Limits to sustained energy intake. XXXIV. Can the heat dissipation limit (HDL) theory explain reproductive aging?

According to the heat dissipation limit (HDL) theory, reproductive performance is limited by the capacity to dissipate excess heat. We tested the novel hypotheses that (1) the age-related decline in reproductive performance is due to an age-related decrease of heat dissipation capacity and (2) the limiting mechanism is more severe in animals with high metabolic rates. We used bank voles (Myodes glareolus) from lines selected for high swim-induced aerobic metabolic rate, which have also increased basal metabolic rate, and unselected control lines. Adult females from three age classes - young (4 months), middle-aged (9 months) and old (16 months) - were maintained at room temperature (20°C), and half of the lactating females were shaved to increase heat dissipation capacity. Old females from both selection lines had a decreased litter size, mass and growth rate. The peak-lactation average daily metabolic rate was higher in shaved than in unshaved mothers, and this difference was more profound among old than young and middle-aged voles (P=0.02). In females with large litters, milk production tended to be higher in shaved (least squares mean, LSM±s.e.: 73.0±4.74 kJ day-1) than in unshaved voles (61.8±4.78 kJ day-1; P=0.05), but there was no significan"t effect of fur removal on the growth rate [4.47±2.29 g (4 days-1); P=0.45]. The results provide mixed support of the HDL theory and no support for the hypotheses linking the differences in reproductive aging with either a deterioration in thermoregulatory capability or genetically based differences in metabolic rate.

Dietary fatty acids and flight-training influence the expression of the eicosanoid hormone prostacyclin in songbirds.

Diet shifts can alter tissue fatty acid composition in birds, which is subsequently related to metabolic patterns. Eicosanoids, short-lived fatty acid-derived hormones, have been proposed to mediate these relationships but neither baseline concentrations nor the responses to diet and exercise have been measured in songbirds. We quantified a stable derivative of the vasodilatory eicosanoid prostacyclin in the plasma of male European Starlings (Sturnus vulgaris, N = 25) fed semisynthetic diets with either high (PUFA) or low (MUFA) amounts of n6 fatty acid precursors to prostacyclin. Plasma samples were taken from each bird before, immediately after, and two days following a 15-day flight-training regimen that a subset of birds (N = 17) underwent. We found elevated prostacyclin levels in flight-trained birds fed the PUFA diet compared to those fed the MUFA diet and a positive relationship between prostacyclin and body condition, indexed by fat score. Prostacyclin concentrations also significantly decreased at the final time point. These results are consistent with the proposed influences of precursor availability (i.e., dietary fatty acids) and regulatory feedback associated with exercise (i.e., fuel supply and inflammation), and suggest that prostacyclin may be an important mediator of dietary influence on songbird physiology.

Mitochondrial metabolism in blood more reliably predicts whole-animal energy needs compared to other tissues.

Understanding energy metabolism in free-ranging animals is crucial for ecological studies. In birds, red blood cells (RBCs) offer a minimally invasive method to estimate metabolic rate (MR). In this study with European starlings Sturnus vulgaris, we examined how RBC oxygen consumption relates to oxygen use in key tissues (brain, liver, heart, and pectoral muscle) and versus the whole organism measured at basal levels. The pectoral muscle accounted for 34%-42% of organismal MR, while the heart and liver, despite their high mass-specific metabolic rate, each contributed 2.5%-3.0% to organismal MR. Despite its low contribution to organismal MR (0.03%-0.04%), RBC MR best predicted organismal MR (r = 0.70). Oxygen consumption of the brain and pectoralis was also associated with whole-organism MR, unlike that of heart and liver. Overall, our findings demonstrate that the metabolism of a systemic tissue like blood is a superior proxy for organismal energy metabolism than that of other tissues.

Repeat Sampling of Female Passerines During Reproduction Reveals Surprising Higher Plasma Oxidative Damage During Resting Compared to Active State.

