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.
Berg L, Koch T, Heerkens T, Bessonov K, Thomsen P, Betts D.Orthopaedic injury is the most common cause of lost training days or premature retirement in the equine athlete. Cell-based therapies are a potential new treatment option in musculo-skeletal diseases. Mesenchymal stromal cells (MSC) have been derived from multiple sources in the horse including bone marrow and umbilical cord blood. The objective of this study was to provide an in vitro comparison of the chondrogenic potential in MSC derived from adult bone marrow (BM-MSC) and umbilical cord blood (CB-MSC). Results: MSC from both sources produced tissue with cartilage-like morphology that stain...
Fintl C, Pearson GT, Mayhew IG, Stewart Lowden C, Hopwood PA, Palgrave CJ, Proudman CJ, Barrie Edwards G, Taylor SE, Hudson NP.Previous immunohistochemical studies targeting the receptor tyrosine kinase (c-Kit) have demonstrated an apparent reduction in the number of gastrointestinal pacemaker cells--the interstitial cells of Cajal (ICC)--in horses with intestinal motility disorders. This study compared the level of transcription of the c-kit gene encoding this receptor in horses with and without such motility disorders. Transcription levels of this gene were also compared to the density of ICC immunohistochemically positive for the c-Kit antigen. Intestinal samples were collected from 18 horses with intestinal diseas...
Oliveira CH, Vasconcelos AB, Souza FA, Martins-Filho OA, Silva MX, Varago FC, Lagares MA.Addition of cholesterol to sperm membranes improved equine sperm stability during semen cryopreservation; however, it also reduced in vivo fertility. The objective of the present study was to determine the effects of adding cholesterol to stallion sperm prior to freezing, and subsequently removing it from frozen-thawed sperm. Semen from 12 stallions was subjected to four treatments: (T1) control, semen was diluted with Kenney extender, centrifuged, and resuspended to 100 x 10(6)spermatozoa/mL in INRA 82 freezing extender, packaged into 0.5-mL straws, cooled to 5 degrees C, and cryopreserved in...
Spizziri BE, Fox MH, Bruemmer JE, Squires EL, Graham JK.Irreversible damage occurs to spermatozoal membranes, during the phase transition, when spermatozoa are cooled from room temperature to 5 degrees C. Some of this damage can be ameliorated by adding cholesterol to the membrane, thereby altering membrane lipid composition. Adding cholesterol-loaded cyclodextrins (CLCs) to stallion spermatozoa prior to freezing, increases cell cryosurvival. However, the fertilizing potential of CLC-treated stallion spermatozoa is unknown. To address this, experiments were conducted which evaluated the ability of CLC-treated stallion spermatozoa to capacitate, acr...
Janicek JC, Wilson DA, Carson WL, Kramer J.To compare stiffness and strength of a dynamic condylar screw plate combined with dorsal broad dynamic compression plate (DCS-bDCP) fixation with double broad dynamic compression plate (dbDCP) fixation used to repair oblique distal fractures of adult equine radii. Methods: Experimental. Methods: Adult equine radii (n=10 pair). Methods: An unconstrained three-dimensional loading-measurement system was used to determine stiffness of a 50 mm long intact, and then DCS-bDCP or dbDCP-plated osteotomized/ostectomized segment of radii when subjected to a nondestructive sequence of compression, torsion...
Sessions DR, Vick MM, Fitzgerald BP.Matrix metalloproteinases (MMP) and tissue inhibitors of MMP (TIMP) regulate tissue remodeling events necessary for ovulation. Thus, changes in MMP and TIMP expression and protein enzyme activity were examined in vivo and in vitro during follicular development and atresia in the horse. Equine granulosa cells and follicular fluid from medium (15 to 29 mm) healthy and atretic follicles and from large (>30 mm) healthy and preovulatory follicles were collected by transvaginal aspiration. The cells were either snap-frozen (in vivo study) or cultured for 48 h (in vitro study) to determine gene ex...
Marchetti B, Gault EA, Cortese MS, Yuan Z, Ellis SA, Nasir L, Campo MS.Bovine papillomavirus type 1 is one of the aetiological agents of equine sarcoids. The viral major oncoprotein E5 is expressed in virtually all sarcoids, sarcoid cell lines and in vitro-transformed equine fibroblasts. To ascertain whether E5 behaves in equine cells as it does in bovine cells, we introduced the E5 open reading frame into fetal equine fibroblasts (EqPalF). As observed in primary bovine fibroblasts (BoPalF), E5 by itself could not immortalize EqPalF and an immortalizing gene, such as human telomerase (hTERT/hT), was required for the cells to survive selection. The EqPalF-hT-1E5 c...
