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.
Vigolo V, Gautier C, Falomo ME, Aurich C.The use of microfluidic technology is increasing in artificial reproduction technologies: With a small amount of semen, it allows for the selection of sperm with the best characteristics of kinetics, morphology and chromatin integrity. The ZyMot Multi (850 μl) is the most popular device of ZyMot Fertility Inc. To date, it was proven to be a valid instrument for sperm selection for in vitro fertilization and intracytoplasmic sperm injection in men. The aim of this study was to test the efficacy of the ZyMot Multi (850 μl) for stallion semen. Frozen-thawed semen from 15 stallions that were...
Sedykh SE, Purvinsh LV, Burkova EE, Dmitrenok PS, Ryabchikova EI, Nevinsky GA.Exosomes are nanovesicles with a 40-150 nm diameter and are essential for communication between cells. Literature data suggest that exosomes obtained from different sources (cell cultures, blood plasma, urea, saliva, tears, spinal fluid, milk) using a series of centrifugations and ultracentrifugations contain hundreds and thousands of different protein and nucleic acid molecules. However, most of these proteins are not an intrinsic part of exosomes; instead, they co-isolate with exosomes. Using consecutive ultracentrifugation, gel filtration, and affinity chromatography on anti-CD9- and anti-C...
Baker ME, Lee S, Clinton M, Hackl M, Castanheira C, Peffers MJ, Taylor SE.Osteoarthritis of the equine distal interphalangeal joint is a common cause of lameness. MicroRNAs from biofluids are promising biomarkers and therapeutic candidates. Synovial fluid samples from horses with mild and severe equine distal interphalangeal joint osteoarthritis were submitted for small RNA sequencing. The results demonstrated that miR-92a was downregulated in equine synovial fluid from horses with severe osteoarthritis and there was a significant increase in COMP, COL1A2, RUNX2 and SOX9 following miR-92a mimic treatment of equine chondrocytes in monolayer culture. This is the first...
Gaitskell-Phillips G, Martín-Cano FE, da Silva-Álvarez E, Tapia JA, Silva A, Gil MC, Ortega-Ferrusola C, Peña FJ.Although recent research has addressed the impact of cryopreservation on the stallion sperm proteome, studies addressing the stallion sperm phosphoproteome are lacking. In the present study, the data set of proteomes of fresh and cryopreserved spermatozoa were reanalyzed, showing that cryopreservation caused significant changes in the phosphoproteome. The phosphoproteins reduced most significantly by cryopreservation were Ca2+binding tyrosine phosphorylation regulated, protein kinase cAMP-activated catalytic subunit beta (CABYR), mitochondria eating protein (SPATA18), A kinase anchoring protei...
Rooney MF, Neto NGB, Monaghan MG, Hill EW, Porter RK.Thoroughbred racehorse performance is largely influenced by a major quantitative trait locus at the () gene which determines aptitude for certain race distances due to a promoter region insertion mutation influencing functional phenotypes in skeletal muscle. To develop an system for functional experiments we established three novel equine skeletal muscle cell lines reflecting the variation in phenotype associated with genotype (CC/II, CT/IN and TT/NN for SNP g.66493737C > T/SINE insertion 227 bp polymorphism). Primary equine skeletal muscle myoblasts, isolated from Thoroughbred horse , we...
Dordas-Perpinyà M, Yanez-Ortiz I, Sergeant N, Mevel V, Bruyas JF, Catalán J, Delehedde M, Briand-Amirat L, Miró J.ProAKAP4 is the precursor of AKAP4 (A-kinase Anchor protein 4), the main structural protein of the fibrous sheath of sperm. The amount of proAKAP4 reflects the ability of spermatozoa to maintain the flagellum activity and functionality up to the site of fertilization and is positively correlated with progressive motility in several mammalian species. The aim of this study was to investigate the relationship between proAKAP4 concentration with horse sperm motility descriptors and spermatic motile subpopulations. For this purpose, a total of 48 ejaculates from 13 different stallions were analyze...
Ferrer MS, Hurley DJ, Norton N, Ellerbrock RE.The objectives of this study were to evaluate the ability of five diagnostic tests to detect polymorphonuclear cells (PMNs) in stallion semen, and to determine the concentration of PMNs that affects sperm motility. We hypothesized that all tests have diagnostic value, and even low concentrations of PMNs affect motility. One ejaculate was obtained from six stallions. Aliquots of 50 × 10 purified sperm were incubated, in triplicate, with six concentrations of purified PMNs: 1) no PMNs, 2) 0.25 × 10 PMN/ml, 3) 0.5 × 10 PMN/ml, 4) 2.5 × 10 PMN/ml, 5) 5 × 10 PMN/ml, 6) 10 × 10 PMN...
