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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.
Use of heavy water (D2O) in developing thermostable recombinant p26 protein based enzyme-linked immunosorbent assay for serodiagnosis of equine infectious anemia virus infection. Thermostabilizing effect of heavy water (D2O) or deuterium oxide has been demonstrated previously on several enzymes and vaccines like oral poliovirus vaccine and influenza virus vaccine. In view of the above observations, effect of heavy water on in situ thermostabilization of recombinant p26 protein on enzyme-linked immunosorbent assay (ELISA) for serodiagnosis of equine infectious anemia virus (EIAV) infection was investigated in the present study. The carbonate-bicarbonate coating buffer was prepared in 60% and 80% D2O for coating the p26 protein in 96-well ELISA plate and thermal stabilit...
Cryopreservation of epididymal stallion sperm. Any event that makes semen collection or mating impossible, such as death, castration, or injury, may terminate a stallion's breeding career. Fortunately, stallion sperm which are capable of fertilization can be harvested from the epididymis, and frozen for future use. However, the fertility of frozen-thawed epididymal sperm has been found to be lower than that of ejaculated sperm. Therefore, this study aimed to optimize the fertility of frozen epididymal stallion sperm by investigating the effects of different cryoprotectants and freezing protocols on sperm quality. Dimethylformamide was test...
Proteomic analysis of tendon extracellular matrix reveals disease stage-specific fragmentation and differential cleavage of COMP (cartilage oligomeric matrix protein). During inflammatory processes the extracellular matrix (ECM) is extensively remodeled, and many of the constituent components are released as proteolytically cleaved fragments. These degradative processes are better documented for inflammatory joint diseases than tendinopathy even though the pathogenesis has many similarities. The aims of this study were to investigate the proteomic composition of injured tendons during early and late disease stages to identify disease-specific cleavage patterns of the ECM protein cartilage oligomeric matrix protein (COMP). In addition to characterizing fragme...
Thiol-disulfide proteins of stallion epididymal spermatozoa. Thiol groups of cysteine residues represent redox centers involved in multiple biological functions. It has been postulated that changes in the redox status of mammalian epididymal spermatozoa contribute to the sperm maturation process. The present work shows the thiol-disulfide protein profile of stallion epididymal spermatozoa achieved by two-dimension electrophoresis and MALDI-TOF/TOF mass spectrometry of proteins labeled with a thiol-reactive fluorescent tag, monobromobimane. Our results have shown the formation of disulfide bonds in several sperm protein fractions during the epididymal ma...
Characterization of arginase expression by equine neutrophils. Neutrophils are the predominant cells recruited in the airways of horses suffering from heaves. These cells have been shown to express arginase in some species. The metabolism of l-arginine is thought to be involved in chronic inflammation, and airway obstruction and remodeling. The aim of this study was to assess the expression, regulation, activity, and functional role of arginase isoforms in equine neutrophils. Arginase I, arginase II, ornithine decarboxylase (ODC) and ornithine aminotransferase (OAT) expression were assessed in resting and stimulated (IL-4, LPS/fMLP, PMA; 5 and 18 h) blood...
Characterization of bovine ruminal and equine cecal microbial populations enriched for enhanced nitro-toxin metabolizing activity. The nitrotoxins 3-nitro-1-propionic acid (NPA) and 3-nitro-1-propanol (NPOH) are produced by a wide variety of leguminous plants, including over 150 different species and varieties of Astragalus potentially grazed by livestock. These toxins are known to be detoxified by at least one ruminal bacterium but detoxification by bacteria from other gut habitats is not known. In the present study, mixed populations of bovine ruminal and equine cecal microbes were enriched for NPA-metabolizing bacteria via consecutive 24-72 h culture in a basal minimal rumen fluid-based medium supplemented with 4.2 mM ...
Case-Study Investigation of Equine Maternity via PCR-RFLP: A Biochemistry Laboratory Experiment. A simple and robust biochemistry laboratory experiment is described that uses restriction fragment length polymorphism (RFLP) of polymerase chain reaction (PCR) products to verify the identity of a potentially valuable horse. During the first laboratory period, students purify DNA from equine samples and amplify two loci of mitochondrial DNA. During the second laboratory period, students digest PCR products with restriction enzymes and analyze the fragment sizes through agarose gel electrophoresis. An optional step of validating DNA extracts through realtime PCR can expand the experiment to th...
