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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.
Utilization of polarized microscopy to differentiate deep penetrating nevus from equine type melanomas. Equine type melanoma can mimic deep penetrating nevus (DPN), making histologic diagnosis challenging. We sought to investigate if the pattern of collagen polarization could be helpful in this setting. A total of 52 specimens were reviewed with polarized microscopy to determine whether refractile collagen was present within melanocytic nests vs. surrounding but not within the nests. Seven of eight (87.5%) equine type melanomas demonstrated refractile collagen within melanocytic nests in part or all of the lesion. In contrast, DPN showed no refractile collagen within the melanocytic nests. Inste...
Effect of inflammatory environment on equine bone marrow derived mesenchymal stem cells immunogenicity and immunomodulatory properties. Mesenchymal stem cells (MSCs) are being investigated for the treatment of equine joint diseases because of their regenerative potential. Recently, the focus mainly has addressed to their immunomodulatory capacities. Inflammation plays a central role in joint pathologies, since the release of proinflammatory mediators to the synovial fluid (SF) leads to the activation of enzymatic degradation of the cartilage. MSCs can modulate the local immune environment through direct or paracrine interaction with immune cells, suppressing their proliferation and re-addressing their functions. Proinflammator...
Differential expression of β-tubulin isotypes in different life stages of Parascaris spp after exposure to thiabendazole. Anthelmintic resistance (AR) to macrocyclic lactones (ML) has been described in Parascaris of horses world-wide. In contrast, benzimidazoles (BZ) are still effective, although reduced efficacy to this drug class was recently reported. The mode of action of BZ is binding to β-tubulin, which prevents polymerisation of microtubules. In this study, β-tubulin gene expression of isotypes 1 and 2 was investigated at seven time points (0, 6, 24, 72, 96 and 120 h) during embryogenesis and in adult worms. In addition, an in ovo larval developmental test was developed to study β-tubulin gene expressio...
Establishment of conditions for ovum pick up and IVM of jennies oocytes toward the setting up of efficient IVF and in vitro embryos culture procedures in donkey (Equus asinus). Most wild and domestic donkey breeds are currently endangered or threatened. Their preservation includes the creation of a Genome Resource Bank. Embryos cryopreservation allows the preservation of genetics from both male and female and is the fastest method to restore a breed. Because embryo production in vivo is limited in equids, our objective was to establish conditions for in vitro production of embryos in donkey using ovum pick up (OPU), IVM, IVF, and in vitro culture of zygotes. Donkey cumulus-oocyte complexes (COCs) were collected by transvaginal ultrasound-guided aspirations OPU in ...
Pharmacokinetics and Pulmonary Distribution of Clarithromycin and Rifampicin after Concomitant and Consecutive Administration in Foals. Drug interactions often result from multiple pharmacokinetic changes, such as after rifampicin (RIF) and clarithromycin (CLA) in the treatment of abscessing lung diseases. Comedication of RIF may interact with CLA disposition by either induction of presystemic elimination processes and/or inhibition of uptake mechanisms because it regulates gene transcription and modulates function of various CYP enzymes, multidrug efflux and uptake transporters for which CLA is a substrate. To distinguish the transcriptional changes from the modulating interaction components upon CLA absorption and pulmonary ...
Extracellular calcium regulates protein tyrosine phosphorylation through calcium-sensing receptor (CaSR) in stallion sperm. Protein tyrosine phosphorylation (PY), a hallmark of sperm capacitation, is inhibited by extracellular calcium in stallion sperm. The objective of this study was to determine the presence and influence of the calcium-sensing receptor (CaSR) in this phenomenon. First, the presence of the CaSR was demonstrated in stallion sperm. We then tested its function in these gametes using its inhibitor NPS2143 or its agonist AC34356. Sperm were capacitated for 4 hr in modified Whitten's medium with 25 mM bicarbonate plus NPS2143 and 2.4 mM calcium or AC34356 alone, followed by analysis of PY. Inhibi...
