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.
The Journal of physiologyJune 15, 1996
Volume 493 ( Pt 3), Issue Pt 3 885-894 doi: 10.1113/jphysiol.1996.sp021431
Ko WH, Chan HC, Chew SB, Wong PY.1. The ionic mechanism involved in Ca(2+)-stimulated electrolyte transport in cultured equine sweat gland epithelial cells was studied using the short-circuit current (ISC) technique. 2. Microscopy revealed that the cultured cells grown on Millipore filters formed polarized monolayers with tight junctions. Monolayers exhibited a mean transepithelial resistance of 333.9 +/- 40.4 omega cm2. 3. Ca(2+)-mobilizing agents, A23187 (1 microM) or thapsigargin (0.01-1 microM), stimulated ISC while forskolin exerted little effect on the ISC. 4. Replacement of external Cl- by gluconate significantly reduc...
Barteri M, Gaudiano MC, Santucci R.The influence of glycerol on the structural properties of Fe(III)-horse heart myoglobin has been investigated by absorbance, CD and SR-SAXS spectroscopy. The results obtained indicate that both the tertiary and the secondary (alpha-helix) conformations of the protein are influenced by glycerol; in particular, an increase of approx. 8% in helical content was observed. Further, analysis of both the acid- and guanidine-induced denaturation transitions points to a glycerol-induced decreased stability of the tertiary structure; conversely, the alpha-helix conformation is found to be stabilized by t...
Taddei N, Stefani M, Magherini F, Chiti F, Modesti A, Raugei G, Ramponi G.Asn41, Thr42, and Thr46 are invariant residues in both muscle and erythrocyte acylphosphatases isolated so far. Horse muscle acylphosphatase solution structure suggests their close spatial relationship to Arg23, the main substrate binding site. The catalytic and structural role of such residues, as well as their influence on muscle acylphosphatase stability, was investigated by preparing several gene mutants (Thr42Ala, Thr46Ala, Asn41Ala, Asn41Ser, and Asn41Gln) by oligonucleotide-directed mutagenesis. The mutated genes were cloned and expressed in Escherichia coli, and the mutant enzymes were...
Benton HP, Cheng TC, MacDonald MH.To determine the response of equine articular cartilage cells to heat and calcium stresses. Methods: Analysis of newly synthesized, [35S]methionine-labeled proteins after treatment of isolated primary equine chondrocytes. Methods: Primary cultures of equine articular chondrocytes were incubated at temperatures ranging from 37 to 42 C for heat stress experiments or incubated in the presence or absence of the intracellular calcium pump inhibitor, thapsigargin, for calcium stress experiments. Patterns of new protein synthesis were determined by incubating with [35S]methionine followed by separati...
Wilhelm KM, Graham JK, Squires EL.Computer-assisted motion analyses (CASA) and flow cytometry were used to evaluate stallion spermatozoa prior to and after cryopreservation. Spermatozoa were pretreated with: (1) Hepes-buffered medium (SHB); (2) phosphatidylserine (PS) liposomes; or (3) liposomes composed of both PS and cholesterol (PSCH) prior to dilution in either SHB or skim milk-egg yolk extender (SMEY). After cooling to 5 degrees C in SHB, PS and PSCH pretreatment (23%). Spermatozoal motion parameters were higher for spermatozoa diluted in SMEY than dilution in SHB. In Experiment 2, motion parameters were compared for sper...
Honess NA, Gibson JS, Cossins AR.The effects of oxygen tension (PO2) upon the K influx pathways of equine red cells have been studied using 86Rb+ as congener for K. Equilibration of cells in 100% nitrogen led to a low and Cl-independent K flux. Change to an atmosphere of 100% air led to a rapid sixfold increase in K flux. The oxygen-activated flux was entirely Cl dependent and was maintained for up to 3 h. Oxygenation-evoked activation was dependent upon PO2 over the physiological range with little effect up to 70% saturation of haemoglobin with oxygen but significant effects between 70 and 100%. K flux at low PO2 was unaffec...
Barlough JE, Madigan JE, DeRock E, Bigornia L.A nested polymerase chain reaction for detecting Ehrlichia equi in horses and ticks (Ixodes pacificus) was developed. A major second-round PCR product of 928 bp could be readily visualized in ethidium bromide-stained agarose minigels. An internal probe was used to verify the identity of the amplified product by non-radioactive (digoxigenin-based) Southern blotting; additional confirmation was provided by DNA sequence analysis. A dilution study testing the sensitivity of the PCR indicated that DNA derived from 3 infected neutrophils was sufficient to generate a PCR signal. The specificity of t...
