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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.
Equine platelet lysate exhibits bacteriostatic effects against gram-negative clinical bacterial isolates. Investigation of alternative antibiotic therapies is critical for the future of medicine. Platelet lysate (PL) is a blood product that has shown antibiotic potential, which could be used to augment or replace current antibiotics. Objective: To compare the effects of equine PL versus antibiotics on growth of clinically isolated antibiotic resistant bacteria. We hypothesized that PL would exert an antimicrobial effect on equine bacterial isolates resistant to antibiotics. Methods: In vitro experimental study. Platelets were collected via apheresis from nine donor horses and underwent two freeze-...
Equine monocyte-derived macrophages revisited: isolation and comprehensive characterization of pro- versus anti-inflammatory polarisation. Macrophages play key roles in tissue homeostasis and regeneration-associated inflammation. Unlike humans, a reliable protocol to obtain and polarise equine monocyte-derived macrophages is lacking. In this study the polarisation of equine macrophages, derived from CD172a peripheral blood monocytes is described. After differentiation, IFN-γ/LPS or IL-4 were used to induce pro- and anti-inflammatory phenotypes, respectively. Evaluation criteria included morphology, mRNA (RT-qPCR) and protein expression (flow cytometry, immunofluorescence), nitric oxide and arginase production, cytokine secretion...
Equine oviduct-specific glycoprotein is modulated by hormones and sperm cells. Oviduct glycoprotein 1 (OVGP1) is a key protein involved in oviductal functions. β-estradiol (E) and progesterone (P), oxytocin (OXT) and tumor necrosis factor-α (TNFα) modulate the equine oviduct function, through prostaglandin regulation. The objective was to evaluate OVGP1 expression within each equine oviduct segment (infundibulum, ampulla isthmus), throughout the estrous cycle. The in vitro effect of (i) E, P, OXT, TNFα; and (ii) spermatozoa, on oviduct OVGP1 transcription and secretion was studied. Gene transcription was assessed by real-time PCR; protein expression by western blot; ...
In vitro inhibitory effects of gentamicin and ceftiofur against Trypanosoma evansi: Promising antibiotic alternatives for equine trypanosomosis in Thailand. infection (Surra) remains a major constraint to equine health and productivity in Thailand. The only available trypanocidal drug, diminazene aceturate (DA), has limited efficacy, poor blood-brain barrier penetration, and toxicity in horses. This study aimed to investigate the inhibitory effects of commonly used equine antibiotics, gentamicin (GMC), ceftiofur (CTF), and trimethoprim-sulfamethoxazole (TS), against (Thai strain isolated from dairy cattle number 953; TEDC 953) to identify potential therapeutic alternatives or adjuncts for equine trypanosomosis. Unassigned: An growth inhibition...
Effect on equine sperm of post-thaw glycerol dilution using two different semen extenders. Glycerol, a penetrating cryoprotectant, is most employed for deep freezing spermatozoa. However, it can induce toxic, chemical, and osmotic effects, altering the lipid structure of the sperm membrane. Rapid glycerol addition has been demonstrated to have fewer toxic effects than its removal. Objective: To minimize both the toxic and osmotic effects of glycerol on equine sperm through post-thaw dilution using two different extenders. Methods: Samples of equine semen frozen with 5% glycerol from nine stallions were thawed, re-diluted 1:2, and incubated for 30 minutes at 22°C in Tyrodes (Tyr) an...
A Simplified 3D-Plasma Culture Method for Generating Minimally Manipulated Autologous Equine Muscle-Derived Progenitor Cells. Musculoskeletal pathologies present challenges in athletic horses, often leading to functional impairment. The slow or limited regenerative capacity of bone, joint, and tendon/ligament injuries, coupled with the limitations of conventional treatments, highlights the need for innovative therapies such as ortho-biologics and mesenchymal stem/stroma cells. Traditional 2D cell culture systems with fetal bovine serum (FBS) fail to replicate the complexity of the in vivo environment, whereas 3D cultures more accurately mimic native tissue architecture and cell-cell interactions. This study describes...
