Toward Transparency and Trust: Assessing and Addressing Key Ethical Concerns in Normothermic Regional Perfusion.

Normothermic Regional Perfusion, or NRP, is a method of donated organ reperfusion using cardiopulmonary bypass or a modified extracorporeal membrane oxygenation (ECMO) circuit after circulatory death while leaving organs in the dead donor's corpse. Despite its potential, several key ethical issues remain unaddressed by this technology.

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.

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.

Integrated molecular and behavioural data reveal deep circadian disruption in response to artificial light at night in male Great tits (Parus major).

Globally increasing levels of artificial light at night (ALAN) are associated with shifting rhythms of behaviour in many wild species. However, it is unclear whether changes in behavioural timing are paralleled by consistent shifts in the molecular clock and its associated physiological pathways. Inconsistent shifts between behavioural and molecular rhythms, and between different tissues and physiological systems, disrupt the circadian system, which coordinates all major body functions. We therefore compared behavioural, transcriptional and metabolomic responses of captive great tits (Parus major) to three ALAN intensities or to dark nights, recording activity and sampling brain, liver, spleen and blood at mid-day and midnight. ALAN advanced wake-up time, and this shift was paralleled by advanced expression of the clock gene BMAL1 in all tissues, suggesting close links between behaviour and clock gene expression across tissues. However, further analysis of gene expression and metabolites revealed that clock shifts were inconsistent across physiological systems. Untargeted metabolomic profiling showed that only 9.7% of the 755 analysed metabolites followed the behavioural shift. This high level of desynchronization indicates that ALAN disrupted the circadian system on a deep, easily overlooked level. Thus, circadian disruption could be a key mediator of health impacts of ALAN on wild animals.

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.

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.

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.

Duets recorded in the wild reveal that interindividually coordinated motor control enables cooperative behavior.

Many organisms coordinate rhythmic motor actions with those of a partner to generate cooperative social behavior such as duet singing. The neural mechanisms that enable rhythmic interindividual coordination of motor actions are unknown. Here we investigate the neural basis of vocal duetting behavior by using an approach that enables simultaneous recordings of individual vocalizations and multiunit vocal premotor activity in songbird pairs ranging freely in their natural habitat. We find that in the duet-initiating bird, the onset of the partner's contribution to the duet triggers a change in rhythm in the periodic neural discharges that are exclusively locked to the initiating bird's own vocalizations. The resulting interindividually synchronized neural activity pattern elicits vocalizations that perfectly alternate between partners in the ongoing song. We suggest that rhythmic cooperative behavior requires exact interindividual coordination of premotor neural activity, which might be achieved by integration of sensory information originating from the interacting partner.

Environmental cues and dietary antioxidants affect breeding behavior and testosterone of male European starlings (Sturnus vulgaris).

Environmental cues, such as photoperiod, regulate the timing of major life-history events like breeding through direct neuroendocrine control. Less known is how supplementary environmental cues (e.g., nest sites, food availability) interact to influence key hormones and behaviors involved in reproduction, specifically in migratory species with gonadal recrudescence largely occurring at breeding sites. We investigated the behavioral and physiological responses of male European starlings to the sequential addition of nest boxes and nesting material, green herbs, and female conspecifics and how these responses depend on the availability of certain antioxidants (anthocyanins) in the diet. As expected, cloacal protuberance volume and plasma testosterone of males generally increased with photoperiod. More notably, testosterone levels peaked in males fed the high antioxidant diet when both nest box and herbal cues were present, while males fed the low antioxidant diet showed no or only a muted testosterone response to the sequential addition of these environmental cues; thus our results are in agreement with the oxidation handicap hypothesis. Males fed the high antioxidant diet maintained a constant frequency of breeding behaviors over time, whereas those fed the low antioxidant diet decreased breeding behaviors as environmental cues were sequentially added. Overall, sequential addition of the environmental cues modulated physiological and behavioral measures of reproductive condition, and dietary antioxidants were shown to be a key factor in affecting the degree of response to each of these cues. Our results highlight the importance of supplementary environmental cues and key resources such as dietary antioxidants in enhancing breeding condition of males, which conceivably aid in attraction of high quality females and reproductive success.

Neuromuscular mechanisms of an elaborate wing display in the golden-collared manakin (Manacus vitellinus).

