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.
Cullier A, Cassé F, Manivong S, Contentin R, Legendre F, Garcia Ac A, Sirois P, Roullin G, Banquy X, Moldovan F, Bertoni L, Audigié F, Galéra P....Osteoarthritis (OA) is a degenerative and heterogeneous disease that affects all types of joint structures. Current clinical treatments are only symptomatic and do not manage the degenerative process in animals or humans. One of the new orthobiological treatment strategies being developed to treat OA is the use of drug delivery systems (DDS) to release bioactive molecules over a long period of time directly into the joint to limit inflammation, control pain, and reduce cartilage degradation. Two vasoactive peptides, endothelin-1 and bradykinin, play important roles in OA pathogenesis. In this ...
Koch DW, Berglund AK, Messenger KM, Gilbertie JM, Ellis IM, Schnabel LV.Tendon injury in the horse carries a high morbidity and monetary burden. Despite appropriate therapy, reinjury is estimated to occur in 50-65% of cases. Although intralesional mesenchymal stem cell (MSC) therapy has improved tissue architecture and reinjury rates, the mechanisms by which they promote repair are still being investigated. Additionally, reevaluating our application of MSCs in tendon injury is necessary given recent evidence that suggests MSCs exposed to inflammation (deemed MSC licensing) have an enhanced reparative effect. However, applying MSC therapy in this context is limited...
Hagen A, Niebert S, Brandt VP, Holland H, Melzer M, Wehrend A, Burk J.Successful translation of multipotent mesenchymal stromal cell (MSC)-based therapies into clinical reality relies on adequate cell production procedures. These should be available not only for human MSC, but also for MSC from animal species relevant to preclinical research and veterinary medicine. The cell culture medium supplementation is one of the critical aspects in MSC production. Therefore, we previously established a scalable protocol for the production of buffy-coat based equine platelet lysate (ePL). This ePL proved to be a suitable alternative to fetal bovine serum (FBS) for equine a...
MacNicol JL, Renwick S, Ganobis CM, Allen-Vercoe E, Weese JS, Pearson W.The equine gastrointestinal (GI) microbiota is intimately related to the horse. The objective of the current study was to evaluate the microbiome and metabolome of cecal inoculum maintained in an anaerobic chamber or chemostat batch fermenter, as well as the fecal slurry maintained in an anaerobic chamber over 48 h. Cecal and fecal content were collected from healthy adult horses immediately upon death. Cecal fluid was used to inoculate chemostat vessels (chemostat cecal, = 11) and vessels containing cecal fluid (anaerobic cecal, = 15) or 5% fecal slurry (anaerobic fecal, = 6) were maintain...
Chodkowski M, Słońska A, Gregorczyk-Zboroch K, Nowak-Zyczynska Z, Golke A, Krzyżowska M, Bańbura MW, Cymerys J.Mitochondria are key cellular organelles responsible for many essential functions, including ATP production, ion homeostasis and apoptosis induction. Recent studies indicate their significant role during viral infection. In the present study, we examined the effects of equine herpesvirus type 1 (EHV-1) infection on the morphology and mitochondrial function in primary murine neurons in vitro. We used three EHV-1 strains: two non-neuropathogenic (Jan-E and Rac-H) and one neuropathogenic (EHV-1 26). The organization of the mitochondrial network during EHV-1 infection was assessed by immunofluores...
Barshick MR, Gonzalez ML, Busse NI, Helsel PJ, Johnson SE.Satellite cell (SC) activation is defined as the time frame during which the stem cell becomes poised to reenter G1 of the cell cycle. The growth factors and events leading to full mitotic activation in equine SCs remain largely unknown. Insulin-like growth factor I (IGF-I), hepatocyte growth factor (HGF), and fibroblast growth factor 2 (FGF2) are sequentially transcribed during the muscle repair and recovery period following strenuous exercise in adult horses. Expression of IGF-I occurs within 24 h of the postexercise recovery period suggesting it may affect early SC actions. As a first step,...
Smith MA, Carpenter AB, Nielsen MK.Essentially all grazing horses are infected with cyathostomin parasites. Adult cyathostomins reside in the large intestine of the horse and larval stages encyst within intestinal mucosa. Manual worm collection from aliquots of intestinal content is the current gold standard for retrieval and enumeration of luminal parasites, however, no research has been conducted to standardize specific parameters for processing and storage of samples. The aims of this study were (1) to evaluate the precision of current standard operating procedures for enumeration of luminal adult cyathostomin populations, (...
