Topic:Adenosine Triphosphate (ATP)
The cholesterol transporter ABCA1 is expressed in stallion spermatozoa and reproductive tract tissues. In the present study, we assessed the presence of the ATP-binding-cassette (ABC) transporter molecules ABCA1 in spermatozoa of adult stallions and in testicular and epididymal tissue of prepubertal and adult stallions. For this purpose, semen samples from six fertile Shetland pony stallions aged 4 to 19 years were collected. Semen was collected from each stallion on three consecutive days. Ejaculates were analyzed immediately after collection, and only ejaculates meeting minimal requirements for fertile stallions were further evaluated. ABCA1 immunosignal was localized after staining of semen ...
Epigallocatechin-3-Gallate (EGCG) Reduces Rotenone Effect on Stallion Sperm-Zona Pellucida Heterologous Binding. Stallion spermatozoa are highly dependent on oxidative phosphorylation for ATP production to achieve normal sperm function and to fuel the motility. The aim of this study was to evaluate the response of equine sperm under capacitating conditions to the inhibition of mitochondrial complex I by rotenone and to test whether epigallocatechin-3-gallate (EGCG), a natural polyphenol component of green tea, could counteract this effect. After 2-h incubation of stallion spermatozoa in modified Tyrode's medium, rotenone (100 nm, 500 nm and 5 μm) and EGCG (10, 20 and 60 μm), alone or in combination, di...
Inhibition of Mitochondrial Complex I Leads to Decreased Motility and Membrane Integrity Related to Increased Hydrogen Peroxide and Reduced ATP Production, while the Inhibition of Glycolysis Has Less Impact on Sperm Motility. 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...
Clinical Research Abstracts of the British Equine Veterinary Association Congress 2015. Selective 5-HT4 receptor agonists such as prucalopride are used as human prokinetics, since activation of 5-HT4 receptors on intestinal cholinergic neurons facilitates acetylcholine release. 5-HT4 receptors, linked to adenylyl cyclase, act via generation of cAMP. None of the 4 in vitro studies on 5-HT in horses provided evidence for neuronal 5-HT4 receptors, but none used the protocol as described in human studies [1-4]. Objective: To investigate whether functional 5-HT4 receptors are present in the equine small intestine. Methods: In vitro organ bath set up, applying electrical field stimulat...
L-carnitine and pyruvate are prosurvival factors during the storage of stallion spermatozoa at room temperature. 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...
Maternal age and in vitro culture affect mitochondrial number and function in equine oocytes and embryos. Advanced maternal age and in vitro embryo production (IVP) predispose to pregnancy loss in horses. We investigated whether mare age and IVP were associated with alterations in mitochondrial (mt) DNA copy number or function that could compromise oocyte and embryo development. Effects of mare age (<12 vs ≥12 years) on mtDNA copy number, ATP content and expression of genes involved in mitochondrial replication (mitochondrial transcription factor (TFAM), mtDNA polymerase γ subunit B (mtPOLB) and mitochondrial single-stranded DNA-binding protein (SSB)), energy production (ATP synthase-couplin...
(D)-Ribose supplementation in the equine: lack of effect on glycated plasma proteins suggesting safety in humans. d-Ribose is a popular dietary supplement for humans and the equine because of its crucial role in cellular bioenergetics. However, as a reducing sugar, it has been suggested that ingestion of d-ribose might promote the formation of glycated proteins in vivo with potential adverse consequences. Objective: The aim of this study was to examine if d-Ribose would promote the formation of glycated proteins in vivo following exercise in training thoroughbred racehorses. Methods: Two groups of horses received the supplement (30 and 50 g d-Ribose daily) for 17 weeks, during which period the horses were...
Regulation of axonemal motility in demembranated equine sperm. Equine in vitro fertilization is not yet successful because equine sperm do not effectively capacitate in vitro. Results of previous studies suggest that this may be due to failure of induction of hyperactivated motility in equine sperm under standard capacitating conditions. To evaluate factors directly affecting axonemal motility in equine sperm, we developed a demembranated sperm model and analyzed motility parameters in this model under different conditions using computer-assisted sperm analysis. Treatment of ejaculated equine sperm with 0.02% Triton X-100 for 30 sec maximized both permeab...
