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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.
In vitro comparison of cortical bone temperature generation between traditional sequential drilling and a newly designed step drill in the equine third metacarpal bone. To compare heat generation and time to finish between a new step drill and sequential drilling in order to create a 6.2 mm pilot hole for insertion of a positive profile transfixation pin into the equine third metacarpal bone. Methods: Nine pairs of equine third metacarpal bones from cadavers of adult horses were used. Maximum temperature rise of the bone was measured continuously at the cis- and trans-cortices 1, 2 and 3 mm from the final pilot hole during creation of a 6.2 mm hole using a step drill and sequential drilling with 4.5, 5.5 and 6.2 mm drill bits. Five holes were drilled into the...
Functional role of alpha2-adrenoceptor subtypes in the cooling-enhanced vasoconstriction of isolated cutaneous digital veins of the horse. 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 ...
The application of in vitro technologies to study the metabolism of the androgenic/anabolic steroid stanozolol in the equine. In this study, the use of equine liver/lung microsomes and S9 tissue fractions were used to study the metabolism of the androgenic/anabolic steroid stanozolol as an example of the potential of in vitro technologies in sports drug surveillance. In vitro incubates were analysed qualitatively alongside urine samples originating from in vivo stanozolol administrations using LC-MS on a high-resolution accurate mass Thermo Orbitrap Discovery instrument, by LC-MS/MS on an Applied Biosystems Sciex 5500 Q Trap and by GC-MS/MS on an Agilent 7000A. Using high-resolution accurate mass full scan analysis o...
Zonal chondrocyte subpopulations reacquire zone-specific characteristics during in vitro redifferentiation. If chondrocytes from the superficial, middle, and deep zones of articular cartilage could maintain or regain their characteristic properties during in vitro culture, it would be feasible to create constructs comprising these distinctive zones. Objective: Zone-specific characteristics of zonal cell populations will disappear during 2-dimensional expansion but will reappear after 3-dimensional redifferentiation, independent of the culture technique used (alginate beads versus pellet culture). Methods: Controlled laboratory study. Methods: Equine articular chondrocytes from the 3 zones were expan...
Cryopreservation does not affect the stem characteristics of multipotent cells isolated from equine peripheral blood. Mammalian adult stem cells show, in vitro, extensive differentiative ability and may represent a versatile tool for tissue regenerative purposes, even after long-term storage. Multipotent stem cells isolated from horse blood have been shown to possess the capacity to differentiate into diverse mesenchymal lineages although their full characterization is still at an early stage. The aim of this study was to examine the effects of cryopreservation on stemness characteristics of adult equine mesenchymal stem cells isolated from peripheral blood (ePB-MSC). Each sample of ePB-MSC was analyzed immed...
Effects of leptin on in vitro maturation, fertilization and embryonic cleavage after ICSI and early developmental expression of leptin (Ob) and leptin receptor (ObR) proteins in the horse. The identification of the adipocyte-derived obesity gene product, leptin (Ob), and subsequently its association with reproduction in rodents and humans led to speculations that leptin may be involved in the regulation of oocyte and preimplantation embryo development. In mice and pigs, in vitro leptin addition significantly increased meiotic resumption and promoted preimplantation embryo development in a dose-dependent manner. This study was conducted to determine whether leptin supplementation during in vitro maturation (IVM) to horse oocytes could have effects on their developmental capacity ...
Effect of different penetration enhancers on diclofenac permeation across horse skin. Diclofenac is a hydrophilic non-steroidal anti-inflammatory drug widely used in humans and animals. Previous reports have shown that this compound has low percutaneous absorption in horses. The effect of five penetration enhancers (10% urea, 15% and 20% oleic acid and 5% and 10% d-limonene) on the percutaneous absorption of diclofenac diethylamine through horse skin was evaluated in vitro using Franz-type diffusion cells. All tested penetration enhancers induced a significant increase in diclofenac diethylamine permeation, with limonene showing the highest enhancing effect at the lowest concen...
Effects of in vitro exposure to hay dust on expression of interleukin-23, -17, -8, and -1beta and chemokine (C-X-C motif) ligand 2 by pulmonary mononuclear cells from horses susceptible to recurrent airway obstruction. To examine gene expression of selected cytokines in pulmonary mononuclear cells isolated from healthy horses and horses susceptible to recurrent airway obstruction (RAO), and to determine whether interleukin (IL)-17 and IL-23 were associated with pulmonary inflammation. Methods: 6 RAO-susceptible and 5 healthy horses. Methods: Bronchoalveolar lavage cells were retrieved from horses that were stabled and fed dusty hay for 24 hours. Lavage cells devoid of neutrophils were incubated for 24 hours with solutions of PBS, hay dust, lipopolysaccharide, or B-glucan. Gene expression of IL-17, IL-23 (p19...
