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.
Reed SA, Johnson SE.Recovery from tendon injury is based on long periods of rest, which results in sub-optimal repair, often replacing tendon with fibrocartilage scar tissue. Recently, the use of stem cells in equine tendon repair has been attempted with variable success. The objective of this work was to determine the expression of scleraxis (scx) and tenascin C (TnC), two markers of tenocytes, in adipose (AdMSC) and umbilical cord blood (UCB) stem cells during culture on various substrata and in response to fibroblast growth factor (FGF) treatment. Equine UCB and AdMSC were cultured on gelatin-coated plasticwar...
Rijkenhuizen AB, Sommerauer S, Fasching M, Velde K, Peham C.There are no studies on the ideal ligature technique for the spermatic cord. Objective: To compare the maximal resistance pressure in the testicular artery and the maximal tensile forces to produce failure of 2 different ligature techniques used for ligation of the equine spermatic cord. Methods: The capabilities of 2 types of ligatures, single knot loop and double knot loop, were assessed using a pressure-resistance test in testicular arteries and with an in vitro mechanical evaluation of the tensile strength by single cycle-to-failure testing. Results: In the pressure-resistance test, the me...
Caliari SR, Harley BA.Biomolecular environments encountered in vivo are complex and dynamic, with combinations of biomolecules presented in both freely diffusible (liquid-phase) and sequestered (bound to the extracellular matrix) states. Strategies for integrating multiple biomolecular signals into a biomimetic scaffold provide a platform to simultaneously control multiple cell activities, such as motility, proliferation, phenotype, and regenerative potential. Here we describe an investigation elucidating the influence of the dose and mode of presentation (soluble, sequestered) of five biomolecules (stromal cell-de...
Scott S, Molesti E, Temperton N, Ferrara F, Böttcher-Friebertshäuser E, Daly J.Standard assays used for influenza serology present certain practical issues, such as inter-laboratory variability, complex protocols and the necessity for handling certain virus strains in high biological containment facilities. In an attempt to address this, avian and human influenza HA pseudotyped retroviruses have been successfully employed in antibody neutralization assays. In this study we generated an equine influenza pseudotyped lentivirus for serological screening. This was achieved by co-transfection of HEK293T cells with plasmids expressing the haemagglutinin (HA) protein of an H3N8...
Klumplerova M, Vychodilova L, Bobrova O, Cvanova M, Futas J, Janova E, Vyskocil M, Vrtkova I, Putnova L, Dusek L, Marti E, Horin P.Insect bite hypersensitivity (IBH) is an allergic dermatitis of horses caused by bites of insects. IBH is a multifactorial disease with contribution of genetic and environmental factors. Candidate gene association analysis of IBH was performed in a group of 89 Icelandic horses all born in Iceland and imported to Europe. Horses were classified in IBH-affected and non-affected based on clinical signs and history of recurrent dermatitis, and on the results of an in vitro sulfidoleukotriene (sLT)-release assay with Culicoides nubeculosus and Simulium vittatum extract. Different genetic markers wer...
Ferris RA, Dern K, Veir JK, Hawley JR, Lappin MR, McCue PM.To develop a broad-range 28S ribosomal DNA quantitative PCR (qPCR) assay for detection of fungal DNA in equine endometrial samples. Methods: 12 fungal samples from a clinical diagnostic laboratory and 29 samples obtained from 17 mares. Methods: The qPCR assay was optimized with commercially acquired fungal organisms and validated with samples obtained from the clinical diagnostic laboratory. Subsequently, 29 samples from 17 mares suspected of having fungal endometritis were evaluated via the qPCR assay and via traditional fungal culture and endometrial cytology. Amplicons from the qPCR assay w...
Lee CM, Kisiday JD, McIlwraith CW, Grodzinsky AJ, Frisbie DD.To develop an in vitro model of cartilage injury in full-thickness equine cartilage specimens that can be used to simulate in vivo disease and evaluate treatment efficacy. Methods: 15 full-thickness cartilage explants from the trochlear ridges of the distal aspect of the femur from each of 6 adult horses that had died from reasons unrelated to the musculoskeletal system. Methods: To simulate injury, cartilage explants were subjected to single-impact uniaxial compression to 50%, 60%, 70%, or 80% strain at a rate of 100% strain/s. Other explants were left uninjured (control specimens). All speci...
Tavela Ade O, de Araújo JV, Braga FR, da Silveira WF, Dornelas e Silva VH, Carretta Júnior M, Borges LA, Araujo JM, Benjamin Ldos A, Carvalho GR....The predatory nematophagous fungi have been used as an alternative control of gastrointestinal nematodes of domestic animals in natural and laboratory conditions. However, it is unclear if the association of some of these species could bring some kind of advantage, from a biological standpoint. In this context, this study consisted of two tests in vitro: in assay A, the assessment of the viability of the association of pellets in sodium alginate matrix containing the fungus Duddingtonia flagrans (AC001) and Monacrosporium thaumasium (NF34) and its predatory activity on infective larvae (L3) of...
