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.
Black JB, Frampton AR.Equine herpesvirus type 1 (EHV-1) is a highly transmissible pathogen that leads to a variety of clinical disease outcomes in infected horses. A major sequela that can occur after an EHV-1 infection is a neurological disease termed equine herpesvirus myeloencephalopathy (EHM). Clinical manifestations of EHM include fever, ataxia, incontinence, and partial to full paralysis, which may ultimately lead to the euthanization of the infected horse. To develop an effective treatment strategy for EHM, it is critical that the specific virus-host interactions that lead to EHM be investigated so that safe...
Petrova V, Vachkova E.Adipose tissue is recognized as the major endocrine organ, potentially acting as a source of mesenchymal stem cells for various applications in regenerative medicine. Athletic horses are often exposed to traumatic injuries, resulting in severe financial losses. The development of adipose-derived stem cells' regenerative potential depends on many factors. The extraction of stem cells from subcutaneous adipose tissue is non-invasive, non-traumatic, cheaper, and safer than other sources. Since there is a lack of unique standards for identification, the isolated cells and applied differentiation p...
Hassall L, Rigsby P, Stickings P.The International Standard for Diphtheria Antitoxin Equine is essential for the standardisation of assays used to determine the potency of therapeutic diphtheria antitoxin products produced from equine serum. This paper describes the production and characterization of the 2nd International Standard for Diphtheria Antitoxin Equine and its calibration in International Units. Calibration was performed by toxin neutralization test in vivo and in vitro (Vero cell assay), and potency was expressed relative to the 1st International Standard to ensure continuity of the International Unit. The candidat...
Smith HL, Berglund AK, Robertson JB, Schnabel LV, McMullen RJ, Gilger BC, Oh A.The objective of the study was to determine the effect of gentamicin on CD3+ T-lymphocyte proliferation and cell viability using an in vitro cell culture model as a means of investigating the mechanism of action of low-dose intravitreal gentamicin injection. Methods: Three adult horses with no evidence of ophthalmic or systemic disease. Methods: Peripheral blood lymphocytes were treated with gentamicin at concentrations 37.5 μg/mL, 112.5 μg/mL, 187 μg/mL, 375 μg/mL, or 750 μg/mL then stimulated to proliferate with concanavalin A (ConA). 4',6-diamidino-2-phenylindole (DAPI) and c...
Bizley SC, Dudhia J, Smith RKW, Williams AC.Oral and parenteral drug delivery in horses can be difficult. Equine-specific transdermal drug formulations offer improved ease of treatment; development of such formulations requires a deeper understanding of the structural and chemical tissue barrier of horse skin. Objective: To compare the structural composition and barrier properties of equine skin. Methods: Six warmblood horses (two males, four females) with no skin diseases. Methods: Routine histological and microscopic analyses were carried out with image analysis for skin from six different anatomical locations. In vitro drug permeatio...
Stage HJ, Trappe S, Söllig K, Trachsel DS, Kirsch K, Zieger C, Merle R, Aschenbach JR, Gehlen H.The investigation of multipotent stem/stromal cells (MSCs) in vitro represents an important basis for translational studies in large animal models. The study's aim was to examine and compare clinically relevant in vitro properties of equine MSCs, which were isolated from abdominal (abd), retrobulbar (rb) and subcutaneous (sc) adipose tissue by collagenase digestion (ASCs-) and an explant technique (ASCs-). Firstly, we examined proliferation and trilineage differentiation and, secondly, the cardiomyogenic differentiation potential using activin A, bone morphogenetic protein-4 and Dickkopf-1. Fi...
Hedia M, Leroy JLMR, Govaere J, Van Soom A, Smits K.The application of trans-vaginal ovum pick up (OPU) and intracytoplasmic sperm injection (ICSI) is well established for commercial in vitro embryo production in horses. These assisted reproductive techniques are especially applied during the non-breeding season of the mare. However, little is known about how the health of the oocyte donor may affect the biochemical composition of the follicular fluid (FF) in small and medium-sized follicles routinely aspirated during OPU. This study aimed to investigate associations between systemic and FF concentrations of interleukin-6 (IL-6), total choleste...
Angel-Velez D, Meese T, Hedia M, Fernandez-Montoro A, De Coster T, Pascottini OB, Van Nieuwerburgh F, Govaere J, Van Soom A, Pavani K, Smits K.In the last decade, in vitro embryo production in horses has become an established clinical practice, but blastocyst rates from vitrified equine oocytes remain low. Cryopreservation impairs the oocyte developmental potential, which may be reflected in the messenger RNA (mRNA) profile. Therefore, this study aimed to compare the transcriptome profiles of metaphase II equine oocytes vitrified before and after in vitro maturation. To do so, three groups were analyzed with RNA sequencing: (1) fresh in vitro matured oocytes as a control (FR), (2) oocytes vitrified after in vitro maturation (VMAT), a...
