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.
Malone ED, Kannan MS, Brown DR.To determine whether substance P (SP) functions as a neurotransmitter in equine jejunum. Methods: Samples of jejunum obtained from horses that did not have lesions in the gastrointestinal tract. Methods: Jejunal smooth muscle strips, oriented in the plane of the circular or longitudinal muscle, were suspended isometrically in muscle baths. Neurotransmitter release was induced by electrical field stimulation (EFS) delivered at 2 intensities (30 and 70 V) and various frequencies on muscle strips that were maintained at low tension or were under contraction. A neurokinin-1 receptor blocker (CP-96...
Morisset S, McClure SR, Hillberry BM, Fisher KE.To compare the in vitro holding power and associated microstructural damage of 2 large-animal centrally threaded positive-profile transfixation pins in the diaphysis of the equine third metacarpal bone. Methods: 25 pairs of adult equine cadaver metacarpal bones. Methods: Centrally threaded positive-profile transfixation pins of 2 different designs (ie, self-drilling, self-tapping [SDST] vs nonself-drilling, nonself-tapping [NDNT] transfixation pins) were inserted into the middiaphysis of adult equine metacarpal bones. Temperature of the hardware was measured during each step of insertion with ...
Greenaway EC, Cunningham FM, Goode NT.Phorbol esters, which activate protein kinase C (PKC), stimulate equine eosinophil superoxide production and adherence. After showing that superoxide production could be inhibited by the nonselective PKC inhibitors, staurosporine and bisindolymaleimide I, the PKC isotypes in equine eosinophils were characterized, because evidence suggests that individual isotypes may play distinct roles in regulating eosinophil function. Western blots demonstrated that equine eosinophils expressed PKC alpha, beta, delta, epsilon, iota, and zeta. However, unlike the equine neutrophil, the majority of the PKC wa...
Byrne KM, Vierck J, Dodson MV.Equine satellite cells are responsible for muscle healing and regeneration in the mature horse. We describe the in vitro cell culture conditions required for clonal populations of equine satellite cells to undergo both proliferation and differentiation. Our hypothesis is that these in vitro conditions model regeneration of muscle and can be used to evaluate potential therapeutics. In this study, 2 areas of satellite cell response were tested: proliferation of clones induced by growth factors, and fusion induced by culture conditions. Equine satellite cell clones showed differences in their res...
Livesey JH, Evans MJ, Mulligan R, Donald RA.To further elucidate the interaction of CRH, AVP and cortisol in the control of ACTH secretion, we used an in vitro perifusion model with dispersed equine anterior pituitary cells. To approximate the in vivo milieu in the horse, CRH was perifused continuously (at 0, 2 and 20 pmol/L) and 5-min pulses of AVP (0, 1, 3 and 10 nmol/L) were given every 30 min in the presence of 0 or 100 nmol/L cortisol. Total (baseline + incremental) ACTH secretion increased as both the CRH (p<0.001) and the AVP (p<0.001) concentration increased and interaction between CRH and AVP was significant (p=0.042). Co...
Prickett TCR , Inder WJ, Evans MJ, Donald RA.The acute-phase cytokine interleukin-1 (IL-1) is known to activate the hypothalamic pituitary adrenal axis, primarily via corticotropin releasing hormone (CRH). The aim of this study was to determine whether IL-1beta could directly stimulate ACTH secretion from perifused equine anterior pituitary cells, and whether CRH pre-incubation affected corticotroph responsiveness. Isolated equine anterior pituitary cells were pre-incubated with media containing 10 nM CRH or vehicle for 20 hours before being loaded onto columns and perifused with 0.02 nM CRH and 100 nM cortisol. Columns were given a 5-mi...
Sabeur K, Vo AT, Ball BA.Angiotensin II is a hormone with a wide array of physiological effects that exerts its effect via interaction with two major subtypes of receptor. The results of this study show that angiotensin II (from 1 to 100 nmol l(-1)) initiates acrosomal exocytosis in equine spermatozoa that have undergone capacitation in vitro in a TALP-TEST (Tyrode's albumin lactate pyruvate; 188.7 mmol TES l(-1), 84.8 mmol Tris l(-1)) buffer with cAMP. The acrosome reaction and sperm viability were assessed with fluorescein isothiocyanate-Pisum sativum agglutinin (FITC-PSA) and Hoechst 33258, respectively. The initia...