Frampton AR, Uchida H, von Einem J, Goins WF, Grandi P, Cohen JB, Osterrieder N, Glorioso JC.To initiate infection, equine herpesvirus type 1 (EHV-1) attaches to heparan sulfate on cell surfaces and then interacts with a putative glycoprotein D receptor(s). After attachment, virus entry occurs either by direct fusion of the virus envelope with the plasma membrane or via endocytosis followed by fusion between the virus envelope and an endosomal membrane. Upon fusion, de-enveloped virus particles are deposited into the cytoplasm and travel to the nucleus for viral replication. In this report, we examined the mechanism of EHV-1 intracellular trafficking and investigated the ability of EH...
Lin M, Zhang C, Gibson K, Rikihisa Y.Neorickettsia risticii is an obligate intracellular bacterium of the trematodes and mammals. Horses develop Potomac horse fever (PHF) when they ingest aquatic insects containing encysted N. risticii-infected trematodes. The complete genome sequence of N. risticii Illinois consists of a single circular chromosome of 879 977 bp and encodes 38 RNA species and 898 proteins. Although N. risticii has limited ability to synthesize amino acids and lacks many metabolic pathways, it is capable of making major vitamins, cofactors and nucleotides. Comparison with its closely related human pathogen N. senn...
Ortega-Ferrusola C, González-Fernández L, Muriel A, Macías-García B, Rodríguez-Martínez H, Tapia JA, Alonso JM, Peña FJ.In an attempt to evaluate the possible relationship between the microbial flora in the stallion ejaculate and its ability to freeze,three ejaculates from five stallions were frozen using a standard protocol. Before freezing, an aliquot was removed for bacteriological analysis. Bacterial growth was observed in all the ejaculates studied. The isolated microorganisms were:Staphylococcus spp. and Micrococcus spp. (in all the stallions), beta-haemolytic Streptococcus (in stallions 3 and 4), Corynebacterium spp. (in stallions 1, 3-5), Rhodococcus spp. (in stallion number 2), Pseudomonas spp. (in sta...
Schmitz A, Theurillat R, Lassahn PG, Mevissen M, Thormann W.CE with multiple isomer sulfated-CD as selector was used for the simultaneous analysis of the stereoisomers of ketamine, norketamine, 5,6-dehydronorketamine and hydroxylated metabolites of norketamine in liquid/liquid extracts of (i) in vitro incubations with ketamine or norketamine and equine liver microsomes and (ii) plasma and urine of ponies receiving a target-controlled infusion of ketamine under isoflurane anesthesia. Hydroxynorketamine metabolites with the hydroxy group at the cyclohexanone ring could be shown to be formed stereoselectively both in vitro and in vivo. Due to the lack of ...
Ueno D, Yabuki A, Obi T, Shiraishi M, Nishio A, Miyamoto A.We investigated the effect of bradykinin (BK) on isolated equine basilar arterial rings with and without endothelium. BK induced concentration-dependent contraction of resting arterial rings and no relaxation when the rings were precontracted by prostaglandin F(2alpha). The maximal response and pD(2) value were 161.2 +/- 28.1% (to 60 mm KCl-induced contraction) and 8.24 +/- 0.25 respectively. The cumulative concentration-response curve for BK was not shifted to the right by des-Arg(9)-[Leu(8)]-BK (a B(1)-receptor antagonist), HOE140 (a B(2)-receptor antagonist) or NPC567 (another B(2)-receptor...
Cehak A, Burmester M, Geburek F, Feige K, Breves G.The aim of this study was to characterize the transport mechanisms of electrolytes and nutrients across the jejunum of nine healthy horses electrophysiologically. The stripped mucosa was mounted in Ussing chambers and tissue conductances (G(t)) and short circuit currents (I(sc)) were continuously monitored. After blocking the sodium and potassium channels with amiloride, tetraethylammonium chloride (TEA) and barium, chloride secretion was stimulated by carbachol and forskolin. Subsequently, chloride channels were inhibited by 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid, 5-nitro-2-(3-ph...
Casaite V, Bruzyte S, Bukauskas V, Setkus A, Morozova-Roche LA, Meskys R.Equine lysozyme (EL) is a calcium (Ca)-binding lysozyme and is an intermediary link between non-Ca-binding C-type lysozyme and alpha-lactalbumin. The feature of lysozymes to assemble into the fibrils has recently gained considerable attention for the investigation of the functional properties of these proteins. To study the structural and functional properties of EL, a synthetic gene was cloned and EL was overexpressed in Escherichia coli as a fused protein. The His-tagged recombinant EL was accumulated as inclusion bodies. Up to 50 mg/l of the recombinant EL could be achieved after purificati...