Evaluation of twenty-two rapid antigen detection tests in the diagnosis of Equine Influenza caused by viruses of H3N8 subtype. Equine influenza (EI) is a highly contagious disease caused by viruses of the H3N8 subtype. The rapid diagnosis of EI is essential to reduce the disease spread. Many rapid antigen detection (RAD) tests for diagnosing human influenza are available, but their ability to diagnose EI has not been systematically evaluated. Objective: The aim of this study was to compare the performance of 22 RAD tests in the diagnosis of EI. Methods: The 22 RAD tests were performed on fivefold serial dilutions of EI virus to determine their detection limits. The four most sensitive RAD tests (ImmunoAce Flu, BD Flu ...
Venezuelan Equine Encephalitis Virus Induces Apoptosis through the Unfolded Protein Response Activation of EGR1. Venezuelan equine encephalitis virus (VEEV) is a previously weaponized arthropod-borne virus responsible for causing acute and fatal encephalitis in animal and human hosts. The increased circulation and spread in the Americas of VEEV and other encephalitic arboviruses, such as eastern equine encephalitis virus and West Nile virus, underscore the need for research aimed at characterizing the pathogenesis of viral encephalomyelitis for the development of novel medical countermeasures. The host-pathogen dynamics of VEEV Trinidad donkey-infected human astrocytoma U87MG cells were determined by car...
Equine-Induced Pluripotent Stem Cells Retain Lineage Commitment Toward Myogenic and Chondrogenic Fates. Induced pluripotent stem cells (iPSCs) hold great potential not only for human but also for veterinary purposes. The equine industry must often deal with health issues concerning muscle and cartilage, where comprehensive regenerative strategies are still missing. In this regard, a still open question is whether equine iPSCs differentiate toward muscle and cartilage, and whether donor cell type influences their differentiation potential. We addressed these questions through an isogenic system of equine iPSCs obtained from myogenic mesoangioblasts (MAB-iPSCs) and chondrogenic mesenchymal stem ce...
Delivery and evaluation of recombinant adeno-associated viral vectors in the equine distal extremity for the treatment of laminitis. Our long-term aim is to develop a gene therapy approach for the prevention of laminitis in the contralateral foot of horses with major musculoskeletal injuries and non-weightbearing lameness. Objective: The goal of this study was to develop a practical method to efficiently deliver therapeutic proteins deep within the equine foot. Methods: Randomised in vivo experiment. Methods: We used recombinant adeno-associated viral vectors (rAAVs) to deliver marker genes using regional limb perfusion through the palmar digital artery of the horse. Results: Vector serotypes rAAV2/1, 2/8 and 2/9 all succes...
Feasibility and Safety of Contrast-Enhanced Ultrasound in the Distal Limb of Six Horses. Vascular alterations play important roles in many orthopedic diseases such as osteoarthritis, tendonitis, and synovitis in both human and equine athletes. Understanding these alterations could enhance diagnosis, prognosis, and treatment. Contrast-enhanced ultrasound (CEUS) could be a valuable method for evaluation of blood flow and perfusion of these processes in the equine distal limb, however no reports were found describing feasibility or safety of the technique. The goal of this prospective, experimental study was to describe the feasibility and safety of distal limb CEUS in a sample of si...
Abnormal coagulation factor VIII transcript in a Tennessee Walking Horse colt with hemophilia A. Hemophilia A is an X-chromosome-linked disorder caused by a deficiency in factor VIII (FVIII). Although foals have been diagnosed with hemophilia A based on deficiency in FVIII activity, causative gene mutations have not been identified. The genomic DNA and cDNA encoding FVIII of a Tennesee Walking Horse colt affected with hemophilia A and the genomic DNA of his dam and a normal unrelated horse were analyzed with no splice site or coding sequence abnormalities identified in any of the horses. Polymerase chain reactions (PCR) were then performed on hepatic cDNA from the affected colt and an unr...