Pusterla N, Vaala W, Bain FT, Chappell DE, Craig B, Schneider C, Barnett DC, Gaughan E, Papich MG.Equine protozoal myeloencephalitis (EPM) has remained a devastating neurological disease of the Americas, especially in young performance horses. Prophylactic treatment strategies with diclazuril have shown to reduce seroprevalence and titer levels to Sarcocystis neurona in healthy horses continuously exposed to the apicomplexan parasite. The goal of this study was to determine if the FDA-labeled dose of 1 mg/kg of 1.56% diclazuril (Protazil) given once weekly to healthy adult horses would achieve steady-state concentrations in plasma known to be inhibitory to S. neurona in cell culture. Five ...
Freitas NPP, Silva BDP, Bezerra MRL, Pescini LYG, Olinda RG, Salgueiro CCM, Nunes JF, Martins JAM, Neto SG, Martins LT.This study investigated platelet-rich plasma (PRP) and adipose stem cell-conditioned medium (ASC-CM) use as a strategy to accelerate tissue healing. Platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) were quantified in fresh and freeze-dried PRP and ASC-CM, and a stability test was performed in the freeze-dried samples (90 and 180 days of storage). A cell proliferation test was performed using equine mesenchymal stem cell culture in reconstituted PRP gel mesh after freeze-drying. In vivo PRP, ASC-CM applications, or their association were performed in induced w...
Morse-Wolfe B, Bleach E, Kershaw C.Stallion spermatozoa are typically cryopreserved at 200 to 300 million sperm/ml; however recent advances such as intracytoplasmic sperm injection (ICSI) requires only one spermatozoon, wasting many, after thawing a whole straw. Cryopreserving at concentrations less than the current standard or refreezing thawed spermatozoa could maximize the use of genetically valuable animals and reduce waste. This investigation aimed to identify if lowering the sperm concentration for cryopreservation affected post-thaw quality after one and two freeze-thaw cycles. Nine ejaculates were collected from three f...
Hernández-Avilés C, Ramírez-Agámez L, Varner DD, Love CC.The current study determined the effect of the egg-yolk (phospholipid source) level (egg yolk [20% EY] vs. skim-milk + egg yolk [SM + 2% EY]), cryoprotectant (glycerol [Gly] vs. glycerol + methylformamide [Gly + MF]), and pre-freeze cooling rate (-0.1 vs. -1 vs. -5 °C/min) on post-thaw stallion sperm quality. In Experiment 1, ejaculates (n = 27) from 9 stallions (3 ejaculates each) with varied sperm quality (High, Average, or Low) were frozen in EY-Gly, SMEY-Gly, EY-Gly + MF, or SMEY-Gly + MF extenders. Sperm in each group were cooled from 22° to 5°C using either -0.1 °C/min or -1 ...
Aguiar CS, Barros CHSC, Machado WM, Allaman IB, Leite AO, Barbosa LP, Snoeck PPDN.The aim of this study was to evaluate the association of different concentrations of Trolox and the addition of a fixed concentration of DHA in the freezing of semen of Mangalarga Marchador stallions. To that end, 16 ejaculates were frozen in the following extenders: E1) BotuCrio (BC; Control); E2) BC + 50 ngml DHA + 30 µM Trolox (BCDHA30T); E3) BC + 50 ngml DHA + 40 µM Trolox (BCDHA40T); E4) BC + 50 ngml DHA + 50 µM Trolox (BCDHA50T). All the tested extenders were similar in preserving different kinematic parameters, cell functional integrity, compacted DNA, and high and intermediate mitoc...
Hainisch EK, Jindra C, Reicher P, Miglinci L, Brodesser DM, Brandt S.Equine sarcoids are common, locally aggressive skin tumors induced by bovine papillomavirus types 1, 2, and possibly 13 (BPV1, BPV2, BPV13). Current in vitro models do not mimic de novo infection. We established primary fibroblasts from horse skin and succeeded in infecting these cells with native BPV1 and BPV2 virions. Subsequent cell characterization was carried out by cell culture, immunological, and molecular biological techniques. Infection of fibroblasts with serial 10-fold virion dilutions (2 × 10-20 virions) uniformly led to DNA loads settling at around 150 copies/cell after four pass...
Junco M, Iglesias LE, Sagüés F, Zegbi S, Guerrero I, Saumell CA.In horses, the nematodes of the Strongylidae family are the most important due to their prevalence and pathogenicity. Sanitary plans include parasite control based on chemical anthelmintics. Among these, the benzimidazole compounds have been used since the 1960s to control the nematode Strongylus vulgaris. Its inappropriate use resulted in the development of resistance in parasites with a shorter biological cycle, such as the small strongyles. Currently, the genera that make up this group show widespread resistance to all chemical treatments available in veterinary medicine, except for macrocy...
