Cell viability refers to the ability of cells to survive and function within their physiological environment. In horses, assessing cell viability is an important aspect of veterinary research, particularly in understanding the effects of various treatments, diseases, and environmental factors on equine cellular health. Techniques such as flow cytometry, trypan blue exclusion, and MTT assays are commonly used to evaluate cell viability in equine studies. These methods help determine the proportion of living cells in a sample, providing insights into cellular responses to different stimuli or conditions. This page compiles peer-reviewed research studies and scholarly articles that explore the methodologies, applications, and implications of cell viability assessments in equine research.
Garvican ER, Cree S, Bull L, Smith RK, Dudhia J.Autologous mesenchymal stem cell (MSC) injection into naturally-occurring equine tendon injuries has been shown to be safe and efficacious and protocols inform translation of the technique into humans. Efficient transfer of cells from the laboratory into tissue requires well-validated transport and implantation techniques. Methods: Cell viability in a range of media was determined over 72 hours and after injection through a 19G, 21G or 23G needle. Viability, proliferation and apoptosis were analysed using TrypanBlue, alamarBlue® and AnnexinV assays. Results: Cell viability was similar in all...
Laverty G, McCloskey AP, Gilmore BF, Jones DS, Zhou J, Xu B.Self-assembling dipeptides conjugated to naphthalene show considerable promise as nanomaterial structures, biomaterials, and drug delivery devices. Biomaterial infections are responsible for high rates of patient mortality and morbidity. The presence of biofilm bacteria, which thrive on implant surfaces, are a huge burden on healthcare budgets, as they are highly resistant to current therapeutic strategies. Ultrashort cationic self-assembled peptides represent a highly innovative and cost-effective strategy to form antibacterial nanomaterials. Lysine conjugated variants display the greatest po...
Fink MK, Giuliano EA, Tandon A, Mohan RR.To evaluate the safety and efficacy of Pirfenidone (PFD) in the treatment of equine corneal fibrosis using an in vitro model. Methods: Healthy donor equine corneas were collected and used to generate primary equine corneal fibroblasts (ECFs) by growing cultures in minimal essential medium supplemented with 10% fetal bovine serum. Equine corneal myofibroblasts (ECMs), used as a model of equine corneal fibrosis, were produced by growing ECF cultures in serum-free medium containing transforming growth factor β1 (1 ng/mL). Trypan blue viability assays and changes in ECF morphology were utilized t...
Álvarez C, Gil L, González N, Olaciregui M, Luño V.The rise of assisted reproduction techniques in equine medicine has fostered investigations that seek to optimize methods to increase fertility rates. Since cryopreservation continues to give low values of viability in stallions, the handling and preservation of the sperm is of vital importance. This reduction of fertility makes it essential for farmers to find new options that ensure reliability in the use of these techniques. The main objective of this study was to assess the effect of INRA 96® (manufactured commercial extender for cooling of Equine semen) as an extender for cryopreservatio...
Chung DH, Jonsson CB, Tower NA, Chu YK, Sahin E, Golden JE, Noah JW, Schroeder CE, Sotsky JB, Sosa MI, Cramer DE, McKellip SN, Rasmussen L, White EL....Alphaviruses present serious health threats as emerging and re-emerging viruses. Venezuelan equine encephalitis virus (VEEV), a New World alphavirus, can cause encephalitis in humans and horses, but there are no therapeutics for treatment. To date, compounds reported as anti-VEEV or anti-alphavirus inhibitors have shown moderate activity. To discover new classes of anti-VEEV inhibitors with novel viral targets, we used a high-throughput screen based on the measurement of cell protection from live VEEV TC-83-induced cytopathic effect to screen a 340,000 compound library. Of those, we identified...
Gallardo Bolaños JM, Balao da Silva C, Martín Muñoz P, Plaza Dávila M, Ezquerra J, Aparicio IM, Tapia JA, Ortega Ferrusola C, Peña FJ.To investigate the mechanisms inducing sperm death after ejaculation, stallion ejaculates were incubated in BWW media during 6 h at 37°C. At the beginning of the incubation period and after 1, 2, 4 and 6 h sperm motility and kinematics (CASA), mitochondrial membrane potential and membrane permeability and integrity were evaluated (flow cytometry). Also, at the same time intervals, active caspase 3, hydrogen peroxide, superoxide anion (flow cytometry) and Akt phosphorylation (flow cytometry) were evaluated. Major decreases in sperm function occurred after 6 h of incubation, although after 1 h ...
