolic mediators stimulated by a leukocyte-reduced platelet-rich gel supernatant (Lr-PRGS) and a leukocyte-reduced plasma supernatant (Lr-PL) at two concentrations (25 and 50%) on normal equine suspensory ligament explants (SLEs) and tendon explants (TEs). SLEs and TEs from six horses were independently incubated for 48 h with Lr-PRGS and Lr-PL at concentrations of 25 and 50%, respectively. Samples were collected from the incubated tissues at 1 h and 48 h, which were employed for ELISA determinat...
Sánchez-Calabuig MJ, Fernández-González R, Hamdi M, Smits K, López-Cardona AP, Serres C, Macías-García B, Gutiérrez-Adán A.Equine embryos exhibit an unusual pattern of glucose tolerance in vitro and are currently cultured in hyperglycaemic conditions. Objective: Our main objective was to analyse the effect of different glucose concentrations on in vitro-produced equine embryo development and quality. Methods: Experiments comparing in vitro and in vivo produced embryos. Methods: Oocytes (n = 641) were collected from post-mortem ovaries, matured in vitro and fertilised by intracytoplasmic sperm injection (ICSI). Embryo culture was divided from Day 0 to Day 4 and from Day 4 to Day 9 in three groups: 5-10 (5 and 10...
Fernandez-Fuertes B, Sánchez JM, Bagés-Arnal S, McDonald M, Yeste M, Lonergan P.This study aimed to determine the effect of bull seminal plasma (SP) and sperm on endometrial function. Bovine endometrial explants were incubated with: ejaculated sperm with or without SP, epididymal sperm, or SP alone. Neither ejaculated nor epididymal sperm induced differential expression of IL1A, IL1B, IL6, IL8, PTGES2, TNFA, and LIF. Interestingly, SP had a detrimental effect on endometrial RNA integrity. Addition of an RNase inactivation reagent to SP blocked this effect, evidencing a role for a SP-RNase. Because bulls deposit the ejaculate in the vagina, we hypothesized that the bovine ...
Anderson RS, Day NK, Good RA.Natural hemagglutinin activity against vertebrate erythrocytes is present in the hemolymph of the cockroach Blabarus craniifer. The hemagglutinin titer against rabbit erythrocytes is high, whereas sheep and horse red cells agglutinate weakly. Hemagglutinin activity was depressed by the complement inhibitor, cobra venom factor. Cockroach hemagglutinin is heat-labile; all activity is destroyed by heating at 56 C for 1 hr. A humoral factor similar to the complement component 3 proactivator is also present in cockroach hemolymph. The formation of the cobra venom factor-hemolymph "complex" is depen...
Meehan M, Burke FM, Macken S, Owen P.Streptococcus equi possesses a haem-uptake system homologous to that of Streptococcus pyogenes and Streptococcus zooepidemicus. The system consists of two ligand-binding proteins (Shr and Shp) and proteins (HtsA-C) with homology to an ABC transporter. The haem-uptake system of S. equi differs from that of S. pyogenes and S. zooepidemicus in that Shr is truncated by two-thirds. This study focused on the SeShr, SeShp and SeHtsA proteins of S. equi. Analysis of shr, shp and shphtsA knockout mutants showed that all three proteins were expressed in vitro and that expression was upregulated under co...
Witonsky S, Buechner-Maxwell V, Santonastasto A, Pleasant R, Werre S, Wagner B, Ellison S, Lindsay D.Equine protozoal myeloencephalitis (EPM) is a common and devastating neurologic disease of horses in the United States. Because some EPM-affected horses have decreased immune responses, immunomodulators such as levamisole have been proposed as supplemental treatments. However, little is known about levamisole's effects or its mechanism of action in horses. Objective: Levamisole in combination with another mitogen will stimulate a macrophage 1 (M1), dendritic cell 1 (DC1), T-helper 1 (CD4 Th1), and T-cytotoxic (CD8) immune response in equine peripheral blood mononuclear cells (PBMCs) in vitro a...