James S, Daffy J, Cook J, Samiric T.Fluoroquinolone antibiotics are associated with increased risk of tendinopathy and tendon rupture, which can occur well after cessation of treatment. We have previously reported that the fluoroquinolone ciprofloxacin (CPX) reduced proteoglycan synthesis in equine tendon explants. This study aimed to determine the effects of CPX on proteoglycan catabolism and whether any observed effects are reversible. Equine superficial digital flexor tendon explant cultures were treated for 4 days with 1, 10, 100 or 300 µg/mL CPX followed by 8 days without CPX. The loss of [S]-labelled proteoglycans and che...
Brito LFC, Loomis PR, Klohonatz KM, Althouse GC.Neat stallion semen can contain a variety of microorganisms, some of which may impair sperm quality and/or cause infection of the mares' reproductive tract. For this reason, antibiotics are commonly added to semen extenders. A combination of gentamicin, tylosin, lincomycin and spectinomycin (GTLS) has been recommended for use, but there are no reports on the use of this mixture in equine semen extender. Penicillin and amikacin (PA) are safe for preserving sperm quality while effectively controlling bacterial growth in equine cooled stored semen, but data on frozen semen are scarce. Therefore, ...
Dobler D, Gerber M, Schmidts TM, Runkel F, Schlupp P.Human nail diseases, mostly caused by fungal infections, are common and difficult to treat. The development and testing of new drugs and drug delivery systems for the treatment of nail diseases is often limited by the lack of human nail material for permeation studies. Animal material is frequently used, but there are only few comparative data on the human nail plate, and there is neither a standardized test design nor a nail bed analogue to study drug uptake into the nail. In this study, a new permeation device was developed for permeation studies, and the permeation behavior of three model s...
Garland A, Wierenga C, McCrae P, Pearson W.The purpose of this study was to determine anti-inflammatory and/or chondroprotective effects of Equine Omega Complete (EOC) on cartilage explants stimulated with lipopolysaccharide (LPS). Explants were aseptically prepared from the intercarpal joints of 17 market-weight pigs and placed in culture at 37°C for a total of 120 hours. For the final 96 hours, explants were conditioned with a simulated digestion extract of EOC (0, 36 or 180 μL/mL), and for the final 48 hours explants were stimulated with LPS (0 or 15µg/mL). Media was removed and replaced every 24 hours. Samples from the final 48 ...
Caruso M, Shuttle S, Amelse L, Elkhenany H, Schumacher J, Dhar MS.Regenerative biological therapies using mesenchymal stem cells (MSCs) are being studied and used extensively in equine veterinary medicine. One of the important properties of MSCs is the cells' reparative effect, which is brought about by paracrine signaling, which results in the release of biologically active molecules, which in turn, can affect cellular migration and proliferation, thus a huge potential in wound healing. The objective of the current study was to demonstrate the in vitro and in vivo potentials of equine allogenic bone marrow-derived MSCs for wound healing. Equine bone marrow-...
Hernández-Avilés C, Ramírez-Agámez L, Varner DD, Love CC.During the fertilization process, the interaction between the sperm and the oocyte is mediated by a process known as acrosomal exocytosis (AE). Although the role of the sperm acrosome on fertilization has been studied extensively over the last 70 years, little is known about the molecular mechanisms that govern acrosomal function, particularly in species other than mice or humans. Even though subfertility due to acrosomal dysfunction is less common in large animals than in humans, the evaluation of sperm acrosomal function should be considered not only as a complementary but a routine test whe...
Thirumalapura NR, Livengood J, Beeby J, Wang W, Goodrich EL, Goodman LB, Erol E, Tewari D.Neorickettsia risticii, an obligate intracellular bacterium, is the causative agent of Potomac horse fever (PHF). Diagnosis of PHF is based on demonstration of serum antibodies, isolation of N. risticii, and/or detection of nucleic acid by a PCR assay. An existing real-time PCR assay targeting the N. risticii 16S rRNA has been validated using blood samples from horses with colitis, and snails; to our knowledge, the performance of the assay for other sample types has not been reported. We describe here a modification of the 16S rRNA gene assay by the addition of a set of primers and probe targe...
Depuydt E, Chiers K, Van Hecke L, Saunders J, Martens A, Pille F, Spaas JH.Injuries to equine tendons and ligaments are career-compromising, causing reduced performance and premature retirement. Promising treatment alternatives have been investigated in the field of mesenchymal stem cells (MSCs). In this study, the tissue adherence and protein expression of tenogenic primed mesenchymal stem cells (tpMSCs) after administration to ex vivo tendon and ligament explants is investigated. First, collagen type I (COL I) and smooth muscle actin (SMA) expression was assessed in cytospins prepared from native MSCs and tpMSCs. Second, equine superficial digital flexor tendon and...
