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.
Messick JB, Rikihisa Y.The effects of equine antiserum, immunoglobulin G (IgG) specific for Ehrlichia risticii, and its Fab fragment on E. risticii binding to, internalization into, and proliferation in P388D1 cells were studied by immunofluorescence flow cytometry. Anti-E. risticii equine serum or IgG inhibited E. risticii at a stage beyond binding and internalization. In contrast, monovalent anti-E. risticii equine Fab fragments inhibited E. risticii binding and internalization into P388D1 cells. In the presence of control equine serum, IgG, or its Fab fragment, E. risticii cells were bound, were internalized and ...
Marquardt J, Heymer J, Heinz H, Adolf GR, Deegen E.Recombinant equine interferon-beta 1 (reqIFN-beta 1) induces an antiviral state in blood mononuclear cells (BMC) of horses. Maximal protection against replication of vesicular stomatitis virus is achieved 6 hours after treatment with IFN in vitro and in vivo. Duration of the protective effect depends on the dose of IFN in vitro and in vivo. Availability of reqIFN-beta 1 in cultures of BMC for up to 48 hours does not prolong the antiviral state. The protective effect on BMC after treatment with IFN has similar duration in vivo and in vitro. Monitoring of the effect of IFN in vivo is, thus, simp...
Ferreira-Dias G, Nequin LG, King SS.Equine endometria representative of Kenney's categories I, II, and III were incubated in vitro with phosphate buffer, Streptococcus pneumoniae, or S zooepidemicus. Endometrial tissues from mares in estrus and diestrus were first categorized according to Kenney's classification, then were tested for adherence of S pneumoniae and S zooepidemicus to the epithelia. Bacteria were not observed when the endometrial tissue was incubated with phosphate buffer or S pneumoniae. There was no statistical difference in attachment of S zooepidemicus to endometrial tissue from mares in estrus or diestrus if e...
Kydd J, Antczak DF, Allen WR, Barbis D, Butcher G, Davis W, Duffus WP, Edington N, Grünig G, Holmes MA.The First International Workshop on Equine Leucocyte Antigens was organized and convened for the purposes of identifying immunologically relevant cell surface molecules of equine leucocytes and establishing a system of nomenclature for those molecules. Participating members of the workshop represented the majority of laboratories world-wide engaged in the tasks of production and characterization of equine leucocyte and lymphocyte markers using monoclonal antibodies. The workshop confirmed the identification of several equine CD molecules described previously by individual laboratories, and in ...
Moore KH, Dunbar BS, Bousfield GR, Ward DN.Inhibin has been characterized from a number of mammals; however, it has not been extensively studied in horses. Western blot analysis was used to examine the size heterogeneity of equine inhibin alpha- and beta-subunits. The distribution of equine inhibin activity from the initial sizing column (S-200, 25 x 94 cm) indicated that the majority of equine inhibin activity was present as larger-molecular-size forms. When the large forms were analyzed by Western blot in nonreducing conditions, alpha-subunit bands were detected at 40,000 M(r), 56,000 M(r), 80,000 M(r), and 90,000 M(r); beta a reacti...
Rich FR, Glisson RR.In vitro failure modes and mechanical properties of the equine cranial cruciate ligament (CCL) were evaluated in 15 stifle joints from 10 ponies. Ponies were from 3 to 25 years of age and weighed from 122 to 208 kg. Femur-CCL-tibia specimens were mounted in 48 degrees of flexion, distracted until a 500-g tensile preload was achieved, and then tested by tensile loading to failure. Specimens failed by complete midsubstance CCL rupture (n = 9), combined tibial insertion avulsion fracture and midsubstance CCL rupture (n = 4), and combined femoral origin avulsion fracture and midsubstance CCL ruptu...
Zhang CH, Grünig G, Davis W, Antczak DF.The regulatory effects of phorbol myristate acetate (PMA) on the expression of the CD4 molecule on horse T cells were investigated. On both peripheral blood lymphocytes and thymocytes, PMA resulted in a rapid and transient down-regulation of equine CD4 expression, but had no such effect on the surface expression of equine CD5, CD8 or major histocompatibility complex (MHC) class I and class II molecules. Over 75% of the surface CD4 molecules per cell were lost after a 4 h exposure to PMA at 37 degrees C. The regulation of equine CD4 expression induced by PMA was temperature dependent and revers...