Goodrich LR, Choi VW, Carbone BA, McIlwraith CW, Samulski RJ.Cell transplantation for the treatment of joint disease is an important clinical tool. Genetic modification of cells before transplantation has shown enhanced healing. Ex vivo genetic modification of joint tissue cells with various adeno-associated virus (AAV) serotypes has not been investigated. The transduction efficiencies of self-complementary AAV serotypes (1-6 and 8) were determined in joint tissue containing chondrocytes and synoviocytes isolated from equine models. When comparing scAAV serotypes for efficient transduction ex vivo, in chondrocytes versus synoviocytes, serotypes 6 and 2,...
Morita H, Shimazu M, Shiono H, Toh H, Nakajima F, Akita H, Takagi M, Takami H, Murakami M, Masaoka T, Tanabe S, Hattori M.We previously isolated five strains of putative lactobacilli from the faeces of a thoroughbred horse (a 4-year-old male). Of the five strains, four were identified as members of existing Lactobacillus species; however, sequence analysis of the 16S rRNA gene revealed that the fifth isolate, DI70(T), showed approximately 97 % identity (1325/1366 bp) with the type strain of Lactobacillus delbrueckii. Therefore, we considered the possibility that DI70(T) represents a novel species of the genus Lactobacillus. Cells of strain DI70(T) were Gram-stain-positive, catalase-negative, non-spore-forming, no...
Crevier-Denoix N, Ravary-Plumioën B, Evrard D, Pourcelot P.A non-invasive ultrasonic (US) technique of tendon force measurement has been recently developed. It is based on the relationship demonstrated between the speed of sound (SOS) in a tendon and the traction force applied to it. The objectives of the present study were to evaluate the variability of this non-linear relationship among 7 equine superficial digital flexor (SDF) tendons, and the reproducibility of SOS measurements in these tendons over successive loading cycles and tests. Seven SDF tendons were equipped with an US probe (1MHz), secured in contact with the skin overlying the tendon me...
Echigoya Y, Sato T, Itou T, Endo H, Sakai T.The species-specific properties of LDH isozymes are essentially determined by M (muscle) and H (heart) subunit proteins encoded by the LDHA and LDHB genes, respectively. In the present study, we molecularly characterized the full-length equine lactate dehydrogenase A (eLDHA) and B (eLDHB) cDNAs. The eLDHA cDNA consisted of a 999-bp open reading frame (ORF), while the eLDHB and newly acquired bat LDHB consisted of a 1002-bp ORF, which is 3 bp shorter than the LDHB ORF of other registered mammals. The alignment of amino acid sequences showed that eLDHA acquired positively charged His 88 and 226,...
Glazar AI, Mullen SF, Liu J, Benson JD, Critser JK, Squires EL, Graham JK.Stallion spermatozoa exhibit osmotic damage during the cryopreservation process. Recent studies have shown that the addition of cholesterol to spermatozoal membranes increases the cryosurvival of bull, ram and stallion spermatozoa, but the exact mechanism by which added cholesterol improves cryosurvival is not understood. The objectives of this study were to determine if adding cholesterol to stallion sperm membranes alters the osmotic tolerance limits and membrane permeability characteristics of the spermatozoa. In experiment one, stallion spermatozoa were treated with cholesterol-loaded cycl...
Hosaka YZ, Takahashi H, Uratsuji T, Tangkawattana P, Ueda H, Takehana K.The aim of this study was to determine the characteristic differences in tendinocytes derived from tendons in the equine forelimb, superficial digital flexor tendon (SDFT), deep digital flexor tendon (DDFT) and common digital extensor tendon (CDET), in morphology, proliferation, collagen production ability and ability for synthesis of matrix metalloproteinases (MMPs). Significant differences were observed in cell number in vivo. The cellular number was largest in the SDFT and smallest in the CDET. The values of in vitro proliferation ratios and ability for synthesis of collagen and MMPs were l...
Radcliffe CH, Flaminio MJ, Fortier LA.Mesenchymal progenitor cells (MPCs) are often characterized using surface markers after expansion and treatment in culture. There are no studies directly comparing gene and protein markers in undifferentiated samples during the very early phases of culture. The goal of this study was to evaluate temporal gene and protein expression changes during establishment of equine MPC cultures. Bone marrow aspirate was obtained from 35 horses and processed by density gradient centrifugation. In freshly isolated bone marrow, mononuclear cells had variable expression of CD44, CD11a/CD18, CD90, and CD45RB c...
Andoh K, Kai K, Matsumura T, Maeda K.Cell lines originating from horses are necessary for isolation and propagation of equine herpesviruses (EHV). Although we established an equine-derived cell line, FHK-Tcl3, propagation ceased after fewer than 40 passages. In this study, FHK-Tcl3 cell propagation continued beyond 40 passages, achieving over 100 passages. FHK-Tcl3 cells were then cloned by limiting dilution at the 100th passage. Cloned cells were termed FHK-Tcl3.1. FHK-Tcl3.1 cells grew well and were propagated every 3 to 4 days by splitting 1:5. In addition, EHV-1, -2 and -4 showed a clear cytopathic effect (CPE) in FHK-Tcl3.1 ...