Wotman KL, Chow L, Martabano B, Pezzanite LM, Dow S.Ocular surface squamous neoplasia (OSSN) is the major cause of corneal cancer in man and horses worldwide, and the prevalence of OSSN is increasing due to greater UVB exposure globally. Currently, there are no approved treatments for OSSN in either species, and most patients are managed with surgical excision or off-label treatment with locally injected interferon alpha, or topically applied cytotoxic drugs such as mitomycin C. A more broadly effective and readily applied immunotherapy could exert a significant impact on management of OSSN worldwide. We therefore evaluated the effectiveness of...
Pezzanite LM, Chow L, Strumpf A, Johnson V, Dow SW.Antimicrobial resistance and biofilm formation both present challenges to treatment of bacterial infections with conventional antibiotic therapy and serve as the impetus for development of improved therapeutic approaches. Mesenchymal stromal cell (MSC) therapy exerts an antimicrobial effect as demonstrated in multiple acute bacterial infection models. This effect can be enhanced by pre-conditioning the MSC with Toll or Nod-like receptor stimulation, termed activated cellular therapy (ACT). The purpose of this review is to summarize the current literature on mechanisms of antimicrobial activity...
Bagge J, Berg LC, Janes J, MacLeod JN.Bone marrow (BM)- and adipose tissue (AT)-derived mesenchymal stromal cells (MSCs) have shown potential as cell-based therapies for cartilage and bone injuries and are used increasingly in human and veterinary practice to facilitate the treatment of orthopedic conditions. However, human and rodent studies have documented a sharp decline in chondrogenic and osteogenic differentiation potential with increasing donor age, which may be problematic for the important demographic of older orthopedic patients. The aim of this study was to identify the effect of donor age on the chondrogenic and osteog...
Catalán J, Martínez-Rodero I, Yánez-Ortiz I, Mateo-Otero Y, Bragulat AF, Nolis P, Carluccio A, Yeste M, Miró J.Follicular fluid is formed from the transudation of theca and granulosa cells in the growing follicular antrum. Its main function is to provide an optimal intrafollicular microenvironment to modulate oocyte maturation. The aim of this study was to determine the metabolomic profile of preovulatory follicular fluid (PFF) in jennies. For this purpose, PFF was collected from 10 follicles of five jennies in heat. Then, PFF samples were analysed by nuclear magnetic resonance (NMR) and heteronuclear single quantum correlation (2D H/C HSQC). Our study revealed the presence of at least 27 metabolites i...
Flores Bragulat AP, Ortiz I, Catalán J, Dorado J, Hidalgo M, Losinno L, Dordas-Perpinyà M, Yánez-Ortiz I, Martínez-Rodero I, Miró J, Gambini A.Equus members exhibit very divergent karyotype, genetic plasticity, and significant differences in their reproductive physiology. Despite the fact that somatic cell nuclear transfer and intracytoplasmic sperm injection (ICSI) has gained relevance in the last few years in horses, few reports have been published exploring ovum pick up (OPU) and in vitro maturation (IVM) of cumulus-oocyte complexes (COCs) in donkeys. Yet, some donkey species and breeds are considered endangered, and these assisted-reproductive technologies could help to preserve the genetic of valuable individuals. In this study...
Wang X, Asgenbaatar N, Shen Y, Yi M, Zhao B, Ren H, Davshilt T, Ulaangerel T, Wang M, Burenbaatar A, Tian S, Li B, Dugarjav M, Bou G.Proliferation of embryonic fibroblasts under the same cell culture conditions, hinny embryonic fibroblasts (HiEFs) was slower than horse embryonic fibroblast (HEFs), donkey embryonic fibroblasts (DEFs) and mule embryonic fibroblasts (MuEFs). The imprinted genes IGF2 and IGF2R are important for cell proliferation. Therefore, we investigated whether the slower proliferation of HiEFs is related to an aberrant gene expression of IGF2 or its receptors or genes influencing the expression of the IGF2 system. Results: Real-time polymerase chain reaction, immunofluorescence and cell starving experiment...
Mainguy-Seers S, Beaudry F, Fernandez-Prada C, Martin JG, Lavoie JP.Extracellular vesicles (EVs) contribute to intercellular communication through the transfer of their rich cargo to recipient cells. The EVs produced by LPS-stimulated neutrophils from healthy humans and horses increase airway smooth muscle (ASM) proliferation, but the roles of neutrophil EVs in asthma are largely unexplored. The aim of this study was to determine whether neutrophil-derived EVs isolated during the remission or exacerbation of asthma influence ASM proliferation differentially. Peripheral blood neutrophils were collected during remission and exacerbation in eight horses affected ...