Grünig G, Barbis DP, Zhang CH, Davis WC, Lunn DP, Antczak DF.Equine peripheral blood lymphocytes (PBL) were enriched by positive selection using panning with a mixture of monoclonal antibodies against putative equine CD4 (Equine Leucocyte Antigen Workshop antibodies WS 1 and WS 72), or CD8 molecules (Workshop antibodies WS 12, WS 49, and WS 74). RNA was extracted from CD4 enriched cells (99% purity), from CD8 enriched cells (69% purity), from peripheral blood lymphocytes, and from neonatal equine thymus. RNA extracted from equine granulocytes and from equine kidney served as negative control. The RNA was electrophoresed in agarose and transferred to nyl...
McD￮ LA, Stover SM, Taylor KT, Les CM.The compressive, bending and torsional mechanical properties of osteotomized adult equine tibiae stabilized with an interlocking intramedullary nail (nail-tibia composite) were compared with those of intact tibiae to determine the clinical applicability of the the nail for repair of tibial fractures in adult horses. The mean yield load, failure load, and stiffness for the nail-tibia composites were significantly less (P < .05) than those for the intact tibiae in all loading configurations. The mean compressive yield load for the nail-tibia composites was greater than the compressive load ca...
Pirhonen A, Linnala-Kankkunen A, Mënpää PH.P1 protamines isolated from ejaculated human, stallion, bull, boar and ram spermatozoa and P2 protamines from human and stallion spermatozoa were subjected, after alkaline phosphatase treatment, to in vitro phosphorylation reactions using cAMP-dependent protein kinase (PKA) and protein kinase C (PKC). All P1 protamines were phosphorylated by PKA, whereas P2 protamines were phosphorylated only by PKC. In addition, human, stallion and boar, but not bull and ram, P1 protamines were phosphorylated by PKC. After phosphoamino acid analysis, the protamines showing positive signals for phosphoserine (...
Platt D, Bayliss MT.The effect of human recombinant interleukin-1 beta (rhIL-1 beta) on proteoglycan metabolism was investigated in cultures of full thickness explants of articular cartilage from horses 3-21 years of age. Proteoglycan synthesis was inhibited at all ages but no alteration in the hydrodynamic size or electrophoretic heterogeneity was observed in proteoglycan isolated from rhIL-1 beta stimulated cartilage. The sulphation pattern of the newly synthesised proteoglycan molecules was, however, significantly affected by the presence of the peptide regulatory factor. Interleukin-1 had no effect on the rat...
Bogumil R, Hunter CL, Maurus R, Tang HL, Lee H, Lloyd E, Brayer GD, Smith M, Mauk AG.The interaction of azide with variants of horse heart myoglobin (Mb) has been characterized by Fourier transform infrared (FTIR), electron paramagnetic resonance (EPR), and UV-VIS absorption spectroscopy and by molecular modeling calculations. Distal histidine variants (His64Thr, His64Ile, His64Lys) and charged surface variants (Val67Arg, Lys45Glu, Lys45Glu/Lys63Glu) were included in this study. All variants, with the exception of Val67Arg, have a lower azide affinity than the wild-type protein. Analysis of the temperature dependence of the FTIR spectra (277-313 K) revealed that the wild-type ...
Ascacio-Martínez JA, Barrera-Saldaña HA.A cDNA encoding horse growth hormone (ecGH) was isolated and sequenced. The coding sequence resembles a typical mammalian GH pre-protein and contains a 3' untranslated region of 101 nucleotides carrying two contiguous polyadenylation signals.
Obi T, Kabeyama A, Nishio A.Isolated equine coronary arteries responded to 5-hydroxytryptamine (5-HT) with relaxations in both endothelium-dependent and endothelium-independent mechanisms. Experiments were designed to characterize the 5-HT receptor subtype mediating these relaxations. Both 5-HT and alpha-methyl-5-HT (alpha-Me-5-HT; 5-HT2 agonist) produced concentration-dependent relaxations in equine coronary arteries precontracted with a thromboxane A2 derivative (ONO11113). The degree of the maximal relaxation induced by alpha-Me-5-HT was about one-half of that induced by 5-HT. In the coronary arteries without endothel...
Morais JF, de Freitas MC, Yamaguchi IK, dos Santos MC, da Silva WD.IgG and F(ab')2 fragments were prepared from horse plasma rich in specific antibodies against Brazilian Bothrops or Crotalus venoms. Both preparations, free of gross contamination with non-immunoglobulin proteins, were able to combine in vitro with their respective antigens, forming immune complexes at antigen excess, equivalence or antibody excess, and activating the C system, through either the classical or the alternative pathways. The IgG preparation was more effective in neutralizing the lethal factors in Bothrops or Crotalus venoms, compared with the F(ab')2 fragments. In contrast, IgG a...