Evidence that antibodies against recombinant SnSAG1 of Sarcocystis neurona merozoites are involved in infection and immunity in equine protozoal myeloencephalitis. Sarcocystis neurona is the principal etiologic agent of equine protozoal myeloencephalitis (EPM). An immunodominant protein of S. neurona, SnSAG-1, is expressed by the majority of S. neurona merozoites isolated from spinal tissues of horses diagnosed with EPM and may be a candidate for diagnostic tests and prophylaxis for EPM. Five horses were vaccinated with adjuvanted recombinant SnSAG1 (rSnSAG1) and 5 control (sham vaccinated) horses were vaccinated with adjuvant only. Serum was evaluated pre- and post-vaccination, prior to challenge, for antibodies against rSnSAG1 and inhibitory effects on...
Arthrodesis of the equine proximal interphalangeal joint: a biomechanical comparison of two parallel headless, tapered, variable-pitched, titanium compression screws and two parallel 5.5 mm stainless-steel cortical screws. To compare the biomechanical characteristics, failure mode, and effects of side (left or right limb) and end (forelimb or hindlimb) of different screws in 2-screw, parallel-screw proximal interphalangeal joint arthrodesis constructs in horses. Methods: In vitro experimental study. Methods: Twenty limbs from 6 cadavers (4 complete limb sets, 2 partial sets-total of 4 forelimb and 6 hindlimb pairs). Methods: Two parallel 5.5 mm cortical (AO) screws were inserted in lag fashion in 1 randomly allocated limb of a pair, and 2 parallel headless, tapered, variable-pitched, titanium compression screws ...
MMP-mediated collagen breakdown induced by activated protein C in equine cartilage is reduced by corticosteroids. The plasma serine protease activated protein C (APC) is synthesized by human chondrocytes at sites of pathological cartilage fibrillation. APC levels are increased in osteoarthritis (OA) synovial fluid, and in vitro APC has been shown to synergize with interleukin-1beta (IL-1) to promote degradation from ovine cartilage. A model of equine cartilage degradation was established and used to explore corticosteroid activities. Intraarticular corticosteroids are a commonly prescribed treatment for joint disease, however their role in disease modification remains unclear. APC synergized with IL-1 or ...
Moxidectin: a review of chemistry, pharmacokinetics and use in horses. This article reviews the current knowledge of the use of moxidectin (MOX) in horses, including its mode of action, pharmacokinetic and pharmacodynamic properties, efficacy, safety and resistance profile.Moxidectin is a second generation macrocyclic lactone (ML) with potent endectocide activity. It is used for parasite control in horses in an oral gel formulation. The principal mode of action of MOX and of other MLs is binding to gamma-aminobutyric (GABA) and glutamate-gated chloride channels. Moxidectin is different from other MLs in that it is a poor substrate for P-glycoproteins (P-gps) and ...
[Construction and in vitro evaluation of an infectious clone of the equine infectious anemia virus vaccine strain EIAV(FDDV) with four reverse-mutated vaccine-specific sites in the S2 gene]. To elucidate the function of the S2 gene in equine infectious anemia virus (EIAV) and its role in the attenuation of the Chinese attenuated EIAV vaccine strains, the S2 in the EIAV vaccine strain EIAV (FDDV) was reverse-mutated and the in vitro replication character of the resultant virus was evaluated. Based on the sequence variation of the S2 gene between the EIAV virulent strains and vaccine strains, all the four vaccine-specific sites in the S2 of an EIAV(FDDV) infectious clone, pFDDV3-8, were reverse-mutated to the sequences of the virulent strain EIAV(DV115). The reverse-mutated molecula...
Sensitization of skin mast cells with IgE antibodies to Culicoides allergens occurs frequently in clinically healthy horses. IgE antibodies are mediators of mast cell degranulation during allergic diseases. The binding of IgE to its high-affinity IgE receptor on mast cell surfaces is called "sensitization" and precedes the development of clinical allergy. Previously, intradermal injection of anti-IgE or the anti-IgG(T) antibody CVS40 induced immediate skin reactions in horses. This suggested that both IgE and IgG(T) sensitize equine skin mast cells. Here, we investigated sensitization to allergen and with IgE or IgG(T) in clinically healthy horses of different age groups. In addition, immediate skin reactions to Cul...
In vitro susceptibility patterns of Aspergillus and Fusarium species isolated from equine ulcerative keratomycosis cases in the midwestern and southern United States with inclusion of the new antifungal agent voriconazole. To evaluate and compare the in vitro susceptibility of Aspergillus and Fusarium spp. isolated from horses with ulcerative keratomycosis, address regional differences in equine keratomycosis isolates, and provide susceptibility data to update prior studies. Methods: Fourteen horses with ulcerative keratomycosis. Methods: Banked fungal isolates from equine corneal ulcers (eight Aspergillus spp. and six Fusarium spp.) were identified at The University of Texas Health Science Center at San Antonio. In vitro minimum inhibitory concentration and susceptibility to natamycin, fluconazole, itraconazole...