Many species perform elaborate physical displays to court mates and compete with rivals, but the biomechanical mechanisms underlying such behavior are poorly understood. We address this issue by studying the neuromuscular origins of display behavior in a small tropical passerine bird, the golden-collared manakin (Manacus vitellinus). Males of this species court females by dancing around the forest floor and rapidly snapping their wings together above their back. Using radio-telemetry, we collected electromyographic (EMG) recordings from the three main muscles that control avian forelimb movement, and found how these different muscles are activated to generate various aspects of display behavior. The muscle that raises the wing (supracoracoideus, SC) and the primary muscle that retracts the wing (scapulohumeralis caudalis, SH) were activated during the wing-snap, whereas the pectoralis (PEC), the main wing depressor, was not. SC activation began before wing elevation commenced, with further activation occurring gradually. By contrast, SH activation was swift, starting soon after wing elevation and peaking shortly after the snap. The intensity of this SH activation was comparable to that which occurs during flapping, whereas the SC activation was much lower. Thus, light activation of the SC likely helps position the wings above the back, so that quick, robust SH activation can drive these appendages together to generate the firecracker-like snap sonation. This is one of the first looks at the neuromuscular mechanisms that underlie the actuation of a dynamic courtship display, and it demonstrates that even complex, whole-body display movements can be studied with transmitter-aided EMG techniques.

Vocal exchanges during pair formation and maintenance in the zebra finch (Taeniopygia guttata).

BACKGROUND: Pair compatibility affects the success of a pair; however, its causes and mechanisms are not fully understood. Vocal exchange may be very important for pair formation, coordinating pair activities, maintaining the pair bond and mate guarding. To investigate the role of vocal exchange in pair formation and pair maintenance, we explored whether new and established pairs of zebra finches differed in their calling relationships. We used individualised backpack microphones to examine the entire daily vocal emission of pairs, with parallel video recording of behaviour. RESULTS: We found that in non-breeding, isolated pairs, a specific type of call, the "stack call", was the most common. Furthermore, all pairs used the stack call for precisely timed antiphonal exchange. We confirmed a difference between new and established pairs in social behaviour, with the former spending less time in physical contact. Notably, we found that this was mirrored by a difference in calling behaviour: members of new pairs converged over time on a more symmetric calling relationship. Additionally, we observed different response rates to partners among individuals, but a repeatable relationship of answering within pairs, which may reflect different degrees of motivation to answer the partner. CONCLUSIONS: Our findings show that there is plasticity in calling behaviour and that it changes during pair formation, resulting in a coordinated stack call exchange with a similar number of answers between partners once the pair is established. It is possible that some of the calling relationship measurements that we present reflect pair compatibility.

Natural melatonin fluctuation and its minimally invasive simulation in the zebra finch.

Melatonin is a key hormone in the regulation of circadian rhythms of vertebrates, including songbirds. Understanding diurnal melatonin fluctuations and being able to reverse or simulate natural melatonin levels are critical to investigating the influence of melatonin on various behaviors such as singing in birds. Here we give a detailed overview of natural fluctuations in plasma melatonin concentration throughout the night in the zebra finch. As shown in previous studies, we confirm that "lights off" initiates melatonin production at night in a natural situation. Notably, we find that melatonin levels return to daytime levels as early as two hours prior to the end of the dark-phase in some individuals and 30 min before "lights on" in all animals, suggesting that the presence of light in the morning is not essential for cessation of melatonin production in zebra finches. Thus, the duration of melatonin production seems not to be specified by the length of night and might therefore be less likely to directly couple circadian and annual rhythms. Additionally, we show that natural melatonin levels can be successfully simulated through a combination of light-treatment (daytime levels during subjective night) and the application of melatonin containing skin-cream (nighttime levels during subjective day). Moreover, natural levels and their fluctuation in the transition from day to night can be imitated, enabling the decoupling of the effects of melatonin, for example on neuronal activity, from sleep and circadian rhythmicity. Taken together, our high-resolution profile of natural melatonin levels and manipulation techniques open up new possibilities to answer various melatonin related questions in songbirds.

Zebra finch mates use their forebrain song system in unlearned call communication.