Obi T, Kabeyama A, Nishio A.In coronary arterial rings isolated from horses, 10(-8)-10(-6) mol/l acetylcholine (ACh) induced concentration-dependent contractions which were potentiated by the removal of endothelium and by pretreatment with L-nitro-arginine (LNAG) or methylene blue (MB). Relatively lower concentrations of ACh (10(-14)-10(-8) mol/l) induced relaxation when the coronary rings were contracted by phenylephrine (PE). ACh-induced contractions in the coronary rings without endothelium were competitively inhibited by each muscarinic subtype selective antagonist in the following order of potency: 4-diphenylacetoxy...
O'Keefe JS, Julian A, Moriarty K, Murray A, Wilks CR.A detection system incorporating the polymerase chain reaction was compared with the use of histopathology and virus isolation to determine the presence of equid herpesvirus type 1 or equid herpesvirus type 4 in equine tissues submitted to a diagnostic laboratory. When the polymerase chain reaction was performed, these tissues had been stored for up to 3 years. Thirty-eight tissues representing 14 cases had been stored embedded in paraffin wax. Analysis of these tissues using the PCR gave predictive values of 1.0 and 0.91 for a positive and negative result respectively, and sensitivity and spe...
Ball JM, Henry NL, Montelaro RC, Newman MJ.A simple, versatile and very inexpensive procedure for cross-linking synthetic peptides to the polystyrene surfaces of micro-well assay plates for use in ELISA was developed. The method is based on the use of poly-L-lysine (PLL) as the anchor protein for synthetic peptides which were then easily and covalently linked to the PLL using glutaraldehyde. The synthetic peptides used for the study were based on the amino acid sequence of the equine infectious anemia virus (EIAV) envelope sequence and evaluated as antigens in an ELISA designed to detect antibodies in serum of EIAV-infected horses and ...
Leigh JA.A protein capable of activating bovine, equine and ovine plasminogen, but not that from human or porcine plasma, was purified from culture filtrates of Streptococcus uberis (strain 0140J). Purification was achieved by ammonium sulphate precipitation followed by molecular exclusion chromatography. The elution position of the native molecule was equivalent to a molecular mass of approximately 57 kDa. However, the molecular mass, as determined by SDS-PAGE, was 29 kDa, suggesting the existence of a dimeric structure. Purified immunoglobulin from three out of five monoclonal antibodies raised to th...
Johnson JU, Oxender WD, Berkhoff HA.Effect of estrogen (E2) and progesterone (P4) on uterine antibacterial activity and immunoglobulin concentrations in mares was studied. In 2 in vitro experiments, 6 mixed-breed mares were ovariectomized, and uterine fluid and blood serum were analyzed. Antibacterial assay methods were used to determine inhibitory effects on Streptococcus zooepidemicus of uterine fluid samples collected on days 3, 5, and 8, and serum obtained on day 8 of treatment. Single radial immunodiffusion methods were used to quantify amounts of IgA and IgG in uterine fluid and serum on days 3, 5, 8, and 14 of treatment. ...
Ko WH, O'Dowd JJ, Pediani JD, Bovell DL, Elder HY, Jenkinson DM, Wilson SM.Changes in intracellular free calcium concentration ([Ca2+]i) were monitored in a cell line that was derived from the equine sweat gland epithelium. ATP and closely related compounds could increase [Ca2+]i with a rank order of potency of UTP > or = ATP > ADP >> AMP = adenosine = alpha,beta-methylene-ATP. The responses to ATP and to UTP were initiated by the release of calcium from an internal store and subsequently sustained by calcium influx. The rise in [Ca2+]i thus seems to be mediated by P2U receptors that are coupled to phosphoinositidase C. Some desensitisation of this respon...
Allen MJ, Jemmerson R, Nall BT.Refolding of surface epitopes on horse cytochrome c has been measured by monoclonal antibody binding. Two antibodies were used to probe re-formation of native-like surface structure: one antibody (2B5) binds to native cytochrome c near a type II turn (residue 44) while the other (5F8) binds to a different epitope on the opposite face of the protein near the amino terminus of an alpha-helical segment (residue 60). The results show that within the first approximately 100 ms of refolding all of the unfolded protein collapses to native-like folding intermediates that contain both antibody binding ...