Dechêne L, Colin M, Demazy C, Fransolet M, Niesten A, Arnould T, Serteyn D, Dieu M, Renard P.Osteoarthritis (OA) is a highly prevalent joint degenerative disease for which therapeutic treatments are limited or invasive. Cell therapy based on mesenchymal stem/stromal cells (MSCs) is therefore seen as a promising approach for this disease, in both human and horses. As the regenerative potential of MSCs is mainly conferred by paracrine function, the goal of this study was to characterize the secreted proteins of muscle-derived MSCs (mdMSCs) in an in vitro model of OA to evaluate the putative clinical interest of mdMSCs as cell therapy for joint diseases like osteoarthritis. An equine ost...
Ball BA, Vo AT, Baumber J.To characterize generation of reactive oxygen species (ROS) by equine spermatozoa. Methods: Multiple semen samples collected from 9 stallions. Methods: Equine spermatozoa were separated from seminal plasma on a discontinuous polyvinylpyrrolidone (PVP)-coated silica gradient and resuspended in a modified Tyrode albumin-lactate-pyruvate medium. Amount of hydrogen peroxide (H2O2) generated was assayed by use of a 1-step fluorometric assay, using 10-acetyl-3,7-dihydroxyphenoxazine as a probe for detection of H2O2 in a microplate assay format. Concentration of H2O2 was determined by use of a fluore...
Clark KC, Kol A, Shahbenderian S, Granick JL, Walker NJ, Borjesson DL.Mesenchymal stem cell (MSC) therapy is being increasingly used to treat dogs and horses with naturally-occurring diseases. However these animals also serve as critical large animal models for ongoing translation of cell therapy products to the human market. MSC manufacture for clinical use mandates improvement in cell culture systems to meet demands for higher MSC numbers and removal of xeno-proteins (i.e. fetal bovine serum, FBS). While serum-free media (SFM) is commercially available, its affects on MSC phenotype and immunomodulatory functions are not fully known. The objective of this study...
Rebordão MR, Carneiro C, Alexandre-Pires G, Brito P, Pereira C, Nunes T, Galvão A, Leitão A, Vilela C, Ferreira-Dias G.Besides the classical functions, neutrophils (PMNs) are able to release DNA in response to infectious stimuli, forming neutrophil extracellular traps (NETs) and killing pathogens. The pathogenesis of endometritis in the mare is not completely understood. The aim was to evaluate the in vitro capacity of equine PMNs to secrete NETs by chemical activation, or stimulated with Streptococcus equi subspecies zooepidemicus (Szoo), Escherichia coli (Ecoli) or Staphylococcus capitis (Scap) strains obtained from mares with endometritis. Ex vivo endometrial mucus from mares with bacterial endometritis wer...
Toribio RE, Kohn CW, Capen CC, Rosol TJ.Parathyroid hormone (PTH) is secreted by the chief cells of the parathyroid gland in response to changes in ionized calcium (Ca(2+)) concentrations. In this study, we measured PTH secretion, and PTH mRNA and calcium-sensing receptor (CaR) mRNA expression by equine parathyroid chief cells in vitro. We also evaluated the effects of interleukin (IL)-1beta, IL-6, and tumor necrosis factor (TNF)-alpha on PTH secretion, and PTH and CaR mRNA expression. The relationship between PTH and Ca(2+) was inversely related. PTH secretion decreased from 100% (day 0) to 13% (day 30). PTH mRNA expression decline...
Li X, Zhou SG, Imreh MP, Ahrlund-Richter L, Allen WR.Inner cell mass (ICM) cells were isolated immunosurgically from day 7-8 horse blastocysts and, after proliferation in vitro for 15-28 passages, three lines of cells were confirmed to be embryonic stem (ES) cells by their continued expression of alkaline phosphatase activity and their ability to bind antisera specific for the recognized stem cell markers, SSEA-1, TRA-1-60, TRA-1-81, and the key embryonic gene Oct-4. When maintained under feeder cell-free conditions in vitro, the three lines of cells differentiated into cells of ectodermal, endodermal, and mesodermal lineages. However, they did ...
Carpenter S, Chesebro B.Similar to other human and animal lentiviruses, equine infectious anemia virus (EIAV) is detectable in vivo in cells of the monocyte-macrophage lineage. Owing to their short-lived nature, horse peripheral blood macrophage cultures (HMC) are rarely used for in vitro propagation of EIAV, and equine dermal (ED) or kidney cell cultures, which can be repeatedly passed in vitro, are used in most studies. However, wild-type isolates of EIAV will not grow in these cell types without extensive adaptation, a process which may attenuate viral virulence. To better define the effect of host cell tropism on...