Mo PM, Picard J, Gummow B.Fungi are ubiquitous in the environment and part of the commensal microflora on the conjunctiva of equine eyes. North Queensland, being tropical, presents an ideal environment for fungi growth. When the cornea is injured, fungi can invade the corneal stroma, resulting in keratomycosis. The objectives of this study were to determine the fungal species specific to the eyes of horses in the Townsville region; to investigate the potential risk factors associated with the presence of fungi; and to test their susceptibility to antifungals to create an empirical guide for treatment. The eyes of forty...
Marsico TV, Valente RS, Annes K, Oliveira AM, Silva MV, Sudano MJ.A wide-ranging review study regarding the molecular characterization of the first cell lineages of the developmental embryo is lacking, especially for the primary events during earliest differentiation which leads to the determination of cellular fate. Here, a systematic review and meta-analysis were conducted according to PRISMA guidelines. MEDLINE-PubMed was searched based on an established search strategy through April 2021. Thirty-six studies fulfilling the inclusion criteria were subjected to qualitative and quantitative analysis. Among the studies, 50 % (18/36) used mice as an animal mod...
Seewald LA, Sabino IG, Montney KL, Delco ML.To evaluate mitochondrial DNA (mtDNA) release from injured chondrocytes and investigate the utility of synovial fluid mtDNA concentration in early detection of posttraumatic osteoarthritis. We measured mtDNA release using four models of osteoarthritis: in vitro interleukin-1β stimulation of cultured equine chondrocytes, ex vivo mechanical impact of bovine cartilage explants, in vivo mechanical impact of equine articular cartilage, and naturally occurring equine intraarticular fracture. In our in vivo model, one group was treated with an intraarticular injection of the mitoprotective peptide S...
Martin-Pelaez S, Rabow Z, de la Fuente A, Draheim P, Loynachan A, Fiehn O, Meyers S, Lyman C, Dini P.Postmortem and pre-euthanasia oocyte retrieval provides the last opportunity to preserve the genetic material in mares. Pentobarbital (PB) is the most common euthanasia agent; however, its effect on the developmental competence of oocytes has not been determined. Here, we evaluated the concentration of PB in equine follicular fluid (FF) and investigated its effect on the developmental competence of oocytes using a bovine IVF model to overcome the low availability of equine oocytes. The concentration of PB was measured by gas-chromatography/mass-spectrometry in FF collected from mare ovaries im...
Vereecke N, Vandekerckhove A, Theuns S, Haesebrouck F, Boyen F.Actinobacillus equuli is mostly associated with disease in horses and is most widely known as the causative agent of sleepy foal disease. Even though existing phenotypic tools such as biochemical tests, 16S rRNA gene sequencing, and Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) can be used to identify members of the Actinobacillus genus, these methods struggle to differentiate between certain species and do not allow strain, virulence, and antimicrobial susceptibility typing. Hence, we performed in-depth analysis of 24 equine Actinobacillus isolate...
Fujiwara Y, Kusakabe KT, Baba K, Sasaki N.Currently, treatment for peripheral nerve injuries in horses primarily relies upon physical therapy and anti-inflammatory drugs. In humans, various treatments using mesenchymal stem cells (MSCs) are being attempted. Therefore, in this study, Schwann-like cell differentiation cultures of equine MSCs were prepared using fetal bovine serum (FBS) and equine platelet lysate (ePL). ePL increased the platelet count to 1 × 10/μl, the optimal concentration for culture. In both groups, an elongated morphology at both ends, characteristic of Schwann cells, was observed under the microscope. Real-time...
Alpoim-Moreira J, Szóstek-Mioduchowska A, Słyszewska M, Rebordão MR, Skarzynski DJ, Ferreira-Dias G.Endometrosis negatively affects endometrial function and fertility in mares, due to excessive deposition of type I (COL1) and type III (COL3) collagens. The pro-fibrotic transforming growth factor (TGF-β1) induces myofibroblast differentiation, characterized by α-smooth muscle actin (α-SMA) expression, and collagen synthesis. In humans, fibrosis has been linked to epigenetic mechanisms. To the best of our knowledge, this has not been described in mare endometrium. Therefore, this study aimed to investigate the in vitro epigenetic regulation in TGF-β1-treated mare endometrial fibroblasts an...
de Moura TCM, Arruda LCP, Cahú TB, Bezerra RS, Carneiro GF, Guerra MMP.Semen cryopreservation ensures the storage of stallion genetics for an unlimited time. The improvement of extenders with new antioxidant substances can optimize the properties of post-thawed semen. The study aimed to investigate the addition effect of medium-molecular-weight carboxymethylchitosan (CQm) derivates to freezing diluent of stallion sperm after freezinf/thawing. Twice a week, five ejaculates of four stallions were obtained, totalizing 20 ejaculates. Semen was diluted in commercial freezing extender (Botucrio) supplemented with CQm: control (0), 0.75, 1.5, and 3 mg/mL. Samples were ...