Efficacy of butylated hydroxytoluene nanoparticles in enhancing the quality and preservation of stallion chilled semen. Encapsulating natural antioxidants presents a robust strategy to neutralize oxidative stress, thereby improving semen preservation. Therefore, this study aimed to evaluate the efficacy of butylated hydroxytoluene nanoparticles (BHT-NPs) in improving stallion semen preservation by investigating sperm quality, redox balance, semen bacteriology, apoptosis, ultrastructure, and acrosome status of chilled stallion semen preserved at 4 °C for 72 h. This in vitro experiment was evidenced by molecular docking analysis. Twenty-five ejaculates from five stallions were collected and extended with 0 (BH...
An in vitro investigation into the effects of postbiotic supplementation on stabilising equine hindgut pH. Dysbiosis of the intestinal microbiome is a precursor to hindgut acidosis. As microbial diversity and fermentation are influenced by diet, including a postbiotic could help maintain healthy gastrointestinal microbes and reduce acidosis risk. Objective: It was hypothesised that including a postbiotic in batch culture would reduce the degree of pH decline and lactate accumulation during fermentation. Methods: In vitro batch cultures with faecal inoculant were prepared with either low starch (LS) or high glucose (G) substrate to represent normal and acidic fermentation. Saccharomyces cerevisiae d...
Naloxone supplementation during vitrification of equine in vitro matured oocytes after overnight holding: insights from a comparative study with the bovine model. Cryopreservation of equine mature oocytes remains a major challenge in assisted reproduction, mainly due to the limited availability of material and logistical constraints requiring a holding phase. To address these issues, this study evaluated whether naloxone (NX), an opioid receptor antagonist with reported antioxidant properties, could improve vitrification outcomes and whether bovine oocytes could serve as a suitable preliminary and supportive model. Two experiments were conducted. In experiment 1, immature (bGV) and mature (bMII) bovine oocytes were vitrified without (VIT) or with NX (VI...
Isolation and differentiation of endometrial mesenchymal stem cells from Arabian mares. This study aimed to extract and isolate endometrial stromal cells from Arabian mares and investigate their growth and differentiation potential. Endometrial biopsies were obtained from three healthy 6-year-old Arabian mares using a standardized, minimally invasive protocol. The isolated cells were characterized using flow cytometry and differentiation analysis. Flow cytometry revealed mesenchymal markers CD90 (95.2%) and CD105 (97.4%) and hematopoietic markers CD34 (1.17%) and CD45 (0.339%). The cells exhibited differentiation potential into adipocytes, osteoblasts, and chondrocytes. The findi...
Transcriptomic Insights into Tumor Necrosis Factor α’s Role in the Fibrosis-Related Processes of Equine Endometrial Fibroblasts. Equine endometrosis is a chronic degenerative condition of the endometrium. A hallmark feature of endometrosis is endometrial fibrosis accompanied by degenerative changes in the adjacent tissue structure. Tumor necrosis factor α (TNFα), a pleiotropic cytokine produced by various immune cells, plays a dual role in coordinating immune responses and regulating cell/tissue homeostasis, making it an important regulator of fibrotic-related disorders. However, the exact role of TNFα in the development of equine endometrial fibrosis remains to be discovered and explained. Therefore, the main aim of...
From clinic to lab: Sperm parameters for equine in vitro embryo production. Equine in vitro embryo production (IVP) offers a valuable opportunity to enhance the efficient utilization of genetically and economically valuable horses. In cases of subfertility, sudden death, emergency euthanasia, or limited sperm availability, IVP becomes the only viable option to produce offspring. Due to steadily improving success rates, IVP has become one of the most sought-after assisted reproductive techniques. The rising global demand has created a clear need for veterinarians and stallion owners to provide IVP-suitable sperm, where the selection of intact sperm for intracytoplasmic...
