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.
Hosaka YZ, Takahashi H, Uratsuji T, Tangkawattana P, Ueda H, Takehana K.The aim of this study was to determine the characteristic differences in tendinocytes derived from tendons in the equine forelimb, superficial digital flexor tendon (SDFT), deep digital flexor tendon (DDFT) and common digital extensor tendon (CDET), in morphology, proliferation, collagen production ability and ability for synthesis of matrix metalloproteinases (MMPs). Significant differences were observed in cell number in vivo. The cellular number was largest in the SDFT and smallest in the CDET. The values of in vitro proliferation ratios and ability for synthesis of collagen and MMPs were l...
Radcliffe CH, Flaminio MJ, Fortier LA.Mesenchymal progenitor cells (MPCs) are often characterized using surface markers after expansion and treatment in culture. There are no studies directly comparing gene and protein markers in undifferentiated samples during the very early phases of culture. The goal of this study was to evaluate temporal gene and protein expression changes during establishment of equine MPC cultures. Bone marrow aspirate was obtained from 35 horses and processed by density gradient centrifugation. In freshly isolated bone marrow, mononuclear cells had variable expression of CD44, CD11a/CD18, CD90, and CD45RB c...
Andoh K, Kai K, Matsumura T, Maeda K.Cell lines originating from horses are necessary for isolation and propagation of equine herpesviruses (EHV). Although we established an equine-derived cell line, FHK-Tcl3, propagation ceased after fewer than 40 passages. In this study, FHK-Tcl3 cell propagation continued beyond 40 passages, achieving over 100 passages. FHK-Tcl3 cells were then cloned by limiting dilution at the 100th passage. Cloned cells were termed FHK-Tcl3.1. FHK-Tcl3.1 cells grew well and were propagated every 3 to 4 days by splitting 1:5. In addition, EHV-1, -2 and -4 showed a clear cytopathic effect (CPE) in FHK-Tcl3.1 ...
Ortega Ferrusola C, González Fernández L, Macías García B, Salazar-Sandoval C, Morillo Rodríguez A, Rodríguez Martinez H, Tapia JA, Peña FJ.The ability of stallion spermatozoa to produce nitric oxide (NO) before (fresh) and after freezing and thawing (FT) was evaluated by means of flow cytometry after loading the sperm suspension with the probe, 4,5-diaminofluorescenin diacetate. The presence of NO synthase (NOS) was investigated by Western blotting using anti-NOS1, anti-NOS3, or anti-universal NOS antibodies (Abs). While NO was detected both in fresh and FT sperm suspensions, its production increased after cryopreservation only when egg yolk was removed from the extender. Anti-NOS1 Ab intensively labeled a single band with an app...
Gobeil PA, Yuan Z, Gault EA, Morgan IM, Campo MS, Nasir L.Equine sarcoids are skin tumours of horses caused by infection with BPV-1 or 2. Maintenance and replication of the viral genome depend upon the viral proteins E1 and E2. We examined the effects of an E2 specific siRNA on E2 and E1 viral gene expression, viral load and cell growth in BPV-1 transformed sarcoid-derived cells. Transfection with E2-siRNA caused a reduction in E2 and E1 mRNA expression as well as viral load, growth inhibition and decreased anchorage-independent growth. siRNA treated cells showed significantly higher apoptosis rates than control cells. Thus sequence specific targetin...
Morrell JM, Johannisson A, Dalin AM, Rodriguez-Martinez H.The objective of the current study was to optimize the volumes of Androcoll-E and sperm sample used in various sizes of centrifuge tube to scale up single-layer centrifugation (SLC) for routine use in the field. Although sperm suspensions of equivalent quality were produced using Androcoll-E in small and large tubes, the sperm yield was much lower in the latter (P<0.001). In contrast, in 200-mL tubes (XL), the yields were approximately 25% higher than those for the small tubes. An increased volume (4.5 mL) of extended ejaculate in small tubes (SLC-Inc) or 15 to 18 mL extended ejaculate on 1...
Mugnier S, Dell'Aquila ME, Pelaez J, Douet C, Ambruosi B, De Santis T, Lacalandra GM, Lebos C, Sizaret PY, Delaleu B, Monget P, Mermillod P....The mechanism of fertilization remains largely enigmatic in mammals. Most studies exploring the molecular mechanism underlying fertilization have been restricted to a single species, generally the mouse, without a comparative approach. However, the identification of divergences between species could allow us to highlight key components in the mechanism of fertilization. In the pig, in vitro fertilization (IVF) and polyspermy rates are high, and spermatozoa penetrate easily through the zona pellucida (ZP). In contrast, IVF rates are low in the horse, and polyspermy is scarce. Our objective was ...
