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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 an in vitro system to simulate equine foregut digestion and the influence of acidity on protein and fructan degradation in the horse’s stomach. The aim of this study was to improve an in vitro system in order to gather optimized information on the digestion of different forages in the horse's upper gastrointestinal tract. Therefore, foregut digestion of several forages was simulated in vitro (Part 1). The effect of different pH values on in vitro fructan degradation of two selected grasses (Part 2) was tested subsequently. Part 1: We hypothesized that our system produces representative results simulating digestive processes in the upper alimentary tract, but neglects microbial fermentation. In vitro digestion of six forages (grass ...
The in vitro biocompatibility of d-(+) raffinose modified chitosan: Two-dimensional and three-dimensional systems for culturing of horse articular chondrocytes. The present study investigated the biocompatibility of chitosan films and scaffolds modified with d-(+)raffinose and their capability to support the growth and maintenance of the differentiation of articular chondrocytes in vitro. Primary equine articular chondrocytes were cultured on films and scaffolds of modified d-(+) raffinose chitosan. Their behavior was compared to that of chondrocytes grown in conventional bi- and three-dimensional culture systems, such as micromasses and alginate beads. Chitosan films maintained the phenotype of differentiated chondrocytes (typical round morphology) a...
Genotyping horse epithelial cells from fecal matter by isolation of polymerase chain reaction products. To show that application of the polymerase chain reaction (PCR) method modified for amplification of a low-copy number DNA samples, ie, the isolation of PCR products (IPCRp), would represent improvement in obtaining genotypes from a fecal DNA compared with previously used genotyping methods. Methods: The DNA from the horse fecal matter was extracted by modified Qiagen DNA Stool Mini Kit protocol. Following the extraction, the DNA genotypes from fecal samples were obtained by the most powerful PCR amplification method, the IPCRp. The IPCRp-based multiplex kit amplified biotin-labeled strands we...
Effects of liposomal-curcumin on five opportunistic bacterial strains found in the equine hindgut – preliminary study. The horse intestinal tract is sensitive and contains a highly complex microbial population. A shift in the microbial population can lead to various issues such as inflammation and colic. The use of nutraceuticals in the equine industry is on the rise and curcumin is thought to possess antimicrobial properties that may help to minimize the proliferation of opportunistic bacteria. Methods: Four cecally-cannulated horses were utilized to determine the optimal dose of liposomal-curcumin (LIPC) on reducing complex (SBEC), K-12, general, , and in the equine hindgut without adversely affecting ce...
Transforming Growth Factor-β2 Downregulates Major Histocompatibility Complex (MHC) I and MHC II Surface Expression on Equine Bone Marrow-Derived Mesenchymal Stem Cells Without Altering Other Phenotypic Cell Surface Markers. Allogeneic mesenchymal stem cells (MSCs) are a promising cell source for treating musculoskeletal injuries in horses. Effective and safe allogeneic therapy may be hindered, however, by recipient immune recognition and rejection of major histocompatibility complex (MHC)-mismatched MSCs. Development of strategies to prevent immune rejection of MHC-mismatched MSCs in vivo is necessary to enhance cell survival and potentially increase the efficacy and safety of allogeneic MSC therapy. The purposes of this study were to evaluate if transforming growth factor-β2 (TGF-β2) downregulated MHC expressi...
In vitro anti-LPS dose determination of ketorolac tromethamine and in vivo safety of repeated dosing in healthy horses. Flunixin meglumine (FM) is a commonly used Nonsteroidal anti-inflammatory drug (NSAID) in horses, but clinical efficacy is often unsatisfactory. Ketorolac tromethamine (KT) demonstrates superior efficacy compared to other NSAIDs in humans, but its anti-inflammatory effects have not been investigated in the horse. Safety of repeated dosing of KT has not been evaluated. The first objective was to conduct a dose determination study to verify that a previously described dosage of KT would inhibit Lipopolysaccharide (LPS)-induced eicosanoid production in vitro, and to compare KT effects of this inh...
