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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.
Does Double Centrifugation Lead to Premature Platelet Aggregation and Decreased TGF-β1 Concentrations in Equine Platelet-Rich Plasma? Blood-derived autologous products are frequently used in both human and equine medicine to treat musculoskeletal disorders. These products, especially the platelet-rich plasma (PRP), may contain high concentrations of growth factors (GFs), and thus improve healing in several tissues. Nevertheless, the procedures for preparation of PRP are currently non-standardized. Several protocols, which are based on distinct centrifugation patterns (rotation speed and time), result in PRPs with different characteristics, concerning platelet and GFs concentrations, as well as platelet activation. The aim of...
Development of a comprehensive protein microarray for immunoglobulin E profiling in horses with severe asthma. Severe asthma in horses, known as severe equine asthma (SEA), is a prevalent, performance-limiting disease associated with increased allergen-specific immunoglobulin E (IgE) against a range of environmental aeroallergens. Objective: To develop a protein microarray platform to profile IgE against a range of proven and novel environmental proteins in SEA-affected horses. Methods: Six SEA-affected and 6 clinically healthy Warmblood performance horses. Methods: Developed a protein microarray (n = 384) using protein extracts and purified proteins from a large number of families including pollen, ba...
Microfluidic nutrient gradient-based three-dimensional chondrocyte culture-on-a-chip as an in vitro equine arthritis model. In this work, we describe a microfluidic three-dimensional (3D) chondrocyte culture mimicking in vivo articular chondrocyte morphology, cell distribution, metabolism, and gene expression. This has been accomplished by establishing a physiologic nutrient diffusion gradient across the simulated matrix, while geometric design constraints of the microchambers drive native-like cellular behavior. Primary equine chondrocytes remained viable for the extended culture time of 3 weeks and maintained the low metabolic activity and high Sox9, aggrecan, and Col2 expression typical of articular chondrocytes...
Technical Note: The use of iSperm technology for on-farm measurement of equine sperm motility and concentration. The iSperm is a newly released semen analysis tool from Aidmics Biotechnology Co. LTD, which allows an iPad Mini to be transformed into a handheld microscope with objective semen analysis software for equine available through the Apple Store (version 4.5.2). The aim of this study was to compare iSperm values for sperm motility and sperm concentration to current acceptable methods for semen analysis and to determine the agreement with these methods using statistical methods. Two ejaculates from each of five Standardbred stallions were used to compare sperm motility (computer-assisted semen anal...
Equine or porcine synovial fluid as a novel ex vivo model for the study of bacterial free-floating biofilms that form in human joint infections. Bacterial invasion of synovial joints, as in infectious or septic arthritis, can be difficult to treat in both veterinary and human clinical practice. Biofilms, in the form of free-floating clumps or aggregates, are involved with the pathogenesis of infectious arthritis and periprosthetic joint infection (PJI). Infection of a joint containing an orthopedic implant can additionally complicate these infections due to the presence of adherent biofilms. Because of these biofilm phenotypes, bacteria within these infected joints show increased antimicrobial tolerance even at high antibiotic concentr...
Fibrinogen in equine pregnancy as a mediator of cell adhesion, an epigenetic and functional investigation. Preimplantation equine embryos synthesize and secrete fibrinogen, which is a peculiar finding as fibrinogen synthesis almost exclusively occurs in the liver. This study investigated the hypothesis that conceptus-derived fibrinogen mediates cell adhesion during fixation. On day 21 of pregnancy, five integrin subunits, including ITGA5, ITGB1, ITGAV, and ITGB1, displayed significantly higher transcript abundance than on day 16 of pregnancy. Endometrial epithelial cells adhered to fibrinogen in an integrin-dependent manner in an in vitro cell adhesion assay. Bilaminar trophoblast and allantochorio...
