Apoptosis is a form of programmed cell death that is essential for maintaining cellular homeostasis and development in horses. This process involves a series of biochemical events leading to characteristic cell changes and death, which are crucial for removing damaged or unnecessary cells without eliciting an inflammatory response. In horses, apoptosis plays a role in various physiological and pathological conditions, including tissue remodeling, immune response regulation, and the progression of certain diseases. Studies on equine apoptosis explore the molecular mechanisms, regulatory pathways, and implications for health and disease management. This page compiles peer-reviewed research studies and scholarly articles that investigate the mechanisms, regulation, and significance of apoptosis in equine biology and medicine.
Thompson JA, Eades SC, Chapman AM, Paulsen DB, Barker SA, McConnico RS.To determine the effects of clenbuterol, at a dosage of up to 3.2 μg/kg for 14 days, PO, on skeletal and cardiac muscle in healthy horses undergoing treadmill exercise. Methods: 12 healthy horses from 3 to 10 years old. Methods: Horses were randomly assigned to a control group (n = 6) or clenbuterol group (6) and received either saline (0.9% NaCl) solution or clenbuterol, PO, every 12 hours for 14 days. Horses were subjected to submaximal treadmill exercise daily during treatment. Muscle biopsy specimens were collected before and after treatment for determination of apoptosis. Echocardiograph...
Macías García B, Ortega Ferrusola C, Aparicio IM, Miró-Morán A, Morillo Rodriguez A, Gallardo Bolaños JM, González Fernández L....Glycerol is, to date, the most widely used cryoprotectant to freeze stallion spermatozoa at concentrations between 2% and 5%. Cryoprotectant toxicity has been claimed to be the single most limiting factor for the success of cryopreservation. In order to evaluate the toxic effects of the concentrations of glycerol used in practice, stallion spermatozoa were incubated in Biggers Whitten and Whittingham (BWW) media supplemented with 0%, 0.5%, 1.5%, 2.5%, 3.5%, and 5% glycerol. In two additional experiments, a hyposmotic (75 mOsm/kg) and a hyperosmotic (900 mOsm/kg) control media were included. Sp...
Lavoie-Lamoureux A, Beauchamp G, Quessy S, Martin JG, Lavoie JP.To compare innate immune responses of peripheral blood leukocytes from healthy and asymptomatic heaves-affected horses. Methods: Heaves-affected horses (n=5-6) and healthy controls (n=4-5) kept under low dust environments (pasture or shavings and pellets). Methods: Blood neutrophil and neutrophil-depleted cell populations were isolated using MACS system. Cells were incubated with or without bacterial products (lipopolysaccharide (LPS), 100 ng/mL and fMLP, 5 ng/mL, 5 h). Cytokine (IL-1β, IL-8, TNF, IL-4, INFγ and IL-10) and receptor (TLR4) mRNA expression was assessed by qPCR. TNF concentrati...
Henson FM, Getgood AM, Caborn DM, McIlwraith CW, Rushton N.To investigate effects of 1% hyaluronic acid-chondroitin sulfate-N-acetyl glucosamine (HCNAG) on the damage repair response in equine articular cartilage. Methods: Articular cartilage from 9 clinically normal adult horses. Methods: Full-thickness cartilage disks were harvested from the third metacarpal bone. Cartilage was single-impact loaded (SIL) with 0.175 J at 0.7 m/s and cultured in DMEM plus 1 % (vol/vol) HCNAG or fibroblastic growth factor (FGF)-2 (50 ng/mL). Histologic and immunohistochemical techniques were used to identify tissue architecture and apoptotic cells and to immunolocalize...
Gallardo Bolaños JM, Miró Morán Á, Balao da Silva CM, Morillo Rodríguez A, Plaza Dávila M, Aparicio IM, Tapia JA, Ortega Ferrusola C, Peña FJ.Apoptosis has been recognized as a cause of sperm death during cryopreservation and a cause of infertility in humans, however there is no data on its role in sperm death during conservation in refrigeration; autophagy has not been described to date in mature sperm. We investigated the role of apoptosis and autophagy during cooled storage of stallion spermatozoa. Samples from seven stallions were split; half of the ejaculate was processed by single layer centrifugation, while the other half was extended unprocessed, and stored at 5°C for five days. During the time of storage, sperm motility (C...
Grosche A, Freeman DE, Morton AJ, Polyak MM, Matyjaszek SA.To assess the effects of ischemia and reperfusion on indicators of oxidative stress, activation of eosinophils, and apoptosis in the large colonic mucosa of horses. Methods: 40 horses. Methods: In 1 or two 20-cm-long segments of the pelvic flexure, ischemia was induced for 1 or 2 hours followed by no reperfusion or 30 minutes and 18 hours of reperfusion in anesthetized horses. Mucosal specimens were collected before (controls; n = 20 horses) and after each period of ischemia, and full-thickness tissue samples were collected after each period of reperfusion. Sections of colonic tissues were sta...