Walbornn SR, Felix M, Schnobrich MR, Bradecamp EA, Scoggin CF, Stefanovski D, Hinrichs K.To determine the effect of stage of estrus cycle (day after ovulation) at the time of transvaginal ultrasound-guided follicle aspiration (TVA) on parameters related to the success of in vitro equine embryo production. 14 healthy mares were used; 11 completed the study and were included for analysis. Mares underwent TVA of all follicles ≥ 5 mm diameter at each of 3 timepoints: 7 days after ovulation, 14 days after ovulation, and S-DSF (subordinate to a dominant stimulated follicle), during estrus at 24 hours after gonadotropin administration. The 3 treatments were assigned to each mare in ran...
Ząbek T, Witarski W, Semik-Gurgul E, Szmatoła T, Kowalska K, Samiec M.Ex vivo expansion of chondrocytes in monolayer (ML) culture for therapeutic purposes is burdened with difficulties related to the loss of cartilaginous phenotype. Epigenetic mechanisms responsible for regulation of gene expression are believed to underlie chondrocyte dedifferentiation. We have inspected the relevance of DNA methylation alterations for passage-related differential expression of NFATC1 gene involved in hard connective tissue turnover and development, NADSYN1 influencing redox metabolism, and JAK3 - an important driver of inflammation. We have assessed relative amount of transcri...
Haikerwal A, Barrera MD, Bhalla N, Zhou W, Boghdeh N, Anderson C, Alem F, Narayanan A.Acutely infectious new world alphaviruses such as Venezuelan Equine Encephalitis Virus (VEEV) pose important challenges to the human population due to a lack of effective therapeutic intervention strategies. Small interfering RNAs that can selectively target the viral genome (vsiRNAs) has been observed to offer survival advantages in several in vitro and in vivo models of acute virus infections, including alphaviruses such as Chikungunya virus and filoviruses such as Ebola virus. In this study, novel vsiRNAs that targeted conserved regions in the nonstructural and structural genes of the VEEV ...
Wells HC, Sizeland KH, Kirby N, Haverkamp RG.Thin, strong scaffold materials are needed for surgical applications. New materials are required, particularly those readily available, such as from non-human sources. Bovine amniotic membrane (antepartum) and equine amniotic membrane (postpartum) were characterized with tear and tensile tests. The structural arrangement of the collagen fibrils was determined by small-angle X-ray scattering, scanning electron microscopy, and ultrasonic imaging. Bovine amnion had a thickness-normalized tear strength of 12.6 (3.8) N/mm, while equine amnion was 14.8 (5.3) N/mm. SAXS analysis of the collagen fibri...
Gupta S, Vohra S, Sethi K, Gupta S, Bera BC, Kumar S, Kumar R.High cytotoxicity and increasing resistance reports of existing chemotherapeutic agents against T. evansi have raised the demand for novel, potent, and high therapeutic index molecules for the treatment of surra in animals. In this regard, repurposing approach of drug discovery has provided an opportunity to explore the therapeutic potential of existing drugs against new organism. With this objective, the macrocyclic lactone representative, ivermectin, has been investigated for the efficacy against T. evansi in the axenic culture medium. To elucidate the potential target of ivermectin in T. ev...
Boger BL, Manfredi JM, Yob C, Weber PSD, Jacobs CC.Diagnosis of equine septic arthritis is not straightforward, and increasing time between onset, diagnosis, and treatment can have serious consequences for quality of life. Defensins are used in diagnosis of human joint infection. The presence of beta defensins (BDs) in equine synovial fluid and their utility as a biomarker of sepsis has not been investigated; therefore, our objectives were to (1) compare in vitro gene expression of lipopolysaccharide (LPS)-stimulated equine neutrophils to unstimulated neutrophils and (2) compare BD protein expression from normal, aseptically inflamed, and sept...
Gupta S, Vohra S, Sethi K, Gupta S, Kumar S, Kumar R.The kinetoplastid protozoan parasite, Trypanosoma evansi causes a fatal disease condition known as Surra in equines throughout the globe. Disease condition being acute in nature, entrust a huge economic and health impact on the equine industry. Till date, quinapyramine methyl sulphate (QPS) is the first line of treatment and a panacea for the T. evansi infection in equines. Still after the >70 years of its discovery, there is no clue about the mode of action of QPS in T. evansi. The establishment of in vitro cultivation of T. evansi in HMI-9 media has provided opportunity to study the alte...