Ortega Ferrusola C, González Fernández L, Macías García B, Salazar-Sandoval C, Morillo Rodríguez A, Rodríguez Martinez H, Tapia JA, Peña FJ.The ability of stallion spermatozoa to produce nitric oxide (NO) before (fresh) and after freezing and thawing (FT) was evaluated by means of flow cytometry after loading the sperm suspension with the probe, 4,5-diaminofluorescenin diacetate. The presence of NO synthase (NOS) was investigated by Western blotting using anti-NOS1, anti-NOS3, or anti-universal NOS antibodies (Abs). While NO was detected both in fresh and FT sperm suspensions, its production increased after cryopreservation only when egg yolk was removed from the extender. Anti-NOS1 Ab intensively labeled a single band with an app...
Gobeil PA, Yuan Z, Gault EA, Morgan IM, Campo MS, Nasir L.Equine sarcoids are skin tumours of horses caused by infection with BPV-1 or 2. Maintenance and replication of the viral genome depend upon the viral proteins E1 and E2. We examined the effects of an E2 specific siRNA on E2 and E1 viral gene expression, viral load and cell growth in BPV-1 transformed sarcoid-derived cells. Transfection with E2-siRNA caused a reduction in E2 and E1 mRNA expression as well as viral load, growth inhibition and decreased anchorage-independent growth. siRNA treated cells showed significantly higher apoptosis rates than control cells. Thus sequence specific targetin...
Morrell JM, Johannisson A, Dalin AM, Rodriguez-Martinez H.The objective of the current study was to optimize the volumes of Androcoll-E and sperm sample used in various sizes of centrifuge tube to scale up single-layer centrifugation (SLC) for routine use in the field. Although sperm suspensions of equivalent quality were produced using Androcoll-E in small and large tubes, the sperm yield was much lower in the latter (P<0.001). In contrast, in 200-mL tubes (XL), the yields were approximately 25% higher than those for the small tubes. An increased volume (4.5 mL) of extended ejaculate in small tubes (SLC-Inc) or 15 to 18 mL extended ejaculate on 1...
Mugnier S, Dell'Aquila ME, Pelaez J, Douet C, Ambruosi B, De Santis T, Lacalandra GM, Lebos C, Sizaret PY, Delaleu B, Monget P, Mermillod P....The mechanism of fertilization remains largely enigmatic in mammals. Most studies exploring the molecular mechanism underlying fertilization have been restricted to a single species, generally the mouse, without a comparative approach. However, the identification of divergences between species could allow us to highlight key components in the mechanism of fertilization. In the pig, in vitro fertilization (IVF) and polyspermy rates are high, and spermatozoa penetrate easily through the zona pellucida (ZP). In contrast, IVF rates are low in the horse, and polyspermy is scarce. Our objective was ...
Wilhelm K, Darinskas A, Noppe W, Duchardt E, Mok KH, Vukojević V, Schleucher J, Morozova-Roche LA.Protein oligomeric complexes have emerged as a major target of current research because of their key role in aggregation processes in living systems and in vitro. Hydrophobic and charged surfaces may favour the self-assembly process by recruiting proteins and modifying their interactions. We found that equine lysozyme assembles into multimeric complexes with oleic acid (ELOA) at the solid-liquid interface within an ion-exchange chromatography column preconditioned with oleic acid. The properties of ELOA were characterized using NMR, spectroscopic methods and atomic force microscopy, and showed...
Scherzer J, Fayrer-Hosken RA, Aceves M, Hurley DJ, Ray LE, Jones L, Heusner GL.We evaluated combinations of two commercial semen extenders and three concentrations of glycerol to determine the combination that yielded the highest post-thaw sperm motility. Methods: A randomised 2 x 3 block design was used. Methods: Semen was collected from four stallions (6 collections per stallion). The sample was diluted with either a dried skim-milk glucose extender (EZ Mixin Original Formula) or a chemically defined, milk-free diluent (INRA 96), and each was used in combination with 2%, 3% or 4% glycerol in standard commercial freezing medium. Sperm motility was assessed by microscopy...
Espino-Solis GP, Calderon-Amador J, Calderon-Aranda ES, Licea AF, Donis-Maturano L, Flores-Romo L, Possani LD.A three-dimensional model of the alphaX I-domain of the horse integrin CD11c from dendritic cells provided information for selecting two segments of the primary structure for peptide synthesis. Peptide 1 contains 20 amino acids and peptide 2 has 17 amino acid residues. The first spans from position Thr229 to Arg248 of an alpha-helix segment of the structure, whereas peptide 2 goes from Asp158 to Phe174 and corresponds to an exposed segment of the loop considered to be the metal ion-dependent adhesion site. Murine polyclonal antisera against both peptides were generated and assayed in periphera...
