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.
Atkinson F, Evans R, Guest JE, Bavin EP, Cacador D, Holland C, Guest DJ.Tendon injuries are a common cause of morbidity in humans. They also occur frequently in horses, and the horse provides a relevant, large animal model in which to test novel therapies. To develop novel cell therapies that can aid tendon regeneration and reduce subsequent reinjury rates, the mechanisms that control tendon tissue regeneration and matrix remodelling need to be better understood. Although a range of chemical cues have been explored (growth factors, media etc.), the influence of the mechanical environment on tendon cell culture has yet to be fully elucidated. To mimic the in vivo e...
Totorikaguena L, Olabarrieta E, Lolicato F, Romero-Aguirregomezcorta J, Smitz J, Agirregoitia N, Agirregoitia E.The cannabinoid (CB) system has been involved in many aspects of reproduction and it is known that the systemic chronic use of exogenous CBs are deleterious to reproductive processes. Even so, it is not known what happens in relation to the physiology of the ovary when CB receptors are absent. The present study investigated the effect of the lack of CB1 and CB2 receptors in mice ovarian morphology, folliculogenesis, oocyte retrieval, and oocyte maturation and evaluated the use of Δ9-tetrahydrocannabinol (THC) on oocyte in vitro maturation (IVM) by comparing classical IVM and two-step IVM by a...
Smith R, Önnerfjord P, Holmgren K, di Grado S, Dudhia J.The diagnosis of tendon injury relies on clinical signs and diagnostic imaging but imaging is subjective and does not always correlate with clinical signs. A molecular marker would potentially offer a sensitive and specific diagnostic tool that could also provide objective assessment of healing for the comparison of different treatments. Cartilage Oligomeric Matrix Protein (COMP) has been used as a molecular marker for osteoarthritis in humans and horses but assays for the protein in tendon sheath synovial fluids have shown overlap between horses affected by tendinopathy and controls. We hypot...
Canesin HS, Ortiz I, Rocha Filho AN, Salgado RM, Brom-de-Luna JG, Hinrichs K.Equine embryo vitrification is still not a well-established technique in equine practice. Notably, little work has been done on the effect of the warming system on viability of vitrified embryos. Our goal was to evaluate the effect of warming without cryoprotectants on in vitro - produced (IVP) embryo viability in culture, quality assessment parameters, and pregnancy after transfer. Equine IVP blastocysts were vitrified using commercial embryo vitrification media and a semi-closed vitrification device. In Exp. 1, we evaluated two warming temperatures (room temperature, RT, ∼22 °C; and 38...
Miranda CMFC, Therrien J, Leonel LCPC, Smith OE, Miglino MA, Smith LC.Equine represents an attractive animal model for musculoskeletal tissue diseases, exhibiting much similarity to the injuries that occur in humans. Cell therapy and tissue bioengineering have been widely used as a therapeutic alternative by regenerative medicine in musculoskeletal diseases. Thus, the aim of this study was to produce an acellular biomaterial of equine skeletal muscle and to evaluate its effectiveness in supporting the in vitro culture of equine induced pluripotency stem cells (iPSCs). Biceps femoris samples were frozen at -20°C for 4 days and incubated in 1% sodium dodecyl s...
J Lacy K, Parlane NA, Riley CB, Gee EK, Roberts JM, McIlwraith CW.CellTrace Violet™ is a commonly used fluorescent dye used with flow cytometry to identify cell proliferation. Activated equine lymphocytes were examined using flow cytometry, microscopy and tritiated thymidine proliferation assays. CellTrace Violet™ was incorporated into the equine lymphocytes effectively. Equine lymphocytes proliferated when activated with pokeweed mitogen, but did not proliferate when previously stained with CellTrace Violet™. Serial dilutions of CellTrace Violet™ did not eliminate the inhibition of activated lymphocytes. Equine lymphocyte viability was greater than ...
De Coster T, Velez DA, Van Soom A, Woelders H, Smits K.Invitro embryo production has evolved rapidly in the horse over the past decade, but blastocyst rates from vitrified equine oocytes remain quite poor and further research is needed to warrant application. Oocyte vitrification is affected by several technical and biological factors. In the horse, short exposure of immature oocytes to the combination of permeating and non-permeating cryoprotective agents has been associated with the best results so far. High cooling and warming rates are also crucial and can be obtained by using minimal volumes and open cryodevices. Vitrification of invivo-matur...