Traditional models of oxidative stress predict accumulation of damage caused by reactive oxygen species (ROS) production as highly correlated with aerobic metabolism, a prediction under increasing scrutiny. Here, we repeat sampled female great tits (Parus major) at two opposite levels of energy use during the period of maximum food provisioning to nestlings, once at rest and once during activity. Our results were in contrast to the above prediction, namely significantly higher levels of oxidative damage during rest opposed to active phase. This discrepancy could not be explained neither using levels of "first line" antioxidant enzymes activity measured from erythrocytes, nor from total nonenzymatic antioxidant capacity measured from plasma, as no differences were found between states. Significantly higher levels of uric acid, a potent antioxidant, were seen in the plasma during the active phase than in rest phase, which may explain the lower levels of oxidative damage despite high levels of physical activity. Our results challenge the hypothesis that oxidative stress is elevated during times with high energy use and call for more profound understanding of potential drivers of the modulation of oxidative stress such as metabolic state of the animal, and thus also the time of sampling in general.

Concerted phenotypic flexibility of avian erythrocyte size and number in response to dietary anthocyanin supplementation.

BACKGROUND: Endurance flight impose substantial oxidative costs on the avian oxygen delivery system. In particular, the accumulation of irreversible damage in red blood cells can reduce the capacity of blood to transport oxygen and limit aerobic performance. Many songbirds consume large amounts of anthocyanin-rich fruit, which is hypothesized to reduce oxidative costs, enhance post-flight regeneration, and enable greater aerobic capacity. While their antioxidant benefits appear most straightforward, the effects of anthocyanins on blood composition remain so far unknown. We fed thirty hand-raised European starlings (Sturnus vulgaris) two semisynthetic diets (with or without anthocyanin supplement) and manipulated the extent of flight activity in a wind tunnel (daily flying or non-flying for over two weeks) to test for their interactive effects on functionally important haematological variables. RESULTS: Supplemented birds had on average 15% more and 4% smaller red blood cells compared to non-supplemented individuals and these diet effects were independent of flight manipulation. Haemoglobin content was 7% higher in non-supplemented flying birds compared to non-flying birds, while similar haemoglobin content was observed among supplemented birds that were flown or not. Neither diet nor flight activity influenced haematocrit. CONCLUSION: The concerted adjustments suggest that supplementation generally improved antioxidant protection in blood, which could prevent the excess removal of cells from the bloodstream and may have several implications on the oxygen delivery system, including improved gas exchange and blood flow. The flexible haematological response to dietary anthocyanins may also suggest that free-ranging species preferentially consume anthocyanin-rich fruits for their natural blood doping, oxygen delivery-enhancement effects.

Measurements of body temperature and oxidative stress reveal differential costs associated with humoral immune function in a passerine bird.

Eco-immunology considers resistance to antigens a costly trait for an organism, but actual quantification of such costs is not straightforward. Costs of the immune response are visible in impaired coloration and reduced growth or reproductive success. Activation of the humoral immune response is a slow, complex and long-lasting process, which makes the quantification of its energetic cost a potential losing game. We implemented near-continuous measurements of body temperature in zebra finches (Taeniopygia guttata) as a proxy for the energetic cost, with a particular focus during activation of the humoral immune response until the peak of antibody release several days later. At the peak of the antibody release we additionally measured oxygen consumption (open-flow respirometry) and markers of oxidative stress (dROMs, OXY). Birds with an activated immune response maintained a higher night-time body temperature during the first 4 nights after an immune challenge in comparison to controls, implying increased night-time energy use. At peak antibody production, we did not find differences in night-time body temperature and oxygen consumption but observed differentiated results for oxygen consumption during the day. Immune-challenged females had significantly higher oxygen consumption compared with other groups. Moreover, we found that activation of the humoral immune response increases oxidative damage, a potential cost of maintaining the higher night-time body temperature that is crucial at the early stage of the immune response. The costs generated by the immune system appear to consist of two components - energetic and non-energetic - and these appear to be separated in time.

Evolution of an increased performance under acute challenge does not exacerbate vulnerability to chronic stress.