Malone ED, Brown DR, Trent AM, Turner TA.To characterize the response of equine jejunal smooth muscle to adrenergic and cholinergic mediators. Methods: Evaluation of myogenic responses, using an in vitro model. Methods: Intestinal tissues were obtained from horses without gastrointestinal tract disorders or systemic disease. Methods: Baseline myogenic tone and amplitude and frequency of contraction were determined for suspended jejunal muscle strips. The level of adrenergic and cholinergic regulation was assessed, using atropine and adrenoceptor antagonists. The response of the muscles to norepinephrine was characterized, using adren...
Hochi S, Kozawa M, Fujimoto T, Hondo E, Yamada J, Oguri N.The study was designed to examine the suitability of immature horse oocytes for vitrification. Immature oocytes derived from slaughtered horse ovaries were transferred to a vitrification solution (EFS; 40% ethylene glycol, 18% Ficoll, and 0.3 M sucrose in modified phosphate-buffered saline) directly (Groups 1 and 4) or were first exposed to 20% ethylene glycol solution for 10 min (Groups 2 and 5) or 20 min (Groups 3 and 6). Oocytes were handled at 20 degrees C (Groups 1, 2, and 3) or 30 degrees C (Groups 4, 5, and 6). After vitrification and warming, their viability was assessed by maturation ...
Miles CA.Ultrasound velocity, attenuation, and backscattering were measured in vitro in samples of equine digital flexor tendon sandwiched between plane, parallel rexolite buffer rods. The buffer rods were coupled to transmitting and receiving transducers (nominally 10 MHz) mounted in-line and facing one another on the jaws of a digital caliper. Six superficial digital flexor (SDF) tendons and six deep digital flexor (DDF) tendons were measured in three orthogonal directions: along the long axis of the tendon (D), and across the tendon in the dorsal-volar (C), and lateral (L) directions. Substantial an...
Koivunen AL, Maisi P, Fang W, Sandholm M.To clarify the role of proteolytic enzymes in the pathogenesis of chronic obstructive pulmonary disease (COPD) in horses, and to investigate new possibilities for treatment of this disease by interfering in the proteolytic process. Methods: Effect of antiproteolytic activity of selected protease inhibitors on tracheal aspirates was studied in vitro, and the inhibition profiles were compared with those of purified proteases. Methods: Respiratory tract secretions with antiproteolytic activity from 9 horses with COPD. Methods: Caseinolytic agar-diffusion assay. Results: The protease-inhibition pr...
Klohnen A, Wilson DG, Hendrickson DA, Cooley AJ, MacWilliams PS.To evaluate the bactericidal properties of chlorhexidine diacetate (CHD) after potentiation with EDTA and Tris buffer (EDTA-Tris), and to find a potentiated CHD concentration that would achieve 90 to 100% killing for all bacteria tested. Methods: 6 adult ponies. Methods: Serial dilutions of CHD, CHD in EDTA-Tris and EDTA-Tris alone were evaluated for bactericidal activity against Staphylococcus aureus, Escherichia coli, and Streptococcus zooepidemicus. The tarsocrural joints of 6 ponies were lavaged with either 1 L phosphate-buffered saline solution (control) or 1 L of 0.0005% CHD in EDTA-Tris...
Lehnert M, de Giuli R, Kunke K, Emerson S, Dalton WS, Salmon SE.The purpose of this study was to evaluate to what extent the ability of various chemosensitisers (CS) to reverse P-glycoprotein-associated multidrug resistance (MDR) is reduced when tested in physiological serum protein concentrations. Utilising drug sensitivity and accumulation assays, the CS were tested in medium containing 10% fetal bovine serum and in 100% horse or human serum. Two RPMI 8226 human myeloma sublines were used which express different levels of P-glycoprotein. The CS were tested at various concentrations, including clinically achievable blood levels. When using the CS at high ...
Christensen P, Whitfield CH, Parkinson TJ.The ability of the glycosaminoglycan, heparin, and the calcium ionophore, A23187, to induce acrosome reaction in equine spermatozoa was assessed using semen from 3 warmblood stallions of known high fertility. After collection of semen, the spermatozoa were washed and incubated in vitro with heparin or A23187. Incubation periods were 0, 4, 6 or 8 h with 0, 1, 10 or 100 microg/ml heparin or 0, 10, 30 or 60 min with 0, 0.01, 0.1, 1 or 10 microM A23187, respectively. Acrosome reactions were determined by staining the spermatozoa with naphthol yellow S plus erythrosin B, and sperm viability was ass...
