Resting membrane potential and intracellular [Na+] at rest, during fatigue and during recovery in rat soleus muscle fibres in situ.

Large trans-sarcolemmal ionic shifts occur with fatiguing exercise or stimulation of isolated muscles. However, it is unknown how resting membrane potential (EM) and intracellular sodium concentration ([Na+]i) change with repeated contractions in living mammals. We investigated (i) whether [Na+]i (peak, kinetics) can reveal changes of Na+-K+ pump activity during brief or fatiguing stimulation and (ii) how resting EM and [Na+]i change during fatigue and recovery of rat soleus muscle in situ. Muscles of anaesthetised rats were stimulated with brief (10 s) or repeated tetani (60 Hz for 200 ms, every 2 s, for 30 s or 300 s) with isometric force measured. Double-barrelled ion-sensitive microelectrodes were used to quantify resting EM and [Na+]i. Post-stimulation data were fitted using polynomials and back-extrapolated to time zero recovery. Mean pre-stimulation resting EM (layer 2-7 fibres) was -71 mV (surface fibres were more depolarised), and [Na+]i was 14 mM. With deeper fibres, 10 s stimulation (2-150 Hz) increased [Na+]i to 38-46 mM whilst simultaneously causing hyperpolarisations (7.3 mV for 2-90 Hz). Fatiguing stimulation for 30 s or 300 s led to end-stimulation resting EM of -61 to -53 mV, which recovered rapidly (T1/2, 8-22 s). Mean end-stimulation [Na+]i increased to 86-101 mM with both fatigue protocols and the [Na+]i recovery time-course (T1/2, 21-35 s) showed no difference between protocols. These combined findings suggest that brief stimulation hyperpolarises the resting EM, likely via maximum Na+-induced stimulation of the Na+-K+ pump. Repeated tetani caused massive depolarisation and elevations of [Na+]i that together lower force, although they likely interact with other factors to cause fatigue. [Na+]i recovery kinetics provided no evidence of impaired Na+-K+ pump activity with fatigue. KEY POINTS: It is uncertain how resting membrane potential, intracellular sodium concentration ([Na+]i), and sodium-potassium (Na+-K+) pump activity change during repeated muscle contractions in living mammals. For rat soleus muscle fibres in situ, brief tetanic stimulation for 10 s led to raised [Na+]i, anticipated to evoke maximal Na+-induced stimulation of the Na+-K+ pump causing an immediate hyperpolarisation of the sarcolemma. More prolonged stimulation with repeated tetanic contractions causes massive elevations of [Na+]i, which together with large depolarisations (via K+ disturbances) likely reduce force production. These effects occurred without impairment of Na+-K+ pump function. Together these findings suggest that rapid activation of the Na+-K+ pump occurs with brief stimulation to maintain excitability, whereas more prolonged stimulation causes rundown of the trans-sarcolemmal K+ gradient (hence depolarisation) and Na+ gradient, which in combination can impair contraction to contribute to fatigue in living mammals.

Plasma Acidosis and Peak Power after a Supramaximal Trial in Elite Sprint and Endurance Cyclists: Effect of Bicarbonate.