The effect of four different freezing conditions and time in frozen storage on the concentration of commonly measured growth factors and enzymes in equine platelet-rich plasma over six months. Platelet-rich plasma (PRP) is a therapeutic biologic that is used for treatment of musculoskeletal pathologies in equine athletes. Due to the expense of PRP kits, and the volumes obtained, freezing aliquots for future dosing is common. Aliquots of PRP are also commonly frozen for later analysis of growth factor concentrations in in vitro research. A variety of freezing methods are used and storage duration until analysis is often not reported. The optimal frozen storage conditions and duration to maintain concentrations of commonly measured growth factors and enzymes in PRP are unknown. Our ob...
Production of Recombinant EAV with Tagged Structural Protein Gp3 to Study Artervirus Minor Protein Localization in Infected Cells. Equine arteritis virus (EAV) is a prototype member of the Arterivirus family, comprising important pathogens of domestic animals. Minor glycoproteins of Arteriviruses are responsible for virus entry and cellular tropism. The experimental methods for studying minor Arterivirus proteins are limited because of the lack of antibodies and nested open reading frames (ORFs). In this study, we generated recombinant EAV with separated ORFs 3 and 4, and Gp3 carrying HA-tag (Gp3-HA). The recombinant viruses were stable on passaging and replicated in titers similar to the wild-type EAV. Gp3-HA was incorpo...
Changes on the Structural Architecture and Growth Factor Release, and Degradation in Equine Platelet-Rich Fibrin Clots Cultured Over Time. The aims of the present study were (1) to describe the microscopic and ultrastructural appearance of equine platelet-rich fibrin (PRF) clots and (2) to determine the release and degradation of transforming growth factor beta 1 (TGF-β) and insulin-like growth factor type I (IGF-I) from PRF clots incubated over 14 days. Whole blood from six horses was collected into plain tubes and centrifuged at 240 g for 8 minutes. Clots were evaluated by histology and by both transmission and scanning electronic microscopy (TEM and SEM). Growth factor concentrations were measured by ELISA at 48-hour inter...
pH-dependent effects of procaine on equine gamete activation†. Procaine directly triggers pH-dependent cytokinesis in equine oocytes and induces hypermotility in stallion spermatozoa, an important event during capacitation. However, procaine-induced hyperactivated motility is abolished when sperm is washed to remove the procaine prior to sperm-oocyte co-incubation. To understand how procaine exerts its effects, the external Ca2+ and Na+ and weak base activity dependency of procaine-induced hyperactivation in stallion spermatozoa was assessed using computer-assisted sperm analysis. Percoll-washed stallion spermatozoa exposed to Ca2+-depleted (+2 mM EGTA) p...
Effect of artificial vagina lubricants on stallion sperm quality. Commercially available vaginal lubricants, typically labeled as non-spermicidal, are used to lubricate equine artificial vaginas prior to semen collection. Improper type or amount of lubricant might affect stallion sperm quality, either after short-time exposure or following cooled storage of extended semen previously exposed to lubricant. The aim of this study was to evaluate stallion sperm quality following exposure to lubricant-containing extender for 1 h (T1h) or 24 h (T24h). Three ejaculates were collected from each of four stallions using a small volume of petrolatum to lubricate a...
EHV-1 Pathogenesis: Current in vitro Models and Future Perspectives. Primary infection and pathogenesis of equine herpesvirus type 1 (EHV-1) require an intricate interaction of virus with the mucosal epithelium, mononuclear cells and the vascular endothelium. Studies on EHV-1 have been facilitated by the development of different models that recapitulate the tissue complexity. The available assays can be categorized into (i) models mimicking the epithelium-peripheral blood mononuclear cell (PBMC) interaction, which include mucosal (nasal and vaginal) explants and equine respiratory epithelial cells (EREC) cultures; and (ii) PBMC-endothelium mimicking models,...
