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.
Longhini ALF, Salazar TE, Vieira C, Trinh T, Duan Y, Pay LM, Li Calzi S, Losh M, Johnston NA, Xie H, Kim M, Hunt RJ, Yoder MC, Santoro D, McCarrel TM....Previously, we showed that mesenchymal stem cells (MSC) can be mobilized into peripheral blood using electroacupuncture (EA) at acupoints, LI-4, LI-11, GV-14, and GV-20. The purpose of this study was to determine whether EA-mobilized MSC could be harvested and expanded in vitro to be used as an autologous cell therapy in horses. Peripheral blood mononuclear cells (PBMC) isolated from young and aged lame horses (n = 29) showed a marked enrichment for MSCs. MSC were expanded in vitro (n = 25) and administered intravenously at a dose of 50 x 106 (n = 24). Treatment resulted in significant improve...
Racine T, Denizot M, Pannetier D, Nguyen L, Pasquier A, Raoul H, Saluzzo JF, Kobinger G, Veas F, Herbreteau CH.There is no vaccine or approved therapy against lethal Ebola virus (EBOV). We investigated a proven technology platform to produce polyclonal IgG fragments, F(ab')2, against EBOV. Horses immunized with nanoparticles harboring surface glycoprotein trimers of EBOV-Zaire/Makona produced anti-Ebola IgG polyclonal antibodies with high neutralization activity. Highly purified equine anti-Ebola F(ab')2 showed strong cross-neutralization of 2 Zaire EBOV strains (Gabon 2001 and Makona) and in vivo 3 or 5 daily F(ab')2 intraperitoneal injections provided 100% protection to BALB/c mice against lethal EBO...
Lopez BS, Hurley DJ, Giancola S, Giguère S, Felippe MJB, Hart KA.Neonatal foals are uniquely susceptible to certain infections early in life. Dendritic cells (DC) are vital in the transition between the innate and adaptive immune response to infection, but DC biology in foals is not fully characterized. Monocyte-derived DC represent a suitable in vitro model similar to DC that differentiate from monocytes recruited from circulation. We hypothesized that foal monocyte-derived DC (MoDC) would exhibit age-dependent phenotypic and functional differences compared to adult horse MoDC. MoDC generated from 9 horses (collected once) and from 8 foals (collected at 1,...
Linardi RL, Dodson ME, Moss KL, King WJ, Ortved KF.Cartilage injury occurs commonly in equine athletes, often precipitating posttraumatic osteoarthritis (PTOA). Orthobiologics such as autologous conditioned serum (ACS) and autologous protein solution (APS) may be useful in decreasing posttraumatic inflammation, thereby preventing PTOA. The objective of this study was to quantify cytokine concentrations in ACS and APS and evaluate the protective effects of ACS and APS on inflamed chondrocytes cultured . We hypothesized that the combination of platelet-derived growth factors (PDGF) and anti-inflammatory cytokines present in APS would be superior...
Fehlberg LK, Lattimer JM, Drouillard JS, Douthit TL.Effects of Co on fiber digestibility in horses are largely unknown. Our objective was to evaluate effects of Co chloride on in vitro gas production, VFA production, and dry matter (DM) disappearance (IVDMD) using cecal fluid from 4 cannulated Quarter Horses. Five grams DM of alfalfa or smooth bromegrass hay were provided as substrate with Co added at 0.0, 0.5, 5.0, 25.0, or 50.0 mg/kg substrate DM. Ten milliliters cecal fluid and 140 mL McDougall's buffer were incubated in duplicate, and pH, VFA concentrations, and IVDMD were measured after 48 hours. Gas production, used as an indicator of...
Silva GB, De La Côrte FD, Brass KE, Palma HE, Gallio M, Cantarelli C, Bertolin K, Krause A, Wergutz J, Fontanari Krause LM, Antoniazzi AQ.Chondrocyte health is altered when exposed to local anesthetics, raising concerns as to the long-term effects of local anesthetics intra-articularly for diagnosis and analgesia. To investigate the drug with the lowest toxic potential, the effect of ropivacaine and mepivacaine on chondrocytes was evaluated. Articular cartilage from normal metacarpophalangeal joints of five equine cadaver specimens was used to establish chondrocyte cultures. Following seven days, chondrocytes were exposed to standard culture medium (DMEM), ropivacaine 7.5 mg/ml (ROP7.5), ropivacaine 10 mg/ml (ROP10), mepivacaine...
Baird A, Dominguez Falcon N, Saeed A, Guest DJ.Horses, like humans, can experience bone fractures and due to their large size and the need to bear weight on all limbs during the recovery period, they can be difficult to treat. Surgical techniques to improve fracture repair are improving, but to date, regenerative medicine technologies to aid fracture healing are not commonly applied in horses. We have previously demonstrated that equine induced pluripotent stem cells (iPSCs) can be differentiated into bone forming osteoblasts in 2D culture. In this study, we report on the use of a thermoplastic, 3D-printed polymer to provide a scaffold for...