Evans PM, Armour MD, Dubielzig RR. To describe the gross and histopathological ocular findings in a horse following suspected lightning injury. Methods: The eyes of a 2-year-old thoroughbred gelding were clinically and histopathologically evaluated following a severe lightning storm following euthanasia because of visual impairment. Results: Severe corneal edema and hydrops were noted clinically oculus dexter. Indirect ophthalmoscopy revealed bilateral symmetrical raised hyporeflective peripapillary geographic lesions. Histopathologic evaluation revealed corneal edema in the right eye with normal corneal endothelium...
Ambruosi B, Uranio MF, Sardanelli AM, Pocar P, Martino NA, Paternoster MS, Amati F, Dell'Aquila ME.Phthalates are ubiquitous environmental contaminants because of their use in plastics and other common consumer products. Di-(2-ethylhexyl) phthalate (DEHP) is the most abundant phthalate and it impairs fertility by acting as an endocrine disruptor. The aim of the present study was to analyze the effects of in vitro acute exposure to DEHP on oocyte maturation, energy and oxidative status in the horse, a large animal model. Cumulus cell (CC) apoptosis and oxidative status were also investigated. Cumulus-oocyte complexes from the ovaries of slaughtered mares were cultured in vitro in presence of...
Deschene K, Céleste C, Boerboom D, Theoret CL.Exuberant granulation tissue (EGT), a fibrotic healing disorder resembling the human keloid, occurs almost exclusively in limb wounds of horses and may be caused in part by a relative state of hypoxia within the wound. Objective: The objectives of this study were therefore to (1) assess the effects of hypoxia on equine dermal fibroblast (EDF) proliferation and apoptosis, (2) study the effects of hypoxia on the expression of key extracellular matrix (ECM) associated proteins and determine if such effects are dependent on hypoxia-inducible factor (HIF), and (3) determine if EDFs from the body or...
Leon PM, Campos VF, Kaefer C, Begnini KR, McBride AJ, Dellagostin OA, Seixas FK, Deschamps JC, Collares T.The gene expression of Bax, Bcl-2, survivin and p53, following in vitro maturation of equine oocytes, was compared in morphologically distinct oocytes and cumulus cells. Cumulus-oocyte complexes (COC) were harvested and divided into two groups: G1 - morphologically healthy cells; and G2 - less viable cells or cells with some degree of atresia. Total RNA was isolated from both immature and in vitro matured COC and real-time reverse transcription polymerase chain reaction (qRT-PCR) was used to quantify gene expression. Our results showed there was significantly higher expression of survivin (P &...
Chiavaccini L, Hassel DM, Shoemaker ML, Charles JB, Belknap JK, Ehrhart EJ.The black walnut extract (BWE) model of equine laminitis is associated with a systemic inflammatory response manifest by increased expression of inflammatory cytokines in the lungs and liver as well as the laminae. The specific role of the gastrointestinal tract in development of this response is unclear and is of utmost importance, as gastrointestinal disease and laminitis are intimately related. We investigated calprotectin expression and epithelial and endothelial apoptosis in the colon of horses exposed to orally administered BWE. Sections of colon from 19 horses including 7 controls not e...
Peña FJ, García BM, Samper JC, Aparicio IM, Tapia JA, Ferrusola CO.We review recent developments in the technology of freezing stallion sperm, paying special attention to the molecular lesions that spermatozoa suffer during freezing and thawing, such as osmotic stress, oxidative damage, and apoptotic changes. We also discuss the applicability of colloidal centrifugation in stallion sperm cryobiology. Increased knowledge about the molecular injuries that occur during cryopreservation may lead to improved protective techniques and thus to further improvements in fertility in the current decade.
Grosche A, Morton AJ, Graham AS, Valentine JF, Abbott JR, Polyak MM, Freeman DE.Intestinal ischaemia and reperfusion (I/R) can activate inflammatory cells in the equine colon, although effects on different types of inflammatory cells have received little attention. Objective: To assess early mucosal injury, the reaction of mucosal neutrophils, eosinophils, mast cells and macrophages, and cyclooxygenase (COX)-1 and -2 expression in response to I/R in the equine large colon. Methods: Large colon ischaemia was induced for 1 h (1hI) followed by 4 h of reperfusion in 6 horses, and mucosal biopsies were sampled before and after ischaemia, and after 1, 2 and 4 h of reperfusion. ...
