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.
Scare JA, Dini P, Norris JK, Steuer AE, Scoggin K, Gravatte HS, Howe DK, Slusarewicz P, Nielsen MK.Ascarid parasites infect a variety of hosts and regular anthelmintic treatment is recommended for all species. Parascaris spp. is the only ascarid species with widespread anthelmintic resistance, which allows for the study of resistance mechanisms. The purpose of this study was to establish an in vitro drug exposure protocol for adult anthelmintic-naïve Parascaris spp. and report a preliminary transcriptomic analysis in response to drug exposure. Live worms were harvested from foal necropsies and maintained in RPMI-1640 at 37 °C. Serial dilutions of oxibendazole (OBZ) and ivermectin (IVM) we...
MacNicol JL, Murrant C, Pearson W.G's Formula is a novel equine feed additive formulated to promote optimal GI function. The objective of this study was to determine whether the addition of a simulated digest of the composite feed additive G's Formula (FA) would alter the contractile response of gastric smooth muscle to acetylcholine (Ach). Smooth muscle strips from porcine stomachs were excised and attached to an isometric force transducer. An experiment was run to compare tissue contraction between tissue exposed to FA (FA; n = 8, simulated digest of FA was added to the bath) and control tissue (CO; n = 8, no additions m...
Hernández-Avilés C, Love CC, Serafini R, Ramírez-Agámez L, Friedrich M, Ghosh S, Teague SR, LaCaze KA, Brinsko SP, Varner DD.In Experiment 1, the effects of glucose concentration in extender (0 mM, 67 mM, 147 mM, 270 mM; G0, G67, G147, and G270, respectively) and storage temperature of extended semen (5, 10, 15 and 20 °C) were evaluated after storage for up to 5 days (T0h to T120h). For all time points tested, mean total (TMOT) and progressive (PMOT) sperm motility were lower in G0 than all other treatment groups (P < 0.05). Mean curvilinear velocity (VCL) was lower in G0 than other treatment groups at all time points tested except T0h (P 0.05). Mean TMOT and PMOT, were lower for semen stored at 20 °...
Valente JSS, Brasil CL, Braga CQ, Zamboni R, Sallis ESV, Albano APN, Zambrano CG, Franz HC, Pötter L, Panagio LA, Reis GF, Botton SA, Pereira DIB.Pythiosis is a rapidly progressing disease that can be lethal to affected individuals due to resistance to available therapeutic protocols. The disease affects mammals, with the largest number of reports in horses and humans. The present study investigated the activity of biogenic silver nanoparticles (bioAgNP) in the treatment of experimental pythiosis. The disease was reproduced in nine female 90-day-old New Zealand rabbits. Animals were divided into three groups: group1 (control, n = 3) daily and topically treated with a nonionized gel-based formulation and 1 ml of sterile distilled wate...
Barrachina L, Cequier A, Romero A, Vitoria A, Zaragoza P, Vázquez FJ, Rodellar C.Antibody production after allogeneic administration of mesenchymal stem cells (MSCs) could impact their clinical application. Proinflammatory priming of MSCs can potentiate their regulatory ability in vivo but increased expression of major histocompatibility complex (MHC) might augment their immunogenicity, potentially leading to immune memory thus limiting repeated allogeneic administration. This study aimed at evaluating the production of cytotoxic allo-antibodies directed against donor's ELA (equine leukocyte antigen) in mismatched and halfmatched horses receiving repeated intraarticular ad...
Menarim BC, Gillis KH, Oliver A, Mason C, Werre SR, Luo X, Byron CR, Kalbfleisch TS, MacLeod JN, Dahlgren LA.Synovial inflammation is a central feature of osteoarthritis (OA), elicited when local regulatory macrophages (M2-like) become overwhelmed, activating an inflammatory response (M1-like). Bone marrow mononuclear cells (BMNC) are a source of naïve macrophages capable of reducing joint inflammation and producing molecules essential for cartilage metabolism. This study investigated the response of BMNC to normal (SF) and inflamed synovial fluid (ISF). Equine BMNC cultured in autologous SF or ISF (n = 8 horses) developed into macrophage-rich cultures with phenotypes similar to cells native to no...
Weber LA, Meißner J, Delarocque J, Kalbitz J, Feige K, Kietzmann M, Michaelis A, Paschke R, Michael J, Pratscher B, Cavalleri JV.Equine malignant melanoma (EMM) is a frequently occurring dermoepidermal tumor in grey horses. Currently available therapies are either challenging or inefficient. Betulinic acid (BA), a naturally occurring triterpenoid, is a promising compound for cancer treatment. To evaluate the potential of BA as a topical therapy for EMM, its anticancer effects on primary equine melanoma cells and dermal fibroblasts and its percutaneous permeation through isolated equine skin were assessed in vitro. Results: BA showed antiproliferative and cytotoxic effects on both primary equine melanoma cells and fibrob...