Wilhelm K, Darinskas A, Noppe W, Duchardt E, Mok KH, Vukojević V, Schleucher J, Morozova-Roche LA.Protein oligomeric complexes have emerged as a major target of current research because of their key role in aggregation processes in living systems and in vitro. Hydrophobic and charged surfaces may favour the self-assembly process by recruiting proteins and modifying their interactions. We found that equine lysozyme assembles into multimeric complexes with oleic acid (ELOA) at the solid-liquid interface within an ion-exchange chromatography column preconditioned with oleic acid. The properties of ELOA were characterized using NMR, spectroscopic methods and atomic force microscopy, and showed...
Scherzer J, Fayrer-Hosken RA, Aceves M, Hurley DJ, Ray LE, Jones L, Heusner GL.We evaluated combinations of two commercial semen extenders and three concentrations of glycerol to determine the combination that yielded the highest post-thaw sperm motility. Methods: A randomised 2 x 3 block design was used. Methods: Semen was collected from four stallions (6 collections per stallion). The sample was diluted with either a dried skim-milk glucose extender (EZ Mixin Original Formula) or a chemically defined, milk-free diluent (INRA 96), and each was used in combination with 2%, 3% or 4% glycerol in standard commercial freezing medium. Sperm motility was assessed by microscopy...
Espino-Solis GP, Calderon-Amador J, Calderon-Aranda ES, Licea AF, Donis-Maturano L, Flores-Romo L, Possani LD.A three-dimensional model of the alphaX I-domain of the horse integrin CD11c from dendritic cells provided information for selecting two segments of the primary structure for peptide synthesis. Peptide 1 contains 20 amino acids and peptide 2 has 17 amino acid residues. The first spans from position Thr229 to Arg248 of an alpha-helix segment of the structure, whereas peptide 2 goes from Asp158 to Phe174 and corresponds to an exposed segment of the loop considered to be the metal ion-dependent adhesion site. Murine polyclonal antisera against both peptides were generated and assayed in periphera...
Nagy A, Bodo G, Dyson SJ, Szabo F, Barr AR.Proximal diffusion of local anaesthetic solution after perineural anaesthesia may lead to the desensitisation of structures other than those intended. However, there is no evidence-based study demonstrating the potential distribution and diffusion of local anaesthetic solution after perineural analgesia in the distal limb. Objective: To document the potential diffusion of local anaesthetic solution using a radiopaque contrast model and to evaluate the influence of walking compared with confinement in a stable after injection. Methods: Radiopaque contrast medium was injected subcutaneously over...
Faleiros RR, Leise BB, Westerman T, Yin C, Nuovo GJ, Belknap JK.C-X-C motif ligand 1 (CXCL1) is an important chemokine of epithelial origin in rodents and humans. Objective: To assess in vivo and in vitro the regulation of CXCL1 in equine laminitis. Methods: Twenty adult horses. Methods: Real-time quantitative polymerase chain reaction (PCR) was used to assess expression of CXCL1 in samples of laminae, liver, skin, and lung from the black walnut extract (BWE) model of laminitis, and in cultured equine epithelial cells (EpCs). Tissue was obtained from control animals (CON, n = 5), and at 1.5 hours (early time point [ETP] group, n = 5), at the onset of leuko...
de Mattos Carvalho A, Alves AL, Golim MA, Moroz A, Hussni CA, de Oliveira PG, Deffune E.The purpose of this work was to isolate and cultivate mesenchymal stem cells (MSC) derived from equine adipose tissue and conduct cellular characterization with the following markers: CD90, CD44 and CD13. Adipose tissue collection was performed at the base of the horses' tails, followed by immediate isolation and cultivation of the MSC and posterior characterization by flow cytometry for the interspecies reaction test using mouse anti-rat CD90 monoclonal antibody (mAb), fluorescein isothiocyanate (FITC), and tests with specific mAb mouse anti-horse CD13 and mouse anti-horse CD44. The technique...
Ryan C, Giguère S, Hagen J, Hartnett C, Kalyuzhny AE.Peripheral blood mononuclear cells (PBMCs) were obtained from 6 foals <1 week of age, 6 foals between 3 and 4 months of age, and 10 adult horses. PBMCs were stimulated with concanavalin A (ConA) or calcium ionomycin-phorbol myristate acetate (CaI-PMA) and the frequency of interferon IFN-gamma and IL-4 secreting cells was measured using an equine-specific ELISPOT assay. The number of IFN-gamma secreting cells was significantly lower in both groups of foals than in adult horses regardless of the mitogen used for stimulation. The number of IFN-gamma secreting cells was significantly higher in ...