Characterization and use of Equine Bone Marrow Mesenchymal Stem Cells in Equine Cartilage Engineering. Study of their Hyaline Cartilage Forming Potential when Cultured under Hypoxia within a Biomaterial in the Presence of BMP-2 and TGF-ß1. Articular cartilage presents a poor capacity for self-repair. Its structure-function are frequently disrupted or damaged upon physical trauma or osteoarthritis in humans. Similar musculoskeletal disorders also affect horses and are the leading cause of poor performance or early retirement of sport- and racehorses. To develop a therapeutic solution for horses, we tested the autologous chondrocyte implantation technique developed on human bone marrow (BM) mesenchymal stem cells (MSCs) on horse BM-MSCs. This technique involves BM-MSC chondrogenesis using a combinatory approach based on the associ...
The development and integrity of equine pre-antral follicles cultured in vitro with follicle-stimulating hormone (FSH) supplementation. This study investigated the effects of different concentrations of FSH (10, 50, 100 and 200 ng/ml) in supplemented MEM+ on the development of equine pre-antral follicles that were cultured in vitro for 2 or 6 days. The ovaries (n = 5) from mares in seasonal anoestrus were collected from a local abattoir. Ten ovarian tissue fragments of approximately 3 × 3 × 1 mm were obtained from each animal. The fragments were cultured in situ for 2 days (D2) or 6 days (D6) in MEM+ or MEM+ supplemented with FSH at four different concentrations, establishing the following 11 groups: control (D0);...
RNA sample preparation applied to gene expression profiling for the horse biological passport. The improvement of doping control is an ongoing race. Techniques to fight doping are usually based on the direct detection of drugs or their metabolites by analytical methods such as chromatography hyphenated to mass spectrometry after ad hoc sample preparation. Nowadays, omic methods constitute an attractive development and advances have been achieved particularly by application of molecular biology tools for detection of anabolic androgenic steroids (AAS), erythropoiesis-stimulating agent (ESA), or to control human growth hormone misuses. These interesting results across different animal spe...
Seasonal changes of DNA fragmentation and quality of raw and cold-stored stallion spermatozoa. In this study annual fluctuations of DNA fragmentation and quality of cold-stored equine sperm were evaluated. Ejaculates were collected weekly during one year from 15 stallions. Ejaculate volume, sperm concentration and total sperm count were determined and semen was then extended and cold-stored for 48 h. Sperm motility was evaluated by CASA before and after 24 as well as 48 h of cold storage. In addition, the percentages of sperm with intact plasma membrane and acrosome (PMAI %) and with low intracellular Ca2+ level were determined in cold-stored semen (24 h, 48 h). SCSA™ was performe...
Development of a Sustained-Release Voriconazole-Containing Thermogel for Subconjunctival Injection in Horses. To determine in vitro release profiles, transcorneal permeation, and ocular injection characteristics of a voriconazole-containing thermogel suitable for injection into the subconjunctival space (SCS). In vitro release rate of voriconazole (0.3% and 1.5%) from poly (DL-lactide-co-glycolide-b-ethylene glycol-b-DL-lactide-co-glycolide) (PLGA-PEG-PLGA) thermogel was determined for 28 days. A Franz cell diffusion chamber was used to evaluate equine transcorneal and transscleral permeation of voriconazole (1.5% topical solution, 0.3% and 1.5% voriconazole-thermogel) for 24 hours. Antifungal activit...
Effect of bone marrow-derived mesenchymal stem cells and stem cell supernatant on equine corneal wound healing in vitro. We aimed to determine and compare the in vitro effects of autologous bone marrow-derived mesenchymal stem cells (BM-MSCs) and mesenchymal stem cell supernatant (MSC-Sp) on the wound healing capacity of equine corneal fibroblasts using a scratch assay. Bone marrow aspirates and eyes were collected from normal, euthanized horses with subsequent isolation and culture of BM-MSCs and corneal stromal cells. Corneal stromal cells were culture-expanded in the culture well of transwell plates and then treated with an autologous BM-MSC suspension (dose: 2.5 × 105/100 μL media with the BM-MSCs con...