Vandergrifft EV, Swiderski CE, Horohov DW.We have cloned equine interleukin 4 (IL-4) cDNA using the polymerase chain reaction (PCR) and primers based on the human IL-4 sequence. The cDNA was amplified from mitogen-stimulated equine peripheral blood mononuclear cells (PBMC). The cloned PCR product shares extensive homology ith IL-4 sequences from other species.
Hough SR, Parks JE.Platelet-activating factor (PAF) acetylhydrolase, which inactivates PAF, has been detected in human and bovine seminal plasma and may represent a mechanism for regulating sperm-derived PAF. This study was designed to characterize further PAF acetylhydrolase in seminal plasma from domestic animal species. Sperm-free seminal plasma from the bull, stallion, rabbit, and rooster was assayed for acetylhydrolase activity based on the release of [3H]acetate from PAF. As reported previously for bull seminal plasma, activity in stallion, rabbit, and rooster seminal plasma was linear with both time and p...
Kim CH, Casey JW.The distribution and replicative status of equine infectious anemia virus (EIAV) DNA in the tissues of a well-characterized inapparent carrier horse were established by using the PCR technique. The EIAV pol region could be amplified in all of the tissues tested, including the cerebellum and periventricular tissue, at concentrations approximately 10(5)-fold less than in the same tissue from an acutely infected horse. Further analysis of the EIAV genome, with primer pairs diagnostic for sequential stages of reverse transcription, suggests that EIAV DNA in the brain, liver, and lymph nodes was in...
Sjostrom L, al-Abdulla IH, Rawat S, Smith DC, Landon J.Commercial antivenoms produced in horses were compared with monospecific antivenoms raised in sheep against Crotalus durissus terrificus, Crotalus atrox, Crotalus adamanteus, Micrurus fulvius fulvius, Naja naja, Naja kaouthia, Echis ocellatus, Vipera lebetina deserti, Vipera berus berus and Vipera ammodytes ammodytes venom. Antibodies raised by immunizing sheep with C. d. terrificus venom were more effective than their equine counterparts in preventing lethal toxicity in mice (ED50), in inhibiting the venom's pharmacological effects (haemolysis, platelet aggregation and coagulation), and in ne...
Holman PJ, Chieves L, Frerichs WM, Olson D, Wagner GG.Babesia equi was continuously cultured through 90 passages in an enriched chemically defined basal medium (HL-1) supplemented with 20% fetal bovine serum and serum replacement factors, including lipid-rich bovine serum albumin, bovine insulin, and human transferrin. Cryopreservation and subsequent recovery of B. equi were easily achieved. Inoculation of a splenectomized and an intact horse with cultured infected erythrocytes resulted in parasitemias and B. equi in vitro reisolation from both animals. In vitro forms of the parasite resembled in vivo forms. After establishment, parasitemias of 1...
Obi T, Kabeyama A, Nishio A.In coronary arterial rings isolated from horse, norepinephrine (NE)(10(-7) - 10(-5) M) induced concentration-dependent contractions which were not influenced by endothelial denudation. Prazosin (alpha 1-antagonist) inhibited the contraction, but yohimbine (alpha 2-antagonist) did not, and propranolol (beta-antagonist) enhanced the contraction. Pretreatment with phentolamine (10(-5) M) (alpha-antagonist) converted the contraction induced by NE to relaxation in coronary rings precontracted with ONO11113 (thromboxane A2 derivative). The relaxation was not influenced by removal of the endothelium,...
Lawrence GL, Gilkerson J, Love DN, Sabine M, Whalley JM.Sets of primers were designed which enabled specific amplification of homologous regions of the glycoprotein C and gene 76 genetic loci of equine herpesviruses 1 and 4 (EHV-1 and EHV-4). The resultant virus-specific polymerase chain reaction (PCR) products arising from each loci could be discriminated easily on the basis of size on an agarose gel, allowing rapid differentiation of the two equine herpesviruses. Specificity of the amplifications were confirmed by Southern hybridization and restriction endonuclease digestion. The PCR test was applied to nasal swab samples from weanling foals and ...
Baxter G.Endotoxin given in vivo has been shown to inhibit endothelial dependent relaxation, and augment adrenergic (norepinephrine) contractions in isolated palmar digital arteries of horses. A study, using tumor necrosis factor (TNF) in vitro, was performed to determine the possible cause of these vascular alterations. Palmar digital arteries were surgically removed from 6 horses under general anesthesia, cut into 4-mm vascular rings (4 segments/horse), suspended in tissue baths, and attached to force displacement transducers for measurement of vascular tension. Four in vitro treatment groups were ev...