The paradoxical relationship between stallion fertility and oxidative stress. The relationship between stallion fertility and oxidative stress remains poorly understood. The purpose of this study was to identify criteria for thoroughbred fertility assessment by performing a logistical regression analysis using "dismount" sperm parameters as predictors and weekly per-cycle conception rate as the dependent variable. Paradoxically, positive relationships between fertility and oxidative stress were revealed, such that samples that produced pregnancies exhibited higher rates of 8-hydroxy-2'-deoxyguanosine release (1490.2% vs. 705.5 pg/ml/24 h) and lower vitality (60.5% vs. 6...
Adenovirus-mediated expression of myogenic differentiation factor 1 (MyoD) in equine and human dermal fibroblasts enables their conversion to caffeine-sensitive myotubes. Several human and animal myopathies, such as malignant hyperthermia (MH), central core disease and equine recurrent exertional rhabdomyolysis (RER) are confirmed or thought to be associated with dysfunction of skeletal muscle calcium regulation. For some patients in whom the genetic cause is unknown, or when mutational analysis reveals genetic variants with unclear pathogenicity, defects are further studied through use of muscle histopathology and in vitro contraction tests, the latter in particular, when assessing responses to ryanodine receptor agonists, such as caffeine. However, since musc...
Sarcoid-derived fibroblasts: links between genomic instability, energy metabolism and senescence. Bovine papillomavirus 1 (BPV-1) is a well recognized etiopathogenetic factor in a cancer-like state in horses, namely equine sarcoid disease. Nevertheless, little is known about BPV-1-mediated cell transforming effects. It was shown that BPV-1 triggers genomic instability through DNA hypomethylation and oxidative stress. In the present study, we further characterized BPV-1-positive fibroblasts derived from sarcoid tumors. The focus was on cancer-like features of sarcoid-derived fibroblasts, including cell cycle perturbation, comprehensive DNA damage analysis, end-replication problem, energy me...
Evidence of purinergic neurotransmission in isolated, intact horse sweat glands. Fluid secretion by sweat glands in response to heat and exercise is underpinned by increases in intracellular calcium. In horses, this is primarily via β2-adrenoceptors, but studies in equine sweat gland cell lines have indicated a possible role for purinergic agonists. Knowledge of equine sweating stimulus-secretion mechanisms in intact glands from healthy animals would allow future comparison to determine whether these mechanisms are affected in equine anhidrosis. Objective: To determine whether purinergic agonists can induce changes in intracellular calcium in intact, freshly isolated equi...
Sex sorting increases the permeability of the membrane of stallion spermatozoa. At present, the only repeatable means of selecting the sex of offspring is the Beltsville semen sorting technology using flow cytometry (FC). This technology has reached commercial status in the bovine industry and substantial advances have occurred recently in swine and ovine species. In the equine species, however, the technology is not as well developed. To better understand the changes induced in stallion spermatozoa during the sorting procedure, pooled sperm samples were sorted: sperm motility and kinematics were assessed using computer assisted sperm analysis, sperm membrane integrity wa...
Extracellular ATP signaling in equine digital blood vessels. The functional distribution of ATP-activated P2 receptors is well characterized for many blood vessels, but not in the equine digital vasculature, which is a superficial vascular bed that displays thermoregulatory functions and has been implicated in ischemia-reperfusion injuries of the hoof. Isolated equine digital arteries (EDA) and veins (EDV) were submitted to isometric tension studies, whereby electric field stimulation (EFS) and concentration-response curves to exogenously applied agonists were constructed under low tone conditions. Additionally, immunofluorescent localization of P2X and...
The influence of casein and urea as nitrogen sources on in vitro equine caecal fermentation. To access the fermentative response of equine caecal microbial population to nitrogen availability, an in vitro study was conducted using caecal contents provided with adequate energy sources and nitrogen as limiting nutrient. Two nitrogen (N) sources were provided, protein (casein) and non-protein (urea). Caecal fluid, taken from three cannulated horses receiving a hay-concentrate diet, was mixed with a N-free buffer-mineral solution. The influence of four N levels (3.7, 6.3, 12.5 or 25 mg of N in casein or urea) was studied using the gas production technique. Total volatile fatty acids (VFA)...
Increased adenosine concentration in bronchoalveolar lavage fluid of horses with lower airway inflammation. Several reports have suggested a role for adenosine in the pathogenesis of chronic airway conditions and this has led to new therapeutic strategies to limit airway inflammation. In this study, detectable levels of adenosine in bronchoalveolar lavage (BAL) samples from 11 horses with non-infectious lower-airway inflammation and 14 healthy controls are reported, with significantly higher values in horses with airway inflammation. Although these increased levels did not correlate with changes in neutrophil percentage in BAL, a positive association between adenosine levels and signs of lower airwa...