Mudge MC.Treatment of acute hemorrhage in the horse involves targeted medical management and also may involve surgical stabilization. This article provides an approach to the initial stabilization and information on available topical hemostats. The practice of blood collection and transfusion is also described, with attention to new information on viability of transfused equine blood, potential negative effects of blood transfusion, and methods of cell salvage.
Paebst F, Piehler D, Brehm W, Heller S, Schroeck C, Tárnok A, Burk J.Horses are an approved large animal model for therapies of the musculoskeletal system. Especially for tendon disease where cell-based therapy is commonly used in equine patients, the translation of achieved results to human medicine would be a great accomplishment. Immunophenotyping of equine mesenchymal stromal cells (MSCs) remains the last obstacle to meet the criteria of the International Society for Cellular Therapy (ISCT) definition of human MSCs. Therefore, the surface antigen expression of CD 29, CD 44, CD 73, CD 90, CD 105, CD 14, CD 34, CD 45, CD 79α, and MHC II in equine MSCs from a...
Gallardo Bolaños JM, Balao da Silva CM, Martín Muñoz P, Morillo Rodríguez A, Plaza Dávila M, Rodríguez-Martínez H, Aparicio IM, Tapia JA....AKT, also referred to as protein kinase B (PKB or RAC), plays a critical role in controlling cell survival and apoptosis. To gain insights into the mechanisms regulating sperm survival after ejaculation, the role of AKT was investigated in stallion spermatozoa using a specific inhibitor and a phosphoflow approach. Stallion spermatozoa were washed and incubated in Biggers-Whitten-Whittingham medium, supplemented with 1% polyvinyl alcohol (PVA) in the presence of 0 (vehicle), 10, 20 or 30 μM SH5, an AKT inhibitor. SH5 treatment reduced the percentage of sperm displaying AKT phosphorylation, w...
Marycz K, Smieszek A, Grzesiak J, Nicpoń JE.Disorders of the locomotive system, especially those occurring due to degenerative changes of the joints, are serious problems in daily veterinary medical practice. Steroid injections are the main way of treating these disorders. However, this approach brings usually only temporary effects of pain relief, and may cause many side effects. Alternative therapies focus on regeneration of damaged tissue using adult mesenchymal stem cells (MSCs). Since 2002, the great plasticity and immunomodulatory properties of MSCs isolated from adipose tissue (AdMSCs) have been used successfully in the treatment...
Mercati F, Pascucci L, Curina G, Scocco P, Tardella FM, Dall'aglio C, Marini C, Ceccarelli P.The transplantation of multipotent mesenchymal stromal cells (MSCs) is a potentially promising therapy for the treatment of tendon and ligament injuries and some forms of articular pathology in horses. This study investigated the effects of storage conditions on MSCs. Equine adipose tissue-derived MSCs (eAd-MSCs) were stored at 4 °C and at room temperature (RT) for 24 and 48 h, and viability, doubling time, expression of CD44 and CD90 antigens, clonogenic/differentiation potentials, and karyotype were subsequently evaluated. The eAd-MSC viability was significantly affected by the storage ...
Murphy C, English AM, Holden SA, Fair S.An unacceptable proportion of stallion sperm do not survive the freeze-thaw process. The hypothesis of this study was that adding cholesterol to a stallion semen extender would stabilise the sperm membrane, resulting in an improved post-thaw semen quality in terms of increased sperm viability, membrane integrity and fluidity, and reduced oxidative stress. Semen was collected from three stallions and diluted in four extenders: TALP; TALP+0.75mg methyl-β-cyclodextrin-cholesterol (MβCD)/mL (MβCD0.75); TALP+1.5mg MβCD-cholesterol/mL (MβCD1.5); and Equipro. Following 15min incubation, samples ...
Caselles AB, Miro-Moran A, Morillo Rodriguez A, Gallardo Bolaños JM, Ortega-Ferrusola C, Salido GM, Peña FJ, Tapia JA, Aparicio IM.Apoptosis in the testis is required to ensure an efficient spermatogenesis. However, sometimes, defective germ cells that are marked for elimination during this process escape elimination in the testes, giving rise to ejaculates with increased percentages of abnormal and apoptotic spermatozoa and a high percentage of apoptotic bodies. Apoptosis markers in the ejaculate have been associated with low fertility, either in animals or humans. Therefore, the goal of this study was to investigate whether fresh equine semen contains apoptotic bodies [initially named Merocyanine 540 (M540) bodies] and ...