Bogard F, Bouchet B, Murer S, Filliard JR, Beaumont F, Polidori G.Cold therapy is commonly used to relieve pain and inflammation and to aid in muscle recovery after exercise in human medicine. A number of applications have also been observed in veterinary practice. In this article, a critical evaluation of equine protocol applied with a new commercial concept of equine whole-body cryostimulation (WBC) was made. With this new concept of WBC, the protocol usually utilized for relieving pain and discomfort in humans has been extended to horses. The investigations described herein focus on the reduction of horse skin temperature when applying human WBC protocols...
Winter RL, Tian Y, Caldwell FJ, Seeto WJ, Koehler JW, Pascoe DA, Fan S, Gaillard P, Lipke EA, Wooldridge AA.Endothelial colony forming cells (ECFCs) may be useful therapeutically in conditions with poor blood supply, such as distal limb wounds in the horse. Encapsulation of ECFCs into injectable hydrogel microspheres may ensure cell survival and cell localization to improve neovascularization and healing. Autologous ECFCs were isolated from 6 horses, labeled with quantum nanodots (QD), and a subset were encapsulated in poly(ethylene) glycol fibrinogen microspheres (PEG-Fb MS). Full-thickness dermal wounds were created on each distal limb and injected with empty PEG-Fb MS, serum, ECFCs, or ECFCs enca...
Kareskoski AM, Palviainen M, Johannisson A, Katila T.For unknown reasons, stallion fertility and sperm longevity during cooled storage of semen vary markedly between individuals. Spermatozoa from individual stallions react differently to the presence, or the removal, of seminal plasma (SP). The aim was to evaluate differences in protein content in stallion seminal plasma with either a positive or a negative effect on sperm chromatin integrity during storage. Stallion semen samples from different ejaculate fractions were stored at 5°C for 24 hr. Sperm survival was assessed after storage using a sperm chromatin structure assay. Protein expressio...
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...
Gandini M, Comino F, Caramello V, Giusto G.To compare three surgical knots for preventing leakage from the vascular bundle during ligation in simulated equine open castrations. Methods: Randomized, case-control, in vitro study. Methods: Testes (N = 60) collected from 30 horses. Methods: Testes were collected from 30 horses and randomly assigned to one of three groups: group G (friction, giant knot), group T (modified transfixing knot), or group S (sliding, strangle knot; n = 20/group). The assigned knot was used to ligate the vascular bundle during open castration. The length of suture material used and the leak pressure of the testicu...
Miró J, Morató R, Vilagran I, Taberner E, Bonet S, Yeste M.Three separate experiments were conducted to improve preservation of stallion epididymal sperm. In the first one, two different cooling extenders (Kenney and Gent) were compared. Sperm viability and motility patterns were assessed in 10 different epididymal sperm samples after 0 hours, 24 hours, 48 hours, 72 hours, and 96 hours of preservation at 4°C. No significant differences were observed in any of the evaluated parameters either between extenders or throughout the storage period. The second set of experiments was designed to determine whether supplementing thawing medium (INRA Freeze...
Mundy LN, Lescun TB, Main RP, Hall Griffin T.To compare heat generation and mechanical bone damage achieved with 2 tapered and 1 cylindrical transfixation pin taps in third metacarpal bones from equine cadavers. Methods: 18 pairs (36 specimens) of third metacarpal bones from euthanized horses with no known metacarpal disease. Methods: In each bone, an investigator drilled 3 holes for placement of a 6.3-mm cylindrical transfixation pin, a 6.3-mm tapered pin using a prototype tapered tap, and a 6.3-mm tapered pin using a revised tapered tap. One bone of each pair was tapped by hand and the other with an electric drill. Temperatures of the ...
Marzano G, Moscatelli N, Di Giacomo M, Martino NA, Lacalandra GM, Dell'Aquila ME, Maruccio G, Primiceri E, Chiriacò MS, Zara V, Ferramosca A.Conventional sperm selection techniques used in ARTs rely on centrifugation steps. To date, the different studies reported on the effects of centrifugation on stallion sperm motility provided contrasting results and do not include effects on mitochondrial functionality and different oxidative parameters. The effects of different centrifugation protocols (300 ×g for 5', 300 ×g for 10', 1500 ×g for 5' and 1500 ×g for 10' vs no centrifugation) on motility and oxidative status in cryopreserved stallion sperm, were analyzed. After centrifugation, almost all motility parameters were significantl...
