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Topic:In Vitro Research
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.
Mechanism of translesion synthesis past an equine estrogen-DNA adduct by Y-family DNA polymerases. 4-Hydroxyequilenin (4-OHEN)-dC is a major, potentially mutagenic DNA adduct induced by equine estrogens used for hormone replacement therapy. To study the miscoding property of 4-OHEN-dC and the involvement of Y-family human DNA polymerases (pols) eta, kappa and iota in that process, we incorporated 4-OHEN-dC into oligodeoxynucleotides and used them as templates in primer extension reactions catalyzed by pol eta, kappa and iota. Pol eta inserted dAMP opposite 4-OHEN-dC, accompanied by lesser amounts of dCMP and dTMP incorporation and base deletion. Pol kappa promoted base deletions as well as ...
A comparison of duck and chicken egg yolk for cryopreservation of stallion sperm. Duck and chicken egg yolk were compared for their protective effects against cold shock during the cryopreservation of stallion sperm in a lactose-EDTA-glycerol cryodiluent. Methods: A completely randomised design was used. Procedure Ejaculates from five stallions (n = 14 ejaculates) were split and diluted to either 20 or 200 x 10(6) sperm/mL in a lactose-EDTA extender containing either duck or chicken egg yolk. The extended semen was then frozen in liquid nitrogen. The percentage of sperm total motility and forward progressive motility were assessed before freezing and at 0 and 1 hr after tha...
Evaluation of activation of protein kinase C during agonist-induced constriction of veins isolated from the laminar dermis of horses. 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 ...
Identification and characterization of equine granzyme B. In the present study we describe the isolation and characterization of putative equine granzyme B for which we propose the designation 'eqGrzmB'. Sequence analysis revealed characteristic features of a GrzmB protease such as the presence of a signal (leader-) peptide and an activation di-peptide. The isolated eqGrzmB is functionally active when expressed in human or in insect cells. Furthermore, exchange of any of three putative active site amino acids, which are highly conserved along granzyme B enzymes, led to a complete loss of enzymatic activity in the newly identified eqGrzmB. Phylogeneti...
Metabolic studies of mesterolone in horses. Mesterolone (1alpha-methyl-5alpha-androstan-17beta-ol-3-one) is a synthetic anabolic androgenic steroid (AAS) with reported abuses in human sports. As for other AAS, mesterolone is also a potential doping agent in equine sports. Metabolic studies on mesterolone have been reported for humans, whereas little is known about its metabolic fate in horses. This paper describes the studies of both the in vitro and in vivo metabolism of mesterolone in racehorses with an objective to identify the most appropriate target metabolites for detecting mesterolone administration. In vitro biotransformation st...
Isolation of mesenchymal stem cells from equine umbilical cord blood. There are no published studies on stem cells from equine cord blood although commercial storage of equine cord blood for future autologous stem cell transplantations is available. Mesenchymal stem cells (MSC) have been isolated from fresh umbilical cord blood of humans collected non-invasively at the time of birth and from sheep cord blood collected invasively by a surgical intrauterine approach. Mesenchymal stem cells isolation percentage from frozen-thawed human cord blood is low and the future isolation percentage of MSCs from cryopreserved equine cord blood is therefore expectedly low. The...
Effect of extracorporeal shock wave therapy on the biochemical composition and metabolic activity of tenocytes in normal tendinous structures in ponies. Extracorporeal shockwave therapy (ESWT) has recently been introduced as a new therapy for tendon injuries in horses, but little is known about the basic mechanism of action of this therapy. Objective: To study the effect of ESWT on biochemical parameters and tenocyte metabolism of normal tendinous structures in ponies. Methods: Six Shetland ponies, free of lameness and with ultrasonographically normal flexor and extensor tendons and suspensory ligaments (SL), were used. ESWT was applied at the origin of the suspensory ligament and the mid-metacarpal region of the superficial digital flexor ten...
Rapid infusion of a phospholipid emulsion attenuates the effects of endotoxaemia in horses. Endotoxaemia currently is associated with a poor prognosis in horses. The results of recent trials in other species indicate that phospholipid emulsions reduce the deleterious effects of endotoxin (LPS). However, in a previous study in horses, a 2 h infusion of emulsion caused an unacceptable degree of haemolysis. Objective: Rapid administration of a lower total dose of emulsion would reduce the effects of LPS and induce less haemolysis; the emulsion would reduce inflammatory effects of LPS in vitro. Methods: Twelve healthy horses received an i.v. infusion either of saline or a phospholipid em...