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 ...
Wu H, Han D, Jiang Z, Zhao D, Liu M, Xu X, Liu X, Yang L, Ji X, Wang M, Zhang S.B cell activating factor (BAFF) and its receptors were regarded as elements of the immune system, regulating the fate of B cell. In recent years, these molecules were identified in a number of normal and pathological tissues, expanding their potential functions beyond the immune system. In this study, on the basis of molecular clone and prokaryotic expression of equine BAFF, we reported that equine adipose-derived stem cell (ASC) expressed BAFF and its receptors, which exhibited the increased expression during ASC adipogenic differentiation in vitro. Moreover, with the addition of recombinant ...
Williams A, Smith JR, Allaway D, Harris P, Liddell S, Mobasheri A.Arthritic diseases are characterized by the degradation of collagenous and noncollagenous extracellular matrix (ECM) components in articular cartilage. The increased expression and activity of matrix metalloproteinases (MMPs) is partly responsible for cartilage degradation. This study used proteomics to identify inflammatory proteins and catabolic enzymes released in a serum-free explant model of articular cartilage stimulated with the pro-inflammatory cytokine interleukin 1β (IL-1β). Western blotting was used to quantify the release of selected proteins in the presence or absence of the cyc...
Lavoie-Lamoureux A, Martin JG, Lavoie JP.Neutrophils are the predominant cells recruited in the airways of horses suffering from heaves. These cells have been shown to express arginase in some species. The metabolism of l-arginine is thought to be involved in chronic inflammation, and airway obstruction and remodeling. The aim of this study was to assess the expression, regulation, activity, and functional role of arginase isoforms in equine neutrophils. Arginase I, arginase II, ornithine decarboxylase (ODC) and ornithine aminotransferase (OAT) expression were assessed in resting and stimulated (IL-4, LPS/fMLP, PMA; 5 and 18 h) blood...
Carter-Arnold JL, Neilsen NL, Amelse LL, Odoi A, Dhar MS.Stem cell therapies are used routinely in equine practice. Most published reports characterise stem cells derived from younger horses; however, middle-aged horses are often in athletic performance, and experience degenerative medical conditions. Thus, mesenchymal stem cells (MSCs) from this group should be investigated. Objective: To describe differences in in vitro adherence, proliferation and potential for differentiation of equine bone marrow-derived MSCs (equine BMMSCs) harvested from middle-aged (10-13 years old) female donors. Methods: Descriptive study of stem cell characteristics. Meth...
Brown BN, Siebenlist NJ, Cheetham J, Ducharme NG, Rawlinson JJ, Bonassar LJ.Normal laryngeal function has a large impact on quality of life, and dysfunction can be life threatening. In general, airway obstructions arise from a reduction in neuromuscular function or a decrease in mechanical stiffness of the structures of the upper airway. These reductions decrease the ability of the airway to resist inspiratory or expiratory pressures, causing laryngeal collapse. We propose to restore airway patency through methods that replace damaged tissue and improve the stiffness of airway structures. A number of recent studies have utilized image-guided approaches to create cell-...
Jülke H, Veit C, Ribitsch I, Brehm W, Ludewig E, Delling U.The purpose of this study was to evaluate the use of three different superparamagnetic iron oxide (SPIO) particles for labeling of ovine and equine bone marrow (BM)-derived multipotent stromal cells (MSCs) in vitro. MSCs were obtained from five adult sheep and horses, respectively. After three passages (p3), cells were labeled with either 1) Molday ION Rhodamine B, 2) Endorem, 3) Resovist, or 4) remained unlabeled as control. Labeling efficiency, marker retention, and long-term detectability in MRI until p7 were evaluated. Further, proliferation capacity and trilineage differentiation as indic...
Hartwig FP, Lisboa FP, Hartwig FP, Monteiro GA, Maziero RR, Freitas-Dell'Aqua CP, Alvarenga MA, Papa FO, Dell'Aqua JA.During the cooling process, sperm may suffer irreversible damage that compromises the fertility rate. Incorporating cholesterol-loaded cyclodextrin (CLC) represents a strategy to increase sperm resistance at low temperatures; however, high levels of cholesterol in the cell membrane can interfere with sperm capacitation. The goals of this study were to determine the CLC concentration and cooling temperature that produce optimal kinetic parameters and viability of sperm from stallions identified as bad coolers (BCs) and good coolers (GCs), as well as the effect of adding CLC on the occurrence of...