Capacitation in the presence of methyl-β-cyclodextrin results in enhanced zona pellucida-binding ability of stallion spermatozoa. While IVF has been widely successful in many domesticated species, the development of a robust IVF system for the horse remains an elusive and highly valued goal. A major impediment to the development of equine IVF is the fact that optimised conditions for the capacitation of equine spermatozoa are yet to be developed. Conversely, it is known that stallion spermatozoa are particularly susceptible to damage arising as a consequence of capacitation-like changes induced prematurely in response to semen handling and transport conditions. To address these limitations, this study sought to develop a...
Equine herpesvirus type 1 (EHV-1)-induced rearrangements of actin filaments in productively infected primary murine neurons. Equine herpesvirus type 1 (EHV-1) causes respiratory disease, abortion and neurological disorders in horses. In the present study, we investigated reorganization of the cytoskeleton in neurons infected with two EHV-1 strains: Jan-E (wild-type strain) and Rac-H (attenuated strain). The studies were performed on primary murine neurons, which are an excellent model for studying neurotropism and neurovirulence of EHV-1. We have demonstrated for the first time that EHV-1 infection causes rearrangements in the actin network of neurons that are dependent on the virus strain and its adaptation to cell...
In vitro effectiveness of the antimicrobial peptide eCATH1 against antibiotic-resistant bacterial pathogens of horses. The equine antimicrobial peptide eCATH1 previously has been shown to have in vitro activity against antibiotic-susceptible reference strains of Rhodococcus equi and common respiratory bacterial pathogens of foals. Interestingly, eCATH1 was also found to be effective in the treatment of R. equi infection induced in mice. The aim of this study was to assess the in vitro activity of eCATH1 against equine isolates of Gram-negative (Escherichia coli, Salmonella enterica, Klebsiella pneumoniae and Pseudomonas spp.) and Gram-positive (R. equi, Staphylococcus aureus) bacteria resistant to multiple cla...
In vitro analysis of equine, bone marrow-derived mesenchymal stem cells demonstrates differences within age- and gender-matched horses. Stem cell therapies are used routinely in equine practice. Most published reports characterise stem cells derived from younger horses; however, middle-aged horses are often in athletic performance, and experience degenerative medical conditions. Thus, mesenchymal stem cells (MSCs) from this group should be investigated. Objective: To describe differences in in vitro adherence, proliferation and potential for differentiation of equine bone marrow-derived MSCs (equine BMMSCs) harvested from middle-aged (10-13 years old) female donors. Methods: Descriptive study of stem cell characteristics. Meth...
Combined use of platelet rich plasma and vitamin C positively affects differentiation in vitro to mesodermal lineage of adult adipose equine mesenchymal stem cells. Repair of injured soft and hard tissues in horses can benefit greatly from the use of regenerative therapies with mesenchymal stem cells (MSC). Vitamin-C and platelet-rich-plasma had been used for in vitro differentiation of MSC. This study was aimed to evaluate the effect of vitamin-C, platelet-rich-plasma and their combination on the in vitro differentiation of adipose horse MSC. We isolated MSC from horse fat and differentiated them in vitro into osteogenic and chondrogenic lineages, as demonstrated by specific staining and RT-qPCR of selected genes. Combining vitamin-C and plasma-rich-plat...
IgE and IgG epitope mapping by microarray peptide-immunoassay reveals the importance and diversity of the immune response to the IgG3 equine immunoglobulin. The presence of whole horse IgG in therapeutic snake antivenom preparations of high purity is a contamination that can cause IgE-mediated allergic reactions in patients. In this study, the immunodominant IgE and IgG-binding epitopes in horse heavy chain IgG3 were mapped using arrays of overlapping peptides synthesized directly onto activated cellulose membranes. Pooled human sera from patients with and without horse antivenom allergies were used to probe the membrane. We have demonstrated that, for both cases, individuals produce antibodies to epitopes of sequential amino acids of horse heavy ...
Effect of Cholesterol and Equex-STM Addition to an Egg Yolk Extender on Pure Spanish Stallion Cryopreserved Sperm. Cholesterol and Equex-STM are frequently added to different commercial and experimental extenders improving postthawing sperm quality. Doses of 125-150 mM of cholesterol from pig liver and 0.5-0.7% of Equex-STM were evaluated in a standard eggyolk extender (Martin et al., 1979). Six ejaculates per stallion from six pure Spanish stallions (6-8 years old) were collected in Martin's extender (B) and different mixtures of 125 mM-0.5% (I), 125 mM-0.7% (II), 150 mM-0.5% (III), and 150 mM-0.7% (IV) were added to original Martin's extender. Samples were frozen in 0.5 mL straws (100 × 10(6...