Nagy A, Bodo G, Dyson SJ, Szabo F, Barr AR.Proximal diffusion of local anaesthetic solution after perineural anaesthesia may lead to the desensitisation of structures other than those intended. However, there is no evidence-based study demonstrating the potential distribution and diffusion of local anaesthetic solution after perineural analgesia in the distal limb. Objective: To document the potential diffusion of local anaesthetic solution using a radiopaque contrast model and to evaluate the influence of walking compared with confinement in a stable after injection. Methods: Radiopaque contrast medium was injected subcutaneously over...
De Lorenzi L, Genualdo V, Iannuzzi A, Di Meo GP, Perucatti A, Mancuso R, Russo M, Di Berardino D, Parma P, Iannuzzi L.A 4-year-old male horse of Friesian breed with normal body conformation, development and libido, and showing an evident ventral penis deviation with hypospadias, underwent both cytogenetic and genetic investigation. Although the karyotype showed normal male arrangement (2n = 64,XY), one telomere of horse (ECA) chromosome 1 was shorter than both the other one and those of a normal horse (control), as revealed by CBA- and RBA-banding, and by Ag-NOR and FISH-mapping techniques using telomere PNA probes. Genetic investigation of the SRY and MAMLD1 coding sequences revealed a normal SRY sequence an...
Elashry MI, Baulig N, Heimann M, Bernhardt C, Wenisch S, Arnhold S.Adipose tissue derived mesenchymal stem cells (ASCs) may be used to cure bone defects after osteogenic differentiation. In this study we tried to optimize osteogenic differentiation for equine ASCs using various concentrations of CaCl in comparison to the standard osteogenic protocol. ASCs were isolated from subcutaneous adipose tissue from mixed breed horses. The osteogenic induction protocols were (1) the standard osteogenic medium (OM) composed of dexamethasone, ascorbic acid and β-glycerol phosphate; (2) CaCl based protocol composed of 3, 5 and 7.5mM CaCl. Differentiation and proliferatio...
Ellenberger MA, Kaeberle ML, Roth JA.A lymphocyte blastogenic assay was developed to serve as an in vitro correlate of cell-mediated immunity to Rhodococcus (Corynebacterium) equi (R equi) in the equine species. Lymphocytes obtained from a group of experimental ponies showed no response in cell culture to R equi heat extract or lysozyme extract antigens. Ponies were assigned to groups for experimental inoculation. Three ponies were inoculated subcutaneously with live R equi, 3 were given live R equi by intranasal and intratracheal routes, and 4 ponies were left untreated. Lymphocytes from all inoculated ponies had a mitogenic res...
Lanovaz JL, Clayton HM, Watson LG.This study was designed to test the impact characteristics of the equine digit in vitro with the objective of providing a better understanding of the role of the digital structures in the attenuation of impact shock. Uni-axial accelerometers were mounted on cadaver digits on the distolateral hoof wall, the proximolateral hoof wall, the dorsal surface of the second phalanx, and the mid-lateral first phalanx. The hoof-mounted accelerometers were aligned with the hoof tubules while the bone-mounted accelerometers were oriented along the longitudinal axis of the bone. Each digit was mounted in a t...
Pamornsakda T, Pojprasath T, Suwimonteerabutr J, Tharasanit T.Equine epididymal sperm are known to be severely sensitive to cryopreservation, in terms of sperm quality and pregnancy rate. The objective of this study was to examine the effects of cholesterol loaded cyclodextrins (CLCs) on the quality of stallion epididymal sperm during cryopreservation. In experiment I, sperm were treated with different concentrations of CLCs: (1) 0mg (control), (2) 1.5mg, (3) 3mg, and (4) 6 mg per 120 × 10(6) sperm. The sperm viability and amount of cholesterol were determined at 15, 30 and 45 min after CLC treatment using viability markers (Ethidium homodimer-1 and Cal...
Zhou T, Yuan XF, Hou SH, Tu YB, Peng JM, Wen JX, Qiu HJ, Wu DL, Chen HC, Wang XJ, Tong GZ.In the early 1970s, the Chinese Equine Infectious Anemia Virus (EIAV) vaccine, EIAV(DLA), was developed through successive passages of a wild-type virulent virus (EIAV(L)) in donkeys in vivo and then in donkey macrophages in vitro. EIAV attenuation and cell tropism adaptation are associated with changes in both envelope and long terminal repeat (LTR). However, specific LTR changes during Chinese EIAV attenuation have not been demonstrated. In this study, we compared LTR sequences from both virulent and attenuated EIAV strains and documented the diversities of LTR sequence from in vivo and in v...
