Reactive oxygen species (ROS) are chemically reactive molecules containing oxygen that are generated as byproducts of normal cellular metabolism in horses. They include free radicals such as superoxide anion, hydroxyl radical, and non-radical molecules like hydrogen peroxide. ROS play a role in cell signaling and homeostasis but can also cause oxidative damage to cells, proteins, and DNA when present in excessive amounts. In horses, ROS are implicated in various physiological and pathological processes, including exercise-induced oxidative stress, inflammatory conditions, and aging. This page compiles peer-reviewed research studies and scholarly articles that explore the generation, regulation, and impact of reactive oxygen species on equine health and performance.
Baumber J, Vo A, Sabeur K, Ball BA.Contaminating leukocytes in the ejaculate are an important source of reactive oxygen species (ROS) in human semen. When present in sufficient numbers, they can have a detrimental influence on sperm function in humans. Unfortunately, there is little published information regarding the importance of leukocytes in stallion semen. The objectives of this study were to determine the production of hydrogen peroxide (H2O2) by activated equine neutrophils and to examine the effect of this ROS production on equine sperm motility in vitro. Motile equine spermatozoa (two ejaculates each from four stallion...
Liu X, Yao J, Pisha E, Yang Y, Hua Y, van Breemen RB, Bolton JL.Excessive exposure to synthetic and endogenous estrogens has been associated with the development of cancer in several tissues. 4-Hydroxyequilenin (4-OHEN), a major metabolite of equine estrogens present in estrogen replacement formulations, has been shown to induce cytotoxic/carcinogenic effects. In the present study, we have found that 4-OHEN caused DNA damage in breast cancer cells, and cells that contain estrogen receptor alpha (S30) are more sensitive to 4-OHEN-mediated DNA damage as compared to estrogen receptor negative cells (MDA-MB-231). For example, concentration-dependent increases ...
McConnico RS, Argenzio RA, Roberts MC.The objective of this project was to determine early tissue biochemical events associated with increased colonic secretion during the acute stage of castor-oil-induced colitis by measuring cecal mucosal and submucosal malondialdehyde (MDA) and prostaglandin E2 (PGE2), levels in ponies. Intestinal tissue (inflamed or healthy) samples were obtained from 4 age- and sex-matched Shetland ponies. Biochemical methods were used to determine MDA and PGE2 levels in intestinal tissue samples from inflamed and healthy equine intestine. Inflamed tissue MDA and PGE2 levels increased with time after castor o...
Baumber J, Ball BA, Gravance CG, Medina V, Davies-Morel MC.The objective of this study was to examine the influence of reactive oxygen species (ROS), generated through the use of the xanthine (X)-xanthine oxidase (XO) system, on equine sperm motility, viability, acrosomal integrity, mitochondrial membrane potential, and membrane lipid peroxidation. Equine spermatozoa were separated from seminal plasma on a discontinuous Percoll gradient, and spermatozoa were incubated with 0.6 mM X and 0.05 U/mL XO for 30 minutes. Catalase (150 U/mL), superoxide dismutase (SOD, 150 U/mL), or glutathione (GSH, 1.5 mM) were evaluated for their ability to preserve sperm ...
Dimock AN, Siciliano PD, McIlwraith CW.Reactive oxygen species (ROS) are capable of degrading many components of the joint in the presence of insufficient antioxidant defences, and as a result have been implicated in the pathogenesis of joint disease in horses. However, to our knowledge, evidence of ROS occurring in diseased joints of horses has not been reported. The objective of this experiment was to compare differences in synovial fluid protein carbonyl content (as a marker of oxidative modification of synovial fluid proteins by ROS) and the antioxidant status of synovial fluid between clinically normal and diseased equine join...
Benbarek H, Grülke S, Deby-Dupont G, Deby C, Mathy-Hartert M, Caudron I, Dessy-Doize C, Lamy M, Serteyn D.We studied the interactions of isolated equine neutrophils with endothelial cells in culture, mimicking a situation of acute inflammation. Our main purpose was to demonstrate that the supernatant of activated neutrophils was sufficient to damage endothelial cells. Equine endothelial cells (from carotid arteries) were covered either with increased numbers of equine neutrophils stimulated by phorbol myristate acetate, or with the supernatant collected after an in vitro stimulation of the neutrophils. Cytotoxicity was estimated by the release of preincorporated 51Cr, and by light microscopy obser...