Rich T, Henderson LB, Becker DL, Cornell H, Patterson-Kane JC.Superficial digital flexor tendon (SDFT) injuries of horses usually follow cumulative matrix microdamage; it is not known why the reparative abilities of tendon fibroblasts are overwhelmed or subverted. Relevant in vitro studies of this process require fibroblasts not already responding to stresses caused by the cell culture protocols. We investigated indicators of replicative damage in SDFT fibroblast monolayers, effects of this on their reparative ability, and measures that can be taken to reduce it. Results: We found significant evidence of replicative stress, initially observing consistent...
Nelis H, D'Herde K, Goossens K, Vandenberghe L, Leemans B, Forier K, Smits K, Braeckmans K, Peelman L, Van Soom A.Equine embryos remain for 6 days in the oviduct and thus there is a need for an in vitro model to study embryo-oviductal interactions in the horse, since this subtle way of communication is very difficult to analyse in vivo. Until now, no equine oviduct explant culture model has been characterised both morphologically and functionally. Therefore, we established a culture system for equine oviduct explants that maintained epithelial morphology during 6 days of culture, as revealed by light microscopy and transmission electron microscopy. We demonstrated the presence of highly differentiated, ta...
Xie L, Zhang N, Marsano A, Vunjak-Novakovic G, Zhang Y, Lopez MJ.Directed differentiation of adult multipotent stromal cells (MSC) is critical for effective treatment strategies. This study was designed to evaluate the capability of equine MSC from bone marrow (BMSC) and adipose tissue (ASC) on a type I collagen (COLI) scaffold to undergo chondrogenic, osteogenic and adipogenic differentiation and form extracellular matrix (ECM) in vitro. Following determination of surface antigen expression, MSC were loaded into scaffolds in a perfusion bioreactor and loading efficiency was quantified. Cell-scaffold constructs were assessed after loading and 7, 14 and 21 d...
Vendrig JC, Coffeng LE, Fink-Gremmels J.Dietary supplementation with oligosaccharides has been proven to be beneficial for health in several mammalian species. Next to prebiotic effects resulting in a modulation of gut micro biota, immunomodulatory effects of oligosaccharides have been documented in vivo. Supplementation with defined oligosaccharide fractions has been shown to attenuate allergic responses and enhance defensive immune responses. Despite the accumulating evidence for immunomodulatory effects, very limited information is available regarding the direct mechanism of action of oligosaccharides. This study aims to elucidat...
Altamura G, Corteggio A, Nasir L, Yuan ZQ, Roperto F, Borzacchiello G.Equine sarcoids are skin tumours of fibroblastic origin affecting equids worldwide. Bovine papillomavirus type-1 (BPV-1) and, less commonly, type-2 are recognized as etiological factors of sarcoids. The transforming activity of BPV is related to the functions of its major oncoprotein E5 which binds to the platelet-derived growth factor β receptor (PDGFβR) causing its phosphorylation and activation. In this study, we demonstrate, by coimmunoprecipitation and immunoblotting, that in equine sarcoid derived cell lines PDGFβR is phosphorylated and binds downstream molecules related to Ras-mitoge...
Ishihara A, Helbig HJ, Sanchez-Hodge RB, Wellman ML, Landrigan MD, Bertone AL.Objective-To determine the efficiency of a novel point-of-care gravitational marrow separator and bone marrow aspiration needle for concentrated bone marrow production and bone marrow-derived mesenchymal stem cell (MSC) separation and assess the effect of repeated bone marrow collections in horses. Animals-8 healthy adult horses. Procedures-Bone marrow aspiration was performed twice (1 month apart) from sternebral bodies with a standard or prototype multidirectional needle. Concentrated bone marrow was obtained by gravitational marrow separation and evaluated for WBC and platelet counts, autom...
Donnelly KS, Giuliano EA, Sharma A, Tandon A, Rodier JT, Mohan RR.To explore (i) the potential of polyethylenimine (PEI) nanoparticles as a vector for delivering genes into equine corneal fibroblasts (ECFs) using green fluorescent protein (GFP) marker gene, (ii) whether PEI nanoparticle-mediated decorin (DCN) gene therapy could be used to inhibit fibrosis in the equine cornea using an in vitro model. Methods: Polyethylenimine-DNA nanoparticles were prepared at nitrogen-to-phosphate (N-P) ratio of 15 by mixing 22 kDa linear PEI and a plasmid encoding either GFP or DCN. ECFs were generated from donor corneas as previously described. Initially, GFP was introduc...