Rahnama S, Vathsangam N, Spence R, Anderson ST, de Laat MA, Bailey S, Sillence MN.Prolonged hyperinsulinemia is thought to be the cause of equine endocrinopathic laminitis, a common and crippling disease of the foot, for which there are no pharmacologic treatments other than pain relief. It has been suggested that insulin causes its effects on the lamellae by activating IGF-1 receptors (IGF-1R), as insulin receptors (InsR) are scarce in this tissue, whereas IGF-1R are abundant and become downregulated after prolonged insulin infusion. As a first step toward confirming this mechanism and beginning to develop a therapeutic anti-IGF-1R monoclonal antibody (mAb) for horses, it ...
Salinas C, Espinosa G, Morales N, Henríquez C, Morán G, Gajardo G, Burgos RA, Uberti B.Obesity and insulin dysregulation (ID) are increasingly prevalent conditions in equid populations worldwide. Immune impairment is well described in humans with metabolic dysfunction and is reported but still incompletely understood in horses. This study evaluated the effect of acute induced transient hyperglycemia on apoptosis, phagocytosis and oxidative burst activity of peripheral blood polymorphonuclear cells (PMN) of lean and obese adult horses with or without insulin dysregulation. Seventeen adult horses were allocated into three groups based on their body condition score (BCS) and metabo...
Hunter BG, Duesterdieck-Zellmer KF, Larson MK.Anecdotal accounts of tiludronate administration via intravenous regional limb perfusion (IVRLP) exist despite a lack of information regarding safety for synovial structures in the perfused area. The objective of this study was to determine whether tiludronate concentrations in synovial structures after IVRLP with low dose (0.5 mg, LDT) or high dose (50 mg, HDT) tiludronate remain below a value demonstrated in vitro to be safe for articular cartilage (<19,000 ng/ml), and to determine effects of tiludronate on synovial fluid cytology variables compared to saline perfused control limbs. Using...
Sirois J, Kimmich TL, Fortune JE.Estrous cycles in mares have several unique characteristics, including the presence of a long period of estrus and the absence of a typical LH surge. Like follicles of other species, equine preovulatory follicles are characterized by their ability to secrete large amounts of 17 beta-estradiol, but it is not clear which follicular cell type is responsible for estradiol synthesis in mares. To better understand the relative roles of theca interna and granulosa cells in follicular steroidogenesis, presumptive ovulatory follicles were obtained from mares during early estrus (first or second day of ...
Nunamaker DM, Butterweck DM, Black J.In vitro local fatigue testing of the third metacarpal bone from Thoroughbred and Standardbred racehorses was performed, using fully reversed cyclic bending. Strain number of cycle data were accumulated and indicated that significant difference did not exist between the 2 breeds. It was concluded that the marked difference in the incidence of fatigue failure in the third metacarpal bone (bucked shins, dorsal metacarpal disease) seen in Thoroughbreds and Standardbreds was not related to any differences between the 2 breeds in mechanical properties of the bone.
Truax RE, Powell MD, Montelaro RC, Issel CJ, Newman MJ.A rapid and simple technique for the cryopreservation and recovery of equine mononuclear cells was developed. Buffy-coat leukocytes were frozen in autologous plasma containing 10% DMSO and mononuclear cells were recovered by gradient sedimentation using a standard Ficoll-Hypaque purification procedure. The total numbers of mononuclear cells recovered from cryopreserved samples were 94%-82% of those recovered from fresh blood samples. The functional capabilities of the mononuclear cells from cryopreserved buffy coat preparations were compared with those of mononuclear cells from fresh samples b...
Yu M, Wang Z, Robinson NE, Derksen FJ.The effects of furosemide on the responses of equine trachealis muscle with and without epithelium to electrical field stimulation (EFS) and exogenous acetylcholine (ACh) were investigated in organ baths. Tissues were pretreated with guanethidine and the parameters used for EFS were those previously demonstrated to activate postganglionic cholinergic neurons. In tissues with intact epithelium, furosemide (100 microM) shifted the frequency-response curve to the right. In the preparations without epithelium, furosemide did not affect the response to EFS. Neither in epithelium-on nor in epitheliu...