PURPOSE: This study aimed to determine whether (i) a plasma acidosis contributes to a reduction of mechanical performance and (ii) bicarbonate supplementation blunts plasma acidosis and arterial oxygen desaturation to resist fatigue during the end spurt of a supramaximal trial in elite sprint and endurance cyclists. METHODS: Elite/world-class cyclists ( n = 6 sprint, n = 6 endurance) completed two randomized, double-blind, crossover trials at 105%V̇O 2peak simulating 3 min of a 4-km individual pursuit, 90 min after ingestion of 0.3 g·kg -1 BM sodium bicarbonate (BIC) or placebo (PLA). Peak power output (PPO), optimal cadence and optimal peak torque, and fatigue were assessed using a 6-s "all-out sprint" before (PPO1) and after (PPO2) each trial. Plasma pH, bicarbonate, lactate - , K + , Na + , Ca 2+ , and arterial hemoglobin saturation (SpO 2 (%)), were measured. RESULTS: Sprint cyclists exhibited a higher PPO, optimal pedal torque, and anaerobic power reserve (APR) than endurance cyclists. The trial reduced PPO (PLA) more for sprint (to 47% initial) than endurance cyclists (to 61% initial). Optimal cadence fell from ~151 to 92 rpm and cyclists with higher APR exhibited a reduced optimal peak torque. Plasma pH fell from 7.35 to 7.13 and plasma [lactate - ] increased from 1.2 to 19.6 mM (PLA), yet neither correlated with PPO loss. Sprint cyclists displayed a lesser plasma acidosis but greater fatigue than endurance cyclists. BIC increased plasma [HCO 3- ] (+6.8 mM) and plasma pH after PPO1 (+0.09) and PPO2 (+0.07) yet failed to influence mechanical performance. SpO 2 fell from 99% to 96% but was unrelated to the plasma acidosis and unaltered with BIC. CONCLUSIONS: Plasma acidosis was not associated with the decline of PPO in a supramaximal trial with elite cyclists. BIC attenuated acid-base disturbances yet did not improve arterial oxygen desaturation or mechanical performance at the end-spurt stage.

Physicochemical Analysis of Mixed Venous and Arterial Blood Acid-Base State in Horses at Core Temperature during and after Moderate-Intensity Exercise.

The present study determined the independent contributions of temperature, strong ion difference ([SID]), total weak acid concentration ([Atot]) and PCO2 to changes in arterial and mixed venous [H+] and total carbon dioxide concentration ([TCO2]) during 37 min of moderate intensity exercise (~50% of heart rate max) and the first 60 min of recovery. Six horses were fitted with indwelling carotid and pulmonary artery (PA) catheters, had PA temperature measured, and had blood samples withdrawn for immediate analysis of plasma ion and gas concentrations. The increase in core temperature during exercise (+4.5 °C; p < 0.001) significantly (p < 0.05) increased PO2, PCO2, and [H+], but without a significant effect on [TCO2] (p > 0.01). The physicochemical acid-base approach was used to determine contributions of independent variables (except temperature) to the changes in [H+] and [TCO2]. In both arterial and venous blood, there was no acidosis during exercise and recovery despite significant (p < 0.05) increases in [lactate] and in venous PCO2. In arterial blood plasma, a mild alkalosis with exercise was due to primarily to a decrease in PCO2 (p < 0.05) and an increase in [SID] (p < 0.1). In venous blood plasma, a near absence of change in [H+] was due to the acidifying effects of increased PCO2 (p < 0.01) being offset by the alkalizing effects of increased [SID] (p < 0.05). The effect of temperature on PO2 (p < 0.001) resulted in an increased arterio-venous PO2 difference (p < 0.001) that would facilitate O2 transfer to contracting muscle. The simultaneous changes in the PCO2 and the concentrations of the other independent acid-base variables (contributions from individual strong and weak ions as manifest in [SID] and [Atot]) show complex, multilevel control of acid-base states in horses performing even moderate intensity exercise. Correction of acid-base variables to core body temperature presents a markedly different physiological response to exercise than that provided by variables measured and presented at an instrument temperature of 37 °C.

A century of exercise physiology: key concepts in muscle cell volume regulation.

Skeletal muscle cells can both gain and lose volume during periods of exercise and rest. Muscle cells do not behave as perfect osmometers because the cell volume changes are less than predicted from the change in extracellular osmolality. Therefore, there are mechanisms involved in regulating cell volume, and they are different for regulatory volume decreases and regulatory volume increases. Also, after an initial rapid change in cell volume, there is a gradual and partial recovery of cell volume that is effected by ion and water transport mechanisms. The mechanisms have been studied in non-contracting muscle cells, but remain to be fully elucidated in contracting muscle. Changes in muscle cell volume are known to affect the strength of contractile activity as well as anabolic/catabolic signaling, perhaps indicating that cell volume should be a regulated variable in skeletal muscle cells. Muscles contracting at moderate to high intensity gain intracellular volume because of increased intracellular osmolality. Concurrent increases in interstitial (extracellular) muscle volume occur from an increase in osmotically active molecules and increased vascular filtration pressure. At the same time, non-contracting muscles lose cell volume because of increased extracellular (blood) osmolality. This review provides the physiological foundations and highlights key concepts that underpin our current understanding of volume regulatory processes in skeletal muscle, beginning with consideration of osmosis more than 200 years ago and continuing through to the process of regulatory volume decrease and regulatory volume increase.