Colla S, Seabaugh KA, Zanotto GM, Selberg K.Techniques for local anesthesia of the tibial (TN) and superficial and deep fibular nerves (FNs) in horses are well established. Ultrasound-guided perineural blocks can identify the nerve location, reduce the anesthetic volume needed and avoid needle misplacement. The aim of this research was to compare the success of blind perineural injection technique (BLIND) to ultrasound-guided technique (USG). Fifteen equine cadaver hindlimbs were divided into two groups. Perineural injection of the TN and FNs was performed using a mixed solution of radiopaque contrast, saline and food dye. BLIND (n = ...
Mawhinney I, Bollard A.Detection of Taylorella equigenitalis (CEMO) in the horse uses genital swabs. These swabs traditionally have been put in Amies charcoal transport medium for detection by culture but are also used for PCR. We determined the suitability of swabs without transport medium (Dry swabs) for CEMO PCR compared to swabs in Amies charcoal transport medium. The experiment was a factorial design using swab type and dilution of organism in culture suspensions, done in two parts. Simulated genital swabs were prepared in the laboratory by dipping in pairs into culture suspensions containing T. equigenitalis w...
Cortez JV, Hardwicke K, Cuervo-Arango J, Grupen CG.The cloning of horses is a commercial reality, yet the availability of oocytes for cloned embryo production remains a major limitation. Immature oocytes collected from abattoir-sourced ovaries or from live mares by ovum pick-up (OPU) have both been used to generate cloned foals. However, the reported cloning efficiencies are difficult to compare due to the different somatic cell nuclear transfer (SCNT) techniques and conditions used. The objective of this retrospective study was to compare the in vitro and in vivo development of equine SCNT embryos produced using oocytes recovered from abatt...
Andrietti ALP, Durgam SS, Naumann B, Stewart M.Mesenchymal stem cells are characterized by their capacities for extensive proliferation through multiple passages and, classically, tri-lineage differentiation along osteogenic, chondrogenic and adipogenic lineages. This study was carried out to compare osteogenesis in equine bone marrow-, synovium- and adipose-derived cells, and to determine whether osteogenic capacity is reflected in the basal expression of the critical osteogenic transcription factors Runx2 and Osterix. Unassigned: Bone marrow, synovium and adipose tissue was collected from six healthy 2-year-old horses. Cells were isolate...
Bourebaba L, Serwotka-Suszczak A, Pielok A, Sikora M, Mularczyk M, Marycz K.Equine metabolic syndrome (EMS) is a multifactorial pathology gathering insulin resistance, low-grade inflammation and past or chronic laminitis. Among the several molecular mechanisms underlying EMS pathogenesis, increased negative insulin signalling regulation mediated by protein tyrosine phosphatase 1 B (PTP1B) has emerged as a critical axis in the development of liver insulin resistance and general metabolic distress associated to increased ER stress, inflammation and disrupted autophagy. Thus, the use of PTP1B selective inhibitors such as MSI-1436 might be considered as a golden therapeut...
Belacic ZA, Sullivan SN, Rice HC, Durgam SS.To investigate the effects of interleukin-1β (IL-1β) and methylprednisolone acetate (MPA) on equine intrabursal deep digital flexor tendon (DDFT) and navicular bone fibrocartilage (NBF) cells in vitro. Methods: Third passage DDFT and NBF cells from 5 healthy donor horses ages 11-17 years euthanized for reasons unrelated to musculoskeletal conditions. Methods: Aggregate cultures were incubated with culture medium alone (control), 10 ng/mL IL-1β, 10 ng/mL IL-1β + 0.05 mg/mL MPA, or 10 ng/mL IL-1β + 0.5 mg/mL MPA for 24 hours. Extracellular matrix (ECM) gene expressions were assessed via rea...