The Journal of physiologyApril 15, 1996
Volume 492 ( Pt 2), Issue Pt 2 347-358 doi: 10.1113/jphysiol.1996.sp021313
Fleischmann BK, Wang YX, Pring M, Kotlikoff MI.1. The relationship between voltage-dependent calcium channel current (I(Ca)) and cytosolic free calcium concentration ([Ca2+]i) was studied in fura-2 AM-loaded equine tracheal myocytes at 35 degrees C and 1.8 mM Ca2+ using the nystatin patch clamp method. The average cytosolic calcium buffering constant was 77 +/- 3 (n = 14), and the endogenous calcium buffering constant component is likely to be between 15 and 50. 2. I(Ca) did not evoke significant calcium-induced calcium release (CICR) since (i)[Ca2+]i scaled with the integrated I(Ca) over the full voltage range of evoked calcium currents, ...
Seidel GE.Principles and procedures for cryopreservation of equine embryos are described. Embryos less than 250 microM in diameter can be cryopreserved successfully if glycerol is used as the cryoprotectant. Cooling is takes place in such a way that most of the water leaves the cells before intracellular ice forms, and glycerol is removed after thawing without undue osmotic swelling of cells. Vitrification procedures also show promise for small embryos. Satisfactory procedures for cryopreserving embryos of more than 250 microM in diameter are not yet available.
Graham JK.Methodologies to capacitate bovine spermatozoa, induce the acrosome reaction, and fertilize bovine oocytes in vitro have been established. The capability to do the same with stallion spermatozoa, however, is not available. Several different methods have been used to capacitate stallion spermatozoa with variable results. More basic research needs to be done to establish in vitro conditions necessary to capacitate and induce an acrosome reaction in stallion spermatozoa. Although much progress can be expected in this area, it is unlikely that the general practitioner will use these technologies i...
Loredo GA, MacDonald MH, Benton HP.To investigate whether recombinant human bone morphogenetic protein-2 (rhBMP-2) regulates glycosaminoglycan (GAG) synthesis and release from equine articular cartilage explant cultures. Methods: Equine articular cartilage explants were maintained in vitro for 7 days in the presence of 0 (control), 1, 10, or 100 ng of rhBMP-2/ml. Synthesis and release of GAG were assessed as measures of production and degradation of the extracellular matrix, respectively. Methods: 6 horses (age range, 2 to 25 years old) without clinically detectable musculoskeletal abnormalities. Methods: Rate of synthesis of G...
Dobrinski I, Suarez SS, Ball BA.Interaction of spermatozoa with oviductal epithelial cells (OEC) in the oviductal isthmus prolongs the life span of spermatozoa. The hypothesis that the interaction of equine spermatozoa with OEC affects their intracellular calcium concentration ([Ca2+]i) was tested in a sperm-OEC coculture model. Changes in [Ca2+]i in spermatozoa loaded with the fluorescent calcium indicator indo-1 acetoxymethylester (AM) were determined for spermatozoa attached to OEC or to Matrigel, as well as for free-swimming spermatozoa incubated without oviductal epithelium. [Ca2+]i was determined before incubation and ...
Vanderwall DK.Tremendous progress has been made in the development of assisted reproductive techniques that may enhance the reproductive efficiency of the horse. However, techniques that involve the manipulation of oocytes and/or embryos may themselves be detrimental to embryo viability and subsequent development. Therefore, an objective method of assessing viability of embryos before and/or after oocyte/embryo manipulation is desirable. At this time, morphologic evaluation is the most widely used method of determining the viability of equine embryos. Although morphologic assessment of embryo quality will n...
Graham JK.This article describes some of the basic methodology for conducting stallion semen evaluations. Not all of these assays will likely be conducted on every semen sample collected. Routine evaluations should include determination of semen volume, sperm concentration, and an estimation of the percentage of progressively motile sperm, at a minimum. Other assays can be used if a seminal problem is discerned at the beginning of the breeding season or after a stallion has recovered from an illness or injury. Additionally, laboratory assays are particularly important to conduct on cryopreserved spermat...
Rodriguez H, Bustos Obregon E.The fertilizing ability of stallion semen was analyzed using fresh and frozen samples, obtained before (June-July) or during (October-November) the breeding season. Thirty ejaculates obtained from 4 stallions were used. The analysis comprises routine seminogram; ATP concentration (Comhaire et al., 1983); subjective and objective motility and sperm velocity (Makler, 1980). Freezing was done following the technique of Martin et al. (1979). Sperm velocity, ATP content and objective motility in ejaculates of subjective motility >50% show values of 14.0 + or - 0.84 mu m s(-1); 4.8 + or - 2.7x10(...