Klohonatz KM, Nulton LC, Hess AM, Bouma GJ, Bruemmer JE.Maternal recognition of pregnancy (MRP) in the mare is an unknown process. In a non-pregnant mare on day 14 post-ovulation (PO), prostaglandin F2α (PGF) is secreted by the endometrium causing regression of the corpus luteum. Prior to day 14, MRP must occur in order to attenuate secretion of PGF. The embryo is mobile throughout the uterus due to uterine contractions from day of entry to day 14. It is unknown what signaling is occurring. Literature stated that infusing oil or placing a glass marble into the equine uterus prolongs luteal lifespan and that in non-pregnant mares, serum exosomes co...
Diaz-Jimenez M, Dorado J, Consuegra C, Ortiz I, Pereira B, Carrasco JJ, Gomez-Arrones V, Domingo A, Hidalgo M.The aim of this study was to assess the effect of different factors affecting vitrification success of donkey sperm: extender, sperm concentration, volume and storage vessel type. In Experiment 1, sucrose supplementations at 0.25 and 0.1 M were compared using two base extenders (containing or not egg-yolk); in Experiment 2, three sperm concentrations were assessed: 100, 200 or 300 million sperm/mL; and in Experiment 3, three different sperm volumes (100, 160 and 200 μL) and two different storage vessels (0.25 and 0.5 mL straws) were assessed. Sperm motility variables (CASA), plasma memb...
Rütten S, Schrödl W, Abraham G.Glucocorticoids are important drugs in the treatment of many inflammatory, autoimmune and allergic diseases in humans and animals. We investigated the effects of hydrocortisone and dexamethasone on TNF-α, IL-1Ra and INF-γ release in stimulated whole blood cell culture from healthy horses. Whole blood cell cultures proved to be useful for the characterization of the anti-inflammatory properties of new drugs. Diluted equine whole blood was exposed to lipopolysaccharide (LPS) and PCPwL (a cocktail consisting of phythemagglutinin E, concanavalin A, pokeweed mitogen and lipopolysaccharide) in the...
Caron JP, Gandy JC, Brown JL, Sordillo LM.Omega-3 fatty acid (n-3 PUFA) supplementation may have beneficial effects in certain chronic diseases, potentially including osteoarthritis. Favorable effects are attributed, in part, to downstream pro-resolving oxylipid metabolites. We investigated the role of n-3 PUFA and docosahexaenoic acid (DHA)-derived oxylipids (docosanoids) on equine synoviocyte metabolism. We hypothesized that n-3 PUFA and selected docosanoids would modulate inflammatory mediator gene expression by recombinant equine (re)IL-1β-stimulated synovial fibroblasts. Synoviocyte monolayer cultures were prepared from grossly ...
Adam EN, Janes J, Lowney R, Lambert J, Thampi P, Stromberg A, MacLeod JN.To determine the chondrogenic potential of cells derived from interzone tissue, the normal progenitor of articular cartilage during fetal development, compared to that of adult bone marrow-derived and adipose-derived mesenchymal cell isolates. The objective of this study was to compare the chondrogenic potential of fetal musculoskeletal progenitor cells to adult cell types, which are currently used therapeutically to facilitate joint cartilage repair in equine clinical practice. The hypothesis tested was that cells derived from interzone tissue have a chondrogenic potential that exceeds that o...
Siemieniuch MJ, Kozdrowski R, Szóstek-Mioduchowska AZ, Raś A, Nowak M.Endometritis is one of the most important causes of infertility in mares. Mares may suffer from endometritis outside the breeding season; however, pathological condition is often undiagnosed in anestrus. The aim of this study was to examine whether the secretion profiles of prostaglandin F2α (PGF2α), prostaglandin E2 (PGE2), and a metabolite of prostacyclin I2 (6-keto-PGF1α) differ in endometrial tissue cultures of heavy draft mares in anestrus with endometritis compared to those without endometritis. The endometrial biopsies were collected from 51 heavy draft mares. Inclusion criteria for ...
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...
Álvarez C, Luño V, González N, Guerra P, Gil L.This study aimed to evaluate the addition of mare colostrum in stallion freezing extenders to improve sperm quality. First, colostrum samples were collected from four mares after the foal's birth and their composition was determined. Ejaculates were collected from nine fertile stallions. Sperm samples were pooled, diluted, and cryopreserved into three experimental extender groups: Lactose-based extender supplemented with mare colostrum (20%), lactose-based extender supplemented with egg yolk (20%), and BotuCrio. The quality of the post-thaw semen samples were evaluated assessing sperm motility...