Handschuh S, Okada CTC, Walter I, Aurich C, Glösmann M.Here, we describe a workflow for high-detail microCT imaging of formalin-fixed and paraffin-embedded (FFPE) equine embryos recovered on Day 34 of pregnancy (E34), a period just before placenta formation. The presented imaging methods are suitable for large animals' embryos with intention to study morphological and developmental aspects, but more generally can be adopted for all kinds of FFPE tissue specimens. Microscopic 3D imaging techniques such as microCT are important tools for detecting and studying normal embryogenesis and developmental disorders. To date, microCT imaging of vertebrate e...
Podico G, Canisso IF.This study aimed to assess the parameters of epididymal sperm harvested by retrograde flushing (RF) followed by slicing float-up (SF). Epididymides from donkeys (n = 18) and horses (n = 28) were subjected to RF with a freezing extender and then SF technique. The retrieved sperm after RF and SF was evaluated for volume, concentration, and total sperm and then cryopreserved separately. Post-thaw total motility (TM) and progressive motility (PM) were evaluated with CASA. Sperm membrane integrity (SMI) and mitochondrial membrane potential (MMP) were assessed with flow cytometry. Sperm concentratio...
Ortiz I, Dorado J, Pereira B, Diaz-Jimenez M, Consuegra C, Gosalvez J, Hidalgo M.The objectives of this study were to evaluate the effect of vitrification on the DNA fragmentation rate of equine cumulus cells and to assess its relationship to oocyte in vitro maturation (IVM) after vitrification. Cumulus cells (CC) from 14 mares were recovered from COCs, previously submitted to vitrification (VIT) and IVM. The DNA fragmentation rate of the cumulus cells (CC-DF) was assessed using a chromatin dispersion test. CC-DF rates between vitrified and control COCs were statistically compared by Student's t-test. The rates of CC-DF from control COCs were lower than in vitrified COCs...
Biegel U, Mevissen M, Schuller S, Ruess K, Christen O, Ayrle H, Koch C, Walkenhorst M.Cancer is a common disease in humans and in companion animals and treatment is challenging. The aim of this systematic review was to identify and assess the potential use of Viscum album L. extracts (VAE) for treatment of neoplastic diseases in companion animals. Peer-reviewed animal, in vivo and in vitro studies were included, considering the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement and A Measurement Tool to Assess Systematic Reviews (AMSTAR). Overall, 6,148 references were identified. Following a predefined protocol, 114 full-text references were ...
Kupczyk P, Rykala M, Serek P, Pawlak A, Slowikowski B, Holysz M, Chodaczek G, Madej JP, Ziolkowski P, Niedzwiedz A.The endocannabinoid system (ECS) is composed of cannabinoid receptors type 1 (CBR1) and type 2 (CBR2), cannabinoid-based ligands (endogenous chemically synthesized phytocannabinoids), and endogenous enzymes controlling their concentrations. Cannabinoid receptors (CBRs) have been identified in invertebrates and in almost all vertebrate species in the central and peripheral nervous system as well as in immune cells, where they control neuroimmune homeostasis. In humans, rodents, dogs, and cats, CBRs expression has been confirmed in the skin, and their expression and tissue distribution become di...
Supokawej A, Korchunjit W, Wongtawan T.The Wingless and Int-1 (WNT) and bone morphogenic protein/growth differentiation factor (BMP/GDF) signalling pathways contribute significantly to the development of the musculoskeletal system. The mechanism by which they contribute is as follows: BMP/GDF signalling usually promotes tendon differentiation, whereas WNT signalling inhibits it. We hypothesised that inhibiting WNT and subsequently stimulating BMP signalling may enhance the tenogenic differentiation of stem cells. The objective of this study was to determine whether a combination of WNT inhibitor (KY02111) and BMP12/GDF7 protein cou...
Amorim GB, Segabinazzi LG, Oliveira OM, Perecmanis S, Arruda R, Canisso IF.Misoprostol, a synthetic PGE1, is becoming a common therapy for mares with suspected uterine tube obstruction. Recently, there have been concerns that uterine administration of misoprostol induces exacerbated uterine inflammation; however, this has not been critically evaluated. This study aimed to assess the inflammatory response and potential systemic reactions after uterine administration of misoprostol, either during prebreeding or immediately after postembryo flushing. Privately owned embryo donor mares (n = 11) were randomly assigned in a crossover design to receive misoprostol (3 mL +...
Song K, Yang GM, Han J, Gil M, Dayem AA, Kim K, Lim KM, Kang GH, Kim S, Jang SB, Vellingiri B, Cho SG.Flavonoids form the largest group of plant phenols and have various biological and pharmacological activities. In this study, we investigated the effect of a flavonoid, 3, 4'-dihydroxyflavone (3, 4'-DHF) on osteogenic differentiation of equine adipose-derived stromal cells (eADSCs). Unassigned: Treatment of 3, 4'-DHF led to increased osteogenic differentiation of eADSCs by increasing phosphorylation of ERK and modulating Reactive Oxygen Species (ROS) generation. Although PD98059, an ERK inhibitor, suppressed osteogenic differentiation, another ERK inhibitor, U0126, apparently increased osteoge...