Whitworth DJ, Ovchinnikov DA, Sun J, Fortuna PR, Wolvetang EJ.In this study we have reprogrammed dermal fibroblasts from an adult female horse into equine induced pluripotent stem cells (equiPSCs). These equiPSCs are dependent only on leukemia inhibitory factor (LIF), placing them in striking contrast to previously derived equiPSCs that have been shown to be co-dependent on both LIF and basic fibroblast growth factor (bFGF). These equiPSCs have a normal karyotype and have been maintained beyond 60 passages. They possess alkaline phosphatase activity and express eqNANOG, eqOCT4, and eqTERT mRNA. Immunocytochemistry confirmed that they produce NANOG, REX1,...
Boere J, Malda J, van de Lest CHA, van Weeren PR, Wauben MHM.The use of extracellular vesicles (EVs) as a potential therapy is currently explored for different disease areas. When it comes to the treatment of joint diseases this approach is still in its infancy. As in joint diseases both inflammation and the associated articular tissue destruction are important factors, both the immune-suppressive and the regenerative properties of EVs are potentially advantageous characteristics for future therapy. There is, however, only limited knowledge on the basic features, such as numerical profile and function, of EVs in joint articular tissues in general and th...
Desancé M, Contentin R, Bertoni L, Gomez-Leduc T, Branly T, Jacquet S, Betsch JM, Batho A, Legendre F, Audigié F, Galéra P, Demoor M.Cartilage engineering is a new strategy for the treatment of cartilage damage due to osteoarthritis or trauma in humans. Racehorses are exposed to the same type of cartilage damage and the anatomical, cellular, and biochemical properties of their cartilage are comparable to those of human cartilage, making the horse an excellent model for the development of cartilage engineering. Human mesenchymal stem cells (MSCs) differentiated into chondrocytes with chondrogenic factors in a biomaterial appears to be a promising therapeutic approach for direct implantation and cartilage repair. Here, we cha...
Moncayo AC, Edman JD, Turell MJ.The effect of eastern equine encephalomyelitis (EEE) virus on the survivorship of Aedes albopictus (Skuse), Anopheles quadrimaculatus Say, and Coquillettidia perturbans (Walker) was determined experimentally. Female mosquitoes were allowed to feed on EEE viremic chicks, and survival rates were compared for infected and uninfected mosquitoes. Additionally, the survival of female Cq. perturbans and An. quadrimaculatus intrathoracically (i.t.) inoculated with EEE was compared with controls receiving diluent inoculations. Infection with EEE significantly reduced survival in Cq. perturbans compared...
Hartmann J, Lindau M.Exocytosis begins with formation of a small fusion pore which then expands allowing rapid release of granular contents. We studied the influence of cytoplasmic free Ca2+ ([Ca2+]i) on the conductance of the initial pore and on the dynamics of subsequent expansion in horse eosinophils using the patch clamp technique. The mean initial conductance is approximately 200 pS independent of [Ca2+]i. This value is close to that previously found in beige mouse mast cells. The pore subsequently expands by 18 nS/s at [Ca2+]i < 10 nM, by 40 nS/s at [Ca2+]i = 1.5 microM and by 90 nS/s at [Ca2+]i = 10 micr...
Paterson YZ, Rash N, Garvican ER, Paillot R, Guest DJ.Autologous mesenchymal stem cells (MSCs) are an attractive concept in regenerative medicine, but their mechanism of action remains poorly defined. No immune response is reported after in vivo injection of allogeneic equine MSCs or embryo-derived stem cells (ESCs) into the equine tendon, which may be due to the cells' immune-privileged properties. This study further investigates these properties to determine their potential for clinical application in other tissues. Methods: Mitomycin C-treated MSCs, ESCs, or differentiated ESCs (dESCs) were cultured with allogeneic equine peripheral blood mono...
Venezuelan equine encephalitis (VEE) virus causes an acute central nervous system infection in human and animals. Melatonin (MLT), minocycline (MIN) and ascorbic acid (AA) have been shown to have antiviral activities in experimental infections; however, the mechanisms involved are poorly studied. Therefore, the aim of this study was to determine the effects of those compounds on the viral titers, NO production and lipid peroxidation in the brain of mice and neuroblastoma cultures infected by VEE virus. Infected mouse (10 LD50) were treated with MLT (500 μg/kg bw), MIN (50mg/kg bw) or AA (50mg...
