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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.
Lidocaine and structure-related mexiletine induce similar contractility-enhancing effects in ischaemia-reperfusion injured equine intestinal smooth muscle in vitro. Postoperative ileus (POI) is a severe complication following small intestinal surgery in horses. It was hypothesised that prokinetic effects of lidocaine, the most commonly chosen drug for treatment of POI, resulted from drug integration into smooth muscle (SM) cell membranes, thereby modulating cell membrane properties. This would probably depend on the structural and lipophilic characteristics of lidocaine. To assess the influence of molecular structure and lipophilicity on prokinetic effects in vitro, the current study compared the effects of lidocaine with four structure-related drugs, nam...
Genome-wide association study implicates testis-sperm specific FKBP6 as a susceptibility locus for impaired acrosome reaction in stallions. Impaired acrosomal reaction (IAR) of sperm causes male subfertility in humans and animals. Despite compelling evidence about the genetic control over acrosome biogenesis and function, the genomics of IAR is as yet poorly understood, providing no molecular tools for diagnostics. Here we conducted Equine SNP50 Beadchip genotyping and GWAS using 7 IAR-affected and 37 control Thoroughbred stallions. A significant (PA and g.11040379C>A (p.166H>N) in exon 4 that were significantly associated with the IAR phenotype both in the GWAS cohort (n = 44) and in a large multi-breed cohort of 265 ho...
Quantification, morphology, and viability of equine preantral follicles obtained via the Biopsy Pick-Up method. A Biopsy Pick-Up (BPU) method was tested to determine the feasibility of retrieving preantral follicles from mare ovaries in vivo. A total of 33 ovarian biopsy procedures were performed on 18 mares during the breeding season. Mares were 5 to 21 years old and biopsies were performed during the estrous and/or diestrous phase, as confirmed by transrectal ultrasonography. Follicles were mechanically isolated using a tissue chopper, counted, and classified as normal or abnormal and primordial or primary. Viability of isolated follicles was determined by Trypan Blue dye. A total of 256 biopsy attemp...
Influence of corticosteroids on interleukin-1β-stimulated equine chondrocyte gene expression. To compare the effects of triamcinolone acetonide (TA) and methylprednisolone acetate (MPA) on expression of selected chondrocyte genes in recombinant equine interleukin-1β (reIL-1β) stimulated articular cartilage explants. Methods: In vitro experiment. Methods: Horses (n = 6). Methods: Articular cartilage explants from 2- to 3- year-old horses were exposed to reIL-1β in the presence and absence of TA and MPA at 10(-7) and 10(-6) M. Resting levels of mRNA of anabolic and catabolic genes of chondrocyte origin were quantified using qPCR after 6- and 12-hour incubations. Genes of interest incl...
Assisted reproduction techniques in the horse. This paper reviews current equine assisted reproduction techniques. Embryo transfer is the most common equine ART, but is still limited by the inability to superovulate mares effectively. Immature oocytes may be recovered by transvaginal ultrasound-guided aspiration of immature follicles, or from ovaries postmortem, and can be effectively matured in vitro. Notably, the in vivo-matured oocyte may be easily recovered from the stimulated preovulatory follicle. Standard IVF is still not repeatable in the horse; however, embryos and foals can be produced by surgical transfer of mature oocytes to th...
Forces applied with a hoof tester to cadaver feet vary widely between users. Hoof testers are commonly used in equine practice. In this study, we determined the intraoperator and interoperator reliability of force application with hoof testers for different groups: experienced veterinarians, novices and farriers. For this purpose, we have developed and validated an instrumented hoof tester. Forces varied significantly between the different regions of the foot for experienced operators applying the highest forces to the heels, then the frog, then the toe, and the lowest forces to the quarters. Novices applied significantly more force to the toe versus the frog. Intraope...
The effect of an immunomodulator (parapoxvirus ovis) on cell-mediated immunity (CMI) in abruptly weaned foals. The weaning process of foals involves a period of considerable stress which likely contributes to an increased risk of infectious disease in these young horses. Mechanisms responsible for this heightened risk of infection remain unknown, although likely due to compromised cell-mediated immunity. Parapoxvirus ovis (PPVO), an immmunomodulator, has been shown to limit the severity of infectious disease outbreaks among horses and has been shown to enhance CMI responses. Thus, an objective of this study was to investigate the effect of PPVO therapy on cell-mediated immune (CMI) responses of abruptl...