Abd-El-Aziz A, Riveroll A, Esparza-Gonsalez B, McD￮ L, Cohen AM, Fenech AL, Montelpare WJ.The aim of this research was to determine the impact of heat stress on cell differentiation in an equine mesenchymal stem cell model (EMSC) through the application of heat stress to primary EMSCs as they progressed through the cell specialization process. A proteomic analysis was performed using mass spectrometry to compare relative protein abundances among the proteomes of three cell types: progenitor EMSCs and differentiated osteoblasts and adipocytes, maintained at 37 °C and 42 °C during the process of cell differentiation. A cell-type and temperature-specific response to heat stress was ...
Catalán J, Yánez-Ortiz I, Tvarijonaviciute A, González-Aróstegui LG, Rubio CP, Barranco I, Yeste M, Miró J.The objective of this study was to determine the relationship of enzymatic (superoxide dismutase, SOD; glutathione peroxidase, GPX; catalase, CAT; and paraoxonase type 1, PON1) and non-enzymatic antioxidants (measured in terms of: Trolox equivalent antioxidant capacity, TEAC; cupric-reducing antioxidant capacity, CUPRAC; and ferric-reducing ability of plasma, FRAP), as well as the oxidative stress index (OSI) in seminal plasma (SP) with the resilience of horse sperm to freeze-thawing. Twenty-one ejaculates (one per individual) were collected and split into two aliquots: the first was used to h...
Bhutia WD, Gupta S, Rani R, Batra K, Sethi K, Kumar S, Kumar R.Trypanosoma evansi is a causative agent of chronic wasting and fatal disease of livestock and wild animals known as surra. In this study, repurposing approach based on drug target was used to investigate the efficacy of kinase inhibitors (Barasertib-HQPA, BAR and Palbociclib isethionate, PAL) and protease inhibitors (Z-pro-prolinal, Z-PRO and Leupeptin hemisulphate, LEU) against T. evansi in HMI-9 medium. BAR, PAL and Z-PRO exhibited IC values of 13.52 µM, 0.6375 µM and 63.20 µM against T. evansi in terms of growth inhibition, in the contrary, LEU failed to exhibit a significant growth i...
Adalbert R, Cahalan S, Hopkins EL, Almuhanna A, Loreto A, Pór E, Körmöczy L, Perkins J, Coleman MP, Piercy RJ.Neurological disorders are prevalent in horses, but their study is challenging due to anatomic constraints and the large body size; very few host-specific in vitro models have been established to study these types of diseases, particularly from adult donor tissue. Here we report the generation of primary neuronal dorsal root ganglia (DRG) cultures from adult horses: the mixed, dissociated cultures, containing neurons and glial cells, remained viable for at least 90 days. Similar to DRG neurons in vivo, cultured neurons varied in size, and they developed long neurites. The mitochondrial movem...
Malek G, Richard H, Beauchamp G, Laverty S.Focal bone microcracks with osteoclast recruitment and bone lysis, may reduce fracture resistance in racehorses. As current imaging does not detect all horses at risk for fracture, the discovery of novel serum biomarkers of bone resorption or osteoclast activity could potentially address this unmet clinical need. The biology of equine osteoclasts on their natural substrate, equine bone, has never been studied in vitro and may permit identification of specific biomarkers of their activity. Objective: (1) Establish osteoclast cultures on equine bone, (2) Measure biomarkers (tartrate resistant ac...
Nogueira BG, Pereira RR, Bitencourt JL, Milan B, Reis WVA, Junior MV, Acácio BR, Sampaio BFB, I L Souza M.The objective of the experiment was to evaluate the effect of the addition of different concentrations of the antioxidants Coenzyme Q10 (CoQ10) and melatonin to equine semen freezing diluent, alone or in combination, during the cryopreservation process. Twenty ejaculates (n = 5 stallions) were divided in groups: Control (C, without the addition of antioxidants), melatonin 0.75 mM (MEL1), melatonin 1.5 mM (MEL2), CoQ10 40 μg/mL (Q1), CoQ10 200 μg/mL (Q2), and CoQ10 40 μg/mL+ 0.75 mM melatonin (Q1 +MEL1). Q1 and Q2 groups demonstrated intact plasma membrane and high mitochondrial me...