Squires EL.Equine oocytes obtained either by transvaginal ultrasound-guided follicular aspiration or from slaughterhouse ovaries can be matured in vitro. This generally requires culture in TCM-199 containing serum and hormones for 30 to 36 hours. With this protocol, approximately 50% to 60% of the oocytes are at metaphase-II at the end of the culture period. At least some of these oocytes appear viable based on production of fertilized eggs either through in vitro fertilization or fertilization in vivo of a recipient mare. The success of producing equine embryos in vitro is still extremely low. More than...
Gregory CR, Latimer KS, Niagro FD, Campagnoli RP, Steffens WL, Ritchie BW.Eastern equine encephalomyelitis (F.EE) virus was detected in infected formalin-fixed horse and emu tissues and in infected chicken embryo fibroblasts. Results of in situ hybridization using a digoxigenin-labeled 40-base DNA probe complementary to a conserved region of the EEE virus RNA compared favorably with results of both virus isolation and serum neutralization tests. This technique may be useful for diagnosis of EEE virus infection in various animal species, especially when fresh tissues are not available for analysis, and also will provide a means for studying the involvement of alphavi...
Bryant CE, Clarke KW.Spirals of endothelially denuded equine saphenous vein were used to study the pre- and post-junctional effects of medetomidine in vitro. The pD2 values were calculated for noradrenaline (6.7 +/- 0.1), phenylephrine (5.6 +/- 0.1), BHT 920 (6.2 +/- 0.2) and UK 14304 (5.7 +/- 0.2). Medetomidine produced a biphasic response, with a pD(2)1 of 8.2 +/- 0.1 and a pD(2)2 of 5.7 +/- 0.1 in the equine saphenous vein (n = 6). Prazosin (10(-7) M) significantly shifted the second phase of the medetomidine concentration-response curve to the right (pD(2)1 was 8.1 +/- 0.2 and pD(2)2 was 5.0 +/- 0.2, P < 0....
Riemersma DJ, van den Bogert AJ, Jansen MO, Schamhardt HC.Strains in the tendons of the m. flexor digitalis superficialis (superficial digital flexor, SDFT) and m. flexor digitalis profundus (deep digital flexor, DDFT) tendons, the accessory ligament of the deep digital flexor muscle (inferior check ligament, ICL) and the m. interosseus medius (suspensory ligament, SL) of 5 ponies were recorded at the walk and trot using mercury-in-silastic strain gauges (MISS), on a hard surface (brick pavement) and on sand. The horses were shod with normal, flat shoes. On pavement, strain in the SDFT, DDFT and SL increased significantly from the walk (2.19%, 1.15% ...
Jarvis GE, Evans RJ.Endotoxin has previously been shown to induce platelet aggregation in equine heparinised whole blood. This study aimed to determine whether platelet-activating factor or products of cyclo-oxygenase metabolism (thromboxane A2 or prostaglandins) were important in mediating the response of platelets to endotoxin. The effects of the following drugs on endotoxin-induced aggregation were investigated: aspirin, flunixin meglumine and carprofen (non-steroidal anti-inflammatory drugs); CV-3988 and WEB2086 (platelet-activating factor receptor antagonists); quinacrine (phospholipase A2 inhibitor). The ef...
Sun Y, MacLean AR, Aitken JD, Brown SM.Equine herpesvirus type 1 (EHV-1) gene 71 encodes a heavily O-glycosylated 192 kDa protein with no identified herpesvirus homologue. Isolation of a deletion mutant in gene 71 (ED71) demonstrated that its protein product is not essential in vitro. To investigate the role of the gene 71 protein in the virus life cycle, ED71 has been characterized in vitro in terms of cellular adsorption, penetration, egress and transmission compared to wild-type and revertant virus. ED71 virions adsorbed to cells less efficiently than wild-type and revertant virus with a consequential effect on virus penetration...
Alape-Giron A, Stiles BG, Gutierrez JM.An ELISA based, non-radioactive acetylcholine receptor (AchR) binding assay was used to detect the alpha-neurotoxins present in Micrurus nigrocinctus nigrocinctus venom. Sera from horses hyperimmunized against M. nigrocinctus venom contain antibodies which inhibit the binding of M. n. nigrocinctus alpha-neurotoxins to AchR and reverse the binding of toxins already complexed with the receptor. This result supports the importance of using antivenom therapeutically in M. n. nigrocinctus envenomations even after the onset of neurological symptoms. M. nigrocinctus antivenoms cross-reacted in an ELI...