Tenogenic induction of equine mesenchymal stem cells by means of growth factors and low-level laser technology. Tendons regenerate poorly due to a dense extracellular matrix and low cellularity. Cellular therapies aim to improve tendon repair using mesenchymal stem cells and tenocytes; however, a current limitation is the low proliferative potential of tenocytes in cases of severe trauma. The purpose of this study was to develop a method useful in veterinary medicine to improve the differentiation of Peripheral Blood equine mesenchymal stem cells (PB-MSCs) into tenocytes. PB-MSCs were used to study the effects of the addition of some growth factors (GFs) as TGFβ3 (transforming growth factor), EGF2 (Epi...
The Impact of Sperm Metabolism during In Vitro Storage: The Stallion as a Model. In vitro sperm storage is a necessary part of many artificial insemination or in vitro fertilization regimes for many species, including the human and the horse. In many situations spermatozoa are chilled to temperatures between 4 and 10°C for the purpose of restricting the metabolic rate during storage, in turn, reducing the depletion of ATP and the production of detrimental by-products such as reactive oxygen species (ROS). Another result of lowering the temperature is that spermatozoa may be "cold shocked" due to lipid membrane phase separation, resulting in reduced fertility. To overcome ...
Combined albumin and bicarbonate induces head-to-head sperm agglutination which physically prevents equine sperm-oviduct binding. In many species, sperm binding to oviduct epithelium is believed to be an essential step in generating a highly fertile capacitated sperm population primed for fertilization. In several mammalian species, this interaction is based on carbohydrate-lectin recognition. D-galactose has previously been characterized as a key molecule that facilitates sperm-oviduct binding in the horse. We used oviduct explant and oviduct apical plasma membrane (APM) assays to investigate the effects of various carbohydrates; glycosaminoglycans; lectins; S-S reductants; and the capacitating factors albumin, Ca(2+) a...
P2Y(1) receptors mediate purinergic relaxation in the equine pelvic flexure. In the equine large intestine, the knowledge of the basic mechanisms underlying motility function is crucial to properly treat motility disorders. P2Y1 receptors are responsible for mediating purinergic colonic relaxation in several species. In vitro experimental studies of the circular muscle from the equine pelvic flexure (n = 6) were performed to characterize inhibitory and excitatory neuromuscular transmission. Electrophysiological studies showed that electrical field stimulation (EFS) evoked biphasic inhibitory junction potentials (IJPs) in smooth muscle cells: a fast IJP (IJPf) follo...
In vitro anticancer activity of Betulinic acid and derivatives thereof on equine melanoma cell lines from grey horses and in vivo safety assessment of the compound NVX-207 in two horses. Betulinic acid, a pentacyclic triterpene, and its derivatives are promising compounds for cancer treatment in humans. Melanoma is not only a problem for humans but also for grey horses as they have a high potential of developing melanoma lesions coupled to the mutation causing their phenotype. Current chemotherapeutic treatment carries the risk of adverse health effects for the horse owner or the treating veterinarian by exposure to antineoplastic compounds. Most treatments have low prospects for systemic tumor regression. Thus, a new therapy is needed. In this in vitro study, Betulinic acid a...
The Equine Embryo Influences Immune-Related Gene Expression in the Oviduct. Although the equine oviduct clearly affects early embryo development and the selective transport of equine embryos through the oviduct indicates a reciprocal interaction, the influence of the embryo on gene expression in the oviduct remains to be determined in the horse. The aim of this study was to examine this by means of RNA sequencing. Four days after ovulation, epithelial cells ipsilateral and contralateral to the ovulation side from five cyclic and five pregnant mares were collected from the oviduct. RNA was extracted, samples were sequenced, and data analysis was performed to determine ...