How do metacarpophalangeal joint extension, collateromotion and axial rotation influence dorsal surface strains of the equine proximal phalanx at different loads in vitro? The biomechanical circumstances that promote sagittal fracture of the equine proximal phalanx (P1) are poorly understood. In order to improve our understanding of equine metacarpophalangeal joint (MCPJ) biomechanics and potential aetiologies of sagittal P1 fractures, the study objectives were to quantify P1 bone strains, collateromotion and axial rotation during MCPJ extension under controlled loading circumstances. Unilateral limbs from six cadavers were instrumented with bone reference markers for measurement of P1 movement relative to third metacarpal bone positions during axial limb loadin...
Identification of protoxins and a microbial basis for red maple (Acer rubrum) toxicosis in equines. The leaves of Acer rubrum (red maple), especially when wilted in the fall, cause severe oxidative damage to equine erythrocytes, leading to potentially fatal methemoglobinemia and hemolytic anemia. Gallic acid and tannins from A. rubrum leaves have been implicated as the toxic compounds responsible for red maple toxicosis, but the mechanism of action and toxic principle(s) have not been elucidated to date. In order to investigate further how red maple toxicosis occurs, aqueous solutions of gallic acid, tannic acid, and ground dried A. rubrum leaves were incubated with contents of equine ileum,...
Flow rates of large animal fluid delivery systems used for high-volume crystalloid resuscitation. Large animal species in states of shock can require particularly high flow rates for volume resuscitation and the ability to deliver adequate volumes rapidly may be a rate-limiting step. The objective of this study was to determine the maximum flow rates of common combinations of IV catheter, extension set, and fluid administration sets. Methods: University veterinary teaching hospital. Methods: In vitro experimental study. Methods: Maximum flow rates were measured using combinations of 4 IV catheters (3 14-Ga and a single 10-Ga), 2 IV catheter extension sets (small bore and large bore), and 2...
Validation of a laboratory method for evaluating dynamic properties of reconstructed equine racetrack surfaces. Racetrack surface is a risk factor for racehorse injuries and fatalities. Current research indicates that race surface mechanical properties may be influenced by material composition, moisture content, temperature, and maintenance. Race surface mechanical testing in a controlled laboratory setting would allow for objective evaluation of dynamic properties of surface and factors that affect surface behavior. Objective: To develop a method for reconstruction of race surfaces in the laboratory and validate the method by comparison with racetrack measurements of dynamic surface properties. Methods...
In vitro mechanical testing of an alternate laryngoplasty system (ALPS) for horses. Mechanical evaluation of a novel alternate laryngoplasty system (ALPS). Methods: Experimental. Methods: Cadaveric horse larynges (n = 10). Methods: Arytenoid and cricoid cartilages were embedded for testing. A novel laryngoplasty system consisting of a metallic anchor, 2 stands of #2 suture material, and a metallic button were evaluated. A traditional laryngoplasty using a single strand of #5 Ethibond was evaluated in parallel. Constructs were evaluated in cyclic testing oscillating from 30 to 50 N for 3600 cycles. Constructs subsequently underwent a single cycle to failure. Distraction during...
Successful isolation of equine mesenchymal stromal cells from cryopreserved umbilical cord blood-derived mononuclear cell fractions. The therapeutic potential of mesenchymal stromal cells for cellular therapy has generated increasing interest in human as well as veterinary medicine. Considerable research has been performed on the cryopreservation of expanded mesenchymal stromal cells, but little information is available on the cryopreservation of the original mononuclear cell fraction. Objective: The present study describes a protocol to expand equine mesenchymal stromal cells after cryopreserving the mononuclear cells of umbilical cord blood. Methods: To this end, mononuclear cells were isolated from 7 umbilical cord blood...
Equine sarcoids: Bovine Papillomavirus type 1 transformed fibroblasts are sensitive to cisplatin and UVB induced apoptosis and show aberrant expression of p53. Bovine papillomavirus type 1 infects not only cattle but also equids and is a causative factor in the pathogenesis of commonly occurring equine sarcoid tumours. Whilst treatment of sarcoids is notoriously difficult, cisplatin has been shown to be one of the most effective treatment strategies for sarcoids. In this study we show that in equine fibroblasts, BPV-1 sensitises cells to cisplatin-induced and UVB-induced apoptosis, a known cofactor for papillomavirus associated disease, however BPV-1 transformed fibroblasts show increased clonogenic survival, which may potentially limit the therapeut...
Single-layer centrifugation reduces equine arteritis virus titre in the semen of shedding stallions. Several countries have adopted strategies for preventing and/or controlling equine viral arteritis based on vaccination and restricting the breeding activities of carrier stallions. However, in some cases, carrier stallions are only identified after they have transmitted virus to a mare. Therefore, a mechanism for separating virus from spermatozoa in the semen of carrier stallions would facilitate control measures for preventing disease transmission. In this study, the use of several modifications of single-layer centrifugation (SLC, SLC with an inner tube and double SLC) through Androcoll-E, ...