Umair M, Beitsma M, de Ruijter-Villani M, Deelen C, Herrera C, Stout TAE, Claes A.The cryotolerance of equine blastocysts larger than 300 μm can be improved by aspirating blastocoele fluid prior to vitrification; however, it is not known whether blastocoele aspiration also enables successful slow-freezing. The aim of this study was therefore to determine whether slow-freezing of expanded equine embryos following blastocoele collapse was more or less damaging than vitrification. Grade 1 blastocysts recovered on day 7 or 8 after ovulation were measured (>300-550 μm, n = 14 and > 550 μm, n = 19) and blastocoele fluid was aspirated prior to slow-freezing in 10...
Ericson KK, Yang TJ.During our studies of cytostatic cytokines in the mixed leukocyte culture, we found that horse serum in the medium control contained a tumor cell growth-inhibitory factor. The fraction isolated by molecular sieving and ion exchange chromatography inhibited the growth and DNA synthesis of the primary culture and passaged cell line of the canine transmissible venereal sarcoma, murine T (L5178Y) and B (P3-X63-Ag8.653) lymphoid tumor cells, murine mammary tumor cells (RIII), bovine lymphoid tumor cells (BL3), and the nontransformed cell line of baby hamster kidney cells. Nontransformed cell lines ...
Foster AP, Cunningham FM, Andrews MJ, Lees P.The effects of the selective platelet activating factor (PAF) receptor antagonist WEB 2170 on PAF-induced responses in equine cells and tissues have been examined and compared with those of WEB 2086. In initial experiments WEB 2170 was shown to inhibit in vitro platelet aggregation in a dose-dependent, competitive reversible manner (pA2 = 7.21). Co-administration of the antagonists with either PAF or histamine also inhibited PAF, but not histamine, induced wheal formation and PAF-induced neutrophil accumulation in vivo in equine skin. Intravenous (i.v.) administration of both drugs at a dose o...
BMC research notesNovember 8, 2012
Volume 5 626 doi: 10.1186/1756-0500-5-626
Boudko SP, Ishikawa Y, Lerch TF, Nix J, Chapman MS, Bächinger HP.Hyperelastosis cutis is an inherited autosomal recessive connective tissue disorder. Affected horses are characterized by hyperextensible skin, scarring, and severe lesions along the back. The disorder is caused by a mutation in cyclophilin B. Results: The crystal structures of both wild-type and mutated (Gly6->Arg) horse cyclophilin B are presented. The mutation neither affects the overall fold of the enzyme nor impairs the catalytic site structure. Instead, it locally rearranges the flexible N-terminal end of the polypeptide chain and also makes it more rigid. Conclusions: Interactions of th...
Wu H, Chen S, Liu M, Xu X, Ji X, Gao K, Tian A, Ke Z, Zhang J, Zhao B, Zhang S.B-cell survival depends on signals induced by B-cell activating factor (BAFF) that binds to the BAFF receptor (BAFF-R). Herein, a BAFF-R homolog was identified in a horse (Equus caballus). The horse BAFF-R gene, located on chromosome 28, spans 1444 base pairs and encodes a 183-amino acid protein. The protein is structurally conserved, in which the DxL motif plays an important role in binding to BAFF. Furthermore, the horse BAFF-R extracellular domain was expressed and purified, which specifically bound to His6-sBAFF and had the capability of blocking the function of His6-sBAFF in vitro. Finall...
Silva GB, De La Côrte FD, Brass KE, Palma HE, Gallio M, Cantarelli C, Bertolin K, Krause A, Wergutz J, Fontanari Krause LM, Antoniazzi AQ.Chondrocyte health is altered when exposed to local anesthetics, raising concerns as to the long-term effects of local anesthetics intra-articularly for diagnosis and analgesia. To investigate the drug with the lowest toxic potential, the effect of ropivacaine and mepivacaine on chondrocytes was evaluated. Articular cartilage from normal metacarpophalangeal joints of five equine cadaver specimens was used to establish chondrocyte cultures. Following seven days, chondrocytes were exposed to standard culture medium (DMEM), ropivacaine 7.5 mg/ml (ROP7.5), ropivacaine 10 mg/ml (ROP10), mepivacaine...
Ferreira-Dias G, Costa AS, Mateus L, Korzekwa A, Redmer DA, Skarzynski DJ.Soon after ovulation, the corpus luteum (CL) starts secreting progesterone (P(4)), a hormone necessary for implantation. The aim of the study was to evaluate whether P(4) exerts an autocrine/paracrine action on luteal angiogenic activity and P(4), prostaglandin E(2) (PGE(2)) and NO production in the mare. Corpora hemorrhagica (CH) and mid-luteal phase CL (MCL) were cultured with (i) no hormone (Control); (ii) P(4); (iii) a P(4) precursor - pregnenolone; or (iv) a P(4) antagonist - onapristone [10(-4) M;10(-5) M; all steroids]. NO production decreased in MCL, with respect to CH, when treated wi...