Schneberger D, Caldwell S, Suri SS, Singh B.Toll-like receptor 9 (TLR9) has been found to be the main receptor to respond to bacterial DNA in a wide variety of species. Recent work has shown that TLR9 is expressed in a diverse set of cells within the lung. However, much of this data has been centered on human and mouse cell culture lines or primary cultures and very little is known of TLR9 expression in intact lung, especially that of the horse. Here we show that TLR9 is expressed in the lungs of horses in a wide variety of cells. In particular, we note expression in pulmonary intravascular macrophages (PIMs), alveolar macrophages, bron...
Tyler CR, Filby AL, Bickley LK, Cumming RI, Gibson R, Labadie P, Katsu Y, Liney KE, Shears JA, Silva-Castro V, Urushitani H, Lange A, Winter MJ....Many factors have been considered in evaluations of the risk-benefit balance of hormone replacement therapy (HRT), used for treating menopausal symptoms in women, but not its potential risks for the environment We investigated the possible environmental health implications of conjugated equine estrogens (CEEs), the most common components of HRT, including their discharge into the environment, their uptake, potency, and ability to induce biological effects in wildlife. Influents and effluents from four U.K. sewage treatment works (STWs), and bile of effluent-exposed fish, were screened for six ...
Weaver MP, Shaw DJ, Munaiwa G, Fitzpatrick DP, Bellenger CR.The objectives of this study were to topographically map pressure distribution across the palmar surface of the navicular bone in response to forces applied by the deep digital flexor tendon (DDFT), and evaluate the effect of raising the heels in vitro . A rig was constructed to hold an equine cadaver limb and apply controlled pressure to the navicular bone from the DDFT. Pressure-sensitive paper was used to quantify and map the resultant pressure. Changes in response to heel wedges (5 degrees and 10 degrees ) were recorded. The areas subject to maximum pressure from the DDFT were: the central...
Cribb NC, Bouré LP, Hanna WJ, Akens MK, Mattson SE, Monteith GJ, Weese JS.To assess the antimicrobial elution characteristics, toxicity, and antimicrobial activity of amikacin-impregnated ferric-hyaluronate implants (AI-FeHAI) for amikacin delivery to the tarsocrural joint of horses. Methods: Experimental study. Methods: AI-FeHAI implants, equine cartilage, and synovium, and horses (n=6). Methods: In vitro study: Five AI-FeHAI were placed in saline solution with daily replacement until implant degradation. Eluent was tested for amikacin concentration and bioactivity. Synovial and cartilage explants were incubated in the presence or absence of AI-FeHAI for 72 hours a...
Kumar S, Kanwar S, Bansal V, Sehgal R.Diphtheria toxin has the capacity to block protein synthesis in cultured mammalian cells, and thus causing cell death. This capacity of diphtheria toxin was utilized for in-vitro neutralization test to determine antibody titer, using Vero cells, which have been found to be susceptible to diphtheria toxin. In the present study, a Vero cell assay was standardized and validated for potency estimation of diphtheria antitoxin serum (DATS). The results obtained by Vero cell assay were compared with in-vivo biological assay. High degree of correlation (+0.98) was found between in-vivo biological assa...
Zhang N, Ding S, Kolbanovskiy A, Shastry A, Kuzmin VA, Bolton JL, Patel DJ, Broyde S, Geacintov NE.The equine estrogens equilin (EQ) and equilenin (EN) are the active components in the widely prescribed hormone replacement therapy formulation Premarin. Metabolic activation of EQ and EN generates the catechol 4-hydroxyequilenin (4-OHEN) that autoxidizes to the reactive o-quinone form in aerated aqueous solutions. The o-quinones react predominantly with C, and to a lesser extent with A and G, to form premutagenic cyclic covalent DNA adducts in vitro and in vivo. To obtain insights into the structural properties of these biologically important DNA lesions, we have synthesized site-specifically...
Choi YH, Harding HD, Hartman DL, Obermiller AD, Kurosaka S, McLaughlin KJ, Hinrichs K.The reported patterns of trophectodermal expression of POU5F1 protein in blastocysts vary among species, and are possibly related to the differences in placental growth and function. This study investigated the pattern of embryonic POU5F1 expression in the horse, a species with delayed placental formation. Immature equine oocytes expressed POU5F1 protein in the cytoplasm and nucleus. Staining for POU5F1 protein in in vitro-produced (IVP) embryos decreased to day 5 of culture, then the nuclear staining increased to day 7. IVP day-7 to -11 blastocysts showed POU5F1 staining in nuclei throughout ...