Benbarek H, Deby-Dupont G, Caudron I, Grülke S, Deby C, Lamy M, Serteyn D.In horses, the mechanisms of lipopolysaccharide (LPS) stimulation of isolated neutrophils to produce reactive oxygen species remain unknown. We re-investigated this problem by monitoring the luminol-enhanced chemiluminescence (CL) produced by LPS-stimulated equine neutrophils. The neutrophils were isolated from horse blood by discontinuous density gradient centrifugation (> or = 99% neutrophils; viability > or = 98%). Increasing concentrations of Escherichia coli (E. coli) LPS (from 0.01-10 microg ml(-1)) were used to activate the neutrophils. When LPS was used directly, without another ...
Pamplona R, Requena JR, Portero-Otín M, Prat J, Thorpe SR, Bellmunt MJ.The non-enzymatic modification of aminophospholipids with lipoperoxidation-derived aldehydes and glycoxidation-derived products have been reported previously. However, it remains unknown whether intracellular membranes are damaged by these glycoxidation-derived products. To investigate this issue, we tested whether aminophospholipids from mitochondrial membranes are damaged by glycoxidative stress the mitochondrion being identified as the major site of reactive-species production in the cell. We have used a selected-ion-monitoring/gas-chromatography/mass-spectrometry assay for carboxymethyleth...
Kooreman K, Babbs C, Fessler J.To evaluate and compare oxidative processes during ischemia and reperfusion of the equine large colon and jejunum. Methods: 2 groups of 6 anesthetized horses undergoing a terminal procedure. Methods: Isolated loops of large colon and jejunum were subjected to 2 hours of ischemia, followed by 2 hours of reperfusion. Tissue specimens were taken after 105 minutes of ischemia and 10, 30, 60, and 120 minutes of reperfusion. Mesenteric arterial and venous blood samples were collected for blood gas analysis at the same times to evaluate ischemia and reoxygenation. Oxidative processes in tissues were ...
Mills PC, Smith NC, Harris RC, Harris P.Allopurinol was administered to six horses in a cross-over study to determine the relative contribution of xanthine oxidase (XO) activity to the formation of reactive oxygen species (ROS) in the horse during intense exercise. Exercise increased the mean (SEM) plasma lipid hydroperioxide concentration to a maximum of 492.7 (33.4) microM within one minute of exercise completion and maximum levels of both oxidised glutathione (GSSG) in haemolysates of red blood cells and the glutathione redox ratio (GRR) occurred 20 minutes after exercise (87.2 [12.2] microM and 8.9 [0.9] per cent, respectively)....
Mills PC, Smith NC, Casas I, Harris P, Harris RC, Marlin DJ.The effects of prolonged variable-intensity and short-term high-intensity exercise on indices of oxidative stress and iron homeostasis were compared in six fit horses under cool [20 degrees C, 40% relative humidity (RH)] or hot/humid (30 degrees C, 80% RH) environmental conditions. The exercise protocols were designed to simulate equine competition, including racing (intense exercise) or the speed and endurance phase of a 3-day event (prolonged exercise). Increased plasma concentrations of lipid hydroperoxides and haemolysate concentrations of oxidised glutathione (GSSG) were measured within 3...
Olszewski MA, Robinson NE, Yu MF, Derksen FJ.During acute bouts of recurrent airway obstruction (heaves) in horses, neutrophils that are capable of increased production of reactive oxygen species accumulate in the airways. In the study reported here, the effect of hydrogen peroxide (H2O2; 1 microM to 0.1M), one of these reactive oxygen species products, on the responses of isolated trachealis muscle of horses was determined. Before and after incubation with H2O2, contractile responses to acetylcholine, electrical field stimulation (EFS), 127 mM KCl, and relaxation responses to isoproterenol and activation of the nonadrenergic noncholiner...
Cardier J, Romano E, Soyano A.Iron and ferritin impair a variety of immunological functions. To evaluate the effect of ferritin iron content on rat lymphocyte proliferative response, isoferritins that differ in their iron content and isoelectric point (pI) were isolated from iron overload rat livers by ultracentrifugation (isoferritins with high iron content and low pI) or crystallization (isoferritins with low iron content and high pI) methods. Additionally, commercial horse splenic ferritin (with a lower pI and higher iron content than rat isoferritins) was also tested. Proliferative response to Con A was decreased in a ...