Wuri L, Agca C, Agca Y.This study compared the morphometric, subcellular characteristics, in vitro fertilisation (IVF) and embryonic developmental potential of metaphase II (MII) mouse oocytes obtained from females superovulated with either anti-inhibin serum-human chorionic gonadotrophin (AIS-hCG) or pregnant mare serum gonadotrophin (PMSG)-hCG. The oocyte's quantity, quality, zona pellucida (ZP) thickness, perivitelline space (PVS), diameter, microtubules, F-actin, cortical granules (CGs) and mitochondrial distribution were determined. Superovulation using AIS-hCG resulted in a higher numbers of oocyte/donor compa...
Höfner L, Luther AM, Waberski D.Ongoing progress in proteomic characterization of seminal plasma has stimulated research on the identification of biomarkers for male fertility and sperm preservability. So far, many studies have evaluated the benefits of reconstituting cryopreserved or sex-sorted semen with seminal plasma. Less information is available about the effect of remaining or added seminal plasma in liquid preserved semen. The interaction between seminal plasma and spermatozoa is species -specific, and within species often complex and ambiguous. This article aims to review the action of seminal plasma on sperm functi...
Szóstek-Mioduchowska A, Słowińska M, Pacewicz J, Skarzynski DJ, Okuda K.Equine endometrial fibrosis (endometrosis) is described as a degenerative chronic condition in the uterus. Its characteristic feature is excessive deposition of extracellular matrix (ECM) components around the endometrial glands and stroma. Although matrix metallopeptidases (MMPs) that mediate ECM turnover are important factors in the process of fibrosis, knowledge of their expression and regulation in endometrosis is limited. In other species, one of the important regulators of MMPs and tissue inhibitors of MMPs (TIMPs) is transforming growth factor (TGF)-β1. The goal of this study was to de...
Papas M, Catalan J, Bonilla-Correal S, Gacem S, Miró J, Yeste M.The aim of this study was to evaluate the response of donkey spermatozoa to oxidative stress induced by hydrogen peroxide, and to determine whether the presence of seminal plasma modulates the sperm response to that stress. Nine ejaculates were collected, extended in skim milk extender and split into two aliquots. Seminal plasma was removed from the first but not second aliquot. Samples were subsequently split into four aliquots supplemented with different concentrations of commercial hydrogen peroxide (0, 100 and 250µM and 50mM). Aliquots were incubated at 37°C under aerobic conditions and ...
do Nascimento AD, Marques JCC, Cezar ARR, Batista AM, Kastelic JP, Câmara DR.Although equine blastocysts ≤ 300 µm in diameter can be successfully vitrified, larger equine blastocysts are not good candidates for cryopreservation. As Na, K-ATPase is involved in maintaining blastocyst expansion, perhaps inhibition of this enzyme would be a viable method of reducing blastocyst diameter prior to cryopreservation. Objectives were to evaluate effects of ouabain-induced inhibition of Na, K-ATPase in equine blastocysts. Sixteen mares were ultrasonographically monitored, given deslorelin acetate to induce ovulation, and inseminated. Embryos (D7 and D9) were harvested and Na, ...
Álvarez-Narváez S, Berghaus LJ, Morris ERA, Willingham-Lane JM, Slovis NM, Giguere S, Cohen ND.The practice of prophylactic administration of a macrolide antimicrobial with rifampin (MaR) to apparently healthy foals with pulmonary lesions identified by thoracic ultrasonography (i.e., subclinically pneumonic foals) is common in the United States. The practice has been associated epidemiologically with emergence of R. equi resistant to MaR. Here, we report direct evidence of multi-drug resistance among foals treated with MaR. In silico and in vitro analysis of the fecal microbiome and resistome of 38 subclinically pneumonic foals treated with either MaR (n = 19) or gallium maltolate (...
Minato E, Kobayashi A, Aoshima K, Fukushi H, Kimura T.Equine herpesvirus-1 (EHV-1), which causes encephalomyelitis in horses, shows endotheliotropism in the central nervous system of horses, and generally does not infect neurons. However, little is known about the mechanism underlying the resistance of neuron to EHV-1, due to the lack of convenient cell culture systems. In this study, we examined EHV-1 infection in immortalized Rn33B rat neuronal cells, which differentiate into neurons when cultured under nonpermissive conditions. Because murine cell lines are resistant to EHV-1 infections due to the lack of functional entry receptors for EHV-1, ...