Conserved arginine residues in the carboxyl terminus of the equine arteritis virus E protein may play a role in heparin binding but may not affect viral infectivity in equine endothelial cells. Equine arteritis virus (EAV), the causative agent of equine viral arteritis, has relatively broad cell tropism in vitro. In horses, EAV primarily replicates in macrophages and endothelial cells of small blood vessels. Until now, neither the cellular receptor(s) nor the mechanism(s) of virus attachment and entry have been determined for this virus. In this study, we investigated the effect of heparin on EAV infection in equine endothelial cells (EECs). Heparin, but not other glycosaminoglycans, could reduce EAV infection up to 93 %. Sequence analysis of the EAV E minor envelope protein reveale...
In Vitro Efficacy of Nonantibiotic Treatments on Biofilm Disruption of Gram-Negative Pathogens and an In Vivo Model of Infectious Endometritis Utilizing Isolates from the Equine Uterus. In this study, we evaluated the ability of the equine clinical treatments N-acetylcysteine, EDTA, and hydrogen peroxide to disrupt in vitro biofilms and kill equine reproductive pathogens (Escherichia coli, Pseudomonas aeruginosa, or Klebsiella pneumoniae) isolated from clinical cases. N-acetylcysteine (3.3%) decreased biofilm biomass and killed bacteria within the biofilms of E. coli isolates. The CFU of recoverable P. aeruginosa and K. pneumoniae isolates were decreased, but the biofilm biomass was unchanged. Exposure to hydrogen peroxide (1%) decreased the biofilm biomass and reduced the CF...
Production of serum amyloid A in equine articular chondrocytes and fibroblast-like synoviocytes treated with proinflammatory cytokines and its effects on the two cell types in culture. To investigate the role of the major equine acute phase protein serum amyloid A (SAA) in inflammation of equine intraarticular tissues. Methods: Articular chondrocytes and fibroblast-like synoviocytes (FLSs) from 8 horses (4 horses/cell type). Methods: Chondrocytes and FLSs were stimulated in vitro for various periods up to 48 hours with cytokines (recombinant interleukin [IL]-1β, IL-6, tumor necrosis factor-α, or a combination of all 3 [IIT]) or with recombinant SAA. Gene expression of SAA, IL-6, matrix metalloproteinases (MMP)-1 and -3, and cartilage-derived retinoic acid-sensitive protein...
Effect of Fibroblast Growth Factor 2 on Equine Synovial Fluid Chondroprogenitor Expansion and Chondrogenesis. Mesenchymal stem cells have been identified in the synovial fluid of several species. This study was conducted to characterize chondroprogenitor (CP) cells in equine synovial fluid (SF) and to determine the effect of fibroblast growth factor 2 (FGF-2) on SF-CP monolayer proliferation and subsequent chondrogenesis. We hypothesized that FGF-2 would stimulate SF-CP proliferation and postexpansion chondrogenesis. SF aspirates were collected from adult equine joints. Colony-forming unit (CFU) assays were performed during primary cultures. At first passage, SF-cells were seeded at low density, with ...
In vitro growth inhibitory efficacy of some target specific novel drug molecules against Theileria equi. The in vitro growth inhibitory efficacies of five drug molecules against Theileria equi were evaluated in in vitro cultured parasites. A continuous microaerophilic stationary-phase culture (MASP) system was established for propagation of T. equi parasites. This in vitro culture system was used to assess the growth inhibitory effect of harmaline hydrochloride dihydrate (HHD), hexadecyltrimethylammonium bromide (HDTAB), hesparidin methyl chalcone (HMC), andrographolide and imidocarb dipropionate against T. equi. The 50% inhibitory concentration value of HHD, HDTAB, HMC, and imidocarb dipropionat...
Development and characterization of a synthetic infectious cDNA clone of the virulent Bucyrus strain of equine arteritis virus expressing mCherry (red fluorescent protein). Strains of equine arteritis virus (EAV) differ in their virulence phenotypes, causing anywhere from subclinical infections to severe disease in horses. Here, we describe the in silico design and de novo synthesis of a full-length infectious cDNA clone of the horse-adapted virulent Bucyrus strain (VBS) of EAV encoding mCherry along with in vitro characterization of the progeny virions (EAV sVBSmCherry) in terms of host-cell tropism, replicative capacity and stability of the mCherry coding sequences following sequential passage in cell culture. The relative stability of the mCherry sequence duri...