Baquero-Perez B, Kuchipudi SV, Nelli RK, Chang KC.Current methods of isolation of muscle satellite cells from different animal species are highly variable making inter-species comparisons problematic. This variation mainly stems from the use of different proteolytic enzymes to release the satellite cells from the muscle tissue (sometimes a single enzyme is used but often a combination of enzymes is preferred) and the different extracellular matrix proteins used to coat culture ware. In addition, isolation of satellite cells is frequently laborious and sometimes may require pre-plating of the cell preparation on uncoated flasks or Percoll cent...
Boerboom D, Pilon N, Behdjani R, Silversides DW, Sirois J.Steroidogenic factor-1 (SF-1, NR5A1a) is a member of the NR5A nuclear receptor subfamily and has been implicated as a key transcriptional regulator of all ovarian steroidogenic genes in vitro. To establish links between the expression of SF-1 and that of the steroidogenic genes in vivo, the objectives of this study were to clone equine SF-1 and examine the regulation of its messenger RNA (mRNA) in follicular cells during human CG (hCG)-induced ovulation. The equine SF-1 primary transcript was cloned by a combination of RT-PCR techniques. Results showed that the transcript was composed of a 5'-...
Galli C, Crotti G, Notari C, Turini P, Duchi R, Lazzari G.Embryo production by in vitro techniques has increased steadily over the years. For cattle where this technology is more advanced and is applied more, the number of in vitro produced embryos transferred to final recipients was over 30,000 in 1998. An increasing proportion of in vitro produced embryos are coming from oocytes collected from live donors by ultrasound-guided follicular aspiration (ovum pick up, OPU). This procedure allows the repeated production of embryos from live donors of particular value and is a serious alternative to superovulation. Ovum pick up is a very flexible technique...
Henson FM, Bowe EA, Davies ME.To identify the effect of fibroblastic growth factor-2 (FGF-2) on the intrinsic damage-repair response in articular cartilage in vitro. Methods: Articular equine cartilage explants, without subchondral bone, had a single impact load of 500 g applied from a height of 2.5 cm. Explants were then cultured in 0, 12, 25, 50 or 100 ng/ml FGF-2 for up to 28 days. Unimpacted discs served as controls for each time-point. Histological and immunohistochemical techniques were used to quantify and characterise the response of putative chondrocyte progenitor cells (CPC) to damage and FGF-2 treatment. Results...
Zerbe H, Schuberth HJ, Engelke F, Frank J, Klug E, Leibold W.In order to investigate pathogenic mechanisms of acute endometritis in cows and mares, we established an in vivo model in both species. Based on the results of an in vitro transmigration system, human recombinant interleukin-8 (rhIL-8; 1.25 microg per mare and 5 microg per cow in 50 ml phosphate-buffered saline) was used to attract polymorphonuclear neutrophil granulocytes (PMNs) into the uteri. Peak numbers of uterine neutrophils were attracted after 6h, in both cows and mares. On average, mares responded more sensitively than cows, with 15 times higher numbers of rhIL-8-attracted uterine neu...
Cheung WK, Working DM, Galuppo LD, Leach JK.Adipose stromal cells (ASC) are a promising alternative to progenitor cells from other tissue compartments because of their multipotential and capacity to retrieve significantly more progenitor cells. Initial cell samples are heterogeneous, containing a collection of cells that may contribute to tissue repair, but the sample becomes more homogeneous with each passage. Therefore, we hypothesized that the osteogenic potential of culture-expanded ASC would differ from uncultured ASC. Methods: Adipose tissue was collected from a yearling colt, and ASC were isolated and expanded using standard prot...
Weiss M, Horzinek MC.In equine dermis cells infected with Berne virus particles were first detected 10 h after infection. Virions were encountered in all parts of the Golgi system and, infrequently, in the rough endoplasmic reticulum. A unique form of budding of preassembled rigid tubular nucleocapsids was demonstrated. Masses of tubular nucleocapsids of a lesser diameter and electron density were prominent in the cytoplasm and the nucleus of infected cells. Within the Golgi system and cytoplasmic cisternae virions appeared as straight or slightly curved rods. Extremely long, aberrant virions (250 nm) were occasio...
Sherman AB, Gilger BC, Berglund AK, Schnabel LV.We aimed to determine and compare the in vitro effects of autologous bone marrow-derived mesenchymal stem cells (BM-MSCs) and mesenchymal stem cell supernatant (MSC-Sp) on the wound healing capacity of equine corneal fibroblasts using a scratch assay. Bone marrow aspirates and eyes were collected from normal, euthanized horses with subsequent isolation and culture of BM-MSCs and corneal stromal cells. Corneal stromal cells were culture-expanded in the culture well of transwell plates and then treated with an autologous BM-MSC suspension (dose: 2.5 × 105/100 μL media with the BM-MSCs con...