Wilkołek P, Szczepanik M, Rodzik B, Sitkowski W, Pluta M, Taszkun I, Gołyński M.Intradermal tests (IDTs) and measurement of specific immunoglobulin E class (sIgE) levels in sera are the most common and reliable methods used in allergological clinical practice. The purpose of this study was to explore the sensitization of pollen allergy in atopic horses with pollinosis and to assess the diagnostic value of the multiple allergen simultaneous tests (MASTs) compared with that of the IDT. Twenty-one Malopolski horses with typical skin hypersensitivity symptoms during pollen seasons were enrolled. Intradermal tests were performed, and allergen-specific IgE concentrations in ser...
Cook NL, Masterson KR, Battaglia D, Beck R, Metcalf ES.Numerous variables affect invitro blastocyst development following intracytoplasmic sperm injection (ICSI). The paternal factor is affected by initial semen quality, processing techniques and final selection of individual spermatozoon for injection. This study investigated whether there was an effect of sperm cryoprotectant agent (CPA) on equine invitro blastocyst production, and reviews recent developments examining how processing equine semen affects ICSI outcomes. Single ejaculates from five stallions were collected and processed in a freezing extender containing either 1M dimethyl sulfoxid...
Claes A, Cuervo-Arango J, Colleoni S, Lazzari G, Galli C, Stout TA.The success of invitro embryo production (IVEP) in horses has increased considerably during recent years, but little is known about the effect of the speed of invitro embryo development. Blastocysts (n=390) were produced by intracytoplasmic sperm injection of IVM oocytes from warmblood mares, cryopreserved, thawed and transferred into recipient mares on Days 3, 4, 5 or 6 after ovulation. The time required for invitro-produced (IVP) embryos to reach the blastocyst stage was recorded (Day 7 vs Day 8). The likelihood of foaling was affected by the speed of invitro embryo development and recipient...
Hinrichs K.Methods for holding of oocytes and embryos during shipment as well as for their cryopreservation can greatly aid equine reproductive management. Oocytes can be held at room temperature overnight or at cooler temperatures for two nights without affecting maturation or embryo development after intracytoplasmic sperm injection. In contrast, methods for cryopreservation of equine oocytes that support high rates of embryo development have not yet been established. Equine embryos may be held overnight at temperatures from 5°C to 19°C without reduction in viability, but longer holding periods, or h...
Balasuriya UBR.The primary goals of this chapter are to discuss common viral RNA isolation and purification methods that are routinely used by various diagnostic laboratories and to highlight the advantages and drawbacks of each method and to identify the most suitable and reliable method to increase the sensitivity and specificity of RT-PCR assays for the detection of equine influenza virus (EIV) in clinical specimens. Our experiences and review of literature show that magnetic bead-based nucleic extraction methods (manual and automatic) work well for isolation and purification of EIV RNA from nasal swab sp...
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...
Fatemikia H, Kamyab M, Movahed A, Sadeghi M, Kim E, Behdani M, Mohammadpour Dounighi N, Shahrivar M, Seyedian R.In this study, the neutralizing abilities of the equine and the recently introduced camelid antivenoms on the hemodynamic parameters (inotropism, chronotropism, and arrhythmogenicity) were assessed following envenomation by venom in rats. Methods: At first, the electrophoretic profiles of both products were obtained by using the SDS-PAGE method (12.5%) and stained with Coomassie blue and silver nitrate. Secondly, different doses of the camelid antivenom (10, 50, and 100 µl) were given intravenously in 10 min before venom injection (400 µg/rat). The neutralizing potencies of camelid and equi...
Lei R, Wang X, Zhang D, Liu Y, Chen Q, Jiang N.Equine piroplasmosis (EP) is a severe disease of horses caused by the tick-borne protozoa Theileria equi (T. equi) and Babesia caballi (B. caballi). Infectious carriers are not always symptomatic, meaning there is a risk to non-enzootic areas. Regulatory tests for EP include sero-epidemiological methods for equine babesiosis, but these lack specificity due to cross-reactivity with other Babesia species. In this study, we present a real-time quantitative recombinase polymerase amplification (qRPA) method for fast simultaneous detection of both T. equi and B. caballi. In this method, primers and...
da Silva LL, Silveira MD, da Costa Garcia CAS, Grudzinski PB, Martins CF, Nardi NB.Mesenchymal stromal cells (MSCs) have attracted great attention for therapeutic applications. Since cells derived from different tissues have different properties, using the right tissue source may impact their efficiency in regenerative medicine. This study describes for the first time the isolation and characterization of MSCs derived from the equine coronary corium, which may be useful for treating diseases such as laminitis. Seven coronary corium samples were used for isolation of cells (ccMSCs). Adherent cells were characterized for morphology, immunophenotype, proliferation and different...