Grosche A, Morton AJ, Graham AS, Sanchez LC, Blikslager AT, Polyak MM, Freeman DE.Ultrastructural changes in the epithelium can provide information on early changes in barrier properties, repair and inflammation in equine colon after ischaemia and reperfusion (I/R). Objective: To describe the morphology and ultrastructure of the epithelium in equine large colonic mucosa after I/R, and the response of inflammatory cells to injury. Methods: Ischaemia was induced for 1 h followed by 4 h of reperfusion in a 40 cm segment of the pelvic flexure in 6 horses. Mucosal biopsies before and after ischaemia, and after 1, 2 and 4 h of reperfusion were fixed in glutaraldehyde/paraformalde...
Thomas CM, Murray R, Sharif M.The purpose of this study was to investigate associations between the extent of chondrocyte apoptosis and expression of the articular cartilage (AC) extracellular matrix (ECM) molecules, cartilage oligomeric matrix protein (COMP) and fibronectin. Methods: Cartilage from four sites (when available) on equine left middle carpal joints (n = 12) were used. Expression of COMP and fibronectin was determined using specific polyclonal antibodies and a biotin-streptavidin/peroxidase method. The intensity of staining for matrix molecules was graded (none, mild, moderate, strong) in each cartilage zone. ...
Lin YZ, Cao XZ, Li L, Li L, Jiang CG, Wang XF, Ma J, Zhou JH.The attenuated equine infectious anemia virus (EIAV) vaccine was the first attenuated lentivirus vaccine to be used in a large-scale application and has been used to successfully control the spread of equine infectious anemia (EIA) in China. To better understand the potential role of cytokines in the pathogenesis of EIAV infection and resulting immune response, we used branched DNA technology to compare the mRNA expression levels of 12 cytokines and chemokines, including IL-1α, IL-1β, IL-4, IL-10, TNF-α, IFN-γ, IP-10, IL-8, MIP-1α, MIP-1β, MCP-1, and MCP-2, in equine monocyte-derived mac...
Maestre AM, Garzón A, Rodríguez D.Toroviruses are gastroenteritis causing agents that infect different animal species and humans. To date, very little is known about how toroviruses cause disease. Here, we describe for the first time that the prototype member of this genus, the equine torovirus Berne virus (BEV), induces apoptosis in infected cells at late times postinfection. Observation of BEV infected cells by electron microscopy revealed that by 24 hours postinfection some cells exhibited morphological characteristics of apoptotic cells. Based on this finding, we analyzed several apoptotic markers, and observed protein syn...
Ranera B, Ordovás L, Lyahyai J, Bernal ML, Fernandes F, Remacha AR, Romero A, Vázquez FJ, Osta R, Cons C, Varona L, Zaragoza P, Martín-Burriel I....Mesenchymal stromal cells (MSCs) represent an attractive source for regenerative medicine. However, prior to their application, fundamental questions regarding molecular characterisation, growth and differentiation of MSCs must be resolved. Objective: To compare and better understand the behaviour of equine MSCs obtained from bone marrow (BM) and adipose tissue (AT) in culture. Methods: Five horses were included in this study. Proliferation rate was measured using MTT assay and cell viability; apoptosis, necrosis and late apoptosis and necrosis were evaluated by flow cytometry. The mRNA expres...
Moran G, Buechner-Maxwell VA, Folch H, Henriquez C, Galecio JS, Perez B, Carrasco C, Barria M.Recurrent airway obstruction (RAO, also known as equine heaves) is an inflammatory condition similar to human asthma caused by exposure of susceptible horses to poorly ventilated stable environments. The disease is characterized by neutrophilic airway inflammation, mucus hypersecretion and reversible bronchoconstriction. This inflammatory process is mediated by several factors, including antibodies, cytokines, resident cells of the airway and inflammatory cellular components that arrive in the respiratory tract. An increasing body of evidence has lent support to the concept that a dysregulatio...
Frey J.RTX toxins are bacterial pore-forming toxins that are particularly abundant among pathogenic species of Pasteurellaceae, in which they play a major role in virulence. RTX toxins of several primary pathogens of the family of Pasteurellaceae are directly involved in causing necrotic lesions in the target organs. Many RTX toxins are known as haemolysins because they lyse erythrocytes in vitro, an effect that is non-specific, but which serves as a useful marker in bacteriological identification and as an easily measurable signal in vitro in experimental studies. More recent studies have shown that...
Wnuk M, Bugno-Poniewierska M, Lewinska A, Oklejewicz B, Zabek T, Bartosz G, Słota E.Recently, the old horse has been proposed as a model to study telomere-dependent senescence, immunosenescence and inflamm-aging. In the present paper, we used 80 Hucul and Anglo-Arabian horses divided into 3 age groups (juvenile, adult, old) to evaluate age-dependent changes at the genomic and DNA level and in cell proliferative potential. The level of positive TUNEL cells (both apoptotic and with DNA fragmentation), oxidative DNA damage (8-oxoG immunostaining), sister chromatid exchange and bleomycin-induced chromatid breaks were significantly increased in the combined old group compared to t...