Bourzac C, Smith LC, Vincent P, Beauchamp G, Lavoie JP, Laverty S.There is a need to assess and standardise equine bone marrow (BM) mesenchymal stem cell (MSC) isolation protocols in order to permit valid comparisons between therapeutic trials at different sites. Objective: To compare 3 protocols of equine BM MSC isolation: adherence to a plastic culture dish (Classic) and 2 gradient density separation protocols (Percoll and Ficoll). Methods: BM aspirates were harvested from the sternum of 6 mares and MSCs isolated by all 3 protocols. The cell viability after isolation, MSC yield, number of MSCs attained after 14 days of culture and the functional characteri...
Orrego A, Issel CJ, Montelaro RC, Adams WV.Five serial passages of a cell-adapted strain of equine infectious anemia (EIA) virus were conducted in Shetland ponies. The 13 recipient ponies became agar-gel immunodiffusion test-positive by 25 days after they were inoculated. The virulence of the cell-adapted strain of EIA virus markedly increased through 3 serial passages, although individual variation within passages was high. The 1st serial-passage recipient remained afebrile through 200 days, whereas a febrile episode occurred about every 185, 44, 35, and 33 days in the 2nd, 3rd, 4th, and 5th serial-passage recipients, respectively. Se...
Sanchez-Garcia J, Fernandez-Funez P.Prion diseases are fatal neurodegenerative diseases caused by misfolding of the prion protein (PrP). These conditions affect humans and animals, including endemic forms in sheep and deer. Bovine, rodents, and many zoo mammals also developed prion diseases during the "mad-cow" epidemic in the 1980's. Interestingly, rabbits, horses, and dogs show unusual resistance to prion diseases, suggesting that specific sequence changes in the corresponding endogenous PrP prevents the accumulation of pathogenic conformations. In vitro misfolding assays and structural studies have identified S174, S167, and ...
Park J, Sutradhar BC, Hong G, Choi SH, Kim G.To compare the chondrotoxicity of bupivacaine, lidocaine, and mepivacaine in equine articular chondrocytes in vitro. Methods: Prospective, experimental study. Methods: Equine articular chondrocytes. Methods: Primary cultured equine chondrocytes were exposed to 0.5% bupivacaine, 2% lidocaine, or 2% mepivacaine for 30 or 60 minutes. After treatment, cell viability was evaluated by trypan blue exclusion and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay in a dose dependent manner. Apoptosis and necrosis of chondrocytes were analyzed with the double stain...
Denoix JM.Knowledge of the normal functional behavior and mechanical properties of the vertebral column is important to understand the pathogenesis of back lesions, to identify the clinical manifestations of back pain, and to ensure a rational approach to physical therapy. The purpose of this article is to present a synthesis of in vivo and in vitro data obtained from different but complementary investigations. Presently, in vivo studies are limited; few gait-specific kinematic and electromyographic investigations are in process. Higher stresses to reach the maximal range of intervertebral motion can be...
Derse D, Carvalho M, Carroll R, Peterlin BM.Transcriptional regulatory mechanisms found in lentiviruses employ RNA enhancer elements called trans-activation responsive (TAR) elements. These nascent RNA stem-loops are cis-acting targets of virally encoded Tat effectors. Interactions between Tat and TAR increase the processivity of transcription complexes and lead to efficient copying of viral genomes. To study essential elements of this trans activation, peptide motifs from Tats of two distantly related lentiviruses, equine infectious anemia virus (EIAV) and human immunodeficiency virus type 1 (HIV-1), were fused to the coat protein of b...
Jackson RE, Bormann CL, Hassun PA, Rocha AM, Motta EL, Serafini PC, Smith GD.To determine the effect of semen storage and separation techniques on sperm DNA fragmentation. Methods: Controlled clinical study. Methods: An assisted reproductive technology laboratory. Methods: Thirty normoozospermic semen samples obtained from patients undergoing infertility evaluation. Methods: One aliquot from each sample was immediately prepared (control) for the sperm chromatin dispersion assay (SCD). Aliquots used to assess storage techniques were treated in the following ways: snap frozen by liquid nitrogen immersion, slow frozen with Tris-yolk buffer and glycerol, kept on ice for 24...
Lovati AB, Corradetti B, Lange Consiglio A, Recordati C, Bonacina E, Bizzaro D, Cremonesi F.The aim of the study was to compare in vitro the stemness features of horse progenitor cells derived from bone marrow (BM-MSCs), amniotic fluid (AF-MSCs) and umbilical cord matrix (EUC-MSCs). It has been suggested that there may be a stem cell population within both umbilical cord matrix and amniotic fluid. However, little knowledge exists about the characteristics of these progenitor cells within these sources in the equine species. This study wanted to investigate an alternative and non-invasive stem cell source for the equine tissue engineering and to learn more about the properties of thes...