Contreras MJ, Treulen F, Arias ME, Silva M, Fuentes F, Cabrera P, Felmer R.Cryopreservation of stallion semen has not reached the level of efficiency and positive results described in other species. This is mainly due to the greater sensitivity of stallion sperm to the freezing process, showing higher rates of oxidative stress and plasma membrane damage, which trigger the activation of several cell damage pathways that ultimately culminate in DNA fragmentation and cell death. Therefore, finding molecules that improve the efficiency of this technique in stallion by preventing oxidative stress and cell damage is required. Thus, the aim of the present study was to evalu...
de Albuquerque PPLF, Santos LHS, Antunes D, Caffarena ER, Figueiredo AS.Infections produced by hepaciviruses have been associated with liver disease in horses. Currently, at least three viruses belonging to the Flaviviridae family are capable of producing a chronic infection in equines: non-primate hepacivirus (NPHV), Theiler's disease-associated virus (TDAV), and equine pegivirus (EPgV). The RNA-dependent RNA polymerases of viruses (RdRp) (NS5 protein), from the flavivirus family, use de novo RNA synthesis to initiate synthesis. The two antiviral drugs currently used to treat hepatitis C (HCV), sofosbuvir and dasabuvir, act on the viral NS5B polymerase as nucleos...
Angel D, Canesin HS, Brom-de-Luna JG, Morado S, Dalvit G, Gomez D, Posada N, Pascottini OB, Urrego R, Hinrichs K, Velez IC.Effects of meiotic stage and cumulus status on development of equine oocytes after vitrification was evaluated. Immature oocytes with corona radiata (IMM); in vitro-matured oocytes with corona radiata (MAT CR+); and in vitro-matured oocytes denuded of cumulus (MAT CR-) were vitrified using the Cryotech® method. Warming medium was equilibrated either in 5% CO2 or Air. IMM oocytes underwent in vitro maturation after warming. Recovery, survival, and maturation rates, and cleavage and blastocyst rates after ICSI, were evaluated. Recovery was higher for oocytes warmed in CO2- than Air-equilibrated...
Schellenberg LM, Bonicelli J, Hochheim I, Regenthal R, Abraham G.The β-adrenergic receptor (β-AR) plays an important role in regulating a variety of cell and organ functions in different animal species and is an important target in asthma pathogenesis and therapy. The β-AR expression and function in equine bronchial epithelial cells (EBEC) were not known but innervation and significant decrease in receptor level were reported in the equine bronchial tissues from asthmatic horses. I-iodocyanopindolol (ICYP) binding studies were undertaken in primary freshly isolated and cultured EBEC to identify the presence of the β-ARs. The receptor distribution was as...
equine sarcoids are the most frequent skin tumors in equidae worldwide. It is well known that delta bovine papillomaviruses are their causative agents. We have recently shown the presence in equine sarcoids of abnormal vessel structures, which could cause a hypoxic condition. The aim of this study was to analyze the expression of hypoxia-inducible factor-1 alpha (HIF-1α) in a subset of BPV positive equine sarcoids and explore the relationship with vascular endothelial growth factor (VEGF) expression. Results: 80% of equine sarcoids showed strong cytoplasmic staining in >60% of neoplastic f...
Gilbertie JM, Schaer TP, Schubert AG, Jacob ME, Menegatti S, Ashton Lavoie R, Schnabel LV.Infectious arthritis is difficult to treat in both human and veterinary clinical practice. Recent literature reports Staphylococcus aureus as well as other gram-positive and gram-negative isolates forming free-floating biofilms in both human and equine synovial fluid that are tolerant to traditional antimicrobial therapy. Using an in vitro equine model, we investigated the ability of platelet-rich plasma (PRP) formulations to combat synovial fluid biofilm aggregates. Synovial fluid was infected, and biofilm aggregates allowed to form over a 2-hour period. PRP was collected and processed into d...
Kadic DTN, Bonilla AG.To develop and assess a needle arthroscopic technique to diagnose conditions of the tarsocrural joint (TCj) in standing sedated horses. Methods: Experimental study. Methods: Six cadaveric hind limbs (phase 1) and six healthy horses (Phase 2). Methods: In phase 1, each TCj was examined with a 1.2-mm-needle arthroscope. Suitability of the needle arthroscope and degree of joint visualization with traditional arthroscopic approaches were assessed. In phase 2, the feasibility of the procedure was assessed in six standing healthy horses. A custom-made splint and base were developed to maintain joint...
Kadivar A, Shams Esfandabadi N, Dehghani Nazhvani E, Shirazi A, Ahmadi E.Protamines substitute DNA-binding histones during late spermatogenesis in sperm nucleus. Stallion sperm contains all three variants of these arginine-rich and positively charged nuclear proteins (P1, P2 and P3). Two variants of protamine-2, that is P2 and P3, constitute approximately 15% of the entire protamine content. Also, the ratio of protamine-1 to protamine-2 varies among different mammalian species, and abnormal protamine ratios and protamine content are correlated with male infertility. In this study, changes in protamine mRNA abundance for all three protamines were investigated in sta...