Hondalus MK, Sweeney CR, Mosser DM.A Rhodococcus equi radiobinding assay has been developed using organisms labeled with 3H-uracil. These labeled organisms resemble their unlabeled counterparts with respect to colony morphology, viability, and buoyant density. Bacteria routinely incorporate between 5 x 10(-3) and 5 x 10(-2) counts per minute per colony forming unit (cfu) which in this assay allows the detection of fewer than 0.2 cfu per macrophage. Once incorporated, greater than 90% of the label remains bacterial associated for at least 4 h postlabeling. The majority of the label is trichloroacetic acid precipitable, partition...
Brandt AM, Kania JM, Gonzalez ML, Johnson SE.Hepatocyte growth factor (HGF) signals mediate mouse skeletal muscle stem cell, or satellite cell (SC), reentry into the cell cycle and myoblast proliferation. Because the athletic horse experiences exercise-induced muscle damage, the objective of the experiment was to determine the effect of HGF on equine SC (eqSC) bioactivity. Fresh isolates of adult eqSC were incubated with increasing concentrations of HGF and the initial time to DNA synthesis was measured. Media supplementation with HGF did not shorten (P > 0.05) the duration of G0/G1 transition suggesting the growth factor does not aff...
Bone reportsAugust 18, 2017
Volume 7 98-107 doi: 10.1016/j.bonr.2017.08.002
Ayodele BA, Mirams M, Pagel CN, Mackie EJ.Chondrocyte hypertrophy makes important contributions to bone development and growth. We have investigated a number of novel cartilage genes identified in a recent transcriptomic study to determine whether they are differentially expressed between different zones of equine foetal growth cartilage. Twelve genes (, , , , , , , , , , and ) were found to be more highly expressed in the zone of hypertrophic chondrocytes than in the reserve or proliferative zones, whereas and were expressed at lower levels in the hypertrophic zone than in the reserve zone. , which encodes vacuolar H ATPase (V-ATP...
Bourebaba N, Sikora M, Qasem B, Bourebaba L, Marycz K.Equine metabolic syndrome (EMS), which encompasses insulin resistance, low-grade inflammation and predisposition to laminitis is a critical endocrine disorder among the most prevalent conditions affecting horses from different breeds. According to the most recent research, low human sex hormone-binding globulin (SHBG) serum levels correlate with an increased risk of obesity, insulin resistance and diabetes, and may contribute to overall metabolic dysregulations. This study aimed to test whether exogenous SHBG could protect EMS affected adipose-derived stromal stem cells (EqASC) from apoptosis,...
Li X, Hu S, Yu Z, He F, Zhao X, Liu R.Nanoplastics (NPs) are emerging pollutants worldwide. Particularly worrisome is that although studies have reported that NPs can amplify the biotoxicity of environmental pollutants, the specific mechanism remains unclear. Here, we found that NPs, even without significant toxicity (cell survival: 99.11%), amplified the hepatocyte toxicity of Cd. Mechanistically, higher Cd uptake (Δ = 23.80%) combined with crucial intracellular desorption behavior of Cd loaded in NPs (desorption rate: 82.70%) were identified as prerequisites for NPs amplifying Cd cytotoxicity. As for toxigenic pathways, the inf...
Iacono E, Lanci A, Gugole P, Merlo B.To use Mesenchymal Stromal Cells (MSCs) in equine patients, isolation and expansion are performed in a laboratory. Cells are then sent back to the veterinary clinic. The main goal of storage conditions during cell transport is to preserve their biological properties and viability. The aim of this study was to evaluate the effects of storage solutions, temperature and time on the characteristics of equine adipose tissue and Wharton's jelly-derived MSCs. We compared two different storage solutions (plasma and 0.9% NaCl), two different temperatures (4 °C and room temperature) and three time fram...
Reisbig NA, Hussein HA, Pinnell E, Bertone AL.OBJECTIVE To create a bioactive synovium scaffold by infusing decellularized synovial-derived extracellular matrix (synECM) with synovial-derived mesenchymal stem cells (synMSCs). SAMPLE Synovium from the femoropatellar and medial femorotibial joints of equine cadavers. PROCEDURES The synMSCs were cultured in monolayer and not treated or cotransduced to enhance expression of green fluorescent protein (GFP) and human bone morphogenetic protein (BMP)-2. The synECM was decellularized with 0.1% peracetic acid and then seeded with synMSCs (0.5 × 10 cells/0.5 mL) by use of a 30% serum gradient. Sam...