Effect of clinically-related factors on in vitro blastocyst development after equine ICSI. Equine intracytoplasmic sperm injection (ICSI) is being used clinically for foal production, but little information is available on factors affecting the efficiency of this procedure. We examined factors that may influence blastocyst development when ICSI is performed clinically, i.e., on oocytes recovered from live mares by transvaginal ultrasound-guided follicle aspiration (TVA), using sperm from the stallion of the client's choice. In a clinical setting, there may be a delay from the time of TVA to isolation of oocytes from the aspirated fluid. In a preliminary study, oocytes from fluid hel...
Evaluation of in vitro inhibitory effect of enoxacin on Babesia and Theileria parasites. Enoxacin is a broad-spectrum 6-fluoronaphthyridinone antibacterial agent (fluoroquinolones) structurally related to nalidixic acid used mainly in the treatment of urinary tract infections and gonorrhea. Also it has been shown recently that it may have cancer inhibiting effect. The primary antibabesial effect of Enoxacin is due to inhibition of DNA gyrase subunit A, and DNA topoisomerase. In the present study, enoxacin was tested as a potent inhibitor against the in vitro growth of bovine and equine Piroplasms. The in vitro growth of five Babesia species that were tested was significantly inh...
The neuropathogenic T953 strain of equine herpesvirus-1 inhibits type-I IFN mediated antiviral activity in equine endothelial cells. Equine herpesvirus-1 (EHV-1) infects equine endothelial cells (EECs) lining the small blood vessels in the central nervous system. However, the effect of type I IFN on EHV-1 replication in the EECs is not well studied. Thus, the primary objective of this study was to investigate the effect of type-I IFN on the replication of the neuropathogenic T953 strain of EHV-1 in vitro in EECs. The initial data showed that the EHV-1 was partly resistant to the biological effect of exogenously supplied recombinant equine IFN-α. Subsequent investigation into the mechanism of resistance showed that EHV-1 in...
Effect of vacuum packing and temperature on survival and hatching of strongyle eggs in faecal samples. Strongyle eggs of helminths of livestock usually hatch within a few hours or days after deposition with faeces. This poses a problem when faecal sampling is performed in the field. As oxygen is needed for embryonic development, it is recommended to reduce air supply during transport and refrigerate. The present study therefore investigated the combined effect of vacuum packing and temperature on survival of strongyle eggs and their subsequent ability to hatch and develop into L3. Fresh faecal samples were collected from calves infected with Cooperia oncophora, pigs infected with Oesophagostomu...
Tissues from equine cadaver ligaments up to 72 hours of post-mortem: a promising reservoir of stem cells. Mesenchymal stem cells (MSCs) harvested from cadaveric tissues represent a promising approach for regenerative medicine. To date, no study has investigated whether viable MSCs could survive in cadaveric tissues from tendon or ligament up to 72 hours of post-mortem. The purpose of the present work was to find out if viable MSCs could survive in cadaveric tissues from adult equine ligaments up to 72 hours of post-mortem, and to assess their ability (i) to remain in an undifferentiated state and (ii) to divide and proliferate in the absence of any specific stimulus. Methods: MSCs were isolated ...
Methods for the cultivation of ciliated protozoa from the large intestine of horses. This paper describes cultivation methods for ciliates from the digestive tract of horses. Members of three different genera were successfully grown in vitro for short periods of time. However, only cells belonging to the genus Blepharocorys, which resides in the horse's large intestine, were maintained for longer periods. This Blepharocorys culture was successfully grown in vitro after inoculation of freshly excreted horse faeces in culture medium containing a population of bacteria. The ciliates survived for over six months, and the density of their population varied between 1.7 × 10(3) and ...
