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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.
Equid herpesvirus 1: platelets and alveolar macrophages are potential sources of activated TGF-B1 in the horse. Cell mediated responses to Equid herpesvirus 1 (EHV-1) are of short duration in vivo and require considerable expansion to be detected in vitro. Raised serum levels of active transforming growth factor B (TGF-B1) have been shown to depress proliferative T cell responses in experimental infections with EHV-1 in ponies. The present work indicates that latent transforming growth factor B (TGF-B1) is present in circulating platelets, lymph node, bronchial epithelium and alveolar macrophages. Activation of platelets in vitro by thrombin resulted in the release of latent TGF-B1 from platelets, with ...
Differential activation of platelets from normal and allergic ponies by PAF and ADP. Altered platelet responsiveness has been demonstrated in human atopic dermatitis. This study has compared the in vitro function of platelets from normal ponies and those with the allergic skin disease, sweet itch. Methods: Ponies with a clinical history of sweet itch and normal ponies were used as blood donors. Methods: PAF and ADP-induced platelet aggregation was measured and TxB2 production quantitated at the time of maximal aggregation; 12-HETE was additionally measured in some samples. Agonist-induced release of 3[H]5-HT was also studied. Results: Although both PAF and ADP caused equine pl...
Cytochemical and electrophoretic study of the stallion epididymal glycoproteins. It has been suggested that proteins produced in specific regions of the epididymis, mostly androgen dependent glycoproteins, are involved in the sperm maturation process. In the present work, the glycoconjugated distribution pattern and the electrophoretic characteristics of the stallion epididymal proteins were examined using lectin probes. The identification in the luminal fluid of some new proteins, probably synthesized and secreted by the epididymis, is an important initial step to investigate their interaction with the stallion sperm membrane. The binding of FITC-lectins (ConA, WGA, LPA, ...
Induction of apoptosis by equine arteritis virus infection. Equine arteritis virus (EAV) is the etiological agent of equine viral arteritis, a contagious viral disease of equids. EAV is the prototype virus of the arteriviruses, a group of small enveloped viruses with positive single-stranded RNA genomes. Because apoptosis or programmed cell death is believed to play an important role in the biogenesis of several cytopathogenic viruses, we examined whether EAV was able to induce cell apoptosis in vitro. To do this, Vero cells were infected with EAV at a multiplicity of infection of 0.1 tissue culture infectious dose (TCID50) per cell, and analyzed at va...
Tissue culture of the enteric nervous system from equine ileum. Ileal samples were harvested fresh from euthanized adult horses. The tissues were microdissected to prepare wholemount preparations for immunohistochemistry and for either explant or dissociated culture systems of the enteric nervous system. Explant culture systems were established using whole-mounts of either the submucous plexus or the muscularis externa (including the myenteric plexus). Dissociated cell cultures could only be obtained from the submucous plexus. Culture systems were maintained for up to 5 days. Immunoreactivity for a neuronal marker (Pan-N) and for glial cell markers (GFAP a...
Effects of different activation treatments on fertilization of horse oocytes by intracytoplasmic sperm injection. The effects of four reagents on the activation and subsequent fertilization of equine oocytes, and the development of these after intracytoplasmic sperm injection, were investigated. Cumulus-oocyte complexes collected from equine ovaries obtained from an abattoir were matured in vitro for 40-44 h in TCM199 medium before being injected, when in metaphase II, with an immobilized stallion spermatozoon. The cumulus-oocyte complexes were then subjected to one of five activation treatments: (a) 10 micromol ionomycin l(-1) for 10 min; (b) 7% (v/v) ethanol for 10 min; (c) 100 micromol thimerosal l(-1)...
Glycosylation in the near-term epitheliochorial placenta of the horse, donkey and camel: a comparative study of interbreeding and non-interbreeding species. Studies from this laboratory have shown great diversity in the glycosylation of tissues comprising the interhaemal barrier of species with different placental types. This diversity may be one of the factors preventing interbreeding between species. Glycan expression within the uterine epithelium and trophoblast of the interhaemal barrier was examined to test this proposition in three species with similar diffuse, microcotyledonary, epitheliochorial allantochorionic types of placenta: the horse (Equus caballus) and donkey (Equus asinus), which can interbreed with each other, and the camel (Came...
Effect of administering a crude equine gonadotrophin preparation to mares on follicular development, oocyte recovery rate and oocyte maturation in vivo. In mares, the shortage of oocytes and the variability in nuclear maturation at a certain time of the oestrous cycle hinders the optimization of methods for in vitro maturation and in vitro fertilization. Increasing the number of small-to-medium-sized follicles available for aspiration in vivo may increase the overall oocyte yield. The aims of the present study were to investigate whether administration of crude equine gonadotrophins affects follicular development, oocyte recovery rate, in vivo oocyte maturation and follicular concentrations of meiosis-activating sterols. During oestrus, all fo...