Tracing Acid-Base Variables in Exercising Horses: Effects of Pre-Loading Oral Electrolytes.

Oral electrolyte supplementation may influence acid-base state during exercise due to the intestinal absorption of administered water and electrolytes used to mitigating sweat losses. This study examined the effect of pre-exercise electrolyte supplementation (3 and 8 L) on plasma acid-base variables at rest, during moderate intensity exercise and during recovery. It was hypothesized that electrolyte supplementation will result in improved acid-base state compared to the alkalosis typical of prolonged exercise. In randomized crossover fashion, four horses were administered 3 L or 8 L of a hypotonic electrolyte solution (PNW) intended to replace sweat losses, or water alone (CON), 1 h before treadmill exercise to fatigue (at 35% of peak VO2) or for 45 min at 50% peak VO2. Blood was sampled at 10-min intervals before, during and after exercise, and analyzed for dependent and independent acid-base variables. Effects of 3 L of supplementation at low exercise intensities were minimal. In the 8 L trials, plasma [H+] decreased (p < 0.05) during exercise and early recovery in CON but not PNW. Plasma TCO2 decreased (p < 0.05) by 30 min after PNW reaching a nadir of 28.0 ± 1.5 mmol/L during the early exercise period (p = 0.018). Plasma pCO2 and strong ion difference [SID] were the primary contributors to changes in [H+] and [TCO2], respectively. Pre-exercise PNW of 8 L intended to fully replenish sweat loses maintained [H+], decreased [TCO2] and mitigated the mild alkalosis during moderate intensity exercise.

In vitro Validation Assessment of a Fecal Occult Blood Protein Test for Horses.

A commercially available equine fecal blood test (FBT) claims to be able to detect the presence of blood proteins (albumin and hemoglobin) in manure. The purpose of this study was to determine the FBT test sensitivity, specificity, positive and negative predictive values (PPV, NPV), lower threshold of detection, time to obtain a visual positive result, effect of temperature and result stability in lab and field conditions. The FBT was assessed for its sensitivity and specificity for detecting pure albumin and hemoglobin at two temperatures over a range of concentrations. Time to result was measured for up to 60 minutes. PPV and NPV were assessed by measuring albumin and hemoglobin in manure from 13 pleasure horses over 25 days. Laboratory tests of hemoglobin alone, albumin alone, and hemoglobin and albumin combined were tested over a range of concentrations from 0.0125 ppm to 50 ppm. In the field study, fresh (within 30 minutes) manure was sampled and tested for proteins using the FBT. The FBT was both sensitive and specific to hemoglobin and albumin. The effect of cold temperature on time to a positive result at 15 minutes was not significant. Results were stable for up to 60 minutes. The field study showed evidence that the appearance of blood proteins in manure was intermittent, and that three tests on consecutive days provided a much better PPV and NPV. It is concluded that this FBT had high specificity, sensitivity, PPV, NPV, was equally functional at low and moderate temperatures, provided a rapid (within 15 minutes) and stable (for up to 60 minutes) reading. Its use in the field is simple and effective.

Pre-loading large volume oral electrolytes: tracing fluid and ion fluxes in horses during rest, exercise and recovery.

