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.
Aguiar FL, Lunardi FO, Lima LF, Rocha RM, Bruno JB, Magalhães-Padilha DM, Cibin FW, Nunes-Pinheiro DC, Gastal MO, Rodrigues AP, Apgar GA, Gastal EL....This study investigated the effect of adding different concentrations of bovine recombinant follicle-stimulating hormone on the IVC of equine preantral follicles enclosed in ovarian tissue fragments. Randomized ovarian fragments were fixed immediately (fresh noncultured control) or cultured for 1 or 7 days in α-MEM(+) supplemented with 0, 10, 50, and 100 ng/mL FSH and subsequently analyzed by classical histology. Culture media collected on Day 1 or Day 7 and were analyzed for steroids (estradiol and progesterone) and reactive oxygen species (ROS). After Day 1 and Day 7 of culture, 50-ng/mL FS...
Balao da Silva CM, Ortega-Ferrusola C, Morrell JM, Rodriguez Martínez H, Peña FJ.To date, the only repeatable method to select spermatozoa for chromosomal sex is the Beltsville sorting technology using flow cytometry. Improvement of this technology in the equine species requires increasing awareness of the modifications that the sorting procedure induces on sperm intactness. Oxidative stress is regarded as the major damaging phenomenon, and increasing evidence regards handling of spermatozoa - including sex sorting - as basic ground for oxidative damage. The aim of this study was to disclose whether the flow cytometric sorting procedure increases the production of reactive...
Martin Muñoz P, Ortega Ferrusola C, Vizuete G, Plaza Dávila M, Rodriguez Martinez H, Peña FJ.Oxidative stress has been linked to sperm death and the accelerated senescence of cryopreserved spermatozoa. However, the molecular mechanisms behind this phenomenon remain poorly understood. Reactive oxygen species (ROS) are considered relevant signaling molecules for sperm function, only becoming detrimental when ROS homeostasis is lost. We hereby hypothesize that a major component of the alteration of ROS homeostasis in cryopreserved spermatozoa is the exhaustion of intrinsic antioxidant defense mechanisms. To test this hypothesis, semen from seven stallions was frozen using a standard tech...
Yamazaki M, Kusano K, Ishibashi T, Kiuchi M, Koyama K.Upon intensive, exhaustive exercise, exercise-induced reactive oxygen species may exceed the antioxidant defence threshold, consequently resulting in muscular damage or late-onset chronic inflammation. Recently, the therapeutic antioxidant and anti-inflammatory effects of molecular hydrogen (H2) for human rheumatoid arthritis have been demonstrated. However, it is also important to clarify the effects of administrating H2 in large animals other than humans, as H2 is thought to reach the target organ by passive diffusion upon delivery from the blood flow, indicating that the distance from the a...
Nogueira BG, Sampaio BF, Souza MI, Costa E Silva EV, Zúccari CE.Biotechnology applied for equine semen increases the levels of reactive oxygen species and reduces the natural antioxidant defence, by both dilution and removal of seminal plasma. Therefore, the aims of this study were to evaluate the effect of adding coenzyme Q10 (CoQ10) and α-tocopherol (α-TOH) to the cooling extender, singly or in combination, on sperm parameters, and their effectiveness in preventing lipid peroxidation (LPO) of equine semen during cooling at 5°C for 72 h. Ten adult stallions of proven fertility were used, using two ejaculates each, subjecting them to the treatments with...
Plaza Davila M, Martin Muñoz P, Tapia JA, Ortega Ferrusola C, Balao da Silva C C, Peña FJ.Mitochondria have been proposed as the major source of reactive oxygen species in somatic cells and human spermatozoa. However, no data regarding the role of mitochondrial ROS production in stallion spermatozoa are available. To shed light on the role of the mitochondrial electron transport chain in the origin of oxidative stress in stallion spermatozoa, specific inhibitors of complex I (rotenone) and III (antimycin-A) were used. Ejaculates from seven Andalusian stallions were collected and incubated in BWW media at 37 °C in the presence of rotenone, antimycin-A or control vehicle. Incubation...
Gibb Z, Lambourne SR, Quadrelli J, Smith ND, Aitken RJ.The spermatozoa of many stallions do not tolerate being cooled, restricting the commercial viability of these animals and necessitating the development of a chemically defined room temperature (RT) storage medium. This study examined the impact of two major modulators of oxidative phosphorylation, pyruvate (Pyr) and L-carnitine (L-C), on the storage of stallion spermatozoa at RT. Optimal concentrations of Pyr (10 mM) and L-C (50 mM) were first identified and these concentrations were then used to investigate the effects of these compounds on sperm functionality and oxidative stress at RT. Mito...
