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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.
Hsp90 mediates insulin-like growth factor 1 and interleukin-1beta signaling in an age-dependent manner in equine articular chondrocytes. Many metabolic processes in chondrocytes thought to contribute to age-related changes in the extracellular matrix are influenced by known roles of Hsp90. Age-related decreases in the level of Hsp90 have been documented in numerous cell types and could contribute to cartilage degeneration. The aim of this study was to investigate the roles of age and Hsp90 in insulin-like growth factor 1 (IGF-1) and interleukin-1beta (IL-1beta) signaling in chondrocytes. Methods: Levels of Hsp90 messenger RNA (mRNA) and protein, with respect to age, were determined by quantitative real-time polymerase chain rea...
In vitro-produced equine embryos: production of foals after transfer, assessment by differential staining and effect of medium calcium concentrations during culture. Viability of equine embryos produced by oocyte maturation, intracytoplasmic sperm injection and embryo culture to the blastocyst stage in vitro was evaluated after transfer of embryos to recipient mares. No pregnancies were produced after transfer of five blastocysts that had been cultured in G media. Transfer of 10 blastocysts cultured in modified DMEM/F-12 medium produced five pregnancies and three live foals; the two lost pregnancies developed only trophoblast (based on transrectal ultrasonography). To evaluate the status of the inner cell mass, equine blastocysts produced in vivo and in vi...
Vaginal lactic acid bacteria in the mare: evaluation of the probiotic potential of native Lactobacillus spp. and Enterococcus spp. strains. Lactic acid bacteria (LAB) are important members of the human vaginal microbiota and their presence is considered beneficial. However, little is known about native vaginal bacteria in other animal species such as the horse. The aim of this work was to quantify the vaginal lactic acid bacteria and lactobacilli of mares and to establish if selected equine vaginal lactic acid bacteria, particularly Lactobacillus and Enterococcus spp. strains, could exhibit potential as probiotics. The vaginal lactic acid bacteria and lactobacilli of 26 mares were quantified by plate counts. Five strains (three La...
Relevance of using a human microarray to study gene expression in heaves-affected horses. Environmental causes of heaves are well described, but the molecular mechanisms of the disease remain unclear. Previous studies have highlighted the implications of variations in gene expression, most using reverse transcription polymerase chain reaction (RT-PCR). This well-known technique limits the number of genes that can be studied in a single assay. Microarray appears to be a valuable tool to by-pass this limitation, but so far there has been no equine-specific microarray available on the market. The present study was performed to determine whether a human microarray could be used to stud...
In vitro equine oocyte maturation in pure follicular fluid plus interleukin-1 and fertilization following ICSI. The interleukin-1 (IL-1) system is thought to be involved in periovulatory events in the mare. Previous in vivo studies have demonstrated that IL-1beta induces oocyte maturation, but depresses the pregnancy rate 14 days after ovulation. To better understand the role of IL-1 in oocyte maturation and fertilization, the effects of IL-1 on the in vitro maturation rate of equine oocytes in pure follicular fluid were evaluated and fertilization rate assessed following intracytoplasmic sperm injection (ICSI). Oocytes collected from slaughterhouse ovaries were cultured in four different media for 30 h...
Comparison of opioid receptor binding in horse, guinea pig, and rat cerebral cortex and cerebellum. To compare the density and binding characteristics of opioid receptor subtypes in horse, rat, and guinea pig cerebral cortex and cerebellum. Methods: Prospective receptor binding study. Methods: Whole brains were obtained from four neurologically normal adult horses during necropsy. Rat and guinea pig brains were obtained commercially. Methods: The cerebellum and cerebral cortex were dissected from each brain, and tissue homogenates prepared. A radioligand binding technique with the highly selective ligands [(3)H]-DAMGO, [(3)H]-U69593, and [(3)H]-DPDPE was used to identify the mu- (mu), kappa-...
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...