Are sainfoin or protein supplements alternatives to control small strongyle infection in horses? The spread of anthelmintic resistance in equine strongyle nematodes has become a major problem, advocating for the development of alternative control for strongyles. Our study consisted of both in vivo and in vitro experiments. We investigate for the first time the efficacy of a short-term consumption of tannin-rich sainfoin (Onobrychis viciifolia) or extra proteins in naturally infected horses. We used 30 horses allocated into three groups of 10 individuals that received for 18 days either (i) a tannin-rich diet with 70% DM sainfoin pellets (Sd), (ii) a protein-rich diet with 52% DM Italian r...
Analysis of Chromosome Segregation, Histone Acetylation, and Spindle Morphology in Horse Oocytes. The field of assisted reproduction has been developed to treat infertility in women, companion animals, and endangered species. In the horse, assisted reproduction also allows for the production of embryos from high performers without interrupting their sports career and contributes to an increase in the number of foals from mares of high genetic value. The present manuscript describes the procedures used for collecting immature and mature oocytes from horse ovaries using ovum pick-up (OPU). These oocytes were then used to investigate the incidence of aneuploidy by adapting a protocol previous...
The antimicrobial activity of bupivacaine, lidocaine and mepivacaine against equine pathogens: An investigation of 40 bacterial isolates. Lameness is the most commonly reported health problem in horses, and lameness investigations which include local anaesthetic injections are routinely performed by equine practitioners. Through this process, bacteria can enter the tissues perforated by the needle and may cause local infections at the injection site. The objective of this in vitro study was to investigate if local anaesthetics at concentrations available in commercially available solutions could inhibit growth and/or kill bacteria that could be inoculated into the synovial space or soft tissues during injection. This study evalu...
Equine Mesenchymal Stromal Cells Retain a Pericyte-Like Phenotype. Mesenchymal stem/stromal cells (MSCs) have been used in human and equine regenerative medicine, and interest in exploiting their potential has increased dramatically over the years. Despite significant effort to characterize equine MSCs, the actual origin of these cells and how much of their native phenotype is maintained in culture have not been determined. In this study, we investigated the relationship between MSCs, derived from adipose tissue (AT) and bone marrow (BM), and pericytes in the horse. Both pericyte (CD146, NG2, and αSMA) and MSC (CD29, CD90, and CD73) markers were detected in ...
Retinoic acid-mediated anti-inflammatory responses in equine immune cells stimulated by LPS and allogeneic mesenchymal stem cells. Retinoic acid (RA), an active metabolite of vitamin A, has shown potential therapeutic immunomodulatory properties. Allogeneic mesenchymal stem cells (MSCs)-based therapy is an effective approach to induce tissue healing and regeneration in many equine orthopedic conditions. However, MSCs-based therapies induced inflammatory responses in vivo. This study aimed to: 1. Determine the effect of RA cell culture treatment on inflammatory responses of lipopolysaccharides (LPS)- and allogeneic MSCs-stimulated peripheral blood mononuclear cells (PBMCs). 2. Determine the effect of RA on stimulated MSCs ...
In vitro comparison of 3 techniques of prosthesis attachment to the muscular process of the equine arytenoid cartilage. To determine the degree and ease of arytenoid abduction achieved with abaxial placement of a FASTak II suture anchor compared to 2 suture patterns with different directions of insertion-caudomedial to craniolateral and medial to lateral. Methods: Ex vivo experimental. Methods: Cadaveric larynges from 10 Standardbred racehorses. Methods: Each larynx was sequentially instrumented with all 3 arytenoid suture attachment in random order: (1) abaxial placement of a FASTak II suture, (2) caudomedial to craniolateral suture, and (3) medial to lateral suture placement. Each construct was abducted at 5N...