Derochette S, Serteyn D, Mouithys-Mickalad A, Ceusters J, Deby-Dupont G, Neven P, Franck T.Excessive neutrophil stimulation and reactive oxygen species (ROS) production are involved in numerous human or horse pathologies. The modulation of the neutrophil NADPH oxidase (NOX) has a great therapeutic potential since this enzyme produces superoxide anion whose most of the other ROS derive. The measurement of NOX activity by cell-free systems is often used to test potential inhibitors of the enzyme. A major drawback of this technique is the possible interferences between inhibitors and the probe, ferricytochrome c, used to measure the activity. We designed the "EquiNox2", a new pharmacol...
Brogan PT, Beitsma M, Henning H, Gadella BM, Stout TA.Short-term storage of equine sperm at 5°C in an extender containing milk and/or egg yolk components is common practice in the equine breeding industry. Sperm motility, viability, DNA integrity and, consequently, fertilizing ability decline over time, partly due to reactive oxygen species (ROS) generation. We investigated whether adding the anti-oxidant d-penicillamine to a commercial milk/egg yolk extender delayed the decrease in semen quality. Semen was recovered on four consecutive days from eight 3-year old Warmblood stallions. On day 5, seven of the stallions were castrated and sperm reco...
Collins JA, Moots RJ, Clegg PD, Milner PI.Mature articular cartilage is an avascular tissue characterized by a low oxygen environment. In joint disease, acidosis and further reductions in oxygen levels occur, compromising cartilage integrity.This study investigated how acidosis and very low oxygen levels affect components of the cellular redox system in equine articular chondrocytesand whether the antioxidants resveratrol and N-acetylcysteine could modulate this system. We used articular chondrocytes isolated from nondiseased equine joints and cultured them in a 3-D alginate bead system for 48h in <1, 2, 5, and 21% O2 at pH 7.2 or ...
Aitken JB, Naumovski N, Curry B, Grupen CG, Gibb Z, Aitken RJ.This study demonstrates for the first time the presence of an L-amino acid oxidase (LAAO) enzyme in equine spermatozoa that is able to generate significant amounts of reactive oxygen species (ROS) and create a state of oxidative stress. RT-PCR analysis revealed that the mRNA for this enzyme was present in the equine testis and spermatozoa, while immunocytochemical studies demonstrated that the mature LAAO protein was located in the sperm head, particularly in the acrosomal and postacrosomal domains. Experimental studies demonstrated that the aromatic amino acids (L-phenylalanine > L-tryptop...
Macías-García B, González-Fernández L, Loux SC, Rocha AM, Guimarães T, Peña FJ, Varner DD, Hinrichs K.Repeatable methods for IVF have not been established in the horse, reflecting the failure of standard capacitating media to induce changes required for fertilization capacity in equine sperm. One important step in capacitation is membrane cholesterol efflux, which in other species is triggered by cholesterol oxidation and is typically enhanced using albumin as a sterol acceptor. We incubated equine sperm in the presence of calcium, BSA, and bicarbonate, alone or in combination. Bicarbonate induced an increase in reactive oxygen species (ROS) that was abolished by the addition of calcium or BSA...
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...
Yeste M, Estrada E, Rocha LG, Marín H, Rodríguez-Gil JE, Miró J.Although cryopreservation of stallion spermatozoa allows long-term preservation of spermatozoa from particular stallions and facilitates international trade, it is understood to inflict damages on sperm cells that may finally reduce their fertilizing ability. In addition, individual differences are known to exist in the sperm ability to withstand freeze-thawing protocols. To date, these differences have mainly been reported on the basis of sperm motility and membrane integrity. For this reason, the present work sought to determine differences between good (good freezability ejaculates: GFE) an...
Johannisson A, Lundgren A, Humblot P, Morrell JM.The decrease in foaling rates after artificial insemination with cooled semen warrants the search for new predictors of fertility. The objectives were to investigate levels of naturally occurring reactive oxygen species (ROS) in cooled, stored stallion semen doses for artificial insemination (AI), and their relationship with parameters of semen quality and with pregnancy rate. Semen was collected from warmblood stallions (n=15) and used to prepare commercial semen doses for AI. Sperm quality was evaluated after cooled transport to the laboratory overnight. The results were correlated with obse...