Padjasek M, Cisło-Sankowska A, Lis-Bartos A, Qasem B, Marycz K.The purpose of this study was to describe the use of PLDLA/TPU matrix enriched with cyclosporine A (CsA) as a therapeutic platform in horses with immune-mediated keratitis (IMMK) with an in vitro evaluation CsA release and degradation of the blend as well as determination of the safety and efficacy of that platform used in the animal model. The kinetics of the CsA release from matrices constructed of thermoplastic polyurethane (TPU) polymer and a copolymer of L-lactide with DL-lactide (PLDLA) (80:20) in the TPU (10%) and a PLDL (90%) polymer blend were studied. Moreover, we used the STF (Simul...
Kang H, Lee GKC, Bienzle D, Arroyo LG, Sears W, Lillie BN, Beeler-Marfisi J.Alveolar macrophages (AMs) are the predominant innate immune cell in the distal respiratory tract. During inflammatory responses, AMs may be supplemented by blood monocytes, which differentiate into monocyte-derived macrophages (MDMs). Macrophages play important roles in a variety of common equine lower airway diseases, including severe equine asthma (SEA). In an experimental model, an inhaled mixture of Aspergillus fumigatus spores, lipopolysaccharide, and silica microspheres (FLS), induced SEA exacerbation in susceptible horses. However, whether equine AMs and MDMs have differing immunopheno...
Langreder N, Schäckermann D, Meier D, Becker M, Schubert M, Dübel S, Reinard T, Figge-Wegener S, Roßbach K, Bäumer W, Ladel S, Hust M.Insect bite hypersensitivity (IBH) is the most common allergic skin disease of horses. It is caused by insect bites of the Culicoides spp. which mediate a type I/IVb allergy with strong involvement of eosinophil cells. No specific treatment option is available so far. One concept could be the use of a therapeutic antibody targeting equine interleukin 5, the main activator and regulator of eosinophils. Therefore, antibodies were selected by phage display using the naïve human antibody gene libraries HAL9/10, tested in a cellular in vitro inhibition assay and subjected to an in vitro affinity...
Verhaar N, Hoppe S, Grages AM, Hansen K, Neudeck S, Kästner S, Mazzuoli-Weber G.α2 agonists are frequently used in horses with colic, even though they have been shown to inhibit gastrointestinal motility. The aim of this study was to determine the effect of dexmedetomidine on small intestinal in vitro contractility during different phases of ischaemia. Experimental segmental jejunal ischaemia was induced in 12 horses under general anaesthesia, and intestinal samples were taken pre-ischaemia and following ischaemia and reperfusion. Spontaneous and electrically evoked contractile activity of the circular and longitudinal smooth muscles were determined in each sample with a...
Hernández-Avilés C, Ramírez-Agámez L, Pearson M, Beckham AMN, Varner DD, Love CC.Analysis of sperm morphology is an important part of the stallion breeding soundness evaluation since it provides an objective measure of a stallion's sperm quality and is one of many factors that estimate a stallion's fertility potential. To describe the effect of sperm quality level on the technique (Differential Interference Contrast - DIC; Phase-contrast - PH; Dip-Quick staining - DQ; and eosin-nigrosin staining - EN; semen samples fixed in buffered-formal saline) and evaluator (three evaluators; using only DIC), stallions were categorized based on sperm quality into three categories: High...
Persistent post-breeding induced endometritis (PPBIE) is considered a major cause of subfertility in mares. It consists of persistent or delayed uterine inflammation in susceptible mares. There are many options for the treatment of PPBIE, but in this study, a novel approach aimed at preventing the onset of PPBIE was investigated. Stallion semen was supplemented with extracellular vesicles derived from amniotic mesenchymal stromal cells (AMSC-EVs) at the time of insemination to prevent or limit the development of PPBIE. Before use in mares, a dose-response curve was produced to evaluate the eff...
Zamith Cunha R, Zannoni A, Salamanca G, De Silva M, Rinnovati R, Gramenzi A, Forni M, Chiocchetti R.The metacarpophalangeal joint undergoes enormous loading during locomotion and can therefore often become inflamed, potentially resulting in osteoarthritis (OA). There are studies indicating that the endocannabinoid system (ECS) modulates synovium homeostasis, and could be a promising target for OA therapy. Some cannabinoid receptors, which modulate proliferative and secretory responses in joint inflammation, have been functionally identified in human and animal synovial cells. Unassigned: To characterize the cellular distribution of the cannabinoid receptors 1 (CB1R) and 2 (CB2R), and the can...
Yuki N, Shimazaki T, Kushiro A, Watanabe K, Uchida K, Yuyama T, Morotomi M.Selective adhesion to only certain epithelia is particularly common among the bacterial members of the indigenous microflora of mammals. We have found that the stratified squamous epithelium of the nonsecreting area of horse stomach is colonized by gram-positive rods. The microscopic features of a dense layer of these bacteria on the epithelium were found to be similar to those reported in mice, rats, and swine. Adhering microorganisms were isolated and identified as Lactobacillus salivarius, L. crispatus, L. reuteri, and L. agilis by DNA-DNA hybridization and 16S rRNA gene sequencing techniqu...