In vitro acute exposure to DEHP affects oocyte meiotic maturation, energy and oxidative stress parameters in a large animal model. 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...
Persistent digital hyperthermia over a 48 h period does not induce laminitis in horses. Persistent digital hyperthermia, presumably due to vasodilation, occurs during the developmental and acute stages of insulin-induced laminitis. The objectives of this study were to determine if persistent digital hyperthermia is the principal pathogenic mechanism responsible for the development of laminitis. The potent vasodilator, ATP-MgCl(2) was infused continuously into the distal phalanx of the left forefoot of six Standardbred racehorses for 48 h via intra-osseous infusion to promote persistent digital hyperthermia. The right forefoot was infused with saline solution and acted as an inter...
Vasodilatory effect of pentoxifylline in isolated equine digital veins. The direct vasodilatory action of pentoxifylline (1-(5-oxohexyl)-3,7-dimethylxanthine) and its signalling pathway was evaluated in equine digital veins. Cumulative concentration-response curves to pentoxifylline (1 nM to 300 μM) were recorded in phenylephrine-precontracted equine digital vein rings under different experimental conditions. Relaxation to pentoxifylline was partially inhibited by endothelium removal, but was unaltered by CGS-15943 (a non-xanthine adenosine receptor antagonist; 3 μM). Nitric oxide synthase (NOS), soluble guanylate cyclase and cyclooxygenase (COX) inhibitors (Nω...
Transcriptome analysis of muscle in horses suffering from recurrent exertional rhabdomyolysis revealed energetic pathway alterations and disruption in the cytosolic calcium regulation. Recurrent exertional rhabdomyolysis (RER) is frequently observed in race horses like trotters. Some predisposing genetic factors have been described in epidemiological studies. However, the exact aetiology is still unknown. A calcium homeostasis disruption was suspected in previous experimental studies, and we suggested that a transcriptome analysis of RER muscles would be a possible way to investigate the pathway disorder. The purpose of this study was to compare the gene expression profile of RER vs. control muscles in the French Trotter to determine any metabolic or structural disruption. T...
Differences in the metabolic properties of gluteus medius and superficial digital flexor muscles and the effect of water treadmill training in the horse. Flexor tendon injury may be due to flexor muscle fatigue, contributing to fetlock joint hyperextension and tendon damage. A water treadmill provides resistance training on flexor tendon muscles, which might reduce the risk of tendon injury. Objective: To determine the effect of water treadmill training on the properties of the gluteal and superficial digital flexor (SDF) muscles and on cardiocirculatory response to a standardised exercise test. Methods: Five healthy unfit horses were trained on a water treadmill for 5 days/week for 4 weeks, starting with 5 min/day increasing to 20 min/day. Bef...
Enhanced IL-6 transcriptional response to adenosine receptor ligands in horses with lower airway inflammation. Accumulation of extracellular adenosine has been closely associated with human asthmatic responses. However, the relevance of adenosine signalling in equine airways has not previously been investigated. Objective: To determine the expression of adenosine receptors (AR) in bronchoalveolar lavage (BAL) cells and assess the reactivity of these cells to AR ligands ex vivo, employing IL-6 as readout of adenosinergic inflammatory signalling. Methods: Eight horses with varying degrees of lower airway inflammation and 10 healthy controls were analysed. Expression of AR-subtypes in each BAL sample was ...
Magnesium disorders in horses. Magnesium (Mg) is an essential macroelement that is required for cellular energy-dependent reactions involving adenosine triphosphate and for the regulation of calcium channel function. Subclinical hypomagnesemia is common in critically ill humans and animals and increases the severity of the systemic inflammatory response syndrome; worsens the systemic response to endotoxins; and can lead to ileus, cardiac arrhythmias, refractory hypokalemia, and hypocalcemia. This article discusses the clinical signs, consequences, and treatment of hypomagnesemia in horses and describes the association of Mg...