Lange-Consiglio A, Romele P, Magatti M, Silini A, Idda A, Martino NA, Cremonesi F, Parolini O.Equine amniotic mesenchymal stromal cells (AMSCs) and their conditioned medium (CM) were evaluated for their ability to inhibit in vitro proliferation of peripheral blood mononuclear cells (PBMCs) with and without priming. Additionally, AMSC immunogenicity was assessed by expression of MHCI and MHCII and their ability to counteract the in vitro inflammatory process. Horse PBMC proliferation was induced with phytohemagglutinin. AMSC priming was performed with 10 ng/ml of TNF-α, 100 ng/ml of IFN-γ, and a combination of 5 ng/ml of TNF-α and 50 ng/ml of IFN-γ. The CM generated from na...
Bruschetta G, D'Ascola A, Medica P, Ferlazzo AM.Serotonin (5-hydroxytryptamine [5-HT]) may induce metabolic effects in different cell types, including leukocytes. In horses, 5-HT is involved in physiological and behavioral functions. Physical exercise is known to increase the amounts of 5-HT both in brain and periphery, but so far, the signal mechanism in response to exercise is not known. The aim of the study was to investigate the effect of a racehorse intensive training session on plasma 5-HT levels, serotonin transporter (SERT), 5HT, 5-HT, 5-HT, 5-HT receptor, interleukin-1 beta, and tumor necrosis factor-alpha expression in horse perip...
Ortiz I, Quiñones-Pérez C, Hidalgo M, Consuegra C, Diaz-Jimenez M, Dorado J, Vega-Pla JL, Crespo F.Dynamic assessment of sperm DNA fragmentation (SDF) has shown to give fuller understanding of stallion semen quality; however, there have been limited attempts to use this parameter to investigate seasonal changes in productive functions. The aims of this study were to: (a) establish a reliable mathematical model to describe the longevity of cooled-stored sperm DNA integrity; (b) to examine the effect of seasonal variations on SDF. Ejaculates were cooled to 5°C, and SDF was analysed after 0, 6 and 24 hr of storage. The coefficient of determination (R ) was calculated after fine-tuning linear...
Canever MF, Miletti LC.The development of new drugs targeting neglected animal diseases is imperative. In Asia and South America, Trypanosoma evansi is a pathogen that affects horses and other species, causing economic losses associated with reduced animal productivity and death. In order to accelerate the identification of drugs with activity against neglected diseases, Medicines for Malaria Venture has developed Pathogen Box®, a library of 400 different molecules. The present work aimed to identify compounds present in the Pathogen Box® library, measuring in vitro activity against T. evansi. Among the 400 compou...
Lewis N, Hinrichs K, Leese HJ, McG Argo C, Brison DR, Sturmey R.Horses are one of the few species, beside humans, in which assisted reproductive technology has important clinical applications. Furthermore, the horse can serve as a valuable model for the study of comparative reproductive biology. Here we present the first comprehensive characterisation of energy metabolism and mitochondrial efficiency in equine cumulus-oocyte complexes (COCs) during in vitro maturation (IVM), as determined using a combination of non-invasive consumption and release assays and mitochondrial function analysis. These data reveal notable species-specific differences in the rate...
Kujawa TJ, van Doorn DA, Wambacq WA, Hesta M, Pellikaan WF.The in vitro gas production technique (IVGPT) has been a valuable tool in ruminant nutrition research for decades and has more recently been used in horse nutrition studies to investigate fermentation activities of the equine hindgut though primarily using feces as inoculum. This study was conducted to evaluate the use of equine rectal content in the IVGPT system as a viable inoculum that can be considered representative of the activities throughout the equine hindgut. Additionally, the study was conducted to measure the effects on fermentation kinetics and end-product production using inoculu...
Pezzanite L, Chow L, Soontararak S, Phillips J, Goodrich L, Dow S.Equine veterinarians frequently inject aminoglycoside antibiotics intra-articularly, either to treat septic arthritis or for prophylaxis with other medications when injecting joints for osteoarthritis. Although aminoglycosides have been demonstrated to be toxic to equine mesenchymal stem cells (MSC), their effects on resident joint cells have not been previously investigated. Moreover, safe and effective intra-articular doses have not been defined. Objective: To determine effects of concentration, duration of exposure, pH and the presence of synovial fluid on the cytotoxic effects of amikacin ...
