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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.
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...
Algometry to measure pain threshold in the horse’s back – An in vivo and in vitro study. The aim of this study was to provide information on algometric transmission of pressure through the dorsal thoracolumbar tissues of the equine back. Using a commercially available algometer, measurements were carried out with six different tips (hemispheric and cylindrical surfaces, contact areas 0.5 cm2, 1 cm2, and 2 cm2). In nine live horses the threshold of pressure that lead to any reaction was documented. In postmortem specimens of five euthanized horses the transmission of algometer pressure onto a pressure sensor placed underneath the dorsal thoracolumbar tissues at the level of the ...
New methods for isolation of keratolytic bacteria inducing intractable hoof wall cavity (Gidoh) in a horse; double screening procedures of the horn powder agar-translucency test and horn zymography. To establish a new system to isolate keratolytic bacteria from the hoof wall cavity () of a racehorse, we invented the horn powder agar-translucency (HoPAT) test and horn zymography (HZ). Using routine bacteriological techniques and these methods, we isolated five strains of keratolytic soil bacteria, which were then identified by means of 16S ribosomal RNA (rRNA) gene sequencing analysis. The findings from the study on the horse suggested that Brevibacterium luteolum played the main role in the local fragility of the hoof, eventually forming a in coordination with four other strains of kerat...
Antiherpetic Drugs in Equine Medicine. Since vaccination may not prevent disease, antiherpetic drugs have been investigated for the therapy of several equine herpesviruses. Drug efficacy has been assessed in horses with disease, but most evidence is in vitro, in other species, or empirical. Oral valacyclovir is most often administered in the therapy of equine herpesvirus type-1 (EHV-1) to protect adult horses from equine herpesvirus myeloencephalopathy, while oral acyclovir is frequently administered for EHV-5 infection in the therapy of equine multinodular pulmonary fibrosis. Other antiherpetic drugs are promising but require furt...
Effect of needle diameter on the viability of equine bone marrow derived mesenchymal stem cells. Mesenchymal stem cells (MSCs) are frequently delivered via needle injection for treatment of musculoskeletal injuries. The purpose of this study was to evaluate the effect of needle diameter on the viability of MSCs. Methods: Equine bone marrow-derived MSCs from 5 horses were suspended in PBS, and held at room temperature for 7 hours to mimic shipping conditions. Two replicate samples for each needle size (20, 22, 23, or 25-gauge [ga]) were aspirated into a 3 mL syringe and re-injected into the holding vial 3 times, to reproduce the resuspension of cells prior to injection in clinical cases. C...
Identification and Expression of Equine MER-Derived miRNAs. MicroRNAs (miRNAs) are single-stranded, small RNAs (21-23 nucleotides) that function in gene silencing and translational inhibition via the RNA interference mechanism. Most miRNAs originate from host genomic regions, such as intergenic regions, introns, exons, and transposable elements (TEs). Here, we focused on the palindromic structure of medium reiteration frequencies (MERs), which are similar to precursor miRNAs. Five MER consensus sequences (MER5A1, MER53, MER81, MER91C, and MER117) were matched with paralogous transcripts predicted to be precursor miRNAs in the horse genome (equCab2) and...
Mapping B lymphocytes as major reservoirs of naturally occurring latent equine herpesvirus 5 infection. Equine herpesvirus 5 (EHV5) is a commonly detected gammaherpesvirus, which, along with the closely related EHV2, constitute the only two known percaviruses that infect horses. Apart from detection in horse populations worldwide and the recent publication of the whole genome, there is little known about the biology and pathogenesis of this virus, with many assumptions made by parallels with EHV2. The long-term survival of gammaherpesviruses within infected hosts involves the establishment and maintenance of latency in selected cell and tissues types, particularly lymphocytes. A latent gammaherp...
Effect of the α2 -receptor agonists medetomidine, detomidine, xylazine, and romifidine on the ketamine metabolism in equines assessed with enantioselective capillary electrophoresis. The combination of ketamine and an α -receptor agonist is often used in veterinary medicine. Four different α -receptor agonists, medetomidine, detomidine, xylazine, and romifidine, which differ in their chemical structure and thus in selectivity for the α -receptor and in the sedative and analgesic potency, are typically employed during surgery of equines. Recovery following anesthesia with ketamine and an α -receptor agonist is dependent on the α -receptor agonist. This prompted us to investigate (i) the inhibition characteristics for the N-demethylation of ketamine to norketamine and (...