Adegbeye MJ, Elghandour MMMY, Barbabosa-Pliego A, Monroy JC, Mellado M, Ravi Kanth Reddy P, Salem AZM.Several concerns exist regarding horse rearing such as environmental pollution, antibiotics resistance, digestive disorders, mycotoxins contamination of animal feed, gut health management, and improvement of feed efficiency. Nanoparticles have the potential to address these issues and thus could be used as feed additive. Citrate reduces and stabilizes gold nanoparticles, alongside biosynthesized silver nanoparticles have the potential to prolong and improve digestive enzyme activity, which would enhance starch digestibility in the stomach. Zinc oxide and selenium nanoparticles could be used to...
Lankveld DP, Bull S, Van Dijk P, Fink-Gremmels J, Hellebrekers LJ.Ketamine is widely used in equine anaesthesia. Beside its anaesthetic and analgesic properties, ketamine possesses a cytokine-modulating activity. However, to date, no data are available regarding the inhibitory effect of ketamine on the cytokine response in horses. In horses, cytokines such as tumour necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) play a pivotal role in the pathogenesis of equine endotoxaemia following gastrointestinal disorders. Hence, the objective of this study was to assess the influence of ketamine on LPS-induced TNF-alpha and IL-6 formation in an equine macro...
Rice NR, Simek S, Ryder OA, Coggins L.Equine infectious anemia virus (EIAV) recently has been shown to possess a high-molecular-weight RNA genome and a virion reverse transcriptase. We completed the demonstration that EIAV is a retrovirus by showing the presence of proviral DNA in equine cells infected in vitro, but not in normal horse DNA. These studies were performed by using a highly representative cDNA probe synthesized by the virion polymerase. It was found that this cDNA reassociated extensively, and with high thermal stability, with either viral RNA or DNA extracted from infected cells, but showed no detectable reassociatio...
Lombana KG, Goodrich LR, Phillips JN, Kisiday JD, Ruple-Czerniak A, McIlwraith CW.Diseases of the musculoskeletal system are a major cause of loss of use and retirement in sport horses. The use of bone marrow-derived mesenchymal stem cells (BMDMSCs) for healing of traumatized tissue has gained substantial favor in clinical settings and can assist healing and tissue regeneration in orthopedic injuries. There are two common sites of harvest of BMDMSCs, the sternum and the ilium. Our objective was to determine if any differences exist in BMDMSCs acquired from the sternum and the ilium. We compared the two harvest sites in their propensity to undergo multilineage differentiatio...
de Mestre AM, Miller D, Roberson MS, Liford J, Chizmar LC, McLaughlin KE, Antczak DF.The objective of this study was to identify transcription factors associated with differentiation of the chorionic girdle, the invasive form of equine trophoblast. The expression patterns of five transcription factors were determined on a panel of conceptus tissues from early horse pregnancy. Tissues from Days 15 through 46 were tested. Eomesodermin (EOMES), glial cells missing homologue 1 (GCM1), heart and neural crest derivatives expressed transcript 1 (HAND1), caudal type homeobox 2 (CDX2), and distal-less homeobox 3 (DLX3) were detected in horse trophoblast, but the expression patterns for...
Fortier LA, Nixon AJ, Lust G.To assess the effects of supraphysiologic concentrations of insulin-like growth factor-1 (IGF-1) on morphologic and phenotypic responses of chondrocytes. Methods: Articular cartilage obtained from 2 young horses. Methods: Chondrocytes were suspended in fibrin cultures and supplemented with 25, 12.5, or 0 mg of IGF-1/ml of fibrin. Chondrocyte morphology and phenotypic expression were assessed histologically, using H&E and Alcian blue stains, immunoreaction to collagen type I and II, and in situ hybridization. Proteoglycan content, synthesis, and monomer size were analyzed. The DNA content w...
Figueiredo LF, Dias-Lopes C, Alvarenga LM, Mendes TM, Machado-de-Ávila RA, McCormack J, Minozzo JC, Kalapothakis E, Chávez-Olórtegui C.A chimeric protein (rCpLi) was constructed expressing three epitopes of rLiD1, a dermonecrotic toxin from the venom of Loxosceles intermedia spider. We have analyzed the neutralization potential of sera obtained by immunization of horses with rCpLi and rCpLi combined with initial doses of venoms and compared these with antivenom traditionally produced in horses using crude Loxosceles gaucho, Loxosceles laeta and L. intermedia venoms as antigens. We have demonstrated by ELISA that horses immunized with three initial doses of crude venom containing mixtures of L. intermedia, L. gaucho and L. lae...