Meeremans M, Van Damme L, De Spiegelaere W, Van Vlierberghe S, De Schauwer C.(1) Background: Tendinopathy is a common injury in both human and equine athletes. Representative in vitro models are mandatory to facilitate translation of fundamental research into successful clinical treatments. Natural biomaterials like gelatin provide favorable cell binding characteristics and are easily modifiable. In this study, methacrylated gelatin (gel-MA) and norbornene-functionalized gelatin (gel-NB), crosslinked with 1,4-dithiotreitol (DTT) or thiolated gelatin (gel-SH) were compared. (2) Methods: The physicochemical properties (H-NMR spectroscopy, gel fraction, swelling ratio, an...
Foster ML, Varner DD, Hinrichs K, Teague S, Lacaze K, Blanchard TL, Love CC.Increasing seminal plasma concentrations in extended stallion semen were utilized to model decreasing sperm motility over time. Level of agreement was determined between flow cytometric measurement of sperm membrane integrity, using a combination of SYBR-14 and propidium iodide, and computer-assisted analysis of sperm motility. Values for total sperm motility (TMOT;%) and membrane integrity (SMI;%) were similar (∼80%) at Time 0 within all sperm treatments. However, TMOT was lower than SMI after 24 and 48 h of storage in treatments with >20% seminal plasma. At Time 0, agreement (bias and a...
Ishikawa S, Horinouchi C, Mizoguchi R, Senokuchi A, Kamikakimoto R, Murata D, Hatazoe T, Tozaki T, Misumi K, Hobo S.The sizes of Japanese native horses have drastically decreased, and protection of these populations is important for Japanese horse culture. Social trials as well as scientific attempts are necessary for maintaining the breed. Mesenchymal stem cells (MSCs) have potential as a cell source for various cell therapies. However, there have been no reports on MSCs of Japanese native horses. We aimed to isolate and characterize MSCs from a Japanese native horse, the Noma horse. Plastic-adherent and self-replicating cells were isolated from a Noma horse's peripheral blood (PB). The isolated cells had ...
Akcay E, Reilas T, Andersson M, Katila T.This study aimed to evaluate stallion sperm survival after 24 h of cooled storage in the presence of seminal plasma (SP) derived from the sperm-rich fractions (SRF) or sperm-poor fractions(SPF) of the ejaculate, without SP, or in the presence of SP from other stallions. Ejaculates were collected from four stallions using an automated phantom, which separated the semen into five cups. Centrifuged and washed spermatozoa from cup 2 (SRF) were mixed with skim milk extender to a concentration of 100 x 10(6) sperm/ml and then 1:1 (v/v) with SP from the stallion's own or another stallions' second (SP...
Ferrante A, Thong YH.Results presented show that highly purified populations of mononuclear (MN) and polymorphonuclear (PMN) leucocytes can be obtained from horse blood by a procedure similar to that previously described for the separation of these leucocytes from human blood. This involved centrifugation of horse blood on a Ficoll-Hypaque medium with a density of 1.095 g/ml. The procedure required approximately 1 h for completion and resulted in the simultaneous preparation of MN (greater than 98% purity) and PMN (greater than 96% purity) leucocytes. Cell viability exceeded 95% and cells retained immunological fu...
Di Francesco P, Cajon P, Desterke C, Perron Lepage MF, Lataillade JJ, Kadri T, Lepage OM.To assess the clinical value and safety of the application of allogeneic equine oral mucosa mesenchymal stromal cells (OM-MSCs) to wounds. Animals. 8 healthy adult horses without front limb skin lesions or musculoskeletal disease. Procedures. Stem cells were isolated from the oral mucosa of a donor horse. Horses were subjected to the creation of eight full-thickness cutaneous wounds, two on each distal forelimb (FL) and two on both sides of the thorax (TH). Each wound was subjected to one out of four treatments: no medication (T1), hyaluronic acid- (HA-) gel containing OM-MSC (T2), HA-gel cont...
Serwotka-Suszczak AM, Marcinkowska KA, Smieszek A, Michalak IM, Grzebyk M, Wiśniewski M, Marycz KM.Insulin resistance (IR) is one of the characteristic features of equine metabolic syndrome (EMS). Presently, the only therapies of choice are caloric restrictions combined with mineral supplementation, which might improve insulin sensitivity. In this study we investigated the effect of Haematococcus pluvialis algae water extract enriched in bioaccumulation process in magnesium ions (Hp_Mg(II)) on equine adipose derived mesenchymal stromal stem cells, in which insulin resistance was induced by palmitic acid (IR-EqASCs). For this purpose, chemical characterization of H. pluvialis was performed w...