Zaniboni A, Merlo B, Zannoni A, Bernardini C, Lavitrano M, Forni M, Mari G, Bacci ML.Sperm mediated gene transfer (SMGT) has been reported to be a powerful tool for producing transgenic livestock with applications in biomedicine and agriculture. To date, two studies have reported the production of transgenic equine embryo with, however, low efficiency in blastocyst production and transgene expression. The aim of the present study was to develop a method which allowed the efficient production of transgene-expressing embryos of the equine species through SMGT. To overcome problems due to in vitro fertilization (IVF) in horses, the ICSI procedure was associated with SMGT. The upt...
Ceusters JD, Mouithys-Mickalad AA, Franck TJ, Derochette S, Vanderplasschen A, Deby-Dupont GP, Serteyn DA.Horses are particularly sensitive to excessive inflammatory reaction where myeloperoxidase, a marker of inflammation, may contribute to mitochondrial dysfunctions. This study investigated the interaction between myeloperoxidase and cultured primary equine skeletal myoblasts, particularly its effect on mitochondrial respiration combined or not with anoxia followed by reoxygenation (AR). We showed that active myeloperoxidase entered into the cells, interacted with mitochondria and decreased routine and maximal respirations. When combined with AR, myeloperoxidase caused a further decrease of thes...
Tappenbeck K, Hoppe S, Hopster K, Kietzmann M, Feige K, Huber K.Postoperative ileus (POI) is a severe complication following small intestinal surgery in horses. It was hypothesised that prokinetic effects of lidocaine, the most commonly chosen drug for treatment of POI, resulted from drug integration into smooth muscle (SM) cell membranes, thereby modulating cell membrane properties. This would probably depend on the structural and lipophilic characteristics of lidocaine. To assess the influence of molecular structure and lipophilicity on prokinetic effects in vitro, the current study compared the effects of lidocaine with four structure-related drugs, nam...
Raudsepp T, McCue ME, Das PJ, Dobson L, Vishnoi M, Fritz KL, Schaefer R, Rendahl AK, Derr JN, Love CC, Varner DD, Chowdhary BP.Impaired acrosomal reaction (IAR) of sperm causes male subfertility in humans and animals. Despite compelling evidence about the genetic control over acrosome biogenesis and function, the genomics of IAR is as yet poorly understood, providing no molecular tools for diagnostics. Here we conducted Equine SNP50 Beadchip genotyping and GWAS using 7 IAR-affected and 37 control Thoroughbred stallions. A significant (PA and g.11040379C>A (p.166H>N) in exon 4 that were significantly associated with the IAR phenotype both in the GWAS cohort (n = 44) and in a large multi-breed cohort of 265 ho...
Haag KT, Magalhães-Padilha DM, Fonseca GR, Wischral A, Gastal MO, King SS, Jones KL, Figueiredo JR, Gastal EL.A Biopsy Pick-Up (BPU) method was tested to determine the feasibility of retrieving preantral follicles from mare ovaries in vivo. A total of 33 ovarian biopsy procedures were performed on 18 mares during the breeding season. Mares were 5 to 21 years old and biopsies were performed during the estrous and/or diestrous phase, as confirmed by transrectal ultrasonography. Follicles were mechanically isolated using a tissue chopper, counted, and classified as normal or abnormal and primordial or primary. Viability of isolated follicles was determined by Trypan Blue dye. A total of 256 biopsy attemp...
Caron JP, Gandy JC, Schmidt M, Hauptman JG, Sordillo LM.To compare the effects of triamcinolone acetonide (TA) and methylprednisolone acetate (MPA) on expression of selected chondrocyte genes in recombinant equine interleukin-1β (reIL-1β) stimulated articular cartilage explants. Methods: In vitro experiment. Methods: Horses (n = 6). Methods: Articular cartilage explants from 2- to 3- year-old horses were exposed to reIL-1β in the presence and absence of TA and MPA at 10(-7) and 10(-6) M. Resting levels of mRNA of anabolic and catabolic genes of chondrocyte origin were quantified using qPCR after 6- and 12-hour incubations. Genes of interest incl...
Hinrichs K.This paper reviews current equine assisted reproduction techniques. Embryo transfer is the most common equine ART, but is still limited by the inability to superovulate mares effectively. Immature oocytes may be recovered by transvaginal ultrasound-guided aspiration of immature follicles, or from ovaries postmortem, and can be effectively matured in vitro. Notably, the in vivo-matured oocyte may be easily recovered from the stimulated preovulatory follicle. Standard IVF is still not repeatable in the horse; however, embryos and foals can be produced by surgical transfer of mature oocytes to th...