Dintinger T, Gaillard JL, Zwain I, Bouhamidi R, Silberzahn P.The results of the measurement of 19-nortestosterone in the testiscular artery and vein of the stallion, the very low levels of this steroid in the peripheral blood of geldings and the similar patterns of increase in the peripheral levels of 19-nortestosterone and testosterone after hCG stimulation, show that 19-nortestosterone, like testosterone, is essentially synthesized in the testis. This testicular origin was confirmed by the ability of testicular tissue to synthesize 19-norandrogens from [4-14C]androgens in vitro. 19-Nortestosterone was 50% conjugated in the peripheral blood and almost ...
Hassan Kadi R, Baghdadi AM.Lactic acid bacteria are microorganisms that can be present in meat, milk and fermented products, as well as fermented drinks and vegetables, and they inhibit the growth of pathogenic and decaying microorganisms. Our aim was isolating, screening, characterization and estimation of bacteriocin/s of Lactic acid bacteria isolated from Horses milk in Jeddah city, Saudi Arabia. That have antagonistic activity against a wide range of pathogenic bacteria. On MRS agar medium 16 LAB isolates were examined using the agar well diffusion method against the pathogenic bacteria (Gram-positive bacteria: S. a...
Grünig G, Witschi U, Winder C, Hermann M, von Fellenberg R.Supernatants of equine respiratory secretions enhanced the migration of equine neutrophils into the lower compartments of Boyden chambers. Checkerboard analysis revealed that the neutrophil migration promoting activity (NMPA) of secretion specimens was in great part caused by chemokinesis, irrespective of the neutrophil score of the specimen. The NMPA of respiratory secretions was correlated neither with the neutrophil score of the secretion specimen nor with the severity of the chronic pulmonary disease. Respiratory secretions collected while horses were kept under low dust or under dusty hou...
Evans JW, Roser JF, Mikuckis GM.Human chorionic gonadotrophin (hCG) can be used to study horse luteinizing hormone (LH) receptors in stallion testicular tissue. hCG was more stable than horse LH during radioiodination when compared by their abilities to bind to testicular receptor sites. During incubation, neither hormone lost binding activity at 4 degrees C. Horse LH lost binding activity during incubation at 25 degrees C and both hormones lost binding activity at 37 degrees C. Both hormones bound to the same receptor sites which are specific for the hormones. The receptor sites were not degraded when incubated at 4 degrees...
Jacob RJ.Preliminary experiments have revealed that several laboratory and wild-type strains of the equine herpesvirus (EHV) triad were temperature-sensitive for growth when assayed at 39 degrees C. The efficiencies of plating (EOP) observed were 10(-2) for both EHV 1 and 2, and 1 X 10(-6) for EHV 3. The EOPs were determined by plaque assays which compared titrations at 34 degrees C and 39 degrees C on equine fetal dermal fibroblast cells. Growth yield experiments, assayed at 34 degrees C, reflected those EOP's, but did not indicate any difference in yields when infected cultures were incubated at 34 d...
Ruggeri E, DeLuca KF, Galli C, Lazzari G, DeLuca JG, Stokes JE, Carnevale EM.Confocal microscopy was used to image stages of equine zygote development, at timed intervals, after intracytoplasmic sperm injection (ICSI) of oocytes that were matured in vivo or in vitro. After fixation for 4, 6, 8, 12, or 16 h after ICSI, zygotes were incubated with α/β tubulin antibodies and human anticentromere antibody (CREST/ACA), washed, incubated in secondary antibodies, conjugated to either Alexa 488 or Alexa 647, and incubated with 561-Phalloidin and Hoechst 33258. An Olympus IX81 spinning disk confocal microscope was used for imaging. Data were analyzed using χ 2 and Fisher's e...
Benbarek H, Deby-Dupont G, Deby C, Serteyn D.The capacity of the two cytokines TNF-alpha and IL-1beta to directly stimulate the oxidative activity of polymorphonuclear neutrophils remains debated. The purpose of this study was to verify if a direct stimulation of equine neutrophils by TNF-alpha and IL-1beta was possible. Equine neutrophils were isolated from blood by discontinuous density gradient centrifugation. The cell viability after isolation was >98%. The neutrophils were used at 1.25 x 10(6) cells by assay, immediately after isolation. The oxidative activity of neutrophils was measured by luminol- or lucigenin-enhanced chemilumine...
MacEachern KE, Smith GL, Nolan AM.Hypoxaemia accompanies dorsal recumbency in the horse and frequently complicates general anaesthesia. The physiology associated with this phenomenon is poorly understood. One possible cause of poor tolerance to dorsal recumbency is an absent or reduced response to hypoxic pulmonary vasoconstriction (HPV). This study compared the HPV response in isolated pulmonary artery vessels from equivalent regions of equine and bovine lung. Methods: Equine and bovine, in vitro study. Methods: Equine and bovine pulmonary arteries were removed from the lungs of euthanased horses and cattle. Measurements of i...