Schauer SN, Sontakke SD, Watson ED, Esteves CL, Donadeu FX.Previous evidence from in vitro studies suggests specific roles for a subset of miRNAs, including miR-21, miR-23a, miR-145, miR-503, miR-224, miR-383, miR-378, miR-132, and miR-212, in regulating ovarian follicle development. The objective of this study was to determine changes in the levels of these miRNAs in relation to follicle selection, maturation, and ovulation in the monovular equine ovary. In Experiment 1, follicular fluid was aspirated during ovulatory cycles from the dominant (DO) and largest subordinate (S) follicles of an ovulatory wave and the dominant (DA) follicle of a mid-cycle...
Maury W.In vivo, equine infectious anemia virus (EIAV) replicates in tissues rich in macrophages, and it is widely believed that the tissue macrophage is the principal, if not sole, cell within the host that replicates virus. No viral replication has been detected in circulating peripheral blood monocytes. However, proviral DNA can be detected in these cells, and monocytes may serve as a reservoir for the virus. In this study, an in vitro model was developed to clarify the role of monocyte maturation in regulating EIAV expression. Freshly isolated, nonadherent equine peripheral blood monocytes were in...
Naskou MC, Sumner SM, Chocallo A, Kemelmakher H, Thoresen M, Copland I, Galipeau J, Peroni JF.Mesenchymal stem cells (MSCs) produced for clinical purposes rely on culture media containing fetal bovine serum (FBS) which is xenogeneic and has the potential to significantly alter the MSC phenotype, rendering these cells immunogenic. As a result of bovine-derived exogenous proteins expressed on the cell surface, MSCs may be recognized by the host immune system as non-self and be rejected. Platelet lysate (PL) may obviate some of these concerns and shows promising results in human medicine as a possible alternative to FBS. Our goal was to evaluate the use of equine platelet lysate (ePL) poo...
Schoster A, Weese JS, Guardabassi L.The gastrointestinal microbiota is extremely important for human and animal health. Investigations into the composition of the microbiota and its therapeutic modification have received increasing interest in human and veterinary medicine. Probiotics are a way of modifying the microbiota and have been tested to prevent and treat diseases. Probiotics are proposed to exert their beneficial effects through various pathways. Production of antimicrobial compounds targeting intestinal pathogens, general immune stimulation, and colonization resistance are among these mechanisms. Despite widespread ava...
Marędziak M, Marycz K, Smieszek A, Lewandowski D, Toker NY.The aim of this study was to evaluate the proliferation rate and morphological changes of adipose-derived mesenchymal stem cells of canine and equine origin (Eq- and CaAdMSC). Investigated cells were exposed to a static magnetic field (MF) with the intensity of 0.5 T. Proliferation activity of cells was determined with the Alamar Blue assay. Obtained results, normalized in respect to the control culture, showed that EqAdMSC exposed to MF maintained a high proliferation status, whereas proliferation activity of CaAdMSC cultured in the presence of MF was decreased. Estimations of population doub...
Mensing N, Gasse H, Hambruch N, Haeger JD, Pfarrer C, Staszyk C.The equine periodontium provides tooth support and lifelong tooth eruption on a remarkable scale. These functions require continuous tissue remodeling. It is assumed that multipotent mesenchymal stromal cells (MSC) reside in the periodontal ligament (PDL) and play a crucial role in regulating physiological periodontal tissue regeneration. The aim of this study was to isolate and characterize equine periodontal MSC. Tissue samples were obtained from four healthy horses. Primary cell populations were harvested and cultured from the gingiva, from three horizontal levels of the PDL (apical, midtoo...
Clutterbuck AL, Smith JR, Allaway D, Harris P, Liddell S, Mobasheri A.This study employed a targeted high-throughput proteomic approach to identify the major proteins present in the secretome of articular cartilage. Explants from equine metacarpophalangeal joints were incubated alone or with interleukin-1beta (IL-1β, 10ng/ml), with or without carprofen, a non-steroidal anti-inflammatory drug, for six days. After tryptic digestion of culture medium supernatants, resulting peptides were separated by HPLC and detected in a Bruker amaZon ion trap instrument. The five most abundant peptides in each MS scan were fragmented and the fragmentation patterns compared to m...