Arndt JL, Pfau T, Day P, Pardoe C, Bolt DM, Weller R.Hoof testers are commonly used in equine practice. In this study, we determined the intraoperator and interoperator reliability of force application with hoof testers for different groups: experienced veterinarians, novices and farriers. For this purpose, we have developed and validated an instrumented hoof tester. Forces varied significantly between the different regions of the foot for experienced operators applying the highest forces to the heels, then the frog, then the toe, and the lowest forces to the quarters. Novices applied significantly more force to the toe versus the frog. Intraope...
Adams AA, Horohov DW.The weaning process of foals involves a period of considerable stress which likely contributes to an increased risk of infectious disease in these young horses. Mechanisms responsible for this heightened risk of infection remain unknown, although likely due to compromised cell-mediated immunity. Parapoxvirus ovis (PPVO), an immmunomodulator, has been shown to limit the severity of infectious disease outbreaks among horses and has been shown to enhance CMI responses. Thus, an objective of this study was to investigate the effect of PPVO therapy on cell-mediated immune (CMI) responses of abruptl...
Singer E, Garcia T, Stover S.The biomechanical circumstances that promote sagittal fracture of the equine proximal phalanx (P1) are poorly understood. In order to improve our understanding of equine metacarpophalangeal joint (MCPJ) biomechanics and potential aetiologies of sagittal P1 fractures, the study objectives were to quantify P1 bone strains, collateromotion and axial rotation during MCPJ extension under controlled loading circumstances. Unilateral limbs from six cadavers were instrumented with bone reference markers for measurement of P1 movement relative to third metacarpal bone positions during axial limb loadin...
Agrawal K, Ebel JG, Altier C, Bischoff K.The leaves of Acer rubrum (red maple), especially when wilted in the fall, cause severe oxidative damage to equine erythrocytes, leading to potentially fatal methemoglobinemia and hemolytic anemia. Gallic acid and tannins from A. rubrum leaves have been implicated as the toxic compounds responsible for red maple toxicosis, but the mechanism of action and toxic principle(s) have not been elucidated to date. In order to investigate further how red maple toxicosis occurs, aqueous solutions of gallic acid, tannic acid, and ground dried A. rubrum leaves were incubated with contents of equine ileum,...
Nolen-Walston RD.Large animal species in states of shock can require particularly high flow rates for volume resuscitation and the ability to deliver adequate volumes rapidly may be a rate-limiting step. The objective of this study was to determine the maximum flow rates of common combinations of IV catheter, extension set, and fluid administration sets. Methods: University veterinary teaching hospital. Methods: In vitro experimental study. Methods: Maximum flow rates were measured using combinations of 4 IV catheters (3 14-Ga and a single 10-Ga), 2 IV catheter extension sets (small bore and large bore), and 2...
Setterbo JJ, Chau A, Fyhrie PB, Hubbard M, Upadhyaya SK, Symons JE, Stover SM.Racetrack surface is a risk factor for racehorse injuries and fatalities. Current research indicates that race surface mechanical properties may be influenced by material composition, moisture content, temperature, and maintenance. Race surface mechanical testing in a controlled laboratory setting would allow for objective evaluation of dynamic properties of surface and factors that affect surface behavior. Objective: To develop a method for reconstruction of race surfaces in the laboratory and validate the method by comparison with racetrack measurements of dynamic surface properties. Methods...
Ahern BJ, Boston RC, Parente EJ.Mechanical evaluation of a novel alternate laryngoplasty system (ALPS). Methods: Experimental. Methods: Cadaveric horse larynges (n = 10). Methods: Arytenoid and cricoid cartilages were embedded for testing. A novel laryngoplasty system consisting of a metallic anchor, 2 stands of #2 suture material, and a metallic button were evaluated. A traditional laryngoplasty using a single strand of #5 Ethibond was evaluated in parallel. Constructs were evaluated in cyclic testing oscillating from 30 to 50 N for 3600 cycles. Constructs subsequently underwent a single cycle to failure. Distraction during...
De Schauwer C, van de Walle GR, Piepers S, Hoogewijs MK, Govaere JL, Meyer E, van Soom A.The therapeutic potential of mesenchymal stromal cells for cellular therapy has generated increasing interest in human as well as veterinary medicine. Considerable research has been performed on the cryopreservation of expanded mesenchymal stromal cells, but little information is available on the cryopreservation of the original mononuclear cell fraction. Objective: The present study describes a protocol to expand equine mesenchymal stromal cells after cryopreserving the mononuclear cells of umbilical cord blood. Methods: To this end, mononuclear cells were isolated from 7 umbilical cord blood...
