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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.
Evaluation of the effects of the R- and S-enantiomers of salbutamol on equine isolated bronchi. Equine obstructive pulmonary disease, also known as heaves or recurrent airway obstruction (RAO) is a common equine pulmonary disease with some similarities to human asthma and COPD, which represents a major cause of morbidity and loss of lung performance. Salbutamol has been widely used for the treatment of human airway diseases and has usually been prepared as the racemic form of the drug. However, recently the R-enantiomer of salbutamol has been introduced into clinical practice in the treatment of asthma in humans and this has been suggested to be an improvement on the racemic form of the ...
Comparison of equine bone marrow-, umbilical cord matrix and amniotic fluid-derived progenitor cells. The aim of the study was to compare in vitro the stemness features of horse progenitor cells derived from bone marrow (BM-MSCs), amniotic fluid (AF-MSCs) and umbilical cord matrix (EUC-MSCs). It has been suggested that there may be a stem cell population within both umbilical cord matrix and amniotic fluid. However, little knowledge exists about the characteristics of these progenitor cells within these sources in the equine species. This study wanted to investigate an alternative and non-invasive stem cell source for the equine tissue engineering and to learn more about the properties of thes...
Different contribution of bovine papillomavirus type 1 oncoproteins to the transformation of equine fibroblasts. Equine sarcoids represent the most common skin tumours in equids worldwide, characterized by localized invasion, rare regression and high recurrence following surgical intervention. Bovine papillomavirus type 1 (BPV-1) and less commonly BPV-2 are now widely recognized as the causative agents of the disease. Fibroblasts isolated from sarcoids are highly invasive. Invasion is associated with a high level of viral gene expression and matrix metalloproteinase upregulation. However, it remains unclear to what extent BPV-1 proteins are involved in the transformation of equine cells. To address this ...
Influence of seminal plasma on fresh and post-thaw parameters of stallion epididymal spermatozoa. Fresh and post-thaw parameters (motility, morphology and viability) of stallion epididymal spermatozoa that have been and have not been exposed to seminal plasma were evaluated, and directly compared to fresh and post-thaw parameters of ejaculated spermatozoa. Six sperm categories of each stallion (n=4) were evaluated for motility, morphology and viability. These categories were fresh ejaculated spermatozoa (Fr-E), fresh epididymal spermatozoa that had been exposed to seminal plasma (Fr-SP+), fresh epididymal spermatozoa that had never been exposed to seminal plasma (Fr-SP-), frozen-thawed eja...
Receptor-mediated enhancement of beta adrenergic drug activity by ascorbate in vitro and in vivo. Previous in vitro research demonstrated that ascorbate enhances potency and duration of activity of agonists binding to alpha 1 adrenergic and histamine receptors. Objective: Extending this work to beta 2 adrenergic systems in vitro and in vivo. Methods: Ultraviolet spectroscopy was used to study ascorbate binding to adrenergic receptor preparations and peptides. Force transduction studies on acetylcholine-contracted trachealis preparations from pigs and guinea pigs measured the effect of ascorbate on relaxation due to submaximal doses of beta adrenergic agonists. The effect of inhaled albuter...
Expansion of mesenchymal stem cells on fibrinogen-rich protein surfaces derived from blood plasma. Mesenchymal stem cells (MSCs) are present in low density in bone marrow and culture expansion is necessary to obtain sufficient numbers for many proposed therapies. Researchers have characterized MSC growth on tissue culture plastic (TCP), although few studies have explored proliferation on other growth substrates. Using adult equine MSCs, we evaluated proliferation on fibrinogen-rich precipitate (FRP) surfaces created from blood plasma. When seeded at 1 × 10(4) cells/cm(2) and passaged five times over 10 days, MSCs on FRP in medium containing fibroblast growth factor 2 (FGF2) resulted in a ...
Use of a defined diluent increases the sex-sorting efficiency of stallion sperm. The low efficiency of flow cytometric sex-sorting of stallion sperm has been attributed to the use of an opaque skim milk-based diluent during Hoechst 33342 (H33342) staining. Three experiments were conducted to formulate an optically clear stallion semen diluent for use during H33342 staining, and to determine whether a clear diluent improved resolution during sorting. For Experiment 1, sperm were incubated at 34 °C in each of five diluents containing either no protein, skim milk, 0.25% Cohn's Fraction V BSA, 0.5% BSA, or 1% BSA, following an 18 h storage (15 °C) period, or shortly after co...
Addition of seminal plasma to post-thawing equine semen: what is the effect on sperm cell viability? Effect of seminal plasma addition after thawing on viability or cryocapacitation is not definitively established. This experiment was performed to verify the effect of adding seminal plasma, autologous or homologous (from an animal with good semen freezability). Five ejaculates from each of four stallions with proven fertility were collected and cryopreserved. The semen was subsequently thawed and divided into the following three treatment groups: no seminal plasma addition after semen thawing (NOSP); the addition of homologous seminal plasma after semen thawing (HSP) and the addition of autol...