KEY POINTS: Exercise results in rapid and large extracellular to intracellular fluid shifts, as well as significant sweating losses of water and ions. It is unknown whether ions within oral electrolyte supplements are taken up by muscle (and other soft tissues) and whether oral supplementation can effectively offset sweating losses. Pre-loading with 8 L of a balanced hypotonic electrolyte supplement attenuated extracellular fluid losses, increased exercise duration and increased sweating fluid and ion losses during submaximal exercise. Supplemented electrolytes appear in skeletal muscle within 1 h after administration. Electrolyte supplementation increased exercise performance, improved maintenance of extracellular fluid volumes, and attenuated body fluid losses while maintaining sweating rates. ABSTRACT: This study used radioactive sodium (24 Na) and potassium (42 K) in a balanced, hypotonic electrolyte supplement to trace their appearance in skeletal muscle, and also quantified extracellular and whole-body fluid and ion changes during electrolyte supplementation, exercise and recovery. In a randomized crossover design, 1 h after administration of 1 to 3 L of water or electrolyte supplement with 24 Na, horses were exercised at 35% VO2max to voluntary fatigue or, after administration of 8 L of water or electrolyte supplement with 42 K were exercised at 50% peak VO2 for 45 min (n = 4 in each trial). Pre-exercise electrolyte supplementation was associated with decreased loss of fluid and electrolytes from the extracellular fluid compartments during exercise and recovery compared with water alone. The improved fluid and ion balance during prolonged exercise was associated with increased exercise duration, despite continuing sweating losses of fluid and ions. Nasogastric administration of radiotracer 24 Na+ and 42 K+ showed rapid absorption into the blood with plasma levels peaking 45 min after administration, followed by distribution into the extracellular space and intracellular fluid of muscle within 1 h. Following exercise, virtually all Na+ remained within the extracellular compartment, while the majority of K+ underwent intracellular uptake by 2 h of recovery. It is concluded that pre-loading with a large volume, balanced electrolyte supplement helps maintain whole-body fluid and ion balance and support muscle function during periods of prolonged sweat ion losses.

Tracing oral Na+ and K+ in sweat during exercise and recovery in horses.

NEW FINDINGS: What is the central question of this study? What are the mechanisms by which equine sweat glands transport sodium, potassium and water into sweat? What is the main finding and its importance? The flux of sodium into sweat does not have an active transport component, the flux of potassium into sweat is partially dependent on an active transport mechanism, and there is no evidence for paracellular transport. ABSTRACT: In two series of experiments, this study used radioactive sodium (Na+ ) and potassium (K+ ) to trace the net flux, and calculate the unidirectional fluxes, of these ions from extracellular fluid into sweat of horses during exercise and recovery. The effect of an oral electrolyte supplement (PNW) on the sweating responses and ion fluxes was also examined. Compared to 8 litres of water (controls), provision of 8 litres of PNW resulted in significantly increased sweating duration (P < 0.001). Two hours before exercise, 99 Tc-labelled diethylene-triamine-pentaacetate (DTPA) was administered i.v. to determine if there was paracellular flux of this molecule in sweat glands during the period of sweating. One hour before beginning moderate-intensity exercise, horses were nasogastrically administered either 24 Na (1-3 litres) or 42 K (8 litres) with water (control) or an electrolyte supplement. Both radiotracers appeared in sweat within 10 min of exercise onset, and the sweat specific activity of both ions increased during exercise (P < 0.001), approaching plasma specific activities. There was no appearance of 99 Tc-DTPA in sweat. The activities of 24 Na and 42 K, together with the concentrations Na+ , K+ and Cl- , argued against significant paracellular flux of these ions into the lumen of sweat glands. The flux analysis for 24 Na indicated a small intracellular pool within sweat gland cells, and no evidence for an active transport component. The flux analysis for 42 K indicated a relatively large intracellular equilibration pool within sweat gland cells, with evidence for an active transport component. The results are discussed with respect to the current understanding of sweat gland epithelial cell ion transport mechanisms at both the basal and the apical membranes. It appears likely that the majority of ions appearing in sweat pass through sweat gland epithelial cells by transcellular mechanisms that include ion transporting pathways as well as apical vesicular exocytosis.

Structured water: effects on animals.