Equine embryology: an inventory of unanswered questions. Carl Hartman's title of 47 years ago is invoked in tribute to his first recovery of a bovine embryo 30 years before that, and his legacy of an emphasis on the value of descriptive and comparative studies in reproductive biology. The horse's qualification as a farm animal has waned since those times but, in a conference understandably dominated by research in ruminants and pigs, there are lessons to be learned from some peculiarities of equine embryonic development. Morphological and physiological features of the conceptus and its interaction with its environment during the first month of pregn...
Effect of yeast culture on in vitro fermentation of a high-concentrate or high-fiber diet using equine fecal inoculum in a Daisy II incubator. Two experiments were conducted to evaluate the use of a closed system, fermentation apparatus (Daisy II incubator) and determine the effects of a yeast culture (YC) preparation (Saccharomyces cerevisiae) on in vitro microbial populations, diet digestion, and fermentation patterns in horses. In Exp. 1, 4 mature horses were fed a pelleted concentrate and alfalfa cubes in a 50:50 (%, as-fed) ratio. Fecal samples were taken from each horse to form the inoculum and placed in 4 separate incubation vessels. Twenty nylon bags (10 with 0.25 g and 10 with 0.50 g of the total mixed diet) were placed in e...
Alginate encapsulation impacts the insulin-like growth factor-I system of monolayer-expanded equine articular chondrocytes and cell response to interleukin-1beta. Alginate hydrogel culture has been shown to reestablish chondrocytic phenotype following monolayer expansion; however, previous studies have not adequately addressed how culture conditions affect the signaling systems responsible for chondrocyte metabolic activity. Here we investigate whether chondrocyte culture history influences the insulin-like growth factor-I (IGF-I) signaling system and its regulation by interleukin-1 (IL-1). Articular chondrocytes (ACs) from equine stifle joints were expanded by serial passage and were either encapsulated in alginate beads or maintained in monolayer cult...
Cooling augments vasoconstriction mediated by 5-HT1 and alpha2-adrenoceptors in the isolated equine digital vein: involvement of Rho kinase. The vasculature of the equine digit fulfils an important role in thermoregulation. In other species, it has been found that cooling may enhance the response of cutaneous vessels to 5-hydroxytryptamine (5-HT) and alpha(2)-adrenoceptor agonists. Translocation of alpha(2)-adrenoceptors to the smooth muscle cell membrane, mediated by Rho kinase, is thought to be involved in the cooling-enhanced response in mouse tail arteries. However, little is known about the effect of cooling on 5-HT receptor function. The present investigation compared the response of 5-bromo-6-(2-imidazolin-2-ylamino) quinoxa...
Equine lysozyme: the molecular basis of folding, self-assembly and innate amyloid toxicity. Calcium-binding equine lysozyme (EL) combines the structural and folding properties of c-type lysozymes and alpha-lactalbumins, connecting these two most studied subfamilies. The structural insight into its native and partially folded states is particularly illuminating in revealing the general principles of protein folding, amyloid formation and its inhibition. Among lysozymes EL forms one of the most stable molten globules and shows the most uncooperative refolding kinetics. Its partially-folded states serve as precursors for calcium-dependent self-assembly into ring-shaped and linear amyloi...
Heterospecific nuclear-transferred embryos derived from equine fibroblast cells and enucleated bovine oocytes. This study was conducted to reconstruct heterogeneous embryos using equine skin fibroblast cells as donor karyoplasts and the bovine oocytes as recipient cytoplast for investigating the reprogramming of equine somatic cell nuclear in bovine oocyte cytoplasm and the developmental potential of the reconstructed embryos. Adult horse skin fibroblast cells serum-starved were used as donor somatic cells. Bovine oocytes matured in vitro were employed as recipient cytoplasts. The fusion of fibroblast cells into recipient cytoplasm was induced by electofusion. The fused eggs were activated by inomycin ...
In vitro development of equine oocytes from preserved ovaries after intracytoplasmic sperm injection. In this study, we evaluated the meiotic competence of equine oocytes from ovaries preserved for one day. We also investigated fertilization, cleavage rate, developmental competence and freezability of equine embryos after intracytoplasmic sperm injection (ICSI). After collection from ovaries, the oocytes were classified into two groups comprised of those having compact cumulus layers (Cp) or those having expanded cumulus layers (Ex). Oocytes with a first polar body were subjected to fertilization by ICSI using frozen-thawed stallion spermatozoa and were then cultured in CR1aa medium. The rates...
IL-4 stimulates the expression of CXCL-8, E-selectin, VEGF, and inducible nitric oxide synthase mRNA by equine pulmonary artery endothelial cells. Little is known concerning the possible contribution of T helper 2 (Th2)-type cytokines to the recruitment of neutrophils into the lung tissue. In the present study, endothelial cells from equine pulmonary arteries were cultured in the presence of recombinant equine (re) IL-4 and reIL-5, and the cytokine mRNA expression of molecules implicated in the chemotaxis and migration of neutrophils was studied using real-time RT-PCR. The functional response of reIL-4-induced endothelial cell stimulation on neutrophil migration was also studied using a chemotaxis chamber. ReIL-4 either increased the exp...