Assessing equine sperm-membrane integrity. The swelling of cells in a hypo-osmotic medium has been described as an important criterion for assessing the functional integrity of the sperm plasma membrane. The resistance of equine spermatozoa to osmolarity changes was studied by extending 98 semen samples collected from nine stallions in media at five osmolarities (300, 200, 150, 100, and 50 mOsmol l(-1)). The response of the cells was measured by the spermatocrit technique and eosin staining. Spermatocrit determines the increase on spermatozoal volume under hypo-osmotic conditions, a sign of functional integrity of sperm plasma membrane...
Concrete use of the joint coordinate system for the quantification of articular rotations in the digital joints of the horse. A method is detailed allowing the computation of three-dimensional (3D) joint angles. Each joint of the equine digit is modelled as a sequence of three single axis rotary joints. The Joint Coordinate System was used; it involves a specific sequence of cardanic angles. The decomposition of the angles was chosen so that the three elementary angles coincide with the flexion/extension, passive abduction/adduction and lateral/medial rotations. The algorithms and kinematic procedures were described for the equine front digital joints. This method was tested in vitro on four forelimbs. For each limb,...
Raman optical activity characterization of native and molten globule states of equine lysozyme: comparison with hen lysozyme and bovine alpha-lactalbumin. Vibrational Raman optical activity (ROA) spectra of the calcium-binding lysozyme from equine milk in native and nonnative states are measured and compared with those of the homologous proteins hen egg white lysozyme and bovine alpha-lactalbumin. The ROA spectrum of holo equine lysozyme at pH 4.6 and 22 degrees C closely resembles that of hen lysozyme in regions sensitive to backbone and side chain conformations, indicating similarity of the overall secondary and tertiary structures. However, the intensity of a strong positive ROA band at approximately 1340 cm(-1), which is assigned to a hydrat...
Techniques for evaluating selected reproductive disorders of stallions. Numerous techniques may be used for evaluation of the different reproductive disorders of the stallion. Approaches may vary from real-time ultrasonography and biopsy for evaluating testicular tumors to use of special assays for evaluating sperm or plasma for presence of antisperm antibodies. This communication addresses techniques used to evaluate five relatively uncommon, but perplexing, disorders of breeding stallions: (1) seminal vesiculitis, (2) hemospermia associated with idiopathic urethral defects, (3) acrosomal dysfunction, (4) abnormal spermatozoal chromatin, and (5) azoospermia.
Effects of interleukin-1beta and tumor necrosis factor-alpha on expression of matrix-related genes by cultured equine articular chondrocytes. To determine the effects of interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) on expression and regulation of several matrix-related genes by equine articular chondrocytes. Methods: Articular cartilage harvested from grossly normal joints of 8 foals, 6 yearling horses, and 8 adult horses. Methods: Chondrocytes maintained in suspension cultures were treated with various doses of human recombinant IL-1beta or TNF-alpha. Northern blots of total RNA from untreated and treated chondrocytes were probed with equine complementary DNA (cDNA) probes for cartilage matrix-related ge...
Streptococcus equi with truncated M-proteins isolated from outwardly healthy horses. The M-protein genes of Streptococcus equi isolated from 17 outwardly healthy horses after 4 strangles outbreaks had ended, including a quarantined animal, were compared with those of S. equi isolates from 167 active cases of strangles across 4 countries. The healthy horses included 16 persistent S. equi carriers, at least one from each of the four outbreaks. These carriers, despite being outwardly healthy, had empyema of the guttural pouch(es), an enlargement of the equine Eustachian tube. A persistent carrier from two of these outbreaks, the quarantined animal and a healthy animal with normal...
Influence of interleukin-1beta and hyaluronan on proteoglycan release from equine navicular hyaline cartilage and fibrocartilage. Proteoglycan (PG) release, in response to recombinant human interleukin-1beta (rh-IL-1beta), was measured in cartilage explants obtained from the equine distal sesamoid bone (navicular bone). Fibrocartilage from the surface of the navicular bone apposing the deep digital flexor tendon and hyaline cartilage from the surface of the navicular bone articulating with the middle phalanx were labelled with 35SO4. Hyaline cartilage from the distal metacarpus was used as a control tissue. Following radiolabel incorporation, the three cartilage types were treated with rh-IL-1beta (100 U/mL) in the prese...