Human chorionic gonadotropin-dependent up-regulation of epiregulin and amphiregulin in equine and bovine follicles during the ovulatory process. Little is known about the expression and regulation of epiregulin (EREG) and amphiregulin (AREG) in ovarian follicles of large monoovulatory animal species. To characterize the gonadotropin-dependent regulation of EREG and AREG mRNAs in equine follicles prior to ovulation, extracts were prepared from equine follicles collected during estrus between 0 and 39h post-hCG and corpora lutea obtained on day 8 of the estrous cycle (day 0=day of ovulation). Results from RT-PCR/Southern blot analyses showed that levels of EREG and AREG mRNAs were very low in follicles obtained at 0h but increased therea...
Influence of drugs on the response characteristics of the LiDCO sensor: an in vitro study. In a previous study, the authors found a large bias (50%) for lithium (LiDCO) compared with thermodilution cardiac output measurement methods in ponies receiving i.v. infusions of xylazine, ketamine, and midazolam. This prompted the authors to examine the effect of drugs on the LiDCO sensor. Methods: Drugs and lithium were dissolved in 0.9% saline to produce the following solutions: saline, saline-lithium, saline-drug, and saline-drug-lithium. The drug concentrations were overlapping the range of clinical interest as estimated from the published literature. These 38°C solutions were pumped th...
O(6)-methylguanine-DNA methyltransferase in equine sarcoids: molecular and epigenetic analysis. Bovine papillomaviruses (BPVs) types 1 and 2 are the only known papillomaviruses able to jump the species. In fact, BPVs 1/2 induce neoplasia in their natural bovine host but infection is also associated to neoplastic skin lesions in equids termed sarcoids. The equine sarcoid is considered to be the most common equine cutaneous tumour worldwide for which no effective therapy is available. Very little is known about the molecular mechanisms underlying tumourigenesis, although genes contributing to sarcoid development have been identified. Several studies associate the development of cancer to t...
Crystal structures of wild-type and mutated cyclophilin B that causes hyperelastosis cutis in the American quarter horse. Hyperelastosis cutis is an inherited autosomal recessive connective tissue disorder. Affected horses are characterized by hyperextensible skin, scarring, and severe lesions along the back. The disorder is caused by a mutation in cyclophilin B. Results: The crystal structures of both wild-type and mutated (Gly6->Arg) horse cyclophilin B are presented. The mutation neither affects the overall fold of the enzyme nor impairs the catalytic site structure. Instead, it locally rearranges the flexible N-terminal end of the polypeptide chain and also makes it more rigid. Conclusions: Interactions of...
Crystal structures of wild-type and mutated cyclophilin B that causes hyperelastosis cutis in the American quarter horse. Hyperelastosis cutis is an inherited autosomal recessive connective tissue disorder. Affected horses are characterized by hyperextensible skin, scarring, and severe lesions along the back. The disorder is caused by a mutation in cyclophilin B. Results: The crystal structures of both wild-type and mutated (Gly6->Arg) horse cyclophilin B are presented. The mutation neither affects the overall fold of the enzyme nor impairs the catalytic site structure. Instead, it locally rearranges the flexible N-terminal end of the polypeptide chain and also makes it more rigid. Conclusions: Interactions of...
Sperm motility is lost in vitro as a consequence of mitochondrial free radical production and the generation of electrophilic aldehydes but can be significantly rescued by the presence of nucleophilic thiols. The prolonged incubation of human spermatozoa in vitro was found to induce a loss of motility associated with the activation of mitochondrial reactive oxygen species generation in the absence of any change in mitochondrial membrane potential. The increase in mitochondrial free radical production was paralleled by a loss of protein thiols and a concomitant rise in the formation of 4-hydroxynonenal, an electrophilic product of lipid peroxidation that was found to directly suppress sperm movement. These results prompted a search for nucleophiles that could counteract the action of such cytotoxic ...
Equine cytochrome P450 2B6–genomic identification, expression and functional characterization with ketamine. Ketamine is an anesthetic and analgesic regularly used in veterinary patients. As ketamine is almost always administered in combination with other drugs, interactions between ketamine and other drugs bear the risk of either adverse effects or diminished efficacy. Since cytochrome P450 enzymes (CYPs) play a pivotal role in the phase I metabolism of the majority of all marketed drugs, drug-drug interactions often occur at the active site of these enzymes. CYPs have been thoroughly examined in humans and laboratory animals, but little is known about equine CYPs. The characterization of equine CYP...