Varner DD, Blanchard TL, Love CL, Garcia MC, Kenney RM.Two experiments were conducted to examine the effects of cooling rate and storage temperature on motility parameters of stallion spermatozoa. In Experiment 1, specific cooling rates to be used in Experiment 2 were established. In Experiment 2, three ejaculates from each of two stallions were diluted to 25 x 10(6) sperm/ml with 37 degrees C nonfat dry skim milk-glucose-penicillin-streptomycin seminal extender, then assigned to one of five treatments: 1) storage at 37 degrees C, 2) storage at 25 degrees C, 3) slow cooling rate to and storage at 4 degrees C, 4) moderate cooling rate to and storag...
Sanders RE, Kearney CM, Buckley CT, Jenner F, Brama PA.To evaluate knot security for 3 knot types created in 3 commonly used 5 metric suture materials incubated in physiological and pathological fluids. Methods: In vitro mechanical study. Methods: Knotted suture loops (n = 5/group). Methods: Loops of 3 different suture materials (glycolide/lactide copolymer; polyglactin 910; polydioxanone) were created around a 20 mm rod using 3 knot types (square [SQ], surgeon's [SK], and triple knot [TK]) and were tested to failure in distraction (6 mm/min) after tying (day 0) and after being incubated for 14 and 28 days in phosphate buffered saline (PBS...
Zielinski J, Mevissen M.Testosterone hydroxylation was investigated in human, canine and equine liver microsomes and in human and canine single CYPs. The contribution of the CYP families 1, 2 and 3 was studied using chemical inhibitors. Testosterone metabolites were analyzed by HPLC. The metabolites androstenedione, 6β- and 11β-hydroxytestosterone were found in microsomes of all species, but the pattern of metabolites varied within species. Androstenedione was more prominent in the animal species, and an increase over time was seen in equines. Testosterone hydroxylation was predominantly catalyzed by the CYP3A subf...
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...
Le Vine H, Cuatrecasas P.A cytosolic, macromolecular factor required for the cholera toxin-dependent activation of pigeon erythrocyte adenylate cyclase and cholera toxin-dependent ADP-ribosylation of a membrane-bound 43,000 dalton polypeptide has been purified 1100-fold from horse erythrocyte cytosol using organic solvent precipitation and heat treatment. This factor, 13,000 daltons, does not absorb to anionic or cationic exchange resins, is sensitive to trypsin or 10% trichloroacetic acid and is not extractable by diethyl ether. Activation of adenylate cyclase by cholera toxin requires the simultaneous presence of AT...
Heinrich A, Riethmüller D, Gloger M, Schusser GF, Giese M, Ulbert S.Equine Arteritis Virus (EAV) belongs to the Arteriviridae and causes viral arteritis in horses. In an attempt to develop novel and save therapies against the infection it was tested whether EAV is susceptible to RNA interference (RNAi) in an equine in vitro system. Horse cells were transfected with chemically synthesized small interfering RNA oligonucleotides (siRNAs) and challenged with EAV. Application of these siRNAs led to a significant protection of the cells, and virus titers decreased drastically. siRNAs derived from DNA plasmids expressing small hairpin RNAs (shRNAs) were also effectiv...
Gygax D, Lischer C, Auer JA.To compare the precision of computer-assisted surgery with a conventional technique (CV) using a special guiding device for screw insertion into the distal sesamoid bone in horses. Methods: In vitro experimental study. Methods: Cadaveric forelimb specimens. Methods: Insertion of a 3.5 mm cortex screw in lag fashion along the longitudinal axis of intact (non-fractured) distal sesamoid bones was evaluated in 2 groups (8 limbs each): CV and computer-assisted surgery (CAS). For CV, the screw was inserted using a special guiding device and fluoroscopy, whereas for CAS, the screw was inserted using ...
Bailey SR, Wheeler-Jones C, Elliott J.5-hydroxytryptamine (5-HT; serotonin) is a potent vasoconstrictor of equine digital blood vessels and has been implicated in the pathogenesis of acute laminitis. Objective: The aims of this study were firstly to examine whether cells of the digital blood vessel wall exhibited an active uptake mechanism for 5-HT and to characterise its efficiency; and secondly, to study the potential inhibitory effect on this process of other amines, produced in the equine caecum. Methods: Confluent monolayers of equine digital vein endothelial cells (EDVEC) and equine digital vein smooth muscle cells (EDVSMC) ...