Côté O, Clark ME, Viel L, Labbé G, Seah SY, Khan MA, Douda DN, Palaniyar N, Bienzle D.Secretoglobin family 1A member 1 (SCGB 1A1) is a small protein mainly secreted by mucosal epithelial cells of the lungs and uterus. SCGB 1A1, also known as club (Clara) cell secretory protein, represents a major constituent of airway surface fluid. The protein has anti-inflammatory properties, and its concentration is reduced in equine recurrent airway obstruction (RAO) and human asthma. RAO is characterized by reversible airway obstruction, bronchoconstriction and neutrophilic inflammation. Direct effects of SCGB 1A1 on neutrophil functions are unknown. We have recently identified that the SC...
Derochette S, Franck T, Mouithys-Mickalad A, Ceusters J, Deby-Dupont G, Lejeune JP, Neven P, Serteyn D.In neutrophils (PMNs), superoxide anion (O2*-), the first reactive oxygen species (ROS) produced to kill pathogenic agents, is generated by NADPH oxidase, an enzymatic complex formed by the translocation of cytosolic subunits to the membrane flavocytochrome b558. In horses, excessive activation of PMNs is often associated with deadly pathologies and the modulation of their ROS production by acting on NADPH oxidase is a prime target to manage inflammation. We developed a cell-free assay to measure the activity of equine NADPH oxidase assembled in vitro, in order to test the effects of natural o...
Morrell JM, Winblad C, Georgakas A, Stuhtmann G, Humblot P, Johannisson A.There are anecdotal reports that equine fertility may decline towards the end of the breeding season. Previous studies have examined differences in sperm quality between the breeding season and non-breeding season but few studies have investigated the proportions of superoxide or peroxide containing spermatozoa at different times during the breeding season. The purpose of this study was to measure the content of these reactive oxygen species (ROS) at the beginning and end of the Swedish breeding season, using flow cytometric analysis of the fluorescence produced after staining with hydroethidi...
Gibb Z, Butler TJ, Morris LH, Maxwell WM, Grupen CG.Excessive reactive oxygen species generation during sex sorting and cryopreservation of stallion sperm leads to DNA fragmentation, lipid peroxidation, and motility loss. In this study we investigated whether antioxidant supplementation during sex sorting and cryopreservation could ameliorate the effects of reactive oxygen species on stallion sperm. In experiment 1, the postthaw characteristics of stallion sperm (N = 9) cryopreserved in the presence or absence of catalase (200 U/mL), cysteine (0.2 mg/mL), or quercetin (0.15 mM) was examined. Motility and acrosome integrity were assessed at 0, 1...
Aktan I, Dunkel B, Cunningham FM.Activated platelets can contribute to host defense through release of products with bactericidal actions such as antimicrobial peptides and reactive oxygen species (ROS), as well as by forming heterotypic aggregates with neutrophils and enhancing their antimicrobial properties. Whilst release of vasoactive mediators from equine platelets in response to stimuli including bacterial lipopolysaccharide (LPS) has been documented, neither ROS production, nor the effects of activated platelets on equine neutrophil ROS production, have been reported. This study first sought evidence that activated equ...
Aitken RJ, Gibb Z, Mitchell LA, Lambourne SR, Connaughton HS, De Iuliis GN.The prolonged incubation of human spermatozoa in vitro was found to induce a loss of motility associated with the activation of mitochondrial reactive oxygen species generation in the absence of any change in mitochondrial membrane potential. The increase in mitochondrial free radical production was paralleled by a loss of protein thiols and a concomitant rise in the formation of 4-hydroxynonenal, an electrophilic product of lipid peroxidation that was found to directly suppress sperm movement. These results prompted a search for nucleophiles that could counteract the action of such cytotoxic ...
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...
Milner PI, Smith HC, Robinson R, Wilkins RJ, Gibson JS.Hypoxia and acidosis are recognized features of inflammatory arthroses. This study describes the effects of IGF-1 and TGF-β(1) on pH regulatory mechanisms in articular cartilage under hypoxic conditions. Acid efflux, reactive oxygen species (ROS), and mitochondrial membrane potential were measured in equine articular chondrocytes isolated in the presence of serum (10% fetal calf serum), IGF-1 (1, 10, 50, 100 ng/ml) or TGF-β(1) (0.1, 1, 10 ng/ml) and then exposed to a short-term (3 h) hypoxic insult (1% O(2)). Serum and 100 ng/ml IGF-1 but not TGF-β(1) attenuated hypoxic regulation of pH hom...