Moncayo AC, Edman JD, Turell MJ.The effect of eastern equine encephalomyelitis (EEE) virus on the survivorship of Aedes albopictus (Skuse), Anopheles quadrimaculatus Say, and Coquillettidia perturbans (Walker) was determined experimentally. Female mosquitoes were allowed to feed on EEE viremic chicks, and survival rates were compared for infected and uninfected mosquitoes. Additionally, the survival of female Cq. perturbans and An. quadrimaculatus intrathoracically (i.t.) inoculated with EEE was compared with controls receiving diluent inoculations. Infection with EEE significantly reduced survival in Cq. perturbans compared...
von Rechenberg B, Guenther H, McIlwraith CW, Leutenegger C, Frisbie DD, Akens MK, Auer JA.To define the release of nitric oxide (NO), prostaglandin E2 (PGE2), and the neutral metalloproteinases (NMPs) in horses with subchondral cystic lesions (SCL) and to study bone resorption triggered by conditioned media of fibrous tissue of SCL in vitro. Methods: Equine explant cultures of fibrous tissue of SCL, and synovial membrane and articular cartilage of normal horses and horses affected with moderate and severe osteoarthritis were performed. NO, PGE2, and NMP concentrations of media samples were measured, and osteoclast formation and activation was studied in vitro. Methods: Experiment 1...
Brinsko SP, Crockett EC, Squires EL.The objective of this study was to determine if centrifugation and partial removal of seminal plasma would improve spermatozoal motility in semen from stallions whose whole ejaculates have poor tolerance to cooling and storage. Stallions were divided into two groups (n = 5/group) based on the ability of their extended semen to maintain spermatozoal motility after cooling and storage. Group 1 stallions ("good coolers") produced semen in which progressive spermatozoal motility after 24 h of cooling and storage was reduced by < or = 30% of progressive motility prior to storage. Group 2 stallions ...
Hurtt AE, Landim-Alvarenga F, Seidel GE, Squires EL.Studies were conducted to compare viability of immature and mature equine and bovine oocytes vitrified in ethylene glycol. Ficoll using open-pulled straws. Oocytes from slaughterhouse ovaries (N=50/group) with >2 layers of compact cumulus cells were vitrified immediately after collection (immature groups) or vitrified after 36 to 40 (equine) or 22 to 24 (bovine) h of maturation (mature groups). Immature oocytes were matured after thawing. Before vitrification, oocytes were exposed to TCM-199 + 10 FCS + 2.5 M ethylene glycol + 18% Ficoll + 0.5 M sucrose (EFS) for 30 sec and then to 5 M ethylene...
Ball BA, Gravance CG, Medina V, Baumber J, Liu IK.To characterize the activity of catalase in equine semen. Methods: 15 stallions of known and unknown reproductive history. Methods: Seminal plasma was collected from raw equine semen by centrifugation, and samples of seminal plasma were frozen prior to assay for catalase activity. Tissue samples (n = 3 stallions) from the bulbourethral gland, prostate gland, vesicular gland, and testis were homogenized, and cauda epididymal fluid was collected for determination of catalase activity. Catalase activity was determined as an enzyme kinetic assay by the disappearance of H2O2 as measured by ultravio...
Raulo SM, Sorsa TA, Maisi PS.To determine whether samples of tracheal epithelial lining fluid (TELF) obtained from horses have elastinolytic activity characteristic of metalloproteinases, to compare elastinolytic activity in TELF obtained from healthy horses and horses with chronic obstructive pulmonary disease (COPD), and to determine whether chemically modified tetracycline-3 (CMT-3) inhibits elastinolytic activity in TELF ANIMALS: 10 horses with COPD and 10 healthy control horses. Methods: Zymography and fluorometry were used to measure elastinolytic activity, and EDTA was used to inhibit elastinolytic activity and ver...
Worster AA, Nixon AJ, Brower-Toland BD, Williams J.To determine the morphologic and phenotypic effects of transforming growth factor beta1 (TGFbeta1) on cultured equine mesenchymal stem cells (MSC) and articular chondrocytes. Methods: Bone marrow aspirates and articular cartilage samples from a 2-year-old and two 8-month-old horses. Methods: After initial isolation and culture, MSC and chondrocytes were cultured in Ham's F-12 medium supplemented with TGF-beta1 at a concentration of 0, 1, 5, or 10 ng/ml. Medium was exchanged on day 2, and cells were harvested on day 4. Medium was assayed for proteoglycan (PG) content. Total RNA was isolated fro...
Martínez AC, Rivera L, Raposo R, García-Sacristán A, Benedito S.The effect of histamine (10(-9)-10(-3) M) on horse penile dorsal artery was evaluated. Precontracted vessels showed a biphasic response (relaxation-contraction) to histamine, while at basal tone, histamine only induced a contractile effect. The H1 receptor agonist, 2-pyridylethylamine (PEA) (10(-9)-10(-3) M), induced concentration-dependent relaxation in precontracted rings and provoked vasoconstriction at basal tone. Mepyramine (10(-9)-10(8) M), an H1 receptor antagonist, competitively antagonized the relaxant response to histamine (pA2 = 9.7) and PEA (pA2 = 9.2). At basal tone, mepyramine (1...