Guignot F, Bezard J, Palmer E.In the mare only a limited number of oocytes can be successfully collected in vivo, so that when large numbers of oocytes are needed for experimentation, ovaries harvested from slaughtered mares must be used. The resulting temperature changes and time intervals mandated by handling and transport of ovaries from the slaughterhouse to the laboratory adversely affect the rate of oocyte recovery and their quality after IVF and maturation. We chose to study the effect of temperature and time in transit of excised ovaries by evaluating rate of oocyte recovery, nuclear maturation stage reached before...
Scott SD, Kinsley R, Temperton N, Daly JM.Pseudotyped viruses (PVs) produced by co-transfecting cells with plasmids expressing lentiviral core proteins and viral envelope proteins are potentially powerful tools for studying various aspects of equine influenza virus (EIV) biology. The aim of this study was to optimise production of equine influenza PVs. Co-transfection of the HAT protease to activate the haemagglutinin (HA) yielded a higher titre PV than TMPRSS2 with the HA from A/equine/Richmond/1/2007 (H3N8), whereas for A/equine/Newmarket/79 (H3N8), both proteases resulted in equivalent titres. TMPRSS4 was ineffective with the HA of...
Squires EL.Marked changes in equine breeding technologies have occurred over the past 25 years. Although there have been numerous reviews on assisted reproduction techniques for horses, few publications include the acceptance and impact of these techniques on the horse industry. In this review, several techniques are discussed, with an emphasis on how they developed in the horse industry and altered equine reproductive medicine. Embryo transfer has become a widely used technology, allowing multiple foals to be produced per year. Embryos can be collected, cooled or frozen, and shipped to a distant facilit...
Davis JL, Papich MG, Morton AJ, Gayle J, Blikslager AT, Campbell NB.The purpose of this study was to determine the pharmacokinetics of etodolac following oral and intravenous administration to six horses. Additionally, in vitro cyclooxygenase (COX) selectivity assays were performed using equine whole blood. Using a randomized two-way crossover design, horses were administered etodolac (20 mg/kg) orally or intravenously, with a minimum 3-week washout period. Plasma samples were collected after administration for analysis using high pressure liquid chromatography with ultraviolet detection. Following intravenous administration, etodolac had a mean plasma half-li...
Walsh DM, Royal HD.Glomerular filtration rate (GFR) was measured in 12 clinically normal horses, using the standard inulin clearance method, and values were compared with values for 2 methods, using a single rapid IV injection of 99mTc-labeled diethylenetriaminepentaacetic acid (99mTc-DTPA). The first 99mTc-DTPA method used a 2-compartment model to calculate GFR blood clearance of the tracer. The second method used sequential digital gamma camera images of the kidneys to determine fractional accumulation of the total dose of the tracer in the kidneys (percentage of injected dose, gamma camera) from 0 to 10 minut...
Ahern BJ, Showalter BL, Elliott DM, Richardson DW, Getman LM.To compare the in vitro biomechanical properties of a 4.5 mm narrow locking compression plate (PIP-LCP) with 2 abaxially located transarticular screws and a 4.5 mm limited contact dynamic compression plate (LC-DCP) with 2 abaxially located transarticular screws using equine pasterns. Methods: Experimental. Paired in vitro biomechanical testing of 2 methods for stabilizing adult equine forelimb PIP joints. Methods: Adult equine forelimbs (n = 8 pairs). Methods: Each pair of PIP joints were randomly instrumented with either a PIP-LCP or LC-DCP plate axially and 2 parasagitally positioned 5.5 mm ...
Herszberg B, Mata X, Giulotto E, Decaunes P, Piras FM, Chowdhary BP, Chaffaux S, Guérin G.Glycogen debranching enzyme (AGL) is a multifunctional enzyme acting in the glycogen degradation pathway. In humans, the AGL activity deficiency causes a type III glycogen storage disease (Cori-Forbes disease). One particularity of AGL gene expression lies in the multiple alternative splicing in its 5' region. The AGL gene was localized on ECA5q14-q15. The sequence of the equine cDNA was determined to be 7.5 kb in length with an open reading frame of 4602 bp. The gene is 69 kb long and contains 35 exons. The equine AGL gene has an ubiquitous expression and presents five tissue-dependent cDNA v...
Galvão A, Skarzynski D, Ferreira-Dias G.In the present work, we investigated the role of Nodal, an embryonic morphogen from the TGFβ superfamily in corpus luteum (CL) secretory activity using cells isolated from equine CL as a model. Expression pattern of Nodal and its receptors activin receptor A type IIB (ACVR2B), activin receptor-like kinase (Alk)-7, and Alk4, as well as the Nodal physiological role, demonstrate the involvement of this pathway in functional luteolysis. Nodal and its receptors were immune localized in small and large luteal cells and endothelial cells, except ACVR2B, which was not detected in the endothelium. Nod...