In Vitro Investigation of Equine Gut Microbiota Alterations During Hypoglycin A Exposure. Hypoglycin A is a plant-derived protoxin that causes atypical myopathy in equids. In atypical myopathy-affected horses, metabolomic and microbiome studies have reported alterations in metabolic markers and faecal microbiota composition, pointing to a potential disruption of microbial homeostasis. However, in vivo observations are strongly confounded by host-related factors, underscoring the need for controlled in vitro approaches. To address this, we used an in vitro static batch fermentation model simulating the equine colon to investigate the direct effects of hypoglycin A on microbiota comp...
In Vitro Activity of Manuka Honey, Either Alone or in Combination With Topical Antibiotics, Against Bacteria Commonly Found in Equine Ulcerative Keratitis. To assess the antibacterial activity of manuka honey against bacterial isolates commonly associated with infected corneal ulcerations in horses, and to investigate possible combined effects of manuka honey and commonly prescribed topical antibiotics. Methods: Four Staphylococcus aureus, including three methicillin-resistant (MRSA), two methicillin-resistant coagulase-negative staphylococci (S. sciuri and S. haemolyticus), and two Streptococcus equi subspecies zooepidemicus isolates from horses with stromal ulcerative keratitis were selected. Minimum inhibitory concentrations (MICs) and minimu...
Transcriptomic profiling reveals similarities between equine IVF and ICSI embryos. In vitro production of equine embryos has been performed using intracytoplasmic sperm injection (ICSI) for the last two decades. Since 2022, a repeatable protocol for conventional in vitro fertilization (IVF) provides a successful alternative. However, little is known about the influence of the fertilization method on embryo quality and the transcriptomic profile. In this study, we aimed to examine differentially expressed genes (DEGs) between ICSI and IVF embryos in the horse. Therefore, ten equine sibling blastocysts, produced in vitro by either ICSI or IVF from three different mares, were s...
Inhibition of interleukin-1 receptor-associated kinase (IRAK)-4 provides partial rescue of interleukin-1 beta induced functional and gene expression changes in equine tenocytes. Interleukin 1 beta (IL-1β) is upregulated following a tendon injury and in vitro studies have shown that it leads to numerous negative effects on tendon cell function and gene expression. IL-1β activates nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and we hypothesised that inhibiting NF-κB activation would mediate the negative effects of IL-1β on equine tendon cells in 3-dimensional (3D) cultures. Results: Here, we tested three inhibitors of NF-κB signalling (Bortezomib, BAY11-7082 and Wedelolactone) along withTJ-M2010-5, an inhibitor of MyD88, which is a critic...
Incubation of Frozen-Thawed Semen Under Capacitating Conditions Supports Successful In Vitro Fertilization and Improves Intracytoplasmic Sperm Injection-Results in Horses. In 2022, a repeatable protocol for in vitro fertilization (IVF) using fresh semen was established in horses. This facilitated successful capacitation of equine semen allowing to explore novel applications. Objective: We aimed to extend this technique to IVF with frozen-thawed semen and intracytoplasmic sperm injection (ICSI), and determine the outcome parameters such as blastocyst production and euploidy rates. Methods: A total of 221 oocytes were subjected to either IVF with frozen-thawed semen, ICSI with frozen-thawed semen incubated under capacitating conditions (ICSI cap) or control ICSI w...
Cellular interactions and gene expression analysis of two equine-derived bone graft materials: an in vitro study. Bone grafting is a surgical procedure that involves the use of bone tissue or bone substitutes to repair damaged bone. In dentistry and maxillofacial surgery, bone graft substitutes from various sources are commonly used. Given their critical role in clinical outcomes, it is essential to thoroughly investigate the biological and mechanical properties of these materials. Methods: In this in vitro study, we evaluated the biological properties of two equine-derived bone graft substitutes in comparison to β-tricalcium phosphate. The materials included one equine-derived graft containing hydrolyze...