Gravance CG, Garner DL, Baumber J, Ball BA.The fluorescent carbocyanine dye, JC-1, labels mitochondria with high membrane potential orange and mitochondria with low membrane potential green. Evaluation of mitochondrial membrane potential with JC-1 has been used in a variety of cell types, including bull spermatozoa; however, JC-1 staining has not yet been reported for equine spermatozoa. The aim of this study was to apply JC-1 staining and assessment by flow cytometry or a fluorescence microplate reader for evaluation of mitochondrial function of equine spermatozoa. Six ejaculates from four stallions were collected and centrifuged thro...
Dunowska M, Holloway SA, Wilks CR, Meers J.Seventeen New Zealand isolates of equine herpesvirus 5 (EHV-5) were compared to the Australian prototype strain. PCR primers were designed to amplify EHV-5 glycoprotein B (gB) gene, and Restriction Fragment Length Polymorphism (RFLP) was used to detect differences between cloned PCR products. EHV-5 isolates from different horses showed a high degree of heterogeneity. However, EHV-5 isolates from individual horses remained homogeneous when examined over a period of time or isolated from different sites. A single EHV-5 gB RFLP profile was detected in isolates from each individual horse but one. ...
John HA, Marrs J, Laffling AJ.In the autonomic nervous system (ANS) of equine grass sickness (GS) cases, some neurones show abnormal changes while neighbouring neurones are unaffected. To test whether noradrenergic neurones showed variable susceptibility to the GS toxin in culture, clonally-derived populations isolated from the same fetal thoracic sympathetic chain ganglion were challenged with plasma from GS cases previously shown to induce ANS damage when injected into normal horses. During the early stages of exposure to toxic plasma, cells within a clonal population showed variable susceptibility ranging from no obviou...
East LM, Trumble TN, Steyn PF, Savage CJ, Dickinson CE, Traub-Dargatz JL.The application of 99mTc-HMPAO labeled white blood cells to support the diagnosis of right dorsal ulcerative colitis was studied in two horses with a history and clinical signs consistent with phenylbutazone toxicity. These images were compared to a reference horse unaffected by right dorsal ulcerative colitis. Blood was collected aseptically in heparinized syringes from the patients for in vitro white blood cell (WBC) radiolabeling. The buffy coat was separated out and radiolabeled with 99mTc-HMPAO. The radiolabeled blood was re-injected i.v. and four images of the right and left side of the ...
Madigan JE, Pusterla N, Johnson E, Chae JS, Pusterla JB, Derock E, Lawler SP.Ehrlichia risticii, the agent of Potomac horse fever (PHF), has been recently detected in trematode stages found in snail secretions and in aquatic insects. Based on these findings, horses could conceivably be exposed to E. risticii by skin penetration with infected cercariae, by ingestion of infected cercariae in water or via metacercariae in a second intermediate host, such as an aquatic insect. In order to test this hypothesis, horses were challenged with infectious snail secretions and aquatic insects collected from a PHF endemic region in northern California. Two horses stood with their f...
Fehr JE, Trotter GW, Oxford JT, Hart DA.To determine relative amounts of mRNA expression of aggrecan, type-II collagen, matrix metalloproteinase (MMP) 1, and MMP3 in articular cartilage and synovial membrane samples from healthy equine joints and joints with osteoarthritis (OA) and to compare results of Northern blot hybridization with results of a reverse transcriptase-polymerase chain reaction (RT-PCR) assay. Methods: Articular cartilage samples from 8 pairs of joints (1 with OA and 1 healthy) from 6 horses and synovial membrane samples from 6 pairs of joints from 5 horses. Methods: RNA was extracted from samples by use of a modif...
Weiss DJ, Evanson OA, Green BT, Brown DR.To study the in vitro effects of cecal contents incubated with corn starch on colonic permeability in horses. Methods: 4 healthy adult ponies. Methods: Mucosal specimens were obtained from the right ventral colon and mounted in Ussing chambers. Changes in short circuit current, conductance, and large-molecule permeability in response to addition of cecal contents and cecal contents incubated with corn starch were evaluated for 120 minutes. Results: Incubation of cecal contents with corn starch for 8 hours resulted in a decrease in cecal content pH and an increase in lactic acid concentration. ...