This review focuses on the effects of structured water (SW) on animals when it is consumed on a daily basis. SW is liquid water that is given altered H-bonding structure by treatment with various forms of energy including magnetic fields and light. While most of the research has been conducted on 'magnetized' water, which has structure of short duration, recent research has examined effects of a SW with stability of at least 3.5 mo. A variety of laboratory and farm animals have been studied over the past 20 yr. Consistent (3 or more studies) responses among animals consuming SW for 1 mo or more include increased rate of growth, reduced markers of oxidative stress, improved glycemic and insulinemic responses in diabetics, improved blood lipid profile, improved semen and spermatozoa quality, and increased tissue conductivity as measured using bioelectrical impedance analysis. While it is known that fluids in and around cells and molecules are structured, it remains unknown if this endogenous water structuring is influenced by drinking SWs. The mechanisms by which SW affects biological systems are unknown and require investigation. Effects of SW, when taken up by biological systems, are likely associated with altered water structuring around biological surfaces, such as proteins and membranes.

Regulation of muscle potassium: exercise performance, fatigue and health implications.

This review integrates from the single muscle fibre to exercising human the current understanding of the role of skeletal muscle for whole-body potassium (K+) regulation, and specifically the regulation of skeletal muscle [K+]. We describe the K+ transport proteins in skeletal muscle and how they contribute to, or modulate, K+ disturbances during exercise. Muscle and plasma K+ balance are markedly altered during and after high-intensity dynamic exercise (including sports), static contractions and ischaemia, which have implications for skeletal and cardiac muscle contractile performance. Moderate elevations of plasma and interstitial [K+] during exercise have beneficial effects on multiple physiological systems. Severe reductions of the trans-sarcolemmal K+ gradient likely contributes to muscle and whole-body fatigue, i.e. impaired exercise performance. Chronic or acute changes of arterial plasma [K+] (hyperkalaemia or hypokalaemia) have dangerous health implications for cardiac function. The current mechanisms to explain how raised extracellular [K+] impairs cardiac and skeletal muscle function are discussed, along with the latest cell physiology research explaining how calcium, ß-adrenergic agonists, insulin or glucose act as clinical treatments for hyperkalaemia to protect the heart and skeletal muscle in vivo. Finally, whether these agents can also modulate K+-induced muscle fatigue are evaluated.

Heat adaptation in humans: the significance of controlled and regulated variables for experimental design and interpretation.

Herein, the principles of homoeostasis are re-visited, but with an emphasis upon repeated homoeostatic disturbances that give rise to physiological adaptation. The central focus is human heat adaptation, and how, for experimental purposes, one might standardise successive adaptation stimuli, and then evaluate and compare the resulting adaptations. To provide sufficient background for that discussion, the principles of physiological control and regulation have been reviewed. The case is presented that, since it is the regulated variables that drive both the effector organs and the processes of physiological adaptation, then it is those variables (e.g., body temperature) that should be used to set and standardise the adaptation stimuli. Alternatively, some have proposed that the same outcome can be achieved through standardising a controlled variable (e.g., heart rate), and so the merits of that proposition are evaluated. Indeed, it can be an effective approach, although some experimental pitfalls are described to highlight its limitations with regard to between-group (e.g., able-bodied versus spinal-injured participants) and between-treatment comparisons (e.g., hot-water versus hot-air adaptation stimuli). The concept of setting the adaptation stimulus relative to an anaerobic or lactate threshold is also critically evaluated. Finally, an appraisal is offered concerning the merits of three different strategies for using deep-body and mean body temperature changes for evaluating thermoeffector adaptations.

Changes in salivary electrolyte concentrations in mid-distance trained sled dogs during 12 weeks of incremental conditioning.

Regular exercise improves the health status of dogs; however, extreme exertion in the absence of adequate fluid and electrolyte replacement may negatively impact health and performance due to dehydration and cardiovascular stress. Unlike humans and horses, dogs thermoregulate predominantly through respiration and salivation, yet there is a dearth of literature defining exercise-induced changes to canine salivary electrolytes. The study objective was to investigate the effects of exercise on salivary electrolyte concentrations, and to determine if adaptations may occur in response to incremental conditioning in client-owned Siberian Huskies. Sixteen dogs were used, with an average age of 4.8 ± 2.5 years and body weight of 24.3 ± 4.3 kg. A 12-week exercise regimen was designed to increase in distance each week, but weather played a role in setting the daily distance. Saliva samples were collected at weeks 0 (pre-run, 5.7 km), 5 (pre-run, 5.7, 39.0 km), and 11 (pre-run, 5.7, 39.0 km). Samples were analyzed for sodium, chloride, potassium, calcium, magnesium, and phosphorous using photometric and indirect ion-selective electrode analysis. When compared across weeks, sodium, chloride, potassium, and calcium concentrations did not differ at any sampling time point; however, phosphorus and magnesium concentrations increased from baseline. Data were then pooled across weeks to evaluate changes due to distance and level of conditioning. Sodium, chloride, and magnesium concentrations increased progressively with distance ran, suggesting that these electrolytes are primarily being lost as exercising dogs salivate. Repletion of these minerals may assist in preventing exercise-induced electrolyte imbalance in physically active dogs.