In vitro reproduction of the life cycle of Pythium insidiosum from kunkers’ equine and their role in the epidemiology of pythiosis. Pythium insidiosum is an important pathogen of mammals' species, including humans. Equine is the main species affected by this oomycete. P. insidiosum requires an aquatic environment to develop its life cycle, and the susceptible hosts are contaminated when they contact the microorganism in swampy areas. The equine pythiosis is characterized by the formation of irregular masses within the cutaneous lesions, called kunkers, which easily detach from the lesion. From these structures, it is possible to isolate P. insidiosum in pure cultures. The present study aimed to reproduce in vitro the life ...
ASVCP guidelines: allowable total error guidelines for biochemistry. As all laboratory equipment ages and contains components that may degrade with time, initial and periodically scheduled performance assessment is required to verify accurate and precise results over the life of the instrument. As veterinary patients may present to general practitioners and then to referral hospitals (both of which may each perform in-clinic laboratory analyses using different instruments), and given that general practitioners may send samples to reference laboratories, there is a need for comparability of results across instruments and methods. Allowable total error (TEa ) is ...
Comparative Labeling of Equine and Ovine Multipotent Stromal Cells With Superparamagnetic Iron Oxide Particles for Magnetic Resonance Imaging In Vitro. The purpose of this study was to evaluate the use of three different superparamagnetic iron oxide (SPIO) particles for labeling of ovine and equine bone marrow (BM)-derived multipotent stromal cells (MSCs) in vitro. MSCs were obtained from five adult sheep and horses, respectively. After three passages (p3), cells were labeled with either 1) Molday ION Rhodamine B, 2) Endorem, 3) Resovist, or 4) remained unlabeled as control. Labeling efficiency, marker retention, and long-term detectability in MRI until p7 were evaluated. Further, proliferation capacity and trilineage differentiation as indic...
Isolation and characterization of equine nasal mucosal CD172a + cells. The nasal mucosa surface is continuously confronted with a broad variety of environmental antigens, ranging from harmless agents to potentially harmful pathogens. This area is under rigorous control of professional antigen presenting cells (APCs), such as dendritic cells (DCs) and macrophages. Mucosal APCs play a crucial role in inducing primary immune responses and the establishment of an immunological memory. In the present study, a detailed characterization of CD172a(+) cells, containing the APCs residing in the equine nasal mucosa was performed for the first time. CD172a(+) cells were isol...
Maintenance of arytenoid abduction following carbon dioxide laser debridement of the articular cartilage and joint capsule of the cricoarytenoid joint combined with prosthetic laryngoplasty in horses: an in vivo and in vitro study. The objective was to evaluate CO2 laser debridement of the cricoarytenoid joint (CAJ) combined with prosthetic laryngoplasty to prevent post-operative loss of arytenoid abduction in seven horses. Horses were assigned to either laser debridement of the left CAJ and laryngoplasty (laser treated, n=5) or control laryngoplasty (sham, n=2), and were evaluated with endoscopic examinations and measurement of right to left angle quotients (RLQ) to assess maintenance of arytenoid abduction. The animals were euthanased at intervals after surgery and larynges were harvested for post-mortem testing, inclu...
Plasma and synovial fluid concentration of doxycycline following low-dose, low-frequency administration, and resultant inhibition of matrix metalloproteinase-13 from interleukin-stimulated equine synoviocytes. To determine whether low-dose, low-frequency doxycycline administration is capable of achieving chondroprotective concentrations within synovial fluid (SF) while remaining below minimum inhibitory concentration 90 (MIC90 ) of most equine pathogens and would be an option in the management of osteoarthritis. Objective: To determine whether low-dose, low-frequency oral administration of doxycycline can attain in vivo SF concentrations capable of chondroprotective effects through reduction of matrix metalloproteinase (MMP)-13 activity, while remaining below MIC90 of most equine pathogens. Method...
In vitro and in vivo evaluation of a new large animal spirometry device using mainstream CO2 flow sensors. A spirometry device equipped with mainstream CO2 flow sensor is not available for large animal anaesthesia. Objective: To measure the resistance of a new large animal spirometry device and assess its agreement with reference methods for volume measurements. Methods: In vitro experiment and crossover study using anaesthetised horses. Methods: A flow partitioning device (FPD) equipped with 4 human CO2 flow sensors was tested. Pressure differences were measured across the whole FPD and across each sensor separately using air flows (range: 90-720 l/min). One sensor was connected to a spirometry ...
A nonsense mutation in the IKBKG gene in mares with incontinentia pigmenti. Ectodermal dysplasias (EDs) are a large and heterogeneous group of hereditary disorders characterized by abnormalities in structures of ectodermal origin. Incontinentia pigmenti (IP) is an ED characterized by skin lesions evolving over time, as well as dental, nail, and ocular abnormalities. Due to X-linked dominant inheritance IP symptoms can only be seen in female individuals while affected males die during development in utero. We observed a family of horses, in which several mares developed signs of a skin disorder reminiscent of human IP. Cutaneous manifestations in affected horses includ...
