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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.
Effect of growth factors on the migration of equine oral and limb fibroblasts using an in vitro scratch assay. The objective of this study was to determine the effect of platelet derived growth factor BB (PDGF), epidermal growth factor (EGF), transforming growth factor β1 (TGFβ1), insulin like growth factor-1 (IGF-1) and fibroblast growth factor-2 (FGF-2) on the proliferation and migration of equine oral mucosa and leg skin fibroblast cell lines, using an in vitro scratch assay. Fibroblasts from the two sites were firstly grown to confluence and then an area of cells removed (cell void area). Cell migration alone (with the addition of the mitosis inhibitor mitomycin-C to the culture media) and prolif...
Efficacy of a single intravenous dose of the neuraminidase inhibitor peramivir in the treatment of equine influenza. Equine influenza A virus (EIV) of the H3N8 subtype is an important pathogen causing acute respiratory disease in horses. Peramivir is a selective inhibitor of the influenza virus neuraminidase (NA). The characteristics of peramivir are not only its capacity for parenteral administration, but also its strong affinity for NA and slow off-rate from the NA-peramivir complex, suggesting that it could lead to a prolonged inhibitory effect and thus allow a lower dosing frequency. The aims of this study were to evaluate the inhibitory efficacy of peramivir against the NA activities of EIV in vitro and...
Evaluation of direct in vivo gene transfer in an equine metacarpal IV ostectomy model using an adenoviral vector encoding the bone morphogenetic protein-2 and protein-7 gene. To evaluate gene transfer in an equine metacarpal IV (MCIV) ostectomy model using adenoviral vectors encoding the human bone morphogenetic protein-2 and protein-7 gene (Ad-BMP-2/-7). Methods: Healthy adult horses (n = 15). Methods: A plate stabilized, critical size 1.5 cm ostectomy was created in left and right MCIV. The ostectomy site was injected with either Ad-green fluorescent protein (Ad-GFP) or Ad-hBMP-2/-7 at completion of surgery; the same treatment was assigned to both the left and right forelimb of each horse (n = 5 horses/group). Bone healing was evaluated radiographically every 2 w...
Effect of scaffold dilution on migration of mesenchymal stem cells from fibrin hydrogels. To evaluate the effect of fibrin concentrations on mesenchymal stem cell (MSC) migration out of autologous and commercial fibrin hydrogels. Methods: Blood and bone marrow from six 2- to 4-year-old horses. Methods: Autologous fibrinogen was precipitated from plasma and solubilized into a concentrated solution. Mesenchymal stem cells were resuspended in fibrinogen solutions containing 100%, 75%, 50%, and 25% of the fibrinogen precipitate solution. Fibrin hydrogels were created by mixing the fibrinogen solutions with MSCs and thrombin on tissue culture plates. After incubation for 24 hours in cel...
Effects of in vitro exposure to autologous blood and serum on expression of interleukin-8, interleukin-1β, and chemokine (C-X-C motif) ligand 2 in equine primary bronchial epithelial cell cultures. To examine the effects of in vitro exposure to solutions of autologous horse blood (AHB) and autologous horse serum (AHS) on expressions of selected cytokine genes in equine primary bronchial epithelial cell (BEC) cultures and to contrast these responses to those induced in BEC cultures by endotoxin and hay dust. Methods: BEC cultures established from bronchi of 6 healthy horses. Methods: 5-day-old BEC cultures were treated with PBS solution, AHB (2 concentrations), AHS, hay dust solution, and lipopolysaccharide solution for 24 hours. Gene expressions of interleukin (IL)-8, IL-1β, chemokine (...
Evaluation of tyrosinase expression in canine and equine melanocytic tumors. To determine the tissue-restricted expression pattern of tyrosinase mRNA in canine and equine melanocytic tumors and relative tyrosinase and major histocompatibility complex (MHC) I mRNA expression in variants of melanocytic tumors. Methods: 39 canine and 8 equine tumor samples and 10 canine and 6 equine normal tissue samples. Methods: RNA was isolated from formalin-fixed, paraffin-embedded tissues. Real-time PCR assays were designed to amplify canine and equine tyrosinase, S18 ribosomal RNA, and major histocompatibility complex I transcripts. Relative expression was determined by use of S18 a...