An adequate stress response plays a vital role in coping with challenges. However, if selection for improved coping with an acute challenge affects the entire stress response system, susceptibility to adverse effects of chronic stressors can be deepened. Here, we used bank voles from lines selected for high swim-induced aerobic metabolism (A) and unselected control (C), and asked if the selection affected sensitivity to chronic mild stress (CMS). The voles were first habituated to daily weighing and feces collection for three weeks, and then for two weeks were exposed to CMS or remained undisturbed. The habituation itself resulted in an increased swim-induced oxygen consumption in both line types, and a decreased body mass. The CMS treatment caused reduction of food consumption in the second week of the experiment, and, in males, a decline in the metabolic rate. Paradoxically, fecal corticosterone metabolites decreased in the CMS-treated group. The response to CMS did not differ between the line types. Thus, the selection for increased performance was not traded off by increased vulnerability to chronic stress. The counter-intuitive results may even lead to a speculation that bank voles-and perhaps also other animals-prefer experiencing unpredictable, unpleasant stressors over the monotony of standard laboratory housing.

Flight training and dietary antioxidants have mixed effects on the oxidative status of multiple tissues in a female migratory songbird.

Birds, like other vertebrates, rely on a robust antioxidant system to protect themselves against oxidative imbalance caused by energy-intensive activities such as flying. Such oxidative challenges may be especially acute for females during spring migration, as they must pay the oxidative costs of flight while preparing for reproduction; however, little previous work has examined how the antioxidant system of female spring migrants responds to dietary antioxidants and the oxidative challenges of regular flying. We fed two diets to female European starlings, one supplemented with a dietary antioxidant and one without, and then flew them daily in a windtunnel for 2 weeks during the autumn and spring migration periods. We measured the activity of enzymatic antioxidants (glutathione peroxidase, superoxide dismutase and catalase), non-enzymatic antioxidant capacity (ORAC) and markers of oxidative damage (protein carbonyls and lipid hydroperoxides) in four tissues: pectoralis, leg muscle, liver and heart. Dietary antioxidants affected enzymatic antioxidant activity and lipid damage in the heart, non-enzymatic antioxidant capacity in the pectoralis, and protein damage in leg muscle. In general, birds not fed the antioxidant supplement appeared to incur increased oxidative damage while upregulating non-enzymatic and enzymatic antioxidant activity, though these effects were strongly tissue specific. We also found trends for diet×training interactions for enzymatic antioxidant activity in the heart and leg muscle. Flight training may condition the antioxidant system of females to dynamically respond to oxidative challenges, and females during spring migration may shift antioxidant allocation to reduce oxidative damage.

An Examination of the Relationship between Urinary Neurotrophin Concentrations and Transcutaneous Electrical Nerve Stimulation (TENS) Used in Pediatric Overactive Bladder Therapy.

This article aims to explore changes in urinary concentrations of selected neurotrophins in the course of TENS therapy in children with overactive bladder (OAB). A two-group open-label prospective study was conducted. The intervention group comprised 30 children aged between 5 and 12 years old with OAB refractory to conservative therapy. They received 12 weeks of TENS therapy in a home setting. The urinary neurotrophins, NGF, BDNF, NT3, NT4, were measured by ELISA at baseline and at the end of the TENS therapy. Total urinary neurotrophins levels were standardized to mg of creatinine (Cr). We compared the results with the reference group of 30 participants with no symptoms of bladder overactivity. The results revealed that children with OAB both before and after TENS therapy had higher NGF, BDNF, and NT4 concentrations in total and after normalization to Cr than the reference group in contrast to NT3. The response to the therapy expressed as a decrease of urinary neurotrophins after TENS depended on the age and the presenting symptoms. In conclusion, children older than 8 years of age with complaints of daytime incontinence responded better to TENS.

Age-Related Changes in the Thermoregulatory Properties in Bank Voles From a Selection Experiment.