Finlay M, Yuan Z, Morgan IM, Campo MS, Nasir L.Bovine papillomavirus type 1 infects not only cattle but also equids and is a causative factor in the pathogenesis of commonly occurring equine sarcoid tumours. Whilst treatment of sarcoids is notoriously difficult, cisplatin has been shown to be one of the most effective treatment strategies for sarcoids. In this study we show that in equine fibroblasts, BPV-1 sensitises cells to cisplatin-induced and UVB-induced apoptosis, a known cofactor for papillomavirus associated disease, however BPV-1 transformed fibroblasts show increased clonogenic survival, which may potentially limit the therapeut...
Morrell JM, Timoney P, Klein C, Shuck K, Campos J, Troedsson M.Several countries have adopted strategies for preventing and/or controlling equine viral arteritis based on vaccination and restricting the breeding activities of carrier stallions. However, in some cases, carrier stallions are only identified after they have transmitted virus to a mare. Therefore, a mechanism for separating virus from spermatozoa in the semen of carrier stallions would facilitate control measures for preventing disease transmission. In this study, the use of several modifications of single-layer centrifugation (SLC, SLC with an inner tube and double SLC) through Androcoll-E, ...
Sayasith K, Lussier J, Doré M, Sirois J.Little is known about the expression and regulation of epiregulin (EREG) and amphiregulin (AREG) in ovarian follicles of large monoovulatory animal species. To characterize the gonadotropin-dependent regulation of EREG and AREG mRNAs in equine follicles prior to ovulation, extracts were prepared from equine follicles collected during estrus between 0 and 39h post-hCG and corpora lutea obtained on day 8 of the estrous cycle (day 0=day of ovulation). Results from RT-PCR/Southern blot analyses showed that levels of EREG and AREG mRNAs were very low in follicles obtained at 0h but increased therea...
Ambrisko TD, Kabes R, Moens Y.In a previous study, the authors found a large bias (50%) for lithium (LiDCO) compared with thermodilution cardiac output measurement methods in ponies receiving i.v. infusions of xylazine, ketamine, and midazolam. This prompted the authors to examine the effect of drugs on the LiDCO sensor. Methods: Drugs and lithium were dissolved in 0.9% saline to produce the following solutions: saline, saline-lithium, saline-drug, and saline-drug-lithium. The drug concentrations were overlapping the range of clinical interest as estimated from the published literature. These 38°C solutions were pumped th...
Altamura G, Strazzullo M, Corteggio A, Francioso R, Roperto F, D'Esposito M, Borzacchiello G.Bovine papillomaviruses (BPVs) types 1 and 2 are the only known papillomaviruses able to jump the species. In fact, BPVs 1/2 induce neoplasia in their natural bovine host but infection is also associated to neoplastic skin lesions in equids termed sarcoids. The equine sarcoid is considered to be the most common equine cutaneous tumour worldwide for which no effective therapy is available. Very little is known about the molecular mechanisms underlying tumourigenesis, although genes contributing to sarcoid development have been identified. Several studies associate the development of cancer to t...
BMC research notesNovember 8, 2012
Volume 5 626 doi: 10.1186/1756-0500-5-626
Boudko SP, Ishikawa Y, Lerch TF, Nix J, Chapman MS, Bächinger HP.Hyperelastosis cutis is an inherited autosomal recessive connective tissue disorder. Affected horses are characterized by hyperextensible skin, scarring, and severe lesions along the back. The disorder is caused by a mutation in cyclophilin B. Results: The crystal structures of both wild-type and mutated (Gly6->Arg) horse cyclophilin B are presented. The mutation neither affects the overall fold of the enzyme nor impairs the catalytic site structure. Instead, it locally rearranges the flexible N-terminal end of the polypeptide chain and also makes it more rigid. Conclusions: Interactions of th...
BMC research notesNovember 8, 2012
Volume 5 626 doi: 10.1186/1756-0500-5-626
Boudko SP, Ishikawa Y, Lerch TF, Nix J, Chapman MS, Bächinger HP.Hyperelastosis cutis is an inherited autosomal recessive connective tissue disorder. Affected horses are characterized by hyperextensible skin, scarring, and severe lesions along the back. The disorder is caused by a mutation in cyclophilin B. Results: The crystal structures of both wild-type and mutated (Gly6->Arg) horse cyclophilin B are presented. The mutation neither affects the overall fold of the enzyme nor impairs the catalytic site structure. Instead, it locally rearranges the flexible N-terminal end of the polypeptide chain and also makes it more rigid. Conclusions: Interactions of th...