Jiang CG, Gao X, Ma J, Lin YZ, Wang XF, Zhao LP, Hua YP, Liu D, Zhou JH.Preliminary studies revealed that the gene of the gp45 transmembrane protein (TM) of the attenuated equine infectious anemia virus (EIAV) vaccine strain EIAV(FDDV13) had a high frequency of a premature stop codon at position 261W, which generated a 154-residue truncation at the C-terminus. EIAV(FDDV-TM36), a recombinant virus with the TM truncated at the intracytoplasmic (CT) domain due to the presence of a stop codon, was constructed based on EIAV(FDDV)3-8, which is a proviral derivative of the vaccine. EIAV(FDDV-TM36) had a significantly reduced replication capability compared to EIAV(FDDV)3...
da Silva CM, Macías-García B, Miró-Morán A, González-Fernández L, Morillo-Rodriguez A, Ortega-Ferrusola C, Gallardo-Bolaños JM, Stilwell G....Lipid peroxidation (LPO) has been claimed as a major factor involved in stallion damage during storage or cryopreservation. Because melatonin is a well-known potent antioxidant, the aim of the present study was to investigate the effect of melatonin during in vitro incubation. Furthermore, we investigated the presence of specific melatonin receptors (MT1 and MT2) using specific polyclonal antibodies and western blotting. Stallion spermatozoa were incubated up to 3 hr at 37°C in the presence of different concentrations of melatonin (0, 50 pm, 100 pm, 200 pm, or 1 μm). At the beginning and at ...
Breuer J, Müller U, Locher L, Spallek A, Recknagel S, Uhlig A, Schusser GF.Proinflammatoric cytokines are released extracellularly during necrosis. These lead to inflammation and destruction of surrounding tissues. The aim of this study was to compare the number of viable, apoptotic and necrotic cells in the bronchoalveolar lavage fluid (BALF) of normal horses and horses with recurrent airway obstruction (RAO) and to determine if fluorescence microscopy is a reliable method for this examination. A group of six normal horses and a group of ten horses with RAO were examined. Samples were assessed using annexin-V and propidium iodide immunofluorescence assay and examine...
Shotton HR, Lincoln J, McGorum BC.Acute equine grass sickness (EGS) is a fatal disease of horses that is thought to be due to ingestion of a neurotoxic agent causing extensive damage to autonomic neurons. The aim of this study was to compare the effects of EGS on neurons in two sympathetic ganglia, the paravertebral cranial cervical ganglion (CCG) and the prevertebral coeliac/cranial mesenteric ganglion (CG/CMG). Specimens from horses with EGS and controls were obtained post mortem and processed using single and double immunofluorescence labelling for PGP 9.5 and HuC/HuD (pan-neuronal markers), TUNEL and caspase 3 (markers for...
García BM, Moran AM, Fernández LG, Ferrusola CO, Rodriguez AM, Bolaños JM, da Silva CM, Martínez HR, Tapia JA, Peña FJ.Cryopreservation introduces extreme temperature and osmolality changes that impart lethal and sublethal effects on spermatozoa. Additionally, there is evidence that the osmotic stress induced by cryopreservation causes oxidative stress to spermatozoa. The main sources of reactive oxygen species in mammalian sperm are the mitochondria. In view of this, the aim of our study was to test whether or not osmotic stress was able to induce mitochondrial damage and to explore the osmotic tolerance of the mitochondria of stallion spermatozoa. Ejaculates from 7 stallions were subjected to osmolalities ra...
Hurley DJ, Berghaus LJ, Hurley KA, Moore JN.To evaluate effects of black walnut extract (BWE) on equine mononuclear cells and determine whether BWE has direct proinflammatory effects. Methods: Mononuclear cells separated from blood samples from 8 horses. Methods: Aqueous BWE was prepared and processed to eliminate contamination with particulates and microbes. A Limulus amoebocyte lysate assay was used to detect lipopolysaccharide (LPS) contamination in the BWE. Mononuclear cells were incubated in minimal essential medium with or without the addition of 0.6% to 10% (vol/vol) BWE. These mononuclear cells were assessed for viability, activ...
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...
Ascenzi P, Ciaccio C, Sinibaldi F, Santucci R, Coletta M.Upon interaction with bovine heart cardiolipin (CL), horse heart cytochrome c (cytc) changes its tertiary structure disrupting the heme-Fe-Met80 distal bond, reduces drastically the midpoint potential out of the range required for its physiological role, binds CO and NO with high affinity, and displays peroxidase activity. Here, the effect of CL on peroxynitrite isomerization by ferric cytc (cytc-Fe(III)) is reported. In the absence of CL, hexa-coordinated cytc does not catalyze peroxynitrite isomerization. In contrast, CL facilitates cytc-Fe(III)-mediated isomerization of peroxynitrite in a d...