Effect of potential oocyte transport protocols on blastocyst rates after intracytoplasmic sperm injection in the horse. Intracytoplasmic sperm injection (ICSI) is used to produce foals from otherwise infertile mares and from stallions with limited sperm stores, but requires expensive equipment and is technically demanding. Methods to transport oocytes to ICSI laboratories would allow collection of oocytes by the referring veterinarian and enable greater application of this technique. Objective: This study was conducted to evaluate protocols that could be used to transport immature and maturing oocytes for ICSI. Methods: In vitro experiment. Methods: Oocytes were recovered by transvaginal ultrasound-guided folli...
Replication of avian influenza viruses in equine tracheal epithelium but not in horses. We evaluated a hypothesis that horses are susceptible to avian influenza viruses by in vitro testing, using explanted equine tracheal epithelial cultures, and in vivo testing by aerosol inoculation of ponies. Results showed that several subtypes of avian influenza viruses detectably replicated in vitro. Three viruses with high in vitro replication competence were administered to ponies. None of the three demonstrably replicated or caused disease signs in ponies. While these results do not exhaustively test our hypothesis, they do highlight that the tracheal explant culture system is a poor pre...
The effect of environmental factors on sister chromatid exchange incidence in domestic horse (Equus caballus) chromosomes. The SCE test is often used as a sensitive and reliable technique in the biomonitoring of genotoxicity of mutagenic and carcinogenic agents. This study analysed the frequency of sister chromatid exchange in domestic horse chromosomes depending on the habitat and age of the analysed horses. The chromosome preparations were obtained from an in vitro culture of peripheral blood lymphocytes stained using the FPG technique. Both the habitat and the age significantly influence SCE frequency. A higher SCE incidence was observed in horses that lived in a large urban agglomeration than in those from the...
Effect of single layer centrifugation using Androcoll-E-Large on the sperm quality parameters of cooled-stored donkey semen doses. The aim of this study was to determine the effect of single layer centrifugation (SLC) using Androcoll-E-Large on donkey sperm quality parameters after 24 h of cool-storage. Ejaculates were collected from Andalusian donkeys and then cooled at 5°C. SLC was carried out after 24 h of cool-storage using Androcoll-E-Large. In the first experiment, all sperm parameters assessed (total and progressive sperm motility, viability, sperm morphology and sperm kinematics VCL, VSL, VAP, LIN, STR, WOB, ALH and BCF) were statistically compared between semen samples processed or not with Androcoll-E-Large. Si...
Molecular characterization and evolutionary analysis of horse BAFF-R, a tumor necrosis factor receptor related to B-cell survival. B-cell survival depends on signals induced by B-cell activating factor (BAFF) that binds to the BAFF receptor (BAFF-R). Herein, a BAFF-R homolog was identified in a horse (Equus caballus). The horse BAFF-R gene, located on chromosome 28, spans 1444 base pairs and encodes a 183-amino acid protein. The protein is structurally conserved, in which the DxL motif plays an important role in binding to BAFF. Furthermore, the horse BAFF-R extracellular domain was expressed and purified, which specifically bound to His6-sBAFF and had the capability of blocking the function of His6-sBAFF in vitro. Finall...
Ovum pick up, intracytoplasmic sperm injection and somatic cell nuclear transfer in cattle, buffalo and horses: from the research laboratory to clinical practice. Assisted reproductive techniques developed for cattle in the last 25 years, like ovum pick up (OPU), intracytoplasmic sperm injection (ICSI), and somatic cell nuclear transfer, have been transferred and adapted to buffalo and horses. The successful clinical applications of these techniques require both the clinical skills specific to each animal species and an experienced laboratory team to support the in vitro phase of the work. In cattle, OPU can be considered a consolidated technology that is rapidly outpacing conventional superovulation for embryo transfer. In buffalo, OPU represents the ...
CatSper and the relationship of hyperactivated motility to intracellular calcium and pH kinetics in equine sperm. In vitro fertilization does not occur readily in the horse. This may be related to failure of equine sperm to initiate hyperactivated motility, as treating with procaine to induce hyperactivation increases fertilization rates. In mice, hyperactivated motility requires a sperm-specific pH-gated calcium channel (CatSper); therefore, we investigated this channel in equine sperm. Motility was assessed by computer-assisted sperm motility analysis and changes in intracellular pH and calcium were assessed using fluorescent probes. Increasing intracellular pH induced a rise in intracellular calcium, w...