Colbath AC, Dow SW, McIlwraith CW, Goodrich LR.Mesenchymal stem cells (MSCs) are widely used for treatment of musculoskeletal diseases in horses, but there is ongoing debate regarding the relative safety and efficacy of allogeneic MSCs, compared with autologous equine MSCs. This review summarises the currently available published data regarding the therapeutic use of autologous and allogeneic MSCs in horses. Arguments that have been advanced against the use of allogeneic MSCs include higher risk of immunological reactions and shorter cell survival times following injection. Arguments favouring the use of allogeneic MSCs include the ability...
Powell CD, Dhanoa MS, Garber A, Murray JMD, López S, Ellis JL, France J.Two models are proposed to describe atypical biphasic gas production profiles obtained from in vitro digestibility studies. The models are extensions of the standard Mitscherlich equation, comprising either two Mitscherlich terms or one Mitscherlich and one linear term. Two models that describe typical monophasic gas production curves, the standard Mitscherlich and the France model [a generalised Mitscherlich (root-) equation], were assessed for comparison. Models were fitted to 25 gas production profiles resulting from incubating feedstuffs with faecal from equines. Seventeen profiles displa...
Bai B, Wang XF, Zhang M, Na L, Zhang X, Zhang H, Yang Z, Wang X.Tetherin is an interferon-inducible type II transmembrane glycoprotein which inhibits the release of viruses, including retroviruses, through a "physical tethering" model. However, the role that the glycosylation of tetherin plays in its antiviral activity remains controversial. In this study, we found that mutation of N-glycosylation sites resulted in an attenuation of the antiviral activity of equine tetherin (eqTHN), as well as a reduction in the expression of eqTHN at the plasma membrane (PM). In addition, eqTHN N-glycosylation mutants colocalize obviously with ER, CD63, LAMP1 and endosome...
Kim DH, Lee HG, Sp N, Kang DY, Jang KJ, Lee HK, Cho BW, Yang YM.To understand the athletic characteristics of Thoroughbreds, high-throughput analysis has been conducted using horse muscle tissue. However, an in vitro system has been lacking for studying and validating genes from in silico data. The aim of this study is to validate genes from differentially expressed genes (DEGs) of our previous RNA-sequencing data in vitro. Also, we investigated the effects of exercise-induced stress including heat, oxidative, hypoxic and cortisol stress on horse skeletal muscle derived cells with the top six upregulated genes of DEGs. Enriched pathway analysis was conduct...
de Albuquerque Lagares M, Silva GCD, Cortes SF, Luz SB, de Resende AC, Alves NC, Wenceslau RR, Stahlberg R.During semen cryopreservation, the sensitivity of equine sperm to oxidative stress is increased by the eliminated seminal plasma. Thus, antioxidant addition to the semen extender can be helpful to the sperm survival after freezing and thawing. This work aimed to test whether coenzyme Q10 (CoQ10) added in different concentrations to the INRA 82 freezing extender has antioxidant function on equine sperm to improve its fertilizing ability. Semen samples from five stallions were frozen with the extenders: (T1) INRA 82, control, (T2) T1+ 5 μM CoQ10, (T3) T1+ 25 μM CoQ10, and (T4) T1+ 50 μM Co...
Johannisson A, Al-Essawe EM, Al-Saffar AK, Karkehabadi S, Lima-Verde I, Wulf M, Aurich C, Morrell JM.The mechanism by which the content of the major groups of seminal plasma proteins in stallion semen changes between the breeding and non-breeding seasons remains unknown. Here, we investigated the proportions of non-heparin-binding, phosphorylcholine-binding, and heparin-binding proteins in seminal plasma with the aim of relating them to sperm quality and testosterone levels in good and bad freezer stallions. Only minor variations in the major protein groups were found between the breeding and non-breeding seasons. In the non-breeding season, a higher content of a subset of non-heparin binding...
Szczerbal I, Nowacka-Woszuk J, Kopp-Kuhlman C, Mackowski M, Switonski M.X monosomy is the most common disorder of sex development in horses. Although cytogenetic analysis is still the gold standard in the diagnosis of equine X monosomy, novel molecular techniques are being sought to quickly and reliably detect this chromosome abnormality. Objective: The goal of this study was to evaluate the usefulness of a novel variant of the PCR technique-namely, droplet digital PCR (ddPCR)-in the detection of X monosomy in mares. Methods: A proof of concept of the usefulness of ddPCR in diagnosing an abnormal number of X chromosomes in mares. Methods: We examined an infertile ...