Alvarado TD, Elghandour MMMY, Ekanem NJ, Alcala-Canto Y, Velázquez AE, Pacheco EBF, Purba RAP, Salem AZM.The present study was conducted to investigate the aqueous extracts of Azadirachta indica (AZN), Cnidoscolus angustidens (CNA), and their combination (MIX) at dosages of 0-, 0.6-, 1.2-, and 1.8- mL for their ability to reduce greenhouse gases and fermentation profiles in an in vitro study using horse feces and a nutrient-dense diet (as substrate). The quantity of greenhouse gas and fermentation profiles were determined in in vitro incubation for 48 h. Extracts of AZN, CNA, and MIX reduced total gas production of the incubated and degraded substrates in a dose-dependent and time-dependent manne...
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...
Tessier L, Bienzle D, Williams LB, Koch TG.Multipotent mesenchymal stromal cells (MSC) have attracted interest for their cytotherapeutic potential, partly due to their immunomodulatory abilities. The aim of this study was to test the robustness of our equine cord blood (CB) MSC isolation protocol, to characterize the CB-MSC before and after cryopreservation, and to evaluate their immunosuppressive phenotype. We hypothesized that MSC can be consistently isolated from equine CB, have unique and reproducible marker expression and in vitro suppress lymphoproliferation. Preliminary investigation of constitutive cytoplasmic Toll-like recepto...
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...
Manivong S, Cullier A, Audigié F, Banquy X, Moldovan F, Demoor M, Roullin VG.The burden of osteoarthritis (OA), one of the major causes of functional disabilities in humans and animals, continues to increase worldwide while no disease-modifying OA drugs (DMOADs) that either slow down or reverse disease progression have been made available. Here, we provide a brief overview of recent advances in: designing new OA drug delivery approaches, focusing on lubrication-based biomaterials and drug delivery systems, such as hydrogels, liposomes, dendrimers, micro- and nanoparticles; using either large (horse) or small (zebrafish) relevant animal models to evaluate new therapeuti...
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...
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...
Ishihara A, Shields KM, Litsky AS, Mattoon JS, Weisbrode SE, Bartlett JS, Bertone AL.This study evaluated healing of equine metatarsal osteotomies and ostectomies in response to percutaneous injection of adenoviral (Ad) bone morphogenetic protein (BMP)-2, Ad-BMP-6, or beta-galactosidase protein vector control (Ad-LacZ) administered 14 days after surgery. Radiographic and quantitative computed tomographic assessment of bone formation indicated greater and earlier mineralized callus in both the osteotomies and ostectomies of the metatarsi injected with Ad-BMP-2 or Ad-BMP-6. Peak torque to failure and torsional stiffness were greater in osteotomies treated with Ad-BMP-2 than Ad-B...
Cabezas J, Rojas D, Navarrete F, Ortiz R, Rivera G, Saravia F, Rodriguez-Alvarez L, Castro FO.Adult stromal mesenchymal stem cells (MSCs) have been postulated as responsible for cell renewal in highly and continuously regenerative tissues such as the endometrium. MSCs have been identified in the endometrium of many species including humans, rodents, pets and some farm animals, but not in horses. The objective of this work was to isolate such cells from the endometrium of mares and to compare their main biological attributes with horse adipose-derived MSCs. Here we successfully isolated and characterized endometrial MSCs (eMSCs) from mares. Said cells showed fibroblast-like morphology, ...
Ortega Ferrusola C, González Fernández L, Salazar Sandoval C, Macías García B, Rodríguez Martínez H, Tapia JA, Peña FJ.In order to evaluate to what extent the changes that occur during cryopreservation involve the mitochondrial permeability transition pore (PT-pore), a specific inhibitor was used during the cryopreservation process of stallion spermatozoa. Four ejaculates from each of 7 stallions were frozen using a standard protocol. Before freezing, each ejaculate was split into three subsamples. The first was supplemented with 2.5 microM bongkrekic acid (BA) and the second with 5 microM BA. The third subsample served as control. The BA significantly reduced the percentage of spermatozoa depicting active cas...
González-Fernández L, Macías-García B, Loux SC, Varner DD, Hinrichs K.Protein tyrosine phosphorylation (PY) is a hallmark of sperm capacitation. In stallion sperm, calcium inhibits PY at pH 7.8). These results show for the first time a role for calcium/calmodulin-dependent kinases in PRKA-dependent sperm PY; a non-PRKA-dependent pathway regulating sperm PY; and the apparent involvement of the FAK family of protein tyrosine kinases downstream in both pathways.