As with many physiological performance traits, the capacity of endotherms to thermoregulate declines with age. Aging compromises both the capacity to conserve or dissipate heat and the thermogenesis, which is fueled by aerobic metabolism. The rate of metabolism, however, not only determines thermogenic capacity but can also affect the process of aging. Therefore, we hypothesized that selection for an increased aerobic exercise metabolism, which has presumably been a crucial factor in the evolution of endothermic physiology in the mammalian and avian lineages, affects not only the thermoregulatory traits but also the age-related changes of these traits. Here, we test this hypothesis on bank voles (Myodes glareolus) from an experimental evolution model system: four lines selected for high swim-induced aerobic metabolism (A lines), which have also increased the basal, average daily, and maximum cold-induced metabolic rates, and four unselected control (C) lines. We measured the resting metabolic rate (RMR), evaporative water loss (EWL), and body temperature in 72 young adult (4 months) and 65 old (22 months) voles at seven ambient temperatures (13-32°C). The RMR was 6% higher in the A than in the C lines, but, regardless of the selection group or temperature, it did not change with age. However, EWL was 12% higher in the old voles. An increased EWL/RMR ratio implies either a compromised efficiency of oxygen extraction in the lungs or increased skin permeability. This effect was more profound in the A lines, which may indicate their increased vulnerability to aging. Body temperature did not differ between the selection and age groups below 32°C, but at 32°C it was markedly higher in the old A-line voles than in those from other groups. As expected, the thermogenic capacity, measured as the maximum cold-induced oxygen consumption, was decreased by about 13% in the old voles from both selection groups, but the performance of old A-line voles was the same as that of the young C-line ones. Thus, the selection for high aerobic exercise metabolism attenuated the adverse effects of aging on cold tolerance, but this advantage has been traded off by a compromised coping with hot conditions by aged voles.

The energy savings-oxidative cost trade-off for migratory birds during endurance flight.

Elite human and animal athletes must acquire the fuels necessary for extreme feats, but also contend with the oxidative damage associated with peak metabolic performance. Here, we show that a migratory bird with fuel stores composed of more omega-6 polyunsaturated fats (PUFA) expended 11% less energy during long-duration (6 hr) flights with no change in oxidative costs; however, this short-term energy savings came at the long-term cost of higher oxidative damage in the omega-6 PUFA-fed birds. Given that fatty acids are primary fuels, key signaling molecules, the building blocks of cell membranes, and that oxidative damage has long-term consequences for health and ageing, the energy savings-oxidative cost trade-off demonstrated here may be fundamentally important for a wide diversity of organisms on earth.

Rest-Phase Hypothermia Reveals a Link Between Aging and Oxidative Stress: A Novel Hypothesis.

In endotherms, growth, reproduction, and survival are highly depended on energy metabolism. Maintenance of constant body temperature can be challenging for endotherms under continuously changing environmental conditions, such as low or high ambient temperatures or limited food. Thus, many birds may drop body temperature below normothermic values during the night, known as rest-phase hypothermia, presumably to decrease energy metabolism. Under the assumption of the positive link between aerobic metabolism and reactive oxygen species, it is reasonable to suggest that low body temperature, a proxy of energy metabolism, will affect oxidative stress of the birds. Aging may considerably affect behavior, performance and physiology in birds and still requires further investigation to understand age-specific changes along the lifespan of the organism. Until today, age-specific rest-phase hypothermic responses and their effect on oxidant-antioxidant status have never been investigated. We exposed 25 zebra finches (Taeniopygia guttata) of three age classes, 12 young birds (1.1-1.3 years old), 8 middle-aged (2.4-2.8 years old), and 5 old birds (4.2-7.5 years old) to day-long food deprivation or provided them normal access to food under thermoneutral conditions. We compared night-time body temperature, measured through implanted data loggers, and quantified plasma oxidative status (uric acid, antioxidant capacity, and d-ROM assay) the following morning. We found age-related differences in night-time body temperature following a day-long food deprivation while all three age groups remained normothermic in the night following a day with access to food. The lowest minimum body temperature (LSM ± SE: 36.6 ± 0.2°C) was observed in old individuals during rest-phase hypothermia. Surprisingly, these old birds also revealed the highest levels of plasma oxidative damage, while young and middle-aged birds maintained higher night-time body temperature and showed lower values of oxidative damage. These results lead us to propose a novel hypothesis on how aging may lead to an accumulation of oxidative damage; the impaired physiological capacity to thermoregulate with advancing age does increase the risk of oxidative stress under challenging conditions. When energy is limited, the risk to encounter oxidative stress is increasing via a compensation to defend normothermic body temperatures.