Little CB, Flannery CR, Hughes CE, Goodship A, Caterson B.To determine whether the focal susceptibility to cartilage degeneration in joints is related to a differential response to cytokine stimulation. Methods: Compare aggrecan and collagen catabolism in in-vitro models of cartilage degradation induced by retinoic acid (RA), interleukin-1 (IL-1), tumor necrosis factor alpha (TNF) and IL-1 plus oncostatin M (OSM). Glycosaminoglycan (GAG) and hydroxyproline (HyPro) quantification and Western immunoblot analyses of aggrecan and collagen degradation products were undertaken in explant cultures of normal cartilage from regions of equine joints with a kno...
Flaminio MJ, Antczak DF.At days 36-38 of gestation, the equine invasive trophoblast cells migrate into the endometrium of the pregnant mare to form the endometrial cups. During their migration, they become surrounded by maternal CD4+ and CD8+ T lymphocytes, and stimulate a cytotoxic antibody response to the paternal major histocompatibility complex class I antigens that they express. Nevertheless, endometrial cup cells remain viable at the site of uterine invasion up to days 80-100 of gestation, suggesting the participation of immunomodulatory mechanisms to the maternal cellular immune response. To determine the effe...
Netolitzky DJ, Schmaltz FL, Parker MD, Rayner GA, Fisher GR, Trent DW, Bader DE, Nagata LP.The complete nucleotide sequence of the 71V-1658 strain of western equine encephalitis virus (WEE) was determined (minus 25 nucleotides from the 5' end). A 5' RACE reaction was used to sequence the 5' terminus from WEE strain CBA87. The deduced WEE genome was 11508 nucleotides in length, excluding the 5' cap nucleotide and 3' poly(A) tail. The nucleotide composition was 28% A, 25% C, 25% G and 22% U. Comparison with partial WEE sequences of strain 5614 (nsP2-nsP3 of the nonstructural region) and strain BFS1703 (26S structural region) revealed comparatively little variation; a total of 149 nucl...
Giraldo CE, Álvarez ME, Carmona JU.To compare five activation methods in equine platelet-rich plasma (PRP) by determination of platelet-derived growth factor BB (PDGF-BB) and transforming growth factor beta 1 (TGF-β1) concentrations in platelet-rich gel (PRG) supernatants. Methods: Platelet-rich plasma from 20 horses was activated by calcium chloride (CC), calcium gluconate (CG), bovine thrombin (BT), and their combinations, BTCC and BTCG. Both growth factor concentrations in PRG supernatants were measured by ELISA and compared with plasma and platelet lysates (PL) over time. Results: Growth factor concentrations were signific...
Nygaard TK, Liu M, McClure MJ, Lei B.Heme is a preferred iron source of bacterial pathogens. Streptococcus equi subspecies equi is a bacterial pathogen that causes strangles in horses. Whether S. equi has a heme acquisition transporter is unknown. Results: An S. equi genome database was blasted with the heme binding proteins Shp and HtsA of Streptococcus pyogenes, and found that S. equi has the homologue of Shp (designated SeShp) and HtsA (designated SeHtsA). Tag-free recombinant SeShp and SeHtsA and 6xHis-tagged SeHtsA (SeHtsAHis) were prepared and characterized. Purified holoSeShp and holoSeHtsA bind Fe(II)-protoporphyrin IX (h...
Phillips T, Ferraz I, Bell S, Clegg PD, Carter SD, Mobasheri A.Glucose serves as the major energy substrate for articular chondrocytes and as the main precursor for the synthesis of extracellular matrix glycosaminoglycans in cartilage. Chondrocytes have been shown to express several glucose transporter (GLUT) isoforms including GLUT1 and GLUT3. The aim of this investigation was to determine the effects of endocrine and cytokine factors on the capacity of equine articular chondrocytes for transporting 2-deoxy-d-[2,6-3H] glucose and on the expression levels of GLUT1 and GLUT3. Chondrocytes maintained in monolayer culture were stimulated for 24 h with TNF-al...
Wilhelm K, Darinskas A, Noppe W, Duchardt E, Mok KH, Vukojević V, Schleucher J, Morozova-Roche LA.Protein oligomeric complexes have emerged as a major target of current research because of their key role in aggregation processes in living systems and in vitro. Hydrophobic and charged surfaces may favour the self-assembly process by recruiting proteins and modifying their interactions. We found that equine lysozyme assembles into multimeric complexes with oleic acid (ELOA) at the solid-liquid interface within an ion-exchange chromatography column preconditioned with oleic acid. The properties of ELOA were characterized using NMR, spectroscopic methods and atomic force microscopy, and showed...