Fedde MR, Wood SC.When horses maximally exercise, splenic contraction and fluid movement out of the vascular compartment greatly increase the hematocrit (up to 0.70). We studied the in vitro rheological characteristics of blood from Thoroughbreds and Quarter Horses to determine the interaction of hematocrit and shear rate on apparent viscosity. We also compared the rheological characteristics of the blood before and after horses received furosemide, a drug commonly used to prevent exercise-induced pulmonary hemorrhage. Although the apparent viscosity of blood with a high hematocrit was high at low shear rates, ...
Miller MM, Sweeney CR, Russell GE, Sheetz RM, Morrow JK.To determine effects of blood contamination on western blot (WB) analysis of CSF samples for detection of anti-Sarcocystis neurona antibodies, and on CSF albumin and IgG concentrations, albumin quotient (AQ), and IgG index in horses. Methods: Prospective in vitro study. Methods: Blood with various degrees of immunoreactivity against S neurona was collected from 12 healthy horses. Cerebrospinal fluid without immunoreactivity against S neurona was harvested from 4 recently euthanatized horses. Methods: Blood was serially diluted with pooled nonimmunoreactive CSF so that final dilutions correspon...
Zintl A, Westbrook C, Mulcahy G, Skerrett HE, Gray JS.In order to explore the feasibility of producing a Babesia divergens live vaccine free of bovine material contaminants the parasite's ability to grow in human, sheep and horse erythrocytes and serum and serum-free medium was investigated. B. divergens was successfully maintained in bovine erythrocytes overlaid with serum-free HL-1 medium. Supplementation of the culture medium with bovine or sheep serum improved parasite growth (monitored by measuring parasitaemia and uptake of tritiated hypoxanthine) whereas horse and human sera reduced parasite growth. As assessed by Giemsa's stained and FITC...
Wagner B, Hillegas JM, Flaminio MJ, Wattrang E.Interferon-alpha (IFN-alpha) is a type I interferon that is secreted during the early stages of the innate immune response and is often induced upon infection with viral pathogens. IFN-alpha production affects multiple downstream events influencing both innate and adaptive immune responses. Here, we describe the expression of an equine rIFN-alpha/IgG4 fusion protein in mammalian cells. The anti-viral activity of rIFN-alpha/IgG4 was found to be 70-fold higher than that of a previously described IFN-gamma/IgG1 as tested by bioassay. The purified rIFN-alpha was subsequently used for the generatio...
Watts AE, Fubini SL, Vernier-Singer M, Golkowski C, Shin S, Todhunter RJ.To determine the effect of nonthermal plasma on Staphylococcus aureus, fibroblasts in monolayer culture, and clean and contaminated skin explants. Methods: Normal skin from euthanized horses. Methods: S aureus organisms were plated and treated with nonthermal plasma followed by bacterial culture to assess viability. Fibroblasts in monolayer culture and the epidermal and dermal surfaces of clean and S aureus-contaminated skin explants were treated. The effects of distance and duration on the response to treatment were compared. Results: Compared with controls, treatment with nonthermal plasma r...
Dini P, Bogado Pascottini O, Ducheyne K, Hostens M, Daels P.In the present study, we examined the effect of holding equine oocytes in Syngro embryo holding medium (EHM) overnight at either 4 °C, 17 °C, or 22 °C to 25 °C, on the time to maturation and developmental competence. We also examined the effect of placing denuded oocyte without extruded polar body back in maturation condition on subsequent maturation rate. In experiment 1, cumulus-oocyte complexes (COCs) were recovered postmortem and placed in EHM at 22 °C to 25 °C for 18 to 20 hours (OH) or placed directly in maturation (DM). The maturation rate was assessed after 22, 24, or 28 hou...
Bugno M, Jablonska Z, Tischner M, Klukowska-Rötzler J, Pienkowska-Schelling A, Schelling C, Slota E.The aim of our study was to diagnose aneuploidy in equine spermatozoa by multicolour fluorescence in situ hybridization (FISH) technique using specific molecular probes for equine sex chromosomes and autosome pair four (EGFR probe) labeled by different fluorochromes. These were applied on decondensed spermatozoa of four stallions. In total, more than 8800 sperm cells were examined. The total frequency of aberrant cells was 0.496%: aneuploidy of XX (0.135%), YY (0.023%), XY (0.102%), diploidy (0.057%), lack of sex chromosome (0.18%). In one stallion the ratio of normal X- and Y-bearing cells wa...