Benbarek H, Grülke S, Deby-Dupont G, Deby C, Mathy-Hartert M, Caudron I, Dessy-Doize C, Lamy M, Serteyn D.We studied the interactions of isolated equine neutrophils with endothelial cells in culture, mimicking a situation of acute inflammation. Our main purpose was to demonstrate that the supernatant of activated neutrophils was sufficient to damage endothelial cells. Equine endothelial cells (from carotid arteries) were covered either with increased numbers of equine neutrophils stimulated by phorbol myristate acetate, or with the supernatant collected after an in vitro stimulation of the neutrophils. Cytotoxicity was estimated by the release of preincorporated 51Cr, and by light microscopy obser...
Lindsay DS, Dubey JP, Kennedy TJ.The present study examined the efficacy of ponazuril in inhibiting merozoite production of Sarcocystis neurona in cell cultures. Ponazuril inhibited merozoite production by more that 90% in cultures of S. neurona treated with 1.0 microg/ml ponazuril and greater than 95% inhibition of merozoite production was observed when infected cultures were treated with 5.0 microg/ml ponazuril. Ponazuril may have promise as a therapeutic agent in the treatment of S. neurona induced equine protozoal myeloencephalitis (EPM) in horses.
Ruitenberg KM, Love DN, Gilkerson JR, Wellington JE, Whalley JM.We have shown previously that equine herpesvirus 1 (EHV-1) glycoprotein D (gD) DNA elicited protective immune responses against EHV-1 challenge in murine respiratory and abortion models of EHV-1 disease. In this study, 20 horses, all with pre-existing antibody to EHV-4 and two with pre-existing antibody to EHV-1, were inoculated intramuscularly with three doses each of 50, 200 or 500microg EHV-1 gD DNA or with 500microg vector DNA. In 8 of 15 horses, inoculation with EHV-1 gD DNA led to elevated gD-specific antibody and nine horses exhibited increased virus neutralising (VN) antibody titres co...
McCann ME, Moore JN, Carrick JB, Barton MH.The effect of intravenous administration of lipid emulsions enriched with omega-3 (n3) and omega-6 (n6) fatty acids on equine monocyte phospholipid fatty acid composition and the synthesis of inflammatory mediators in vitro was evaluated. In a randomized crossover design, horses were infused intravenously with 20% lipid emulsions containing n3 or n6 fatty acids. Monocytes were isolated from the horses before and 0 h, 8 h, 24 h, and 7 days after lipid infusion. Monocyte fatty acid analysis demonstrated incorporation of the parenteral n3 and n6 fatty acids in monocyte phospholipids immediately a...
Forsell PK, Lindberg A, Karlsson S, Lindgren JA, Claesson HE.It has been demonstrated that equine neutrophils, but not eosinophils, require exogenous arachidonic acid for calcium ionophore A23187-induced leukotriene synthesis. Because cytosolic phospholipase A(2) (cPLA(2)) plays an essential role in leukotriene formation in leukocytes, we investigated the presence of a functional cPLA(2) in equine neutrophils. To determine whether cPLA(2) from neutrophils was catalytically active, we purified the enzyme >6,500 fold with 3% recovery from equine neutrophils. The full-length cDNA sequence encoded a 749-amino acid protein. The deduced amino acid sequence...
Yasunaga S, Maeda K, Matsumura T, Kondo T, Kai K.A type-specific enzyme-linked immunosorbent assay (ELISA) using equine herpesvirus types 1 (EHV-1) and 4 (EHV-4) glycoprotein G was applied for sero-epizootiology of EHV infections in Japan. Recently, an inactivated EHV-1 vaccine has been administered to racehorses for prevention of upper respiratory disease. To examine the effect of the vaccination on the result of the ELISA, 6 horses were experimentally inoculated three times intramuscularly or intranasally with inactivated EHV-1 vaccine. Sera collected from these horses were used to the type-specific ELISA and complement-fixation (CF) test....
Zheng YH, Sentsui H, Kono Y, Ikuta K.An attenuated equine infectious anemia virus (EIAV), named V26, was previously obtained after 50 passages of the Japanese virulent strain V70 in primary macrophage culture. To clarify the differences between both viruses, their full-length sequences were determined. There were higher mutations in S2 (6.15% amino acid difference) and LTR (10.7% nucleotide difference). The presumed initiation codon of the S2 gene was absent from the sequence of V26. There was a large insertion within the long-terminal repeat (LTR) U3 hypervariable region of V26. In addition, there were minor mutations in gag (1....
Graham AS, Grosche A, Morton AJ, Polyak MM, Freeman DE.To induce ischemia and reperfusion injury in the large colon mucosa of horses in vivo and evaluate the recovery and effects of components of an organ transplant solution on mucosal recovery in vitro. Methods: 6 healthy horses. Methods: Horses were anesthetized, and ischemia was induced for 60 minutes in the pelvic flexure, which was followed by reperfusion for 240 minutes. Ischemic (n = 4 horses), reperfused (6), and adjacent control (6) colonic mucosae were isolated for in vitro testing and histologic examinations. Tissues were mounted in Ussing chambers with plain Krebs Ringer bicarbonate (K...