Wauters J, Martens A, Pille F, Dumoulin M, Gasthuys F, Sys S, Meyer E.Synovial fluid samples from culture-confirmed infected joints (n=13), joints with pronounced non-infectious synovitis (n=11) and healthy joints (n=14) were collected from 24 equine patients and seven slaughterhouse horses. The samples from the joints with non-infectious synovitis and healthy joints served as negative controls. After isolation, counting and identification of neutrophils, the percentage viability, and the proportion apoptotic and necrotic neutrophils were determined by flow cytometry. Viability was significantly higher in infected samples compared to the controls. A significant ...
Shakeel M, Yoon M.This study investigated the effect of heat stress on stallion testicular cells (TCs) and the effect of insulin-like growth factor (IGF)-1 on TC viability, proliferation, and apoptosis, including different stages of germ cells. TCs were divided into control or treatment groups with 0.01, 0.1, 1, 10, and 100 ng/mL of recombinant human IGF-1 (rhIGF-1) for 24 h at 34 °C and 37 °C. The population and viability were measured before and after treatment. The effects of rhIGF-1 on TC viability, proliferation, and apoptosis were determined using RT-qPCR. Proliferating cell nuclear antigen (PCNA) a...
Samie KA, Kowalewski MP, Schuler G, Gastal GDA, Bollwein H, Scarlet D.In horses, the mechanisms behind ovarian follicle growth and oocyte maturation remain largely unknown. In other species, oocyte-secreted factors growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) have been related to the acquisition of developmental competence and to interaction with granulosa cells for the regulation of follicle development. This study assessed the expression and localization of GDF9 in the equine ovary, and its possible relationship with granulosa cell function. Results: Using custom-made antibodies, GDF9 protein was localized in oocytes from th...
Anderson SL, Duke-Novakovski T, Robinson AR, Townsend HGG, Singh B.The objective of this study was to determine the effect of pulmonary intravascular macrophage depletion on systemic inflammation and ex vivo neutrophil apoptosis using an experimental model of intestinal ischemia and reperfusion injury in horses. Neutrophils were isolated before and after surgery from horses that were randomized to three treatment groups, namely, sham celiotomy (CEL, = 4), intestinal ischemia and reperfusion (IR, = 6), and intestinal ischemia and reperfusion with gadolinium chloride treatment to deplete pulmonary intravascular macrophages (PIMs, IRGC, = 6). Neutrophil apopt...
Hollanders B, Mougin A, N'Diaye F, Hentz E, Aude X, Girard A.A new two-step gradient technique has been used in the separation of the different classes of lipoproteins from the serum of cows, horses, dogs, pigs, rabbits and rats. Total lipoproteins were first isolated at d 1.21 then floated through a d 1.006 to d 1.21 gradient. Collection by mean of a gradient fractionator provided directly comparable lipoprotein profiles, allowed the determination of the exact density range of each lipoprotein class and the fraction by fraction analysis of composition. Cholesterol and apo AI recoveries were high. Horse, dog, rabbit and pig exhibited three distinct lipo...
Kozai K, Hojo T, Tokuyama S, Szóstek AZ, Takahashi M, Sakatani M, Nambo Y, Skarzynski DJ, Okuda K.Regression of the corpus luteum (CL) is characterized by a decay in progesterone (P4) production (functional luteolysis) and disappearance of luteal tissues (structural luteolysis). In mares, structural luteolysis is thought to be caused by apoptosis of luteal cells, but functional luteolysis is poorly understood. 20α-hydroxysteroid dehydrogenase (20α-HSD) catabolizes P4 into its biologically inactive form, 20α-hydroxyprogesterone (20α-OHP). In mares, aldo-keto reductase (AKR) 1C23, which is a member of the AKR superfamily, has 20α-HSD activity. To clarify whether AKR1C23 is associated wi...
Faleiros RR, Stokes AM, Eades SC, Kim DY, Paulsen DB, Moore RM.To determine and compare the number, type, location, and distribution of apoptotic epidermal cells in the laminae of clinically normal horses and horses with laminitis. Methods: Formalin-fixed samples of digital lamellar tissue from 47 horses (including clinically normal horses [controls; n = 7], horses with acute [4] and chronic [7] naturally acquired laminitis, and horses with black walnut extract-induced [11] or carbohydrate overload-induced [18] laminitis). Methods: Blocks of paraffin-embedded lamellar tissues were stained for DNA fragmentation with the terminal deoxynucleotidyl transferas...