Madewell BR, Munn RJ.Abnormal irregularly branched and anastomosing tubules within cisternae of endoplasmic reticulum were observed by transmission electron microscopy in tumour cells comprising connective tissue neoplasms (sarcoids) from three horses and a mule. These tubuloreticular inclusions were also observed in cultured tumour cells from one of these horses examined, but were not detected in fibroblasts (fibrocytes), epidermis, or vascular endothelial cells in skin biopsy specimens from five clinically healthy horses, nor in one additional equine connective tissue neoplasm.
Ledbetter EC, Irby NL, Schaefer DM.To describe in vivo corneal confocal microscopy of horses with microscopic corneal foreign bodies and to correlate findings with clinical, cytological, and histopathologic evaluations of clinical cases and foreign body morphologies observed in vitro with the confocal microscope. Methods: Five horses with microscopic corneal foreign bodies. Methods: Sedated and anesthetized horses were examined with a modified Heidelberg Retina Tomograph II and Rostock Cornea Module. Confocal microscopy images were compared with images from cytologic and histopathologic corneal samples. To establish microscopic...
Swardson CJ, Lichtenstein DL, Wang S, Montelaro RC, Kociba GJ.To characterize infection of bone marrow-derived macrophages (BMDM) with equine infectious anemia virus (EIAV) by determining virus production, effects on viability, and induction of cytokines. Methods: BMDM obtained from bone marrow of 6 clinically normal adult horses. Methods: BMDM were infected with EIAV at a multiplicity of infection of 8. Cell viability, percentage of cells with detectable viral protein, reverse transcriptase activity, and concentrations of infective virus (focus-forming units/ml), interleukin 6, and tumor necrosis factor-alpha were measured in culture supernatant samples...
Coyne CP, Kelly AB, Hornof WJ, O'Brien TR, Philp MS, Lamb JF.A method is presented for the in vitro isolation and radiolabeling of equine platelets with the isotope indium 111 (111In: half-life = 2.8 days, gamma = 173 keV, 89%; 247 keV, 94%). The technique described involves complexing 111In with the lipid-soluble chelating agent, 2-mercaptopyridine-N-oxide (merc), in an aqueous medium. 111In-merc platelet-labeling efficiencies in autologous plasma pretreated with or without ferric citrate reagent were 82 +/- 7% and 24 +/- 12%, respectively. Mean intravascular survivals of 111In-merc-radiolabeled platelets in 8 healthy horses according to simple linear,...
Veeraragavan K, Singh K, Wachter E, Hochstrasser K.A trypsin inhibitor was isolated from pregnant mares' urine by adsorption on bentonite and elution with aqueous pyridine followed by batch DEAE-cellulose treatment and column chromatography. Final purification to an electrophoretically homogenous glycoprotein was achieved by gel permeation chromatography. This equine urinary trypsin inhibitor (E-UTI) is acid- and heat-stable, has a molecular weight of 22 to 23 kDa, an isoelectric point of 4.55, forms a 1:1 molar complex with trypsin and has serine as its N-terminal amino acid. The N-terminal amino acid sequence of this protein is almost identi...
Dumoulin M, Martens A, Van den Abeele AM, Boyen F, Oosterlinck M, Wilderjans H, Gasthuys F, Haesebrouck F, Pille F.This study evaluated the Etest for direct antimicrobial susceptibility testing (AST) of bacteria from equine synovial specimens, incubated in BACTEC enrichment bottles. Ninety-four culture-positive broths were inoculated onto agar to directly determine the minimum inhibitory concentrations (MICs) of 13 antimicrobials, using the Etest (direct Etest). Results were compared with those obtained with the agar dilution reference method, the standard Etest, and the disc diffusion method, after subculture and standardisation of the inoculum. For categorical comparison of AST results, MICs were transla...
Gray AW, Davies ME, Jeffcott LB.We report on novel methods to isolate osteoclasts (OC s) and generate osteoclast-like cells (OCL s) from the bone and bone marrow of the equine femur. OC s were successfully isolated from bone scrapings taken from the endosteal surface of the femurs of three horses. OCL s were generated from bone marrow cells taken from the same animals. The validity of using the formation of OCL s as a method for studying OC differentiation and activity was confirmed by the similar characteristics of these two cells. In particular, they both were multinuclear, expressed the enzyme tartrate resistant acid phos...