Auer DE.The role of oxygen-derived free radicals is considered critical to the etio-pathogenesis of equine inflammatory joint disease. In vivo, the superoxide radical in the joint may be derived either from activated polymorphonuclear leukocytes or from an ischemia/reperfusion cycle. In the presence of ferrous iron, it may generate the highly reactive hydroxyl radical (OH *). Predisposing factors may include synovitis, exercise-induced ischemia and minor traumatic injury to the joints. Unlike other inflammatory mediators, oxygen-derived free radicals may damage tissue directly and these reactive speci...
Peterman BF, Morton RA.The research explores how different binding ions affect the oxidation speed of horse heart ferrocytochrome c, a protein, by potassium ferricyanide at a constant ionic strength. Studying the Ion Effect […]
Folin M, Gennari G, Jori G.The irradiation of horse and sperm-whale Fe” or Fez’ myoglobins with visible light
showed that axial ligands that render the heme diamagnetic (e.g. 02, CO or CN-) endow the
hemoproteins with a marked photosensitivity. In contrast, high-spin myoglobins are unaffected by
visible light. These findings appear to be of general validity for all hemo-proteins and are in agreement
with the involvment of the triplet state of the heme as the reactive intermediate. In all cases, the overall
photoprocess occurs within a very narrow spatial range, leading to specific modification of these
photoox...
Rifkind JM.The reduced glutathione in the erythrocyte was found to inhibit the autoxidation of purified horse hemoglobin. It was observed that much higher concentrations of oxidized glutathione also stabilize hemoglobin. The stabilization by oxidized glutathione most likely involves the formation of a mixed disulfide with the reactive β-93 sulfhydryl groups on the hemoglobin. A similar effect is also observed with N-ethyl- maleimide and HgCl2 which also react with the sulfhydryl groups. The apparent stabilization by reduced glutathione is partially due to the reduction of ferrihemoglobin formed by autox...
Vieira LA, Gadea J, García-Vázquez FA, Avilés-López K, Matás C.After injury or death of a valuable male, recovery of epididymal spermatozoa may be the last chance to ensure preservation of its genetic material. The objective of this research was to study the effect of sperm storage, at 4°C up to 96h, in the epididymides obtained from castrated horses and its effect on different functional sperm parameters. Aims were to study the effect of (1) sperm storage on viability and chromatin condensation; (2) pre-incubation of recovered epididymal sperm in the freezing extender, prior cryopreservation, on viability and chromatin condensation; and (3) freezing-tha...
Dimock AN, Siciliano PD, McIlwraith CW.Reactive oxygen species (ROS) are capable of degrading many components of the joint in the presence of insufficient antioxidant defences, and as a result have been implicated in the pathogenesis of joint disease in horses. However, to our knowledge, evidence of ROS occurring in diseased joints of horses has not been reported. The objective of this experiment was to compare differences in synovial fluid protein carbonyl content (as a marker of oxidative modification of synovial fluid proteins by ROS) and the antioxidant status of synovial fluid between clinically normal and diseased equine join...
Umair M, Claes A, Buijtendorp M, Cuervo-Arango J, Stout TAE, Henning H.Artificial insemination with chilled stallion semen is hampered by a limited period of maximum fertility maintenance (24-48 h). This study used multiparametric flow cytometry to simultaneously measure reactive oxygen species (ROS) production, mitochondrial function or [Ca ] and plasma membrane fluidity in viable, acrosome-intact spermatozoa, with the aim of providing insight into changes in sperm function during storage at 5°C. High proportions of viable and acrosome-intact spermatozoa (71 ± 8%) remained after 96 h of storage demonstrating that the basic integrity of the cells was we...
Teti G, Mazzotti E, Gatta V, Chiarini F, Alfieri ML, Falconi M.Osteoarthritis (OA) is described as a chronic degenerative disease characterized by the loss of articular cartilage. Senescence is a natural cellular response to stressors. Beneficial in certain conditions, the accumulation of senescent cells has been implicated in the pathophysiology of many diseases associated with aging. Recently, it has been demonstrated that mesenchymal stem/stromal cells isolated from OA patients contain many senescent cells that inhibit cartilage regeneration. However, the link between cellular senescence in MSCs and OA progression is still debated. In this study, we ai...