Atkinson F, Evans R, Guest JE, Bavin EP, Cacador D, Holland C, Guest DJ.Tendon injuries are a common cause of morbidity in humans. They also occur frequently in horses, and the horse provides a relevant, large animal model in which to test novel therapies. To develop novel cell therapies that can aid tendon regeneration and reduce subsequent reinjury rates, the mechanisms that control tendon tissue regeneration and matrix remodelling need to be better understood. Although a range of chemical cues have been explored (growth factors, media etc.), the influence of the mechanical environment on tendon cell culture has yet to be fully elucidated. To mimic the in vivo e...
Cho HJ, Entz SC, Deregt D, Jordan LT, Timoney PJ, McCollum WH.A potent ELISA antigen was prepared from equine arteritis virus (EAV) by differential centrifugation of EAV-infected cell culture fluid, followed by solubilization of the preparation by Triton X-100 treatment. Using this antigen and a mouse monoclonal antibody against the G(L) protein of EAV, a reliable blocking ELISA (bELISA) was developed for the detection of EAV antibodies in equine sera. The bELISA was evaluated using a total of 837 test serum samples. The relative sensitivity (n = 320) of the bELISA compared to the serum neutralization (SN) test was 99.4%. The bELISA appears to be a highl...
Neuhauser S, Bollwein H, Siuda M, Handler J.Cryopreservation of epididymal sperm allows the saving of genetic material in case of unexpected death or emergency castration. The aim of the present study was the comparison of five different combinations of extenders commercially available for equine frozen semen processing for cryopreservation of epididymal sperm. Epididymal sperm were harvested from gonads of 10 healthy stallions after routine castration by retrograde flush technique. Then, samples were split and diluted with (1) INRA96 + INRA Freeze, (2) BotuSemen + BotuCRIO, (3) EquiPlus + Gent Freeze, (4) EquiPlus + EquiPlus Freeze...
Dean PW, Nelson JK, Schumacher J.Cartilage retention strengths of laryngoplasty prostheses were compared in larynges of 2-, 3-, and 4-year-old horses, using doubled polyester and expanded polytetrafluoroethylene prostheses. Bilateral laryngoplasties were performed on each of 15 (seven 2-year-old, two 3-year-old, and six 4-year-old) larynges, which were collected at an abbatoir. Prostheses were secured to a mechanical testing machine, and tension causing arytenoid cartilage abduction was applied, until total failure of the cartilage or prosthesis resulted. Tension caused cricoid cartilage failure in 1 specimen, and muscular pr...
Al-Essawe EM, Johannisson A, Wulf M, Aurich C, Morrell JM.Freezing and thawing processes induce structural and functional damage to sperm plasma membranes and internal organelles. Adding seminal plasma (SP) has been found to minimize or repair the cryoinjuries in some species. The objective of this study was to investigate whether adding SP from stallions of known freezability after thawing could repair cryoinjuries. Semen was collected from warmblood stallions (n = 8, three ejaculates/stallion) and processed by Single Layer Centrifugation (SLC) to remove SP prior to freezing. Pooled SP (5%) from bad freezer (BF) or good freezer (GF) stallions wa...
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...
Terrasa AM, Guajardo MH, Marra CA, Zapata G.Oxidative stress is a possible risk factor for eye diseases. Lipid peroxidation is one of the major events induced by oxidative stress and is particularly active in polyunsaturated fatty acid (PUFA)-rich biomembranes. This work evaluated endogenous lipid antioxidants, in vitro non-enzymatic lipid peroxidation of rod outer segment membranes (ROS), the fatty acid composition during oxidative damage of total lipids from equine retina and ROS, and the protective action of alpha-tocopherol (alpha-Toc). The major lipid soluble antioxidant was alpha-Toc followed by retinoids and carotenoids. The reti...