Ball AN, Phillips JN, McIlwraith CW, Kawcak CE, Samulski RJ, Goodrich LR.Optimizing the environment of complex bone healing and improving treatment of catastrophic bone fractures and segmental bone defects remains an unmet clinical need both human and equine veterinary medical orthopaedics. The objective of this study was to determine whether scAAV-equine-BMP-2 transduced cells would induce osteogenesis in equine bone marrow derived mesenchymal stem cells (BMDMSCs) in vitro, and if these cells could be cryopreserved in an effort to osteogenically prime them as an "off-the-shelf" gene therapeutic approach for fracture repair. Our study found that transgene expressio...
Di Salvo A, Chiaradia E, Della Rocca G, Giorgi M, Mancini F, Marenzoni ML, Conti MB, Nannarone S.Intra-articular administration of analgesics is performed to ensure good perioperative pain management avoiding undesirable systemic effects. To evaluate the effect of intra-articular injection of tramadol on postoperative pain after arthroscopy in horses and to determine whether tramadol had a local effect. Before the in vivo study, an in vitro test was performed aiming to evaluate the viability of equine chondrocytes after exposure to various concentrations of tramadol. The concentration identified as most appropriate was used to treat the horses' joints. Twelve horses affected by osteochond...
Van Cleemput J, Poelaert KCK, Laval K, Nauwynck HJ.Equine herpesvirus type 5 (EHV5) is a ubiquitous, yet obscure pathogen in the horse population and is commonly associated with fatal equine multinodular pulmonary fibrosis (EMPF). To date, little is known about the precise pathogenesis of EHV5. Here, we evaluated the dynamics of EHV5 infection in representative ex vivo and in vitro equine models, using immunofluorescence staining and virus titration. EHV5 was unable to infect epithelial cells lining the mucosa of nasal and tracheal explants. Similarly, primary equine respiratory epithelial cells (EREC) were not susceptible to EHV5 following in...
Ibrahim S, Szóstek-Mioduchowska A, Skarzynski D.Bacterial infections of the genital tract are the major cause of reproductive failure in the mares. MiRNAs are important regulators of gene expression, mostly through transcriptional and translational regression. We hypothesized that LPS induced aberrant expression of miRNAs and their targets, which are involved in regulation of uterine homeostasis. Three groups of primary endometrial epithelial and stromal cells, and endometrial tissue explants were cultured. The 1 group was kept as control, while the 2 and 3 groups were challenged with low (0.5 μg/mL) or high (3.0 μg/mL) doses of Lipop...
Tassone S, Renna M, Barbera S, Valle E, Fortina R.Successful studies on in vitro digestibility measurement of feedstuffs with fecal inoculum have been reported for horses, whereas data on donkeys are currently lacking. In this study, we evaluated the use of the Daisy Incubator for in vitro digestibility measurement of feedstuffs using donkey feces as source of microbial inoculum. The method was tested using seven feedstuffs commonly used in donkey diets (alfalfa, bromegrass, ryegrass, and timothy hays; wheat bran and wheat straw; barley grains). Feces were obtained from four female donkeys, and incubations were carried out at 1-week interva...
Lee JJ, Hsieh CL, Widman J, Mingala C, Ardeza Villanueva M, Feng H, Divers T, Chang YF.Current serological tests cannot discriminate between bactericidal Borrelia burgdorferi antibodies from others that are merely a response to Borrelia antigenic stimulation. Objective: To develop a sensitive and convenient luminescence-based serum bactericidal assay (L-SBA) to identify serum borreliacidal activity. Methods: Prospective validation study and method comparison. Methods: Serum samples were obtained either from archives of the Animal Health Diagnostic Center at Cornell University (N = 7) or from a vaccination trial (N = 238). Endogenous complement-inactivated serum sample was in...
Schwarzbach SV, Melo CF, Xavier PLP, Roballo KC, Cordeiro YG, Ambrósio CE, Fukumasu H, Carregaro AB.Osteoarthritis (OA) is one of the main locomotor disorders in horses. Although nonsteroidal anti-inflammatory drugs are the first-line treatment for OA, opioids could also be used. In previous studies, opioids showed promising anti-inflammatory and analgesic effects. In this study, we aimed to investigate the effects of two opioids (morphine and methadone) against inflammation in lipopolysaccharide (LPS)-stimulated synoviocytes by analyzing microsomal prostaglandin E synthase-1 (mPGES-1) and prostaglandin-endoperoxide synthase 2 (PTGS2) expression. Synoviocytes were obtained from the joints at...