Does selection for behavioral and physiological performance traits alter glucocorticoid responsiveness in bank voles?

One of the key elements of an animal's Darwinian fitness is its ability to adequately respond to and cope with challenging situations. Glucocorticoid hormones, such as corticosterone, affect an organism's ability to overcome such challenges. We hypothesized that changes in the glucocorticoid response curve contribute to the evolution of increased performance during challenging conditions, and tested it on bank voles (Myodes glareolus) from a multidirectional artificial selection experiment, which involves lines selected for high aerobic exercise metabolism achieved during swimming (A - Aerobic), predatory behavior towards a cricket (P - Predatory) and ability to maintain body mass on a low-quality herbivorous diet (H - Herbivorous), as well as unselected control lines (C - Control). We elicited a glucocorticoid response either by restraining the animal or by maximum pharmacological stimulation, and measured plasma corticosterone levels at baseline, during the response and during the recovery phase. Response-level corticosterone was higher in females, and recovery from maximal level was faster than that of males. Selection did not affect baseline or stress-induced corticosterone levels, but it decreased the maximum corticosterone level in Aerobic and Predatory lines, reducing the difference between stress-induced and maximum levels. Recovery from restraint-induced corticosterone level tended to be slower in the Herbivorous than in the other lines, an effect that was stronger in females than in males. In conclusion, successful selection for increased performance in challenging conditions was not associated with changes in absolute values of the glucocorticoid response to stress, but can affect other characteristics of the glucocorticoid response curve.

Dietary antioxidants attenuate the endocrine stress response during long-duration flight of a migratory bird.

Glucocorticoids (GCs) are metabolic hormones that promote catabolic processes, which release stored energy and support high metabolic demands such as during prolonged flights of migrating birds. Dietary antioxidants (e.g. anthocyanins) support metabolism by quenching excess reactive oxygen species produced during aerobic metabolism and also by activating specific metabolic pathways. For example, similar to GCs' function, anthocyanins promote the release of stored energy, although the extent of complementarity between GCs and dietary antioxidants is not well known. If anthocyanins complement GCs functions, birds consuming anthocyanin-rich food can be expected to limit the secretion of GCs when coping with a metabolically challenging activity, avoiding the exposure to potential hormonal detrimental effects. We tested this hypothesis in European starlings (Sturnus vulgaris) flying in a wind tunnel. We compared levels of corticosterone, the main avian GC, immediately after a sustained flight and at rest for birds that were fed diets with or without an anthocyanin supplement. As predicted, we found (i) higher corticosterone after flight than at rest in both diet groups and (ii) anthocyanin-supplemented birds had less elevated corticosterone after flight than unsupplemented control birds. This provides novel evidence that dietary antioxidants attenuate the activation of the HPA axis (i.e. increased secretion of corticosterone) during long-duration flight.

Cellular Turnover: A Potential Metabolic Rate-Driven Mechanism to Mitigate Accumulation of DNA Damage.

Oxidative stress, the imbalance of reactive oxygen species and antioxidant capacity, may cause damage to biomolecules pivotal for cellular processes (e.g., DNA). This may impair physiological performance and, therefore, drive life-history variation and aging rate. Because aerobic metabolism is supposed to be the main source of such oxidative risk, the rate of oxygen consumption should be positively associated with the level of damage and/or antioxidants. Empirical support for such relationships remains unclear, and recent considerations suggest even a negative relationship between metabolic rate and oxidative stress. We investigated the relationship between standard metabolic rate (SMR), antioxidants, and damage in blood plasma and erythrocytes for 35 grass snakes (Natrix natrix). Reactive oxygen metabolites (dROMs) and nonenzymatic antioxidants were assessed in plasma, while two measures of DNA damage and the capacity to neutralize H2O2 were measured in erythrocytes. Plasma antioxidants showed no correlation to SMR, and the level of dROMs was positively related to SMR. A negative relationship between antioxidant capacity and SMR was found in erythrocytes, but no association of SMR with either measure of DNA damage was detected. No increase in DNA damage, despite lower antioxidant capacity at high SMR, indicates an upregulation in other defense mechanisms (e.g., damage repair and/or removal). Indeed, we observed a higher frequency of immature red blood cells in individuals with higher SMR, which indicates that highly metabolic individuals had increased erythrocyte turnover, a mechanism of damage removal. Such DNA protection through upregulated cellular turnover might explain the negligible senescence observed in some ectotherm taxa.