Sarcocystis neurona manipulation using culture-derived merozoites for bradyzoite and sporocyst production. Equine protozoal myeloencephalitis (EPM) remains a significant central nervous system disease of horses in the American continents. Sarcocystis neurona is considered the primary causative agent and its intermediate life stages are carried by a wide host-range including raccoons (Procyon lotor) in North America. S. neurona sarcocysts mature in raccoon skeletal muscle and can produce central nervous system disease in raccoons, mirroring the clinical presentation in horses. The study aimed to develop laboratory tools whereby the life cycle and various life stages of S. neurona could be better stu...
The effect of flash-freezing temperature on stallion sperm DNA structure. The effect of flash-freezing storage temperature on stallion sperm DNA has not been evaluated. Commonly, sperm are flash-frozen at various temperatures to preserve sperm DNA prior to analysis. It is unclear whether the temperature at which sperm are frozen and stored may affect the results of DNA assays. In this study, the neutral comet assay was used to evaluate the effect of flash-freezing storage temperature (freezer [-60 °C], dry ice [-78.5 °C], liquid nitrogen [-196 °C]) compared to fresh sperm DNA structure. In addition, intra- and inter-assay and intra- and inter-stallion variabil...
Dynamics of 5-methylcytosine and 5-hydroxymethylcytosine during pronuclear development in equine zygotes produced by ICSI. Global epigenetic reprogramming is considered to be essential during embryo development to establish totipotency. In the classic model first described in the mouse, the genome-wide DNA demethylation is asymmetric between the paternal and the maternal genome. The paternal genome undergoes ten-eleven translocation (TET)-mediated active DNA demethylation, which is completed before the end of the first cell cycle. Since TET enzymes oxidize 5-methylcytosine to 5-hydroxymethylcytosine, the latter is postulated to be an intermediate stage toward DNA demethylation. The maternal genome, on the other ha...
Evaluation of equine oocyte developmental competence using polarized light microscopy. The purpose of this study was to observe -matured equine oocytes with an objective computerized technique that involves the use of a polarized light microscope (PLM) in addition to the subjective morphological evaluation obtained using a classic light microscope (LM). Equine cumulus-oocyte complexes (COCs, = 922) were subjected to different maturation times (24, 36 or 45 h), however, only 36-h matured oocytes were analyzed using CLM. The 36-h matured oocytes that reached maturity were parthenogenetically activated to evaluate the quality and meiotic competence. Average maturation perce...
Evaluation of contractile phenotype in airway smooth muscle cells isolated from endobronchial biopsy and tissue specimens from horses. OBJECTIVE To develop a method to maintain the initial phenotype of airway smooth muscle (ASM) cells isolated from equine endobronchial biopsy specimens in long-term cell culture. SAMPLE Endobronchial tissue specimens (8 to 10/horse) collected from the lungs of previously healthy horses at necropsy (n = 12) and endobronchial biopsy specimens collected from standing, sedated, heaves-affected horses in clinical remission of the disease (5) and control horses (4). PROCEDURES A sampling protocol was developed to recover and maintain a contractile phenotype in ASM cells from endobronchial specimens ...
Cartilage oligomeric matrix protein neoepitope in the synovial fluid of horses with acute lameness: A new biomarker for the early stages of osteoarthritis. Clinical tools to diagnose the early changes of osteoarthritis (OA) that occur in the articular cartilage are lacking. Objective: We sought to identify and quantify a novel cartilage oligomeric matrix protein (COMP) neoepitope in the synovial fluid from the joints of healthy horses and those with different stages of OA. Methods: In vitro quantitative proteomics and assay development with application in synovial fluids samples obtained from biobanks of well-characterised horses. Methods: Articular cartilage explants were incubated with or without interleukin-1β for 25 days. Media were analysed...
Ovarian fragment sizes affect viability and morphology of preantral follicles during storage at 4°C. The method of transportation and the conditions imposed on the ovarian tissue are pivotal aspects for the success of ovarian tissue cryopreservation (OTC). The aim of this study was to evaluate the effect of the size of the ovarian tissue (e.g. whole ovary, biopsy size and transplant size) during different times of storage (0, 6, 12 and 24 h) on the structural integrity of equine ovarian tissue transported at 4°C. Eighteen pairs of ovaries from young mares (<10 years old) were harvested in a slaughterhouse and processed to simulate the fragment sizes (biopsy and transplant size groups) o...