Ahmed A, Siman-Tov G, Keck F, Kortchak S, Bakovic A, Risner K, Lu TK, Bhalla N, de la Fuente-Nunez C, Narayanan A.Venezuelan equine encephalitis virus (VEEV), a new world alphavirus belonging to the Togaviridae family, causes periodic disease outbreaks in humans and equines with high associated mortality and morbidity. VEEV is highly infectious via the aerosol route and so has been developed as a biological weapon (Hawley and Eitzen, 2001). Despite its current classification as a category B select agent, there are no FDA approved vaccines or therapeutics to counter VEEV infections. Here we utilize a naturally occurring host defense peptide, LL-37, as a therapeutic strategy to inhibit VEEV multiplication i...
Costa D, Leiva M, Naranjo C, Ríos J, Peña MT.To evaluate microbiological, histological, and ultrastructural characteristics of short-term cryopreserved (STC) equine corneoscleral tissue (7 years). Methods: Thirty-four healthy equine globes. Methods: After a decontamination protocol, globes were enucleated and stored at -20°C in broad-spectrum antibiotics. Corneoscleral tissue was evaluated at different storage periods: 1 month-1 year (20 eyes) and 7-9 years (12 eyes). Two eyes were used as controls. Microbiologic study included direct (blood, McConkey, and Sabouraud agars) and enrichment (brain-heart infusion broth) cultures. Cryopr...
Vafaei F, Kohram H, Zareh-Shahne A, Ahmad E, Seifi-Jamadi A.This study was aimed to evaluate the effect of permeable cryoprotectants in combination with trehalose or sucrose on the freezing capacity of stallion sperm. For this purpose, the ejaculates (n = 24) were collected from four healthy mature Turkmen stallions. The ejaculates were pooled and diluted with one of the extenders containing a combination of 5% of permeating (dimethylacetamide [DMA]; dimethylformamide [DMF] or glycerol) and 50 mM of nonpermeating cryoprotectant agents (CPAs) (sucrose or trehalose) to a final concentration of 200 × 10 spermatozoa/mL. The extended samples were cryopr...
Cuervo-Arango J, Claes AN, Beitsma M, Stout TAE.The in vitro production of embryos by ovum pickup (OPU) and intracytoplasmic sperm injection (ICSI) is gaining popularity among horse breeders and veterinarians. Various collection media are available for flushing follicles during OPU. The objective of this study was to determine whether the type of flushing media used to aspirate follicles and collect oocytes influences the outcome of a commercial equine OPU-ICSI program. Two commercial embryo flushing media (EFM1 and EFM2) supplemented with heparin were compared with a flushing media designed specifically for the collection of oocytes (oocy...
Consuegra C, Crespo F, Dorado J, Diaz-Jimenez M, Pereira B, Ortiz I, Arenas R, Morrell JM, Hidalgo M.Stallion sperm was vitrified using straws in comparison with spheres and conventional freezing. Vitrification was performed plunging 30 μL of sperm (spheres) or 0.5 mL straws into liquid nitrogen (LN) and conventional freezing using 0.5 mL straws frozen in LN vapors. Sperm vitrified in straws were submitted to different warming procedures (42°C/20 seconds; 60°C/15 seconds) and single-layer centrifugation (SLC). Total (TM, %) and progressive sperm motility (PM, %), plasma membrane (IMS, %) and acrosome integrity (AIS, %) were statistically compared between treatments (mean ± SEM). Signif...
Leemans B, Stout TAE, De Schauwer C, Heras S, Nelis H, Hoogewijs M, Van Soom A, Gadella BM.In contrast to various other mammalian species, conventional in vitro fertilization (IVF) with horse gametes is not reliably successful. In particular, stallion spermatozoa fails to penetrate the zona pellucida, most likely due to incomplete activation of stallion spermatozoa (capacitation) under in vitro conditions. In other mammalian species, specific capacitation triggers have been described; unfortunately, none of these is able to induce full capacitation in stallion spermatozoa. Nevertheless, knowledge of capacitation pathways and their molecular triggers might improve our understanding o...
Valberg SJ, Soave K, Williams ZJ, Perumbakkam S, Schott M, Finno CJ, Petersen JL, Fenger C, Autry JM, Thomas DD.Sarcolipin (SLN), myoregulin (MRLN), and dwarf open reading frame (DWORF) are transmembrane regulators of the sarcoplasmic reticulum calcium transporting ATPase (SERCA) that we hypothesized played a role in recurrent exertional rhabdomyolysis (RER). Objective: Compare coding sequences of SLN, MRLN, DWORF across species and between RER and control horses. Compare expression of muscle Ca2+ regulatory genes between RER and control horses. Methods: Twenty Thoroughbreds (TB), 5 Standardbreds (STD), 6 Quarter Horses (QH) with RER and 39 breed-matched controls. Methods: Sanger sequencing of SERCA reg...