An in vitro study to compare 5 different techniques for injection of the navicular bursa in the horse. Numerous different techniques for injection of the navicular bursa have been described but there is little conformity between these descriptions. A literature review was performed and techniques for injection of the navicular bursa were categorised into 5 approaches. This study was performed to evaluate the reproducibility of each technique and to assess which technique was consistently most successful. Five inexperienced operators performed each technique on 5 cadaver forelimbs. Once a needle had been placed, a radiograph was taken to confirm placement was in accordance with the description o...
Molten globule structure of equine beta-lactoglobulin probed by hydrogen exchange. The molten globule structure of equine beta-lactoglobulin has been inferred from the hydrogen exchange protection of the backbone amide protons. In order to make it possible to measure the hydrogen exchange kinetics of the individual backbone amide protons, the uniformly (15)N-labeled recombinant protein was expressed in Escherichia coli and the NMR peak assignment was obtained for most of the backbone protons. The chemical shift and NOE results obtained under the condition where the protein assumes the native structure are fully consistent with the known secondary structure of bovine beta-lac...
Cross reactivity between human erythropoietin antibody and horse erythropoietin. Erythropoietin (EPO) is the primary hormone of erythropoiesis. Administration of recombinant human erythropoietin (rhuEPO) to improve racing performance in the horse represents a new form of blood doping, which has been associated with increased mortality. While immunoassay kits have become plentiful, very few commercial hormone assays are made specifically for equine research. There is a strong degree of sequence homology reported for EPO among species, which has allowed antibodies designed for human EPO research to be used to determine EPO concentration in other species. The objective of the...
Evaluation of the role of keratan sulphate as a molecular marker to monitor cartilage metabolism in horses. The role of keratan sulphate (KS) as a metabolic marker of cartilage was evaluated using an in vitro model of equine articular cartilage. Articular cartilage was harvested from clinically healthy 6-month-old foals (n = 3). Chondrocytes were centrifuged and cultured as pellets. Chondrocyte pellets were stimulated by insulin-like growth factor-I alpha (IGF-I alpha) or interleukin-1 alpha (IL-1 alpha) for 2 weeks. The concentrations of sulphated glycosaminoglycans (GAG) and KS in the culture media were measured by a 1,9-dimethyl-methylene blue (DMMB) colorimetric assay and an inhibition enzyme-li...
Effects of polysulfated glycosaminoglycan and hyaluronan on prostaglandin E2 production by cultured equine synoviocytes. To investigate effects of the anti-arthritic agents hyaluronan and polysulfated glycosaminoglycan (PSGAG) on inflammatory metabolism in cultured equine synoviocytes. Methods: Synoviocytes cultured from samples obtained from the metacarpophalangeal joints of 4 horses. Methods: Equine synoviocytes were grown in monolayer culture. Synoviocytes were stimulated with lipopolysaccharide (LPS) and simultaneously treated with various concentrations of hyaluronan or PSGAG for 48 hours. Three hyaluronan preparations were compared. Prostaglandin E2 (PGE2) concentrations in culture medium were measured, us...
Identification and phylogenetic comparison of Salem virus, a novel paramyxovirus of horses. A virus that could not be identified as a previously known equine virus was isolated from the mononuclear cells of a horse. Electron microscopy revealed enveloped virions with nucleocapsid structures characteristic of viruses in the Paramyxoviridae family. The virus failed to hemabsorb chicken or guinea pig red blood cells and lacked neuraminidase activity. Two viral genes were isolated from a cDNA expression library. Multiple sequence alignments of one gene indicated an average identity of 45% as compared to Morbillivirus N protein sequences. A weaker relationship was found with Tupaia paramy...
Susceptibility of equine erythrocytes to oxidant-induced rheologic alterations. To evaluate the rheologic responses of equine versus human RBC to oxidant stress induced by superoxide anions. Methods: Equine blood samples were obtained from 8 healthy, 3- to 6-year-old various breed horses of either sex; human blood samples were obtained from 8 healthy adults. Methods: Washed RBC were exposed to superoxide anions generated by the xanthine oxidase (XO)-hypoxanthine system (XO activity of 0 to 0.1 U/ml). Deformability of RBC was assessed by ektacytometry, and RBC aggregation was measured in autologous plasma or 3% solution of dextran 70 via a defined-shear photometric techniq...
Primary nucleotide structure of predominant and alternate splice forms of equine insulin-like growth factor I and their gene expression patterns in tissues. To isolate, clone, and determine primary nucleotide sequence of equine insulin-like growth factor I (IGF-I) and to examine IGF-I gene expression in tissues and cartilage from horses. Methods: Horses of various ages. Methods: Total RNA was isolated from tissues and purified. Complementary DNA (cDNA) was derived by reverse transcription and polymerase chain reaction (PCR) amplification and subcloned to plasmid vectors for sequencing and comparison with other species. Total RNA from various tissues was probed with radiolabeled cDNA or complimentary RNA constructs by use of northern blotting, tube...