Wong DM, Moore RM, Brockus CW.Oxygen is essential to aerobic life, but it is also associated with the production of highly reactive compounds that can pose danger to physiologic systems when the oxygen concentration is excessive. Reactive oxygen species (ROS) are required for normal physiologic processes, but when produced in excess, they can overwhelm endogenous antioxidants, resulting in significant cellular damage and, eventually, cell death. Ischemic events can initiate numerous pathophysiologic mechanisms leading to increased production of ROS, loss of cellular energy production, and lipid peroxidation. Although reper...
Echigoya Y, Morita S, Itou T, Sakai T.To evaluate effects of extracellular lactate on viability, shape change, lactate metabolism, and reactive oxygen species (ROS) production in equine polymorphonuclear leukocytes (PMNs). Methods: PMNs isolated from equine venous blood samples. Methods: PMNs were incubated with 0 to 300mM lactate for 30 minutes before each experiment. Viability was assessed via trypan blue exclusion. Shape change was assessed via flow cytometry and light microscopy. Relative quantification of monocarboxylic acid transporter and lactate dehydrogenase lactate dehydrogenase (LDH) isotype mRNAs was performed with a r...
Borer KE, Bailey SR, Harris PA, Elliott J.The underlying pathophysiological triggers for equine acute laminitis are unknown, although digital vasoconstriction, ischaemia, hypoxia and reperfusion injury may be involved. The contractile responses of isolated equine digital arteries (EDAs), harvested from the hindlimbs of normal horses postmortem at an abattoir, were studied acutely (up to 3 h) under hyperoxic (95% oxygen, 5% CO2 ) and hypoxic (95% nitrogen, 5% CO2 ) conditions in organ baths. Phenylephrine (PHE; 10(-6) m), 5-hydroxytryptamine (5-HT; 10(-7) m) and high potassium (K(+) ; 118 mm) caused contraction in EDAs which was signif...
Venugopal C, Mariappan N, Holmes E, Kearney M, Beadle R.To determine and compare the reactive oxygen and nitrogen species (ROS and RNS) in pulmonary tissues of horses affected with recurrent airway obstruction (RAO) and clinically healthy horses, and to evaluate the effectiveness of potential therapeutic agents in reducing ROS and RNS in the tissues of these horses. Objective: We hypothesised that RAO-affected horses would have high levels of reactive species and that the test agents would reduce them. The objectives were as follows: 1) to determine the level of ROS and RNS in pulmonary tissues (bronchial and arterial rings) of RAO-affected and cli...
Macías-García B, González-Fernández L, Gallardo-Bolaños JM, Peña FJ, Johannisson A, Morrell JM.The aim of this study was to elucidate if SLC after 24 h storage selects the subpopulation of spermatozoa that better withstands osmotic shock. To test this hypothesis, viability, mitochondrial membrane potential (MMP) and superoxide anion (O(2)(·-)) production of uncentrifuged (UC) and single layer centrifugation (SLC) - selected spermatozoa were analyzed following SLC after storage of the semen. An aliquot of the extended ejaculate (100×10(6) spermatozoa/mL) was centrifuged through a single layer of a silane-coated silica based colloid formulation optimized for equine spermatozoa (Androcol...
Ceusters JD, Mouithys-Mickalad AA, de la Rebière de Pouyade G, Franck TJ, Votion DM, Deby-Dupont GP, Serteyn DA.To culture equine myoblasts from muscle microbiopsy specimens, examine myoblast production of reactive oxygen species (ROS) in conditions of anoxia followed by reoxygenation, and assess the effects of horseradish peroxidase (HRP) and myeloperoxidase (MPO) on ROS production. Methods: 5 healthy horses (5 to 15 years old). Methods: Equine skeletal myoblast cultures were derived from 1 or 2 microbiopsy specimens obtained from a triceps brachii muscle of each horse. Cultured myoblasts were exposed to conditions of anoxia followed by reoxygenation or to conditions of normoxia (control cells). Cell p...
de Rebière de Pouyade G, Salciccia A, Ceusters J, Deby-Dupont G, Serteyn D, Mouithys-Mickalad A.The endothelium plays an active role in ischemia/reperfusion injuries. Herein, we report the effect of a single or successive cycles of anoxia/reoxygenation (A/R) on the mitochondrial respiratory function of equine endothelial cells (cultured from carotids) monitored by high resolution oxymetry, and on their production of reactive oxygen species (ROS). ROS were measured by electron paramagnetic resonance (ESR) using POBN and DMPO spin traps, and by gas chromatography (GC) of ethylene released by ROS-induced α-keto-γ-(methylthio)butyric acid (KMB) oxidation. The oxygen consumption significant...