Subpopulations of equine blood lymphocytes expressing regulatory T cell markers. Several distinct T lymphocyte subpopulations with immunoregulatory activity have been described in a number of mammalian species. This study performed a phenotypic analysis of cells expressing regulatory T cell (Treg) markers in the peripheral blood of a cohort of 18 horses aged 6 months to 23 years, using antibodies to both intracellular and cell surface markers, including Forkhead box P3 (FOXP3), CD4, CD8, CD25, interferon gamma (IFNγ) and interleukin 10 (IL-10). In peripheral blood, a mean of 2.2 ± 0.2% CD4+ and 0.5 ± 0.1% CD8+ lymphocytes expressed FOXP3. The mean percentage of CD4+FOXP...
Use of in vitro technologies to study phase II conjugation in equine sports drug surveillance. Within equine drug surveillance, there is significant interest in analyzing intact phase II conjugates of drugs in urine, but progress has been limited by a lack of reference material. Methods: In this study, in vitro techniques using equine liver fractions were employed to produce glucuronide and sulfate conjugates of stanozolol, 16β-hydroxystanozolol and nandrolone, the glucuronide conjugate of morphine and the glutathione metabolite of chlordinitrobenzene for the first time in equine sports drug surveillance. Results: The glucuronide conjugate of the synthetic progestagen altrenogest was a...
Comparative human-horse sequence analysis of the CYP3A subfamily gene cluster. Cytochrome P450 enzymes (CYP450s) represent a superfamily of haem-thiolate proteins. CYP450s are most abundant in the liver, a major site of drug metabolism, and play key roles in the metabolism of a variety of substrates, including drugs and environmental contaminants. Interaction of two or more different drugs with the same enzyme can account for adverse effects and failure of therapy. Human CYP3A4 metabolizes about 50% of all known drugs, but little is known about the orthologous CYP450s in horses. We report here the genomic organization of the equine CYP3A gene cluster as well as a compara...
Genomic structure, polymorphism and expression of ACCN1 and ACCN3 genes in the horse. A category of cation gate proteins was shown to be present in sensory neurons and act as receptors of protons present in tissues such as muscles. The Amiloride-sensitive Cation Channel, Neuronal (ACCN) gene family is known to play a role in the transmission of pain through specialized pH sensitive neurons. Muscles from horses submitted to strenuous exercises produce lactic acid, which may induce variable pain through ACCN differential properties. The sequences of the equine cDNAs were determined to be 2.6 kb in length with an open reading frame of 1539 bp for ACCN1 and 2.1 kb in length with an...
Cardiolipin modulates allosterically peroxynitrite detoxification by horse heart cytochrome c. Upon interaction with bovine heart cardiolipin (CL), horse heart cytochrome c (cytc) changes its tertiary structure disrupting the heme-Fe-Met80 distal bond, reduces drastically the midpoint potential out of the range required for its physiological role, binds CO and NO with high affinity, and displays peroxidase activity. Here, the effect of CL on peroxynitrite isomerization by ferric cytc (cytc-Fe(III)) is reported. In the absence of CL, hexa-coordinated cytc does not catalyze peroxynitrite isomerization. In contrast, CL facilitates cytc-Fe(III)-mediated isomerization of peroxynitrite in a d...
Effects of non-steroidal anti-inflammatory drugs on proliferation, differentiation and migration in equine mesenchymal stem cells. In equine medicine, stem cell therapies for orthopaedic diseases are routinely accompanied by application of NSAIDs (non-steroidal anti-inflammatory drugs). Thus, it has to be analysed how NSAIDs actually affect the growth and differentiation potential of MSCs (mesenchymal stem cells) in vitro in order to predict the influence of NSAIDs such as phenylbutazone, meloxicam, celecoxib and flunixin on MSCs after grafting in vivo. The effects of NSAIDs were evaluated regarding cell viability and proliferation. Additionally, the multilineage differentiation capacity and cell migration was analysed. N...
Antimicrobial disposition in pulmonary epithelial lining fluid of horses, part III. cefquinome. Cefquinome concentrations, following intravenous and aerosol administration to horses, in pulmonary epithelial lining fluid (PELF) were examined and compared to plasma concentrations. Single dose of cefquinome sulphate (1 mg/kg) was administered intravenously to six horses followed by a single aerosol administration (225 mg) with a wash-out period of 14 days between treatments. After each drug administration, cefquinome concentrations in plasma and PELF, obtained by intrabronchial cotton swabs, were determined. After intravenous administration, cefquinome concentrations in plasma declined fast...