Equine adipose-derived stem cells modulate in vitro neutrophil extracellular trap release by polymorphonuclear neutrophils. Neutrophil extracellular trap (NET) are thin and long web-like structures composed of DNA and antimicrobial proteins released by activated polymorphonuclear neutrophils (PMN) as part of the innate immune response. Adipose-derived stem cells (ADSCs) represent an accessible, abundant and minimal invasive source of mesenchymal stem cells (MSCs), with high regenerative potential, immunomodulatory and anti-inflammatory properties. Although recognized immunomodulatory properties of ADSCs, their interaction with PMN and their role on NET formation remains poorly characterized. The present study aimed...
Neutralisation of the Immunoglobulin-Cleaving Activity of Streptococcus equi Subspecies equi IdeE by Blood Sera from Ponies Vaccinated with a Multicomponent Protein Vaccine. subspecies () is the cause of strangles, one of the most prevalent diseases of horses worldwide. The disease is characterised by fever and the formation of abscesses in the lymph nodes of the head and neck, which can restrict the airway. A multicomponent subunit vaccine, Strangvac, has been shown to effectively reduce clinical signs of strangles and to reduce its incidence. The aim of this study was to determine the immune response against the immunoglobulin-cleaving endopeptidase IdeE, a key protective component within the vaccine and the ability of antibodies to neutralize the proteolytic...
Th1 and Th2 cells in equine endometrosis and their interactions with endometrial fibroblasts. Mare endometrosis is a chronic degenerative condition of the endometrium, primarily characterized by fibrosis, involving interactions among fibroblasts, immune cells, and epithelial cells regulated by cytokines and growth factors. T helper (Th)1 and Th2 cells seem to play a pivotal role in fibrosis. However, their roles in equine endometrial fibrosis remain unknown. This study explores Th1 and Th2 cell distribution across different stages of endometrium histopathological Kenney and Doig categories; and evaluated their secretome effects on non-fibrotic endometrium derived fibroblast functional ...
Generation of equine induced pluripotent stem cells from cells of embryonic, perinatal and adult tissues. Regenerative therapies are quickly expanding to application in equine patients because of their importance as sporting and companion animals. Furthermore, aligning with a One Health concept, veterinary medicine offers a unique platform for preclinical studies. While mesenchymal stem/stromal cells (MSCs) therapies are already used in treating horses, strategies involving induced pluripotent stem cells (iPSCs) are poorly developed. iPSCs present great potential for therapy and disease modelling, but their consistent generation in horses requires further investigation into the source of somatic c...
In Vitro Effects of PRP, Ozonized PRP, Hyaluronic Acid, Paracetamol, and Polyacrylamide on Equine Synovial Fluid-Derived Mesenchymal Stem Cells. Musculoskeletal disorders are a major cause of lameness in horses, often necessitating innovative regenerative strategies to restore joint function and improve quality of life. This study investigated the effects of platelet-rich plasma (PRP), ozonized PRP, hyaluronic acid, paracetamol, and polyacrylamide hydrogel (NOLTREX) on the behavior of mesenchymal stem cells (MSCs) derived from equine synovial fluid. Synovial fluid samples were collected under strict cytological criteria to ensure viability, followed by in vitro expansion and phenotypic characterization of MSCs. Cultures were supplement...
Newly isolated bacteriophages show efficacy and phage-antibiotic synergy in vitro against the equine genital pathogens Klebsiella pneumoniae and Pseudomonas aeruginosa. Bacterial infections of the genital tract are a severe problem in equine reproduction. Biofilms produced by Klebsiella (K.) pneumoniae and Pseudomonas (P.) aeruginosa present further concerns in such cases as they can limit the success of antibiotic treatments. Alternative treatment approaches are urgently needed for treating bacterial equine genital tract infections and thus, reduce antibiotic use. The present study reports on the bactericidal efficacy of both, novel K. pneumoniae- and P. aeruginosa-specific phages, in a biofilm model and in conjunction with antibiotic drugs as tested in vitr...