Optimization of RNA extraction protocol for long-term archived formalin-fixed paraffin-embedded tissues of horses. A suitable RNA extraction protocol was established to gain high quality RNA from formalin-fixed paraffin-embedded tissues to perform reliable molecular assays either applicable for using FFPE tissue archives or tissues with harsh formalin-fixation. Eighteen FFPE samples from the central nervous system of horses, stored up to 11 years, were used as archive cases. To test the influence of the fixation period, brain, liver, kidney, and skeletal muscle tissue fragments from another horse, were treated either with water or tris-acetate-EDTA buffer after fixation under different timepoints with 10...
Storage procedures and time influence the detectability of Clostridium difficile toxin A but not toxin B in porcine fecal specimens. Storage procedures are known to affect the detectability of toxins in equine and human feces. We assessed the impact of different storage conditions on the detectability of toxins in swine feces. Specimens were inoculated with toxins, 112 ng/g of toxin A (TcdA) and 16 ng/g of toxin B (TcdB) and subjected to the following 3 storage treatments: 4°C, -30°C, repetitive freezing at -30°C and thawing. Toxin determination was assessed at 1, 2, 7, 14, and 21 d with ELISA. A decrease in concentrations of TcdA with time was observed for samples stored at 4°C and repetitive freezing-thawing ( ≤0....
Cryopreservation of Andalusian donkey (Equus asinus) spermatozoa: Use of alternative energy sources in the freezing extender affects post-thaw sperm motility patterns but not DNA stability. The aim of this study was to compare the effect of three sugars and Equex paste in a freezing extender for donkey sperm cryopreservation. Ejaculates (n = 18) were collected from six Andalusian donkeys of proven fertility were pooled (two ejaculates per pool) and cryopreserved using a freezing extender containing three different sugars (glucose, fructose and sorbitol), with or without the addition of Equex paste. Sperm quality was assessed before and after freezing-thawing for motility, morphology, plasma membrane integrity, acrosome integrity and DNA integrity. The use of sorbitol in the f...
Aging Markers in Equine Red Blood Cells. Detection of hematopoietic activity in horses is a challenge due to the lack of cells carrying reticulocyte markers such as RNA remnants or CD71 in the circulation. In this study, we fractionated equine red cells according to their density and analyzed the cells forming low (L), medium (M), and high (H) density fractions for markers of aging such as membrane loss, oxidation, and alterations in the intracellular free Ca levels. Cells forming L and M fraction were highly heterogeneous in projected areas and shapes, and had higher propensity to swell in response to hypo-osmotic challenge than the...
Analysis of the equine “cumulome” reveals major metabolic aberrations after maturation in vitro. Maturation of oocytes under in vitro conditions (IVM) results in impaired developmental competence compared to oocytes matured in vivo. As oocytes are closely coupled to their cumulus complex, elucidating aberrations in cumulus metabolism in vitro is important to bridge the gap towards more physiological maturation conditions. The aim of this study was to analyze the equine "cumulome" in a novel combination of proteomic (nano-HPLC MS/MS) and metabolomic (UPLC-nanoESI-MS) profiling of single cumulus complexes of metaphase II oocytes matured either in vivo (n = 8) or in vitro (n = 7). Re...
Ultrastructural morphology is distinct among primary progenitor cell isolates from normal, inflamed, and cryopreserved equine hoof tissue and CD105+K14+ progenitor cells. The equine hoof dermal-epidermal interface requires progenitor cells with distinct characteristics. This study was designed to provide accurate ultrastructural depictions of progenitor cells isolated from inflamed tissue and normal tissue before and after cryopreservation and following selection of cells expressing both keratin (K) 14 (ectodermal) and cluster of differentiation (CD) 105 (mesodermal). Passage 3 cell ultrastructure was assessed following 2D culture and after 3D culture on decellularized hoof tissue scaffolds. Outcome measures included light, transmission electron, and scanning e...