Development of an equine-tropic replication-competent lentivirus assay for equine infectious anemia virus-based lentiviral vectors. The release of lentiviral vectors for clinical use requires the testing of vector material, production cells, and, if applicable, ex vivo-transduced cells for the presence of replication-competent lentivirus (RCL). Vectors derived from the nonprimate lentivirus equine infectious anemia virus (EIAV) have been directly administered to patients in several clinical trials, with no toxicity observed to date. Because EIAV does not replicate in human cells, and because putative RCLs derived from vector components within human vector production cells would most likely be human cell-tropic, we previous...
Characteristics of stallion epididymal spermatozoa at collection and effect of two refrigeration protocols on the quality of the frozen/thawed sperm cells. Cryopreservation of epididymal spermatozoa is a useful tool to preserve genetic material of valuable stallions after emergency castration or unexpected death. For that, testicles and epididymides are generally sent refrigerated to the laboratory. Collection of epididymal spermatozoa is a simple procedure that reduces the volume of the material to be shipped, and may improve the quality of the chilled epididymal sperm cells. In the present study we compared the characteristics of frozen/thawed epididymal spermatozoa after refrigeration of the epididymis or after direct refrigeration of the exte...
Equine spermatozoa stored in the epididymis for up to 96h at 4°C can be successfully cryopreserved and maintain their fertilization capacity. After injury or death of a valuable male, recovery of epididymal spermatozoa may be the last chance to ensure preservation of its genetic material. The objective of this research was to study the effect of sperm storage, at 4°C up to 96h, in the epididymides obtained from castrated horses and its effect on different functional sperm parameters. Aims were to study the effect of (1) sperm storage on viability and chromatin condensation; (2) pre-incubation of recovered epididymal sperm in the freezing extender, prior cryopreservation, on viability and chromatin condensation; and (3) freezing-tha...
Profound re-organization of cell surface proteome in equine retinal pigment epithelial cells in response to in vitro culturing. The purpose of this study was to characterize the cell surface proteome of native compared to cultured equine retinal pigment epithelium (RPE) cells. The RPE plays an essential role in visual function and represents the outer blood-retinal barrier. We are investigating immunopathomechanisms of equine recurrent uveitis, an autoimmune inflammatory disease in horses leading to breakdown of the outer blood-retinal barrier and influx of autoreactive T-cells into affected horses' vitrei. Cell surface proteins of native and cultured RPE cells from eye-healthy horses were captured by biotinylation, an...
Ovulation-inducing factor in seminal plasma: a review. Ovulation in mammals involves pulsatile release of GnRH from the hypothalamus into the hypophyseal portal system with subsequent release of LH from the anterior pituitary into systemic circulation. Elevated circulating concentrations of LH induce a cascade of events within the mature follicle, culminating in follicle rupture and evacuation. The broad classification of species as either spontaneous or induced ovulators is based on the type of stimulus responsible for eliciting GnRH release from the hypothalamus. In spontaneously ovulating species (e.g., human, sheep, cattle, horse, pigs), relea...
Effect of cytokines and ovarian steroids on equine endometrial function: an in vitro study. Regulation of immune-endocrine interactions in the equine endometrium is not fully understood. The aims of the present study were to: (1) investigate the presence of tumour necrosis factor alpha (TNF), interferon gamma (IFNG), Fas ligand (FASLG) and their receptors in the mare endometrium throughout the oestrous cycle; and (2) assess endometrial secretory function (prostaglandins), angiogenic activity and cell viability in response to TNF, oestradiol (E2), progesterone (P4) and oxytocin (OXT). Transcription of TNF and FASLG mRNA increased during the early and late luteal phase (LP), whereas IF...
Equine cellular therapy–from stall to bench to bedside? Pioneering clinical stem cell research is being performed in the horse, a recipient of cutting edge veterinary medicine as well as a unique animal model, paving the way for human medical applications. Although demonstrable progress has been made on the clinical front, in vitro characterization of equine stem cells is still in comparatively early stages. To translate the promising results of clinical stem cell therapy in the horse, advances must be made in the characterization of equine stem cells. Aiming to improve communication between veterinarians and other natural scientists, this review g...
Equine arteritis virus induced cell death is associated with activation of the intrinsic apoptotic signalling pathway. Equine arteritis virus (EAV) causes a respiratory and reproductive disease in horses, equine viral arteritis. Though cell death in infection with EAV is considered to occur by apoptosis, the underlying molecular mechanism has not been extensively elucidated. We investigated the expression of mRNA of pro-apoptotic and caspase genes during EAV infection in BHK21 cells, a well-established cell type for EAV replication. Using a SYBR Green real-time PCR, mRNA of p53, Bax, caspase 3 and caspase 9 were found up-regulated in a time dependent manner in EAV infected cells. Western blot analysis for casp...