Yánez-Ortiz I, Catalán J, Delgado-Bermúdez A, Carluccio A, Miró J, Yeste M.In donkeys, the use of frozen-thawed sperm for artificial insemination (AI) leads to low fertility rates. Furthermore, donkey sperm produce a large amount of reactive oxygen species (ROS), and post-AI inflammation induces the formation of neutrophil extracellular traps (NETosis), which further generates many more ROS. These high ROS levels may induce lipid peroxidation in the sperm plasma membrane, thus affecting its integrity. Enzymatic and non-enzymatic antioxidants, mainly found in the seminal plasma (SP), are responsible for maintaining the redox balance. However, this fluid is removed pri...
Vieira LA, Matás C, Torrecillas A, Saez F, Gadea J.Seminal plasma (SP) plays a crucial role in sperm protection and functionality. However, the effect of SP on the sperm cryopreservation is dependent on the stallion and SP composition. The use of epididymal spermatozoa incubated in the presence of SP could help the identification of the components of SP that are able to confer protection upon the spermatozoa during freezing. The aims of this study were (i) to identify SP components involved in the potential protection of epididymal spermatozoa during the freeze-thawing process and (ii) to identify and evaluate the proteins likely related to sp...
Snider C, Grant D, Grant SA.Post-traumatic osteoarthritis (PTOA) is a progressive articular degenerative disease that degrades articular cartilage and stimulates apoptosis in chondrocyte cells. An injectable decellularized, extracellular matrix (ECM) scaffold, that might be able to combat the effects of PTOA, was developed where the ECM was conjugated with 20 nm gold nanoparticles (AuNP) and supplemented with curcumin and hyaluronic acid (HA). Porcine diaphragm ECM was decellularized and homogenized; AuNPs were conjugated using chemical crosslinking followed by mixing with curcumin and/or HA. Injection force testing and...
McConnico RS, Argenzio RA, Roberts MC.The objective of this project was to determine early tissue biochemical events associated with increased colonic secretion during the acute stage of castor-oil-induced colitis by measuring cecal mucosal and submucosal malondialdehyde (MDA) and prostaglandin E2 (PGE2), levels in ponies. Intestinal tissue (inflamed or healthy) samples were obtained from 4 age- and sex-matched Shetland ponies. Biochemical methods were used to determine MDA and PGE2 levels in intestinal tissue samples from inflamed and healthy equine intestine. Inflamed tissue MDA and PGE2 levels increased with time after castor o...
Ertmer F, Oldenhof H, Schütze S, Rohn K, Wolkers WF, Sieme H.If the physiological balance between production and scavenging of reactive oxygen species (ROS) is shifted towards production of ROS this may result in accumulation of cell damage over time. In this study stallion spermatozoa were incubated with xanthine and xanthine oxidase (X-XO) to artificially generate defined levels of superoxide and hydrogen peroxide resulting in sub-lethal oxidative damage. The effects of X-XO treatment on various sperm characteristics were studied. Special emphasis was placed on sperm osmotic tolerance pre-freeze and its correlation with cryosurvival, given that cryopr...
Folin M, Gennari G, Jori G.The irradiation of horse and sperm-whale Fe” or Fez’ myoglobins with visible light
showed that axial ligands that render the heme diamagnetic (e.g. 02, CO or CN-) endow the
hemoproteins with a marked photosensitivity. In contrast, high-spin myoglobins are unaffected by
visible light. These findings appear to be of general validity for all hemo-proteins and are in agreement
with the involvment of the triplet state of the heme as the reactive intermediate. In all cases, the overall
photoprocess occurs within a very narrow spatial range, leading to specific modification of these
photoox...