Connelly C, Norton NA, Hurley DJ, Hart KA, Meichner K, Gogal RM.Peripheral blood is commonly sampled to assess the health status of human and veterinary patients. Venous blood collection is a minimally invasive procedure, and in the horse, the common collection site is the jugular vein. Post blood collection, sample processing for leukocyte enrichment can vary by research laboratory with the potential to yield different effects on the enriched cells and their function. The focus of the present study was to compare a common blood dilution-leukocyte enrichment technique using a Histopaque gradient medium (His) to a modified leukocyte buffy coat syringe-lymph...
Mari G, Bucci D, Love CC, Mislei B, Rizzato G, Giaretta E, Merlo B, Spinaci M.The aim of this study was to compare the effect of presorting centrifugation (cushioned [CC] or single-layer colloid [SLC]), with simple dilution (SD), on the quality of sex-sorted stallion semen before and after sorting and after freezing and thawing. Four ejaculates from each of two fertile stallions were collected 1 week apart and evaluated for percent total sperm motility (TM), percent viable acrosome-intact sperm (VAI), and DNA quality (percentage of DNA fragmentation index). Freezing caused, independently from CC and SLC treatments, a significant decrease of TM (P < 0.05) and VAI (...
Stewart F, Maher JK.The number of genes encoding the common alpha-subunit and hormone-specific beta-subunits of the equine gonadotrophins (FSH, LH and CG) were investigated in the horse (Equus caballus), donkey (E. asinus) and 2 horse x donkey hybrids (the mule and hinny). The Southern technique, involving restriction enzyme digestion, blotting and DNA hybridization to 32P-labelled DNA probes was used to estimate the copy number for each gene and to assess the extent to which equids resemble primates, the only other animals that secrete a CG during pregnancy. These methods indicated that, in common with mammals, ...
Ghassab S, Dulin J, Bertone AL.To compare clotting efficiency of platelet-rich plasma (PRP) and concentrated platelet-poor plasma (cPPP) to citrated whole blood after activation by autologous thrombin, bovine thrombin, or calcium chloride (CaCl2 ). Methods: Experimental study. Methods: Healthy adult horses (n = 6). Methods: PRP and cPPP were prepared by commercial devices. Using thromboelastography, clotting variables were compared after activation of citrated autologous blood, PRP, and cPPP by autologous thrombin, bovine thrombin, or CaCl2 , respectively. Results: PRP had the greatest clot strength and quickest clot rate, ...
Galuppo LD, Stover SM, Jensen DG, Willits NH.To compare drilling, tapping, and screw-insertion torque, force, and time for the 4.5-mm AO and 6.5-mm Acutrak Plus (AP) bone screws, and to compare the mechanical shear strength and stiffness of a simulated complete lateral condylar fracture of the equine third metacarpal bone (MC3) stabilized with either an AO or AP screw. Methods: In vitro biomechanical assessment of screw-insertion variables, and shear failure tests of a bone-screw-stabilized simulated lateral condylar fracture. Methods: Eight pairs of cadaveric equine MC3s Methods: Metacarpi were placed in a fixture and centered on a biax...
Buntenkötter K, Osmers M, Schenk I, Schänzer W, Machnik M, Düe M, Kietzmann M.Although acetylsalicylic acid (ASA) is not frequently used as a therapeutic agent in horses, its metabolite SA is of special interest in equestrianism since it is a natural component of many plants used as horse feed. This led to the establishment of thresholds by horse sport organizations for SA in urine and plasma. The aim of this study was to investigate plasma and urine concentrations of salicylic acid (SA) after oral administration of three different single dosages (12.5 mg/kg, 25 mg/kg and 50 mg/kg) of acetylsalicylic acid (ASA) to eight horses in a cross-over designed study. Results:...
Liu IK, Cheung AT, Walsh EM, Miller ME, Lindenberg PM.The functional competence of peripheral blood and uterine-derived polymorphonuclear leukocytes (PMN) from 12 mares were analyzed for chemotactic responsiveness using a chemotactic chamber (filter) assay and for deformability by cell elastimetry analysis. Peripheral blood PMN obtained from control mares and from 8 mares experimentally inoculated via the uterus with 1 x 10(9) Streptococcus zooepidemicus had similar normal chemotactic responsiveness and were highly deformable before and at 12 hours after inoculation. Uterine PMN obtained 12 hours after uterine inoculation with S zooepidemicus fro...