CCL2 and CCL5 driven attraction of CD172a+ monocytic cells during an equine herpesvirus type 1 (EHV-1) infection in equine nasal mucosa and the impact of two migration inhibitors, rosiglitazone (RSG) and quinacrine (QC). Equine herpesvirus type 1 (EHV-1) causes respiratory disease, abortion and neurological disorders in horses. Besides epithelial cells, CD172a monocytic cells become infected with EHV-1 in the respiratory mucosa and transport the virus from the apical side of the epithelium to the lamina propria en route to the lymph and blood circulation. Whether CD172a monocytic cells are specifically recruited to the infection sites in order to pick up virus is unknown. In our study, equine nasal mucosa explants were inoculated with EHV-1 neurological strains 03P37 and 95P105 or the non-neurological strains ...
Testosterone metabolism of equine single CYPs of the 3A subfamily compared to the human CYP3A4. Cytochrome P450 enzymes (CYPs) are responsible for the phase I metabolism of drugs, xenobiotics and endogenous substances. Knowledge of single CYPs and their substrates is important for drug metabolism, helps to predict adverse effects and may prevent reduced drug efficacy in polypharmacy. In this study, three equine isoenzymes of the 3A subfamily, the equine flavoprotein NADPH-P450 oxidoreductase (POR), and the cytochrome b5 (CYB5) were cloned, sequenced and heterologously expressed in a baculovirus expression system. Testosterone, the standard compound for characterization of the human CYP3A...
Revealing the influence of glucocorticoid treatment on the excretion of anabolic-androgenic steroids in horses through in vitro digestive simulations and an in vivo case study. Anabolic-androgenic steroids (AAS) are strictly forbidden in equine sports because of their stimulating effect on muscle growth and performance. Nevertheless, low levels of AAS have been found in some horses, untreated with AAS. Glucocorticoids (GC), used as an anti-inflammatory therapy and structurally related to AAS, might play a role in this phenomenon. In order to unravel this possible correlation the influence of glucocorticoid treatment on the excretion of AAS was studied both in vivo and in vitro. In vivo effects were investigated by analysing urine samples collected from a gelding trea...
Characterisation and intracellular labelling of mesenchymal stromal cells derived from synovial fluid of horses and sheep. Multipotent mesenchymal stromal cells (MSCs) derived from synovial fluid (SF) are considered to be a promising cell type for therapeutic applications in joint disease. However, despite their potential relevance for clinical and experimental studies, there is insufficient knowledge about SF-derived MSCs isolated from horses and sheep. In this study, cells were recovered from healthy SF and bone marrow (BM) of sheep, and from healthy and osteoarthritic SF of horses. Ovine SF-MSCs were used to assess the efficiency of intracellular labelling with quantum dots (QDs). Colony forming units, generati...
Response to cooling of pony stallion semen selected by glass wool filtration. The aim of this study was to compare the sperm separation technique using filtration through glass wool compared with just diluted cooled semen. Eighteen ejaculates were collected from 6 pony stallions of the Brazilian pony breed. Evaluations were done on pH, osmolarity, total motility, membrane functionality (HOST), membrane integrity (CFDA/PI), morphology and mitochondrial viability (MTT) in fresh, 24 and 48 h of cooled semen at 5°C. After dilution, the half of the extended semen was cooled (control group). The other half was cooled after filtration trough glass wool (filtered group). Reta...
Stemness Signature of Equine Marrow-derived Mesenchymal Stem Cells. Application of competent cells such as mesenchymal stem cells (MSCs) for treatment of musculoskeletal disorders in equine athletes is increasingly needed. Moreover, similarities of horse and human in size, load and types of joint injuries, make horse as a good model for MSCs therapy studies. This study was designed to isolate and characterize stemness signature of equine bone marrow-derived mesenchymal stem cells (BM-MSCs). Methods: BM of three mares was aspirated and the mononuclear cells (MNCs) were isolated using density gradient. The primary MNCs were cultured and analyzed after tree passa...
In vitro phase I metabolism of selective estrogen receptor modulators in horse using ultra-high performance liquid chromatography-high resolution mass spectrometry. Selective estrogen receptor modulators (SERMs) are chemicals that possess the anti-oestrogenic activities that are banned 'in' and 'out' of competition by the World Anti-Doping Agency (WADA) in human sports, and by the International Federation of Horseracing Authorities (IFHA) in horseracing. SERMs can be used as performance-enhancing drugs to boost the level of androgens or to compensate for the adverse effects as a result of extensive use of androgenic anabolic steroids (AASs). SERMs have indeed been abused in human sports; hence, a similar threat can be envisaged in horseracing. Numerous an...