Choi YH, Hartman DL, Fissore RA, Bedford-Guaus SJ, Hinrichs K.We evaluated the effect of different activation methods on blastocyst development after equine nuclear transfer. All activation treatments were followed by incubation in 2 mM 6-dimethylaminopurine for 4 h. In Experiment 1, reconstructed oocytes were injected with sperm extract for 0.1, 0.2, 0.4, 0.8, or 1.6 sec using a FemtoJet injection device, then treated with ionomycin. The blastocyst rate (9.8%) for 0.1-sec injection was significantly higher than that for 0.2 sec (0%) or 0.8 sec (1.4%). In Experiment 2, injection of murine PLCzeta cRNA before or after ionomycin treatment did not increase ...
Koch TG, Thomsen PD, Betts DH.A robust methodology for the isolation of cord blood-derived multipotent mesenchymal stromal cells (CB-MSCs) from fresh umbilical cord blood has not been reported in any species. The objective of this study was to improve the isolation procedure for equine CB-MSCs. Methods: Pre-culture separation of red and white blood cells was done using either PrepaCyte?-EQ medium or Ficoll-Paque? PREMIUM density medium. Regular FBS and MSC-qualified FBS were compared for their ability to support the establishment of putative primary MSC colonies. Conclusions: Our results indicate that PrepaCyte-EQ medium i...
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...
Capponi L, Schmitz A, Thormann W, Theurillat R, Mevissen M.To investigate cytochrome P450 (CYP) enzymes involved in metabolism of racemic and S-ketamine in various species and to evaluate metabolic interactions of other analgesics with ketamine. Methods: Human, equine, and canine liver microsomes. Methods: An analgesic was concurrently incubated with luminogenic substrates specific for CYP 3A4 or CYP 2C9 and liver microsomes. The luminescence signal was detected and compared with the signal for negative control samples. Ketamine and norketamine enantiomers were determined by use of capillary electrophoresis. Results: A concentration-dependent decrease...
Stewart AA, Barrett JG, Byron CR, Yates AC, Durgam SS, Evans RB, Stewart MC.To compare viability and biosynthetic capacities of cells isolated from equine tendon, muscle, and bone marrow grown on autogenous tendon matrix. Methods: Cells from 4 young adult horses. Methods: Cells were isolated, expanded, and cultured on autogenous cell-free tendon matrix for 7 days. Samples were analyzed for cell viability, proteoglycan synthesis, collagen synthesis, and mRNA expression of collagen type I, collagen type III, and cartilage oligomeric matrix protein (COMP). Results: Tendon- and muscle-derived cells required less time to reach confluence (approx 2 weeks) than did bone marr...
Gregg K, Polejaeva I.Prevention and regulation of equine infectious anemia virus (EIAV) disease transmission solely depend on identification, isolation, and elimination of infected animals because of lack of an effective vaccine. Embryo production via the somatic cell nuclear transfer (SCNT) technology uses oocytes collected mainly from untested animals, which creates a potential risk of EIAV transmission through infected embryos. The current review examines the risk of EIAV disease transmission through SCNT embryo production and transfer. Equine infectious anemia virus is a lentivirus from the family Retroviridae...
Glover ID, Henry GM, Townsend NB, Coles GC.The Stomacher is very widely used in food and medical research for extracting tissues. To determine whether nematode larvae were disrupted by the Stomacher, L3 larvae of Haemonchus contortus were homogenised for up to 40 min at full power but no larval disruption occurred. Therefore, tissue from the mucosa and submucosa of the caecum of horses collected from a licenced abattoir was treated to determine whether inhibited cyathostomin larvae could be extracted. The optimum time on full power for a 10-g sample was 20 min, and in three out of five caecal samples from different horses, significantl...
Hobbs SJ, Mather J, Rolph C, Richards J.Radial strain in normal hooves has been found to vary with strain gauge location, limb posture and sample limb but reported magnitudes were considered to be low. More accurate measurement of radial strain may enhance the understanding of hoof function. Objective: To explore in vitro radial hoof strain in relation other kinetic and kinematic variables that may be related. Methods: Five normal forelimbs were removed at the proximal articular surface of the third metacarpal bone (McIII). The limbs were loaded using a modified Instron test machine. Six calibrated infrared cameras captured movement...