Mfitilodze MW, Hutchinson GW.In a series of laboratory studies the optimum conditions for the development and survival of the free-living stages of strongyle parasites occurring in horses in tropical north Queensland were determined. No differences in behaviour were noted between the strongyle species. Development to the infective stage occurred only between 10 and 35 degrees C. The rate was affected by temperature, taking 15-24 days and 3 days, respectively, at the lowest and highest temperatures for the developing stages to reach the infective third stage. Yields of infective larvae were very low outside the range 20-33...
Martin Muñoz P, Ortega Ferrusola C, Vizuete G, Plaza Dávila M, Rodriguez Martinez H, Peña FJ.Oxidative stress has been linked to sperm death and the accelerated senescence of cryopreserved spermatozoa. However, the molecular mechanisms behind this phenomenon remain poorly understood. Reactive oxygen species (ROS) are considered relevant signaling molecules for sperm function, only becoming detrimental when ROS homeostasis is lost. We hereby hypothesize that a major component of the alteration of ROS homeostasis in cryopreserved spermatozoa is the exhaustion of intrinsic antioxidant defense mechanisms. To test this hypothesis, semen from seven stallions was frozen using a standard tech...
Adams AP, Santschi EM, Mellencamp MA.A silver chloride-coated nylon wound dressing (Ag-WD) was evaluated in vitro for antimicrobial activity against five common equine wound pathogens. Methods: Bacterial susceptibility study. Methods: Equine wound pathogens: Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Streptococcus equi subspecies zooepidemicus, and Staphylococcus aureus. Methods: An inoculum of each pathogen was incubated directly with Ag-WD and quantitated after 24 to 48 hours of incubation. To determine if bactericidal activity of Ag-WD was contact dependent, an inoculum of E. coli and Staphylococcus aureu...
Schuh EM, Friedman MS, Carrade DD, Li J, Heeke D, Oyserman SM, Galuppo LD, Lara DJ, Walker NJ, Ferraro GL, Owens SD, Borjesson DL.OBJECTIVE-To optimize the isolation and culture of mesenchymal stem cells (MSCs) from umbilical-cord blood (UCB), identify variables that predicted successful MSC isolation, and determine whether shipping, processing, and cryopreservation altered MSC viability, recovery rates, and expansion kinetics. SAMPLE POPULATION-UCB samples from 79 Thoroughbred and Quarter Horse mares. PROCEDURES-UCB samples were processed to reduce volume and remove RBCs. Nucleated cells (NCs) were cryopreserved or grown in various culture conditions to optimize MSC monolayer expansion and proliferation. Donor and UCB-s...
Carossino M, Loynachan AT, James MacLachlan N, Drew C, Shuck KM, Timoney PJ, Del Piero F, Balasuriya UB.Equine arteritis virus (EAV) is the causative agent of equine viral arteritis, a respiratory and reproductive disease of equids. EAV infection can induce abortion in pregnant mares, fulminant bronchointerstitial pneumonia in foals, and persistent infection in stallions. Here, we developed two RNA in situ hybridization (ISH) assays (conventional and RNAscope(®) ISH) for the detection of viral RNA in formalin-fixed paraffin-embedded (FFPE) tissues and evaluated and compared their performance with nucleocapsid-specific immunohistochemistry (IHC) and virus isolation (VI; gold standard) techniques...
Sung SE, Hwang M, Kim AY, Lee EM, Lee EJ, Hwang SK, Kim SY, Kim HK, Jeong KS.Mesenchymal stem cells could potentially be used in the clinical treatment of muscle disorders and muscle regeneration. Adipose-derived stem cells (ADSCs) can be easily isolated from adipose tissue, as opposed to stem cells of other tissues. We believe that cell therapy using ADSCs could be applied to muscle disorders in horses and other species. We sought to improve the myogenic differentiation potential of equine ADSCs (eqADSCs) using a MyoD lentiviral vector. MyoD lentiviruses were transduced into eqADSCs and selected using puromycin. Cells were cultured in differentiation media containing ...
Lopez MJ, Jarazo J.According to Greek mythology, Prometheus' liver grew back nightly after it was removed each day by an eagle as punishment for giving mankind fire. Hence, contrary to popular belief, the concept of tissue and organ regeneration is not new. In the early 20th century, cell culture and ex vivo organ preservation studies by Alexis Carrel, some with famed aviator Charles Lindbergh, established a foundation for much of modern regenerative medicine. While early beliefs and discoveries foreshadowed significant accomplishments in regenerative medicine, advances in knowledge within numerous scientific di...
Bubenickova F, Postlerova P, Simonik O, Sirohi J, Sichtar J.Seminal plasma (SP) is the natural environment for spermatozoa and contains a number of components, especially proteins important for successful sperm maturation and fertilization. Nevertheless, in standard frozen stallion insemination doses production, SP is completely removed and is replaced by a semen extender. In the present study, we analyzed the effects of the selected seminal plasma protein groups that might play an important role in reducing the detrimental effects on spermatozoa during the cryopreservation process. SP proteins were separated according to their ability to bind to hepar...