Matczuk AK, Chodaczek G, Ugorski M.Equine arteritis virus (EAV) is a prototype member of the Arterivirus family, comprising important pathogens of domestic animals. Minor glycoproteins of Arteriviruses are responsible for virus entry and cellular tropism. The experimental methods for studying minor Arterivirus proteins are limited because of the lack of antibodies and nested open reading frames (ORFs). In this study, we generated recombinant EAV with separated ORFs 3 and 4, and Gp3 carrying HA-tag (Gp3-HA). The recombinant viruses were stable on passaging and replicated in titers similar to the wild-type EAV. Gp3-HA was incorpo...
Wattle OS.To determine whether there is a change in the expression of cytokeratins in the epidermal cells of the non-weight-bearing parts of the limb in horses with acute laminitis and thus determine whether the morphologic changes that develop in the periople and chestnut (torus carpeus) of horses early in acute laminitis are caused by inhibition of keratinocyte differentiation. Methods: 8 horses with acute laminitis. Methods: Tissue specimens were obtained from the chestnuts of all 8 horses and from the stratum externum of the hoof wall of 3 horses. Tissue specimens were obtained within 48 hours of th...
Watson ED.Incubation of blood neutrophils with uterine flushings collected from ovariectomised mares treated with oestradiol, stimulated migration under agarose, whereas flushings from mares treated with progesterone or oily vehicle, inhibited migration. After intra-uterine infusion of bacteria, however, flushings from oestradiol-treated and vehicle-treated mares inhibited migration, whereas progesterone treatment stimulated migration. Migration of uterine-derived neutrophils under agarose was less than that of blood neutrophils and was not influenced by treatment with ovarian steroids. Uterine suscepti...
Hinrichs K.In vitro embryo production is not yet successful in the horse, largely due to low rates of fertilization in vitro. However, methods to produce embryos from isolated oocytes have been developed. Oocytes may be recovered from living mares by aspiration of the dominant preovulatory follicle by trans-abdominal puncture, and from both preovulatory and immature follicles by trans-vaginal ultrasound-guided puncture. Transfer of in vivo-matured oocytes to the oviducts of bred recipient mares has resulted in good pregnancy rates (75-85%). Little work has been done on transfer of horse oocytes matured i...
Casey PJ, Robertson KR, Liu IK, Espinoza SB, Drobnis EZ.Subfertility in stallions is common, and methodologies are needed to increase the fertility in these animals. In other species, removal of the dead sperm from semen increases the quality and fertility of semen. With horse semen we evaluated 48 combinations of column separation techniques using micro-spin chromatography columns. The greatest improvement in motility was observed with glass wool, whereas glass beads exhibited the greatest recovery of motile sperm. Although centrifugation time did not influence recovery rate or percent motility, a column length of 2 cm was superior for recovery of...
Redding WR, Booth LC.Equine fibroblasts and Staphylococcus aureus were exposed for 30 minutes to six dilutions of chlorhexidine gluconate, a chlorous acid-chlorine dioxide irrigation solution, a chlorous acid-chlorine dioxide disinfectant, and phosphate buffered saline controls. Cell viability was determined by trypsinizing the cells, staining them with trypan blue, and counting cells that did not take the stain. All fibroblasts were killed when exposed to 1.0% and 0.5% chlorhexidine. The survival rate of fibroblasts increased linearly with decreasing concentrations of chlorhexidine gluconate, with a peak survival...
Noble P, Lumay G, Coninx M, Collin B, Magnée A, Lecomte-Beckers J, Denoix JM, Serteyn D.An equine fetlock joint pendulum test was studied and the influence of post mortem time and intra-articular lipid solvent on the viscous frictional response examined. Fresh equine digits (group 1, n=6 controls; group 2, n=6 lipid solvent) were mounted on a pendulum tribometer. Assuming that pendular joint damping could be modelled by a harmonic oscillator fluid damping (HOFD), damping time (τ), viscous damping coefficient (c) and friction coefficient (μ) were monitored for 5h under experimental conditions (400N; 20°C). In all experiments, pendular joint damping was found to follow an expone...
Junco M, Iglesias LE, Sagüés F, Zegbi S, Guerrero I, Saumell CA.In horses, the nematodes of the Strongylidae family are the most important due to their prevalence and pathogenicity. Sanitary plans include parasite control based on chemical anthelmintics. Among these, the benzimidazole compounds have been used since the 1960s to control the nematode Strongylus vulgaris. Its inappropriate use resulted in the development of resistance in parasites with a shorter biological cycle, such as the small strongyles. Currently, the genera that make up this group show widespread resistance to all chemical treatments available in veterinary medicine, except for macrocy...