Bruschetta G, D'Ascola A, Medica P, Ferlazzo AM.Serotonin (5-hydroxytryptamine [5-HT]) may induce metabolic effects in different cell types, including leukocytes. In horses, 5-HT is involved in physiological and behavioral functions. Physical exercise is known to increase the amounts of 5-HT both in brain and periphery, but so far, the signal mechanism in response to exercise is not known. The aim of the study was to investigate the effect of a racehorse intensive training session on plasma 5-HT levels, serotonin transporter (SERT), 5HT, 5-HT, 5-HT, 5-HT receptor, interleukin-1 beta, and tumor necrosis factor-alpha expression in horse perip...
Bailey SR, Elliott J.Cumulative concentration response curves to 5-hydroxytryptamine (5-HT; 10(-10)-10(-4) mol/l) were constructed using isolated rings of equine digital, facial, tail and coronary arteries (endothelium intact). 5-HT was 17.7 and 41 times more potent as a vasoconstrictor of digital arteries than facial and tail arteries respectively. Removal of the endothelium increased the vasoconstrictor potency of 5-HT in the facial artery by 3.7-fold (P<0.05) but did not alter the sensitivity of digital arteries to 5-HT. Coronary arteries failed to contract to 5-HT. Coronary arteries pre-contracted with U440...
Bridges CG, Edington N.Intrinsic phagocytosis and killing of C. albicans by equine monocytes and polymorphonuclear leucocytes (PMN) was examined during Equid Herpesvirus 1 (EHV-1) (subtypes 1 or 2) and Adenovirus infections. Monocyte function increased during EHV-1 subtype 2 and Adenovirus infection. Conversely, there was an impairment of monocyte ingestion during EHV-1 subtype 1 infection which was ascribed to virus replication in peripheral blood mononuclear cells. PMN phagocytosis was not decreased in any of the infections studied. The raised levels of haemolytic complement in animals which subsequently developed...
Nemoto M, Kizaki K, Yamamoto Y, Oonuma T, Hashizume K.In vitro cell studies might be a useful tool for studying tendon pathology, but no suitable in vitro models exist for tendon disorders. The purpose of this study was to confirm whether cell scratch culture using tendon-derived fibroblasts can provide a suitable in vitro tendon disorder model. Extracellular matrix components were examined immunohistochemically in tendon tissue, and then their related gene expression levels were analyzed by conventional reverse transcription polymerase chain reaction (RT-PCR) and/or quantitative real-time RT-PCR in tissues and cells. Collagen type I (Col I), col...
Kings MA, de Weck AL.Pharmacological histamine releasing agents, such as compound 48/80, poly-L-lysine, adrenocorticotrophic hormone (ACTH; beta 1-24 available commercially as Synacthen), catecholamines, purine bases, etc., are well known to induce histamine release from rat peritoneal mast cells and mast cells of other species; and to a lesser extent from peripheral blood leucocytes. It is reported in this paper that several of these potent histamine-releasing agents induce little or no histamine release from horse leucocytes. In particular the calcium ionophore A 23187 induced no histamine release. On the other ...
Weiss R, Abel D, Scholtysik G, Straub R, Mevissen M.The effects of 5-hydroxytryptamine (5-HT), HTF 919, a new 5-HT(4) agonist, and the antagonists SB 203-186 (5-HT(4)) and tropisetron (5-HT(3)) on intestinal motility were tested in vitro on isolated preparations of horse ileum and pelvic flexure. Concentration-response curves were created by cumulative application of the agonists with or without preincubation of the antagonists. The 5-HT preparation induced a concentration-dependent contraction in equine ileum and pelvic flexure. The results indicate that 5-HT receptors are present in all parts of equine intestine investigated in this study. Tr...
Chopineau M, Martinat N, Galet C, Guillou F, Combarnous Y.Horse LH/CG (eLH/CG) and donkey LH/CG (dkLH/CG) are strictly LH-specific in their respective homologous species. However, both bind to the FSH receptors from non-equid species, whereas the zebra hormone (zbLH/CG) does not. The FSH/LH ratio of eLH/CG and of the alphadkbetae hybrid is about tenfold higher than that of dkLH/CG and of the alphaebetadk hybrid, showing that the betae subunit contains the structural features responsible for the high FSH activity of eLH/CG. Only six amino acid positions (51, 94, 95, 102, 103 and 106) are unique to the betae subunit when compared with the betadk and be...
Murata D, Yamasaki A, Matsuzaki S, Sunaga T, Fujiki M, Tokunaga S, Misumi K.Dedifferentiated fat (DFAT) cells have been shown to be multipotent, similar to mesenchymal stem cells (MSCs). In this study, we aimed to establish and characterize equine DFAT cells. Equine adipocytes were ceiling cultured, and then dedifferentiated into DFAT cells by the seventh day of culture. The number of DFAT cells was increased to over 10 million by the fourth passage. Flow cytometry of DFAT cells showed that the cells were strongly positive for CD44, CD90, and major histocompatibility complex (MHC) class I; moderately positive for CD11a/18, CD105, and MHC class II; and negative for CD3...