Osheroff N, Feinberg BA, Margoliash E, Morrison M.Iodination of horse cytochrome c with the lactoperoxidase-hydrogen peroxide-iodide system results initially in the formation of the monoiodotyrosyl 74 derivative. This singly modified protein was obtained in pure form by ion exchange chromatography and preparative column electrophoresis. It shows an intact 695 nm absorption band, the midpoint potential of the native protein, a nuclear magnetic resonance spectrum which indicates an undisturbed heme crevice structure, a normal reaction with antibodies directed against native horse cytochrome c, and circular dichroic spectra in which the only cha...
Spinaci M, Nerozzi C, Mislei B, Blanco-Prieto O, Mari G, Galeati G, Bucci D.The growing and widespread use of glyphosate-based herbicides (GBHs) has raised an intense public debate about the impact of environmental contamination on animal and human health, including male fertility. The aim of this study was to deepen the impact of glyphosate (Gly) and GBHs on mammalian sperm investigating the effect of in vitro exposure of stallion spermatozoa to Gly and to its commercial formulation Roundup® (R). Spermatozoa were incubated at 37 °C with different Gly or R concentrations (from 0.5 to 720 μg/mL Gly or R at the same Gly-equivalent concentrations). After 1 h of in...
Sinibaldi F, Fiorucci L, Mei G, Ferri T, Desideri A, Ascoli F, Santucci R.Recombination of two fragments of horse cytochrome c (the heme-containing N-fragment, residues 1-56, and the C-fragment, residues 57-104), which are substantially unstructured at neutral pH, gives rise to a 1:1 fragment complex with a compact conformation, in which the alpha helical structure and the native Met80-Fe(III) axial bond are recovered. With respect to the native protein, the ferric complex shows a less rigid atomic packing and a decreased stability [Delta(DeltaG(o))D = 14.7 kJ.mol(-1)], ascribed to perturbations involving the Trp59 microenvironment and, to a lower extent, the heme p...
Hardy GW, Darbre A, Merrett M.3-Phosphoglycerate kinase was isolated from horse muscle and subjected to the action of cyanogen bromide. The resulting peptides were separated using gel filtration combined with either ion exchange chromatography on phosphocellulose in 6 M urea or high voltage paper electrophoresis. The sequence of the largest peptide, CB5, has been determined by a combination of automated and manual Edman degradation carried out on the intact peptide and derivatives obtained by proteolytic digestion. The isolation of two peptides derived from CB5 by cleavage of the bond between Asp109 and Pro110 facilitated ...
Frippiat T, Paindaveine C, Duprez JN, Delguste C, Mainil J, Art T.Antimicrobial resistance is increasing in both human and veterinary medicine. Bacteria can be part of the etiology of respiratory disorders in horses. Bactericidal activity of silver has been largely described and silver is currently used in veterinary therapeutic applications such as wound dressings. The aim of this study was to assess the in vitro bactericidal effects of nebulized silver nanoparticles (AgNP) on 2 common equine respiratory bacteria, Streptococcus equi subsp. zooepidemicus and Actinobacillus equuli subsp. equuli. Firstly, antimicrobial susceptibility of AgNP was determined ove...
Altamura G, Strazzullo M, Corteggio A, Francioso R, Roperto F, D'Esposito M, Borzacchiello G.Bovine papillomaviruses (BPVs) types 1 and 2 are the only known papillomaviruses able to jump the species. In fact, BPVs 1/2 induce neoplasia in their natural bovine host but infection is also associated to neoplastic skin lesions in equids termed sarcoids. The equine sarcoid is considered to be the most common equine cutaneous tumour worldwide for which no effective therapy is available. Very little is known about the molecular mechanisms underlying tumourigenesis, although genes contributing to sarcoid development have been identified. Several studies associate the development of cancer to t...
Bird J, Herd RP.The ability of two species of nematophagous fungi, Arthrobotrys oligospora and Arthrobotrys flagrans (syn. Trichothecium flagrans, Duddingtonia flagrans), to control the development of infective larvae in feces from naturally infected horses was assessed in vitro. The horses were from a farm where it had been previously established that cyathostomes accounted for 100% of the strongyle egg output. The feces from these naturally infected horses were mixed with spores of each fungal species at four concentrations: 0 (control), 1, 10, and 100 spores per egg. Five replicates for each group were inc...