Garnica-Galvez S, Korntner SH, Skoufos I, Tzora A, Diakakis N, Prassinos N, Zeugolis DI.The use of macromolecular crowding in the development of extracellular matrix-rich cell-assembled tissue equivalents is continuously gaining pace in regenerative engineering. Despite the significant advancements in the field, the optimal macromolecular crowder still remains elusive. Herein, the physicochemical properties of different concentrations of different molecular weights hyaluronic acid (HA) and their influence on equine adipose-derived stem cell cultures were assessed. Within the different concentrations and molecular weight HAs, the 10 mg/mL 100 kDa and 500 kDa HAs exhibited the high...
Leutenegger CM, von Rechenberg B, Huder JB, Zlinsky K, Mislin C, Akens MK, Auer J, Lutz H.Specific amplification and quantitation of nucleic acid sequences by the polymerase chain reaction (PCR) has been extensively used for the detection of viral infection and gene expression. Although successful amplification of DNA and RNA sequences extracted from paraffin embedded tissue have been described, there are presently no reports available regarding RNA analysis from bone and calcified tissues embedded in hydrophobic acrylic resin. Here we describe a general method for quantitation of specific mRNA sequences extracted from undecalcified bone sections, fixed in paraformaldehyde, and emb...
Walter J, Huwiler F, Fortes C, Grossmann J, Roschitzki B, Hu J, Naegeli H, Laczko E, Bleul U.Maturation of oocytes under in vitro conditions (IVM) results in impaired developmental competence compared to oocytes matured in vivo. As oocytes are closely coupled to their cumulus complex, elucidating aberrations in cumulus metabolism in vitro is important to bridge the gap towards more physiological maturation conditions. The aim of this study was to analyze the equine "cumulome" in a novel combination of proteomic (nano-HPLC MS/MS) and metabolomic (UPLC-nanoESI-MS) profiling of single cumulus complexes of metaphase II oocytes matured either in vivo (n = 8) or in vitro (n = 7). Re...
Robertson AT, Caughman GB, Gray WL, Baumann RP, Staczek J, O'Callaghan DJ.Equine herpesvirus type 1 (EHV-1) gene expression is coordinately regulated in an alpha, beta, gamma fashion. Viral alpha gene products include a 6.0-kb immediate early (IE) mRNA species (W. L. Gray et al., 1987, Virology 158, 79-87) and at least four closely related IE polypeptides (IEPs) (G.B. Caughman et al., 1985, Virology 145, 49-61). In this report, we describe results obtained from a series of in vitro translation experiments which were performed in an effort to characterize the IEPs and identify the mechanism by which individual IE protein species are generated. Our data indicate that ...
Swiderski CE, Klei TR, Horohov DW.Quantification of cytokine mRNA using reverse transcription coupled with the polymerase chain reaction (RT-PCR) has become a corner stone of the study of cytokine regulation. Quantitative competitive RT-PCR (QCRT-PCR) is commonly accepted as a reliable method for quantifying differences in mRNA levels but is both labor- and reagent-intensive. A noncompetitive polymerase chain reaction method that utilizes cytokine-specific, plasmid-derived, standard curves was developed for the quantification of equine cytokine mRNA. The assay can be performed on minute samples of cellular material, utilizes s...
Gil L, Olaciregui M, Luño V, Malo C, González N, Martínez F.In recent years, there has been an increased interest in new preservation techniques that facilitate sperm storage and distribution, with freeze-drying (FD) having been proposed as an alternative method for sperm preservation and maintenance of genetic resources in different animal species. FD is a method in which frozen material is dried by sublimation of ice, thereby involving a direct transition from a solid (ice) to a vapour (gas) phase. One of the main advantages of FD is that nitrogen and dry ice are no longer required for the storage and shipment of frozen sperm, which can be stored at ...
Finno CJ, Spier SJ, Valberg SJ.The recent development of equine genome maps by the equine genome community and the complete sequencing of the horse genome performed at the Broad Institute have accelerated the pace of genetic discovery. This review focuses on genetic diseases in the horse for which a mutation is currently known, including hyperkalemic periodic paralysis, severe combined immunodeficiency, overo lethal white syndrome, junctional epidermolysis bullosa, glycogen branching enzyme deficiency, malignant hyperthermia, hereditary equine regional dermal asthenia, and polysaccharide storage myopathy. Emphasis is placed...
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...