Na+, K(+)-ATPase activity and ATP concentration in horses of the Wielkopolski breed in relation to age. This study aimed at determining relationships between the age of the Wielkopolski horses, ATP in whole blood and in the erythrocytes, and between erythrocyte Na+, K(+)-ATPase activity, and serum concentrations of mineral components. ATP was measured in whole blood and in erythrocytes by HPLC method. Serum concentrations of Ca2+ and Mg2+ were measured spectrophotometrically, while Na+ and K+ by flame photometry. In horses aged from 4 to 48 months, a dynamic decrease in ATP activity was found. Erythrocyte Na+, K(+)-ATPase activity decreased proportionally with the decrease in ATP activity. The r...
Immunotherapy for pythiosis: Effect on NTPDase activity in lymphocytes of an experimental model. NTPDase (EC 3.6.1.5) occurs in lymphocytes and plays an important role in immune function, in that hydrolyzes extracellular nucleoside tri- and/or diphosphates to form AMP. Pythium insidiosum causes the disease pythiosis, a pyogranulomatous disease of horses, dogs, cattle, cats and humans. Most antifungal drugs are ineffective against this pathogen, and immunotherapy, a treatment approach that relies on the injection of P. insidiosum antigen, has been successfully used in humans and horses to manage this disease. In this study, we investigated NTPDase activity in lymphocytes from rabbits inocu...
Inhibition of the mitochondrial permeability transition pore reduces “apoptosis like” changes during cryopreservation of stallion spermatozoa. In order to evaluate to what extent the changes that occur during cryopreservation involve the mitochondrial permeability transition pore (PT-pore), a specific inhibitor was used during the cryopreservation process of stallion spermatozoa. Four ejaculates from each of 7 stallions were frozen using a standard protocol. Before freezing, each ejaculate was split into three subsamples. The first was supplemented with 2.5 microM bongkrekic acid (BA) and the second with 5 microM BA. The third subsample served as control. The BA significantly reduced the percentage of spermatozoa depicting active cas...
De novo fatty acid synthesis and NADPH generation in equine adipose and liver tissue. The lipogenic capacities of equine liver and adipose tissue explants were investigated in vitro. Preference for glucose or acetate as the primary carbon source for de novo fatty acid synthesis was determined using (14)C labeled substrates. Additional aims included determining the relative contribution of NADPH generating pathways to reducing equivalent generation and comparing the lipogenic activity of two adipose depots, mesenteric and subcutaneous harvested from the crest region of the neck. Mesenteric adipose tissue had greater lipogenic activity than subcutaneous adipose tissue, and liver ...
Changes in Adenylate Nucleotides Concentration and Na, K-ATPase Activities in Erythrocytes of Horses in Function of Breed and Sex. The aim of this study was to examine the relationships between the concentrations of ATP, ADP, AMP (HPLC methods), total nucleotide pool (TAN), adenylate energy charge (AEC) and Na(+), K(+)-ATPase erythrocytic activities (by Choi's method) of horses as a function of breed and sex. The studies were conducted on 54 horses (stallions and mares) of different constitution types: breathing constitution (Wielkopolska and Hanoverian breed) and digestive constitution (Ardenian breed). Horse erythrocytes, independently of examined breed, present low ATP concentration in comparison to other mammal specie...
Sarcoplasmic Reticulum Ca(2+)-ATPase Activity and Glycogen Content in Various Fiber Types after Intensive Exercise in Thoroughbred Horses. To find a new parameter indicating muscle fitness in Thoroughbred horses, we examined time-dependent recovery of glycogen content and sarcoplasmic reticulum (SR) Ca(2+)-ATPase activity of skeletal muscle after intensive treadmill running. Two repeated 50-sec running sessions (13 m/sec) were performed on a flat treadmill (approximately 90%VO2max). Muscle samples of the middle gluteal muscle were taken before exercise (pre) and 1 min, 20 min, 60 min, and 24 hr after exercise. Muscle fiber type composition was determined in the pre muscle samples by immunohistochemical staining with monoclonal an...
Infectious entry of equine herpesvirus-1 into host cells through different endocytic pathways. We investigated the mechanism by which equine herpesvirus-1 (EHV-1) enters primary cultured equine brain microvascular endothelial cells (EBMECs) and equine dermis (E. Derm) cells. EHV-1 colocalized with caveolin in EBMECs and the infection was greatly reduced by the expression of a dominant negative form of equine caveolin-1 (ecavY14F), suggesting that EHV-1 enters EBMECs via caveolar endocytosis. EHV-1 entry into E. Derm cells was significantly reduced by ATP depletion and treatments with lysosomotropic agents. Enveloped virions were detected from E. Derm cells by infectious virus recovery a...