Saukko AEA, Nykänen O, Sarin JK, Nissi MJ, Te Moller NCR, Weinans H, Mancini IAD, Visser J, Brommer H, van Weeren PR, Malda J, Grinstaff MW....To prevent the progression of posttraumatic osteoarthritis, assessment of cartilage composition is critical for effective treatment planning. Posttraumatic changes include proteoglycan (PG) loss and elevated water content. Quantitative dual-energy computed tomography (QDECT) provides a means to diagnose these changes. Here, we determine the potential of QDECT to evaluate tissue quality surrounding cartilage lesions in an equine model, hypothesizing that QDECT allows detection of posttraumatic degeneration by providing quantitative information on PG and water contents based on the partitions of...
Rötting AK, Freeman DE, Constable PD, Eurell JA, Wallig MA.To study the effects of phenylbutazone, indomethacin, prostaglandin E2 (PGE2), glutamine, and butyrate on restitution of oxidant-injured right dorsal colon of horses in vitro. Methods: Right dorsal colon from 9 adult horses euthanatized for reasons other than gastrointestinal tract disease. Methods: Mucosal segments from the right dorsal colon were injured via exposure to HOCl and incubated in Ussing chambers in solutions containing phenylbutazone, indomethacin, indomethacin and PGE2, glutamine, and butyrate. Transepithelial resistance and mucosal permeability to mannitol were measured, and al...
Papaioannou KZ, Murphy RP, Monks RS, Hynes N, Ryan MP, Boland MP, Roche JF.An objective double-staining method was developed to evaluate viability and mitochondrial function of stallion spermatozoa using flow cytometry. Sperm viability was assessed by propidium iodide (PI) exclusion, and mitochondrial function was measured by the intensity of rhodamine 123 (R123) fluorescence. Flow cytometry estimates of sperm viability measured by PI were equivalent (P > 0.05) to estimates made using Hoechst 33258 stain and fluorescent microscopy (% dead: 25 +/- 2.4 vs 21.5 +/- 3.5). The use of both PI and R123 was validated by addition of various proportions of freeze-shocked (m...
Archambault D, St-Laurent G.Equine arteritis virus (EAV) is the etiological agent of equine viral arteritis, a contagious viral disease of equids. EAV is the prototype virus of the arteriviruses, a group of small enveloped viruses with positive single-stranded RNA genomes. Because apoptosis or programmed cell death is believed to play an important role in the biogenesis of several cytopathogenic viruses, we examined whether EAV was able to induce cell apoptosis in vitro. To do this, Vero cells were infected with EAV at a multiplicity of infection of 0.1 tissue culture infectious dose (TCID50) per cell, and analyzed at va...
Love CC, Love LB, Varner DD, Hinrichs K.The relationship of holding time in media at room temperature (approximately 22 degrees C) to initial chromatin configuration and rate of in vitro maturation (IVM) of equine oocytes was determined. Only oocytes having a complete, compact cumulus were used in this study. Oocytes were removed from ovaries 3.5-8 h after slaughter and were put into one of four treatment groups: (1) immediate/fix (IF) = immediate fixation following removal from the ovary; (2) delay/fix (DF) = fixation after oocytes were held 1-4 h in medium at room temperature; (3) immediate/mature (IM) = immediate placement into m...
Bergqvist AS, Johannisson A, Bäckgren L, Dalin AM, Rodriguez-Martinez H, Morrell JM.This study was designed to evaluate the effect of single layer centrifugation (SLC) and subsequent cold storage on stallion sperm capacitation-like status and acrosome reaction. Three stallions were included in the study, with three ejaculates per stallion. The samples were examined 4, 24 and 72 h after collection, extension and SLC, with storage at 6°C. Sperm capacitation-like status was investigated using the fluorescent dye chlortetracycline (CTC). There was no difference in capacitation-like status between colloid-selected and non-selected spermatozoa. Sperm motility decreased significant...
van Duijkeren E, van Klingeren B, Vulto AG, Sloet van Oldruitenborgh-Oosterbaan MM, Breukink HJ, van Miert AS.The in vitro activity of trimethoprim (TMP) and 9 sulfonamides and their combinations in 6 concentration ratios was tested against 62 Salmonella strains isolated from horses over a 3-year period in the Netherlands, using the agar-dilution method. Most of the isolates were S typhimurium strains (n = 52); the others were S heidelberg (n = 3), S hadar (n = 2), S thompson (n = 2), S enteritidis (n = 1), S infantis (n = 1), and S derby (n = 1). The minimal TMP concentration at which 50% of the Salmonella strains were inhibited (MIC50) was 0.12 micrograms/ml. Sulfachlorpyridazine (SCP; MIC50, 16 mic...