Quintero-Moreno A, Miró J, Teresa Rigau A, Rodríguez-Gil JE.The aim of this study was to test the presence of separate sperm subpopulations, with specific motility characteristics, in stallion ejaculates by using a computer-assisted semen motility analysis (CASA) system. Motility data were analyzed with a hierarchical clustering of variables based on a correlation or covariance matrix to select like parameters of sperm motility descriptors that better explain the kinetics of spermatozoa. The statistical analyses clustered the whole motile sperm population in both fresh and 24 h stored ejaculates into four separate groups. There were significant differe...
da Silva CM, Macías-García B, Miró-Morán A, González-Fernández L, Morillo-Rodriguez A, Ortega-Ferrusola C, Gallardo-Bolaños JM, Stilwell G....Lipid peroxidation (LPO) has been claimed as a major factor involved in stallion damage during storage or cryopreservation. Because melatonin is a well-known potent antioxidant, the aim of the present study was to investigate the effect of melatonin during in vitro incubation. Furthermore, we investigated the presence of specific melatonin receptors (MT1 and MT2) using specific polyclonal antibodies and western blotting. Stallion spermatozoa were incubated up to 3 hr at 37°C in the presence of different concentrations of melatonin (0, 50 pm, 100 pm, 200 pm, or 1 μm). At the beginning and at ...
Álvarez-Narváez S, Berghaus LJ, Morris ERA, Willingham-Lane JM, Slovis NM, Giguere S, Cohen ND.The practice of prophylactic administration of a macrolide antimicrobial with rifampin (MaR) to apparently healthy foals with pulmonary lesions identified by thoracic ultrasonography (i.e., subclinically pneumonic foals) is common in the United States. The practice has been associated epidemiologically with emergence of R. equi resistant to MaR. Here, we report direct evidence of multi-drug resistance among foals treated with MaR. In silico and in vitro analysis of the fecal microbiome and resistome of 38 subclinically pneumonic foals treated with either MaR (n = 19) or gallium maltolate (...
O'Callaghan DJ, Hyde JM, Gentry GA, Randall CC.Infection of exponential-phase suspension cultures of mouse fibroblast cells (L-M) with equine abortion virus (EAV) resulted in inhibition of cell growth and marked alterations in host metabolic processes. The synthesis of deoxyribonucleic acid (DNA) and ribonucleic acid was inhibited within 4 hr after infection and was suppressed by more than 90% by the time of maximal virus replication (14 to 18 hr). The overall rate of protein synthesis, however, was similar in uninfected and virus-producing cells as determined by measurements of net protein and isotope incorporation. The time course of vir...
Hotham WE, Thompson C, Szu-Ting L, Henson FMD.Osteoarthritis (OA) in the horse is an economic and welfare issue and there are no current disease modifying drugs available. Stem cells have been suggested as a therapeutic intervention for OA, originally on the basis of their regenerative capacity. However, it is hypothesised that mesenchymal stem cells (MSC) exert their effects via paracrine factors including the production of extracellular vesicles that can themselves recapitulate the MSC effects in the joint. Objective: To isolate extracellular vesicles from bone marrow MSC and investigate their anti-inflammatory effects on chondrocytes. ...
Menendez MI, Clark DJ, Carlton M, Flanigan DC, Jia G, Sammet S, Weisbrode SE, Knopp MV, Bertone AL.To evaluate healing of surgically created large osteochondral defects in a weight-bearing femoral condyle in response to delayed percutaneous direct injection of adenoviral (Ad) vectors containing coding regions for either human bone morphogenetic proteins 2 (BMP-2) or -6. Methods: Four 13mm diameter and 7mm depth circular osteochondral defects were drilled, 1/femoral condyle (n=20 defects in five ponies). At 2 weeks, Ad-BMP-2, Ad-BMP-6, Ad-green fluorescent protein (GFP), or saline was percutaneously injected into the central drill hole of the defect. Quantitative magnetic resonance imaging (...
Alhassan A, Govind Y, Tam NT, Thekisoe OM, Yokoyama N, Inoue N, Igarashi I.The sensitivity of LAMP, PCR and in vitro culture methods for the detection of Theileria equi and Babesia caballi was evaluated using tenfold serially diluted culture parasites. On day 1 post-culture, both T. equi and B. caballi parasites could only be observed at 1% parasite dilution from the in vitro culture method, whereas LAMP could detect up to 1 x 10(-3)% of both T. equi and B. caballi parasite dilutions, whilst PCR could detect 1 x 10(-3)% T. equi and 1 x 10(-1)% B. caballi parasite dilutions. On day 7 post-culture, the detection limit for T. equi and B. caballi in the in vitro culture ...