Mapping the Metabolic Fate of Suzetrigine in Equine and Camel Models: A Step Toward Reliable Antidoping Detection. Effective pain management remains a persistent challenge, with opioids limited by tolerance, dependence, respiratory depression, and misuse. This has created demand for safer, nonaddictive alternatives. Suzetrigine (VX-548, Journavx) is a novel analgesic with promising efficacy, but its potential misuse in competitive racing requires investigation. Limited information is available on its metabolism and detection, emphasizing the need for metabolic characterization to aid antidoping strategies. Methods: In vitro metabolism of suzetrigine was evaluated using equine liver microsomes and homogeniz...
Ex vivo biomechanical testing of a three-dimensional printed titanium plate and spacer construct and 4.5 mm locking compression plate for ventral cervical fusion of C4-C5 in the horse. To compare the biomechanical properties of a three-dimensional (3D) printed titanium plate and spacer (3DM) and a 4.5 mm broad locking compression plate (LCP) for intervertebral fusion of C4-C5 in the horse. Methods: Ex vivo experimental study. Methods: Twenty-four equine cadaveric cervical spines. Methods: Twenty-four cadaver equine cervical spines were randomly assigned to ventral intervertebral fusion with either 3DM (n = 12) or LCP (n = 12) at C4-C5. Four-point bending tests were performed in flexion (3DM n = 6, LCP n = 6) and extension (3DM n = 6, LCP n = 6) in a sin...
Differential effects of flunixin meglumine and meloxicam on TNF- α production in LPS-stimulated equine neutrophils in vitro. Systemic inflammatory response syndrome (SIRS) induced by endotoxemia is usually secondary to colitis and is a major cause of high morbidity and mortality in horses. Non-steroidal anti-inflammatory drugs (NSAIDs), such as flunixin meglumine (FM) and meloxicam (MX), are used to improve clinical outcomes in SIRS/endotoxemia. These NSAIDs suppress tumor necrosis factor-alpha (TNF-α) levels; however, the underlying mechanisms remain unclear. The aim of this study was to investigate the inhibitory effects of FM and MX on TNF-α in lipopolysaccharide (LPS)-stimulated peripheral blood mononuclear ce...
Identification of Candidate Biomarkers Detected in the Urine of Racehorses After Anabolic Agent Administration: Use of Orthogonal Methods for Structural Elucidation. Biomarker identification by mass spectrometry represents a key step in the workflow of nontargeted metabolomic studies. Given the complexity of the data, this step, which must be carried out by a trained specialist, is time-consuming, and the biomarkers discovered are not always identified. While this stage is not an obstacle to the development of new screening and classification tools, it is nonetheless crucial to a better understanding of the results obtained. For this reason, the aim of this study was to perform structural elucidation of candidate biomarkers, which had previously been displ...
Lipopolysaccharide reduces progesterone and cytokines in equine follicular fluid without affecting oocyte development in vitro. Lipopolysaccharide (LPS) in follicular fluid impairs steroid production and oocyte developmental competence in cows and mice. This study assessed LPS concentrations in equine follicular fluid and their association with steroid and some cytokine levels. Additionally, we evaluated whether LPS exposure during in vitro maturation (IVM) affects equine oocyte developmental competence. In experiment 1, follicular fluid from large follicles (>30 mm in diameter) was collected from 16 slaughterhouse mares, and concentrations of LPS, estradiol, progesterone, TNF-α, and IL-6 were measured. In experim...
In vitro and in vivo effects of Acacia mearnsii De Wild extract for cyathostomin control in horses. Anthelmintic resistance has led to the use of organic extracts as alternative methods of parasite control. Objective: The study aimed to assess the effects of Acacia mearnsii extract (tannin) on the control of cyathostomins in horses, both in vitro and in vivo. Methods: Thirty Pantaneiro horses naturally infected with cyathostomins were sourced from two distinct farms, designated as Farms A and B. At the start of the study, all third-stage larvae were cyathostomins, and an egg hatchability test (EHT) was performed using fecal samples from horses on both farms. Two randomized 28-day field studi...