Den Hartog SM, Back W, Brommer H, van Weeren PR.Insight into the loading pattern of the articular cartilage surface during the complete stride is important as biomechanical factors play a pivotal role in the pathogenesis of joint trauma and osteoarthritis (OA). Objective: To determine the loading pattern in the equine MCP articulation in vitro during simulated walk. Methods: Eight cadaveric limbs from mature Dutch Warmblood horses were loaded in a pneumatic loading device in 6 different positions (A1-A6). The pressure distribution on the articular surface of the proximal phalanx (P1) was measured at 7 sites (S1-7) using intra-articularly pl...
Mangal D, Uboh CE, Soma LR.Oxidative metabolites of arachidonic acid (AA) are implicated in inflammation. Thus, we evaluated cycloxygenases (COXs) and lipoxygenases (LOs) mediated metabolism of AA to eicosanoids in equine plasma. Eicosanoids were extracted from plasma by two liquid-liquid extraction (LLE) steps; first was by chloroform/isopropanol and second by methyl-tert-butyl ether. For identification and quantification of 25 eicosanoids, a highly specific, selective and sensitive stable isotope dilution liquid chromatography (LC) multiple reaction monitoring (MRM) mass spectrometric (MS) method was developed. To avo...
Orino K, Miura T, Muto S, Watanabe K.Canine and equine ferritin H and L subunit cDNA clones were obtained using reverse transcriptase-polymerase chain reaction (RT-PCR) and TA cloning from various tissues. Canine liver and spleen ferritin H subunit cDNA clones contained an open reading frame for the same 182-amino acid protein as that reported in canine brain ferritin H subunit cDNA although there were substitutions in the 3'-noncoding regions. Ferritin L subunit cDNA clones from canine liver, spleen, and kidney showed identical coding sequences encoding the 174-amino acid protein except for a single nucleotide substitution in ki...
Seo JP, Tsuzuki N, Haneda S, Yamada K, Furuoka H, Tabata Y, Sasaki N.A three dimensional scaffold is essential in mesenchymal stem cells (MSCs) delivery in cell-based therapy for facilitating cell adherence, migration, proliferation, and differentiation. The objectives of this study were to evaluate the possibility of β-tricalcium phosphate incorporated gelatin sponges (Gelatin/β-TCP sponge) as scaffolds for equine MSCs and to examine the effects of seeding density and seeding method on the proliferation of equine MSCs in the Gelatin/β-TCP sponges. Mononuclear cells and MSCs isolated from bone marrow were seeded into Gelatin/β-TCP sponges at different densi...
Iniguez P, Zientara S, Marault M, Machin IB, Hannant D, Cruciere C.A random hexapeptide fusion-phage library was screened to isolate phages that bind to antibodies present in horse sera positive for equine arteritis virus (EAV). Analysis of the peptide sequences displayed by isolated phages identified seven groups. 25% of the isolated phages used as antigens in an ELISA test were specifically recognised by a pool of sera which was positive for EAV in virus neutralisation test (VN). Five of these, when used as antigen in ELISA, detected greater than 50% of sera (n = 30) containing antibodies to EAV as detected by VN. When these five phages were pooled together...
Hussein H, Boyaka P, Dulin J, Russell D, Smanik L, Azab M, Bertone AL.Selective inhibition of Cathepsin K (CatK) has a promising therapeutic potential for diseases associated with bone loss and osseous inflammation, such as osteoarthritis, periodontitis, and osteoporosis. In horses, stress-related bone injuries are common and accompanied by bone pain and inflammation resulting in excessive bone resorption and periostitis. VEL-0230 is a highly selective inhibitor of CatK that significantly decreased bone resorption and increased bone formation biomarkers. The goal of this study was to demonstrate the presence of CatK in equine bone and a simultaneous influence on...
The measurement of cell proliferation and cell viability using 5'bromo-2'deoxy-uridine (BrdU) labelling has been described in several cell types and species. The aim of this study was to adapt this technique to equine embryos and to compare the index of DNA replication (S-phase) between equine and caprine embryos. Seventeen equine embryos were recovered at day 6.5 post-ovulation and 20 caprine embryos were recovered at day 7 after the onset of estrus. Equine embryos were incubated during 1h at 39 degrees C in PBS containing 1mM of BrdU. Embryos were then treated in 0.05% trypsin during 15 min ...
Ellse L, Burden F, Wall R.Equine pediculosis is a significant health and welfare issue, particularly in elderly and chronically debilitated animals. Currently infestation is controlled predominantly using topically applied pyrethroid insecticides, allowing limited scope for the rotation of drugs and increasing the risk of selection for resistance. Here the insecticidal efficacies of two pyrethroid-based products against the louse Bovicola (Werneckiella) ocellatus collected from donkeys were examined in vitro. The products were cypermethrin (Deosect™, Pfizer Ltd., 5% (w/v) cypermethrin, cutaneous spray) and permethrin...