Deschene K, Céleste C, Boerboom D, Theoret CL.Exuberant granulation tissue (EGT), a fibrotic healing disorder resembling the human keloid, occurs almost exclusively in limb wounds of horses and may be caused in part by a relative state of hypoxia within the wound. Objective: The objectives of this study were therefore to (1) assess the effects of hypoxia on equine dermal fibroblast (EDF) proliferation and apoptosis, (2) study the effects of hypoxia on the expression of key extracellular matrix (ECM) associated proteins and determine if such effects are dependent on hypoxia-inducible factor (HIF), and (3) determine if EDFs from the body or...
Dhar M, Neilsen N, Beatty K, Eaker S, Adair H, Geiser D.Two studies report variability in proliferation and limited adipocyte differentiation of equine peripheral blood-derived adult mesenchymal stem cells, thus casting doubt on their adipogenic potential. Peripheral blood can be a valuable source of adult mesenchymal stem cells if cell culture conditions permissive for their adherence, proliferation and differentiation are defined. Hyperbaric oxygen treatment has been reported to mobilise haematopoietic progenitor stem cells into the peripheral blood in humans and mice, but similar experiments have not been done in horses. Objective: To optimise c...
Ferris RA, McCue PM, Borlee GI, Loncar KD, Hennet ML, Borlee BR.In this study, we evaluated the ability of the equine clinical treatments N-acetylcysteine, EDTA, and hydrogen peroxide to disrupt in vitro biofilms and kill equine reproductive pathogens (Escherichia coli, Pseudomonas aeruginosa, or Klebsiella pneumoniae) isolated from clinical cases. N-acetylcysteine (3.3%) decreased biofilm biomass and killed bacteria within the biofilms of E. coli isolates. The CFU of recoverable P. aeruginosa and K. pneumoniae isolates were decreased, but the biofilm biomass was unchanged. Exposure to hydrogen peroxide (1%) decreased the biofilm biomass and reduced the CF...
Malisauskas M, Zamotin V, Jass J, Noppe W, Dobson CM, Morozova-Roche LA.The calcium-binding equine lysozyme has been found to undergo conversion into amyloid fibrils during incubation in solution at acidic pH. At pH 4.5 and 57 degrees C, where equine lysozyme forms a partially unfolded molten globule state, the protein forms protofilaments with a width of ca. 2 nm. In the absence of Ca(2+) the protofilaments are present as annular structures with a diameter of 40-50 nm. In the presence of 10 mM CaCl(2) the protofilaments of equine lysozyme are straight or curved; they can assemble into thicker threads, but they do not appear to undergo circularisation. At pH 2.0, ...
Zembowicz A, Knoepp SM, Bei T, Stergiopoulos S, Eng C, Mihm MC, Stratakis CA.Pigmented epithelioid melanocytoma (PEM) is a recently described entity comprising most cases previously described as "animal-type melanoma" and epithelioid blue nevus (EBN) occurring in patients with the multiple neoplasia syndrome Carney complex (CNC). Mutations of the protein kinase A regulatory subunit type 1alpha (R1alpha) (coded by the PRKAR1A gene) are found in more than half of CNC patients. In this study, we investigated whether PEM and EBN are related at the molecular level, and whether changes in the PRKAR1A gene status and the expression of the R1alpha protein may be involved in th...
McClain AK, McCarrel TM.Platelet-rich plasma (PRP) is a therapeutic biologic that is used for treatment of musculoskeletal pathologies in equine athletes. Due to the expense of PRP kits, and the volumes obtained, freezing aliquots for future dosing is common. Aliquots of PRP are also commonly frozen for later analysis of growth factor concentrations in in vitro research. A variety of freezing methods are used and storage duration until analysis is often not reported. The optimal frozen storage conditions and duration to maintain concentrations of commonly measured growth factors and enzymes in PRP are unknown. Our ob...
Esteves CL, Sheldrake TA, Dawson L, Menghini T, Rink BE, Amilon K, Khan N, Péault B, Donadeu FX.Mesenchymal stem/stromal cells (MSCs) have been used in human and equine regenerative medicine, and interest in exploiting their potential has increased dramatically over the years. Despite significant effort to characterize equine MSCs, the actual origin of these cells and how much of their native phenotype is maintained in culture have not been determined. In this study, we investigated the relationship between MSCs, derived from adipose tissue (AT) and bone marrow (BM), and pericytes in the horse. Both pericyte (CD146, NG2, and αSMA) and MSC (CD29, CD90, and CD73) markers were detected in ...