Karyotypic relationships in Asiatic asses (kulan and kiang) as defined using horse chromosome arm-specific and region-specific probes. Cross-species chromosome painting has been applied to most of the species making up the numerically small family Equidae. However, comparative mapping data were still lacking in Asiatic asses kulan (Equus hemionus kulan) and kiang (E. kiang). The set of horse arm-specific probes generated by laser microdissection was hybridized onto kulan (E. hemionus kulan) and kiang (E. kiang) chromosomes in order to establish a genome-wide chromosomal correspondence between these Asiatic asses and the horse. Moreover, region-specific probes were generated to determine fusion configuration and orientation of...
Differential modulation of lipopolysaccharide-induced expression of inflammatory genes in equine monocytes through activation of adenosine A2A receptors. Adenosine is an endogenous nucleoside that has potent receptor-mediated immunomodulatory effects on macrophage/monocyte function. In this study, we determined the effects of an adenosine A(2A) receptor agonist, ATL313, on the expression of mRNAs for four pro-inflammatory mediators, IL-1beta, IL-8, COX-2, and TNF-alpha, and the mRNA and protein for the anti-inflammatory cytokine, IL-10 in equine monocytes incubated with lipopolysaccharide (LPS). The results indicate that ATL313 significantly reduces LPS-induced expression of COX-2 and TNF-alpha, enhances the expression of IL-10 and IL-8, but do...
The effects of oral glucosamine on joint health: is a change in research approach needed? Oral glucosamine (GlcN) has been widely studied for its potential therapeutic benefits in alleviating the pain and disability of osteoarthritis (OA). Its popularity has grown despite ongoing controversy regarding its effectiveness vs placebo in clinical trials, and lack of information regarding possible mechanisms of action. Here, we review the state of knowledge concerning the biology of GlcN as it relates to OA, and discuss a framework for future research directions. Methods: An editorial "narrative" review of peer-reviewed publications is organized into four topics (1) Chemistry and pharmac...
A pilot in vitro biomechanical comparison of locking compression plate fixation and kerf-cut cylinder fixation for ventral fusion of fourth and fifth equine cervical vertebrae. The mechanical properties of equine cervical vertebrae joined by implants have not yet been reported. Locking compression plates (LCP) may provide a useful alternative system to the commonly used stainless steel kerf-cut cylinders (KCC) currently used for fixation of cervical vertebrae in horses. Objective: The objectives of this study were to establish a method for biomechanical testing of equine C4-C5 articulations and to compare the biomechanical properties of cadaveric spines stabilised with KCC and LCP. Methods: Twenty-four equine cadaveric cervical spines were size measured from radiogra...
Chondrogenic potential of mesenchymal stromal cells derived from equine bone marrow and umbilical cord blood. Orthopaedic injury is the most common cause of lost training days or premature retirement in the equine athlete. Cell-based therapies are a potential new treatment option in musculo-skeletal diseases. Mesenchymal stromal cells (MSC) have been derived from multiple sources in the horse including bone marrow and umbilical cord blood. The objective of this study was to provide an in vitro comparison of the chondrogenic potential in MSC derived from adult bone marrow (BM-MSC) and umbilical cord blood (CB-MSC). Results: MSC from both sources produced tissue with cartilage-like morphology that stain...
Comparative analysis of c-kit gene expression and c-Kit immunoreactivity in horses with and without obstructive intestinal disease. Previous immunohistochemical studies targeting the receptor tyrosine kinase (c-Kit) have demonstrated an apparent reduction in the number of gastrointestinal pacemaker cells--the interstitial cells of Cajal (ICC)--in horses with intestinal motility disorders. This study compared the level of transcription of the c-kit gene encoding this receptor in horses with and without such motility disorders. Transcription levels of this gene were also compared to the density of ICC immunohistochemically positive for the c-Kit antigen. Intestinal samples were collected from 18 horses with intestinal diseas...
Cholesterol addition protects membrane intactness during cryopreservation of stallion sperm. Addition of cholesterol to sperm membranes improved equine sperm stability during semen cryopreservation; however, it also reduced in vivo fertility. The objective of the present study was to determine the effects of adding cholesterol to stallion sperm prior to freezing, and subsequently removing it from frozen-thawed sperm. Semen from 12 stallions was subjected to four treatments: (T1) control, semen was diluted with Kenney extender, centrifuged, and resuspended to 100 x 10(6)spermatozoa/mL in INRA 82 freezing extender, packaged into 0.5-mL straws, cooled to 5 degrees C, and cryopreserved in...