Schmitz A, Thormann W, Moessner L, Theurillat R, Helmja K, Mevissen M.Ketamine, an injectable anesthetic and analgesic consisting of a racemic mixture of S-and R-ketamine, is routinely used in veterinary and human medicine. Nevertheless, metabolism and pharmacokinetics of ketamine have not been characterized sufficiently in most animal species. An enantioselective CE assay for ketamine and its metabolites in microsomal preparations is described. Racemic ketamine was incubated with pooled microsomes from humans, horses and dogs over a 3 h time interval with frequent sample collection. CE data revealed that ketamine is metabolized enantioselectively to norketamine...
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...
Li Y, Yao J, Chang M, Nikolic D, Yu L, Yager JD, Mesecar AD, van Breemen RB, Bolton JL.Catechol-O-methyltransferase (COMT) plays an important role in the inactivation of biologically active and toxic catechols. It has been shown that COMT is genetically polymorphic with a wild-type and variant form where a valine has been substituted with a methionine. Several, but not all, epidemiological studies have shown that women, homozygous with the variant form, have an increased risk of developing breast cancer. Previously, we showed that 4-hydroxyequilenin (4-OHEN), a cytotoxic/genotoxic equine catechol estrogen metabolite, is both a substrate of COMT and an irreversible inhibitor of t...
McD￮ LA, Anderson GI, Wright GM, Ryan DA.Bone cell cultures were evaluated to determine if osteogenic cell populations at different skeletal sites in the horse are heterogeneous. Osteogenic cells were isolated from cortical and cancellous bone in vitro by an explant culture method. Subcultured cells were induced to differentiate into bone-forming osteoblasts. The osteoblast phenotype was confirmed by immunohistochemical testing for osteocalcin and substantiated by positive staining of cells for alkaline phosphatase and the matrix materials collagen and glycosaminoglycans. Bone nodules were stained by the von Kossa method and counted....
Madden CR, Shih DS.Sequential passage of the tissue culture-adapted prototype strain of EIAV in fetal donkey dermal (FDD) cell cultures generated a virus stock which exhibits cytopathic effects in FDD cell cultures. In this study, the effects of the long terminal repeat (LTR) region on virus replication and cytopathogenicity were examined. The FDD-adapted virus LTR was found to contain a number of base pair mutations and a large insertion within the U3 region in comparison with the previously characterized LTR, lambda12. Transient gene expression studies showed that basal promoter activity, in FDD cell cultures,...
Meyers SA, Overstreet JW, Liu IK, Drobnis EZ.Mammalian sperm that have completed capacitation are capable of undergoing the acrosome reaction in response to a number of biological and chemical stimuli. In the present report, we have investigated the ability of progesterone to stimulate acrosome reactions of stallion sperm capacitated in vitro. Motile sperm were selected by a two-layer Percoll gradient centrifugation and were incubated in TALP medium modified by the 1:1 (v/v) addition of TEST-yolk medium for 5 hours at 39 degrees C, under 5% CO2 in humidified air. Sperm incubated in vitro in TALP-TEST medium had a higher percentage of acr...
Golke A, Cymerys J, Słońska A, Dzieciatkowski T, Chmielewska A, Tucholska A, Bańbura MW.Real-time cell electronic sensing (RT-CES) based on impedance measurements is an emerging technology for analyzing the status of cells in vitro. It allows label-free, real time monitoring of the biological status of cells. The present study was designed to assess dynamic data on the cell processes during equine herpesvirus type 1 (EHV-1) infection of ED (equine dermal) cells and primary murine neuronal cell culture. We have demonstrated that the xCELLigence system with dynamic monitoring can be used as a rapid diagnostic tool both to analyze cellular behavior and to investigate the effect of v...
Ortgies F, Klewitz J, Görgens A, Martinsson G, Sieme H.Reasons for low in vitro fertilisation rates in the horse include the difficulties in inducing capacitation and/or hyperactivation of stallion spermatozoa. The aim of this study was to analyse the effect of noncapacitating and capacitating modified Whitten's (MW) and modified Tyrode's medium (MT) and treatment with procaine (5 mmol), pentoxifylline (3.5 mmol) and trolox (120 mmol) on motility (CASA), capacitation, acrosomal status, viability and mitochondrial membrane potential of stallion spermatozoa (n = 4). While there was no influence of MW and MT on sperm motility, a significant increase ...