Effect of a solution of hyaluronic acid-chondroitin sulfate-N-acetyl glucosamine on the repair response of cartilage to single-impact load damage. To investigate effects of 1% hyaluronic acid-chondroitin sulfate-N-acetyl glucosamine (HCNAG) on the damage repair response in equine articular cartilage. Methods: Articular cartilage from 9 clinically normal adult horses. Methods: Full-thickness cartilage disks were harvested from the third metacarpal bone. Cartilage was single-impact loaded (SIL) with 0.175 J at 0.7 m/s and cultured in DMEM plus 1 % (vol/vol) HCNAG or fibroblastic growth factor (FGF)-2 (50 ng/mL). Histologic and immunohistochemical techniques were used to identify tissue architecture and apoptotic cells and to immunolocalize...
Isolation, characterization and differentiation of mesenchymal stem cells from amniotic fluid, umbilical cord blood and Wharton’s jelly in the horse. Mesenchymal stem cells (MSCs) have been derived from multiple sources of the horse including umbilical cord blood (UCB) and amnion. This work aimed to identify and characterize stem cells from equine amniotic fluid (AF), CB and Wharton's Jelly (WJ). Samples were obtained from 13 mares at labour. AF and CB cells were isolated by centrifugation, while WJ was prepared by incubating with an enzymatic solution for 2 h. All cell lines were cultured in DMEM/TCM199 plus fetal bovine serum. Fibroblast-like cells were observed in 7/10 (70%) AF, 6/8 (75%) CB and 8/12 (66.7%) WJ samples. Statistically ...
In vivo osteoinductivity of gelatin β-tri-calcium phosphate sponge and bone morphogenetic protein-2 on an equine third metacarpal bone defect. This study evaluated the therapeutic effects of a gelatin-β-TCP sponge (sponge) incorporating BMP-2 (BMP-2/sponge) on bone regeneration in equines. Six bone defects were created in third metacarpals of five thoroughbred horses, and a total of six treatments were applied in a randomized manner. The treatments were BMP-2/sponge, BMP-2/gelatin hydrogel sheet (sheet), free BMP-2, bFGF/sheet, plain sponge, and plain sheet. The defects were monitored for 16 weeks by radiography and then examined by histological analysis. Radiographic evaluation scores of bone regeneration revealed significantly gre...
Effect of Hoechst 33342 on stallion spermatozoa incubated in KMT or Tyrodes modified INRA96. The only known means of effectively separating populations of X and Y bearing sperms is the Beltsville sexing technology. The technology implies that each individual sperm is interrogated for DNA content, measuring the intensity of the fluorescence after staining the spermatozoa with Hoechst 33342. Because there are no data regarding the effect of the staining on stallion sperm, ejaculates were incubated up to 90 min in presence of 0, 4.5, 9, 22.5, 31.5, 45, 54, 67.5, 76.5 and 90 μM of Hoechst 33342, in two media, KMT or INRA-Tyrodes. After 40 and 90 min of incubation, motility (CASA) and mem...
Fermentation of six different forages in the semi-continuous fermentation technique Caesitec. The aim of the present study was to compare carbohydrate degradation of forages which store carbohydrates either predominantly as fructan or starch, in horses' hindgut. The effects of an abrupt change from hay-based feeding to green fodder-based feeding on the caecal flora were tested with the in vitro hindgut simulation technique 'Caesitec'. Six trials with different forages (English ryegrass, tall fescue, grass mixture-horses, grass mixture-cows, lucerne, white clover) were conducted. During a 4-day stabilisation period, samples were taken once a day before loading the fermenters with hay. A...