Malisauskas M, Ostman J, Darinskas A, Zamotin V, Liutkevicius E, Lundgren E, Morozova-Roche LA.In amyloid diseases, it is not evident which protein aggregates induce cell death via specific molecular mechanisms and which cause damage because of their mass accumulation and mechanical properties. We showed that equine lysozyme assembles into soluble amyloid oligomers and protofilaments at pH 2.0 and 4.5, 57 degrees C. They bind thioflavin-T and Congo red similar to common amyloid structures, and their morphology was monitored by atomic force microscopy. Molecular volume evaluation from microscopic measurements allowed us to identify distinct types of oligomers, ranging from tetramer to oc...
Merchant KA, Thompson DE, Xu QH, Williams RB, Loring RF, Fayer MD.Two-dimensional (2D) infrared vibrational echoes were performed on horse heart carbonmonoxymyoglobin (MbCO) in water over a range of temperatures. The A(1) and A(3) conformational substates of MbCO are found to have different dephasing rates with different temperature dependences. A frequency-frequency correlation function derived from molecular dynamics simulations on MbCO at 298 K is used to calculate the vibrational echo decay. The calculated decay shows substantial agreement with the experimentally measured decays. The 2D vibrational echo probes protein dynamics and provides an observable ...
Hillmann A, Ahrberg AB, Brehm W, Heller S, Josten C, Paebst F, Burk J.Multipotent mesenchymal stromal cells (MSCs) have gained tremendous attention as potential therapeutic agents for the treatment of orthopedic diseases. Promising results have been obtained after application of MSCs for treatment of tendon and joint disease in the equine model, making it appear favorable to use these results as a basis for the translational process of the therapy. However, while the horse is considered a highly suitable model for orthopedic diseases, knowledge is lacking regarding the level of analogy of equine MSCs and their human counterparts. Therefore, the aim of this study...
Cordeiro Rde A, de Oliveira JS, Castelo-Branco Dde S, Teixeira CE, Marques FJ, Bittencourt PV, Carvalho VL, Bandeira Tde J, Brilhante RS, Moreira JL....Candida tropicalis has been associated with invasive candidiasis, being the first or second most common non-Candida albicans Candida species isolated in humans with candidemia and candiduria, as well as being frequently isolated from healthy animals. This study aimed to characterize C. tropicalis isolates (n = 64) obtained from several animal species regarding antifungal susceptibility and production of virulence factors. The isolates were obtained from the microbiota of healthy animals (goats, n = 25; sheep, n = 6; psittacines, n = 14; rheas, n = 6; horses, n = 2; sirenians, n = 5; shrimp, n ...
de Vries AA, Glaser AL, Raamsman MJ, de Haan CA, Sarnataro S, Godeke GJ, Rottier PJ.Equine arteritis virus (EAV) is an enveloped, positive-stranded RNA virus belonging to the family Arteriviridae of the order Nidovirales. The unsegmented, infectious genome of EAV is 12,704 nt in length [exclusive of the poly(A) tail] and contains eight overlapping genes that are expressed from a 3'-coterminal nested set of seven leader-containing mRNAs. To investigate the importance of the overlapping gene arrangement in the viral life-cycle and to facilitate the genetic manipulation of the viral genome, a series of mutant full-length cDNA clones was constructed in which either EAV open readi...
Schuurman W, Gawlitta D, Klein TJ, ten Hoope W, van Rijen MH, Dhert WJ, van Weeren PR, Malda J.If chondrocytes from the superficial, middle, and deep zones of articular cartilage could maintain or regain their characteristic properties during in vitro culture, it would be feasible to create constructs comprising these distinctive zones. Objective: Zone-specific characteristics of zonal cell populations will disappear during 2-dimensional expansion but will reappear after 3-dimensional redifferentiation, independent of the culture technique used (alginate beads versus pellet culture). Methods: Controlled laboratory study. Methods: Equine articular chondrocytes from the 3 zones were expan...
Branly T, Bertoni L, Contentin R, Rakic R, Gomez-Leduc T, Desancé M, Hervieu M, Legendre F, Jacquet S, Audigié F, Denoix JM, Demoor M, Galéra P.Articular cartilage presents a poor capacity for self-repair. Its structure-function are frequently disrupted or damaged upon physical trauma or osteoarthritis in humans. Similar musculoskeletal disorders also affect horses and are the leading cause of poor performance or early retirement of sport- and racehorses. To develop a therapeutic solution for horses, we tested the autologous chondrocyte implantation technique developed on human bone marrow (BM) mesenchymal stem cells (MSCs) on horse BM-MSCs. This technique involves BM-MSC chondrogenesis using a combinatory approach based on the associ...