Portier K, Crouzier D, Guichardant M, Prost M, Debouzy JC, Kirschvink N, Fellmann N, Lekeux P, Coudert J.To evaluate whether a period of hyperoxia or after a period of hypoxia produced changes attributable to reactive oxygen species in anaesthetized horses. Methods: Prospective randomized experimental study. Methods: Six healthy (ASA I) geldings, aged 4.5-9.5 years and weighing 510-640 kg(-1). Methods: After 30 minutes breathing air as carrier gas for isoflurane, horses were assigned randomly to breathe air as carrier gas (CG0.21) or oxygen as carrier gas (CG1.00) for a further 90 minutes. After an interval of 1 month each horse was re-anaesthetized with the other carrier gas for the 90 minute te...
Zholobenko A, Mouithys-Mickalad A, Modriansky M, Serteyn D, Franck T.A recent study showed that silymarin, a standardized extract of S. marianum might be used in the prevention of equine laminitis. We investigated the effects of quercetin and some compounds found in silymarin (silybin, taxifolin and dehydrosilybin) on reactive oxygen species (ROS) production and myeloperoxidase (MPO) release by stimulated equine neutrophils (PMNs) and on MPO activity. All compounds (tested between 100 nm and 100 μm) inhibited superoxide anion production by stimulated PMNs in a dose-dependent manner. Dehydrosilybin and quercetin inhibited superoxide production and MPO release...
Catalán J, Yánez-Ortiz I, Tvarijonaviciute A, González-Arostegui LG, Rubio CP, Yeste M, Miró J, Barranco I.This study investigated whether the activities of the antioxidant components of donkey seminal plasma (SP)-both enzymatic (superoxide dismutase (SOD), catalase-like (CAT), glutathione peroxidase-like (GPX), and paraoxonase type 1 (PON1)) and non-enzymatic (measured in terms of total thiol, copper-reducing antioxidant capacity (CUPRAC), ferric-reducing ability of plasma (FRAP), and Trolox equivalent antioxidant capacity (TEAC))-and oxidative stress index (OSI) are related to sperm cryotolerance. For this purpose, 15 ejaculates from jackasses (one per individual) were collected and split into tw...
Wong DM, Moore RM, Brockus CW.Oxygen is essential to aerobic life, but it is also associated with the production of highly reactive compounds that can pose danger to physiologic systems when the oxygen concentration is excessive. Reactive oxygen species (ROS) are required for normal physiologic processes, but when produced in excess, they can overwhelm endogenous antioxidants, resulting in significant cellular damage and, eventually, cell death. Ischemic events can initiate numerous pathophysiologic mechanisms leading to increased production of ROS, loss of cellular energy production, and lipid peroxidation. Although reper...
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...
Aguiar FL, Lunardi FO, Lima LF, Rocha RM, Bruno JB, Magalhães-Padilha DM, Cibin FW, Nunes-Pinheiro DC, Gastal MO, Rodrigues AP, Apgar GA, Gastal EL....This study investigated the effect of adding different concentrations of bovine recombinant follicle-stimulating hormone on the IVC of equine preantral follicles enclosed in ovarian tissue fragments. Randomized ovarian fragments were fixed immediately (fresh noncultured control) or cultured for 1 or 7 days in α-MEM(+) supplemented with 0, 10, 50, and 100 ng/mL FSH and subsequently analyzed by classical histology. Culture media collected on Day 1 or Day 7 and were analyzed for steroids (estradiol and progesterone) and reactive oxygen species (ROS). After Day 1 and Day 7 of culture, 50-ng/mL FS...
Moreno-Irusta A, Dominguez EM, Marín-Briggiler CI, Matamoros-Volante A, Lucchesi O, Tomes CN, Treviño CL, Buffone MG, Lascano R, Losinno L....Sperm chemotaxis may facilitate the finding of the oocyte. Only capacitated spermatozoa can orient their movement by chemotaxis, which as well as capacitation, is regulated in part by the cAMP-PKA pathway. Reactive oxygen species (ROS) are produced during sperm capacitation which is closely related to chemotaxis. Then, the ROS participation in the chemotactic signaling can be expected. Here we studied the role of ROS in the chemotaxis signaling of equine spermatozoa which produce high quantities of ROS because of their energy metabolism. The level of capacitated and chemotactic spermatozoa was...