Wilkołek P, Szczepanik M, Sitkowski W, Adamek Ł, Pluta M, Taszkun I, Gołyński M.New techniques in equine veterinary allergy testing include multiple allergen simultaneous tests (MASTs) that cover insect bite hypersensitivity (IBH), respiratory and atopic allergic diseases. The in vivo intradermal test (IDT) method is one of the most widely used techniques in veterinary practice, but recently, in vitro methods have been introduced as possible alternatives. This study was designed to assess the correlation between IDTs and MASTs based on IgE determination in serum. The study was performed on Malopolski breed horses with IBH (n = 26). IDTs were performed, and allergen-spe...
Squires E.Numerous reproductive technologies have been developed in the past several decades, which have dramatically changed the way mares are bred. This review will focus on embryo recovery and transfer, cooled-shipped embryos, embryo freezing, oocyte freezing, oocyte collection and transfer, intracytoplasmic sperm injection (ICSI), and sexed semen. Embryo transfer procedures have been constant for many years and the costs have not changed. The major change has been the ability to store embryos at 5 C for 12-24 hours and transport them to recipient stations. Embryo freezing has become more common us...
Martineau D, Bowser PR, Wooster G, Forney JL.Sixty-seven adult walleye fish were examined by light and transmission electron microscopy. The fish were affected by a mesenchymal tumor previously termed Walleye Dermal Sarcoma that commonly affects up to 27% of the population seasonally. Biopsies from 24 fish were collected, and complete postmortem examinations were performed on 43 fish. Grossly, the tumors had the appearance of randomly distributed, often clustered, spherical nodules, 2-5 mm in diameter with a smooth and often ulcerated surface. The tumors arose from the superficial surface of scales and consisted of fibroblast-like cells ...
Berghaus LJ, Giguère S, Sturgill TL, Bade D, Malinski TJ, Huang R.The objectives of this study were to determine the plasma and pulmonary disposition of gamithromycin in foals and to investigate the in vitro activity of the drug against Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) and Rhodococcus equi. A single dose of gamithromycin (6 mg/kg of body weight) was administered intramuscularly. Concentrations of gamithromycin in plasma, pulmonary epithelial lining fluid (PELF), bronchoalveolar lavage (BAL) cells, and blood neutrophils were determined using HPLC with tandem mass spectrometry detection. The minimum inhibitory concentration of gamithr...
Guasti PN, Souza FF, Scott C, Papa PM, Camargo LS, Schmith RA, Monteiro GA, Hartwig FP, Papa FO.During ejaculation, a large amount of seminal plasma proteins interact with the sperm membrane, leading to a series of biochemical and structural changes implicated in sperm function and gamete interaction. However, the roles of the majority of these proteins remain unknown. This study aimed to investigate the proteome and functionality of the major equine proteins of seminal plasma and the sperm membrane. Seminal plasma and enriched-membrane proteins (150 μg) were separated by two-dimensional gel electrophoresis, and the respective maps were analyzed. Protein identification was performed by...
Martin F, Dube F, Karlsson Lindsjö O, Eydal M, Höglund J, Bergström TF, Tydén E.Parascaris univalens is a pathogenic parasite of foals and yearlings worldwide. In recent years, Parascaris spp. worms have developed resistance to several of the commonly used anthelmintics, though currently the mechanisms behind this development are unknown. The aim of this study was to investigate the transcriptional responses in adult P. univalens worms after in vitro exposure to different concentrations of three anthelmintic drugs, focusing on drug targets and drug metabolising pathways. Methods: Adult worms were collected from the intestines of two foals at slaughter. The foals were natu...
Reisinger N, Schaumberger S, Nagl V, Hessenberger S, Schatzmayr G.The pathogenesis of laminitis is not completely identified and the role of endotoxins (lipopolysaccharides, LPS) in this process remains unclear. Phytogenic substances, like milk thistle (MT) and silymarin, are known for their anti-inflammatory and antioxidant properties and might therefore have the potential to counteract endotoxin induced effects on the hoof lamellar tissue. The aim of our study was to investigate the influence of endotoxins on lamellar tissue integrity and to test if MT and silymarin are capable of inhibiting LPS-induced effects in an in vitro/ex vivo model. In preliminary ...
Espinosa G, Plaza A, Schenffeldt A, Alarcón P, Gajardo G, Uberti B, Morán G, Henríquez C.Polymorphonuclear neutrophils (PMN) are the largest leukocyte population in the blood of most mammals including horses, and play an important defensive role in many infectious diseases. However, the mechanisms that increase PMN as one of the main cellular subsets in the defense against pathogens could also be involved in the pathophysiology of dysregulated inflammatory conditions. Mesenchymal stem/stromal cells (MSCs) are a heterogeneous population with a modulatory potential on the inflammatory response and are known to interact with nearly all cells of the immune system, including PMN. In th...