Watts AE, Ackerman-Yost JC, Nixon AJ.To compare in vitro three-dimensional (3D) culture systems that model chondrogenesis of bone marrow-derived mesenchymal stem cells (MSCs). Methods: MSCs from five horses 2-3 years of age were consolidated in fibrin 0.3% alginate, 1.2% alginate, 2.5×10(5) cell pellets, 5×10(5) cell pellets, and 2% agarose, and maintained in chondrogenic medium with supplemental TGF-β1 for 4 weeks. Pellets and media were tested at days 1, 14, and 28 for gene expression of markers of chondrogenic maturation and hypertrophy (ACAN, COL2B, COL10, SOX9, 18S), and evaluated by histology (hematoxylin and eosin, Tolu...
Worster AA, Nixon AJ, Brower-Toland BD, Williams J.To determine the morphologic and phenotypic effects of transforming growth factor beta1 (TGFbeta1) on cultured equine mesenchymal stem cells (MSC) and articular chondrocytes. Methods: Bone marrow aspirates and articular cartilage samples from a 2-year-old and two 8-month-old horses. Methods: After initial isolation and culture, MSC and chondrocytes were cultured in Ham's F-12 medium supplemented with TGF-beta1 at a concentration of 0, 1, 5, or 10 ng/ml. Medium was exchanged on day 2, and cells were harvested on day 4. Medium was assayed for proteoglycan (PG) content. Total RNA was isolated fro...
Raabe O, Shell K, Goessl A, Crispens C, Delhasse Y, Eva A, Scheiner-Bobis G, Wenisch S, Arnhold S.Mesenchymal stem cells are regarded as common cellular precursors of the musculoskeletal tissue and are responsible for tissue regeneration in the course of musculoskeletal disorders. In equine veterinary medicine extracorporeal shock wave therapy (ESWT) is used to optimize healing processes of bone, tendon and cartilage. Nevertheless, little is known about the effects of the shock waves on cells and tissues. Thus, the aim of this study was to investigate the influence of focused ESWT on the viability, proliferation, and differentiation capacity of adipose tissue-derived mesenchymal stem cells...
Alghamdi AS, Lovaas BJ, Bird SL, Lamb GC, Rendahl AK, Taube PC, Foster DN.Bovine semen is naturally deposited in the vagina and spermatozoa migrate through the cervix into the uterus leaving the bulk of seminal plasma (SP) behind. In equine, both spermatozoa and SP are deposited directly in the uterus and SP reduces sperm binding to neutrophils and prevents the formation of DNA-based neutrophil extracellular traps (NETs). We investigated the role of bovine SP on sperm-neutrophil binding using the four most common bovine semen extenders. Contrary to equine, bovine spermatozoa removed from SP had low binding to neutrophils for up to 3h, but as little as 10% SP increas...
Böer U, Lohrenz A, Klingenberg M, Pich A, Haverich A, Wilhelmi M.Decellularized equine carotid arteries (dEAC) may represent a reasonable alternative to alloplastic materials in vascular replacement therapy. Acellularity of the matrix is standardly evaluated by DNA quantification what however may not record sufficiently the degree of matrix immunogenicity. Thus, our aim was to analyze dEAC with a low DNA content for residual cellular proteins. A detergent-based decellularization protocol including endonuclease treatment resulted in dEAC with 0.6 ± 0.15 ng DNA/mg dry weight representing 0.33 ± 0.14% of native tissue DNA content. In contrast, when matrices ...
Bowles DE, Holden VR, Zhao Y, O'Callaghan DJ.To assess the role of the equine herpesvirus type 1 (EHV-1) ICP0 protein (EICP0) in gene regulation, a variety of molecular studies on the EICP0 gene and gene products of both the attenuated cell culture-adapted Kentucky A (KyA) strain and the Ab4p strain were conducted. These investigations revealed that (i) the ICP0 open reading frame (ORF) of the KyA virus strain is 1,257 bp in size and would encode a protein of 419 amino acids, and in comparison to the ICP0 gene (ORF63) of the Ab4p strain of 1,596 bp (E. A. Telford, M. S. Watson, K. McBride, and A. J. Davison, Virology 189:304-316, 1992), ...
Jörg A, Henderson WR, Murphy RC, Klebanoff SJ.Horse eosinophils purified to greater than 98% generated slow reacting substance (SRS) when incubated with the calcium ionophore A23187. On a per cell basis, eosinophils generated four to five times the SRS produced by similarly treated horse neutrophils. Eosinophil SRS production was inhibited by 5,8,11,14-eicosatetraynoic acid and augmented by indomethacin and arachidonic acid, suggesting that it was a product(s) of the lipoxygenase pathway of arachidonic acid metabolism. Compounds with SRS activity were purified by high-pressure liquid chromatography (HPLC) and identified by ultraviolet spe...