Stress coping and evolution of aerobic exercise performance: corticosterone levels in voles from a selection experiment.

The locomotor performance achieved in a challenging situation depends not only on physiological limitations, such as the aerobic exercise capacity, but also on behavioral characteristics, such as adequate coping with stress. The stress response is mediated largely by the hypothalamic-pituitary-adrenal (HPA) axis, through modulated release of glucocorticoids. We used a unique experimental evolution model system to test the hypothesis that the evolution of an increased aerobic exercise performance can be facilitated by modification of the glucocorticoid-related stress-coping mechanisms. Bank voles (Myodes glareolus) from 'aerobic' (A) lines, selected for 22 generations for high maximum swim-induced rate of oxygen consumption (V̇O2,swim), achieved a 64% higher V̇O2,swim than those from unselected, control lines. The temporal pattern of exercise during the swimming trial also evolved, and the A-line voles achieved V̇O2,swim later in the course of the trial, which indicates a modification of the stress response characteristics. Both V̇O2,swim and the average metabolic rate measured during the trial tended to increase with baseline corticosterone level, and decreased with the post-exercise corticosterone level. Thus, increased baseline corticosterone level promotes high metabolic performance, but a high corticosterone response to swimming acts as an inhibitor rather than stimulator of intense activity. However, neither of the corticosterone traits differed between the A-selected and control lines. Thus, the experiment did not provide evidence that evolution of increased aerobic performance is facilitated by the modification of glucocorticoid levels. The results, however, do not exclude the possibility that other aspects of the HPA axis function evolved in response to the selection.

Experimental evolution of personality traits: open-field exploration in bank voles from a multidirectional selection experiment.

Evolution of complex physiological adaptations could be driven by natural selection acting on behavioral traits. Consequently, animal personality traits and their correlation with physiological traits have become an engaging research area. Here, we applied a unique experimental evolution model-lines of bank voles selected for (A) high exercise-induced aerobic metabolism, (H) ability to cope with low-quality herbivorous diet, and (P) intensity of predatory behavior, that is, traits shaping evolutionary path and diversity of mammals-and asked how the selection affected the voles' personality traits, assessed in an open field test. The A- and P-line voles were more active, whereas the H-line voles were less active, compared those from unselected control lines (C). H-line voles moved slower but on more meandering trajectories, which indicated a more thorough exploration, whereas the A- and P-line voles moved faster and on straighter trajectories. A-line voles showed also an increased escape propensity, whereas P-line voles tended to be bolder. The remarkable correlated responses to the selection indicate a common genetic underlying mechanism of behavioral and physiological traits, and support the paradigm of evolutionary physiology built around the concept of correlated evolution of behavior and physiology.

The effect of monoamines reuptake inhibitors on aerobic exercise performance in bank voles from a selection experiment.