Hagen A, Niebert S, Brandt VP, Holland H, Melzer M, Wehrend A, Burk J.Successful translation of multipotent mesenchymal stromal cell (MSC)-based therapies into clinical reality relies on adequate cell production procedures. These should be available not only for human MSC, but also for MSC from animal species relevant to preclinical research and veterinary medicine. The cell culture medium supplementation is one of the critical aspects in MSC production. Therefore, we previously established a scalable protocol for the production of buffy-coat based equine platelet lysate (ePL). This ePL proved to be a suitable alternative to fetal bovine serum (FBS) for equine a...
Krakowski L, Obara J, Wąchocka A, Piech T, Bartoszek P, Kostro K, Tatara MR.The objective of the study was to assess apoptosis and DNA defragmentation in equine semen diluted and chilled to +4°C. Semen was collected from nine fertile stallions, including four Arabian thoroughbreds and five coldbloods. Examinations were carried out immediately after semen collection (0) and at five storage times (24, 48, 72, 96 and 120 h). The basic semen evaluation was performed in terms of volume, sperm concentration, viable sperm percentage, progressive motility and morphology. Using flow cytometry, DNA defragmentation and cell membrane integrity of spermatozoa were determined. Th...
Lima-Neto JF, Fernandes CB, Alvarenga MA, Golim MA, Landim-Alvarenga FC.This experiment aimed to study equine fibroblasts in culture analyzing and the cell cycle and viability of cells pre- and post-freezing. Skin fragments were obtained from 6 horses and cultured in DMEM high glucose + 10% FCS in 5% CO(2) until the beginning of confluence. Two passages were performed before freezing. Cells subjected to serum starvation (0.5% FCS) were analyzed for viability and cell cycle at 24, 48, 72, 96, 120, 144 and 168 h of culture. For the confluent groups, cells were analyzed at the moment they achieved confluence. Cellular viability was assisted with Hoescht 33342 and pro...
Wilson JM, Caceci T, Potter GD, Kraemer DC.Embryos were recovered non-surgically at about Day 6 after ovulation from 15 Quarter horse-type mares and were evaluated for morphological changes which may occur because of exposure to the cryoprotectant and/or cryopreservation. Electron microscopy was used to elucidate the fine structure of intracellular organelles which, if damaged, could cause cellular death. The horse embryo does not totally re-expand in the 10% glycerol freezing medium, nor will it completely re-expand in the isotonic holding medium following glycerol removal whether or not the embryo has been frozen. Embryos in this stu...
Fedorka CE, El-Sheikh Ali H, Scoggin KE, Loux SC, Ball BA, Troedsson MHT.Ascending placentitis is the leading cause of abortion in the horse. The pleiotropic cytokine tumor necrosis factor (TNF) is an upstream regulator of this disease, but little is understood regarding its function in pregnancy maintenance or placental infection. To assess this, RNA sequencing was performed on chorioallantois and endometrium of healthy pregnant mares at various gestational lengths (n = 4/gestational age), in addition to postpartum chorioallantois, and diestrus endometrium to assess expression of TNF, TNFR-1, and TNFR-2. Additionally, ascending placentitis was induced via trans-...
Souza C, Villarino NF, Farnsworth K, Black ME.Electroporation is a method used to deliver poorly permeant chemotherapeutic drugs to tumor cells, potentiating the cytotoxic effects of drugs and overall clinical response. Despite existing evidence of the potential benefits of electroporation to enhance the antitumoral effects of drugs, there is a lack of understanding about the effects of electroporation on equine tumor cells. This study investigated the combined effects of electroporation and bleomycin, cisplatin, and carboplatin on an equine sarcoid cell line (EqS04b). The use of electroporation increases the cytotoxic effects of bleomyci...
Journal of proteomicsAugust 5, 2020
Volume 228 103927 doi: 10.1016/j.jprot.2020.103927
Chiaradia E, Pepe M, Sassi P, Mohren R, Orvietani PL, Paolantoni M, Tognoloni A, Sforna M, Eveque M, Tombolesi N, Cillero-Pastor B.Osteochondrosis is a developmental orthopedic disease affecting growing cartilage in young horses. In this study we compared the proteomes of equine chondrocytes obtained from healthy and osteochondrotic cartilage using a label-free mass spectrometry approach. Quantitative changes of some proteins selected for their involvement in different functional pathways highlighted by the bioinformatics analysis, were validated by western blotting, while biochemical alterations of extracellular matrix were confirmed via Raman spectroscopy analysis. In total 1637 proteins were identified, of which 59 wer...
Albornoz A, Morales N, Uberti B, Henriquez C, Burgos RA, Alarcon P, Moran G.Neutrophils participate in innate immunity as the first line of host defence against microorganisms. However, persistent neutrophil activity and delayed apoptosis can be harmful to surrounding tissues; this problem occurs in diverse inflammatory diseases, including asthma-affected horses. Previous studies in horses with acute lung inflammation indicated that treatment with tamoxifen (TX), a selective oestrogen receptor modulator, produces a significant decrease in bronchoalveolar lavage fluid (BALF) neutrophil content. The aim of this study was to investigate the effect of tamoxifen and its me...