Skive B, Rohde M, Molinari G, Braunstein TH, Bojesen AM. subsp. () is an opportunistic pathogen of several species including humans. is found on mucus membranes of healthy horses, but can cause acute and chronic endometritis. Recently was found able to reside in the endometrium for prolonged periods of time. Thus, we hypothesized that an intracellular phase may be part of the pathogenesis and investigated if was able to invade and survive inside epithelial cells. HEp-2 and HeLa cell lines were co-cultured with two strains (1-4a and S31A1) both originating from the uterus of mares suffering from endometritis. Cells were fixed at different time...
Baird A, Lindsay T, Everett A, Iyemere V, Paterson YZ, McClellan A, Henson FMD, Guest DJ.Bone fractures occur in horses following traumatic and non-traumatic (bone overloading) events. They can be difficult to treat due to the need for the horse to bear weight on all legs during the healing period. Regenerative medicine to improve fracture union and recovery could significantly improve horse welfare. Equine induced pluripotent stem cells (iPSCs) have previously been derived. Here we show that equine iPSCs cultured for 21 days in osteogenic induction media on an OsteoAssay surface upregulate the expression of osteoblast associated genes and proteins, including , , , , and We al...
Brophy PM, MacKintosh N, Morphew RM.Anthelmintics are the cornerstone of parasitic helminth control. Surprisingly, understanding of the biochemical pathways used by parasitic helminths to detoxify anthelmintics is fragmented, despite the increasing global threat of anthelmintic resistance within the ruminant and equine industries. Reductionist biochemistry has likely over-estimated the enzymatic role of glutathione transferases in anthelmintic metabolism and neglected the potential role of the cytochrome P-450 superfamily (CYPs). Proteomic technologies offers the opportunity to support genomics, reverse genetics and pharmacokine...
Martin DH, Dietz WH, Alvaerez O, Johnson KM.One hundred and fifty-eight strains of Venezuelan equine encephalomyelitis virus were typed antigenically and classified epidemiologically as either epizootic or enzootic. Plaque sizes for 148 of these strains were determined, and the pH requirements for hemagglutination (HA) of goose erythrocytes of 131 were studied. Only antigenic variant group IABC strains could be classified epidemiologically as epizootic. In vitro these strains were characterized by the formation of small plaques in Vero cells and a relatively narrow pH range for optimum HA reactivity. Experimental studies in horses confi...
Grubman MJ, Lewis SA.Proteins present in purified African horsesickness virus (AHSV) and in infected cells were analyzed by SDS-polyacrylamide gel electrophoresis. Twelve viral proteins were identified, one minor and four major structural proteins, three major and two minor nonstructural proteins, as well as variable amounts of two additional structural proteins. Cell-free translation of total AHS virion RNA in a rabbit reticulocyte system resulted in the synthesis of proteins which were qualitatively and quantitatively similar to those found in infected cells. The in vivo and in vitro synthesized proteins were vi...
Trimeche A, Yvon JM, Vidament M, Palmer E, Magistrini M.The supplementation of the freezing diluent with 3 amino acids (glutamine, proline and histidine) and 1 amino acid-related compound (betaine) in preserving stallion spermatozoa diluted in INRA82 extender containing 2.5% (v/v) glycerol and 2% (v/v) egg yolk (control extender) during freezing and thawing was studied at 0, 40, 80, 120 and 160 mM in 20 split ejaculates (10 stallions x 2 ejaculates; Experiment 1). Glutamine and proline were studied at 0, 10, 20, 30, 40, 50, 60, 70 and 80 mM in 20 split ejaculates (10 stallions x 2 ejaculates; Experiment 2). In each experiment, spermatozoa were eval...
Felix MR, Turner RM, Dobbie T, Hinrichs K.Methods for standard in vitro fertilization have been difficult to establish in the horse. We evaluated whether prolonged sperm pre-incubation would support subsequent fertilization. Fresh sperm were pre-incubated with penicillamine, hypotaurine, and epinephrine (PHE) for 22 h. Co-incubation of cumulus-oocyte complexes (COCs) for 6 h yielded 43% fertilization; culture of presumptive embryos yielded 21% blastocysts. Sperm incubated similarly, but without PHE, did not fertilize oocytes. Use of extended semen in the system yielded 54% blastocysts and was applied in subsequent experiments. Trans...
Elashry MI, Gegnaw ST, Klymiuk MC, Wenisch S, Arnhold S.Cell-based therapies have become a promising approach to promote tissue regeneration and the treatment of musculoskeletal disorders. Bone regeneration maintains bone homeostasis, mechanical stability and physical performance. Mechanical stimulation showed to induce stem cell differentiation into the osteogenic fate. However, the effect of various osteogenic protocols on the osteogenic commitment of equine adipose-derived stem cells is not fully elucidated. Here we examined the influence of fluid-based shear stress (FSS) via mechanical rocking to assess whether mechanical stimulation promotes o...