Foundational insights into the estimation of whole-body metabolic rate.

Since 2013, this journal has promoted the publication of thematic reviews (Taylor in Eur J Appl Physiol 113:1634, 2013), where leading groups were invited to review the critical literature within each of several sub-topics. The current theme is historically based, and is focussed on estimating the metabolic rate in humans. This review charts the development of our understanding of those methods, from the discovery of oxygen and carbon dioxide, to the introduction of highly sophisticated modern apparatus to examine the composition of expired gas and determine respiratory minute volume. An historical timeline links the six thematic vignettes on this theme. Modern advances have greatly enhanced data collection without significant decrements in measurement accuracy. At the same time, however, conceptual errors, particularly steady-state requirements, are too often ignored. Indeed, it is recognised that we often neglect the past, leading to errors in research design, experimental observations and data interpretation, and this appears to be increasingly prevalent within the open-access literature. Accordingly, the Editorial Board, in recognition of a widening gap between our experimental foundations and contemporary research, embarked on developing a number of thematic review series, of which this series is the first. The intent of each accompanying overview is to introduce and illuminate seminal investigations that led to significant scientific or intellectual breakthroughs, and to thereby whet the appetite of readers to delve more deeply into the historical literature; for it is only when the foundations are understood that we can best understand where we are now, and in which directions we should head.

A time-course evaluation of inflammatory and oxidative markers following high-intensity exercise in horses: a pilot study.

Exercise is a physiological stress resulting in reactive oxygen species and inflammatory mediators, the accumulation of which are thought to contribute to degenerative articular diseases. The horse is of particular interest in this regard as equine athletes are frequently exposed to repetitive bouts of high-intensity exercise. The purpose of this study was to provide a detailed description of the response of articular and systemic oxidative and inflammatory biomarkers following high-intensity, exhaustive exercise in horses. A group of horses (Ex) underwent repeated bouts of high-intensity exercise, at a target heart rate of 180 beats/min, until voluntary exhaustion. Baseline plasma and synovial fluid (SF) samples were taken 24 h before exercise and then at 0.5, 1, 2, 4, 8, and 24 h following exercise cessation. This time course was repeated in a group of nonexercised control horses (Co). Plasma and SF samples were analyzed for prostaglandin E2 (PGE2), nitric oxide (NO), total antioxidant status (TAS), and glycosaminoglycans (GAG). The Ex group had significantly higher plasma NO at 0.5, 1, and 2 h; and higher plasma PGE2 at 0.5 and 1 h compared with Co. SF PGE2 and GAG were also higher in Ex horses at 8 h compared with Co. It is concluded that high-intensity exercise in horses results in a rapid increase in systemic oxidative and inflammatory markers from 0.5 to 2 h after exercise, which is followed by local articular inflammation and cartilage turnover at 8 h postexercise. NEW & NOTEWORTHY In horses, the influence of exercise systemically and within the articular space remains unclear and requires further detailed characterization. In this study, we identify that an acute bout of high-intensity exercise in horses induces systemic inflammation and oxidative stress within 30 min of exercise cessation, which lasts for ~2 h. Articular inflammation and cartilage turnover were also be observed within the equine carpal joint 8 h following exercise completion.

Reduced Dental Plaque Formation in Dogs Drinking a Solution Containing Natural Antimicrobial Herbal Enzymes and Organic Matcha Green Tea.