Love LB, Choi YH, Love CC, Varner DD, Hinrichs K.Two experiments were conducted to determine the effects of storage on equine ovaries or isolated oocytes. Ovaries were collected at an abattoir and were maintained at room temperature during collection and transport (3-9h total). After arrival at the laboratory, ovaries were divided into three groups: immediate oocyte collection (control), storage at room temperature overnight (15-18 h) before oocyte collection, or storage at 4 degrees C overnight before oocyte collection. Collected oocytes were cultured in maturation medium for 24h. There was a significant increase in the proportion of oocyte...
Zerpa H, Berhane Y, Woodcock H, Elliott J, Bailey SR.A decrease in environmental temperature can directly affect the contractility of cutaneous vasculature, mediated in part by alpha(2)-adrenoceptors. Most of the cellular mechanisms underlying the cooling-enhanced contractility to alpha(2)-adrenoceptor agonists have been reported in cutaneous arteries but little information is available on cutaneous veins. To investigate the cellular mechanisms associated with the cooling-enhanced contraction to UK-14304 (alpha(2)-adrenoceptor agonist), isolated equine digital veins (EDVs) were studied at 30 degrees C and 22 degrees C. The effects of inhibitors ...
Rodríguez H, Torres C, Valdés X, Guerra H, Pastor LM, Maccallini G, Bustos-Obregón E.In the female genital tract, spermatozoa must undergo capacitation and acrosome reaction prior to fertilization. A number of factors may induce physiological acrosome reaction assayed in vitro. The aims of this study are to determine the inductive effect of the preovulatory follicular fluid on the sperm acrosomal status in the equine, once some characteristics of the follicular fluid during folliculogenesis had been evaluated. The spermatozoa were obtained from cauda epididymes of adult stallion. Follicular fluid was taken from mare ovarian follicles classified according to their diameter. In ...
LeBlanc PH, Broadstone RV, Derksen FJ, Robinson NE.Distal airway segments (ID, 3 to 4 mm; length, 5 mm) from 2 groups of horses were isolated and suspended in tissue baths filled with Krebs solution, aerated with 5% CO2 in oxygen and maintained at 37 degrees C. Responses to exogenous acetylcholine, isoproterenol, or electrical field stimulation were compared. Control horses (n = 30) had no history of recurrent airway obstruction, whereas principal horses (n = 15) had recurrent airway obstruction and were studied during an acute episode of airway obstruction. Although the distal airways contracted in response to the cumulative half-logarithmic ...
Chang CK, Ward B, Ebina S.Carbon monoxide- and oxygen-binding rates and affinities were measured for horse heart myoglobins reconstituted with synthetic hemes lacking peripheral methyl and vinyl groups. There is an apparent correlation between heme size and ligand specificity, i.e. larger m values (ratios of CO vs O2 association rates, l'/k') with smaller hemes. However, this correlation broke down with the most dealkylated heme. This is interpreted as resulting from protein conformational changes altering the steric crowdedness at the O2-binding site. Spectral properties and autoxidation rates also corroborate this vi...
McD￮ LA, Stover SM, Taylor KT.To determine cyclic biomechanical properties of gap osteotomized adult equine tibiae stabilized with an equine interlocking nail (EIN). Methods: In vitro experimental biomechanical investigation. Methods: Thirteen adult equine cadaveric tibiae. Methods: Adult equine tibiae with transverse, midshaft, 1-cm gap osteotomies, stabilized with an equine interlocking nail, underwent cyclic biomechanical testing in vitro under axial compression, 4-point bending, and torsion. Different specimens were subjected to different load levels that represented estimated in vivo loads at 2 Hz for 740,000 cycles. ...
Rideout MI, Burns SJ, Simpson RB.Sterile filtrates were prepared from equine isolates of Klebsiella pneumoniae (genitalium), Proteus mirabilis, Pseudomonas aeruginosa, Escherichia coli, Streptococcus zooepidemicus, Streptococcus equisimilis, Actinobacillus equuli, and Corynebacterium equi and mixed individually with extended stallion semen. When diluted in the extended semen the filtrates represented bacterial populations of 0.5 x 10(6), 1 x 10(6), 2 x 10(6) and 4 x 10(6) cells/ml. pH values were recorded for each filtrate. Specimens were monitored for percentage motile spermatozoa at 30-min intervals until they reached 10% o...
Ramos MI, Hermosura ME, Nakabayashi T.Horse, calf and bovine serum were successfully used as human serum substitutes in the in vitro cultivation of Plasmodium falciparum. Positive results were obtained only after gradually adapting the parasites to the substitute serum. Adapted lines were established within 4-5 weeks. 10% horse serum was observed to be the best substitute with growth rates comparable or even surprising that obtained in human serum. Pure calf or bovine serum supported stable growths of 20-30% less which was enhanced to comparable levels after addition of 1% glucose-peptone to the medium. Direct transfers of adapted...