Effects of FSH addition to an enriched medium containing insulin and EGF after long-term culture on functionality of equine ovarian biopsy tissue. The effect of FSH supplementation on an enriched cultured medium containing insulin (10 ng/mL) and EGF (50 ng/mL) was investigated on in vitro culture of equine ovarian biopsy tissue. Ovarian tissue fragments were collected from mares (n = 10) and distributed in the following treatments: noncultured control, cultured control, and cultured + FSH. Both treated groups were cultured for 7 or 15 days. The end points evaluated were: follicular morphology, estradiol levels in the culture medium, fluorescence intensity for TUNEL, EGFR and Ki-67 detection, and gene expression of GDF-9, BMP-15, a...
Secreted factors from equine mesenchymal stromal cells diminish the effects of TGF-β1 on equine dermal fibroblasts and alter the phenotype of dermal fibroblasts isolated from cutaneous fibroproliferative wounds. The prevalence of cutaneous fibroproliferative disorders (CFPDs) is high and almost exclusively occurs in humans (keloids and hypertrophic scars) and horses (exuberant granulation tissue), making the horse a valuable translational model for studies on prevention and treatment of human CFPDs. CFPDs arise as a result of dysregulated wound healing characterized by persistently high levels of cytokines, such as transforming growth factor beta 1 (TGF-β1), that contribute to excessive extracellular matrix deposition, and the physical disorganization of dermal fibroblasts (DF). The mesenchymal strom...
Effects of hyaluronan alone or in combination with chondroitin sulfate and N-acetyl-d-glucosamine on lipopolysaccharide challenge-exposed equine fibroblast-like synovial cells. OBJECTIVE To investigate effects of hyaluronic acid (HA) or HA combined with chondroitin sulfate (CS) and N-acetyl-d-glucosamine (NAG) by use of a lipopolysaccharide (LPS) in vitro method. SAMPLE Monolayer cultures of synovial cells from 4 adult horses. PROCEDURES Synovial cell cultures were untreated or treated with HA alone or HA-CS-NAG for 24 hours, subsequently unchallenged or challenge-exposed with 2 LPS concentrations (20 and 50 ng/mL) for 2 hours, and retreated with HA or HA-CS-NAG for another 24 hours. Cellular morphology of cultures was evaluated at 0, 24 (before LPS), 26 (after LPS),...
Development and application of a quantitative PCR assay to study equine herpesvirus 5 invasion and replication in equine tissues in vitro and in vivo. Equine herpesvirus 5 (EHV-5) infection is associated with pulmonary fibrosis in horses, but further studies on EHV-5 persistence in equine cells are needed to fully understand viral and host contributions to disease pathogenesis. Our aim was to develop a quantitative PCR (qPCR) assay to measure EHV-5 viral copy number in equine cell cultures, blood lymphocytes, and nasal swabs of horses. Furthermore, we used a recently developed equine primary respiratory cell culture system to study EHV-5 pathogenesis at the respiratory tract. PCR primers and a probe were designed to target gene E11 of the EH...
Equine ovarian tissue viability after cryopreservation and in vitro culture. Ovarian tissue cryopreservation allows the preservation of the female fertility potential for an undetermined period. The objectives of this study were to compare the efficiency of cryoprotective agents (CPAs; dimethyl sulfoxide, DMSO; ethylene glycol, EG; and propylene glycol, PROH) using slow-freezing and vitrification methods, and evaluate the viability of cryopreserved equine ovarian tissue after 7 days of culture. Fresh and cryopreserved ovarian fragments were evaluated for preantral follicle morphology, stromal cell density, EGFR, Ki-67, Bax, and Bcl-2 protein expression, and DNA fragmen...
Isolation and characterization of equine native MSC populations. In contrast to humans in which mesenchymal stem/stromal cell (MSC) therapies are still largely in the clinical trial phase, MSCs have been used therapeutically in horses for over 15 years, thus constituting a valuable preclinical model for humans. In human tissues, MSCs have been shown to originate from perivascular cells, namely pericytes and adventitial cells, which are identified by the presence of the cell surface markers CD146 and CD34, respectively. In contrast, the origin of MSCs in equine tissues has not been established, preventing the isolation and culture of defined cell population...