Eldridge-Panuska WD, di Brienza VC, Seidel GE, Squires EL, Carnevale EM.Experiments were conducted to determine viability of equine embryos in vivo after vitrification. In a preliminary study (Experiment 1), embryos were exposed in three steps to vitrification solutions containing increasing concentrations of ethylene glycol and glycerol (EG/G); the final vitrification solution was 3.4 M glycerol + 4.6 M ethylene glycol in a base medium of phosphate-buffered saline. Embryos were warmed in a two-step dilution and transferred into uteri of recipients. No pregnancies were observed after transfer of blastocysts >300 microm (n = 3). Transfer of morulae or blastocyst...
Schumann B, Winkler J, Mickenautsch N, Warnken T, Dänicke S.Both deoxynivalenol (DON), zearalenone (ZEN), and their metabolites are known to modulate immune cells in various species whereby viability and proliferation are influenced. Such effects were rarely examined in horses. Therefore, one aim of the present study was to titrate the inhibitory concentrations of DON, 3-acetyl-DON (3AcDON), de-epoxy-DON (DOM-1), ZEN, and α- and β-zearalenol (ZEL) at which viability and proliferation of equine PBMC were reduced by 50 % (IC50) and 10 % (IC10) in vitro. For evaluation of practical relevance of the in vitro findings, a further aim was to screen horses...
Agnieszka N, Joanna K, Wojciech W, Adam O.The aim of this study was determine the viability and developmental competence of equine oocytes after IVM and vitrification using the Rapid-I method, as part of an effort to develop an effective equine oocyte vitrification protocol. Equine oocytes were collected by scraping ovarian follicles of slaughtered mares. A total of 1052 ovaries were used in this study, from which 3135 oocytes were obtained. Of the 2853 oocytes retrieved, 2557 underwent in vitro maturation for approximately 36 h. After in vitro culture, 1202 oocytes (47%) had a first polar body. To evaluate the toxicity of the solu...
Price S, Aurich J, Davies-Morel M, Aurich C.This study was undertaken to investigate the effects of storage of stallion semen in a defined milk protein extender at 5 and 15 degrees C under either anaerobic or aerobic conditions, with or without addition of the antibiotic gentamicin. Semen samples were collected from eight fertile stallions and stored for 96 h (day 0-4) and assessed daily for motility, velocity and membrane integrity (viability) using a CASA system. Samples for bacteriology assessment were taken on day 2 of storage. No significant (p > 0.05) differences in motility, velocity or viability were observed between treatmen...
Scare JA, Steuer AE, Shaffer CL, Slusarewicz P, Mousley A, Nielsen MK.In vitro maintenance of helminth parasites enables a variety of molecular, pharmaceutical and immunological analyses. Currently, the nutritional and environmental in vitro requirements of the equine ascarid parasite, Parascaris spp., have not been determined. Additionally, an objective method for assessing viability of Parascaris spp. intestinal stages does not exist. The purpose of this study was to ascertain the in vitro requirements of intestinal stages of Parascaris spp., and to develop a viability assessment method. A total of 1045 worms were maintained in a total of 212 cultures. Worms o...
Ishihara A, Helbig HJ, Sanchez-Hodge RB, Wellman ML, Landrigan MD, Bertone AL.Objective-To determine the efficiency of a novel point-of-care gravitational marrow separator and bone marrow aspiration needle for concentrated bone marrow production and bone marrow-derived mesenchymal stem cell (MSC) separation and assess the effect of repeated bone marrow collections in horses. Animals-8 healthy adult horses. Procedures-Bone marrow aspiration was performed twice (1 month apart) from sternebral bodies with a standard or prototype multidirectional needle. Concentrated bone marrow was obtained by gravitational marrow separation and evaluated for WBC and platelet counts, autom...
Ball BA, Sabeur K, Allen WR.Sperm-mediated gene transfer has been reported as a method for production of transgenic animals in a variety of species, and this technique represents a possible method for production of transgenic equids. Objective: To evaluate the uptake of exogenous DNA (enhanced green fluorescent protein; pEGFP) by equine spermatozoa and to assess the ability of transfected spermatozoa to introduce this transgene into early equine embryos. Methods: To evaluate incorporation of pEGFP into equine spermatozoa, washed spermatozoa were incubated with 32P-pEGFP, with or without lipofection. Spermatozoa were also...