Lewis SR, Ellison SP, Dascanio JJ, Lindsay DS, Gogal RM, Werre SR, Surendran N, Breen ME, Heid BM, Andrews FM, Buechner-Maxwell VA, Witonsky SG.Sarcocystis neurona is the most common cause of Equine Protozoal Myeloencephalitis (EPM), affecting 0.5-1% horses in the United States during their lifetimes. The objective of this study was to evaluate the equine immune responses in an experimentally induced Sarcocystis neurona infection model. Neurologic parameters were recorded prior to and throughout the 70-day study by blinded investigators. Recombinant SnSAG1 ELISA for serum and CSF were used to confirm and track disease progression. All experimentally infected horses displayed neurologic signs after infection. Neutrophils, monocytes, an...
Grosche A, Morton AJ, Graham AS, Valentine JF, Abbott JR, Polyak MM, Freeman DE.Intestinal ischaemia and reperfusion (I/R) can activate inflammatory cells in the equine colon, although effects on different types of inflammatory cells have received little attention. Objective: To assess early mucosal injury, the reaction of mucosal neutrophils, eosinophils, mast cells and macrophages, and cyclooxygenase (COX)-1 and -2 expression in response to I/R in the equine large colon. Methods: Large colon ischaemia was induced for 1 h (1hI) followed by 4 h of reperfusion in 6 horses, and mucosal biopsies were sampled before and after ischaemia, and after 1, 2 and 4 h of reperfusion. ...
Chodkowska KA, Ciecierska A, Majchrzak K, Ostaszewski P, Sadkowski T.Gamma-oryzanol (GO) is a popular supplement for performance horses, dogs, and humans. Previous studies indicated that GO supplementation decreases creatine kinase activity and lactate level after exercise and may affect oxidative stress in Thoroughbred horses. GO may change genes expression in equine satellite cells (ESC). The purpose of this study was to evaluate the effect of GO on miRNA, gene expression, oxidative stress, and cell damage and viability in differentiating ESC pretreated with hydrogen peroxide (H₂O₂). ESCs were obtained from a young horse's skeletal muscle. ESCs were pre-i...
Bone reportsJune 21, 2018
Volume 9 19-26 doi: 10.1016/j.bonr.2018.06.003
Semevolos SA, Duesterdieck-Zellmer KF, Larson M, Kinsley MA.Osteochondrosis (OC) is a naturally occurring disease of the articular-epiphyseal cartilage and subchondral bone layers, leading to pain and decreased mobility. The objective of this study was to characterize gene and protein expression of apoptotic markers in chondrocytes surrounding cartilage canals and along the osteochondral junction of osteochondrosis (OC)-affected and normal cartilage, using naturally occurring disease in horses. Paraffin-embedded osteochondral samples (6 OC, 8 normal controls) and cDNA from chondrocytes captured with laser capture microdissection (4 OC, 6 normal control...
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...
Hegre OD, Marshall S, Hickey GE.Plasma amine oxidase activities (benzylamine oxidase and spermine oxidase) were determined in the sera of a number of species of various ages. Benzylamine oxidase (BZO) activity, measured spectrophotometrically, was present in bovine, equine, and ovine species examined. Generally its activity in serum increased with the age of the animal. Spermine oxidase activity (SPO) was estimated by a bioassay of in vitro toxicity and did not necessarily correlate with BZO. Cytotoxicity in the presence of spermidine was found only in the sera of the ruminant species examined. Serum activity tended to rise ...
Rowe EL, White NA, Buechner-Maxwell V, Robertson JL, Ward DL.To identify apoptosis in equine intestines and determine whether apoptosis is associated with gastrointestinal tract disease or a specific tissue layer of intestine. Methods: 38 horses that underwent surgery or were euthanatized for small or large intestine obstruction, strangulation, or distension and 9 control horses euthanatized for reasons other than gastrointestinal tract disease or systemic disease. Methods: Specimens were collected at surgery from intestine involved in the primary lesion and distant to the primary lesion site or at necropsy from several sites including the primary lesio...
Meng Q, Li S, Liu L, Xu J, Liu Y, Zhang Y, Zhang X, Shao Y.Equine Infectious Anemia Virus (EIAV), like other lentiviruses, has a transmembrane glycoprotein with an unusually long cytoplasmic tail (CT). Viral envelope (Env) proteins having CT truncations just downstream the putative membrane-spanning domain (PMSD) are assumed to exist among all wild-type budded virions, and also in some cell-adapted strains. To determine whether CT-truncated Env proteins can cause particularly deleterious effects on the Env expressing cells and/or their neighboring cells, plasmids encoding codon-optimized env gene including full-length (pE863) or CT-truncated (pE686* a...