In vitro Characteristics of Heterogeneous Equine Hoof Progenitor Cell Isolates. Damage to an ectodermal-mesodermal interface like that in the equine hoof and human finger nail bed can permanently alter tissue structure and associated function. The purpose of this study was to establish and validate culture of primary progenitor cell isolates from the ectodermal-mesodermal tissue junction in equine hooves, the stratum internum, with and without chronic inflammation known to contribute to lifelong tissue defects. The following were evaluated in hoof stratum internum cell isolates up to 5 cell passages (P): expansion capacity by cell doublings and doubling time; plasticity ...
Enhanced chondrogenic differentiation of equine bone marrow-derived mesenchymal stem cells in zirconia microwell substrata. In human cartilage tissue engineering, three-dimensional zirconia substrata have the potential advantage of producing many uniform cell clusters of controlled size without xenobiotic material, allowing easy clinical application. The objective of this study was to evaluate the possibility of using zirconia porous three-dimensional microwell substrata for chondrogenic differentiation of equine bone marrow-derived mesenchymal stem cells (BMMSCs) in vitro. In regular medium, 8 × 10, 2 × 10, and 5 × 10 equine BMMSCs from five thoroughbred horses were cultured on zirconia microwell su...
Characterization of the in vitro CYP450 mediated metabolism of the polymorphic CYP2D6 probe drug codeine in horses. Despite their widespread popularity as sport and companion animals and published and anecdotal reports of vast difference in drug disposition and pharmacokinetics between individuals, studies describing equine drug metabolism are limited. It has been theorized that similar to humans, members of the CYP2D family in horses may be polymorphic in nature leading to differences in metabolism of substrates. This study aims to build on the limited current knowledge regarding P450 mediated metabolism in horses by describing the metabolism of the polymorphic CYP2D6 probe drug codeine in vitro. Codeine, ...
In Vitro and In Vivo Efficiency of Extenders Added to Thawed Stallion Semen. Addition of extenders to thawed semen could improve fertility. Objective: To determine the efficiency of extenders to increase viability of thawed semen, measured by sperm parameters in vitro and pregnancy rates after artificial insemination (AI). Methods: Sperm motility and acrosin activity were measured during a thermoresistance test (TRT). Results: Progressive motility decreased (P<0.05) after 30 min in thawing semen treated with saline solution (SS) and only after 60 min with Tyrode's solution (TS) or freezing diluent (FD). The total motility decreased (P<0.05) after 60 min in t...
Polyphasic Discrimination of Trichophyton tonsurans and T. equinum from Humans and Horses. The anthropophilic dermatophyte Trichophyton tonsurans and its zoophilic counterpart T. equinum are phylogenetically closely related. The barcoding marker rDNA internal transcribed spacer (ITS) shows limited variation between these two species. In the current study, we combined molecular approaches with phenotypic data to determine the species boundaries between T. tonsurans (n = 52) and T. equinum (n = 15) strains originating from humans (n = 40), horses (n = 26), and a mouse (n = 1). Culture characteristics and physiology on Trichophyton agar media 1 and 5 were evaluated....
Supplemental Antibiotics in a Commercial Extender for Stallion Semen. Commonly marketed semen extenders contain various antibiotic types and concentrations to control bacterial growth from stallion's external genitalia. An experiment was conducted to test the effects of supplemental amikacin disulfate (1,000 μg/mL) + potassium penicillin G (1,000 IU/ML: INRA-AP), or ticarcillin-clavulanate (1,000 μg/mL: INRA-TIM) in the INRA 96 extender, on sperm function and antimicrobial activity, compared with extender without supplemental antibiotics (INRA-C). In freshly extended semen (Time 30m), no differences were observed among the three treatment groups for sperm m...
The influence of trimming of the hoof wall on the damage of laminar tissue after loading: An in vitro study. Laminitis is associated with failure of the suspensory apparatus of the distal phalanx (SADP) connecting the distal phalanx to the hoof wall. The specific aim of this study was to examine in vitro whether thinning of the hoof wall leading to increased deformability influences the damage of the laminar tissue created by loading of the hoof. Paired cadaver forelimbs from twelve horses were used. For each pair, the hoof wall from one hoof was thinned by 25%; this was ascertained by radiography. The contralateral hooves were used as controls. In a material testing machine, hooves were loaded in a ...