Long terminal repeat sequences from virulent and attenuated equine infectious anemia virus demonstrate distinct promoter activities. In the early 1970s, the Chinese Equine Infectious Anemia Virus (EIAV) vaccine, EIAV(DLA), was developed through successive passages of a wild-type virulent virus (EIAV(L)) in donkeys in vivo and then in donkey macrophages in vitro. EIAV attenuation and cell tropism adaptation are associated with changes in both envelope and long terminal repeat (LTR). However, specific LTR changes during Chinese EIAV attenuation have not been demonstrated. In this study, we compared LTR sequences from both virulent and attenuated EIAV strains and documented the diversities of LTR sequence from in vivo and in v...
Combinatorial selection of a RNA thioaptamer that binds to Venezuelan equine encephalitis virus capsid protein. A phosphorothioate RNA aptamer (thioaptamer) targeting the capsid protein of Venezuelan equine encephalitis virus (VEEV) was isolated by in vitro combinatorial selection. The selected thioaptamer had a strong binding affinity (approximately 7nM) and high specificity for the target protein. For the binding to the protein, the overall tertiary structure of the thioaptamer is required. We introduce two theoretical methods to examine the effect of phosphorothioate modification on the enhancement of binding affinity and one experimental method to examine the nature of the multiple bands of thioapta...
Time- and dose-dependent effects of roundup on human embryonic and placental cells. Roundup is the major herbicide used worldwide, in particular on genetically modified plants that have been designed to tolerate it. We have tested the toxicity and endocrine disruption potential of Roundup (Bioforce on human embryonic 293 and placental-derived JEG3 cells, but also on normal human placenta and equine testis. The cell lines have proven to be suitable to estimate hormonal activity and toxicity of pollutants. The median lethal dose (LD(50)) of Roundup with embryonic cells is 0.3% within 1 h in serum-free medium, and it decreases to reach 0.06% (containing among other compounds 1.2...
A preliminary study of the short circuit current (Isc) responses of sweat gland cells from normal and anhidrotic horses to purinergic and adrenergic agonists. The causal factors of equine anhidrosis have not yet been elucidated but defective electrolyte transport mechanisms in the gland are likely to be involved. To investigate this possibility, experiments were performed on cultured equine sweat gland epithelia from five free-sweating UK horses (3 intact males, 2 mares, aged 2-4 years) and from three free-sweating Singapore horses (1 intact male, 2 mares, aged 3-5 years) and three anhidrotic (Singapore) horses (1 intact male, 1 gelding, 1 mare, aged 3-6 years). Cultured cells from each animal were grown on permeable supports and loaded into Ussing ...
Cytokine and chemokine gene expression of IL-1beta stimulated equine articular chondrocytes. To evaluate mRNA expression of several proinflammatory and anti-inflammatory cytokines and chemokines in equine unstimulated and interleukin-1beta (IL-1beta)-stimulated chondrocytes. Methods: In vitro experiment using equine chondrocyte cultures. Methods: Whole articular cartilage from metacarpophalangeal joints (n=5 horses; 10 fetlocks). Methods: Chondrocyte monolayer cultures were established from digested adult equine articular cartilage and stimulated with 5 ng/mL of recombinant human IL-1beta. RNA was extracted from the cells 24 hours after stimulation. IL-1beta, IL-4, IL-6, IL-8, tumor n...
Clinical and pathological studies on intoxication in horses from freshly cut Jimson weed (Datura stramonium)-contaminated maize intended for ensiling. Spontaneous intoxication in 34 horses after ingesting freshly harvested maize that was to be used for ensiling and heavily contaminated with young Datura stramonium plants, is described. The clinical status of all horses was monitored for 7 days, and included body (rectal) temperature, respiratory and heart rates, colour and moistness of visible mucosae, changes in pupil size, appetite, thirst, general behaviour, locomotion, sensory perceptions, urination and defaecation. The intoxication was accompanied by altered clinical status, namely mild hyperthermia, tachycardia, polypnoea, dyspnoea and...
Potential risk of equine herpes virus 1 (EHV-1) transmission by equine embryo transfer. The objective of this study was to determine whether the 10 wash cycles proposed by the International Embryo Transfer Society (IETS) for bovine embryos efficiently decontaminated equine embryos exposed to equine herpes virus 1 (EHV-1) in vitro. Donor mares and stallions were individually screened and shown to be negative for the virus by PCR detection of EHV-1 DNA in blood leukocytes, semen, and uterine lavages in which embryos were recovered. Twenty embryos were recovered and randomly assigned to one of two groups: 10 embryos were exposed for 24h to infectious EHV-1 at 10(6)TCID(50)/ml, and 1...