Al-Kass Z, Brown A, Johannisson A, Ntallaris T, Morrell JM.Although single layer centrifugation (SLC) selects robust spermatozoa from stallion semen, the effect of individual variation has not been studied in detail. The objective of this study was to determine the variation among stallions in the effects of SLC on sperm quality during cooled storage for up to 48 hr. Semen samples from seven stallions (18 ejaculates) were split, with one portion being used for SLC and the other serving as a control (CON). Sperm quality (kinematics, reactive oxygen species (ROS) production, membrane integrity (MI) and chromatin integrity) were analysed at 0, 24 and 48...
Ceusters JD, Mouithys-Mickalad AA, de la Rebière de Pouyade G, Franck TJ, Votion DM, Deby-Dupont GP, Serteyn DA.To culture equine myoblasts from muscle microbiopsy specimens, examine myoblast production of reactive oxygen species (ROS) in conditions of anoxia followed by reoxygenation, and assess the effects of horseradish peroxidase (HRP) and myeloperoxidase (MPO) on ROS production. Methods: 5 healthy horses (5 to 15 years old). Methods: Equine skeletal myoblast cultures were derived from 1 or 2 microbiopsy specimens obtained from a triceps brachii muscle of each horse. Cultured myoblasts were exposed to conditions of anoxia followed by reoxygenation or to conditions of normoxia (control cells). Cell p...
Rocha CC, Kawai GKV, de Agostini Losano JD, Angrimani DSR, Rui BR, de Cássia Bicudo L, da Silva BDCS, Alonso MA, Mendes CM....Semen biotechniques may impair sperm quality due to excessive production of reactive oxygen species (ROS). Additionally, products of the oxidative reaction, especially involving lipids (e.g., malondialdehyde - MDA), may be even more harmful to sperm. Carnosine, previously reported to be present in seminal plasma of several species, may be a key factor on sperm tolerance to biotechniques by counterattacking the deleterious influence of MDA. Therefore, the aim of this study was to measure the levels of carnosine present in equine seminal plasma and relate these findings with sperm function and o...
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 ...
Sotgia S, Taras A, Zinellu A, Cherchi R, Mangoni AA, Carru C, Bogliolo L.The dependence of a stallion's spermatozoa on oxidative phosphorylation for energy requirements results in an unconventional relationship between reactive oxygen species (ROS) production and fertility. In such a scenario, antioxidant activity must be finely controlled and not affect the essential functions of ROS. Some in vivo evidence suggests that the naturally occurring antioxidant ergothioneine (ERT) interferes with the critical roles of ROS/reactive nitrogen species (RNS) in pro-oxidant states but not in healthy tissues. The measurement of ERT in seminal plasma collected from 14 stallions...
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...
Péters F, Franck T, Pequito M, de la Rebière G, Grulke S, Salccicia A, Verwilghen D, Chiavaccini L, Deby-Dupont G, Serteyn D.The previous experiments have shown that some phenothiazines have antioxidant and anti-inflammatory properties in vitro. In this study the inhibition of the production of reactive oxygen species (ROS) by neutrophils was studied in two groups of horses, which received a dose of 0.1 mg/kg of either acepromazine or promethazine intravenously. Blood samples were collected before (T0) and 0.5, 1, 3 and 5 h after drug administration. The chemiluminescence (CML) response of neutrophils was measured ex vivo in the presence of luminol for a period of 10 min and the maximum CML value (peak value) record...
Derochette S, Serteyn D, Mouithys-Mickalad A, Ceusters J, Deby-Dupont G, Neven P, Franck T.Excessive neutrophil stimulation and reactive oxygen species (ROS) production are involved in numerous human or horse pathologies. The modulation of the neutrophil NADPH oxidase (NOX) has a great therapeutic potential since this enzyme produces superoxide anion whose most of the other ROS derive. The measurement of NOX activity by cell-free systems is often used to test potential inhibitors of the enzyme. A major drawback of this technique is the possible interferences between inhibitors and the probe, ferricytochrome c, used to measure the activity. We designed the "EquiNox2", a new pharmacol...
Marañón G, Manley W, Cayado P, García C, de la Muela MS, Vara E.Colic could be accompanied by changes in the morphology and physiology of organs and tissues, such as the intestine. This process might be, at least in part, due to the accumulation of oxidative damage induced by reactive oxygen (ROS) and reactive nitrogen species (RNS), secondary to intestinal ischemia. Glutathione (GSH), being the major intracellular thiol, provides protection against oxidative injury. The aim of this study was to investigate whether ischemia-induced intestinal injury could be related with alterations in GSH metabolism. Results: Ischemia induced a significant increase in lip...