Unlearned calls are produced by all birds whereas learned songs are only found in three avian taxa, most notably in songbirds. The neural basis for song learning and production is formed by interconnected song nuclei: the song control system. In addition to song, zebra finches produce large numbers of soft, unlearned calls, among which "stack" calls are uttered frequently. To determine unequivocally the calls produced by each member of a group, we mounted miniature wireless microphones on each zebra finch. We find that group living paired males and females communicate using bilateral stack calling. To investigate the role of the song control system in call-based male female communication, we recorded the electrical activity in a premotor nucleus of the song control system in freely behaving male birds. The unique combination of acoustic monitoring together with wireless brain recording of individual zebra finches in groups shows that the neuronal activity of the song system correlates with the production of unlearned stack calls. The results suggest that the song system evolved from a brain circuit controlling simple unlearned calls to a system capable of producing acoustically rich, learned vocalizations.

Behavioural and physiological effects of population density on domesticated Zebra Finches (Taeniopygia guttata) held in aviaries.

Zebra Finches (Taeniopygia guttata) are highly social and monogamous birds that display relatively low levels of aggression and coordinate group life mainly by means of vocal communication. In the wild, small groups may congregate to larger flocks of up to 150-350 birds. Little is known, however, about possible effects of population density on development in captivity. Investigating density effects on physiology and behaviour might be helpful in identifying optimal group size, in order to optimise Zebra Finch wellbeing. A direct effect of population density on development and reproduction was found: birds in lower density conditions produced significantly more and larger (body mass, tarsus length) surviving offspring than birds in high density conditions. Furthermore, offspring in low density aviaries produced slightly longer song motifs and more different syllables than their tutors, whereas offspring in high density aviaries produced shorter motifs and a smaller or similar number of different syllables than their tutors. Aggression levels within the populations were low throughout the experiment, but the number of aggressive interactions was significantly higher in high density aviaries. Baseline corticosterone levels did not differ significantly between high- and low density aviaries for either adult or offspring birds. On day 15 post hatching, brood size and baseline corticosterone levels were positively correlated. On days 60 and 100 post hatching this correlation was no longer present. The results of this study prove that population density affects various aspects of Zebra Finch development, with birds living in low population density conditions having an advantage over those living under higher population density conditions.

Flexible seasonal timing and migratory behavior: results from stonechat breeding programs.

Rigid schedules of long-distance migrants could be among candidate traits for adaptive migratory syndromes. This prediction was tested on stonechats, passerines that differ widely in migratory behavior and seasonal schedules. Stonechats in Europe are short-distance migrants and multiclutched, whereas African residents and Siberian long-distance migrants usually raise single broods. In captivity, all subspecies displayed endogenous cycles of reproductive development and molt. The subspecies differed in time afforded to life cycle stages. Under conducive aviary conditions, African stonechats were multibrooded, whereas Siberian stonechats did not add clutches. This difference in flexibility was exclusively related to the length of breeding windows. Stonechats also differed in premigratory preparations. Postjuvenile molt started early in Siberian stonechats, but in European and African stonechats, depended strongly on hatching date. In contrast, all subspecies shortened molt duration at the same rate when hatched from late broods. Plasticity of Zugunruhe timing was identical in Siberian and European subspecies and nearly compensated for hatching late. The stonechat data suggest a refined understanding of temporal plasticity in long-distance migrants. Overall, plasticity was not reduced, but was differently organized. Apparently rigid migrant schedules were related to short breeding cycles and inflexible molt onset. Short windows for breeding and juvenile development could provide safety measures for timely departure. Once molt was initiated, temporal plasticity of long-distance migrants matched that of less migratory conspecifics. In addition to adjusting endogenous programs, stonechats differed in implementing them in the field. Modifying the conditions under which programs are expressed may be an efficient way to enhance seasonal plasticity.

Basal metabolic rate, food intake, and body mass in cold- and warm-acclimated Garden Warblers.

We address the question of whether physiological flexibility in relation to climate is a general feature of the metabolic properties of birds. We tested this hypothesis in hand-raised Garden Warblers (Sylvia borin), long-distance migrants, which normally do not experience great temperature differences between summer and winter. We maintained two groups of birds under cold and warm conditions for 5 months, during which their body mass and food intake were monitored. When relatedness (siblings vs. non-siblings) of the experimental birds was taken into account, body mass in cold-acclimated birds was higher than in warm-acclimated birds. BMR, measured at the end of the 5-month temperature treatment, was also higher in the cold- than the warm-acclimated group. Migrant birds thus seem to be capable of the same metabolic cold-acclimation response as has been reported in resident birds. The data support the hypothesis that physiological flexibility is a basic trait of the metabolic properties of birds.