Differentiation and genomic and antigenic variation among fetal, respiratory, and neurological isolates from EHV1 and EHV4 infections in The Netherlands. Ten monoclonal antibodies (MAbs) were produced against equine herpes virus type 1 (EHV1). Two appeared type-specific, while the other eight were directed against epitopes common to both EHV1 and EHV4. Two MAbs directed against the glycoprotein gp2 recognized linear epitopes, as demonstrated by Western blotting. With pools of type-specific MAbs, 282 field isolates were typed in an immunoperoxidase monolayer assay (IPMA). From a total of 254 fetal or neonatal isolates, 244 (96%) were typed as EHV1, whereas 14 out of 15 (93%) respiratory tract isolates were typed as EHV4. Surprisingly, 3 out of 1...
Osteoclastic resorption of equine cranial and postcranial bone in vitro. To address possible differences in the resorbability of cranial and postcranial bone, slices of equine frontal bone and leg (first phalanx or third metacarpus) were seeded with embryonic chick bone cells and cultured for 20-24h. After removing the cells and drying the specimens, the areas and volumes of more than 800 resorption pits in each set were measured using a video-rate reflection confocal microscope system. Relative mineralization densities were determined by quantitative electron backscattering analysis. The mean mineralization density was greater in the leg bone, but the mean depths ...
Genetic manipulation of equine arteritis virus using full-length cDNA clones: separation of overlapping genes and expression of a foreign epitope. Equine arteritis virus (EAV) is an enveloped, positive-stranded RNA virus belonging to the family Arteriviridae of the order Nidovirales. The unsegmented, infectious genome of EAV is 12,704 nt in length [exclusive of the poly(A) tail] and contains eight overlapping genes that are expressed from a 3'-coterminal nested set of seven leader-containing mRNAs. To investigate the importance of the overlapping gene arrangement in the viral life-cycle and to facilitate the genetic manipulation of the viral genome, a series of mutant full-length cDNA clones was constructed in which either EAV open readi...
Meiotic competence in horse oocytes: interactions among chromatin configuration, follicle size, cumulus morphology, and season. Horse oocytes were collected from an abattoir over a 15-mo period. After classification of follicle size and cumulus morphology, oocytes were either fixed immediately (0 h) or matured in vitro (24 h). There was no effect of season on the number of antral follicles present on the ovaries, or on oocyte maturation rate for any class of oocyte. The proportion of oocytes having condensed chromatin at 0 h increased with increasing follicle size. The oocyte maturation rate also increased with follicle size, and for follicles </= 20-mm diameter, was higher for oocytes initially having expanded cumu...
Intracellular microelectrode recording to characterize inhibitory neuromuscular transmission in jejunum of horses. To evaluate electrical activity of jejunal circular muscle in horses and characterize electrical responses to stimulation by intrinsic inhibitory neurons. Methods: Portions of jejunum obtained from horses euthanatized for reasons other than gastrointestinal tract disease. Methods: Isolated circular muscle preparations were perfused with oxygenated modified Krebs solution. Glass microelectrodes were used for intracellular recording of membrane potentials from single smooth muscle cells. Electrical activity and responses to electrical field stimulation (EFS) of intrinsic neurons in the presence ...
Use of specific sugars to inhibit bacterial adherence to equine endometrium in vitro. To determine whether specific sugars inhibit adhesion of Streptococcus zooepidemicus, Pseudomonas aeruginosa, and Escherichia coli to equine endometrial epithelial cells in vitro. Methods: Endometrial biopsy specimens collected during estrus from 7 healthy mares. Methods: Endometrial specimens on glass slides were incubated for 30 minutes at 4 C with suspensions of S. zooepidemicus, P. aeruginosa, or E. coli in phosphate-buffered saline solution (PBSS) alone or with various concentrations of D-(+)-mannose, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, D-(+)-glucose, galactose, or N-acetyl-...
Metabolic and mitogenic activities of insulin-like growth factor-1 in interleukin-1-conditioned equine cartilage. To determine response of interleukin-1alpha (IL-1alpha)-conditioned equine articular cartilage explants to insulin-like growth factor-1 (IGF-1). Sample Population-Cartilage from the trochlea and condyles of the femur of a clinically normal 4-year-old horse. Methods: Effects of IGF-1 (0 to 500 ng/ml) after addition of IL-1alpha were evaluated by assessing matrix responses, using a sulfated glycosaminoglycan (GAG) assay, matrix 35SO4 GAG incorporation, and release of GAG. Mitogenic response was assessed by 3H-thymidine incorporation into DNA and fluorometric assay of total DNA concentration. Res...