Enders AC, Lantz KC, Liu IK, Schlafke S.Twelve blastocysts, collected 7-12 days after ovulation (Day 0), were examined by light and electron microscopy to investigate the nature of the relationship of the polar trophoblast (Rauber's layer) to the inner cell mass. On Day 7, the polar trophoblast was intact and formed a flattened layer overlying the epiblast cells of the inner cell mass. As blastocysts enlarged to greater than 1 mm in diameter, small discontinuities appeared in the polar trophoblast, where epiblast cells intruded onto the surface. At this time, trophoblast cells adhered closely to adjacent and underlying epiblast cell...
Mudge MC.Treatment of acute hemorrhage in the horse involves targeted medical management and also may involve surgical stabilization. This article provides an approach to the initial stabilization and information on available topical hemostats. The practice of blood collection and transfusion is also described, with attention to new information on viability of transfused equine blood, potential negative effects of blood transfusion, and methods of cell salvage.
Wilhelm KM, Graham JK, Squires EL.Computer-assisted motion analyses (CASA) and flow cytometry were used to evaluate stallion spermatozoa prior to and after cryopreservation. Spermatozoa were pretreated with: (1) Hepes-buffered medium (SHB); (2) phosphatidylserine (PS) liposomes; or (3) liposomes composed of both PS and cholesterol (PSCH) prior to dilution in either SHB or skim milk-egg yolk extender (SMEY). After cooling to 5 degrees C in SHB, PS and PSCH pretreatment (23%). Spermatozoal motion parameters were higher for spermatozoa diluted in SMEY than dilution in SHB. In Experiment 2, motion parameters were compared for sper...
Len JA, Jenkins JA, Eilts BE, Paccamonti DL, Lyle SK, Hosgood G.The objectives of this study were to determine the effects of centrifugation on equine sperm total and progressive motility, viability, and acrosomal integrity. We hypothesized that although high centrifugation forces would be detrimental to equine Equus caballus sperm, recovery rates would increase. Ejaculates from six stallions were collected, extended to a concentration of 25x10(6) cells/mL, and subjected for 10min to (1) no centrifugation (NC) or (2) centrifugation at 400xg, (3) 900xg, or (4) 4500xg. Before and after centrifugation (Day 0), and after 24h of cooling (Day 1), sperm motility ...
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...
Kwirant LADA, De La Corte FD, Cantarelli C, Cargnelutti JF, Martins M, Cabral MW, Maciel N, Rubin MIB.Equine platelet-rich plasma (PRP) has been used in horses to repair bone, articular and tendinous lesions, laminitis, and even endometritis. However, platelets have a very limited lifespan, which makes it difficult to prepare and use PRP, except in loco. With the aim to produce PRP with higher platelet viability for clinical purposes, the effects of the cryoprotectants dimethyl sulfoxide (DMSO) and trehalose were evaluated on cooled (4°C) and cryopreserved (-196°C) equine PRP. The protocols of cooling and cryopreservation were performed independently, comparing the following treatments: fres...
Pereira BC, Ortiz I, Dorado JM, Diaz-Jimenez MA, Consuegra C, Gosalvez J, Hidalgo M.DNA fragmentation of cumulus cells could be used as an indicator of oocyte vitrification success as an indirect indicator of the quality of the oocyte. This study was designed to compare the DNA fragmentation of post-mortem equine cumulus cells before or after vitrification in the absence of permeable cryoprotectant agents. Cumulus-oocyte complexes (COCs; n = 56) were recovered from slaughterhouse ovaries and subjected to in vitro maturation (42 hr/38.2°C/5%CO ) before (control group) or after a permeable cryoprotectant-free vitrification method using 1 M sucrose (vitrification group). Af...
Sedgwick AD, Morris T, Russell BA, Lees P.Percoll gradients have been used to separate relatively pure populations of viable equine polymorphonuclear (PMN) and mononuclear (MN) cells. In preliminary studies, a continuous density gradient of 70% Percoll solution was used to separate two distinct leucocyte-rich bands. After measurement of the density of each band on the continuous gradient, discontinuous Percoll gradients, using 60% and 75% Percoll solutions, were used to provide a rapid means of separating PMN and MN cells. The yield of viable cells per ml of blood was 3.0 X 10(6) and 3.2 X 10(6) for MN and PMN cells, respectively. Cor...