Gil MC, Ferrusola CO, Anel-López L, Ortiz-Rodriguez JM, Alvarez M, de Paz P, Anel L, Peña FJ.Spermatozoa undergo apoptotic changes during the cryopreservation process. These changes, recently termed spermptosis, resemble the cryopreservation induced delayed onset of cell death observed after thawing of somatic cells. Due to its importance in cryobiology, methods to easily identify spermptotic cells are warranted. In this study, a well-validated method for identification of spermatozoa with caspase 3 activity was compared with use of the combination of Hoechst 33342 (H-42) and ethidium homodimer (Eth-1). Live, dead and apoptotic spermatozoa assessed with each method were compared using...
Souza SS, Alves BG, Alves KA, Brandão FAS, Brito DCC, Gastal MO, Rodrigues APR, Figueireod JR, Teixeira DIA, Gastal EL.Heterotopic and orthotopic ovarian tissue autotransplantation techniques, currently used in humans, will become promising alternative methods for fertility preservation in domestic and wild animals. Thus, this study describes for the first time the efficiency of a heterotopic ovarian tissue autotransplantation technique in a large livestock species (i.e., horses) after ovarian fragments were exposed or not to a cooling process (4°C/24 h) and/or VEGF before grafting. Ovarian fragments were collected in vivo via an ultrasound-guided biopsy pick-up method and surgically autografted in a subcutan...
Roels S, Tilmant K, Van Daele A, Van Marck E, Ducatelle R.Melanocytic tumours are a well-known clinical and pathological entity in horses, but further phenotypic characterization of these tumours is lacking. Six melanocytic tumours from five horses (two metastatic and four benign) were examined by Ki67, PCNA and p53 immunostaining, DNA nick end labelling (Tunel) and Feulgen staining. The stainings were evaluated using quantitative image analysis. The resulting parameters of growth fraction (Ki67), S-phase index (PCNA), p53 index, apoptotic index, DNA index, nuclear diameter, ploidy balance, proliferation index (Feulgen) and hyperploidy were analysed....
Leon PM, Campos VF, Kaefer C, Begnini KR, McBride AJ, Dellagostin OA, Seixas FK, Deschamps JC, Collares T.The gene expression of Bax, Bcl-2, survivin and p53, following in vitro maturation of equine oocytes, was compared in morphologically distinct oocytes and cumulus cells. Cumulus-oocyte complexes (COC) were harvested and divided into two groups: G1 - morphologically healthy cells; and G2 - less viable cells or cells with some degree of atresia. Total RNA was isolated from both immature and in vitro matured COC and real-time reverse transcription polymerase chain reaction (qRT-PCR) was used to quantify gene expression. Our results showed there was significantly higher expression of survivin (P &...
Bourebaba L, Bourebaba N, Galuppo L, Marycz K.Nowadays, regenerative medicine techniques are usually based on the application of mesenchymal stem cells (MSCs) for the repair or restoration of injured damaged tissues. However, the effectiveness of autologous therapy is limited as therapeutic potential of MSCs declines due to patient's age, health condition and prolonged in vitro cultivation as a result of decreased growth rate. For that reason, there is an urgent need to develop strategies enabling the in vitro rejuvenation of MSCs prior transplantation in order to enhance their in vivo therapeutic efficiency. In presented study, we attemp...
Walters M, Skovgaard K, Heegaard PMH, Fang Y, Kharaz YA, Bundgaard L, Skovgaard LT, Jensen HE, Andersen PH, Peffers MJ, Jacobsen S.MicroRNAs, a class of small noncoding RNAs, serve as post-transcriptional regulators of gene expression and are present in a stable and quantifiable form in biological fluids. MicroRNAs may influence intra-articular responses and the course of disease, but very little is known about their temporal changes in osteoarthritis. Objective: To identify miRNAs and characterise the temporal changes in their abundance in SF from horses with experimentally induced osteoarthritis. We hypothesised that the abundance of miRNA would change during disease progression. Methods: In vivo experiments. Methods: R...
Sahoo DK, Wong D, Paital B, Ruby RE, Patel A.The progression of inflammation during sepsis represents a multifaceted biological cascade that requires effective therapeutic interventions to improve survival. In septic neonatal foals, oxidative stress (OS) arises due to a compromised antioxidant defense system. Oxidative stress may disrupt the functionality of redox-sensitive organelles, such as the endoplasmic reticulum (ER). Endoplasmic reticulum stress disorder affects multiple cellular signaling pathways, including redox balance, inflammation, and apoptosis, and contributes to the pathogenesis of sepsis. The study aimed to elucidate wh...