Adipogenic differentiation of adult equine mesenchymal stromal cells. Equine adipose tissue-derived mesenchymal stem cells (ASCs) have only recently been investigated for their adipogenic, chondrogenic, and osteogenic differentiation potential. This chapter will briefly outline the molecular mechanisms leading to adipogenesis and the methods of equine adipose tissue harvest, ASC isolation, and adipogenic differentiation. The reader is also directed to other reported methods of adipogenesis for ASCs and mesenchymal stem cells (MSCs) from other tissues.
Computed tomography to identify preoperative guidelines for internal fixation of the distal sesamoid bone in horses: an in vitro study. To assess the reliability of computed tomography (CT) to identify the direction of implant insertion for cortical screws along the longitudinal axis of intact (nonfractured) distal sesamoid bones. Methods: In vitro study. Methods: Cadaveric paired equine forelimbs (n=16). Methods: Insertion of a cortical screw in lag fashion along the longitudinal axis of intact (nonfractured) distal sesamoid bones was evaluated in 2 groups (3.5 and 4.5 mm) of 8 paired limbs. In each group, the direction of the distal sesamoid bone was determined by CT (Equine XTC 3000 pQCT scanner). Screw placement was verifi...
Hydrolyzed fish collagen induced chondrogenic differentiation of equine adipose tissue-derived stromal cells. Adipose-derived stromal cells (ADSCs) are multipotent cells which, in the presence of appropriate stimuli, can differentiate into various lineages such as the osteogenic, adipogenic and chondrogenic. In this study, we investigated the effect of transforming growth factor beta 1 (TGF-β1) in comparison to hydrolyzed fish collagen in terms of the chondrogenic differentiation potential of ADSCs. ADSCs were isolated from subcutaneous fat of horses by liposuction. Chondrogenesis was investigated using a pellet culture system. The differentiation medium was either supplemented with TGF-β1 (5 ng/ml)...
Developmental validation of feline, bovine, equine, and cervid quantitative PCR assays. Accurate DNA quantification is essential for optimizing DNA testing and minimizing sample consumption. Real-time quantitative polymerase chain reaction (qPCR) assays have been published for human and canine nuclear DNA, and the need for quantifying other forensically important species was evident. Following the strategy employed for the canine qPCR assay, we developed individual assays to accurately quantify feline, bovine, equine, and cervid nuclear DNA. Each TaqMan-based assay incorporates a genus-specific probe targeting the Melanocortin-1 Receptor gene and includes a piece of synthetic DNA...
Identification of two novel equine papillomavirus sequences suggests three genera in one cluster. The number of recognized papillomavirus (PV) species and potential PV genera has dramatically been increasing throughout the past decade. It seems that every host species might potentially harbour a large set of PVs, while the PVs of each species appear to belong to only a few genera. In horses at least three conditions beside the equine sarcoid have been described, being supposedly PV induced namely classical equine papillomas, genital papillomas and aural plaques. We were able to identify the DNA of novel equine PVs (EcPVs) in the two latter disorders where PV involvement had been predicted....
Effect of different workload and hydrocortisone in vitro on platelet aggregation in athletic horse. The objective of the present study was to evaluate the platelet response to different workload in athletic horse and the influence of hydrocortisone on this response. For this purpose, six Sella Italiana gelding athletic horses, ranging in age from 8 to 10 years and clinically healthy, were subjected to four different exercises: horse-walker, treadmill, obstacle course of 1.00 m and obstacle course of 1.25 m. From all the horses, citrated plasma samples were collected at rest, immediately after exercise and 30 min after the end of the exercise to assess the maximum degree of platelet aggregati...
Insulin-like growth factor-I (IGF-I) protects cultured equine Leydig cells from undergoing apoptosis. Leydig cells located in the interstitial space of the testicular parenchyma produce testosterone which plays a critical role in the maintenance and restoration of spermatogenesis in many species, including horses. For normal spermatogenesis, maintaining Leydig cells is critical to provide an optimal and constant level of testosterone. Recently, an anti-apoptotic effect of IGF-I in testicular cells in rats has been reported, but a similar effect of IGF-I on equine Leydig cells remains to be elucidated. If IGF-I also protects stallion testicular cells from undergoing apoptosis, then IGF-I may ha...
Validation and usefulness of the Sperm Quality Analyzer V equine for equine semen analysis. Routine semen analysis includes evaluation of concentration combined with seminal volume, morphology and motility. Subjective analysis of these parameters is known to be inaccurate, imprecise and subject to variability. Automated semen analysis could lead to an increased standardization in and between laboratories but for that to happen automated devices need to be validated. A new device, the sperm quality analyzer V equine (SQA-Ve) version 1.00.43, was evaluated for its repeatability and agreement with light microscopy (LM), for raw and extended equine semen. Results were compared with compu...