Sailer BL, Jost LK, Evenson DP.Sperm from four mammalian species were analyzed by the sperm chromatin structure assay (SCSA) and the terminal deoxynucleotidyl transferase assay (TdTA) using flow cytometry. The SCSA quantitates the susceptibility of sperm nuclear DNA to in situ acid denaturation, while the TdTA quantitates the presence of endogenous DNA strand breaks in sperm nuclear chromatin. Correlations were seen between the percentage of sperm cells showing susceptibility to in situ acid denaturation and the percentage of cells showing the presence of DNA strand breaks for humans (r = 0.56, P = 0.004), rams (r = 0.84, P...
Yin X, Hu Z, Gu Q, Wu X, Zheng YH, Wei P, Wang X.Human tetherin is a host restriction factor that inhibits replication of enveloped viruses by blocking viral release. Tetherin has an unusual topology that includes an N-terminal cytoplasmic tail, a single transmembrane domain, an extracellular domain, and a C-terminal glycosylphosphatidylinositol anchor. Tetherin is not well conserved across species, so it inhibits viral replication in a species-specific manner. Thus, studies of tetherin activities from different species provide an important tool for understanding its antiviral mechanism. Here, we report cloning of equine tetherin and charact...
Wagner B, Miller DC, Lear TL, Antczak DF.This report contains the first map of the complete Ig H chain constant (IGHC) gene region of the horse (Equus caballus), represented by 34 overlapping clones from a new bacterial artificial chromosome library. The different bacterial artificial chromosome inserts containing IGHC genes were identified and arranged by hybridization using overgo probes specific for individual equine IGHC genes. The analysis of these IGHC clones identified two previously undetected IGHC genes of the horse. The newly found IGHG7 gene, which has a high homology to the equine IGHG4 gene, is located between the IGHG3 ...
Mancini IAD, Schmidt S, Brommer H, Pouran B, Schäfer S, Tessmar J, Mensinga A, van Rijen MHP, Groll J, Blunk T, Levato R, Malda J, van Weeren PR.Recent research has been focusing on the generation of living personalized osteochondral constructs for joint repair. Native articular cartilage has a zonal structure, which is not reflected in current constructs and which may be a cause of the frequent failure of these repair attempts. Therefore, we investigated the performance of a composite implant that further reflects the zonal distribution of cellular component both in vitro and in vivo in a long-term equine model. Constructs constituted of a 3D-printed poly(ϵ-caprolactone) (PCL) bone anchor from which reinforcing fibers protruded into ...
Reed SA, Johnson SE.Mesenchymal stem cells (MSCs) offer promise as therapeutic aids in the repair of tendon, ligament, and bone damage suffered by sport horses. The objective of the study was to identify and characterize stem-like cells from newborn foal umbilical cord blood (UCB). UCB was collected and MSC isolated using human reagents. The cells exhibit a fibroblast-like morphology and express the stem cell markers Oct4, SSEA-1, Tra1-60 and Tra1-81. Culture of the cells in tissue-specific differentiation media leads to the formation of cell types characteristic of mesodermal and endodermal origins. Chondrogenic...
Delco ML, Goodale M, Talts JF, Pownder SL, Koff MF, Miller AD, Nixon B, Bonassar LJ, Lundgren-Åkerlund E, Fortier LA.Early intervention with mesenchymal stem cells (MSCs) after articular trauma has the potential to limit progression of focal lesions and prevent ongoing cartilage degeneration by modulating the joint environment and/or contributing to repair. Integrin α10β1 is the main collagen type II binding receptor on chondrocytes, and MSCs that are selected for high expression of the α10 subunit have improved chondrogenic potential. The ability of α10β1-selected (integrin α10high) MSCs to protect cartilage after injury has not been investigated. To investigate integrin α10high MSCs to prevent postt...
Les CM, Keyak JH, Stover SM, Taylor KT, Kaneps AJ.The purpose of this study was to investigate the relationships between data obtained from quantitative computed tomography and mechanical properties in the equine metacarpus, as measured in vitro in bone specimens. Three hundred and fifty-five bone specimens from the metacarpi of 10 horses were machined into right cylinders aligned with the long axis of the bone. A computed tomographic scan of the specimens, along with a Cann-Genant K2HPO4 calibration standard, was obtained. The specimens then were compressed to failure, and the elastic modulus, yield stress, yield strain, strain energy densit...
Mobasheri A, Lewis R, Maxwell JE, Hill C, Womack M, Barrett-Jolley R.Chondrocytes possess the capacity to transduce load-induced mechanical stimuli into electrochemical signals. The aim of this study was to functionally characterize an ion channel activated in response to membrane stretch in isolated primary equine chondrocytes. We used patch-clamp electrophysiology to functionally characterize this channel and immunohistochemistry to examine its distribution in articular cartilage. In cell-attached patch experiments, the application of negative pressures to the patch pipette (in the range of 20-200 mmHg) activated ion channel currents in six of seven patches. ...