Sequence variations in equine candidate genes For XX and XY inherited disorders of sexual development. Inherited disorders of sexual development (DSD) cause sterility and infertility in horses. Mutations causing such disorders have been identified in other mammals, but there is little information on the molecular causes in horses. While the equine genome sequence has made it possible to identify candidate genes, additional tools are needed to routinely screen them for causative mutations. In this study, we designed a screening panel of polymerase chain reaction primer pairs for 15 equine genes. These are the candidate genes for testicular or ovotesticular XX DSD and XY DSD, the latter of which ...
Inflammation and immune response of intra-articular serotype 2 adeno-associated virus or adenovirus vectors in a large animal model. Intra-articular gene therapy has potential for the treatment of osteoarthritis and rheumatoid arthritis. To quantify in vitro relative gene transduction, equine chondrocytes and synovial cells were treated with adenovirus vectors (Ad), serotype 2 adeno-associated virus vectors (rAAV2), or self-complementary (sc) AAV2 vectors carrying green fluorescent protein (GFP). Using 6 horses, bilateral metacarpophalangeal joints were injected with Ad, rAAV2, or scAAV2 vectors carrying GFP genes to assess the in vivo joint inflammation and neutralizing antibody (NAb) titer in serum and joint fluid. In vit...
In vitro potential of equine DEFA1 and eCATH1 as alternative antimicrobial drugs in rhodococcosis treatment. Rhodococcus equi, the causal agent of rhodococcosis, is a severe pathogen of foals but also of immunodeficient humans, causing bronchopneumonia. The pathogen is often found together with Klebsiella pneumoniae or Streptococcus zooepidemicus in foals. Of great concern is the fact that some R. equi strains are already resistant to commonly used antibiotics. In the present study, we evaluated the in vitro potential of two equine antimicrobial peptides (AMPs), eCATH1 and DEFA1, as new drugs against R. equi and its associated pathogens. The peptides led to growth inhibition and death of R. equi and ...
The biology of equine mesenchymal stem cells: phenotypic characterization, cell surface markers and multilineage differentiation. Mesenchymal stem cells (MSCs) are multipotent stem cells that can give rise to a range of connective tissue cells including osteoblasts, chondrocytes and adipocytes. MSCs have been isolated from humans and a variety of animal species including rodents, dogs, horses and rabbits. There is currently no consensus on how these cells are identified and characterized. This is partly due to the lack of standardized specific cell surface markers for MSCs. The aim of this review is to examine the literature on equine MSCs and establish whether there is a well-defined phenotype for these cells. Equine MS...
Comparative Analysis of the Immunomodulatory Properties of Equine Adult-Derived Mesenchymal Stem Cells(). Mesenchymal stem cells (MSCs) derived from bone marrow (BM), adipose tissue (AT), umbilical cord blood (CB), and umbilical cord tissue (CT) are increasingly being used to treat equine inflammatory and degenerative lesions. MSCs modulate the immune system in part through mediator secretion. Animal species and MSC tissue of origin are both important determinants of MSC function. In spite of widespread clinical use, how equine MSCs function to heal tissues is fully unknown. In this study, MSCs derived from BM, AT, CB, and CT were compared for their ability to inhibit lymphocyte proliferation and ...
Comparison of equine tendon- and bone marrow-derived cells cultured on tendon matrix with or without insulin-like growth factor-I supplementation. To compare in vitro expansion, explant colonization, and matrix synthesis of equine tendon- and bone marrow-derived cells in response to insulin-like growth factor-I (IGF-I) supplementation. Methods: Cells isolated from 7 young adult horses. Methods: Tendon- and bone marrow-derived progenitor cells were isolated, evaluated for yield, and cultured on autogenous cell-free tendon matrix for 7 days. Samples were analyzed for cell viability and expression of collagen type I, collagen type III, and cartilage oligomeric matrix protein mRNAs. Collagen and glycosaminoglycan syntheses were quantified ov...