Janda J, Lehmann M, Luttmann W, Marti E.Interleukin-3 is a growth and differentiation factor for various hematopoietic cells. IL-3 also enhances stimulus-dependent release of mediators and cytokine production by mature basophils. Function of IL-3 has not been studied in horses because of lack of horse-specific reagents. Our aim was to produce recombinant equine IL-3 and test its effect on sulfidoleukotriene and cytokine production by equine peripheral blood leukocytes (PBL). Equine IL-3 was cloned, expressed in E. coli and purified. PBL of 19 healthy and 20 insect bite hypersensitivity (IBH)-affected horses were stimulated with Culi...
Kumar J, Chaudhury A, Bera BC, Kumar R, Kumar R, Tatu U, Yadav SC.The present immuno-diagnostic method using soluble antigens from whole cell lysate antigen for trypanosomosis have certain inherent problems like lack of standardized and reproducible antigens, as well as ethical issues due to in vivo production, that could be alleviated by in vitro production. In the present study we have identified heat shock protein 70 (HSP70) from T. evansi proteome. The nucleotide sequence of T. evansi HSP70 was 2116 bp, which encodes 690 amino acid residues. The phylogenetic analysis of T. evansi HSP70 showed that T. evansi occurred within Trypanosoma clade and is most c...
Zerpa H, Berhane Y, Elliott J, Bailey SR.Cooling-enhanced contractile responses in cutaneous arteries have been reported to involve the activation of alpha(2C)-adrenoceptors, but their role in cutaneous veins is not clearly understood. The aim was to pharmacologically characterize the subtype of postsynaptic alpha(2)-adrenoceptors in the equine digital vein mediating contraction at two temperatures. The increase in isometric tension of endothelium-denuded equine digital vein in response to UK-14304 was studied in the absence and presence of relatively selective alpha(2)-adrenoceptor antagonists at temperatures of either 30 degrees C ...
Consuegra C, Crespo F, Dorado J, Diaz-Jimenez M, Pereira B, Sánchez-Calabuig MJ, Beltrán-Breña P, Pérez-Cerezales S, Rizos D, Hidalgo M.The aim of this study was to evaluate the fertilizing capacity of frozen or vitrified stallion sperm after assessing different warming procedures. In Experiment 1, different warming procedures were compared after sperm vitrification: immersion in extender at 43 °C (C), or in a water bath at 37 °C/30 s (W37), 43 °C/10 s (W43) or 60 °C/5 s (W60). With the W60 treatment, there were greater values (P < 0.05) for VCL (83.93 ± 3.6 μm/s) and ALH (3.00 ± 0.2 μm) than freezing and with the C group, and greater values (P < 0.001) for PM (35.33 ± 2.5 %) than with the W43 treatment. In Experiment...
Khoshtariya DE, Dolidze TD, Shushanyan M, van Eldik R.Horse muscle myoglobin (Mb) was tightly immobilized at Au-deposited ~15-Å-thick mixed-type (1:1) alkanethiol SAMs, HS-(CH₂)₁₁-COOH/HS-(CH₂)₁₁-OH, and placed in contact with buffered H₂O or D₂O solutions. Fast-scan cyclic voltammetry (CV) and a Marcus-equation-based analysis were applied to determine unimolecular standard rate constants and reorganization free energies for electron transfer (ET), under variable-temperature (15-55 °C) and -pressure (0.01-150 MPa) conditions. The CV signal was surprisingly stable and reproducible even after multiple temperature and pressure cycl...
Taddei N, Buck M, Broadhurst RW, Stefani M, Ramponi G, Dobson CM.The stability and equilibrium unfolding behaviour of horse muscle acylphosphatase have been studied by denaturing the protein under various conditions of temperature, pH, and urea concentration. Far-ultraviolet circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy indicate that this small monomeric protein unfolds reversibly and cooperatively. Thermodynamic parameters, the Gibbs free energy delta G and enthalpy delta H of unfolding, have been estimated for denaturation of the protein from NMR and CD data as 19 kJ mol-1 and 350 kJ mol-1, respectively. CD and 1H-NMR results s...
Vallejo AN, Miller NW, Clem LW.This work was undertaken to investigate whether or not antigen processing and presentation are important in channel catfish in vitro secondary immune responses elicited with structurally defined proteins, namely, pigeon heart cytochrome C (pCytC), hen egg lysozyme, and horse myoglobin. The use of in vitro antigen-pulsed and fixed B cells or monocytes as antigen presenting cells (APC) resulted in autologous peripheral blood leukocytes (PBL) responding with vigorous proliferation and antibody production in vitro. In addition, several long-term catfish monocyte lines have been found to function a...