Luethy D, Owens SD, Stefanovski D, Nolen-Walston R, Giger U.Assessment of blood compatibility, typically by tube agglutination (TUBE) and hemolysis crossmatch or, less commonly, by blood typing and alloantibody screening, often is performed before blood transfusion in horses. In contrast, gel column (GEL) and immunochromatographic strip (STRIP) techniques are preferred for compatibility testing in dogs and cats. Objective: To determine the accuracy of novel and standard crossmatch and typing methods. Methods: Thirty-eight healthy horses, previously blood typed and alloantibody screened. Methods: TUBE and GEL crossmatches were performed on 146 different...
Kisiday JD, Hale BW, Almodovar JL, Lee CM, Kipper MJ, Wayne McIlwraith C, Frisbie DD.Mesenchymal stem cells (MSCs) are present in low density in bone marrow and culture expansion is necessary to obtain sufficient numbers for many proposed therapies. Researchers have characterized MSC growth on tissue culture plastic (TCP), although few studies have explored proliferation on other growth substrates. Using adult equine MSCs, we evaluated proliferation on fibrinogen-rich precipitate (FRP) surfaces created from blood plasma. When seeded at 1 × 10(4) cells/cm(2) and passaged five times over 10 days, MSCs on FRP in medium containing fibroblast growth factor 2 (FGF2) resulted in a ...
Van Dael H, Haezebrouck P, Morozova L, Arico-Muendel C, Dobson CM.Despite their homologous structure, c-type lysozymes and alpha-lactalbumins have been found to differ profoundly in their unfolding behavior, in that the alpha-lactalbumins readily enter a partially unfolded collapsed state (the "molten globule"), whereas lysozymes unfold cooperatively to a highly unfolded state. The calcium-binding property of lysozyme from equine milk provides an evolutionary link between the two families of proteins. We demonstrate here that equine lysozyme undergoes a two-stage unfolding transition upon heating or in the presence of guanidine hydrochloride that is highly d...
Pagl R, Aurich JE, Müller-Schlösser F, Kankofer M, Aurich C.A problem of semen extenders based on milk or egg yolk is the fact that these biological products consist of a variety of substances. Extenders containing only components with clearly protective effects on spermatozoa would thus be an advantage. In this study, we have compared the effects of an extender containing defined caseinates and whey proteins only (EquiPro, defined milk protein extender) with skim milk extender on equine spermatozoa during cooled storage. The defined milk protein extender was used with and without the antioxidant N-acetyl cysteine (NAC). In a second experiment, semen w...
Varner DD, Gibb Z, Aitken RJ.Stallion fertility is a vast subject, with a wide array of permutations that can impact reproductive performance in either positive or negative ways. This review is intended to address a mere segment of the male fertility issue, but the very essence of the male contribution to fertilisation, that of the spermatozoon. Spermatozoal ultrastructure and form-to-function are detailed and spermatozoal metabolism is discussed, with specific reference to distinctive characteristics of stallion spermatozoa. Lastly, methods for assessment of spermatozoal function are considered, with emphasis on spermato...
Pereira A, Braekman JC, Dumont JE, Boeynaems JM.The incorporation of iodide into proteins (PBI) and lipids (LBI) of horse thyroid slices was measured in various conditions. Their dependency on the concentration of extracellular iodide was strikingly different. For PBI the relationship was biphasic with a decrease above 10 microM, likely to correspond to the Wolff-Chaikoff effect. On the contrary, LBI increased as a function of iodide concentration up to 100 microM. Methimazole (MMI) inhibited the incorporation of iodide into both LBI and PBI, but higher concentrations of MMI were required to depress LBI as compared to PBI. The inhibition of...
Squires EL, Carnevale EM, McCue PM, Bruemmer JE.Recent studies demonstrated that zwitterionic buffers could be used for satisfactory storage of equine embryos at 5 degrees C. The success of freezing embryos is dependent upon size and stage of development. Morulae and blastocysts <300 microm can be slowly cooled or vitrified with acceptable pregnancy rates after transfer. The majority of equine embryos are collected from single ovulating mares, as there is no commercially available product for superovulation in equine. However, pituitary extract, rich in FSH, can be used to increase embryo recovery three- to four-fold. Similar to human medic...
Haag KT, Magalhães-Padilha DM, Fonseca GR, Wischral A, Gastal MO, King SS, Jones KL, Figueiredo JR, Gastal EL.A Biopsy Pick-Up (BPU) method was tested to determine the feasibility of retrieving preantral follicles from mare ovaries in vivo. A total of 33 ovarian biopsy procedures were performed on 18 mares during the breeding season. Mares were 5 to 21 years old and biopsies were performed during the estrous and/or diestrous phase, as confirmed by transrectal ultrasonography. Follicles were mechanically isolated using a tissue chopper, counted, and classified as normal or abnormal and primordial or primary. Viability of isolated follicles was determined by Trypan Blue dye. A total of 256 biopsy attemp...