Responses of equine tendon- and bone marrow-derived cells to monolayer expansion with fibroblast growth factor-2 and sequential culture with pulverized tendon and insulin-like growth factor-I. To compare in vitro expansion of equine tendon- and bone marrow-derived cells with fibroblast growth factor-2 (FGF-2) supplementation and sequential matrix synthesis with pulverized tendon and insulin-like growth factor-I (IGF-I). Methods: Cells from 6 young adult horses. Methods: Progenitor cells were expanded in monolayers with FGF-2, followed by culture with autogenous acellular pulverized tendon and IGF-I for 7 days. Initial cell isolation and subsequent monolayer proliferation were assessed. In pulverized tendon cultures, cell viability and expression of collagen types I and III and carti...
Development and laboratory evaluation of two lateral flow devices for the detection of vesicular stomatitis virus in clinical samples. Two lateral flow devices (LFD) for the detection of vesicular stomatitis (VS) virus (VSV), types Indiana (VSV-IND) and New Jersey (VSV-NJ) were developed using monoclonal antibodies C1 and F25VSVNJ-45 to the respective VSV serotypes. The performance of the LFDs was evaluated in the laboratory on suspensions of vesicular epithelia and cell culture passage derived supernatants of VSV. The collection of test samples included 105 positive for VSV-IND (92 vesicular epithelial suspensions and 13 cell culture antigens; encompassing 93 samples of subtype 1 [VSV-IND-1], 9 of subtype 2 [VSV-IND-2] and 3...
Expression, refolding and preliminary X-ray crystallographic analysis of equine MHC class I molecule complexed with an EIAV-Env CTL epitope. In order to clarify the structure and the peptide-presentation characteristics of the equine major histocompatibility complex (MHC) class I molecule, a complex of equine MHC class I molecule (ELA-A1 haplotype, 7-6 allele) with mouse β(2)-microglobulin and the cytotoxic T lymphocyte (CTL) epitope Env-RW12 (RVEDVTNTAEYW) derived from equine infectious anaemia virus (EIAV) envelope protein (residues 195-206) was refolded and crystallized. The crystal, which belonged to space group P2(1), diffracted to 2.3 Å resolution and had unit-cell parameters a = 82.5, b = 71.4, c = 99.8 Å, β = 102.9°. T...
The ability of selected pupal parasitoids (Hymenoptera: Pteromalidae) to locate stable fly hosts in a soiled equine bedding substrate. The ability of Spalangia cameroni Perkins, Spalangia endius Walker, and Muscidifurax raptorellus Kogan and Legner to locate and attack stable fly hosts was evaluated under laboratory conditions. Postfeeding third-instar stable fly larvae were released and allowed to pupate in two arena types: large 4.8 liter chambers containing a field-collected, soiled equine bedding substrate; or 120-ml plastic cups containing wood chips. At the time of fly pupariation, parasitoids were released and permitted 72 h to locate and attack hosts. On average, parasitism rates of freely accessible stable fly pupae ...
The impact of cushioned centrifugation protocols on semen quality of stallions. The objective was to determine if decreased cushion-fluid volume and increased sperm number during centrifugation, or if sperm concentration of extended semen following centrifugation, affected stallion sperm quality. Three ejaculates from each of three stallions were subjected to cushioned centrifugation (1,000g for 20 min). Cushion-fluid volume was set at 1 or 3.5 ml, and sperm number per centrifuge tube was set 1 billion or 3 billion. Following centrifugation, sperm pellets were resuspended in semen extender containing 20% seminal plasma (v/v) with sperm concentrations of 25 or 250 million/...
Cytokines tumor necrosis factor-α and interferon-γ participate in modulation of the equine corpus luteum as autocrine and paracrine factors. Knowledge on the regulation of corpus luteum (CL) function in the mare is scarce. In this study, the presence of cytokines tumor necrosis factor alpha (TNF) and interferon gamma (IFNG), and their receptors (TNFRI, TNFRII and IFNRI), was investigated in equine CL throughout the luteal phase. The effects of TNF and IFNG on secretory function and viability of luteal cells were defined in vitro. Cytokine ligands and receptors were present in steroidogenic and endothelial cells. Protein expression for TNF was greater in mid-phase and regressing CL, while TNFRI was increased in regressing CL and TNF...