Equine adipose mesenchymal stem cells (eq-ASCs) appear to have higher potential for migration and musculoskeletal differentiation. Equine adipose-derived mesenchymal stem cells (eq-ASCs) possess excellent regeneration potential especially for treatment of musculoskeletal disorders. Besides their common characteristics, MSCs harvested from different species reveal some species-specific and donor-dependent behaviors. Hence, the molecular analysis of MSCs may shed more light on their future clinical application of these cells. This study aimed to investigate some behavioral aspects of eq-ASCs in vitro which may influence the efficacy of stem cell therapy. For this purpose, MSCs of a donor horse were isolated, characterized a...
In vitro effects of poly(ADP-ribose) polymerase inhibitors on the production of tumor necrosis factor-α by interferon- γ – and lipopolysaccharide-stimulated peripheral blood mononuclear cells of horses. To evaluate effects of poly(ADP-ribose) polymerase-1 (PARP1) inhibitors on the production of tumor necrosis factor-α (TNF-α) by interferon-γ (IFN-γ)- and lipopolysaccharide (LPS)-stimulated peripheral blood mononuclear cells (PBMCs) of horses as an in vitro model of inflammation in horses. Methods: 1,440 samples of PBMCs from 6 healthy research horses. Methods: From heparinized whole blood samples, PBMC cultures were obtained. An initial dose-response trial on 48 PBMC samples from 2 horses (24 samples each) was used to determine concentrations of IFN-γ and LPS for use as low- and high-lev...
In vitro evaluation of the knot-holding capacity and security, weight, and volume of forwarder knots tied with size-3 polyglactin 910 suture exposed to air, balanced electrolyte solution, or equine abdominal fat. To evaluate the effect of exposure to a balanced electrolyte solution (BES), or equine abdominal fat on the knot-holding capacity (KHC), relative knot security (RKS), weight, and volume of forwarder knots versus surgeon's knots. Methods: 315 knots tied and tested in vitro. Methods: United States Pharmacopeia size-3 polyglactin 910 suture exposed to air (dry [control]), equine abdominal fat (fat-exposed), or BES (BES-exposed) was used to tie forwarder knots with 2, 3, and 4 throws and surgeon's knots with 5, 6, 7, and 8 throws. A universal materials testing machine was used to test the tensile ...
Effects of blood-derived products and sodium hyaluronate on equine synovial fluid cells and on synovial fluid from osteochondrotic joints of horses after arthroscopy and administration of treatment. To compare effects of platelet-rich plasma (PRP), interleukin-1 receptor antagonist protein (IRAP), autologous processed plasma (APP), and sodium hyaluronate treatments on synovial fluid cells in vitro and on synovial fluid obtained from osteochondrotic joints of horses. Methods: Synovial fluid cells from 8 healthy equine tibiotarsal joints (in vitro experiment) and synovial fluid samples from 40 tibiotarsal joints of 25 horses with osteochondrosis dissecans (in vivo experiment). Methods: Effects of various treatments on concentrations of prostaglandin (PG) E2, interleukin (IL)-1β, tumor necr...
Transcriptome analysis reveals that fertilization with cryopreserved sperm downregulates genes relevant for early embryo development in the horse. Artificial insemination with cryopreserved spermatozoa is a major assisted reproductive technology in many species. In horses, as in humans, insemination with cryopreserved sperm is associated with lower pregnancy rates than those for fresh sperm, however, direct effects of sperm cryopreservation on the development of resulting embryos are largely unexplored. The aim of this study was to investigate differences in gene expression between embryos resulting from fertilization with fresh or cryopreserved sperm. Embryos were obtained at 8, 10 or 12 days after ovulation from mares inseminated post-...