Nagata LP, Hu WG, Masri SA, Rayner GA, Schmaltz FL, Das D, Wu J, Long MC, Chan C, Proll D, Jager S, Jebailey L, Suresh MR, Wong JP.The efficacy of a DNA vaccine against western equine encephalitis (WEE) infection in mice was evaluated. The 26S structural region was expressed, in vitro from an internal T7 promoter using a rabbit reticulysate transcription/translation system; and from a CMV promoter after transfection into Vero cell monolayers. The proteins synthesized were reactive with anti-WEE virus (WEEV) antibodies, both in western blot analysis and histochemical staining, respectively. When the DNA vaccine plasmid, pVHX-6, was administered intraepidermally to mice, followed by challenge in a lethal mouse model, the le...
Lewis N, Hinrichs K, Leese HJ, McG Argo C, Brison DR, Sturmey R.Horses are one of the few species, beside humans, in which assisted reproductive technology has important clinical applications. Furthermore, the horse can serve as a valuable model for the study of comparative reproductive biology. Here we present the first comprehensive characterisation of energy metabolism and mitochondrial efficiency in equine cumulus-oocyte complexes (COCs) during in vitro maturation (IVM), as determined using a combination of non-invasive consumption and release assays and mitochondrial function analysis. These data reveal notable species-specific differences in the rate...
Dell'Aquila ME, Cho YS, Minoia P, Traina V, Lacalandra GM, Maritato F.The aim of this study was to compare the effect of the addition of follicular fluid (FF) collected from preovulatory follicles with that of oestrous mare serum (EMS) (acting as the control) to TCM-199 medium on the in-vitro maturation, fertilization and development of equine cumulus-enclosed oocytes. Oocytes (<30 mm in diameter) were obtained from the ovaries of slaughtered mares. After in-vitro maturation in the presence of the two supplements, their fertilization, cleavage and developmental potential were compared after conventional in-vitro fertilization (IVF) or intracytoplasmic sperm inje...
Thompson RE, Meyers MA, Veeramachaneni DNR, Pukazhenthi BS, Hollinshead FK.Organoids are a type of three-dimensional (3D) cell culture that more closely mimic the in vivo environment and can be maintained in the long term. To date, oviductal organoids have only been reported in laboratory mice, women, and cattle. Equine oviductal organoids were generated and cultured for 42 days (including 3 passages and freeze-thawing at passage 1). Consistent with the reports in mouse and human oviductal organoids, the equine oviductal organoids revealed round cell clusters with a central lumen. Developing a 3D model of the mare oviduct may allow for an increased understanding of t...
Garvican ER, Vaughan-Thomas A, Redmond C, Gabriel N, Clegg PD.The plasma serine protease activated protein C (APC) is synthesized by human chondrocytes at sites of pathological cartilage fibrillation. APC levels are increased in osteoarthritis (OA) synovial fluid, and in vitro APC has been shown to synergize with interleukin-1beta (IL-1) to promote degradation from ovine cartilage. A model of equine cartilage degradation was established and used to explore corticosteroid activities. Intraarticular corticosteroids are a commonly prescribed treatment for joint disease, however their role in disease modification remains unclear. APC synergized with IL-1 or ...
Falomo ME, Ferroni L, Tocco I, Gardin C, Zavan B.Endometriosis is a degenerative process due to a chronic inflammatory damage leading to extracellular matrix components deposition and glandular fibrosis. It is known that mesenchymal stem cells secrete a wide range of bioactive molecules, some of them modulating the immune inflammatory response, and others providing regeneration and remodeling of injured tissue. We have performed in vitro experiments in order to analyze the capability of allogenic equine adipose-derived stem cells (ADSCs) to infiltrate mares' endometrial tissues and to stimulate the expression of cytokines and metallopeptidas...
Allen GP, Yeargan MR, Bryans JT.The effect of in vitro and in vivo serial virus passage on the genetic stability of equine herpesvirus 1 (EHV-1) was investigated by restriction endonuclease analysis of the viral DNA. DNAs of EHV-1 isolates at different passage levels in cultured cells or in Syrian hamsters were compared by electrophoresis of the DNA cleavage fragments produced by restriction endonuclease digestion. No changes were observed in the restriction profile of the DNAs of EHV-1 strains after 100 sequential passages in cultured equine cells. However, serial passage of the virus in hamsters or in cells of non-equine o...
Willingham-Lane JM, Berghaus LJ, Giguère S, Hondalus MK.The soil-dwelling, saprophytic actinomycete is a multihost, facultative intracellular pathogen of macrophages. When inhaled by susceptible foals, it causes severe bronchopneumonia. It is also a pathogen of pigs, which may develop submaxillary lymphadenitis upon exposure. isolates obtained from foals and pigs possess conjugative plasmids housing a pathogenicity island (PAI) containing a novel family of genes of unknown function called the virulence-associated protein or family. The PAI regions of the equine and swine plasmids differ in gene composition, with equine isolates possessing six ...