Johnson NL, Galuppo LD, Stover SM, Taylor KT.To compare screw insertion variables and pullout mechanical properties between AO 6.5-mm cancellous and 7.3-mm cannulated bone screws in foal femoral bone. Methods: A paired, in vitro mechanical study. Methods: Seven pairs of femora from immature (1-7 months) foals. Methods: The 6.5 cancellous and 7.3-mm cannulated screws were inserted at standardized proximal and distal metaphyseal, and mid-diaphyseal locations. Insertion torque, force, and time to drill, tap (6.5-mm cancellous), guide wire insertion (7.3-mm cannulated), and screw insertion were measured. Screw pullout properties (yield and f...
Goudet G, Belin F, Bézard J, Gérard N.The intrafollicular content of LH receptor, alpha-inhibin, and aromatase are known good indicators of follicular status. We investigated the amounts of these proteins in granulosa and cumulus cells in relation to oocyte competence for in vitro maturation, follicular growth, and estrous cycle stage in the mare. Follicular punctures were performed 34 h after an injection of crude equine gonadotropins, either during the follicular phase, at the end of the follicular phase, or during the luteal phase. The cumulus-oocyte complex, granulosa cells, and follicular fluid of follicles larger than 5 mm w...
Ferreira LVO, Kamura BDC, Oliveira JPM, Chimenes ND, Carvalho M, Santos LAD, Dias-Melicio LA, Amorim RL, Amorim RM.Schwann cells (SCs) are essential for the regenerative processes of peripheral nerve injuries. However, their use in cell therapy is limited. In this context, several studies have demonstrated the ability of mesenchymal stem cells (MSCs) to transdifferentiate into Schwann-like cells (SLCs) using chemical protocols or co-culture with SCs. Here, we describe for the first time the in vitro transdifferentiation potential of MSCs derived from equine adipose tissue (AT) and equine bone marrow (BM) into SLCs using a practical method. In this study, the facial nerve of a horse was collected, cut into ...
Van Erck E, Votion D, Kirschvink N, Genicot B, Lindsey J, Art T, Lekeux P.The objective of this paper was to determine if changes in ventilation patterns could influence the outcome of respiratory function measurements performed with our impulse oscillometry system (IOS) in horses. In a first study, IOS tests were performed in vitro on six isolated equine lungs. Lung inflation levels were controlled by modifying depressurisation inside an artificial thorax and different ventilation patterns were imposed. In a second in vivo study, transient variations in breathing pattern were evaluated both with the IOS and a current reference technique (CRT) in five healthy mature...
Clulow J, Gibb Z.The ability to store stallion spermatozoa between the events of semen collection and insemination has facilitated improved rates of gain in selective breeding programs by enabling the transport of spermatozoa. While cryopreservation is the only viable means of storing spermatozoa for long intervals, the higher costs and reduced fertility of cryopreserved spermatozoa have led to most breeders opting to use liquid stored spermatozoa. Stallion spermatozoa is commonly cooled during liquid storage (approximately 4-5 °C), and there has been an enormous body of research dedicated to development of ...
Cawdell-Smith AJ, Todhunter KH, Perkins NR, Bryden WL.Equine amnionitis and fetal loss (EAFL) is an unusual form of abortion in mid- to late-gestation mares, first identified in Australia in 2004. It has been shown that both whole processionary caterpillars (Ochrogaster lunifer) and their shed exoskeletons can induce abortion in mares during midgestation. These abortions exhibited gross pathology and bacteriology results consistent with field cases of EAFL. Objective: To determine whether exposure of mares to the shed exoskeletons of processionary caterpillars can induce abortion in the preplacentation (<35 days' gestation) and early plac...
Kwok WH, Leung GN, Wan TS, Curl P, Schiff PJ.Androsta-1,4,6-triene-3,17-dione (ATD) is an irreversible steroidal aromatase inhibitor and is marketed as a supplement. It has been reported to effectively reduce estrogen biosynthesis and significantly increase the levels of endogenous steroids such as dihydrotestosterone and testosterone in human. ATD abuses have been reported in human sports. Its metabolism in human has been studied, and the in vitro metabolic study of ATD in horses has been reported, however, little is known about its biotransformation and elimination in horses. This paper describes the in vitro and in vivo metabolism stu...