Khan FA, Chenier TS, Foster RA, Hewson J, Scholtz EL.Emerging research suggests that the nitric oxide system may play a role in persistent breeding-induced endometritis (PBIE) in the mare. Differences in uterine nitric oxide (NO) levels between mares susceptible or resistant to PBIE and a dose-dependent inhibitory effect of NO on uterine contractility have been demonstrated. The objectives of this study were to investigate the difference in total nitric oxide synthase (NOS) activity of the endometrium between susceptible and resistant mares and the effect of a specific inducible nitric oxide synthase (iNOS) inhibitor on the endometrial NOS activ...
Marlo TL, Giuliano EA, Tripathi R, Sharma A, Mohan RR.To explore the impact of equine corneal fibroblast (ECF) to myofibroblast (ECM) differentiation by altering the expression of the Smad genes either individually or in combination. Specifically, we sought to examine the ECF differentiation after (a) silencing of Smad2, 3, and 4 profibrotic genes individually and (b) overexpression of antifibrotic Smad7 gene and in a combination with pro- and antifibrotic Smad genes. Methods: Equine corneal fibroblast primary cultures were generated as previously described. ECFs were transfected with individual plasmids which silenced gene expression of either S...
Nakagawa K, Tokushima A, Fujiwara K, Ikeguchi M.The secondary structure in the molten globule state (an equilibrium analogue of a burst-phase folding intermediate) of equine beta-lactoglobulin was investigated by changes in the circular dichroic spectrum induced by a series of site-directed proline substitutions. The results challenge the structural picture obtained from previous hydrogen/deuterium exchange experiments. A stable non-native alpha-helix was found to exist in the region corresponding to the eighth strand (H strand) in the native structure, where the backbone amide protons are the most strongly protected from exchange. Therefor...
Vereecke N, Vandekerckhove A, Theuns S, Haesebrouck F, Boyen F.Actinobacillus equuli is mostly associated with disease in horses and is most widely known as the causative agent of sleepy foal disease. Even though existing phenotypic tools such as biochemical tests, 16S rRNA gene sequencing, and Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) can be used to identify members of the Actinobacillus genus, these methods struggle to differentiate between certain species and do not allow strain, virulence, and antimicrobial susceptibility typing. Hence, we performed in-depth analysis of 24 equine Actinobacillus isolate...
Moore-Colyer M, Tuthill P, Bannister I, Daniels S.Traditions and the economic advantage when producing big athletic Thoroughbred (TB) yearlings for sale encourage owners to feed high levels of cereals. Such diets can predispose gastric ulceration and developmental orthopedic diseases, impacting negatively on future careers. This study aimed to (1) determine if an all-fiber creep feed (total mixed fiber ration [TMFR] would sustain equal growth rates in TB foals versus a cereal-based stud cube (SC) and (2) compare pH and lactate produced by SC and TMFR in in vitro foregut and hindgut conditions. Eight TB foals in matched-pairs were fed SC or T...
Yuan Z, Gault EA, Campo MS, Nasir L.Equine sarcoids represent the most common skin tumours in equids worldwide, characterized by extensive invasion and infiltration of lymphatics, rare regression and high recurrence after surgical intervention. Bovine papillomavirus type 1 (BPV-1) activity is necessary for the transformation phenotype of equine fibroblasts. Among the many changes induced by BPV-1, matrix metalloproteinase 1 (MMP-1) upregulation contributes to the invasiveness of equine fibroblasts. However, it is not yet known how BPV-1 proteins regulate equine MMP-1 expression. To elucidate this mechanism, the equine MMP-1 prom...
Anzai T, Sheoran AS, Kuwamoto Y, Kondo T, Wada R, Inoue T, Timoney JF.Streptococcus equi causes equine strangles. The acute disease has many of the hallmarks of an acute response including high fever, elevated plasma fibrinogen and neutrophilia, affects known to be mediated by proinflammatory cytokines. The objective of this study was to screen-culture supernatants from equine clinical isolates of S. equi and S. zooepidemicus for stimulation of mitogenic responses by horse peripheral blood mononuclear cells. Mitogenicity comparable to that of concanavalin A was detected in culture supernatants of S. equi strains but not in those of S. zooepidemicus. Mitogenicity...