Boes KM, Sink CA, Camus MS, Werre SR.Method validation studies characterize the performance of new laboratory methods relative to established methods using quality guidelines in order to define the new method's performance characteristics and to identify differences that could influence data interpretation. We investigated the performance of an in-clinic dry chemistry analyzer (Catalyst One, IDEXX) for measuring 19 routine plasma biochemistry analytes in dogs, cats, and horses. We analyzed 2 levels of quality control material (QCM) in duplicate twice daily for 5 d to determine the coefficient of variation (CV), percent bias, ob...
Lagares MA, Petzoldt R, Sieme H, Klug E.The swelling of cells in a hypo-osmotic medium has been described as an important criterion for assessing the functional integrity of the sperm plasma membrane. The resistance of equine spermatozoa to osmolarity changes was studied by extending 98 semen samples collected from nine stallions in media at five osmolarities (300, 200, 150, 100, and 50 mOsmol l(-1)). The response of the cells was measured by the spermatocrit technique and eosin staining. Spermatocrit determines the increase on spermatozoal volume under hypo-osmotic conditions, a sign of functional integrity of sperm plasma membrane...
Sieme H, Oldenhof H.In modern livestock breeding, cryopreserved semen is routinely used for artificial insemination. Sperm cryopreservation secures future reproduction, and insemination doses can be easily shipped. Processing of semen for cryopreservation can be done with minimal efforts and relatively low costs. In this chapter we describe the entire cryopreservation process for stallion and bull sperm including dilution of sperm in primary and freezing extender, cooling and packaging in straws, freezing in liquid nitrogen vapor, cryogenic storage, and thawing. Special emphasis is given on preparation of commonl...
Roepstorff L, Johnston C, Drevemo S.The objective of this study was to validate a simple method to measure the mediolateral expansion of the heels and to apply this method in an in vivo experiment. It was also the aim to quantify the mediolateral expansion in different areas of the heel using an in vitro model. In the in vitro study, 5 right and 5 left distal forelimb specimens from 5 Standardbreds were mounted vertically in a custom-made compressive test machine. The heel expansion was measured using optical kinematic analysis and a potentiometer system. Specimens were tested unshod, with frog pressure and with the weightbearin...
Even KM, Gaesser AM, Ciamillo SA, Linardi RL, Ortved KF.Joint injury often leads to cartilage damage and posttraumatic inflammation, which drives continued extracellular matrix degradation culminating in osteoarthritis. Mesenchymal stem cells (MSCs) have been proposed as a biotherapeutic to modulate inflammation within the joint. However, concerns have been raised regarding the immunogenicity of MSCs cultured in traditional fetal bovine serum (FBS) containing media, and the potential of xenogenic antigens to activate the immune system causing rejection and destruction of the MSCs. Xenogen-free alternatives to FBS have been proposed to decrease MSC ...
Zhang XY, Zhu FX, Olszewski MA, Robinson NE.The beta 2-agonists currently used as bronchodilators are racemic mixtures of R- and S-enantiomers. In the present study, we examined the effects of enantiomers of the beta 2-agonists albuterol and formoterol on acetylcholine (ACh) release from equine trachealis parasympathetic nerves. ACh release was evoked by electrical field stimulation (20 V, 0.5 ms, 0.5 Hz) and measured by high-performance liquid chromatography coupled with electrochemical detection. We also tested the effects of enantiomers of albuterol and formoterol on equine tracheal smooth muscle (TSM) contraction in response to exog...
Martoriati A, Lalmanach AC, Goudet G, Gérard N.A growing body of evidence suggests that the ovary is a site of inflammatory reactions, and thus, ovarian cells could represent sources and targets of the interleukin-1 (IL-1) system. The aim of the present work was to investigate the expression of IL-1alpha, IL-1beta, IL-1ra, IL-1R1, and IL-1R2 genes in equine cumulus cells and oocytes. Moreover, the influence of IL-1beta on in vitro maturation of cumulus-oocytes complexes (COCs) was examined. COCs were collected using ultrasound-guided follicular puncture in vivo. Oocytes and cumulus cells were isolated from preovulatory and subordinate foll...
Kim DS, Kim DW, Kim MY, Nam SH, Choi SH, Kim RN, Kang A, Kim A, Park HS.A conjoined gene is defined as one formed at the time of transcription by combining at least part of one exon from each of two or more distinct genes that lie on the same chromosome, in the same or opposite orientation, which translate independently into different proteins. We comparatively studied the extent of conjoined genes in thirteen genomes by analyzing the public databases of expressed sequence tags and mRNA sequences using a set of computational tools designed to identify conjoined genes on the same DNA strand or opposite DNA strands of the same genomic locus. The CACG database, avail...