Hart KA, Sherlock CE, Davern AJ, Lewis TH, Robertson TP.N-butylscopolammonium bromide (NBB) is an anticholinergic agent used to treat spasmodic colic in horses. Intestinal smooth muscle spasm also occurs in horses with intraluminal intestinal obstructions, such as ileal impactions. The antispasmodic effects of NBB may be useful in managing ileal impactions, but the effects of NBB on equine ileal smooth muscle are unknown. Objective: To investigate the effects of NBB on spontaneous and induced contraction of the equine ileum in an ex vivo model. Methods: Ex vivo biomechanical study assessing contractile properties in isolate equine ileal smooth musc...
Richer CL, Romagnano A.Both dynamic G-banding and cell synchronization produced by bromodeoxyuridine (BrdU), were applied to equine chromosomes. BrdU incorporated during the first half of the S-phase is taken up into the R-bands that are early replicating. These bands, which have incorporated BrdU, cannot contract as usual and remain elongated; only the other regions of the chromosome, i.e., the G-bands, contract normally and are sharply defined. BrdU also can be used for cell synchronization. The addition of BrdU in a high concentration, 15 hours before harvest, and its removal 11 hours later, has two effects: init...
Apostolakos JM, Lazaro L, Williams RJ.This article is a critical analysis of a study, "Minimally Manipulated Bone Marrow Concentrate Compared with Microfracture Treatment of Full-Thickness Chondral Defects: A One-Year Study in an Equine Model," by Chu et al. (. 100(2):138-146, 2018). The investigation compared two interventions in the management of full-thickness chondral defects in an equine model: autologous bone marrow concentrate without concomitant microfracture treatment versus microfracture treatment alone. This review analyzes the methodology and results of their investigation and examines how their findings may influence ...
Sato F, Yamashita S, Kugo T, Hasegawa T, Mitsui I, Kijima-Suda I.To determine the full-length complementary DNA (cDNA) sequence of equine erythropoietin (EPO) and to develop region-specific antibodies to differentiate equine EPO (eEPO) and human EPO (hEPO). Methods: RNA and lysate extracted from renal tissues of an adult Thoroughbred. Methods: Full-length cDNA was determined by use of a reverse transcriptase-polymerase chain reaction assay and a rapid amplification of cDNA ends method. The deduced amino acid sequence was compared with sequences of EPO reported for other species. Furthermore, 4 synthetic peptides were designed in 2 distinctive parts of the e...
Yang Q, Lopez MJ.The equine hoof dermal-epidermal interface requires progenitor cells with distinct characteristics. This study was designed to provide accurate ultrastructural depictions of progenitor cells isolated from inflamed tissue and normal tissue before and after cryopreservation and following selection of cells expressing both keratin (K) 14 (ectodermal) and cluster of differentiation (CD) 105 (mesodermal). Passage 3 cell ultrastructure was assessed following 2D culture and after 3D culture on decellularized hoof tissue scaffolds. Outcome measures included light, transmission electron, and scanning e...
Zweygarth E, Ahmed JS, Rehbein G, Voigt WP.The capacity of equine peripheral blood lymphocytes (PBL) to proliferate in the presence of Babesia equi-transformed lymphoblastoid stimulator cells was tested in an autologous as well as in an allogenic one way mixed lymphocyte reaction (MLR). It was found that both autologous and allogeneic responder lymphocytes incorporated high amounts of 3H-thymidine. The incorporation of 3H-thymidine was lower in MLR using as stimulator cells lymphocytes from which the cell line had previously been established, than when using parasitized culture cells as stimulator. Proliferation of PBL was achieved onl...
Coruzzi G, Poli E, Montanari C, Bertaccini G.1. Uterine motility was studied in vitro in the myometrial tissue obtained from pregnant and non-pregnant mares. 2. The spontaneous contractions of the preparations were not modified by tetrodotoxin, by anticholinergics, antiadrenergics, histamine H1 and H2 blockers, antiserotoninergic and opioid antagonists; but disappeared in Ca2+ and Na+ free medium. 3. beta 2-adrenergic stimulants like salbutamol and hexoprenaline and the calcium channel blockers nifedipine and verapamil were effective inhibitors of the amplitude of phasic contractions (ID50S for salbutamol and nifedipine were 7.7 nM and 1...
Miller L, Woodward EM, Campos JR, Squires EL, Troedsson M.The objectives of this study were to (i) verify localization of SP22 on fresh, cooled, and frozen/thawed equine spermatozoa and to (ii) determine SP22 mRNA and protein expression in equine testicular and epididymal tissues. Immunocytochemistry and Western blots were performed on the spermatozoa samples. Northern blots and Western blots were performed on the tissue samples. The immunocytochemistry revealed the presence of SP22 in all samples tested. The fresh spermatozoa stained predominantly over the equatorial segment as did the samples cooled for 1 and 2 days. The samples cooled for 3 days, ...