Smith HL, Berglund AK, Robertson JB, Schnabel LV, McMullen RJ, Gilger BC, Oh A.The objective of the study was to determine the effect of gentamicin on CD3+ T-lymphocyte proliferation and cell viability using an in vitro cell culture model as a means of investigating the mechanism of action of low-dose intravitreal gentamicin injection. Methods: Three adult horses with no evidence of ophthalmic or systemic disease. Methods: Peripheral blood lymphocytes were treated with gentamicin at concentrations 37.5 μg/mL, 112.5 μg/mL, 187 μg/mL, 375 μg/mL, or 750 μg/mL then stimulated to proliferate with concanavalin A (ConA). 4',6-diamidino-2-phenylindole (DAPI) and c...
Robertson TP, Moore JN, Noschka E, Lewis TH, Lewis SJ, Peroni JF.To determine the effects of the protein kinase C (PKC) inhibitor, Ro-31-8220, on agonist-induced constriction of laminar arteries and veins obtained from horses. Methods: Laminar arteries and veins obtained from 8 adult mixed-breed horses. Methods: Laminar arteries and veins were isolated and mounted on small vessel myographs for the measurement of isometric tension. Concentration-response curves were then obtained for the vasoconstrictor agonists phenylephrine, 5-hydroxytryptamine, prostaglandin F(2), and endothelin-1. All responses were measured with or without the addition of Ro-31-8220 (3 ...
Barrey E, Valette JP, Jouglin M, Picard B, Geay Y, Robelin J.The content in slow and fast myosin heavy chains (MHC 1 and MHC 2) of 5 equine muscles was determined using an enzyme-linked immunosorbent assay. The results obtained with this immunoenzymatic method were compared with complementary techniques: electrophoresis and immunohistochemistry. Slices of masseter, diaphragm, tensor faciae latae, semitendinosus and cutaneus trunci were obtained from a 12-year-old saddle horse after slaughter. Muscular proteins were specifically extracted to be analysed by ELISA. The technique used 2 complimentary monoclonal antibodies (MAb). MAb 1 was prepared from a hu...
Art T, Lekeux P.In order to study the in vitro mechanical properties of the equine trachea submitted to the compressive pressures observed in vivo, the pressure-volume relationship was determined in intra- and extra-thoracic tracheal segments taken post mortem from 29 healthy horses (one to 15 years old; 352 to 651 kg). At the same time, the cross-sectional lumen area (X-SA) at the mid-point of the segment was measured using a slit-lamp transillumination and photographic measurement by endoscopy. The tracheal specific compliance (Cs) as well as the relative changes in X-SA and in the sagittal and transverse d...
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...
Bauer SM, Santschi EM, Fialkowski J, Clayton MK, Proctor RA.To quantify the adhesion of Staphylococcus aureus to 4 equine bone surfaces passivated in a balanced polyionic solution (Plasmalyte) or hyperimmune equine plasma (Polymune plasma). Methods: In vitro comparative study. Methods: Third metacarpal bone (MC3) surface explants from 9 equine cadavers. Methods: Approximately 1 cm(2) sections of periosteum were removed from MC3 and stapled to sterile stainless steel screens. Three bone surface explants were cut using a surgical saw to present 1 cm(2) surfaces of subperiosteal bone, cut cortical bone, or endosteum. Duplicate explants of each surface wer...
Ingvast-Larsson C.A comparison between the relaxant effects of clenbuterol and theophylline on horse tracheal smooth muscle has been made in vitro. Rat tracheal smooth muscle was also investigated as a reference. The tracheal preparations were initially contracted with carbachol since the smooth muscle did not spontaneously develop tone. The response of the carbachol-contracted preparations to theophylline was the same in the two species. The response to clenbuterol varied. In only five out of eleven horses were the tracheal smooth muscles sensitive to clenbuterol (mean pD2 = 7.92 M). In the remaining six horse...
McClure SR, Watkins JP, Ashman RB.Breaking strength (torque at failure) of equine third metacarpal bones, with transfixation pins placed parallel in the frontal plane and 30 degrees divergent from the frontal plane, was determined in vitro. Two transfixation pins were placed through the distal metaphysis, using a jig designed to drill the holes in the assigned configuration. Paired metacarpal bones II through IV from 12 horses were tested in torsion. The torsional moment of the force applied through the transfixation pins at failure was compared for each limb. Metacarpal bones with divergent pins were significantly (P = 0.030)...