Sato K, Wakai T, Seita Y, Takizawa A, Fissore RA, Ito J, Kashiwazaki N.A sperm-specific phospholipase C (PLC), PLCzeta (PLCζ), is thought to underlie the initiation of calcium ([Ca(2+) ]i ) oscillations that induce egg activation in mammals. In large domestic species, only bovine, porcine and recently equine PLCζ have been cloned, and the physiological functions of these molecules have not been fully characterized. Here, we evaluated the physiological functions of equine PLCζ (ePLCζ) in mouse oocytes. ePLCζ was cloned from testis using RT-PCR. The expression of ePLCζ messenger RNA was confirmed in testis but not in other tissues. Microinjection of ePLCζ co...
Lang HM, Schnabel LV, Cassano JM, Fortier LA.Mesenchymal stem cells (MSCs) are frequently delivered via needle injection for treatment of musculoskeletal injuries. The purpose of this study was to evaluate the effect of needle diameter on the viability of MSCs. Methods: Equine bone marrow-derived MSCs from 5 horses were suspended in PBS, and held at room temperature for 7 hours to mimic shipping conditions. Two replicate samples for each needle size (20, 22, 23, or 25-gauge [ga]) were aspirated into a 3 mL syringe and re-injected into the holding vial 3 times, to reproduce the resuspension of cells prior to injection in clinical cases. C...
Orth MW, Peters TL, Hawkins JN.Glucosamine and chondroitin sulphate in many animal and human trials has improved joint health. In vitro studies are beginning to clarify their mode of action. The objective of this research was to: 1) determine at what concentrations glucosamine-HCl (GLN) and/or chondroitin sulphate (CS) would inhibit the cytokine-induced catabolic response in equine articular cartilage explants and 2) to determine if a combination of the 2 was more effective at inhibiting the catabolic response than the individual compounds. Articular cartilage was obtained from carpal joints of horses (age 1-4 years). Carti...
Olivera R, Moro LN, Jordan R, Luzzani C, Miriuka S, Radrizzani M, Donadeu FX, Vichera G.The demand for equine cloning as a tool to preserve high genetic value is growing worldwide; however, nuclear transfer efficiency is still very low. To address this issue, we first evaluated the effects of time from cell fusion to activation (<1h, n = 1261; 1-2h, n = 1773; 2-3h, n = 1647) on in vitro and in vivo development of equine embryos generated by cloning. Then, we evaluated the effects of using different nuclear donor cell types in two successive experiments: I) induced pluripotent stem cells (iPSCs) vs. adult fibroblasts (AF) fused to ooplasts injected with the pluripotency-inducin...
Donadeu FX, Fahiminiya S, Esteves CL, Nadaf J, Miedzinska K, McNeilly AS, Waddington D, Gérard N.Several aspects of equine ovarian physiology are unique among domestic species. Moreover, follicular growth patterns are very similar between horses and humans. This study aimed to characterize, for the first time, global gene expression profiles associated with growth and preovulatory (PO) maturation of equine dominant follicles. Granulosa cells (GCs) and theca interna cells (TCs) were harvested from follicles (n = 5) at different stages of an ovulatory wave in mares corresponding to early dominance (ED; diameter ≥22 mm), late dominance (LD; ≥33 mm) and PO stage (34 h after administration...
Kankofer M, Kolm G, Aurich J, Aurich C.Sperm cell membranes are susceptible to peroxidative damage by an excess of reactive oxygen species (ROS). Antioxidative defence systems consisting of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and catalase (CAT) physiologically control the balance between ROS production and neutralization. In the present study the hypothesis was tested that lipid peroxidation occurs during storage of semen at 5 degrees C and that semen extender has positive effects on the antioxidative potential of equine semen. The aim of the study was to determine the activity of GSH-Px, SOD and CAT and the...
Harlow BE, Donley TM, Lawrence LM, Flythe MD.The goal was to determine the effect of starch source (corn, oats and wheat) and concentration on: (i) total amylolytic bacteria, Group D Gram-positive cocci (GPC), lactobacilli and lactate-utilizing bacteria, and (ii) fermentation by equine microbiota. Results: When faecal washed cell suspensions were incubated with any substrate amylolytics increased over time. However, at 24 h there were 10 and 1000-fold more amylolytics with corn than wheat or oats respectively. Predominant amylolytics isolated were Enterococcus faecalis (corn, wheat) and Streptococcus bovis (oats). GPC increased with any ...