Waters LJ, Hanrahan JP, Tobin JM, Finch CV, Parkes GMB, Ahmad SA, Mohammad F, Saleem M.Three mesoporous silica excipients (Syloid® silicas AL-1 FP, XDP 3050 and XDP 3150) were formulated with a model drug known for its poor aqueous solubility, namely phenylbutazone, in an attempt to enhance the extent and rate of drug dissolution. Although other forms of mesoporous silica have been investigated in previous studies, the effect of inclusion with these specific Syloid® silica based excipients and more interestingly, with phenylbutazone, is unknown. This work reports a significant enhancement for both the extent and rate of drug release for all three forms of Syloid® silica at a ...
Earing JE, Cassill BD, Hayes SH, Vanzant ES, Lawrence LM.The objective of this study was to determine if in vitro methodologies developed for the Ankom Daisy(II) incubator could produce accurate estimates of in vivo equine DM digestibility (DMD) and NDF digestibility (NDFD) when equine feces were used as the inoculum source. Four mature geldings were utilized in a 4 × 4 Latin square design experiment with a 2 × 2 factorial arrangement of dietary treatments (timothy hay, alfalfa hay, timothy hay plus oats, and alfalfa hay plus oats), in which the geldings were individually housed and fed. During each 5-d total fecal collection period, feces were co...
ROCKEY JH, KLINMAN NR, KARUSH F.Anti-p-azophenyl-beta-lactoside (Lac) antibody has been isolated from equine antiserum by specific precipitation with Lac-HSA (human serum albumin) and Lac-Hy (hemocyanin). Antibody was separated from antigen by chromatography on DEAE cellulose and zone electrophoresis in solvents containing lactose. Six antigenically distinct immunoglobulins have been identified in purified equine anti-Lac antibody: 7S beta(2A)-globulin, 19S gamma-globulin, a 10S gamma(1) globulin, and three antigenically distinct 7S gamma-globulins. The specific hapten-binding activity of the 7S beta(2A)-antibody and of the ...
Livolant F.The fine structure of chromatin in sperm heads was investigated by different microscopic techniques: in vivo examinations in the polarizing microscope, thin sections and freeze-fracture replicas observed by transmission electron microscopy. The freeze-fractured chromatin appears to be formed of superimposed lamellae, each one 330 A thick. These lamellae are parallel to the flattening plane of the sperm head. This situation was already described in other mammal spermatozoa and in particular in the bull and the rabbit. This work presents a new interpretation of this lamellated aspect. The chroma...
Nambo Y, Kaneko H, Nagata S, Oikawa M, Yoshihara T, Nagamine N, Watanabe G, Taya K.Physiological roles of inhibin in mares were investigated by means of passive immunization using an antiserum to inhibin that had been raised in a castrated goat. Eight mares were given an intravenous injection of either 100 mL (n = 4) or 200 mL (n = 4) of inhibin antiserum 4 d after a single intramuscular injection of PGF2 alpha on Day 8 after ovulation, 4 control mares were treated with 100 mL castrated goat serum in the same manner. Jugular vein blood samples were collected after treatment with the serum until 192 h post treatment. Follicular growth and ovulations were monitored by ultrasou...
Bolaños JM, Morán AM, da Silva CM, Dávila MP, Muñoz PM, Aparicio IM, Tapia JA, Ferrusola CO, Peña FJ.To investigate the role of the processed autophagy marker light chain 3 (LC3B) protein in sperm survival in stallion semen processing during cooled storage, split ejaculates were diluted in two different extenders, KMT and INRA 96, and LC3B processing and sperm quality evaluated during incubation at 5°C for five days. After 3 days of incubation there was a drop in total motility in both extenders, although the percentage of progressive motile sperm was greater (P<0.05) in samples extended in INRA96. On Day 5 of cooled storage all sperm parameters decreased significantly independent of the ...
Bailey SR, Rycroft A, Elliott J.Acute laminitis can be induced experimentally in horses by the administration of carbohydrate, resulting in fermentation within the cecum and ischemia-reperfusion of the digits. The products of fermentation that trigger acute laminitis are as yet unknown; however, compounds such as amines might play a role due to their potential vasoactive properties. The objectives of this study were to quantify the amines present in equine cecal contents and to use a model of carbohydrate overload in vitro to test the hypothesis that carbohydrate fermentation is associated with increased amine production. Ce...