Donovan DC, Jackson CA, Colahan PT, Norton NN, Clapper JL, Moore JN, Hurley DJ.To evaluate the effects of a standardized exercise test to exhaustion in horses on leukocyte function ex vivo. Methods: 6 Thoroughbred geldings. Methods: Blood samples were obtained from each horse before exercise; at exhaustion (termed failure); and at 2, 6, 24, 48, and 72 hours after exercise to evaluate hematologic changes, rate of leukocyte apoptosis, and leukocyte production of reactive oxygen species (ROS) ex vivo. To assess leukocyte function, leukocyte ROS production in response to stimulation with lipopolysaccharide, peptidoglycan, zymosan, and phorbol myristate acetate was evaluated....
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 […]
Salinas C, Espinosa G, Morales N, Henríquez C, Morán G, Gajardo G, Uberti B.Obesity is a highly prevalent condition in horses. Dysfunctional neutrophil activity has been reported in metabolically healthy obese humans, but minimal data exist regarding horses. The present study evaluated the effect of obesity on apoptosis, phagocytosis and oxidative burst activity of peripheral blood neutrophils from lean and obese non-insulin dysregulated horses. Seven lean (BCS, body condition score 4-6/9) and five obese (BCS 8-9) horses were enrolled in the study. All animals underwent two metabolic tests (OGT, oral glucose test; IRT, insulin response test) before their selection to ...
Brogna R, Fan J, Sieme H, Wolkers WF, Oldenhof H.Even though dried sperm is not viable, it can be used for fertilization as long as its chromatin remains intact. In this study, we investigated drying- and temperature-induced conformational changes of nucleic acids and stallion sperm chromatin. Sperm was diluted in preservation formulations with and without sugar/albumin and subjected to convective drying at elevated temperatures on glass substrates. Accumulation of reactive oxygen species was studied during storage at different temperatures, and the sperm chromatin structure assay was used to assess DNA damage. Fourier transform infrared spe...
Wu S, Canisso IF, Yang W, Ul Haq I, Liu Q, Han Y, Zeng S.This study aimed to investigate the effects of different concentrations of 1,2-bis-(o-aminophenoxy)-ethane-N,N,N0 N0-tetraacetic acid, tetra-acetoxymethyl ester (BAPTA-AM), an intracellular calcium chelating agent, on stallion semen cooling and freezing-thawing. After collection, semen was extended (1:1 v/v) on a skim milk-based extender, centrifuged and resuspended at 400 million/ml into cooling or freezing extenders containing 0, 5, 25, 50, 100 and 200 μΜ BAPTA-AM. Motility parameters were assessed after cooling in Equitainer at 5°C for 12, 24, 48, 72 and 120 hr and after freezing-tha...
Sandersen C, Mouithys-Mickalad A, de la Rebière G, Deby G, Serteyn D, Franck T.To investigate the effect of acepromazine (ACP) on reactive oxygen species (ROS) production by stimulated equine neutrophils. Methods: Ex vivo biochemical experiments. Methods: Isolated neutrophils from healthy untreated horses. Methods: Neutrophils were incubated with ACP at concentrations of 10(-4), 10(-5) or 10(-6) M and then stimulated with phorbol-myristate-acetate (PMA) before measurement of lucigenin-enhanced chemiluminescence (CL). In a second experiment neutrophils were incubated in the presence of α-keto-γ methylthiobutyric acid (KMB) and treated with ACP at concentrations of 10(-4...
Serpa PBS, Woolcock A, Taylor SD, Pires Dos Santos A.Oxidative stress refers to the accumulation of reactive oxygen species (ROS). Most assays for ROS detection are costly, laborious, and usually use indirect markers. The use of 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) is a possible alternative. This substance becomes a fluorochrome when oxidized by ROS, with the resultant fluorescence proportional to ROS concentration. Erythrocytes are highly exposed to ROS, resulting in cell damage and consequently impaired oxygen delivery. The effects of this exposure in physiologic and pathologic conditions necessitate an improvement in ROS detec...
Ortiz-Rodríguez JM, Martín-Cano FE, Gaitskell-Phillips G, Silva A, Tapia JA, Gil MC, Redondo E, Masot J, Ortega-Ferrusola C, Peña FJ.Spermatozoa are redox-regulated cells, and stallion spermatozoa, in particular, present an intense mitochondrial activity in which large amounts of reactive oxygen species (ROS) are produced. To maintain the redox potential under physiological conditions, sophisticated mechanisms ought to be present, particularly in the mitochondria. In the present study, we investigated the role of the SLC7A11 antiporter. This antiporter exchanges intracellular glutamate for extracellular cystine. In the spermatozoa, cystine is reduced to cysteine and used for GSH synthesis. The importance of the antiporter f...