Petrov R, MacDonald MH, Tesch AM, Van Hoogmoed LM.To determine rate and degree of cooling for the superficial digital flexor tendon (SDFT) during a standard cryotherapy application in horses and evaluate in vitro effects of cooling on survival of tendon cells. Methods: 6 limbs of 5 adult horses and cultured cells obtained from SDFT of 3 adult horses during necropsy. Methods: In vivo data were acquired by use of a thermocouple temperature probe inserted into the SDFT of a forelimb of each standing sedated horse. After baseline temperatures were recorded, a commercial compression splint with circulating coolant was placed on each selected limb,...
Cehak A, Krägeloh T, Zuraw A, Kershaw O, Brehm R, Breves G.Fructo-oligosaccharides are commonly administered as prebiotics to horses in order to reduce the risk of disruption of microbial populations in the hindgut. Their microbial degradation to SCFA already begins in the stomach potentially resulting in increased gastric concentrations of SCFA such as butyric acid. The impact of butyric acid on the squamous mucosa is postulated to be detrimental, its effects on the glandular mucosa are yet unknown. Thus, the aim of this study was to determine the effects of butyric acid exposure on the functional integrity and morphology of the equine nonglandular a...
Armstrong S, Lees P.To examine effects of carprofen (enantiomers and a racemic mixture) on the metabolism of equine chondrocytes. Methods: Cartilage from clinically normal horses. Methods: Effects of carprofen on proteoglycan neosynthesis, glycosaminoglycan (GAG) release and prostaglandin (PG) E2 production by unstimulated chondrocyte monolayers and cartilage explants were examined, as were similar variables in monolayers and explants exposed to carprofen and recombinant human interleukin 1beta (IL-1). Carprofen (enantiomers and racemic mixture) was used alone or along with IL-1 on monolayers and explant cultures...
Baxter G.Endotoxin given in vivo has been shown to inhibit endothelial dependent relaxation, and augment adrenergic (norepinephrine) contractions in isolated palmar digital arteries of horses. A study, using tumor necrosis factor (TNF) in vitro, was performed to determine the possible cause of these vascular alterations. Palmar digital arteries were surgically removed from 6 horses under general anesthesia, cut into 4-mm vascular rings (4 segments/horse), suspended in tissue baths, and attached to force displacement transducers for measurement of vascular tension. Four in vitro treatment groups were ev...
Draper ACE, Wilson Z, Maile C, Faccenda D, Campanella M, Piercy RJ.A glutamic acid to lysine (E40K) residue substitution in superoxide dismutase 1 (SOD1) is associated with canine degenerative myelopathy: the only naturally occurring large animal model of amyotrophic lateral sclerosis (ALS). The E40 residue is highly conserved across mammals, except the horse, which naturally carries the (dog mutant) K40 residue. Here we hypothesized that in vitro expression of mutant dog SOD1 would recapitulate features of human ALS (ie, SOD1 protein aggregation, reduced cell viability, perturbations in mitochondrial morphology and membrane potential, reduced ATP production,...
Carnevale EM, Metcalf ES.Intracytoplasmic sperm injection (ICSI) is used to produce equine embryos invitro. The speed of embryo development invitro is roughly equivalent to what has been described for embryos produced invivo. Morphological evaluations of ICSI-produced embryos are complicated by the presence of debris and the dark nature of equine embryo cytoplasm. Morulas and early blastocysts produced invitro appear similar to those produced invivo. However, with expansion of the blastocyst, distinct differences are observed compared with uterine embryos. In culture, embryos do not undergo full expansion and thinning...
Hendricks KE, Hansen PJ.Apoptosis is common during spermatogenesis. Here, it was tested whether apoptosis could be induced in sperm after ejaculation. There were several lines of evidence to indicate that sperm are resistant to induction of apoptosis. First, incubation of bull sperm at temperatures characteristic of normothermia (38.5 degrees C) or heat shock (40 and 41 degrees C) for 4h did not increase the proportion of sperm positive for the TUNEL reaction. There was also no reduction in mitochondrial polarity caused by exposure to 40 or 41 degrees C. Incubation at 38.5 degrees C (least-squares mean+/-SEM=4.0+/-1....
Ferrer MS, Canisso IF, Ellerbrock RE, Podico G, Lister BN, Hurley DJ, Kline K, Palomares RA.Freezing cooled-transported semen allows veterinarians and breeders to collect and process the semen of stallions on farm, and then ship the semen to a semen freezing center. There, however, is a lack of standardization of shipping and freezing protocols. The objectives were to optimize and simplify protocols to freeze cooled-shipped semen. In Experiment 1, cooled-transported semen was centrifuged at room temperature or 5 °C before freezing. Sperm variables (motility, membrane integrity, acrosome integrity, membrane fluidity) were evaluated before and after freezing. Centrifugation temperat...