Zhao J, Poelaert KCK, Van Cleemput J, Nauwynck HJ.Equine herpesvirus type 1 (EHV-1) causes respiratory disease, abortion and neurological disorders in horses. Besides epithelial cells, CD172a monocytic cells become infected with EHV-1 in the respiratory mucosa and transport the virus from the apical side of the epithelium to the lamina propria en route to the lymph and blood circulation. Whether CD172a monocytic cells are specifically recruited to the infection sites in order to pick up virus is unknown. In our study, equine nasal mucosa explants were inoculated with EHV-1 neurological strains 03P37 and 95P105 or the non-neurological strains ...
Egerbacher M, Edinger J, Tschulenk W.To study chondrotoxic effects of enrofloxacin (ENR) and ciprofloxacin hydrochloride (CFX) on canine and equine articular chondrocytes in culture and to compare the effects with that of cultivation in Mg2+-free medium. Methods: Chondrocytes from articular cartilage of 4- and 6 -month old dogs and 2- to 4- year-old horses. Methods: Chondrocytes were cultivated with 10, 40, 80, and 160 microg of CFX/ml, 10, 50, 100, and 150 microg of ENR/ml, or in Mg2+-free medium. A live-to-dead test was performed to test cytotoxic effects. Morphologic changes were evaluated by electron microscopy. An attachment...
Dordas-Perpinyà M, Yanez-Ortiz I, Sergeant N, Mevel V, Bruyas JF, Catalán J, Delehedde M, Briand-Amirat L, Miró J.ProAKAP4 is the precursor of AKAP4 (A-kinase Anchor protein 4), the main structural protein of the fibrous sheath of sperm. The amount of proAKAP4 reflects the ability of spermatozoa to maintain the flagellum activity and functionality up to the site of fertilization and is positively correlated with progressive motility in several mammalian species. The aim of this study was to investigate the relationship between proAKAP4 concentration with horse sperm motility descriptors and spermatic motile subpopulations. For this purpose, a total of 48 ejaculates from 13 different stallions were analyze...
McPartlin LA, Littell J, Mark E, Nelson JL, Travis AJ, Bedford-Guaus SJ.Efficient in vitro capacitation of stallion sperm has not yet been achieved, as suggested by low sperm penetration rates reported in in vitro fertilization (IVF) studies. Our objectives were to evaluate defined incubation conditions that would support changes consistent with capacitation in stallion sperm. Protein tyrosine phosphorylation events and the ability of sperm to undergo acrosomal exocytosis under various incubation conditions were used as end points for capacitation. Sperm incubated 4-6h in modified Whitten's (MW) with the addition of 25 mM NaHCO3 and 7 mg/mL BSA (capacitating mediu...
Ealy AD, Eroh ML, Sharp DC.The equine embryo must signal its presence to the uterus for pregnancy to continue to term. Mobility of the conceptus throughout the uterus is crucial for its survival, and this action presumably permits the conceptus to transmit its antiluteolytic signal to the endometrium. Studies were completed to establish whether this unidentified antiluteolytic signal targets prostaglandin G/H synthase 2 (PGHS2), a rate limiting enzyme in converting arachidonic acid to prostaglandins (PGs). In the first study, quantitative RT-PCR was used to determine the relative abundance of PGHS2 mRNA in endometrium d...
Oldenhof H, Zhang M, Narten K, Bigalk J, Sydykov B, Wolkers WF, Sieme H.Nonviable freeze-dried sperm have intact chromatin and can be used for fertilization via intracytoplasmic sperm injection. Freeze-dried sperm preferably should be stored at 4°C or lower, because DNA damage accumulates during storage at room temperature. Disaccharides are known to protect biomolecules both during freezing and drying, by forming a glassy state. Their use is challenging because cellular membranes are normally impermeable for disaccharides. In the current study, we demonstrate that membrane impermeable compounds, including lucifer yellow and trehalose, are taken up by stallion sp...
Yuan Z, Gobeil PA, Campo MS, Nasir L.Papillomaviruses are DNA viruses that cause tumours of the skin in humans and animals. The natural host of bovine papillomavirus is cattle, but also equids, resulting in tumours termed sarcoids. Matrix metalloproteinase 1 (MMP-1) expression is up-regulated in sarcoid fibroblasts and tumours. We extended our observation to other MMPs and determined whether MMPs induced invasion of sarcoid fibroblasts. Collagenase (MMP-1) and Gelatinase (MMP-2, MMP-9) were over-expressed in sarcoid fibroblasts and tumours. The fibroblasts were invasive in a 3D/matrigel invasion assay system. Inhibition of MMP by...