The results of an exploratory, multicenter clinical study confirmed the hypothesis that a novel, natural, and safe oral care product (OCP) reduced the rate of plaque formation on teeth of dogs consuming the OCP (antimicrobial plant-derived enzymes, organic matcha green tea, cultured dextrose, sodium bicarbonate, and ascorbic acid) compared to controls. Healthy dogs without periodontitis, of varying breeds, sex, and age, were recruited and enrolled, using nonrandomized stratification methods, into a control and treatment groups. Treatment group dogs drank only water into which OCP was suspended, for 28 days. Control group dogs drank their normal household water. On day 0 all teeth were cleaned by a veterinarian and gingivitis was assessed. On days 14, 21, and 28 plaque index, plaque thickness, gingivitis, freshness of breath, and general health were assessed. Over the 28 days of study, dogs on the OCP had significant reduction in plaque index and plaque thickness compared to controls. By day 14 OCP reduced plaque formation by 37%; the 28-day reduction in plaque index and coverage averaged 22% with no measurable gingivitis or calculus. Conclusion. Using the OCP attenuated dental plaque formation when consumed as normal drinking water and in the absence of other modes of oral care.

Seventy day safety assessment of an orally ingested, l-glutamine-containing oat and yeast supplement for horses.

We describe a safety assessment of an oral supplement designed to nutritionally support the gastrointestinal system of horses. The supplement comprised a mixture of essential (l-threonine) and conditionally essential (l-glutamine) amino acids, polar lipids, oat bran rich in beta glucans and yeast extract. Young (1-2years) horses of both sexes were allocated to control (n=7) and treatment groups (n=7) and studied for 9weeks. Horses in the treatment group received the supplement daily for 8weeks. After 8weeks of supplementation, horses were studied for one additional week. Outcome measures included body mass, weight gain, results of clinical examination, hematology and plasma chemistry. There were no adverse events associated with supplementation and horses in both groups showed normal weight gain, clinical signs, hematology and chemistry. l-Glutamine, which is not yet listed as GRAS, was considered with respect to its potential for nutritional support and safety when ingested orally. It is concluded that this oral supplement, when ingested by horses at twice the recommended daily level, was safe and does not pose a health risk when used in accordance with good feeding practice.

Inward flux of lactate⁻ through monocarboxylate transporters contributes to regulatory volume increase in mouse muscle fibres.

Mouse and rat skeletal muscles are capable of a regulatory volume increase (RVI) after they shrink (volume loss resultant from exposure to solutions of increased osmolarity) and that this RVI occurs mainly by a Na-K-Cl-Cotransporter (NKCC)-dependent mechanism. With high-intensity exercise, increased extracellular osmolarity is accompanied by large increases in extracellular [lactate⁻]. We hypothesized that large increases in [lactate⁻] and osmolarity augment the NKCC-dependent RVI response observed with a NaCl (or sucrose)-induced increase in osmolarity alone; a response that is dependent on lactate⁻ influx through monocarboxylate transporters (MCTs). Single mouse muscle fibres were isolated and visualized under light microscopy under varying osmolar conditions. When solution osmolarity was increased by adding NaLac by 30 or 60 mM, fibres lost significantly less volume and regained volume sooner compared to when NaCl was used. Phloretin (MCT1 inhibitor) accentuated the volume loss compared to both NaLac controls, supporting a role for MCT1 in the RVI response in the presence of elevated [lactate⁻]. Inhibition of MCT4 (with pCMBS) resulted in a volume loss, intermediate to that seen with phloretin and NaLac controls. Bumetanide (NKCC inhibitor), in combination with pCMBS, reduced the magnitude of volume loss, but volume recovery was complete. While combined phloretin-bumetanide also reduced the magnitude of the volume loss, it also largely abolished the cell volume recovery. In conclusion, RVI in skeletal muscle exposed to raised tonicity and [lactate⁻] is facilitated by inward flux of solute by NKCC- and MCT1-dependent mechanisms. This work demonstrates evidence of a RVI response in skeletal muscle that is facilitated by inward flux of solute by MCT-dependent mechanisms. These findings further expand our understanding of the capacities for skeletal muscle to volume regulate, particularly in instances of raised tonicity and lactate⁻ concentrations, as occurs with high intensity exercise.