Müller M, Raabe O, Addicks K, Wenisch S, Arnhold S.In equine medicine, stem cell therapies for orthopaedic diseases are routinely accompanied by application of NSAIDs (non-steroidal anti-inflammatory drugs). Thus, it has to be analysed how NSAIDs actually affect the growth and differentiation potential of MSCs (mesenchymal stem cells) in vitro in order to predict the influence of NSAIDs such as phenylbutazone, meloxicam, celecoxib and flunixin on MSCs after grafting in vivo. The effects of NSAIDs were evaluated regarding cell viability and proliferation. Additionally, the multilineage differentiation capacity and cell migration was analysed. N...
Wagner B, Radbruch A, Rohwer J, Leibold W.In this study we describe the generation of monoclonal antibodies (mAbs), which recognize different epitopes of the equine IgE constant heavy chain. Equi-murine recombinant IgE (rIgE), composed of the murine V(H)186.2 heavy chain variable region, linked to the equine IgE constant heavy chain and expressed together with the murine lambda(1) chain in J558L cells was used to immunize BALB/C mice. A total of 17 different mAbs were obtained, which recognized the rIgE heavy chain constant region. None of the mAbs reacted with monoclonal equine isotypes IgM, IgG1 (IgGa), IgG3 (IgG(T)), IgG4 (IgGb) or...
Wood PL, Scoggin K, Ball BA, Troedsson MH, Squires EL.Using a nontargeted lipidomics analysis of equine sperm and seminal plasma, we were able to characterize a diverse array of individual lipids including ethanolamine and choline ether lipids and seminolipids essential to membrane raft function. We also detected, for the first time in sperm, the presence of (O-acyl)-ω-hydroxy-fatty acids (OAHFA) with up to 52 carbon chain lengths, which were localized to the head and not the tail of sperm. The only previous identification of OAHFAs has been in meibomian glands and their sebaceous secretions. The identities of these lipid amphiphiles were valida...
Garvican ER, Cree S, Bull L, Smith RK, Dudhia J.Autologous mesenchymal stem cell (MSC) injection into naturally-occurring equine tendon injuries has been shown to be safe and efficacious and protocols inform translation of the technique into humans. Efficient transfer of cells from the laboratory into tissue requires well-validated transport and implantation techniques. Methods: Cell viability in a range of media was determined over 72 hours and after injection through a 19G, 21G or 23G needle. Viability, proliferation and apoptosis were analysed using TrypanBlue, alamarBlue® and AnnexinV assays. Results: Cell viability was similar in all...
Glaser AL, de Vries AA, Dubovi EJ.Three murine monoclonal antibodies (MAbs) that neutralize equine arteritis virus (EAV) infectivity were identified and characterized. The antibodies, 93B, 74D(B) and 38F, recognized the major envelope glycoprotein (GL) encoded by open reading frame (ORF) 5 in immunoblots and by immunoprecipitation. All three MAbs were used to compare the Bucyrus isolate of EAV and MAb neutralization-resistant (NR) escape mutants with the vaccine virus and 19 independent field isolates of EAV by virus neutralization. The different abilities of the MAbs to neutralize virus isolates indicated that they recognize ...
Yeste M, Estrada E, Rocha LG, Marín H, Rodríguez-Gil JE, Miró J.Although cryopreservation of stallion spermatozoa allows long-term preservation of spermatozoa from particular stallions and facilitates international trade, it is understood to inflict damages on sperm cells that may finally reduce their fertilizing ability. In addition, individual differences are known to exist in the sperm ability to withstand freeze-thawing protocols. To date, these differences have mainly been reported on the basis of sperm motility and membrane integrity. For this reason, the present work sought to determine differences between good (good freezability ejaculates: GFE) an...
Bastos RG, Suarez CE, Laughery JM, Johnson WC, Ueti MW, Knowles DP.Members of the CCp protein family have been previously described to be expressed on gametocytes of apicomplexan Plasmodium parasites. Knocking out Plasmodium CCp genes blocks the development of the parasite in the mosquito vector, making the CCp proteins potential targets for the development of a transmission-blocking vaccine. Apicomplexans Babesia bovis and Babesia bigemina are the causative agents of bovine babesiosis, and apicomplexan Theileria equi causes equine piroplasmosis. Bovine babesiosis and equine piroplasmosis are the most economically important parasite diseases that affect world...