Cholesterol-loaded-cyclodextrins and fertility potential of stallions spermatozoa. Irreversible damage occurs to spermatozoal membranes, during the phase transition, when spermatozoa are cooled from room temperature to 5 degrees C. Some of this damage can be ameliorated by adding cholesterol to the membrane, thereby altering membrane lipid composition. Adding cholesterol-loaded cyclodextrins (CLCs) to stallion spermatozoa prior to freezing, increases cell cryosurvival. However, the fertilizing potential of CLC-treated stallion spermatozoa is unknown. To address this, experiments were conducted which evaluated the ability of CLC-treated stallion spermatozoa to capacitate, acr...
An in vitro biomechanical comparison of dynamic condylar screw plate combined with a dorsal plate and double plate fixation of distal diaphyseal radial osteotomies in adult horses. To compare stiffness and strength of a dynamic condylar screw plate combined with dorsal broad dynamic compression plate (DCS-bDCP) fixation with double broad dynamic compression plate (dbDCP) fixation used to repair oblique distal fractures of adult equine radii. Methods: Experimental. Methods: Adult equine radii (n=10 pair). Methods: An unconstrained three-dimensional loading-measurement system was used to determine stiffness of a 50 mm long intact, and then DCS-bDCP or dbDCP-plated osteotomized/ostectomized segment of radii when subjected to a nondestructive sequence of compression, torsion...
Characterization of matrix metalloproteinase-2 and matrix metalloproteinase-9 and their inhibitors in equine granulosa cells in vivo and in vitro. Matrix metalloproteinases (MMP) and tissue inhibitors of MMP (TIMP) regulate tissue remodeling events necessary for ovulation. Thus, changes in MMP and TIMP expression and protein enzyme activity were examined in vivo and in vitro during follicular development and atresia in the horse. Equine granulosa cells and follicular fluid from medium (15 to 29 mm) healthy and atretic follicles and from large (>30 mm) healthy and preovulatory follicles were collected by transvaginal aspiration. The cells were either snap-frozen (in vivo study) or cultured for 48 h (in vitro study) to determine gene ex...
Bovine papillomavirus type 1 oncoprotein E5 inhibits equine MHC class I and interacts with equine MHC I heavy chain. Bovine papillomavirus type 1 is one of the aetiological agents of equine sarcoids. The viral major oncoprotein E5 is expressed in virtually all sarcoids, sarcoid cell lines and in vitro-transformed equine fibroblasts. To ascertain whether E5 behaves in equine cells as it does in bovine cells, we introduced the E5 open reading frame into fetal equine fibroblasts (EqPalF). As observed in primary bovine fibroblasts (BoPalF), E5 by itself could not immortalize EqPalF and an immortalizing gene, such as human telomerase (hTERT/hT), was required for the cells to survive selection. The EqPalF-hT-1E5 c...
Equine herpesvirus type 1 (EHV-1) utilizes microtubules, dynein, and ROCK1 to productively infect cells. To initiate infection, equine herpesvirus type 1 (EHV-1) attaches to heparan sulfate on cell surfaces and then interacts with a putative glycoprotein D receptor(s). After attachment, virus entry occurs either by direct fusion of the virus envelope with the plasma membrane or via endocytosis followed by fusion between the virus envelope and an endosomal membrane. Upon fusion, de-enveloped virus particles are deposited into the cytoplasm and travel to the nucleus for viral replication. In this report, we examined the mechanism of EHV-1 intracellular trafficking and investigated the ability of EH...
Analysis of complete genome sequence of Neorickettsia risticii: causative agent of Potomac horse fever. Neorickettsia risticii is an obligate intracellular bacterium of the trematodes and mammals. Horses develop Potomac horse fever (PHF) when they ingest aquatic insects containing encysted N. risticii-infected trematodes. The complete genome sequence of N. risticii Illinois consists of a single circular chromosome of 879 977 bp and encodes 38 RNA species and 898 proteins. Although N. risticii has limited ability to synthesize amino acids and lacks many metabolic pathways, it is capable of making major vitamins, cofactors and nucleotides. Comparison with its closely related human pathogen N. senn...
Does the microbial flora in the ejaculate affect the freezeability of stallion sperm? In an attempt to evaluate the possible relationship between the microbial flora in the stallion ejaculate and its ability to freeze,three ejaculates from five stallions were frozen using a standard protocol. Before freezing, an aliquot was removed for bacteriological analysis. Bacterial growth was observed in all the ejaculates studied. The isolated microorganisms were:Staphylococcus spp. and Micrococcus spp. (in all the stallions), beta-haemolytic Streptococcus (in stallions 3 and 4), Corynebacterium spp. (in stallions 1, 3-5), Rhodococcus spp. (in stallion number 2), Pseudomonas spp. (in sta...