Anderson SL, Townsend HGG, Singh B.OBJECTIVE To evaluate the effect of lipopolysaccharide (LPS) on apoptosis of equine neutrophils in vitro. SAMPLE Venous blood samples from 40 adult horses. PROCEDURES Neutrophils were isolated from blood samples and cultured with or without LPS from Escherichia coli O55:B5 for 12 or 24 hours. Neutrophil apoptosis was assessed by use of cytologic examination, annexin V and propidium iodide staining quantified with flow cytometry, coincubation with inducers of intrinsic and extrinsic apoptosis or a toll-like receptor (TLR) 4 inhibitor, and measurement of caspase-3, -8, and -9 activities. RESULTS...
Billinghurst RC, Buxton EM, Edwards MG, McGraw MS, McIlwraith CW.To develop an antibody that specifically recognizes collagenase-cleaved type-II collagen in equine articular cartilage. Methods: Cartilage specimens from horses euthanatized for problems unrelated to the musculoskeletal system. Methods: A peptide was synthesized representing the carboxy- (C-) terminus (neoepitope) of the equine type-II collagen fragment created by mammalian collagenases. This peptide was used to produce a polyclonal antibody, characterized by western analysis for reactivity to native and collagenase-cleaved equine collagens. The antibody was evaluated as an antineoepitope anti...
Liu N, Yu W, Guo X, Chen J, Xia D, Yu J, Li D.Lytic polysaccharide monooxygenases (LPMOs) belonging to the auxiliary activity 9 family (AA9) are widely found in aerobic fungi. These enzymes are O-dependent copper oxidoreductases that catalyze the oxidative cleavage of cellulose. However, studies that have investigated AA9 LPMOs of aerobic fungi in the herbivore gut are scare. To date, whether oxidative cleavage of cellulose occurs in the herbivore gut is unknown. Results: We report for the first time experimental evidence that AA9 LPMOs from aerobic thermophilic fungi catalyze the oxidative cleavage of cellulose present in the horse gut t...
Albihn A, Waelchli RO, Samper J, Oriol JG, Croy BA, Betteridge KJ.A novel xenogeneic transplantation approach was used to determine whether it is embryonic or maternal tissue that produces the material that gives rise to the mucin-like glycoprotein of the equine embryonic capsule. Endometrial biopsy samples and conceptuses from six mares at days 13-15 after ovulation were prepared as 1 mm(3) grafts of endometrium, trophoblast and capsule for transplantation, alone or in combination, into various sites in 88 immunodeficient (severe combined immunodeficient or RAG2/gamma(c) double mutant) mice. The overall recovery rate of grafts was over 50%, reaching 100% wi...
Seeber PA, Soilemetzidou SE, East ML, Walzer C, Greenwood AD.Direct collection of samples from wildlife can be difficult and sometimes impossible. Non-invasive remote sampling for the purpose of DNA extraction is a potential tool for monitoring the presence of wildlife at the individual level, and for identifying the pathogens shed by wildlife. Equine herpesviruses (EHV) are common pathogens of equids that can be fatal if transmitted to other mammals. Transmission usually occurs by nasal aerosol discharge from virus-shedding individuals. The aim of this study was to validate a simple, non-invasive method to track EHV shedding in zebras and to establish ...
Coli A, Nocchi F, Lamanna R, Iorio M, Lapi S, Urciuoli P, Scatena F, Giannessi E, Stornelli MR, Passeri S.The amnion is a particular tissue whose cells show features of multipotent stem cells proposed for use in cellular therapy and regenerative medicine. From equine amnion collected after the foal birth we have isolated MSCs (mesenchymal stem cells), namely EAMSCs (equine amnion mesenchymal stem cells), from the mesoblastic layer. The cells were grown in α-MEM (α-modified minimum essential medium) and the effect of EGF (epidermal growth factor) supplementation was evaluated. To assess the growth kinetic of EAMSCs we have taken into account some parameters [PD (population doubling), fold increas...
Schlusselhuber M, Guldbech K, Sevin C, Leippe M, Petry S, Grötzinger J, Giguère S, Cauchard J.The equine antimicrobial peptide eCATH1 previously has been shown to have in vitro activity against antibiotic-susceptible reference strains of Rhodococcus equi and common respiratory bacterial pathogens of foals. Interestingly, eCATH1 was also found to be effective in the treatment of R. equi infection induced in mice. The aim of this study was to assess the in vitro activity of eCATH1 against equine isolates of Gram-negative (Escherichia coli, Salmonella enterica, Klebsiella pneumoniae and Pseudomonas spp.) and Gram-positive (R. equi, Staphylococcus aureus) bacteria resistant to multiple cla...