The establishment of in vitro culture and drug screening systems for a newly isolated strain of Trypanosoma equiperdum. Dourine is caused by Trypanosoma equiperdum via coitus with an infected horse. Although dourine is distributed in Equidae worldwide and is listed as an internationally important animal disease by the World Organization for Animal Health (OIE), no effective treatment strategies have been established. In addition, there are no reports on drug discovery, because no drug screening system exists for this parasite. A new T. equiperdum strain was recently isolated from the genital organ of a stallion that showed typical symptoms of dourine. In the present study, we adapted T. equiperdum IVM-t1 from...
Influence of long-term equine herpesvirus type 1 (EHV-1) infection on primary murine neurons-the possible effects of the multiple passages of EHV-1 on its neurovirulence. Equine herpesvirus 1 (EHV-1), like other members of the Alphaherpesvirinae subfamily, is a neurotropic virus causing latent infections in the nervous system of the natural host. In the present study, we have investigated EHV-1 replication (wild-type Jan-E strain and Rac-H laboratory strain) during long-term infection and during the passages of the virus in cultured neurons. The studies were performed on primary murine neurons, which are an excellent in vitro model for studying neurotropism and neurovirulence of EHV-1. Using real-time cell growth analysis, we have demonstrated for the first tim...
Electrophilic aldehyde products of lipid peroxidation selectively adduct to heat shock protein 90 and arylsulfatase A in stallion spermatozoa. Oxidative stress is a major determinant of mammalian sperm function stimulating lipid peroxidation cascades that culminate in the generation of potentially cytotoxic aldehydes. The aim of this study was to assess the impact of such aldehydes on the functionality of stallion spermatozoa. The impact of exposure to exogenous acrolein (ACR) and 4-hydroxynonenal (4HNE) was manifested in a highly significant dose- and time-dependent increase in mitochondrial reactive oxygen species (ROS), total cellular ROS, a decrease in sperm motility, and a time-dependent increase in lipid peroxidation. Notably, ...
The 5-HT4 receptor agonist prucalopride does not facilitate cholinergic neurotransmission in circular and longitudinal smooth muscle preparations of equine mid-jejunum. Postoperative ileus (POI) remains an important cause of death in horses. The recently developed selective 5-HT receptor agonists such as prucalopride target 5-HT receptors on myenteric cholinergic neurons to enhance acetylcholine release and GI motility. No clearcut in vitro evaluation whether highly selective 5-HT receptor agonists enhance submaximal cholinergic neurotransmission towards the muscle layer has been performed in horses. Objective: To identify functional 5-HT receptors in equine jejunum. Methods: In vitro experimental study. Methods: Circular and longitudinal smooth muscle strips...
Three-Dimensional Coculture of Meniscal Cells and Mesenchymal Stem Cells in Collagen Type I Hydrogel on a Small Intestinal Matrix-A Pilot Study Toward Equine Meniscus Tissue Engineering. Meniscal injuries are the most frequently encountered soft tissue injuries in the equine stifle joint. Due to the inherent limited repair potential of meniscal tissue, meniscal injuries do not only affect the meniscus itself but also lead to impaired joint homeostasis and secondary osteoarthritis. The presented study compares 3D coculture constructs of primary equine mesenchymal stem cells (MSC) and meniscus cells (MC) seeded on three different scaffolds-a cell-laden collagen type I hydrogel (Col I gel), a tissue-derived small intestinal matrix scaffold (SIS-muc) and a combination thereof-for ...
Exogenous lactobacilli mitigate microbial changes associated with grain fermentation (corn, oats, and wheat) by equine fecal microflora ex vivo. Cereal grains are often included in equine diets. When starch intake exceeds foregut digestion starch will reach the hindgut, impacting microbial ecology. Probiotics (e.g., lactobacilli) are reported to mitigate GI dysbioses in other species. This study was conducted to determine the effect of exogenous lactobacilli on pH and the growth of amylolytic and lactate-utilizing bacteria. Feces were collected from 3 mature geldings fed grass hay with access to pasture. Fecal microbes were harvested by differential centrifugation, washed, and re-suspended in anaerobic media containing ground corn, whe...