Pharmacokinetics and in vitro effects of tegaserod, a serotonin 5-hydroxytryptamine 4 (5-HT4) receptor agonist with prokinetic activity in horses. Tegaserod, a serotonin agonist, has been shown to have prokinetic effects in horses, but pharmacokinetic information is not currently available. The pharmacokinetics and in vitro effects of tegaserod were evaluated. Tegaserod increased the contractile activity of smooth muscle preparations of the equine pelvic flexure. Pertinent pharmacokinetic parameters for a single IV and oral dose were determined. Therapeutic plasma concentrations of tegaserod were achieved by a single oral dose at 0.27 mg/kg. These findings indicate that further clinical studies are warranted to investigate potential bene...
Storage of equine red blood cells as a concentrate. The study was undertaken to determine how equine red blood cells (RBCs) survive in storage bags designed for use with human RBCs. Separated RBCs were stored in a routine manner for 35 days and examined every 7 days for storage lesions. Measured parameters included haematology, haemolysis, pH, potassium, lactate, adenosine triphosphate (ATP) and 2,3-diphosphoglycerate (2,3-DPG). All tests were performed in vitro. Haematology did not change significantly. Haemolysis increased during storage but did not exceed human limits. pH and 2,3-DPG decreased, while lactate, potassium and ATP increased. RBC...
Modification of host erythrocyte membranes by trypsin and chymotrypsin treatments and effects on the in vitro growth of bovine and equine Babesia parasites. In the present study, we investigated the effects of protease pretreatments of host erythrocytes (RBC) on the in vitro growth of bovine Babesia parasites (Babesia bovis and B. bigemina) and equine Babesia parasites (B. equi and B. caballi). The selected proteases, trypsin and chymotrypsin, clearly modified several membrane proteins of both bovine and equine RBC, as demonstrated by SDS-PAGE analysis; however, the protease treatments also modified the sialic acid content exclusively in bovine RBC, as demonstrated by lectin blot analysis. An in vitro growth assay using the protease-treated RBC sh...
Restricted and selective tropism of a Venezuelan equine encephalitis virus-derived replicon vector for human dendritic cells. Dendritic cells (DCs) consist of heterogeneous phenotypic populations and have diverse immunostimulatory functions dependent on both lineage and functional phenotype, but as exceptionally potent antigen-presenting cells, they are targets for generating effective antigen-specific immune responses. A promising replicon particle vector derived from Venezuelan equine encephalitis virus (VEE) has been reported to transduce murine footpad DCs. However, the receptive DC subset, the degree of restriction for this tropism, and the extent of conservation between rodents and humans have not been well cha...
Identification and functional analysis of sequence variants in the long control region and the E2 open reading frame of bovine papillomavirus type 1 isolated from equine sarcoids. BPV-1 DNA is the predominant viral type detected in equine sarcoids and represents the only reported natural cross species infection of papillomaviruses. In this study, nucleotide variations in the LCR and the E2 regions of equine sarcoid-associated BPV-1 were characterised by sequence analysis. Variants particular to sarcoid BPV-1 were identified in both the LCR and E2 sequence. The functionality of the most common LCR variant was examined in equine and bovine cells. These studies showed that the activity of the variant LCR was higher in equine cells than bovine cells; the activity of the var...
Comparative in vitro biomechanical evaluation of two soft tissue defect products. A soft tissue defect is often an unavoidable consequence of various surgical procedures or a result of trauma. Recently, intraoperative use of xenograft as a patch to the soft tissue defect has become popular with various products available in the market. In this study, mechanical properties of the OrthADAPTtrade mark Bioimplants (Pegasus Biologics, Irvine, CA), new xenograft products composed of collagen from equine pericardium, were evaluated individually and against an existing bioimplant product. The OrthADAPTtrade mark Bioimplants have three subtypes which differ in the degree of crosslin...
Movement between the equine ilium and sacrum: in vivo and in vitro studies. Sacroiliac disease (SID) is an important cause of loss of performance in horses, yet little is known about the biomechanics of movement of the sacroiliac joint (SIJ). Objective: (a) To document the presence of equine SIJ motion by measuring the change in cross-sectional area (CSA) of the dorsal portion of the dorsal sacroiliac ligament (DSIL) during application of manual forces to the pelvis, and to ascertain if this varied between horses with SID and normal horses; (b) To determine the amount and direction of motion available at the equine SIJ using 3-D orientation sensors, and how motion is ...