Exercise performance depends on both physiological abilities (e.g., muscle strength) and behavioral characteristics (e.g., motivation). We tested the hypothesis that evolution of increased aerobic exercise performance can be facilitated by evolution of neuropsychological mechanisms responsible for motivation to undertake physical activity. We used a unique model system: lines of bank voles Myodes glareolus selected for high swim-induced aerobic metabolism ("aerobic" A lines). In generation 21, voles from the 4 A lines achieved a 57% higher "voluntary maximum" swim-induced aerobic metabolism (VO2swim) than voles from 4 unselected, "control" C lines. In C lines, VO2swim was 9% lower than the maximum forced-exercise aerobic metabolism (VO2run; P = 0.007), while in A lines it was even higher than VO2run, although not significantly (4%, P = 0.15). Thus, we hypothesized that selection changed both the aerobic capacity and the neuronal mechanisms behind motivation to undertake activity. We investigated the influence of reuptake inhibitors of dopamine (DARI), serotonin (SSRI), and norepinephrine (NERI) on VO2swim. The drugs decreased VO2swim both in C and A lines (% decrease compared with saline: DARI 8%, P < 0.001; SSRI 6%, P < 0.001; NERI 8%, P < 0.001), but the proportional response differed between selection directions only for NERI (stronger effect in C lines: P = 0.008) and the difference was marginally non-significant for SSRI (P = 0.07) and DARI (P = 0.06). Thus, the results suggest that all the 3 monoamines are involved in signaling pathways controlling the motivation to be active and that norepinephrine could have played a role in the evolution of increased aerobic exercise performance in our animal model.

Is Experimental Evolution of an Increased Aerobic Exercise Performance in Bank Voles Mediated by Endocannabinoid Signaling Pathway?

The level of physical activity achieved in a given situation depends on both physiological abilities and behavioral characteristics (motivation). We used a unique animal model to test a hypothesis that evolution of an increased aerobic exercise performance can be facilitated by evolution of motivation to undertake physical activity, mediated by brain endocannabinoid system. Bank voles (Myodes glareolus) from "aerobic" A lines selected for 22 generations for high swim-induced aerobic metabolism (VO2swim) achieved 65% higher "voluntary maximum" VO2swim than voles from unselected, "control" C lines. In C lines, VO2swim was 24% lower than the maximum forced-running aerobic metabolism (VO2run), while in A lines VO2swim and VO2run were practically the same. Thus, the selection changed both the aerobic capacity and motivation to exercise at the top performance level. We applied a pharmacological treatment manipulation to test a hypothesis that the endocannabinoid signaling pathway 1) affects the voles performance in the aerobic exercise trials, and 2) has been modified in the selection process. Administration of the CB1 receptor antagonist (Rimonabant) did not affect the level of metabolism, but administration of the endocannabinoid reuptake inhibitor (AM404) decreased VO2swim both in A and C lines (4%, p = 0.03) and tended to decrease VO2run (2%, p = 0.07). The significant effect of AM404 suggests the involvement of endocannabinoids in signaling pathways controlling the motivation to be active. However, the response to AM404 did not differ between A and C lines (interaction effect, p ≥ 0.29). Thus, the results did not provide a support to the hypothesis that modifications of endocannabinoid signaling have played a role in the evolution of increased aerobic exercise performance in our experimental evolution model system. SUMMARY STATEMENT: The results corroborated involvement of endocannabinoids in the regulation of physical activity, but did not support the hypothesis that modification of endocannabinoid signaling played a role in the evolution of increased aerobic exercise performance in our experimental evolution model.

More than just the numbers-contrasting response of snake erythrocytes to thermal acclimation.

Acclimation to lower temperatures decreases energy expenditure in ectotherms but increases oxygen consumption in most endotherms, when dropped below thermoneutrality. Such differences should be met by adjustments in oxygen transport through blood. Changes in hematological variables in correspondence to that in metabolic rates are, however, not fully understood, particularly in non-avian reptiles. We investigated the effect of thermal acclimation on a snake model, the grass snakes (Natrix natrix). After 6 months of acclimation to either 18 °C or 32 °C hematocrit, hemoglobin concentration, erythrocyte number, and size were assessed. All variables revealed significantly lower values under warm compared to cold ambient temperature. Our data suggest that non-avian reptiles, similarly as birds, reduce erythrocyte fraction under energy-demanding temperatures. Due to low deformability of nucleated erythrocytes in sauropsids, such reduced fraction may be important in decreasing blood viscosity to optimize blood flow. Novel findings on flexible erythrocyte size provide an important contribution to this optimization process.