Antimicrobial activity of tulathromycin and 14 other antimicrobials against virulent Rhodococcus equi in vitro. This study determined the antimicrobial activity of tulathromycin against Rhodococcus equi in vitro. Ninety-eight virulent isolates of R. equi from equine clinical cases were examined, of which 20 isolates were macrolide resistant. A custom 96-well antimicrobial susceptibility testing plate was used, allowing 14 additional antimicrobials to be tested against R. equi. Isolates were cultured with various concentrations of antimicrobials, and minimal inhibitory concentration (MIC) values were determined. Tulathromycin was found to have poor activity in vitro against R. equi isolates susceptible o...
Analysis of CD14 expression levels in putative mesenchymal progenitor cells isolated from equine bone marrow. A long-term goal of mesenchymal progenitor cell (MPC) research is to identify cell-surface markers to facilitate MPC isolation. One reported MPC feature in humans and other species is lack of CD14 (lipopolysaccharide receptor) expression. The aim of this study was to evaluate CD14 as an MPC sorting marker. Our hypothesis was that cells negatively selected by CD14 expression would enrich MPC colony formation compared with unsorted and CD14-positive fractions. After validation of reagents, bone marrow aspirate was obtained from 12 horses. Fresh and cultured cells were analyzed by flow cytometry ...
Comparison of the osteogenic potential of equine mesenchymal stem cells from bone marrow, adipose tissue, umbilical cord blood, and umbilical cord tissue. To determine the optimal osteogenic source of equine mesenchymal stem cells (eMSCs) and optimize collection of and expansion conditions for those cells. Methods: 10 adult Quarter Horses and 8 newborn Thoroughbred foals. Methods: eMSCs were isolated from bone marrow (BM), adipose tissue, and umbilical cord blood and tissue, and the osteogenic potential of each type was assessed. Effects of anatomic site, aspiration volume, and serum type on eMSC yield from BM were investigated. Results: BM-eMSCs had the highest overall expression of the osteogenic genes Cbfa1, Osx, and Omd and staining for ALP ...
Expression of genes associated with inflammation induced by ex vivo exposure to lipopolysaccharide in peripheral blood leukocytes from horses with gastrointestinal disease. To investigate the effect of ex vivo exposure to lipopolysaccharide (LPS) on the expression of inflammatory genes in leukocytes from horses with gastrointestinal (Gl) disease and determine whether the pattern or magnitude of the response to LPS correlated with the type of disease and outcome. Methods: 49 horses with Gl disease and 10 healthy horses Methods: Leukocytes were isolated from blood samples and submitted to 3 protocols: immediate freezing, freezing after 4-hour incubation in medium, and freezing after 4-hour incubation in medium containing LPS. Expression of 14 genes associated with ...
Evaluation of the osteogenic and chondrogenic differentiation capacities of equine adipose tissue-derived mesenchymal stem cells. To evaluate the proliferative behavior, telomere length, immunophenotype, and differentiation capacity of equine adipose tissue-derived mesenchymal stem cells (AT-MSCs). Methods: 6 adult racing horses treated for articular Injury but otherwise healthy. Methods: AT-MSCs were Isolated from horses and expanded In Dulbecco modified Eagle medium enriched with fetal bovine serum and antimicrobials. Expression of cell surface antigens and telomere length were Investigated via flow cytometry Differentiation of MSCs Into chondrocytes, osteoblasts, and adipocytes was Induced In vitro by specific stimuli...
The use of in vitro technologies coupled with high resolution accurate mass LC-MS for studying drug metabolism in equine drug surveillance. The detection of drug abuse in horseracing often requires knowledge of drug metabolism, especially if urine is the matrix of choice. In this study, equine liver/lung microsomes/S9 tissue fractions were used to study the phase I metabolism of eight drugs of relevance to equine drug surveillance (acepromazine, azaperone, celecoxib, fentanyl, fluphenazine, mepivacaine, methylphenidate and tripelennamine). In vitro samples were analyzed qualitatively alongside samples originating from in vivo administrations using LC-MS on a high resolution accurate mass Thermo Orbitrap Discovery instrument and by...
The relationship between in vivo limb and in vitro tendon mechanics after injury: a potential novel clinical tool for monitoring tendon repair. Highly prevalent superficial digital flexor tendon (SDFT) injury results in compromised tendon function through fibrosis and high frequency of re-injury due to altered biomechanical function. This study investigated the consequences of SDF tendinopathy on limb mechanics in relation to the mechanical properties of injured tendon. Objective: To develop and validate a noninvasive in vivo assessment of tendon mechanics to investigate the effect of recent SDFT injury on limb stiffness index, providing an objective method to assess quality of healing. Objective: Limb stiffness index would reduce as ...