Mathematical modelling of tissue formation in chondrocyte filter cultures. In the field of cartilage tissue engineering, filter cultures are a frequently used three-dimensional differentiation model. However, understanding of the governing processes of in vitro growth and development of tissue in these models is limited. Therefore, this study aimed to further characterise these processes by means of an approach combining both experimental and applied mathematical methods. A mathematical model was constructed, consisting of partial differential equations predicting the distribution of cells and glycosaminoglycans (GAGs), as well as the overall thickness of the tissue....
[Regenerative therapy for tendon and ligament disorders in horses. Terminology, production, biologic potential and in vitro effects]. Conventional treatments of equine tendon injuries lead to an unsatisfactory healing process that usually results in a relatively high recurrence rate. Therefore, in recent years so-called regenerative therapeutics were studied scientifically in vitro and in laboratory animals. These include substances that ideally lead to the formation of replacement tissue, which in contrast to the low quality scar, has similar functional properties as the original intact tendon. Currently, a plethora of different substrates is either commercially available or can be produced in practice with the help of kits...
Clarithromycin is absorbed by an intestinal uptake mechanism that is sensitive to major inhibition by rifampicin: results of a short-term drug interaction study in foals. Pulmonary penetration of clarithromycin (CLR) in epithelial lining fluid (ELF) and bronchoalveolar lavage cells (BALCs) can be influenced by CYP3A4, by P-glycoprotein, and, according to our hypothesis, by a member of the organic anion-transporting protein (OATP) family, for which rifampicin (RIF) is inhibiting in single doses but inducing after long-term coadministration. To assess the partial inhibitory effect, we measured absorption and pulmonary distribution of CLR after short-term (2.5-day) coadministration of RIF, after which up-regulation is not expected. The drug interaction study was p...
Effect of progesterone on the in vitro response of peripheral blood mononuclear cells stimulated by Escherichia coli in mares. Escherichia coli(E. coli) isolated from the uterus of a Thoroughbred mare with bacterial endometritis was used to evaluate the effect of progesterone (P(4)) on the immune response of mares. Peripheral blood mononuclear cells (PBMCs) were collected from 10 nonpregnant clinically healthy adult mares (range, 4-12 years) during diestrus, four Thoroughbreds and six Hokkaido native horses. Cell proliferation and expression of cytokine mRNA, including interferon (IFN)-γ, tumor necrosis factor (TNF)-α and interleukin (IL)-10, of PBMCs stimulated with E. coli and P(4) were examined in vitro. P(4) was...
Influence of counting chamber type on CASA outcomes of equine semen analysis. Sperm motility is considered to be one of the key features of semen analysis. Assessment of motility is frequently performed using computer-assisted sperm analysis (CASA). Nevertheless, no uniform standards are present to analyse a semen sample using CASA. Objective: We hypothesised that the type of counting chamber used might influence the results of analysis and aimed to study the effect of chamber type on estimated concentration and motility of an equine semen sample assessed using CASA. Methods: Commonly used disposable Leja chambers of different depths were compared with disposable and re...
Evaluation of chondrocyte behavior in a new equine collagen scaffold useful for cartilage repair. Association of biomaterials with autologous cells can provide a new generation of implantable devices for cartilage repair. An ideal scaffold should possess a preformed three-dimensional shape, fix the cells to the damaged area and prevent their migration into the articular cavity. Furthermore, the constructs should have sufficient mechanical strength to facilitate handling in a clinical setting and stimulate the uniform spreading of cells and a phenotype re-differentiation process. The aim of this study was to verify the ability of an equine collagen membrane to support the growth of human ch...
Characterization and differentiation of equine tendon-derived progenitor cells. Mesenchymal stem cells have been recently investigated for their potential use in regenerative medicine. Population of adult stem cells were recently identified in human and lab animal tendons, but no detailed investigations have been made in the equine species. The aim of our study is to identify a progenitor cell population from tendon tissue (TSPCs) in the horse superficial digital flexor tendon that are able to be highly clonogenic, to grow fast and to differentiate in different induced cell lineages as well as bone marrow derived progenitor cells (BM-MSCs). The hypothesis that TSPCs posse...