Balao da Silva CM, Ortega Ferrusola C, Gallardo Bolaños JM, Plaza Dávila M, Martín-Muñoz P, Morrell JM, Rodriguez Martínez H, Peña FJ.Flow cytometry is considered the only reliable method for the separation of X and Y chromosome bearing spermatozoa in equines. The MoFlo SX DP sorter is highly efficient, allowing the production of foals of the desired sex. However, to achieve acceptable pregnancy rates the currently used protocol requires working with fresh semen obtained close to, or at, the sorting facility. An alternative protocol was tested during two consecutive breeding seasons. Fresh stallion semen was cooled for 20 h, during which staining with Hoechst 33342 took place. On the following day, this sample was flow sort...
Davis MS, Bayly WM, Hansen CM, Barrett MR, Blake CA.Exercise is associated with the development of oxidative stress, but the specific source and mechanism of production of pro-oxidant chemicals during exercise has not been confirmed. We used equine skeletal muscle mitochondria to test the hypothesis that hyperthermia and acidosis affect mitochondrial oxygen consumption and production of reactive oxygen species (ROS). Skeletal muscle biopsies were obtained at rest, after an acute episode of fatiguing exercise, and after a 9-wk conditioning program to increase aerobic fitness. Mitochondrial oxygen consumption and ROS production were measured simu...
Taylor SD, Hart KA, Vaughn S, Giancola SC, Serpa PBS, Santos AP.Ascorbic acid (AA) is an antioxidant that might be beneficial for adjunctive treatment of sepsis in horses. The optimal dose and effects on oxidative status are unknown. Objective: Ascorbic acid administration will increase plasma AA concentrations and decrease determinants of reactive oxygen metabolites (dROM), basal and stimulant-induced intraerythrocytic reactive oxygen species (ROS) concentrations, and stimulant-induced neutrophil ROS production, and increase plasma antioxidant capacity (PAC) in a dose-dependent manner. Methods: Eight healthy horses. Methods: Randomized placebo-controlled ...
Vaughn SA, Norton NA, Hurley DJ, Hart KA.Reactive oxygen species (ROS) are the end-products of physiologic functions in health. Oxidative stress occurs when endogenous antioxidants are insufficient to neutralize ROS in the system. As a result, ROS can damage DNA, RNA, proteins, lipids, and cell organelles. To obtain accurate measurements of plasma oxidative stress, levels of both oxidants and antioxidants must be measured. This study validates a commercially available, semi-quantitative, photometric analytical system that measures systemic determinants of reactive oxygen metabolites (dROM) and plasma antioxidant capacity (PAC) in sto...
Riccio AV, Costa BK, Alonso MA, Affonso FJ, França DS, Nichi M, Belli CB, McLean AK, Boakari YL, Fernandes CB.After parturition, a rapid transition occurs from the intrauterine to the extrauterine milieu, exposing neonates to physiological circumstances characterized by oxidative conditions that instigate the generation of reactive oxygen species. These free radicals play pivotal roles in physiological processes; however, an imbalance between their production and the removal of antioxidants can result in severe cellular damage. The main objective of this study was to compare the oxidative and antioxidant profiles in mule and horse neonates immediately post-parturition, as well as at subsequent time po...
Quiroga J, Cortes B, Sarmiento J, Morán G, Henríquez C.This study explores Neutrophil Extracellular Trap (NET) formation in equine neutrophils, which is crucial for eliminating infections and is implicated in various equine inflammatory diseases. We investigated the molecular pathways involved in NET release by equine neutrophils in response to stimuli. We use PMA, A23187, LPS, PAF, OZ, and cytokines, observing NET release in response to PMA, PAF, and A23187. In contrast, LPS, OZ, and the cytokines tested did not induce DNA release or did not consistently induce citrullination of histone 4. Peptidyl-arginine deiminase inhibition completely halted ...
Catalán J, Yánez-Ortiz I, Torres-Garrido M, Ribas-Maynou J, Llavanera M, Barranco I, Yeste M, Miró J.Cryopreservation is a stressful process for sperm, as it is associated with an increased production of reactive oxygen species (ROS). Elevated ROS levels, which create an imbalance with antioxidant capacity, may result in membrane lipid peroxidation (LPO), protein damage and DNA fragmentation. This study aimed to determine whether the membrane LPO and DNA fragmentation of frozen-thawed horse sperm relies upon antioxidant activity, including enzymes (superoxide dismutase (SOD), glutathione peroxidase (GPX), catalase (CAT) and paraoxonase type 1 (PON1)); non-enzymatic antioxidant capacity (Trolo...