Horohov DW, Adams AA, Chambers TM.It is widely recognized that advanced age is associated with alterations in immunological responses that likely contribute to increased morbidity and mortality in the elderly population. This decreased efficacy of the immune system with age is referred to as 'immunosenescence' and has been reported for a number of species. Similar age-related changes are seen in horses and are manifested as decreased responsiveness to vaccination in vivo and diminished proliferative responses to mitogens in vitro. The underlying mechanism responsible for these impaired immunological responses remains unknown. ...
Seabaugh KA, Thoresen M, Giguère S.Extracorporeal shockwave therapy (ESWT) and platelet-rich plasma (PRP) are common treatments for soft tissue injuries in horses. Shockwave triggers cell specific responses to promote healing. Growth factors released from PRP also promote healing. It has been hypothesized that greater growth factor release would amplify the healing process. The combination of ESWT and PRP could promote healing in injured tendons and ligaments in the horse. The objective of this study was to determine if application of shockwaves to PRP samples increases the concentration of transforming growth factor-β1 (TGF-...
Daniels SP, Leng J, Swann JR, Proudman CJ.Anthelmintic treatment is a risk factor for intestinal disease in the horse, known as colic. However the mechanisms involved in the onset of disease post anthelmintic treatment are unknown. The interaction between anthelmintic drugs and the gut microbiota may be associated with this observed increase in risk of colic. Little is known about the interaction between gut microbiota and anthelmintics and how treatment may alter microbiome function. The objectives of this study were: To characterise (1) faecal microbiota, (2) feed fermentation kinetics in vitro and (3) metabolic profiles following m...
Raabe ML, Issel CJ, Montelaro RC.We have previously demonstrated a high propensity for enhancement of virus replication and disease resulting from experimental immunization of ponies with a baculovirus recombinant envelope (rgp90) vaccine from equine infectious anemia virus (EIAV). The current studies were undertaken to examine the correlation between the observed in vivo vaccine enhancement and in vitro assays for antibody-dependent enhancement (ADE) of EIAV replication. Toward this goal an optimized EIAV in vitro enhancement assay was developed using primary equine macrophage cells and used to evaluate the enhancement prope...
Durgam SS, Altmann NN, Coughlin HE, Rollins A, Hostnik LD.There is increased incidence of tendon disorders and decreased tendon healing capacity in people with diabetes mellitus (DM). Recent studies have also suggested pathological ossification in repair tendon of people with DM. Therefore, the objective of this study is to investigate the effects of insulin supplementation, an important pathophysiologic stimulus of DM, on tendon progenitor cell (TPC) proliferation and in vitro osteogenic capacity. Passage 3 TPCs were isolated from collagenase-digested, equine superficial digital flexor tendons. TPC proliferation was measured via MTT assay after 3 da...
Mekuria ZH, El-Hage C, Ficorilli NP, Washington EA, Gilkerson JR, Hartley CA.Equine herpesvirus 5 (EHV5) is a commonly detected gammaherpesvirus, which, along with the closely related EHV2, constitute the only two known percaviruses that infect horses. Apart from detection in horse populations worldwide and the recent publication of the whole genome, there is little known about the biology and pathogenesis of this virus, with many assumptions made by parallels with EHV2. The long-term survival of gammaherpesviruses within infected hosts involves the establishment and maintenance of latency in selected cell and tissues types, particularly lymphocytes. A latent gammaherp...
Sarmiento J, Perez B, Morales N, Henriquez C, Vidal L, Folch H, Galecio JS, Morán G.A reduction in inflammatory cell apoptosis is an important concept in the maintenance of inflammation and a potential target for the resolution of inflammation in many inflammatory diseases. Dysregulation of apoptosis has been implicated in a range of diseases, including tumors, neurodegenerative disorders and autoimmunity, and may also be implicated in allergic asthma. In horses, recurrent airway obstruction (RAO) is an asthma-like condition that is characterized increased survival neutrophil bronchial. Tamoxifen is a synthetic, non-steroidal, anti-estrogen agent that is widely used for treat...
Silva BC, Sandes SH, Alvim LB, Bomfim MR, Nicoli JR, Neumann E, Nunes AC.The aim of this study was to verify the suitable use of candidate 'probiotics' selected by in vitro tests and the importance of in vivo assays to nominate micro-organisms as probiotics and alternative prophylactic treatments for Salmonella Typhimurium infection. Results: Thirty-three lactic acid bacteria (LAB) isolated from foal's faeces were assessed based on the main desirable functional in vitro criteria. Based on these results, Pediococcus pentosaceus strain 40 was chosen to evaluate its putative probiotic features in a mouse model of Salmonella infection. Daily intragastric doses of Pe...