Müller C, Heidenreich E, Franzreb M, Frankenfeld K.Current purification of the glycoprotein equine chorionic gonadotropin (eCG) from horse serum includes consecutive precipitation steps beginning with metaphosphoric acid pH fractionation, two ethanol precipitation steps, and dialysis followed by a numerous of fixed-bed chromatography steps up to the specific activity required. A promising procedure for a more economic purification procedure represents a simplified precipitation process requiring only onethird of the solvent, followed by the usage of magnetic ion exchange adsorbents employed together with a newly designed 'rotor-stator' type Hi...
Pepin K, Momose F, Ishida N, Nagata K.Heat shock protein 90 (Hsp90), a molecular chaperone, is ubiquitous and involved in numerous cellular processes. To contribute to the relatively small collection of vertebrate Hsp90 sequences in the gene data bank, we cloned and sequenced horse (Equus caballus) Hsp90 alpha and beta cDNAs. This enabled identification of horse-specific primers for development of a convenient PCR-based method that could monitor horse stress tolerance. We analyzed the sequence data comparatively and phylogenetically with other Hsp90 cDNA sequences, and identified vertebrate-specific and isoform-specific conserved ...
Reyner CL, Wagner B, Young JC, Ainsworth DM.To examine gene expression of selected cytokines in pulmonary mononuclear cells isolated from healthy horses and horses susceptible to recurrent airway obstruction (RAO), and to determine whether interleukin (IL)-17 and IL-23 were associated with pulmonary inflammation. Methods: 6 RAO-susceptible and 5 healthy horses. Methods: Bronchoalveolar lavage cells were retrieved from horses that were stabled and fed dusty hay for 24 hours. Lavage cells devoid of neutrophils were incubated for 24 hours with solutions of PBS, hay dust, lipopolysaccharide, or B-glucan. Gene expression of IL-17, IL-23 (p19...
Roberts MC, Clarke LL, Johnson CM.A reproducible, reversible model of colitis induced in ponies by administering castor oil (2.5 ml/kg bodyweight [bwt] per os) was characterised by abdominal pain, fever, watery diarrhoea, dehydration, hypovolaemia, toxaemia, leucopenia, decreased serum Cl, Na and K levels and metabolic acidosis. The signs were most severe between 24 and 48 h post induction, stabilisation was frequently observed after 72 h, although diarrhoea could persist beyond 96 h. Morphological and in vitro transport studies (right ventral colon) were conducted on tissues from animals destroyed at 24, 48 and 72 h. In the c...
Gambini A, Jarazo J, Karlanian F, De Stéfano A, Salamone DF.The current limitations for obtaining ovaries from slaughterhouses and the low efficiency of in vivo follicular aspiration necessitate a complete understanding of the variables that affect oocyte developmental competence in the equine. For this reason, we assessed the effect on equine oocyte meiotic competence and the subsequent in vitro cloned embryo development of 1) the time interval between ovary collection and the onset of oocyte in vitro maturation (collection-maturation interval time) and 2) the pregnancy status of the donor mares. To define the collection-maturation interval time, coll...
León G, Lomonte B, Gutiérrez JM.Early adverse reactions occur in a number of patients treated with heterologous antivenoms and have been associated with anticomplementary activity (ACA). In order to reduce the ACA of equine whole IgG antivenoms produced by caprylic acid fractionation, three different fractionation protocols were compared: (a) routine caprylic acid fractionation; (b) caprylic acid fractionation followed by beta-propiolactone treatment; and (c) caprylic acid fractionation followed by ion-exchange chromatography using a quaternary ammonium membrane. The three protocols yielded products with similar physicochemi...
Choi SJ, Anderson GB, Roser JF.In vitro production of free estrogens and estrogen conjugates by intact Day 12.5, 13.5 and 14.5 equine embryos was measured at 2-h intervals over a 24-h culture period. Production of free estrogens was higher for Day 14.5 than Day 12.5 embryos. Differences in production of conjugated estrogens were not significant, but a trend toward increased production with increased age of embryo was apparent. No trend toward increased free and conjugated estrogen production per cell was observed with age. Embryo diameter and number of cells increased with age but varied considerably within groups. The amou...
Mangal D, Uboh CE, Soma LR.Oxidative metabolites of arachidonic acid (AA) are implicated in inflammation. Thus, we evaluated cycloxygenases (COXs) and lipoxygenases (LOs) mediated metabolism of AA to eicosanoids in equine plasma. Eicosanoids were extracted from plasma by two liquid-liquid extraction (LLE) steps; first was by chloroform/isopropanol and second by methyl-tert-butyl ether. For identification and quantification of 25 eicosanoids, a highly specific, selective and sensitive stable isotope dilution liquid chromatography (LC) multiple reaction monitoring (MRM) mass spectrometric (MS) method was developed. To avo...