Chiaradia E, Gaiti A, Scaringi L, Cornacchione P, Marconi P, Avellini L.To better define the species-specific antioxidant systems and to ascertain the influence of the intracellular redox status on the immune system of different animal species, we determined lymphocyte glutathione peroxidase (GSHPx) activity, plasmatic glutathione levels (GSH) and the effect of H2O2 on the responsiveness of lymphocytes to proliferative stimuli. Among the three species considered, sheep presented the lowest plasmatic GSH and the highest lymphocyte GSHPx activity. On the contrary, dogs showed an inverted pattern (high GSH - low GSHPx). Horses displayed intermediate values for both p...
Muko R, Matsuda H, Oikawa MA, Shin T, Matsuda K, Sato H, Sunouchi T, Tanaka A.Histidine-rich glycoprotein (HRG) is an abundant plasma protein that has been identified in most mammals. We first identified whole genome sequence of equine HRG (eHRG) and succeeded to purify eHRG from plasma of horses. Since HRG interacts with various ligands, this protein is thought to be involved in immune response, coagulation, and angiogenesis. Systemic inflammatory response syndrome (SIRS) is characterized as a non-specific, clinical, pro-inflammatory immune response that damage organs and tissues in the host. Recent reports revealed that blood HRG levels in human patients with SIRS are...
Borer KE, Bailey SR, Harris PA, Elliott J.The underlying pathophysiological triggers for equine acute laminitis are unknown, although digital vasoconstriction, ischaemia, hypoxia and reperfusion injury may be involved. The contractile responses of isolated equine digital arteries (EDAs), harvested from the hindlimbs of normal horses postmortem at an abattoir, were studied acutely (up to 3 h) under hyperoxic (95% oxygen, 5% CO2 ) and hypoxic (95% nitrogen, 5% CO2 ) conditions in organ baths. Phenylephrine (PHE; 10(-6) m), 5-hydroxytryptamine (5-HT; 10(-7) m) and high potassium (K(+) ; 118 mm) caused contraction in EDAs which was signif...
Papas M, Catalán J, Recuero S, Morrell JM, Yeste M, Miró J.This study sought to determine whether single layer centrifugation (SLC) of fresh donkey semen with Equicoll has any impact on sperm quality parameters and on the modulation of endometrial reaction following semen deposition using an in vitro model. Seventeen ejaculates from five jackasses were obtained using an artificial vagina and diluted in a skim-milk extender. Samples were either selected through SLC (Equicoll) or non-treated (control). Two experiments were performed. The first one consisted of incubating selected or non-selected spermatozoa at 38 °C for 180 min. Integrity and lipid dis...
Giaretta E, Mislei B, Martínez-Pastor F, Nesci S, Spinaci M, Galeati G, Nerozzi C, Mari G, Tamanini C, Bucci D.Equine spermatozoa highly rely on oxidative phosphorylation for their energy management. The present work aimed to characterize the role of mitochondria on horse sperm motility and ROS production by incubating spermatozoa with specific inhibitors of the different mitochondrial complexes. Equine spermatozoa were incubated 1 h and 3 h at 37 °C with: complex I inhibitor rotenone (5 μM, ROT), complex II inhibitor dimethyl-malonate (10 mM, DMM), complex III inhibitor antimycin A (1.8 μM, ANTI), the uncoupling agent carbonyl cyanide m-chlorophenyl hydrazine (5 μM, CCCP), ATP synthase inhi...
Milner PI, Smith HC, Robinson R, Wilkins RJ, Gibson JS.Hypoxia and acidosis are recognized features of inflammatory arthroses. This study describes the effects of IGF-1 and TGF-β(1) on pH regulatory mechanisms in articular cartilage under hypoxic conditions. Acid efflux, reactive oxygen species (ROS), and mitochondrial membrane potential were measured in equine articular chondrocytes isolated in the presence of serum (10% fetal calf serum), IGF-1 (1, 10, 50, 100 ng/ml) or TGF-β(1) (0.1, 1, 10 ng/ml) and then exposed to a short-term (3 h) hypoxic insult (1% O(2)). Serum and 100 ng/ml IGF-1 but not TGF-β(1) attenuated hypoxic regulation of pH hom...
